key: cord- - zbp uqf authors: rasche, andrea; saqib, muhammad; liljander, anne m.; bornstein, set; zohaib, ali; renneker, stefanie; steinhagen, katja; wernery, renate; younan, mario; gluecks, ilona; hilali, mosaad; musa, bakri e.; jores, joerg; wernery, ulrich; drexer, jan felix; drosten, christian; corman, victor max title: hepatitis e virus infection in dromedaries, north and east africa, united arab emirates, and pakistan, – date: - - journal: emerg infect dis doi: . /eid . sha: doc_id: cord_uid: zbp uqf a new hepatitis e virus (hev- ) was recently found in dromedaries and human from the united arab emirates. we screened , dromedary samples from pakistan, the united arab emirates, and african countries. hev- is long established, diversified and geographically widespread. dromedaries may constitute a neglected source of zoonotic hev infections. h epatitis e virus (hev) is a major cause of acute hepatitis worldwide ( ) . four hev genotypes belonging to the species orthohepevirus a are commonly found in humans (hev- through hev- ). genotypes and seem to be restricted to humans, whereas genotypes and also occur in domesticated and wild animals. zoonotic transmission by ingestion of contaminated meat, mainly from pigs, is the most likely zoonotic source of infection ( ) . recently, hev sequences were reported from dromedaries sampled in the united arab emirates (uae) in and were classified as a new orthohepevirus a genotype, hev- ( , ) . afterwards a human patient also from the uae who had chronic hepatitis after liver transplantation was shown to carry hev- ( , ) . until now, knowledge on hev- and its zoonotic potential relied on these studies, which provide no insight into the prevalence and distribution of hev- . to determine the geographic distribution of hev- , we conducted a geographically comprehensive study of hev- prevalence in dromedaries by testing , specimens sampled in countries during the past decades. serum and fecal samples were collected from dromedary camels in the uae, somalia, sudan, egypt, kenya, and pakistan during - ( - ). a total of , serum samples and fecal samples were tested for hev rna by using reverse transcription pcr (rt-pcr) as previously described ( ) . seventeen samples were positive for hev rna: ( . %) of , serum samples and ( . %) of fecal samples (table) . positive samples originated from uae, somalia, kenya, and pakistan and dated to (figures , ). viral loads were measured by using real-time rt-pcr ( ) calibrated on the basis of the world health organization international standard for hev rna ( ) . viral rna concentrations ranged from . × to . × iu/g in feces and . × to . × iu/ml in serum. we sequenced a -nt fragment of the rna-dependent rna polymerase gene of all positive samples for phylogenetic analyses. all camel hev clustered in a monophyletic clade with the human hev- sequence (figure ), supporting the classification of camel-associated hev to a separate orthohepevirus a genotype ( ) . african viruses from somalia and kenya formed a monophyletic clade, whereas viruses from uae and pakistan were intermixed ( figure ). distances based on nucleotide identities were calculated for all sequences from this study and reference strain from each orthohepevirus a genotype as defined by smith et al. ( ) . this subset of references comprised genbank accession nos. m (hev- ), m (hev- ), af (hev- ), aj (hev- ), ab (hev- ), ab (hev- ), and kj (hev- ). nucleotide diversity was remarkable among viral sequences from dromedaries, reaching a maximum distance of . %, compared with a maximum distance of . % among all genotypes. the internal distance among the african viruses was . %, compared with . % distance within viruses from uae and pakistan. the african viruses were . %- . % distant from uae and pakistan viruses, which corresponds to the distance threshold of %- % author that separates the prototype hev- sequence from hev- and hev- prototype sequences. this finding suggests that hev- is a strongly diversified clade of viruses that might need to be further subclassified. hev- was recently shown to belong to the same serotype as hev- - ( ) . therefore, we conducted a preliminary serologic analysis with a subset of specimens ( per country) by adapting a human hev elisa (eu-roimmun, lübeck, germany) for application with camel serum. serum was tested at a : dilution. the signalto-noise ratio was optimized by normalizing the optical density (od) of test samples against ods of a reference serum included in every run (online technical appendix figure, http://wwwnc.cdc.gov/eid/article/ / / - -techapp .pdf). for confirmation of elisa results and to determine an appropriate elisa cutoff, we tested samples covering the complete range of od ratios by adapting the recomline immunoblot (mikrogen, neuried, germany). thirtytwo samples reacted against > of the presented antigens and were therefore ranked positive in the immunoblot. all tested samples with elisa od ratios > . were positive by immunoblot, whereas only of tested samples below this value were positive by immunoblot (online technical appendix figure) . subsequently we set an elisa cutoff of . . using this cutoff, we found ( %) of the serum samples originating from all countries were positive (table) , which is comparable with the seroprevalences typically observed in pigs that are known zoonotic reservoirs for hev- in developed countries ( ) . the percentage of elisa-positive serum samples ranged from % in kenya to % in egypt but did not differ significantly among all countries (p = . , yates' χ test). these results suggest a wide occurrence and high prevalence of hev in dromedaries. we investigated hev- infection in dromedaries. the broad spatial extent, the high diversity of hev- in dromedaries, and the detection of hev-rna in a sample collected in suggest a long evolutionary history of hev- in dromedaries. our study has some limitations. first, although most tested dromedaries seemed healthy, no detailed health information from the rna-positive animals was available. second, we studied limited genome fragments that prevented formal classification into genome subtypes ( ) . third, although we used different antibody detection methods, the antibody prevalence in camels should be confirmed by larger studies including virus neutralization studies to determine potential genotype variability. investigations of camelids other than dromedaries could help to further elucidate the geographic and evolutionary origin of hev- . furthermore, other wild or domestic ungulates with close contact to dromedaries could be investigated to assess the host range of hev- . human infection with hev is common in all studied areas ( ). on the basis of clinical observations and hev antibody detection tools, several hev outbreaks mainly linked to water contamination or poor hygienic circumstances have been described for pakistan, sudan, somalia, and egypt. for kenya and uae, data about hev prevalence is scarce ( ) . in large parts of the middle east, human infections are unlikely to be caused by contact with swine or consumption of pork for cultural reasons. even in saudi arabia, where pork is absent in diet, blood donors have antibodies at proportions of up to . % ( ). thus, most hev infections in the middle east are assumed to be caused by nonzoonotic genotypes and . however, our study and previous studies ( ) showed that hev- and other human genotypes form serotype, suggesting a lack of discrimination in seroprevalence studies. the human hev seroprevalence in the middle east region might in fact be caused by hev- infection. furthermore, human hev- infections might contribute to the hev prevalence in all studied areas, where camel products are frequent parts of human diet ( ) . a foodborne transmission scenario is further suggested by the fact that of positive serum in the study was actually sampled in a slaughterhouse, documenting that meat from infected animals can enter the food chain ( ) . detections of hev- rna in feces in this and a previous study ( ) point at feces or feces-contaminated camel products, such as milk, as putative additional sources of human infection. considering the importance of dromedaries as livestock animals ( ) , risk groups, such as slaughterhouse workers, should be screened for hev- infection. orthohepevirus c (genbank accession no. gu ) as an outgroup. the phylogenetic tree was calculated with mega . (http://www.megasoftware.net) by using the neighbor-joining algorithm and a nucleotide percentage distance substitution model. bootstrap values (%) of , repetitive analyses > are shown next to the nodes. new camel hev sequences obtained in this study are in red. scale bar represents the genetic distance. all sequences obtained in this study are deposited in genbank (accession nos. km -km and ku -ku ). uae, united arab emirates. the global prevalence of hepatitis e virus infection and susceptibility: a systematic review new hepatitis e virus genotype in camels, the middle east consensus proposals for classification of the family hepeviridae chronic infection with camelid hepatitis e virus in a liver transplant recipient who regularly consumes camel meat and milk antibodies against mers coronavirus in dromedary camels mers coronavirus neutralizing antibodies in camels antibodies against mers coronavirus in dromedary camels bats worldwide carry hepatitis e virus-related viruses that form a putative novel genus within the family hepeviridae in silico and in vitro interrogation of a widely used hev rt-qpcr assay for detection of the species orthohepevirus a world health organization international standard to harmonize assays for detection of hepatitis e virus rna consensus proposals for classification of the family hepeviridae characterization of self-assembled virus-like particles of dromedary camel hepatitis e virus generated by recombinant baculoviruses age-related and regional differences in the prevalence of hepatitis e virus-specific antibodies in pigs in germany proposed reference sequences for hepatitis e virus subtypes camel meat and meat products wallingford (uk): cab international we thank monika eschbach-bludau, sebastian brünink, and tobias bleicker for providing excellent technical assistance.this study was supported by the european commission (project compare), the german research foundation (project dr / - ). a.l. and v.c.m were supported by the centrum of international migration and development (contract no. ). the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.mrs. rasche is a doctoral student at the institute of virology, bonn, germany. her primary research interests include detection and characterization of novel zoonotic hepatitis viruses. key: cord- -g f bdlp authors: neske, florian; blessing, kerstin; ullrich, franziska; pröttel, anika; kreth, hans wolfgang; weissbrich, benedikt title: wu polyomavirus infection in children, germany date: - - journal: emerg infect dis doi: . /eid . sha: doc_id: cord_uid: g f bdlp nan u of hotstartaq polymerase. the cycling conditions were cycles ( °c for s, °c for s, and °c for min) after a preheating step of min at °c. all pcr products of positive reactions by agarose gel electrophoresis with ethidium bromide staining were sequenced completely in both directions for confi rmation of sequence specifi city. one negative control was extracted and amplifi ed for every npa samples. a plasmid containing the cloned pcr product was used as positive control. the sensitivity of the wupyv pcr was . copies per reaction as determined by probit analysis, which corresponds to copies per ml of sample. the study was approved by the ethics committee of the medical faculty at the university of würzburg. during the study period, , npa of hospitalized children with febrile respiratory tract diseases were received for viral diagnostic evaluation. the median age of the patients was . years (mean age . years; range days- years), and . % were boys. dna of , npa from , children was available for retrospective testing. of these, ( . %) samples from children were positive by wupyv pcr and subsequent sequencing. the median age of the wupyvpositive children was . years (mean . years; range months- . years) ( figure) , and % were boys. of the children with wupyv-positive npa, . % were > years of age, although children in this age group constituted . % of the total population. infections with wupyv were found year round, but most occurred in the winter months. yearly frequencies (july-june) of wupyv-positive results varied from . % to . % during the observation period. these variations were not statistically signifi cant. in ( . %) of the wupyv-positive samples, co-infections with other respiratory viruses were detected, most frequently with adenovirus (n = ) and fl ua (n = ), followed by hbov (n = ) and rsv (n = ). the co-infections bronchitis, wheezing bronchitis, and pneumonia. in the context of the previous reports of wupyv detection in australia and north america ( ), our data suggest a worldwide distribution of wupyv. most of the wupyv-positive children were < years of age, and wupyv dna was rarely found in children > years of age. this age distribution is compatible with wupyv infection occurring in day nurseries and kindergartens. in keeping with the fi ndings of gaynor et al. ( ), we observed a high number of co-infections. the true number of co-infections in our study is probably higher than the reported . % because we did not test for several respiratory pathogens, such as coronaviruses, rhinoviruses, enteroviruses, and the human metapneumovirus. hypotheses to account for the detection of wupyv in respiratory samples include the following: wupyv is a persisting asymptomatic virus that is detected by chance, wupyv is a persisting virus that is reactivated by an infl ammatory process, or wupyv is a predisposing or aggravating factor of respiratory diseases. further studies are necessary to determine whether wupyv is a human pathogen. to the editor: outbreaks of hepatitis e virus (hev) have been documented in many geographic regions and nonindustrialized countries ( - ); they have been primarily associated with fecal contamination of drinking water ( ). in the central african republic (car), economic indicators (car ranks / countries on the united nations development program human development index), political instability, geographic situation, a deteriorating health network, and a very poor epidemiologic surveillance system all contribute to the country's epidemic susceptibility. in july , ministry of health (moh) and médecins sans frontières (msf) teams working in the begoua commune health center, north of car's capital bangui, reported an increased number of patients from the yembi i neighborhood who were showing signs of jaundice and extreme fatigue. patients suspected of having hepatitis e were defi ned as those with clinical jaundice (yellow discoloration of the sclera) and symptoms of malaise, anorexia, abdominal pain, arthralgia, and fever. confi rmed cases were those in which patients' serum samples were positive for hev immunoglobulin (ig) m or igg. initially, pairs of serum and stool samples were collected from jaundiced patients. fecal samples were stored at - °c and sent to the national reference center of enterically transmitted hepatitis, hospital val de grâce (paris, france) for hev marker testing; serum samples were tested at the bangui pasteur institute for yellow fewer (yf) igm by mac-elisa. the hev epidemic was confi rmed by the detection of hev markers: hev igg (enzyme immuno assay, hev, abbott laboratories, abbott park, il, usa), hev igm (abbott laboratories), amplifi cation of rna ( ), and the absence of yf igm. the hev genome was detected in of the fecal samples. genotyping and sequencing showed that one of these was genotype , prevalent in africa; the others were related to genotype (mexico-like) (genbank accession nos. dq , dq ) ( , ). data suggest that the epidemic began in the yembi i neighborhood, then spread to the rest of the begoua commune and fi nally to bangui or surrounding areas (figure) . of suspected hev case-patients recorded in the msf hospital between july and october , , ( %) lived in the begoua commune ( in the yembi i neighborhood). the attack rate for the begoua commune ( , inhabitants) was . %. of suspected case-patients serologically tested for igg and igm anti-hev antibodies, ( %) had igm antibodies, including / pregnant women ( . % of all confi rmed cases). most patients reported jaundice ( . %) and choluria ( . %);other reported symptoms polyomaviruses and human diseases identifi cation of a third human polyomavirus identifi cation of a novel polyomavirus from patients with acute respiratory tract infections frequent detection of bocavirus dna in german children with respiratory tract infections we thank the team of the viral diagnostic laboratory for skillful and dedicated assistance. weissbrich* *university of würzburg, würzburg, germany; and †university hospital of würzburg, würzburg, germany key: cord- -hfnht wa authors: berto, a.; pham, h. a.; thao, t. t. n.; vy, n. h. t.; caddy, s. l.; hiraide, r.; tue, n. t.; goodfellow, i.; carrique-mas, j. j.; thwaites, g. e.; baker, s.; boni, m. f. title: hepatitis e in southern vietnam: seroepidemiology in humans and molecular epidemiology in pigs date: - - journal: zoonoses public health doi: . /zph. sha: doc_id: cord_uid: hfnht wa viral pathogens account for a significant proportion of the burden of emerging infectious diseases in humans. the wellcome trust-vietnamese initiative on zoonotic infections (wt-vizions) is aiming to understand the circulation of viral zoonotic pathogens in animals that pose a potential risk to human health. evidence suggests that human exposure and infections with hepatitis e virus (hev) genotypes (gt) and results from zoonotic transmission. hypothesising that hev gt and gt are circulating in the vietnamese pig population and can be transmitted to humans, we aimed to estimate the seroprevalence of hev exposure in a population of farmers and the general population. we additionally performed sequence analysis of hev in pig populations in the same region to address knowledge gaps regarding hev circulation and to evaluate if pigs were a potential source of hev exposure. we found a high prevalence of hev gt viral rna in pigs ( . % in faecal samples and . % in rectal swabs) and a high hev seroprevalence in pig farmers ( . %) and a hospital-attending population ( . %) in southern vietnam. the hospital population was recruited as a general-population proxy even though this particular population subgroup may introduce bias. the detection of hev rna in pigs indicates that hev may be a zoonotic disease risk in this location, although a larger sample size is required to infer an association between hev positivity in pigs and seroprevalence in humans. emerging infectious diseases have an important impact on human health. it is well established that viruses account for a significant proportion of emerging infections in humans and the majority are have a zoonotic origin, as highlighted by the recent ebola epidemic in africa and the repeated mers cov outbreaks in the middle east. hepatitis e virus (hev) is a public health concern, as it causes an estimated million human infections annually, with over three million symptomatic hev cases and , deaths worldwide (kamar et al., ) . mortality in immunocompromised patients and pregnant women can approach % for those infected with genotype (kamar et al., ) . hev infections are responsible for > % of cases of acute viral hepatitis in endemic countries (kamar et al., ) . hev is generally associated with a self-limiting hepatitis, as the infection clears within months from onset of symptoms. recently, it was observed that infection with hev gt can become chronic in immunocompromised patients, such as organ transplant recipients or those infected with hiv (galante et al., ) . hev is the sole member of the hepeviredae family of the orthohepevirus a genus. it has only one serotype and four genotypes (gts), although a reclassification into seven gts has been proposed (smith et al., ) . of the four genotypes, gt and gt are able to sustain human-to-human transmission and sporadically cause large human outbreaks in endemic regions due to faecal contamination of the water supply. genotypes and are typically zoonotic; infection can occur from eating undercooked pork or venison, via direct exposure to animal faeces, and sporadically through blood transfusion or liver transplantation (berto et al., ; christou & kosmidou, ; lu, li, & hagedorn, ; renou et al., ; shen et al., ; sridhar, lau, & woo, ; tedder et al., ) . there is now evidence that hev gts and infections mainly originate in swine, and local zoonotic transmission of hev has been recorded worldwide (christou & kosmidou, ) . recent initiatives to increase and improve the surveillance for hev disease in humans have shown that it may be more common than hepatitis a; however, the true incidence of hev infections in humans is still not known (kamar et al., ; da silva et al., ) . it has been shown that seroprevalence of hev in human populations varies between % and % in blood donors, depending on the geographical location (dalton et al., ; scotto et al., ; slot et al., ; vollmer et al., ) . furthermore, it is possible that the majority of patients with unexplained and undiagnosed hepatitis may be hev positive as hev is generally not considered as a potential cause especially in developing countries, due to the lack of diagnostic tests (kamar et al., ; da silva et al., ) . the seroprevalence of hev in pigs in various european countries varies between . % and % (berto et al., ; mccreary et al., ) , and antibodies against hev have also been detected in several other species worldwide, such as rabbit, rats, cattle, sheep, goats, chickens and dogs (drexler et al., ; johne et al., ; sun et al., ) . to date, there are only two published studies describing the circulation of hev in vietnam. one reported a hev seroprevalence rate of % in patients with elevated alanine aminotransferase (alt) (buchy et al., ) , while the second reported that nine of human serum samples obtained from patients with acute sporadic hepatitis in hanoi were infected with hev genotype (dieu ngan et al., ) . vietnam, along with other southeast asian countries, is considered a global hotspot for zoonotic infections (jones et al., ) . the wt-vizions (wellcome trust-vietnamese initiative on zoonotic infections) programme is aiming to gather data on the circulation of viral zoonotic pathogens in animals that pose a risk to human health. hypothesising that hev gt and gt are common zoonotic pathogens in vietnam, we aimed to estimate (i) the hev seroprevalence in a hospital-attending population, as a proxy for the general population, (ii) the hev seroprevalence in individuals working in close contact with pigs (farmers, family members of farmers, animal workers, veterinarians and abattoir workers) and (iii) the prevalence of hev infection in pigs. all collected farm samples (faeces/rectal swabs and human plasma) were collected from dong thap province (southern vietnam). furthermore, we retrospectively tested for presence of anti-hev igg in a hospital population sample of , human serum samples collected between and from dong thap. the wt-vizions is a descriptive observational farm-based and community-based study of zoonotic infection, diseases in domestic livestock and pathogens circulating among individuals with significant occupational or residential exposures to domestic livestock, wildlife and/or animal products. the study enrolled cohort members from approximately different sites in dong thap province. the wt-vizions study design aimed at having an average of - cohort members per farm and - cohort members per abattoir/market rabaa et al., ) . selection of dong thap province for this substudy was coordinated with a complementary long-term seroepidemiology study from dong thap provincial hospital (dtph), with residual samples collected from the hospital haematology laboratory; this was part of an ongoing seroepidemiology study being conducted in southern vietnam (boni et al., ; nhat et al., ) . additionally, dong thap province was selected because the high density of domestic livestock and/or farmed wildlife populations, the diversity of local agroecosystems, and pre-existing frameworks for collaborative research between oxford university clinical research unit and sub-departments of preventive medicine and animal health (pmc and sdah, respectively). the vizions study team, working with the sdah, identified the individual farms and abattoirs within dong thap province. the provincial coordinator (pc) of dong thap organised a first site visits to confirm whether inclusion criteria were met. at each study site, the person with primary management responsibility was identified (farmer, farm manager or abattoir manager), informed of the study objectives, and asked to obtain written informed consent to sample animals and human blood. the study sites were included in the study if they fulfilled the following criteria: permission from local authorities (peoples' committee and institutional management bodies), farmers provided written permission, minimum two consenting participants per site and access to a cell phone with texting capacity. sites were excluded if they were difficult to reach, if the participant was less than year of age, or if the participant had any known immunosuppressive condition or ongoing receipt of any immunosuppressive therapy. the study was approved by the university of oxford tropical research ethics committee (oxtrec no. ) and the scientific and ethical committee of the hospital for tropical diseases in ho chi minh city. written informed consent was required from patients and parents or legal guardians if children under age of prior to participation in the study. pig faeces samples from pigs were collected on farms during , while pig rectal swabs were collected in - from farms. from each farm up to individual animals were sampled. as this study was a part of the wt-vizions project, which aimed to assess the circulation of many different pathogens in addition to hev, the number of animals to sample from each species was a function of the relative number of animals of each species present on the farm. for the above reason, a scoring system was developed to assign different weight to each species and calculate the number of individuals to sample. therefore, for each farm between and , pigs were randomly sampled depending on the total number of different animals species present on the farm rabaa et al., ) . the total number of pigs for each farm was not recorded. the age of the pigs varied between weeks and years, but the precise age of the individual sampled pig was not recorded, although the majority of the animals were adult pigs. in all farms, samples of a minimum of g of faeces were collected aseptically in a sterile plastic container and resuspended in ml of viral transport medium (vtm); rectal swabs were placed in tubes containing ml of vtm. the collected samples were maintained at °c (max. hr), subsequently transported to oucru and frozen at − °c until processing. human plasma samples were collected from sampling sites: the farms sampled in - from which pig rectal swabs were tested, additional farms from which no pig rectal swabs were available, three rodent trading sites, two poultry markets, two pheasant trading sites and one abattoir. an average of five human plasma samples were collected from each site ( in total), and the plasma sampling on the farms with pig rectal swabs was concurrent with the pig sampling. at enrolment, participants were asked to consent to a ml whole blood specimen collection ( ml for children aged < years). blood was allowed to clot at room temperature, transported at ambient temperature and centrifuged within hr. berto et al. page zoonoses public health. author manuscript; available in pmc july . europe pmc funders author manuscripts the primary coordinator coordinated the storage and shipment of human specimen materials to oucru laboratory in hcmc. all specimens were separated into three equal aliquots and preserved at − °c until processing. serum samples from the hospital population were collected from dtph from the hospital haematology laboratory, as part of an on-going seroepidemiology study currently being conducted in southern vietnam. in this study, samples are collected at each collection time point (between three and six per year), in order to allow for the lower range of seroprevalences to be inferred accurately; with this design, a % seroprevalence can be inferred with a % confidence interval from . % to . %. in each collection, a minimum of samples were required from each of the age bands - , - and +. for the subsample used in this study, , samples were selected. the sampling design was approximately per year from to , with a : gender ratio. thirty samples were chosen from each of the age bands - , - , - and - . fifteen samples were chosen from the age bands . - and - . the entire sample set is smaller than the intended , as some age groups did not have enough samples. each collected specimen contained approximately . ml of serum. samples for this seroepidemiology study are stored at − °c and transported on dry ice twice yearly to ho chi minh city where they are stored at − °c as part of a central serum bank for southern vietnam (boni et al., ; nhat et al., ; thuy et al., ) . plasma from the farmers and farmers' family members, veterinarians, animal workers and abattoir workers (n = , farms plus eight other sites) and serum from the hospital population (n = , ) from dong thap (figure ) were screened for anti-hev antibody (igg) using the wantai elisa kit (sanbio, china), following the manufacturer's instructions (izopet et al., ) . all faecal samples and rectal swabs from pigs were screened for hev rna using quantified pcr (qpcr) to detect hev rna as described by jothikumar, cromeans, robertson, meng, and hill ( ) . the qpcr-positive samples were amplified using a nested pcr targeting a bp fragment of orf as previously described by li et al. ( ) . all pcr amplicons ( bp) were visualised on % agarose gels under ultraviolet (uv) light and sequenced by sanger sequencing using an abi sequencer in both forward and reverse directions using the primers from the second round nested pcr. the hev sequences were aligned with a subset of publicly available reference sequences from all seven recently new proposed classification of hev genotypes (smith et al., ) , yielding a -reference sequences data set (both orf and whole genome) and vietnamese sequence data set from pigs ( bp). all sequences were aligned with mega . maximum-likelihood phylogeny was inferred using raxml v . . (stamatakis, ) with bootstrap replicates. the tree was visualised and annotated using figtree (version . . ). a standard loess curve (local polynomial regression fitting; smoothing parameter = . ) was used to construct an age-seroprevalence curve with % confidence intervals. a spearman rank correlation coefficient was used to determine the correlation between the hev rna-positive pigs and the hev seropositivity of farmers. all confidence intervals in the text are presented as exact binomial confidence intervals. the figure ). all sequences obtained from the hev pig samples (genbank accession numbers from kx to kx ) were found to belong to gt and clustered into three main clades ( figure ). all vietnamese pig sequences clustered separately from the previously identified gt sequences circulating worldwide and the subgenotypes did not cluster with any previously known subgenotype (figure ). the anti-hev igg unadjusted seroprevalence was lower in the farmer cohort ( . %; % ci: . %- . %) than the general population ( . %, % ci: . %- . %) in dong thap (table , figure ). there was no difference in seroprevalence by gender in either sample set. although the seroprevalence in both populations increased with age, the prevalence of anti-hev igg was higher in children in the hospital population than in children enrolled in the farm cohort study (figure ). breaking the comparisons into year-age bands, the hospital population had higher hev seroprevalence for individuals aged - (both p < . ; one-way test on elisa optical density); hev seroprevalence was indistinguishable between the two groups when considering individuals aged > years (all p > . ) (figure ). hev seroprevalence did not vary through time in the hospital population cohort. due to the small sample size (n = ) of farms with both molecular diagnostics on pigs and farmer plasma samples, there was insufficient statistical power to determine if there was a positive correlation between hev rna-positive pigs and seropositive farmers (spearman pvalue = . ). this study aimed to measure the prevalence of faecal hev shedding on pig farms and the seroprevalence of hev in the human population in southern vietnam (dong thap province). we report an observed prevalence of hev rna in pigs in southern vietnam that is similar to countries in europe and asia (liu et al., ) . the hev prevalence observed in pig rectal swab samples was . %, which is lower in comparison with previous studies, but the sensitivity of pcr screening is considerably lower using rectal swabs than faecal samples. we additionally found that all sequences obtained from pig samples were hev gt . despite all samples being collected in the same province, these data show that hev sequences clustered into three distinct clades within gt , and the clusters were not associated with the farm or year of collection. further, although the sequences clustered with hev gt , they did not cluster with previously known subgenotypes. these data confirm the substantial genetic variability that exists within the various hev genotypes (cao, dianjun, & meng, ) . the hev seroprevalence identified in the farmer cohort ( . %) was comparable to other asian and european countries (drexler et al., ; johne et al., ; sun et al., ) . the retrospective serology from the general population identified an unadjusted seroprevalence of . %. despite using the same elisa as other studies, this figure is lower when compared to previous data from vietnam ( %) and other endemic regions such as nepal ( . %) and bangladesh ( . %) (izopet et al., ; tran et al., ) ; nevertheless, the results show that a large number of individuals in southern vietnam are exposed to hev. while the participants studied in the farmer cohort were healthy individuals, the samples from the "general-population proxy" are best described as a sample from the potentially hospital-attending population. the same type of comparison is performed in other epidemiological studies where blood donors, for logistic reasons (e.g., easier recruitment), are recruited as a general-population proxy even though this particular population subgroup may introduce bias. as our study assessed the hev seroprevalence in the hospital-attending population, it is important to highlight this distinction because some socioeconomic or occupational groups may have more reason to attend a hospital than others. thus, these samples may represent individuals that have higher disease risk or vulnerability, to hev or to infections in general, and hence may not be representative of the population at large. in particular, children in this sample are likely to be more vulnerable than children in a true general-population sample. the observed low seroprevalence in young children from the farmer cohort is in agreement with previous published data from endemic and non-endemic countries, such as nepal, bangladesh and france (izopet et al., ) , contrary to what was observed in the same age group in the hospital population sample in dong thap. despite not knowing the exact representativeness of the general-population samples, we believe that the results of this study provide unique and valuable insights into the epidemiology of hev in the south of vietnam that could be used to investigate associations of hev seroprevalence and putative risk factors by a nationwide cross-sectional study. in particular, despite hev gt being a zoonotic infection, a higher seroprevalence was not found in adult farmers than in other adults. this suggests that additional hev risk factors need to be identified and evaluated. confounders associated with the high hev seroprevalence detected in both farmers and the hospital-attending population may be due to other risk factors rather than just direct contact with pigs. one potential risk factor might be consumption of internal pig organs, which is common in vietnam. other risk factors associated with high seroprevalence in human may be due to household flooding, drinking contaminated water or poor household hygiene. future population-based studies, using random controls chosen by geographic area, should be performed to confirm or contradict these results. farmers do not have a higher hev seroprevalence than adults in the dtph sample; however if they did, the relative risk of zoonotic hev genotypes (i.e., gt or gt ) would be impossible to estimate using this seroprevalence assay. our initial hypothesis was that individuals living on pig farms would have higher hev seroprevalence than the general population. we did not find evidence to support this hypothesis. however, our study had certain limitations; first no viral characterisation was performed in the two human cohorts, as no individuals presented with symptoms of liver diseases or jaundice during the study. therefore, we could not link human hev genotypes to pig hev genotypes; only presence of anti-hev igg was measured, and direct evidence of hev zoonotic infection was not identified. furthermore, it was not possible to determine the genotype of hev exposure in humans because hev has only one serotype, and the elisa cannot distinguish the different gts. our farm-based individuals work and live in close contact with pigs, and dong thap is a semi-rural region with a high density of pig farms; therefore, we speculate that the majority of the individual anti-hev igg-positive individuals have been exposed to hev gt rather than other hev genotypes (only hev gt was identified in the pigs in our sample). in conclusion, this study demonstrates for the first time that hev is circulating in the pig population in southern vietnam, and also that the human population in the same location has high seroprevalence to hev. therefore, this study provides data that may be used for hev risk factor assessment to prevent and reduce hev infection and transmission. more studies, incorporating testing blood donors or an alternative healthy general-population sample, are required to better understand hev dynamics in human and pig populations in vietnam in order to assess the implication for human health. seroprevalence in both general population and framers cohort groups analysed in this study. the graphs represent the total seroprevalence observed in the general population (left panel) and in the farmer cohort (right panel) stratified by age. y-axis shows the seroprevalence percentage of people anti-hev igg positive, while the x-axis indicates the age groups; individuals were grouped into -year-age bands for maximum informativeness and clarity. the red line shows the inferred seroprevalence curve, and the pink shaded area shows the % confidence band. the grey dots show the seroprevalence for each -year-age group, and the size of the dot is proportional to the sample size. prevalence and transmission of hepatitis e virus in domestic swine populations in different european countries hepatitis e virus in pork liver sausage, france. emerging infectious diseases population-level antibody estimates to novel influenza a/h n prevalence of hepatitis a, b, c and e virus markers among patients with elevated levels of alanine aminotransferase and aspartate amino-transferase in phnom penh (cambodia) and nha trang (central vietnam) molecular biology and replication of hepatitis e virus the baseline characteristics and interim analyses of the high-risk sentinel cohort of the vietnam initiative on zoonotic infections (vizions). regional animal health laboratory hepatitis e virus in the western world -a pork-related zoonosis. clinical microbiology and infection: the official publication of the european society of clinical microbiology and infectious diseases autochthonous hepatitis e in southwest england: natural history, complications and seasonal variation, and hepatitis e virus igg seroprevalence in blood donors, the elderly and patients with chronic liver disease first report of human psittacosis in vietnam bats worldwide carry hepatitis e virus-related viruses that form a putative novel genus within the family hepeviridae relevance of chronic hepatitis e in liver transplant recipients: a real-life setting adhikary d. hepatitis e virus seroprevalence in three hyperendemic areas: nepal, bangladesh and southwest france detection of a novel hepatitis elike virus in faeces of wild rats using a nested broad-spectrum rt-pcr global trends in emerging infectious diseases broadly reactive one-step real-time rt-pcr assay for rapid and sensitive detection of hepatitis e virus design and application of a set of universal pcr primers for genotyping of hepatitis e virus. bing du xue bao. chinese journal of virology/ [bian ji, bing du xue bao bian ji wei yuan hui seroprevalence and molecular characteristics of hepatitis e virus in household-raised pig population in the philippines phylogenetic analysis of global hepatitis e virus sequences: genetic diversity, subtypes and zoonosis papers & articles excretion of hepatitis e virus by pigs of different ages and its presence in slurry stores in the united kingdom structure of generalpopulation antibody titer distributions to influenza a virus the vietnam initiative on zoonotic infections (vizions): a strategic approach to studying emerging zoonotic infectious diseases possible zoonotic transmission of hepatitis e from pet pig to its owner global policy report on the prevention and control of viral hepatitis in who member states seroprevalence of hepatitis e virus among blood donors in a district of southern italy cloning of full genome sequence of hepatitis e virus of shanghai swine isolate using race method prevalence of hepatitis e virus antibodies in individuals exposed to swine in mato grosso, brazil. memórias do instituto oswaldo cruz silent hepatitis e virus infection in dutch blood donors proposed reference sequences for hepatitis e virus subtypes hepatitis e: a disease of reemerging importance raxml version : a tool for phylogenetic analysis and post-analysis of large phylogenies genetic identification of avian hepatitis e virus (hev) from healthy chicken flocks and characterization of the capsid gene of avian hev isolates from chickens with hepatitis-splenomegaly syndrome in different geographical regions of the united states hepatitis e risks: pigs or blood-that is the question tetanus in southern vietnam: current situation prevalence of hepatitis virus types b through e and genotypic distribution of hbv and hcv novel approach for detection of hepatitis e virus infection in german blood donors key: cord- -xq iq ai authors: frossard, jean-pierre; grierson, sylvia; cheney, tanya; steinbach, falko; choudhury, bhudipa; williamson, susanna title: uk pigs at the time of slaughter: investigation into the correlation of infection with prrsv and hev date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: xq iq ai hepatitis e virus (hev) and porcine reproductive and respiratory syndrome virus (prrsv) and are both globally prevalent in the pig population. while hev does not cause clinical disease in pigs, its zoonotic potential has raised concerns in the food safety sector. prrs has become endemic in the united kingdom (uk) since its introduction in , and continues to cause considerable economic losses to the swine industry. a better understanding of the current prevalence and diversity of prrsv and hev in the uk, and their potential association, is needed to assess risks and target control measures appropriately. this study used plasma, tonsil, and cecal content samples previously collected from pigs in abattoirs in england and northern ireland to study the prevalence of several pathogens including prrsv and hev. the diversity of prrsv strains detected in these samples was analyzed by sequencing open reading frame (orf ), revealing no substantial difference in prrsv strains from these clinically unaffected pigs relative to those from clinical cases of disease in the uk. despite the potential immuno-modulatory effect of prrsv infection, previously demonstrated to affect salmonella and hev shedding profiles, no significant association was found between positive prrsv status and positive hev status. hepatitis e virus (hev) is the cause of hepatitis e in humans, typically a self-limiting hepatitis but more serious in those with pre-existing liver conditions and in the immunocompromised [ ] . in pigs, hev infection alone does not cause clinical disease. hev genotypes hev- and hev- are the cause of sporadic cases of hepatitis e in developed countries, and are ubiquitous in the pig population worldwide [ ] . hepatitis e is a foodborne zoonosis, for which pork or pork products from infected pigs is one of the risks identified in europe [ , ] and consumption of processed pork products in the united kingdom (uk) has been shown to be associated with an increased risk of acquiring hev [ ] . hence, there is a need to better understand factors influencing hev entering the food chain. porcine reproductive and respiratory syndrome virus (prrsv) was first confirmed in the uk in and is now considered endemic [ ] . the economic and welfare impacts of the disease are considerable, as both the breeder and grower segments of the pig industry are affected [ ] . all prrsv infections in the uk characterized to date have been identified as being caused by genotype virus, but the genetic diversity of the virus is continually increasing [ ] . the phylogenetic analyses of uk prrsv sequences have previously been based on data from samples submitted for diagnostic purposes, originating from clinical cases of prrs, thereby possibly introducing a bias in our coverage of circulating strains. it is therefore possible that prrsv strains circulating in apparently healthy pigs in the uk may represent a different subset from those causing disease. prrsv infection has been suggested to modulate pig immune responses, thereby rendering pigs more susceptible to other infections [ ] [ ] [ ] . for example, previous studies have shown significant associations between prrsv presence and salmonella shedding [ ] . salines et al. [ ] reported that experimental co-infection of hev and prrsv affected the dynamics of hev infection. however, a direct immune-regulation in infected pigs could not be confirmed for genotype prrsv [ ] , rather suggesting a role for co-infection viruses influencing each other more directly. moreover, less pathogenic strains of genotype prrsv seem to cause a more persistent infection than highly pathogenic ones which are better resolved by the immune response [ ] . notably, viruses closely related to the modified-live vaccine used in the uk have previously been found circulating on farms with clinical prrs [ ] . in , an abattoir-based study was undertaken to estimate the prevalence of various pathogens including hev and prrsv in uk-reared pigs at slaughter and seroprevalence for prrsv was . % ( / ) [ ] , while hev seroprevalence was . % ( / ). approximately . % of pigs were hev viremic ( / ) and around one in five pigs had evidence of an active hev infection ( / ), with hev rna detected in serum or cecal contents [ ] . to follow up these studies, we report here on ( ) the investigation of prrsv active infection (rna in tonsil) using the same abattoir survey sample-set and ( ) an analysis of the correlation of prrsv and hev infection in these pigs, which could be of significance for the control of both diseases and in informing farming practices for reducing hev in the food chain. the overall study design for the abattoir survey has been described previously [ ] . briefly, qualifying pigs were sampled at abattoirs in england and northern ireland, between january and may . these pigs originated from farms, with between and pigs from each. the majority of pigs were from farms in england ( . %), followed by northern ireland ( . %), scotland ( . %), and wales ( . %), which is representative of the uk pig population [ ] . of the samples collected from each selected carcass along the processing line, those relevant for the investigation of hev and prrsv were a blood sample (whole blood with ethylenediaminetetraacetic acid (edta)), tonsil, and the whole cecum. antibody to prrsv had been detected in of plasma samples, with additional samples being inconclusive [ ] . only of these plasma samples were also analyzed for the presence of antibodies to hev, with of these being sero-positive for prrsv. tonsil samples from all seropositive or inconclusive pigs were then tested by a real-time reverse transcription polymerase chain reaction (rt-pcr) to detect prrsv nucleic acid. the tonsils of four other pigs for which plasma samples-and hence enzyme-linked immunosorbent assay (elisa) results-were missing were also tested by pcr, while for five seropositive animals the tonsil samples were not available, so a total of tonsil samples were analyzed; only of these animals also had matching hev pcr data. rna was extracted from the tonsil samples using the magna pure lc dna isolation kit ii (tissue) following the manufacturer's instructions (roche diagnostics, burgess hill, uk), setting the sample volume to µl and the elution volume to µl. a real-time rt-pcr assay targeting the nucleocapsid (orf ) gene of prrsv and differentiating genotypes and was used [ ] with a stratagene mx p qpcr system (agilent genomics, wokingham, uk). sequencing of the prrsv open reading frame (orf ) gene was subsequently performed on nucleic acids from tonsil samples testing positive in the diagnostic prrsv pcr to characterize the viruses present [ ] . the pcr amplicons were purified using the beckman ampure solid phase reversible immobilisation (spri) technique (beckman coulter ltd., high wycombe, uk). cycle sequencing was performed using forward and reverse primers and abi bigdye chemistry (applied biosystems ltd., warrington, uk) at the end of which the dye terminators were removed using beckman cleanseq spri (beckman coulter ltd., high wycombe, uk). samples were sequenced on an abi capillary electrophoresis dna analyser (applied biosystems ltd., warrington, uk) and the raw data analysed by abi seqscape software (applied biosystems ltd., warrington, uk). the resulting sequences were aligned with reference sequences from genbank, and with other uk prrsv orf sequences from samples submitted to the animal and plant health agency (apha) for prrs diagnostic testing between and , using the clustalw algorithm [ ] in mega [ ] . the phylogenetic tree was generated in mega using the neighbour-joining method [ ] , with the evolutionary distances being computed using the maximum composite likelihood method [ ] . the sequences obtained were deposited in genbank with accession numbers mf to mf . existing data for prrsv seropositivity [ ] and hev seropositivity and active infection [ ] were collated with data obtained in this study for prrsv active infection. associations between the two viruses, or antibodies to them, in the same carcasses were investigated using χ tests, with stata v. (statacorp, college station, tx, usa). collated data for prrsv seropositivity and hev seropositivity and active infection (rna in plasma and/ or cecal contents) were available for pigs. collated data for prrsv active infection (rna in tonsil) and seropositivity and active infection for hev were available for pigs. prrsv rna was detected in of tonsil samples. this corresponds to . % of the prrsv seropositive finisher pigs showing active prrsv infection at slaughter. importantly, all pcr-positive samples were of genotype (european), which is endemic in the uk, and no genotype virus, which is exotic to the uk, was detected. while the elisa-positive pigs tested by pcr originated from farms in counties, pcr-positive pigs were only identified from farms in eight of those counties. almost two-thirds of the pcr-positive pigs were from farms in east anglia or east riding and north lincolnshire. while seropositivity had been found to vary significantly between age groups (p = . ) with the highest level found in pigs aged less than six months ( . %) and lowest in those aged > months ( . %) [ ] , the prevalence of prrsv rna-positive tonsils was similar across the age groups (table ) . sequencing of the orf prrsv gene was undertaken on of the tonsil samples from which prrsv rna was detected. two pcr-positive tonsils were not suitable for sequencing as there was insufficient viral nucleic acid in the samples. six samples did not yield useable sequence data. all of the sequences confirm that the viruses belong to prrsv genotype . only three of the sequences may be considered to possibly originate from the currently licensed attenuated vaccine, with greater than % similarity between the sample and vaccine strain orf sequences ( . %, . %, and %). the phylogenetic trees (figure ) illustrate the genetic diversity of the orf genes from the samples in this study in comparison to the vaccine virus licensed in the uk at the time and published reference sequences representing the different genotypes and subtypes ( figure a ) and in more detail, in the context of previously sequenced viruses specifically from uk pigs between and (unpublished data) ( figure b ). in the within-uk analysis, there is no clear association between geographic origin and the clade in which the prrsv strains belong. all of the sequences are found in clades where other uk strains were already identified, and no distinct clustering is observed. analyses were performed to identify potential associations between prrsv serology or pcr status (prrsv rna detected in tonsil sample) and hev serology or pcr status (hev rna detected in serum or cecal contents). these are summarized in tables and . of the six animals that were pcr positive for both hev and prrsv, one was less than six months old, three were six months of age, and two were greater than six months of age. there was no evidence from this study that prrsv infection, as detected by serology or pcr, increased the likelihood of hev infection in pigs at the time of slaughter. the only associations identified suggested that for the pigs in this study, prrsv seropositive animals were less likely ( . % vs. . %, p = . ) to be hev pcr positive in plasma or cecum (table ) ; prrsv seropositive animals were less likely ( . % vs. . %, p = . ) to be hev pcr positive in plasma (table ) ; prrsv pcr positive animals were less likely ( . % vs. . %, p = . ) to be hev seropositive (table ) . it has been suggested that infection with prrsv renders pigs more susceptible to secondary bacterial [ , , , ] or viral [ , ] infections. in this study, we further investigated the active prrsv infection at slaughter age and the association between prrsv infection and an active hev infection in pigs entering the food chain in the uk. since the majority of uk pig farms that vaccinate against prrsv use a live vaccine [ ] it was considered that prrsv in both the vaccinated and naturally infected pigs may modulate hev infection. the abattoir survey had found a prevalence of antibodies to prrsv in slaughter-age pigs of . % [ ] . antibody to vaccine and field prrsv cannot be distinguished but vaccination of rearing pigs is less common than that of breeding pigs in the uk and is generally performed when there is an expectation of field prrsv challenge during the rearing period. therefore, seropositivity in finishers is considered a reasonable indicator of the presence of prrsv infection on the respective rearing units. from the prrsv seropositive pigs in the study, the prevalence of prrs viral rna in tonsils, where prrsv may persist up to days post infection [ ] , was . %. as tonsils from most seronegative pigs were not tested by pcr, it is possible that detection of a few prrsv-positive pigs in the early stages of infection were missed, although prrsv rna was not detected in any of the tonsils from seronegative pigs in a pilot study (data not shown). as the pigs were not showing obvious clinical signs, this is a significant finding, highlighting that a proportion of healthy pigs from prrsv-infected units may be infectious at slaughter, and if still shedding virus, may be able to transmit the virus on to other farms, for example through contaminated vehicles [ ] , underlining the need for good biosecurity during and after transport to slaughter. these findings triggered the genetic characterization of the viruses in order to further evaluate the potential risks associated with their presence. overall, the sequences from the abattoir samples did not cluster separately from those from clinical cases submitted for diagnostic testing, and they appear to be representative of the overall diversity of prrsv strains circulating in the uk. none of them was from an eastern european subtype of genotype prrsv. of the successfully-sequenced viruses, three showed greater than . % similarity to the modified-live vaccine used in the uk at the time. these 'vaccine-like' viruses may derive from vaccine virus, and have been identified in previous years, although at a lower rate [ ] , possibly because most samples previously sequenced were from disease outbreaks, whereas these were detected in clinically normal pigs. the other viruses showed between . % and . % similarity to the vaccine sequence. although the degree of genetic difference of a field prrs virus from the vaccine strain cannot alone predict the degree of protection that would be afforded by the vaccine to infection by the field virus, nor allow for a determination of the strain's pathogenicity, these results further illustrate the diverse nature of field prrsv in the uk. interestingly, some of the viruses from this study are sufficiently similar to one another to be potentially linked epidemiologically, even when they originate from pigs from different geographic regions. conversely, for two pigs from the same farm, the viruses detected in them did not show a great degree of similarity. there was no relation between four prrsv sequences from animals co-infected with hev (no sequence data was available for the other two), as they each grouped into different clusters in the phylogenetic analysis, one being homologous with the porcilis prrs vaccine strain sequence. the sequences from the abattoir samples did not cluster separately from those from clinical cases submitted for diagnostic testing, and they appear to fit within the overall diversity of prrsv strains previously found to be circulating in the uk. the existence of infected slaughter-age pigs with the potential, if shedding, to transmit the virus to susceptible pigs is thereby confirmed. we found no evidence in this study that exposure to, or infection with prrsv enhanced hev infection rates in pigs entering the food chain. indeed, pigs exposed to prrsv were less likely to be viremic (p = . ). the age-range of the six co-infected pigs identified reflected that of the overall population sampled. the calculated virus load in plasma for hev showed no association with either prrsv serology or pcr status (data not shown). several other studies have specifically investigated prrsv and hev co-infection [ , , ] , some by experimental infection. one report of an association of co-infection with disease was restricted to investigation of a single pig [ ] . martelli et al. [ ] found no association between these pathogens in an investigation of diagnostic submissions. the abattoir survey had investigated the presence of a number of other pathogens in clinically healthy pigs, but no evidence was found that the prrsv-seropositive pigs were more likely to carry salmonella or yersinia or have antibodies to toxoplasma [ ] . interestingly, salines et al. [ ] reported that experimental prrsv and hev co-infection did affect the hev infection dynamics in five-week old pigs with no maternal antibody for these two endemic viruses. the simultaneous co-infection resulted in delays to both the latent period ( . vs. . days with hev alone) and the humoral response ( . vs. . days) to hev, as well as increasing the infectious period ( . vs. . days), in association with an increased hev viral load. in contrast, several other studies have failed to demonstrate any association between prrsv infection and the dynamics of other viral infections [ , , ] . while the present study failed to show any positive association overall between prrsv and hev infections, this may be an age-dependent effect, and variation in the timing of the respective infections may also affect their outcomes. future investigations of natural prrsv and hev co-infections should perhaps be directed towards younger pigs, since these were under-represented in this study, and may provide different outcomes. a field study showed that the majority of pigs were infected by weeks of age [ ] . experimental infections to further characterize prrsv and hev co-infections must consider non-simultaneous infections, as they may be more relevant to the situation in the field. other viral co-infections such as porcine circovirus type (pcv- ) and prrsv or hev also remain to be investigated, with at least one report of fatal disease associated with hev and pcv- [ ] . an active hev infection in pigs entering the food chain is a potential risk to public health and there is a need to better understand factors that may influence this. the uk abattoir survey had found that one in five pigs had an active hev infection as they entered the food chain [ ] . this same finding of active hev infections in slaughter-age pigs is found worldwide [ ] [ ] [ ] [ ] . there was no evidence from the current study that prrsv infection adversely affected the proportion of hev infected pigs entering 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virus in swine and effluent samples from slaughterhouses in brazil genetic characterization and serological prevalence of swine hepatitis e virus in shandong province the authors declare no conflict of interest. the funding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results. key: cord- -hkxlq os authors: anang, saumya; kaushik, nidhi; surjit, milan title: recent advances towards the development of a potent antiviral against the hepatitis e virus date: - - journal: j clin transl hepatol doi: . /jcth. . sha: doc_id: cord_uid: hkxlq os hepatitis e virus (hev) is one of the leading causes of acute viral hepatitis. it also causes acute liver failure and acute-on-chronic liver failure in many patients, such as those suffering from other infections/liver injuries or organ transplant/chemotherapy recipients. despite widespread sporadic and epidemic incidents, there is no specific treatment against hev, justifying an urgent need for developing a potent antiviral against it. this review summarizes the known antiviral candidates and provides an overview of the potential targets for the development of specific antivirals against hev. hepatitis e virus (hev) is a positive-sense, single-strand rna virus that causes acute and chronic viral hepatitis, fulminant hepatitis, acute liver failure and acute-on-chronic liver failure in infected individuals. it is known to be transmitted through the fecal-oral route, transfusion of infected blood products or through the vertical route. [ ] [ ] [ ] [ ] [ ] [ ] zoonotic transmission due to consumption of infected meat products, resulting in sporadic cases, is particularly frequent in developed countries. the disease symptoms include jaundice, nausea, vomiting, fever and sore muscles. though the infection is acute in normal individuals, it becomes chronic in immunocompromised patients, such as organ transplant recipients, individuals infected with the human immunodeficiency virus, and patients undergoing chemotherapy. [ ] [ ] [ ] [ ] [ ] [ ] the disease worsens in pregnancy, with mortality rates reaching as high as to %. , , recent reports have described extra-hepatic manifestations, such as guillain-barre syndrome, neurological amyotrophy, arthritis, pancreatitis and glomerulonephritis, in several hev infected patients. [ ] [ ] [ ] drave et al. have also demonstrated the replication of hev in human neuronal-derived cell lines. out of the eight recognized genotypes of hev, genotypes and were responsible for about . million infections in , including . million symptomatic cases, , fatalities and , still births. outbreaks of hev have been reported from different parts of the world. several parts of eastern and the central india, including orissa, chhattisgarh, maharashtra and nellore, witnessed hev outbreaks between - . [ ] [ ] [ ] [ ] many parts of africa have also been affected by frequent hev epidemics. outbreaks were also reported from egypt, uganda, sudan, ethiopia, chad, niger and kenya. an outbreak has even been reported from australia, caused by a locally-acquired hev. the recent increase in organ transplantation and exposure to the disease due to growing trade and travel has further expanded the incidence of hev infection, thereby intensifying the need for research of antivirals against hev. hev-induced acute hepatitis is usually self-limiting. the disease is generally cured in - weeks, without the need of any medication. during severe acute and chronic infections, a reduction in immunosuppressant dose or administration of pegylated-interferon-alpha (peg-ifn-a), ribavirin or a combination of both is the available therapeutic option. reduction in immunosuppressant dose helps in virus clearance in approximately % of organ transplant patients. ribavirin monotherapy has been found to be effective in the treatment of chronic hev infection. [ ] [ ] [ ] [ ] ribavirin inhibits host inosine monophosphate dehydrogenase, thereby depleting cellular gtp pools and blocking viral replication during hev infection. another possible mechanism of ribavirin action on hev is attributed to its ability to induce mutations in the viral genome (fig. ) . the g r mutation in the hev polymerase increases the replication competence of the hev genome and has been shown to confer resistance against ribavirin treatment in some patients. however, subsequent studies revealed that the g r mutation does not lead to absolute ribavirin resistance. [ ] [ ] [ ] [ ] a major limitation of ribavirin therapy is attributed to the undesirable consequences of the treatment, such as: (i) it's not being a treatment option during pregnancy, owing to its teratogenicity; (ii) its potential to cause hemolytic anemia and decreased hemoglobin, requiring direct supervision of hemolytic parameters during its application; and (iii) its potential to cause insomnia, dyspnea, lack of concentration and irritability. hev enters a permissive cell supposedly through a receptor-dependent process, aided by heparan sulfate proteoglycans (hspgs) and other unknown factors. the viral genome is released, orf gets translated and processed into different functional domains, followed by replication. multiple copies of capped (green box) genomic (g)rna and subgenomic (sg)rnas are thus produced. sgrnas synthesize viral capsid (orf ) and orf proteins. orf , grna and other viral and/or host factors mediate assembly of new virions, which are released out of the cell through an endosomal sorting complex required for transport (escrt)-dependent process involving the viral orf protein. the green asterik indicates the steps that can be targeted for antiviral development. a, b, c and d represent the unknown factors present in the viral replication complex. note that orf is present only in the case of genotype hev. known antivirals have been indicated at the appropriate steps. the mode of pegylated-interferon-alpha (peg-ifn-a) action is represented through the illustration of the inteferonalpha (ifn-a) signaling pathway. peg-ifn-a induces the production of interferon-stimulated proteins (isgs) and interferon-inducible transmemebrane proteins (ifitm), which activate the canonical antiviral signaling pathways that results in the inhibition of hev entry and/or replication. also of note is the fact that although ribavirin treatment for chronic hev-infected organ transplantation recipients is effective in the majority of cases, it does not reach a % success rate. further, in a study involving a chronic hev-infected burkitt's lymphoma patient treated with chemotherapy, months of ribavirin treatment failed to eliminate the hev. peg-ifn-a has been used in patients with liver transplant, kidney transplant, human immunodeficiency virus infection and leukemia who are chronically infected with hev. [ ] [ ] [ ] [ ] the mechanism by which peg-ifn-a clears hev is not clearly understood. however, all the types of ifn, including ifn-a (type i), ifn-g (type ii), and ifn-l (type iii) inhibit hev replication and ifn-a subtypes a and b exert the strongest antiviral activity against hev in mammalian cell culture. hev is also equipped with multiple strategies to restrict the ifn response, leading to moderate and delayed anti-hev effects in vitro and in patients treated with ifn-a. the hev x and papain-like cysteine protease domains inhibit ifn (type i) induction, while hev orf is known to inhibit ifn-a signaling by inhibiting phosphorylation of stat . , interestingly, orf also inhibits phosphorylation and nuclear translocation of stat as well as expression of its target genes in cells treated with epidermal growth factor. further studies using suitable in vivo models should decipher the significance of the crosstalk between host interferon signaling and the viral interferon restriction factors. the common side effect associated with ifn treatment is flu-like symptoms. among the more serious adverse effects are neuropsychiatric disorders, neurologic disturbances, myelosuppression, cardiovascular disorders, altered liver function, renal insufficiency and gastrointestinal manifestations. further, months of treatment with peg-ifn-a- a is reported to result in sustained virolgical response in out of chronic hev-infected liver transplant patients. in summary, peg-ifn-a treatment appears to be a promising therapeutic option against hev infection. nevertheless, additional studies involving large cohorts of patients should provide a better understanding of its therapeutic benefits. several laboratories have been focusing on identifying suitable drug targets and developing antivirals against hev. summarized below is the outcome of recent efforts to identify potent antivirals against hev. inosine monophosphate dehydrogenase is an essential enzyme in the purine biosynthesis pathway. several inosine monophosphate dehydrogenase inhibitors, such as mycophenolic acid, ribavirin and -ethynyl- -b-d-ribofuranosylimidazole- carboxamide, inhibit hev replication. , the combination of mycophenolic acid and ribavirin acts more effectively to inhibit hev replication than mycophenolic acid or ribavirin alone. further, mycophenolate mofetil, a prodrug of mycophenolic acid, exhibited frequent hev clearance in heart transplant patients, providing protection from chronification. dihydroorotate dehydrogenase and orotidine- '-monophosphate decarboxylase are essential enzymes in the pyrimidine biosynthesis pathway. dihydroorotate dehydrogenase inhibitors, such as brequinar, leflunomide and orotidine- '-monophosphate decarboxylase inhibitor -azauracil, also inhibit hev replication in mammalian cell culture models. these compounds deserve further validation as antivirals against hev. '-c-methylcytidine is a nucleoside analogue that efficiently inhibits hev replication in the cell culture system. it was also shown that '-c-methylcytidine retained anti-hev activity even after long-term exposure to the virus, implying its potential use to combat development of drug resistance. however, '-c-methylcytidine showed an antagonistic effect when tested in combination therapy with ribavirin. further in vivo evaluation of this compound should provide insights about its anti-hev effects. sofosbuvir, a prodrug of a uridine nucleoside analogue that acts as a direct-acting antiviral against hepatitis c virus (hcv) rna-dependent rna polymerase in its active form, was reported by dao thi et al. to inhibit hev genotype replication in vitro and to exert additive effect when combined with ribavirin. however, those data were not fully reproducible by wang et al. and, moreover, sofosbuvir treatment failed to clear hev viremia in an immunosuppressed patient with chronic hcv and hev without ribavirin. therefore, usage of sofosbuvir as an anti-hev therapeutic needs further validation (discussed further in the rna-dependent rna polymerase section of this manuscript). the zhang laboratory developed hev-specific ppmos and evaluated their efficacy in inhibiting viral replication. out of the four ppmos tested, ppmo hp was most effective in reducing viral replication in mammalian cell culture. ppmo hp specifically inhibits viral translation by targeting a highly conserved sequence in the start region of orf of genotype and genotype hev. treatment of cells with , and mm of ppmo hp reduced luciferase expression by . %, . % and . %, respectively, in a luciferase reporter based hev replicon system. the antiviral activity of ppmo hp was specific, dose-responsive and potent. hence, its further validation as a potential hev-specific antiviral is warranted. e has been identified as an inhibitor of hev replication in hepatocytes. e inhibits genotype hev replication by ; %, without producing any detectable cytotoxicity. interestingly, e also inhibits hcv and dengue virus replication. the mechanism by which e inhibits viral replication remains to be explored. mg is a cell permeable inhibitor of the host s proteasome complex, which is responsible for degradation of ubiquitinated proteins. it also inhibits serine and cysteine proteases with lower efficiency. it is also known to induce c-jun n-terminal kinase-dependent apoptosis, to inhibit nfkb activity and to block b-secretase cleavage. karpe et al. reported significant inhibition of hev replication-related luciferase activity in cells treated with mg . however, it was subsequently shown that journal of clinical and translational hepatology vol. | - mg also reduced the cellular rna and protein levels, indicating its effect to be nonspecific. zinc a recent report by kaushik et al. has demonstrated the antiviral activity of zinc against hev. zinc is an essential micronutrient, which plays a crucial role in multiple cellular processes. it also acts as a broad-spectrum antimicrobial against several pathogens. , zinc salts were shown to block the replication of both genotype and genotype hev by inhibiting the activity of viral rna-dependent rna polymerase in cultured human hepatoma cells. further, zinc salts did not affect virus entry into the host cell. zinc also displayed moderate cooperativity with ribavirin in inhibiting viral replication. these data indicate the possible therapeutic usage of zinc in controlling hev infection. however, considering the complexities involved in serum/plasma and intracellular zinc homeostasis, the efficacy of zinc in inhibiting hev replication in vivo remains to be evaluated. moreover, the detailed mechanism(s) underlying the inhibitory action of zinc on hev replication needs to be investigated. the following stages of the hev life cycle are potential targets for the development of specific antivirals (fig. ) . the specific receptor by which hev enters the host cell is unknown. however, it has been demonstrated that heparin sulfate proteoglycans may serve as attachment receptors to facilitate hev entry into the host cells. the hev capsid protein orf also interacts with heat shock protein and glucoseregulated protein (grp ). grp or heat shock protein may be involved in the intracellular transport of the virus. , grp has also been shown to interact with the envelope protein of the japanese encephalitis virus, facilitating its entry into the host cells. it remains to be tested whether grp and orf interaction mediates hev entry. inhibitors of receptor binding or intracellular transport of the virus are supposed to block viral life cycle at a very early stage. among the nonstructural proteins encoded by the hev orf , methyltransferase is responsible for capping of the viral genome. addition of a -methylguanosine cap at the ′terminus of the viral genome confers stability and protects the viral rna from the host innate immune effectors. uncapped hev rna is inefficient in replication. moreover, in contrast to the host methyltransferases wherein guanyltransferase donates a gmp moiety to the rna, followed by cap methylation by guanine- -methyltransferase activity; hev methyltransferase follows a reverse order, thereby restricting its activity to the viral rna. therefore, inhibition of hev methyltransferase activity appears to be a potent antiviral strategy. it is noteworthy that neplanocin a and -deaza-adenosine, the two known inhibitors of influenza virus methyltransferases, interfere with virus replication. neplanocin a is also a potent inhibitor of vaccinia virus replication. inhibitors against dengue virus methyltransferases have also been screened. direct-acting inhibitors of hev rna-dependent rna polymerase function: rna-dependent rna polymerase is the most important factor in the life cycle of all rna viruses and, therefore, rna-dependent rna polymerase inhibitors are supposed to be potent antivirals. one such antiviral against hcv is sofosbuvir, which acts by inhibiting the activity of hcv rnadependent rna polymerase. dao thi et al. indicated the effectiveness of sofosbuvir in inhibiting hev replication; however, subsequent studies failed to observe its potent inhibitory effect. [ ] [ ] [ ] nevertheless, optimization of the sofosbuvir structure that improves its inhibitory effect on hev rna-dependent rna polymerase is an attractive area of investigation. knowledge of hev rna-dependent rna polymerase structure might expedite the above study. apart from sofosbuvir-like molecules, new chemical entities should be explored to identify potent inhibitors of hev rna-dependent rna polymerase activity. other inhibitors of hev rna-dependent rna polymerase function: our earlier studies showed that the interaction between host eef a and viral rna-dependent rna polymerase is important for optimal rna-dependent rna polymerase activity. we recently reported the construction and characterization of the host-virus protein-protein interaction network of hev. using a yeast two-hybrid cdna library screening-based approach, host proteins were identified to be the direct interaction partners of g- hev rna-dependent rna polymerase and of them could also associate with g -hev rna-dependent rna polymerase. notably, host translation regulatory factors, such as eif a , eef a and eif a, directly associated with the rna-dependent rna polymerase protein of both genotype and genotype hev. further in silico analysis of the functional significance of the protein-protein interaction network revealed distinct protein-protein interaction clusters in the secondary network, representing enrichment of proteins involved in different host processes, such as translation initiation, the ubiquitin proteasome pathway and the oxidative phosphorylation pathway. depletion of the translation regulatory factors by gene silencing technique resulted in significant reduction of viral replication and pull-down studies under similar conditions revealed the assembly of a multiprotein complex consisting of the translation regulatory factors, rna-dependent rna polymerase and many other virus and host factors. remarkably, eef a was identified to be the most important host factor for maintaining the integrity of the above multiprotein complex, thereby suggesting it to be an attractive target for antiviral discovery. additionally, inhibitors against other host translation factors present in the complex such as eif a and eif a are also supposed to block viral replication. targeting a combination of direct and indirect inhibitors of rna-dependent rna polymerase function might prove to be an apt antiviral strategy against hev. inhibitors of helicase function: hev helicase is a nucleoside triphosphatase with the ability to unwind rna duplexes in the ′ to ′ direction, thus playing a role in hev replication. due to the common properties shared between the helicases encoded by viruses and their host, designing inhibitors against helicases is challenging. nevertheless, potent inhibitors of helicase encoded by the herpes simplex virus, severe acute respiratory syndrome coronavirus, hcv, dengue virus, japanese encephalitis virus, west nile virus and human papillomavirus have been reported. release of the progeny virions from infected cells leads to the infection of neighboring uninfected cells, thus amplifying the unwanted consequences. antivirals that prevent the release of the progeny virus will prevent further infection, thereby minimizing progression of the disease. release of the newly assembled virus from an infected cell is a complicated process involving multiple protein-protein interactions between the virus and host factors. a thorough understanding of such interactions will help in decoding the mechanism underlying virus release. an inhibitor of human immunodeficiency virus release has been identified, which acts by blocking the interaction between the viral gag and host tumor susceptibility gene encoded proteins. interaction between hev orf and host tumor susceptibility gene -encoded protein is also known to mediate the release of genotype hev. , an inhibitor against the above interaction may prove to be a potent antiviral against hev. apart from that, detailed investigation of hev release mechanism should identify additional targets for antiviral development. the advantages of using antivirals, particularly to cut off the disease in an infected person and providing treatment to poor responders to vaccines, such as immune-compromised patients, warrants the need for development of specific drugs against hev. the antivirals will also prove to be useful for patients with acute, chronic or fulminant hev infections. as summarized in table and fig. , a number of promising antiviral candidates have been identified through the efforts of several researchers, which should be further characterized to identify one or more potent inhibitor(s) of hev. a combinatorial therapy targeting crucial virus-encoded factors at different stages of viral life cycle as well as inhibition of virus-host interactions should be a potent antiviral strategy against hev. the recent finding of hev inhibitory activity of zinc also appears to be an attractive area for further investigation. zinc directly inhibits hev rna-dependent rna polymerase activity in vitro and displays moderate cooperativity with ribavirin in inhibiting viral replication in mammalian cell culture models of hev infection. therefore, even a moderate increase in the level of bioavailable zinc may significantly improve the therapeutic benefits when combined with ribavirin therapy. in summary, recent studies have identified multiple leads, which should be pursued further to develop a potent antiviral against hev. the dbt-rgyi grant and ramalingswamy fellowship to ms is gratefully acknowledged. sa and nk are supported by senior research fellowships from the department of science & technology and the university grants commission, government of india, respectively. the authors have no conflict of interests related to this publication. wrote all sections of the manuscript except the zinc section, and generated the figure and the table (sa), wrote the zinc section 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(review) heparan sulfate proteoglycans are required for cellular binding of the hepatitis e virus orf capsid protein and for viral infection role of heat-shock protein in hepatitis e virus capsid trafficking homology model and potential virus-capsid binding site of a putative hev receptor grp grp is an important host factor for japanese encephalitis virus entry and replication in mammalian cells virus-specific mrna capping enzyme encoded by hepatitis e virus conventional and unconventional mechanisms for capping viral mrna in vitro replication of hepatitis e virus (hev) genomes and of an hev replicon expressing green fluorescent protein the methyltransferase inhibitor neplanocin a interferes with influenza virus replication by a mechanism different from that of -deazaadenosine a potent inhibitor of s-adenosylhomocysteine hydrolase and of vaccinia virus multiplication in mouse l cells development of specific dengue virus '-o-and n -methyltransferase assays for antiviral drug screening ns a inhibitors in the treatment of hepatitis c endoplasmic reticulum stress induced synthesis of a novel viral factor mediates efficient replication of genotype- hepatitis e virus hostvirus protein interaction network reveals the involvement of multiple host processes in the life cycle of hepatitis e virus rna '-triphosphatase activity of the hepatitis e virus helicase domain understanding helicases as a means of virus control herpes simplex virus helicase-primase inhibitors are active in animal models of human disease opportunistic intruders: how viruses orchestrate er functions to infect cells inhibition of hiv budding by a genetically selected cyclic peptide targeting the gag-tsg interaction enhanced alpha microglobulin secretion from hepatitis e virus orf -expressing human hepatoma cells is mediated by the tumor susceptibility gene a psap motif in the orf protein of hepatitis e virus is necessary for virion release from infected cells key: cord- -nsq z authors: denner, joachim; mueller, nicolas j. title: preventing transfer of infectious agents date: - - journal: int j surg doi: . /j.ijsu. . . sha: doc_id: cord_uid: nsq z xenotransplantation using pig cells, tissues and organs may be associated with the transfer of porcine infectious agents, which may infect the human recipient and in the worst case induce a disease (zoonosis). to prevent this, a broad screening program of the donor animals for putative zoonotic microorganisms, including bacteria, viruses, fungi and others, using sensitive and specific detection methods has to be performed. as long as it is still unknown, which microorganism represents a real risk for the recipient, experience from allotransplantation should be brought in. due to the fact that pigs can be screened long before the date of transplantation, xenotransplantation will become eventually safer compared with allotransplantation. screening and selection of animals free of potential zoonotic microorganisms, caesarean section, vaccination and/or treatment with chemotherapeutics are the strategies of choice to obtain donor animals not transmitting microorganisms. in the case of porcine endogenous retroviruses (pervs) which are integrated in the genome of all pigs and which cannot be eliminated this way, selection of animals with low virus expression and generation of genetically modified pigs suppressing perv expressions may be performed. xenotransplantation using pig cells, tissues and organs has to overcome three hurdles before being applied in the clinic for the treatment of organ failure: immunological rejection, physiological incompatibility and transfer of infectious agents. the microbiological safety of xenotransplantation is an important issue, however it can be managed easily. the risk of infection is also known in allotransplantation. numerous infectious agents have been transmitted together with human donor transplants, including human cytomegalovirus (hcmv), human immunodeficiency virus- (hiv- ) and rabies virus [ ] . since xenotransplantation allows screening the donor animals beforehand, most risks can be excluded by careful testing and xenotransplantation finally will be a microbiologically safer technology compared with allotransplantation. like all animals, pigs carry numerous microorganisms in their digestive tract and on their skin, and therefore cells, tissues and organs to be used for transplantation should be removed under aseptic conditions. the number of microorganisms present in the tissues and organs of interest should be zero [ ] . in some reviews concerning the microbiological safety of xenotransplantation numerous microorganisms are listed which were thought to induce zoonoses when transmitted to the human recipient [ ] . zoonosis means that the microorganisms not only infect the new host, but cause a disease. in general, bacteria, fungi, parasites and viruses may be transmitted. however, at present it is rather difficult to classify most of the porcine microorganisms into pathogenic and non-pathogenic for human recipients. in addition, when a microorganism is pathogenic in the pig it does not mean that it is also pathogenic in humans and vice versa. the risk of transmission is certainly higher when pharmaceutical immunosuppression to prevent immunological rejection of the transplant will be applied. therefore it is still unclear which microorganisms should be monitored. the auckland island pigs which had been used in the first clinical trials performed by the new zealand company lct were screened regularly for bacteria, viruses and toxoplasma (table ) [ ] . the g€ ottingen minipigs which are used for numerous biomedical investigations are screened regularly for bacteria, viruses, three fungi and four parasites (http//www.minipigs.dk/). an additional screening of the g€ ottingen minipigs involved perv [ ] , hepatitis e and other microorganisms [ , ] . in general, most microorganisms found in pigs to be used for xenotransplantation may be eliminated by specified pathogen free (spf) or designated pathogen-free (dpf) breeding of the animals. in the case there is a bacterial or fungal infection in the donor pig, treatment with antibiotics or chemotherapeutics may be successful. at the moment hepatitis e virus (hev), porcine cytomegalovirus (pcmv), porcine circoviruses (pcv), porcine lymphotropic herpes viruses (plhv), and porcine endogenous retroviruses (pervs) are thought to pose the main risk for reasons to be discussed below and therefore these microorganisms will be analysed in the next chapters in more details. in most cases hev causes self-limiting hepatitis in humans. whereas hev of the genotype (gt) and gt are found in people, are transmitted mainly by contaminated water and are causing a high mortality during pregnancy, hev gt and gt are swine viruses and do not cause a disease in pigs, however, when they infect humans they may cause in rare cases a zoonotic disease (for review see [ , ] ). a severe hepatitis after infection with hev gt and gt was observed only in the case of other underlying liver diseases. importantly, neurological disorders have also been described for hev gt and gt . note, that only hev gt / may pose a risk when xenotransplantation using pig cells, tissues and organs is performed, not gt or gt . usually hev gt / are transmitted by contaminated meat or direct contact with infected pigs. hev gt rna was detected in pig liver at grocery stores and infectious virus could be isolated [ , ] . hev transmission by shellfish and vegetables possibly contaminated by pig manure as well as by blood transfusion and allotransplantion was also reported. a chronic infection was more likely to develop in immunosuppressed patients, including hiv- infected individuals [ , ] . sensitive pcr-based methods have been developed to determine a hev infection and to genotype the virus. detection of hev and its elimination from pigs seems not to be easy. first, the virus is heterogeneous, e.g., subtypes of genotype exist, what makes it difficult to design efficient pcr or real-time pcr. second, the virus load seems to be very low so that even highly sensitive pcrs may be unable to detect the virus. although hev gt / are widely distributed, the prevalence in pigs, especially in multitransgenic pigs generated for usage in xenotransplantation, is not well studied. in contrast, the non-transgenic auckland island pigs, generated by living cell technologies (lct) in new zealand are better table microorganisms tested in auckland island pigs used for islet cell transplantation [ ] . [ ] . although hev is very common in pigs in new zealand, the auckland island pigs were free of all hev [ ] . another well investigated breed is the g€ ottingen minipig produced by ellegaard in denmark. these animals are used worldwide for numerous biomedical investigations. the herd was established by entry of animals obtained by caesarean sectioning and colostrum deprivation. despite this, hev was found in one study in % of the animals ( of ) [ ] , in another, using real-time pcr and western blot analysis detecting antibodies against hev, hev was found in only very few animals [ ] . the result suggested a transplacental mother-to-piglet transmission of the virus. this observation may help to explain how the virus entered the pig herd despite caesarean sectioning and other precautions. it remains unclear, whether the absence of the virus in all older g€ ottingen minipigs is due to the elimination of the virus possibly by the immune system or due to the limits of the detection methods. in order to eliminate hev from a herd, a hev elimination program was proposed ( fig. ) [ ] . elimination should include selection of hev negative animals using highly sensitive real-time pcr. since it is not clear, whether the animals are truly negative, or carry hev in concentrations below the sensitivity of the detection method, a treatment step should be included using ribavirin, a guanosine analogue used to stop viral rna synthesis. although there are no data on the treatment of hev infection with ribavirin in pigs, ribavirin has been successfully used for the treatment of other virus infections in pigs [ ] . another strategy may include a vaccination step, e.g. using a vaccine based on a recombinant orf- fragment of hev gt that has been approved by the chinese fda [ ] . since hev gt - represents only one serotype, the induced antibodies should be protecting from infection with all genotypes [ ] . immunisation of pigs with orf- of pig hev gt resulted in effective protection [ ] , indicating that pigs can be immunized and mount an effective antiviral immune response. as human cytomegalovirus (hcmv) causes severe transplant rejection in allotransplantation [ , ] , considerable concern is warranted on the potential pathogenicity of porcine cytomegalovirus (pcmv) in the setting of xenotransplantation. pcmv is endemic in the world pig population, it is acquired early in life and pcmv infection results in seroconversion and lifelong latent infection [ ] . pcmv spreads by both vertical and horizontal transmission [ , ] . active infection causes fatal systemic failure in piglets less than weeks of age. the clinical symptoms of infected piglets include pneumonia and inclusion body rhinitis with a high mortality rate. pcmv-infected sows are prone to abortion, with pathological changes including edema in the heart and other organs [ ] . pcmv can remain latent in adult pigs. the ubiquitous nature of herpesviruses, including pcmv, means that these viruses should be a major focus in the development of xenotransplantation. herpesviruses are able to infect other species. porcine cells can be infected by hcmv [ ] , indicating that the pig transplants may be infected when the recipient is hcmv positive, and pcmv can infect human cells [ ] . entry of hcmv into porcine endothelial cells depends on both the cellular vascular origin and the viral strain [ ] . when pcmv was transmitted by the pig transplant into baboons, the baboon cmv was activated causing invasive disease and consumptive coagulopathy, the pcmv was mainly replicating in the pig transplant causing ureteric necrosis in one transplant [ e ]. when baboons received pig kidneys from pcmv-infected pigs, the survival time was . days in comparison to . or days when organs from uninfected animals were transplanted [ ] . in a similar experiment with cynomolgus monkeys the difference was . days versus . days [ ] . alone the presence of the virus had such an important influence on transplant survival. when cd -transgenic large white pigs were analyzed, all animals were found positive for pcmv, however under specified pathogen free (spf) or designated pathogen-free (dpf) conditions pcmv-free animals were obtained [ ] , indicating that selection of pcmv-free animals by caesarean delivery and spf breeding is possible. on the other hand, due to reactivation of the baboon cmv after pig cardiac xenotransplantation with pharmaceutical immunosuppression a lethal outcome in some cases was observed despite prophylactic treatment with the antiviral drugs ganciclovir or valganciclovir [ ] . the auckland island pigs, already used in clinical islet cell transplantation were shown to be free of pcmv [ ] . as mentioned above, pcmv can be eliminated easily by caesarean delivery and dpf or spf breeding of the herd [ , ] . in addition, early weaning of the piglet from the sow can eradicate cmv [ ] . to be on the safer site, a treatment with the antiviral drugs ganciclovir, cidofovir, foscarnet, acyclovir, valaciclovir, a prodrug of acyclovir, or valganciclovir can be included into the elimination protocol ( fig. ) [ ] . concerning vaccination against hcmv, despite the urgent need for allotransplant recipients, no success was reported, although first attempts to use the major envelope glycoprotein gb have demonstrated efficacy against hcmv infection and on hcmv-induced disease [ e ]. immunization studies with the gb protein of pcmv should be performed in pigs. porcine lymphotropic herpesvirus (plhv) , , and are common porcine viruses, however the prevalence and importance of these viruses for xenotransplantation is not well studied [ ] . phylogenetic analyses showed that all three phlv clustered together with ruminant gammaherpesviruses, but the plhv- is more distantly related to plhv- and plhv- [ ] . the transmission of phlv in pigs is not well understood. plhv may be fig . . schematic presentation of the proposed virus elimination program. the original herd was screened for the presence of a putative zoonotic virus (grey, positive animals; pink, negative animals). negative animals (that may be actually positive, but below the detection limit) were selected and using caesarean delivery, and treatment with antiviral drugs and/or vaccines, truly negative animals could be obtained and used for further breeding and xenotransplantation. transmitted by pre-partum cross-placental vertical transfer and post-partum horizontal transmission, however, cross-placental transfer is not the common way [ ] . between % up to % of animals in different herds in germany, ireland, france, spain and the united states were infected with one of the plhv [ e ]. in contrast to pcmv, early weaning cannot eradicate phlv [ ] . circoviruses belong to the smallest viruses replicating autonomously in mammalian cells [ ] . porcine circovirus (pcv ) has not been linked with any disease, whereas pcv is the causing agent of post-weaning multisystemic wasting syndrome (pmws), a multifactorial disease in pigs [ ] . this means that the presence of the virus is necessary for the disease but requires additional factors. the onset of the disease and the severity of the symptoms are influenced by the status of the immune system and genetic predisposition [ ] . characteristic clinical signs of pmws are wasting, respiratory dysfunction, enlargement of inguinal lymph nodes, diarrhoea and a generalised depletion of lymphocytes. although humans ingest pcv-contaminated foods and are exposed to pcv through other sources, serological evidence indicated that no transmission of pcv to humans took place [ ] . also, a contamination of a human vaccine with pcv was shown not to transmit pcv to humans [ , ] . retrospective testing confirmed the presence of pcv dna in rotarix, an oral live-attenuated human rotavirus vaccine for children, beginning with the initial stages of its development and in vaccine lots used in clinical studies conducted preand post-licensure [ ] . when human cell lines have been infected with pcv and pcv , pcv persisted in most cell lines without causing any visible changes, while pcv -transfected cells showed a cytopathogenic effect [ ] . most importantly, the infection was non-productive [ , ] . porcine endogenous retroviruses are the result of a transspecies transmission of a retrovirus and integration into the genome of all pigs. perv-a and perv-b are polytropic viruses, infecting also human cells (for review see [ ] ). perv-c infects only pig cells, it is present in most, but not all pigs. whereas transformed human cells lacking some intracellular restrictions factors such as abopec can be infected easily, human primary cells can be infected only by high-titre perv-a and recombinant perv-a/c after adaptation on human cells leading to an increased number of transcription factor binding sites in their long terminal repeats [ e ]. since perv-c are present in many pigs, but not all, the selection of pigs not carrying perv-c proviruses automatically prevents generation of high-titre recombinant perv-a/ c. until now, no transmission of pervs was observed, neither in first clinical trials enrolling more than patients, nor in preclinical pig to non-human primate transplantation, nor in infection experiments in small animals or non-human primates with or without pharmaceutical immunosuppression (for review see [ ] ). however, most of the patients in the clinical trials were not exposed for a long time to the xenotransplant and with some exceptions associated with parallel kidney allotransplantations, no pharmaceutical immunosuppression was applied. in addition, no transmission of perv was observed in numerous pig to nonhuman primate transplantations [ ] . however, it is meanwhile clear that non-human primates are not a suitable model to study this question since non-human primates carry e in contrast to humans e a mutated receptor for perv allowing only infection at a low efficiency [ ] . therefore, the question, whether pervs may be transmitted during xenotransplantation is still open and an elimination of infectious proviruses is advised, since retroviruses may induce tumours and immunodeficiencies. pervs are closely related to other gammaretroviruses such as the feline leukaemia virus (felv), the murine leukaemia virus (mulv) and the koala retrovirus (korv) [ ] . all these related viruses induce severe immunodeficiencies and tumours in the infected host. gammaretroviruses used as vectors in gene therapy for the treatment of a severe combined immunodeficiency in children were shown to induce leukaemia in the patients [ ] , indicating that transspecies transmission of gammaretroviruses into human may induce tumour development. highly sensitive and specific methods have been developed to screen for the prevalence and expression of perv in donor pigs. whereas expression of perv in german landrace pigs is relatively low, expression in yucatan minipigs is high [ ] . in these animals viral protein may be detected in different tissues of the animals [ ] and infectious virus was found released from stimulated peripheral blood mononuclear cells (pbmcs) [ ] . crossing of landrace pigs with minipigs increased the expression rate of perv [ ] . when g€ ottingen minipigs were screened, a relatively low expression and no release of virus particles were observed [ ] . the elimination programs for hev and pcmv were described in detail above [ , ] . the elimination programs for other microorganisms (with exception of perv, see below) will be similarly, including screening of the donor pigs using specific and sensitive methods, treatment with antiviral drugs and immunisation (fig. ) . for some porcine microorganisms effective vaccines are available, for example for circoviruses (ingelvac circoflex ® ) and mycoplasma (ingelvac circoflex ® ). several other vaccines are available to treat pigs and prevent transmission of the infectious agents (table ) [ ]. however, vaccination is only necessary, when the infectious agent is present in the pig herd and pose a risk for the transplant recipient. in the case of bacteria it has to be considered whether the donor pig (and in some cases also the recipient) should be treated with antibiotics. since pervs are present in the genome of all pigs and cannot be eliminated by the way other microorganisms can be eliminated easily, other strategies were developed to prevent transmission of these infectious agents. first, animals with a low copy number of perv and a low expression rate should be selected. second, animals not carrying perv-c should be selected in order to prevent perv-a/ c recombination. third, transgenic pigs have been generated expressing small interfering rna, specifically inhibiting the expression of perv and therefore lowering the probability of perv transmission [ e ].since in the genome of pigs multiple perv proviruses were found, sometimes more than , an elimination of all perv copies by genome editing using zinc finger nucleases (zfns) may be difficult. we selected zfn specific for a sequence of the highly conserved pol region of the virus. however first attempts to eliminate pervs in the pig genome failed, obviously due to the strong toxic effect when zfns were cutting the genome at multiple sites and therefore destabilising the genome [ ] . a lower concentration of the zfn and a gradual elimination may be alternative strategies. in addition, new attempts using crispr/cas (clustered regularly interspaced short palindromic repeats/crisprassociated) for gene editing should be undertaken with the goal to eliminate at least the replication competent proviruses. fourth, and last but not least, transmission of pervs may be prevented by a vaccine. when immunising different animal species (goats, rats, mice, hamster, guinea pigs) with the recombinant transmembrane envelope protein p e and the surface envelope protein gp of perv, always effective neutralising antibodies were induced [ e ]. the results of the immunisation with the transmembrane envelope protein are of great interest. in all cases neutralising antibodies were obtained and one epitope recognised was localised in the membrane proximal external region (mper), the other in the fusion peptide proximal region [ , ] . the epitope in the mper was not only localised similarly as a corresponding epitope recognised by antibodies broadly neutralising hiv- isolated from hiv- infected individuals in the transmembrane envelope protein gp of hiv- , but despite the evolutionary difference between perv and hiv- three identical amino acids were detected in the epitopes [ , ] . immunisation with the surface envelope protein gp induced higher titres of neutralising antibodies compared with immunisation with the transmembrane envelope protein p e [ ] . since there is no animal model which allows analysing the efficiency of the perv vaccine in vivo, the related felv was studied. cats were immunised with recombinant p e and gp of felv. neutralising antibodies were detected and the epitopes recognised were very similarly located compared with the epitopes recognised by the antibodies neutralising perv [ , ] . when the immunised cats were challenged with infectious felv, the animals were protected, suggesting that in the case of perv the vaccine may also work [ ] . these data indicate that immunisation of the transplant recipient may be successful in the case other prevention strategies do not work. elimination programs: screening, selection, treatment and isolation of donor pigs transmission of infection with human allografts: essential considerations in donor screening the international xenotransplantation association consensus statement on conditions for undertaking clinical trials of porcine islet products in type diabetesechapter : source pigs microbiological safety of the first clinical pig islet xenotransplantation trial in new zealand screening pigs for xenotransplantation: prevalence and expression of porcine endogenous 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postweaning multisystemic wasting syndrome characterization of the dna polymerase loci of the novel porcine lymphotropic herpesviruses and in domestic and feral pigs early weaning of piglets fails to exclude porcine lymphotropic herpesvirus molecular biology of porcine circovirus: analyses of gene expression and viral replication the serological evidence in humans supports a negligible risk of zoonotic infection from porcine circovirus type porcine circovirus diseases detection of porcine circovirus type in commercial pig vaccines using polymerase chain reaction investigation of a regulatory agency enquiry into potential porcine circovirus type contamination of the human rotavirus vaccine, rotarix: approach and outcome infection studies on human cell lines with porcine circovirus type and porcine circovirus type infection barriers to successful xenotransplantation focusing on porcine endogenous retroviruses genetic alterations of the long terminal repeat of an ecotropic porcine 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endogenous retroviruses (perv) in different organs of a pig production of transgenic pigs that express porcine endogenous retrovirus small interfering rnas knockdown of porcine endogenous retrovirus (perv) expression by perv-specific shrna in transgenic pigs long-term effects of perv-specific rna interference in transgenic pigs inhibition of porcine endogenous retroviruses by rna interference: increasing the safety of xenotransplantation attempts to knock out multiple porcine endogenous retrovirus (perv) sequences in the pig genome by zinc finger nucleases (zfn) neutralizing antibodies against conserved domains of p e of porcine endogenous retroviruses (pervs): basis for a vaccine for xenotransplantation? development of sensitive methods for detection of porcine endogenous retrovirus-c (perv-c) in the genome of pigs increased titers of neutralizing antibodies after immunization with both envelope proteins of the porcine endogenous retroviruses (pervs) novel neutralising antibodies targeting the n-terminal helical region of the transmembrane envelope protein p e of the porcine endogenous retrovirus (perv) neutralising antibodies against the transmembrane protein of feline leukaemia virus (felv) increased neutralizing antibody response after simultaneous immunization with leucogen and the feline leukemia virus transmembrane protein immunization with the transmembrane protein of a retrovirus, feline leukemia virus: absence of antigenemia following challenge supplementary data related to this article can be found at http:// dx.doi.org/ . /j.ijsu. . . . key: cord- -cpwl ych authors: andries, k.; pensaert, m. title: propagation of hemagglutinating encephalomyelitis virus in porcine cell cultures date: - - journal: j vet med b infect dis vet public health doi: . /j. - . .tb .x sha: doc_id: cord_uid: cpwl ych summary: this study reports some cultural characteristics of the vw strain of hemagglutinating encephalomyelitis virus (hev) in primary pig kidney (ppk) cells, primary pig testicle (ppt) cells, secondary pig thyroid (spth) cells and the cell lines pk‐ (pig kidney), sk− (swine kidney) and st (swine testicle). a growth curve, based on cytopathic effect (cpe), immunofluorescence (if), hemadsorption (hads) and hemagglutination (ha), showed that spth and ppk cells were most susceptible for cultivation and quantitation of the virus. for the detection of replication in tubes inoculated with small amounts of virus, cpe, hads and ha appeared to be useful and sensitive criteria. repeated virus quantitation trials revealed a high variation in titration end‐points, even in the most susceptible cell types. the optimal procedure for the isolation of hev from clinical specimens is preferably to inoculate the material on spth or ppk cells and to make a blind passage if the ha test at days post inoculation is negative. zusammenfassung: züchtung von hemagglutinating encephalomyelitis virus (hev) in porcinen zellkulturen kulturelle eigenschaften des hev‐stammes vw in primären schweinenierenzellen (ppk), primären schweinehodenzellen (ppt), sekundären schweineschilddrüsenzellen (spth) und in den zellinien pk‐ (schweinenieren), sk− (schweinenieren) und st (schweinehoden) werden beschrieben. vermehrungskurven, die mit hilfe des cytopatischen effektes (cpe), der immunofluoreszenz (if), der hämadsorption (hads) und der hämagglutination als kriterien für die virusreplikation erstellt wurden, zeigten, daß spth und ppk‐zellen am empfindlichsten für die züchtung und titration des virus sind. zum nachweis der virusvermehrung in röhrchen, die mit kleinen virusmengen beimpft wurden, waren cpe, hads und ha empfindliche und brauchbare kriterien. wiederholte virustitrationen zeigten eine hohe variation der titerendpunkte auch in hochempfänglichen zellkulturen. die optimale methode für die isolierung von hev von klinischem material ist die verimpfung auf spth oder ppk mit anschließender blindpassage, wenn der ha‐test sieben tage p. inf. negativ ist. rÉsumÉ: culture du virus hémagglutinant de l'encéphalomyélite (hev) dans des cultures de cellules de porc on décrit les propriétés de culture de la souche hev vw dans des cellules primaires de reins de pores (ppk), dans des cellules primaires de testicules de porcs (ppt), dans des cellules secondaires de glandes tyroïdes de porcs (spth) et dans les lignées cellulaires pk‐ (reins de porcs), sk− (reins de porcs) et st (testicules de porcs). les courbes de multiplication établies avec l'effet cytopathique (cpe), l'immunofluorescence (if), l'hémadsorption (hads) et l'hémagglutination (ha) comme critères pour la réplication du virus ont montré que les cellules spth et ppk étaient les plus sensibles pour la culture et la titration du virus. cpe, hads et ha furent les critères sensibles et utilisables pour la mise en évidence de la multiplication virale en tubes inoculés avec de petites quantités de virus. des titrations du virus répétées ont montré une forte variation du titre final également dans les cultures cellulaires hautement réceptrices. la méthode optimale pour l'isolement de hev à partir d'un matériel clinique est l'inoculation sur spth ou ppk avec passages à l'aveugle si le test ha est négatif jours après l'infection. resumen: propagación del virus hemoaglutinante de la encefalomielitis (hev) en los cultivos de células de cerdos se describen las propiedades culturales de la estirpe hev vw en células renales primarias de cerdo (ppk), células testiculares primarias de cerdo, células tiroideas secundarias de cerdo (spth) y en las líneas celulares pk‐ (riñones de cerdo), sk− (riñones porcinos) y st (testículos de cerdo). las curvas de multiplicación, las cuales se establecieron con ayuda del efecto citopático (cpe), la inmunofluorescencia (if), la hemoadsorción (hads) y la hemoaglutinación como criterios para la replicación virósica, mostraban que las células spth y ppk son las más sensibles para el cultivo y titulación del virus. para la puesta en evidencia de la multiplicación virósica en tubitos, los cuales se inocularon con cantidades pequeñas de virus, eran cpe, hads y ha criterios sensibles y útiles. titulaciones repetidas de virus mostraban una variación elevada de los puntos finales de títulos incluso en cultivos celulares harto receptibles. el método óptimo para el aislamiento de hev a partir de material clínico consiste en la inoculación a spth o ppk con pase ciego inmediato si la prueba ha es negativa días después de la infección. hemagglutinating encephalomyelitis virus (hev) is a coronavirus ( , ) that causes vomiting and wasting ( ) or encephalomyelitis ( ) in young pigs. the virus was first isolated in primary pig kidney cells by greig et al. ( ) , who described a cytopathic effect characterized by the appearance of multinucleated cells. the hemadsorption and hemagglutination tests were also used for the demonstration of viral growth. with the immunofluorescence test, hev was shown to pro agate in several other porcine cell cultures such as ibrs, cell line ( ) and sk cell line ( ) . non-porcine cell cultures were shown to have little susceptibility for growth of hev ( , ) . it was the purpose of the present study to determine which is the most sensitive cell culture system for the isolation of hev and the optimal test for the demonstration of viral growth. since non-porcine cell cultures have been shown to support virus growth poorly, only porcine cell cultures were selected for this study: primary pig kidney cells, primary pi testicle cells, secondary pig thyroid cells and the cell lines pk- (pig kidney), sk- (swine kidney) and st (swine testicle). a viral growth curve was made in the different cell cultures using the following criteria: cytopathic effect, immunofluorescence, hemadsorption and hemagglutination. also, the optimal time to use the different criteria in relation to the virus concentration inoculated was determined. finally, using the optimal detecting system, attempts were made to determine whether repeated virus quantitation trials gave reproducible results and whether a blind passage is necessary. virus two stodrs of hev were used. a tissue culture stock was prepared from the th passage on primary pig kidney (ppk) cells of vw virus, a belgian isolate of hev adult thyroid gland, em g ryonic lung, testicle cell line, pk- cell line ( ) ( ) . the supernatant of the infected culture was centrifuged for min. at , g. and stored in ml. portions at - oc. the stock had a titer of approximately ' o/o tissue culture infective doses (tcid,,) per ml. when titrated on secondary pig thyroid cells. a lung homogenate stock was prepared in gnotobiotic piglets which were inoculated at the age of days with the tissue culture stock mentioned above. the piglets were killed days after inoculation and their lungs were ground with ten broecks homogenizers to a o/o (w/v) suspension in phosphate buffered saline (pbs). after low speed centrifugation, the infective supernatant fluid was also stored in ml. portions at - oc. primary pig kidney (ppk) cells were prepared from kidneys taken from to week old pigs, according to standard procedures primary pig testicle (ppt) cells were prepared from testicles of to week old pigs. the cells were grown in the same medium as used for ppk cells, but the medium was replaced after days. at the time they were inoculated, the fully sheeted monolayers were nahco,. prescription bottles ( cm*. growth surface) were inoculated with , cells per ml. and put in a per cent c incubator. the cells were refed after to days when they became confluent. the monolayers were trypsinized day later into secondary cells, using the same medium, the same cell concentration and the c incubator as described for the primary pig thyroid cells. the spth cells were refed after days and inoculated after days. after the inoculation, the cells were fed as described for the ppk cells but were incubated without co,. pk- cells were fully sheeted after days when seeded at a concentration of , cells/ml. the medium consisted of mem supplemented with . o/o lah and per cent cs for growth medium or . o/o lah and per cent bfs for maintenance medium. sk- cells ( ) and st-cells'* were seeded at a concentration of , cells/ml. the growth and maintenance medium were the same as for pk- cells. fully sheeted monolayers were present after days and were then used for inoculation with virus. a growth curve of hev was prepared with the cell culture virus stock inoculated on spth, ppk, pk- , sk- , ppt and pt cells. for each cell type, tubes were inoculated with a multiplicity of infection of approximately . and left for h at o c . the monolayers were washed three times with pbs to remove non-adsorbed virus and, after addition of medium, the cells were incubated at oc. at daily intervals from to days after the inoculation, the monolayers were examined for the occurrence of cytopathic effect. each day, the nutrient medium of tubes was pooled, entrifuged ( g. for lomin.), and collected for titration of infectivity and hemagglutination (ha). the hemadsorption (hads) test was performed on the remaining monolayers using mem with . o/o chicken red blood cells. after incubation for min. at oc, the monolayers were washed once with pbs and examined for evidence of hads. the erythrocytes for the hads and hatests were collected from chickens between four and twelve weeks of age, according to girard et al. ( ) . virus .growth was detected by immunofluorescence (if) on coverslips in leighton tubes whirh were inoculated using the same virus dilution as described above. two infected coverslips were fixed daily in cold acetone and stained with a fluorescent * provided by norden laboratories inc., smith kline corporation of philadelphia, pennsylvania, u. s. a. antibody conjugate prepared from a hyperimmune hev antiserum as previously described ( ) . control as well as infected cell cultures were counterstained with . / evans blue for min. the virus infectivity of the supernatant fluid of the tubes was assayed using microtiter plates. serial -fold dilutions in amounts of . ml. were added to . to determine the optimal time at which different criteria could be used when varying virus concentrations were inoculated, serial -fold dilutions of the tissue culture stock were inoculated in amounts of . ml. in tubes with spth, ppk, pk- or sk- cells. the virus was allowed to adsorb for one hour. at , , , , , and days after the inoculation, four inoculated tubes of each cell type and of each dilution were randomly selected. monolayers of spth and ppk were examined for the presence of cpe and hads. supernatant fluid of spth, ppk, sk- and pk- was tested for ha activity. titration end-points (tcid,,) were calculated daily for each criterion for the respective cell type. the ha test was done with the centrifuged ( g. for min.) supernatant fluid, from which . ml. was mixed with an equal volume of a . / suspension of washed chicken erythrocytes in pbs. to examine the reproducibility of infectivity titrations, not only the cell culture adapted stock but also the virus-containing lung homogenate was titrated repeatedly. the test was performed on spth, ppk, pk- and sk- monolayers. during each of the trials, all cell types were inoculated with the same dilution series for each stock. the titration end-points were calculated on the seventh day after inoculation, using the ha test. six infectivity titrations were made on ppk cells, using a series of different dilutions of the tissue culture stock each time. each tube in which no hemagglutinating activity was detected after days of incubation was subjected to two cycles of freezing and thawing and was individually centrifuged for min. at g. the supernatant was inoculated onto a fresh monolayer of ppk cells. presence of virus was again examined using the ha test days after the inoculation. figs. to show the virus growth curves based on the following criteria: cytopathic effect consisted of the formation of syncytia which were easily visible in vivo with an inverted microscope. syncytia were small in ppk cells but could be very large in spth cells. soon after their appearance, the syncytia degenerated and holes, surrounded by opaque irregularly shaped tsyncytial debris, were formed in the monolayer. after the release of this ,sycytial debris from the monolayer, part of it was conserved into balloonlike structures. these structures soon represented the only evidence of viral induced cell degeneration, since the formation of new syncytia had stopped. recording of the cpe in the growth curves was performed using indices to , which stand for approximately , , , syncytia per tube or syncytia in the entire monolayer, respectively. the presence of balloon-like structures was not taken into account. hemadsorption, in the early stages of infection, was characterized by the presence of attached red blood cells in groups of (ppk cells) up to (spth cells). later, the hemadsorption was more diffuse and large groups of red blood cells were no longer seen. recording was performed using the indices to which stand for approximately , , , and adsorbed erythrocytes, respectively, per field, using a x objective and counting about fields. recording of cytoplasmic fluorescence was performed using the indices to which stand for a few cells, a few small foci, a few large foci, many large foci or almost all the cells positive, respectively. in spth and ppk cells, the decrease in fluorescence as shown in figs. and was mainly due to degeneration and loss of infected cells. recording of the infectivity and hemagglutination titers was also done using indices to , which stand for lo to lo tcid,, and , , , , ha units per . ml. of supernatant fluid, respectively. the following pattern of virus growth in different cell types ( fig. to ) was observed: pk-lli cells: peaks of if, infective virus and ha were reached at pid , and , respectively. cpe was not clear in unstained cultures, although hematoxylin eosin staining revealed that a few small giant cells were present on pid . hemadsorption did occur, but to such a low degree that it could not be taken into account. sk- cells: ha, hads or cpe were not observed in this cell line. infectious virus was, however, produced with a peak on pid to . small foci of fluorescence were found with a peak on pid . ppt and st cells: no ha, hads or cpe were observed in these cells. no fluorescence was found in st cells. a few ppt cells were positive by immunofluorescence at pid only. the presence of infectious virus was not examined. both ppt and st cells were excluded from the further experiments because it was clear from these first studies that their sensitivity for hev was very low. the period during which maximal infectivity titers were recorded with a given criterion was considered as the optimal period to apply this criterion. the titers calculated daily using the different criteria are presented in table . it can be seen that maximal titers on spth cells were obtained when reading was done on the basis of the cpe between pid and , on the basis of hads between pid and , or on the basis of ha between pid and . using ppk cells, reading the cpe between pid and gave about the same results as reading by hads between pid and or reading by ha between pid and . the ha test applied on pid appeared to be optimal for both pk- and sk- cells. either refractory (st cells), or allowed viral growth only to such a slight extent (ppt, sk- and pk- cells) that they cannot be considered for practical use. in spth and ppk cells, all the criteria tested (if, cpe, hads, ha) were useful to demonstrate viral replication. when the different criteria were correlated, it was observed that in spth cells peaks of all criteria were reached at h post inoculation. the amount of if, hads and cpe decreased quickly, while the h a titer remained high until days post inoculation. in ppk cells a similar correlation was observed except that the peaks had a tendency to appear somewhat later and, especially for cpe, reached lower levels. hemagglutinating activity in the supernatant fluid was the last criterion to become positive and persisted longest in these cells also. the daily calculation of the infectivity titers revealed that cpe, hads and ha became positive even in the tubes which were inoculated with the highest virus dilutions. although cpe and hads may be more sensitive to detect the beginning of viral growth, virus replication was finally high enough for h a to become ositive, even in tubes inoculated with a low virus conis simple to perform and remains clearly positive for several days. the comparative infectivity titrations provided further evidence that secondary thyroid and primary kidney cells were more susceptible than the established cell lines. comparable results were obtained by pirtle ( ) , who made one titration of hev in spth cells, embryonic porcine kidney cells and pk- cells, using the if test after hours of inoculation as criterion. in the present studies, the repeated quantitation trials revealed an unacceptable variation in the titration end-points ( table ) . similar difficulties in which considerable variation was encountered have also been described for two other coronaviruses, infectious bronchitis virus ( ) and transmissible gastroenteritis virus ( ). it was not clear whether this variation was related to the condition of the cells at the time of inoculation, to dilution faults or to the presence of aggregates in the virus stock. however, we have observed several times that ppk cells are reliable for virus isolation trials only when the cells were used during rapid growth. if the cells were inoculated when fully sheeted days after planting, the results of virus isolation were much more reproducible than when fully sheeted cells were used after a slow start. if no attention is given to this point, the results of virus isolation in ppk cells varied from week to week. the final infectivity titer of the virus stocks titrated on ppk cells was not significantly increased after carrying out a blind passage. however, in virus isolation trials starting from diagnostic specimens, such a blind passage is recommended since tissue homogenates from pigs infected with vw often contain very small amounts of infectious virus ( ). we conclude that the optimal procedure for isolation of hev from clinical specimens is preferably to inoculate the material on spth or ppk cells and to make a blind passage if the ha test at pid is negative. hemagglutination inhibition or seroneutralisation tests using specific antisera should then be performed for further virus identification. centration. of the t k ree tested criteria, h a was finally preferred because it this study reports some cultural characteristics of the vw strain of hemagglutinating encephalomyelitis virus (hev) in primary pig kidney (ppk) cells, primary pig testicle (ppt) cells, secondary pig thyroid (spth) k. andries and m. pensaert cells and the cell lines pk- (pig kidney), sk- (swine kidney) and st (swine testicle). a growth curve, based on cytopathic effect (cpe), immunofluorescence (if), hemadsorption (hads) and hemagglutination (ha), showed that spth and ppk cells were most susceptible for cultivation and quantitation of the virus. for the detection of replication in tubes inoculated with small amounts of virus, cpe, hads and h a appeared to be useful and sensitive criteria. repeated virus quantitation trials revealed a high variation in titration end-points, even in the most susceptible cell types. the optimal procedure for the isolation of hev from clinical specimens is preferably to inoculate the material on spth or ppk cells and to make a blind passage if the ha test at days post inoculation is negative. sibles et utilisables pour la mise en kvidence de la multiplication virale en tubes inoculks avec de petites quantitks de virus. des titrations du virus rkpktkes ont montrk une forte variation du titre final kgalement dans les cultures cellulaires hautement rkceptrices. la mkthode optimale pour i'isolement de hev a partir d'un matkriel clinique est l'inoculation sur spth ou ppk avec passages i l'aveugle si le test h a est ncgatif jours aprks l'infection. propagaci n del virus hemoaglutinante de la encefalomielitis (hev) en s cultivos de cclulas de cerdos se describen las propiedades culturales de la estirpe hev vw en cklulas renales primarias de cerdo (ppk), cklulas testiculares primarias de cerdo, cklulas tiroideas secundarias de cerdo (spth) y en las lineas celulares pk- (riiiones de cerdo), sk- (riiiones porcinos) y st (testiculos de cerdo). las curvas de multiplicaci n, las cuales se establecieron con ayuda del efecto citopitico (cpe), la inmunofluorescencia (if), la hemoadsorci n (hads) y la hemoaglutinacibn como criterios para la replicaci n vidsica, mostraban que las cklulas spth y ppk son las mis sensibles para el cultivo y titulacibn del virus. para la puesta en evidencia de la multiplicaci n vir sica en tubitos, s cuales se inocularon con cantidades pequeiias de virus, eran cpe, hads y ha criterios sensibles y tiles. titulaciones repetidas de virus mostraban una variaci n elevada de s puntos finales de titulos incluso en cultivos celulares harto receptibles. el mktodo ptimo para el aislamiento de hev a partir de material clinico consiste en la inoculaci n a spth o ppk con pase ciego inmediato si la prueba h a es negativa dias despuks de la infecci n. viral encephalomyelitis of swine in ontario -experimental and natural transmission pathogenicity of hemagglutinating encephalomyelitis (vomiting and wasting disease) virus of pigs, using different routes of inoculation pensaert: virus isolation and imrnunofluorescence in different organs of pigs infected with hemagglutinating encephalomyelitis virus vomiting and wasting disease of piglets isolement en france et identification de la maladie du vomissement et du dcpcrissement des porcelets (corona-like virus) an electron microscopic study of haemagglutinating encephalomyelitis virus of pigs h stability studies with avian infectious bronchitis virus (corona-virus) strains encephalomyelitis of swine caused by a hemagglutinating virus : a hemagglutinating virus producing encephalomyelitis in baby pigs encephalomyelitis of swine caused by a hemagglutinating virus in vitro differentiation and p h sensitivity of field and cell culture attenuated strains of transmissible gastroenteritis virus : establishment, viral susceptibility and biological characteristics of a swine kidney cell line sk- diagnostic procedures for viral and riqettsia diseases fluorescent antibody technique in the study of three porcine viruses transmissible gastroenteritis virus, vomiting and wasting virus and the parvovirus e / characteristics of a coronavirus causing vomition and wasting in pigs titration of two porcine respiratory viruses in mammalian cell cultures by direct fluorescent antibody staining a simple method of estimating fifty per cent endpoints the study was supported by the belgian research institute for industry and agriculture (instituut tot aanmoediging van het wetenschappelijk onderzoek in nijverheid en landbouw), brussels, belgium. the technical assistance of mrs. gerry van leeuwe is gratefully admowledged.zusammenfassung zuqtung von hemagglutinating encephalomyelitis virus (hev) in porcinen zellkulturen kulturelle eigenschaften des hev-stammes vw in primaren schweinenierenzellen (ppk), primaren schweinehodenzellen (ppt), sekundaren schweineschilddrusenzellen (spth) und in den zellinien pk- (schweinenieren), sk- (schweinenieren) und st (schweinehoden) werden beschrieben.vermehrungskurven, die mit hilfe des cytopatischen effektes (cpe), der immunofluoreszenz (if), der hamadsorption (hads) und der hamagglutination als kriterien fur die virusreplikation crstellt wurden, zeigten, daf spth und ppk-zellen am empfindlichsten fur die zuchtung und titration des virus sind. zum nachweis der virusvermehrung in rohrchen, die mit kleinen virusmengen beimpft wurden, waren cpe, hads und h a empfindliche und brauchbare kriterien.wiederholte virustitrationen zei ten eine hohe variation der titerendpunkte a u k in hochempfanglichen zel p kulturen. die optimale methode fur die isolierung von hev von klinischem material ist die verimpfung auf spth oder ppk mit anschliei ender blindpassage, wenn der ha-test sieben tage p. inf. negativ ist. culture du virus himagglutinant de l'enciphalomyclite (hev) dans des cultures de cellules de porc on dkcrit les propriktes de culture de la souche hev vw dans des cellules primaires de reins de porcs (ppk), dans des cellules primaires de testicules de porcs (ppt), dans des cellules secondaires de glandes tyroi'des de porcs key: cord- -gs ebo authors: yin, xin; feng, zongdi title: hepatitis e virus entry date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: gs ebo hepatitis e virus (hev) infection is a major cause of acute hepatitis worldwide. it is transmitted enterically but replicates in the liver. recent studies indicate that hev exists in two forms: naked, nonenveloped virions that are shed into feces to mediate inter-host transmission, and membrane-cloaked, quasienveloped virions that circulate in the bloodstream to mediate virus spread within a host. both virion types are infectious, but differ in the way they infect cells. elucidating the entry mechanism for both virion types is essential to understand hev biology and pathogenesis, and is relevant to the development of treatments and preventions for hev. this review summarizes the current understanding of the cell entry mechanism for these two hev virion types. hepatitis e virus (hev) infection is a common cause of acute hepatitis worldwide [ ] . each year about million individuals are infected with hev, resulting in~ , deaths. in recent years, increased cases have been reported in developed countries. in addition to humans, hev also infects a wide range of animal species [ ] . at least six different hev genotypes have been shown to be able to infect humans. most cases in developed countries are caused by zoonotic transmission of genotype hev through consumption of contaminated pork and game meat. in addition, genotype hev infections frequently cause persistent infections in individuals with a weakened immune system resulting in an increased risk for accelerated liver cirrhosis. hev infection-related extrahepatic manifestations including various neurologic problems have also been described [ ] . despite these public health concerns, direct antivirals are not available for hev. ribavirin, a broad spectrum nucleoside analog, has been used in treating patients with chronic hepatitis e [ , ] , but resistance mutations have been described [ , ] . there is an hev vaccine available in china that offers long-term protections against clinically symptomatic acute hepatitis caused by both genotype and hev infections [ , ] . this vaccine is currently under evaluation in a phase clinical trial in the united states (https://clinicaltrials.gov/ct /show/nct ) and in a phase clinical trial in bangladesh (https://clinicaltrials.gov/ct /show/nct ). since its discovery in the s, hev has been considered as nonenveloped [ ] . while this is true for virions in the bile and feces, it is now recognized that virions circulating in the bloodstream exist in a membrane associated, "quasienveloped" form (ehev) [ , ] . ehev particles are infectious, but they do not have classic envelope proteins thus infect cells via an unusual mechanism [ , ] . elucidating the entry mechanisms for both virion types is critical for understanding the unique life cycle of hev and its pathogenesis. knowledge obtained from studying hev entry may also aid in the development of antiviral drugs, which is crucial for patients with chronic hepatitis e experiencing ribavirin failure and in severe liver disease in pregnant women from developing countries. this review will begin with a discussion about the structural differences between the two hev virion types, then discuss the current understanding regarding the entry mechanism for each virion type. hev was discovered in by dr. mikhail balayan, a russian virologist who intentionally infected himself by ingesting pooled stool samples collected from hev-infected russian soldiers deployed in afghanistan [ ] . he subsequently developed acute hepatitis. by examining his fecal material using electron microcopy (em), dr. balayan obtained the first morphologic evidence of hev virions, which appeared as nonenveloped icosahedral particles, - nm in diameter, with a "spiky" surface [ ] . due to its similar structural appearance to the calicivirus, hev was initially misclassified within the caliciviridae family. in , hev was molecularly cloned and its full genome was sequenced [ ] . the significant sequence divergence of hev from other known families of viruses led to its reclassification into a new family, hepeviridae. the . kb single-stranded positive sense hev rna genome was shown to be polyadenylated and contain three open reading frames (orfs) (figure a ). the ' two-thirds of the genome encodes orf , a large multi-domain polyprotein that is involved in the genomic replication. the rest of the genome encodes orf and orf , both of which are translated from a subgenomic rna generated during the virus replication cycle. orf is the only capsid protein of hev. the orf protein is unique to hev and plays a role in virion egress [ ] . a new orf, orf , was recently discovered within the coding region of orf , but appears to be restricted only to genotype hev [ ] . viruses , , x for peer review of the development of antiviral drugs, which is crucial for patients with chronic hepatitis e experiencing ribavirin failure and in severe liver disease in pregnant women from developing countries. this review will begin with a discussion about the structural differences between the two hev virion types, then discuss the current understanding regarding the entry mechanism for each virion type. hev was discovered in by dr. mikhail balayan, a russian virologist who intentionally infected himself by ingesting pooled stool samples collected from hev-infected russian soldiers deployed in afghanistan [ ] . he subsequently developed acute hepatitis. by examining his fecal material using electron microcopy (em), dr. balayan obtained the first morphologic evidence of hev virions, which appeared as nonenveloped icosahedral particles, - nm in diameter, with a "spiky" surface [ ] . due to its similar structural appearance to the calicivirus, hev was initially misclassified within the caliciviridae family. in , hev was molecularly cloned and its full genome was sequenced [ ] . the significant sequence divergence of hev from other known families of viruses led to its reclassification into a new family, hepeviridae. the . kb single-stranded positive sense hev rna genome was shown to be polyadenylated and contain three open reading frames (orfs) ( figure a ). the ' two-thirds of the genome encodes orf , a large multi-domain polyprotein that is involved in the genomic replication. the rest of the genome encodes orf and orf , both of which are translated from a subgenomic rna generated during the virus replication cycle. orf is the only capsid protein of hev. the orf protein is unique to hev and plays a role in virion egress [ ] . a new orf, orf , was recently discovered within the coding region of orf , but appears to be restricted only to genotype hev [ ] . the orf capsid protein of hev contains amino acids (a.a) that are divided into structural domains: a shell (s) domain, a middle (m) domain, and a protruding (p) domain comprised of a.a. - , - , and - , respectively [ ] . each virion consists of copies of an orf dimer, forming a t = icosahedron. recombinant orf proteins expressed in the bacterial and insect cells can self-assemble into virus-like particles (vlps), of which the crystal structures have been determined [ ] [ ] [ ] [ ] . p , a t = vlp composed of a.a. - and produced in e. coli, is the main component of the vaccine hecolin currently marked in china. according to the d structure, the s domains form the base of the icosahedron, and the dimeric p domains form the "spikes" extending from the virion surface that are believed to be responsible for receptor binding and a main target for neutralizing antibodies [ ] . the enteric route of transmission, em evidence of naked virions in the feces, and the lack of coding capacity for envelope proteins all suggest that hev is a nonenveloped virus. however, recent studies show that the virus released from infected cells and circulating in the blood adopts a membrane associated, "quasienveloped" form, named "ehev" [ , , ] (figure b ). these particles band at a much lower buoyant density (~ . g/cm ) in isopycnic gradient centrifugation than the the orf capsid protein of hev contains amino acids (a.a) that are divided into structural domains: a shell (s) domain, a middle (m) domain, and a protruding (p) domain comprised of a.a. - , - , and - , respectively [ ] . each virion consists of copies of an orf dimer, forming a t = icosahedron. recombinant orf proteins expressed in the bacterial and insect cells can self-assemble into virus-like particles (vlps), of which the crystal structures have been determined [ ] [ ] [ ] [ ] . p , a t = vlp composed of a.a. - and produced in e. coli, is the main component of the vaccine hecolin®currently marked in china. according to the d structure, the s domains form the base of the icosahedron, and the dimeric p domains form the "spikes" extending from the virion surface that are believed to be responsible for receptor binding and a main target for neutralizing antibodies [ ] . the enteric route of transmission, em evidence of naked virions in the feces, and the lack of coding capacity for envelope proteins all suggest that hev is a nonenveloped virus. however, recent studies show that the virus released from infected cells and circulating in the blood adopts a membrane associated, "quasienveloped" form, named "ehev" [ , , ] (figure b ). these particles band at a viruses , , of much lower buoyant density (~ . g/cm ) in isopycnic gradient centrifugation than the traditional, nonenveloped virions (~ . g/cm ) [ ] , and are insensitive to neutralizing anti-capsid antibodies in standard neutralization assays [ ] . under em, these particles look like enveloped viruses, with hev capsids encased in limiting host-derived membranes [ ] . unlike naked hev particles, the ehev particles contain both orf and orf proteins. however, both of them are hidden within the host-derived membranes and can only be detected by specific antibodies upon disruption of the membrane by detergent [ ] . ehev is the only form detected in blood [ ] , suggesting that it mediates hev spread within the host. the biogenesis of ehev has been reviewed elsewhere [ , ] . the prevalent model involves viral hijacking of the cellular endosomal sorting complex required for transport (escrt) machinery, producing an exosome-like vesicle containing the viral capsid and orf protein. usurping escrt components is a common mechanism for the budding of classic enveloped viruses [ ] . the hev orf protein plays a key role in this process. a proline-serine-alanine-proline (psap) late domain motif located at the c-terminus of orf specifically interacts with the cellular tsg (tumor susceptibility gene- ) protein, an escrt-i protein, which then recruits escrt-ii and -iii complexes to promote the budding of the hev capsid into multivesicular bodies (mvb) [ ] . subsequent fusion of mvb with the plasma membrane leads to the release of single membrane-encased ehev particles. consistent with the biogenesis pathway for exosomes, ehev membranes contain tetraspannins such as cd , cd , and cd [ , ] . certain host proteins are likely copackaged during ehev biogenesis, but their identity and roles in the hev life cycle remain largely unexplored. a recent study used a quantitative proteomic approach to identify proteins associated with quasienveloped hepatitis a virus (ehav) [ ] . it has revealed that the ehav particles are highly enriched for components of the endolysomal system such as cd , dpp (dipeptidylpeptidase , cd ), alix, and epcam (epithelial cell-adhesion molecule), but lack lc- (an autophagosomal protein)-related peptides, consistent with an endosomal exosome-like origin of ehav. this approach may be also useful for identifying ehev-associated proteins. the nonenveloped hev particles found in the feces likely originated from ehev released through the bile canaliculi, but their membrane are stripped by the detergent action of bile [ ] . the lack of lipid membrane renders the nonenveloped virions much more stable in the environment to facilitate transmission to new hosts. the production of two virion types is integral to hev biology. thus, it is important to understand how each virion type plays a role in the infection process. the available experimental evidence suggests that they enter and infect target cells through distinct pathways. the naked, nonenveloped hev particles are present in the bile and feces of infected patients or experimentally infected monkeys [ , , ] . these particles are stable in the environment and ideal for transmission to new hosts. although hev is primarily transmitted enterically, how it penetrates the gut barrier to reach the bloodstream remains enigmatic. no compelling evidence exists that hev replicates in the human gut, although it is possible that hev infects only a rare cell type in the gut, as is the case for norovirus [ ] . regardless of the origin of the first cell to be infected, whether a hepatocyte or a yet-to-be-identified cell type in the gut, naked hev is necessary for establishing the first round of infection. despite being highly hepatotropic in vivo, under in vitro conditions, hev is able to infect a range of cell types other than hepatocytes, including a (human lung epithelial cells), caco- (human colon epithelial cells), human neuronal-derived cells, and human placenta cells [ ] [ ] [ ] [ ] [ ] [ ] [ ] . this widened cell tropism in vitro is not unique to hev, as hepatitis a virus (hav), which is known to only infect hepatocytes, can also infect many nonhepatic cell types in vitro [ ] . it should be noted that cancer cell lines often bear genetic abnormalities and altered phenotypes as compared to their corresponding primary cells in vivo, thus results obtained from such cells must always be interpreted with caution. study of the hev life cycle has been hampered by the lack of efficient cell culture systems for hev [ ] . the particle to focus-forming unit (ffu) ratio for hev ranges from to , , prohibiting the use of single particle imaging techniques to track the hev entry process. most published studies have used fluorescently labeled vlps to study the early steps of cell entry including initial cell attachment and internalization. the recent development of hev infection systems has provided the first opportunity to study the entire hev life cycle in detail [ , ] . below we summarize the current knowledge regarding the entry process of naked hev particles. the receptor for hev is unknown. however, a number of host factors have been shown to be involved in cell attachment and/or entry of naked hev. these factors are summarized below. hsgps are glycans present on the cell surface that are involved in cell attachment of many nonenveloped and enveloped viruses. hsgps, particularly syndecans, have been shown to play a role in the binding of hev vlps to human hepatoma cells [ ] . treatment of cells with heparinase reduced vlp binding and hev infection. however, hsgps are not essential for cell attachment and infection by ehev particles [ ] . grp , also known as bip (binding immunoglobulin protein), is a molecular chaperone in the er. however, presence of grp on the cell surface has also been described and implicated in the attachment and entry of both enveloped and nonenveloped viruses [ ] [ ] [ ] [ ] . grp binds to p vlps in both co-immunoprecipitation and a cell model [ ] . asgpr is a protein receptor present on the basolateral membrane of hepatocytes that binds glycoproteins that lack sialic acid modifications. a direct interaction has been shown between the ectodomain of both asgr and asgr and hev orf by coimmunoprecipitation, pull-down, and elisa [ ] . ectopic expression of asgrp in hela cells increased hev binding, whereas depletion of asgpr in plc/prf/ cells lowered hev binding but not virion release. both anti-asgpr antibody and purified asgpr ectodomain also reduced hev binding to plc/prf/ cells. atp synthase is largely a mitochondrial protein, but a small fraction is expressed on the cell surface and is implicated in other viral infections [ ] . atp b was identified as a binding partner on the p vlp using a pull down and mass spectrometric approach [ ] . the role of atp b in hev entry was validated using antibody and sirna mediated approaches, and infectious hev from the stool of a hepatitis e patient. integrin α was recently identified as an entry factor for hev in plc/prf/ cells [ ] . hev permissive and nonpermissive subclones of plc/prf/ have been identified, and compared to each other by microarray. overexpression of integrin α in nonpermissive cells rendered cells permissive for hev, while knocking out integrin α in permissive cells abrogated permissiveness. for unknown reasons, none of these subclones supported infection by ehev particles. although these results are encouraging, independent studies will be needed to validate the role of these factors in hev and ehev cell attachment and entry in the context of infection, preferably in primary human hepatocytes. using gfp-and firefly luciferase-tagged vlps and naked hev particles, kapur et al. showed that hev is internalized by cells in a clathin-, dynamin-dependent pathway [ ] . an independent study using fitc-labeled vlps subsequently confirms these results, and additionally shows that hev-like particles initially traffic to rab -positive compartments, then to acidic lysosomal compartments where they are degraded [ ] . the same study has also identified membrane cholesterol, the pi k pathway, and actin as important factors for hev internalization and infection, but low ph is not required. more recently, using naked hev purified from cell lysates, we have shown that hev infectivity is dependent on clathrin and dynamin , consistent with others. however, we found that the infectivity of naked hev is not affected by inhibiting rab and rab , or by lysosomotropic agents such as bafilomycin and ammonium chloride [ ] . these results suggest that the uncoating of naked hev particles occurs before reaching the rab + compartment, and the co-localization of capsids to rab observed by others likely represent empty capsids that remain in the endocytic pathway after genome release. as a cytoplasmically replicating rna virus, hev has to deliver its genome across the endosomal membrane to access the cytoplasm for translation and replication. how this process takes place remains poorly understood. studies with other nonenveloped rna viruses have demonstrated that upon receptor binding the virions undergo structural rearrangements to cause the externalization of hydrophobic peptides [ ] . these hydrophobic peptides subsequently insert into the endosomal membrane, creating a channel/pore through which the genome traversed the endosomal membrane to enter the cytoplasm. viral capsids remaining in the endosome are ultimately degraded in the lysosomes. since naked hev particles do not colocalize with rab and rab , hev capsids may undergo substantial conformational changes during the uncoating process. the presence of a quasienvelope raises several questions about the entry mechanism for ehev. how does ehev bind to cells and how is its cell tropism determined? does ehev entry involve membrane fusion? and does ehev have a different uncoating mechanism from naked hev? while there are no definitive answers for these questions, available evidence suggests that the ehev quasienvelope is degraded by lysosomal enzymes prior to uncoating. if this model holds true, it would present a novel entry mechanism for viruses. since quasi-enveloped hev particles do not have viral proteins on the surface of their envelope, they must use different attachment factors and/or cellular receptors to initiate the viral entry. as with exosomes, the ehev membrane has phosphatidylserine, which may bind to its receptor such as tim- on the target cells [ , ] . our previous study found that the cellular uptake of the ehev virions is less efficient than naked hev due to inefficient cell attachment, indicating that the undetermined molecules on the ehev surface mediate the cell attachment with slower kinetics [ ] . unlike the naked hev virion, the attachment of ehev to the hepatocytes does not require hspgs [ ] . the less specific cell binding by ehev may provide an explanation for the detection of hev beyond the liver [ ] . in addition, the exosome-like nature could facilitate its penetration of immunologically privileged sites such as the central nervous system. since hev infection has been associated with various types of extrahepatic manifestations [ ] , a better understanding of the tropism and replicative capacity of ehev in relevant cells/tissues will help differentiate between virus-mediated and immune-mediated effects in these conditions and shed light on hev pathogenesis. similar to naked hev particles, ehev enters hepatocytes mainly through the clathrin-and dynamin-dependent pathway [ , ] . following the endocytic uptake, ehev particles are transported sequentially into rab + and rab + endosomal compartments, and eventually reach the lysosome, where the uncoating is thought to take place [ , ] . treatment of cells with lysosomotropic agents such as bafilomycin a and nh cl dramatically reduces ehev infectivity, indicating that the endosomal acidification is required for ehev entry. however, the low ph itself is not sufficient for ehev cell entry, since extracellular exposure to low ph did not alter the density or infectivity of ehev [ ] . the study of ehav has provided additional insights into the intracellular trafficking of quasienveloped viruses [ ] . confocal imaging experiments using fluorescently labelled ehav particles suggests that intact ehav is transported to the lysosome, but similarly labelled exosomes produced by uninfected cells do not colocalize with lysosomal markers. it is speculated that the ehav membrane may contain a signal that drives its continued trafficking towards the lysosome [ ] . whether ehev uses a similar mechanism for trafficking to the lysosome is unknown, but would be an interesting area for future investigation. the presence of a quasienvelope would require the hev capsid to penetrate two layers of membranes in order to deliver the viral genome into the cytoplasm. one possible way to achieve this is through fusion of the viral membrane with the cellular membrane. however, membrane fusion would result in the intact capsid, rather than the genome, entering the cytoplasm, likely a dead end for hev. a second, more likely scenario is by removing the quasienvelope, so that the capsid will be exposed and available for binding to a receptor. lipid membrane degradation in lysosomes is a complex process [ ] . a critical step for lipid membrane degradation is the niemann-pick disease type c (npc )-mediated extraction of cholesterol from lipids. depletion of npc reduced ehev infection by % without significantly affecting hev infectivity [ ] . moreover, cells pretreated with a specific inhibitor of lysosomal acid lipase (lal), an enzyme essential in lipid metabolism because it hydrolyzes cholesteryl esters and triglycerides in lysosomes [ ] , displayed a dose-dependent reduction in ehev infectivity, while no reduction was observed for non-enveloped hev infectivity [ ] . these results provide evidence that the ehev membrane is degraded in the endolysosomes, rather than fusing with the host membrane ( figure ) . a similar requirement of npc and lal has also been described for ehav entry [ ] . once ehev loses its membrane, the capsid would be free to interact with its receptor on the endosomal membrane. at the simplest level, the capsid binds to the same receptor for naked hev and uncoat via the same mechanism. many cell surface proteins cycle between the plasma membrane and endosomes, serving as attractive candidates if this is the case. alternatively, a different receptor may be used for ehev capsids, and it is not uncommon for viruses to switch receptors during the entry process [ , ] . an unexplored question is the role of orf in ehev entry. orf has been shown to interact with orf [ ] . thus, the presence of orf may interfere with receptor binding. in addition, orf possesses an ion channel activity that is required for hev egress [ ] . it will be interesting to know whether this activity also plays a role in ehev entry. once ehev loses its membrane, the capsid would be free to interact with its receptor on the endosomal membrane. at the simplest level, the capsid binds to the same receptor for naked hev and uncoat via the same mechanism. many cell surface proteins cycle between the plasma membrane and endosomes, serving as attractive candidates if this is the case. alternatively, a different receptor may be used for ehev capsids, and it is not uncommon for viruses to switch receptors during the entry process [ , ] . model for cellular entry of naked and quasi-enveloped hev virions. naked hev virions initially bind nonspecifically to hspgs, followed by a specific interaction with a putative cell receptor. receptor binding triggers virion internalization via clathrin-mediated endocytosis and virions subsequently uncoat in a rab − early endocytic compartment. quasienveloped hev virions attach to cells less efficiently than naked virions, but are similarly internalized via clathrin-mediated endocytosis. virions are routed through early (rab +) and late (rab +) endocytic compartments and ultimately reach the lysosome. the quasi-envelope becomes slowly degraded by lysosomal enzymes, allowing the exposure of the capsid which subsequently penetrates the endosomal membrane to release its genome into the cytoplasm. the presence of two distinct virion types has challenged our view about the fundamental biology and pathogenesis of hev. the ability of hev to infect its target cells both in the presence and absence of a viral membrane is a paradigm shift in virology and an exciting area for future investigations. the available evidence suggests that naked and quasienveloped hev particles use different mechanisms for cell entry, and that entry of ehev requires lysosomal degradation of the viral membrane. identifying the cellular receptor for both virion types will be key to elucidating their entry mechanisms. since ehev is the only form detected in the bloodstream, understanding how ehev spreads has the potential for identifying targets for intervention. with the recent development of more efficient culture systems for hev, it is anticipated that our understanding of hev entry will be improved within the next few years. funding: this work is supported by grants from the national institute of allergy and infectious diseases (ai and ai ), the gilead science research scholars program in liver disease, and internal startup funds from the nationwide children's hospital (to z.f.). the authors declare no conflict of interest. hepatitis e virus: advances and challenges the current host range of hepatitis e viruses ribavirin therapy inhibits viral replication on patients with chronic hepatitis e virus infection ribavirin for chronic hepatitis e virus infection in transplant recipients a mutation in the hepatitis e virus rna polymerase promotes its replication and associates with ribavirin treatment failure in organ transplant recipients hepatitis e virus mutations associated with ribavirin treatment failure result in altered viral fitness and ribavirin sensitivity efficacy and safety of a recombinant hepatitis e vaccine in healthy adults: a large-scale, 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epithelial tight junctions the phosphorylated form of the orf protein of hepatitis e virus interacts with its non-glycosylated form of the major capsid protein, orf hepatitis e virus orf is a functional ion channel required for release of infectious particles key: cord- - rsk g l authors: kinast, volker; burkard, thomas l; todt, daniel; steinmann, eike title: hepatitis e virus drug development date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: rsk g l hepatitis e virus (hev) is an underestimated disease, leading to estimated million infections and up to , deaths annually. infections are mostly asymptomatic but can reach mortality rates up to % in pregnant women or become chronic in immunocompromised patients. the current therapy options are limited to the unspecific antivirals ribavirin (rbv) and pegylated interferon-α (pegifn-α). rbv leads to viral clearance in only % of patients treated, and is, similar to pegifn-α, contraindicated in the major risk group of pregnant women, emphasizing the importance of new therapy options. in this review, we focus on the urgent need and current efforts in hev drug development. we provide an overview of the current status of hev antiviral research. furthermore, we discuss strategies for drug development and the limitations of the approaches with respect to hev. with approximately million infected people per year, hepatitis e virus (hev) leads to more cases of acute hepatitis than any other human hepatotropic virus, such as hepatitis a virus (hav), hepatitis b virus (hbv), hepatitis c virus (hcv) and hepatitis d virus (hdv). hev is a quasi-enveloped positive strand rna virus ( figure a ) that is classified as a member of the genus orthohepevirus within the family of hepeviridae [ ] . the genotypes (gt) and of hev are obligate human pathogens and are primarily transmitted via contaminated drinking water. recent outbreaks of acute hepatitis linked to hev have, amongst others, been reported in nigeria [ ] , chad [ ] , and bangladesh [ ] . by contrast, the zoonotic gt and , which are endemic especially in europe and the americas, can cause in addition to acute infections also chronic infections in immunocompromised individuals. the most common infection routes are thought to be the consumption of undercooked meat of or contact with infected animals, such as pigs, wild boars, and deer, which constitute the virus reservoir. furthermore, the transfer of contaminated blood products is a man-made safety hazard, especially for risk groups such as immunocompromised patients [ ] . recent cases of human infections with gt [ ] and rat hev [ , ] extended the spectrum of hev gts, which are capable of jumping over the species barrier and are able to infect humans. acute hev infections are self-limiting in most cases, but especially for gt , they are linked to high mortality rates up to % in pregnant women [ ] . it is hypothesized that immunological and hormonal changes are responsible for the high mortality [ ] . hev has also been reported to cause a variety of extrahepatic manifestations, for instance, guillain-barré syndrome or pancreatitis (reviewed in [ ] , see figure b ). in total, . million estimated cases of acute illness and , - , deaths per year make this pathogen a non-negligible health burden. however, the current therapeutic options against hev are limited to the off-label use of the unspecific antivirals ribavirin (rbv) and pegylated interferon-α (pegifn-α). the treatment algorithm for figure . schematic representation of hepatitis e virus (hev) particle and the major clinical manifestations. (a) hev particle and genomic organization. the hev genome is composed of a single-stranded rna genome of~ . kb and is encapsulated in an icosahedral capsid. hev virions can occur in both a non-enveloped and in an enveloped form. the viral rna, which is capped with -methylguanosine ( mg) at the ´noncoding region and polyadenylated at the noncoding region, comprises three open reading frames (orf). furthermore, gt is believed to contain an additional orf (orf ). orf encodes the replicase proteins, including a methyltransferase (mt), cysteine protease (pro), helicase (hel), and rna polymerase (pol), as well as three regions without a reported enzymatic function (y, hypervariable region (hvr), and x). orf encodes the capsid protein, whereas orf encodes a viroporin. (b) major clinical manifestations. the majority of hev infections are asymptomatic. gt and gt infections can become chronic in immunosuppressed individuals, with high risk for developing severe complications, such as liver cirrhosis. hev has also been reported to cause a variety of extrahepatic manifestations, like guillain-barré syndrome. infections with hev gt cause acute hepatitis, with high mortality rates up to % in pregnant women. identification of novel therapy options can encompass several strategies. while some rely on de novo identification of compounds, another approach is to reuse already existing compounds, a process which is termed drug repurposing. drug repurposing can generally be described as the idea of using drugs or drug candidates for another than their primary indication. the pharmacokinetic and pharmacodynamic profile, including undesirable effects, is often already elucidated in vivo in animals and humans. the current first choice of anti-hev treatment, rbv, is an example for a repurposed drug that was originally developed for treatment of respiratory syncytial virus (rsv) in infants [ ] . rbv has not only been administered for antiviral therapy against rsv but also against hcv, influenza, viral hemorrhagic fevers [ ] and hev [ ] . recently, drug repurposing has gained increased attention in the scientific community with reviews covering either specific viruses [ ] or viral diseases in general [ , ] . surprisingly, there are only a few publications, in which this approach is used as a strategy to find an antiviral against hev. however, given the availability of public fda-approved compound libraries and the anticipated benefits of this approach over de novo development, publications screening for antiviral activity of already approved drugs are anticipated. de novo drug development can rely on screens, where compound libraries are tested for their capacity to interfere with the viral life cycle. both the target and the mode of action of the substance do not need to be identified. for structure-guided development, the target and ideally its crystal structure is already identified, which enables to specifically design antivirals. independent from the approach used to identify a prospect compound, the candidates have to be validated in vitro and in vivo. there are several in vitro models available, including different cell culture models as well as primary human hepatocytes and induced pluripotent stem cells. for a detailed overview, please see a recent review by meister et al. [ ] . similarly, there are several small animal models as rabbits, rats, ferrets, and birds, which are used for the different hev strains (reviewed in [ ] ). this review aims to give an overview over the current state of efforts to establish additional treatment options against hev. antiviral candidates are grouped according to the strategy that was used to identify them. before the concluding remarks, vaccination is also briefly discussed in terms of its potential to replace antiviral therapy options. '-c-methylcytidine ( -cmc), also known as nm , is a nucleoside analogue (na) originally developed against hcv. when first applied against hev in vitro, the compound demonstrated an inhibitory effect against hev replication (ic of µm/l) [ ] . furthermore, it inhibited the replication of both a luciferase replicon based on kernow c as well as the full-length virus without showing signs of resistance upon prolonged incubation [ ] . the effect could be reverted by addition of cytidine triphosphate, but not guanosine triphosphate to the cells. applying it together with rbv yielded in a moderate antagonistic effect, suggesting a convergent mechanism of inhibition. when assessed for treatment of hcv, insufficient oral bioavailability was reported. therefore, a prodrug termed nm was developed [ ] . however, the development was discontinued due to adverse toxic effects, which has been a problem for several 'methyl nucleosides [ ] . the toxicity correlates with the property to serve as a substrate for the mitochondrial dna polymerase and thus leading to the termination of mitochondrial rnas [ ] . the example of nm emphasizes that -cmc, although efficacious against hev in vitro, will probably need modifications to rule out undesired effects, in particular against the mitochondrial dna polymerase. recently, netzler and colleagues tested a collection of compounds with a reported inhibitory effect on the rna-dependent rna polymerase (rdrp) of different classes of viruses [ ] . both the class of nas as well as non-nucleoside inhibitors (nni) were considered in their study, covering compounds from late-preclinical stage to fda-approved drugs. they identified two compounds, gpc-n and nitd , inhibiting a gt derived subgenomic replicon. gpc-n had a half maximal effective concentration (ec ) of . µm and a therapeutic index (ti) of > (gpc-n ) whereas nitd had an ec . µm and a ti > . gpc-n has been described as an nni of the rdrp of multiple genera within the picornaviridae family [ ] . nitd has been reported to act as an antiviral on numerous viruses, amongst others hcv [ ] , west nile virus [ ] or zika [ ] . nitd has shown toxicity in vivo [ ] . so far, no clinical trials have been registered for both of these compounds. nishiyama and colleagues used an fda approved drug library for an in vitro screen against a gt strain containing a gaussia luciferase reporter [ ] . ciprofloxacin (cpfx) and ifn-λ - showed the best activity against the reporter genome. they also used an infection model, where cells infected with a full-length gt strain at a plateau phase were co-cultivated with naïve cells. all ifn-λ subtypes, but not cpfx, showed robust reduction of hev rna. sofosbuvir is a nucleotide prodrug, which can be incorporated into hcv rna by the ns b rdrp acting as a chain terminator [ ] . its antiviral potential against hev was demonstrated using gt hev replicons in huh and hepg cells. the ic values were in a low micromolar range, to orders of magnitude less potent than against hcv replicons [ ] . sofosbuvir showed an additive effect together with rbv. the inhibitory effect was confirmed by the same group in induced pluripotent stem-cell derived hepatocyte-like cells (ipsc-hlcs) for all four common gts [ ] and in cell culture for gt [ ] as well as gt [ ] by other groups. however, it was also described that both gt and gt replicons as well as full-length gt virus were not inhibited in hek t, u- mg, and huh cells [ ] . very recently, li et al. reported that a gt strain was moderately inhibited at µm by an isopropyl ester of sofosbuvir, psi [ ] . similar to the situation in vitro, the data on sofosbuvir's efficacy in vivo have been inconclusive. three case studies stated that sofosbuvir failed to clear hev in patients when administered together with rbv [ ] [ ] [ ] , while three reported successful treatment together with rbv [ ] [ ] [ ] . a multicenter phase ii study addressed whether sofosbuvir monotherapy is efficacious against hev. a total of mg sofosbuvir was administered once daily over a course of weeks to nine patients who had previously failed rbv therapy [ ] . none of the patients cleared the virus, although it reduced hev rna levels by at least one order of magnitude in five out of nine patients during the study period and significantly reduced alanine aminotransferase (alt) levels. with its antiviral efficacy being only moderate, it is probably not suited for use as monotherapy. however, it could further be investigated in combination with rbv. compound screening allows for the rapid testing of a large number of chemical substances and extracts with the aim of identifying new compounds. in the context of viral infections, important drugs identified with this method include maraviroc and etravirine [ ] , both of which target hiv as well as daclatasvir, which targets hcv [ ] . prerequisite for compound screening is a suitable assay, reflecting the physiological conditions of infection. importantly, the screening method has the benefit of not necessarily requiring profound knowledge about the viral lifecycle or specific targets. assays using purified enzymes, subgenomic replicons or full virus already have successfully been used in hcv research. there have been two reports about the antiviral activity of ethanol extracts of plants against hev. one extract was prepared from the plant lysimachia mauritiana and showed activity against a pshev -luc replicon in huh . cells, as well as while a full-length gt virus on rna and protein level in a cells [ ] . however, identification of the compound(s) responsible for the antiviral effect was not performed. the same group reported the antiviral effect of an ethanol extract of another plant, liriope platyphylla, against hev [ ] . both a pshev -luc-replicon as well as the c full-length genome were inhibited by the extract in huh . cells. by performing activity-guided fractionation and multicolumn chromatography, spicatoside a could be identified as the active compound, and its activity as a pure compound was demonstrated. no testing for toxicity, resistance induction or in vivo efficacy was conducted. zinc is an essential micronutrient that has been reported to reduce replication of, amongst others, hiv [ ] and coronavirus [ ] in high concentrations. in a study investigating the influence of different salts on hev replication, it was demonstrated that zinc salts inhibited replication of both gt and replicons as well as a gt clinical isolate in porf -huh cells [ ] . the effect was likely due to an inhibition of the rdrp, as an inhibitory effect on the protein was observed in vitro. a mild effect could be observed at µm, being strongest at µm. it will be of interest to investigate if zinc supplementation will prove an effective strategy against hev in patients. zinc levels in plasma have been reported to be usually around - µm depending on the study, as described in a meta-analysis [ ] . the authors also found that zinc supplementation increased plasma levels, with every doubling of the dose leading to a % increase [ ] . these data imply that zinc levels are highly regulated, suggesting that effective plasma levels in patients might be at best challenging to reach. there has been no evaluation of zinc monotherapy against hev in vivo so far. however, a case study [ ] investigated the influence of intra-erythrocyte zinc levels on the outcome of rbv treatment. they showed that treatment successes could not be attributed to increased zinc levels. although only comprising four patients in total, this suggests that it might not lead to viral clearance in immunocompromised individuals when combined with rbv. e is part of a small molecule compound library belonging to the diversity set ii of national cancer institute developmental therapeutic program. it was initially identified as an inhibitor of the rdrp of hcv, inhibiting hcv a replicon with an ec of . µm [ ] . the compound also inhibited hev, both the replicon p -luc as well as full-length p in huh or huh s - cells. interestingly, it did not show inhibitory effect on purified rdrp for both hcv and hev, suggesting that it does inhibit replication either by a affecting a host factor or that it does undergo modification within the host. no testing resistance induction or in vivo efficacy was performed. different from screening approaches, target-or structure-guided development aims at identification of suitable targets for an intervention first. prerequisite is that the function of a target is characterized. both the virus itself and the host factors necessary for the viral life cycle can be targeted ( figure ). only one protein of hev has been crystallized so far, which is a truncated version of the capsid protein porf [ , ] . although some conclusions regarding the involvement of certain domains in cell binding could be drawn, and heparan sulfates seem to be important for hev attachment to the host cell surface [ ] , this knowledge has not led to the establishment of a therapy concept yet. the scarcity of antiviral candidates directly acting on the virus can be explained by the lack of knowledge about hevs molecular virology. due to their small genomes, viruses depend on host factors for completion of their life cycle. this dependence on the host is a potential point to intervene. host-acting antivirals have the potential to interfere with the distinct steps of the viral life cycle, from blocking the entry receptor over preventing the formation of the replication complex to hamper the viral maturation by inhibiting cellular proteins. a detailed functional analysis of the host factor and its role in the context of hev infection is desirable and would help to optimize chemical intervention. additionally, direct acting antiviral specifically can target viral enzymes (e.g., helicase, polymerase, protease) without affecting host components. notably, the nucleoside analog ribavrin is reported to exert antiviral effects by targeting both the virus and the host [ ] . a hammerhead ribozyme is a small catalytically active rna molecule, capable of cleavage or self-cleavage [ ] . hammerhead ribozymes have been designed to target, amongst others, severe acute respiratory syndrome coronavirus (sars-cov) [ ] or hcv [ ] . however, there has only been one report so far describing the use of this technique in vivo [ ] , which dates back already more than a decade. in , it was reported that a hammerhead ribozyme could be designed to specifically cleave rna of hev gt in the 'cis-acting element [ ] . when expressed from a vector in hepg cells, they could decrease replication of a psgi-hev- 'luc construct. questions about delivery of the agent to target site in vivo in patients, as well as potential off-target effects, might remain unsolved, considering that there has been no follow-up study so far. peptide conjugated phosphorodiamidate morpholino oligomer (ppmo) are uncharged nucleic acid analogs containing a morpholine backbone connected by phosphorodiamidate linkage, rendering them resistant to many enzymes like nucleases, esterases, and proteases [ ] . ppmos bind rna via watson-crick base pairing and interfere with viral translation by steric blockage. ppmos complementary to sequences in sar have been shown to inhibit replication of a psk-e replicon in huh s - cells. they also reduced porf levels in hepg /c cells infected with kernow c and huh s - cells infected with psk-e [ ] . so far, there has been no follow-up on this study. mg , an inhibitor of the proteasome with low nanomolar inhibitory constant [ ] has been suggested as an antiviral against hev. it reduced rna and protein levels of a hev gt replicon in huh s - cells [ ] . however, when others reproduced the experiment in huh cells, they also found reduced expression of several housekeeping genes as well as overall rna levels [ ] . it is therefore assumed that mg inhibitory effect on hev is unspecific. some viruses can bind the cellular protein tumor susceptibility gene (tsg ) to hijack the escrt machinery for egress, for instance, hiv- [ ] . it was demonstrated that porf of hev can bind tsg via a psap motif in the viral protein and does colocalize with it in hev-transfected cells [ ] . consequently, porf is essential for viral egress, while having a neglectable effect on cell binding and replication [ ] . the interaction with tsg is mediated via the psap motif in the porf [ ] , the same motif that p gag of hiv- uses to engage with the cellular protein [ ] . cyclic peptides (cp) that had been developed to abrogate interaction of p gag and tsg and inhibited viral release of hiv virus like particles (vlps) [ ] were tested for their activity against hev [ ] . one of the inhibitors, cp , inhibited interaction of tsg and porf both in a yeast-three hybrid screen as well as in a pulldown assay. viral release of both gt and gt hev from huh porf or huh cells, respectively, was inhibited without showing significant toxicity. inhibition of viral release by targeting porf /tsg interaction is an interesting mechanism for antiviral development, with cp providing a starting point for further compound development and characterization. in general, targeting production of nucleotide synthesis to deplete or imbalance nucleotide pools is a strategy employed against several viruses [ , ] . the compounds rbv and mycophenolic acid (mpa), both of which target enzymes involved in nucleotide synthesis, are either already used as treatment against hev or have been reported for their potential to inhibit the virus. as its ester mmf, mpa inhibits the inosine- -monophosphate dehydrogenase (impdh), which is a pivotal enzyme in the purine nucleotide synthesis and is part of immunosuppressive regimens in organ transplant recipients. in a german study, a tendency was identified for its use in heart-transplant recipients being linked to clearance of hev infection without development of chronicity [ ] . in huh cells, mpa inhibited replication of a p -luc replicon, an effect that could be reverted by supplementation of guanosine, suggesting inhibition of impdh as inhibitory mechanism [ ] . in the same study, an additive effect with rbv was found. these results could not be confirmed in patients. a french study assessed the effect of immunosuppression by mmf on rbvs' potential to lead to a sustained virological response (svr) [ ] . they did not find evidence for an additive effect of mmf on rbv treatment. because of its immunosuppressive effect, mmf monotherapy to treat hev infections seems unlikely. given the lack of evidence for an additive effect with rbv, it will probably also not be used to treat hev infections together with rbv. although mmf seems unsuited as a drug against hev, targeting the impdh or enzymes involved in the purine synthesis pathway might still be a starting point for further studies. in a proof-of-concept study, wang and colleagues [ ] tested custom designed inhibitors of the impdh. all inhibitors decreased replication of a p -luc replicon, emphasizing the potential of this target. furthermore, inhibitors of pyrimidine synthase like brequinar and leflunomide inhibited the replicon. this is especially interesting, since both drugs have been tested in clinical trials and are therefore better characterized than newly developed inhibitors. targeting nucleotide synthesis, especially of pyrimidines, might therefore be an interesting approach to tackle hev, with brequinar and leflunomide already providing starting points for testing in vivo. it would be of interest to characterize these compounds better to evaluate their potential as hev antiviral. silvestrol is a natural compound belonging to the class of cyclopenta[b]benzofuran compounds, which are exclusively found in plants of the aglaia genus [ ] . silvestrol has been shown to target the translation initiation factor a (eif a) to rna, thereby preventing ribosome loading onto mrna and blocking translation [ ] . originally described in the context of cancer treatment, silvestrol has been reported to inhibit several viruses in vitro [ ] [ ] [ ] . silvestrol reduced viral titers, the number of infected a cells, as well as viral protein levels in infected cells in vitro [ ] . another study confirmed the inhibitory effect of silvestrol on gt replicons in hepg cells with an ic of around nm for gt hev [ ] . the inhibitory effects were consistent for different patient isolates covering the hev gt - , which were evaluated in ipsc-hlcs. silvestrol reduced viral titers in feces in xenograft mice infected with hev gt , therefore demonstrating its effectiveness in vivo. importantly, silvestrol was effectively inhibiting a p -luc replicon harboring the g r mutant, which confers rbv resistance [ , ] . these data demonstrate that silvestrol might provide a therapy option for otherwise untreatable rbv resistant cases. characterization of potential resistance barriers and a structure-activity relationship of the compound will be important next steps for further development of a promising candidate. vaccination may be an important strategy to reduce the global burden of the virus. in china, there is a vaccine licensed under the name hecolin ® . it consists of the amino acids - of the capsid e protein of hev gt , produced in e. coli [ ] . three doses at , , and months were tested in healthy patients - years of age [ ] . long-term studies following the same cohort up to months after vaccination showed an efficacy of . % in the intention-to-treat-analysis as well cross-protection against hev gt [ ] . there are currently several open questions about the vaccine, thereunder its efficacy and safety in risk groups, the cross-reactivity against other hev gts, and long-term protection after more than months. it is unknown whether hecolin ® is safe for pregnant women and their fetuses, which are a population especially at risk of fatal outcomes of hev gt infection. results obtained from pregnant women that had unintendedly been enrolled by mistake in the phase iii study suggest that the vaccine might be safe for them [ ] . however, because the study was not designed for that purpose, this finding is not a definitive conclusion. a study that does not exclude pregnant women is currently running in bangladesh and expected to finish in (clinicaltrials.gov identifier: nct ). it is also unknown if the vaccine is protective in the second risk group of immunosuppressed or immunocompromised patients. a french study investigated re-infections with hev in organ transplant recipients seropositive for anti-hev igg before transplantation [ ] . they showed that an igg titer up to . who units/ml does not guarantee protection from a re-infection up to one year after transplantation. the data from the long-term study on hecolin ® [ ] indicate that the geometric mean of igg titers months after the first dose is already below that value in individuals seronegative at baseline. notably, the igg titers of individuals, which were anti-hev igg positive before vaccination, fell below the threshold of . who units/ml after months. this raises the question whether chronic hev infections in immunocompromised patients can be attacked with the current vaccine. there are also no data on safety and efficacy in the risk group of patients with chronic liver disease. it is unknown if the vaccine confers cross-protection against hev gts , , or rat hev, although the cross-reactivity of the vaccine against gt hev is promising. the vaccine schedule requires six months; it would be to clarify if the vaccine can be used short-term to combat an outbreak. these limitations and uncertainties make research on antiviral therapy an important issue, regardless of the vaccine's benefits. the recently evolving attention on hev also led to an increased focus on finding a satisfactory therapy. however, sofosbuvir is the only candidate in clinical trials to date (figure ) . preliminary results indicate that it will not be a breakthrough in hev therapy options. apart from that, the nature of repurposed drugs might make them appealing to use for clinical trials. however, a -cmc derivative was discontinued due to toxic effects, while gpc-n or nitd never moved to clinical trials, which might indicate that they have adverse effects that could hinder development to an antiviral. the hits identified from other screens are either not well characterized yet, including toxicity, or are difficult to dose in vivo. poor characterization is also an issue for several of the target-based compounds, while one is an immunosuppressant. inhibition of porf and tsg might be an interesting target for drug development, as demonstrated using cp . silvestrol is a promising candidate, since it is comparably well characterized and shows efficacy in vivo and an additive effect with rbv. despite some challenges like scaling up its production, silvestrol is the most promising candidate at the moment. although the vaccine can be of great use in reducing hev burden, there are too many open questions, especially about their efficacy in risk groups and outbreak scenarios to solely rely on it to combat hev. an ultimate goal would be to discover specific agents only targeting the viral enzymes as, for instance, the hev protease or polymerase. these so-called direct acting antivirals (daas) are highly specific and were a breakthrough in hcv therapy with high cure rates and enhanced efficacy. being virus-specific, they might also be much more suited for the use in immunocompromised patients and especially pregnant patients, for whom there is no therapy yet. of note, none of the drug candidates presented in this paper has an approval for use in pregnant women. integral for the discovery of daa was in the case of hcv and will be in the case of hev the crystallization of the respective enzyme structures. this will enable structure-guided design of potent inhibitors by fitting the compound and the viral enzyme to complementary surfaces. as mentioned above, the structure of porf has been determined, but without further implications for drug development yet. regarding the polyprotein from orf , it is still debated whether it is cleaved into fragments upon translation or not. some studies argue that it is not cleaved [ ] [ ] [ ] [ ] , while others report cleavage into several fragments [ , ] . recently, a study was published suggesting that the proteases factor x and thrombin cleave the polyprotein and that silencing of these reduced psk-hev -luc replication [ ] . the assignment of functional domains to the porf polyprotein was done in , according to similarity to proteins in related viruses that had known functions [ ] . of these seven domains, the functionality could be confirmed with biochemical assays for four: methyltransferase [ ] , papain-like cysteine protease [ ] , helicase [ ] , and rdrp [ ] . the exact function of the other domains as well as the exact borders of each functional domain are not known to date. until it is not completely understood how the polyprotein is processed and how this does affect the structure and enzymatic activity of the functional domains, studies to identify antivirals based on structural insights seem at best unlikely. to date, the de novo development of candidate structures into a potential licensed drug is only a future perspective, underlined by the fact that none of the anti-hev candidates has been designed based on a structure of an hev protein. there are also only a few host factors known; therefore, the identification of novel host factors will be another cornerstone in combating hev. all -omics approaches to decipher the altered cellular environment during infection, as well as functional studies using cdna, shrna and sirna libraries to overexpress or silence host proteins, are powerful and successful tools to discover novel host factors. this will both give new starting point for drug discovery as well as potentially refine in vitro and in vivo models. overview of molecules/extracts with antiviral activity against hev. the depicted molecules/extracts are classified according to the strategy that was used to identify them. so far, the antiviral activity against hev of only four drugs (sofosbuvir, pegifn-α, ribavirin and silvestrol) was approved in experimental settings beyond in vitro cell culture systems. both drugs in the clinics are used off-label and therefore have not been tested in clinical trials against hev. funding: e.s. was supported by the german ministry of education and research (bmbf) through a ginaico grant gw and a silvir grant ggnatm . proposed reference sequences for hepatitis e virus subtypes a new hepatitis e virus genotype strain identified from an outbreak in nigeria a large outbreak of hepatitis e virus genotype infection in an urban setting in chad likely linked to household level transmission factors an outbreak of hepatitis e in an urban area of bangladesh abravanel, f. hev and 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processing for hepatitis e virus replication protein porf the in vitro-synthesized rna from a cdna clone of hepatitis e virus is infectious expression and processing of the hepatitis e virus orf nonstructural polyprotein activities of thrombin and factor xa are essential for replication of hepatitis e virus and are possibly implicated in orf polyprotein processing computer-assisted assignment of functional domains in the nonstructural polyprotein of hepatitis e virus: delineation of an additional group of positive-strand rna plant and animal viruses virus-specific mrna capping enzyme encoded by hepatitis e virus hepatitis e virus (hev) protease: a chymotrypsin-like enzyme that processes both non-structural (porf ) and capsid (porf ) protein ntpase and ' to ' rna duplex-unwinding activities of the hepatitis e virus helicase domain the ' end of hepatitis e virus (hev) genome binds specifically to the viral rna-dependent rna polymerase (rdrp) this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license we would like to thank yannick brüggemann and patrick behrendt for insightful comments. the authors declare no conflict of interest. key: cord- -klhmed authors: bassal, r.; wax, m.; shirazi, r.; shohat, t.; cohen, d.; david, d.; abu-mouch, s.; abu-ghanem, y.; mendelson, e.; ben-ari, z.; mor, o. title: seroprevalence of hepatitis e virus in dromedary camels, bedouins, muslim arabs and jews in israel, – date: - - journal: epidemiol infect doi: . /s sha: doc_id: cord_uid: klhmed hepatitis e virus (hev) is an emerging cause of viral hepatitis worldwide. recently, hev- has been shown to infect camels and humans. we studied hev seroprevalence in dromedary camels and among bedouins, arabs (muslims, none-bedouins) and jews and assessed factors associated with anti-hev seropositivity. serum samples from dromedary camels (n = ) were used to determine camel anti-hev igg and hev rna positivity. human samples collected between and from > years old bedouins (n = ), non-bedouin arabs (n = ) and jews (n = ), were randomly selected using an age-stratified sampling design. human hev igg levels were determined using wantai igg elisa assay. of the samples obtained from camels, . % were anti-hev positive. among the human populations, bedouins and non-bedouin arabs had a significantly higher prevalence of hev antibodies ( . % and . %, respectively) compared with the jewish population ( . %). seropositivity increased significantly with age in all human populations, reaching . % and . % among ⩾ years old, in bedouins and non-bedouin arabs, respectively. the high seropositivity in camels and in ⩾ years old bedouins and non-bedouin arabs suggests that hev is endemic in israel. the low hev seroprevalence in jews could be attributed to higher socio-economic status. hepatitis e virus (hev) is an emerging pathogen and one of the causes of viral hepatitis in the world [ ] . the infection is mostly silent, but when symptoms do appear, illness is usually selflimiting [ ] . hev infection can also become chronic in immunocompromised individuals, such as those receiving organ transplants or chemotherapy, as well as among individuals with hiv infection [ ] . moreover, extra hepatic manifestations of hev infection, mostly neurological, are increasingly recognised [ ] . currently, eight hev genotypes are known, all belonging to a single serotype [ ] . of those, hev- and hev- have been identified in dromedary ( -humped) and bactrian ( -humped) camels, respectively [ , ] . hev- , identified in a liver transplant recipient had been linked to the consumption of camel products [ ] . human hev infection, investigated using seroprevalence studies, was found to be more prevalent in older ages [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , lower socio-economic status [ ] , poorer residence areas [ , [ ] [ ] , among sheltered homeless adults [ ] or uneducated people [ ] , specific nationalities (for example, higher in mixed race donors and ethnic groups within china [ , ] , or in immigrants from afghanistan [ ] ), drinking water from wells or rivers [ ] , consumption of meat products [ , , , ] especially pork [ , ] and following blood transfusions [ ] . the majority of the population in israel comprises jews ( . %) and arabs ( . %), of which . % are muslims [ ] . while almost all israeli-arabs were born in israel, jews compose a mixed population of israeli-born and non-israeli-born individuals. previously, we have shown that the overall hev seroprevalence in israel was . % and higher in arabs ( . %) compared with jews ( . %); with most of the anti-hev positive jews being immigrants not born in israel [ ] . however, an assessment of anti-hev prevalence in specific subgroups of arabs has not been done. the bedouins in israel compose a unique nomadic muslim arab population with cultural, historical and social uniqueness. while non-bedouin muslim arabs live in villages spread around the country, mostly in the northern and central part of israel and are not physically related to camels, many of the bedouins who live in southern israel own dromedary camels and consume camel products [ ] . this study aimed to assess hev seroprevalence in camels (dromedary), in bedouins living in the southern part of israel in the vicinity of camels, in non-bedouins arabs (muslims living in northern and central israel) and in israeli-born jews and to assess the factors associated with anti-hev seropositivity. camel serum samples (n = ) from female dromedary camels aged (n = ), (n = ) and and above years (n = ) were collected between and from bedouin households residing in the southern district of israel (as part of the middle east respiratory syndrome (mers) national control programme) and were used for determining hev seroprevalence in the local -humped camels. human serum samples were collected from: bedouins living in the southern district, in tribes that own dromedary camels (overall ; tested for the first time in this study and nine added from our previous study [ ] ), non-bedouin arabs (overall ; from the present study and from our previous study [ ] ) and from jews ( samples presented in our previous study [ ] ). all human samples were randomly selected using an age-stratified sampling design from the stored sera bank of the israeli centre for disease control. the samples were selected from those collected between and , > years old who were born in israel. the selection was enabled by the availability of basic demographic information including age, gender, place of residence (city), birth country and population group (bedouins; non-bedouin arabs; jews [ ] ) for each sample in this sera bank. the socio-economic status was allocated on the basis of the given address using the socio-economic residential classification published by the israeli central bureau of statistic [ ] . this socio-economic status is based on variables including demographic characteristics, education, lifestyle, etc. and was divided into low ( - ) vs. high ( - ). sera collection was approved by the legal department of the israeli ministry of health. total anti-hev antibodies were assessed in camel samples using hev-ab elisa kit (wantai, biologic pharmacy enterprise, beijing, republic of china) which detects total antibodies and is suitable for detection of anti hev antibodies in non-human sera. hev rna in camel sera was assessed with the realstar hev kit (altona diagnostics gmbh, hamburg, germany) which, according to the manufacturer, should detect all hev genotypes. human samples from the current study were tested with anti-igg hev elisa kit (wantai, biologic pharmacy enterprise, beijing, republic of china) which recognises human antibodies against all hev genotypes with . % sensitivity and . % specificity [ ] . assays were performed according to the manufacturer's instructions. all serological equivocal results were considered as negative. in the previous study, we used the dsi-anti-hev-igg kit (diagnostic systems italy, saronno, italy) [ ] . merging the current and previous study data were applicable after comparing the kits performances using positive and negative samples, revealing . % concordance between the kits. hev rna was not assessed in human sera due to lack of sufficient serum material. descriptive analysis was done for the study populations. prevalence rates of anti-hev antibodies in camels and in human sera were calculated by dividing the number of samples positive to anti-hev antibodies by the total number of samples tested in each group. for the human samples, we calculated the prevalence rates in each of the studied populations by age group, gender and socio-economic status. we used the cochran-armitage trend test to evaluate trends in binomial proportions and the χ test to compare between population groups. logistic regression analyses were applied to assess the factors associated with anti-hev seropositivity. interaction was assessed for each variable associated with hev seropositivity. statistical significance was evaluated using -sided tests with an alpha level of . . all analyses were performed using sas enterprise guide (version . hf , sas institute inc., cary, nc, usa). of the samples obtained from camels, . % ( % ci . - . %) were anti-hev igg positive. the seroprevalence among camels and above, and years old were . % ( / . % of the samples obtained from non-bedouin arabs ( % ci . - . %) and . % of the samples obtained from jews ( % ci . - . %) were anti-hev igg positive. hev seropositivity was significantly higher in bedouins and in non-bedouin arabs compared to jews (p-value < . ). the difference in seropositivity rates between bedouins and non-bedouin arabs was statistically significant (p-value = . ). table demonstrates the odds for being hev positive, by population group, demonstrating significantly higher odds among the older age groups of both arab populations. a single variable analysis demonstrated that seropositivity was significantly higher among arabs (bedouins and non-bedouin); older age groups ( - and ⩾ years) and lower socioeconomic status (table ) . no significant interaction was identified. these associations remained statistically significant in the multivariable analysis, except for the lower socio-economic status ( table ) . in a sensitivity analysis performed for the classification of the equivocal samples as positive or negative, no significant difference was observed. hev prevalence among dromedary camels, has been studied in the past and following literature review we realise that our finding ( . %) is higher than the . % seroprevalence rates reported in ethiopia [ ] , but similar to the . % seropositivity among dromedary camels recently reported in egypt, a nearby country [ ] . together, these results suggest that the -humped camels in our region are highly seropositive for hev. r. bassal et al. both bedouins ( . %) and non-bedouin arabs ( . %) were characterised by significantly higher hev seroprevalence rates compared with the overall low seropositivity rates ( . %) observed in jews. together with the high seroprevalence of hev in camels, these results indicate endemicity of hev in israel. worldwide, the seroprevalence of hev documented in human populations varies between high (⩾ . %), medium ( . - . %) and low (< . %). high hev seroprevalence rates were reported in uganda ( . %) [ ] , poland ( . % and . %) [ , ] , bolivia ( . %) [ ] , jordan ( . %) [ ] and south africa ( . %) [ ] . medium hev seroprevalence rates were reported in china ( . %) [ ] , belgium ( . %) [ ] , portugal ( . %) [ ] and the mediterranean region ( . %) [ ] . low hev seroprevalence rates were reported in new zealand ( . %) [ ] , scotland ( . %) [ ] and iceland ( . %) [ ] . accordingly, within israel, high, medium and low hev seroprevalence rates were observed in bedouins, non-bedouin arabs and jews. the higher seroprevalence rate in bedouins could be attributed to their low socio-economic status but could also result from exposure to camels, especially as the latter were highly seroprevalent (possibly a consequence of hev- infection [ ] ). as camel samples were hev rna negative and fecal specimens were not available for analysis, this hypothesis could not be further explored. in jordan, a nearby country, an overall of . % anti-hev prevalence rate was determined in the local population and owning camels was associated with increased odds of hev seropositivity [ ] . future studies should aim to assess the hev rna prevalence in camel's feces and in their fresh blood samples, as well as in similar samples obtained from bedouins who own camels. with the recent report suggesting that hev originated in asia, most likely from a human ancestor that existed ∼ - years ago and that the split of camel-infecting genotypes occurred during camel domestication, hev circulation between camels and bedouins could be expected [ ] . the high hev seropositivity rate observed in non-bedouin arabs, most of whom live in northern parts of israel, could be attributed to exposure to hev- which is possibly circulating in the country and was recently identified in local sewage facilities mainly in the north of israel [ ] . as both judaism and islam forbid the consumption of pork, we find it unlikely that this is the main risk for the identified hev seroprevalence in the muslim population. the low hev seroprevalence observed in israeli-born jews may be associated with the overall higher socio-economic status characterising this population, living in better sanitary conditions than the bedouins and most of the non-bedouin arabs, rendering hev infection less conceivable. the variability observed between different population groups and in different studies, may be explained by exposure to potential risk factors associated with hev infection as lifestyle habits (dietary), environmental conditions, geographic location, occupation, religion (as the prohibition of consuming pork among jews and islam) but also to co-morbidities. differences may also be explained by the assays used for antibodies detection, demonstrating wide variation in the ability to detect hev antibodies (sensitivity and specificity) [ ] . the association of hev seropositivity with age observed in our study has also been reported by others [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] and may be explained not only by the cumulative lifetime exposure to hev, but also by cohort effect, where certain population groups were more likely to be exposed to the virus during their life. the major limitation of our study is the lack of data on characteristics that might be important to exposure to hev, such as actual exposure to animals, consumption of camel meat and the quality of the local water source. additionally, the cross-sectional nature of the data cannot establish a temporal relationship between risk factors and outcome, thus limits the interpretation of the results. finally, hev rna from human samples could not be assessed due to limited serum volumes. however, for the first time, we have investigated samples from local camels and from specific human populations, with fair distribution by age groups and gender. based on this cohort, our results suggest that hev is endemic in israel and that specific population groups like bedouins and non-bedouin arabs are at higher risk of hev infection. further studies are needed to determine the hev genotypes circulating among dromedary camels and the specific arabs populations. author orcids. r. bassal, - - - . hepatitis e: a disease of reemerging importance hepatitis e virus: advances and challenges easl clinical practice guidelines on hepatitis e virus infection hepatitis e virus genotypes and evolution: emergence of camel hepatitis e variants new hepatitis e virus genotype in camels, the middle east chronic infection with camelid hepatitis e virus in a liver 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israel municipal authorities ranking by the socio-economic index wantai hepatitis e virus diagnostics. wantai hepatitis e virus diagnostics hev-igg elisa serological evidence of hepatitis e virus infection in dromedary camels in ethiopia hepatitis e virus infection in dromedaries hepatitis e virus seroprevalence and correlates of anti-hev igg antibodies in the rakai district clinical burden of hepatitis e virus infection in a tertiary care center in flanders hepatitis e virus seroprevalence rate among eastern mediterranean and middle eastern countries; a systematic review and pooled analysis hepatitis e virus (hev) in scotland: evidence of recent increase in viral circulation in humans low prevalence of hepatitis e in iceland: a seroepidemiological study origin and dispersal of hepatitis e virus hepatitis e virus genotype in sewage and genotype in acute hepatitis cases laboratory challenges in the diagnosis of hepatitis e virus acknowledgments. the authors would like to thank nadia pekurovski, israel center for disease control, for her assistance in data collection and the laboratory assistant. this research received no specific grant from any funding agency, commercial or not-for-profit sectors.conflict of interest. none. key: cord- - vk enj authors: schultze, beate; wahn, kurt; klenk, hans-dieter; herrler, georg title: isolated he-protein from hemagglutinating encephalomyelitis virus and bovine coronavirus has receptor-destroying and receptor-binding activity date: - - journal: virology doi: . / - ( ) - sha: doc_id: cord_uid: vk enj abstract bovine coronavirus (bcv) and hemagglutinating encephalomyelitis virus (hev) from swine were found to grow to high titers in mdck i cells, a subline of madin darby canine kidney cells. virus grown in these cells was used to isolate and purify the he-protein. this protein has been shown recently to have acetylesterase activity and to function as the receptor-destroying enzyme of bcv. here we show that hev contains this enzyme, too. the glycoproteins were solubilized by treatment of virions with octylglucoside. following centrifugation through a sucrose gradient the surface proteins s and he (hemagglutinin-esterase) were obtained in purified form. after removal of the detergent by dialysis, he formed rosettes as shown by electron microscopy. the purified he protein retained acetylesterase activity and was able to function as a receptor-destroying enzyme rendering red blood cells resistant against agglutination by both coronaviruses. he protein released from the viral membrane failed to agglutinate red blood cells. however, it was found to recognize glycoconjugates containing n-acetyl- -o-acetylneuraminic acid as indicated by a binding assay with rat serum proteins blotted to nitrocellulose and by its ability to inhibit the hemagglutinating activity of bcv, hev, and influenza c virus. the purified enzyme provides a useful tool for analyzing the cellular receptors for coronaviruses. it has been reported recently that bovine coronavirus (bcv) contains a receptor-destroying enzyme (vlasak et al., a) . this is the first example of such an enzyme present on a positive-stranded rna virus . up to then, receptor-destroying enzymes have been known as structural components only of negative-stranded rna viruses : influenza viruses and paramyxoviruses (hirst, ) . influenza a and b viruses as well as paramyxoviruses inactivate their receptors by means of a neuraminidase which releases terminal sialic acid from glycoconjugates (klenk et al ., ) . the receptordestroying enzyme of influenza c virus, on the other hand, cleaves not a glycosidic linkage but rather an ester linkage . it has been identified as an acetylesterase (herrler et at, c) , which is able to release acetate from various synthetic substrates (vlasak et at, ; schauer et al., ) . the receptor-destroying activity, however, is due to the release of the acetyl group from position c- of n-acetyl- -o-acetylneuraminic acid (neu , acz ) (herrler et al., c) . the -o-acetyl residue is crucial for the ability of influenza c virus to recognize a glycoprotein as a receptor (herrler et at, c ; rogers et al., ) . ' to whom requests for reprints should be addressed . the intriguing observation of a sequence similarity between the glycoprotein hef of influenza c virus and a protein sequence derived from an open reading frame within the genome of mhv-a (mouse hepatitis virus) (luytjes et at, ) led to the finding that bcv has the same type of receptor-destroying enzyme as influenza c virus (vlasak et al., a) . the acetylesterase activity of bcv has been shown to be a function of the glycoprotein he (vlasak et al., b) , the amino acid sequence of which has been derived from the nucleotide sequence of the cloned mrna (parker et al., ) . interestingly, this protein is present only on some coronaviruses, while others, such as avian infectious bronchitis virus, porcine transmissible gastroenteritis virus, and feline infectious peritonitis virus, are lacking a corresponding protein . the genome of mhv-a contains an open reading frame coding for an he protein, which is, however, not expressed in infected cells (luytjes et al., ; shieh et al., ) . other murine coronaviruses contain such a protein (sugiyama and amano, ; shieh et al., ) and the he protein of the strain mhv-jhm has been shown to have acetylesterase activity pfleiderer et all ) . we describe the isolation and purification of he from bcv and from porcine hemagglutinating encephalomyelitis virus (hev) . the activities of the purified glycoprotein are reported . schultze et al . strain l- of bcv was obtained from dr . rott (giessen, frg) . strain nt- of hev was provided by dr . hess (koblenz . frg) . mdck i cells, a subline of madin-darby canine kidney cells, were maintained as described previously (herrler et al., a) . growth and purification of virus bcv and hev were grown in mdck i cells as reported recently (schultze et al., ) . virus was harvested from the supernatant hrp .i . after clarification of the medium by low-speed centrifugation ( rpm, min), virus was sedimented by ultracentrifugation at , rpm for hr in a sw rotor . the pellet was resuspended in pbs and layered on a sucrose gradient ( - % w/w in pbs) . after centrifugation in a sw ti rotor at , rpm for min, the virus band was collected, diluted with pbs, and sedimented under the same centrifugation conditions . the virus pellet was resuspended in pbs and used for (i) analysis by polyacrylamide gel electrophoresis ; (ii) determination of the esterase activity ; (iii) hemagglutination and hemagglutination-inhibition assays ; and (iv) purification of the viral glycoproteins . purified coronavirus suspended in µl pbs was incubated in the presence of % n-octylglucopyranoside for min at room temperature . after centrifugation for min at , g, the supernatant was further incubated for min at °. following centrifugation for min at , rpm in a tla . rotor, the supernatant was layered onto a sucrose gradient ( - % w/w, on a cushion of . ml % sucrose) in pbs containing % octylglucoside . after centrifugation for hr at , rpm in a sw . rotor, fractions of . ml were collected from the bottom of the tube . the samples were analyzed by sds-polyacrylamide gel electrophoresis and assayed for acetylesterase activity. for hemagglutination and hemagglutination-inhibition assays, as well as for treatment of cell surface receptors, the fractions were dialyzed against pbs/h ( : ) . if the enzyme activity should be preserved for longer periods, the purified acetylesterase was stored at - ° in the presence of mg bovine serum albumin per milliliter. hemagglutination assays were performed as described previously (herrler et al., a) . the hemag-glutination titer indicates the reciprocal value of the maximum dilution that caused complete agglutination . hemagglutination-inhibition assays were performed as described previously (herrler et al., b) . the esterase activity of purified virions or proteins was determined by incubation with µg p-nitrophenyl acetate (pnpa) in ml pbs at room temperature . the substrate was dissolved in / vol ethanol . using a kinetics program, the release of acetate was monitored by determining the optical density at nm for min at intervals of min . the background level of substrate incubated in the absence of esterase was subtracted from the samples . the amount of esterase which cleaves umol of p-nitrophenyl acetate in min at room temperature was defined as unit of enzyme . labeling of esterases with [ h]dfp was performed as described previously (herrler et al., b) . purified virions or proteins in µl of pbs were incubated with µl of h-labeled dfp ( . ci/mmol) on ice . after min the samples were prepared for electrophoresis . analysis of proteins by sds-polyacrylamide gel electrophoresis was performed as described previously (herrler et at, a) . samples containing µl of a % suspension of chicken erythrocytes were incubated with µl of the gradient fraction containing purified he protein (see above) . prior to use the acetylesterase was dialyzed to remove octylglucoside . after incubation for hr at °, the red blood cells were washed twice with pbs and suspended in ml of the same buffer. these erythrocytes and control cells, which had been incubated with pbs, were used to determine the ha titer of bcv, hev, and influenza c virus . solid-phase assay for virus binding different dilutions of rat serum ( : , : , and : in pbs) in µl were applied to nitrocellulose and air-dried . excess protein-binding sites were blocked with % nonfat dry milk in pbs overnight at °. the nitrocellulose strips were washed three times for min with pbs/ . % tween and incubated for hr at ° with pbs, purified acetylesterase from influenza c virus ( mu), or . n naoh, all subsequent steps were done at °. following three washes with pbs/ tween, the nitrocellulose was incubated for hr with bcv, he protein from bcv (untreated or pretreated with dfp), or influenza a virus (wsn) . after being washed with pbs/tween, strips were incubated with rabbit antiserum directed against bcv (dilution : ) or against influenza a virus (pr , dilution : ) . the nitrocellulose was again washed three times and then incubated for hr with biotinylated anti-rabbit immunoglobulins from donkey . after being washed, the strips were incubated with streptavidin-biotinylated horseradish peroxidase complex ( hr) and washed again . bound bcv, he protein, or wsn was detected by incubation of the nitrocellulose with pbs, -chloro- -naphtol, and h o ( : : ) . for negative staining, samples were applied to piooforrn-coated copper grids, stained with % uranylacetate, and examined in a siemens-elmiskop . growth of bcv and hev in mdck i cells mdck i cells are a subline of madin-darby canine kidney cells which differs from other sublines in both functional and morphological characteristics (richardsonetal ., ) . strain johannesburg/ / of influenza c virus was found to grow to high titers in this cell line, while another subline-mdck ii-was resistant to infection because of a lack of cell surface receptors (herrler and klenk, ; szepanski et al ., ) . due to the similarity of the erythrocyte receptors for bcv isolation of the acetylesterase of coronaviruses and influenza c virus, we tried to grow bcv in mdck i cells . strain l- of bcv, which had been grown previously in bovine cell cultures (storz and rott, ) , was able to replicate in mdck i cells without adaptation . hemagglutination titers of hau/ml were determined in the supernatant hr p .i . the same growth kinetics was observed with strain nt- of hev, which had been grown previously in porcine cell cultures (hess and bachmann, ) . these titers are equally high as or higher than those reported for bcv and hev in other cell lines . both mdck-grown coronaviruses were found to have acetylesterase activity . comparing virus suspensions with the same ha titer, the ability of both viruses to release acetate from p-nitrophenyl acetate was similar, hev being somewhat more active than bcv (fig . ) . as expected from previous studies with influenza c virus and bcv (muchmore and varki, ; vlasak et al., b) , the esterase activity of the mdck-grown coronaviruses was abolished after treatment with dfp . this compound inhibits serine esterases and proteases by covalently attaching to the active-site serine . up to the time of virus harvest no cytopathic changes were detectable in the monolayer of mdck i cells infected with either bcv or hev . virions released into the medium were purified by centrifugation into a sucrose gradient and analyzed by sds-polyacrylamide gel electrophoresis . as shown in fig . for hev, following coomassie staining of the gel, the major bands visible are the known structural polypeptides : the nucleocapsid protein n, the membrane protein m, the surface protein s, and the hemagglutinin-esterase protein he . the latter polypeptide is detected under nonreducing conditions as a disulfide-linked dimer, (he) z . several other coronaviruses require two rounds of gradient centrifugation for a satisfying degree of purification . using the mdck cell system a single gradient centrifugation step is sufficient to obtain both bcv and hev in purified form . the acetylesterase was isolated from coronaviruses bya modification of the procedure used forthe purification of the influenza c glycoprotein (harrier et al ., a) . purified hev or bcv were treated with octylglucoside to solubilize the components of the lipid envelope . the viral polypeptides n and m were sedimented by centrifugation (not shown) . the glycoproteins remaining in the supernatant (s and he) were centrifuged into a sucrose gradient containing detergent . to determine the location of the viral proteins, the gradient fractions were analyzed by sds-polyacrylamide gel electrophoresis . as shown in fig . for hev, the peak of the he protein was detected in fraction . igg was found to have the same sedimentation behavior (not shown) . the s-protein was well separated from he with the majority being present in fraction . an identical separation was obtained with the glycoproteins of bcv (compare figs . and ) . the clear separation of both coronavirus glycoproteins was also evident when the individual fractions were analyzed for acetylesterase activity using p-nitrophenyl acetate as a substrate (fig . ) . the peak fraction of the esterase activity of both bcv and hev coincided with the heprotein (fraction ) ; no enzymatic release of acetate was detectable with fraction . the purification procedure preserved not only the acetylesterase activity of he, but also the sensitivity of this enzyme to treatment with dfp, which binds covalently to serine hydrolases . as shown in fig . , upon incubation with [ h]dfp, purified he becomes radioactively labeled . figure also fig . . esterase activity of sucrose gradient fractions after centrifugation of the glycoproteins he and s from bcv (circles) and hev (squares) . compare fig . for a polypeptide analysis of the gradient fractions . illustrates why the initial treatment of virus with detergent was performed in two steps with intermittent centrifugation . if the first centrifugation was omitted, part of the m-protein remained in the supernatant which during gradient centrifugation cosedimented with he (compare lanes e and f) . in order to analyze the effect of purified he-protein on cells, it was necessary to remove octylglucoside . the gradient fraction containing he was dialyzed and analyzed by electron microscopy . removal of the detergent resulted in rosette formation of the glycoprotein (shown in fig . for bcv-he) as has been reported for other viral surface glycoproteins . analysis of the purified he-protein for receptorbinding and receptor-destroying activity the purified he protein was analyzed for its ability to function as a receptor-destroying enzyme . as shown in table , incubation of erythrocytes with purified acetylesterase from either bcv or hev rendered the cells resistant to agglutination by both coronaviruses as well as by influenza c virus . on the other hand, agglutination by influenza a virus was not affected . this result indicates that the specificity of the coronavirus esterase as a sialate -o-acetylesterase is preserved during purification of the he protein . next we analyzed whether purified he has receptorbinding activity . as shown in table , the gradient fraction containing he was unable to agglutinate chicken red blood cells . however, it was found to have a low hemagglutination-inhibition activity ; i .e ., purified he was able to prevent intact virions (bcv or hev) from agglutinating erythrocytes . this finding suggests that, following purification, he is still able to attach to coronavirus receptors on red blood cells . however, this interaction appears not to be sufficient for agglutination of erythrocytes . the ability of purified he to bind to neu , acz containing receptors was further analyzed by a solid-phase binding assay . rat serum is known to be a potent hemagglutination-inhibitor of influenza c virus (styk, ) as well as of bcv and hev (schultze et al ., ) . the inhibitory activity of rat serum is mainly due to al -macroglobulin (herrler et al., b ; kitame at al., ) , which has been shown to contain a substantial amount of neu , ac z (herrler at at, c) . bcv and purified he from this virus were analyzed for their ability to bind to rat serum which had been spotted on a nitrocellulose filter . as shown in fig . (left lane), attachment of bcv to rat serum could be demonstrated . purified he gave a positive result in this assay only at the highest concentration of rat serum tested (compare sections designated bcv and he) . if he was pretreated with dfp, however, to inactivate the esterase activity, binding of he protein to rat serum was as efficient as binding of whole virus (compare sections designated bcv and he-dfp) . to find out whether attachment of bcv and he-protein was mediated by neu , ac z , the serum proteins blotted on the nitrocellulose were preincubated with either acetylesterase or sodium hydroxide . both treatments are known to release -o-acetyl residues from neus, ac . preincubation of the serum proteins at alkaline ph completely abolished binding of both bcv and he protein, while attachment of influenza a virus (strain wsn) could still be demonstrated (fig . , middle lane) . pretreatment of serum proteins with acetylesterase from influenza c virus (hef) drastically reduced binding of bcv, he, or dfp-treated he protein (fig . , right lane) . these results indicate that purified he protein is able to recognize neus, ac as a receptor determinant for attachment to glycoproteins . binding of he is, however, less efficient than binding of whole virions and can be increased by inactivation of the esterase . coronaviruses grow most readily in cells from their natural host, although adaptation to cells from other species is possible (siddell at al., ) . in our attempts to set up a cell culture system for two hemagglutinating coronaviruses, bcv and hev, it turned out that both viruses can be grown to high titers in mdck i . binding of bcv, he-protein from bcv, and influenza avirus (strain wsn) to rat serum proteins . after different dilutions ( : , : , and : , from top to bottom for each binding assay) of rat serum were blotted to nitrocellulose, the serum proteins were incubated with pbs (left lane), sodium hydroxide (middle lane), or acetylesterase from influenza c virus (hef, right lane) . the samples were then incubated with virus (bcv, wsn) or purified he-protein, which had been either untreated (designated he) or pretreated with dfp (designated he-dfp) . binding was detected by an enzyme-linked immunoassay using rabbit antiserum directed against bcv or influenza virus . cells without prior adaptation . this subline of madin-darby canine kidney cells has been shown recently to be sensitive to infection by strain johannesburg/ / of influenza c virus (szepanski et at, ) . this virus initiates infection of cultured cells by attaching to neu , acz containing receptors, which area major determinant of the cell tropism of influenza c virus (harrier and klenk, ) . neu , ac serves also as a receptor determinant for attachment of bcv and hev to red blood cells (vlasak et al., a ; schultze et at, ) . it remains to be shown whether the same type of receptors is also used for infection of cells . another question to be answered is whether the restricted tropism of coronaviruses is determined by the cellular receptors or at a later stage of the infectious cycle . with mhv-a evidence has been presented indicating a crucial role of cellular receptors for the cell tropism . this virus strain has been reported to use a to -kda protein as a receptor (boyle et al ., ) . mdck cells provide a promising system to analyze the initial stage of infection with other coronaviruses such as bcv and hev, which appear to recognize different receptors on the cell surface . this cell line is also known for its polarized organization, which is reflected -among other characteristics-by a polarity of the virus infection . with vesicular stomatitis virus it has been shown that virus entry and release is restricted to the basolateral domain of the plasma membrane . influenza a virus, on the other hand can infect mdck cells from both the apical and the basolateral side, whereas budding of the virus is restricted to the apical portion of the plasma membrane (rodriguez-boulan and sabatini, ; fuller et at, ) . coronaviruses are known to bud into intracellular vesicles (tooze at at, ) . it will be interesting to analyze how viruses with this type of maturation are released by polarized cells . the amount and the purity of both bcv and hev grown in mdck i cells were sufficient to attempt the isolation of the viral glycoproteins . using the protocol described it was possible to get a complete separation of the coronavirus proteins he, , and m . a similar method using triton x- rather than octylglucoside had been used previously to purify the glycoproteins of hev (callebaut and pensaert, ) . as triton x- cannot be removed by dialysis, these authors used butanol precipitation to obtain detergent-free glycoprotein . he prepared in this way was unable to agglutinate red blood cells . at that time the esterase activity as well as the ability of this virus to recognize neu , ac as a receptor determinant was unknown . using octylglucoside as a detergent we obtained both he and s in purified form . the isolated s-protein was also subject to a biochemical and functional analysis (manuscript in preparation) . the sedimentation behavior of the isolated he protein was similar to that of igg . considering the molecular weight of the monomeric glycoprotein (parker et at, ) , a dimeric structure is suggested for the purified he protein . dinners of he are also detected following polyacrylamide gel electrophoresis under nonreducing conditions (see fig . ), indicating that the two monomers are held together by disulfide bonds . we cannot, however, exclude the possibility that the functional protein is embedded in the lipid envelope as a multimeric structure, e .g ., a tetramer, which is dissociated into dimers upon detergent treatment and/or sucrose gradient centrifugation . for the g-protein of vesicular stomatitis virus it has been reported that gradient centrifugation results in the dissociation of the trimeric glycoprotein into monomers (doms et al ., ) . of the two functions which have been assigned to the he-protein (hemagglutinin and esterase) the esterase could be detected easily in the purified protein . the acetylesterase activity was evident both in the color test with the synthetic substrate pnpa (fig . ) and in the ability of he to inactivate the erythrocyte receptors for bcv, hev, and influenza cvirus (table ) . the hemagglutinin function of he could not be demonstrated with the purified glycoprotein . however, he was still able to recognize neu , ac z containing receptors as indicated by the binding-assay with rat serum proteins (fig . ) and bythe low hemagglutination-inhibition activity of the purified glycoprotein (table ) . this interaction was, however, not sufficient for agglutination of erythrocytes . this situation is reminiscent of influenza c virus, the purified glycoprotein (hef) of which also has esterase and hemagglutination-inhibition but no hemagglutinating activity (herrler et al., a) . the purified hemagglutinin of influenza a viruses, on the other hand, has been shown to form rosettes which are able to agglutinate red blood cells (laver and valentine, ) . the failure of purified he protein to cause hemagglutination is not due to a lack of rosette formation (fig . ) . the receptor-destroying activity of he counteracts the receptor-binding activity . this is evident from the result of the solid phase binding assay . binding of bcv-he was found to be more efficient after inactivation of the esterase by dfp (fig . ) . the binding assay has not been applied to hev-he, but because of the similarity of both he proteins in other tests (see figs . - and tables and ) it is likely that the results obtained with bcv-he are also valid for hev-he . dfp-treated he was still unable to agglutinate red blood cells . there was, however, some interaction between erythrocytes and the purified coronavirus glycoprotein . erythrocytes incubated with dfptreated he did not pour, when the microtiter plate was placed in a semivertical position (not shown), which is in contrast to control cells . the lack of ha activity in the purified he protein might be due to a conformational change during the purification procedure, which affects the receptor-binding activity more than the receptor-destroying activity . another possibility is that the individual glycoproteins have only a low affinity for neu , ac z containing receptors . in this case the multivalent attachment of he-proteins present in the viral membrane may increase the binding activity and enable agglutination of erythrocytes . in any case, the purified acetylesterases of bcv and hev are useful analytical tools to study the importance of neu , ac z in general, and specifically the cellular receptors for coronaviruses . the technical assistance of birgit doll and heldrun will is gratefully acknowledged . we thank drs . r . rot : and r . g . hess for providing genetic resistance to mouse hepatitis virus correlates with absence of virus-binding activity on target tissues characterization and isolation of structural polypeptides in haemagglutinating encephalomyelitis virus role for adenosine triphosphate in regulating the assembly and transport of vesicular stromatitis virus g protein trimers vesicular stomatitis virus infects and matures only through the basolateral surface of the polarized epithelial cell line neuraminic acid is involved in the binding of influenza c virus to erythrocytes rat a,-macroglobulin inhibits hemagglutination by influenza c virus the receptor-destroying enzyme of influenza c virus is neuraminate-o-acetylesterase the surface receptor is a major determinant of the cell tropism of influenza c virus the glycoprotein of influenza c virus is the hemagglutinin, esterase and fusion factor . l serine of the glycoprotein hef is located at the active site of the acetylesterase of influenza c virus erbrechen and kommern der ferkel : vorkommen and verbreitung in soddeutschland the relationship of a new strain of virus to those of the mumps-ndv-influenza group isolation and characterization of influenza c virus inhibitor in rat serum uber die enzymetische wirkung von influenza-virus morphology of the isolated hemagglutinin and neuraminidase subunits of influenza virus sequence of mouse hepatitis virus a mrna : indications for rna-recombination between coronaviruses and influenza c virus selective inactivation of influenza c esterase : a probe for detecting -o-acetylated sialic acids cloning and in vitro expression of the gene for the e haemagglutinin glycoprotein of bovine coronavirus functional analysis of the coronavirus mhv-jhm surface glycoproteins in vaccinia virus recombinants identification of two strains of mdck cells which resemble separate nephron tubule segments asymmetric budding of viruses in epithelial monolayers : a model system for study of epithelial polarity influenza c virus uses - -acetyl-n-acetylneuraminic acid as a high affinity receptor determinant for attachment to cells isolation and characterization of sialate ( )- -acerylesterase from influenza c virus hemagglutinating encephalomyelitis virus attaches to n-acetyl- - -acetylneuraminic acid-containing receptors on erythrocytes: comparison with bovine coronavirus and influenza c virus identification of a newtranscriptional initiation site and the corresponding functional gene h in the murine coronavirus rna genome the structure and replication of coronaviruses coronaviruses : structure and genome expression reactivity of antibodies in human serum with antigens of an enteropathogenic bovine coronavirus non-specific inhibitors in normal rat serum for the influenza c virus hemagglutination and structural polypeptides of a new coronavirus associated with diarrhea in infant mice analysis of a mutant of influenza c virus with a change in the receptor s pecificity replication of coronavirus mhv-a in sac-cells : determination of the first site of budding of progeny virions the influenza c virus glycoprotein (he) exhibits receptor-binding (hemagglutinln) and receptor-destroying (esterase) activities human and bovine coronaviruses recognize sialic acid containing receptors similar to those of influenza c viruses the e protein of bovine ooronavirus is a receptor-destroying enzyme with acetylesterase activity biosynthesis, structure, and biological activities of envelope protein gp of murine coronavirus key: cord- -mprsdi e authors: zhu, zhongyu; bossart, katharine n; bishop, kimberly a; crameri, gary; dimitrov, antony s; mceachern, jennifer a; feng, yang; middleton, deborah; wang, lin-fa; broder, christopher c; dimitrov, dimiter s title: exceptionally potent cross-reactive neutralization of nipah and hendra viruses by a human monoclonal antibody date: - - journal: j infect dis doi: . / sha: doc_id: cord_uid: mprsdi e we have previously identified neutralizing human monoclonal antibodies against nipah virus (niv) and hendra virus (hev) by panning a large nonimmune antibody library against a soluble form of the hev attachment-envelope glycoprotein g (sg(hev)). one of these antibodies, m , which exhibited the highest level of cross-reactive neutralization of both niv and hev g, was affinity maturated by light-chain shuffling combined with random mutagenesis of its heavy-chain variable domain and panning against sg(hev). one of the selected antibody fab clones, m . , had affinity of binding to sg(hev) that was equal to or higher than that of the other fabs; it was converted to igg and tested against infectious niv and hev. it exhibited exceptionally potent and cross-reactive inhibitory activity with % inhibitory concentrations below . and . μg/ml, respectively. the virus-neutralizing activity correlated with the binding affinity of the antibody to sg(hev) and sg(niv). m . bound a soluble form of niv g (sg(niv)) better than it bound sg(hev), and it neutralized niv better than hev, despite being originally selected against sg(hev). these results suggest that m . has potential as a therapeutic agent for the treatment of diseases caused by henipaviruses. it could be also used for prophylaxis and diagnosis, and as a research reagent we have previously identified neutralizing human monoclonal antibodies against nipah virus (niv) and hendra virus (hev) by panning a large nonimmune antibody library against a soluble form of the hev attachment-envelope glycoprotein g (sg hev ). one of these antibodies, m , which exhibited the highest level of cross-reactive neutralization of both niv and hev g, was affinity maturated by light-chain shuffling combined with random mutagenesis of its heavychain variable domain and panning against sg hev . one of the selected antibody fab clones, m . , had affinity of binding to sg hev that was equal to or higher than that of the other fabs; it was converted to igg and tested against infectious niv and hev. it exhibited exceptionally potent and cross-reactive inhibitory activity with % inhibitory concentrations below . and . g/ml, respectively. the virus-neutralizing activity correlated with the binding affinity of the antibody to sg hev and sg niv . m . bound a soluble form of niv g (sg niv ) better than it bound sg hev , and it neutralized niv better than hev, despite being originally selected against sg hev . these results suggest that m . has potential as a therapeutic agent for the treatment of diseases caused by henipaviruses. it could be also used for prophylaxis and diagnosis, and as a research reagent. hendra virus (hev) and nipah virus (niv) are highly pathogenic paramyxoviruses that have recently emerged from flying fox populations to cause serious disease outbreaks in humans and livestock in australia, malaysia, singapore, bangladesh, and india [ ] . hev emerged in queensland, australia, in , killing human and horses [ ] and the virus was responsible for at least other sporadic outbreaks involving horses and humans between and [ ] . the closely related niv emerged in - in peninsular malaysia, resulting in the death of more than people and the culling of more than one million pigs [ ] . since then, several outbreaks of niv infection have been recorded in bangladesh and india [ , , ] . several important observations have been made during these most recent outbreaks, such as a higher incidence of acute respiratory distress syndrome, higher rates of person-to-person transmission, and higher case fatality rates ( %- %), compared with the malaysian outbreak (with case fatality rates of ϳ %) in which the virus was initially discovered [ - ] . there are currently no therapeutic modalities for treating niv or hev infections, and a vaccine for prevention of disease in human or livestock populations does not exist. the first antiviral antibody-based drug approved by the u.s. food and drug administration is a humanized antibody against respiratory syncytial virus (manufactured by medimmune), which has proven to be highly successful in reducing respiratory syncytial virus infection in infants and immunocompromised patients; this antibody has been recently improved [ ] . in this context, the development of neutralizing human mabs (hmabs) against henipaviruses could have important implications for prophylaxis and passive immunotherapy. one of the challenges in the development of human antibodies for antiviral applications is the heterogeneity and mutability of rna viruses. it is, therefore, important to select antibodies that recognize epitopes that are highly conserved among different virus variants. previously, we reported the isolation and characterization of potent neutralizing recombinant hmabs that targeted the viral envelope glycoprotein g by use of a highly purified, oligomeric, soluble hev g glycoprotein (sg hev ) [ ] as the antigen for the screening of a large, naive human phagedisplayed antibody library [ ] . one of these antibodies, m , exhibited cross-neutralizing activity against both hev and niv. in this article, we report the identification and characterization of a novel antibody, m . , derived from m by light-chain shuffling and heavy-chain variable domain random mutagenesis. this antibody exhibits exceptional potency against both, niv and hev, and being fully human antibody, it could be directly used for prophylaxis or treatment of humans exposed to or infected by hev or niv. such an antibody could also be used for improved diagnosis and as a research tool for better understanding of virus-host interaction. fine mapping of the hmabdefined epitope may also provide information useful for the rational development of candidate vaccines and small molecule drugs. hela-usu cells have been described elsewhere [ , ] . vero cells were provided by the australian animal health laboratory. the human glioblastoma cell line u -mg was provided by adam p. geballe, fred hutchinson cancer research center, seattle. hela-usu and u cells were maintained in dulbecco's modified eagle medium (dmem [quality biologicals]) supplemented with % cosmic calf serum (hyclone), and mmol/l l-glutamine (dmem- ). vero cells were maintained in eagle minimal essential medium (emem) (invitrogen) supplemented with % fetal calf serum (invitrogen australia pty. ltd), mmol/l hepes (invitrogen), and mmol/l l-glutamine (invitrogen) (emem- ). all cell cultures were maintained at °c in a humidified % co atmosphere. generation of phage-displayed fab libraries and selection of affinity-matured fabs. the original human fab phage display library, from which the antibodies m -m were identified [ ] , was used as the source for the light-chain variable domain (vl) repertoire in the shuffled library. the phagemid preparation from the original library was first digested with ncoi and spei, followed by electrophoresis on an agarose gel to delete the entire heavy-chain variable domain (vh) repertoire. the gene encoding the vh of m was amplified by error-prone polymerase chain reaction (pcr) (stratagene) to introduce random mutations and then fused with the ch gene fragment by use of splicing by overlapping extension pcr. the fused fragment was digested with ncoi and spei and purified from gel; it was then ligated into the purified backbone vector to create the vls-shuffled fab repertoire. escherichia coli tg cells were transformed with the ligation mixtures via electroporation. the transformed tg cells were plated on ϫ yeast extract-tryptone ( yt) agar plates containing g/ml ampicillin and % glucose ( ytag). after incubation overnight at °c, all of the colonies grown on the plates were scraped into ml of ytag medium, mixed with . ml of % glycerol (final concentration, %), aliquoted, and stored at Ϫ °c as the library stock. two rounds of biopanning were performed on sg hev conjugated magnetic beads as described for the original library panning [ ] . eight clones were identified as affinity-maturated antibodies, and m . was selected for further characterization. selected fabs and m . igg expression and purification was performed as described elsewhere [ ] . elisa binding assay. the sg hev glycoprotein ( ng per well) was coated in a -well plate in l pbs. serially diluted antibodies were added into the well after washing with ϫpbs with . % tween . after incubation for hour at room temperature and washing, horseradish-peroxidase conjugated secondary antibody was added and incubated for another hour. after washing, plates were developed and read at nm in an elisa reader. stable cell line construction and fermentation. linearlized m . pdr construct was transfected into cho k cells with polyfectin in accordance with the protocol from qiagen. a stable antibody-producing cell line was selected by screening in glutamine acid-free culture medium with mol/l methionine sulphoximine. it was adapted to grow in suspension in serum-free medium. the antibody was produced by fermentation in a -l fermentor and purification was performed with protein a. expression and immunoprecipitation of alanine hev g mutants. alanine mutations were introduced at specific residues in myc-tagged hev g using site-directed mutagenesis (stratagene). expression and immunoprecipitation of all hev g mutants were as performed as described elsewhere [ ] . binding and competition analysis using multiplex microsphere assays. multiplexed microsphere assays were performed as described elsewhere [ ] . cell fusion assays. the inhibition assay of cell fusion by fabs and iggs was performed as described elsewhere [ ] . virus neutralization assays. all live virus experiments were conducted under strict biocontainment procedures in a biosafety level laboratory. a total of ϫ vero cells were added per well with l emem- in -well plates and incubated overnight at °c in a humidified % co atmosphere. the foci assay was performed as described elsewhere [ ] . antibody pharmacokinetics in plasma and biological activity. four juvenile (Ͻ months), male ferrets were anesthetized by mask induction with isofluorane and maintained on % isofluorane and % oxygen. a baseline blood sample was collected from an axillary vein, and a -g intravenous catheter was placed in the left jugular vein. the antibody m . was administered via the catheter by slow infusion over minutes; ferrets received mg of m . and ferrets received mg of m . . the catheter was withdrawn, and animals were allowed to recover from the anesthetic. subsequently, the ferrets were anes-thetized on days , , , , , , , , and by intramuscular injection with ketamine (ketamil; ilium) and medetomidine (domitor; novartis) (reversed with atipamazole [antisedan; novartis]). blood was collected from an axillary or jugular vein while animals were under anesthesia. after collection of the final blood sample, the animals were euthanized by means of an intravenous barbiturate overdose. for all samples, serum was aliquoted and stored at Ϫ °c. ferret serum was diluted : and assayed by use of the binding multiplex microsphere assay described above. an m . standard curve ranging from ng/ml to . ng/ml and all ferret serum samples were assayed simultaneously. ferret serum m . concentrations were extrapolated from the standard curve using nonlinear regression analysis (graphpad software; graphpad). half-lives were estimated from the slopes of the natural logarithms of the antibody concentration as function of time by using the formulas ta ϭ . /a[ln ( ) respectively, a and b are measured as the slopes of ln (m . concentration, g/ml) dependent on time. serum collected on days , , and was evaluated in virus neutralization assays. all sera were diluted : , : , : and : and assayed in duplicate using the foci assay. complete neutralization was defined as no viral foci in either well at a particular dilution. previously, we reported the isolation of henipavirus-neutralizing recombinant hmabs by screening of a large nonimmune phage-displayed fab library against a soluble form of the hev g glycoprotein (sg hev ). one of these antibodies, m , exhibited the highest level of crossreactivity and relatively better binding to niv g than to hev g. we reasoned that improving m 's binding to hev g could further increase its cross-reactive neutralizing activity by increasing its affinity to hev g. to mature in vitro m , we constructed an antibody library-which contained approximately ϫ clones-by light-chain shuffling combined with heavychain vh random mutations introduced by error-prone pcr. two rounds of panning against sg hev conjugated to magnetic beads demonstrated sufficient enrichment (data not shown), and random colonies were screened by phage elisa. the best binders were selected for sequencing analysis. they represented different clones, which were designated m . -m . . although there were no amino acid sequence changes, silent mutations occurred in the vh regions in of the clones (data not shown), indicating that the error-prone pcr had worked. seven of the different light chains were from v subfamily iii, which is the same as that of m ; clone (m . ) was from the v subfamily i. a sequence analysis of these light chains showed that all clones contained mutations in all complementarity-determining regions; of the clones from subfamily iii, m . had the largest number of mutations (data not shown). all clones were expressed as fabs, purified, and analyzed by elisa for binding to the selecting antigen sg hev . the elisa data confirmed that all fab clones displayed a higher level of binding to sg hev than did the parental m fab (figure ). clone m . , which had binding affinity equal to or higher than that of the other clones, was selected for further characterization and converted to an igg format. binding of igg m . to cognate antigens. to investigate the binding of igg m . to hev g and niv g and its ability to block receptor-g interactions, we used multiplex mi- crosphere assays that we recently developed [ ] . as shown in figure a , m . binds to both sg hev and sg niv in a dosedependent fashion. at relatively low concentrations (in the range of ng/ml or less), the binding reached % of its maximum, indicating strong binding to both soluble g proteins. furthermore, the multiplex assays also demonstrated that m . is highly efficacious in blocking the binding of ephrin-b ligand (efnb ) to both sg hev and sg niv (figure b). it is important to note that the increase in affinity did not alter the specificity of the fab; it could still bind to both g proteins very well. it is also interesting to note that m . binds sg niv better than sg hev , a result that was also reflected in its slightly better efficiency at blocking efnb -sg niv binding. these results suggest that m . is a cross-reactive, high-affinity binder to the soluble forms of both hev g and niv g glycoproteins. hev env-mediated cell fusion in both formats, as fab and as igg . as expected, fab m . was significantly more potent than fab m , and igg m . was more potent than fab m . ( figure ) . a comparison with a previously identified antibody, m , which is specific for hev and the most potent inhibitor of infectious hev [ ] , suggested that m . and m had comparable activity in both fab and igg formats ( figure b) . interestingly, and similar to the multiplexed results, the inhibitory activity against niv g-mediated cell fusion was higher than that against hev g-mediated fusion although m . was selected by using sg hev . these results suggest m . possesses exceptional cross-reactivity and potency against hev and niv env-mediated cell fusion and syncytia formation. potent cross-reactive neutralization of live viruses. igg m . exhibited exceptionally potent and cross-reactive inhibitory activity against infectious niv and hev with ic values fig. . immunofluorescence-based syncytia assay of hendra virus (hev) and nipah virus (niv) infection. vero cells were plated into -well plates and grown to % confluence. virus and antibodies were premixed for min at °c prior to addition to the cell monolayers. cells were incubated in the presence of antibody-virus mixtures for h, fixed in methanol, and immunofluorescently stained for p protein prior to digital microscopy. all images were obtained at an original magnification of ϫ. a, hev without antibody; b, hev with m at g/ml; c, hev with m at g/m; d, hev with . at g/ml; e, hev with . at g/ml; f, niv without antibody; g, niv with m at g/ml; h, niv with m at g/ml; i, niv with . at g/ml; j, niv with . at g/ml. below . and . g/ml, respectively (table ) . these data suggest a strong correlation between binding to the antigens, inhibition of fusion, and neutralization of infectious virus and confirm the exceptional potency and cross-reactivity of m . . igg m . was also evaluated in the sensitive vero cell-fociimmunostaining assay overnight side by side with igg m ( figure ) . in this case, not only was the amount of virus per well high ( tcid for hev and tcid for niv), but the antibody-virus mixtures were incubated on vero cell monolayers overnight. if no antibody was present (panels a and f), there was massive coalesced syncytia for both viruses. in the presence of m , hev was neutralized to localized foci at g/ml, which was further reduced to individual infected cells at g/ ml. importantly, if this assay was carried out as a standard cytopathic effect-based neutralization assay, these wells would essentially look uninfected. by comparison, m . neutralized % of hev and niv at either g/ml or g/ml. this extended neutralization window demonstrates the exceptional potency of m . and may have important implications for postexposure therapeutic efficacy. to characterize the epitope of the affinity-maturated m . -compared with m -on the hev g glycoprotein, a panel of hev g alanine-scanning mutants constructed in a previous study [ ] were expressed and tested for binding to m and m . by immunoprecipitation. the binding of hev g mutants d a, g a, k a, and k a to both m and m . was almost absent, whereas the mutations g a and d a significantly decreased the binding of both antibodies, although they did so to varying degrees ( figure ). interestingly, one mutation, k a, almost completely eliminated the binding of the hevspecific antibody m but did not have any effect on the binding of the cross-reactive antibody m . . bishop et al. [ ] showed that all mutations-d a, g a, k a and k a-that eliminated the binding of both mabs in this study are detrimental for the binding of hev g to the receptor ephrin-b . these results suggest that the m . epitope overlaps the receptor-binding domain of hev g and the epitope of m . in vivo plasma half-life and biological activity. as hmab m . has the potential to be a potent henipavirus therapeutic agent, we next assessed its in vivo half-life and toxicity. we chose to use ferrets for these studies because they have been shown to be susceptible to niv-mediated disease (k. bossart, j. bingham, and d. middleton; unpublished data) and have become important animal models for several other human respiratory viruses, including sars coronavirus and highly pathogenic avian influenza virus. ferrets received of different doses of m . ( or mg), as detailed above. animals were closely observed for at least hours after recovery, and no adverse effects were noted. blood samples were collected over a -day period, and antibody concentrations were measured. a typical antibody concentration over time is shown in figure , which shows slopes in a logarithmic scale. half-lives calculated from these slopes did not vary with antibody dose. average distribution and elimination half-lives of . days and . days, respectively, were calculated, with relatively small individual differences. to determine whether the relatively short elimination half-life was the result of immune responses, we tested the ferret serum for anti-m . ferret antibodies. we were not able to detect such antibodies in ferret serum samples after administration of m . (data not shown). to demonstrate that m . measured in plasma was biologically active, serum collected on days , , and was evaluated using virus neutralization assays, as described above (data not shown). importantly, for all ferrets, niv was completely neutralized by : diluted serum collected on day after antibody administration. when serum samples collected on day were as- sayed, samples demonstrated complete virus neutralization, and a third serum sample demonstrated % neutralization. although negative on day , the fourth serum sample showed % neutralization on day . taken together, these data demonstrate that m . can remain biologically active in vivo for at least days. the major finding of this study is the identification of a novel, exceptionally potent, cross-reactive neutralizing hmab, m . . this antibody has significantly improved potency, compared with the parent antibody m and with other hmabs identified and characterized in our previous study [ ] . importantly, the substantial gain in potency was achieved without decreasing cross-reactivity. to our knowledge, this is the first fully human antibody that is capable of potently neutralizing both infectious hev and niv. an interesting observation made during the present study was that m . had better binding to sg niv than to sg hev , despite the fact that sg hev was used as the selector antigen during the original library screening [ ] and in the maturation panning procedures. the better binding to sg niv correlated with better neutralizing activity against niv, compared with hev. further studies are required to understand the mechanism underlying this unexpected observation. although the epitope mapping by alanine-scanning mutagenesis indicated that m . and m share most of the residues on the hev g glycoprotein that affect their binding, a dramatic difference was observed for the k a mutation, which completely abolished the binding of m to hev g, but did not affect m . 's binding. it was previously shown by bishop et al. [ ] that k a had no effect on the binding of hev g to the henipavirus receptors ephrin-b and ephrin-b . thus, the m epitope may contain contact site(s) located outside the receptor binding site. because the m . epitope does not include this site, one could hypothesize that it overlaps the receptor binding site to larger extent than the m epitope does. such a hypothesis is in agreement with m . 's much higher observed degree of cross-reactivity, compared with that of m . thus, one could further hypothesize that the m . epitope closely mimics the receptor binding site and, therefore, that the generation of escape mutants in the presence of this antibody would be less likely, compared with m and indeed any other known hmab. we have previously made similar observation for our potent cross-reactive neutralizing hmab m , which is predicted to be effective against all sars coronavirus isolates with known sequences [ ] . the m binding site overlaps extensively with the receptor binding site on the sars coronavirus spike (s) glycoprotein, as shown by the crystal structure of the m -s complex [ ] . thus, targeting the conserved and functionally important receptor binding site that is critical for virus entry into cells is a promising strategy for the development of cross-neutralizing antibodies. the igg m . was well tolerated in ferrets, and no any adverse effects were noted for the relatively short time ( days) of the experiment. the antibody pharmacokinetics consisted of phases. the estimated distribution half-life of ϳ . days is typical for iggs. the elimination half-life was significantly shorter than that for human iggs in humans (typically - weeks). we hypothesized that this could be the results of immune responses, specifically the elicitation of anti-human igg antibodies, which typically develop after - weeks. however, our attempts to detect such ferret antibodies against m . did not result in any measurable quantities above the background (data not shown). this could be because of the low levels of such antibodies during the relatively short period of observation. further studies are required to clarify the answer to this question. we also found that m . demonstrates reasonable stability and retains its biological activity in vivo. it has been previously shown that serum from hamsters immunized with vaccinia viruses that expressed niv envelope glycoproteins can protect the animals from challenge with niv [ ] . this important study by guillaume et al. [ ] further supports the notion that biologically active m . would be able to protect animals and humans from henipavirus infections. in summary, m . appears to be close to an ideal candidate for further development into an immunotherapeutic agent for henipavirus infection because it possesses many of the properties desired in such a therapeutic modality. it is a fully human mab; it retains its biological activity in vivo; it does not cause toxicity in ferrets; it has a distribution half-life typical for iggs, and its elimination half-life is likely to be significantly longer in humans; it cross-neutralizes both hev and niv; it has a muchimproved potency, compared with m , its parental antibody; and it targets the g glycoprotein region, which largely overlaps with the receptor binding site. this mab may also prove useful in the development of diagnostics, small molecule drugs, and vaccines, and as a research reagent. hendra and nipah viruses: different and dangerous a morbillivirus that caused fatal disease in horses and humans nipah virus: a recently emergent deadly paramyxovirus nipah virus encephalitis reemergence emerging viruses: coming in on a wrinkled wing and a prayer emerging infectious diseases. nipah virus (or a cousin) strikes again fatal fruit bat virus sparks epidemics in southern asia nipah virus encephalitis reemergence nipah virus outbreak(s) in bangladesh person-to-person transmission of nipah virus in a bangladeshi community ultra-potent antibodies against respiratory syncytial virus: effects of binding kinetics and binding valence on viral neutralization receptor binding, fusion inhibition, and induction of cross-reactive neutralizing antibodies by a soluble g glycoprotein of hendra virus potent neutralization of hendra and nipah viruses by human monoclonal antibodies ephrin-b ligand is a functional receptor for hendra virus and nipah virus identification of hendra virus g glycoprotein residues that are critical for receptor binding neutralization assays for differential henipavirus serology using bio-plex protein array systems potent cross-reactive neutralization of sars coronavirus isolates by human monoclonal antibodies structure of severe acute respiratory syndrome coronavirus receptor-binding domain complexed with neutralizing antibody nipah virus: vaccination and passive protection studies in a hamster model we wish to thank tim hancock from the australian animal health laboratory for his help with the in vivo ferret work. key: cord- -ro wm kf authors: yang, yifei; shi, ruihan; she, ruiping; mao, jingjing; zhao, yue; du, fang; liu, can; liu, jianchai; cheng, minheng; zhu, rining; li, wei; wang, xiaoyang; soomro, majid hussain title: fatal disease associated with swine hepatitis e virus and porcine circovirus co-infection in four weaned pigs in china date: - - journal: bmc vet res doi: . /s - - -z sha: doc_id: cord_uid: ro wm kf background: in recent decades, porcine circovirus (pcv ) infection has been recognized as the causative agent of postweaning multisystemic wasting syndrome, and has become a threat to the swine industry. hepatitis e virus (hev) is another high prevalent pathogen in swine in many regions of the world. pcv and hev are both highly prevalent in pig farms in china. case presentation: in this study, we characterized the hev and pcv co-infection in – month-old piglets, based on pathogen identification and the pathological changes observed, in hebei province, china. the pathological changes were severe, and general hyperemia, hemorrhage, inflammatory cell infiltration, and necrosis were evident in the tissues of dead swine. pcr was used to identify the pathogen and we tested for eight viruses (hev, porcine reproductive and respiratory syndrome virus, pcv , classical swine fever virus, porcine epidemic diarrhea virus, transmissible gastroenteritis coronavirus, porcine parvovirus and pseudorabies virus) that are prevalent in chinese pig farms. the livers, kidneys, spleens, and other organs of the necropsied swine were positive for hev and/or pcv . immunohistochemical staining showed hev- and pcv -antigen-positive signals in the livers, kidneys, lungs, lymph nodes, and intestine. conclusion: hev and pcv co-infection in piglets was detected in four out of seven dead pigs from two pig farms in hebei, china, producing severe pathological changes. the natural co-infection of hev and pcv in pigs in china has rarely been reported. we speculate that co-infection with pcv and hev may bring some negative effect on pig production and recommend that more attention should be paid to this phenomenon. the rapid development of the pig industry in china accompanies with outbreaks of epidemic diseases in recent years. hepatitis e virus (hev) has been identified on pig farms in many regions of the world, including china [ ] [ ] [ ] . hev seropositivity rates of . % and % have been reported in pig herds of large-scale and family-scale farms in china, respectively [ ] . increasing evidence indicates that hev can infect both humans and animal [ ] . to date, most studies of hev based on prevalence surveys, and research into hev-associated mortality during natural infection was limited. mao et al. reported that co-infection with hev and porcine reproductive and respiratory syndrome virus (prrsv) could lead to high mortality in swine [ ] , and they speculated that co-infection with hev and other pathogens could cause serious disease. it has been demonstrated that hev and porcine circovirus (pcv ) could cause infectious hepatitis, but swine naturally co-infected with hev and pcv in china has rarely been reported [ , , ] . pcv infection occurs in many countries and poses a considerable threat to the swine industry [ ] . although the recently research showed that infection of pcv could be effectively reduced by utilizing pcv vaccine [ ] , prevention of pcv in the pig production should be paid more attention. in the present study, pathogen identification and the observation of pathological changes demonstrated a natural co-infection with hev and pcv in the swine on two pig farms in hebei province, china. this discovery may provide a new perspective for clinical research. medical history and clinical symptoms from november to december , an outbreak of an unknown disease occurred at two small-scale pig farms ( pigs in farm a and pigs in farm b), operating for a short time in hebei province, china. all of the piglets fed in both pig farm a and b were aged - months. pig farm a reported the deaths of piglets (mortality rate was . %), and pig farm b the deaths of pigs (mortality rate was . %). the affected animals on both farms presented with symptoms of fever, dyspnea, diarrhea, and anorexia. in pig farm a, the veterinary administrated timicosin and doxycycline to treat the pigs. and in pig farm b, florfenicol was administrated. however, the swine did not respond to antibiotic treatment. necropsies were performed on seven dead piglets: three from farm a (pigs , , and ) and four from farm b (pigs , , , and ). the tissues examined included the liver, spleen, lung, kidney, heart, intestine, and lymph nodes. all tissues used for histological examination were fixed in . % (w/v) glutaraldehyde-polyoxymethylene solution for h. the fixed tissues were routinely processed, embedded in paraffin, sectioned ( μm thickness), and stained with hematoxylin and eosin. portions of the liver, spleen, kidney, brain and lung tissues were used for pathogen detection and stored at − °c until required. seven dead piglets were necropsied and diagnosed. scattered hemorrhagic spots were observed on the surface of the skin ( figure a ). the right ventricle was dilated so that the ratio of the transverse/longitudinal diameters was increased ( figure b) . hyperemia, hemorrhage, and necrosis were present in large local areas of the lung ( figure c ). a transparent gelatinous exudate was observed in the trachea ( figure d ). the liver was enlarged and the surface was a dark red color ( figure e ). it was difficult to strip the kidney capsule, and all the kidneys showed varying degrees of enlargement ( figure f ). the lymph nodes and spleens were swollen to varying degrees ( figure g , h). hemorrhage and infarction were observed in the spleen ( figure h ). the mesenteric lymph nodes were enlarged and hyperemic ( figure i ). the pathological changes in various tissues were determined with microscopy. the lesions observed in the lung, liver, heart, kidney, lymph node, spleen and intestinal tract tissues were similar in all the pigs necropsied. the heart lesions were characterized as viral myocarditis (figure a ). the epicardium was predominantly infiltrated by lymphocytes, with a small number of neutrophils ( figure b ). granular myocardial degeneration, edema, and lymphocyte and neutrophil infiltration in the myocardium were observed ( figure c) . a hepatic examination revealed features characteristic of hepatitis in a number of liver samples, including congestion, vacuolization, and necrosis, ( figure e ). lymphocyte and neutrophil infiltration, particularly in the portal area, was clearly observed ( figure f ). examination of the lungs demonstrated large areas of hyperemia, hemorrhage, and lymphocyte and neutrophil infiltration, with very little normal histological structure. the bronchioles contained exfoliated alveolar epithelial cells and pink liquid exudate ( figure g ,h). enlargement of the glomerulus and focal lymphocyte infiltration were observed in the kidneys. the renal tubule epithelial cells showed granular degeneration and necrosis, and congestion and hemorrhage were present in the kidneys. the renal tubule epithelial cells shed off from the basilar membrane. the glomerulus contained albuminoid droplets of exudate ( figure i ,j). the organs of immune system were severely underdeveloped, and malformed splenic white pulp was responsible for the reduced numbers of lymphocytes ( figure d ). poorly developed lymph nodes were also evident. the majority of capillaries were expanded and hyperemia was present. the lymphoid nodules were smaller than normal, resulting from fibrosis, necrosis, and lymphocyte depletion ( figure k , l). examination of the intestine revealed necrosis, and coagulation of the intestinal villi. the submucosal layer was exposed due to the loss of mucosal layer. epithelial cell shedding and secretion from the intestinal glands into the gut cavity were increased ( figure m , n). the main pathological changes observed in the various organs of the seven necropsied pigs are summarized in table . pcr was used to detect any viruses in the liver, lung, spleen, brain and kidney samples (table ). viral pathogens responsible for suspicious diseases in swine were investigated: hev, pcv , classical swine fever virus . other tissues were also tested using pcr. the liver, spleen, kidney, lung, and brain of pig no. , , , , and were positive for hev rna and these tissues were pcv dna positive in pig no. , , , , , and . the co-infection rate for hev and pcv was . % ( / ). the livers, lungs, kidneys, and spleens of the necropsied pigs were negative for pedv, tgev, csfv, prrsv, prv, and ppv. immunohistochemical (ihc) staining confirmed the presence of hev and pcv antigens in several tissues and organs. hev antigen was detected in the livers, kidneys, lung, intestine and lymph nodes of all five hev-positive swine (pig no. , , , , ). granular or diffuse positive staining was seen in the hepatic sinusoid and the cytoplasm of hepatocytes ( figure a ). the nuclei and cytoplasm of the renal tubular epithelial cells ( figure b ) and lung cells ( figure c ) were positive for hev antigen. the staining for hev antigen in the lymph nodes was intense in the lymphocytes and macrophages ( figure d ). the staining for hev antigen in the intestinal tissue was intense in the lamina propria and gut-associated lymphoid tissue ( figure e ). the negative control is shown in figure . hev antigen was negative in the two hev rna negative swine (pig no. and ). the lungs, livers, kidneys, lymph nodes, and intestine were tested for pcv antigen with ihc staining. the tissue distribution of the pcv antigen was similar in all pcv dna positive pigs (pig no. , , , , , ). in the liver, pcv antigen was detected within the hepatocytes and küpffer cells ( figure a ); in the kidneys, the positive signals were in the tubular epithelial cells ( figure b) ; and for the lungs, pcv -antigen positive signals were in the alveolar and septal macrophages, and fibroblast-like cells in the lamina propria of the airways ( figure c ). pcv antigen was intense in the lymphocytes and macrophages in the lymph nodes ( figure d ), and the mucous layer and lamina propria of the intestine ( figure e ). the negative control is shown in figure . pcv antigen was negative in the pcv dna negative pig (pig no. ). hepatitis e virus infections are a major cause of acute hepatitis in developing countries, and because of the zoonotic transmission of hev, they are also an emerging health problem in industrialized countries. swine are considered to be a major reservoir of the hev transmitted to humans [ , ] . four main genotypes have been identified in hev. genotypes and have only been found in humans, whereas genotypes and have been recovered from both humans and pigs [ ] . smith et al. recently proposed a taxonomic scheme, which divided the family hepeviridae into the genera orthohepevirus (all mammalian and avian hepatitis e virus (hev) isolates) and piscihepevirus (cutthroat trout virus) [ ] . the livers of pigs naturally infected or intravenously inoculated with hev display focal lymphocytic infiltration and swollen, vacuolated hepatocytes [ ] . the livers of the seven pigs investigated in the present study had significant lymphocytic infiltration in the portal area, and large localized areas of fibrosis, necrosis, and vacuolization. ihc staining showed that the orf protein of hev was distributed across multiple organs, particularly in the liver and kidneys. this result was not unexpected because the liver is the target organ of hev, and the kidney plays an integral role in maintaining extracellular fluid homeostasis. the previous study also demonstrates that hev has been found in liver and kidney after experimental infection in domestic pigs [ ] . a pcr assay specific for hev orf confirmed that the pigs were hev positive. isolates chn-hb-hd-l , chn-hb-hd-l , hb-l , chn-hb-hd-l , and chn-hb-hd-l were shown to belong to genotype , the most prevalent hev genotype in china. according to smith et al. [ ] , the hev strains isolated in our case were classified to orthohepevirus a. pcv is the primary causative agent of pmws which was first described in canada in [ ] . in recent years, pmws has become a serious economic problem for the swine industry in china. according to the data from a prevalence survey, more than . % of piglet stool samples were pcv positive [ ] . the disease predominantly affects pigs between and weeks of age and is characterized by growth retardation, diarrhea, dyspnea, jaundice, and enlargement of the inguinal lymph nodes. in our study, infected swine aged - months displayed clinical symptoms consistent with previous reports of the disease [ ] . in this study, the clinical and pathological changes observed were consistent with typical pcv infection. hemorrhage, hyperemia, edema, necrosis, and lymphocyte infiltration were observed in all organs, most notably the lungs. histological changes consistent with lobar pneumonia were also evident in the lungs, and normal lung histology was rarely seen. the alveolar walls were thickened, with substantial lymphocyte, erythrocyte, and exudate infiltration. exfoliated alveolar epithelial cells and pink liquid exudate were observed within the bronchioles. pcv has a small, nonenveloped icosahedral virion, and a single-stranded circular dna genome, , - , nt in length. the genome has two major orfs encoded in the antisense direction [ ] . isolates hbhd-l , hbhd-l , hbhd-l , hbhd-l , hbhd-l , and hbhd-l recovered in this study were closely related to the genotype pcv d strains hnf (kj ), sd-zb (kj ), wsec (kj ), gxyq (kj ), tdbs (kj ) (figure ) . the genotype pcv d represented a novel genotype and a shift from pcv a to pcv b as the predominant genotype in china in recent years [ ] . a genetic analysis, combined with the observed pathological changes, indicated that the pcv isolates detected in this study were probably high prevalent in china [ ] . ihc staining of tissues for the orf protein of pcv revealed that the antigen was observed in the lungs, liver, lymph node, intestine and kidneys, further evidence of pcv infection. further pathological changes typical of pcv infection were observed in this study. significant immune-system- organ dysplasia was apparent, with the characteristic histopathological findings of lymphoid depletion and histiocytic replacement in the lymphoid tissues. combined with the positive pcv orf signals in lymph node in ihc, these results suggest that the systemic immune function of these pigs had been disrupted. ihc staining for hev and pcv antigens revealed a diffuse labeling pattern in the intestine, with the greatest reactivity observed in the cytoplasm of cells in the mucous layer and lamina propria. this observation is consistent with the viral invasion pathways. the transmission of hev occurs via the fecal-oral route, so hev may invade the animal through the intestinal mucous layer, with infection progressing to the lamina propria. we have investigated the mucosal immunity in the intestines of rabbits [ ] and gerbils (data not shown) experimentally infected with hev, and both studies demonstrated a strong hev orf positive signals in intestinal. in the present study, naturally infected swine exhibited significant necrosis of the intestinal epithelial cells and also showed hev orf positive signals in intestine. therefore, hev invasion of the intestine may proceed rapidly and widely, consistent with the diffuse labeling pattern observed in the intestine in this study. we also tested for other suspicious pathogens in this study. according to the medical history, the sick piglets failed to respond to antibiotic treatment (timicosin and doxycycline in pig farm a, and florfenicol in pig farm b), indicating that bacterial infection was unlikely. prrsv, csfv, pedv, tgev, prv, and ppv are high prevalent in swine in china and across the globe. although infections with these viruses may present with similar clinical symptoms, including fever, diarrhea, depressed, and decrease of feed intake. pcr confirmed that all seven pigs were negative for these viruses. presence of signs and lesions such as lymphoid depletions, hepatitis, nephritis, etc. resemble the microscopic characteristic of pmws. nevertheless, no typical microscopic lesions such as granulomatous inflammation or intracytoplasmic inclusion bodies were observed in lymphoid tissue, liver, spleen, and other tissues [ ] . the mortality associated with pcv infection is generally around % (range %- %), but can reach % [ ] . in our case, seven pigs were detected and the pathogens had been identified. however, the true reason for the high mortality in the whole pig farm a and b still need more exploring. the occurrence of this disease may not be only a matter of pmws caused by pcv infection. the previous study showed swine hev infection can be a significant factor to the development of hepatitis regardless of the pmws status [ ] . in addition, j. ellis [ ] claimed that the severity of hepatic lesions in pcv- infected pigs may be enhanced by co-infection with swine hepatitis e virus. it may indicate that the significance of hev is hardly negligent. further investigation about the mechanistic basis for the pathogenesis of the clinical syndrome that associated with pcv and hev coinfection needs to be conducted. hev infection in humans and animals is common, but the natural occurrence of hev and pcv co-infection in pigs in china, reported here, has rarely been seen. the experimental infection of domestic pigs with hev did not cause death [ ] . therefore, the hev-infected swine observed in this study requires further investigation. based on these results, we believe that considerable attention should be directed towards co-infections of hev and pcv in swine. according to the comparison of histopathological changes between these cases in table , no specific characteristics are demonstrated, which is really thoughtprovoking. it is very significant to explore the reasons for the similar pathological changes in the hev and/or pcv infected pigs. in order to reveal the mechanism for the similar pathological changes in the hev and/or pcv infected pigs, further tests about hev and pcv co-infection, single hev infection and single pcv infection in pigs have been in the planning. we hope to reveal the mechanism of the similar pathological changes and also discover the similarity and differences between the natural cases and the experimental infected pigs. to our best knowledge, theses following reasons are speculated to explain the similar pathological changes: a) the individual differences in pigs. different pigs may have different reaction to the attack of the viruses; b) pig no. may once have been infected with pcv and then pcv were neutralized by the antibody. but lesions in the tissues hadn't recovered when it died; c) hev is rna virus without envelope. it may have degraded in tissues in pig no. and before testing. d) the complicate in natural infected cases. in conclusion, co-infection of hev and pcv were identified in four out of seven dead weaned pigs from two pig farms in china. severe pathological changes and high mortality were observed in the infected animals. our results indicate that co-infection with hev and pcv may bring some negative effect on the swine industry in china, and this phenomenon requires further investigation. what's more, further research is required to demonstrate the role of co-infection of hev and pcv in swine and whether these two viruses exert a synergistic effect. written informed consent was obtained from the owners of the two farms for the publication of this report and any accompanying images. according to the gross and histopathological lesions, eight suspected viruses were detected. total rna and dna were extracted from liver, lung, kidney, brain, and spleen specimens using the ultrapure™ rna kit and the general allgen kit (cwbio, beijing, china), according to the manufacturers' instructions. the extracted rna prevalence of hepatitis e virus in swine fed on kitchen residue detection of hepatitis e virus (hev) in italian pigs displaying different pathological lesions a novel virus in swine is closely related to the human hepatitis e virus prevalence of hepatitis e virus in swine under different breeding environment and abattoir in beijing, china phylogenetic analysis of global hepatitis e virus sequences: genetic diversity, subtypes and zoonosis one case of swine hepatitis e virus and porcine reproductive and respiratory syndrome virus co-infection in weaned pigs nucleotide sequence of porcine circovirus associated with postweaning multisystemic wasting syndrome in pigs association of hepatitis e virus (hev) and postweaning multisystemic wasting syndrome (pmws) with lesions of hepatitis in pigs porcine circoviruses: a review commercial pcv a-based vaccines are effective in protecting naturally pcv b-infected finisher pigs against experimental challenge with a mutant pcv prevalence and transmission of hepatitis e virus in domestic swine populations in different european countries molecular virology of hepatitis e virus consensus proposals for classification of the family hepeviridae comparative pathogenesis of infection of pigs with hepatitis e viruses recovered from a pig and a human evidence of extrahepatic sites of replication of the hepatitis e virus in a swine model isolation of circovirus from lesions of pigs with postweaning multisystemic wasting syndrome complete genome sequence of a highly prevalent porcine circovirus isolated from piglet stool samples in china detection of a novel strain of porcine circovirus in pigs with postweaning multisystemic wasting syndrome porcine circovirus type (pcv ): genetic variation and newly emerging genotypes in china genetic variation analysis of chinese strains of porcine circovirus type detection and localization of rabbit hepatitis e virus and antigen in systemic tissues from experimentally intraperitoneally infected rabbits postweaning multisystemic wasting syndrome: a review of aetiology, diagnosis and pathology porcine circovirus type and porcine circovirus-associated disease porcine circovirus- and concurrent infections in the field experimental hepatitis e infection in domestic pigs molecular variation in the nucleoprotein gene (orf ) of the porcine reproductive and respiratory syndrome virus (prrsv) multiplex pcr for detection and typing of porcine circoviruses genetic diversity of the envelope glycoprotein e of classical swine fever virus: recent isolates branched away from historical and vaccine strains a multiplex rt-pcr assay for rapid and differential diagnosis of four porcine diarrhea associated viruses in field samples from pig farms in east china from to development of multiplex pcr for simultaneous detection of six swine dna and rna viruses we thank dr. liu jue (beijing municipal key laboratory for the prevention and control of infectious diseases in livestock and poultry, beijing, china) for kindly supplying the anti-pcv orf antibody for the study. this work was supported by the national natural science foundation of china (grant nos. , ). the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. the authors declare that they have no competing interests.authors' contributions yy, rshi, and rshe designed the study. yy and rshi wrote article. yy, rshi, fd, rn, wl, mc, xw, and mhs performed the laboratory experiments. yy, rshi, cl, jm, yz, and jl analyzed and interpreted the data. all authors have read, commented upon, and approved the final article. key: cord- -qbf lv d authors: nucera, eleonora; aruanno, arianna; rizzi, angela; centrone, michele title: latex allergy: current status and future perspectives date: - - journal: j asthma allergy doi: . /jaa.s sha: doc_id: cord_uid: qbf lv d allergy to natural rubber latex (nrla) from hevea brasiliensis is a relevant public health issue, in particular in healthcare workers and groups at risk. clinical manifestations of nrla can range from mild skin disorders to life-threatening systemic reactions. prevention measures remain the gold-standard treatment for patients suffering from nrla, but the only etiological therapy able to influence the natural history of nrla is specific desensitization. this review aims to underline the epidemiological, clinical and diagnostic aspects of nrla, and carries out a complete and wide-ranging review of the current literature on nrla management and immunotherapy. natural rubber latex (nrl) is an intracellular cytosol secreted from a rubber tree, hevea brasiliensis (hev b), which functions as a protective sealant. the milky substance is first ammoniated to prevent bacterial contamination and coagulation and then multiple chemicals are added: accelerators, antioxidants and secondary preservatives. because of its excellent elastic properties, nrl is widely used in the manufacture of medical devices and in a variety of everyday articles such as catheters, gloves, condoms and balloons. the first recorded reactions to nrl date back to , when two german doctors, stern and grimm, respectively described a case of urticaria caused by a dental prothesis and a case of professional asthma. [ ] [ ] [ ] immediate-type allergic reactions to nrl were epidemiologically silent until , when nutter reported a case of urticaria after contact with gloves. thereafter, reports linked to latex exposure increased exponentially in the late s and s, simultaneously with the increasing use of latex devices. in general, latex protein components are responsible for type i latex-specific allergy and the accelerators and antioxidants are agents of type iv allergic reactions (contact dermatitis), although rare cases of delayed allergy to latex proteins have been reported. undiagnosed latex allergy is potentially very serious for patients and is increasingly recognized as a significant contributor to morbidity and mortality during medical and surgical procedures. the aim of this review is to underline the epidemiological, clinical and diagnostic aspects of natural rubber latex allergy (nrla), and to carry out a complete and large review of the literature on nrla management. a literature search (pubmed) for articles until june was conducted using the following key words: "latex", "latex allergy", "latex hypersensitivity", "management", "primary and secondary prevention", "immunotherapy", "desensitization", "treatment" and "anaphylaxis". the following eligibility criteria were used for article inclusion: population: patients with latex allergy and/or at risk for anaphylaxis; intervention: any approaches or protocols that incorporated a strategy for latex allergy and anaphylaxis management; comparator: any studies irrespective of whether there was a comparator included in the study design; outcomes: any related to prevalence, diagnostics, and treatments including primary prevention and immunotherapy; and study design: experimental studies and observational studies. we excluded basic science or animal (non-human) studies. the search focused on english-language articles. overall, approximately articles were reviewed and of these are included here for reference. the current prevalence rate of latex allergy changes according to the population considered. the highest risk population is represented by patients undergoing repeated surgical interventions (such as spina bifida patients). these groups have a higher chance of being exposed to latex allergens and therefore have a higher risk of developing allergy. the current prevalence of latex allergy and sensitization among susceptible patients are . % and . %, respectively. in particular, the prevalence of latex sensitivity among the spina bifida pediatric population was between % and %. [ ] [ ] [ ] healthcare workers (hcws) are the occupational group most affected by nrla owing to their frequent use of latex gloves. the current prevalence of latex allergy and sensitization among hcws worldwide are . % and . %, respectively. other occupational workers, including rubber industry workers, hairdressers and housekeepers, are also at high risk for latex allergy, although reports have suggested that general populations who have not had occupational contact with latex products can also develop latex sensitization and latex allergy. data analysis from limited studies suggests that the current average latex allergy prevalence among the general population worldwide is . %. although the use of latex gloves in surgery became routine after , gloves were not consistently used in other areas of patient care until the late s. the emergence of human-to-human transmission of infectious pathogens, such as hepatitis c and hiv, produced a dramatic increase in the use of latex gloves and other latex devices in all areas of patient care. for these reasons, in the late s and s there was a considerable increase in latex allergy. airborne antigen exposure is an important source of latex sensitization among hcws. the addition of cornstarch powder to improve the fit of gloves has been shown to increase this latex protein aerosolization; for this reason, the use of powder-free latex gloves markedly reduces the risk of sensitization. , latex absorption through the skin is another major route of sensitization in hcws, especially when the skin is damaged. moreover, susceptible patients can also be latex exposed during surgical procedures. non-medical rubber products, such as car tires, have little allergen in them owing to prolonged heating during manufacture and chemical solvent use; consequently, it does not seem necessary to avoid contact. patients need to be mostly concerned about products made by a dipping method with low heat and minimal vulcanization time (gloves, condoms, etc), which have a high allergenic risk. the most frequent clinical manifestations of latex allergy are related to type i hypersensitivity mediated by immunoglobulin e (ige), and can involve the skin (itching, swelling, pruritus and contact urticaria), the respiratory system (sneezing, wheezing and rhinitis) and the eyes (conjunctivitis). clinical manifestations can also be systemic, such as bronchospasm, hypotension, cardiorespiratory collapse and shock. anaphylactic shock is potentially fatal and occurs most commonly in an intraoperative context. an important clinical manifestation of nrla is the "latex-fruit syndrome" due to latex proteins that have clinical cross-reactivity with multiple vegetable foods. the ingestion of many fruits or vegetables (table ) can cause clinical symptoms ranging from itching and pruritus of the oral cavity (oral allergy syndrome [oas]) to anaphylaxis. [ ] [ ] [ ] type iv hypersensitivity reactions type iv hypersensitivity reactions typically develop - hours after exposure. these reactions are generally seen as eczematous dermatitis at the contact site, and are localized and uncomfortable, but not life threatening. delayed reactions are usually caused by accelerators and antioxidants (e.g. carbamates, thiurams) added during the manufacturing process of nrl. only a few cases of delayed allergy to nrl proteins have been reported in the literature. , relevant allergens nrl secreted by hevea brasiliensis contains more than polypeptides, detected by electrophoresis. , fifteen allergens (hev b - ) have been characterized and listed by the world health organization/international union of immunologic societies allergen nomenclature committee (www.allergen.org) ( table ). ige reactivity to hev b and hev b , membrane-bound elongation proteins, seems to be predominant in patients with spina bifida and urological congenital anomalies, while hev b and hev b (with the domains hev b . and hev b . ) were recognized as major allergens in hcws. , in particular, the isolation and characterization of hev b -specific ige has resulted in an increase in the sensitivity of the serological assay, and improved allergy diagnostics. the other allergens appear to be minor contributors to a genuine sensitization to latex; some of them belong to the families of defense proteins, such as lipid transfer protein (hev b ) and profilin (hev b ), and are responsible for cross-reactivity with fruits and vegetables. hev b , a serine protease inhibitor, is the last hev b allergen to be discovered. [ ] [ ] [ ] [ ] [ ] [ ] the diagnosis of nrla is formulated on the basis of an accurate medical history, physical examination, and in vivo and in vitro tests. the medical history is the cornerstone for establishing an accurate diagnosis of latex allergy and for identifying risk factors and a correlation between latex exposure and the appearance of symptoms. it is also fundamental to investigate some key points, such as history of atopy, food allergies (particularly bananas and kiwi fruit), and undiagnosed reactions or complications during dental work or surgical procedures. skin-prick tests (spts) must be carried out in a hospital setting by trained allergist experts in test technique and in interpreting the results. the overall risk of inducing anaphylactic reactions by spts is less than . %. the first systemic reactions to spts were published in , when four of patients with latex allergy reported anaphylactic reactions; since then, further cases have been reported in the literature. [ ] [ ] [ ] in highly sensitive patients, such as those with spina bifida, latex allergens may induce systemic reactions; in this condition, spts are considered a risk and in vitro tests should be performed in order to complete the diagnosis. a patch test is used to identify type iv hypersensitivity reactions. rare cases of delayed allergy to nrl proteins have been reported, so this method is helpful in differentiating allergic contact dermatitis from irritant contact dermatitis generally caused by accelerators and antioxidants. the allergens that have most commonly shown positive reactions are carbamates, thiurams mix, -mercaptobenzothiazole and , -diphenylguanidine. irritant contact dermatitis occurs when an exogenous substance without previous sensitization causes direct damage to the skin; most cases of hand dermatitis, in particular in hcws, have this underlying mechanism and may be clinically similar to delayed allergies. serological assays have been developed for the diagnosis of ige-mediated latex allergy and include ige testing immunocap measures. traditional latex-specific ige are based upon the quantification of ige directed against crude natural allergen extract. the hev b allergens, available in recombinant form, can be identified with immunocap (thermo fisher scientific, uppsala, sweden). sensitization to some components (e.g. hev b , hev b , hev b . and hev b . ) is associated with severe clinical phenotypes and is expressed as genuine latex allergy whereas sensitization to other components (e.g. hev b ) generally results in milder symptoms or is asymptomatic. , the basophil activation test (bat) is a flow-cytometrybased functional assay that assesses the degree of cell activation after exposure to a stimulus. this test could be useful to evaluate, in vitro, what happens in vivo following the exposure of the immune system to latex. provocation tests are important to test the target tissue's responsiveness to the allergen under controlled conditions. it is necessary to carefully evaluate the opportunity to carry out provocation tests if there is a positive anamnesis for anaphylactic reactions or in patients with important comorbidities. several methods of performing challenge tests have been reported (cutaneous, mucous-oral, sublingual, conjunctival, nasal, bronchial and vaginal), although some of them (e.g. vaginal test) have a low sensitivity and many limitations related to the procedure. , cutaneous challenge was performed by donning a latex glove and recording local symptoms. for sublingual, conjunctival, nasal and bronchial tests, latex solutions were prepared with latex extract, starting with the highest dilution and progressively increasing the concentration to reach the threshold dose. the management of groups at risk for latex allergy and hcws is based on a step-by-step process through four possible strategies: preventive measures, symptomatic treatment, immunotherapy and anti-ige therapy. , preventive measures primary prevention of latex allergy (nrl) means the reduction of exposure of nrl to prevent sensitization in susceptible workers and at-risk populations. , however, these measures are focused on the use of gloves and, in particular, the total replacement of latex gloves with powder-free low-protein (pflp) latex gloves or synthetic gloves made of alternative material. , since , the substitution of powdered nrl gloves with nonpowdered nrl sterile gloves in the operating room has resulted in a marked decrease in the number of new cases of latex sensitization. in , korniewicz et al showed that, although the initial cost of conversion may be high, it could help to reduce long-term healthcare costs. in fact, the resulting health expenditure was lower than the level of hcws' compensation claims for latex-related disability. furthermore, there have been efforts by the international glove industry to develop innovative protocols in order to reduce the allergenic content, satisfying both consumer demand and regulatory requirements. these include deproteinization and purification obtained by the addition of proteolytic and/or surfactant enzymes, chlorination process and high-temperature post-washing. regarding the use of alternative synthetic gloves, manufacturing companies have produced accelerator-free gloves using different materials (polychloroprene, nitrile and polyisoprene thermoplastic elastomers) or after washing in a strong alkaline solution. a survey of dental practitioners, published in , confirmed that routine use of latex-containing products in uk dental offices was low and that examination gloves in nitrile were replacing the general practice of using nrl gloves. nevertheless, at the time, the evidence did not support a total ban on the use of latex gloves. furthermore, with the exception of a few clinical studies, these latex-free gloves do not seem to have the same characteristics in terms of elasticity, tactile quality, providing a protective barrier against infections, resistance to permeability and cost accessibility. , in , raulf emphasized that most studies demonstrating a decline in the prevalence of latex sensitization following the introduction of powder-free latex gloves have been conducted on healthcare professionals in highly industrialized countries (europe and north america). in contrast, in developing countries and in those areas where primary prevention policies are not implemented, latex allergy continues to be a serious public health problem. another countermeasure is technological research into potential alternative sources of natural rubber gloves, starting from plant species such the mexican shrub guayule, which is not botanically related to hevea brasiliensis, but has a protein content of less than % and no crossreactivity with hevea latex allergens. , in the usa the food and drug administration (fda) has approved guayule gloves for use in the general population and has recognized and labeled these gloves as hevea latex free. in march , the fda released guidance, titled "process for making available guidance documents related to coronavirus disease " (available from https://www.govinfo.gov/content/pkg/fr- - - /pdf/ - .pdf), aimed at increasing the supply of other personal protective equipment (ppe) important in the fight against coronavirus disease (covid- ) pneumonia: medical gowns, other apparel and gloves. the fda recommends that hcws follow current centers for disease control and prevention (cdc) guidance regarding ppe that should be used during the covid- pandemic. in order to help to ensure the availability of these devices, the fda does not intend to object to the distribution and use of patient examination and surgeon's gloves that do not comply with the regulatory requirements (device registration and listing, premarket notification -where applicable -and quality system regulation compliance), provided that these devices are marketed using labeling that ) expressly delineates the uses for which they are appropriate, ) warns against uses that may create excessive risk, ) meets applicable barrier protection/flammability/sterility standards, and ) does not indicate a use that may increase an undue risk in light of the public health emergency. another crucial strategy of primary prevention in the workplace is the creation of a latex allergy task force and the development of appropriate facility policies, awareness and educational initiatives among hcws. as early as , allmers et al demonstrated that a joint program of education and regulation in german hospitals was followed by a remarkable change in glove purchase patterns. since children with spina bifida represent the population with the highest risk of developing latex allergy, primary prevention is intended to avoid latex exposure in subjects (not yet sensitized) from birth. numerous primary prevention studies have demonstrated its effectiveness, with the possibility of reducing the sensitization and, therefore, the appearance of allergic symptoms. [ ] [ ] [ ] in , stinkens et al highlighted the heterogeneity of recommendations concerning patient safety in the operating room, provided by some scientific societies. ideally, a latex-safe environment should be used in all healthcare facilities. the guidelines of the american society of anesthesiologists recommend that high-risk patients are scheduled first on the day of surgery to be treated in an operating room left unused for - hours, with possible and foreseeable management implications, including postponed surgery, increased patient discomfort and additional financial costs. in contrast, the guidelines of the australasian society of clinical immunology and allergy suggest that the effect of this specific scheduling is negligible when all powdered latex gloves are removed from the operating room and replaced by pflp latex or synthetic gloves. according to stinkens et al, patients with a history of latex allergy can be treated safely without specific scheduling when all powdered latex gloves are substituted with pflp latex gloves. to date, only a few prevalence studies concerning other occupational workers exposed to latex outside the healthcare setting have been conducted, and there is a lack of data on the effects of primary preventive strategies. [ ] [ ] [ ] secondary prevention of latex allergy has focused on procedures that prevent the development of reactions in sensitized/allergic patients, and include premedication before carrying out any risky procedures and providing latex-safe environments. however, this is not always possible because of the ubiquity of latex products and the cross-reactions to latex and fruit and vegetables. a meta-analysis of studies published between january and september highlights that avoidance of nrl powder gloves in the workplace reduces both symptoms and markers of sensitization in latex-allergic subjects, regardless of co-workers' use of non-latex gloves or pflp latex gloves. moreover, the authors concluded that there was limited evidence that latex-allergic health care workers can continue to use pflp gloves with no worsening in their symptoms, provided that their co-workers use pflp latex, or non-latex gloves. on the other hand, only a small case series of nine patients has described the efficacy of accelerator-free medical gloves in the secondary prevention of allergic contact dermatitis (acd) caused by rubber accelerators in hcws. gentili et al showed that an effective and exemplary example of secondary prevention of latex allergy is feasible for infants born with spina bifida. previously, a prospective study by reider's group investigated the effectiveness of secondary prevention strategies in the hitherto largest cohort of subjects at high risk for latex allergy for a comparatively long follow-up period. the authors attributed a significant decrease in latex-specific ige in latex-sensitized patients with hydrocephalus to medical more than home prophylaxis, which was "unrealistic in everyday life". to date, the use of non-latex gloves, catheters and alternative products, usually made of silicone, plastic or vinyl, can be considered the best recommendation for secondary prevention of latex allergy, derived from the scientific literature. moreover, individuals who have experienced allergic reactions during surgical or medical procedures should consider wearing a medicalert bracelet or necklace, carrying auto-injectable epinephrine and sterile non-latex gloves for emergency use, and discussing latex allergy with all healthcare and community providers, including school, day care and camp. in addition, consultation with an allergologist with experience in the management of latex allergy is recommended to fully evaluate the risks and the possible need for preoperative treatment with special medications to suppress the potential for severe allergic reaction. moreover, the avoidance of all latexcontaining items, especially in the operating room, is strongly recommended. finally, regarding prevention, all patients should have a list of substitute latex-safe products for hospital and home duties, cross-reacting fruits and occult sources of nrl exposure. indeed, the concept of "latex-safe" environments versus "latex-free" environments has turned out to be safe, practical and ideal for patients with latex allergy. the management of exposed and symptomatic individuals requires pharmacological treatment depending on the type of reaction that is present -from a mild sensitivity to a life-threatening allergic reaction (anaphylaxis). if the clinical manifestations consist of irritant dermatitis, removal of the latex and cleaning of the area are the first step. the application of topical steroids is used to reduce inflammation, and evaluation by a dermatologist is recommended. delayed type iv hypersensitivity reactions require the same treatment. in case of severe, life-threatening, generalized or systemic hypersensitivity reaction, defined as anaphylaxis, patients should managed and treated according to dedicated guidelines. [ ] [ ] [ ] immunotherapy more than a hundred years ago, noon and freeman published the first works on allergen-specific immunotherapy (ait) using grass pollen extracts. , since then, ait has been performed by a large number of modalities and has proven effective and safe in the treatment of allergic diseases, although in some areas of the world (e.g. the usa) no standardized nrl reagent is available. administration of the allergen at increasing doses results in a shift of t-helper cell polarization from the th to th cell phenotype; this switch is mediated by t-regulatory cells with the production of interleukin- , tumor necrosis factor-alpha and other chemical mediators. although not all mechanisms of action of specific immunotherapy have been clarified, ait remains the only etiological and decisive therapy able to modify the natural course of allergic diseases by inducing long-term immunological tolerance (table ). the first research tried to desensitize patients with latex allergy belonging to at-risk groups (such as health operators) by the percutaneous route. patriarca et al in suggested a progressively increasing exposure to latex, obtained by wearing latex gloves daily. after the desensitizing treatment, a maintenance latex exposure of at least minutes in both hands three times a week was recommended. the proposed percutaneous route seems absolutely safe (although few patients have been treated), and no side effects were highlighted. in , pereira et al reported the first experience with subcutaneous immunotherapy (scit) for latex. a -yearold woman, professionally exposed to latex devices, underwent scit for latex up to the maximum tolerated dose. although treatment was effective, systemic reactions related to its administration were reported. one year later, a randomized, multicenter, double-blind placebo-controlled trial was performed in patients by leynadier et al; desensitization was effective but the frequency of systemic reactions was higher in the active group, even during the maintenance phase. the evidence from these studies, with the experience of a high rate of adverse reactions (even anaphylaxis), was confirmed by further studies; tabar et al even reported systemic reactions in . % of patients in the active group versus . % in the placebo group. , for all these reasons, the subcutaneous route of latex immunotherapy administration was largely abandoned. in the early s, researchers assessed the potential use of sublingual immunotherapy (slit) for latex. the published literature regarding latex slit began in , when our group described the case report of a patient with nrla who became able to tolerate -hour mucosal and -hour cutaneous latex challenge tests after a -day rush build-up with latex slit. from this case, in the following years, numerous works have pursued the study of the sublingual route of administration. , in , patriarca et al studied patients in a randomized double-blind placebo-controlled trial. both groups of patients underwent a -day rush protocol followed by slit maintenance for months. after maintenance, tolerance of latex exposure (confirmed by latex-specific challenges) significantly improved in all patients in the slit group compared with the placebo group, and only mild local reactions were reported. although this was a small trial, these preliminary data suggested that slit could be safe and efficacious for the treatment of latex allergy. another study, by cisterò bahima et al in , enrolled latex-allergic patients treated with a commercial extract for sublingual administration (slit-latex; alk abello). according to a rush schedule, the maintenance dose was administered for weeks. a significant improvement in skin reactivity upon cutaneous exposure was reported, but . % of patients experienced at least one systemic reaction. this evidence, together with further, although rare, reports of anaphylaxis, suggest that the induction phase should be performed under medical supervision. nettis et al, in , enrolled adult patients with asthma or urticaria randomized to an ultrarush doubleblind, placebo-controlled latex slit protocol with the slit-latex extract. after a -month maintenance phase, patients underwent cutaneous challenge and a statistically significant improvement was again noted compared with the placebo group. only three patients in the active group reported local side effects, confirming the overall safety of latex slit. other data in the literature also support the long-term safety and efficacy of nrl immunotherapy in children. [ ] [ ] [ ] the interest in rush protocols has grown over the years; our group in compared two different rush induction protocols ( or days) in terms of safety and tolerability, showing no systemic or local reactions in the -day protocol patients. buyukoturk et al studied hcws who remained symptomatic despite attempted avoidance in . this work further confirms the safety and efficacy of latex slit. additional confirmation of safety and efficacy was given by lasa luaces et al in . they studied latexallergic children and, after months of slit, also underline immunological changes to predict clinical efficacy or safety outcomes. although some serological changes were observed (the bat with latex showed a reduction in reactivity after months), this trial did not show significant statistical changes in ige and igg . the most recent study regarding latex slit is a large observational case series of adult patients with nrla who underwent slit for years of treatment. after desensitization, in this study there was a marked reduction in serum levels of latex-specific ige and, according to the literature, a reduction in symptoms and scores on provocation tests, while igg levels did not change. on the other hand, the literature also describes cases that show a lack of effectiveness of the treatment; for example, morfin maciel et al describe the case of a boy who, despite an ait for latex, experienced anaphylaxis after an airport inspection with latex gloves. gastaminza et al showed no significant difference in specific provocation tests or in vitro testing after a year of slit-latex, with the exception of a reduction in the percentage of basophil activation both in the active group after years of therapy and in the placebo group after year of treatment. long-term latex slit complications are less often described in literature; significant is the case of a -yearold woman who developed solid food dysphagia, heartburn and dyspepsia after years of the maintenance phase. the esophageal endoscopy and biopsies showed an eosinophilic esophagitis that improved after months of slit interruption; this case seems to confirm what has already been highlighted in the literature for other oral or sublingual immunotherapies. , anti-ige therapy omalizumab has been shown to be clinically efficacious in the treatment of patients with allergic asthma and chronic spontaneous urticaria (csu). , therefore, some authors have studied the role of omalizumab in nrla treatment. leynadier et al, in a randomized, double-blind, placebo-controlled study, showed a statistically significant reduction in conjunctival and cutaneous responses to latex after omalizumab treatment. di leo at al also reported an improvement in latex-induced contact urticaria in a patient with uncontrolled chronic spontaneous urticaria. our case report confirms these findings; in fact, an asthmatic patient who was allergic to latex, during the omalizumab treatment, did not report clinical symptoms after any accidental contact with latex. the use of omalizumab can also be extended as an adjunct to latex immunotherapy; its use in conjunction with immunotherapy has shown promising results in the treatment of venom and food allergy, especially in the reduction of adverse reactions. nrla still represents a substantial health, social and financial problem for society today. although the prevalence of sensitization to nrl has significantly declined in developed countries over the past few years, nrla remains a relevant issue, especially among certain professional categories. the lack of data and preventive measures in developing countries means that the problem is still underestimated. another important aspect is the heterogeneity of the recommendations concerning primary prevention. it would be desirable that international guidelines (in particular concerning patient safety in the operating room) be drawn up in the coming years. during , the covid- pandemic has sparked a great deal of interest in how people might avoid becoming infected; for the general population it would seem that a simple solution is to wear disposable gloves, which are often made of latex. however, the widespread use of latex gloves during the covid- pandemic by both hcws and lay people could potentially worsen or induce nrla, as suggested by the first published epidemiological studies. the production and the use of alternative synthetic gloves at this time does not seem to be completely regulated because in order to help to ensure the availability of these devices, the fda does not intend to object to the distribution and use of gloves that do not comply with the regulatory requirements. given these considerations, an imminent increase in nrla patients could be expected. the authors believe that an international call to raise awareness of latex allergy by the world population may help to mitigate the risk of an increase in the incidence of nrla. this is the first step toward not forgetting the lessons learned in the s and s. in addition, despite there being reasonable data to support the use of latex slit, there is no commercially available latex extract in the usa, and in europe the production of slit has been slowed down by the manufacturer, alk abello. because of these problems, determining the effectiveness and safety of the percutaneous route, although highlighted only in one case series, may be the strategy to follow to desensitize allergic patients, even if more randomized trials are needed. the use of omalizumab, already supported by some scientific data, may provide an additional opportunity for therapeutic management, in association with immunotherapy. further studies on a larger scale are needed to validate this new possible use of omalizumab and to evaluate the persistence of latex tolerability after discontinuation. prevention remains the gold-standard treatment for patients suffering from nrla. however, the only etiological and decisive therapy able to influence the natural history of nrla is specific desensitization. rush protocols have provided evidence to suggest that effective doses of latex slit can be safely and rapidly achieved. regrettably, there are several limitations to latex studies. the complexity of the clinical manifestations of nrla continues to limit the power of these studies because patients with differing symptoms are often grouped together when exposed to specific challenge tests. in addition, sample sizes remain small. there is also a lack of long-term data (one case report) to suggest sustained efficacy after the cessation of slit. the current results show that the nrl slit is the only etiological method to resolve nrla in at-risk patients who cannot avoid this allergen. moreover, latex slit is characterized by a very low incidence of adverse reactions, good patient compliance and a high success rate. at the end of the immunotherapy, almost all patients are able to wear latex gloves, undergo medical examinations or surgery, and stay in environments where latex is present. although the tolerability of slit has allowed it to remain as the only potential immunotherapy modality, its safety continues to be closely monitored. for these reasons, further investigations in this field are necessary, especially regarding long-term tolerability, safety and efficacy, and the maintenance dosage to be adopted, as the literature still shows a wide variation. history of latex allergy Überempfindlichkeit gegen kautschuk als ursache von urticaria und quinckeschem odem uberempfindlichtkeit gegen kautschuk als uorsache von urticaria und quinckeschem ödem contact urticaria to rubber current prevalence rate of latex allergy: why it remains a problem? longitudinal study on specific ige against natural rubber latex, banana and kiwi in patients with spina bifida effects of latex avoidance on latex sensitization, atopy and allergic diseases in patients with spina bifida cornstarch powder on latex products is an allergen carrier management of latex hypersensitivity in the perioperative setting latex-fruit syndrome: frequency of cross-reacting ige antibodies latex allergy in fruit-allergic patients position paper of the eaaci: food allergy due to immunological cross-reactions with common inhalant allergens allergic contact dermatitis from natural rubber latex without immediate hypersensitivity delayed hypersensitivity to natural rubber latex: does it exist or not? contact dermatitis latex allergy: frequent occurrence of ige antibodies to a cluster of latex proteins in patients with spina bifida and histories of anaphylaxis a two-dimensional electrophoretic analysis of latex peptides reacting with ige and igg antibodies from patients with latex allergy characterization of major latex allergen associated with hypersensitivity in spina bifida patients in vivo sensitization to purified hevea brasiliensis proteins in health care workers sensitized to natural rubber latex identification, cloning, and sequence of a major allergen (hev b ) from natural rubber latex (hevea brasiliensis) isolated hevein-like domains, but not -kd endochitinases, are responsible for ige mediated in vitro and in vivo reactions in latex-fruit syndrome class i chitinases as potential panallergenes involved in the latex-fruit syndrome the clinical meaning of positive latex sige in patients with food/pollen adverse reactions on the allergenicity of hev b among health care workers and patients with spina bifida allergic to natural rubber latex the new latex allergen hev b : ige-binding properties of a recombinant serine protease inhibitor natural rubber latex allergy: spectrum, diagnostic approach, and therapy systemic reactions from skin testing: literature review skin and serologic testing in the diagnosis of latex allergy anaphylaxis following prick testing with natural rubber latex systemic reactions on spt to latex diagnostics of occupational type i allergies-comparison of skin prick test solutions from different manufacturers for selected occupational allergens an -year retrospective review of patch testing with rubber allergens: the mayo clinic experience hevea latex associated allergies: piecing together the puzzle of the latex ige reactivity profile allergen-specific ige to recombinant latex allergens in occupational allergy diagnostics basophil activation test and specific ige measurements using a panel of recombinant natural rubber latex allergens to determine the latex allergen sensitization profile in children in vivo diagnosis of allergic diseases-allergen provocation tests challenge tests in the diagnosis of latex allergy specific inhalation challenge in the diagnosis of occupational asthma: consensus statement clinical value of conjunctival allergen challenge in diagnosing allergic conjunctivitis related to latex latex allergy: position paper management of natural rubber latex allergy latex medical gloves: time for a reappraisal prevention of ige sensitization to latex in health care workers after reduction of antigen exposures occupational latex allergy: the current state of affairs conversion to low-protein, powder-free surgical gloves: is it worth the cost? am assoc occup health nurses the manufacture of gloves from natural rubber latex latex and synthetic rubber glove usage in uk general dental practice: changing trends current state of occupational latex allergy assessment of the risk of type i latex allergy sensitization or reaction during use of products made from latex derived from guayule and other alternative rubber producing species identification and practical management of latex allergy in occupational settings rubber: new allergens and preventive measures cdc's recommendations on the use of ppe during the covid- outbreak enforcement policy for gowns, other apparel, and gloves during the coronavirus disease (covid- ) public health emergency. guidance for industry and food and drug administration staff process for making available guidance documents related to coronavirus disease primary prevention of natural rubber latex allergy in the german health care system through education and intervention efficacy of latex avoidance for primary prevention of latex sensitization in children with spina bifida reduction of latex sensitization in spina bifida patients by a primary prophylaxis programme (five years experience) safety of a powder-free latex allergy protocol in the operating theatre: a prospective, observational cohort study american society of anesthesiologists committee on occupational health of operating room personnel latex allergy in housekeeping personnel personal exposure to inhalable dust and the specific latex aero-allergen, hev b . , in latex glove manufacturing in thailand use of protective gloves by hairdressers: a review of efficacy and potential adverse effects occupational management of type i latex allergy bensefa-colas l. accelerator-free gloves as alternatives in cases of glove allergy in healthcare workers secondary prevention of latex allergy in children: analysis of results outcome of a latex avoidance program in a high-risk population for latex allergy -a five-year follow-up study prevention of latex allergy among health care workers and in the general population: latex protein content in devices commonly used in hospitals and general practice emergency treatment of anaphylactic reactions. guidelines for healthcare providers. working group of the resuscitation council (uk) cardiac arrest in special circumstances section collaborators. european resuscitation council guidelines for resuscitation : section . cardiac arrest in special circumstances eaaci task force clinical epidemiology of anaphylaxis: experts' perspective on the use of adrenaline autoinjectors in europe prophylactic inoculation against hay fever further observation on the treatment of hay-fever by hypodermic inoculation of pollen vaccine years of hyposensitization: history of allergen-specific immunotherapy (asit) desensitization to latex by percutaneous route latex allergy desensitization by exposure protocol: five case reports specific immunotherapy for occupational latex allergy specific immunotherapy with a standardized latex extract versus placebo in allergic healthcare workers specific immunotherapy with a standardized latex extract in allergic workers: a double-blind, placebo-controlled study specific immunotherapy with standardized latex extract versus placebo in latex-allergic patients latex rush desensitization sublingual immunotherapy for other indications: venom large local, latex, atopic dermatitis, and food latex immunotherapy: state of the art sublingual desensitization: a new approach to latex allergy problem tolerance and effects on skin reactivity to latex of sublingual rush immunotherapy with a latex extract anaphylaxis by latex sublingual immunotherapy double-blind, placebo-controlled study of sublingual immunotherapy in patients with latex-induced urticaria: a -month study sublingual immunotherapy with a latex extract in paediatric patients: a double-blind, placebo-controlled study natural rubber latex allergy in children: clinical and immunological effects of -years sublingual immunotherapy sublingual desensitization in children with congenital malformations and latex sublingual immunotherapy for latex allergy: tolerability and safety profile of rush build-up phase latex sublingual immunotherapy: can its safety be predicted? component-resolved immunologic modifications, efficacy, and tolerance of latex sublingual immunotherapy in children latex immunotherapy: evidence of effectiveness failure of sublingual immunotherapy to treat latex allergy. a report of a case randomized, double-blind, placebo controlled clinical trial of sublingual immunotherapy in natural rubber latex allergic patients eosinophilic esophagitis during latex desensitization relation between eosinophilic esophagitis and oral immunotherapy for food allergy: a systematic review with meta-analysis roles of omalizumab in various allergic diseases two decades with omalizumab: what we still have to learn effect of omalizumab in healthcare workers with occupational latex allergy use of omalizumab in uncontrolled chronic spontaneous urticaria also improved latex-induced contact urticaria efficacy of omalizumab in reducing latex allergy the use of omalizumab in allergen immunotherapy the adverse skin reactions of health care workers using personal protective equipment for covid- the latex story sublingual immunotherapy with natural rubber latex: a case report with -year follow-up the authors are responsible for the content and the writing of this paper. the authors declare that no funding was received for the present review. the authors declare that they have no conflict of interest. key: cord- -hjxph jm authors: petrović, t.; d'agostino, m. title: viral contamination of food date: - - journal: antimicrobial food packaging doi: . /b - - - - . -x sha: doc_id: cord_uid: hjxph jm a review of the relevant foodborne viruses is presented. published data from scientific journals as well as the data presented in official reports and published on the internet were used for this review. in the review, information is given for the main foodborne viruses, implicated virus species, and food matrices involved, some history data are given, as well as modes of transmission, and sources of the virus presence in food. results of surveys on the presence of viruses in different kind of foods commodities (fresh produces and shellfish) and in some cases connections to caused outbreaks are presented. also, possible zoonotic infection and implicated viruses that could be transmitted through food are given. human norovirus followed by hepatitis a virus are the most common foodborne viruses, which are transmitted by food consumed raw, such as shellfish, fresh vegetables, and berry fruit. in developed countries, hepatitis e virus is increasingly being recognized as an emerging viral foodborne pathogen that includes zoonotic transmission via pork products. the existing knowledge gaps and the major future expectations in the detection and surveillance of foodborne viruses are mentioned. , and over persons in japan contracted foodborne gastroenteritis due to astv (oishi et al., ) . more recently, there was an outbreak in germany, predominantly in schools and childcare settings, linked to nov in frozen strawberries that were imported from china (mäde et al., ) . outbreaks have been documented to be caused by different kind of food items (e.g., deli meat, vegetables, berries, shellfish, and a great variety of rte foods like sandwiches, bread rolls, bakery products, cold meat, pastries, and ice cubes) (efsa, ) . the food types that are at highest risk of contamination are foods requiring either intensive manual handling, including manual handling under poor hygienic conditions, or close-to-fork and final-product manual handling. dishes containing fresh (or freshly frozen) fruits and vegetables have been the source of numerous outbreaks of foodborne illness (koopmans and duizer, ; efsa, ) . filter-feeding shellfish are a particular risk, as they concentrate viruses present in water during their growth, and numerous outbreaks linked to the consumption of shellfish have been reported (koopmans and duizer, ; efsa, ) . foods at greatest risk of virus contamination at the preharvest stage are shellfish, soft berry fruits, herbs, and salad vegetables. preharvest contamination of fruits and vegetables, including strawberries (niu et al., ) , raspberries (reid and robinson, ; ramsay and upton, ) , blueberries (calder et al., ) , lettuce (pebody et al., ) , and green onions (cdc, ) were reported and have resulted in outbreaks of disease in countries such as finland and new zealand, where populations have low or no immunity to the disease (pebody et al., ; calder et al., ) . the source of contamination in these outbreaks was reported to be either infected fruitpickers or contaminated irrigation waters (greening, ) . postharvest contamination of raw food may occur as a result of human handling by workers and consumers, contaminated harvesting equipment, transport boxes, contaminated aerosols, wash and rinsing water, or cross-contamination during transportation and storage (harris et al., ) . recontamination after cooking or processing and inadequate sanitation has also been associated with outbreaks of enteric virus infections (richards, ) . foods at risk from contamination by food handlers include a wide range of foods that are subjected to too much handling and are subsequently consumed cold or uncooked. these include bread and bakery goods (kuritsky et al., ) , lightly cooked or raw shellfish, delicatessen meats, sandwiches (parashar et al., ; daniels et al., ) , salads, herbs, fresh fruits, and cold desserts. poor food handling was shown to be a key risk factor in the transmission of noroviruses and rotaviruses in the netherlands (de wit et al., ) . an expert meeting convened under the auspices of the food and agriculture organization (fao) of the united nations and the world health organization (who) reviewed available evidence and grouped viruses according to their ability to cause high morbidity, severe disease, or a significant ability to cause foodborne outbreaks (fao/who, ) . in the fao/who document, the common pathogens such as nov, group a hrv, and hav were ranked as priority hazards. in the category of emerging hazards, hev, nipah viruses, h n avian influenza viruses, and sars coronavirus were considered to be of greatest concern. the meeting discussion resulted in several virus-commodity combinations for which prevention and control measures should be considered. those combinations are: for nov and hav in bivalve molluscan shellfish; for nov and hav in fresh produce; for nov and hav in prepared foods; for rotaviruses in water for food preparation; and emerging viruses in selected commodities. nov is one of the most widely recognized viral agents associated with foodborne outbreaks of nonbacterial and often epidemic gastroenteritis and is considered to be the most common cause of foodborne disease worldwide (greening, ; efsa, ) . nov is shed in huge quantities in the stool and vomit of infected persons, and it has been estimated that the infectious dose may be as few as virus particles (teunis et al., ) . novs are primarily transmitted through the fecaloral route, by consumption of fecally contaminated food or water, or by direct person-to-person spread that is still the major mode for nov transmission. secondary spread is person-to-person spread, but may also occur by airborne transmission. according to efsa ( ) caliciviruses (including nov) cause approximately % of epidemic nonbacterial outbreaks of gastroenteritis around the world and are responsible for many foodborne outbreaks of gastroenteritis. the majority of viral gastroenteritis outbreaks in europe have been attributed to novs, where they were reported to be responsible for more than % of nonbacterial gastroenteritis outbreaks between and koopmans et al., ) . estimations based upon analysis of questionnaire data suggested that in the netherlands approximately - % of community cases of nov gastroenteritis were attributed to foodborne consumption (efsa, ) . also, european data from the beginning of this century show that about % of the nov outbreaks are foodborne (ecdc, ) . this makes nov as common a cause of foodborne gastroenteritis as campylobacter and a more common cause than salmonella (de wit et al., ) . a european-wide surveillance network for nov outbreaks, divine-net, has noted that europe has been faced with an increased nov activity during the second half of the first decade of the twenty-first century. the new nov variants of gii. - had most likely been the dominating circulating strains. the role of foods, such as oysters and imported raspberries, as vectors for nov transmission, had been stressed, because both food commodities have been associated in several nov outbreaks in many countries (petrović, ) . hav is the etiological agent of one of the most common types of hepatitis worldwide, and hav as a serious foodborne infection is a notifiable disease in most developed countries. approximately . million people worldwide become infected with hav annually (issa and mourad, ) . the incidence of infection varies among regions of the world, with the highest rate in developing countries where sewage treatment and hygiene practices are poor and where more than % of children have been reported to be infected, usually asymptomatically, by years of age (cliver, ; greening, ) . conversely, the number of reported cases of hav infection has declined substantially in countries with effective vaccination. the major mode of transmission for hav is directly or indirectly from the human reservoir, mainly as a consequence of traveling to endemic regions, engaging in risky sexual practices, or consuming contaminated water or food (efsa, ). food (pebody et al., ; lees, ; greening, ) and drinking water (tallon et al., ) are considered major vehicles of hav transmission to humans. hav can, via sewage discharge, contaminate watercourses, soil, and consequently food crops (bosch, ; cook and rzeźutka, ) . the other main source of produce-associated hav infection is from food handlers and food processors. hav is distinguished from other viral agents by its prolonged ( - -week) incubation period. since hav is shed before symptoms become apparent and there are often more than infectious virus particles excreted per gram of feces, hav-infected produce harvesters and food handlers can become a source of contamination without their knowledge. in areas with poor hygiene practices, this can present a high risk to human health. foodborne outbreaks of hav are relatively uncommon in developing countries where there are high levels of immunity in the local population, but foreigners in these regions can be susceptible if they are not vaccinated (greening, ) . hev is usually the result of a waterborne infection in developing countries and is suspected to be spread zoonotically in industrialized countries (bosch et al., ) . the disease is endemic in many parts of the world, mostly in the indian subcontinent, northwest china, and central asia. in these regions, hev is transmitted mainly through the fecal-oral route, especially by the consumption of fecally contaminated drinking water, and sewage is a major source for contamination of surface water (greening, ; fao/who, ) . foodborne outbreaks of hev are most common in developing countries as a consequence of inadequate environmental sanitation (greening, ) . hev is unusually reported in industrialized countries and when it is reported, it is mostly as sporadic cases in humans who have traveled to endemic countries. recently, some human hev infection in nonendemic countries could not be explained by the contact of those patients with the virus in the endemic regions. although originally it was believed that hev did not occur in industrialized countries, in recent years it has been identified in europe, asia, australia, and the united states; however, it rarely is a cause of overt disease in these countries (clemente-casares et al., ; emerson and purcell, ) . in contrast to nov and hav, hev has been identified also as a zoonosis (efsa, ) . hev has been detected in the feces of a wide range of domestic animals (meng et al., ; vasickova et al., ; greening, ; petrović et al., ) . it has been found to be highly prevalent in pigs in several countries where hev in humans is rare, including spain, new zealand, the netherlands, serbia, japan, and canada (emerson and purcell, ; lupulović et al., ; petrović et al., ) . also, recent studies have revealed quite variable seroprevalence rates among europe's population and a possible porcine zoonotic transmission has been postulated (meng, ; petrović et al., ) . moreover, the human hev strains described in industrialized countries appear to be closely related to the swine hev strains found in the same countries. although rare, the importance of hev transmission via food is increasingly being recognized in the european union (eu) (efsa, ) . hrv is the leading cause of severe diarrhea among infants and young children. in adults, the disease caused by hrv is considered to be mild (greening, ) . it is estimated that hrv causes more than million cases of diarrhea in children less than years of age annually worldwide (glass and kilgore, ) . hrv infection is a particularly serious problem in developing countries where up to , deaths occur annually among children. in the united states, hrv had been estimated to cause about four million infections per year, resulting in almost , hospitalizations and more than deaths annually . it was estimated that only % of hrv cases was foodborne (mead et al., ) . hrv causes disease in both humans and animals, especially domestic animals (greening, ) . outbreaks associated with food and water have been reported in a number of countries . in countries with a seasonal climate change, hrv is more common during the winter months. in tropical regions, outbreaks can occur both in the cooler and drier months and throughout the year, especially where transmission is related to contaminated water supplies and where no sewage treatment systems exist (ansari et al., ) . hrv is stable in the environment, so infection can occur through consumption of contaminated water or food and contact with contaminated surfaces (greening, ) . evs of concern for water and foodborne spread include polioviruses, coxsackie a and b viruses, and echo (enteric cytopathic human orphan) viruses. they are transmitted by the fecal-oral route and are excreted in feces, but generally do not cause gastroenteritis. they can cause a range of other diseases, including viral meningitis, myocarditis and poliomyelitis (greening, ) . polioviruses were the first viruses that have been confirmed to be foodborne (jubb, ; , but virulent wild-type strains are now very rare because of global immunization campaigns. outbreaks of foodborne illness associated with coxsackie viruses and echo viruses have been reported (cliver, ; . enteroviral infection is most common in summer and early autumn, and many infections are asymptomatic. although evs are regularly detected in the environment, there have been very few recorded foodborne outbreaks associated with these viruses. evs, including echo viruses and coxsackie a and b viruses, have been isolated from shellfish, but no outbreaks associated with the consumption of shellfish have been reported (greening, ) . astvs are distributed worldwide and they have been isolated from different animal species like cats, dogs, pigs, sheep, cattle, and birds, as well as from humans. the main feature of astv infection in both humans and animals is a self-limiting gastroenteritis (greening, ) . astvs are a common cause of human gastroenteritis, with most cases of infection detected in young children less than year of age . although astvs cause a mild infection in adults, they have been associated with gastroenteritis in immunocompromised persons. transmission is through the fecal-oral route via water, food, and person-to-person contact . hadvs are widespread within nature, infecting birds and mammals, including humans. they commonly cause respiratory disease but may also cause other illnesses such as gastroenteritis and conjunctivitis. in children under years of age, the enteric hadvs are the second most prevalent cause of gastroenteritis (after hrv) (allard et al., ) . hadvs can be transmitted from person to person by direct contact, or via fecal-oral, respiratory, or environmental routes. most hadv infections in normally healthy individuals are mild or subclinical, but can be associated with respiratory, ocular, and gastrointestinal disease. all virus serotypes are shed enterically in feces, but of the many types of hadvs, only hadv serotypes and are generally associated with fecal-oral spread and cause gastroenteritis (greening, ) . the virus is shed in large numbers in feces and respiratory secretions for long period, even for months or years after the infection. enteric hadv infections are common all year round. these viruses have been identified in a variety of environmental samples, including wastewater, sludge, in marine, surface, and drinking waters, and shellfish, but no foodborne or waterborne outbreaks associated with the enteric hadv have been reported (greening, ) . food may be contaminated by viruses during all stages of the food supply chain. the presence of viruses in food can be the result and consequence of the environmental contamination during primary production-contaminated irrigation waters by sewage as well as manure, which in turn contaminate produce on the field, during the processing and storage phases-by water contaminated with viruses, and from contact virus transmission from humans, such as infected food handlers (involving fecal-oral and aerosol spread of fecal material and vomit). transmission of zoonotic viruses (e.g., hev) can also occur by consumption of products of animal origin (efsa, ). the relative contribution of different sources (shellfish, fresh produce, food handler including asymptomatic shedders, food-handling environment) to foodborne illness has not yet been determined (efsa, ) . food handlers are very often the reason for virus transmission. transmission could occur via infected food handlers with clinical symptoms, but also from infected food handlers who have recovered from illness and no longer display any symptoms, but may still be shedding high numbers of nov. in addition, transmission could occur via infected food handlers with asymptomatic infections and food handlers who come in contact with sick people (koopmans and duizer, ) . although most outbreaks can be traced to infected food handlers at the end of the food chain, the food contamination could occur anywhere (e.g., seasonal workers during berry harvesting or people on recreational boats near shellfish harvesting areas). fresh fruits and vegetables can become contaminated by enteric viruses, possibly through the use of contaminated fertilizers or irrigation water supplies (grohmann and lee, ) . an increased number of foodborne viral outbreaks are being recorded in several countries. reasons for this include the improved diagnostic methods for virus detection and the increased marketing of fresh and frozen foods that have led to a worldwide availability of high-risk foods (efsa, ) . in , a total of foodborne outbreaks were reported in the eu, and it was at the same level as in . overall, , human cases, hospitalizations, and deaths were recorded. the largest number of reported foodborne outbreaks was caused by salmonella ( . % of all outbreaks), followed by viruses ( . %), bacterial toxins ( . %), and campylobacter ( . %). during , eu member states reported a total of foodborne outbreaks caused by viruses (efsa and ecdc, ) . overall, the number of reported viral foodborne outbreaks increased by more than % compared to and . only a few ( . %) reported viral outbreaks were verified (efsa and ecdc, ); however, the number of verified viral outbreaks also increased by . %, from outbreaks in to in . for out of the total of verified foodborne virus outbreaks, the implicated foods were fruit/berries and juices, and products thereof. these outbreaks were reported by finland and sweden and involved human cases (efsa and ecdc, ) . the panel on biological hazards (biohaz) identified nov, hav, and hev as viruses of significance for foodborne transmission (efsa, ) . data from systemic virus surveillance in foods are missing mainly because there are no systemic surveillances on national or wider levels, and the existing data were collected partly from research projectbased studies and mainly from studies after the outbreak occasions. in the european rapid alert system for food and feed (rasff) online database (http://ec.europa.eu/food/food/rapidalert/rasff_portal_database_en.htm) up to december , , presence of enteric viruses in fruit and vegetables were found in a total of cases (alerts). mostly nov was detected ( / ; . %) in fruit ( / ), most often frozen raspberries ( / ), and just in one case in lettuce (from france). out of alerts of hav presence in different kind of fruits, hav-positive fruit (dates) cases from to originated from algeria, and hav fruit (different kinds of berries) alert cases from until april originated from different, mostly european countries. nov-positive raspberries originated from serbia ( / ), poland ( / ), china ( / ), and chile ( / ). nov outbreaks linked to fresh soft red fruits and leafy greens have been reported. between and , foodborne outbreaks of infectious enteric disease were reported in england and wales. from that number, ( . %) were associated with the consumption of salad vegetables or fruit. the pathogens most frequently reported were salmonellas ( . %) and nov ( . %) (long et al., ) . in denmark, at least linked outbreaks of gastroenteritis with a total of cases were reported in january . lettuce of the lollo bionda type grown in france was found to be the vehicle of virus transmission (ethelberg et al., ) . baert et al. ( ) reported that during from a total of reported foodborne outbreaks in belgium, were caused by nov, affecting persons. the major implicated foods were sandwiches ( / ). furthermore, baert et al. ( ) summarized the data collected from international outbreaks between and reported by eurosurveillance, morbidity and mortality weekly reports and from internationally available peer-reviewed scientific journals. as a result, foodborne and waterborne outbreak events due to nov, epidemiological and/or laboratory confirmed, from to have been reported. further analysis revealed that in . % of the cases, the food handler was responsible for the outbreak, followed by water ( . %), bivalve shellfish ( . %), and raspberries ( . %). maunula et al. ( ) the nov detected in patients' stool samples from six outbreaks were sequenced and epidemiologically linked to the single batch of frozen raspberries originating from serbia. these molecular investigations showed that the apparently independent outbreaks were the result of one contamination event of frozen raspberries (müller et al., ) . out of examined fruit products, despite a good bacteriological quality, stals et al. ( ) found nov gi and/or gii in / , / , / , and / of the tested raspberries, cherry tomatoes, strawberries, and fruit salad samples, respectively. the level of detected nov genomic copies ranged between . and . log per g. baert et al. ( ) reported the results of the study where in total, samples of leafy greens, samples of fresh soft red fruits, and samples of other types of fresh produce (tomatoes, cucumber, and fruit salads) were analyzed in those three countries. nov was detected in . % (n = ), . % (n = ), and % (n = ) of leafy greens tested in canada, belgium, and france, respectively. soft red fruits were found positive in . % (n = ) of the samples tested in belgium and in . % (n = ) of the samples tested in france. also, . % (n = ) of the other fresh produce types, analyzed in belgium, were found to be nov-positive. hav has often been associated with the consumption of contaminated fresh-cut vegetables and fruit (efsa, ). at the beginning of , cases of hav were reported in and around jefferson county, kentucky (rosenblum et al., ) . a case-control study found that eating green salad was strongly associated with acquiring hepatitis. rosenblum et al. ( ) concluded that this outbreak of hav was the first recorded outbreak in the united states apparently associated with fresh produce contaminated before distribution to restaurants. in , a total of cases of hav were reported from schools in michigan and cases from schools in maine. most of the patients ate lunch in schools, and preliminary analysis established a strong association between illness and consumption of food items containing frozen strawberries originating from mexico (hutin et al., ) . forty-three cases of serologically confirmed hav occurred among individuals who ate at a restaurant in ohio in . a case-control study was conducted that determined foods containing green onions, which were eaten by ( %) of case patients were associated with illness (dentinger et al., ) . in , a large hav outbreak connected to one restaurant in pennsylvania was described by wheeler et al. ( ) . out of identified patients, died and at least were hospitalized. identical sequences of hav strains from all tested patients were identified. mild salsa, which contained green onions grown in mexico, was identified as the source of the hav (wheeler et al., ) . petrignani et al. ( ) reported the connection between hav infection with cases in the netherlands at the beginning of , and that semi-dried tomatoes in oil was the source of the outbreak. all the examined patients were infected by an identical hav strain not previously detected in the netherlands. in october , semi-dried tomatoes originating from turkey were identified as the source of several hav outbreaks in australia (more than cases) and france ( cases) (efsa, ). gillesberg lassen et al. ( ) described a foodborne outbreak of hav in denmark from october to april . a case-control study identified frozen berries eaten in smoothies as the potential vehicle. in the following weeks, finland, norway, and sweden also identified an increased number of hav patients without travel history. most cases reported having eaten frozen berries at the time of exposure. in total, cases were notified in the four countries. according to information obtained in the case-control study, different kinds of berries were suspected to be the source of hav, but no specific type of berry, brand, or origin of berries was identified. during , more than cases of hav were reported by eu member states as potentially linked to an ongoing outbreak (wenzel et al., ; ecdc, ) . epidemiological, microbiological, and environmental investigations indicate frozen berries as the vehicle of infection for this outbreak and suggested that it could be linked to a single source (ecdc, ) . frequent zoonotic transmission of hev has been suspected. norder et al. ( ) sequenced the orf genome region of hev strains originating from human blood sera collected between and and found that patients infected in europe were infected by genotype . in order to find the connection between human and swine hev, norder et al. ( ) additionally sequenced the hev strains originating from piglets from herds in sweden and denmark. phylogenetic analyses of the genotype strains showed geographical clades and high similarity between strains from patients and pigs from the same area, so the authors concluded that autochthonous hev cases are present in scandinavia. also, bouquet et al. ( ) assessed the genetic identity of hev strains found in humans and pigs in france. hev sequences identified in patients with autochthonous hev infection were compared with sequences amplified from pig livers collected in slaughterhouses. a similarity of > % was found between hev sequences of human and swine origins, indicating that consumption of some pork products, such as raw liver, is a major source of exposure for autochthonous hev infection (bouquet et al., ) . recently, there has been increasing evidence of foodborne transmission of hev. tei et al. ( ) concluded that consumption of uncooked deer meat was a major epidemiological risk factor for hev infection in the city of kasai in japan. in their study, from the total of examined volunteer subjects with experience of eating raw deer meat, ( . %) of the subjects and only ( . %) of the controls had measurable serum anti-hev igg levels. in addition, the studies of yazaki et al. ( ) and tamada et al. ( ) suggest that consumption of undercooked pig liver and undercooked wild boar meat may have been the cause of some cases of hev in japan. wild boar liver is often eaten raw in japan, and this has also been linked to some hev cases (matsuda et al., ) . numerous survey studies have estimated the prevalence of hev rna in marketed livers. hev rna was detected in . % of livers from supermarkets in japan (yazaki et al., ) , and in % of packages in the netherlands (bouwknegt et al., ) . feagins et al. ( ) examined packages of commercial pig liver sold in local grocery stores in the united states for the presence of hev rna, and found ( %) positive for hev rna. subsequent experimental infection of pigs inoculated with positive pig livers homogenates demonstrated that hev in pig livers was infective. leblanc et al. ( ) examined the presence of hev in the tissues of adult pigs, randomly selected from an experimental herd at slaughter in canada. hev rna was detected in out of the animals tested. even although no hev rna was detected in any of the muscle tested, . % of liver samples obtained at the slaughterhouse tested positive for hev rna. in a chinese abattoir, li et al. ( ) found that . % of liver samples tested were positive for hev rna. during , the centre for food safety in honk kong obtained a total of fresh pig liver samples from pigs slaughtered in a local slaughterhouse. among the collected samples, out of ( %) roaster liver samples were found positive for hev, while none of the pork liver samples were found positive. partial orf sequences of some hev isolates from roaster pigs were found to be the same as those from seven local human cases from , as well as local cases recorded in the past. this study suggests the possibility that, apart from contaminated water or food such as raw or undercooked shellfish, pigs also could be one of the sources of human hev in endemic regions (anon, ) . available data suggests that the consumption of raw/undercooked sausage meat is a potential route of hev transmission. in the united kingdom, grierson et al. ( ) detected hev in out of ( . %) tested sausages, and the presence of hev was found at all three points of the pork food supply chain: production, processing, and point of sale. in another study in the united kingdom, berto et al. ( ) detected hev in out of ( . %) and in ( %) of tested sausages and livers. hev rna was also detected at each of three sites (production, processing, and point of sale) in the pork food supply chain. an autochthonous hev infection was recently described in portugal in a patient who recalled eating traditional homemade pork sausages made of raw meat about weeks prior to the development of the clinical manifestations of acute hepatitis (duque et al., ) . renou et al. ( ) presented the case of the direct evidence of foodborne transmission of hev after consumption of uncooked "figatellu" sausage in france, with % identity between the sequences from the patient and the food product. di evaluated the prevalence of hev in the pork production chain in the czech republic, italy, and spain during . hev rna was detected in at least one of the samples (feces, liver, or meat) from ( %) out of examined slaughtered pigs at slaughterhouses. pig feces showed highest hev rna presence ( %), followed by liver ( %) and meat ( %). out of sausages sampled at the processing and point of sale (supermarkets) stages, hev was detected only in spain ( %, / ). hev sequencing confirmed only g hev strains. the efsa biohaz has published a scientific opinion urging for measures to prevent hev from entering the food chain (efsa, ). the biohaz opinion states that in contrast to nov and hav, hev has been identified as a zoonotic virus that can be very effectively transmitted between pigs, and can be transmitted to humans through consumption of products of animal origin, especially through consumption of meat; however, there are no measures in place to control the spread of the virus (efsa, ). eleven foodborne outbreaks consisting of cases of rotaviral gastroenteritis were reported in new york between and (greening, ) . from that number, seven outbreaks have been associated with food-service premises, and the foods included salad, cold foods, shepherd's pie, and water or ice . large-scale outbreaks of rotaviral gastroenteritis have been reported in japanese primary schools with more than cases recorded for one outbreak (matsumoto et al., ) . school lunches prepared at a central facility were suspected as the vehicle of infection, but no hrv was isolated from food or water. lettuce at a market was found to be contaminated with hrv and hav at a time when there was a high incidence of rotaviral diarrhea in the costa rican community (hernandez et al., ) . recently, mayr et al. ( ) described an hrv outbreak in a mother-and-child sanatorium. in total, food samples from the sanatorium kitchen were taken and tested for hrv. hrv particles were isolated from potato stew. out of samples of packaged leafy greens, tested by mattison et al. ( ) , ( %) were found and confirmed to be positive for nov, and only ( . %) was found positive for hrv group a. additionally, brassard et al. ( ) described the presence of hrv as one of the detected pathogenic human and zoonotic viruses on strawberries. probably one of the most recognized routes of foodborne transmission of enteric viral infections is through the consumption of shellfish grown in sewage-polluted marine environments (okoh et al., ) . the most common route for transmission is accidental contamination after heavy rainfall, when extra loads cause an overflow and there is a release of untreated sewage into the aquatic environment. current water treatment practices are unable to provide virus-free wastewater effluents. consequently, human pathogenic viruses are routinely introduced into marine and estuarine waters (bosch and le guyader, ) . shellfish, which includes mollusks such as oysters, mussels, cockles, clams, and crustaceans such as crabs, shrimps, and prawns are filter-feeders that result in the bioconcentration of environmentally stable, positive-stranded rna viruses, such as nov, hav, and ev in their edible tissues, digestive glands, and gills . shellfish can filter some - l of water per hour and in that process, they concentrate infectious agents that are present in the marine environment (grohmann and lee, ) . by this process, oysters can concentrate viruses up to times compared to the surrounding water (burkhardt and calci, ) . a major public health concern posed by virus-contaminated bivalves is that shellfish are often eaten raw, like oysters and clams, or lightly cooked, like most other molluscan shellfish, just steamed for a few minutes (bosch and le guyader, ) . hav has contributed to numerous foodborne outbreaks that are often associated with raw or lightly cooked shellfish (richards, ) . contamination generally occurs either preharvest or during food handling. the first recorded outbreak of shellfish-associated viral disease resulted from storing clean oysters in a fecally contaminated harbor while awaiting sale (gard, ) . that hav outbreak resulted in more than cases. the largest foodborne outbreak of hav occurred in china in when approximately , people were infected during a -month period after consumption of partially cooked, hav-contaminated clams harvested from a growing area contaminated by raw sewage (halliday et al., ) . a few of the documented shellfish-associated outbreaks include oysters in australia (conaty et al., ) , oysters in brazil (coelho et al., ) , mussels in italy (croci et al., ) , and clams in spain (bosch et al., ) . sewage was generally the source of pollution in most of these outbreaks. contamination of shellfish with hav is still common in italy, spain, and other european countries (greening, ) . foodborne nov outbreaks often result from preharvest contamination of foods such as shellfish (christensen et al., ) . berg et al. ( ) described three oyster-related gastroenteritis outbreaks attributed to nov that occurred in louisiana between and . traceback and environmental investigations revealed that the overboard disposal of sewage by oyster harvesters into oysterbed waters was the most likely source of contamination in at least two of the outbreaks. christensen et al. ( ) described the outbreak in which more than people in denmark became ill from consumption of imported oysters during the new year of / . nov and ev were identified from both oyster and patients' fecal samples. bosch et al. ( ) provide examples of large outbreaks (with more than cases) described in literature connected to the viruses in shellfish. in presented outbreaks from to , mostly nov in oysters, cockles, and clams ( / ) was the causative agent followed by hav in cockles and clams ( / ). in recently conducted studies, nov has been detected in - % of oyster samples collected from europe and the united states by random sampling at market places and oyster farms (boxman et al., ; costantini et al., ) . boxman ( ) published a detailed review about human enteric virus presence and prevalence in bivalve mollusks that were collected from european waters or markets from to . rna of enteric viruses have been detected in shellfish from commercial and noncommercial harvesting areas, as well as in products available on the market for direct consumption and in shellfish associated with disease outbreaks. the presented data suggest a high prevalence of different human enteric viruses, but mostly nov, hav, ev, hadv, and hrv were found in shellfish samples collected from growing areas, as well as from the market in different countries. the viruses were present in shellfish from polluted areas, in depurated shellfish and even in shellfish classified in category class a, as well as those ready for human consumption. the relation with the e. coli most probable number (mpn) that is in use for classification of growing areas and to determine whether shellfish products can be presented for human consumption could not be confirmed in this study. up to february , , in rasff online database notifications of enteric viruses in shellfish on the european market (http://ec.europa.eu/food/food/rapidalert/rasff_portal_database_en.htm), boxman ( ) found alerts on the (suspected) presence of viruses. twenty-eight alerts have been reported on nov in food notified by different eu countries between and , and alerts have been reported on the (suspected) presence of hav in food between and . the majority of these alerts on nov in food concerned oysters ( times), followed by scallops (one report). half of the notified batches of oysters were of french origin, followed by oysters derived from the united kingdom, and ireland. all alerts on the (suspected) presence of hav in food were reported by italy and spain and were only involving shellfish: oysters (five reports), small bivalve animals (four reports), and scallops (one report). half these products were of french origin, whereas the other half was shellfish from peru (boxman, ) . after this period and the data described by boxman ( ) until december , , new nov-positive shellfish alerts were published in the rasff online database. analyzing the alert reports on shellfish, nov presence was mostly connected to oysters ( / ; . % cases) from france ( / ), ireland ( / ), the netherlands ( / ), and spain ( / ); in three cases connected to mussels from the netherlands ( / ) and spain ( / ); in three cases connected to clams from portugal, united kingdom, and vietnam; and in one case connected to raw shell scallops from chile. in the efsa report ( ), from a total of foodborne outbreaks reported in eu member states during , crustaceans, shellfish, mollusks, and products thereof were the most frequently implicated food items. for those outbreaks that were verified, nov was the most frequent cause, followed by hav (efsa, ). in the recent united kingdom food safety authority project-based study, nov was detected in . % oyster samples ( / ), with similar prevalence in the two species of oysters tested ( . % ( / ) for crassostrea gigas and . % ( / ) for ostrea edulis). clear seasonality was observed with a positivity rate of . % ( / ) for samples taken between october and march compared with . % ( / ) for samples taken between april and september (anon, ) . in the first report on the presence of human enteric viruses in shellfish from portugal, approximately different kinds of shellfish, organized in batches, were collected between march and february (mesquita et al., ) . viral contamination was detected throughout the year in all shellfish species and in all collection areas, independently of classification of their harvesting areas. nov was detected in % of the batches, followed by ev in %, and hav in %. overall, % of all analyzed batches were found to be contaminated by at least one of the studied viruses, while the simultaneous presence of two and three viruses was detected in % and % batches, respectively. the special problem was the fact that viruses were detected in six of the eight shellfish batches from the a-class harvesting areas (one nov, three ev, and two hav) (mesquita et al., examined the prevalence of different enteric viruses in commercial mussels at the retail level in three european countries (finland, greece, and spain). a total of mussel samples from different origins were analyzed for virus presence. samples were positive in % of cases. hadv was found to be the most prevalent virus detected ( %), and the prevalence of nov gg ii, hev, and nov gg i were %, %, and . %, respectively. presence of hav was not detected. epidemiological evidence of astv transmission by foods is limited, but infections via contaminated seafood like shellfish and water have been reported (oishi et al., ; . one large outbreak of acute gastroenteritis was reported in japan involving thousands of children and adults from different schools in (oishi et al., ) . the outbreak was traced to food prepared by a common supplier for school lunches and astv type was identified as causative agent. there are several japanese reports of astv genomes identified in shellfish with the evidence of their contribution in foodborne outbreaks of gastroenteritis, mainly after the consumption of contaminated oysters (kitahashi et al., ) . hadvs have been identified in a variety of environmental samples, including wastewater, sludge, and in marine, surface, and drinking waters, as well as in shellfish, but no foodborne nor waterborne outbreaks associated with the enteric hadvs have been reported (greening, ) . swine manure could be a source of hev contamination of coastal waters with subsequent contamination of shellfish (smith, ) . said et al. ( ) reported that the small genotype hev outbreak on a cruise ship returning to the united kingdom in was connected to the consumed shellfish. zoonotic viral infections are generally not transmitted by food; however, there are a few reports on transmission of some emerging viruses via food. this transmission is likely to be rare, relative to other transmission routes, and will probably be restricted to a few food products or items and occasions. for example, highly pathogenic avian influenza (hpai) virus in undercooked poultry or eggs, hev in porcine organs, or muscle tissue and nipah virus in date palm sap are postulated to be foodborne. another emerging virus for which this mode of transmission may be relevant is severe acute respiratory syndrome coronavirus (sars-cov) (fao/who, ; newell et al., ) . all mentioned viruses are zoonotic, and limited epidemiological data exist that support their transmission by the consumption of contaminated foods. each of these viruses is capable of causing significant illness and mortality in humans. they are present in the intestinal tracts of infected humans and animals, and are shed into the environment through feces that can contain high levels of virus (newell et al., ) . sars-cov was spread into the human population through the preparation and consumption of food animals that appear to be infected from another reservoir, probably bats (lau et al., ) . infectious h n avian influenza virus has been found in duck meat, and the consumption of duck blood has resulted in the infection of humans (tumpe et al., ) . almost all reported cases of avian influenza (ai) virus infection in humans that have been recently caused by hpai viruses belonging to the h or h subtypes were transmitted directly from infected birds to humans. other routes of infection, such as consumption of edible tissues from infected avian species or contact with contaminated water, have been suggested as possible sources of infection, but have not yet been proven (efsa, ). transmission of hev through food of animal origin is already documented (yazaki et al., ; tei et al., ; li et al., ; meng, ; said et al., ) and explained in detail previously. nipah virus was shown to affect people slaughtering pigs. whether eating produce from infected pigs can transmit the nipah virus is not known (fao/who, ) . nipah virus was shown to affect children eating fruits contaminated with urine from bats shedding the virus, and three outbreaks in bangladesh have been linked to consumption of fresh local sweet delicacy, which had been contaminated by bats (luby et al., ) . besides those mentioned, there is evidence of transmission of the ebola virus through bushmeat mainly by ingesting the meat of fruit bats. this mode of ebola virus transmission has been found as a route of virus transmission from wildlife to human population (leroy et al., ) . it is important to stress that, for most of the aforementioned emerging foodborne pathogens, contaminated foods is not a usual or even a likely vehicle of transmission, but the potential for foodborne transmission should be considered in epidemiological studies (fao/who, ; newell et al., ) . recently, the european food safety authority (efsa) biohaz stressed that except for tick-borne encephalitis virus, which can be shed by infected dairy animals and subsequently infect humans via milk; and hev, which can be transmitted through consumption of undercooked meat, viral foodborne infections are limited to the recycling of human viruses back to humans (efsa, ). food and environmental virology is a relatively young scientific discipline and consequently there is little published data on virus presence and prevalence in different matrices. there are just a few existing data on virus presence and prevalence in different foods. the data available originates mainly from research project-based studies, and in most cases were from studies conducted after the outbreak occurred. data from systemic virus surveillance in foods are missing mainly because there is no systemic surveillance either on a national or wider level (petrović, ) . another important data gap relates to the lack of knowledge regarding the prevalence of disease caused by viruses in foods in comparison with other possible transmission routes. also, the relative contribution of different sources (shellfish, fresh produce, food handler including asymptomatic shedders, and food handling environment) to foodborne illness has not been determined. most countries have some level of reporting of foodborne illness outbreaks, but few of these systems include viral foodborne illness (greening, ; newell et al., ) . due to the high rate of secondary transmissions, small initial foodborne events may rapidly present person-to-person outbreaks, if the initial introduction event was not recognized (efsa, ). some case-based surveillance exists for hav and evs, but usually it is not focused on detecting foodborne transmission as a source of the infection (newell et al., ; efsa, ) . as a result, national statistics on foodborne viral disease are not readily available and, where present, it likely reflects significant underreporting (mead et al., ; greening, ; fao/who, ) . routine harmonized surveillance of viral outbreaks and of virus occurrence in different foods would be recommended to aid source attribution studies. estimates of the proportion of illness caused by foodborne viruses that can be connected to consumption of contaminated food are based upon very few studies, and according to the efsa biohaz (efsa, ) would require the addition of systematic strain typing to routine surveillance, or more systematic studies to provide more reliable data for burden estimates. testing for viruses in food products is difficult, and there is considerable debate over interpretation of findings. as a consequence, data from food-product monitoring are at the least inconsistent (efsa, ). a problem for the detection, study, as well as for the control of most of the foodborne viruses is that some enteric viruses replicate poorly (hav) or not at all (nov) in cultured cells (atmar and estes, ). in addition, there are no laboratory animal models available for experimental studies of virus inactivation. for these reasons, detection methods currently rely on virus genome detection by molecular techniques such as reverse transcription polymerase chain reaction (rt-pcr). the application of molecular techniques such as real-time (rt)-pcr has enabled relatively rapid, sensitive, and specific detection of viral genome sequences. the problem of this methodology is the fact that the positive signal does not provide information on virus infectivity; rather it indicates the presence of the viral genomic segment. so, inactivated virus particles that pose no threat to public health may still contain intact rna and give a positive result (koopmans and duizer, ; stals et al., ) . the positive results of nov presence in food are of special concern in the absence of linked outbreaks. consequently, a potential risk for infection cannot be excluded, but the actual risk from rt-pcr nov-positive produce remains unknown. for this reason, studies should be designed determining the probability of infection related to the presence or levels of nov genomic copies . a lack of appropriate detection methods for confirmation of viruses as the etiological agent in food is also the reason for underreporting of foodborne virus outbreaks (baert et al., ) . although protocols are available for the detection of hav and nov as the viruses that are most frequently associated with foodborne outbreaks, few laboratories use them when investigating the causes of foodborne diseases, because the methods are considered to be too expensive and too time-consuming for the routine screening of foods (lopman et al., ) . from , an international organization for standardization (iso) methods (technical specifications) for the detection of hav and nov in foods exists: "horizontal methods for determination of hepatitis a virus and norovirus in food using real-time rt-pcr (iso ts - , and iso ts - , )," but still they are very expensive and time-consuming and not adequate for wide surveillance studies. currently, methods used for monitoring of foods using e. coli as microbiological criteria do not correlate consistently with presence or absence of viruses in foods. also, current safety standards for determining food quality typically do not specify what level of viruses should be considered acceptable (okoh et al., ) . as a consequence, the food industry and food safety authorities, at present, lack the tools that enable them to monitor virological quality control in contrast with the situation that exists for bacteriological contamination (efsa, ) . despite the fact that viruses are one of the most common pathogens transmitted via food, no systematic inspection and legislation exist regarding the presence of viruses in the food chain that would set up virological criteria for food safety (koopmans and duizer, ; okoh et al., ) . accordingly, the education of food-industry managers, producers, distributors, and consumers about hygienic regulations and conditions of food production and processing are essential (vasickova et al., ) . commission regulation (ec) / on microbiological criteria for foodstuffs lays down food safety criteria; however, no specific criteria are set for viruses. at the time of this writing, no routine monitoring of viruses in foodstuffs is performed; however, it would be highly beneficial to have such surveillance, including a system where data from food and environmental monitoring could be epidemiologically compared with data from outbreaks in the population (petrović, ) . molecular epidemiology and surveillance of food samples are necessary to elucidate the public health hazards associated with exposure to foodborne viruses and for the estimation of the true size of food-related cases (ecdc, 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associated with the consumption of processed pork products multiple norovirus outbreaks linked to imported frozen raspberries other food-borne viruses rotavirus a review, foodborne viruses and fresh produce a review of hepatitis e virus screening of fruit products for norovirus and the difficulty of interpreting positive pcr results recovery and sequence analysis of hepatitis a virus from spring water implicated in an outbreak of acute viral hepatitis consumption of wild boar linked to cases of hepatitis e zoonotic transmission of hepatitis e virus from deer to human beings consumption of uncooked deer meat as a risk factor for hepatitis e virus infection: an age-and sex-matched case-control study norwalk virus: how infectious is it? characterization of a highly pathogenic h n avian influenza a virus isolated from duck meat viruses as a cause of food-borne diseases: a review of the literature hepatitis a outbreak in europe: imported frozen berry mix suspected to be the source of at least one infection in austria in an outbreak of hepatitis a associated with green onions enteric viruses in the aquatic environment sporadic acute or fulminant hepatitis e in hokkaido, japan, maybe food-borne, as suggested by the presence of hepatitis e virus in pig liver as food the authors would like to acknowledge funding from the project no. tr of ministry of education, science, and technological development of the republic of serbia. special thanks go to dr. nigel cook, fera, united kingdom, who introduced the research interest for food and environmental virology to the authors of this book chapter. key: cord- -km qcmlh authors: fehr, anthony r.; jankevicius, gytis; ahel, ivan; perlman, stanley title: viral macrodomains: unique mediators of viral replication and pathogenesis date: - - journal: trends in microbiology doi: . /j.tim. . . sha: doc_id: cord_uid: km qcmlh viruses from the coronaviridae, togaviridae, and hepeviridae families ​all contain genes that encode a conserved protein domain, called a macrodomain; however, the role of this domain during infection has remained enigmatic. the recent discovery that mammalian macrodomain proteins enzymatically remove adp-ribose, a common post-translation modification, from proteins has led to an outburst of studies describing both the enzymatic activity and function of viral macrodomains. these new studies have defined these domains as de-adp-ribosylating enzymes, which indicates that these viruses have evolved to counteract antiviral adp-ribosylation, likely mediated by poly-adp-ribose polymerases (parps). here, we comprehensively review this rapidly expanding field, describing the structures and enzymatic activities of viral macrodomains, and discussing their roles in viral replication and pathogenesis. macrodomains were discovered in togaviruses, coronaviruses, and the hepatitis e virus over years ago. they were called the 'x' domain because they had no known function. about years later, several macrodomain structures were determined. they consist of central b-sheets surrounded by a-helices and bind to adp-ribose and its derivatives. they were named after the structurally homologous domain of the macroh a histone. originally described as adp-ribose- -phosphatases, both cellular and viral macrodomains enzymatically remove mono-and poly-adp-ribose from proteins, supporting the notion that protein adp-ribosylation is a component of the antiviral response. chikungunya virus and hepatitis e virus macrodomains are critical for replication, while the coronavirus macrodomain both blocks the innate immune response and separately promotes in vivo replication. recently, more defined roles for the marylating parps have been described, including a prominent role in the cellular stress response. parp regulates the er-stress response [ ] , and several parps are present in cytoplasmic stress granules (parps , , , , and a) [ ] . adp-ribosylation of argonaute proteins in stress granules by an unknown parp inhibits rna interference [ , ] . finally, parps have both antiviral and proviral activities (box ). as with any ptm, adp-ribose must be removed in a timely manner to prevent an untoward response and to recycle adp-ribose components ( figure ). the cellular enzymes that remove par from proteins are par glycohydrolase (parg), and to a lesser extent adp-ribosyl hydrolase [ , ] . the importance of removing par is demonstrated by the embryonic lethality of mice with genetic deletion of parg [ ] . however, parg only cleaves between the unique glycosidic bonds of par [ ] , and thus it was unclear how the terminal adp-ribose moiety was removed from the modified protein. this conundrum was resolved by the discovery that cellular macrodomain-containing proteins, including terminal adp-ribose glycohydrolase (targ ), macrod , and macrod removed this terminal adp-ribose [ ] [ ] [ ] . the human genome contains at least macrodomain-containing genes, but the substrate specificity and physiological roles of these proteins are largely unknown. almost three decades ago, bioinformatics approaches identified a unique conserved gene domain within three viral families, the togaviridae, coronaviridae, and hepeviridae [ ] [ ] [ ] . all of these families are positive-sense rna viruses and cause disease in humans and animals (box ). the domain was named the 'x' domain as its structure and function was unknown. these domains were defined as 'macrodomains' after crystal structures of both cellular and viral macrodomains revealed a core fold homologous to the nonhistone part of the macroh a protein [ ] [ ] [ ] . these macrodomains were shown to have phosphatase activity and were named adp-ribose- -phosphatases (adrps) [ , [ ] [ ] [ ] [ ] . despite the demonstrated adrp activity, it was unclear why viruses would devote a gene product to converting adp-ribose- -phosphate to adp-ribose. the discovery that macrodomains de-adp-ribosylate proteins strongly suggested that their major role was in reversing antiviral adp-ribosylation. however, this hypothesis has yet to be proven. as all three viral families that contain macrodomains include established human pathogens, understanding how box . the interplay between parps and virus infections several studies have found parps to have both proviral and antiviral activities. using parp inhibitors, several dna viruses, such as herpesviruses, poxviruses, and adenoviruses, have been found to require adp-ribosylation for optimal replication [ ] [ ] [ ] . this is not surprising given the importance of parps in cellular dna replication and repair. the hsv- icp protein enhances parylation by degrading par-glycohydrolase, which removes par from proteins [ ] . furthermore, tiparp was shown to adp-ribosylate tbk- and decrease ifn-b production, leading to enhanced replication of several rna and dna viruses [ ] . the best known antiviral parp, parp , also known as zap (zincantiviral protein) restricts the replication of several rna viruses by targeting viral or host rna for degradation [ ] . parp contains mutations in its parp domain, making it enzymatically inactive, indicating that its antiviral activity does not require adp-ribosylation [ ] . however, parp is known to be important for stress granule adp-ribosylation, and it also contributes to the adp-ribosylation of the overexpressed pa and pb proteins of influenza a virus which leads to their ubiquitination and proteasomal degradation [ , , ] . thus, while it does not directly adp-ribosylate proteins, it appears that parp participates in this process, possibly by binding to active parps and recruiting them to target proteins. similarly, parps , , and directly inhibit alphavirus replication, but in a parp-independent manner, as catalytically inactive mutants still blocked replication [ ] . overexpression of parp has also been shown to inhibit the replication of several negative-strand rna viruses, including vesicular stomatitis virus (vsv) [ ] . parp represses the transcription of integrated retroviruses in a chicken lymphoblastoid cell line, again in a parp-independent manner [ ] . in addition, multiple parps are interferon-stimulated genes (isgs), and in silico analyses indicate that parps , , , and are rapidly evolving, a hallmark of antiviral defense proteins [ ] . however, despite these data, antiviral adpribosylation of cellular or viral targets have yet to be identified. macrodomains contribute to replication and pathogenesis is important. here we comprehensively review the structures and enzymatic activities of these viral macrodomains, describe their potential functions, and discuss future challenges in understanding how macrodomains impact virus biology. macrodomains are conserved, $ amino acid protein domains present in all kingdoms of life. based on phylogeny, macrodomains are divided into different classes. most rna virus macrodomains fall into the macrod-like family, which includes human macrodomains macrod and macrod [ ] . the exceptions are the sars coronavirus (sars-cov) unique macrodomains (mac /mac formely known as sud domains), which we will not further discuss as they are distinct from the conserved macrodomain (mac ). macrodomains adopt a globular, mixed a/b/a sandwich fold, first shown when the structure of the archaeal macrodomain af [ ] hepatitis e virus (hev) is an emerging pathogen and is probably the most common cause of acute viral hepatitis in the world, with as many as million infections per year. mortality rates for acute infections are $ % for normal adults but as high as % for pregnant women in their third trimester, also often resulting in stillbirths of the fetus. hev, like hcv, can also lead to chronic hepatitis and cirrhosis [ ] . in developing countries, hev is spread by the fecal-oral route, while in developed countries hev is generally acquired through animal reservoirs, most likely pigs. treatment for hev infection includes either monotherapy with ribavirin or combination therapy of pegylated ifn and ribavirin. additionally, two vaccines have been developed for hev that have shown to be effective [ ] . the togaviridae are divided into two genera, the alphaviruses and the rubiviruses. rubella virus, a rubivirus, causes a congenital syndrome characterized by multiorgan birth defects. however, this disease has largely disappeared in the usa and other developed countries since an effective vaccine was developed. alphaviruses, which include chikungunya virus and ross river virus, are transmitted by mosquitoes and can infect both vertebrate and invertebrate species. in humans, these infections can lead to a number of different pathologies, the most common being severe arthritis or rash [ , ] . there are no alphavirus vaccines currently available. coronaviruses (covs) cause a wide variety of diseases in both humans and mammals. covs such as porcine epidemic diarrhea virus (pedv), bovine cov, and infectious bronchitis virus (ibv) cause severe disease in veterinary animals. while originally only thought to be a cause of the common cold in humans, recent epidemics have demonstrated that covs can also cause severe human disease. severe acute respiratory syndrome cov (sars-cov) and middle east respiratory syndrome cov (mers-cov) have emerged in the last years, causing serious respiratory disease in humans with high mortality rates ( - %). both viruses probably originated in bats and were transmitted to humans through animal reservoirs. mers-cov is endemic in camels in the middle east and will likely continue to cause lethal disease for many years [ ] . there are no approved vaccines or antiviral agents for human covs; however, several vaccines have been licensed for cov-mediated veterinary diseases [ ] . including those of alphaviruses and coronaviruses (covs), but not from hepatitis e virus, have been determined, with pdb entries available (www.rcsb.org). the globular macrodomain contains a conserved cleft that has been shown to bind adp-ribose [ ] , a feature that has been confirmed for viral macrodomains by numerous biochemical studies [ , [ ] [ ] [ ] . some of the most conserved residues of macrodomains are located at the surface and near the adpribose binding cleft ( figure ). based on adp-ribose-macrodomain complex structures, these conserved residues provide high specificity and affinity for adp-ribose. the distal ribose moiety is especially tightly coordinated by key residues found in loop (between b-sheet and a-helix ) and loop (between b-sheet and a-helix ) ( figure ). loop contributes backbone contacts to the adp-ribose a-phosphate and and oh groups of the distal ribose. in addition, the conserved asparagine residue (n in sars-cov) makes a hydrogen bond to the oh group. in cov macrodomains, the conserved histidine residue (h ) contributes to adp-ribose binding through a coordinated water. alphavirus macrodomains have a conserved cysteine (c ) that could fulfil a similar role, whereas human macrod contains an aspartic acid (d ) that makes a hydrogen bond with the oh group. loop contributes backbone contacts primarily to the b-phosphate of adp-ribose. in addition, isoleucine (i ) and phenylalanine (f ) residues provide van der waals contacts and are thought to direct orientation of the distal ribose [ , , , ] . in some macrodomains, tyrosine replaces phenylalanine and forms a hydrogen bond to the oh of adp-ribose. macrodomains with a threonine before the g residue contribute an additional hydrogen bond to the ʹ oh of the proximal ribose (e.g., t in chikungunya virus (chikv)). besides the two loops, other notable adp-ribose binding residues are d (n in sindbis virus (sinv) and s in hepatitis e virus (hev)), which contacts the amino group of adenine, and n (r in chikv and f in macrod ), which interacts with the adenine ring via p-charge or p-p interactions. depending on the adp-ribose contacting residues, the affinities for adp-ribose by different macrodomains vary, greatly with dissociation constants ranging from . to above mm [ , , , ] . binding of adp-ribose is not the sole property of macrodomains. the first enzymatic activity ascribed to macro-d macrodomains was the dephosphorylation of adp-ribose- -phosphate (a by-product of trna splicing reactions) identified through a biochemical-genomic screen in yeast [ ] . this phosphatase activity was also detected for viral macrodomains [ , , , , ] . however, the enzymatic activity of macrodomains is low, the activities vary across different virus species, and no easy explanation exists as to why adp-ribose- phosphate hydrolysis would benefit these viruses. therefore, other functions of viral macrodomains were considered. they include but are not limited to: binding adp-ribosylated proteins, poly-adp-ribose, rna or other nucleic acids [ ] . the discovery that macrod-like macrodomains are de-adp-ribosylating enzymes [ , ] suggested that viral macrodomains could have the same activity. indeed, li et al. were the first to show both de-mar-ylating and de-par-ylating activities for macrodomains from members of all three macrodomain-containing virus families [ ] . fehr et al. confirmed and additionally tested the effects of several mutations on the sars-cov macrodomain de-adpribosylating activity [ ] . eckei et al. demonstrated de-adp-ribosylating activity of macrodomains from different alphaviruses, addressing both de-mar-ylating and de-par-ylating activities [ ] ; however, it is still unknown which features of the macrodomain limit its activity to de-mar-ylation [ ] and which are required for de-par-ylation [ , ] . finally, mcpherson et al. showed de-mar-ylating activity for the chikungunya virus macrodomain and determined that this macrodomain removes adp-ribose from acidic residues but not from lysines [ ] . together, these four studies strongly suggest that de-adp-ribosylation is likely the biochemical function of viral macrodomains. adp-ribosylated proteins are linked to adp-ribose through the position, reminiscent of adp-ribose- -phosphate, explaining how macrodomains could have both activities. these latest studies have shifted the focus to de-adp-ribosylation, but other potential macrodomain functions should not be neglected, as the catalytic mechanism of de-adp-ribosylating macrodomains is not fully understood and the residues impacting de-adp-ribosylation often affect adp-ribose binding and other catalytic activities (e.g., adpribose- -phosphate hydrolysis). macrodomain mutations experimentally shown to disrupt catalytic function are summarized in table . the corresponding residues in different viral macrodomains are identified by sequence alignment and their relation to the adp-ribose substrate are visualized in figure . the mutational approaches interfere with macrodomain function by (i) reducing adp-ribose binding affinity (e.g., d a (sars-cov), t a (chikv), y f (chikv)), (ii) introducing steric hindrance (e.g., g v (sars-cov), g a (hev), g e (chikv)), or (iii) displacing potential catalytic water and reducing conformational strain on the distal ribose (e.g., g e+v r +y n (chikv)). the most common studied mutation is substitution of the highly-conserved asparagine to alanine (n a in sars-cov). this asparagine coordinates the oh of distal ribose and was proposed to be essential for catalysis. while the n a mutation abolishes the catalytic activity of sars-cov macrodomain [ , , ] , it reduces but does not abolish the catalytic activity of other cellular and viral macrodomains [ , , ] . such apparent discrepancy could be explained by reduced adp-ribose binding by the asparagine-to-alanine mutant since viral macrodomains, especially the covs, have a lower affinity for adp-ribose ( mm in sars-cov compared to . mm in human macrod ). the loss of a single hydrogen bond between the cov macrodomain and adp-ribose could reduce its affinity to a point where the macrodomain enzymatic activity is nondetectable. an alternative explanation is that different catalytic mechanisms exist among macro-d type macrodomains. despite these uncertainties, the strict conservation of this asparagine and the effects of its mutation on in vitro activity and virus fitness make it a valuable tool for studying viral macrodomains. the fact that viral macrodomains are generally required for virulence (see section below) suggests that small-molecule inhibition of macrodomains could be a novel therapeutic approach to preventing severe alphavirus, hepatitis e virus, or coronavirus-induced disease. one concern in considering the development of such molecules is the potential difficulty in specifically targeting the viral macrodomains but not the cellular macrod or macrod proteins. while such concern is warranted, there is potential for exploiting differences between human and viral macrodomains. residues contributing to distal ribose oh coordination differ between human and viral macrodomains. in addition, the space around the proximal ribose and the surface residues contacting the adenine-ring might be additional areas of interest for specific small-molecule design. the potential for virus escape by mutating at these differing structural information is available, uniprot retrieved sequences of the macrodomains were used and are indicated as (seq.). secondary structure elements are schematically depicted above the alignment, and the numbering is for a generic macrodomain (i.e., not including additional helices or sheets present in some but not all macrodomains). asterisks and red boxes indicate highly conserved, mostly substituted or key catalytic determinant residues of viral macrodomains. magenta-shaded boxes depict the degree of conservation. g e | d | q À reverted to wt (g e) [ ] g a | s + | +/À # virulence & replication [ ] v a | f +/À [ ] v e À [ ] residues could hamper the feasibility of inhibitor design and usability, but these concerns could be addressed experimentally. the observation that even a % reduction in viral macrodomain activity greatly reduced virus fitness suggests that viruses would struggle to escape potential inhibitors [ ] . if the risk of off-target effects in humans is too high, such inhibitors could be used in veterinary medicine. in recent years, significant progress has been made in deciphering the role of viral macrodomains, largely due to the advent of reverse genetic methods for these viruses. in this section, we review the known and potential roles for viral macrodomains. hev is a nonenveloped positive-sense rna virus with a genome of . kb that contains three open reading frames: orf , orf , and orf . the hev macrodomain is a part of the multidomain orf gene product of hev (figure ) [ ] . in a study on the orf gene products of hev, nan et al. found that orf inhibited poly i:c-dependent induction of interferon-b (ifnb) [ ] . using overexpressed proteins, only the papain-like cysteine protease and the macrodomain could block ifn-b expression. importantly, the hev macrodomain inhibited both ikke overexpression-induced ifn-b expression and interferon regulatory factor- (irf- ) phosphorylation. since the protein kinase ikke phosphorylates irf- , these data suggest that the macrodomain targets irf- . however, macrodomain inhibition of ikke-induced ifn-b expression was significantly less than its inhibition of poly i:c-induced ifn-b expression, suggesting that other mechanisms are involved. in a study by ojha and lole, the hev macrodomain was found to interact with the light-chain subunit of human ferritin (ftl), which led to a reduction of secreted ferritin [ ] . ferritin is an iron-binding protein that helps the cell store free iron. ferritin complexed with iron can activate nf-kb-dependent ifn production, suggesting that this interaction could be a mechanism of innate immune suppression [ ] . neither of these studies addressed whether the ability of the hev macrodomain to bind or hydrolyze adp-ribose was important for these observations. utilizing full-length replicon-based reverse genetic systems with either a gfp or luciferase reporter, parvez et al. and li et al. tested the role of the macrodomain in hev replication [ , ] . in these studies, several amino acids in the active site were mutated (n , n , h , g , g , g ) and tested for their ability to replicate. in the parvez study, all mutations produced wild-type levels of viral rna, but only n and g mutants were viable as measured by gfp expression. li et al. found that replication strongly correlated with the enzymatic activity of each mutation they created. these results demonstrate that the hev macrodomain is important for replication and acts after rna replication, likely prior to translation of viral proteins. another study found that the hev macrodomain interacted with the viral methyltransferase and orf using yeast- -hybrid and coimmunoprecipitation methods [ ] . it will be of interest to determine if these proteins are adp-ribosylated and whether these interactions are important for replication. togaviruses are positive-sense enveloped viruses with - kb rna genomes. the first twothirds of the genome encodes four nonstructural proteins while the final one-third of the genome encodes structural genes. the macrodomain is located at the n-terminus of nsp ( figure ) [ ] . in , park and griffin published a study focusing on two asparagine mutants (n a and n a) in the adp-ribose binding pocket of the sinv macrodomain [ ] . these mutant viruses replicated normally in bhk- cells but were modestly attenuated in neuronal cells and significantly attenuated in -week-old mice. upon replication in neurons, the n a mutation changed to threonine or aspartic acid, and a second site mutation of a glutamic acid at position to a glycine appeared. most macrodomains have a glycine at this position, suggesting that this mutation may enhance macrodomain activity. it was unclear how these mutations affected this protein, as neither mutant affected par binding and it was unknown whether alphaviruses' macrodomains had de-adp-ribosylating activity. however, recently it was demonstrated that chikv contains these activities [ , ] , making it likely that these mutations within the adp-ribose-binding pocket of the sinv macrodomain attenuated its enzymatic activity. as opposed to sinv, mutations in the active site of the chikv macrodomain had severe effects on virus replication [ ] . three recombinant viruses with mutations that severely affected both adp-ribose binding and hydrolase activity were unrecoverable as they quickly reverted to wild type (d a, g e, g e). the g e mutation even reverted when transfected into an aedes albopictus cell line, suggesting that antiviral adp-ribosylation also occurred in mosquito cells. other mutations that partially ablated hydrolase activity and adp-ribose binding (t a, g s, g a) replicated at to logs lower efficiency than wild-type virus in a neuronal cell line . finally, one mutation, y a, was identified that had enhanced adp-ribose binding but decreased hydrolase activity. this mutation was also significantly attenuated in nsc- cells, demonstrating that adp-ribosyl hydrolase activity alone is important for replication. in this study, de-adp-ribosylating activity, replication, and virulence did not perfectly correlate, suggesting a role for adp-ribose binding in virulence. of note, an additional mutant, r a, which had normal levels of adp-ribose binding and hydrolase activity in vitro, quickly reverted in tissue culture cells. this mutation could affect alternative functions of the macrodomain. for example, studies have found that mutations in the adp-ribose binding domain of the veev macrodomain could compensate for mutations in other regions of the genome; however, it is unclear whether these activities were related to de-adp-ribosylating activity or other functions [ ] [ ] [ ] . additionally, lulla et al. found that c-terminal amino acids of the semliki forest virus (sfv) macrodomain are required in the processing of the nsp / cleavage site, which could explain the defect of the r a mutation [ ] . covs are large, positive-sense enveloped rna viruses with $ kb genomes. the n-terminal two-thirds of the cov genome contains nonstructural proteins (nsps) while the c-terminal one-third of the genome contains the structural and accessory genes. the conserved macrodomain is located at the n-terminus of the multidomain nsp protein (figure ) [ ] . in sars-cov and possibly other covs, two additional macrodomains (mac /mac ) immediately follow the conserved macrodomain (mac ) [ ] . mac and mac do not bind or hydrolyze adpribose; in contrast, they bind g quadruplexes [ ] . using a sars-cov replicon system, kusov et al. found that mac was essential for replication [ ] . most studies of the conserved cov macrodomain (mac ) function have used recombinant viruses with the highly-conserved asparagine mutated to an alanine. this mutation has small to no effects on cov replication in cell culture [ , , , , ] . also, deletion of the macrodomain had no effect on replication of a sars-cov replicon [ ] . in marked contrast, several studies have found that the cov macrodomain is essential for viral pathogenesis. eriksson et al. first showed that the macrodomain was required for murine hepatitis virus (mhv) strain a -induced hepatitis [ ] , while fehr et al. demonstrated that mutation of the macrodomain of an encephalitic strain of mhv and of mouse-adapted (ma) sars-cov resulted in attenuation in vivo [ , ] . in all cases, the mutation was associated with reductions in viral load. it was also shown by both eriksson et al. and kuri et al. that the macrodomain could repress ifn production; however, it was unclear if this occurred in vivo and whether it was important for pathogenesis [ , ] . fehr et al. found that the sars-cov macrodomain repressed ifn and cytokine production both in mice and in a bronchial epithelial cell line [ ] . a coinfection with wild type and the macrodomain mutant sars-cov demonstrated that the enhanced interferon response was partially protective in mice. also, the reduced viral load observed in sars-cov-infected mice was independent of the ifn response, as viral loads were not increased in ifnar À/À mice infected with macrodomain mutant virus. in combination, these studies led to the conclusion that the sars-cov macrodomain promotes replication in vivo and independently suppresses the ifn response, with both functions playing a role in pathogenesis. despite the discovery that viral 'x' domains closely resemble cellular macrodomains, the function of these domains remained an enigma until recent discoveries showed that viral macrodomains remove adp-ribose from proteins. while this activity has not been demonstrated during infection, the publications discussed in this review strongly suggest that this is the case. as such, the primary focus of macrodomain research will likely shift from basic functional and biochemical studies to identifying antiviral parps and the protein targets of viral macrodomains (see outstanding questions). while identifying adp-ribosylated proteins has proven difficult in the past, new methods for detecting adp-ribosylation should lead to important discoveries in this field [ ] . these studies will significantly enhance our understanding of these proteins, which could lead to new approaches for treating and preventing disease caused by macrodomain-expressing pathogens. do viral macrodomains counteract antiviral adp-ribosylation? which parp(s) is responsible for antiviral adp-ribosylation? do invertebrates have antiviral parp activity that is counteracted by macrodomains? what are the adp-ribosylated protein targets of viral macrodomains? are targets mono-or poly-adp-ribosylated? how does adp-ribosylation impact these targets? what are the determinants of mono-versus poly-adpribose binding/removal? do all viruses require macrodomain enzymatic activity or is mar/par binding sufficient to promote virus fitness/virulence? what effects do mutations in viral macrodomains have on de-adp-ribosylation kinetics? how do viral macrodomains acquire specificity? do regions outside of the adp-ribose binding pocket contribute to interactions with targets? can a macrodomain from one virus family substitute for a different one? is the development of small-molecule inhibitors specific for viral macrodomains to repress viral pathogenesis feasible? could viruses escape these inhibitors? would these inhibitors also affect mammalian enzymes, leading to significant side effects? nicotinamide mononucleotide activation of new dna-dependent polyadenylic acid synthesizing nuclear enzyme adp-ribosylation: new facets of an ancient modification a family of killer toxins. exploring the mechanism of adp-ribosylating toxins toward a unified nomenclature for mammalian adp-ribosyltransferases family-wide analysis of poly(adp-ribose) polymerase activity readers of poly(adp-ribose): designed to be fit for 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morgane; andraud, mathieu; rose, nicolas title: combining network analysis with epidemiological data to inform risk-based surveillance: application to hepatitis e virus (hev) in pigs date: - - journal: prev vet med doi: . /j.prevetmed. . . sha: doc_id: cord_uid: q kpmx animal movements between farms are a major route of pathogen spread in the pig production sector. this study aimed to pair network analysis and epidemiological data in order to evaluate the impact of animal movements on pathogen prevalence in farms and assess the risk of local areas being exposed to diseases due to incoming movements. our methodology was applied to hepatitis e virus (hev), an emerging foodborne zoonotic agent of concern that is highly prevalent in pig farms. firstly, the pig movement network in france (data recorded in ) and the results of a nation-wide seroprevalence study (data collected in farms in ) were modelled and analysed. the link between network centrality measures of farms and hev seroprevalence levels was explored using a generalised linear model. the in-degree and ingoing closeness of farms were found to be statistically associated with high hev within-farm seroprevalence (p < . ). secondly, the risk of a french département (i.e. french local administrative areas) being exposed to hev was calculated by combining the distribution of farm-level hev prevalence in source départements with the number of movements coming from those same départements. by doing so, the risk of exposure for départements was mapped, highlighting differences between geographical patterns of hev prevalence and the risk of exposure to hev. these results suggest that not only highly prevalent areas but also those having at-risk movements from infected areas should be monitored. pathogen management and surveillance options in the pig production sector should therefore take animal movements into consideration, paving the way for the development of targeted and risk-based disease surveillance strategies. developing risk-based surveillance programmes for animal diseases is essential to support both strategic and operational decision-making in the field of animal and veterinary public health (reist et al., ) . indeed, mobilising resources towards targeted high-risk populations improves the sensitivity and cost-effectiveness of surveillance systems (stärk et al., ) . the sub-populations to be targeted are usually chosen based on epidemiological studies assessing the probability of occurrence of the hazard in the sub-population (e.g. farms with specific risk factors) and/or the consequences of the disease potentially being introduced in this sub-population (e.g. economic effects, spread to other herds or countries) (stärk et al., ) . however, most current pathogen surveillance programmes do not quantitatively include the risk related to animal movements, even though these are a major transmission route between farms. the exposure of farms or areas to pathogens is therefore closely related to the movement network's features. as such, animal movement data have been increasingly studied using social network analysis (sna) methods, with farms being considered as nodes, and animal movements between farms as links (wasserman and faust, ; bigras-poulin et al., ; bigras-poulin et al., ; martínez-lópez et al., ; natale et al., ; ribbens et al., ; nöremark et al., ; lindstrom et al., ; rautureau et al., ; buttner et al., ; dorjee et al., ; guinat et al., ; thakur et al., ) . although in most studies network analyses have been motivated by the consequences of animal trade on the epidemiology of animal diseases (keeling, ; lloyd-smith et al., ; bigras-poulin et al., ; martínez-lópez et al., ; rautureau et al., ; buttner et al., ) , the specific role of animal shipments in pathogen transmission and/or exposure has only scarcely been documented and rarely quantified, especially in the swine sector (ortiz-pelaez et al., ; green et al., ; martin et al., ; porphyre et al., ; frössling et al., ; nicolas et al., ; beaunee et al., ; lee et al., ; salines et al., b; sintayehu et al., ) . analysing contact patterns related to pig trade could provide new insight into infection dynamics, pathogen spread and risk factors, helping to design risk-based surveillance programmes. hepatitis e is an emerging foodborne zoonosis of concern for which pigs have been recognised as a major reservoir in industrialised countries (dalton et al., ; pavio et al., ; adlhoch et al., ; efsa et al., ) . indeed, several human hepatitis e cases have been related to the consumption of raw or undercooked products containing pig liver (colson et al., ; moal et al., ; motte et al., ) . hev is highly prevalent in pig farms and is likely to spread between farms through the introduction of infected pigs, especially due to the pyramidal structure of the pig production sector (salines et al., a) . to date, no continuing hev surveillance programmes have ever been implemented in industrialised countries (salines et al., a) . the aim of our study was therefore to combine network analysis with disease epidemiology and propose methods to quantify the epidemiological role of animal movements on two different scales: firstly by measuring the impact of animal movements on pathogen prevalence at the farm level; and secondly by assessing the risk of french départements being exposed to diseases due to incoming movements from infected areas. our methodology was applied to hepatitis e virus (hev) in the pig production sector. . . data . . . movement data . . . . pig movement database. as described by salines et al. ( b) , pig movement data were obtained from the national swine identification database (bdporc), managed by swine industry professionals and recognised by the french ministry for agriculture. all pig movements between farms and to slaughterhouses, rendering plants and trade operators are systematically recorded in this database. movements of pigs are reported at the batch level: groups of animals are sent off production sites (loadings, further denoted l) and dispatched either to other production units or to slaughterhouses (unloadings, further denoted u). a single truck can load and unload animals at several production sites: one round corresponds to a series of movements by a truck, from the first loading operation to the last unloading event leaving the truck empty. . . . . design of the movement network ( fig. ) . movement data recorded from january to december were modelled into a onemode directed network aggregated on a one-year basis: holdings were considered as nodes, and movements between two nodes were considered as directed links. all movements between two given holdings during the time period were aggregated into a single link. in-between movements forming a round were replaced with direct movements between holdings, meaning that intermediate transit movements by a truck through a farm without any animal unloading were excluded. all sites corresponding to unloading operations were assumed to be linked to all prior loading sites for the same round. for example, assuming successive loadings at sites l and l followed by an unloading operation at site u , then holding u was linked to l and l . as described by rose et al. ( ) , a nation-wide study was undertaken in to collect representative hev prevalence data accounting for the production level diversity throughout the country. in short, previous data had indicated a farm-level prevalence close to % (rose et al., ) ; the number of herds required to estimate % with % relative precision and % confidence, was . this number was increased to to anticipate uncontrolled events. the herds to be sampled were determined by random selection of a list of slaughter dates and times from a database table. the observed minimum withinherd prevalence in this same preliminary study was close to % (rose et al., ) and this value was retained as the minimum within-herd target prevalence to be detected. given the sensitivity and specificity of the commercial serological tests (rose et al., ) , this led to sampling of pigs in batches with less than pigs, pigs in batches of - pigs and pigs in batches with more than pigs. finally, sera and livers were randomly sampled from pig farms located in different french départements, corresponding to between and individual serum samples per farm and between and liver samples per farm collected at the slaughterhouse. serum samples were tested with the anti-hev total immunoglobulin for human diagnosis, eiagen hev ab kit ® by adaltis (ingen, france) adapted to pig serum. . . . farm centrality indicators and within-farm hev seroprevalence . . . . farm centrality indicators. only farms out of the sampled in the prevalence study were recorded in the movement database. using the pig movement network, several centrality measures were calculated for each of the farms: the in-degree, i.e. the number of different holdings from which a holding receives animals; the out-degree, i.e. the number of different holdings to which a holding sends animals; the ingoing and outgoing closeness, which focus on how close a farm is to all the others in the network through incoming or outgoing links; the betweenness, i.e. the number of geodesics going through a node; the average monthly ingoing contact chain (icc), i.e. the number of holdings in contact with a given holding (called the root) through time-respecting paths reaching the root within a month; the average monthly outgoing contact chain (occ), i.e. the number of holdings in contact with a root through time-respecting movements of animals leaving the root within a month; and the node loyalty, measuring the fraction of preserved links of a node for a pair of two consecutive network configurations over time, with the time window in our case being a half-year. all continuous variables were categorised according to the form of their distribution, with categories containing at least % of the sample size. . . . . within-farm hev seroprevalence. the hev seroprevalence of each of the farms was defined as the number of hev-seropositive pigs in relation to the total number of pigs sampled in the farm. the individual sensitivity and specificity of the test (rose et al., ) were used to correct the apparent seroprevalence estimates (rogan and gladen, ) . . . . . statistical model. a univariable analysis was conducted to assess the statistical link between each explanatory variable (i.e. the farms' centrality metrics) and the outcome (i.e. the unbiased withinfarm hev seroprevalence). to do so, a generalised estimating equation (gee) logistic regression was performed using proc genmod in sas . . with the "farm" effect being included as a repeated statement (sas, ) . factors associated with the outcome (p < . ) were then subjected to bivariable analysis. the objective was to identify strong correlations between each explanatory variable to prevent multicollinearity. if variables did not show strong collinearity (p > . ), they were included in a multivariable model. we also investigated the role of farm type as a potential confounding factor, by testing the link between farm type and the explanatory variables and the outcome with chi-squared tests and logistic regression, respectively. . . . . departmental farm-level hev seroprevalence (fig. ) . hev prevalence was defined at the département level as the number of farms having at least one hev-seropositive pig out of the total number of farms sampled in the département. the standard deviation for farmlevel hev prevalence was calculated thanks to an exact binomial test and weighted with a correction factor reflecting the sampling rate (i.e. the proportion of sampled farms among the total number of farms in the département). for each of the départements where data were available, uncertainty regarding the farm-level hev prevalence estimate was represented by a beta distribution using the estimate and the confidence interval to define the parameters of the distribution (). . . . . estimation of the risk of exposure at departmental level. an indicator of the risk of a département being exposed to hev was computed as follows: first, for each département, an hev farm-level prevalence value was randomly sampled from the beta distribution; the corresponding number of hev-positive farms in the département was then derived from this selected prevalence value and the individual status of the herds was randomly assigned. source herds were then randomly selected according to the actual number of movements leaving the source département, leading to a number of infected outgoing movements. lastly, the indicator of the risk of a département being exposed to hev was calculated as the number of positive movements it had received from source départements divided by its total number of external incoming movements. to stabilise the outputs of the procedure, the whole calculation was repeated , times, resulting in a risk distribution of hev exposure for each département. the exposure risk model was implemented in r (ihaka, ) . the farms' mean in-and out-degrees were . (range: - ) and . (range: - ), respectively. mean ingoing and outgoing closeness were . . − and . . − , respectively, with little variability. mean betweenness was . (range: - ). mean monthly ingoing and outgoing contact chains were . (range: - ) and . (range: - ), respectively. mean node loyalty was . (range: - ). in the studied farms, hev unbiased seroprevalence ranged from % to % hev-seropositive pigs (mean: %, median: %). the univariable analysis showed that two of the eight analysed centrality indicators were statistically associated with the outcome (table ) : high in-degree and ingoing closeness for farms were significantly and positively associated with high within-farm hev seroprevalence. since in-degree and ingoing closeness were correlated (chi-squared test, p < . ), they were not included in a multivariable model. farm type was associated with all explanatory variables (p < . ) but not with within-farm hev seroprevalence (p > . ). departmental farm-level hev prevalence distributions were plotted (see examples in supplementary file , figure a) . due to the varying number of sampled farms depending on the département (fig. ) , quite a few départements exhibited large farm-level prevalence distributions (e.g. département a in supplementary file , fig. a) . distributions of the risk indicator of french départements being exposed to hev were plotted (see examples in supplementary file , fig. b) . the median risk of exposure for each département was mapped (fig. ) . geographical patterns of hev prevalence and hev exposure risk showed major differences (figs. and fig. ). understanding the features of movement networks is crucial to analyse infection dynamics, pathogen occurrence and risk factors and to support risk-based surveillance strategies. although network studies have often been motivated by the outcome of animal movements on pathogen epidemiology (keeling, ; rautureau et al., ; buttner et al., ; thakur et al., ) , the specific role of animal shipments in pathogen transmission and/or exposure has rarely been quantified, especially in the swine sector. the primary advantage of our study lies in combining epidemiology and network analysis to quantify both the impact of animal movements on pathogen prevalence within farms and the risk of areas being exposed to diseases due to between-area movements. hev was chosen as a pathogen for implementation. indeed, pig movements are likely to play a pivotal role in hev epidemiology (salines et al., a) , although they have only scarcely been explored to date (nantel-fortier et al., ) . we assessed the role of pig shipments in relation to within-farm hev seroprevalence level and to the risk of exposure of french départements to hev. pig movement data originated from the french national swine identification database (bdporc), in which all pig shipments are systematically recorded. the information provided by this database is recognised by the french ministry for agriculture and can therefore be considered trustworthy. moreover, a thorough cleaning stage was carried out to manage incorrect or incomplete data. the quality of data in terms of accuracy, reliability, and comprehensiveness guaranteed the robustness of our results (salines et al., b) . the random selection process for tested farms and for individual pigs tested from each farm (rose et al., ) ensured reliable estimates for the seroprevalence values used in our study. moreover, the within-farm apparent seroprevalence estimates were corrected for serological test characteristics, providing true seroprevalence estimates. eight production sites surveyed in ceased activity before , limiting movement data availability to only out of the farms. this is consistent with the observed overall decrease in the number of pig farms in france (salines et al., b) . on the département scale, the model involving the random sampling of farm-level hev prevalence from beta distributions − with a weighted confidence interval − made it possible to take into account the low precision of some prevalence figures in quite a few départements where a low number of farms had been sampled. temporal variability of both pig movements and hev seroprevalence was a limitation of our study. indeed, one should note that movement and summary statistics as obtained thanks to a generalised estimating equation (gee) univariable logistic regression with the "farm" effect being included as a repeated statement. *statistically significant effect. m. salines et al. preventive veterinary medicine ( ) - prevalence data were not simultaneously collected. however, the french pig movement network has been found to be stable over time (salines et al., b) , so we can assume that combining the prevalence data with the pig movement data is still consistent. moreover, % of the farms included in our study showed a loyalty equal to (i.e. they exchanged animals with the same suppliers/buyers over the year), reflecting the stability of their movements. regarding hev prevalence, our data were dated ( ) and hev prevalence is likely to vary over time. however, a more recent study also conducted in france reported similar prevalence figures ( % seroprevalence in feurer et al. ( ) vs % in rose et al. ( ) ). aggregating movement data on a yearly basis also appeared to be relevant due to the absence of seasonality in the french pig network salines et al., b) and provided indicators representing the overall activity of farms over a year. a possible improvement to the network model may involve weighting links depending on the number of animals exchanged. in the recent literature, several farm connectivity indicators were identified as risk factors for disease occurrence and spread (martin et al., ; frössling et al., ; lee et al., ; sintayehu et al., ) . our study found that the farms' in-degree was positively associated with high within-farm hev seroprevalence. this is consistent with several studies conducted in livestock production sectors showing that farms having a high in-degree were more likely to be infected with a pathogen (martin et al., ; frössling et al., ; lee et al., ; sintayehu et al., ) . since repeated animal shipments to a farm from the same supplier were aggregated into a single link, the association between hev seroprevalence and in-degree not only indicates that the hev seroprevalence of farms increases with the number of incoming shipments, but it also proves that buying animals from several suppliers is linked to higher hev seroprevalence. our results also showed that the greater the ingoing closeness of a pig farm, the higher its hev seroprevalence. a high value for the ingoing closeness centrality of a given farm indicates that the farm can be reached by its trade partners in only a few movements. farm centrality in the network therefore appears to be a factor in vulnerability to hev. this is consistent with the findings of previously published papers (lee et al., ; sintayehu et al., ) . as lee et al. ( ) demonstrated for prrsv, we found that the odds of having higher within-herd hev seroprevalence was increased more by ingoing closeness than by in-degree, meaning that the level of connectivity with all other holdings in the network is a better predictor of hev infection than the number of directly connected farms. unlike for other pathogens (lee et al., ) , no significant association was found between hev within-farm seroprevalence and outdegree or outgoing closeness. the absence of an effect for these centrality indices was expected since hev is mainly transmitted by infected pigs introduced into a naïve population. introduction into a farm due to the sole transit of a possibly contaminated truck loading pigs in the farm for an outgoing shipment is therefore extremely unlikely. unlike sintayehu et al. ( ) regarding bovine tuberculosis, our statistical model did not show any significant effect of a herd's betweenness on within-herd hev seroprevalence. production units with high betweenness centrality play a key role in the spread of disease throughout the network since they can build so-called bridges between distinct network components. since we explored the role of centrality metrics in hev occurrence in farms, and not in their ability to transmit hev to other farms, the lack of an effect for betweenness was also expected. ingoing and outgoing contact chain values were not found to have a significant effect on hev seroprevalence either. again, as we did not investigate a farm's potential for spreading hev, the lack of a link between occ and hev seroprevalence is coherent. an association between icc and hev seroprevalence could have been expected. this kind of association has indeed been demonstrated in other studies, but frössling et al. ( ) showed that this link was pathogen-dependent: indeed, high icc was fig. . median risk of french départements being exposed to hev through external incoming pig movements ( , simulations). an indicator of the risk of a french département being exposed to hev was calculated as the number of infected movements it had received from source départements divided by its total number of external incoming movements. found to be a risk factor in the occurrence of bovine coronavirus but not for bovine respiratory syncytial virus. to the best of our knowledge, the exposure of a geographical area to a pathogen due to animal movements has never been quantified. the choice of the departmental level for our study was policy-oriented; indeed, french départements are local administrative areas and surveillance programmes are often designed and implemented on this scale. due to the low precision of hev farm-level prevalence data in quite a few départements, the distribution of the risk of exposure was large in these départements and the results in these départements therefore lack precision. nevertheless, the outputs of the procedure used to assess the risk of hev exposure were stabilised thanks to a high number of simulations. given the form of the risk distribution, the median appeared the most appropriate metric for the risk of exposure. high variability in the median risk of exposure to hev was observed depending on the french département, confirming the relevance of designing targeted and differentiated surveillance strategies based on the area's risk level. moreover, the discrepancy between the departmental observed prevalence figures and the departmental risk levels provides justification for monitoring not only highly prevalent areas but also those having atrisk movements coming from infected areas. confounding factors may bias our results. indeed, we had limited data regarding farm and département characteristics. for instance, no detailed data was available regarding farm size, pig density or farm management practices, but we checked that farm type (breeding, farrowing-to-finishing, etc.) was not a confounding factor. several research teams have recently developed farm-level risk scores based on animal movements. for instance, schärrer et al. ( ) introduced a cumulative score taking several parameters into account, including the icc, the number of animals per incoming movement, the type of pasture and the number of weeks per year with movements. another study proposed a method for calculating a diseasespecific relative ratio for the increased probability of infection due to the introduction of animals (frössling et al., ) . ribeiro-lima et al. ( ) also identified farms with a higher risk of bovine tuberculosis infection using a model based on a risk score at movement level. a further stage in our study could be to build a farm-level risk score including both risk factors linked to pig movements and other farm-specific risk factors for hev that have previously been identified (walachowski et al., ) . such a score would make it possible to target only high-risk farms for more effective surveillance. combining network analysis with epidemiological data demonstrated that direct network connectivity and farm centrality in the network are related to the within-herd hev seroprevalence level and that some areas are more at risk for hev due to their pig movements. more generally, the methods we proposed prove that farm-or area-level parameters derived from animal movements can support the risk-based selection of farms for surveillance programmes or the implementation of differentiated surveillance strategies depending on the area's 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sha: doc_id: cord_uid: n irdy x emerging viral infections represent a public health risk pointed out by the spreading of pathogens with potential zoonotic risk. moreover, the risk of zoonosis has probably been underestimated in occupational settings. a literature review between and was performed to identify evidences concerning the epidemiological associations between some emerging viruses and occupational diseases. observational studies and case-reports were selected and analyzed. west nile virus (wnv) disease, crimean-congo hemorrhagic fever (cchf) disease and hepatitis e virus (hev) infection were included in the review for their potential zoonotic transmission. the most important risk factor for acquiring wnv infection and cchf infection is the exposure to infected mosquitoes and ticks, respectively; therefore, outdoor workers are at risk of infection. hev is responsible for epidemics and endemics of acute hepatitis in humans, that can become infected through waterborne, foodborne and zoonotic transmission routes. a total of , and eligible studies for wnv, cchf virus (ccfhv) and hev, respectively, were analyzed by year, country, study design, risk group and outcomes. the occupational risk groups mainly included farm and agricultural workers, veterinarians, slaughterers, animal handlers, healthcare workers and soldiers. these findings support the need to develop effective interventions to prevent transmission of emerging viruses. it is well known that most infectious diseases in humans originate in animals, and the frequency of such diseases, named zoonoses, has been increasing over time (belay et al. ) . a joint consultation of who/fao/oie held in (who ) defined emerging zoonosis as 'a zoonosis that is newly recognized or newly evolved, or that has occurred previously but shows an increase in incidence or expansion in geographical, host or vector range'. drivers responsible for the emergence of zoonotic diseases include climate and environmental changes, human behavior, farming and trading practices, vector distribution and characteristics of the pathogens. many zoonosis emerge from wildlife species, e.g. severe acute respiratory syndrome coronavirus raised from bats and transmitted to civets before affecting humans. examples of zoonotic virus of other origin include west nile virus (wnv), chikungunya virus and crimean-congo hemorrhagic fever virus (cchfv), responsible for diseases with a high impact on public health (wang and crameri ; belay et al. ) . hepatitis e virus (hev) had been considered a sanitation problem in resource limited countries; however, the zoonotic form has emerged in industrialized countries with high seroprevalence, as detected in swine abattoir workers (ukuli and mugimba ) . recent epidemiological data on zoonosis are also of concern regarding the occupational medicine. mitigating the impact of viral emerging zoonotic diseases of occupational health importance is difficult because of several work and economic conditions worldwide, and requires multisectoral collaboration and interdisciplinary partnerships. in fact, control and prevention strategies for most zoonosis are effective according to a one health approach at the human-animal-ecosystem interface. in this study, a review was carried out to assess and summarize the scientific evidences concerning the epidemiological associations between some emerging viruses and occupational diseases. wnv and cchfv were included as examples of pathogens responsible for vector-borne infections, transmitted by mosquitoes and ticks, respectively, and both viruses are spreading in europe and neighboring countries at an increasing rate (marcantonio et al. ) . hev infection was also included in the review since growing evidences show that zoonotic transmission through contact with infected animals or consumption of contaminated food is responsible for most of the autochthonous cases in industrialized countries (clemente-casares et al. ) . the purpose was to identify which occupational sector, job, population at risk are more vulnerable to three emerging zoonotic viruses and main clinical outcomes, according to the selected papers, in order to provide evidence for policy makers and stakeholders involved in occupational safety and health. wnv is a neurotropic member of the family flaviviridae, genus flavivirus, maintained in enzootic cycles involving several species of birds, which act as amplifying reservoir host, and mosquitoes belonging principally to the culex pipiens complex, although other species would also support the spread of the virus (marcantonio et al. ) . a study conducted in italy between and detected wnv in three mosquito species belonging to two genera: culex pipiens s.l., culex modestus and ochlerotatus caspius (mancini et al. . humans, horses and other mammals are incidental or dead-end host. first isolated in uganda in , starting from the s wnv has spread rapidly across all the continents. climate change (warmer temperature and higher cumulative rainfall) could be one of the drivers that contribute to the changing pattern of transmission of several vector-borne diseases (riccardo et al. ) . following transmission via mosquito bites, wnv replicates in keratinocytes and in the skin dendritic cells (dcs), langherans cells (lcs), which then migrates to the local lymphnodes from where the virus disseminate to the kidney, spleen and other visceral organs. in about % of all infected patients, the wnv infection evolves to severe neurologic disease, including encephalitis, meningitis, acute flaccid paralysis and death. the virus entry to the central nervous system can be either via blood stream as well as via trans-neural pathways. infection can also happen by blood transfusion, organ, tissue and cell transplants. according to the above, although most human infections are subclinical, symptoms can vary from a self-limiting fever to severe neurological disease (ulbert ) . it has been demonstrated, by in vitro and in mouse models in vivo, that wnv infection induces innate cell immune response through activation of the toll like receptors (tlr ) and retinoic acid-inducible gene i (rig-i pathways) and induction of type i interferon (ifn-i) as well as of ifn λ. type i ifn receptors signaling in astrocytes regulates the permeability of the blood-brain barrier and protects the cerebellum from neuroinvasive infection by wnv. therefore, the negative regulation of ifn responses, by either host and viral factors, can contribute to the pathogenesis of wnv. in particular, ns protein of wnv, secreted upon infection, represses tlr -induced ifn in mouse and human cells, thus favoring virus spreading in the cns (luo and wang ) . cchfv belongs to the genus orthonairovirus, family nairoviridae. ticks of the genus hyalomma are considered both main vector and natural reservoir; direct contact with fluids, tissue or blood of infected animals are also considered transmission routes of the infection to humans. cchfv is maintained and transmitted in a vertical and horizontal transmission cycle involving a variety of wild and domestic animals that act as amplification hosts without showing signs of illness. despite these animals have been considered reservoirs of the virus, they develop only a transient viremia, while the virus can persist in ticks for their entire lifespan, and can be vertically transmitted to the next generation. therefore, ticks are considered both vector and reservoir for the virus (gargili et al. ) . nosocomial transmission may occur through direct contact with human infected blood or body fluids or contaminated medical equipment or supply. first recognized in , human cchfv infections have been reported in over countries in asia, middle east, south-eastern europe and africa. clinical symptoms usually comprise mild and nonspecific febrile illness; in some cases, severe hemorrhagic disease can develop (wang and crameri ) . the pathogenesis of cchfv infection in humans is mainly based on immunopathogenetic mechanisms, mediated by either innate or adaptive immune responses. the rig-i pathway is an immune sensor of cchfv rna. studies in human patients have shown that tlrs, in particular tlr , , and polymorphisms correlate with the severity and outcome of the disease in some geographical areas (turkey) (hawman and feldmann ) . the virus itself is able to antagonize innate immune signaling through deubiquitinatin and cleavage of proteins involved in innate immunity, such as isg , mediated by a specific domain (otu, ovarian tumor-like deubiquinase domain) in the l segment of cchfv. with regard to the role of the adaptive immunity responses to cchfv in human pathogenesis, it is not completely clear, due to the lack of suitable model in vivo. the evidence obtained from the recently developed cynomologus animal model suggested that neither the antibodies titer nor their neutralizing activity seem to correlate to the outcome of cchfv infection. the role of t cell responses, such as cytolitic activity in hepatic injury and severity of the cchfv associated haemorragic disease, seems not necessary but needs further studies. hepatitis e is an acute disease caused by hev, classified in the family hepeviridae, genus orthohepevirus a. genotypes hev- and hev- are restricted to humans and circulate in endemic area (asia and africa) causing outbreaks following the ingestion of contaminated water. in non-endemic areas (industrialized countries), hev- and hev- are linked to travel in endemic areas. in the last years, an increasing number of autochthonous infections, linked to the zoonotic transmission of the genotypes hev- and hev- , have been described (kamar et al. ) . there are evidences for the presence of autochthonous cases of hev infections in italy since (stroffolini et al. ) . the virus is transmitted via oral-fecal route, as well as by zoonotic transmission through direct contact with infected animals or food. swine is the principal reservoir of hev, mainly belonging to genotypes and , with prevalence of anti hev antibodies ranging from % to % (huang et al. ) . other reservoirs are wild board, rabbits, deer, mongooses, yaks and camels, infected with different genotypes (nan and zhang ) . vertical transmission from mother to fetuses (sharma et al. ) , and bloodborne transmission of the virus has been reported (al-sadeq, majdalawieh and nasrallah ). the virus probably replicates in extra-hepatic sites, such as intestinal tract, lymphnodes, colon, to reach the hepatocytes where it replicates in cytoplasm and then is released into the bloodstream and bile. the main liver damage by hev is mediated by t cells and natural killer (nk) cells. the virus is shed in the stool (lhomme et al. ) . hepatitis e is usually a self-limiting illness, in most cases ( %) the infection is asymptomatic, as the hev is non cytopathic, with mortality rate of - % worldwide (who ). sometimes symptoms of acute hepatitis can manifest. hev infection is associated with a number of extrahepatic manifestations, including kidney and a range of neurological injuries, in particular, guillain-barré syndrome, neuralgic amyotrophy and encephalitis/myelitis (dalton et al. ) . during pregnancy, hev infection can take a fulminant course, resulting in fulminant hepatic failure, membrane rupture, spontaneous abortions and stillbirths. studies from various developing countries have shown a high incidence of hev infection in pregnancy, with a fatality rate of up to % (pérez-gracia, suay-garcía and mateos-lindemann ). usually mild illness occurs in adult healthy individuals, whereas chronic severe disease occur in immunocompromised patients (transplant recipients, hiv immunocompromised patients) (kamar et al. ) and in pregnant women, in which hev- and hev- are likely to cause serious medical complications including liver failure, increased risk of miscarriage and premature delivery (khuroo and kamili ) . chronic hev infection is defined as detection of hev rna in serum or stool for longer than months and is typically associated to the genotype of hev. it is thus evident that clinical features of hev infection range from asymptomatic or acute liver failure to chronic infection without clinical symptoms but with increase in liver enzymes. one of the critical point in hev infection is clinical diagnosis of acute and chronic infection that is achieved by means of serologic and molecular tests, that are often non-specific. initially an anti-hev igm assay is used, whose positivity is confirmed by evidence of rising the igg titers. although the igm appear in the early phase of clinical illness and last for to months in % of patients, serology may be negative in a considerable proportion of acutely infected patients. anti-hev igg increase during the convalescent phase but it is not clear how long they persist. hev rna can be detected in stool about week before the onset of illness and persists up to weeks thereafter; serum viral rna can persist up to weeks in those who resolve the acute infection and for years in patients who develop chronic infection. the serological assays are easy to perform and relatively low expensive and several commercial and in-house elisa assays are available; however, due to the cross-reactivity with other viruses and to the genotype variability of hev, the sensitivity and specificity as well as performance of the serological tests are low and poor, providing inconclusive results. this is even more complicated in the case of immunosuppressed patients due to their delayed seroconversion upon hev infection. the detection of hev rna by pcr and rt-pcr is therefore needed to confirm serological screening in persistent infection, especially in blood and organ donations (al-sadeq et al. ). the aim of this review is to identify observational studies that show evidence of association between human anti-hev, anti-wnv and anti-cchfv antibody seropositivity (igg and/or igm) and certain occupational groups at risk of exposure. for this review, we included studies meeting the following eligibility criteria. r observational studies and case-reports (including crosssectional, seroprevalence, retrospective, case-control and case-report). r outdoor working population of all ages, sex and ethnic groups. r well-defined and quantitative information source for the selected etiologic agents: hepatitis e (he), west nile (wn), crimean-congo hemorrhagic fever (cchf) viruses. r outcome measures: seroprevalence of anti-hev, anti-wnv and anti-cchfv igg and/or igm among occupationally exposed populations. r studies on humans only. we excluded studies that did not report original results (reviews, letters and comments) or did not provide sufficient data (e.g. lack of information about the number of cases and controls or about the used method). exploratory studies were not included. studies were identified by searching electronic database (pubmed, january to october ) and scanning reference lists of articles (from reviews not included). the following medical subject headings (mesh) terms were used: occupational groups; occupational medicine; industry; occupational diseases; disease; employment; occupational health; occupations; workplace; occupational exposure; workload and work. when building the search syntax, for prompt identification of studies conducted in the occupational setting, we referred to the strings developed precisely for this purpose by mattioli et al. (mattioli et al. ) the choice of this strategy allows either to assess diseases, which produce only a few articles or to explore scarcely studied disease in more depth, that is the case of the present review on emerging viruses among occupational populations. two pairs of authors independently screened titles and abstracts of studies obtained by the search strategy. each potentially relevant study located in the search was obtained in full text and assessed for inclusion independently by the two groups. measure of inter-reviewer agreement was assessed via cohen's κ statistics (landis and koch ) . disagreements between authors were resolved by consensus. data were collected from each relevant study. extracted information included: r source (first author and year of publication); r general study details (citation, study design and year of publication); r setting (country/region considered, study population and job); r exposure measurement details (methodology including diagnostic tools used); r outcome data; r main findings. we reviewed the scientific literature to give an overview of the evidence available from the last years regarding the occupational risk of exposure to three emerging zoonotic viral infections: wnv disease, cchf disease and hepatitis e infection. reports or studies were original papers suitable for inclusion; therefore, full-texts were analyzed and the following information was extracted (as applicable to study): type of study, geographical location, study population (number of cases, patients, control or risk groups), antibody positivity rates of exposed and control subjects, statistical significance comparing risk groups vs. non-risk groups, preventive measures. a total of studies on wnv were collected and examined to determine if the inclusion criteria were met; were discarded because did not meet the criteria. two articles were reviews, therefore excluded; three studies whose abstracts were not found were also excluded. the full text of the remaining studies were searched and analyzed: one full text was not available, the remaining fully met the inclusion criteria and were included in the systematic review. see flow diagram fig. (moher et al. ). there was a significantly good measure for interreviewer agreement (cohen's k = . , p < . ). summary data of selected studies are in table . of the paper included in our review, were case report studies described in usa, brazil and south africa; the remaining were epidemiological studies conducted in turkey, italy, spain and south africa. we found that main transmission pathways were direct contact with animal's body fluids and indirect contact by infected mosquito bite. the first pathway was described in one veterinarian exposed to infected horse brain while performing an autopsy (venter and swanepoel ) , and in one laboratorist who acquired the infection after a needlestick injury that exposed her to cell-culture fluid containing wnv strain spu / (venter et al. ). wnv infection acquired by mosquito bite occurred in one security guard (smith ) and in one ranch worker (vieira et al. ) . both zoonotic transmission pathways were also responsible for infections in farmers, agricultural workers and veterinarians enrolled in the epidemiological studies included in the review, with serologic igg positivity ranging from . % (barzon et al. ) to . % (karakoç et al. ) . a total of studies on cchfv were identified for inclusion in the review; subsequently were excluded because did not meet the criteria. reviews and studies based on questionnaires were also excluded, abstract was not found for one study. full text of the remaining papers were analyzed and totally met the inclusion criteria; therefore, included in the review. see flow diagram fig. (moher et al. ). there was 'moderate measure' for inter-reviewer agreement (cohen's k = . , p < . ). summary data from selected studies are reported in table . of the paper included in our study, were case reports (from spain, saudi arabia, india, turkey, iran and russia), cross-sectional studies (from greece and madagascar), retrospective studies (from india and turkey), epidemiological studies (from turkey, afghanistan, ghana, tunisia, greece, south africa, iran, malaysia and saudi arabia). cchfv can follow different pathways while infecting humans: in the selected papers, worker's categories who acquired the virus by direct contact with infected animal's fluids or tissue included slaughterhouse workers with history of being splashed with fluids of animal viscera or of cutting their hands or other parts of the body (mofleh and ahmad ; akuffo et al. ; cikman et al. ; wasfi et al. ; mostafavi et al. ; vawda et al. ) , and butchers because of contact with infected meat (mofleh and ahmad ; mostafavi et al. ) . cchfv seroprevalence among slaughterhouse workers and butchers ranged from . % (vawda et al. ) to . % (mostafavi et al. ) . vector borne transmission of cchfv following infected tick bite was found in agricultural and animal husbandry, farmers (duran et al. ; sisman ) and in military personnel deployed in areas where the virus is endemic (memish et al. ; newman et al. ; mostafavi et al. ). human to human as well as nosocomial cchfv transmission may occur through percutaneous or permucosal exposure to blood or body fluids from infected subjects. health care workers (hcws) are at risk for contracting infection during patient care, as reported in papers included in the review (ergonul et al. ; mardani et al. ; mardani et al. ; naderi et al. ; gozel et al. ; guner et al. ; ozsoy et al. ; leblebicioglu et al. ; yildirmak, tulek and bulut ; negredo et al. ) . a probable cchfv transmission occurred in hcws after aerosol generating medical procedures in russia (pshenichnaya and nenadskaya ) . a total of studies regarding hev were identified for inclusion in the review. subsequently, studies were discarded because in univariate analysis serologic positivity in the high-risk group was more statistically significant than in the low-risk group ( % versus %, p = . ). in multivariate analysis, being in an occupational risk group (or = . , ci . - . , p = . ) was found to be a risk factor for wnv serologic positivity. did not meet the criteria. reviews and studies based on questionnaires were also excluded. for three studies the abstracts could not be retrieved and were not considered. the full text of the remaining studies were analyzed and fully met the inclusion criteria and were included in the review. see flow diagram fig. (moher et al. ). there was significantly good measure for inter-reviewer agreement (cohen's k = . , p < . ). summary data from selected studies are reported in table . the majority were epidemiological ( ) and cross-sectional ( ) studies from africa (uganda, nigeria, madagascar, ghana and burkina faso), asia (india, china, korea, indonesia, taiwan and thailand), europe (italy, germany, portugal, norway, finland, france, united kingdom, spain and netherlands), brazil and cuba. three were retrospective studies (from switzerland, italy and spain), two case studies (australia and spain) and one casecontrol study (china). in occupational settings zoonotic transmission of hev implies direct contact with swine, principal reservoir of hev or other animals (wild boar, deer). indirect contact in areas where animals live and roam or with objects or surfaces contaminated with hev stools is also considered a transmission route, as well as contact with pig and slaughterhouse sewage. articles selected in the review comprised mainly swine workers, including farmers and slaughterers, and veterinarians as occupational categories at risk of exposure to hev; to a lesser extent food handlers (appuhamy et al. ; cui et al. ), workers exposed to wastewater (tschopp et al. ; albatanony and el-shafie ; martins et al. ) and forestry workers (carpentier et al. ; dremsek et al. ) . seroprevalence data for anti hev igg ranged between . % for a group of abattoir workers in sardinia (masia et al. ) and . % among farmers in uk (meader et al. ) and . % among pig farmers in germany (krumbholz et al. ) and % among butchers in burkina faso (traoré et al. ) . significant risk factors for anti-hev igg positivity were age, amount of years of occupational exposure and direct swine contact. hunting has been considered a possible risk factor for acquiring hev infection, as reported in a study conducted in hunters from estonia that revealed the presence of hev-specific igg in . % of the samples (ivanova et al. ) . others found an anti-hev prevalence significantly higher in okinawa wild boar hunters ( . %) than in the residents (male . % and female . %) (p < . ) (toyoda et al. ). however, the retrieved studies were not included in this review because we did not consider hunters as an occupational category. infections continue to represent a global threat to human health. some emerging viruses with potential zoonotic transmission seem to pose a risk not only for the general population but also for workers in specific settings and activities. wnv, cchfv and hev were key topics of our review, which aims to identify possible association between occupational exposure and increasing risk for acquiring these infections. the search of pertinent articles has excluded sources other than pubmed, considered exhaustive for the aim of summarizing current available evidences of occupational risks for the three selected viruses. this is a limit of our study; however, this intends to be a pilot study in the field, suitable for more detailed research in the future. the number of patients with a history of contact with animals or animal blood was significantly higher than that in the control group (p < . ). the number of patients with a history of tick bites was higher than that in the control group but the difference was not statistically significant (p > . ). vawda et al. ( ) south epidemiological study animal husbandry workes; subjects exposed or bitten by ticks, healthy subjects not exposed to livestock or ticks about . % workers igg positive, . % subjects exposed or bitten by ticks and . % subjects not exposed. statistically significant difference between prevalence of cchf in livestock workers or subjects exposed to cchfv and those unexposed or residing in city (p < . ). high seroprevalence in erzincan, where the disease is endemic. educational and training programs targeted at high risk group should be developed and implemented. mohd shukri et al. cchf causes severe disease and has a mortality risk of about % in turkey. high-risk groups are working in agriculture and animal husbandry in rural areas, especially those living at an altitude of m or higher, in may, june and july. hcws also have a higher risk. more patients infected by contact with meat and body fluids died that those whose contact was through animal husbandry or ticks (p = . ). butchers are routinely exposed to the blood and other body fluids of animals, which suggest exposure to higher doses of the virus. semi-finished products processing workers and . %: less exposed group). age ( - years), working years ( - and > ), raw seafood processing workers and semi-finished products processing workers significantly associated with hev infection. no difference in seroprevalence between exposed and unexposed. symptoms compatible with hepatitis reported in / pig exposed. temmam et al. madagascar, cross-sectional study a total of slaughterhouse workers. . % significantly higher seropositivity for working years < (p = . ). egypt, na cross-sectional study a total of workers at wastewater treatment plants (wwtps) and not exposed workers. wwtps workers: %; not exposed workers: %. significantly higher seropositivity (p < . ) in wwtps than in not exposed. adjei et al. ( ) ghana, cross-sectional study a total of were swine exposed (feeding the pigs, cleaning barns, assisting the sows at birth and butchering on the farm). total . % ( . % anti-hev igg; . % anti-hev igm, p < . ). significantly higher seroprevalence (p < . ) among swine exposed in the same farm setting for < months than > months (or: . ; % ci: . - . ). significantly, higher seroprevalence in subjects with swine contact ( . - . %) compared with that in non-exposed humans ( . - . %). total . % workers with close swine contact and . % workers without swine contact. significantly higher seropositivity for age range of - years and - working years in pig farms (p = . ). highest anti-hev rate for swine contact and not keeping animal at home ( . %). carpentier et al. higher seroprevalence in game and fishing keepers and rangers ( . %) and in silviculturists ( . %) compared to controls (not statistically significant). woodcutters at higher risk ( . %; multivariate analysis: or: . (p = . )). silva et al. ( ) brazil, epidemiological study a total of swine exposed and blood donors. differences in positivity rates of anti-hev between the groups (p < . ). significantly higher positivity in pig farm than in slaughterhouse workers (p < . ), and both significantly higher than general population (p < . ). meader et al. ( ) u n i t e d kingdom, kingdom, , kingdom, , epidemiological study full-text articles excluded, (n = ) -reviews ( ) -no full text available ( ) studies included in qualitative synthesis (n = ) wnv transmission cycles through birds and mosquitoes, and mammals represent dead-end host of the infection, acquired through mosquito bites. the virus is able to amplify or replicate to high titre within birds, usually wetlands birds, which in turn transmit the infection to mosquitoes, primarily belonging to the culex genus. mosquitoes can reinfect birds, perpetuating enzootic infection, or can bridge the infection to mammals, humans and horses principally, representing a public health concern (ahlers and goodman ). pointing out environmental conditions that favor wnv circulation and transmission to humans is quite difficult, mainly due to the complexity of its biological cycle. factors contributing to the current epidemiological picture, characterized by an increasing rate of spread in europe and neighboring countries, are several and include urbanization, variation in land use and climate changes (marcantonio et al. ) . temporal extension of transmission season may increase the risk of exposure to the infection. in fact, in , early wnv transmission has been observed in italy and in other countries in south and south eastern europe, with a high number of cases (riccardo et al. ) . moreover, changes in daily work activities caused by increased heat, as longer rest periods in the middle records identified through database searching (n = ) of the day and augmented work at dawn and dusk, could correspond to period when vectors are most active, therefore increasing the risk of disease transmission (vonesch et al. ) . outdoor workers, such as farmers and agricultural workers, may be at increased risk of wnv infection. furthermore, workers in many other occupations could be at potential risk of exposure to wnv-infected humans and animals, their blood or other fluids and tissues. they comprise laboratory diagnosticians, researchers and technicians, veterinarians, wildlife rehabilitators, wildlife biologists, ornithologists, zoo and aviary curators, healthcare workers, emergency response and public safety personnel (niosh . few studies on the occupational risk caused by wnv have been performed; in fact, we identified only ten articles eligible for inclusion. the four case reports we included in the review concerned one us security guard who attended an off-site work event (smith ) , one brazilian ranch worker (vieira et al. ) , one veterinarian and one laboratorist, both in south africa, (venter et al. ; venter and swanepoel ) , all affected by the neuroinvasive illness that required hospitalization. in the first two cases, mosquito bites were responsible for the transmission of the virus; the veterinarian and the laboratorist acquired infection by needle stick injuries. the seroprevalence/seroepidemiological studies included in the study showed very low positivity for anti-wnv igg in italy and spain, from % (spataro et al. ; remoli et al. ) to . % (bernabeu-wittel et al. ) , mainly regarding farmers, agricultural workers, veterinarians and foresters. higher levels were reported in south africa ( . % for veterinarians) (van eeden, swanepoel and venter ) and turkey ( . % for farmers and agricultural workers). in south africa the virus is an endemic zoonotic agent and occurs where the principal records identified through database searching (n = ) additional records identified through other sources (n = ) records after duplicates removed (n = ) records screened (n = ) full-text articles assessed for eligibility (n = ) full-text articles excluded (n = ) -no full text available ( ) -reviews ( ) -no sufficient data (e.g. lack of information about occupational exposures, or about seroprevalence in exposed groups) ( ) studies included in qualitative synthesis (n = ) vector (culex univittatus) and avian host are present (van eeden, swanepoel and venter ). world health organization (who ) as well as experts in europe are calling for greater awareness of wnv infection, since the number of cases rises because of demographic, environmental and social factors (holt ) . wnv outbreak in animals precede human cases; therefore, an active surveillance system to detect cases in birds and horses is essential to provide early warning for veterinary and human health authorities. in italy entomological, veterinary and human surveillance systems for wnv infection have been implemented starting from , when the disease was first detected in horses in tuscany region. starting from , human cases have been reported in northeastern italy, an area now considered endemic for the virus: this is not unexpected since the geographical position of the country favors the distribution of arthropods as possible vectors of human pathogens. the few numbers of studies conducted in italian workers could be explained by the difficulty in recruiting workers at risk of exposure to the infection, since they are mainly seasonal, often foreigners and cultural and language barriers could limit their participation to the studies (remoli et al. ) . gloves and other protective clothing should be worn while handling sick animals or their tissues. physicians should be alerted to detect clinical cases and educational programs raising awareness about the disease and the risk factors should be implemented. healthcare workers caring for patients with suspected or confirmed wnv infection or handling their specimens, should implement standard infection control precautions; laboratorists should use effective personal protective equipment and apply biosafety measures (who ). vaccines are not yet available for humans; treatment is supportive for patients with neuroinvasive disease (who ) . it should be taken into account that although most wnv infections are subclinical, assessing the occupational risk is important not only for protecting workers' health, but also for providing information on the potential spread of the virus. cchf is the most widely spread tick-borne viral infection of humans and is endemic in extensive geographical areas comprising many countries in africa, southeastern europe, asia and the middle east. human infection can occur either by tick bites, mainly belonging to the genus hyalomma, or by direct contact with blood or tissues of viremic humans or livestock (sargianou, papa ) . in recent years, the incidence of cchfv infection has increased rapidly in countries of the world health organization eastern mediterranean region (who emr) and in central asia, probably due to a combination of biological, environmental and social factors, and enhanced awareness and diagnostic capability. weather conditions may influence the timing of activation and densities of ticks, being ectothermic organisms. migrating birds are sources of blood-meals for immature ticks, contributing to the dispersal of infected vectors and potential emergence of disease foci. long-distance movement of livestock may also contribute to dispersal of cchfv-infected ticks (al-abri ss et al. ; gargili et al ) . in endemic areas, farmers, veterinarians, livestock market workers, abattoir workers and other personnel engaged in activities in contact with animals and/or animal products are considered at risk for acquiring cchfv infection, as well outdoor workers who could be exposed to infected ticks. healthcare workers are at risk of exposure to the virus, when nursing infected patients with severe bleeding and hemorrhages without strict barrier procedures. nosocomial transmission may therefore occur through direct contact with infected blood or body fluids, or through contaminated medical equipment or supply (ecdc ) . case reports attesting cchfv transmission through direct contact with infected blood or tissue of animals regarded mainly farmers (yadav et al. ) and livestock workers (mardani et al. ; mardani, namazee ) . traditional slaughtering and butchery performed in some country (iran) can be considered activities at risk (fazlalipour et al. ) . most articles sharing this transmission pathway are seroprevalence studies, carried out in africa, asia and europe, and mainly regarding farmers, slaughterers, butchers and veterinarians (gunes et al. ; sidira et al. ; akuffo et al. ; cikman et al. ; wasfi et al. ; mostafavi et al. ; vawda et al. ) . the low seroprevalence for anti-cchfv igg antibodies ( . %) found in south africa among workers exposed to or in contact with animals seems to suggest that the virus is uncommon in this area (vawda et al. ) . the higher seroprevalence detected in iran ( . % among butchers and slaughterhouse workers) could be caused by the minimal use of personal ppe during daily work, as admitted by workers who completed a questionnaire (mostafavi et al. ) . a statistically significant difference between prevalence of cchfv igg antibodies in livestock workers and unexposed subjects was found in turkey. cchfv is endemic in central and north-eastern anatolia and southern black sea regions of this country and several cases are emerging in other zones (cikman et al. ) . a cross sectional study conducted in greece showed that an agro-pastoral occupation, contact with sheep and goats, tick bites and increasing age were significantly associated with cchfv seropositivity. another cross sectional study performed in madagascar (andriamandimby et al. ) showed that here the percentage of cchf infection is very low among at risk professionals because of the lack of ticks of the genera hyalomma in this country. four retrospective studies were conducted in india (mourya et al. ) and turkey (duran et al. ; guner et al. ; leblebicioglu et al. ) on patients with a history of occupational exposure, suspected to have cchfv infection, through a retrospective analysis of clinical and laboratory data. in endemic areas, hemorrhagic manifestations including melena, low platelet count and raised alanine aminotransferase may provide a suspicion of cchfv infection. in turkey, people living and actively working in rural areas (including housewives occupied in agriculture and animal husbandry) are particularly subjected to the infection. it was observed that public awareness about cchfv has decreased the incidence of the disease (duran et al. ) . nosocomial transmission of cchfv to hcws has been reported from different countries. the evidence that hcws are at risk of exposure to cchfv while caring infected patients is also supported by most case reports selected for the review (mardani et al. ; naderi et al. ; celikbas et al. ; ozsoy et al. ; pshenichnaya and nenadskaya ; yildirmak, tulek and bulut ; negredo et al. ). in the differential diagnosis of subjects with hemorrhagic signs, physicians should consider cchfv infection if these patients have recently returned from any area where the virus is endemic or prevalent. of interest the concern regarding transmission of cchfv via respiratory contact, as supposed by a case report from russia (pshenichnaya and nenadskaya ) and one from turkey (yildirmak, tulek and bulut ) , suggesting that airborne precautions could be essential during aerosol generating procedures. high mortality rate has been attested during nosocomial outbreaks; in some cases, ribavirin has been considered an effective treatment for the infection and could be used for postexposure prophylaxis (celikbas et al. ) . in our review, we included seroprevalence studies regarding seropositive hcws from turkey (gozel et al. ) , greece (maltezou, maltezos and papa ) and iran (mardani et al. ). needle-stick injury, interventions for gastrointestinal bleeding, unprotected handling of infected materials, and emergency surgical interventions have been reported as high-risk activities for viral transmission. military personnel that travel to and work in environments where they could be exposed to endemic or emerging infections, that are not present or prevalent in their native country, can be considered at high risk of contracting cchfv. we selected articles regarding afghan national army recruits (todd et al. ) uk military personnel deployed to afghanistan (newman et al. ) , and military units from saudi arabian provinces. in these groups, seroprevalences were . %, % and . %, respectively. cchfv infection has important public health implication due to the potential of humanto-human transmission; therefore, enhanced surveillance for tick vectors and cchfv cases is essential. control and prevention of the infection in ticks and animal is quite difficult since the tick-animal-tick cycle usually goes unnoticed and the infection in animals is usually not apparent. educational and training programs addressed to workers with potential exposure to the virus aiming at increasing their knowledge, attitude and practice should be developed and implemented as preventive measures. moreover, the use of approved acaricides on clothing and tick repellent on exposed skin and clothing, and wearing protective clothing are suggested for reducing the risk of tick to human transmission. wearing gloves and other protective clothing while handling animals or their tissues in areas where cchfv is endemic could minimize the risk of animal to human transmission (who ; ecdc ) . in healthcare settings, implementation of standard infection control precautions by healthcare workers caring for patients with suspected or confirmed cchfv infection or handling their specimens, should be recommended. hev is a major cause of epidemic viral hepatitis in developing countries and of sporadic and cluster cases in industrialized countries. according to the who, approximately one-third of the world population has been exposed to hev, through the ingestion of contaminated water and food or the direct contact with infected animals, and in minor cases by blood-borne transmission (sinakos et al. ) . serological evidence of prior exposure to the virus has been found in most areas, with higher seroprevalence rates (proportion of people who test positive for igg antibodies to hev) in regions with lower standards of sanitation and thus higher risk for transmission. however, presence of these antibodies does not imply presence or increased risk of disease. traditionally, industrialized countries were considered nonendemic, with most hev infections in these regions being sporadic and considered to be imported. nevertheless, in recent years, enhanced surveillance has detected an increasing number of non-travel-associated hev infections. genotypes and of hev are distributed worldwide, have a much wider host range and are considered to be zoonotic: hev- is the principal genotype circulating in commercial swine herds, hev- , typical of the asian continent, is believed to have recently introduced in europe (hakze-van der honing et al. ; monne et al. ) . since the high pathogenicity of genotype , other studies should be performed to better understand to which extent this genotype has spread across europe. the hev- and hev- genotypes circulate also in europe and they have a high level of nucleotide identity between swine and human strains (di bartolo et al. ) . in the present review, available studies were carried out in africa, asia, europe and latin america. recently, piggish reservoirs and growing evidence of zoonotic transmission of hev have been reported in these countries, suggesting the possibility of occupational transmission to humans. exposed groups comprise swine farmers (organized-mixed feed feeders and unorganized-swill feeders), slaughterhouse workers, sewage workers and veterinarians (bansal et al. ) . in this review, an increased risk was found also among food handlers (appuhamy et al. ; cui et al. ), workers exposed to wastewater (tschopp et al. ; albatanony and el-shafie ; martins et al. ) and forestry workers (carpentier et al. ; dremsek et al. ) . seroprevalence results were higher in individuals exposed to swine and/or wild animals, and increased with age and amount of years of occupational exposure. humans with occupational exposure to wild animals and environmental sources of domestic animal wastes or with unexplained hepatitis showed an increased seroprevalence of anti-hev antibodies. poor environmental conditions in farms, occupation and low socioeconomic status might be risk factors in hev infection. wild boar stools may be responsible for a further source of hev infection for people in close contact with the forestry environment. forestry workers have already been identified to be at risk for hev infection, as well as woodcutters. the finding of hev and hev-related rna in a rising number of different animal species suggests a possible role for unidentified animal reservoirs, up to now as risk factors associated with hev seropositivity in humans in areas where hev is not endemic. such reservoirs should be further explored by means of suitable diagnostic tools (carpentier et al. ) . the fact that some european countries, such as germany, have classified hepatitis e as a notifiable infectious disease for several years and therefore have well defined records, whereas other countries, for example italy, started in , could explain difference in prevalence found in countries with a similar socioeconomic and health status (masia et al. ). moreover, variations in seropositivity rates reported in studies from around the world, could be ascribed by the use of immunological assays with different sensitivity (meader et al. ) . the usefulness of such data for epidemiological purposes may also be limited due to variable and possible sub-optimal performance of available serological assays, and possible disappearance of the antibody with the passage of time among those exposed to the virus. at population level, transmission of hev and hepatitis e disease can be reduced by maintaining quality standards for public water reserves and proving appropriate removal systems for human feces. at individual level, infection risk can be diminished by maintaining hygienic habits such as hand washing with safe water, particularly before handling food; preventing consumption of water and/or ice of unknown pureness and adhering to who safe food procedures (who ). standard biosecurity measures, including regular cleaning and disinfection, should be put in place to limit contamination of swine facilities. a vaccine against hev, licensed in china in , prevent symptomatic hev- infections, but does not provide sterilising immunity. the vaccine seems to be safe in pregnant women, but the long-term efficacy in immunosuppressed and in subjects with chronic liver disease has to be determined. an important role of the vaccine could be the prevention of hev outbreaks, e.g. in african refugee camps or other emergency settings (easl ). in the absence of an effective vaccine against hev, prevention for swine workers, farmers, butchers and veterinarians relies on the 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indonesia with identification of swine hev genotype nosocomial infection of cchf among health care workers in rajasthan crimean-congo hemorrhagic fever in migrant worker returning from oman to india crimean-congo hemorrhagic fever: transmission to visitors and healthcare workers hepatitis e virus (hev) seroprevalence in the general population of the republic of korea in - : a nationwide cross-sectional study none declared. key: cord- -qqhgmqrg authors: nan, yuchen; zhang, yan-jin title: molecular biology and infection of hepatitis e virus date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: qqhgmqrg hepatitis e virus (hev) is a viral pathogen transmitted primarily via fecal-oral route. in humans, hev mainly causes acute hepatitis and is responsible for large outbreaks of hepatitis across the world. the case fatality rate of hev-induced hepatitis ranges from . to % in young adults and up to % in infected pregnant women. hev strains infecting humans are classified into four genotypes. hev strains from genotypes and are zoonotic, whereas those from genotypes and have no known animal reservoirs. recently, notable progress has been accomplished for better understanding of hev biology and infection, such as chronic hev infection, in vitro cell culture system, quasi-enveloped hev virions, functions of the hev proteins, mechanism of hev antagonizing host innate immunity, hev pathogenesis and vaccine development. however, further investigation on the cross-species hev infection, host tropism, vaccine efficacy, and hev-specific antiviral strategy is still needed. this review mainly focuses on molecular biology and infection of hev and offers perspective new insight of this enigmatic virus. hepatitis e virus (hev) is a positive-sense, single-stranded rna virus, and is classified in the genus orthohepevirus, the family hepeviridae (smith et al., ) . the hev-caused hepatitis e is generally a self-limiting disease with a case fatality rate from . to % in young adults but up to % in infected pregnant women in their third trimester of gestation (jameel, ; nan, ) . world health organization (who) estimates that there are million infections with over million symptomatic cases and , deaths annually across the world (who, ) . hev is primarily transmitted via fecal-oral route. hev infection was previously thought to be a public health problem only for the developing countries. indeed, hepatitis e is highly endemic in east and south asia, as well as africa according to the who (who, ) . hev strains infecting humans are classified into four genotypes. hev strains from genotypes and are zoonotic, whereas, those from genotypes and have no known animal origin. discovery of hev from swine and other species suggests that genotypes and hev has a wide host range (christensen et al., ; dalton et al., ; meng, ; pavio et al., ) . currently, hepatitis e is frequently recognized in industrialized countries, where it was not thought to be endemic (kwo et al., ; erker et al., ; schlauder et al., ; worm et al., ; kabrane-lazizi et al., ; mizuo et al., ; sadler et al., ) . moreover, along with isolation of hev from the pig, chicken, mongoose, rabbit, rat, ferret, bat, fish, and deer (meng et al., ; haqshenas et al., ; li et al., c; cossaboom et al., ; smith et al., ) , cross-species infection of hev from animal reservoirs to humans is thought to be the major cause of sporadic cases of hepatitis e in the industrialized countries (pavio et al., ) . although previously thought to only cause acute infections, hev is found in chronic infections reported both in immune compromised and immunocompetent individuals (hoofnagle et al., ; grewal et al., ) . in addition, extrahepatic manifestations, such as neurological disorders and kidney injury in hev infected patients have been documented (kamar et al., (kamar et al., , b van eijk et al., ; dalton et al., ; geng et al., ) . taken together, current knowledge for hev implies a significant underestimation of hev infection as a public health concern. in the following sections, recent progress in hev biology, functions of viral proteins, cell culture system, epidemiology, viral pathogenesis, treatment, and vaccine development are reviewed and perspective new insights are discussed. hepatitis e was initially designated as enterically transmitted non-a, non-b hepatitis (et-nanbh) due to similar clinical presentations to hepatitis a and b in patients, but the prospective causative agent was initially unknown (balayan et al., ) . early research implied that an rna virus was the potential pathogen for the et-nanbh. by analysis of a cdna library from infectious bile sample, a portion of a highly conserved rnadependent rna-polymerase (rdrp) motif, commonly found in rna viruses, was identified (reyes et al., ). this new virus was designated as hev, which was responsible for the outbreak of et-nanbh. the complete sequence of hev genome was published year later (tam et al., ) . sequence analysis indicated that hev contains a . kb single-stranded positive-sense rna genome, which is capped and poly-adenylated (ahmad et al., ) . there are three partially overlapped open reading frames (orfs) in an order of sequences encoding non-structural proteins (nsps) followed by structural protein (tam et al., ; tsarev et al., ; figure ) . hev orf encodes a non-structural polyprotein that consists of replicase proteins needed for hev replication. orf encodes the capsid protein, which is the major structural protein of the hev virions, which are non-enveloped particles of - nm in diameter (mori and matsuura, ) . orf encodes a small multifunctional protein with a molecular mass of kda (vp ). there are also short untranslated regions (utrs) in both the and -end of the genome. recently, an orf was identified from genotype hev solely ; figure ). expression of orf is cap-independent and driven by a putative ires-like element between and nt of the hev genome . the orf of hev can be translated directly from the genomic rna, whereas orf and orf are translated soly from the sub-genomic rna in alternative frames (graff et al., ) . in an earlier report, three rna species were detected in liver tissue of experimentally infected macaques, with sizes of . , . , and kb (tam et al., ) . the . and kb rna species were thought to be sub-genomic rnas for translation of orf and orf , respectively. however, a later study in huh cells only identified one capped . kb sub-genomic rna, which is a bicistronic mrna for translation of both orf and orf (graff et al., ) . transcription of this sub-genomic rna initiates at nucleotide position in the sar strain, which is located downstream of the first two methionine codons of the initially presumed orf . the same conclusion was drawn from another in vitro study of genotype hev infection in plc/prf/ hepatoma cells . the hev genome contains two cis-reactive elements (cres) that are essential for the viral replication (cao et al., ; parvez, b) . the first cre overlaps the end of orf and the utr and is essential for hev replication. the second cre locates in the intergenic region of the hev genome and forms a stem-loop structure that may be the promoter for synthesis of the . -kb subgenomic rna (cao et al., ) . hepatitis e virus was initially classified as a member of the caliciviridae family. however, sequence analysis of hev orf indicated no similarity to caliciviruses, or other picorna-like viruses. on the other hand, there is limited but significant similarity to the alphavirus-like superfamily of rna viruses, specifically, the rubella virus (berke and matson, ) . consequently, hev was classified into the family hepeviridae (berke and matson, ; emerson and purcell, ) . although hev strains are highly diverse and heterogenic, only one serotype of hev exists. classification of hev strains is under transition due to the different criteria used (smith et al., (smith et al., , . recently, a new proposal for the classification of the family hepeviridae was published (smith et al., ) . in this proposal, the family hepeviridae contains two genera: orthohepevirus (all mammalian and avian hev isolates) and piscihepevirus (trout hev; table ). within the genus orthohepevirus, four different species (a, b, c, d) are designated to include isolates from different hosts (smith et al., ) . all four previously recognized hev genotypes ( - ) that infect humans belong to the orthohepevirus a virus (smith et al., ) . the previously recognized hev genotypes - classification system was based on complete genomic sequences (lu et al., ) . hev genotype is the most conserved among the four genotypes. there is only one full-length genotype sequence available (smith et al., ) . both hev genotypes and are restricted to humans with no known animal reservoirs, whereas genotypes and are zoonotic with an expanded host range ( table ; meng, ; ahmad et al., ) . therefore, genotypes and hev strains are highly diverse (lu et al., ; smith et al., ) . since the constant discovery of new hev or hev-related isolates from rabbit, rat, ferret, bat, moose, farmed mink, camel, and wild boar (lorenzo et al., ; zhao et al., ; johne et al., ; geng et al., ; takahashi et al., ; drexler et al., ; raj et al., ; krog et al., ; lin et al., ; woo et al., ) , four genotypes are no longer satisfying classification of expanding hev isolates. in the new classification system, some hev strains from wild boars in japan with unique viral nucleotide sequences are designated as genotypes and , while hev from camel is classified as genotype of orthohepevirus a (smith et al., ) . figure | schematic illustration of hepatitis e virus (hev) genome, subgenomic rna, and orfs. orf (nt - ) is labeled above the genomic rna box. orf (nt - ) and orf (nt - ) are encoded by the same subgenomic rna. the newly identified isre like sequence (nt - ) and orf (nt - ) which are overlapped with orf are listed as well. moreover, the numbers above or below the rna boxes indicate nucleotide numbers of the cdna of hev sar (genbank accession number af ) genomic rna. the genotypes that infect humans include , , , , and ( table ; smith et al., ) . hepatitis e virus-like virus isolated from avian species is called avian hev, which shares less than % nucleotide identity but common antigen epitopes in the capsid protein with mammalian hev (haqshenas et al., (haqshenas et al., , huang et al., ) . currently, avian hev is classified into the species orthohepevirus b (smith et al., ) . hev strains from rat, ferret and bat are classified into the species of orthohepevirus c and d, respectively (smith et al., ) . the cutthroat trout virus (ctv) is identified as an hev-like virus in retrospective studies. ctv shares even lower sequence identity with mammalian and avian hev and is now classified as a member of the genus piscihepevirus (batts et al., ; smith et al., ) . for the four genotypes ( - ) of hev infecting humans, there are differences in their geographic distributions. genotype hev mainly includes strains from asia and africa including the sar isolate, while genotype contains a mexican strain and variants from africa. genotypes , including human and swine hev, is mainly found in the industrialized countries (purcell and emerson, ) . the genotype is previously thought to be found only in china (purcell and emerson, ) , however, recent reports show that genotype hev strains are also isolated in other countries, including india, indonesia, japan, vietnam, spain, france, and italy (okamoto, ; midgley et al., ; lapa et al., ) . for details about molecular epidemiology and viral evolution of hev, please refer to the article by purdy and khudyakov ( ) . the orf is the largest orf in the hev genome and has nt in length according to the sar strain (tsarev et al., ; emerson et al., ) . it starts at the end of the genome after a nt non-coding region and can be translated directly from the hev genome. orf encodes a amino acid (aa) polyprotein, which is needed for hev replication. bioinformatics analysis for the protein sequence encoded by orf found eight putative domains according to their similarity to counterparts in the other viruses . moreover, the orf sequence is highly related to the group of rubi-like viruses including rubivirus, betatetravirus, benyvirus, and omegatetravirus (koonin and dolja, ; liu et al., ). these functional domains include methyltransferase domain (met), y domain (y), papain-like cysteine protease (pcp or plp), hypervariable region (hvr), proline-rich region (pro), x domain, helicase domain (hel) and rdrp (figure ) . in recent publications, the proline-rich region is frequently named together with hvr as the hvr. the current data are conflicting about whether the hev orf product functions as a single polyprotein or needs to be further processed into smaller units by viral or cellular proteases ropp et al., ; sehgal et al., ; suppiah et al., ; perttila et al., ) . one study using a vaccinia-derived expression system demonstrated that the orf polyprotein could be cleaved by the pcp within it (ropp et al., ) . more than years after that publication, the same group showed a lack of processing of the orf polyprotein in hek t cells (suppiah et al., ) . two fragments were found in another study on in vitro translation of full-length orf , but they were not observed in pulse-chase assay in human cells and their production was not dependent on the predicted protease domain in orf product (perttila et al., ) . furthermore, in escherichia coli and a cell-free system based on hepg cells, orf was expressed as a kda protein without further processing detected . on the other hand, other studies demonstrated contrasting results. transfection of hepg cells with in vitro transcribed rna from hev cdna produced cleaved products with sizes of , , and kda for the met, hel, and rdrp domains, respectively (panda et al., ) . another study focusing on the analysis of the orf functional domains also observed proteolytic processing of the hev orf fragment in insect cells (magden et al., ) . in a later study, the orf product expressed in insect cells by baculovirus expression system was shown to exist as smaller fragments and this proteolytic processing could be inhibited by e- d, a cell-permeable cysteine protease inhibitor (sehgal et al., ) . a recent publication reported that the refolded pcp domain expressed in e. coli is able to process orf polyprotein in vitro (paliwal et al., ) . moreover, based on an hev-sar replicon system in s - cells (a subclone of huh cells with improved hev replication; graff et al., ) , the putative catalytic aa residues in the orf protease domain are indispensable for hev replication (parvez, ) . overexpression of orf from hev sar strain in s - cells also resulted in cleaved products (parvez, ) . thus, despite the lack of conclusive data, the majority of studies so far are in favor of the polyprotein proteolysis. the cleaved orf products could be possibly detected in the hevinfected cells if effective and specific antibodies against the domains are available. a recent study employing yeast two hybrid (y h) demonstrated intraviral interactome within the domains from orf , further supporting the proteolysis of orf polyproteins (osterman et al., ) . moreover, orf could be a determining factor for host tropism as a recombinant hev harboring orf of a genotype hev strain and the rest genome from genotype strain replicates in transfected porcine kidney cells (chatterjee et al., ) . therefore, orf products involve in determination of hev host tropism and should be further investigated. it is possible that proline rich region or hvr may be involved in host tropism determination since other domains are more conserved among the four hev genotypes. the met domain is the first one at the n-terminus of the orf -encoded polyprotein. as the hev genome is capped and the capping is crucial for its infectivity, a viral-specific frontiers in microbiology | www.frontiersin.org methyltransferase was expected zhang et al., ) . based on sequence analysis, the region in aa residues - was assumed to be a putative methyltransferase . the hev met domain is similar to that of tricornaviruses, which belong to the alpha-like supergroup of rna viruses (van der poel et al., ) . there are an invariant his residue, an aspxxarg signature and an invariant tyr residue in methyltransferase motifs i, ii, and iv, respectively . expression of hev orf cdna (aa residues - ) in insect cells yields a kda protein (p ), along with a kda protein that is believed to be the proteolytic product of p (magden et al., ) . in vitro assays shows that the p possesses guanine- -methyltransferase and guanylyl transferase activity (magden et al., ) . the second domain after the methyltransferase is the y domain, which is assumed to start from aa residue and ends at aa . it is highly similar to that of the rubella virus . currently, there is no information available for the function of this y domain in either hev or the rubella virus. papain cysteine protease domain is downstream of the y domain. the pcp domain demonstrates moderate similarity to the protease domain in the rubella virus . in the rubella virus, the pcp domain is responsible for the proteolytic processing of its nsp (marr et al., ) . mutation of the catalytic residue within the pcp (cys ) abolishes its protease activity and results in inhibition of the nsp processing. it is also involved in trans and cis cleavage of the rubella virus nsp (liang et al., ) . however, regarding the function of the hev pcp domain, the current data are incomplete and controversial. in the vaccinia-mediated orf expression system, mutation of the putative catalytic core (cys ) of hev pcp had no effect on proteolytic processing of the orf product (ropp et al., ) . another putative catalytic site of his in pcp is not conserved among different hev strains. later studies showed controversial data for the processing of the hev orf product ropp et al., ; sehgal et al., ; suppiah et al., ) . this leads to the speculation that whether hev pcp is a real cysteine protease. recently, parvez demonstrated that the mutation of six cystine residues (c a, c a, c a, c a, c a, c a) and three histidine residues (h l, h l, h l) in the pcp domain completely abolished hev rna replication in a sar -based replicon system in s - cells. notably, of these essential cys and his residues, c and h were previously predicted as putative catalytic residues in the pcp domain (parvez, ) . furthermore, the pcp domain expressed in the e. coli c strain (resistant to toxic protein expression) possesses protease activity (paliwal et al., ) . the purified protein cleaves both hev orf and orf products that are in vitro translated. protease inhibitor assay indicates the hev pcp domain is a chymotrypsin-like protease (paliwal et al., ) . this observation suggests that hev pcp is a real protease for hev orf polyprotein processing. in recent years, the connection between ubiquitination and innate immunity signaling has been demonstrated (zeng et al., ; mao et al., ; liu et al., ) , and the antiviral function of some ubiquitin-like molecules, such as interferon-stimulated gene (isg ) and small ubiquitin-like modifier (sumo), has been described (liu et al., ) . some studies indicate that viral coded cysteine proteases possess deubiquitinase activity to inhibit host innate immunity, such as arterivirus papain-like protease (van kasteren et al., ) and pcp from porcine reproductive and respiratory syndrome virus (prrsv; li et al., ; sun et al., ) . similar research performed on the hev pcp domain suggests that it acts as an antagonist to isg function to inhibit host innate immunity when expressed together with met as the met-pcp protein (karpe and lole, ) . moreover, study from our laboratory demonstrated that the pcp domain from hev genotype sar strain is able to inhibit ubiquitination of rig-i and tbk , therefore resulting in the inhibition of rig-i mediated signaling in innate immune responses (nan et al., b) . between the pcp domain and the x domain, there are hvr and pro domains. these two regions were first named as hvr due to the extreme divergence in sequence between nt and (corresponding to residues aa - ) when the hev sar was compared with two other strains (tsarev et al., ) . in a later study, aa - in this region were designated a prolinerich region, which could be found in rubella virus as well. it was also considered to serve as a hinge between the x domain and its upstream domains because multiple proline residues in a protein or polypeptide may result in an unstable tertiary structure tsai et al., ; dosztanyi et al., ; dunker et al., ) . the length and sequence of hvr and pro is highly variable among different hev strains (pudupakam et al., ; smith et al., ) . currently, there is some confusion regarding the nomenclature of those two regions. some of the recent publications designated the region of aa - as the hypervariable domain, which was originally referred to proline-rich region and left out the immediately upstream domain (aa - ; pudupakam et al., pudupakam et al., , , whereas others still designate the aa - as the proline-rich region (purdy, ) . current research mainly focuses on the pro region and pays less attention to the upstream hvr domain. as a result, the function of hvr is unknown. however, data gained from the rubella virus shows that deleting part of the hvr domain along with part of the pro region renders the mutant non-viable (tzeng et al., ) . the pro domain is considered to be an intrinsically disordered region (idr) with flexibility for insertion and deletion (purdy, ; purdy et al., ) . data from its counterpart in the rubella virus indicates that this region is not required for viral replication (tzeng et al., ) . as expected, deletion and mutation of this region in hev indicates that it is not required for viral replication and infectivity, but it plays a role in replication efficiency in vitro (pudupakam et al., (pudupakam et al., , . it was also demonstrated that the pro domain is interchangeable between genotypes with genotype-specific differences (pudupakam et al., ) . more interestingly, a remarkable hev strain kernow-c , which was originally isolated from an hiv-positive patient with chronic hev infection, contains an insertion of a nt gene fragment of human ribosomal protein s in the pro region (shukla et al., ) . this recombinant virus was adapted in culture cells and is able to propagate in cells from different species. it was speculated that insertion of the s fragment occurred in the host but was selected in cultured cells. this speculation needs further verification as direct detection of the inserted fragment from the host sample was not successful. experimental insertion of the s fragment into the pro domain of the sar strain also generated a viable chimeric virus (shukla et al., ) . the s sequence insertion in hev correlates with novel nuclear/nucleolar trafficking capabilities to the orf protein of hev kernow c- p and the enhanced replication of this strain (kenney and meng, a,b) . although the pro domain is considered highly diverse, some motifs are found in the idr. based on computer analysis and comparison with other idrs, purdy et al. identified several linear motifs (lms), including two protease cleavage sites, three ligand binding sites and two kinase phosphorylation sites across all four genotypes . the putative protein-protein interactions of the pro domain were proposed in the same report as well, but need experimental verification. nevertheless, this report provides some assumptions about the disorder-to-order state of the pro domain. in another study, alignment of the pro domain from different genotypes indicated the sequence is more conserved in genotypes and than genotypes and (purdy, ) . adaptation to a wide host range for genotypes and is a possible reason. the authors also assessed the diversity of the pro domain due to the higher rate of substitutions at the first and second codon positions, leading to a shift in translation to be more proline, alanine, serine, and threonine rather than histidine, phenylalanine, tryptophan, and tyrosine. this pattern matches the aa usage in proline-rich idrs (purdy, ) . furthermore, the c-terminus of this domain can tolerate more mutations than the n-terminus. recently, the heterogeneity of the pro and x domains is implicated in hev persistence, which was revealed in an investigation into the association between the genetic heterogeneity of hev quasispecies in orf and the outcome of infection in solid-organ transplant patients ). the x domain is located immediately downstream of the pro domain. in hev, its function is unknown. the hev x domain homologs in other viruses such as rubella virus, alpha virus and coronavirus, are commonly identified as domain flanking the pcp domain (gorbalenya et al., ; koonin et al., ) . it is also known as macro domain, due to its similarity with non-histone domain of the histone macroh a. macro domain has been identified in a variety of bacterial, archaeal, and eukaryotic organisms (pehrson and fried, ; pehrson and fuji, ) . early studies of the human macro domain indicate that it is enriched in inactive mammalian x chromosomes, suggesting a role in gene silencing and inactivation (costanzi and pehrson, ) . the macro domain inhibits transcription and binds to the transcription activator nf-κb (perche et al., ; angelov et al., ) . crystal structure analysis identifies a dna binding motif in the macro domain, suggesting that it might interact with nucleic acids (allen et al., ) . a biochemical functional analysis indicates that the macro domain is involved in the downstream processing of adp-ribose -phosphate, a side product of cellular pre-trna splicing (martzen et al., ) . furthermore, the macro domain is found in association with proteins involved in poly(adp-ribose) polymerization, adpribosylation and atp-dependent chromatin remodeling (aguiar et al., ) . information about the function of viral macro domains is limited. adp-ribose -phosphatase activity has been demonstrated in the macro domain from coronavirus (martzen et al., ; putics et al., putics et al., , saikatendu et al., ) . crystal structure analysis and in vitro assays on the macro domain of the sars virus indicate that the viral macro domain has relatively poor adp-ribose -phosphohydrolase activity, but can bind free adp-ribose and poly(adp-ribose) efficiently (egloff et al., ) . in another report, the macro domains from semliki forest virus, hev, sars virus and yeast were compared with the human macro domain (neuvonen and ahola, ) . the viral macro proteins bind poly (adp-ribose) and poly (a), but have a low affinity for monomeric adp-ribose. this implies that viral macro domains are functionally different from human homolog and may participate in cellular pathways involving rna rather than adp-ribose derivatives. however, a recent study shows that viral macro domains (hev, coronavirus, and venezuelan equine encephalitis virus) can reverse protein adp-ribosylation by acting on adp-ribosylated substrates through the hydrolytic activity of their macro domains (li et al., ) . furthermore, other studies indicate that the expression of viral macro domain in liver cells inhibits apoptosis since it is functionally related to poly(adp-ribose) polymerase- (parp- ; allen et al., ; chen et al., ) , suggesting a role in apoptosis during viral infection. recently, a highly conserved "glycinetriad" (gly -gly -gly ) was identified downstream of the macro domain of hev, which is homologous to the rubella virus protease-substrate (g -g -g ; parvez, ) . mutagenesis study indicates that g v and g v mutations in the macro domain are lethal for sar replication in s - cells. further analysis identified the n-terminus residues asn , asn , his , gly -gly -and gly formed a potential catalytic-site homolog of coronavirus adp-ribose- -monophosphatase, which has essential role in viral replication (parvez, a) . as mentioned above, a recent report suggests that the quasispecies heterogeneity in the macro domain might facilitate hev persistence in solid-organ transplant patients . study from our laboratory demonstrates that x domain of hev sar strain inhibits the phosphorylation of irf , which is a key transcription factor for type i ifn induction (nan et al., b) . moreover, except interacting with light chain subunit of human ferritin and inhibiting ferritin secretion, the x domain interacts with hev met and vp ojha and lole, ) . the rna helicase domain is downstream of the x domain. it is encoded by many positive-stranded rna viruses and is essential for their replication (kadare and haenni, ) . helicases are motor proteins that are able to unwind nucleic acid strands by using energy from atp hydrolysis (kadare and haenni, ) . helicases can be divided into six superfamilies (sf - ; singleton et al., ) . rna virus coded helicases are mainly classified into sf and sf . helicases sf and sf contain seven signature motifs (i, ia, ii, iii, iv, v, and vi) that form the core of the enzyme (kadare and haenni, ) . the hev helicase belongs to helicase superfamily sf and is proposed to possess both ntpase and rna unwinding activities kadare and haenni, ) . in vitro experiments demonstrate that the hev helicase purified from e. coli expression has both of the activities. it drives the hydrolysis of rntps but also dntps at a lower efficiency, as well as unwinds rna duplexes with overhangs (karpe and lole, a) . rna -triphosphatase activity has also been observed in the hev helicase domain, which is proposed to function along with methyltransferase for catalyzing rna capping (karpe and lole, b) . recently, a mutagenesis study on hev helicase demonstrated that motifs i, iv, and vi are dispensable, while motifs i and iii are crucial and unique for hev helicase function (mhaindarkar et al., ) . a recent study shows that a v a substitution in the helicase domain of a swine hev strain is potentially associated with increased virulence (ward et al., ) . however, no human infection was reported to be associated with this strain. the last domain of hev orf polyprotein is the rdrp. all positive-stranded rna viruses code an rdrp, which is necessary for viral replication (o'reilly and kao, ) . the rdrp from all positive-sense rna viruses are classified into three large supergroups. all rdrp domains contain approximately amino acid residues, with the central and c-terminal parts showing high similarity between each other (koonin, ) . rdrp from hev belongs to supergroup iii and has the highest similarity to the domains in rubella virus and beet necrotic yellow vein virus (bnyvv; koonin et al., ) . all eight conserved motifs can be found in hev rdrp, including an mg + binding sequence (gdd), which is essential for rdrp activity. the purified hev rdrp is able to bind the end of hev rna, and needs two stem-loop structures at the end of the poly(a) stretch for this binding (agrawal et al., ) . expression of the rdrp in mammalian cells as a gfp fusion protein indicates that it localizes in endoplasmic reticulum (er), which could be a potential replication site for hev (rehman et al., ) . recent studies indicate that the emergence of g r mutation in the hev rdrp is possibly due to ribavirin-induced mutagenesis and is associated with treatment failure of ribavirin monotherapy in solid-organ transplant patients (debing et al., b; lhomme et al., ; todt et al., ) . the capsid protein is the major component of hev virions. orf is nt in length beginning from nt downstream of orf and ending at nt upstream of the poly-a tail (reyes et al., ; figure ) . the deduced full-length orf product has aa residues with a predicted molecular mass of kda (robinson et al., ) . recombinant orf protein can bind to the region of hev genome . it was first shown that the orf product exists as an kda protein, which carries n-terminal linked glycans and a potential er-directing signal about aa from its n terminus (jameel et al., ) . this kda protein can be further processed and has the potential to form non-covalent homodimers. a further study from the same group demonstrated asn in orf product to be the major site for glycosylation (zafrullah et al., ; figure ) . a mutagenesis study indicated that the n-terminal signal peptide is required for its cell surface expression via er transition, but glycosylation of the capsid protein is not required (zafrullah et al., ) . since glycosylation of the capsid protein in non-enveloped viruses is not common, it is not known whether these modifications have biological significance for hev infection. mutations in the putative glycosylation sites (aa , aa , aa ) prevent formation of viral particles and infection of rhesus macaques but without an effect on genome replication in cells (graff et al., ) . mutation in the first two glycosylation sites prevents virion assembly, while mutation of the third site allows virion particle formation and rna encapsulation (graff et al., ) . hev particles released from cultured cells have a lipid component (quasi-enveloped), which can be removed by detergent treatment (qi et al., ) . the relationship between glycosylation of orf and lipid envelope of viral particles needs to be clarified. on the other hand, data acquired from studies using insect cells provide a different conclusion regarding orf expression and processing. when expressed in insect cells, orf product can be an insoluble full length protein of about kda, and a soluble form of . kda, a processed product of the intact form (mcatee et al., ) . another group shows that when orf is expressed in sf cells, a kda product is detected while lacking the first aa residues of the putative orf polypeptide (zhang et al., ) . further studies in two different insect cell lines (sf and tn ) show a soluble form of orf product with a molecular mass of kda, which lacks the first aa and the last aa of orf polypeptide but retains the ability to form virus-like particles (vlps; li et al., li et al., , d . vlp assembly is thought to involve dimer formation, and the c-terminus of the recombinant orf protein is believed to be responsible for homo-oligomerization (tyagi et al., a; xiaofang et al., ; li et al., a) . a . -Å resolution crystal structure obtained from hev vlp indicates that the truncated hev capsid protein has three domains designated as s (shell, aa - ), m (middle, aa - ), and p (protruding, aa - ; yamashita et al., ; figure ) . the vlp is composed of subunits of the truncated capsid protein, forming icosahedral , , and -fold axes (yamashita et al., ) . mutational analyses indicate that the protruding domain is involved in binding to the susceptible cells and contains neutralization epitopes (yamashita et al., ) . moreover, the hev vlp can be used as a delivery system to display foreign epitopes on its surface (xing et al., ) . the orf product expressed in insect cells is reactive with anti-hev antibodies (tsarev et al., ) . genetic analysis of orf showed over % similarity among the four major hev genotypes in mammalian hosts (mori and matsuura, ) . amino acid alignment indicates that divergences are mainly in the first aa of the n terminus, which is not a component of the virions (mori and matsuura, ) . a study manipulating a phage display system for overlapping peptides and truncated orf proteins maps the major neutralizing domain to residues - , which matches the location of the p domain (schofield et al., ; meng et al., ; zhou et al., ) . both conformational and linear neutralizing epitopes have been identified from the hev capsid protein tang et al., ) . these data provide valuable information for vaccine development. the orf truncated proteins generated by baculovirus or bacterial expression systems have been tested in clinical trials (shrestha et al., ; zhu et al., ) . however, a recent study that evaluated cross-protection against heterologous hev indicates that vaccination of pigs with truncated capsid proteins derived from swine, rat and chicken hev only elicits partial protection against a genotype mammalian hev (sanford et al., ) . study of avian hev capsid protein indicates that the n-terminal aa residues react with swine and human anti-hev sera (wang et al., b) . moreover, a recent study suggests that antigenic composition and immunoreactivity differed between hev recombinant capsid proteins from different genotypes (behloul et al., ) , which raises the concerns about the efficacy of the current hev subunit vaccine marketed in china. on the other hand, experiments based on the newly identified quasi-enveloped hev particles indicate that lipid membrane protects the virions from neutralizing antibodies against the capsid protein (yin et al., ) . additionally, as a structural protein, the hev capsid protein has been found to interact with some cellular proteins and plays a role in cell signaling. in one study, the capsid protein activates the pro-apoptotic gene chop, and increases the expression of hsp , hsp b and hsp , and interacts with hsp (john et al., ) . in addition, the capsid protein interacts with β-trcp, a component of the ubiquitination complex that inhibits iκbα ubiquitination-mediated nf-κb activation (surjit et al., ) . however, these data are all based on overexpression of orf in mammalian cells, and need to be further verified in whole virus infection. the orf is the smallest among the three orfs of hev and overlaps with orf by approximately nt in a different reading frame. however, it does not overlap with orf (graff et al., ) . the overlapping region with orf (nt - ) was found to be the most conserved region between the sar and bur strains (tsarev et al., ). an early study proposed that orf encodes a protein with aa and comes from a different subgenomic rna other than that encoding orf (tam et al., ) . however, a later study based on an hev replicon shows that orf is translated from the bicistronic subgenomic rna and initiates at the third aug of the presumed orf at nt for sar and the product is a protein with or aa and molecular size of kda (vp ), which is aa shorter than the earlier predicted version (graff et al., ) . this observation has been confirmed by another study using a different hev strain (huang et al., ) . sequence analysis has indicated that vp is unique and has no similarity to any other proteins known. it contains two hydrophobic domains in its n-terminal half and two proline-rich domains in its c-terminal portion (kannan et al., ; holla et al., ) . a phosphorylation site (ser ) was identified in the first proline-rich domain and can be phosphorylated by map kinase (zafrullah et al., ) . furthermore, two psap motifs have been identified in genotype vp , with the first psap motif located at aa - and the second at aa - , whereas genotypes , , and have only one psap motif at aa - (nagashima et al., b) . the second psap motif is needed for hev virion release. interestingly, among the four genotypes that infect humans, only genotype vp has an additional prolinerich region that contains a pxxp motif in aa - , which is linear and surface-oriented (nan et al., a (nan et al., ,b, . the unique motif reacts with a genotype vp -specific monoclonal antibody. this proline-rich region contains residues pmsplr, a typical motif (pxxpx+) (+ is either arginine or lysine, x can be any aa) for class ii src homology (sh ) domains. sh domains are known to bind to proline-rich sequences containing a core pxxp motif flanked by a positively charged residue (baumann et al., ; raeder et al., ) . sh domains comprise of about residues and proteins containing sh domains typically play a role in signaling pathways involved in cell growth, differentiation and other regulatory functions (zarrinpar et al., ) . the next proline-rich region spanning aa - are rpsapplp, containing an additional residue than the typical motif (+xxpxxp) for class i sh domains. but interestingly only the pxxp motif in the second proline-rich region is known to interact with sh domains (korkaya et al., ) . the function of the pxxp motif in the first proline-rich region (aa - ) of vp of genotype hev is unknown. it might play a role in cellular signaling as proline-rich motifs are also involved in interacting with other domains besides sh (zarrinpar et al., ) . although the full function of hev vp has not been defined yet, some studies have suggested that vp plays multiple roles during hev infection. early studies focusing on vp antigenicity and epitope mapping demonstrated that the last aa of vp are an immunodominant region, and a synthesized peptide from that region is reactive with anti-hev serum from a recovered patient (semiletov et al., ; dement'eva et al., ) . however, another study mapping the t cell epitopes in orf and orf products indicated that no t cell proliferation was observed when cells were stimulated with peptides from vp (aggarwal et al., ) . a recent study based on genotype hev shows that continuous amino acid motif, vdlp, at the c-terminus of genotype hev vp , is a core sequence of a vp epitope . although vp is dispensable for viral replication in cultured cells , it is indispensable for hev infection in vivo, implying an important role for vp in host invasion (graff et al., ; huang et al., ) . a yeast two-hybrid system is employed to screen for the interaction partners for vp . the vp can bind to inactive mitogen-activated protein kinase (mapk) phosphatase and lead to activation of the mapk (kar-roy et al., ), suggesting that vp can modulate host gene expression since mapk is related to cell signaling and gene expression. another study shows that vp inhibits the nuclear translocation of stat and down-regulates stat -mediated gene expression, such as acute-phase response proteins (chandra et al., ) . the vp can also increase the expression of glycolytic pathway enzymes by increasing the phosphorylation and transactivation activity of p /cbp (moin et al., ) . furthermore, microarray analysis of huh cells with vp expression suggests that liver-specific genes can be modulated, as vp is able to modulate the phosphorylation of hepatocyte nuclear factor (chandra et al., ) . the vp can also up-regulate mitochondrial voltagedependent anion channel genes, which can protect cells from mitochondrial depolarization and death (moin et al., ) . this result implies that vp is able to inhibit the mitochondrial apoptosis pathway. the pro-survival role of vp is also demonstrated in another study showing vp delays the trafficking and degradation of the activated hepatocyte growth factor receptor to prolong endomembrane growth factor signaling (chandra et al., ) . additional interacting molecules have been identified for vp by yeast two-hybrid screens, including α- -microglobulin, bikunin, and bikunin precursor protein (ambp), fibrinogen β chain and hemopexin (tyagi et al., (tyagi et al., , ratra et al., ratra et al., , . moreover, a recent study screening for intraviral protein interactions identified the met, pcp, x, helicase, and rdrp domains as interacting partners for vp as well (osterman et al., ) . besides yeast two-hybrid screens, overexpression of vp coding plasmid in mammalian cells was also employed to elucidate the function of vp . the vp associates with the cytoskeleton fraction when expressed in cells, and deletion of the n-terminal hydrophobic domain of vp abolishes this association (zafrullah et al., ) . in a more detailed study, gfp-tagged vp is found to interact with microtubules to form a filamentous pattern in cells and modulate the microtubule dynamics (kannan et al., ) . vp leads to an elevation of acetylated α-tubulin, indicating increased microtubule stability (kannan et al., ) . since there are two hydrophobic domains located in the n-terminus of vp , truncation analysis indicated that both the hydrophobic domains are required for its association with the microtubules. moreover, salt extraction studies have suggested that the vp -microtubule interaction is electrostatic and motor protein dynein is needed for the interaction (kannan et al., ). an earlier study showed that vp cannot be co-precipitated with tubulin by anti-tubulin antibody (zafrullah et al., ) . these results suggest that vp may associate with microtubules through interaction with another protein. this microtubule-like distribution of vp suggests that it may play a role in promoting virus egress, as the pul protein of herpesvirus can interact with dystonin, an important cytoskeleton cross-linker involved in microtubulebased transport, in order to promote capsid transport on microtubules during egress (pasdeloup et al., ) . besides cellular proteins, vp has been shown to interact with viral helicase, pcp and methytransferase from hev orf , which suggests a regulatory function for vp in orchestrating the formation of the replicase complex (osterman et al., ) . more interestingly, another study using monoclonal antibody against vp to capture hev particles showed that vp can associate with virions and support virus release (takahashi et al., ) . the requirement of vp for virion release was later confirmed by a cell culture-adapted genotype hev strain with vp deletion . studies in caco- cells and huh cells for the sar , a genotype hev strain, showed that the intact psap motif spanning aa - in vp is required for virion release (emerson et al., ; nagashima et al., b) . for avian hev, the psap motif in vp has also been found to play a role in virus release . the psap motif in vp is required for the formation of membrane-associated hev particles with the vp protein itself associated with lipids. this process is mediated by the cellular tsg protein (nagashima et al., a,b) . replacement of the vp psap motif with heterologous late domain motifs (pppy, ypdl, and psaa) affects the virus release (kenney et al., ) . the specific interaction between vp and tsg as well as involvement of endosomal sorting complex required for transport (escrt), which commonly participates in budding of many enveloped viruses, leads to the biogenesis of membrane-associated, "quasi-enveloped" hev particles (hurley, ; feng et al., ; nagashima et al., ; yin et al., ) . therefore, vp is associated with virion during egress and anti-vp antibodies are able to capture hev virions from the serum and cell culture supernatant, but not fecal samples from patients (takahashi et al., ) . a possible explanation is that viral particles could lose lipid-associated vp after passing through the gut (takahashi et al., ) . the role of vp in virus release may be one of its functions during hev replication in vivo, indispensable for viral spread during infection. on the other hand, as a small phosphorylated protein, vp can be phosphorylated at ser by mapk when expressed in cos and huh cells (zafrullah et al., ) . a later study indicates that the ser phosphorylation site is required for the interaction of the capsid protein and vp as vp can interact with the capsid protein in a yeast two-hybrid screen, especially for the non-glycosylated capsid protein (tyagi et al., ) . this finding also supports a role for vp in hev structural assembly. however, a mutagenesis study shows that hev lacking the phosphorylation site in vp is able to replicate its genome in cultured cells, and to infect rhesus monkeys similarly to wild type hev in viremia and seroconversion (graff et al., ) . these data suggest that phosphorylation of vp is not necessary for genome replication or for the production of infectious virions. moreover, in addition to phosphorylation and interaction with the capsid protein, vp can form a homodimer via the aa domain located in the c-terminus (tyagi et al., b) . on the other hand, vp has been reported to activate mapk-jnk / in hepatoma cells (parvez and al-dosari, ) . besides the functions mentioned above, vp also plays a role in the interferon induction and signaling. data from our laboratory show that vp is able to enhance rig-i activation, which leads to enhanced rig-i signaling (nan et al., a) . the vp extends rig-i half-life and interacts with the n-terminal portion of rig-i to enhance its activation by polyi:c. interestingly, there is a genotype difference in the enhancement of rig-i: genotypes and vp but not genotypes and vp have the role, implicating that vp may relate to hev virulence and pathogenesis. on the other hand, another study demonstrate that vp of a genotype hev strain is able to interact with the stat (signal transducer and activator of transcription) to inhibit interferon-α mediated signaling in a (human lung adenocarcinoma epithelial cell line; dong et al., ) . in summary, as the product of the smallest orf of hev, vp has multiple functions and plays an indispensable role in infectivity in experimentally infected animal models. however, it is not required for hev replication in cultured cells. our current knowledge indicates that vp is a multifunctional protein in interacting with many cellular proteins, modulating host gene expression and involved in virion release. recently, a novel orf (nt - ) was identified from genotype hev . unlike other orfs in hev, translation of orf is driven by an ires-like sequence located in nt - of hev genome . the orf product interacts with multiple viral proteins to form a protein complex consisting of viral rdrp, helicase and x, and the orf product stimulated viral rdrp activity to promote viral replication. expression of the orf was verified in a cellfree system and antibodies against this protein were determined from hev-infected patients . however, analysis of hev sequences from other genotypes suggests orf is not conserved across genotypes . therefore, more investigation is needed to elucidate the exact function of orf . due to the lack of an effective in vitro cell culture system for hev, the replication cycle of hev is largely unknown. the capsid protein is believed to bind to an unidentified cellular receptor to initiate viral entry. hev-vlps generated from recombinant orf protein attach to cells via heparin sulfate proteoglycans (hspgs; kalia et al., ). moreover, one study based on a viral overlay protein binding assay (vopba) suggests that a protein with molecular weight about kda could be the candidate receptor for hev entry; but mass spectrometry revealed that this virus binding band contained different proteins . another study suggests that aa - located in the c-terminal region of the capsid protein (m domain) may be the putative receptor binding site of hev virions (he et al., ) . moreover, structure and sequence analyses suggest that the putative binding motif of the capsid protein is conserved among all four major mammalian hev genotypes (guu et al., ) . heat shock cognate protein (hsc ), hspgs and grp are found to be involved in either cell surface binding with hev capsids or intra-cellular transport in different models and are potential cellular receptors or essential factors for hev proliferation (kalia et al., ; yu et al., ; cao and meng, ) . however, further investigation is needed to confirm if these molecules truly act as receptors for hev. after binding with its receptor, hev particles are internalized via a dynamin- , clathrin, and membrane cholesterol-dependent pathway (kapur et al., ; holla et al., ) . in addition, a recent report suggests the quasienveloped hev particles enter cells via a distinct pathway that involves in degradation of the lipid membrane in the lysosome (yin et al., ) . after entry into permissive cells, the hev capsid is uncoated by unknown mechanisms. in one study utilizing vlp from the truncated capsid protein hev , an hsp -specific inhibitor (geldanamycin) blocks the intracellular transport of the hev vlp without affecting its entry . this suggests that hsp may play a role in the intracellular transport of hev particles. after uncoating, the hev orf translation is followed. hev genomic rna replication relies on the replicase encoded by orf . along with the generation of the sub-genomic rna, translation of orf and orf occurs, followed by virion packing and egress. for the release of hev particles, multivesicular body (mvb) pathway and escrt machinery in the cytoplasm are used (nagashima et al., ) . since the discovery of hev, many efforts have been made to develop a rigorous in vitro cell culture system. however, the cell culture system of hev is still limited and relatively ineffective, especially for genotype hev. an early study tried to use primary hepatocytes from macaques with serum-free medium for hev propagation; however, hev replication was limited and the detection of hev in the medium relied on pcr amplification (tam et al., ) . a group from japan reports that hev isolate a is able to replicate in a cells; however, pcr was also used to detect viral rna in the cell culture supernatant (huang et al., ) , instead of immunofluorescence assay to detect viral proteins. another group also showed that the a cell line could be used effectively for passaging two chinese hev isolates (wei et al., ) . on the other hand, as the commonly employed method for single-stranded, positive-sense rna virus, transfection of capped rna from an hev cdna infectious clone via in vitro transcription to plc/prf/ (hepatocellular carcinoma) and huh cells demonstrates limited replication of hev . although cell lysates from the rna transfected cells is infectious in rhesus monkeys, cell to cell spread of the virus in cultured cells is not observed . s - cell line, a subclone of huh hepatoma cell line, has improved replication efficiency of hev for the sar strain (graff et al., ; shukla et al., ) . but this assay still relies on transfection of the cells with full length hev rna. a recent report suggests that replication efficiency of genotype hev in human hepatoma cell lines (huh , huh . , and hepg /c a) is affected by innate immune response . a japanese group reports that a genotype isolate from acute hepatitis patient propagates in plc/prf/ and a cells (tanaka et al., ; okamoto, ) . after a cells were seeded in a six-well plate and inoculated with hev at . × and . × rna copies per well, hev rna reached the highest titer of copies/ml at days post-inoculation. however, plc/prf/ cells could only support efficient growth as a with a higher moi ( . × viral rna copies per well). moreover, in this hev cell culture system, hev infected cells need to be maintained at . • c and cultured with a mixed cell culture medium ( % dulbecco's modified eagle medium and % medium ) supplemented with % (v/v) fetal bovine serum and mm mgcl . the same group also reports that a genotype hev from a fulminant hepatitis patient can grow in plc/prf/ and a cells and reach a titer of . × copies/ml within - days incubation period ). moreover, a human hepatoma-derived cell line heparg and a porcine embryonic stem cell-derived cell line picm- , which have morphological and functional properties similar to primary hepatocytes, were shown to support hev replication (rogee et al., ) . however, the hev replication level in these two cell lines is very low and requires month incubation (rogee et al., ) . in hev kernow-c p cell culture system, the recombinant virus with human s gene insertion was presumed as a minor species in the host but was selectively adapted to cells after six passages (shukla et al., ) . replication of kernow-c p is . -fold higher in hepg /c a human hepatoma cells than in huh . , plc/prf/ , a , caco- or rhesus kidney cells in a -day incubation period, suggesting that hepg /c a cell line is the most permissive. moreover, this hev isolate is also able to infect a variety of non-primate cells, including cow, mouse, chicken, cat, dog, and rabbit cells, albeit with lower efficiency. although it is still unclear how the insertion of s occurred in hev infected patient, okamoto's group demonstrated that two cell adapted hev strains hev je - f (genotype ) and hev jf / f (genotype ) did not shown any recombination with cellular s gene after and generations of passages in plc/prf/ and a cells, respectively (okamoto, ) , which suggested the recombination and insertion of s may occur in patients rather than in cultured cell. besides human hev isolates, animal hev strains from domestic pigs, wild boars, rabbits, and rats can be propagated in human hepatoma cell lines as well takahashi et al., ) . a recent report demonstrates that pluripotent stem cell derived hepatocytes support hev replication in vitro (helsen et al., ) . in summary, the current cell culture systems for hev have limitations. so far, only one report shows limited replication of a genotype hev strain from serum sample in cell culture without rna transfection (takahashi et al., ) . on the other hand, although several groups have demonstrated that genotypes or hev strains can be adapted to cultured cells and are able to reinfect new cells, a long incubation time is needed in comparison to other rna viruses with good cell culture systems. moreover, the cell culture adapted kernow-c virus may have a different phenotype compared with its parental wild type virus as this cell culture adapted virus contains host gene sequence. the hev is primarily transmitted via fecal-oral route. the most common source of infection is contaminated drinking water in developing countries. for a long time, hepatitis e was thought to be a public health problem only for developing countries. however, hepatitis e is now frequently recognized in industrialized countries where it was not thought to be endemic previously (kwo et al., ; erker et al., ; schlauder et al., ; worm et al., ; kabrane-lazizi et al., ; mizuo et al., ; sadler et al., ) . world health organization estimates that there are million hev infections annually across the world. among these cases, there are over million symptomatic cases and , deaths (who, ) . hepatitis e is highly endemic in east and south asia. data indicates over % of global hepatitis e deaths occur in this region. in east asia, large outbreaks of hepatitis e have only been described in china. hepatitis e accounts for - % of acute hepatitis cases in this region. the seroprevalence of anti-hev antibodies in the region varies from to %, indicating that hepatitis e is hyperendemic in this region. in south asia, outbreaks of hepatitis e have been reported in most countries in this region, but variable in scale (who, ) . hev accounts for - % of sporadic acute hepatitis and fulminant liver failure in this region. in particular, the rates of fulminant liver failure are usually higher in pregnant patients. a recent paper reported that hev infection causes % acute viral hepatitis and % fulminant hepatic failure in pregnant women in one area in india . however, the seroprevalence rates of prior exposure to hev are relatively low, ranging from to % in most studies. in the developed countries, such as north america, western europe and japan, no outbreaks have been reported. these areas are considered as low or non-endemic for hev. however, sporadic cases of hepatitis e have been reported. transmission of hev from animal reservoirs to humans is assumed to be the major cause of those sporadic cases. a series of cases of hev infection in people who ate undercooked deer meat - weeks before the onset of disease have been reported (tei et al., ; yazaki et al., ; li et al., c) . hev rna recovered from the leftover deer meat was found to be identical in sequence to the hev rna recovered from the patients (takahashi et al., ) . consumption of shellfish is considered a risk factor in a documented case (koizumi et al., ) . thus, foodborne infection may occur from the consumption of uncooked/undercooked products from infected animals. moreover, blood transfusion and solid organ transplant mediated hev transmission are reported wedemeyer et al., ; sue et al., ) . igm and igg against hev are detected in recipients of blood transfusions (wedemeyer et al., ) . hepatitis e virus genotype is responsible for most endemic and epidemic cases of hepatitis e in asia, and genotype is prevalent in central america and africa (purcell and emerson, ) . there is no known animal reservoir for hev genotypes and (wedemeyer et al., ) . genotypes and are zoonotic and can cause hev infections in the developed countries. for the detailed geographical distribution of hepatitis e virus genotypes, please refer to these reviews (dalton et al., ; kamar et al., a) . hepatitis e virus infection mainly causes acute hepatitis with a case fatality rate from . to % in young adults (jameel, ) . remarkably, case fatality rate resulting from hev-related fulminant liver failure can reach up to % in infected pregnant women in their third trimester of gestation (jameel, ) . generally, hev has an incubation period of - weeks (purcell and emerson, ) . the initial symptoms of acute hepatitis e are unspecific and flu-like, such as myalgia, arthralgia, and weakness. after this short prodromal phase, a period of symptoms such as vomiting, itching, uncolored stools, darkened urine and jaundice could last for days to several weeks accompanied by increased levels of liver transaminases, bilirubin, alkaline phosphatase, and γ-glutamyltransferase (hoofnagle et al., ; wedemeyer et al., ) . current case reports indicate that most cases are selflimited and do not result in chronic hepatitis (hoofnagle et al., ). an investigation on pregnancy outcomes in hepatitis e shows higher hev loads in pregnant women with acute viral hepatitis and fulminant hepatic failure, and higher levels of tnf-α, il- , ifn-γ, and tgf-β than non-pregnant women, which suggests that high cytokine levels are correlated with severe liver injury in hev infection . a recent study highlights the role of tlr and ifn-γ in hev pathogenesis. patients with high levels of tlr and robust ifn-γ response are observed in self-limiting acute viral hepatitis cases, and are able to limit the disease and recover uneventfully (majumdar et al., ) . however, patients with lower expression of tlr and ifn-γ progress to acute liver failure (majumdar et al., ) . hev can cause chronic infection as well. although chronic hev infection was initially reported only in immunocompromised persons, such as organ transplant recipients, patients receiving cancer chemotherapy and hiv-infected persons (hoofnagle et al., ) , latest reports show that chronic hev infection also occurs in an immunocompetent individual with systemic lupus erythematosus (sle; grewal et al., ) . however, since this kind of cases are rare, data available so far are not sufficient to consider this patient as an immunocompetent individual . in organ transplant recipients, the chronic course leads to persistent increases in levels of alanine aminotransferase, significant histological activity and fibrosis in some cases (wedemeyer et al., ) . hiv-infected individuals have higher positive rate of anti-hev antibody than individuals without hiv infection (wedemeyer et al., ) . besides hepatitis, extrahepatic manifestations have been documented. neurological disorders, such as polyradiculopathy, guillain-barré syndrome, bilateral brachial neuritis, encephalitis and proximal myopathy, and neuralgic amyotrophy are reported in patients with acute and chronic hev infections (kamar et al., van den berg et al., ; van eijk et al., ; dalton et al., ; drave et al., ) . the kidney injury caused by hev infection is reported and also documented in monkeys infected experimentally with hev as well (kamar et al., (kamar et al., , b geng et al., ) . furthermore, a recent report provides evidence that extrahepatic replication of hev in the placenta of infected mothers, which may be associated with fetal mortality (bose et al., ) . it also raises the concern for the vertical transmission of hev to fetus and newborn by an infected mother (krain et al., ) . a report suggests the association between the outcome of hev infection in solid-organ transplant patients and the genetic heterogeneity of hev quasispecies in orf . analysis of the viral genetic heterogeneity indicates that both nucleotide complexity and genetic distance of the orf proline-rich domain in patients whose infection became chronic are higher than the patients who cleared the virus . although diagnostic tests for hev are commercially available, none of them have been formally approved in the united states by the food and drug administration (fda; hoofnagle et al., ) . current tests mainly target anti-hev antibodies, including igg and igm. however, several assays are based on antigens expressed by a single hev genotype, especially genotype , and might be limited for the detection of all hev genotypes. indeed, there are variations in sensitivity, specificity and agreement in the results of these assays, which may account for the discrepancies among positive rates of anti-hev antibodies in various populations (mast et al., ; herremans et al., ; drobeniuc et al., ) . it is also notable that a recent study demonstrates the false positive result in hev igm test due to cross reaction with ebv and cmv, which heavily affects the accuracy of hev serology testing (hyams et al., ) . only . % of the total samples with the positive hev igm were pcr positive for hev rna. the cross reactivity of igm against hev, ebv, and cmv is very high. these data suggest that to confirm hev infection in patients, clinical features, blood alt level and pcr testing should be all included in addition to serological test alone. on the other hand, although hev rna can also be detected in blood and stool for several weeks after acute hev infection, in addition to a narrow detectable window of hev viremia (hyams et al., ) , current hev rna tests are still experimental since they have not been standardized yet (wedemeyer et al., ) . furthermore, diagnostic support for igm and igg anti-hev detection in clinical samples using commercially available kits and pcr assay for detection of hev rna in serum and stool samples are also available from the division of viral hepatitis in the centers for disease control and prevention (cdc, ). hepatitis e virus infection mainly causes a self-limited disease and most infected individuals are able to clear it spontaneously. although the case fatality rate in adults is . - %, the rate can increase to % in pregnant women during their third trimester of gestation in south asia (jameel, ) . therefore, antiviral therapy is needed. although no specific treatment has been approved for hev, off-label application of ribavirin as monotherapy for hev has demonstrated promising results in both acute and chronic hepatitis e patients (kamar et al., ; mallet et al., ; gerolami et al., ) . in vitro assay showed that ribavirin could inhibit replication of genotypes - hev through the depletion of intracellular gtp pools in hev infected cells (debing et al., a) . for immunosuppressed patients, a reduction of immunosuppression has shown efficacy in the treatment of chronic hev infection (wedemeyer et al., ) . moreover, application of pegylated interferon in combination with ribavirin has been reported as a treatment for chronic hev infection but only shown moderately synergistic effect (wedemeyer et al., ; debing et al., a) . however, due to the evidence of embryolethality and teratogenicity revealed by animal study, ribavirin has been assigned to pregnancy category x by the fda and contraindicated in women who are pregnant and in the male partners of women who are pregnant (sayed et al., ) . moreover, ribavirin-induced g r mutation was reported and associated with treatment failure of ribavirin monotherapy in solid-organ transplant patients (debing et al., b; lhomme et al., ; todt et al., ) . therefore, viral specific treatment for hev is needed. our laboratory has successfully tested application of peptideconjugated morpholino oligomers (ppmos) as novel anti-hev compounds (nan et al., ) . ppmos are water soluble, nuclease-resistant single-stranded dna analogs containing a backbone of morpholine rings and phosphorodiamidate linkages along with conjugation of arginine-rich cell penetrating peptide for facilitating cell delivery (summerton, ; abes et al., ) . ppmos bind to mrna by watson-crick base pairing and interfere with translation through steric blockade of the augtranslation initiating region. antisense morpholino oligomers are currently tested in clinical trials for treating duchenne muscular dystrophy in humans and has been documented as effective against numerous types of viral infections in experimental animal models (anthony et al., ; mendell et al., ; moulton, ) . importantly, upon systemic administration, ppmos distribute to liver, remain pharmacologically viable, and are effective at reducing viral titers (amantana et al., ; burrer et al., ; paessler et al., ) . in our study, ppmo hp targeting utr of hev genotype sar strain demonstrates strong inhibition of hev replication (nan et al., ) . since the utr of hev genome is highly conserved among different hev genotypes infecting humans, the ppmo hp may be an hevspecific inhibitor with antiviral activity across multiple hev genotypes (nan et al., ) . these qualities, along with the in vitro efficacy against hev (nan et al., ) , make ppmos be appealing for consideration as a novel inhibitor of hev infections. another nucleic-acid based strategy, sirna, has also been reported to be effective in inhibiting hev replication. an sirna targeting hev rdrp was reported to inhibit hev replication in a cells and in piglets . in another report, sirna targeting a cis-acting element and viral nucleotide sequences coding for helicase and rdrp are effective against hev in hepg cells (kumar et al., ) . however, it is generally acknowledged that sirna needs considerable improvements in their delivery to relevant targets in vivo before they can be considered for clinical applications involving systemic delivery against virus infections. current prevention for hev relies on sanitary measures, such as providing clean water, and appropriately cooked food to avoid transmission from undercooked food (kamar et al., a) . since in vitro culturing of hev is limited and ineffective, hev vaccine development mainly focuses on the expression of the capsid protein as a subunit vaccine. the capsid protein shares over % identity among the four major hev genotypes in mammalian hosts (mori and matsuura, ) . the capsid protein from genotype hev expressed by baculovirus or bacterial vectors has been tested in clinical trials. the first candidate was a kda protein expressed in insect cells. in a phase trial in nepal, the vaccine is well-tolerated and highly immunogenic, with % efficacy for protection against hepatitis e (shrestha et al., ) . the second vaccine, hev , encompasses aa - of orf product, is a kda truncated protein expressed in e. coli (li et al., b) . this vaccine is well-tolerated with an efficacy of % protection after three doses in a population tested in china, which included both men and women aged - years (zhu et al., ) . the hev vaccine was approved and marketed in china in . whether it will be endorsed in other countries or how effective it is against all other genotypes of hev infecting humans remains unknown. moreover, a study shows that hev vaccine could protect rabbits against homologous and heterologous hev challenge (liu et al., ; zhang et al., ) . a recent report demonstrates that a hybrid protein fusing protruding (p) domains from capsid proteins of both norovirus (nov) and hev induces a higher antibody titer than either p domain alone . subunit vaccine candidates containing antigens of hev, rotavirus, and astrovirus are reported as well (xia et al., ) . more than years have passed since the discovery and complete genome sequencing of hev. our understanding of hev is still limited, though ongoing research continues to reveal more and more information about this virus. currently, we know that hev is not only a public health concern in developing countries as previously thought, but also a concern with a more complicated scenario in the developed countries. more and more animal reservoirs are revealed and we now understand that genotypes and hev are zoonotic and foodborne pathogens. however, the cross-species transmission and host tropism of different hev genotypes are still elusive. current data imply certain viral proteins such as orf product plays a role in the host tropism of hev. further investigation is needed to elucidate the basic biology of hev. on the one hand, although approved in china, the hev vaccine is still unavailable to most of the world, despite the fact that serum surveillance indicates a high prevalence rate of hev throughout the world. moreover, recent discoveries about the antigenicity variation between hev genotypes and quasienveloped viral particles hidden from neutralizing antibody suggest new challenges and questions about the efficacy of the approved vaccine. further investigation about the vaccine efficacy against multiple hev genotypes or seeking for an improved vaccine is needed. in addition, virus specific treatment for hev infection is not available yet. although, the off-label using of pegylated ifns and antiviral drugs for general purposes have demonstrated efficacy against hev, safety is still a concern as no validation has yet been conducted for these treatments. therefore, a hev-specific treatment such as ppmos is needed. due to the absence of a suitable animal model and a simple cell culture system, many details about this virus and its infection, such as its biology, pathogenesis, strain variances, genotype differences, molecular mechanisms and vaccine efficacy for cross protection are still incomplete. however, current 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hepatitis e virus capsid trafficking an elisa for putative neutralizing antibodies to hepatitis e virus detects antibodies to genotypes , , , and efficacy and safety of a recombinant hepatitis e vaccine in healthy adults: a large-scale, randomised, double-blind placebo-controlled, phase trial the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © nan and zhang. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -a uhy ex authors: huang, fen; zhou, junfang; yang, zhibiao; cui, li; zhang, wen; yuan, congli; yang, shixing; zhu, jianguo; hua, xiuguo title: rna interference inhibits hepatitis e virus mrna accumulation and protein synthesis in vitro date: - - journal: vet microbiol doi: . /j.vetmic. . . sha: doc_id: cord_uid: a uhy ex hepatitis e virus (hev) is a zoonotic pathogen to which several species, including human beings, pigs and rodents, are reported to be susceptible. to date, vaccines developed against hev still need to be improved and a structural gene (orf ), which encodes a capsid protein with high sequence conservation found across hev genotypes, is a potential candidate. to exploit the possibility of using rna interference (rnai) as a strategy against hev infection, four small interference rna (sirna) duplex targeting orf gene were constructed. a challenge against hev infection by rnai was performed in a cells. real-time quantitative polymerase chain reaction (real-time qpcr) and western blot assay demonstrated that four hev specific sirnas (si-orf - , si-orf - , si-orf - and si-orf - ) were capable of protecting cells against hev infection with very high specificity and efficiency. the results suggest that rnai is a potent anti-hev infection prophylaxis strategy. hepatitis e virus (hev) is a zoonotic pathogen to which several species, including human beings, pigs and rodents, are reported to be susceptible. to date, vaccines developed against hev still need to be improved and a structural gene (orf ), which encodes a capsid protein with high sequence conservation found across hev genotypes, is a potential candidate. to exploit the possibility of using rna interference (rnai) as a strategy against hev infection, four small interference rna (sirna) duplex targeting orf gene were constructed. a challenge against hev infection by rnai was performed in a cells. real-time quantitative polymerase chain reaction (real-time qpcr) and western blot assay demonstrated that four hev specific sirnas (si-orf - , si-orf - , si-orf - and si-orf - ) were capable of protecting cells against hev infection with very high specificity and efficiency. the results suggest that rnai is a potent anti-hev infection prophylaxis strategy. ß elsevier b.v. all rights reserved. ). moreover, immune escapes can also occur as new virulent hev variants evolve, as the case with severe acute respiratory syndrome coronavirus (sars-cov) evolved from human coronavirus (lin et al., ; saif, ) . in this respect, the mutational events in cell cultures that have been reported during the primary propagation and consecutive passages (p , p , and p ) reported in (lorenzo et al., ; tanaka et al., ) are particularly worrisome. in this paper, rna interference (rnai) was used as a way of circumventing some of these issues regarding vaccines, such as high specificity and efficiency. rnai is a phenomenon in which small double-stranded rna molecules induce sequence-specific degradation of homologous singlestranded rna (hannon, ) . rnai is a powerful tool to investigate gene function through specific suppression of a particular mrna, and has been employed in therapeutic studies of human diseases, including cancer, neurogenerative diseases and viral infectious diseases. similar strategies were capable to reduce significantly sars-cov replication (akerstrom et al., ) and hepatitis c virus mrna accumulation (liu et al., ) , which suggests that rnai may be one of the most useful antiviral therapy strategies currently being investigated. as the field of rnai has expanded, much research has been reported for many genes of interest, including oncogenes and viral genes, indicating their successful silencing in both cells and animal models (akerstrom et al., ; kirchhoff, ; kleinman et al., ; liu et al., ) . there have also been a number of clinical trials using rnai strategies, including one on agerelated macular degeneration (amd) (kleinman et al., ) , and more applications are expected. the hev used in this study was characterized as genotype hev (genbank accession no. ef ) and was isolated from swine feces from the shanghai, china. this was used as template for a reverse transcription nested polymerase chain reaction pcr (rt-npcr) to amplify the hev orf gene. the orf gene encodes the major structural or capsid protein of hev (riddell et al., ) . the external forward primer and reverse primer were -cgattttgcgctt-gagcttga- (p ) and -tggagaccgagcgcacggcac- (p ), respectively. the internal forward primer and reverse primer were -ctcggcgggctcccgacag- (p ) and -aggtgcgaggacaccaacggcag- (p ), respectively. rt-npcr analysis was conducted using an amv reverse transcriptase xl kit for rt-pcr (takara, tokyo, japan) according to the manufacturer's directions. the rt-pcr protocol performed included a reverse transcription phase at c for min and c for s. two microliters of the cdna synthesized were then amplified by nested pcr at c for min, followed by c for s, c for s and c for min, and repeated for cycles. the pcr products were detected by electrophoresis on agarose gel containing . mg/ml ethidium bromide. the amplified dna fragment was inserted into the multicloning site of a eukaryotic expression vector pegfp-n (clontech, a gift from dr. wei liu) which contains the reporter gene of enhanced green fluorescence protein (egfp), using standard cloning procedures with the restriction sites of ecori and bamhi. the egfp gene was located downstream of the target genes. the recombinant plasmid was named pegfp-orf . four nt sirnas with dna ends corresponding to the target genes ( fig. ) were designed according to qiagen's guideline (http://www.qiagen.com). the sirna duplexes have been designed using the hiperformance design algorithm licensed from novartis ag, integrated with a stringent in-house homology analysis tool. the highestranking sirna duplexes generated by the algorithm were chosen as representing the best combination of activity and specificity. scrambled sirna, constructed from a random sequence heterology with the hev sequence, served as a negative control for identifying the specificity of hev sirna ( table ). the synthesized non-modified sirnas were diluted with rnase-free buffer to obtain a mm solution. the solution was denatured by heating at c for min, incubated at c for min, then either used immediately or stored at À c in an rnase-free environment. a (human lung carcinoma, atcc, va, usa) cell line was used in this study and maintained as described previously (huang et al., ) . a cells were trypsinized and transferred to ml antibiotic-free growth medium in -well plates at . - .  /well. cells were cultured h before transfection. one microgram of pegfp-orf scramble sirna sense -r(uuc ucc gaa cgu guc acg u)dtdt- antisense -r(acgugacacguucggagaa)dtdt- recombinant plasmid was diluted in ml of serum-free dmem medium, and mixed gently with . ml each of the five nm sirnas and . ml lipofectamine (invitrogen, ca, usa) according to the manufacturer's instructions in ml of serum-free dmem medium for min, and then co-transfected to the cells. meanwhile, each sirnas were either co-transfected with pegfp vector or transfected alone into a cells to test the interferon response. the optimal combination of four parameters for each cell line, including the highest transfection efficiency, the lowest non-specific effects, the conditions for the most efficient delivery of sirna, and the concentration of lipofectamine , was first determined in preliminary experimentation. four to six hours post-transfection, the culture medium was replaced with fresh complete medium and the cells were incubated at c for h for mrna detection, and h for protein detection. to determine transfection efficiency, the egfp fluorescence intensity of transfected cells was monitored with an inverted fluorescence microscope (nikon te , tokyo, japan) before real-time qpcr analysis. the efficiency of suppression of the hev orf gene by various hev specific sirnas was evaluated by fluorescence microscopy, real-time qpcr and western blot. specificity of the inhibition was confirmed by co-transfection of the pegfp-orf recombined plasmid with scrambled sirna. hev has been successfully cultured in a cells as previously described (huang et al., ) . for hev infection challenge, a cells were transfected with each of sirnas (si-orf - , si-orf - , si-orf - , si-orf - , and scrambled sirna) as described previously h before the virus challenge. the viral challenge was performed as the previously described (huang et al., ; tanaka et al., ) . the virus at a viral count of -  /ml as calculated by viral genomic titer determined by real-time quantitative pcr (li et al., ; kasorndorkbua et al., ) was inoculated into a cells, and cpes were observed daily. the cells were harvested h postinoculation, when the cpe was observed, for real-time qpcr analysis (downstream of the target in orf ), and h for western blot assay. a cells ( . -  ) per well seeded in -well plates were incubated as described above except that the amounts of sirnas and virus/pegfp in each well were onefourth of those in -well plates. forty-eight hours post-inoculation with viruses, the culture medium was removed, and the cells were lysed by freeze-thaw three times. the total rna was extracted by trizol (invitrogen, ca, usa) according to the manufacturer's directions and reverse transcribed into cdna using an amv reverse transcriptase xl kit. the synthesized first strand cdna ( ml) was added as a template for real-time qpcr using sybr premix ex taq tm (perfect real-time, takara, tokyo, japan) according to the manufacturer's directions. the forward primer for orf was -cgcacct-cactctgcgctg- ( - nt), and reverse primer was -attggaaagcgcagccctg- ( - nt). the mixtures were reacted at c for s, followed by c for s and c for s repeated for cycles. the product was expected to be bp. the housekeeping gene gapdh served as a loading control. the forward primer of gapdh was -tgggctacactgagcaccag- , and reverse primer was -aagtggtcgttgagggcaat- . the pcr protocol was same as the cells except repeated for cycles. the real-time qpcr analysis was performed in the abi prism real-time pcr system (abi, ct, usa). all procedures were performed in triplicate and data are expressed as means ae s.d. the infected a cells were harvested h postinoculation and lysed with buffer ( mm tris-hcl, ph . , mm nacl, . % sodium azide, % triton x- , mg/ ml aprotinin, and mg/ml pmsf). equivalent amounts of total protein were separated by % sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) and transferred onto a nitrocellulose membrane. after blocking non-specific-binding sites with % skim milk, the membrane was incubated with primary antibodies at c overnight. the primary antibodies used in this experimental were rabbit polyclonal anti-hev (abr, ca, usa, : dilution), and a rabbit polyclonal anti-gapdh for loading control (proteintech group, co, usa, : dilution). after washing with tbs buffer, the blots were incubated with hrp conjugated goat anti-rabbit igg (promega, wi, usa, : dilution) at room temperature for h. the bands were then exposed to x-ray films with supersignal west pico trial kit (pierce, rockford, usa). the bp orf fragment of hev ( - nt) was amplified by rt-npcr and introduced into the pegfp-n vector with the restriction sites of ecori and bamhi to yield pegfp-orf . the recombinant plasmid was identified by digestion with restriction enzymes (ecori and bamhi) and sequence. a cells transfected with pegfp-orf recombinant plasmid with each sirnas were observed by fluorescence microscope at - h post-transfection (fig. pegfp-orf ). the efficiency of transfection was confirmed by the highly expressed reporter gene egfp protein. fluorescence assays showed an obvious reduction effect induced by all hev specific sirnas except the scrambled one (fig. ) , which suggested that a high efficiency and specificity of inhibition effects by sirna had been achieved. to further study the effect of sirnas on protecting a cells against hev destruction, a mts assay was performed. the od values (mean ae s.d.) of solutions in wells treated with each sirnas (si-orf - , si-orf - , si-orf - , and si-orf - ) were shown in fig. . for the absorbance at nm is directly proportional to the number of living cells in culture, the number of living cells in a well treated with each of hev specific sirnas was more than that treated with scrambled sirna or untransfected controls cells. interferon response was not observed in a cells transfected with each sirnas alone or co-transfected with pegfp vector. the reduction of mrna level of hev was evaluated by real-time qpcr. the percentage of hev mrna in cells previously transfected with sirna over that in cells inoculated with hev alone was calculated according to pfaffl method (pfaffl, ) . the suppression of hev mrna h post-inoculation was decreased about . -fold, . fold, . -fold and . -fold in cells transfected with nm/well sirnas (si-orf - , si-orf - , si-orf - and si-orf - , respectively), while no significant changes were noted in cells transfected with scrambled sirna (fig. a) . the gapdh transcription level was rarely changed in either untransfected or transfected cells (data not shown). to optimize the inhibitory effect of sirna on hev transcription, a viral mrna yield reduction assay was conducted by transfection a cells with each sirnas at indicated doses. as shown in fig. b , transfection of nm/ well of each sirnas just induced an approximately % reduction in viral mrna. however, with the dosage of sirnas increasing, the hev mrna decreased. when the fig. . each of sirnas co-transfected with pegfp-orf /pegfp in a cells. the expression of hev orf protein was observed h post-transfection. hev specific sirnas induced an obvious reduction, whereas the expression level of egfp protein co-transfected with scrambled sirna showed no significant change. each of sirnas (si-orf - or si-orf - ) was co-transfected with pegfp vector to test the interferon response. pegfp-orf : pegfp-orf recombinant plasmid transfection alone; pegfp+ si-orf - : pegfp vector co-transfected with si-orf - ; pegfp+ si-orf - : pegfp vector co-transfected with si-orf - ; scrambled sirna: pegfp-orf recombinant plasmid co-transfection with scrambled sirna; si-orf - : pegfp-orf recombinant plasmid and si-orf - co-transfection; si-orf - : pegfp-orf recombinant plasmid and si-orf - co-transfection; si-orf - : pegfp-orf recombinant plasmid and si-orf - co-transfection; si-orf - : pegfp-orf recombinant plasmid and si-orf - co-transfection. pictures were taken at h post-transfection with a nikon te fluorescence microscope. fig. . protection of a cells from hev infection by sirnas. rnai inhibited a cells growth as determined by mts assay. cells transfected with hev specific sirnas, scrambled sirna, and untransfected (infected) cells were served as controls. absorbance was read at nm and the results were obtained in triplicate wells. od value shown is the mean ae s.d. amount of sirna reached nm/well, the highest inhibition of hev transcription was obtained. the data indicated that the suppression of hev transcription by sirna was dose-dependent. to analyze further the efficiency of suppression of the hev protein synthesis by sirna, a cells were collected for western blot analysis. only the cells infected with hev expressed the expected $ kda protein band , while uninfected a cells showed no protein band reacting with anti-hev antibody. western blot showed that hev putative structural protein expression was reduced significantly in cells transfected with hev specific sirnas (fig. -si-orf - to si-orf - ) compared with cells infected with hev alone or transfected with scrambled sirna (fig. -hev and -scrambled) . gapdh was used as a loading control in this experiment. in conclusion, the results demonstrated that highly efficient inhibition of hev transcription by hev specific sirnas in a cells was obtained. hev is a major cause of enterically transmitted acute hepatitis of adults and is highly lethal in pregnant women. the hev orf gene is highly conserved in four genotypes and is the most important structural region of the virus. evidence from epidemiological, animal transmission and vaccine studies had indicated that the orf gene is a suitable region for hev treatment studies. therefore, the hev orf gene was chosen as the target for sirnas gene silencing in the current study. rna interference has showed great promise as an antiviral therapy since its discovery in mammalian cells (elbashir et al., ) . its application in vitro and in vivo has shown it to be a new and efficient approach for inhibition of viral infection. however, as also shown in this study, different sirna sequences have different interference efficacies, which depending on the characteristics of the target rna, including local rna folding and the accessibility of the sirna-binding site on the target rna (kurreck, ; shao et al., ) . the highest potential efficiency of interference is considered a high knockdown that avoids the degradation of untargeted genes (off-target effects) with the fewer sirnas (ladunga, ) . rna interference in viral infection has been the focus of numerous studies (liu et al., ; mungall et al., ) , and an effective and specific interference in hev transcription was obtained in a cells in the current study. rnai, its exquisite specificity in seeking targets, which determines the exclusive protection against hepatitis e virus. hev specific sirnas were capable of preventing hev infection in a cells. antiviral activity was demonstrated both in mrna transcription level by real-time qpcr and in protein expression level by western blot. these findings indicate that this protocol may be an effective antiviral strategy for protecting host cells against viral invasion. the sirna is very effective in vitro; however, there are many obstacles to application of rnai in vivo as a therapy. the main obstacles are prevention of degradation of sirna and avoidance of side effects to the host induced by sirna itself. couzin ( ) and grimm and kay ( ) mentioned the toxicity of both sirna or shrna expression vectors in vitro and in vivo, and cell apoptosis was observed when vectors were introduced at a high concentration. although the rnai pathway is a promising treatment for cancer, virus and hepatitis therapy, there are serious safety issues that first need to be addressed (mcbride et al., ) . there needs to be a focus on decreasing the toxicity that accompanies sirna treatment. in conclusion, the present study demonstrated that hev specific sirnas could specifically and effectively suppress hev transcription and translation in a cells. these results indicate that rnai can be a potential antiviral therapy for suppression hev infection. further study is required to determine the effectiveness and the safety of rna interference as a means of protection against hev infection in vivo. viral 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virus vaccine: mission accomplished? development and evaluation of an efficient cell-culture system for hepatitis e virus mutational events during the primary propagation and consecutive passages of hepatitis e virus strain je - f in cell culture zoonotic transmission of hepatitis e virus from deer to human beings elisa for antibody to hepatitis e virus (hev) based on complete open-reading frame- protein expressed in insect cells: identification of hev infection in primates serological evidence of hepatitis e virus infection in different animal species from the southeast of brazil key: cord- -no ucyq authors: murkey, jamie a.; chew, kara w.; carlson, margrit; shannon, chelsea l.; sirohi, deepika; sample, hannah a.; wilson, michael r.; vespa, paul; humphries, romney m.; miller, steve; klausner, jeffrey d.; chiu, charles y. title: hepatitis e virus–associated meningoencephalitis in a lung transplant recipient diagnosed by clinical metagenomic sequencing date: - - journal: open forum infect dis doi: . /ofid/ofx sha: doc_id: cord_uid: no ucyq hepatitis e virus (hev) infection uncommonly causes chronic hepatitis and neurologic disease. we describe a case of genotype a hev meningoencephalitis diagnosed by metagenomic next-generation sequencing, illustrating the power of an unbiased molecular approach to microbial testing and the first reported case of hev infection presumably acquired through lung transplantation. the precision diagnosis of acute infectious diseases (pdaid) study for the diagnosis of hospitalized patients with suspected infectious causes of meningoencephalitis was launched in june . the goal of this multihospital, nationwide study is to evaluate the utility and cost-effectiveness of a clinical metagenomic next-generation sequencing (mngs) assay for pathogen detection as compared with conventional microbiological testing (supplementary methods). the mngs approach does not define targets a priori; rather, any and all viruses, bacteria, fungi, and parasites are identified in clinical samples on the basis of sequence homology to genbank microbial reference databases. the mngs assay has been validated in a clinical laboratory improvement amendments (clia) laboratory [ , ] , and results are relayed to the treating clinical team(s) and reported in the patient's electronic medical record. here we present a case of hev meningoencephalitis diagnosed by clinical mngs in an immunocompromised patient enrolled in the pdaid study. we demonstrate the power of clinical mngs in elucidating the cause of uncommon and unexpected infections and identify a case of chronic hev infection most likely transmitted through the transplanted lungs the patient had received years prior. the case patient is a -year-old woman with a history of idiopathic pulmonary fibrosis status post bilateral lung transplant in , migraines, hypercoagulability, and multiple sclerosis (ms) on chronic immunosuppression who was admitted to university of california, los angeles medical center in october with days of fever, headache, nausea, vomiting, neck stiffness, and photophobia. the patient had been hospitalized days prior to admission at an outside hospital complaining of the "worst headache of my life. " during that hospitalization, she was treated with a variety of abortive migraine medications with only partial response and diagnosed with tacrolimus toxicity (initial tacrolimus level of . ng/ml; . ng/ml on discharge) and acute kidney injury. of note, her symptoms occurred in the setting of > years of ms-attributed episodic leg pain and swelling, years of occasional word-finding difficulty and slurred speech, year of recurrent episodes of dizziness and falls, and month of lower extremity weakness. in addition, the patient had an acute episode of encephalopathy in and a first-time seizure of unclear etiology in march . the patient is a resident of orange county, california. she denied sick contacts, pets or other animal exposure, insect bites, and eating shellfish or game meats. she reported travel to the mountains in utah in august , the caribbean in , and throughout europe decades before admission. her outpatient medications included immunosuppressive medications (tacrolimus, mycophenolate mofetil, and prednisone for lung transplant; teriflunomide for ms), antimicrobial prophylaxis (trimethoprim/sulfamethoxazole and acyclovir), anticoagulation, and pain medications, including an intrathecal morphine pump. upon admission, the patient was noted to be sleepy but fully oriented and in moderate distress from pain; she had a fever of . °c but otherwise had normal vital signs. physical exam was remarkable for right leg tenderness from deep venous thrombosis. initial exams showed stable pancytopenia (white blood cell and platelet counts of . × /l and /l, respectively, and hemoglobin of . g/dl), elevated transaminases (alanine aminotransferase and aspartate aminotransferase of u/l and u/l, respectively), elevated international normalized ratio of . , and tacrolimus level of . ng/ml. magnetic resonance imaging revealed baseline periventricular, subcortical, and juxtacortical t /flair white matter intensities associated with her ms but no acute changes (table ) . empiric antimicrobials were initiated with vancomycin, ceftazidime, acyclovir, and voriconazole. tacrolimus was initially held but subsequently resumed. given the patient's ongoing symptoms, a lumbar puncture was performed on day , revealing a lymphocytic pleocytosis ( table ) . all clinical microbiologic studies returned negative (table ) . by day , the patient was clinically improved except for mild persistent headache. the differential diagnosis included viral meningoencephalitis or tacrolimus toxicity. the patient was identified as a possible pdaid case based on the unknown etiology of her meningoencephalitis. a cerebrospinal fluid sample from day of her hospitalization was analyzed by clinical mngs testing at university of california, san francisco. dna and rna sequencing libraries yielded and reads, respectively. analysis using the sequence-based ultra-rapid pathogen identification (surpi+) clinical bioinformatics pipeline detected hev. assembly yielded a . % complete viral genome with approximately % pairwise identity to the closest matched reference in genbank (supplementary figure a and b) . phylogenetic analysis assigned the genome to genotype a, most closely related to viral strains from japan and southeast asia (supplementary figure c) . the diagnosis of hev-associated meningoencephalitis was communicated to the patient. review of the patient's electronic medical record showed normal transaminase levels before lung transplantation, with persistent low-level elevations after transplantation in (supplementary figure a and b) . the patient was readmitted weeks after being discharged with new decompensated liver disease, encephalopathy, asterixis, ascites, and cirrhosis by abdominal ultrasound and liver elastography. the hev mngs results prompted immediate follow-up clinical testing demonstrating serum hev immunoglobulin m (igm) positivity, negative immunoglobulin g (igg), and plasma hev viremia ( international units [iu]/ml) ( table , supplementary figure c ), and treatment with ribavirin and low-dose diuretics. hepatitis e virus rna declined, transaminases normalized, and ascites, edema, and mentation improved, but the patient subsequently had readmissions for headache, nausea, and vomiting attributed to tacrolimus toxicity or side effects from ribavirin, necessitating multiple ribavirin treatment interruptions. the case was reported to the united network for organ sharing donor safety net. testing of stored donor serum was positive for hev igg and igm antibody but negative for hev rna. the donor was reported to be a -year-old woman with methamphetamine use from central california without clear risk factors for hev infection or abnormal transaminase levels. pretransplant samples from the case patient were not available. we present a case of genotype a hev infection in a lung transplant recipient with ms on immunosuppressive therapy. the patient's symptoms and lymphocytic pleocytosis on admission were consistent with acute viral meningitis. tacrolimus toxicity may have also contributed to her presentation because she improved rapidly after tacrolimus discontinuation. the patient's persistent low-level transaminitis for several years before admission and subsequent evidence of cirrhosis suggest that she was likely chronically infected with hev. although initial testing for hev igg was negative, serologies can be unreliable in immunosuppressed patients [ ] . her prior episodes in and of seizure and "encephalopathy" are also suggestive of chronic neuroinvasive hev disease with intermittent flares. although the development of chronic hev infection is infrequent in the general population, solid organ transplant (sot) recipients are at greater risk [ ] . in series of sot recipients with hev infection, > % developed chronic hepatitis [ ] . in some of these patients, cirrhosis can develop within several years [ ] . neurologic manifestations of hev infection, including inflammatory polyradiculopathy, encephalitis, and guillain-barré syndrome, have been seen in . % of cases [ ] . the timing of neurological manifestations after hev infection has not been well described, but ranged from - months in review of cases in immunosuppressed patients. in animal models, hev is able to cross the blood-brain barrier, replicate in the central nervous system, and cause neuronal necrosis and myelin degeneration [ ] . hepatitis e virus infection is infrequently considered as an infectious cause of meningoencephalitis and specific diagnostic testing is not routinely done, underscoring the benefit of an unbiased approach such as mngs for pathogen detection [ ] . hepatitis e virus infection may be detected through serological testing, but concurrent blood and stool hev rna testing is recommended, especially in immunosuppressed patients [ ] . clinicians should consider hev in the differential diagnosis for sot or other immunosuppressed patients with unexplained hepatitis, particularly those taking calcineurin inhibitors [ ] . the treatment of chronic hev infection includes reduction in immunosuppression, enabling viral clearance in approximately % of sot patients [ ] . ribavirin monotherapy has achieved sustained virologic response in approximately % of patients and was initiated in this patient [ ] . new antivirals such as sofosbuvir may have a future role in treatment [ ] . notably, our patient appears to have contracted hev from her donor. supporting evidence includes the positive anti-hev igg/igm testing of the donor's serum and the patient's persistent low-level transaminase elevations that began after transplant. of only published reports of donor-derived hev transmission, liver transplant recipient developed cirrhosis and death from septic shock within months of transplantation, and renal transplant recipients (with the same donor) developed cholestatic hepatitis at and months after transplantation [ , ] . this is the first report of presumptive hev transmission through lung transplantation. transplant centers and clinicians should be aware of the potential for hev infection in donors judged to be at elevated risk. supplementary materials are available at open forum infectious diseases online. consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author. hepatitis e virus and chronic hepatitis in organ-transplant recipients hepatitis e and pregnancy: understanding the pathogenesis hepatitis e virus in blood components: a prevalence and transmission study in southeast england professional practice committee and committee on laboratory practices of the microbiology resource committee of the college of american pathologists. validation of metagenomic next-generation sequencing tests for universal pathogen detection neurobrucellosis: unexpected answer from metagenomic next-generation sequencing hepatitis e virus reinfections in solid-organ-transplant recipients can evolve into chronic infections hepatitis e virus infection among solid organ transplant recipients, the netherlands factors associated with chronic hepatitis in patients with hepatitis e virus infection who have received solid organ transplants hepatitis e virus and neurologic disorders evidence of hepatitis e virus breaking through the blood-brain barrier and replicating in the central nervous system a cloud-compatible bioinformatics pipeline for ultrarapid pathogen identification from next-generation sequencing of clinical samples treatment of hev infection in patients with a solid-organ transplant and chronic hepatitis liver transplant from a donor with occult hev infection induced chronic hepatitis and cirrhosis in the recipient evidence of hepatitis e virus transmission by renal graft we would like to thank the patient for participating in the pdaid research study. permission to publish this case report was granted by the patient through written informed consent.financial support. this work was supported by the california initiative to advance precision medicine; an award from abbott laboratories, inc; and philanthropic grants from the sandler, bowes, and schwab foundations.potential conflicts of interest. c. y. c. is the director of the ucsf-abbott viral diagnostics and discovery center and receives research support from abbott laboratories, inc.all other authors report no conflicts of interest. all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. key: cord- -nnefbeo authors: tam, albert w.; smith, matthew m.; guerra, martha e.; huang, chiao-chain; bradley, daniel w.; fry, kirk e.; reyes, gregory r. title: hepatitis e virus (hev): molecular cloning and sequencing of the full-length viral genome date: - - journal: virology doi: . / - ( ) - sha: doc_id: cord_uid: nnefbeo abstract we have recently described the cloning of a portion of the hepatitis e virus (hev) and confirmed its etiologic association with enterically transmitted (waterborne, epidemic) non-a, non-b hepatitis. the virus consists of a single-stranded, positive-sense rna genome of approximately . kb, with a polyadenylated ' end. we now report on the cloning and nucleotide sequencing of an overlapping, contiguous set of cdna clones representing the entire genome of the hev burma strain [hev(b)]. the largest open reading frame extends approximately kb from the fend and contains the rna-directed rna polymerase and nucleoside triphosphate binding motifs. the second major open reading frame (orf ) begins by downstream of the first and extends approximately kb to the termination codon present by from the ' terminal stretch of poly(a) residues. orf contains a consensus signal peptide sequence at its amino terminus and a capsid-like region with a high content of basic amino acids similar to that seen with other virus capsid proteins. a third open reading frame partially overlaps the first and second and encompasses only bp. in addition to the . -kb full-length genomic transcript, two subgenomic polyadenylated messages of approximately . and . kb were detected in infected liver using a probe from the ' third of the genome. the genomic organization of the virus is consistent with the fend encoding nonstructural and the ' end encoding the viral structural gene(s). the expression strategy of the virus involves the use of three different open reading frames and at least three different transcripts. hev was previously determined to be a nonenveloped particle with a diameter of – nm. these findings on the genetic organization and expression strategy of hev suggest that it is the prototype human pathogen for a new class of rna virus or perhaps a separate genus within the caliciviridae family viral hepatitis results from infection with one of at least four very different viral agents. available serological tests allow the diagnosis of acute hepatitis due to infection with hepatitis a virus (hav) and hepatitis b virus (hbv). hbv is required for propagation of the delta agent, or hepatitis d virus (hdv); this co-infection results in a high proportion of cases progressing to chronic active hepatitis. the clinical and diagnostic exclusion of hav and hbv led to the recognition of other viral hepatitides that were formerly grouped together as non-a, non-b hepatitis (nanbh) (prince et a/., ; feinstone et a/., ; tabor, ) . nanbh is caused by more than one viral agent and can be transmitted by either parenteral or fecal/oral routes (bradley, a; reyes and baroudy, ) . the cloning of a blood-borne agent, termed hepatitis c virus (hcv) by us and others led to the development of a specific assay for circulating antibody to hcv (choo etal., ; kuo et al., ; kubo eta/, ; maeno et al., ; reyes et a/., d) . this assay predomi-nantly detects infections at the chronic stage, but has facilitated the identification of hcv as the cause of up to % of parenterally transmitted nanbh. a second epidemiologically distinct form of nanbh was shown to occur in both epidemic and sporadic patterns in developing countries and is referred to as enterically transmitted non-a, non-b hepatitis (et-nanbh) due to its water-borne mode of virus transmission and presumed enteric route of infection (khuroo, ; wong et a/., ) . et-nanbh has been documented in india, pakistan, burma, ussr, costa rica, mexico, and countries in africa, where epidemic outbreaks can generally be traced to fecal contamination of drinking water (bradley and maynard, ; bradley, b) . the causative viral agent was previously shown to passage successfully in cynomolgus macaques (cyno) and tamarins with typical liver enzyme elevations and recovery of morphologically similar -to -nm viruslike particles from the feces of clinical specimens and experimental animals (balayan et al,, ; anjaparidze et al., ; bradley et al., ; arankalle et al., ) . we recently reported the isolation of a partial cdna clone from the virus responsible for et-nanbh, and have termed the newly identified agent the hepatitis e virus (hev) (reyes et a/., ) . the clone was from a burma isolate of hev and hybridized with cdna made from five other distinct geographic isolates. these molecular epidemiological findings are consistent with the available serologic data based on the use of immune electron microscopy and immunofluorescence blocking studies that indicate a single major agent is responsible for the majority of et-nanbh seen worldwide (purcell and ticehurst, ; bradley et al., a; krawczynski and bradley, ) . we now report on the molecular cloning and sequencing of the complete hev (burma; b) viral genome together with the deduced amino acid sequences of viral-encoded proteins general perspectives on the genetic organization of the virus, as deduced from sequence and open reading frame analyses, indicate that hev bears some similarity to the caliciviridae but may represent a new class of nonenveloped rna virus. rna purification. total cellular rna was isolated from normal and hev(b)-infected cyno livers by the guanidinium-lici precipitation method (cathala et al., ) and pofy(a)+ rna was selected by one round of oligo(dt) cellulose chromatography (aviv and leder, ) . cdna library construction and screening. synthesis and screening of the infectious bile cdna library had previously been described (reyes et al., ) . oligo(dt)-, random hexamer-, and hev sequence-specific oligomer-primed (primer a, see fig. ) cdna were synthesized using a commercially available cdna synthesis kit (boehringer-mannheim biochemicals, indianapolis, in), ligated to ecorl linker-adapters and cloned into x gtl (stratagene, san diego, ca). g-tailed cdna was made essentially as described before (tam et al., ) . briefly, first strand cdna primed with hev sequence-specific primer c (see fig. ) was tailed with dgtp using terminal deoxynucleotidyl transferase. the modified cdna was then amplified in a polymerase chain reaction (pcr) (saiki et al., ; mullis and faloona, ) employing the same synthetic hev primer and an oligo(dc) primer, both of which contained an ecorl cloning site at the ' end. all four cdna libraries were screened with appropriate synthetic oligomer probes (applied biosystems, foster city, ca) described under results. hybridizations were generally performed in duplicate (using p kinased probes) at " in % formamide, x ssc, x denhardt's ( . % ficoll, . % polyvinylpyrrolidone, . % bsa), mm sodium phosphate, ph . , and @g/ml salmon sperm dna. after an overnight hybridization, filters were washed three times with . x ssc and . % sds at - " depending on the length of the oligomer probe. primer extension analysis. primer extension studies were carried out using oligonucleotide primers kinased to a specific activity greater than x ' cprn/pg with [t-~~p]atp (icn radiochemicals, irvine, ca) essentially as described (mcknight et a/., ) . extension products were separated on a % pofyacrylamide- m urea sequencing gel that was subsequentty dried and autoradiographed. the sequences forthe hev primers used in these studies are: primer a: '-cccgataagcagcctcaagcctc- ' primer b: '-ccgcgtacacactaaccccccggc-caataat-tcacgctgg- ' primer c: '-caagctggcgaggttgcattagg- ' primer d: '-acagcaticgccagggcagagtt- ' northern blot analysis. four micrograms of hev(b)infected cyno liver poly(a)+ rna was electrophoresed on a . % agarose gel containing . m formaldehyde and transferred onto a nitrocellulose filter. the filter was hybridized under high stringency conditions with a radiolabeled betg- ecorl fragment insert ( x lo* wmhg). dna nucleotide sequencing. dna sequencing was performed by the dideoxynucleotide method @anger et a/., ) using -deaza-dgtp (pharmacia, piscataway, nj). all sequencing reactions were carried out on both strands using bluescript plasmid (stratagene, san diego, ca) subclones obtained from hev xgtlo phage clones. appropriate overlapping subfragments were exploited wherever possible, or adjoining dissimilarend subclones were employed for unambiguous orientation. sequencing primers were commercially available or synthesized based on derived hev sequences. -deaza-dgtp eliminated areas of compression due to the high g + c content of the viral genome (see results) . computer analyses of nucleotide and amino acid sequences. computer programs for manipulation of nucleic acid and protein sequences were obtained from lntelligenetics (mountain view, ca). a partial hev cdna clone, et .l, was isolated by differential screening of a cdna library constructed from infectious bile collected from a third-passage cyno inoculated with subpassaged fecal suspensions originally derived from burma patients with weff-defined et-nanbh (reyes et al., ) . bife was chosen as the rna source for cdna synthesis because it contained relatively large numbers of virus particles when hev cdna clones were identified from libraries made from randomly primed cyno bile (solid square), or from cyno liver after priming by oligo-dt (solid circle), random sequence hexamers (open circle) and hev-sequence specific oligonucleotides (open square). the designations given to the various clones are indicated together with their sizes and relative position and overlap along the - . kb genome. a and b represent synthetic oligonucleotides used for the generation and screening, respectively, of specifically primed cdna libraries, the anchor pcr strategy using g-tailing and pcr (primer c) was used in the synthesis of primer extension libraries for the extreme ' end. the procedure yielded numerous clones by hybridization with primer d, of which bet-expcr is a representative example. primer extension studies confirmed the ' extent of the viral genome (see fig. ). the bet clone contained a long stretch of poly(a) residues at its ' end indicating its position at the ' terminus of the viral genome. compared with fecal preparations. it was also expected that the lower sequence complexity would enhance the sensitivity of the differential (plus/minus) screening protocol used for clone identification. et .l contained a . -kb ecorl fragment that was exogenous to both human and cyno genomic dna and specifically hybridized to cdna derived only from infected sources (reyes et al., ) . oligonucleotides based on the end sequences of et . were used as hybridization probes to rescreen the original bile-derived cdna library. the largest identified clone, betg- , contained a . -kb ecorl insert. restriction mapping revealed that the original et .l clone was contained within the larger betg- ( fig. ; fry et a/., ) . the same end-probe strategy was used with oligonucleotides derived from betg- to screen oligo(dt)primed and random hexamer-primed hev(b)-infected cyno liver cdna libraries. a collection of overlapping clones was identified from both libraries (fig. ) . one of the oligo(dt)-primed clones, bet contained two ecorl fragments that comprised . kb in total length. the authenticity of the ecorl site was strengthened by its presence in another clone, bet , isolated from the random-primed cdna library. a long poly(a) stretch of - - adenosine residues was located at the ' end of bet confirming the original observation that genomic rna could be selected on oligo-dt cellulose . this result indicated that the ' end of the viral genome was present in the bet clone. the ' end of the viral genome was isolated from a cdna library made by primer extension using a synthetic -bp oligonucleotide complementary to the ' end of clone bet (primer a, see fig. ). one of two positive clones identified by an oligonucleotide probe (primer b), located 'to the specific primer, was clone bet-spl , this clone contained a single large insert of . kb. with the acquisition of bet-spl, the composite cdna map (omitting overlaps) spanned approximately . kb from the ' end of bet-spl to the polyadenylated 'end of clone bet ; in good agreement with the maximum length of hev rna as detected on northern blots. the ' end of bet-spl was therefore believed to be in close proximity to the putative ' end of the viral genome. primer extension studies using poly(a)-selected rna from infected cyno liver were performed in order to firmly establish the distance from the existing 'end of bet-spl to the end of the genome (fig. ) . two specific oligonucleotide primers (primers c and d, see fig. ) were synthesized and bp from the 'end of bet-spl and used to prime cdna synthesis after p labeling their ' ends with polynucleotide kinase. the resulting extension products for each synthesis reaction were, respectively, and bp longer than the expected product, thereby suggesting that the ' end of bet-spl was about nucleotides from the ' end of the virus (fig. ) . after several failed attempts at cloning the remaining ' end sequences by oligonucleotide hybridization of specifically primed cdna libraries, an alternative expansion/enrichment procedure of pcr amplification of specifically primed g-tailed cdna was applied (tam et al., ). an atiquot of the amplified material was fcorl digested, electrophoresed, blotted, and probed with a ' internal hev oligomer (primer d). this hybridization study confirmed the amplification of the &aired hev extension products (data not shown). this same dna, after preparative gel etectrophoresis, was recovered and ligated into xgtl . the specific priming procedure (followed by pcr amplification) resulted in a high percentage (over %) of hev-positive recombinants in the enriched library. bet-expcr is a representative clone from over analyzed; all of these were bp in length and therefore in agreement with the primer extension experiment. the isolation of bet-expcr completed the hev genomic cdna cloning. the entire nucleotide and deduced amino acid sequence of hev are presented in fig. . the nucleotide composition of the hev genomic rna is % a, % c, % g, and % u, conferring an overall g -i-c content of %. sequence homology to any nucleotide sequences contained in the genbank database could not be detected when the hev sequence was searched in either the forward or reverse orientation. only two regions were identified that had homology with previously described nonstructural gene elements present in other positive strand rna viruses (see below; . using the cod rny sequence analysis program, the - . -kb of hev sequence, exclusive of the ' poly(a) tract, was analyzed for the presence of open reading frames (orf) in the six possible translation frames (fig. ) . the identification of the rna-dependent rna polymerase in the original et .l clone (reyes et al., ) and strand-specific probe hybridization (reyes et al., b) established the positive-sense orientation of the hev genome. a representation of the potential orfs and stop codons in the three positive-polarity frames is presented in fig. a . two large potential orfs were found in the first and second reading frames. orfl begins at the ' end of the viral genome after bp of apparent noncoding sequence at the ' end, and then extends bp before termination at nucleotide position . the second major orf (orf ) begins at nucleotide position and extends bp before terminating bp upstream of the poly(a) tail. the termination of orfl and the transition into orf was confirmed by sequencing the region in question five times using two different hev sequencespecific primers. the sequence of a second clone in this region yielded the same results. furthermore, cdna clones isolated directly from infected human (huang et al., , data not shown). a third positive-polarity reading frame of bp (orf ) overlaps both orfl and orf and was found by independent experiments to encode an immunoreactive epitope recognized by sera from hev-infected humans and animals (yarbough et af., ; reyes et a/., b) . no orfs greater than bp were identified by computer search of the negative-polarity rna strand (fig. b) . the nucleotide frequencies at each codon position were also analyzed and a comparison was made with two other hepatotropic positive-strand rnaviruses (ta-ble ). the overall frequencies for hev and hcv are similar (- % g + c), but differ markedly from that of hav ( % g + c). the relatively high g + c content results in a higher overall frequency of codons containing g + c throughout the hev coding sequence. this contrasts with the cg dinucleotide discrimination in the second and third position that has been noted in human coding sequences (nussinov, ) . there appears to be a slight selection for codons ending in c, which is also seen with hcv; however, the discrimination against codons ending in a is far more apparent (- %) and is a unique fea?ure of hev when compared to hav and hcv. the third position discrimination a lluiulllluul u jul uului u lll uuuul jui i b iu uuuu~iu ~ p i uj l iuiu i i uii i u uuulii ~ui.~uu~~ computer generated open reading frame analysis of the entire hev nucleotide sequence is presented in both the forward (a) and reverse directions (b). the positions of all termination codons are depicted by arrows. the three forward orfs are numbered , , and and those on the opposite strand similarly labeled. the forward (positive-sense) orientation was defined by strand-specific hybridization of genomic rna (reyes et al.. b) and the identification of consensus sequence motifs related to nonstructural gene products in orfl (kamer and argos, ) . the horizontal line running through the various orfs indicates that orf with the highest probability of encoding protein as predicted by the algorithm devised by shepherd based on the rny codon analysis in all three orfs (shepherd, ) . the hydrophilic@ plot of orf is presented in (c). the dotted line plotted at the - value on they-axis represents the midpoint with hydrophobic domains above and hydrophilic domains below the dotted line. note the large hydrophobic region at the beginning of the sequence that marks the putative signal sequence highlighted in fig. . against a is shared by the structural orf region of another positive-strand rna virus, rubella virus, where the frequency of a is only % (frey and marr, ) . hev and hcv are also similar in their apparent preference for g in the first coding position ( and %, respectively). the computer translation of the partial nucleotide sequence from clone et . led to the detection of a conserved amino acid motif recognized in all positivestrand rna viruses (reyes er a/., ; fry et a/., ). the canonical gly-asp-asp (gdd) tripeptide (amino acids -l , identified by asterisks in fig. ) is believed to encode a portion of the rna-dependent rna polymerase (rdrp) gene critical to viral replication (kamer and argos, ) . translation of the complete orfl revealed a second region ' to the rdrp gene bearing similarity to another nonstructural gene product (fry et a/., ) . two well-conserved sequence motifs have been found in association with purine nucleoside triphosphate (ntp)-binding activity (gentry, ; strauss and strauss, ) . the first, site a (g/axxxxgks/t), is represented in the hev sequence by gvpgsgks at amino acid position - (underlined in fig. ) . a version of the second ntp- binding motif, site b (dead), occurs approximately amino acids downstream ( ') from site a and is represented in hev by the partially conserved amino acid sequence deap at position -l (underlined in fig. ). the latter site is believed to interact with the mg+ cation of the mg-ntp complex for rna-or dna-dependent ntpase activity. a superfamily of helicases involved in replication, recombination, and dna repair has been described with consensus features similar to those described here for ntp-binding (gorbalenya eta/., ). these nonstructural genesimilarities are seen in other geographically distinct isolates of hev and may indicate a putative helicase function for this region. the localization of an ntp-binding domain and the rdrp gene to orfl is consistent with a genomic organization where the nonstructural genes are expressed from the ' end of the viral genome. translation of the second major open reading frame, orf , indicated a novel polypeptide not present in the pir protein database. the hydropathicity plot of the sequence indicated a large hydrophobic domain at the amino terminus of orf followed by a hydrophilic electropositive peak (fig. ~ ). the hydrophobic region marks a typical signal sequence (amino acids to ) and contains a potential cleavage site (paippp) as predicted by the lntelligenetics eukaryotic secretory signal sequence program. in orf , between residues and , nearly % of the amino acids are arginine conferring a high isoelectric point (pl = . ) to the first half of the orf polypeptide. the basic charge of capsid proteins is believed to indicate their involvement in the encapsidation of the genomic transcript by effectively neutralizing the electronegatively charged rna (dalgarno ef al,, ; rice et a/., ) . the mechanism of capsid assembly in hev, and the exact nature of the membrane targeting (if any) of the orf polypeptide, will require further study. such studies will be facil-itated by the availability of an appropriate in vitro propagation system for hev and immunospecific anti-hev reagents. the utilization of orf was substantiated by the independent isolation of a cdna clone by immunoscreening of a hgtl cdna expression library made from the hev (mexico) isolate (yarbough et a/., ) . that xgt clone mapped to the ' end of orf . these same experiments identified a second cdna epitope clone that was localized to orf : the third positive-polarity open reading frame that overlaps both orfl and orf . the fact that sera from acutely infected humans and animals detected hev antigens encoded by orf and orf confirmed their expression and established that the virus utilized all three positive-polarity reading frames. the presence of a consensus s&al sequence motif in orf , together with the immunodominant seroreactivity of an identified epitupe (yarbough et a/., ) suggested that the viral structural protein(s) were encoded by this region of the genome. the mechanism by which orf and orf are expressed was suggested by a northern mot hybridization using the betg- clone as probe ( fig. ). in addition to the previously identified poly(a) transcript of - . kb, the probe also hybridized to subgenomic messages of . and . kb present in the infected cyno liver. it is of note that et .l did not originally identify these subgenomic messages (reyes et al., ) and other northern blot studies using probes located ' to et .l also did not hybridize to these viral-specific transcripts (data not shown). these same subgenomic messages were identified in poly(a)-selected r#a from hev(m)-infected cyno liver when the epitope-encoding clones were used as probes (yarbough et al., ) . the orf epitope is located at the extreme ' end of that reading frame (yarbough ez a/., ) , therefore indicating that these messages may be co-terminal with the ' end of the viral genomic transcript. it is pos- fig. . northern blot analysis of hev (burma)-infected cyno liver rna. three hev transcripts were detected using the . -kb ecorl insert from betg- as probe. numbers to the left represent the sizes of the three hybridizing rna species as determined relative to rna size markers. hev cdna probes were negative against similarly preoared rna from uninfected liver (data not shown). sible that these polyadenylated subgenomic messages are used in the expression of orf and orf . et-nanbh has been well-documented in both sporadic and epidemic outbreaks throughout the developing world. hepatitis e virus has been established as the major causative agent of et-nanbh by the association of hev-specific sequences with human specimens derived from six geographically diverse epidemics and also through the detection of these same sequences in various specimens derived from experimentally infected animals (reyes et al., ) . hev viral particles recovered from infected patients are similar to those recovered from infected primates. the virus contains a single-strand, positive-sense rna genome of approximately . kb. the nucleotide sequence described here comprises bases excluding the poly(a) tail. if the ' stretch of adenosine residues (at least - nucleotides) is included, the determined sequence agrees well with the genome size originally estimated by northern hybridization studies (reyes et al., ) . open reading frame analysis of the nucleotide sequence revealed two major positive-polarity orfs. a portion of orfl appears to encode the rdrp gene of the virus. the highly conserved amino acid residues, including the invariant gdd tripeptide found in all posi-tive-strand animal and plant rna viruses, can be located in the deduced amino acid sequence (reyes et a/., ; . additional evidence for the encoded polyprotein having a function in viral replication is provided by the presence of conserved motifs involved in purine ntpase activity found in a variety of cellular and viral helicases (geider and hoffman-berling, ) . these helicases promote the unwinding of dna, rna, or dna-rna duplexes required for genome replication, recombination, repair, and transcription. the deduced amino acid sequence of orf suggests that it encodes a capsid-like peptide following the canonical signal sequence at its 'end. orf would appear to be the major orf encoding the viral structural protein(s). an identified immunoreactive epitope in orf indicates that the virus utilizes all three positive-polarity frames for encoding viral proteins (yarbough et al., ; reyes et a/., c) . this pattern of gene expression employed by hev has not been described in the various families of single-stranded positive-sense, nonenveloped rna viruses affecting humans or animals. among the enveloped rna viruses, the structural proteins of rubella virus and certain alphaviruses are found in a different reading frame from those encoding the nonstructural proteins and are also expressed from a subgenomic 'end transcript (ou eta/., ) . the presence of hev-specific subgenomic rnas localized to the ' one-third of the genome suggests that these may be the transcripts from which these ' end orfs are expressed and is indicative of a unique expression strategy among nonenveloped positive-sense rna viruses infecting humans. the mechanism by which these subgenomic transcripts are generated is unknown. the differential abundance of the various messages (i.e., . kb > kb > . kb; see fig. ) does, however, suggest active transcriptional regulation rather than genomic rna fragmentation as the means by which these subgenomic messages are generated. this would in turn imply the existence of an internal rna initiation sequence and expression from the anti-genomic strand. experiments are currently in progress to map the 'ends of these subgenomic transcripts. we at this time, however, cannot exclude other mechanisms of expression for orf and orf including frameshifting or internal translation initiation, although there is little evidence for the latter among other positive-sense rna viruses (march and haenni, ) . it is also possible that complex rna splicing could account for these subgenomic messages although there is evidence that northern hybridization probes from the extreme ' end failed to detect hybridization to the . -and . -kb messages (a. w. tam, unreported) . . hev genomic organization: the proposed organization of the hev genome is presented with the nonstructural genes encoded by the ' orfl and the structural genes located at the ' end of the genome (orf and possibly orf ). the genomic organization, nature of the virus particle (enveloped or nonenveloped), presence of subgenomic messages, and the presence of a 'terminal poly(a) addition is compared for the various positive-sense, single-stranded rna virus families. the relative locations of the various virus sequence motifs is &so indicated, including the: hel, putative helicase motif or ntp binding domain; pol, rna-directed rna polymerase; ss, signal sequence; ire, immunoreactive epitope. s, structural gene coding region; ns, nonstructural gene coding region. it is postulated from the proposed genomic organization of hev, as presented in fig. , that the nonstructural viral proteins are translated from the full-length genomic rna. the ' nonstructural/ structural genomic organization of hev is similar to that found in the alphavirus, rubivirus, and coronavirus families (see fig. ). there is an absence of any significant homology with these enveloped viruses at both the nucleotide and amino acid levels (excluding the canonical amino acid residues noted above for the nonstructural gene products). immune electron microscopy has clearly established that the virions of hev are -to -nm nonenveloped viral particles and are therefore clearly distinguished from these enveloped viruses. picornaviruses are small nonenveloped, single-stranded, positive-sense, polyadenylated rna viruses. the various members of the picornaviridae, however, exhibit vastly different genomic organization (siddell, ) . hev has been shown to be unrelated antigenically and biophysically to picornaviruses (arankalle, ) . it was previously hypothesized that hev is calicivirus-like based on the biophysical characterization of viral particles (bradley and balayan, ; bradley et a/., a,b) . recently the nucleotide sequence of a large portion of the nonstructural gene region of feline calicivirus (fcv) has become available for comparison to hev (neill, ) . although having a similar overall genomic organization to that of hev with '-nonstructural and '-structural genes, it is clear that fcv shares a higher degree of similarity with picornaviruses in the recognized nonstructural gene motifs. the proposed gene order for the nonstructural polypeptides in fcv is c (ntp-binding), c (cysteine protease), followed by the d gene (rdrp). the distance between the ntp-binding site motif a and the gdd triplet of the rorp is amino acids in fcv compared to rhe amino acids in hev. in addition there is no evidence in the hev sequence for an intervening cysteine prateaselike region as recognized in fcv. these finding would further suggests that hev represents either the prototype member of an as yet unclassified novel virus family or perhaps a separate genus within the calciviridae. it is too early, however, to propose dlfinitive classification of hev beyond the hypothesis presented here based on the proposed genetic organization and expression strategy of the virus. fecal-oral transmitted non-a, non-b hepatitis induced in monkeys aetiological association of a virus-like particle with enterically transmitted non-a, non-b hepatitis purification of bioiogi&fy active globin messenger rna by chromatography on al&o-thymidyfic acidcellulose evidence for a virus in non-a, non- hepatitis transmitted via the fecal-oral route 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coding for virion proteins c and e and the carboxy terminus of the nonstructural proteins of rubella virus: comparison with alphaviruses hepatitis e virus (hev): strain variation in the nonstructural gene region encoding consensus motifs for an rna-dependent rna polymerase and an atpigtp binding site, ~%%%%, i ress locating a nucleotide bi ding site in the thymidine kinase of vaccinia virus and of herpes mplex virus by scoring triply aligned protein sequences. hoc two related supedemilies of putative helicases involved in replication, recombination, repair and expression of dna and rna genomes hepatitis e virus: cloning of a mexican strain and comparison to the burmese strain primary structural comparison of rna-dependent polymerases from plant, animal, and bacterial viruses study of an epidemic of non-a, non-b hepatitis: possibility of another human hepatitis virus distinct from posttransfusion non-a, non-b type the detection and classification of membrane-spanning proteins enterically transmitted non-a, non-b hepatitis: identification of virus-associated antigen in experimentally infected cynomolgus macaques a cdna fragment of hepatitis c virus isolated from an implicated donor of post-transfusion non-a, non-b hepatitis in japan an assay for circulating antibodies to a major etiologic virus of human non-a, non-b hepatitis a cdna clone closely associated with non-a, non-b hepatitis analysis of transcriptional regulatory signals of the hsv thymidine kinase gene: identification of an upstream control region organization of plant virus genomes that comprise a single rna molecule specific synthesis of dna in vitro via a polymerase catalysed chain reaction. ln nucleotide sequence of a region of the feline calicivirus genome which encodes picornavirus-like rna-dependent rna polymerase, cysteine protease and c polypeptides eukaryotic dinucleotide preference rules and their implications for degenerate codon usage sequence studies of several alphavlrus genomic rnas in the region containing the start of the subgenomic rna long-incubation post-transfusion hepatitis without serological evidence of exposure to hepatitis b virus enterically transmitted non-a, non-b hepatitis: epidemiology and clinical characteristics molecular biology of non-a, non-b hepatitis agents: the hepatitis c and hepatitis e viruses molecular cloning of the hepatitis e virus. /n "viral hepatitis and liver disease hepatitis e virus (hev): epitope mapping and detection of strain variation hepatitis e virus (hev): the novel agent responsible for enterically transmitted non-a, non-b hepatitis isolation of a cdna from the virus responsible for enterically transmitted non-a, non-b hepatitis nucleotide sequence of yellow fever virus: implications for flavivirus gene expression and evolution enzymatic amplification of b-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia dna sequencing with chain terminating inhibitors. froc method to determine the reading frame of a protein from the purinelpyrimidine genome sequence and its possible evolutionary justification. hoc the organization and expression of coronavirus genomes evolution of rna viruses the three viruses of non-a, non-b hepatitis construction of cdna libraries from small numbers of cells using sequence independent primers. nucleic acids ffes epidemic and endemic hepatitis in india: evidence for a non-a, non-b hepatitis virus aetiology hepatitis e virus: identification of type common epitopes, j viral we thank dr. michael lovett for his review and comments on this manuscript and appreciate the expert assistance of r. cuevas, j. fernandez, and the genelabs visual arts department. key: cord- -o kc x z authors: saraswat, shweta; chaudhary, meenakshi; sehgal, deepak title: hepatitis e virus cysteine protease has papain like properties validated by in silico modeling and cell-free inhibition assays date: - - journal: front cell infect microbiol doi: . /fcimb. . sha: doc_id: cord_uid: o kc x z hepatitis e virus (hev) has emerged as a global health concern during the last decade. in spite of a high mortality rate in pregnant women with fulminant hepatitis, no antiviral drugs or licensed vaccine is available in india. hev-protease is a pivotal enzyme responsible for orf polyprotein processing leading to cleavage of the non-structural enzymes involved in virus replication. hev-protease region encoding – amino acids of genotype- was amplified, expressed in sf cells and purified in its native form. the recombinant enzyme was biochemically characterized using sds-page, western blotting and immunofluorescence. the enzyme activity and the inhibition studies were conducted using zymography, ftc-casein based protease assay and orf polyprotein digestion. to conduct orf digestion assay, the polyprotein, natural substrate of hev-protease, was expressed in e. coli and purified. cleavage of kda orf polyprotein by the recombinant hev-protease lead to appearance of non-structural proteins viz. methyltransferase, protease, helicase and rna dependent rna polymerase which were confirmed through immunoblotting using antibodies generated against specific epitopes of the enzymes. ftc-casein substrate was used for kinetic studies to determine km and vmax of the enzyme and also the effect of different metal ions and other protease inhibitors. a % inhibition was observed with e- which was validated through in silico analysis. the correlation coefficient between inhibition and docking score of inhibitors was found to have a significant value of r( ) = . . the predicted d model showed two domain architecture structures similar to papain like cysteine protease though they differed in arrangements of alpha helices and beta sheets. hence, we propose that hev-protease has characteristics of “papain-like cysteine protease,” as determined through structural homology, active site residues and class-specific inhibition. however, conclusive nature of the enzyme remains to be established. is one of the most important viruses responsible for water born epidemics (kamar et al., ) . it is primarily transmitted through the faeco-oral contaminated drinking water. hev was discovered in in an outbreak of unexplained hepatitis in soviet soldiers in afghanistan. although, hev is more prevalent in developing countries due to poor sanitation and water supplies (cao and meng, ) however, cases of hev infection in industrialized countries like europe, usa and japan are becoming more common (minuk et al., ; bendall et al., ; mushahwar, ) . hev causes self-limiting acute infection in approximately million people annually, with a global mortality rate of % (jameel, ; nan and zhang, ) . this mortality rate remarkably increases up to % in the infected pregnant women in their third trimester due to fulminant liver failure (navaneethan et al., ; aggarwal and naik, ) . infection with hev represents an important global public health problem due to significant morbidity and mortality (gupta and agarwala, ) . currently a vaccine has been developed but licensed only in china, thus there is no vaccine or therapeutics available against hev infection elsewhere. also, there is no accepted treatment for hev but the treatments of both interferon and/or ribavirin as a combinatorial therapy have been used successfully to treat chronic hev infection (kamar et al., ) , though it has some side effects. genetically, hev genome is a non-enveloped single-stranded positive sense rna of ∼ . kb long and contains three partially overlapping open reading frames orf , orf , and orf (tam et al., ; tsarev et al., ; ahmad et al., ) . hev orf translates into a small phospho-protein that modulates some of the host-regulatory functions including establishment of infection and virion egress (graff et al., ; chandra et al., ; yamada et al., ). orf forms a amino acid ( kda) protein and its processed form constitutes the viral capsid. orf is the largest orf, , bases long and translated into , amino acids, which encode the non-structural polyprotein of ∼ kda, essential for viral replication . computational analysis of orf has identified seven putative domains (koonin et al., ) . these include an active methyltransferase domain (met), y domain (y) (parvez, ) , papain-like cysteine protease (pcp) (parvez, ; paliwal et al., ) , a proline -rich region that contains a hypervariable region (h), x -domain (x), helicase (hel), and an rna dependent rna polymerase (rdrp) from n-to c-terminal (koonin et al., ; parvez, ) . except pcp and y domain, all other putative domains have been partially characterized and their functions have been predicted bioinformatically and some of them even experimentally (agrawal et al., ; magden et al., ; karpe and lole, a,b) . a recent report has identified an additional orf in genotype- hev, which is presumed to play an essential role in viral replication (nair et al., ) . various attempts have been made to study orf processing and validate proteolytic activity of the pcp domain but not much success has been achieved. expression of orf in cell free system and the bacteria showed a kda polyprotein while the same construct, expressed using vaccinia virus showed two fragments of kda and kda in hepg cell (ropp et al., ) . in another study, transfection of infectious hev rna into hepg cells showed processed orf fragments, of , , and kda using anti-mett, anti-hel and anti-rdrp antibodies, respectively (panda et al., ) . similarly expression of orf using baculovirus system showed processing of orf as eight fragments that was inhibited by cell permeable cysteine protease inhibitor (e- d) (sehgal et al., ) but this could not be concluded whether the orf processing was due to hev-protease or an host-encoded protease. in recent report the role of host factor xa and thrombin was postulated to initiate the hev orf processing but it still needs further validation (kanade et al., ) . recently, mutation in conserved cysteine and histidine residues in the putative protease inhibits its proteolytic activity indicating its role in orf processing and viral replication (paliwal et al., ) . the hev-protease has been reported to act as papain-like cysteine proteases with conserved catalytic dyad (cys and his) (koonin et al., ; parvez and khan, ) , but it was also reported as a chymotrypsin like protease in another study (paliwal et al., ) .the main goal of our study has been to validate the nature of hev-protease and established its role in the polyprotein processing. since, other viral proteases have been identified as a potent antiviral target in literature (lv et al., ) hence, hev-prorease was thought to be a putative inhibitor for hev replication. therefore, we report the expression of a soluble, catalytically active recombinant hev protease using baculovirus system. further, we developed highly sensitive and reliable cell free assays to screen the cysteine protease activity. also a d model of the hev protease was generated to identify possible active sites and the residues responsible for binding of inhibitors. we considered the hev-protease to be a papain like cysteine protease. collectively, this study significantly advances our understanding of the structure and function of hev protease. a bp segment encoding hev-protease was amplified using psk-hev- clone as template by pcr ( figure a) . the purified hev-protease amplicon was cloned in pfastbac/ct-topo vector in frame with xhis at c-terminal, under polyhedrin (ph) promoter ( figure b) . screening of selected clones by pcr using gene specific primers confirmed integration of desired sequence ( figure c) . further, dna sequencing confirmed the clone in correct reading frame with % similarity to the pcp sequences of the genotype- of hev (supplementary figure , data sheets , ) . transformation of pfastbac-pcp in dh bac cells resulted in the formation of recombinant bacmid carrying hev-protease sequence under the ph promoter and the recombination was confirmed through pcr ( figure d ). hence, a recombinant baculovirus containing the hev-protease expression cassette was formed by transfecting represent immunoflorescence of uninfected and infected sf cells after staining with hev-protease epitope specific primary antibody and goat anti-rabbit igg-alexa fluor secondary antibody; (c,f) shows composite image of dapi-and alexa fluor -stained uninfected and infected sf cells. the recombinant bacmid into sf insect cells. transfection leads to cell enlargement, granulation, and vacuole formation after h. integration of hev-protease was confirmed by pcr from baculovirus genomic ( figure e ). in order to confirm the expression of hev-protease in infected sf cells, an indirect immunofluorescence assay was performed using hev-protease epitope specific antibody. the baculovirusinfected sf cells produced fluorescence after staining with hev-protease antibody (table , figure ). the highest expression of hev-protease was obtained at • c at moi after h of infection. sds-page analysis showed expression of ∼ kda hev-protease on sds-page ( figure a ) which was confirmed by immunoblotting using anti hev-protease antibody ( figure b ). the expressed protein was solubilized using % chaps and % dmso. purification was performed by ni + -nta affinity chromatography under native conditions. the eluted protein was > % pure by gel analysis (figure c) . the activity of purified hev-protease was determined by negative staining using native gelatin zymography. gelatin is a non-specific substrate for protease and forms a clear zone on zymography assay due to its digestion. increasing quantity of hev-protease ( , , , and ng) resulted with increased intensity of clear zone due to digestion of gelatin (figure , lane - ). digestion of gelatin was not seen in negative control (figure , lane , ), indicating specificity of the assay. further, protease activity was confirmed by digestion assay using orf as a substrate. orf has been reported to cleave in putative domains of met, pcp, hel, and rdrp (sehgal et al., ) . for the first time we showed digestion of orf polyprotein into smaller fragments. after digestion, kda polyprotein breaks in to and kda products which further breaks into smaller product of , , , and kda (figure , lane - ). no autolysis has been seen in full length orf even after h of incubation at • c ( figure a , lane ) validating the specificity of proteolysis of orf by hev-protease in digestion assay. to confirm the processing of orf , digested samples were immunoblotted using anti-met, anti-protease, anti-rdrp and anti-hel antibodies ( table ) next, ftc-casein was used as a substrate to determine the activity of hev protease. the cleavage of ftc-casein results tca-soluble, ftc-peptides in the presence of active protease. protease activity was quantified by measuring absorbance at nm. as seen in figure a significant protease activity could be detected as compared to the mock reaction. protease activity was proportional to the quantity of the substrate. signal saturation was obtained at h ( figure b ). further experiments were carried out to estimate kinetic parameters of hev protease using the above conditions reaction velocity v (mm/h) was found to be different for each substrate concentration s (mm). accordingly, different v and s the lineweaver-burk graph was plotted against /v and /s to calculate km and vmax. through the slope and the interception of the plot of /v vs. /s, the exact values of km and vmax were determined to be . mm and . mm/h, respectively ( figures c,d) . buffer and assay conditions were optimized to find activity at ph - . . higher ph impaired the protease activity significantly in comparison to lower ph ( figure a ). the optimal temperature for protease activity was found to be - • c ( figure b ). the effect of glycerol (anti-chemotropic agent) was also observed. at - % glycerol concentration, slight increase in protease activity was seen. however, further increase in glycerol concentration resulted in the decrease of protease activity ( figure c ). the results with different na+ concentrations indicated that the optimal concentration was around - mm and that the enzyme is relatively insensitive to na + ( figure d ) analysis of the effect of different divalent cations like ca + , cu + , mg + , mn + , ni + , zn + , fe + , and pb + on the activity of hev protease revealed that ca + , mg + , pb + , and fe + had no effect, however, zn + (p = . ), ni + , pb + (p = . ) and mn + (p = . ) significantly decreased the protease activity ( figure e ). the effect of different protease inhibitors was tested using ftc-casein protease assay in the presence of inhibitors. the enzyme was completely resistant to serine proteases inhibitors (pmsf, aebsf, aprotinin), aspartic proteases (pepstatin), aminopeptidases (bestatin), metallo-endoproteases (phosphoramidon). the enzyme was moderately inhibited by cysteine protease inhibitors (antipain, leupeptin, alln, chymostatin, and e- ) ( figure a ). minimum relative protease activity seen with e- suggested it to be a papain like cysteine protease. further, activity of hev protease on orf digestion was tested in the presence of twelve different protease inhibitors in independent reactions ( figure b) . it was observed that hev protease was strongly inhibited by e- ( %) and chymostatin ( %). moderate inhibition was seen with leupeptin, alln and antipain whereby no inhibition was observed with phosphoramidon, pmsf, aebsf, aprotinin, pepstatin ( figure b ). to further know the active site residue involved in the interaction between hev-protease with inhibitors, its d structure has been modeled which also showed the nature of the hevprotease. the predicted model showed two domains architecture, n-terminal helix and c-terminal β-sheet domain similar to papain-like cysteine proteases (verma et al., ) but differ in arrangement of secondary structure elements. an active site (catalytic pocket) for substrate binding was present between two domains. the residues present in the predicted active site cleft were gln , thr , cys , val , asp , leu , pro , ile , glu , arg , his , asn , leu , met , lys , leu , phe , and gly . a "cysteine(cys)-histidine(his)-asparagine(asn)" catalytic triads has also been seen between n-terminal helical domain and a c-terminal βsheet domain.cys -his -asn residues were found to be involved in this catalytic triad, which is the main characteristic of the "papain" family. one di-sulfide bond observed between cys and cys necessary for stability and the proper folding of the protein. residues present in ctc motif (cys and cys ) and chc motif (cys and cys ) may be involved in zinc metal ion coordination and catalysis ( figure a) . it is reported that the zn binding site present on the opposite side of active site (herold et al., ) , which even validated by crystal structure of hepatitis c virus ns proteases (stempniak et al., ; barbato et al., ; arasappan et al., ) picornavirus a (yu and lloyd, ) pl pro of hcov- e (ziebuhr et al., ) , p of rubv (zhou et al., ) , and lpro of fmdv (guarne et al., ) . the diagrammatic representation indicates presence of chc motif, opposite to the predicted active site, could be zinc binding site of hev-protease (figure ). the inhibition efficiency of all the inhibitors used in protease assay was also proved by docking studies to know the interaction between inhibitors and predicted active site residues of hevprotease. results summarized in table showed that most of the active compounds had good agreement between the docking score and experimental results (r ≈ . ). it suggests that the parameters of docking simulations and quality of structural model are good in reproducing experimental course of these compounds in the modeled hev pcp. the observed difference among e docking scores and interacting residues may be due to difference in binding affinity of different figure | optimal reaction conditions for the protease activity of hev-protease (a) effects of ph on protease activity. hev protease and substrate were incubated with various buffers of ph ranging from to . the relative activity was calculated at different ph at ph . as %. (b) effects of temperature ( - • c), the relative activity was calculated by taking activity at • c as %. (c) effect of glycerol and (d) nacl concentration on protease activity. hev protease and substrate were incubated in a buffer of mm tris (ph . ) containing the indicated concentrations ( , , , , , , and mm) of nacl. (e) effects of various divalent cations ( mm) on protease activity (*p < . , **p < . , ***p < . ) with respect to control. inhibitors as observed in inhibitory efficiency. four inhibitors (e , chymostatin, leupeptin, and alln) showed good ligandreceptor interactions that correlated better with the enzyme inhibition activity of recombinant hev pcp protein with glide docking ( table ). docking result shows that cysteine protease inhibitors e , chymostatin, leupeptin, and alln (docking score − , − . , − . , − . respectively) have strong binding affinity. result of e docking shows that strong h-bond interactions formed with backbone oxygen of leu and lys and side chain oe atom of glu and nine hydrophobic interactions observed with met , gln , cys , ala , val , ile , his , asn , leu , arg , leu , residues ( table ) . all inhibitors bound to the hev pcp active site in a manner similar to the inhibitor e (figure ) . in chymostatin five h-bond interactions formed, two with backbone oxygen of met and leu and one nitrogen atom of asn , whereas two side chain h-bond interactions were observed with oxygen atom of glu and thr and eight hydrophobic interactions were observed with val , his , leu , phe , glu , leu , glu , leu residues. leupeptin had an h-bond with backbone oxygen of asn and twelve hydrophobic interactions observed with gln , met , thr , ala , val , ile , ile , phe , his , leu , arg , leu residues. inhibitor alln showed two side chain interactions with nitrogen atom of glu and oxygen atom of glu , respectively. eight hydrophobic interactions observed with val , his , leu , arg , leu , asn residues. among the remaining inhibitors, pmsf phosparamidon and aebsf which were found to be inactive in the hev pcp enzyme inhibition assay, ligand-receptor interaction analysis showed that inhibitor pmsf and phosparamidon did not formed h-bond interactions ( table ) . three inhibitors aebsf, bestatin and pepstatin showed week h-bond and hydrohobic interactions. interestingly, two inhibitors edta and antipain which were inactive in experimental results remain undocked. (table ) . viral proteases are good targets for development of antiviral therapeutics due to their crucial role in viral polyprotein processing. thus, as a pre-requisite for screening of small molecule inhibitors (smis) against hev protease, we expressed and purified a soluble, catalytically active recombinant viral protease with good yield and homogeneity in baculovirus expression system. further, we developed a robust cell free assay with high sensitivity, selectivity and reproducibility to screen targeted compounds with anti-hev protease activity. we validate hev cysteine protease as a papain like protease on the basis of in silico d modeling and inhibition with specific inhibitors. positive-strand rna viruses are supposed to encode proteases to process their polyproteins usually required for enzymatic activities, interactions with other proteins, subcellular localization, and the viral assembly (debing et al., ) . processing of hev orf by its protease domain remains controversial due to unavailability of well-characterized native hev protease. it was hypothesized that a putative region between residues and of hev-orf encodes protease (koonin et al., ; parvez and khan, ) but the nature of protease, its biochemical and biophysical characterization need to be understood. in some reports, role of cysteine protease in hev processing has been identified but possibility of other proteases could not be ruled out (ropp et al., ; sehgal et al., ) . in previous study hev protease was expressed from different genotype of hev in e. coli (accession no: fj region - ) containing extra amino acid (sfdasqstm) at the cterminal region have amino acids. this refolded protein was also found to be active and classified as chymotrypsin like protease (paliwal et al., ) . in the present study a amino acid long protein of accession no: af _ region - of genotype , similar to the one predicted by putative domain of hev (koonin et al., ; parvez, ) has been expressed using baculovirus expression system and purified in native condition. these constructs having significant variation, could result in difference as mentioned by paliwal et al. ( ) and our study. with the present data it cannot be interpreted the nature of protein with dual properties of papain like cysteine protease or chymotrypsin like properties. further studies will be required to validate this fact. in the present study, recombinant hev protease was expressed using the baculovirus expression system since it is considered to be an efficient insect expression system with the ability to carry post-translational modifications (chambers et al., ) . previously it has been reported that baculovirus system can express toxic proteins and proteases of different organisms (keyvani-amineh et al., ; sali et al., ) . the cell free assay was developed to quantify the activity of hev protease since no robust protocols for protease digestion have been described so far. for development of cell free assays, three different approaches have been used to measure protease activity i.e., gelatin zymography, ftc-casein based assay and digestion of full lengthorf polyprotein. in pevious report gelatin zymography based assays has been used for hepatitis e viruses (paliwal et al., ) , whereby a clearing zone on the slide due to digestion of gelatin was formed. similar to that, a clear zone was observed on zymogram when the native hev protease was used. to determine the conditions for the optimal activity of the protease, it has been reported that cysteine protease activates at lower ph (sundararaj et al., ) . in our study the protease activity has been shown at lower ph which is typical of a cysteine protease. digestion of gelatin in zymography gave the first direct evidence of protease activity by the expressed putative protease domain ( - aa) of porf leading us to express porf in bacteria to be used as the substrate. the natural activity of hev-protease could be studied by proteolysis of orf polyprotein which was expressed via e. coli. bl -de cells. hev-protease cleaves orf polyprotein in a time dependent manner. after h of incubation, bands of ∼ and ∼ kda were seen, which further cleaved into smaller products with time and after h, multiple band of smaller size were seen due to proteolysis of orf . digestion of orf by cysteine protease resulted in ∼ kda rdrp-hel, and ∼ kda met-pcp fragments which were further processed into ∼ kda rdrp, ∼ kda met, and ∼ kda hel fragments with higher incubation time. the above obtained fragments were similar in molecular mass to the ones observed in previous immunoprecipitation studies with replicon transfected hepg cells (panda et al., ) and after expression of orf polyprotein in cell free system as well as in mammalian cells (ropp et al., ) . in addition, ∼ kda intermediately processed form of the rdrp and∼ kda met protein has also been seen in previous study after digestion of kda rdrp-hel and kda met-pcp fragments by immunoprecipitation (paliwal et al., ) . similar size of products also has been seen in self-digestion of baculovirus derived orf polyprotein (sehgal et al., ) . these results suggest role of hev-protease in orf polyprotein processing and hev life cycle. for the first time, enzyme kinetics of native active hev protease has been determined in this study using ftc-casein substrate based assay to validate enzymatic activity and kinetics of hev-protease. the values of km and vmax were found to be . mm and . mm/h, respectively. the optimal temperature for protease activity was found to be - • c which corresponds well to the appropriate environments for hev growth in the cells (emerson et al., ; tanaka et al., ; johne et al., ) . activity of hev protease was found to be ph dependent. the protease activity was found to be optimal at low ph of which is the crucial factor to activate papain like cysteine proteases (richau et al., ; sundararaj et al., ) . a number of reports have shown that cysteine proteases can be auto catalytically activated in vitro from zymogen to mature enzyme even at reduced ph (lowther et al., ; sundararaj et al., ; verma et al., ) indicating it to be a cysteine protease. in an attempt to validate the nature of the protease we used various inhibitors to monitor its activity. twelve inhibitors were used to study the inhibition and it was found that the enzyme was completely resistant to the inhibitors of serine proteases (pmsf, aebsf, aprotinin), aspartic proteases (pepstatin) and metalloproteases (edta) while it was moderately inhibited by cysteine protease inhibitor leupeptin, chymostatin and e- . hev protease was strongly inhibited by e- ( %) and chymostatin ( %), which had been reported to inhibit papain like cysteine protease (saha et al., ) . to confirm these results, modeled hev protease structure has been docked with above inhibitors which shows that cysteine protease inhibitors e , chymostatin, leupeptin, and alln have good docking score and strong binding affinity whereas remaining inhibitors pmsf, phosparamidon, aebsf, bestatin and pepstatin showed no or weak interactions and edta and antipain remain undocked. correlation coefficient was found . which showed good agreement between in vitro and in silico studies, suggesting strongly that the hevprotease is a papain like cysteine protease. besides, in silico modeled structure of hev-protease was found to be similar with papain like cysteine proteases as it has structurally similar n-terminal helix domain and c-terminal β-sheet domain and conserved cys-his-asn triad at the active site (koonin et al., ; coulombe et al., ) . cys and his form a catalytic triad with asn which is present in active site between nterminal helical domain and a c-terminal β-sheet domain of hev-protease. a histidine residue, presents in the active site act as proton donor and increase the nucleophilicity of the cysteine residue. nucleophilic cysteine, act up on the carbon of the reactive peptide bond, release of an amine or amino terminus fragment of the substrate and producing the first tetrahedral thioester intermediate (coulombe et al., ) . this intermediate is now stabilized by hydrogen bonding between highly conserved glutamine residue and substrate oxyanion. subsequently, thioester bond is hydrolyzed and form a carboxylic acid moiety from the remaining substrate fragment (verma et al., ) . earlier, the papain like proteases are characterized by the presence of two domains, an α-helix and a β-sheet domain forming a deep cleft which contains cys-his-asn residues along with gln acting as a substrate-binding site (turk et al., ) . similar observation has been recorded in our predicted d model showing two domain architecture structures where the cys-his-and asn triad is formed along with gln at the active site. further, according to our findings, the cys and his forms a catalytic triad with asn present in active site between nterminal helical domain and a c-terminal β-sheet domain of hev-protease. further, our findings are also in agreement with previous study where hev-protease has been categorized as a papain like cysteine protease (koonin et al., ; parvez and khan, ) . in this study hev protease was found to have these characteristics and on the basis of structural similarity with papain like protease it has been characterized as papain like cysteine protease. these findings are in the agreement of previous study where hev-protease has been categorized as a papain like cysteine protease (koonin et al., ; parvez and khan, ) . effect of different divalent cations like ca + , cu + , mg + , mn + , ni + , zn + , fe + , and pb + were studied on activity of hev protease. out of these divalent cations zn + remarkably decreased protease activity. divalent cations like zinc and its conjugates have been identified as protease inhibitors for sars-cov proteases (hsu et al., ) , also zinc have been found to be essential for structural stability, folding of ns protease of hcv (urbani et al., ) and catalytic activity of rubella protease (liu et al., ) . in another report, zn has been found to be an essential structural cofactor for human corona virus papain like protease (herold et al., ) . thus, we were interested in testing the influence of metal conjugated compound on hev protease activity. further, in silico studies have been performed to determine the zinc binding sites present in hev protease. two zinc binding sites have been identified, as "zn + binding motif " cys -his -cys (chc) and cys -thr -cys (ctc) and these findings are in agreement with previous report (parvez and khan, ) who studied that mutation in cys and cys leads to loss of protease activity (parvez, ; paliwal et al., ) . these residues present in the zinc binding site may play role in its structural stability and proper folding. it was reported that zinc binding site mediates proteolytic activity of corona viral papain like protease (berg and shi, ) . these findings support our report where the loss of protease activity has been found in the presence of zinc, which may act as a cofactor and confer the structural stability. most of the rna viral proteases are categorized either as chymotrypsin like or papain-like proteases (neurath, ) . chymotrypsin like proteases have been found to exhibited with three different catalytic triads ser-his-asp, cys-his-asp, and cys-his-glu (bazan and fletterick, ; gorbalenya et al., ; matthews et al., ) , whereas papain like proteases are found to have the conserved cys-his dyad which are assisted by unique asn and asp residues (gorbalenya et al., ) . in both the chymotrypsin-like or papain-like proteases, zn + is located at the site opposite to the active center (herold et al., ) . therefore, the classification of hev protease not only depends on the involvement of cysteine and histidine but also on structure and sequence homology. papain like cysteine proteases are very stable enzymes and often are found in proteolytically harsh environments (richau et al., ) . besides catalytic dyad/triad prediction we find that hev-protease activity was found to be optimal at low ph of which is the crucial factor to activate cysteine proteases. it is already reported that cysteine protease activates at lower ph (sundararaj et al., ) . in our study the protease activity has been shown at lower ph which is typical of a cysteine protease. on the basis of structural homology, active site residue and class specific inhibition we have classified hev-protease as a "papain-like cysteine protease." spodoptera frugiperda (sf ) cells (invitrogen, california, us) were used for generation, propagation and titration of recombinant baculovirus. sf cells were maintained in sf iii insect cell medium (gibco, newyork, us) and supplemented with % fetal bovine serum (gibco, newyork, us) at • c in refrigerated incubator. hev cdna clone psk-hev- genotype genbank accession no. af was used as a reference virus in this study. all the virus experiments were performed in bsl- facility in shiv nadar university, dadri, india. the hev protease domain (nts - t of hev; genbank accession no. af ) was pcr-amplified by ′ ctcaga attcatggctcagtgtaggcgctg ′ and ′ tctagc ggccgc gagattgtggcgctc tgg ′ forward and reverse primer from full length hev cdna clone psk-hev- using pfu dna polymerase (neb, massachusetts, us). amplified product was cloned into pfastbac/ct-topo vector (invitrogen, california, us) following manufacture's protocol and transformed into one shot tm mach tm t r chemically competent e. coli (invitrogen, california, us). positive transformants were screened by pcr and confirmed by dna sequencing. further, pfastbac/ct-hev protease construct was transformed into max efficiency tm dh bac tm competent e. coli (invitrogen, california, us) that contains baculovirus shuttle vector or bacmid. positive colonies were confirmed by pcr amplification using puc/m reverse and forward primers. the recombinant bacmid was purified using purelink hipure plasmid miniprep kit (invitrogen, california, us) following manufacture's protocol. recombinant bacmid containing hev protease gene was transfected in sf cells using cellfectin tm ii reagent (invitrogen, california, us). briefly × sf cells /well were seeded in six well plates. five-hundred nanogram bacmid dna was mixed with cellfectinii reagent and transfected into sf cells. after h incubation at • c, complete sf iii medium was added and plate incubated at • c for h. supernatant was collected and titrated using plaque assay. further, plaques were purified and amplified in sf cells while the recombinant baculovirus was confirmed through pcr of genomic dna of the infected sf cells. in order to demonstrate the expression of hev-protease in sf cells, × sf cells were seeded in six well plates and infected at moi (multiplicity of infection) with recombinant baculovirus clone having hev-protease gene. after h of infection, cells were fixed with chilled methanol for min, followed by permeabilisation with . % triton x- for min. the cells were blocked for h at room temperature with % bsa in pbst (pbs with . % triton x ). infected sf cells were probed with hev-protease epitope specific antibodies constructed by genscript against - peptide region of hev-protease (table ) for h at room temperature. after three washing with pbs, the cells were stained with goat anti-rabbit igg-alexa fluor (biogenix) for min at room temperature. subsequently, the cells were washed with pbs and their nuclei were stained with dapi ( ′ , -diamidino- -phenylindole) (sigma) for min at room temperature. the cells were washed and fluorescence was visualized using a fluorescence microscope (olympus ix , germany). expression of hev-protease was performed in sf cells as described previously (sehgal et al., ) . briefly × sf cells were seeded in t tissue culture flask and incubated at • c to attach the cells. cells were washed with incomplete sf iii medium and infected with the recombinant baculovirus at moi of . the cells were incubated at • c for min followed by addition of ml sf iii medium (invitrogen, california, us) containing % fbs and incubated at • c for h. the infected sf cells were daily observed for cytopathic effect (cpe) and harvested after h of infection, when the cpe was ∼ %. the harvested cells were centrifuged at , × g for min at • c, washed using pbs and stored at − • c until further use. extraction of hev-protease from sf cells was performed, as described earlier (ramya et al., ) with some modifications. in order to solubilise the hev-protease, × infected sf cells were resuspended in ml resuspension buffer ( mm tris-hcl ph . , mm nacl, % dmso and % chaps) and incubated on ice for min. the cells were sonicated for min ( s on s off) at % amplitude on ice and incubated for h at • c in end to end rotator. cell lysate was centrifuged at , x g for h at • c. solubilized protein was purified through immobilized metal affinity chromatography (imac) using ni-nta super flow chelating agarose column (qiagen, germany). the column was equilibrated with buffer a ( mm tris-hcl, mm nacl, . % chaps and mm imidazole ph . ), followed by washing with buffer b ( mm tris-hcl, mm nacl, . % chaps and mm imidazole ph . ). the protein fraction was eluted in buffer c ( mm tris-hcl, mm nacl, . % chaps and mm imidazole ph . ) and analyzed on % sds-page. theelutes were pooled and dialyzed against pbs and analyzed by western blot using hev-protease epitope specific antibodies (genscript, us) ( table ) . activity of purified recombinant hev-protease was determined by gelatin zymography, as described earlier (saitoh et al., ) . briefly % sds-page having mg/ml gelatin was polymerized in mm thickness plate. different quantities of recombinant hev-protease ( - µg), cell lysate of uninfected cells and trypsin were loaded as negative and positive control, respectively. the gel was run at volt until separation, washed twice for min at • c with wash buffer ( . % triton x- , mm tris-hcl, ph . , mm cacl and µm zncl ) to remove sds. the gel was rinsed twice with distilled water and incubated at • c in incubation buffer ( % triton x , mm tris-hcl, ph . , mm cacl and µm zncl ) for h. after incubation, gel was stained by coomassie brilliant blue r (cbbr) ( . g in % methanol, % acetic acid, and % distilled water). a clear zone was visualized after destaining with % methanol, % acetic acid, and % distilled water. the role of hev-protease in orf polyprotein processing was determined by digestion assay. briefly, full length orf polyprotein was expressed in e. coli. bl cells and purified by ni-nta chromatography and characterized by western blotting using hev epitope specific antibodies ( table ) . the kda protein was used as a substrate for hev-protease. to set up digestion assay, ng of orf polyprotein was incubated with ng of hev-protease in . m tris-hcl (ph . ) and incubated at • c for - h. further, to check internal protease activity in full length orf , it was incubated without hev-protease for h. after incubation, protein was fractioned on sds-page and size of digested products was checked by western blotting using epitope specific antibodies against all four enzymes present on orf viz. methyl transferase, cysteine protease, helicase and rdrp (table ) . ftc-casein based assay was performed to check the activity of protease as described earlier (twining, ) . fifty micrometer ftc-casein was incubated with µm hev-protease for - h at • c. similar reaction was set up with trypsin as a positive control and without any protease. protease cleaves ftc-casein into smaller, tca-soluble, ftc-peptides. . % tca was added to the reaction mixture, to precipitate any remaining undigested ftc-casein. the supernatant was collected following centrifugation at , g at • c and the ftc-peptides were quantified by measuring the absorbance at nm in spectrophotometer. the intensity of color estimated by the assay was found to be proportional to the total protease activity present in the sample. biochemical properties of hev-protease were characterized through kinetic studies under varying reaction conditions like ph ( - ), temperature ( - • c), salt concentration ( − mm nacl), presence of glycerol ( - %) and various enzyme and substrate concentration described previously (ahmed et al., ) . finally apparent kinetic parameters (v max and k m ) of the hev-protease were determined by ftc-casein based protease assay. the initial velocity was measured using varying substrate concentration and the reciprocal of initial velocity and substrate concentration were plotted using lineweaver-burk curve to determine briefly, different concentration of the substrate ( . - . mm) was incubated with µm hevprotease in x incubation buffer ( mm tris-hcl, ph . , mm cacl ) at • c. to measure enzyme kinetics, absorbance was taken in multiplate reader (bio-rad) at nm for - h and initial velocity of each reaction was determined. finally, the reaction rate was calculated using michaelis-menten equation (michaelis and menten, ) vmax is the maximum rate of reaction and km is the substrate concentration at which reaction rate is half of the obtained vmax. further, the effect of various activators or divalent cations on the protease activity was examined by supplementing the reaction with mm of cucl , nicl , mgcl , cacl , zncl , mncl , fecl , and pbcl as described above. similar reaction was also performed in the presence of various types of protease inhibitors (protease inhibitor set; g biosciences) to determine the nature of protease by inhibition assay, briefly, µl ( µm) hev protease was pre-incubated with × protease inhibitors aebsf, alln, antipain dihydro chloride, aprotonin, bestatin, chymostatin, sodium edta, e- , leupeptin, pepstatin, phosphoramidon and pmsf and the inhibition assay was performed using ftc-casein. inhibition of protease activity was measured as relative absorbance at nm. for relative inhibition activity, same reaction was performed without any inhibitors. in order to validate the inhibition, orf digestion was performed in presence of these inhibitors. a protease-negative control with ng porf was incubated in parallel under the same conditions to monitor porf autolysis. these reactions were incubated for h at • c for over-digestion. all the reactions and controls were resolved by % sds-page. the d model of hev pcp (aal . , region - ) was generated using homology modeling, fold recognition and threading techniques (zhang, ; roy et al., ; yang et al., ) . initially i-tasser was used for modeling of amino acid residues; unfortunately only c-terminal region could be modeled using nu as a template and n-terminal region remain unmodeled. integrated protein structure and function prediction server intfold version . (roche et al., ; mcguffin et al., mcguffin et al., , was used for modeling of n-terminal residues - amino acids of orf . finally easy modeler was used for final model generation (kuntal et al., ) . the generated model was energy minimized in water using opls (optimized potencials for liquid simulations) force field with the convergence threshold of . by using macromodel of maestro (macromodel, ) , to remove steric clashes between atoms and to improve overall structural quality of predicted models (sastry et al., ; protein preparation wizard, ) . d model was validated on the basis of stereochemical and geometric consideration and docking studies (glide, ) . the top ranked model was further validated and analyzed based on their ramachandran plot, verify d and errat analysis (bowie et al., ; lüthy et al., ; laskowski et al., ; colovos and yeates, ) . to identify possible binding sites, site map of maestro was used for binding site prediction (halgren, (halgren, , sitemap, ) . further, receptor grid generation was done using glide module. docking study was done in using grid based ligand docking with energetic (glide) (friesner et al., , halgren et al., ) . the d model of hev pcp was docked with inhibitors retrieved from pubchem search. eleven known protease inhibitors were docked against hev-pcp structural model. the different conformations of the compounds were docked flexibly and maximum , poses per compound were generated (ligprep, ) . the analysis of the poses, complexes and the binding affinities between the receptor and ligands was analyzed using schrodinger's software glide and correlation coefficient between inhibitory concentration and docking score was calculated using "correl" function of ms excel (microsoft corp., usa). for enzyme kinetics, initial velocities were calculated using the linear regression function in the microsoft office excel software. data was analyzed and plotted using michaelis-menten equation with graph pad prism to obtain kinetic parameters. all the assays were performed in triplicate and results were graphed, with error bars indicating the sd. inhibition assay data was analyzed using student's t-test p < . was considered statistically significant. each experiment was performed thrice in triplicate. all datasets generated for this study are included in the article/supplementary material. ds conceived the study, designed the experiments and prepared the manuscript. ss designed, carried out the experiments performed the data analysis and drafted the manuscript. mc designed, carried out the insilco work and data analysis. all the authors reviewed the manuscript. this study was funded by department of science and technology, india serb grant emr/ / , pi: ds and npdf-serb grant pdf/ / , pi: ss. macromodel epidemiology of hepatitis e: current status the ′ end of hepatitis e virus (hev) genome binds specifically to the viral rna-dependent rna polymerase (rdrp) molecular virology of hepatitis e virus purification and 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system for hepatitis e virus characterization of a prototype strain of hepatitis e virus determination of protease cleavage site motifs using mixture-based oriented peptide libraries fluorescein isothiocyanate-labeled casein assay for proteolytic enzymes the metal binding site of the hepatitis c virus ns protease. a spectroscopic investigation cysteine proteases: modes of activation and future prospects as pharmacological targets orf protein of hepatitis e virus is essential for virion release from infected cells the i-tasser suite: protein structure and function prediction characterization of the roles of conserved cysteine and histidine residues in poliovirus a protease i-tasser server for protein d structure prediction identification of a ca +-binding domain in the rubella virus nonstructural protease human coronavirus e papain-like proteases have overlapping specificities but distinct functions in viral replication we thank dr. s. emerson for providing pskhev plasmid. the authors would like to thank shiv nadar university for its support and providing infrastructure to carry out this work. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/ . /fcimb. . /full#supplementary-material key: cord- -xdpb ukt authors: lhomme, sebastien; garrouste, cyril; kamar, nassim; saune, karine; abravanel, florence; mansuy, jean-michel; dubois, martine; rostaing, lionel; izopet, jacques title: influence of polyproline region and macro domain genetic heterogeneity on hev persistence in immunocompromised patients date: - - journal: j infect dis doi: . /infdis/jit sha: doc_id: cord_uid: xdpb ukt hepatitis e virus (hev) can chronically infect immunocompromised patients. the polyproline region (ppr) and the macro domain of orf protein may modulate virus production and/or the host immune response. we investigated the association between the genetic heterogeneity of hev quasispecies in orf and the outcome of infection in solid-organ transplant patients. both sequence entropy and genetic distances during the hepatitis e acute phase were higher in patients whose infection became chronic than in those who cleared the virus. hence, great quasispecies heterogeneity in the regions encoding the ppr and the macro domain may facilitate hev persistence. hepatitis e virus (hev) can chronically infect immunocompromised patients. the polyproline region (ppr) and the macro domain of orf protein may modulate virus production and/or the host immune response. we investigated the association between the genetic heterogeneity of hev quasispecies in orf and the outcome of infection in solidorgan transplant patients. both sequence entropy and genetic distances during the hepatitis e acute phase were higher in patients whose infection became chronic than in those who cleared the virus. hence, great quasispecies heterogeneity in the regions encoding the ppr and the macro domain may facilitate hev persistence. keywords. hepatitis e; chronic infection; orf ; polyproline region; macro domain. hepatitis e virus (hev) is a major cause of enterically transmitted non-a, non-b hepatitis. it is also responsible for large outbreaks of waterborne acute hepatitis in tropical and subtropical countries as well as sporadic infections worldwide. hepatitis e is a zoonotic disease in industrialized countries, with pigs, wild boar, and deer being the major animal reservoirs of hev [ ] . hev, genus hepevirus, family hepeviridae, is an unenveloped, single-stranded, positive-sense rna virus. like all rna viruses, hev exists as a mixture of closely related variants defining a quasispecies. the approximately . kb genome of hev has a coding region consisting of open reading frames (orfs). orf encodes the capsid protein; orf encodes a small protein implicated in virus egress [ ] , whereas orf encodes a nonstructural protein that contains several putative functional domains. these include at least enzyme activities: methyltransferase, cystein protease, helicase, and rna-dependent rna polymerase [ ] . homologies with other plant and animal positive strand rna viruses have been used to identify other domains: the y domain, the polyproline region (ppr), and the macro domain. the ppr is genetically very diverse and could correspond to an intrinsically disordered region involved in virus adaptation [ ] . in addition, the ppr does not seem to be required for hev replication in vitro or in vivo [ ] . nonstructural virus genes that are not essential for replication are usually involved in modulating host immune responses [ ] . the genomes of several virus families, including all members of coronaviridae, rubella virus, and alphaviruses (togaviridae), and hev, have macro domains. the macro domain of the mouse hepatitis virus (mhv) influences the pathogenicity of the virus [ ] . hev can lead to chronic hepatitis in solid-organ transplant (sot) patients [ ] . but our knowledge of the factors associated with the development of chronic infection in sot patients exposed to hev is far from complete. our working hypothesis was that the genetic heterogeneity of the ppr or the macro domain play a role in the outcome of hev infection in immunocompromised patients, as the ppr could modulate the host immune response and the macro domain could influence virus pathogenicity. we therefore analysed the characteristics of hev quasispecies at the acute phase of hepatitis e in groups of sot patients, one whose infection became chronic and the other who cleared the virus. we studied sot patients who became acutely infected with hepatitis e between january and june . the infection was diagnosed by the detecting hev rna using the real time polymerase chain reaction (pcr) and immunoglobulin m/immunoglobulin g anti-hev antibodies by a commercial enzyme-linked immunosorbent assay [ ] . each patient was assigned to one of groups according to the outcome of the infection. the first group (group i; patients) had chronic infections, defined by persistently elevated liver enzyme activity and serum that was positive for hev rna for more than months after diagnosis. the second group (group ii; patients) had resolving infections. serum samples were collected from all patients at the acute phase of their infection and stored at − °c. each patient underwent an exhaustive clinical and laboratory examination, including the immunosuppressive drugs used, the hepatic enzyme activities, and white blood cell count. the serum concentration of hev rna was measured by real-time pcr [ ] . virus genotype was determined by sequencing a nucleotide fragment within the orf gene. the sequences were compared to reference hev strains (genbank) as described elsewhere [ ] . nucleic acids were extracted from serum samples and analysed by -step rt-pcr with the sense primer hevorf -s and anti-sense primer hevorf -a using the super script iii enzyme (invitrogen, cergy-pontoise, france). the sequences of the primers are listed in supplementary table . the sequence amplified included the ppr (nucleotide [nt] - ) and the macro domain (nt - ), using the genome b l as a reference. the pcr products were purified using qiaprep (qiagen, courtaboeuf, france) miniprep kits and stored at − °c. in total, ng of the amplified sequence were directly ligated into µl of pcr vector (kit topo ta cloning; invitrogen) containing a gene conferring resistance to ampicilline. recombinant plasmids were used to transform escherichia coli-competent cells, and transformants were grown on ampicillin-coated plates at °c for hours. the complementary dna (cdna) clones ( from each patient sample) were analysed by pcr with the primers hevorf -s and hevorf -a (supplementary table ). the pcr products of these amplifications were purified (quick-spin columns; qiagen) and sequenced using the fluorescent dye terminator method for big dyeterminator cycle sequencing (applied biosystems, paris, france) with the primers hevorf -s , hevorf -s , hevorf -s , hevorf -a and hevorf -a (supplementary table ) on an applied biosystems abi xl analyzer. sequences were aligned using clustal x and compared with those of hev strains obtained from genbank. we quantified the complexity of the hev quasispecies by calculating the shannon entropy: s = −Σ i ( p i ln p i ), where pi is the frequency of each sequence in the viral quasispecies. the normalized entropies for both nucleotides and amino-acids, sn, were calculated using sn = s/ln n, where n is the total number of sequences analysed. we quantified diversity as the mean genetic distance calculated for all pairs of nucleotide sequences using mega . . the numbers of synonymous (ks) substitutions per synonymous site and nonsynonymous (ka) substitutions per nonsynonymous site were calculated with the jukes-cantor correction for multiple substitutions using mega . . the ka/ks ratio is an indicator of the positive (> ) or negative (< ) selection pressure on a quasispecies [ ] . proportions were compared by fisher exact test. quantitative variables were compared with the nonparametric mann-whitney test. correlations between complexities or diversities of quasispecies were estimated by calculating spearman rank correlation coefficient. a p-value below . was considered to be statistically significant. the sequences have been deposited in the genbank database under accession numbers kc to kc . the clinical and biological characteristics of the patients are summarized in supplementary table . there was no significant difference between patients with chronic infections and those with resolving infections in terms of gender, age, or immunosuppressant treatment. the alt activities of individuals with a chronic infection tended to be lower. patients whose infection became chronic had lower total, cd , cd , and cd lymphocyte counts, but the differences were not significant. the serum hev rna concentrations at the acute phase of the groups of patients were similar. they were all infected with hev genotype f strains, except one whose infection was genotype c. we compared the sequence heterogeneity of regions of orf in patients with chronic hev infection and those with resolving infections. the mean nucleotide sequence entropy (nt complexity) of the ppr was higher in group i [ ] . the viral determinants associated with the persistence of such an infection are poorly documented. we therefore investigated the influence of the genetic heterogeneity of hev quasispecies on the outcome of infection, focusing on the ppr and the macro domain. both the complexity and diversity of the ppr and the macro domain were higher in viral population of the patients whose infection became chronic than in those who cleared the virus. it has been shown that the quasispecies heterogeneity in the orf region encoding the capsid protein during the acute phase of infection is associated with the development of a chronic hev infection [ ] . our data support the finding that great genetic heterogeneity of the quasispecies in patients whose infection become chronic seems to favor the appearance of variants that can persist, as reported for hcv infections [ ] . although the diversity of the region preceding the ppr was higher in patients with chronic infection, nt and aa complexities were not different in the groups (data not shown), suggesting that the higher heterogeneity in chronic patients was not a general effect seen across the entire region studied. this great genetic heterogeneity could also reflect an inadequate control of the viral replication, but no correlation was found between quasispecies heterogeneity and viral concentrations. ppr appears to be dispensable for in vitro hev replication, but it is required for in vivo infection, suggesting that it is involved in infecting cells with innate immunity [ ] . although the role of the macro domain in the replication of hev is unknown, it does not seem to be essential for the replication of coronavirus in cell culture [ ] . it was recently suggested that genes that are dispensable for virus replication are involved in modulating the host immune response, like down-regulating interleukin β (il- β) or tumor necrosis factor α (tnf-α) secretion [ ] . the macro domain is also involved in the inflammation caused by the mouse hepatitis virus (mhv), and the substitution of a strictly conserved amino-acid residue is responsible for reducing the secretion of inflammatory cytokines [ ] . the great quasispecies heterogeneity in patients whose infection became chronic may include some variants that reduce inflammatory cytokine production, which could facilitate hev persistence. this could explain the lower serum concentrations of interleukin (il- ) receptor antagonist and tnf-α found in patients whose infection became chronic [ ] . we find that the complexities and diversities of the ppr and the macro domain are correlated. these regions may well have evolved together as the protein encoded by orf does not seem to be cleaved [ ] . we also studied the correlations between the orf and orf as this study and our previous one on orf diversity [ ] were carried out on the same patients (except for ). we also find that the complexity and diversity of the regions in orf and orf that were studied are correlated (data not shown), suggesting that they, too, have evolved together. finally, we find no differences in the ka/ks ratios of the studied regions, even though the ka/ks ratios of the ppr seemed to be higher in patients who cleared the virus. alternatively, the ka/ks ratio may not allow us to infer differences in selection pressure. in both regions, ka/ks ratios indicate negative selection, probably due to structural or functional constraints. a limitation of our study is the small number of patients in each group and an implication of immunological factors in the evolution toward chronicity cannot be excluded. we conclude that the high genetic heterogeneity of the ppr and the macro domain at the acute phase of an hev infection is associated with persistence of the virus. this association may be due to the appearance of mutants able to modulate the host immune response. further investigation is now needed to confirm this association. supplementary materials are available at the journal of infectious diseases online (http://jid.oxfordjournals.org/). supplementary materials consist of data provided by the author that are published to benefit the reader. the posted materials are not copyedited. the contents of all supplementary data are the sole responsibility of the authors. questions or messages regarding errors should be addressed to the author. molecular virology of hepatitis e virus the hepatitis e virus polyproline region is involved in viral adaptation deletions of the hypervariable region (hvr) in open reading frame of hepatitis e virus do not abolish virus infectivity: evidence for attenuation of hvr deletion mutants in vivo immunodominant epitopes in nsp of porcine reproductive and respiratory syndrome virus are dispensable for replication, but play an important role in modulation of the host immune response mouse hepatitis virus liver pathology is dependent on adp-ribose- ′′-phosphatase, a viral function conserved in the alpha-like supergroup hepatitis e virus and chronic hepatitis in organ-transplant recipients characteristics of autochthonous hepatitis e virus infection in solid-organ transplant recipients in france genotype diversity and quantification of hepatitis e virus rna hepatitis e virus genotype diversity statistical methods for detecting molecular adaptation hev quasispecies and the outcome of acute hepatitis e in solid-organ transplant patients the outcome of acute hepatitis c predicted by the evolution of the viral quasispecies adp-ribose- ′′-monophosphatase: a conserved coronavirus enzyme that is dispensable for viral replication in tissue culture early secretory pathway localization and lack of processing for hepatitis e virus replication protein porf financial support. this work was supported by institut national de la santé et de la recherche médicale u .potential conflicts of interest. all authors: no reported conflicts. all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. key: cord- - pw qje authors: dryden, kelly a.; tihova, mariana; nowotny, norbert; matsui, suzanne m.; mendez, ernesto; yeager, mark title: immature and mature human astrovirus: structure, conformational changes, and similarities to hepatitis e virus date: - - journal: journal of molecular biology doi: . /j.jmb. . . sha: doc_id: cord_uid: pw qje abstract human astroviruses (hastvs) are a major cause of gastroenteritis. hastv assembles from the structural protein vp and undergoes a cascade of proteolytic cleavages. cleavage to vp is required for release of immature particles from cells, and subsequent cleavage by trypsin confers infectivity. we used electron cryomicroscopy and icosahedral image analysis to determine the first experimentally derived, three-dimensional structures of an immature vp virion and a fully proteolyzed, infectious virion. both particles display t = icosahedral symmetry and nearly identical solid capsid shells with diameters of ~ Å. globular spikes emanate from the capsid surface, yielding an overall diameter of ~ Å. while the immature particles display dimeric spikes, the mature capsid only displays spikes, located on the icosahedral -fold axes. loss of the peripentonal spikes likely plays an important role in viral infectivity. in addition, immature hastv bears a striking resemblance to the structure of hepatitis e virus (hev)-like particles, as previously predicted from structural similarity of the crystal structure of the astrovirus spike domain with the hev p-domain [dong, j., dong, l., méndez, e. & tao, y. ( ). crystal structure of the human astrovirus capsid spike. proc. natl. acad. sci. usa , – ]. similarities between their capsid shells and dimeric spikes and between the sequences of their capsid proteins suggest that these viral families are phylogenetically related and may share common assembly and activation mechanisms. human astroviruses (hastvs) are an important cause of gastroenteritis in children, the elderly, and immunocompromised adults. the virus was first identified in in infants who developed diarrhea in hospital nurseries in the united kingdom. , hastv is a small, single-stranded, positive-sense rna virus with an~ . -kb genome. the ′-terminal two-thirds of the genome [open reading frame (orf)- a and orf- b] encodes two major nonstructural proteins, a serine protease whose structure has been solved by x-ray crystallography and an rnadependent rna polymerase. the ′-terminal onethird of the genome (orf- ) encodes the structural protein vp , which is translated as an -to -kda capsid precursor protein. vp has a highly conserved n-terminal domain and a variable c-terminal domain among astrovirus serotypes. to date, eight serotypes have been identified of which type is the most prevalent, and type has been studied extensively. while their genomes are fairly well conserved, - the mature virions of different serotypes vary somewhat in the exact size and number of structural proteins that form the capsid (reviewed by krishna ) . the current consensus is that morphogenesis of infectious particles relies on a series of proteolytic cleavages of the capsid precursor protein (fig. ). vp assembles in infected cells and undergoes c-terminal cleavages by caspases to generate vp (a -to -kda protein) for release from the cells as immature virus particles. generation of infectious particles is dependent upon further trypsin cleavages usually resulting in three structural proteins, vp , vp , and vp , each of which range in actual size and name depending on the serotype. - vp contains the conserved n-terminal domain and is ascribed to the capsid, while the overlapping vp and vp subunits contain the variable c-domain and are ascribed to the viral spikes. , vp is generated by additional trimming at the n-terminus of vp . astroviruses were initially named for the distinctive star-like appearance of their viral surface observed in~ % of the fecally shed viral particles evaluated by negative-stain electron microscopy (em). em ultrastructural analysis of infectious hastv generated in cell culture showed spherical particles with a surface that was studded with spikes and an external diameter of~ Å. a recent milestone was determination of a highresolution x-ray structure of the p-domain of the astrovirus spike vp (ordered residues - ). similarities in sequence and domain organization of hepatitis e virus (hev) and hastv enabled the building of a homology model of hastv. in this study, we sought to gain direct insight into the structure and assembly of astrovirus by electron cryomicroscopy (cryoem) of immature and proteolytically activated particles. we examined ( ) immature hastv- particles composed of the -kda intermediate protein, which has undergone caspase but not trypsin cleavage and is therefore not infectious, and ( ) fully cleaved, mature infectious particles of hastv- . the sequences of the two strains are extremely similar, with % identity in the conserved domain and % identity in the variable domain. therefore, we expect that our three-dimensional ( d) reconstructions are representative of the astroviridae family. although the capsid shells of both immature and mature hastvs are nearly identical, there is a striking difference in the apparent stoichiometry of the surface spikes. immature, uncleaved particles, which are strikingly similar in appearance to hev-like particles (hev-lp), trypsin cleavage of the coat protein between the conserved (white boxes) and variable (shaded boxes) domains is required for viral maturation. the specific pattern of peptide products varies between serotypes. in these experiments, we examined uncleaved particles of hastv- formed by vp and fully cleaved particles of hastv- . have spikes, whereas mature, cleaved particles only display spikes, all located at the icosahedral -fold axes. loss of the peripentonal spikes likely plays an important role in viral infectivity. astrovirus particles have t = icosahedral symmetry purification of hastv has been very challenging, and sufficient material was only available to perform cryoem once for immature hastv- and mature hastv- . icosahedral image analysis of electron cryomicrographs (fig. ) yielded d maps at~ Å resolution ( fig. a and b ). both particles display two concentric layers of density that conform to t = icosahedral lattice symmetry. a nearly identical inner layer forms a solid capsid shell Å thick with an outer diameter of~ Å. this capsid shell has a chiseled appearance assembled from protein subunits in which trimeric facets form a plateau ( fig. a and b, bottom) . the trimeric facets are in close contact across the icosahedral -fold ( f) symmetry axes, with depressions at the -fold ( f) and -fold ( f) symmetry axes and a groove across the local f axes. distal from the solid capsid shell is an outer layer of globular spike-like densities. the spikes are~ Å in diameter, with a somewhat elliptical shape, yielding an outer particle diameter of~ Å. the stoichiometry suggests that the spikes are dimers with contributions from adjacent trimer facets (fig. a , color code and labels). thin linker densities connect the spikes to the capsid shells and are visible in d reconstructions of negatively-stained particles ( fig. s ) or when the isosurface is lowered significantly in the cryoem maps. the lack of well-defined linker density may be the result of the narrowness of the linker, minor heterogeneity in maturation cleavage, or wobbling of the spike head, which would obscure the connections during icosahedral symmetry averaging. although the capsid shells of both immature and mature hastvs are nearly identical, there is a striking difference in the apparent stoichiometry of the surface spikes. immature, uncleaved particles have spikes (fig. a, top) . thirty are located at the icosahedral f symmetry axes, and sixty surround the f vertices, centered at the local f axes. mature, cleaved particles only display spikes, all located at the icosahedral f axes (fig. b , top). the packing of the viral proteins displays two arrangements for the dimer interaction of the spikes-one that places the dimer densities in close proximity (cc dimers) and one that spans nearly twice the distance between the contributing subunits (ab dimers) (fig. a , bottom labels). this difference in separation may expose different cleavage sites during maturation. proteolytic cleavage is a common mechanism for activation of both enveloped and nonenveloped viruses, including influenza virus, , coronavirus, rotavirus, reovirus, and alphaviruses. a recurring theme is that cleavage triggers a conformational change in a surface spike or other cell-attachment domain. for astrovirus, the uncleaved, immature t = particle contains copies of vp , in which the viral capsid is attributed to the n-terminal domains and the spikes are attributed to dimers of the variable c-terminal domain. similarly, trypsin cleavage of the immature particles produces three products, of which we correspondingly attribute vp to the capsid shell and the two different c-terminal products, vp and vp , to the spikes. sds-page suggests that trypsin cleaves virtually all of the vp molecules (fig. s ). in addition, the d reconstruction in fig. b suggests that trypsin cleavage results in release of two-thirds of the spikes in the mature particles. however, we note that the bands of vp and vp on sds-page gels display intensities similar to that of vp , suggesting that trypsin cleavage might lead rather to conformational flexibility and that the products of proteolysis, or a significant percentage of them, could remain noncovalently attached to the capsid shell. limitations in material have precluded careful quantitative analysis to resolve this question. however, we note that the trypsintreated particles in fig. b have noticably less surface density than the particles in fig. a . if the cleaved proteins remain noncovalently associated with the capsid shell, then one would still expect to see surface density in the images of individual particles, even if the polypeptides do not conform to icosahedral symmetry. consequently, our working hypothesis is that trypsin cleavage leads to loss of peripentonal spikes, which confers infectivity, possibly by exposing additional receptor binding sites on the capsid surface. the structures of immature hastv- and hev-lp are remarkably similar the size, shape, and architecture of immature hastv- are very similar to those reported for a t = hev-lp (fig. c) . in the case of hev-lp, a t = x-ray crystal structure allowed detailed modeling of the protein subunits, which led to the proposal that the capsid is assembled from dimers around the f axes and across the icosahedral f axes. the hev capsid protein is divided into three domains, defined as the shell (s-domains), intermediate (m-domains), and protruding densities (p-domains). dong et al. identified the hev p-domain as structurally similar to their crystal structure of the astrovirus dimer (construct p - ), which represents most of vp by dali alignment, and from which they proposed a phylogenetic relationship. they also used the hev t = x-ray structure to create a homology model of the astrovirus core sequences (residues - ) and docked their crystal structure of residues - in place of the hev p-domain. our experimental results support the proposed relationship between hastv and hev. both in silico was docked into the d cryoem density map of hastv- (b). the close-up view at the right shows a -Å-thick central slab of the density maps. with no scaling or translation, the maps align very well, except that the hastv spikes extend to a higher radius (arrow) than those of hev. density maps are in gray scale, and for clarity in the enlarged views, the interior density was set to zero at a radius of Å. the backbone chains are color coded by domain as defined in (a) for hev; s-domain (green), m-domain (blue), and p-domain (red). atomic models match exceedingly well with the hastv density maps and dock without any scaling: the hev-lp model into our vp immature structure (figs. c and a ) and the hastv model into our mature structure (figs. d and b) . the similarity reinforces the supposition that vp forms the capsid shell and that vp /vp complexes comprise the spikes. the one noticeable difference between the structures is that the astrovirus spikes extend to a radius greater than that of hev-lp (fig. a, arrow) . in comparison, structures from the caliciviridae family, many of which are also t = viruses that assemble as dimers, do not dock nearly as well, with differences in capsid features and orientation of their p-domains (data not shown). furthermore, although the conservation is low, the top score for viral proteins from a naïve national center for biotechnology information blast (basic local alignment search tool) search of the protein database for the hastv capsid protein identified similarity with the hev capsid protein (e = e − ) (fig. ) . the sequence in the hastv conserved domain, amino acids - , has % identity with the capsid s-domain of hev. aaa ) . a pair of proline residues in hastv were aligned with the hev hinge (red ellipse), and a long spacer inserted for hastv to account for the greater distance from the capsid observed for its spike. yellow highlights identical residues, and orange highlights similar residues. regions of the hastv vp sequence can also be aligned to the hev m-and p-domains with moderate identity (~ %) (fig. b) . when the hev capsid protein is used as the reference sequence, the search identifies the conserved domain from multiple astrovirus species, with the strongest scores for bat and bovine astroviruses (e b e − ). it is also interesting to note that, while most astroviruses cause gastroenteritis in their host, duck astrovirus causes fatal hepatitis. possible similarities in assembly and activation of hastv and hev although the astrovirus polyprotein is larger bỹ residues, there may also be biological similarities between hastv and hev. for instance, in vitro assembly of expressed hev capsid protein requires deletion of the c-terminal residues, which may be analogous to caspase cleavage of vp and removal of~ amino acids to generate vp (fig. ) . as there is no cell culture system currently capable of propagating hev, little is known about its infectivity. nevertheless, it is reasonable to speculate that a cleavage cascade similar to hastv may be required for both assembly and activation. furthermore, by analogy with the hev-lp model, we may be able to infer that the differences between the ab and cc dimers of immature astrovirus is relevant for assembly, in addition to activation. in the case of hev-lp, there is a measureable difference in the hinge angles between the ab and cc dimers, which may explain the similar differences observed in hastv. it was proposed for hev that the flatter angle of the cc dimers is required to make t = rather than t = particles and only happens in the presence of rna. , the need for rna as a structural component may account for the inability, to date, to assemble stable astrovirus-like particles from expressed proteins. both systems may provide insights for the other. production and purification of hastv- the astrovirus strain yuc was isolated from a natural infection and adapted to grow in caco- cells. to propagate the virus, we washed the cells twice with minimal essential medium without fetal bovine serum and inoculated them with yuc , previously treated with μg/ml of trypsin for h, followed by a -h treatment with soybean trypsin inhibitor at the same concentration. infected cells were incubated at °c and harvested day post-infection. the cells were lysed by three freeze-thaw cycles and extracted once with an equal volume of genetron (trichlorotrifluoroethane). from this point, every step was carried out at °c, and solutions were maintained in an ice-water bath. the aqueous phase obtained after centrifugation at g for min was pelleted via ultracentrifugation for h at , g (beckman sw rotor). the pellet was resuspended in tne buffer [ mm tris-hcl (ph . ), . m nacl, and mm ethylenediaminetetraacetic acid] and layered onto a cesium chloride solution ( . g/ml). a density gradient was formed during ultracentrifugation for - h at , g (beckman sw . rotor), and the opalescent band corresponding to viral particles was collected and diluted in tne buffer. the final pellet was isolated by ultracentrifugation of this suspension for . h at , g (beckman sw . rotor), which was resuspended in tne buffer and stored at °c. infectivity assays and protein analysis of trypsin-treated virus were performed to ensure the identity of the purified particles. production and purification of hastv- monolayers of a continuous line of colon carcinoma cells (caco- ) were infected with hastv serotype (oxford strain) that was initially adapted to grow in a continuous line of monkey kidney epithelial cells (llcmk ). virus propagation required the addition of trypsin ( μg/ml, type ix; sigma) to the culture medium (rpmi ). at h post-infection, cells were lysed by three freeze-thaw cycles, and the lysates were partially purified by fluorocarbon extraction and differential centrifugation. the viral pellets were solubilized in tnmc buffer [ mm tris-hcl (ph ), mm nacl, mm mgcl , and mm cacl ]. the suspensions were then layered on a cesium chloride density gradient ( . g/ml). following isopycnic centrifugation, the fraction containing peak enzyme immunoassay activity corresponded to a density of . g/ml. this fraction was diluted -fold with tnmc buffer, and viral particles were concentrated by ultracentrifugation for . h at °c ( , g). samples were prepared for cryoem by standard methods. in brief, -μl aliquots from each viral preparation were applied to freshly glow-discharged, holey carbon grids, blotted to near dryness, and flash frozen in a slurry of liquid ethane. grids were maintained at liquid nitrogen temperatures in a gatan cryo-holder and examined using an fei cm electron microscope (eindhoven) under low electron dose conditions as previously described. images were recorded on kodak so- low-density film at nominal magnifications of , or , ×. micrographs with minimal astigmatism and drift were digitized on a zeiss microdensitometer (z/i imaging) at a -μm sampling interval corresponding to . or . Å resolution at the specimen. particles were digitally extracted from the micrographs using the program x d. a linear background gradient was subtracted from the masked particle images, and the contrast was normalized. underfocus values were calculated using robem ‡ and ranged from − . to − . μm. correlation methods (ppft) were used to derive particle orientations, and the d reconstructions were generated by fourier-bessel inversion. initial models were determined from cycles of the random model calculation. additional rounds of image processing restricted the data search to the outer radii (r = - Å), but definition of the surface spikes did not improve. the data sets were randomly divided into two groups of particle images, and the two resulting maps were compared by fourier shell correlation analysis, from which the resolution for both hastv- and hastv- maps was estimated to bẽ Å using a correlation coefficient cutoff value of . . the final d hastv- and hastv- maps were calculated from (of ) and (of ) particles, respectively, with selection based on their correlation coefficients. the handedness of both maps was set by matching the capsid features to those of the hev structure. the amino acid sequences of hastv- (genbank id: caa ), hastv- (genbank id: caa ), and human hev (genbank id: aaa ) were analyzed using the programs blast § and clustalw ∥. blast searches were run using the default selections and with multiple database options [nonredundant protein sequences, protein data bank (pdb) sequences, and swiss protein sequences]. density maps calculated from pdb models pdb files containing full t = icosahedrally related copies of the hev (pdb id: iyo ) or hastv (kindly provided by dr. yizhi tao ) asymmetric unit (or spiketruncated sequences) were generated using the viperdb utility generateoligomers. density maps of the models were then calculated from the pdb files with the eman program pdb mrc using a step-size equivalent to the cryoem maps and truncated at Å resolution. the hev cryoem map (emd id: ) was low-pass filtered with the eman program proc d. contour levels were set to encompass the expected volume of copies of vp (hastv- ), or copies of vp plus copies of vp , based on a protein density of . g/cm . figures of the maps and docking of pdb coordinates were performed using chimera. letter: viruses and gastroenteritis in infants letter: nm particles in faeces in infantile gastroenteritis structural and biochemical analysis of human pathogenic astrovirus serine protease at . Å resolution molecular analysis of a serotype human astrovirus genome subgenomic rna sequence of human astrovirus supports classification of astroviridae as a new family of rna viruses the complete sequence of a human astrovirus identification of structural domains involved in astrovirus capsid biology caspases mediate processing of the capsid precursor and cell release of human astroviruses characterization of a human astrovirus serotype structural protein (vp ) that contains an epitope involved in virus neutralization proteolytic processing of the astrovirus capsid proteolytic processing of a serotype human astrovirus orf polyprotein crystal structure of the human astrovirus capsid spike ultrastructure of human astrovirus serotype structure of hepatitis e virion-sized particle reveals an rnadependent viral assembly pathway activation of influenza a viruses by trypsin treatment enhancement of the infectivity of influenza a and b viruses by proteolytic cleavage of the hemagglutinin polypeptide cleavage of spike protein of sars coronavirus by protease factor xa is associated with viral infectivity proteolytic enhancement of rotavirus infectivity: molecular mechanisms proteolytic digestion of reovirus in the intestinal lumens of neonatal mice membrane fusion process of semliki forest virus ii: cleavage-dependent reorganization of the spike protein complex controls virus entry astrovirus-like particles associated with hepatitis in ducklings structure of the hepatitis e virus-like particle suggests mechanisms for virus assembly and receptor binding three-dimensional structure of the rotavirus haemagglutinin vp by cryo-electron microscopy and difference map analysis methods for reconstructing density maps of "single" particles from cryoelectron micrographs to subnanometer resolution a model-based approach for determining orientations of biological macromolecules imaged by cryoelectron microscopy ab initio random model method facilitates d reconstruction of icosahedral particles viperdb : an enhanced and web api enabled relational database for structural virology eman: semiautomated software for high-resolution singleparticle reconstructions ucsf chimera-a visualization system for exploratory research and analysis we gratefully acknowledge dr. yizhi tao for providing her model of astrovirus vp . we also thank dr. barbie ganser-pornillos for helpful discussions and yunuen acevedo for technical support. molecular graphics images were produced using the ucsf chimera package from the resource for biocomputing, visualization, and informatics at the university of california, san francisco [supported by national institutes of health (nih) p rr ]. this work was supported by funding from the austrian science fund (j ) to n.n., dgapa-unam ( ) and conacyt ( ) to e.m., nih r ai to s.m.m., and nih r gm to m.y. supplementary data to this article can be found online at http://dx.doi.org/ . /j.jmb. . . key: cord- -c xyev authors: proença-módena, josé luiz; buzatto, guilherme p.; paula, flávia e.; saturno, tamara h.; delcaro, luana s.; prates, mirela c.; tamashiro, edwin; valera, fabiana c.p.; arruda, eurico; anselmo-lima, wilma t. title: respiratory viruses are continuously detected in children with chronic tonsillitis throughout the year date: - - journal: int j pediatr otorhinolaryngol doi: . /j.ijporl. . . sha: doc_id: cord_uid: c xyev objective: to evaluate the oscillations on the viral detection in adenotonsillar tissues from patients with chronic adenotonsillar diseases as an indicia of the presence of persistent viral infections or acute subclinical infections. study design: cross-sectional prospective study. setting: tertiary hospital. methods: the fluctuations of respiratory virus detection were compared to the major climatic variables during a two-year period using adenoids and palatine tonsils from children with adenotonsillar hypertrophy and clinical evidence of obstructive sleep apnoea syndrome or recurrent adenotonsillitis, without symptoms of acute respiratory infection (ari), by taqman real-time pcr. results: the rate of detection of at least one respiratory virus in adenotonsillar tissue was %. the most frequently detected viruses were human adenovirus in . %, human enterovirus in . %, human rhinovirus in . %, human bocavirus in . %, human metapneumovirus in . % and human respiratory syncytial virus in . %. although increased detection of human enterovirus occurred in summer/autumn months, and there were summer nadirs of human respiratory syncytial virus in both years of the study, there was no obvious viral seasonality in contrast to reports with ari patients in many regions of the world. conclusion: respiratory viruses are continuously highly detected during whole year, and without any clinical symptomatology, indicating that viral genome of some virus can persist in lymphoepithelial tissues of the upper respiratory tract. objective: to evaluate the oscillations on the viral detection in adenotonsillar tissues from patients with chronic adenotonsillar diseases as an indicia of the presence of persistent viral infections or acute subclinical infections. study design: cross-sectional prospective study. setting: tertiary hospital. methods: the fluctuations of respiratory virus detection were compared to the major climatic variables during a two-year period using adenoids and palatine tonsils from children with adenotonsillar hypertrophy and clinical evidence of obstructive sleep apnoea syndrome or recurrent adenotonsillitis, without symptoms of acute respiratory infection (ari), by taqman real-time pcr. results: the rate of detection of at least one respiratory virus in adenotonsillar tissue was %. the most frequently detected viruses were human adenovirus in . %, human enterovirus in . %, human rhinovirus in . %, human bocavirus in . %, human metapneumovirus in . % and human respiratory syncytial virus in . %. although increased detection of human enterovirus occurred in summer/autumn months, and there were summer nadirs of human respiratory syncytial virus in both years of the study, there was no obvious viral seasonality in contrast to reports with ari patients in many regions of the world. conclusion: respiratory viruses are continuously highly detected during whole year, and without any clinical symptomatology, indicating that viral genome of some virus can persist in lymphoepithelial tissues of the upper respiratory tract. ß elsevier ireland ltd. all rights reserved. we have previously reported high rates of detection of respiratory virus genomes in tonsils and adenoids from patients with chronic adenotonsillar diseases, suggesting a significant association of viruses, particularly picornaviruses, with severe tonsillar hypertrophy [ ] . however, no conclusive evidence of productive -acute or persisting -viral infection, as opposed to virus latency, has been established. in general, peaks of respiratory virus detection in children with ari occur with marked seasonal variations in temperate and subtropical regions [ , ] . in tropical areas, the seasonal pattern of viral detection is more difficult to be analysed, due to the heterogeneity of data in several parts of the world. however, respiratory viruses have been mainly observed during the rainy seasons [ ] . in southeast brazil, a region of transition between tropical and subtropical climates, peaks of viral ari tend to occur during cooler months [ ] [ ] [ ] . the present paper reports analyses of over time variations in rates of detection of respiratory viruses in tissues and secretions removed from children undergoing tonsillectomy while in the absence of ari symptoms. the rationale was that if detection of respiratory viruses in hypertrophic tonsillar tissues oscillated with variations in temperature and rainfall, in a way similar to what occurs among ari patients, this would suggest an association with acute subclinical respiratory viral infections, rather than prolonged asymptomatic harbouring of viral nucleic acids in the tissues. respiratory virus genomes were searched in adenoids (ad), palatine tonsils (pt) and nasopharyngeal secretions (nps) obtained from all children ( males) aged - years (mean . years) who underwent adenotonsillectomy to treat adenotonsillar hypertrophy with clinical evidence of obstructive sleep apnoea syndrome [ ] or recurrent adenotonsillitis according to paradise criteria [ ] . patients were treated at the division of otorhinolaryngology of the school of medicine of ribeirão preto, university of são paulo, between may and june . patients with signs/symptoms of acute respiratory infections within the last four weeks prior to surgery and patients with immunodeficiencies were excluded from the study. indeed, the exclusion of these patients was a safety criterion for surgery. all clinical samples obtained in this study were maintained in a preservative solution (rna later -invitrogen, carlsbad, ca, usa) at À c until nucleic acid extraction. the study was conducted according to the principles expressed in the declaration of helsinki and was approved by the university hospital clinical research ethics committee (file number / ). a written informed consent was obtained from all parents and guardians prior to enrolment. the detailed description of each pcr, including the primers sequences, can be obtained in a previously published paper [ ] . in the present study were included patients, extending the observation period to two years, allowing that the seasonal pattern of viral circulation was determined in these patients. the analysis of the seasonality of respiratory viruses in adenotonsillar tissues was performed cross matching the virus presence to the temperature and rainfall. ribeirão preto is a city in the state of são paulo, southeast brazil with a population of , , located at s and w, m above sea level. the climate is a transition between tropical and subtropical conditions, with annual average temperature of c, with dry mild winters and hot rainy summers. during this study, the mean monthly minimal and maximal daily temperatures were respectively . c (range, . - . ) and . c (range, . - . ). rainfall is the major climate variable, with yearly rainy seasons between november and march and dry season from june to september. the mean monthly accumulated rainfall throughout the study was mm, ranging from to mm. accumulated rainfall and mean seasonal temperatures were obtained from the site of the integrated center of agrometeorological information of são paulo sate (http:// www.ciiagro.sp.gov.br). rates of detection of respiratory viruses in adenoids, tonsils and respiratory secretions were determined for children. genomes of at least one respiratory virus were detected in over % of the patients, without discernible seasonal variations (fig. ). remarkably, high rates of virus detection were obtained from all three kinds of clinical samples throughout the study. the frequencies of virus detection ranged from . % to . % in adenoids, and from . % to . % in secretions and palatine tonsils. of the three sample kinds, adenoid tissue yielded the highest frequencies of virus detection during almost the whole study, except for autumn months (march-june) of , when nasal secretions yielded higher rates of positivity (fig. ) . the present analysis is based on results from a total of patients, covering a -year period, and raises new issues made clear upon inclusion of additional cases to a study that was underway [ ] . overall, the most frequent viruses were human adenovirus (hadv) detected in . %, followed by human enterovirus (hev) in . %, human rhinovirus (hrv) in . %, human bocavirus (hbov) in . %, human metapneumovirus (hmpv) in . %, human respiratory syncytial virus (hrsv) in . %, influenza virus (flu) in . %, human parainfluenzavirus (hpiv) in . %, and human coronavirus (hcov) in . %. the frequencies of hadv, hbov and hrsv were higher in adenoids, whereas hrv was more frequently detected in nasal secretions and hev in palatine tonsils. the rates of viral co-infections and the agreement between results from different tissues were high. in this -year study period, two or more viruses were detected in . % of the patients, and % of them had the same virus detected in adenoids and palatine tonsils. overall, hadv detection rates fluctuated from summer troughs of approximately . % up to peaks of greater than % in spring- and autumn- , without clearly seasonal periods (fig. ). hbov detection rates were usually above % ( . - %) without discernible seasonality (fig. ) . detection frequencies of hadv and hbov were consistently higher in adenoids than in other samples. rates of detection of the picornaviruses hrv and hev were opposite in the summer, while the rate of hev detection was at its [ ( f i g . _ ) t d $ f i g ] peak, hrv was at its lowest ( figs. and ) . the overall hev detection rates were composed mostly by results obtained from adenoids and palatine tonsils, while detection in respiratory secretions was found in a smaller proportion of the hev-positive patients (fig. ) . although peaks of hev detection occurred in summer/autumn months, the rates of hev positivity were always above %, indicating that a great proportion of tonsil tissues harbour hev, independently of the season of the year. contrary to hev, hrv overall rates showed no clear seasonal variations, and were mostly composed by results obtained from secretions, with correspondingly lower rates of positivity in tonsillar tissues (fig. ) . rates of detection of the paramyxoviruses hmpv and hrsv varied from % to % during the study period (figs. and ). hrsv detection reached maximum level in spring/winter months, mostly composed by detection in respiratory secretions, with summer nadirs in both years of the study (fig. ) . differently, hmpv overall detection rates did not vary significantly during the study period (fig. ) , and the nadir observed in the summer of probably reflects the low number of samples analysed in that season. flu, hpiv and hcov were detected in very low frequencies, with sporadic cases distributed during the -year study period without seasonal pattern. several studies have shown that some respiratory viruses circulate seasonally, with a typically increase of the viral incidence in colder months, mainly in temperate regions [ ] , but also in subtropical regions [ ] [ ] [ ] . in tropical regions the results are more difficult to interpret, with several studies indicating higher viral circulation in rainy seasons [ , [ ] [ ] [ ] , while others show that respiratory viruses are prevalent year-round [ ] . in salvador for instance, a tropical city in the northeast of brazil, the presence of viral infections was significantly associated with precipitation during the rainy season in patients with community-acquired pneumonia [ ] . it is broadly accepted that respiratory viruses spread by shedding in secretions from acutely symptomatic patients [ ] , but respiratory viruses are also frequently detected in asymptomatic individuals [ ] [ ] [ ] , raising the hypothesis that the viral shedding by people without acute symptoms can be important to the viral dissemination. in fact, high frequencies of detection of respiratory viruses have also been observed in secretions and tissues from patients with chronic adenotonsillar diseases [ , , , ] , but no analysis of seasonality had been done in that particular setting. although the presently reported analyses confirmed previously published findings that found high rates of viral detection in patients with chronic adenotonsillar diseases, the analysis of the fluctuations in viral detection rate during these years showed that most of respiratory viruses have no obvious seasonal pattern, supporting the notion that such high frequency of virus genome detection can be related to virus persistence in lymphoepithelial tissues of the upper respiratory tract. the discovery of hadv was consequence of its recovery from adenoid explants [ ] , and several studies have documented that several adenovirus species, especially adenovirus c, can persist in mucosal lymphoid tissues, possibly by maintenance of the quiescent viral genomes in non-dividing lymphocytes [ , ] . in fact, hadv causes persistent/latent infection in tonsillar t-cell subpopulations [ ] , and can infect continuous b-cell and myeloid cell lineages in vitro [ ] , suggesting that several different cell populations in tonsils may carry the virus genome. therefore, it was no surprise that in the present study hadv was the most frequent respiratory virus detected in adenoids, and the second most frequent in palatine tonsils. in addition, the lack of seasonal trends in rates of hadv detection, both in tissues and secretions, is confirmatory that most of the high hadv frequency is attributable to persistence. in tropical regions, adenovirus is frequently associated with the rainy season in patients with acute respiratory infections [ ] . hbov is a parvovirus that occurs worldwide in association with respiratory and gastrointestinal disorders [ , ] . in addition to the general propensity of parvoviruses to persist and even endogenise into host genomes [ ] , at least two lines of evidence support persistence of hbov in humans. only around % of patients who are pcr-positive for hbov have mrna for a viral structural protein detectable as a marker of active viral replication [ ] and hbov episomes have been found in human clinical samples, including tissue biopsies [ ] [ ] [ ] . therefore, it is not surprising that in the present study hbov was frequently detected, without discernible seasonality, suggesting that, at least part of [ ( f i g . _ ) t d $ f i g ] this high frequency could be attributed to virus persistence, mainly in adenoids. while persistence and latency of dna viruses in lymphoepithelial tissues have long been known, the same is not the case for rna viruses. in the present study, hev was the second most frequently detected agent at overall frequencies consistently above %. although there are very few studies about seasonality of hev in tropical regions, the observed trend for an increase in hev detection towards the summer is in keeping with what happens in temperate regions of the world [ ] . however, the high rates of hev positivity were consistently due to detection in tissue fragments, which was less frequently accompanied by shedding in secretions, supporting the idea that hev can persist in adenoids and tonsils in a high proportion of patients. while confirmation of hev persistence in tonsillar tissues will require detailed molecular investigation, the present findings of such consistently and non-seasonal hev detection over time, coupled with the already reported higher frequencies of hev detection in the most highly hypertrophic tonsils [ ] indicates that perhaps hev persistence can be associated with the pathogenesis of chronic adenotonsillar diseases. hrv, the other picornavirus included in the present analyses, is frequently detected in acute respiratory infections of children and adults, usually with marked seasonal variation, especially in subtropical and temperate areas [ , ] , as well as in asymptomatic patients [ ] . in tropical regions, the literature results are controversial. in trinidad, west indies, hrv was prevalent throughout the year, without seasonal association [ ] , whilst in salvador hrv was associated with relative humidity (p = . ) [ ] . in the present analysis hrv was frequently detected in all seasons, mostly in secretions, rather than in tissues. it is interesting that hrv and hev are both picornaviruses, currently classified in the same genus, with very similar replication cycles, and yet showed dissimilar frequencies of detection in tissues. hrv was detected in lower frequencies in lymphoid tissues as compared to hev, suggesting the existence of other sites of infection as sources of hrv shedding into secretions, such as nasal epithelium. this is remarkable, since the patients with hypertrophic adenotonsillar diseases had no acute nasal symptoms at the time of surgery. although the overall rates of detection of hmpv and hrsv in the present analyses were lower than those of other viruses, they were still frequently higher than %. moreover, hrsv rates were frequently higher than %, with significant contribution of positivity in adenotonsillar tissues, again suggesting that these tissues may be regarded as sites of persistence of paramyxoviruses. pertaining to this issue, it is interesting that phylogenetic studies of respiratory syncytial virus have pointed to the existence of reservoirs to maintain the virus during inter-seasonal periods, thus creating potential for its reintroduction in the susceptible population [ ] . it is therefore reasonable to think that adenoids and tonsils of children with chronic adenotonsillar diseases would be natural reservoirs of respiratory viruses and that, by shedding viruses in respiratory secretions, these children would become sources of infection for their siblings and schoolmates. to the best of our knowledge, this has been the most comprehensive study so far conducted on the variations of respiratory virus detection rates over time in children with chronic adenotonsillar diseases in a subtropical/tropical region. although further studies are in order to clarify whether these findings result from long term virus shedding consequent to persistence, or to current asymptomatic viral infections, the lack of obvious seasonal patterns of respiratory viruses in hypertrophic adenotonsillar tissues supports the view that some respiratory viruses may persist in adenoids and tonsils. in the absence of results from molecular markers of active viral replication, the mere detection of viral genomes is not enough to establish that such infections were productive. however, the existence of genomes of numerous viruses in adenotonsillar tissues is an exciting finding, and deserves further investigation. in addition to its obvious epidemiological importance as possible sources of community respiratory virus outbreaks, persistence of these viruses could have pathogenic potential in the development of tonsillar hypertrophy, functioning as chronic stimuli for inflammation. alternatively, for reasons not yet understood, respiratory viruses could more readily establish an asymptomatic carrier states in patients with chronic tonsillar hypertrophy. however, the lack of control group, with samples of tonsils obtained from healthy patients, makes any inference about the development of disease difficult to be explored in the present study. thereby, studies about viral persistence in adenoids and tonsils, mainly those including tissue samples from healthy subjects, can bring new insights to the understanding of poorly understood chronic tonsillar diseases that affect large numbers of children worldwide. respiratory viral infections in infants: causes, 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latitudinal variations in seasonal activity of influenza and respiratory syncytial virus (rsv): a global comparative review detecting respiratory viruses in asymptomatic children risk factors for wheezing in a subtropical environment: role of respiratory viruses and allergen sensitization picornavirus infections in children diagnosed by rt-pcr during longitudinal surveillance with weekly sampling: association with symptomatic illness and effect of season frequent detection of respiratory viruses by real-time pcr in adenoid samples from asymptomatic children detection of viruses in human adenoid tissues by use of multiplex pcr group name proposed for new respiratory-tract viruses latent species c adenoviruses in human tonsil tissues modeling adenovirus latency in human lymphocyte cell lines andersson, cloning of a human parvovirus by molecular screening of respiratory tract samples human bocavirus, a respiratory and enteric virus widespread endogenization of densoviruses and parvoviruses in animal and human genomes detection of human bocavirus mrna in respiratory secretions correlates with high viral load and concurrent diarrhea bocavirus episome in infected human tissue contains non-identical termini detection of head-to-tail dna sequences of human bocavirus in clinical samples detection of a bocavirus circular genome in fecal specimens from children with acute diarrhea in beijing enterovirus surveillance -united states respiratory picornaviruses and respiratory syncytial virus as causative agents of acute expiratory wheezing in children acute viral respiratory infection in children under years: epidemiological study in two centers in distribution and seasonality of rhinovirus and other respiratory viruses in a cross-section of asthmatic children in trinidad, west indies phylodynamics and dispersal of hrsv entails its permanence in the general population in between yearly outbreaks in children the authors thank maria cecília onofre and helder g. de souza for secretarial assistance; lú cia lopes, jamila mendonça de souza and maria lú cia silva for expert technical support. in addition, the authors thank fapesp (fundação de amparo à pesquisa do estado de são paulo) for financial support (grant number / - ). the authors have no conflicts of interest to disclose and have no financial disclosures to make. key: cord- -ihc ly authors: li, yan‐chao; bai, wan‐zhu; hirano, norio; hayashida, tsuyako; taniguchi, takahide; sugita, yoichi; tohyama, koujiro; hashikawa, tsutomu title: neurotropic virus tracing suggests a membranous‐coating‐mediated mechanism for transsynaptic communication date: - - journal: j comp neurol doi: . /cne. sha: doc_id: cord_uid: ihc ly swine hemagglutinating encephalomyelitis virus (hev) has been shown to have a capability to propagate via neural circuits to the central nervous system after peripheral inoculation, resulting in acute deadly encephalomyelitis in natural host piglets as well as in experimental younger rodents. this study has systematically examined the assembly and dissemination of hev n in the primary motor cortex of infected rats and provides additional evidence indicating that membranous‐coating‐mediated endo‐/exocytosis can be used by hev for its transsynaptic transfer. in addition, our results suggested that this transsynaptic pathway could adapted for larger granular materials, such as viruses. these findings should help in understanding the mechanisms underlying coronavirus infections as well as the intercellular exchanges occurring at the synaptic junctions. j. comp. neurol. : – , . © wiley periodicals, inc. abstract swine hemagglutinating encephalomyelitis virus (hev) has been shown to have a capability to propagate via neural circuits to the central nervous system after peripheral inoculation, resulting in acute deadly encephalomyelitis in natural host piglets as well as in experimental younger rodents. this study has systematically examined the assembly and dissemination of hev n in the primary motor cortex of infected rats and provides additional evidence indicating that mem-branous-coating-mediated endo-/exocytosis can be used by hev for its transsynaptic transfer. in addition, our results suggested that this transsynaptic pathway could adapted for larger granular materials, such as viruses. these findings should help in understanding the mechanisms underlying coronavirus infections as well as the intercellular exchanges occurring at the synaptic junctions. j. comp. neurol. : - , . swine hemagglutinating encephalomyelitis virus (hev) is a positive, nonsegmented, single-stranded rna coronavirus belonging to the betacoronaviruses, together with mouse hepatitis virus, bovine coronavirus, human coronavirus oc , and severe acute respiratory syndrome coronavirus (masters, ; de groot et al., ) . as the first member of the group of coronaviruses found to invade the central nervous system (cns), hev was initially isolated from encephalomyelitic piglet brains in in canada by greig et al. ( ) . subsequent studies demonstrated that hev first oronasally infects the epithelial cells lining the respiratory tract and small intestine and thereafter is delivered retrogradely via peripheral nerves to the central neurons in charge of peristaltic function of the digestive tract, resulting in the so-called vomiting diseases (andries and pensaert, ) . early electron microscopic (em) and virological analysis showed that glial cells were not infected by hev in primarily cultivated astrocytes (yagami et al., ) or in the brains of infected piglets or mice (meyvisch and hoorens, ; yagami et al., ) , indicating that neurons are the major target of hev in the cns. however, previous in vivo studies concentrated mainly on the later stage of infection, and it is not clearly understood how this virus is replicated within and released from the central neurons. recently we examined hev-infected rat dorsal root ganglia (drg) and found that hev in the cell bodies of infected sensory neurons budded from endoplasmic reticulum-golgi intermediate compartments and was assembled either individually within small vesicles or in groups within large vesicles. the progeny virions were released from the sensory neurons mainly by the large, smooth-surfaced, vesicle-mediated secretory pathway (li et al., ) . in the present study, systematic electron microscopy (em) and morphological analysis were employed to obtain detailed insights into the replication and dissemination of hev in the cns. a plaque-cloned hev n strain (hirano et al., ) was used, which has previously been demonstrated to be confined exclusively to neurons (hirano et al., ; bai et al, ; li et al., ) . preliminary em analysis of hev-infected motor cortices showed that the pyramidal cells remained largely intact at day postinfection (p.i.) and that both the cytoplasmic and the perineuronal architectures maintained their normal morphology. destructive changes in the infected neurons did not become prominent until day p.i., which was coincident with the recruitment of immune cells to the infected areas. therefore, the brains collected at day p.i. were used in this experiment to obtain detailed insights into the replication and dissemination of hev in the cns. hev n strain, also called the north american strain, was initially isolated from the nasal cavity of apparently healthy swine in iowa, during a routine survey for viruses harbored in the respiratory tract by mengeling et al. ( ) . this virus was obtained from dr. w.l. mengeling (national animal disease laboratory, ames, ia). specificpathogen-free male wistar rats ( weeks old, $ - g), serologically negative for mhv infections, were purchased from slc (hamamatsu, japan). cell culture and plaque assay hev n was passaged times in primary porcine kidney cell cultures (hirai et al., ) and more than times in suckling mouse brains by intracerebral inoculation. the mouse-brain-adapted hev n strain was plaque-purified three times in an established swine kidney cell line and thereafter was propagated times in the same cell line until use. the viral supernatant from infected cell culture, with a titer of pfu/ . ml as assayed by plaque method, was kept at À c and was used for all experiments. swine kidney cell culture and plaque assay of the virus were carried out as described by hirano et al. ( ) . four-week-old mice were inoculated intraperitoneally twice with . ml hev n strain at an interval of weeks; at week after the last inoculation, mice were killed to collect the blood. the antiserum was heated at c for minutes, and the specificity was examined by neutralization and hemagglutinating inhibition tests (yagami et al., ; hirano et al, ). thirteen rats were inoculated by injecting ll viral culture supernatant ( pfu) into the right hind foot pad with a -ml syringe. three rats were inoculated with virusfree cell culture supernatant as vehicle controls. all the rats were reared separately in cages, with food and water freely available. all the experiments with active virus were carried out in biosafety level containment and performed in accordance with the nih guide for the care and use of laboratory animals. three hev-infected rats were perfused at day p.i. through the heart with ml physiological saline under halothane anesthesia (fluothane; takeda pharmaceutical, osaka, japan), followed by - ml % paraformaldehyde in . m phosphate buffer (pb; ph . ). the brains were dissected out, postfixed in fresh fixatives for - hours, and then washed in a series of cold sucrose solutions of increasing concentration. the samples were embedded in oct compound (tissue-tek; sakura finetek japan, tokyo, japan), frozen on dry ice, then cut into lm-thick transverse sections with a freezing microtome. the cryosections were preincubated with % normal goat serum in . m pb containing . % triton x- for minutes at room temperature, then incubated with the mouse anti-hev antibody overnight at c. after several washes with . m pb, the sections were reacted with alexa -conjugated goat anti-mouse igg antibody (molecular probes, eugene, or) in . m pb for minutes at room temperature. the specificity of the primary antibody has been reported previously (hirano et al., ) and was verified in this study by replacing the antiviral antibody with . m pb. the sections were mounted onto glass slides with immu-mount (shandon, pittsburgh, pa) and examined with a laser scanning confocal microscope (fv ; olympus, tokyo, japan). ten infected rats and three vehicle-injected rats were perfused at day p.i. through the heart with ml physiological saline under halothane anesthesia, followed by - ml of fixatives containing % glutaraldehyde and % tanic acid in . m pb. the brains were dissected out, postfixed in fresh fixatives for - hours, and then left in . m pb containing % sucrose overnight at c. serial coronal sections of rat brains about lm thick were cut on a dsk microslicer (dtk- , zero- ; dosaka em, kyoto, japan). the sections were fixed in % osmium tetroxide in . m pb for hours at c, dehydrated in increasing concentrations of ethanol, and embedded in epon . ultrathin sections about nm thick were cut on an ultracut e ultramicrotome (reichert-jung, wien, austria) and collected on formvar-coated grids. the ultrathin sections were stained with uranyl acetate and lead citrate and then examined with a transmission electron microscope (technai fei). high-resolution em images were recorded on image plates (fujifilm, tokyo, japan), which were scanned with fujifilm fdl and converted into digital images with the software image gauge . (fujifilm). the brightness and contrast of each image file were uniformly adjusted with this software. the dimensions of cells and virions were measured on electron micrographs with olympus soft imaging . (olympus soft imaging solutions gmbh, münster, germany). final pictures were prepared in adobe photoshop . (adobe systems, san jose, ca). the involved neural circuits and the temporal patterns of infected neuron groups after inoculation have been characterized previously (hirano et al., ; bai et al., ; unpublished data) . these experiments showed that footpad inoculation of hev in -week-old rats consistently resulted in the ipsilateral infection of drg ($l -l ) and spinal motoneurons at lumbar levels at day p.i. and in the contralateral infection of layer v of the hind limb area in the primary motor cortex at day p.i. infected rats showed a relatively constant onset of encephalomyelitis, with an average survival of about days. fatal infection with hev can be protected against by cutting the proximal segment of the sciatic nerve within hour after inoculation (hirano et al., ) . moreover, no anterograde propagation of hev has been found along spinal ascending pathways throughout the periods examined (bai et al., ; unpublished data) . consistent with the results in the previous experiments, the present study showed that at day p.i. hev-positive cells were observed in only a certain population of neurons with different sizes in layer v of the primary motor cortex (fig. a,b) . the fluorescence signals for hev showed a punctate pattern and were located in the cell bodies and the primary dendrites of pyramidal cells (fig. c ). by contrast, less intense staining was also seen in the putative axons (arrows in fig. c ). usually, it is not easy to examine virus particles ultrastructurally in vivo, even if the viral antigen has been labeled by immunostaining, because virions are very small, and viral structures or vesicular membrane cannot be identified clearly because of the immunostaining reaction. therefore, the ultrastructural analysis was focused mainly on the layer v pyramidal neurons in the hind limb representation of motor cortex, and only unstained sections were used. ultrastructural examinations showed that virus particles were distributed in axons ( fig. f-h) , perikarya (figs. e, c,d), and dendrites ( fig. a,b ,e) but not in the nucleus (fig. e ) and were undetectable in control materials (figs. g, e-h). under em, intracellular virus was identified as spherical particles inside vesicles or as electron-dense materials in the process of budding into the intracellular membranous cisternae, as reported previously (clarke and mcferran, ; mengeling et al., ; yagami et al., ) . enveloped virus particles were spherical, with an electronlucent or -dense center, and the outer surfaces of the viral envelopes were often covered by a layer of welldefined projections, forming a typical ''corona'' profile ( fig. f , ). the number of virus particles per cell varied greatly from cell to cell, suggesting that the severity of infection was different among neurons even at the same postinoculation time. with our preparations, we encountered a total of hev-infected pyramidal cells and eight longitudinally cut axons belonging to the infected pyramidal cells. more than photographs, both lower and higher magnifications, have been taken of each cell, and all the axons and cells have been thoroughly examined and analyzed. hev particles were observed in the axon hillocks, the initial segments (fig. f) , and the distal myelinated axons (fig. g,h) . the number of virus particles varied from axon to axon, but all the virus particles in the axoplasm were enclosed within smooth-surfaced vesicles, some of the journal of comparative neurology | research in systems neuroscience which were found closely associated with microtubules (arrows in fig. f,h) . such microtubule-associated vesicles have been observed in all the axons examined, suggesting that the virus was being transported within the vesicles along the microtubules in the axons. in pyramidal cells, hev replication occurred mainly in the perikaryon and dendrites. the earliest sign of viral assembly observed inside infected cells was an electrondense crescent segment indenting into the external the journal of comparative neurology | research in systems neuroscience membrane of er cisternae in the perikaryal cytoplasm ( fig. a) . large crescent segments were observed bulging into the lumen of ers by incorporating the lipid bilayer of the er (fig. b) . as virus particles were internalized into the lumen of ers, the lipid bilayer of ers seemed to be gradually pinched off and finally fused around the virus particles (fig. c,d) . with the progression of infection, large numbers of virions were packed individually in vesicles in the trans-golgi networks (fig. e) . some of the vesicles were decorated with small spinule coats around their outer surfaces (arrows in fig. e) , which were about - nm long and exhibited the morphology characteristic of clathrin (fig. g ). such coated vesicles were observed throughout the cytoplasm of the perikarya and dendrites and appeared abundant near the golgi complexes (fig. d,e) . the diameters of virions and virion-containing vesicles in two representative infected pyramidal cells were measured under  , magnification on electron micrographs. hev virions ranged from to nm in diameter ( . . nm, mean sd, n ¼ ), whereas the virion-containing vesicles ranged from to nm in diameter ( . . nm, mean sd, n ¼ ). morphological analysis suggested that coated vesicles played a pivotal role in both the egress and the subsequent entry of hev. the coated vesicles, with one virion within one vesicle, were distributed beneath the cell surface of infected cells, and some of them were found apparently fusing with the plasma membrane (fig. a) . through the openings at the fusion sites, progeny virions seemed to be released outside, whereas the membranous coating still remained at the release sites (fig. b) . no virus particles were observed in the adjacent axonal terminals, so the extracellular virus particles were thought to be the progeny virions that had been liberated from the infected pyramidal cells. in the synaptic clefts, virus particles were found trapped in the invaginations of the presynaptic membrane, where new membranous coating was seen on the cytoplasmic side (fig. c) . coated vesicles enclosing a single virion were further found in the axonal terminals adjacent to infected pyramidal cells (fig. d) . extracellular virions were not enclosed by any vesicular structures, whereas vesicle-enclosed virus particles were otherwise observed in the axonal terminals touching on the infected neurons, so it seemed that the virions within the synapses had acquired new vesicular membrane after entry. possibly because of the accumulation of extracellular virions, the synaptic clefts became aberrantly dilated and had lost their normal integrities. on the other hand, infected astrocytes were not seen, even in the late stage of infection (data not shown). transsynaptic transfers of hev were demonstrated undoubtedly in nine pyramidal cells. although only nine pyramidal cells were counted, the other cells were not included in the analysis not because hev showed different transfer means in them but because the pre-or postsynaptic invaginations were not very clear because of the sectioning directions or because the virus particles were not located exactly nearby the pre-or postsynaptic invaginations. a shows an infected pyramidal cell, in which virus particles are observed within vesicles in the perikaryal cytoplasm. near the cell surface, a virus-containing coated vesicle is found attaching to the plasma membrane (arrow; see also the inset for details). this coated vesicle is apparently fusing with the plasma membrane, and there is an opening at the fusion site. notably, no virus is found in the adjacent neuropils. b shows the primary dendrite of an infected pyramidal cell. several progeny virions are found extracellularly near the fusion sites, where a layer of coats still remain on the plasma membrane (arrows). the virion labeled by right arrow is enlarged in the inset. no virus is found either in the adjacent axonal terminals or in the surrounding glial processes. c shows extracellular virions in the dilated synaptic clefts, and some of them are trapped in the coated invaginations of the presynaptic membrane (arrows; see also the inset for details). d shows a virion within a coated vesicle (arrow; see also the inset for details) in the axon terminal adjacent to an infected pyramidal cell. e-h: electron photographs from vehicle controls showing coated structures in the pre-and postsynaptic cytoplasm. e shows a coated invagination (arrow) in the postsynaptic membrane of a pyramidal cell. the bulbous portion of the invagination communicates with the extracellular synaptic cleft via a thin neck (see the inset for details). f shows a coated invagination (arrow) in the presynaptic membrane, which is further enlarged in the inset. g shows a coated vesicle (arrow) in the postsynaptic cytoplasm, which is further enlarged in the inset. h shows coated vesicles (arrows) present in both the pre-and the postsynaptic cytoplasm. the coated vesicle in the presynaptic region is further enlarged in the inset. i-l: hev in spinal motoneurons. three infected rats were perfused at day p.i. with % paraformaldehyde in . m pb. spinal segments at the level of $l -l were dissected and examined by em. i and k show two a-motoneurons in the ventral horn of spinal cord. the boxed areas in i and k are enlarged in j and l, respectively. j shows a virus-containing coated vesicle (arrow) in the motoneurons attaching to the membrane. virus particles without any vesicular structures are found extracellularly in the synaptic cleft. m, neuronal cell body; t, axonal terminal. in l, coated vesicles (arrowheads) containing virus particles are seen both in the neuronal cytoplasm (m) and in an adjacent axonal terminal (t). in addition, many virions are accumulated in the dilated synaptic clefts, and some of them are seen trapped in the coated invaginations (arrows) of the axonal membrane. scale bars ¼ nm in h (applies to a-h); nm in inset h (applies to insets a-h); lm in i,k; nm in j,l. in uninfected pyramidal cells from the controls, the trans-golgi network and some small vesicles in golgi regions often possessed membranous decorations on parts of their surface, but no peri-golgi vesicles containing viruses were observed (fig. h) . coated vesicles were frequently encountered in either the pre-or the postsynaptic cytoplasm (fig. g,h) . coated invaginations were also found in pre-and postsynaptic membranes (fig. e,f) . they were continuous with the synaptic cleft by a thin neck. however, no virus particles were found within either the coated vesicles or the invaginations. examples of coated vesicles and invaginations were found in the pre-or postsynaptic regions in all the samples from the controls. the diameters of coated vesicles, from three representative pyramidal cells, were measured under  , magnification on electron micrographs. they were . . nm (mean sd, n ¼ ) in diameter, significantly smaller than the virus-containing vesicles (p < . , t-test). to make clear whether the findings in the motor cortex could be generalized to other parts of the cns, the spinal cords from three paraformaldehyde-fixed rats used for another experiment were also examined by em. a-motoneurons located in the ipsilateral ventral horn were constantly infected at day p.i. both the replication and the dissemination of hev in the motoneurons were similar to those found in the pyramidal cells ( fig. i-l) . the present study examines the assembly and dissemination of coronavirus hev n in the rat cns, specifically the motor cortex. hev virions initially budded from endoplasmic reticulum-golgi intermediate compartments in the neurons and were assembled within coated vesicles through golgi complexes. progeny hev virions were exocytosed from the host neuronal cells, possibly by use of the coated vesicles, and entered into the next-order neurons by endocytosis, again with the help of the coated vesicles (fig. ) . to the best of our knowledge, this means of dissemination used by hev in the cns is largely different from the means reported for the well-known neurotropic viruses herpesviruses and rhabodoviruses. herpesviruses, including herpes simplex virus- and pseudorabies virus, also exploit exocytosis for egress from the host neuronal cells, but their subsequent entry into the synapse-linked neurons occurs exclusively by fusion of the virus envelope with the axonal membrane (card et al., ; diefenbach et al., ) . because of the direct penetration by fusion of herpesviruses, only virus nucleocapsids can gain access to the axoplasm (dolivo et al., ; marchand and schwab, ; card et al., ) . on the other hand, rhabodoviruses, such as vesicular stomatitis virus and rabiesvirus, are released from the host neuronal cells by directly budding from the membrane of the neurons and subsequently endocytosed within vesicles by adjacent axonal terminals (iwasaki and clark, ; dal canto et al., ; charlton and casey, ; superti et al., ; lewis and lents, ; sun et al., ) . the general features of hev development observed in this study are largely consistent with those reported in previous investigations of coronavirus morphogenesis (hobman, ; de haan and rottier, ; masters, ) . these previous studies have demonstrated that coronavirus morphogenesis takes place predominantly at smooth-walled, tubulovesicular membranes located intermediately between the rough er and the golgi complex. immature virions in pre-golgi compartments and golgi cisternae are further subject to a postbudding maturation process in the trans-golgi network and are finally assembled in vesicles with or without clathrin coating (chasey and alexander, ; tooze et al., ; salanueva et al., ) . studies on mouse hepatitis virus and transmissible gastroenteritis virus in vitro showed that progeny virions, collected in groups within one large vesicle lacking clathrin coats, were transported along the constitutive exocytic route to the host cell surface and were liberated by the fusion of the vesicle membrane with the plasma membrane (tooze et al., ; salanueva et al., ) . our recent study on hev in rat drg showed that this is also true for hev in the sensory neurons, where the progeny virions were released in groups by the large-vesicle-mediated fusion (li et al., ) . on the other hand, some coronaviruses, such as infectious avian bronchitis virus (beaudette strain), have been reported to be able to leave the host cells by coated vesicle-mediated exocytosis (chasey and alexander, ) . this is the case for hev in the pyramidal cells, insofar as the present study showed that both the entry and the egress of hev were mediated by coated vesicles, which differs from the large vesicles reported previously in that they are much smaller, bear a fuzzy coat, and usually contain only one virion. given that coronaviruses depend on assistance from the host cell in virtually all stages of the infection, it seems conceivable that the means used even by the same virus for dissemination may differ within different cell types (hobman, ; masters, ) . the cell bodies of drg neurons relay sensory information from the periphery to the cns and are surrounded by a unique glial envelope formed by satellite cells (hanani, ) . because no synaptic structures are present in the drgs, the released hev virions from the sensory neurons were subsequently phagocytosed by the adjacent satellite cells, which are now known not merely to give mechanical or nutrient support to the neurons but also to have a scavenging function to control the extracellular microenviroment around the neurons (hanani, ; li et al., ) . in comparison, the neurons in the cns are linked functionally together by synaptic junctions, where nerve impulses are transmitted from one nerve cell to another. increasing data suggest that synapses may also function as a preferential site of interneuronal exchange (charlton and gray, ; waxman and pappas, ; grafstein, ; waxman et al., ; davis and mckinnon ; trojanowski and schmidt, ; luo and dessem, ; spacek and harris ) . many em investigations have demonstrated the presence of coated vesicular invaginations as a general feature of both the pre-and postsynaptic membranes (charlton and gray, ; waxman and pappas, ; waxman et al., ; spacek and harris ) . moreover, studies utilizing a variety of tracing mol-ecules have shown significant transsynaptic labeling after intracellular injection, indicating that interneuronal transfer indeed occurs at the synaptic junctions (grafstein, ; davis and mckinnon ; trojanowski and schmidt, ; luo and dessem, ) . however, the transsynaptic process is complicated and not easy to study, partially because of the small but complex structure of the synapse and the lacking of effective tracing methods. coronaviruses, which are about nm in diameter, have the largest rna genomes ( - kb) known to date and can be recognized by em without labeling (masters, ) . moreover, their life cycle has been shown to rely virtually completely on the cellular events preexisting in the host cells (hobman, ; pelkmans and helenius, ; masters, ) . therefore, coronaviruses are re-emerging as a useful molecular and cellular tool for analyzing a variety of complex cellular processes (tooze et al., ; pelkmans and helenius, ) . overall, the present study on hev has provided additional evidence that membranous-coating-mediated endo-/exocytosis can be used for the transsynaptic exchanges of coronaviruses. in addition, our results suggested that the transsynaptic pathway has the capability of being adapted for larger granular materials, such as viruses. our study contributes high-resolution morphological data relevant for understanding the mechanisms underlying coronavirus infections as well as the intercellular exchanges occurring at the synaptic junctions. immunofluorescence studies on the pathogenesis of hemagglutinating encephalomyelitis 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system. ii. experimental rabies in mouse rabies virus entry into cultured rat hippocampal neurons coronavirus infection of rat dorsal root ganglia: ultrastructural characterization of viral replication, transfer, and the early response of satellite cells transneuronal transport of intracellularly injected biotinamide in primary afferent axons binding, uptake and retrograde axonal transport of herpes virus suis in sympathetic neurons the molecular biology of coronaviruses characteristics of a coronavirus (strain n) of pigs an electron microscopic study of experimentally induced hev encephalitis insider information: what viruses tell us about endocytosis structural maturation of the transmissible gastroenteritis coronavirus trans-endocytosis via spinules in adult rat hippocampus role of clathrin-mediated endocytosis during vesicular stomatitis virus entry into host cells entry pathway of vesicular stomatitis virus into different host cells sorting of progeny coronavirus from condensed secretory proteins at the exit from the trans-golgi network of att cells interneuronal transfer of axonally transported proteins: studies with hrp and hrp conjugates of wheat germ agglutinin, cholera toxin and the b subunit of cholera toxin pinocytosis at postsynaptic membranes: electron microscopic evidence coordinated micropinocytotic activity of adjacent neuronal membranes in mammalian central nervous system pathogenesis of haemagglutinating encephalomyelitis virus (hev) in mice experimentally infected by different routes neurotropism of mouse-adapted haemagglutinating encephalomyelitis virus key: cord- -ce wa authors: wang, zheng; malanoski, anthony p; lin, baochuan; kidd, carolyn; long, nina c; blaney, kate m; thach, dzung c; tibbetts, clark; stenger, david a title: resequencing microarray probe design for typing genetically diverse viruses: human rhinoviruses and enteroviruses date: - - journal: bmc genomics doi: . / - - - sha: doc_id: cord_uid: ce wa background: febrile respiratory illness (fri) has a high impact on public health and global economics and poses a difficult challenge for differential diagnosis. a particular issue is the detection of genetically diverse pathogens, i.e. human rhinoviruses (hrv) and enteroviruses (hev) which are frequent causes of fri. resequencing pathogen microarray technology has demonstrated potential for differential diagnosis of several respiratory pathogens simultaneously, but a high confidence design method to select probes for genetically diverse viruses is lacking. results: using hrv and hev as test cases, we assess a general design strategy for detecting and serotyping genetically diverse viruses. a minimal number of probe sequences ( for hrv and for hev), which were potentially capable of detecting all serotypes of hrv and hev, were determined and implemented on the resequencing pathogen microarray rpm-flu v. / (tessarae rpm-flu). the specificities of designed probes were validated using hrv and hev strains. all strains were successfully detected and identified at least to species level. hrv strains and hev strains could be further differentiated to serotype level. conclusion: this study provides a fundamental evaluation of simultaneous detection and differential identification of genetically diverse rna viruses with a minimal number of prototype sequences. the results demonstrated that the newly designed rpm-flu v. / can provide comprehensive and specific analysis of hrv and hev samples which implicates that this design strategy will be applicable for other genetically diverse viruses. human febrile respiratory illness (fri) results in significant annual health and economic burden worldwide, but the diversity and number of pathogens make differential diagnosis very challenging. thus, it represents a useful example where many organisms ranging from bacteria (haemophilus influenzae) to fairly conserved viruses (respiratory syncytial virus) to genetically diverse viruses, i.e. influenza a virus, human rhinoviruses (hrv), and human enteroviruses (hev) need to be detected for successful differential diagnosis. several technologies, mass-code™ multiplex rt-pcr system [ ] , electrospray ionization mass spectrometry analysis of pcr amplicons [ ] , luminex ® xmap™ [ ] , and various microarray-based approaches [ ] [ ] [ ] [ ] [ ] , are currently under development as diagnostic platforms to effectively and simultaneously detect and identify large numbers of diverse viral and bacterial respiratory pathogens. one high-density resequencing microarray platform, the respiratory pathogen microarray version (rpm v. ) , has been successfully demonstrated to identify a much broader range of pathogens (including bacteria and dna and rna viruses) in a single test at sensitivities and specificities that are similar to or improved over those of other technologies [ , ] . in addition, the rpm v. platform has the demonstrated capability to discriminate among known and previously unknown strains and variants of targeted pathogens [ , ] . while promising, the rpm v. platform was a proof-ofconcept microarray for the detection of common respiratory pathogens primarily encountered among military basic trainees. it did not provide comprehensive coverage of all potential respiratory pathogens and the design methodology used was not appropriate for genetically diverse viruses. the design methodology for the rpm v. microarray consisted of applying selection rules developed for long oligonucleotide microarrays. these rules were not optimal but worked for bacterial organisms and fairly conserved viruses since previous studies had shown a single sequence on a resequencing microarray could reliably detect and serotype strains with as much as to % variation [ , [ ] [ ] [ ] . their application to cover more diverse viral organisms was less successful. for example, the ' untranslated region ( 'utr) sequence chosen for hrv on the rpm v. only provided identification of the prototype hrv- and very little coverage of other hrv serotypes. the 'utr sequences, which are relatively conserved among hrv and hev, have been used in pcr and de novo sequencing for tentative viral identification or serotype classification in lieu of the much more variable capsid proteins that actually determine serotypes [ , ] . however, the 'utr sequences still have ~ to % nucleotide sequence variations among different serotypes so require more than one prototype sequence for proper identification and serotyping. serotyping hrv and hev is important to fri differential diagnosis because even though these "common cold" viruses generally only induce mild symptoms, they can cause a wide variety of other severe illnesses, such as aseptic meningitis [ ] , bronchitis and asthma [ ] . new resequencing pathogen microarray designs, versions . and . (rpm-flu v. / ), have been constructed to address the shortcomings of the previous design. the use of μm feature allows microarrays with greater coverage, currently , of common respiratory organisms and high human health risk zoonotic pathogens (bacteria and viruses). a new approach to select a minimal number of prototype sequences that can be used to detect all and correctly identify many of the relevant strains of genetically diverse viruses such as hrv and hev was developed. due to the great genetic diversity of hrv and hev, in order to ensure that designed probes (referred to as probe sequences) generated from selected database sequences (referred to as prototype regions) would detect and discriminate all serotypes of hrv and hev, a predictive model was used to assist the microarray design [ ] . this in silico model developed for predicting resequencing microarray hybridization patterns shows good concordance in the overall percentage of base calls predicted versus experimental results. thus it is possible to use this model for evaluating the performance of database sequences as potential prototype regions. in this study, we report on results of this algorithm applied to the 'utr sequences of hrv and hev and confirm that using ~ % of the rpm-flu v. / microarray ( , hrv and hev nucleotides of total , nucleotides on array) is sufficient to detect and differentiate many hrv and hev serotypes. in silico modeling figure illustrates the procedures used for the selection of hrv and hev probe sequences. first, sequences that contain the specified target region ( 'utr) and meet any selection criteria applied were downloaded from a database (currently genbank). downloaded sequences were trimmed to cover the same region using pair-wise sequence alignment. these sequences were treated as target sequences (what would be detected by the microarray) and also as prototype sequences (the potential probe sequences tiled on the microarray). each downloaded sequence was treated as a prototype used to generate probe sequences (fig . step - ) and the remaining sequences were treated as target sequences (fig . step [ ] [ ] . sets of -mer probes ( perfect match and mismatches in the th position) were generated from a prototype sequence and correspond to what would actually appear on a resequencing microarray. the other sequences were treated as a target one at a time and generated overlapping fragments from to bases long with a near neighbor Δg energy less than - . . these fragments have been shown to have strong binding strength and produce unique base calls. the generated probes and sequence fragments were the input to the in silico model [ ] for simulation which compared the fragments to the probe sets and determined the base calls a target sequence would generate. the predicted base calls were assembled into a simulated resequencing microarray result. the simulated result of a target sequence for the current prototype sequence was then run through the previously developed cibsi analysis algorithm [ ] with the following criteria. a sequence was considered detected by the cur-rent prototype sequence if at least one region of or more contiguous nucleotides was predicted to consist of a, c, g, and t base calls and no ambiguous base calls (ns). as shown in figure "yes" for "cibsi would identify" updated the list of sequences that could be detected by the current prototype sequence. this procedure was applied for every downloaded sequence. for example, if we collect sequences "a-z" from genbank, we will first use sequence "a" as a prototype sequence, then use sequences "b-z" each in turn to generate target sequences for in silico schematic of algorithm representing the prototype sequences selection process figure schematic of algorithm representing the prototype sequences selection process. a collection of database sequences covering a specified region are processed together. each sequence is treated as probe sequence that the other sequences are tested against. the numbers of these sequences detected by the probe sequences are determined. a group of sequences that are predicted to detect all the sequences is then selected. simulation. after the completion of the simulation and cibsi analysis, a list of sequences from the pool of sequences "b-z" that can be detected by prototype sequence "a" will be generated. then the cycle begins again with sequence 'b" as prototype sequence, while sequences "a, c-z" each in turn is used as target sequences to generate the list for sequence "b". the cycle will continue until we generate the list for all download sequences (sequences 'a-z"). after this is completed, the second stage of the process will be undertaken. the number of sequences that a sequence (as prototype) is predicted to detect will be sorted and ordered. the sequence that was predicted to detect the most other sequences (as targets) was selected as a probe sequence to be used in the microarray design. it was then removed from the list of sequences. all the target sequences detected by that prototype sequence were also removed from the list of sequences. this procedure was repeated until the list of sequences was empty. when two or more prototype sequences were predicted to detect the same maximal number of target sequences, one was randomly selected hence the method was non-deterministic. the process was repeated with different random seeds and the number of required probe sequences did not vary significantly while the sequences used in the microarray design could change. application: hrv and hev the described design method could be applied to any group of sequences and a minimum set of prototype regions would be determined. the group of sequences used for hrv probe design was chosen using different criteria than those used to select the hev sequences in the hev probe design due to differences in the available sequences for each in genbank. at the time of this design, only eight hrv serotypes had complete genomes sequenced. these genome sequences and all complete and partial 'utr sequences available for hrv in genbank were retrieved in april and a total of sequences were used in the predictive modeling. a set of sequences with lengths between and bp were predicted to provide detection of all those input sequences (additional file ). because hev is better characterized with complete genome sequences of all recognized serotypes, the design algorithm was applied to one complete genome sequence of each serotype. in addition, the design algorithm was applied to the d region. the design procedure generated to sequences for hev detection using 'utr region, and sequences were predicted to detect all hev d regions. it was decided to use corresponding 'utr sequences of the genomes that the d targets were selected from so that the same serotype was targeted by both target regions (additional file ). these 'utr regions were predicted to still provide complete and now redundant coverage. to assess the performance of rpm-flu v. / chip design, known hrv serotype strains obtained from atcc were tested. of the strains, had corresponding 'utr sequences tiled on the microarray and were called prototype serotypes, which were used to verify the accuracy of the designed hrv probes. the remaining strains, representing near neighbor serotypes, were selected from diverse clades based on phylogenetic classification of hrv serotypes [ , ] . these strains were used to investigate the capability of the microarray to detect other hrv serotypes that did not have their sequences tiled on the microarray. overall, the selected strains covered every single clade of hrv serotypes based on phylogenetic analysis of the p -p regions of 'utr sequences [ ] . one metric of the hybridization in a reference region is to divide the number of bases reported as a, c, g, or t by the total number of bases for that region (probe length), which we refer to as the base call rate and proportionally reflects the hybridization strength or homology between the prototype and target sequences. a hybridization profile ( fig. a) using the base call rates clearly showed a unique pattern for each serotype. the closely related serotypes with less nucleotide divergences had similar hybridization profiles across the tiled regions, so it is possible to assign species (hrva and hrvb) based only on the hybridization patterns. the brighter red spots (higher base call rates) along the diagonal suggested that stronger hybridizations between the tiled probes and 'utrs from the prototype serotypes. it is also of note that hrv does not fall into either of two major clusters which agrees with other findings that it should really be classified as a hev [ , ] . to validate the accuracy of the array clustering, 'utr of each serotype was amplified by type-specific rt-pcr and subjected to conventional sequencing. the phylogenetic tree derived from the de novo 'utr sequences (additional file ) confirms the hrva and hrvb classification. pair-wise sequence comparisons also indicated that the average nucleotide divergence of to database entries, this information could be integrated into the final identification. for these samples with high base call rates, the best hit would have tens to hundreds more matched bases than the next best hit. the remaining atcc strains represent near neighbor serotypes, which 'utr sequences share > % identifies to those of prototypes. in all but the case of hrv , the information was sufficient to identify the correct serotypes. in these samples, the difference in the number of base calls matching in the best hit and the next best hit was more variable and on average was fewer. the confidence of the identification depends on the accuracy of the base call. since the best hit always had at least one base that matched the resequencing results and was a mismatch for the next best hit, it was hybridization profiles of hrv and hev serotypes from rpm-flu v. / microarrays figure hybridization profiles of hrv and hev serotypes from rpm-flu v. / microarrays. (a) hrv serotypes were classified into two clusters corresponding to species hrva and hrvb; (b) hev serotypes (including hrv ) were classified into two clusters corresponding to heva/b and hevc/d species. base call rates (number of base calls/probe length in each tile) generated from viral samples (rows) and prototype probes (columns) were calculated and clustered using dchip software. rows standardized base call rates. positive hybridization was represented by red color. higher base call rates were shown as brighter red colors. negative hybridization (no base call) was represented by green color. the sample hrv is underlined. possible to establish the lowest confidence level that the best hit was the correct identification. the resequencing microarray's accuracy for determining the base call has been established under a variety of conditions [ ] . using this information, there is a . n that the next best hit is the most similar sequence in the database because a base is misidentified by the resequencing microarray where n is the number of mismatches between the next best hit and the matches for the best result. with . % being the largest level of uncertainty seen for these samples, it was deemed acceptable to treat the best hit as the correct identification. for hrv , several database sequences representing different serotypes had the same score and since no further information was available it was only possible to determine that a hrv species b was present. the 'utr sequences from the tested strains generated with de novo sequencing were subjected to in silico predictive modeling analysis and the result of this was used as input in the cibsi analysis program. for the case of hrv , the in silico model predicted a larger fraction of base calls being made than were observed in the experiment. for all other samples there was a good correspondence on base call fractions between model and experiment as expected. this leads us to suspect there was a processing error or sample degradation leading to the less accurate identification. a panel of hev serotypes, including serotypes from all four hev species, was similarly used to validate the specificity of rpm-flu v. / for hev detection and identification. these serotypes were originally typed based on vp sequences (personal communication -steve oberste) prototype serotypes having strong hybridization signals were designated in bold characters. the strain not identified at serotype level by microarray was underlined. *serotype identities were made by searching 'utr sequences of hrv isolates against genbank; () indicates the highest percentage of identity to the sequence in genbank. and the majority of them belonged to members of hevb. the hybridization profile ( figure b) shows distinct clusters in a similar fashion to the hrv samples based on serotypes. in this case, heva and hevb make up one cluster, while hevc and hevd (including hrv ) comprise a second cluster. this finding is consistent with the previously described clusters for hev utr sequences [ , ] . the redundancy of the targets that was a consequence of how they were selected is apparent in the more uniform response observed within each cluster to the various strains. analysis of sequences reported from rpm-flu / array analysis indicated that two levels of identifications were obtained from strains ( table ). serotype level identification was made for of the strains, in which cases correlated with typing made by the vp genes. for example, hev and coxsackievirus a (cava ), known to cause hand-foot-mouth disease, were unambiguously recognized as hev and cava respectively using rpm-flu v. / and analysis program. two strains were identified by rpm-flu v. / as hev and hev , results in agreement with the conventional sequencing of each 'utr. however, the strains as provided by cdc were identified as hev and cavb respectively, based on the vp region. specific serotypes could not be identified for the remaining sixteen samples using the sequence read generated from the array. nevertheless these samples were easily categorized into the respective species. due to amplification problems only a subset of strains were successfully de novo sequenced, which showed - % variations in 'utr sequences. the base call rates obtained by in silico predictions based on the de novo sequences were similar to the microarray results and the identifications agreed in all but one case. this study demonstrated the use of an algorithm for the design of probe sets based on an in silico predictive model [ ] , developed by our group, that minimized the probes needed for detection and identification of most serotypes of hrv and hev. the potential of using resequencing microarray for simultaneous detection and identification of highly diverse respiratory pathogens, such as hrv and hev, was also demonstrated. the conserved nature of the ' utr regions of hrv and hev genomes and the capabil- ities of the resequencing microarray allow serotype level identification of near-neighbor serotypes of hrv and hev, when long (> nucleotides) sequences are read from the array. identifications can be still made for shorter length sequences to the species level particularly when the array has one or more such sequences derived from different probes. the utility of the resequencing microarray is related to the target selection, the optimized prototype sequences represented on the array. in the case of rpm-flu v. / , the selection of hrv targets has proved to be very robust. the 'utr has been shown in this study to be a good choice for serotyping hrv on rpm, as it performed similarly well on other platforms [ , ] . all hrv variants tested in this study could be detected and identified at least to the species level. the limited number of hrv sequences available in genbank during the time of design of rpm-flu v. / rendered a few of the targets represented on rpm-flu v. / are shorter than bp. in the past year, complete genome sequences from more serotypes and another two divergent hrv'x's have been reported [ , , ] . it will be worthwhile to update the design for the next generation of the chip. in the case of hev, the rpm-flu v. / assay identified only of strains tested at serotype level. several strains not producing serotype identifications might have been indicative of assay protocol issues or probe design. the fact that the in silico model prediction was also not serotype specific indicates it was most likely a design issue. this was further confirmed by agreement in base calls made from the resequencing microarray and from conventional resequencing. although all the strains of hev have complete genome sequences, there are also many partial sequence submissions for each strain in gen-bank that were ignored for the hev design. a re-examination of the 'utr regions showed up to % difference in sequences grouped in the same serotype. this indicates that a redesign of the hev prototype regions is needed where selection of a minimal set of prototype regions would be based on all available 'utr sequence data (complete and partial) and not a subset of genome sequences. comparing the identifications made from de novo sequencing to the identifications made by cdc (sources of the samples) illustrated another shortcoming of using the 'utr region for hev that did not occur for hrv. oberste et al. demonstrated that typing based upon hev vp capsid gene sequences showed excellent correlation with serotype determined by classical antigenic methods [ ] . thus amplification and sequencing of the partial vp amino-terminal coding region has been accepted as a standard molecular typing method for hev but such is not the case for the 'utr region [ ] [ ] [ ] [ ] [ ] . our results show that the 'utr region did not correlate as closely as vp -based typing to antigenic type definitions for hev unlike how it performed for hrv. while the 'utr region is sufficient to accurately identify the groupings, a design using vp as the probe region is needed to provide serotyping identifications that will match classical methods. the current rpm design can detect and identify a more comprehensive set of viral and bacterial respiratory pathogens in parallel, including detailed discrimination of certain serotypes of hrv and hev. this study showed that most shortcomings in the design were a result of not including adequate reference sequences for the initial design. the selection of vp and d regions also showed that incorporation of primer design considerations must be contemplated sooner in the design process than it has been currently done to prevent the selection of regions that cannot be used. future development will address these limitations by reducing hev probe redundancy and lack of coverage, by updating or confirming the hrv probes to be derived from newly available hrv sequences, and by involving primer design earlier in the overall design process. a powerful feature of the expanded rpm-flu v. / resequencing pathogen microarray is that the nucleotide sequences generated from hybridization of the sample rna/dna and array-bound probe sets in conjunction with previously developed sequence analysis algorithm cibsi can be easily interpreted to make serotype or strain identifications. this feature and the platform's high resolution and high throughput aspects undoubtedly have great potential for use as a diagnostic tool, and therefore, efforts are currently underway to test the utility of this array on more clinical samples. the results presented also validated the usefulness of the design methodology and it is currently being applied to assist in a new microarray application associated with other genetically diverse viruses. a panel of cultured enterovirus (hev) prototype strains was purchased from center for disease control and prevention (cdc, atlanta, ga). the prototype strains of rhinoviruses (hrv) and hev with known titers were purchased from the american type culture collection (atcc, manassas, va). total nucleic acids were extracted from μl cultured samples by using the mas-terpure™ dna purification kit (epicentre technologies, madison, wi) and dissolved in μl of nuclease-free water. all 'utr sequences of hrv and hev with approximately bp sizes were downloaded from genbank. potential pcr primer pairs that are able to amplify - bp fragments from hrv and hev were automatically selected by a perl script primer search program developed by our group using the rules described in previous publication [ , ] . the multiplex reverse transcription polymerase chain reaction (rt-pcr) protocols for rpm-flu v. / were carried out as previously described [ ] with the following modifications. for the rt step, primer ln was replaced by primer nln (a random mer with the unique linker sequence), pg each of two internal controls nac and triosephosphate isomerase (tim), and μl of the extracted viral nucleic acids were used. the μl rt reaction product was subjected to the multiplex pcr reaction. platinum taq dna polymerase (invitrogen life technologies, carlsbad, ca) was replaced by gotaq ® dna polymerase (promega corporation, madison, wi) in the pcr reaction. primer nl instead of primer l was used with - nm each of 'utr primers in the multiplex pcr. the amplification reaction was carried out in a peltier thermal cycler -ptc dna engine tetrad (mj research inc., reno, nv) with an initial incubation at °c for min, then preliminary denaturation at °c for min followed by cycles of °c for s, - °c for s (incremental increase of °c per cycle), and °c for s, then cycles of °c for s and °c for s. microarray hybridization and processing, and the image scanning were performed according to the manufacture's recommended protocol (affymetrix inc., santa clara, ca) using a genchip resequencing assay kit (affymetrix) with modification as previously described [ ] . after scanning, gcos software was used to reduce the raw image (.dat) file to a simplified file format (.cel file) with intensities assigned to each of the corresponding probe positions. gdas software was then used to produce nucleotide reads (a, c, g and t) or base calls, comparing the respective intensities for the sense and antisense probe sets. the sequences from base calls made for each tiled region of the resequencing microarray were exported from gdas as the fasta-formatted files. base call rate refers percentage of number of base calls generated from the full length of probe in each tile. final pathogen identification for the rpm-flu v. / assay was performed using computer-implemented biological sequence identifier (cibsi) version . software [ ] , an automatic pathogen identification algorithm based on nucleic acid sequence alignment, which was developed and tested in detail in previous studies [ , ] . the ncbi blast and taxonomy databases used for cibsi analysis was downloaded in december . heat-map and clustering dendrogram was made with dchip (dna-chip analyzer, http://www.dchip.org). the rows of the imported data (base call rates) were standardized and clustered. clustering distance was -correlation with average linkage, and gene ordering by cluster tightness. 'utr sequences were amplified from hrv-or hev cdna with specific primers. amplified products were purified and sent to macrogen usa (gaithersburg, md) for automated sanger/electrophoresis-based sequencing using corresponding specific primers. phylogenetic analysis of 'utr sequences was performed by using neighbor-joining method in mega software http://www.megasoft ware.net. all nucleotide sequences used in this study are available at genbank (accession nos. eu -eu ). diagnostic system for rapid and sensitive differential detection of pathogens rapid identification and strain-typing of respiratory pathogens for epidemic surveillance applications of luminex xmap technology for rapid, high-throughput multiplexed nucleic acid detection detection of respiratory viruses and subtype identification of influenza a viruses by greenechipresp oligonucleotide microarray microarray-based detection and genotyping of viral pathogens pan-viral screening of respiratory tract infections in adults with and without asthma reveals unexpected human coronavirus and human rhinovirus diversity microarray-based detection of genetic heterogeneity, antimicrobial resistance, and the viable but nonculturable state in human pathogenic vibrio spp broad-spectrum respiratory tract pathogen identification using resequencing dna microarrays use of resequencing oligonucleotide microarrays for identification of streptococcus pyogenes and associated antibiotic resistance determinants using a resequencing microarray as a multiple respiratory pathogen detection assay application of broad-spectrum, sequence-based pathogen identification in an urban population identifying influenza viruses with resequencing microarrays amplicon sequencing and improved detection of human rhinovirus in respiratory samples goossens h: improved detection of rhinoviruses by nucleic acid sequence-based amplification after nucleotide sequence determination of the ' noncoding regions of additional rhinovirus strains enteroviruses: polioviruses, coxsackieviruses, echoviruses, and newer enteroviruses lower airways inflammation during rhinovirus colds in normal and in asthmatic subjects a model of base-call resolution on broad-spectrum pathogen detection resequencing dna microarrays a diverse group of previously unrecognized human rhinoviruses are common causes of respiratory illnesses in infants genome-wide diversity and selective pressure in the human rhinovirus human rhinovirus and enterovirus represent a unique serotype with rhinovirus and enterovirus features evidence for frequent recombination within species human enterovirus b based on complete genomic sequences of all thirty-seven serotypes automated identification of multiple micro-organisms from resequencing dna microarrays highthroughput variation detection and genotyping using microarrays classification of enteroviruses based on molecular and biological properties complete genome sequences of all members of the species human enterovirus a assessing unmodified -mer oligonucleotide probe performance on glass-slide microarrays new complete genome sequences of human rhinoviruses shed light on their phylogeny and genomic features molecular evolution of the human enteroviruses: correlation of serotype with vp sequence and application to picornavirus classification typing of human enteroviruses by partial sequencing of vp improved molecular identification of enteroviruses by rt-pcr and amplicon sequencing molecular strategy for 'serotyping' of human enteroviruses molecular characterization of human enteroviruses in clinical samples: comparison between vp , vp , and rna polymerase regions using rt nested pcr assays and direct sequencing of products molecular identification and typing of enteroviruses isolated from clinical specimens the funding for this research was provided in part by the office of naval research via the nrl base program. partial support from tessarae, llc (potomac falls, va) through cooperative research and development agreement that help make this research possible is also gratefully appreciated.the opinions and assertions contained herein are those of the authors and are not to be construed as those of the u.s. navy or military service at large. zw conceived and designed the study, performed microarray experiments, analyzed data and wrote the manuscript; am designed microarray probes, analyzed data and wrote the manuscript; bl assisted in data analysis and preparing the manuscript; ck, nl and kb performed microarray experiments; dt helped to generate heatmap; ct assisted in data analyses; ds initiated the project and helped to prepare the manuscript. additional key: cord- - tszqh authors: xu, kai; rockx, barry; xie, yihu; debuysscher, blair l.; fusco, deborah l.; zhu, zhongyu; chan, yee-peng; xu, yan; luu, truong; cer, regina z.; feldmann, heinz; mokashi, vishwesh; dimitrov, dimiter s.; bishop-lilly, kimberly a.; broder, christopher c.; nikolov, dimitar b. title: crystal structure of the hendra virus attachment g glycoprotein bound to a potent cross-reactive neutralizing human monoclonal antibody date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: tszqh the henipaviruses, represented by hendra (hev) and nipah (niv) viruses are highly pathogenic zoonotic paramyxoviruses with uniquely broad host tropisms responsible for repeated outbreaks in australia, southeast asia, india and bangladesh. the high morbidity and mortality rates associated with infection and lack of licensed antiviral therapies make the henipaviruses a potential biological threat to humans and livestock. henipavirus entry is initiated by the attachment of the g envelope glycoprotein to host cell membrane receptors. previously, henipavirus-neutralizing human monoclonal antibodies (hmab) have been isolated using the hev-g glycoprotein and a human naïve antibody library. one cross-reactive and receptor-blocking hmab (m . ) was recently demonstrated to be an effective post-exposure therapy in two animal models of niv and hev infection, has been used in several people on a compassionate use basis, and is currently in development for use in humans. here, we report the crystal structure of the complex of hev-g with m . , an m . derivative, and describe niv and hev escape mutants. this structure provides detailed insight into the mechanism of hev and niv neutralization by m . , and serves as a blueprint for further optimization of m . as a therapeutic agent and for the development of entry inhibitors and vaccines. henipaviruses, hendra virus (hev) and nipah virus (niv) [ ] , are recently emerged, highly pathogenic paramyxovirus zoonoses whose major reservoirs in nature are several species of pteropid fruit bats [ , ] . hev causes lethal respiratory disease and encephalitis in horses and severe respiratory disease or late onset encephalitis in humans. in total, there have now been hev spillover events in australia including cases of human infection with fatalities since [ ] [ ] [ ] [ ] [ ] [ ] . niv subsequently emerged in peninsular malaysia in - , causing a large outbreak of respiratory disease in pigs and encephalitis among pig farmers, and was later shown to be closely related to hev [ ] . similar to hev, nearly annual outbreaks of niv infection have been observed. these niv outbreaks have been associated with significantly higher case fatality rates in people, up to %, and several outbreaks have also been linked to the consumption of raw date palm sap contaminated with virus as well as humanto-human transmission [ ] [ ] [ ] . to date, there have been reported cases of niv infection in people with fatalities [ , , ] . the unusual broad species tropism, high morbidity and mortality rates, as well as the lack of any licensed therapeutics, have rendered the henipaviruses biological safety level- (bsl- ) pathogens and potential biological threats to humans and livestock. an often utilized approach to antivirus drug design is to block viral entry via small molecules, peptides and neutralizing monoclonal antibodies (mabs) that bind to the viral surface glycoproteins. a unique feature of the majority of paramyxoviruses is that they require two surface glycoproteins for host cell entry: a class i fusion (f) glycoprotein and an attachment glycoprotein, which can be a hemagglutinin-neuraminidase (hn), hemagglutinin (h), or as in the case for henipaviruses a g glycoprotein that has neither hemagglutinating nor neuraminidase activities [ ] . the henipavirus g glycoprotein engages the host cell membrane protein receptors ephrin-b and -b , and this initial interaction is believed to be sufficient to trigger the f-mediated fusion event between the viral envelope and the host cell membrane leading to virus entry [ , , ] . in the absence of available vaccines or antiviral drugs, neutralizing hmabs offer the possibility for effective pre-and/or post-exposure treatment for many important human viral infections. previously, several hmabs, m -m , were isolated using a recombinant soluble hendra virus g (hev-g) glycoprotein as the antigen for panning of a large naïve antibody library [ ] . among the hmabs, m and its derivatives (m . - ) generated by heavy chain random mutations and light chain shuffling, showed improved binding to hev-g; clone m . had equal or higher binding affinity than the other clones and was selected for further characterization and converted to an igg format [ ] . the m . hmab was able to cross-react with both niv-g and hev-g in vitro with % inhibitory concentrations (ic )of less than ng/ml and ng/ml for nipah virus and hendra virus, respectively, and is capable of neutralizing all available isolates of hev and niv [ , ] . in animal disease models, m . has been shown capable of protecting ferrets against a lethal niv challenge [ ] , as well as african green monkeys (agm) against a lethal hev challenge [ , ] , in time frames of to even hours post viral exposure, respectively. in light of the experimental success of this post-exposure treatment of both niv and hev infection, m . has since been administered on a compassionate use basis to two individuals in australia with a high risk of hev exposure during the hev spillover, and again in in another person exposed to hev. in , m . was used again by compassionate use protocol in an individual with a laboratory exposure to niv in united states. in all these cases, none of these individuals showed symptoms of hev or niv infection at the time of m . administration, and all individuals remain in good health to date. altogether, as a fully human mab, m . shows promise as a potential prophylactic or therapeutic agent against henipavirus infection, and appears to be suitable for controlled safety trials in humans. to fully characterize the binding epitope [ ] , as well as the binding and recognition mechanism, we determined and here present the crystal structure of the complex between the globular head domain of hev-g and the fab domain of m . , a close derivative of m . , featuring an identical heavy chain and a similar light chain. the structure reveals the molecular mechanism of neutralization and cross-reactivity and provides a basis for further improvement of m . , including efficacy enhancement and escape mutant prevention. additionally, the presented structural information may aid the development of a henipavirus vaccine or other specific entry inhibitors. the head domain of hev-g (residues - ) was produced using the baculovirus expression system, and the fab domain of m . was expressed in hb cells. the protein complex was generated by mixing hevsg with hmab in a : . molar ratio followed by a hour incubation and purification by size-exclusion chromatography (sec). the peak fractions containing the complex were collected and used for crystallization. we obtained two crystal forms of the m . /hev-g complex and used molecular replacement to determine the structures at . Å resolution (in space group p ) and at . Å resolution (in space group i ) (table s ). there is one : complex per asymmetric unit in both crystal forms. the overall structures in the two crystal forms are very similar ( figure s ) and the region containing the hev-g molecules and the complementarity determining region- of the heavy chain (cdr-h ) of the fabs can be superimposed with an r.m.s.d of . Å for ca atoms, while the two fab structures, excluding just cdr-h , can be superimposed with an r.m.s.d of . Å for ca atoms. the difference in the two structures is in the angle between cdr-h and the rest of the fab, which consequentially causes a slight difference in the buried m . /hev-g interface area: Å in crystal form i , and Å in crystal form p . however, most interface residues in the core region are the same in both crystal forms. for the remainder of this report and figures, we use the p structure. the fab binds a similar area on hev-g ( figure a ) as ephrin-b , which is composed of a hydrophobic central cavity and a hydrophilic rim. interestingly, cdr-h of the fab approaches the hev-g central cavity in a similar angle and from the same direction as the g-h loop of ephrin-b ( figure b) . the interface involves fab residues mostly located on cdr-h , as well as three cdr-h residues (l , g and i ), one cdr-h residue (n ) and one cdr-l residue (r ) (figure ). the long protruding cdr-h ( residues) adopts a b-hairpin conformation, including a stalk and a tip (q -y ). the central hydrophobic hev-g/fab contacts since their initial emergence, henipaviruses have continued to cause spillover events in both human and livestock populations, posing significant biothreats. currently there are no licensed or approved therapies for treatment of henipavirus infection and the human case mortality rates average . %. we used x-ray crystallography to determine the high-resolution structures of the hendra virus g glycoprotein in complex with a cross-reactive neutralizing human monoclonal antibody. the structures provide detailed insight into the mechanism of hev and niv neutralization by this potent and clinically-relevant human monoclonal antibody that is currently in development for use in humans. this monoclonal antibody was recently shown to be an effective post-exposure therapy in nonhuman models of lethal hendra virus infection. indeed, it has already been used in four people on a compassionate use request, three in australia and one in the united states, as a therapeutic agent. furthermore, we identified and characterized two escape mutants generated in vitro and evaluated their mechanism of escape. our results serve as a blueprint for further optimization of this antibody and for the development of novel entry inhibitors and vaccines. this report also supports the additional pre-clinical data required for eventual licensure by detailing the antibody's mechanism of henipavirus neutralization. are formed by embedding the tip of cdr-h into the hydrophobic hev-g receptor-binding cavity. a simulated annealing omit electron density map of this region is illustrated in figure s . among the eight residues on the tip of cdr-h , l is surrounded by q , a , n , y , i , i and e of hev-g; p is surrounded by t , a , p , t , q of hev-g; p is surrounded by e , w , y of hev-g; s is surrounded by l and w of hev-g; and y is surrounded by t , c , t , s and e of hev-g ( figure a and c ). in addition, the side chain of q , h and p of the fab are stacked together, with q , which forms the top layer of the stack and hydrogen bonds to q of hev-g. the surrounding hydrophilic hev-g/fab contacts involve residues on cdr-h , cdr-l and cdr-h . notably, r on cdr-l forms salt-bridges with e and d of hev-g; hydrogen bonds are also formed between n on cdr-h and e of hev-g, as well as between r , e , y , y and y on cdr-h and y , s , t , q and t of hev-g, respectively ( figure a) . all hev-g residues engaged by m . are listed in figure s . interestingly, fab binding does not induce any significant conformational changes in hev-g and the r.m.s.d in ca positions between unbound and fab-bound hev-g is . Å . some previously identified hev-g mutations that were reported to affect the m or m . binding [ , ] are not part of the m . epitope ( figure s ) suggesting that those mutations might affect the overall hev-g structure. the hev-g head domain and fab are both strictly monomeric, but, interestingly, when these two proteins are mixed together, they first form a hetero-dimeric complex that oligomerizes further in solution. indeed the sec assays indicate that hours after mixing more than % of the complex migrates at a position corresponding to twice its original size in the gel-filtration column. an explanation of this phenomenon is provided by the crystal packing of the complex, where two copies of fab and hev-g assemble into a heterotetramer ( figure s ). it should be noted that the same : heterotetrameric fab/hev-g complex assembly is observed in both crystal forms. as illustrated in figure s , the heterotetrameric m . /hev-g assembly is generated by a two-fold crystallographic symmetry axis in which the two fab molecules contact each other burying approximately Å surface area on each side, while the two hev-g molecules remain separate. importantly, there is an additional contact area between the fab light chains elbow region and the hev-g molecule of the interacting complex, in which a further , Å are buried in each binding partner ( figure a , blue region). thus, a total Å surface area is occluded on each side of the interface between the two : complexes. within the : heterotetrameric complex, m . and hev-g form a more extensive interacting interface, rendering a more stable assembly. as shown in figs. a and b, the total hev-g surface region involved in the hev-g/m . interaction is almost the same as the one in the hev-g/ephrin-b , b interaction. however, the physiological relevance of this tetrameric assembly, and resulting cross-linking of mab/g complexes on the viral membrane, needs to be studied further. ephrin-b and m . both interact with the receptor-binding surface of hev-g, which includes a central hydrophobic cavity and surrounding hydrophilic rim. cdr-h of m . resembles the g-h loop of ephrin-b in both its shape and the insertion angle into the hev-g cavity ( figure ) . most of the g-h loopcontacting residues of hev-g also participate in the cdr-h binding (figure c, d). unlike ephrin, the binding of m . does not cause any significant conformational changes in hev-g. interestingly, although the tips of the g-h loop and cdr-h both target the pockets in the hev-g central cavity, each of them uses slightly different residues and anchoring strategies. as shown in figure d , from left to right, f , p , l and w on the ephrin g-h loop insert into four hydrophobic hev-g pockets, occupying half of the hev-g cavity; while l , p and p on the m . cdr-h insert into the first three of these same pockets ( figure c ). among these, ephrin p and p of cdr-h are strikingly similar. additionally, the insertion of cdr-h further embeds s and y into the other half of the hev-g cavity. the side chain of h of cdr-h extends in the same direction as w of the g-h loop but does not reach the fourth pocket. instead, it is embedded in a groove defined by q , w , and e of hev-g. a hydrogen bond between q of cdr-h and q of hev-g further locks the h in this position, preventing the withdrawal of cdr-h from the binding cavity. since the s contacting resides on hev-g are l and w , mutation of s to a or v could presumably enhance the interaction between m . and hev-g. in summary, cdr-h binds to hev-g utilizing a very high affinity lock-and-key mode without inducing conformational changes in hev-g. structure of hendra g with a neutralizing antibody plos pathogens | www.plospathogens.org comparison of m . binding to niv-g and hev-g although m mab was originally isolated against hev-g, it demonstrated a more potent neutralization capacity against niv than hev. indeed, in vitro binding measurements using biolayer infetrometry (table s ) document that both m . and m . display higher binding affinities for niv-g than for hev-g. due to the high similarity between niv-g and hev-g, presumably m . binds to niv-g and hev-g in a very similar manner, but an examination of the structure highlights some small structural differences that may explain the increased affinity of m for niv-g. upon superimposition of niv-g to the m . -bound hev-g, we found that only three residues at the mab contacting regions are different. among them, t and y in hev-g, which are part of the binding pockets for p and p of cdr-h , are replaced by two hydrophobic residues, valine and phenylalanine, in niv-g ( figure ). increasing hydrophobicity in this area very likely strengthens the predominantly hydrophobic interaction between the g protein and m . , enhancing its neutralizing activity. the panel of m derivatives was generated by light chain shuffling and heavy chain random mutagenesis. among them, m . was reported to have an equal or slightly higher affinity to henipavirus g glycoproteins in comparison to the others [ ] . biolayer interferometry (table s ) , on the other hand indicates that m . actually has slightly higher binding affinities to both niv-g (k d : . nm) and hev-g (k d : . nm) than m . (k d : . nm and nm, respectively). the primary sequences of m . and m . are overall highly similar, featuring an identical heavy chain (which provides all but one of the binding residues) and different light chain amino acid that are not part of the m . /hev-g interface ( figure s ) (but indirectly account for the small differences in binding affinities). as all hev-g contacting residues in m . are conserved in m . , the structural information obtained from the m . /hev-g complex could also be applied to explain the mechanism of m . neutralization. in the neutralization assay we performed, the efficiency of the m . mab to wild type niv and hev is three folds higher than that of the m . fab and m . fab, highlighting the importance of dimerization conferred by the fc region. notably, the efficiency of the m . fab is within the same range or slightly lower than that of the m . fab (figure ) , suggesting that the neutralization efficiency may also be affected by the other factors, such as protein stability or flexibility. to further detail and characterize the binding of the hmab to the virus, infectious niv and hev were used to generate antibody neutralization escape mutants by incubating and culturing high titers of virus in the presence of hmabs m . (fab fragment) or m . (fab fragment and mab). after passages, the resulting virus stocks were plaque purified and tested for neutralization efficacy. the g and f glycoprotein genes from a minimum of five plaques of each escape variant were sequenced in order to identify mutations associated with the antibody escape phenotype. for the niv escape mutant, all ten plaques of both the m . and m . escape mutants contained a single amino acid change at location v i. hev mutants that escaped m . neutralization all contained a single amino acid mutation at location d n ( figure s ). the m . and m . cloned virus stocks of these escape mutants of niv and hev, in contrast to wild-type niv and hev, were no longer neutralized by m . at antibody concentrations exceeding mg/ml (figure ). in addition, the cloned virus stocks of the escape mutants were then analyzed in single round growth assays in comparison to wild-type hev and niv on vero e , hela-usu-ephrin-b and hela-usu-ephrin-b cells ( figure ). both neutralization escape mutants grew as efficiently and to equal titers as the wild-type virus in vero e cells. noticeably however, during passaging, the m . and m . neutralization resistant viruses were relatively slow in developing cytopathic effects (cpe). whole genome sequence was performed to identify any additional mutations in these two escapes, as compared to their parent strains (sequencing report is attached in supporting materials as report s ). the hev escape variant contains another silent mutation in the p gene, in addition to the d n mutation in the g gene; while the niv escape variant contains mutations in the n gene ( end), m gene and l gene, in addition to the v i mutation in the g gene. thus, the escape is certainly due to the mutation in the g gene in case of the d n hev escape mutant. whole genome analysis of the parent virus stocks and the escape mutants and identification of all snps, indicates that the likely reason for the slow appearance of cpe in the presence of m . was the need for resistant virus amplification to levels sufficient for cell-cell fusion. combined with binding affinity measurements (table s ) , the hev-g/m . structure provides clues to the escape mechanism of the escape mutants ( figure c and d) . interestingly, the affinity of the g proteins to both antibodies and ephrin-b was increased by the v i mutation in niv-g, and decreased by the d n mutation in hev-g. residue v i is located at the bottom of the niv-g cavity, interacting with p of ephrin-b , b and p of m . . the additional methyl group of i would likely result in a more intimate interaction with both the cdr-h and g-h loops resulting in a lower k d value due to a decreased dissociation (k off ) rate. furthermore, ephrinb binding benefits slightly more than both antibodies from the v i substitution. intriguingly, d is located on the b s -s loop of the g protein, which is outside of the receptor/mab binding region, suggesting the d n mutation affects the fab/g-protein interaction through an indirect pathway ( figure s ). d forms salt-bridges with two residues on b s , r and k . the d n mutation would likely cause a conformational change in b s , causing re-arrangement of several m . -contacting residues including i , y and i , thus hindering the insertion with both the cdr-h and g-h loops. indeed, the observed association rates (k on , table s ) of this mutant to both antibodies and ephrinb decreased similarly. however, such a conformational change would affect the overall ephrin binding less, and it seems a similar rearrangement takes place even in the wild-type g protein ( figure s ) [ , ] . indeed, i , y and i are the pocket-forming residues for f of ephrinb ( figure d ). the elimination of the two salt-bridges resulting from the d n substitution might even have a slight stabilizing effect on the g-h loop insertion. accordingly, the observed dissociation rate (k off ) for binding of the hev-g mutant (d n) to ephrin-b was slightly decreased, while the dissociation rates for binding the antibodies were increased. in summary, both mutations in the g protein favor ephrin-b binding as compared to mab binding, consistent with their neutralization-escape phenotypes. targeting the viral surface spike proteins has been a powerful strategy in the development of neutralizing mabs. similar to m targeting the henipavirus g proteins, a number of potent antibodies have been developed against the spike (s) glycoprotein of the sars-associated coronavirus, the hemagglutinin glycoprotein of influenza virus and the envelope glycoprotein of hiv. the epitopes recognized by these antibodies are often functionally associated with the viral entry mechanism (e.g. attachment and membrane fusion) to reduce the occurrence of escape mutants. for instance in hiv, the epitopes targeted by neutralizing antibodies are located in four regions: receptor binding site (rbs), fusion associated membrane-proximal external region (mper) region, conserved glycan structures and glycan associated loop regions, while in influenza, the epitopes are located in the fusion associated stem region and sialic acid binding pocket region [ ] [ ] [ ] . in henipaviruses, as in all members of the paramyxovirus family, the attachment and fusion functions are exerted by two different proteins, which renders as possible epitope locations the rbs, the fusion-related regions, and sites associated with transducing the fusion-triggering signal from the attachment to the fusion proteins. in the past years, crystal structures of complexes between viral rbs and neutralizing antibodies have been determined, including m and r targeting the sars s glycoprotein rbs [ , ] , ch and c targeting the influenza virus sialic acid binding pocket [ , ] , and b , hj , vrc , nih - , a , bnc , vrc-pg and vrc-ch targeting the hiv- cd -binding site (reviewed in [ ] ). interestingly, in many of the examples above, the antibody cdr region mimics the conformation of the binding region of the cellular receptor (either protein or carbohydrate). amongst them, most similar to our m . /hev-g structure are the structures of ch and c targeting the influenza virus sialic acid binding site, which is a conserved shallow groove. all three mabs use only cdrh to bind their target groove, but compared to ch , c contacts a larger conserved region in the rbs, without interacting with the surrounding variable regions, which accounts for its greater neutralization breadth. the same strategy could be applied to further improve m . / . the m . / antibodies feature a long cdr-h ( residues in kabat numbering), adapting a b-hairpin, providing an interesting example of how antibodies circumvent obstacles in reaching the targeted epitope. one of the challenges in viral epitope targeting is that the epitopes are sometimes hidden, either behind heavy glycosylation or deep in a cavity. another example of a long cdr-h forming a b-hairpin is the antibody against hiv ( residues) [ ] . the extreme cases in this category are the hiv neutralizing antibodies pg and pg , which contain a -residue axe-shaped cdr-h [ , ] . of the many tested therapeutic strategies to prevent and/or treat infection and disease caused by the henipaviruses in a variety of well-characterized animal models, few have been effective [ , ] . recently, the only post-exposure therapeutic option that is highly effective in animal models with clear potential for future approved human use applications has been the hmab m . [ , ] . the reported success of m . in a nonhuman primate model of hev infection has been particularly encouraging, and the m . exhibited an excellent distribution half-time (, day) and elimination half-time (, days) in the agm. no evidence of hev-specific pathology was observed in any of the m . -treated animals and no infectious hev could be recovered. this study revealed that hmab m . prevented wide-spread hev dissemination in virus challenged subjects, and was the first successful post-exposure in vivo therapy against hev and the first in a nonhuman primate [ ] . during the hev spillover occurrence in queensland, australia, there were two individuals that were considered to be at high risk of hev infection [ ] . the m . hmab was requested by australian health authorities and administered to the two individuals as a compassionate use therapeutic option even though no human safety testing has been carried out and it was not recommended for use in humans. in this instance, m . was administered to the individuals prior to any hev diagnosis or onset of clinical disease [ ] with doses (, mg/kg) sufficient to achieve a high serum concentration, and to date both individuals remain healthy and no evidence of hev infection has been reported. the antibody appeared well tolerated when administered which was not unexpected since m . is a human mab. the m . hmab is now in further pre-clinical development stages in both the united states and australia. as part of our continued characterization of m . we sought to provide the molecular details of its ephrin receptor blocking activity by determining the crystal structure of a (nearly identical) m . derivative, m . , which possesses the same crossreactive neutralizing and henipavirus g binding activity with an identical heavy chain sequence and g glycoprotein binding loop in the cdr domain. the structure reported here of the hev-g/ m . complex reveals the molecular mechanism underling the exceptional cross-reactivity and neutralizing potency of these antibodies. the binding of the hmab to the g glycoprotein involves a single loop of its heavy chain with hydrophobic amino acid residues occupying the same pockets in g that the ephrin receptors engage during receptor binding. it is clear now that the central cavity on the henipavirus g glycoprotein receptor-binding face is vital for viral attachment and infection. from the crystal structure of the m . /g protein complex, we know that blocking access to this cavity is a feasible and efficient way of inhibiting henipavirus attachment and infection. specific peptide or small-molecule inhibitors for the viral attachment glycoprotein can be designed based on the structural data. for example, the existing pockets in the g glycoprotein cavity can be used as targets in structure-based computational screens. another approach would be to screen compound libraries using a protein interaction primary assay, and then optimize the initial hits to better fit the binding cavity. the data presented here are consistent with the initial steps of the henipavirus entry models proposed earlier based on the analysis of the g glycoprotein and the ephrin receptor/g glycoprotein complex structures [ , [ ] [ ] [ ] . of further importance, the new hmab . /g complex structure provides important information and leads for potential antibody improvement in two regards: increasing the antibody's affinity to the g glycoprotein in order to obtain even higher efficiency, and manipulating the interacting interface in order to reduce the potential of occurrence of escape mutants. the difficulty of the second aspect lies in the observation that the affinity of the attachment proteins to their receptors is not strictly correlated with the infection efficiency of henipaviruses. thus, mutations that affect the henipavirus g glycoprotein binding affinities to ephrin receptors and mabs to similar degrees could still allow potential escape. we indeed observed that even though there was a remarkable overlap between the m . epitope and the receptor binding region of henipavirus g, two escape mutant variants of hev and niv, containing g glycoprotein mutations d n and v i respectively, were identified. in vitro manipulation, such as repeated passaging of virus and allowing replication in the presence of a neutralizing antibody is routinely used as an approach to generate escape variants that can then be examined as a means to map epitopes and detail mab neutralization mechanisms. however, it should be emphasized that the appearance of m . escape variants has not been observed in any of the in vivo efficacy testing against hev or niv to date, and this is likely explained by the fact that very high doses of mab are utilized, similar to mab dosing used in people in the prophylactic treatment of rsv infection with f (synagis/palivizumab) [ ] . in addition, the effectiveness of m . appears to be by virtue of its ability to slow the progression and dissemination of virus within the challenged host, allowing the host an effective window in which to mount its own innate and adaptive immune response that eventually prevents lethal disease outcome. taken together, the success of hmab m . in vivo as an effective post-exposure treatment against henipavirus disease in two different well-characterized animal models (the ferret and nonhuman primate), along with the new detailed structural findings on its viral g glycoprotein binding features that help explain its superior cross-reactive neutralizing activity, will facilitate efforts aimed at obtaining approved human use application to treat accidental exposure to hev or niv infection. soluble head domain (amino acid residues - ) of hev-g was cloned into a pgp vector and expressed in the baculovirus expression system (bd biosciences). the plasmid was transfected into sf cells using cellfectin (invitrogen) and baculo-gold linearized baculovirus dna (bd biosciences). the virus was then amplified in sf cells for three rounds to reach the proper titer before applying to hi cells for final expression (in : volume infection ratio). the infected hi cells were harvested hours after infection. the cell media containing the hev-g protein was purified using ion-exchange and size-exclusion chromatography (ge biosciences). soluble fab was expressed and purified as described [ , ] . the hev-g/m . complex was obtained by mixing the two proteins in a : . molar ratio and was passed through a superdex column (ge biosciences). the fractions containing both proteins were collected and concentrated to mg/ml in hbs buffer ( mm hepes ph . , mm kcl). the initial crystallization condition was obtained with wizard iii (emerald biosystems) and pro-complex (qiagen) screens using robot screening (ttp labtech's mosquito). after several rounds of optimization using hanging drop vapor diffusion at room temperature, two crystals forms were obtained in conditions: % peg , . m (nh ) citrate, and % peg , . m hepes ph . , . m mgcl . crystals were frozen in liquid nitrogen with % glycerol as cryo-protectant. diffraction data were collected at beamline ne-cat id- of the advanced photon source at argonne national laboratory. data images were processed using program hkl . the structures were determined by molecular replacement with pdb id d (niv-g) and rz (hmab against gp of hiv virus). phaser [ ] , coot [ ] and phenix refine in the program suite phenix [ ] were used for structure determination, model building and refinement. the details of the crystallographic analysis are presented in table s . neutralization resistant niv and hev mutants were generated by incubating tcid of each virus with mg or mg of mabs m . (niv) and m . (niv or hev) respectively, in ml media for h at uc and then inoculated onto vero e cells in the presence of mabs at the same concentration. the development of cytopathic effect (cpe) was monitored over h and progeny viruses harvested. mab treatment was repeated two additional times with cpe developing slowly with each passage. passage viruses were plaque purified in the presence of mabs and neutralization resistant viruses were isolated. experiments were performed in duplicate and the glycoprotein and fusion protein genes of individual plaques from each experiment were sequenced. the neutralization titers between wild type and the neutralization resistant virus were determined by micro neutralization assay. briefly mab m . and fabs m . and m . were serially diluted two-fold, and incubated with tcid of the wild type (wt) and neutralization resistant niv or hev for h at uc. virus and antibodies were then added to a -well plate with vero e /well in wells per antibody dilution. wells were checked for cpe days post infection and the % neutralization titer was determined as the mab concentration at which at least % of wells showed no cpe. growth curves were performed by inoculating cell cultures with niv, hev and their escape mutants at a multiplicity of infection (moi) of for h, after which the cells were washed times with pbs and overlaid with medium. virus samples were obtained at various time points after infection and stored at uc until viral titers were determined by tcid . the binding kinetics of the wild type or mutant g proteins to both antibodies (m . and m . ) or to ephrin-b were measured by bio-layer interferometry on a blitz instrument (fortebio). ni-nta biosensors were used to immobilize the hexa-histidine fused antibodies and ephrin-b proteins. kinetic parameters (k on and k off ) and affinities (k d ) were calculated from a non-linear fit of the blitz instrument data using the blitz software. whole genome sequencing and analysis ml of each virus was mixed with ml trizol ls and rna was extracted following the manufacturers guidelines. illumina truseq cdna libraries were prepared from total rna, omitting the polya selection step. each library was subjected to half a miseq run using a cycle kit, paired end sequencing. a quality control tool for high throughput sequence, fastqc, a java stand-alone program was downloaded from babraham bioinformatics institute: http://www.bioinformatics. babraham.ac.uk/projects/fastqc/ and each fastq file was checked for quality. resulting wt hev and wt niv reads were mapped to their respective reference genomes, nc_ and nc_ , using clc genomics workbench v . . , using default parameters. consensus sequence was extracted for each and used as the reference genome to which the reads resulting from sequencing the mutant samples were mapped. consensus sequence for each mutant was extracted and aligned to the wt using clc genomics workbench v . . , and default parameters. henipavirus g attachment glycoprotein sequences were aligned in clc main workbench v . . using default parameters (gap open cost = ; gap extension cost = ). all molecular representations were produced with pymol (delano scientific llc). figures were prepared using adobe illustrator, adobe photoshop. figure s comparison of the fab/hev-g structures in the two crystal forms. a: the m . /hev-g complex structures in the two crystal forms were superimposed using the fab as a reference. b: the complex structures were superimposed using hev-g as a reference. fab and hev-g are colored in blue and grey in the p hendra and nipah viruses: different and dangerous fields virology the natural history of hendra and nipah viruses infection of humans and horses by a newly described morbillivirus human hendra virus encephalitis associated with equine outbreak human hendra virus infection causes acute and relapsing encephalitis hendra and nipah: lethal zoonotic paramyxoviruses henipavirus vaccine development new insights into the hendra virus attachment and entry process from structures of the virus g glycoprotein and its complex with ephrin-b nipah virus outbreak with person-to-person transmission in a district of bangladesh date palm sap linked to nipah virus outbreak in bangladesh person-to-person transmission of nipah virus in a bangladeshi community recurrent zoonotic transmission of nipah virus into humans nipah encephalitis, human -bangladesh: (jipurhat). international society for infectious diseases modes of paramyxovirus fusion: a henipavirus perspective entry and fusion of emerging paramyxoviruses henipavirus mediated membrane fusion, virus entry and targeted therapeutics potent neutralization of hendra and nipah viruses by human monoclonal antibodies exceptionally potent cross-reactive neutralization of nipah and hendra viruses by a human monoclonal antibody a neutralizing human monoclonal antibody protects against lethal disease in a new ferret model of acute nipah virus infection a neutralizing human monoclonal antibody protects african green monkeys from hendra virus challenge a novel model of lethal hendra virus infection in african green monkeys and the effectiveness of ribavirin treatment host cell recognition by the henipaviruses: crystal structures of the nipah g attachment glycoprotein and its complex with ephrin-b crossneutralization of influenza a viruses mediated by a single antibody loop structural insights into key sites of vulnerability on hiv- env and influenza ha human antibodies that neutralize hiv- : identification, structures, and b cell ontogenies structural basis of neutralization by a human anti-severe acute respiratory syndrome spike protein antibody, r structure of severe acute respiratory syndrome coronavirus receptor-binding domain complexed with neutralizing antibody broadly neutralizing human antibody that recognizes the receptor-binding pocket of influenza virus hemagglutinin crystal structure of human antibody reveals conserved features of quaternary structure-specific antibodies that potently neutralize hiv- crystal structure of pg and chimeric dissection with somatically related pg : structure-function analysis of two quaternary-specific antibodies that effectively neutralize hiv- structure and function of broadly reactive antibody pg reveal an h subdomain that mediates potent neutralization of hiv- therapeutics and vaccines against hendra and nipah viruses henipavirus outbreaks to antivirals: the current status of potential therapeutics hendra virus, equine -australia ( ): (queensland) human exposure structural basis of nipah and hendra virus attachment to their cell-surface receptor ephrin-b crystal structure and carbohydrate analysis of nipah virus attachment glycoprotein: a template for antiviral and vaccine design dimeric architecture of the hendra virus attachment glycoprotein: evidence for a conserved mode of assembly a systematic review of compliance with palivizumab administration for rsv immunoprophylaxis phaser crystallographic software coot: model-building tools for molecular graphics phenix: a comprehensive python-based system for macromolecular structure solution the authors wish to thank nick fera, uniformed services university, for preparation of the m . fab protein; and the staff of the ne-cat beamline id- at the advanced photon source of argonne national laboratory, for crystal data collection. key: cord- -abxv mkz authors: izopet, jacques; dubois, martine; bertagnoli, stéphane; lhomme, sébastien; marchandeau, stéphane; boucher, samuel; kamar, nassim; abravanel, florence; guérin, jean-luc title: hepatitis e virus strains in rabbits and evidence of a closely related strain in humans, france date: - - journal: emerg infect dis doi: . /eid . sha: doc_id: cord_uid: abxv mkz hepatitis e virus (hev) strains from rabbits indicate that these mammals may be a reservoir for hevs that cause infection in humans. to determine hev prevalence in rabbits and the strains’ genetic characteristics, we tested bile, liver, and additional samples from farmed and wild rabbits in france. we detected hev rna in % ( / ) of bile samples from farmed rabbits (in ) and in % ( / ) of liver samples from wild rabbits (in – ). full-length genomic sequences indicated that all rabbit strains belonged to the same clade (nucleotide sequences . %– . % identical to hev genotypes – ). comparison with hev sequences of human strains and reference sequences identified a human strain closely related to rabbit strain hev. we found a -nt insertion in the x domain of open reading frame of the human strain and all rabbit hev strains. these findings indicate that the host range of hev in europe is expanding and that zoonotic transmission of hev from rabbits is possible. h epatitis e virus (hev) is a major cause of acute hepatitis in many developing countries in asia and africa, where it is transmitted by the fecal-oral route because of poor sanitation practices ( ) . acute hepatitis e is also increasingly reported in industrialized countries, where the transmission is mainly zoonotic ( ) . the initial discovery of hev transmission from domestic pigs ( ) has been followed by evidence that other mammals, such as wild boars and deer, are also potential reservoirs of hev ( ) . although the course of hev infection is generally self-limiting and asymptomatic (or symptomatic with acute hepatitis), fulminant hepatitis can occur in pregnant women and in persons with underlying liver disease ( ) ( ) ( ) . hev infections can also become chronic in immunocompromised patients, such as recipients of solid-organ transplants ( ) ( ) ( ) , those with hematologic diseases ( , ) , and patients infected with hiv ( ) ( ) ( ) . hev, genus hepevirus, family hepeviridae, is a positive-sense, single-stranded, nonenveloped rna virus ( ) . the hev genome is ≈ . kb long and contains open reading frames (orfs) as well as ′ and ′ untranslated regions: orf encodes nonstructural proteins, orf encodes the capsid protein, and orf encodes a small phosphoprotein. phylogenetic analysis of hev sequences has led to the recognition of major genotypes that infect mammals from a variety of species. hev and hev are restricted to humans and transmitted through contaminated water in developing countries. hev and hev infect humans, pigs, and other mammals and are responsible for sporadic cases of hepatitis e in developing and industrialized countries ( ) . hev is distributed worldwide, whereas hev largely is found in asia. although hev and hev infections have been linked to the consumption of raw or undercooked meats, such as pig liver sausages or game meats ( , ) , the full spectrum of animals that are reservoirs of hev is still unknown. recent studies have characterized new hev genotypes in isolates from rats in germany ( ) , wild boars in japan ( ) , and farmed rabbits in the people's republic of china ( , ) . because the potential risk for zoonotic transmission strain in humans, france of hev from rabbits in france is unknown, and cases of autochthonous hepatitis e are commonly reported in this country ( , ) , we investigated the prevalence of hev in farmed and wild rabbits. we also looked for a genetic link between hev strains circulating in rabbits and hev strains circulating in humans in france. bile specimens (n = ) were collected in september from rabbits raised on farms in western france, in the departments of maine et loire (n = ), vendée (n = ), deux-sèvres (n = ), calvados (n = ), and loire atlantique (n = ), the main geographic areas of rabbit farming in france. we sampled rabbits from each farm when they were slaughtered at - days of age (table ) . all rabbits were healthy and intended for human consumption. all samples were immediately stored at − °c. liver specimens (n = ) were collected during september -november from populations of wild rabbits, established in warrens; each population was considered epidemiologically independent. the populations were located in several departments of mainland france: dordogne (n = ), finistère (n = ), deux-sèvres (n = ), loire-atlantique (n = ), haute-garonne (n = ), charentes (n = ), morbihan (n = ), and pyrénées-orientales (n = ) ( table ). the number of rabbits sampled in a given warren ranged from to . they were > months of age, apparently healthy, and intended for human consumption. each rabbit was eviscerated within a few hours of its death, and a sample of its liver was taken and immediately frozen at − °c. necropsies were performed on a group of rabbits from the same warren in haute-garonne (w ), and samples of their intestine and cecum were taken, in addition to samples from the liver. serum specimens were collected from immunocompetent and immunocompromised patients who had received a diagnosis of hepatitis e from the department of virology at toulouse university hospital. all samples were stored at − °c ( , ) . samples ( μl of rabbit bile and mg of liver, intestine, and cecum) were disrupted with trizol (invitrogen, saint aubin, france). rna was extracted with qiaamp viral rna mini kits (qiagen, courtaboeuf, france). we used -step real-time reverse transcription pcr on the light cycler instrument (roche diagnostics, meylan, france) to amplify a -bp fragment. the primers and probes targeted the orf region: forward primer hevorf -s: ′-ggtggtttctggggtgac- ′, reverse primer hevorf -as: ′aggggttggttggatgaa - ′, and probe ′-fam-tgattctcagcccttcgc-tamra- ′ ( ) . each -μl reaction mix contained μl of superscript iii platinum one-step quantitative rt-pcr system (invitrogen), μl of rna, primers ( nmol/l) and probes ( nmol/l), and u of rnase out (invitrogen). reverse transcription was carried out at °c for min, followed by denaturation at °c for min. dna was amplifi ed with pcr cycles at °c ( s) and °c ( s). hev rna was quantifi ed by using a transcribed rna standard constructed from a genotype f hev strain (genbank accession no. eu ). the limit of detection was copies/ml. two fragments, one within orf ( bp) and the other within orf , encompassing the hypervariable region and x domain (≈ , bp), were amplifi ed and sequenced in both directions by the dideoxy chain termination method (prism ready reaction ampli taq fs and dye deoxy primers; applied biosystems, paris, france) on an abi xl capillary dna analyzer (applied biosystems, foster city, ca, usa). the primers used for the orf fragment were the following: forward primer hevorf -s: ′-gacagaattratttcgtcggctgg- ′ and reverse primer hevorf -as: ′-tgytggttrtcataatcc tg- ′. the primers used for the orf fragment were the following: forward primer hevorf -s: ′-tgacggcyacygtkgarcttg- ′ and reverse primer hevorf -as: ′-acatcracatccccctgy tgtatrga- ′. the whole genomes of rabbit strains (w - and w - ) and human strain (tls- -human) were amplifi ed by overlapping rt-pcr. the primers are listed in table . the genotype was determined by using reference strains as previously described ( ) . phylogenetic analyses were performed with genotype information on reference sequences based on the hev classifi cation proposed by lu et al. ( ) . sequences were aligned by using clustalw (mega , www.megasoftware.net; bioedit version . , www.mbio.ncsu.edu/bioedit/bioedit). phylogenetic trees were created by the neighbor-joining (kimura -parameter) method with a bootstrap of , replicates. the partial sequences of orf and the full-length sequences reported in this study have been deposited in genbank. the accession numbers are jq and jq for orf , and jq to jq for the full-length sequences of w - , w - , tls- -human, tr (genotype c), and tr (genotype e), respectively. all bile specimens from the farmed rabbits and the liver specimens from the wild rabbits were tested for hev rna (table ) . samples from farms ( %) and warrens ( %) tested positive for hev rna. hev rna was found in a bile samples ( %) from farmed rabbits. the median hev rna concentration in the bile samples was . × copies/ml (range copies/ml- copies/ml). a total of liver samples ( %) from wild rabbits were positive for hev rna; median hev rna concentration was . × copies/g (range , copies/g- . × copies/g). we tested the liver, intestine, and cecum samples from wild rabbits from the same warren (w ) in triplicate to obtain a clear picture of the tissue distribution of hev in infected rabbits. hev rna was detected in all the tissues from rabbits (nos. , , , ) , in the liver and intestine of rabbit (no. ), and in the liver only of rabbit (no. ) ( table ) . the virus loads in the liver (mean . log copies/g), intestine (mean . log copies/g), and cecum (mean . log copies/g) were not signifi cantly different. phylogenetic analyses, conducted on the basis of a -nt fragment within orf of the hev strains from rabbits, hev strains from humans circulating in france, and hev reference sequences (hev , hev , hev , hev , rabbit hev, rat hev, wild-boar hev) indicated that the new orf sequences from rabbit hevs were clustered. one cluster contained orf sequences from previously characterized hev from farmed rabbits from china, orf sequences from hevs from farmed rabbits in france, and orf sequences from hevs from wild rabbits in france (figure ). this cluster also contained an orf sequence from a strain from a person in france (tls- -human) (figure ). this strain was found in a serum sample from a -year-old man with an elevated alanine aminotransferase level ( iu/l, reference < iu/l). phylogenetic analysis based on a , -nt fragment within orf , indicated that the orf sequences from hev strains from rabbits in france (n = ) or china (n = ) and the orf sequence from the human strain tls- human formed a distinct genetic group among sequences of hev genotypes - (data not shown). the cluster of rabbit hev sequences was also distinct from the hev sequences from wild-boar and rat hev genotypes that were characterized recently. comparison of the orf sequences from rabbit hev strains with reference orf sequences from hev genotypes - showed an insertion of nt in the x domain of the orf of all the rabbit hev strains. this insertion was also found in the tls- -human strain. the deduced amino acid sequences corresponding to this insertion, located between amino acids and (burmese strain, m ), were not very similar, except for conserved amino acids at the c-terminal end. we obtained the full-length genomic sequences of hev strains from wild rabbits in france and the tls- -human strain. the phylogenetic tree, constructed by the neighbor-joining method using the full-length genomic sequences (including the sequences of genotypes f, e, and c, which were circulating in france), revealed that the hev genomes from the rabbit strains and the tls- -human strain belonged to the same clade. this clade was clearly separated from genotypes - , found in other mammals and from the new hev genotypes found in wild boars and rats (figure ) . the length of the rabbit strain w - genome, excluding the poly(a) tract at the ′ terminus, was , nt. the length of the rabbit strain w - was , nt, and that of the tls- -human strain was , nt. the nucleotide sequences of the rabbit strains and the tls- -human strain were . %- % identical ( table ). the nucleotide sequences of the rabbit or tls- human strains were . % to . % identical to those of genotype , . % to . % identical to those of genotype , . % to . % identical to those of genotype , and . % to . % identical to those of genotype . these comparisons therefore indicate that the sequences of the rabbit hev strains and the tls- -human strain are distinct from all known strains of hev genotypes - and from the newly described hev genotypes from wild boars and rats. we found that farmed and wild rabbits in france are naturally infected with hev. we also characterized a human hev strain that is closely related to rabbit hev strains; this fi nding thus supports the potential of zoonotic transmission from rabbits to humans. the hevs found in farmed rabbits in several geographic areas of china have been identifi ed ( , ) . hev was also recently found in farmed rabbits in virginia, usa ( ) . our study results show that rabbits in europe are infected with hev and that some farmed rabbits and wild rabbits in france are infected. we found hev rna in % of the farmed rabbits and in % of the wild rabbits. however, the ages of the rabbits and the tissues tested (bile samples from farmed rabbits and liver samples from wild rabbits) may explain the observed difference in hev prevalence. nevertheless, previous studies have shown that the virus loads in liver and bile samples from swine infected with hev are similar ( , ) . although the greater prevalence of hev in wild rabbits could be linked to their older age, we could not test for a relationship between the prevalence of hev and rabbit age because we did not know the rabbits' precise ages. our analysis of the distribution of hev in the tissues of infected wild rabbits showed hev rna not only in the liver, but also in the intestine and cecum; our analysis also showed that the virus loads from these organs were not signifi cantly different. this fi nding suggests that emerging infectious diseases • www.cdc.gov/eid • vol. extrahepatic sites of hev replication exist in rabbits, as has been demonstrated for hev in pigs ( ) . however, because the intestine and cecum samples may have been contaminated with blood, our results need to be confi rmed in future studies using methods that ensure that tissues other than the liver are not contaminated with blood. to determine whether rabbits could be a reservoir for viruses that cause human infection, we analyzed partial and complete nucleotide sequences of the rabbit hev strains and compared these sequences with those of human hev strains circulating in france. analysis of orf showed that the sequences from rabbit hev strains formed clusters, one of which included the sequences of hev genotypes and . the bootstrap values were very low because the fragments analyzed were small. in contrast, phylogenetic analyses based on orf and the full-length genome indicated that all the rabbit strains from china and france belong to the same clade. one human strain, tls- -human, clustered with the rabbit strains and appeared to be somewhat different from the major hev genotypes found in mammals and the newly described hev genotypes from rats and wild boars. although the full-length sequences of the genomes of the rabbit strains and the tls- -human strain are more similar to that of hev than to those of hev , hev , and hev , they do not seem to belong to the established hev genotype found in humans and swine, as recently suggested ( , ) . differences in the classifi cation of rabbit hev could be because the full-length genomic sequences were used as the reference for phylogenetic analyses. genotype is highly diverse, with identifi ed subtypes ( ) . we included in our analysis the full-length genomes of subtypes f, c, and e, which account for ≈ %, %, and % of the human and swine hev strains circulating in france ( , ) . we also included the other full-length genomes representative of hev subtypes, but subtypes d, h, and i are not yet available in genbank. our data indicate that the genomes of rabbit hev strains or tls- -human were < % identical with hev , regardless of which method was used to align the sequences. this fi nding is compatible with the defi nition of a new genotype, as previously proposed ( , ) . we found a -nt insertion in the x domain of the orf of the human strain tls- -human and of all the rabbit hev strains. this insertion, also found in the rabbit hev strains from china ( ), is not present in any known strain of hev genotypes - or in the new hev genotypes from rats and wild boars. the x domain corresponds to a macro domain found in the nonstructural polyproteins of several positive-stranded viruses such as togaviruses and coronaviruses ( ) ( ) ( ) . this domain can bind polyadenosine diphosphate-ribose regions and could play a role in the replication or transcription of virus rna. whether the insertion in the x domain infl uences the function of the hev macro domain warrants further investigation. several determinants, including this insertion, could be essential for specifying the host range, zoonotic transmission, and pathogenesis of rabbit hev strains ( ) . what rabbit hev strains contribute to the epidemiology of hepatitis e in humans is not clear. hev is endemic to southwestern france, and the annual incidence of locally acquired hev infections has been estimated as . % ( , ) . a case-control study found that the only factor independently associated with hev infection was the consumption of game meat, mostly wild boar, deer, and wild rabbit ( ) . however, molecular data from various studies in france indicate that most hev strains identifi ed belong to genotypes f, c, or e, which are prevalent in pigs and wild boars ( , , ) . a recent study showed the same proportions of genotypes f, c, and e in human and pig populations ( ) . although this fi nding could indicate that rabbit hev strains are less readily transmitted to humans than hev genotype strains, the primers used for pcr amplifi cation were not specifi cally designed for rabbit hev strains. therefore, the true prevalence of hev rna among rabbits and humans may have been underestimated. in addition, genotyping rabbit hev may have been diffi cult because reference sequences have become available only recently. the immunocompetent or immunocompromised status of the patient that became infected with a rabbit hev strain, as well as the source of his contamination, is unknown because of the lack of medical follow-up. molecular and epidemiologic studies are needed to determine the prevalence of rabbit hev strains among immunocompetent and immunocompromised patients. in conclusion, we have shown that in france, farmed and wild rabbits can be infected with hev. phylogenetic analysis, based on full-length genomes and a molecular signature in the x domain of orf , indicates that rabbit hev strains could be a new genotype. our identifi cation of *hev, hepatitis e virus; hev (m - a-burma, d - b-japan, x - c-india, ay - e-chad, ay - d-morocco); hev (m - a-mexico) ; hev (af - a-usa, ay - j-canada-sw, ab - b-japan, tls-tr - c, tls-tr - e, ab - e-japan-sw,eu - f-france,af - g-kyrgyzstan-sw); hev (ab - c-japan, ab - g-china, ay - d-china-sw). a human hev strain that is closely related to rabbit hev strains reinforces the potential zoonotic risk for infection with this virus. further studies are needed to demonstrate cross-species transmission directly and to evaluate the contribution of the rabbit reservoir to human hev infection and disease. hepatitis e: an emerging awareness of an old disease hepatitis e: an emerging infection in developed countries a novel virus in swine is closely related to the human hepatitis e virus hepatitis e virus: animal reservoirs and zoonotic risk locally acquired hepatitis e in chronic liver disease clinical course and outcome of sporadic acute viral hepatitis in pregnancy fulminant 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norway rats, germany analysis of the full-length genome of a hepatitis e virus isolate obtained from a wild boar in japan that is classifi able into a novel genotype the serological prevalence and genetic diversity of hepatitis e virus in farmed rabbits in china a novel genotype of hepatitis e virus prevalent among farmed rabbits in china characteristics of autochthonous hepatitis e virus infection in solid-organ transplant recipients in france acute hepatitis e in south-west france over a -year period a broadly reactive one-step real-time rt-pcr assay for rapid and sensitive detection of hepatitis e virus hepatitis e virus genotype diversity, france phylogenetic analysis of global hepatitis e virus sequences: genetic diversity, subtypes and zoonosis hepatitis e virus in rabbits detection of hepatitis e virus in liver, mesenteric lymph node, serum, bile and faeces of naturally infected pigs affected by different pathological conditions hepatitis e virus load in swine organs and tissues at slaughterhouse determined by real-time rt-pcr evidence of extrahepatic sites of replication of the hepatitis e virus in a swine model zoonotic hepatitis e: animal reservoirs and emerging risks close similarity between sequences of hepatitis e virus recovered from humans and swine phylogenetic analysis of the full genome of rabbit hepatitis e virus (rbhev) and molecular biologic study on the possibility of cross species transmission of rbhev structural and functional basis for adp-ribose and poly(adpribose) binding by viral macro domains computer-assisted assignment of functional domains in the nonstructural polyprotein of hepatitis e virus: delineation of an additional group of positive-strand rna plant and animal viruses differential activities of cellular and viral macro domain proteins in binding of adp-ribose metabolites hepatitis e virus infection without reactivation in solid-organ transplant recipients hepatitis e virus antibodies in blood donors a national survey of acute hepatitis e in france we thank pascal bihannic, thierry delhorme, christian bernard, olivier galaup, francis berger, and jacky aubineau for their help with obtaining samples from wild rabbits.dr izopet is chief of the virology laboratory of toulouse university hospital. his research interests include the molecular virology of hev and its pathogenesis. key: cord- -dc t vh authors: todt, daniel; walter, stephanie; brown, richard j. p.; steinmann, eike title: mutagenic effects of ribavirin on hepatitis e virus—viral extinction versus selection of fitness-enhancing mutations date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: dc t vh hepatitis e virus (hev), an important agent of viral hepatitis worldwide, can cause severe courses of infection in pregnant women and immunosuppressed patients. to date, hev infections can only be treated with ribavirin (rbv). major drawbacks of this therapy are that rbv is not approved for administration to pregnant women and that the virus can acquire mutations, which render the intra-host population less sensitive or even resistant to rbv. one of the proposed modes of action of rbv is a direct mutagenic effect on viral genomes, inducing mismatches and subsequent nucleotide substitutions. these transition events can drive the already error-prone viral replication beyond an error threshold, causing viral population extinction. in contrast, the expanded heterogeneous viral population can facilitate selection of mutant viruses with enhanced replication fitness. emergence of these mutant viruses can lead to therapeutic failure. consequently, the onset of rbv treatment in chronically hev-infected individuals can result in two divergent outcomes: viral extinction versus selection of fitness-enhanced viruses. following an overview of rna viruses treated with rbv in clinics and a summary of the different antiviral modes of action of this drug, we focus on the mutagenic effect of rbv on hev intrahost populations, and how hev is able to overcome lethal mutagenesis. hepatitis e virus (hev) was first described as novel agent responsible for enterically transmitted non-a, non-b hepatitis by reyes and colleagues in [ ] . this was years after the first documented epidemic outbreak ( ) ( ) ) of a retrospectively identified hev infection-transmitted via the fecal-oral route-in new delhi, india [ ] . hev is a nonenveloped single-stranded rna virus with a . kb genome of positive orientation. three open reading frames (orfs) encode for: ( ) the nonstructural proteins (orf ), comprising a methyltransferase, a papain-like cysteine protease, a helicase, and an rna-dependent rna polymerase (rdrp), connected by a y-domain and a hypervariable region (hvr); ( ) the capsid protein (orf ); and ( ) small proteins whose functions are not yet completely understood (orf ) [ ] . the viral subgenomic rna is comparable to mammalian mrnas, flanked by a -methylguanine cap and a -poly(a) tail. hev has recently been taxonomically reassigned to the genus orthohepevirus in the family of hepeviridae [ ] . differences in the sequences of isolates led to the current classification into seven genotypes, four of which infect humans. hev- and hev- (i.e., genotypes and ) are solely human pathogens and are mainly transmitted orally by feces-contaminated drinking water. in , rbv was described as a broad-spectrum antiviral against several dna and rna viruses [ ] . since then, numerous studies have reported on the in vitro antiviral properties of rbv. figure provides an overview of a selection of rna viruses against which rbv was shown to be active: hepatitis c virus (hcv, flaviviridae), dengue virus (denv, flaviviridae), respiratory syncytial virus (rsv, paramyxoviridae), influenza a and b virus (orthomyxoviridae), chikungunya virus (chikv, togaviridae), poliovirus (picornaviridae), hantaan virus (bunyaviridae), and lassa virus (arenaviridae) [ , ] ( figure ). for further reading we would like to refer to other reviews like [ ] [ ] [ ] . studying multiple viruses from the family flaviviridae, crance et al. investigated the in vitro antiviral properties of rbv against flaviviruses including denv, japanese encephalitis virus (jev), and yellow fever virus (yfv). inhibition of virus replication was observed for all tested flaviviruses [ ] . furthermore, effectiveness of rbv could be confirmed in vivo for yfv using a hamster model by administering early upon infection [ , ] . however, these effects could not be confirmed in a nonhuman primate model [ ] . therefore, further studies are required to evaluate the possible application of rbv as a treatment option for yfv. here, the dosage as well as the time points of treatment represent the major hurdles, which need to be overcome [ ] . additionally, hemorrhagic fever-causing viruses, which are categorized into the families of arena-, bunya-, and togaviridae, were demonstrated to be susceptible to inhibition by rbv ( figure ). for example, for lassa virus the antiviral efficiency of rbv was proven both in vitro and in vivo in guinea pigs and monkeys [ , ] . hantaviruses (i.e., hantaan virus) and phleboviruses (i.e., rift valley fever virus, rvf) were also shown to be susceptible to rbv treatment [ ] . in a mouse model for hantaan virus, an increase of survival and milder signs of disease were described [ ] . in experimental rvf infections of mice and hamsters, rbv led to a prevention of death, delay of death, or the onset of milder symptoms, depending on the time point of administration [ ] . in general, higher doses of rbv were needed to inhibit flaviviruses compared to arena-, bunya-, and hantaviruses [ ] . for chikv, rbv also demonstrated antiviral effects, although to a lesser extent when compared to interferon-α (ifn-α). nevertheless, a synergistic effect of rbv and ifn-α could be demonstrated in vitro [ , ] . moreover, the antiviral properties of rbv could be shown for members of the picornaviridae: both foot-and-mouth disease virus (fmdv) [ , ] and poliovirus (pv) [ ] were inhibited by rbv (figure ). demonstrated antiviral effects, although to a lesser extent when compared to interferon-α (ifn-α). nevertheless, a synergistic effect of rbv and ifn-α could be demonstrated in vitro [ , ] . moreover, the antiviral properties of rbv could be shown for members of the picornaviridae: both foot-and-mouth disease virus (fmdv) [ , ] and poliovirus (pv) [ ] were inhibited by rbv ( figure ). figure . antiviral properties of ribavirin (rbv) against rna viruses. the broad-spectrum antiviral properties of rbv have been reported for several rna viruses. depicted is a selection of the different viral families and the respective genus and species. viruses for which rbv was clinically approved are highlighted with an orange box. viruses for which lethal mutagenesis or increased mutation rate was proposed as a possible rbv mechanism are indicated in blue. while displaying broad antiviral activity against a wide range of rna viruses, clinical data on the application of rbv are still limited and restricted to only a few viruses. initially, rbv was considered as a treatment option for influenza a and b virus infections. however, clinical trials showed inconclusive data; although some studies reported an improvement of symptoms of influenza virus infection, results were generally inconsistent [ , ] . due to lack of conclusive data from clinical trials, coupled with the development of alternative antiviral therapies, rbv has never been approved for the treatment of influenza virus. nonetheless, lassa virus, hcv, and rsv are prominent examples of viruses for which rbv has received approval as an antiviral compound for clinical application [ ] . rbv was shown to be effective in treating patients suffering from lassa fever [ ] and can be administered orally, intravenously, or as pre-or post-exposure prophylaxis [ ] ., in , rbv was approved by the food and drug administration (fda) as a treatment option for hcv [ ] and was, in combination with pegylated ifn-α, the standard treatment for chronic hcv infection for over two decades [ ] . it has been shown that after failure of a monotherapy with ifn-α alone, a combination therapy with rbv is more effective than subsequent repetition of ifn-α monotherapy [ ] . however, the sustained virological response (svr) rates varied among genotypes, and dual therapy was associated with severe side effects [ ] . nowadays, rbv is no longer the standard-ofcare anti-hcv therapy, and has been replaced by direct-acting antivirals (daas). initial trials for the treatment of rsv infection showed a reduced duration of hospitalization and requirement of mechanical ventilation [ , ] . a routine use of rbv in rsv-infected children is not recommended; however, treatment can be considered for individual cases [ ] . while displaying broad antiviral activity against a wide range of rna viruses, clinical data on the application of rbv are still limited and restricted to only a few viruses. initially, rbv was considered as a treatment option for influenza a and b virus infections. however, clinical trials showed inconclusive data; although some studies reported an improvement of symptoms of influenza virus infection, results were generally inconsistent [ , ] . due to lack of conclusive data from clinical trials, coupled with the development of alternative antiviral therapies, rbv has never been approved for the treatment of influenza virus. nonetheless, lassa virus, hcv, and rsv are prominent examples of viruses for which rbv has received approval as an antiviral compound for clinical application [ ] . rbv was shown to be effective in treating patients suffering from lassa fever [ ] and can be administered orally, intravenously, or as pre-or post-exposure prophylaxis [ ] . in , rbv was approved by the food and drug administration (fda) as a treatment option for hcv [ ] and was, in combination with pegylated ifn-α, the standard treatment for chronic hcv infection for over two decades [ ] . it has been shown that after failure of a monotherapy with ifn-α alone, a combination therapy with rbv is more effective than subsequent repetition of ifn-α monotherapy [ ] . however, the sustained virological response (svr) rates varied among genotypes, and dual therapy was associated with severe side effects [ ] . nowadays, rbv is no longer the standard-of-care anti-hcv therapy, and has been replaced by direct-acting antivirals (daas). initial trials for the treatment of rsv infection showed a reduced duration of hospitalization and requirement of mechanical ventilation [ , ] . a routine use of rbv in rsv-infected children is not recommended; however, treatment can be considered for individual cases [ ] . taken together, since its first description as an antiviral in , rbv has been shown to be active against a broad range of rna viruses. however, due to limited clinical trial data supporting its in vivo efficacy, clinical applications are currently limited to a minority of viruses. the broad antiviral effect of rbv against numerous rna viruses suggests different modes of action for the molecule; indeed, several antiviral mechanisms have been described in the past [ , ] and are summarized in figure a . among the indirect mechanisms, a t-cell-mediated effect was described for hcv ( figure a) . here, the balance of t helper cells was changed by switching from a t helper type phenotype to a t helper type [ ] . in a study by hultgren et al., an inhibition of in vitro t-cell proliferation as well as a change in secreted cytokines was observed [ ] . simultaneously, alanine transaminase (alt) levels in serum were reduced with no change in hcv titers [ ] . furthermore, an early switch of a t helper type immune response to a t helper type immune response was associated with disease progression and the development of chronicity [ ] . thus, rbv restored the t helper phenotype needed for balanced expression and secretion of cytokines produced from type and t helper cells [ ] . another example where an immunomodulatory effect was described for rbv is in rsv infection. it was proposed that a t helper type cytokine response initiated the cascade leading to airway hyper-reactivity, which in turn can be blocked by rbv treatment [ ] (figure a) . another indirect mode of action for rbv is the inhibition of the cellular inosine monophosphate dehydrogenase (impdh), which was already proposed in [ ] (figure a ). after uptake into the cell, rbv is phosphorylated to rbv mono-, di-, and triphosphate (rmp, rdp, and rtp, respectively). rmp represents a good mimic of inosine monophosphate (imp) and thereby inhibits the synthesis of imp to xanthosine monophosphate (xmp) by impdh. consequently, no guanosine monophosphate (gmp), and subsequently guanosine triphosphate (gtp), can be synthesized. in vitro, replication of measles virus in vero cells could be blocked by the addition of xmp, gmp, and to a lesser extent, also imp [ ] , which underlines the mode of action of rmp. a linear correlation of the depletion of gtp pools and in vitro antiviral activity of rbv against human parainfluenza virus and yfv was confirmed [ ] . furthermore, the addition of guanosine to cell cultures restored the antiviral activity of rbv against gb virus b (gbv-b) [ ] . in contrast, in vitro experiments with lassa virus and hantaan virus indicated that rbv did not primarily act via depletion of gtp pools for these two viruses [ , ] . moreover, experiments with influenza a virus showed no linear correlation of intracellular gtp pools and viral replication with increasing concentrations of rbv [ ] . additionally, the authors did not observe a complete restoration of influenza a virus replication after addition of guanosine [ ] . no effect of guanosine or gmp on the antiviral effect of rbv against influenza a virus in mice could be demonstrated [ ] . overall, these data suggest that other mechanisms for the mode of action of rbv exist. the influence of rbv on the expression of ifn-stimulated genes (isg) is controversial in the literature. most studies, both in vivo and in vitro, come from the hcv and rsv fields. rbv is able to increase the antiviral effects of an ifn-based therapy and restore ifn-responsiveness in hcv-infected livers [ ] [ ] [ ] [ ] . also, a direct, ifn-independent upregulation of isgs has been proposed [ , ] . however, a recent study with hcv patients receiving rbv monotherapy showed a downregulation of abnormally preactivated isgs through chromatin remodeling and modulation of histone methylation, resulting in a higher liver susceptibility to ifn by lowering the baseline expression of certain isgs [ ] . some rna viruses, as well as cellular mrnas, harbor a -methylguanosine cap structure at the end [ ] . the rbv-induced reduction of gtp pools within the cell was proposed to also have an effect on the capping efficiency of rna viruses (figure a ). for example, denv encodes for a -o-methyltranferase at the n-terminus of the ns polymerase, termed ns mtase dv . ns mtase dv binds gtp and catalyzes the formation of a cap structure [ ] . after rbv treatment, less gtp is present and rtp was shown to compete for binding to the ns mtase dv , thereby blocking the synthesis of the cap [ ] . likewise, rbv directly and strongly inhibited the viral mrna guanylyltransferase of vaccinia virus and thus prevented capping of nascent viral rna [ , ] . however, this mechanism is controversially discussed in literature [ ] [ ] [ ] [ ] , and not all rna viruses display a -methylguanosine cap structure at the end. therefore, this mode of action cannot account exclusively for the observed effects of rbv. another suggested mechanism is the direct impact of rbv treatment on the function of viral polymerases (figure a,b) . here, rtp is thought to directly inhibit viral rna replication by being recognized by the viral polymerase and thereby leading to chain termination or preventing the binding of other nucleotides important for elongation [ ] . in a cell-free system, rtp was shown to inhibit the rna polymerase of influenza a virus [ ] . moreover, inhibition of viral rna synthesis of vesicular stomatitis virus (vsv) in the presence of rmp, rdp, and rtp was described with the triphosphorylated form being the least active [ ] . this would argue against a mode of action that is based on the incorporation of rtp in the nascent viral rna in vsv. in the same study, an inhibitory effect of rdp on la crosse virus rna synthesis was also reported [ ] . interestingly, crotty et al. could demonstrate that rtp is indeed employed by pv rdrp, and that integrated rbv acts as mutagen [ ] . another example of an effect of rtp on the viral polymerase is the case of reovirus. rankin et al. proposed that rtp binds close to the catalytic site of the transcriptase, thereby affecting the helicase function and subsequently lowering the binding affinity of viral rna [ ] . as a consequence, elongation of the viral rna is inhibited. interestingly, no effect on the capping activity was demonstrated [ ] . the nucleotide binding site of the polymerase is highly conserved among hcv genotypes, supporting this proposed mechanism [ ] . indeed, in vitro analysis showed a minor decrease of hcv replication [ , , ] . however, in clinical trials with rbv monotherapy, only a mild decrease of hcv replication was noticed [ , ] . in recent years, a mutagenic effect of rbv via its incorporation into newly synthesized rna genomes, leading to viral extinction was described for several rna viruses ( figure b ). in contrast to dna viruses, the major characteristic of rna viruses is the occurrence of a cloud of related but genetically distinct variants in infected patients, often referred to as a quasispecies. however, the term "quasispecies" refers to a particular mutation-selection balance, with natural selection acting on the group rather than on the individual [ , ] . it is not simply a surrogate for genetic heterogeneity [ ] . while quasispecies behavior has been demonstrated experimentally in artificially expanded poliovirus populations in infected mice [ ] , evidence is lacking for quasispecies' behavior in many viruses, including hev. these diverse intra-host viral populations are the result of the lack of proofreading activity of rdrp. however, due to this high variation, viral isolates are close to the error threshold, which would lead to reduction in viral fitness [ ] . incorporation of rbv into newly synthesized rna genomes thereby increases the frequency of mutations in the population, pushing the virus over an error threshold and resulting in viral extinction. this mechanism of action for rbv has been described, at least in vitro, for fmdv [ ] , poliovirus [ ] , hcv [ ] , gbv-b [ ] , hantaan virus [ ] , and hev [ , ] . ever since the first reports by sidwell et al. describing rbv as a broad-spectrum antiviral [ ] , there have been multiple discussions about its mechanisms of action. of course one has to always keep in mind that in vitro data, where most of the proposed models arose from, cannot just be translated into in vivo situations. remarkably enough, monotherapy with rbv is only potently effective against lassa virus [ ] and hev [ , ] . future studies should address questions regarding the biocompatibility of rbv and its availability in the targeted liver to investigate if intracellular concentrations can account for the different proposed mechanisms-for example, to outcompete cellular nucleoside triphosphates (ntps) for misincorporation. in summary, several mechanisms have been postulated for rbv activity. among these, there are indirect, immunomodulatory mechanisms and effects on impdh. furthermore, mechanisms on the virus itself were described by inhibition of the capping efficiency, the viral polymerase, and a mutagenic effect on newly synthesized rna genomes. rna viruses do not exist as a clonal population of genomes within the infected host, but rather diversify into a swarm of related but non-identical genome sequences [ ] . this heterogeneous viral population-also referred to as mutant cloud, mutant swarm, or mutant spectra-is capable of better adapting to changing environmental conditions and rapidly evolving, during passage from host to host, due to its high heterogeneity. the concept of quasispecies was mainly developed by manfred eigen and peter schuster [ ] . by demonstrating viral heterogeneity for fmdv [ , ] and vsv [ , ] domingo and colleagues and holland and colleagues were the first to extrapolate this concept to virology [ , ] . these viral populations are the product of very high replication rates found in rna viruses, coupled with a lack of an rdrp proofreading function. for hcv, it is estimated that between and new virions are produced in one infected individual per day [ , ] . estimates for hev do not currently exist, although comparably high replication rates can be assumed. there is data on -end repair mechanisms identified in small rna viral polymerases [ ] . in coronaviruses, for example, a -to- exoribonuclease (exon) domain within the nonstructural protein was identified as being essential for high-fidelity replication [ , ] ; for hcv, pyrophosphorolytic and ntp-mediated nucleotide excision activity of the ns b rdrp have been described as viral mechanisms for removing misincorporated bases [ , ] . despite these reports, most rna viruses, and most likely also hev, do not have any real proofreading capability, causing an error-prone replication of viral genomes. together with the short generation times, this results in highly diverse intra-host populations [ ] . as expected, hev also exists as a heterogeneous population within infected individuals [ , , [ ] [ ] [ ] . early publications relied on the classical tools for detecting diversification of viral genomes, including restriction fragment length polymorphism (rflp) and haplotype profiling [ , ] or clonal sequencing [ ] to characterize hev intra-host diversity. recently, next-generation sequencing (ngs) methods have been utilized to study the distribution of snvs in hev genomes over time [ , ] . hev and most other rna virus populations exist in close proximity to the so-called genomic error threshold, which defines a maximum error rate that still guarantees the maintenance and transmission of the genetic information of the master sequence [ , ] . a replication and, most importantly, mutation rate beyond this extinction threshold causes a sharp reduction in the efficiency of transmission of the genetic information contained in the population master sequence to the next generation of viral progeny, a phenomenon sometimes referred to as error catastrophe [ ] : the majority of genomes in the population are nonfunctional. broad-spectrum antiviral agents like rbv can cause increased mutation rates, and potentially can result in the extinction of the virus population in a process called lethal mutagenesis [ ] . however, the mutated viral intra-host populations can acquire mutations accounting for drug resistance or decreased sensitivity to rbv as a direct consequence of the boosted complexity of the mutational spectra. this has been shown for several viruses like hcv [ ] , fmdv [ ] , pv [ ] , sindbis virus [ ] , and also for hev [ ] [ ] [ ] . the dynamics of hev populations in patients under rbv therapy is not fully understood, but recent studies and reports from other rna viruses point to a dichotomy of opposing outcomes resulting from rbv therapy: rbv-induced lethal mutagenesis resulting in viral extinction versus the accumulation of mutations beneficial to the virus in the population, which can lead to therapeutic failure [ , ] . as a consequence of the emergence of rbv-resistant mutations and subsequent treatment failure, clinicians could draw back on combination therapies to overcome or avoid this phenomenon. possible combinations are one mutagen and a conventional antiviral drug or using several rna mutagens in combination or sequence as proposed by perales and domingo [ ] [ ] [ ] . hev is one of the pathogenic viruses that can currently only be treated with rbv as an off-label drug. ifn-α as an alternative therapy has been evaluated in small patient cohorts with limited success and considerable side effects [ , ] . in addition, in vitro data suggests careful assessment of ifns when treating hev [ , ] . considering high mortality rates of over % for genotype- -infected pregnant women [ , ] , the urgent need for extensive research in the field of novel anti-hev treatment regimens is required. patients who fail to achieve sustained virological responses after rbv therapy for hev have no further treatment options: this is particularly of importance in a solid organ transplant setting, as a reduction of immunosuppression beyond a certain level will lead to the rejection of the allograft [ , ] , and hepatitis caused by hev cannot be impeded. recently, two independent studies were able to correlate rbv treatment failure with the emergence of novel single-nucleotide variations in the viral genome during treatment [ , ] . both research groups identified a variant previously described, g r [ ] , as well as other new variants, k n, d g, k r, v i, and y f, all in the polymerase region of orf . in addition, todt et al. also determined nine additional snvs in orfs and [ ] . in both studies, k n mutations emerged in several patients; additionally, an overall increase in viral intra-host heterogeneity could be shown [ , ] . the authors demonstrated significant increases in the number of sites exhibiting snvs, synonymous as well as nonsynonymous, in viral populations after the first administration of rbv in nine patients. this phenomenon was observed for all orfs of the hev genome. interestingly, this increase in heterogeneity was reversible with a decline in the number of snv sites when rbv treatment was stopped. strikingly, none of the described variants that became dominant in the viral populations under treatment resulted in a decreased sensitivity to rbv when cloned into an hev subgenomic reporter replicon in tissue culture. only g r mutations altered the viral replication efficacy, increasing replication rates [ , ] , while rbv sensitivity was unmodified [ , , ] . why rbv treatment fails in some patients, while others are able to clear the virus under rbv monotherapy, remains an open question. rbv has been shown to block hev replication through a depletion of cellular gtp pools in cell culture model systems [ ] , in addition to the strong mutagenic effect of rbv on the hev genome in vivo described above. rbv inhibits the impdh, thus causing a two-fold reduction of the intracellular gtp pools and increasing ctp and utp concentrations at the same time [ , ] . hev genome replication is a cyclic process of alternating synthesis of negative-strand rna and positive-strand rna [ ] . during the replication process, the extrinsically administered, rtp is randomly incorporated into the nascent negative-stranded rna as a result of pairing with either of the pyrimidine bases cytidine or uracil ( figure b, upper panel) . this negative-stranded antigenome rna then serves as a template for subsequent production of positive-stranded genomic rna. the rdrp subsequently incorporates, again randomly, a cytidine or uracil at rbv residues located in the antigenome template ( figure b, middle panel) . these stochastic incorporations lead to nucleotide substitutions in the newly synthesized viral genomes. additionally, rbv will also be incorporated in the positive-stranded rna genome, leading to increased amounts of replication-defective viral genomes packaged into the capsid, ultimately leading to an increase in frequency of replication-defective virions. additionally, new antigenome templates can be produced from defective positive-stranded genomes, so misincorporations are amplified in the replication process ( figure b, lower panel) . this results in the fixation of transitional substitutions in nascent rnas. transitional purine-to-purine (g<>a) or pyrimidine-to-pyrimidine (c<>t) nucleotide substitutions are preferentially enriched during rbv monotherapy, leading to the observed synonymous exchanges as well as to the amino acid replacements favorable for the survival of the viral population [ , ] . whether rbv also inhibits the hev methyltransferase comparably to the direct inhibition of the vaccinia virus guanylyltransferase (see above), or if the rtp is incorporated as a cap analog [ , ] (thus impacting correct translation) has not been investigated yet. the mutagenic effect of rbv-based therapy can have divergent effects on hev populations, which may impact the therapy success. on the one hand, rbv increases the mutation rate in the viral genome, driving the population towards its extinction threshold. in contrast, the increased variability in the viral population can result in selection of variants with improved replication fitness which become dominant in the viral population and are associated with therapeutic failure. these advantageous variants could be (i) a downregulation of the replication machinery, thus preventing the accumulation of more mutations, as shown from in vitro data when reverse engineering the k n variant into hev cell culture systems [ ] , and (ii) an increase in viral polymerase fidelity as hypothesized by debing et al. for the k n variant-a mutant with a substitution in the f -motif of the rdrp-which could hinder the incorporation of rbv into the viral genome. in fact, the lab of esteban domingo was able to dissect a multistep process of viral adaption to a mutagenic nucleoside analog in fmdv that led to an extinction escape by changing the fidelity of the polymerase [ ] . hev is a life-threatening infection when immunosuppressed individuals fail to achieve an svr during rbv treatment. currently, clinicians do not have alternative therapy regimens available. recent studies have suggested that the heterogeneous viral population is able to acquire snvs that decrease rbv sensitivity [ , ] . their data supports a conclusion whereupon the mutagenic effect of the broad-spectrum antiviral agent leads to increased heterogeneity in the intra-host viral population introducing a race between the virus trying to gain and accumulate beneficial variations and the mutagenic potential of rbv intended to drive the virus beyond an error threshold and thus into lethal mutagenesis resulting in viral extinction. hepatitis-e virus (hev)-the novel agent responsible for enterically transmitted non-a, non-b hepatitis infectious hepatitis in delhi ( - ): a critical study-epidemiology update on hepatitis e virology: implications for clinical practice international committee on taxonomy of viruses hepeviridae study group consensus proposals for classification of the family hepeviridae history and global burden of viral hepatitis pathogenesis and treatment of hepatitis e virus infection viral hepatitis in pregnancy-a study of its effect on maternal and foetal outcome hepatitis e virus infection in the hiv-positive patient hepatitis e virus infection in the hiv-positive patient world health organization hepatitis e (fact sheet) hepatitis e: an old infection with new implications hepatitis e in germany-an under-reported infectious disease hepatitis e virus and neurological injury guillain-barre syndrome associated with preceding hepatitis e virus infection evidence of hepatitis e virus breaking through the blood-brain barrier and replicating in the central nervous system hepatitis e virus infection as a direct cause of neuralgic amyotrophy guillain-barre syndrome following hepatitis e extra-hepatic replication and infection of hepatitis e virus in neuronal-derived cells selection of hepatitis c virus resistant to ribavirin foot-and-mouth disease virus mutant with decreased sensitivity to ribavirin: implications for error catastrophe a single mutation in poliovirus rna-dependent rna polymerase confers resistance to mutagenic nucleotide analogs via increased fidelity sindbis virus mutants resistant to mycophenolic acid and ribavirin a mutation in the hepatitis e virus rna polymerase promotes its replication and associates with ribavirin treatment failure in organ transplant recipients hepatitis e virus mutations associated with ribavirin treatment failure result in altered viral fitness and ribavirin sensitivity in vivo evidence for ribavirin-induced mutagenesis of the hepatitis e virus genome broad-spectrum antiviral activity of virazole: -beta-d-ribofuranosyl- , , -triazole- -carboxamide ribavirin and lethal mutagenesis of poliovirus: molecular mechanisms, resistance and biological implications mechanisms of action of ribavirin against distinct viruses approved antiviral drugs over the past years ribavirin-current status of a broad spectrum antiviral agent interferon, ribavirin, -azauridine and glycyrrhizin: antiviral compounds active against pathogenic flaviviruses treatment of yellow fever comparison of the inhibitory effects of ribavirin and interferon alfacon on a yellow fever virus infection in syrian golden hamsters prospects for treatment of viral hemorrhagic fevers with ribavirin, a broad-spectrum antiviral drug molecular approaches for the treatment of hemorrhagic fever virus infections chikungunya: a re-emerging virus in vitro inhibition of chikungunya and semliki forest viruses replication by antiviral compounds: synergistic effect of interferon-alpha and ribavirin combination curing of foot-and-mouth disease virus from persistently infected cells by ribavirin involves enhanced mutagenesis action of mutagenic agents and antiviral inhibitors on foot-and-mouth disease virus the broad-spectrum antiviral ribonucleoside ribavirin is an rna virus mutagen oral ribavirin treatment of influenza a and b ribavirin small-particle aerosol treatment of influenza intervention strategies for emerging viruses: use of antivirals effective therapy with ribavirin new opportunities for field research on the pathogenesis and treatment of lassa fever the role of ribavirin in the combination therapy of hepatitis c virus infection mechanisms of antiviral treatment efficacy and failure in chronic hepatitis c interferon and ribavirin vs. interferon alone in the re-treatment of chronic hepatitis c previously nonresponsive to interferon: a meta-analysis of randomized trials new options in the treatment of respiratory syncytial virus disease ribavirin for respiratory syncytial virus infection of the lower respiratory tract in infants and young children ribavirin for the treatment of chronic hepatitis c virus infection: a review of the proposed mechanisms of action the antiviral compound ribavirin modulates the t helper (th) /th subset balance in hepatitis b and c virus-specific immune responses the expanding universe of t-cell subsets: th , th and more reduced long-term respiratory morbidity after treatment of respiratory syncytial virus bronchiolitis with ribavirin in previously healthy infants: a preliminary report mechanism of action of -β-d-ribofuranosyl- , , -triazole- -carboxamide (virazole), a new broad-spectrum antiviral agent the predominant mechanism by which ribavirin exerts its antiviral activity in vitro against flaviviruses and paramyxoviruses is mediated by inhibition of imp dehydrogenase ribavirin induces error-prone replication of gb virus b in primary tamarin hepatocytes depletion of gtp pool is not the predominant mechanism by which ribavirin exerts its antiviral effect on lassa virus activity of ribavirin against hantaan virus correlates with production of ribavirin- -triphosphate, not with inhibition of imp dehydrogenase mode of action of ribavirin: effect of nucleotide pool alterations on influenza virus ribonucleoprotein synthesis lack of interference of guanosine with ribavirin aerosol treatment of influenza a infection in mice effect of ribavirin on viral kinetics and liver gene expression in chronic hepatitis c ribavirin regulates hepatitis c virus replication through enhancing interferon-stimulated genes and interleukin ribavirin improves early responses to peginterferon through improved interferon signaling hepatic gene expression during treatment with peginterferon and ribavirin: identifying molecular pathways for treatment response ribavirin treatment up-regulates antiviral gene expression via the interferon-stimulated response element in respiratory syncytial virus-infected epithelial cells ribavirin enhances interferon signaling via stimulation of mtor and p activities ribavirin restores ifnalpha responsiveness in hcv-infected livers by epigenetic remodelling at interferon stimulated genes viral and cellular enzymes involved in synthesis of mrna cap structure a structural basis for the inhibition of the ns dengue virus mrna -o-methyltransferase domain by ribavirin -triphosphate the broad spectrum antiviral nucleoside ribavirin as a substrate for a viral rna capping enzyme the broad spectrum antiviral agent ribavirin inhibits capping of mrna ribavirin is not a functional mimic of the -methyl guanosine mrna cap the antiviral drug ribavirin does not mimic the -methylguanosine moiety of the mrna cap structure in vitro the application and mechanism of action of ribavirin in therapy of hepatitis c inhibition of influenza virus ribonucleic acid polymerase by ribavirin triphosphate effect of phosphorylated ribavirin on vesicular stomatitis virus transcription studies on the mechanism of the antiviral activity of ribavirin against reovirus mechanism of action of ribavirin in the combination treatment of chronic hcv infection mechanisms of action of interferon and ribavirin in chronic hepatitis c: summary of a workshop antiviral action of ribavirin in chronic hepatitis c what is a quasispecies? historical origins and current scope the rna virus quasispecies: fact or fiction? quasispecies diversity determines pathogenesis through cooperative interactions in a viral population rna virus mutations and fitness for survival viral rna mutations are region specific and increased by ribavirin in a full-length hepatitis c virus replication system ribavirin causes error catastrophe during hantaan virus replication treatment of hev infection in patients with a solid-organ transplant and chronic hepatitis treatment of hepatitis e virus the hypercycle. a principle of natural self-organization. part a: emergence of the hypercycle nucleotide sequence heterogeneity of the rna from a natural population of foot-and-mouth-disease virus multiple genetic variants arise in the course of replication of foot-and-mouth disease virus in cell culture high multiplicities of infection favor rapid and random evolution of vesicular stomatitis virus rapid evolution of rna genomes meeting on 'quasispecies: past, present and future hepatitis c viral dynamics in vivo and the antiviral efficacy of interferon-alpha therapy viral dynamics in hepatitis b virus infection a novel -end repair mechanism in an rna virus discovery of an rna virus → exoribonuclease that is critically involved in coronavirus rna synthesis implications of altered replication fidelity on the evolution and pathogenesis of coronaviruses ntp-mediated nucleotide excision activity of hepatitis c virus rna-dependent rna polymerase pyrophosphorolytic excision of nonobligate chain terminators by hepatitis c virus ns b polymerase evidence for hepatitis e virus quasispecies the application of single strand conformation polymorphism (sscp) analysis in determining hepatitis e virus intra-host diversity hepatitis e virus quasispecies and the outcome of acute hepatitis e in solid-organ transplant patients theories of lethal mutagenesis: from error catastrophe to lethal defection counteracting quasispecies adaptability: extinction of a ribavirin-resistant virus mutant by an alternative mutagenic treatment potential benefits of sequential inhibitor-mutagen treatments of rna virus infections antiviral strategies based on lethal mutagenesis and error threshold pegylated interferon-alpha for treating chronic hepatitis e virus infection after liver transplantation treatment of chronic hepatitis e in liver transplant recipients with pegylated interferon alpha- b antiviral activities of different interferon types and subtypes against hepatitis e virus replication disparity of basal and therapeutically activated interferon signalling in constraining hepatitis e virus infection viral hepatitis during pregnancy incidence and severity of viral hepatitis in pregnancy the impact of hepatitis e in the liver transplant setting hepatitis e virus infection as a cause of graft hepatitis in liver transplant recipients mutation in the hepatitis e virus polymerase and outcome of ribavirin therapy ribavirin inhibits in vitro hepatitis e virus replication through depletion of cellular gtp pools and is moderately synergistic with alpha interferon metabolism of -amino- -beta-d-ribofuranosylimidazole- -carboxamide and related five-membered heterocycles to -triphosphates in human blood and l y cells -beta-d-ribofuranosyl- , , -triazole- -carboxamide; a cytostatic agent hepatitis e virus replication involves alternating negative-and positive-sense rna synthesis a multi-step process of viral adaptation to a mutagenic nucleoside analogue by modulation of transition types leads to extinction-escape acknowledgments: this work was supported by the german ministry for education and research (bmbf) through a ginaico grant gw . e.s. was further supported by the helmholtz centre for infection research. the authors declare no conflict of interest. key: cord- -st gyozv authors: taherkhani, reza; farshadpour, fatemeh; makvandi, manoochehr title: design and production of a multiepitope construct derived from hepatitis e virus capsid protein date: - - journal: j med virol doi: . /jmv. sha: doc_id: cord_uid: st gyozv the aim of this study was to design a high density multiepitope protein, which can be a promising multiepitope vaccine candidate against hepatitis e virus (hev). initially, conserved and antigenic helper t‐lymphocyte (htl) epitopes in the hev capsid protein were predicted by in silico analysis. subsequently, a multiepitope comprising four htl epitopes with high‐affinity binding to the hla molecules was designed, and repeated four times as high density multiepitope construct. this construct was synthesized and cloned into pet‐ a (+) vector. then, it was transformed and expressed in escherichia coli bl cells. the high density multiepitope protein was purified by ni‐nta agarose and concentrated using amicon filters. finally, the immunological properties of this high density multiepitope protein were evaluated in vitro. the results showed that the high density multiepitope construct was successfully expressed and purified. sds‐page and western blot analyses showed the presence of a high density multiepitope protein band of approximately kda. approximately mg of the purified protein was obtained from each liter of the culture media. moreover, the purified multiepitope protein was capable of induction of proliferation responses, ifn‐γ elispot responses and ifn‐γ and il‐ cytokines production in a significant level in peripheral blood mononuclear cells (pbmcs) isolated from hev‐recovered individuals compared to the control group. in conclusion, the newly produced multiepitope protein can induce significant t helper type responses in vitro, and can be considered as a novel strategy for the development of hev vaccines in the future. j. med. virol. : – , . © wiley periodicals, inc. hepatitis e virus (hev) is a positive-stranded rna virus which has been classified as the member of the genus hepevirus in the family hepeviridae [yamashita et al., ]. at least four major hev genotypes have been identified, which have different geographical distributions and host ranges [xing et al., ] . despite classification into four geographically distinct genotypes, the hev strains antigenically belong to a single serotype and share cross-reacting epitopes due to the high degree of sequence conservation [wang and zhuang, ; xing et al., ] . genotype includes most of human hev strains which is endemic in asia and north africa, genotype is identified in human and includes the mexican strain and few strains of african countries. genotypes and have been identified in pigs and other animals; however, humans can be infected with these two genotypes too. genotype predominates in industrialized countries, and genotype is endemic in asia, particularly china, taiwan, and japan yamashita et al., ] . hev causes acute hepatitis in one-third of the world's population [zhu et al., ] , with mortality rate of - % in the general population [xing et al., ] . however, sporadic and epidemic outbreaks of the acute hepatitis usually lead to a self-limited disease, but the infection is potentially fatal and causes fulminant hepatitis in susceptible individuals such as pregnant women and patients with underlying liver disease [zhu et al., ; xing et al., ] , and has high mortality rate of up to % among pregnant women [xing et al., ] . therefore hev is a significant public health problem worldwide especially in endemic areas and primary prevention of the hev infection seems essential. currently, no protective vaccine against hev infection has been licensed and primary prevention remains limited [wang and zhuang, ] . development of an effective vaccine, which can significantly reduce the prevalence of this disease, is a desirable goal. killed and live attenuated vaccines are not available due to the lack of a permissive cell culture system for hev replication . therefore the main focus for vaccine design is on molecular approaches, especially expression of viral proteins in different expression systems jimenez de oya et al., ] . the hev genome ($ . kb) contains three partially overlapping open reading frames (orfs), including orf , orf and orf [zhou et al., ; jimenez de oya et al., ] . orf , which encodes capsid protein, is highly conserved and immunogenic among hev species . due to hydrophobicity and insolubility of full-length capsid protein ( kda) [tsarev et al., ; zhang et al., ] , new generation vaccines such as multiepitope vaccines are of particular interest. therefore, the present study was undertaken to design a high density multiepitope protein compromising four htl epitopes with high-affinity binding to the hla molecules using the in silico analysis, and to evaluate the immunological properties of this protein in vitro. overall, this study represents a novel strategy for the development of hev vaccines in the future. construct from hev orf protein the amino acid sequences of the orf from standard strains of hev genotype including pakistan/ sar- strain (accession number p ), china strain (accession number q ), burma strain (accession number p ), myanmar strain (accession number q ) and india strain (accession number q ) were obtained from uniprot knowledgebase (www. uniprot.org). the sequences were aligned by mega software version . (biodesign institute, tempe, az) [tamura et al., ] , and then the conserved sequences among these strandard strains were determined. the highly conserved hev-derived hla-dr-restricted htl epitopes of these strains were predicted by syfpeithi online software (http://www.syfpeithi.de/ home.htm). hla-dr binding affinity of the predicted epitopes was determined by iedb analysis resource online program (http://tools.immuneepitope.org/analyze/html/mhc_ii_binding.html). genetic diversity of mhc molecules is very high in human population; therefore htl epitopes with high population coverage are needed for this study. four epitopes with highaffinity to the most common hla-dr alleles in iran and world population were selected. therefore it is predicted that this construct could cover the most world population. the four htl epitopes ( amino acids) derived from hev capsid protein including hla-drb à (a - ), hla-drb à (b - ), hla-drb à (c - ), hla-drb à and hla-drb à (dr ) (d - )restricted epitopes were binded to each other as a linear multiepitope (abcd). to avoid creation of junctional epitopes, the construct was designed with amino acid spacer sequences (gly-pro-gly-pro-gly) between sequential htl epitopes [livingston et al., ; depla et al., ] . the four times repeated multiepitope was constructed as high density multiepitope (fig. a) . based on e. coli codon usage, the nucleotide sequence of the high density multiepitope gene was optimized using genscript rare codon analysis tool (genscript, piscataway, nj) for efficient expression. in order to simplify the purification of the target protein, a sequence encoding -his tag was added at the c-terminal of the optimized gene followed by adding two stop codons (taa and tag) to terminate protein synthesis. two restriction-enzyme digestion sites, ndei and xhoi, were placed in the both ends of the codon-optimized gene to subclone it into the pet a (þ) vector. ndei digestion site (catatg) with starting codon (atg) was inserted at the n-terminal of the construct. the xhoi digestion site (ctcgag) was inserted at the c-terminal of the construct after the second termination codon. in silico digestion was performed using clone manager basic software version (sci-ed software, cary, nc), and was checked by vector nti software (invitrogen, carlsbad, ca). finally, the optimized high density multiepitope sequence was synthesized and cloned into the commercial cloning vector, pbluescript ii sk (þ) by biomatik company (biomatik corporation, cambridge, canada). both pbluescript ii sk (þ) vector carrying the high density multiepitope gene (pbluescript ii sk-high density multiepitope) and the pet- a (þ) plasmid (novagen, madison, wi) were digested by ndei and xhoi restriction enzymes (new england biolab, ipswich, ma). after ligation by t dna ligase (new england biolab, ipswich, ma), the recombinant plasmid pet a-high density multiepitope was generated and transformed into e. coli dh a competent cells (novagen) by electroporation [chassy et al., ] . the subcloning was confirmed by colony pcr, restriction-enzyme digestion, and dna sequencing. the recombinant plasmid was then transformed into competent e. coli bl (de ) cells to enhance protein expression (novagen). e. coli bl (de ) cells containing the recombinant plasmid were cultured overnight, then inoculated in terrific broth (himedia, mumbai, india) supplemented with kanamycin ( mg/l) in a : volumetric ratio, and grown with shaking at rpm at ˚c until the optical density at nm (od ) reached . . the bacterial culture was induced by adding various concentrations of iptg ( . - mm) (sigma-aldrich, st. louis, mo), and grown with shaking for different induction times ( , , , , and hr) at different induction temperatures ( ˚c, ˚c, and ˚c) to determine the optimal conditions of protein expression. after the large-scale protein expression, the target protein was purified by ni-nta (qiagen, hilden, germany) under denaturing conditions using m urea . the purified protein was dialyzed in pbs containing % glycerol with a linear urea gradient of , , and m at ˚c for hr in order to decrease the amount of urea, and concentrated using amicon ultra- centrifugal filter unit (emd millipore, billerica, ma). sds-page and western blot were performed to confirm the presence of the target protein [laemmli, ; hao et al., ; taherkhani et al., ] . concentration of the protein was measured by the bradford method [bradford, ] . to remove bacterial endotoxin contamination in the purified protein, toxin eraser endotoxin removal kit was used according to the manufacturer procedure (genscript). the truncated orf protein (aa - ) of hev strain sar- ( fig. b) was expressed in e. coli bl host cells using the recombinant plasmid pet- a-orf (aa - ) and purified by ni-nta (qiagen) as described previously . this protocol was approved by the ethical committee of ahvaz jundishapur university of medical science. a statement on ethical committee approval and the informed consent was taken from all participants. ten recovered individuals from acute hepatitis e infection ( males and females; mean age ae sd, . ae . years) with positive anti-hev igg antibody were enrolled in this study. the control group consisted of healthy individuals ( males and females; mean age ae sd, . ae . years) with no history of acute hepatitis, and with negative anti-hev igm/igg antibodies. the commercial elisa kits (hev igg and hev igm elisa kits, dia.pro, milan, italy) were used for all the participants. all the participants were negative for hepatitis b surface antigen, anti-hav igm antibody and anti-hcv antibodies (hbs ag elisa kit, hav igm elisa kit, hcv ab elisa kit, dia.pro) with normal alt level. peripheral blood mononuclear cells (pbmcs) of the each individual were isolated from ml heparinized venous blood by density gradient centrifugation using ficoll-hypaque (lymphoflot, biotest, dreieich, germany). the isolated pbmcs were washed and resuspended in complete rpmi medium (invitrogen). the viability of the cells was assessed by trypan blue staining. the cell proliferation assay was performed using non-radioactive mtt ( -( , -dimethylthiazol- -yl)- , diphenyl tetrazolium bromide) assay [majumdar et al., ] . in brief, approximately  cells/well of pbmcs of each sample in rpmi and % fcs were added to four wells of round-bottom -well plates in total volume of ml/well, stimulated with ml/well of truncated orf protein ( mg/ml), high density multiepitope ( mg/ml) and phytohemagglutinin (pha) ( mg/ml) (sigma-aldrich) separately, and incubated at ˚c for days. the unstimulated well of each sample was used as negative control. rpmi ( ml) was used as blank. after days, ml of mtt solution ( mg/ml) was added to each well and incubated at ˚c for hr. then, ml of isopropanol containing . n hcl was added to each well and mixed thoroughly. finally, the absorbance of the wells was read at nm with a reference wavelength of nm by an elisa reader (tecan sunrise elisa reader; tecan trading ag, mannedorf, switzerland). the results were showed as proliferation index (pi), calculated as od stimulated sample/od unstimulated sample. the elispot assay for hev-specific ifn-g-secreting cells was carried out by human ifn-g elispot ready-set-go kit (ebioscience, san diego, ca) according to manufacturer's protocol. briefly, a -well pvdf bottomed elispot plate (millipore, bedford, ma) was coated with ml/well of capture antibody solution and incubated overnight at ˚c. approximately  cells/well of pbmcs of each sample suspended in rpmi and % fcs along with mg/ml high density multiepitope protein and mg/ ml truncated orf- protein separately were added to wells in total volume of ml/well. for negative control unstimulated pbmcs was used. for positive control, mg/ml of pha (sigma-aldrich) was used. after -hr incubation at ˚c, cells and medium were decanted from the plate and the plate was washed three times with elispot wash buffer. ml of biotinylated detection antibody was added to each well and incubated overnight at ˚c. after incubation, the plate was emptied and washed as described above. ml/well of avidin-horseradish peroxidase reagent was added, and the plates were incubated at room temperature for min. after incubation, the plates were washed three times with elispot wash buffer and two times with x pbs; then, ml/well of freshly prepared aec ( -amino- ethyl carbazole) substrate solution (sigma-aldrich) was added to the wells and incubated at room temperature until dark purple spots appears. the reaction was stopped by washing wells three times with ml/well distilled water. plate was air-dried and the number of ifn-g-producing cells in each well was counted by a dissection stereoscope (nikon, tokyo, japan) and expressed as spot-forming cells (sfcs) per cells. at a concentration of  cells/well, pbmcs of each sample in rpmi and % fcs were added to the four wells of -well plates, and stimulated with truncated orf protein ( mg/ml), high density multiepitope protein ( mg/ml), and pha ( mg/ml) separately at ˚c. the unstimulated pbmcs of each sample were used to evaluate spontaneous production of cytokines. culture supernatants were collected after hr ( hr for il- ). levels of four cytokines (il- p , ifn-g, il- and il- ) were measured in duplicate by commercial elisa kits (ebioscience) according to the manufacturer's instructions. the elisa results were expressed as picograms per milliliter (pg/ml). statistical analysis was performed using spss software (spss, chicago, il) and p values of less than . were considered statistically significant. discrete variables were compared using the pearson's chi-square test or fisher's exact test. intergroup comparisons (recovereds vs. controls) were performed using independent t test or mann-whitney u test. intragroup comparisons (high density multiepitope versus orf ) were made by paired t test or wilcoxon test. all data were presented as mean ae se. the high expression of the protein was induced by iptg at a final concentration of mm at ˚c for hr. sds-page and western blotting analysis of the resultant protein showed a protein band of approximately kda corresponding to high density multiepitope (fig. ) . approximately mg of purified protein was obtained from each liter of culture media. the cell proliferative responses in hev-recovered group following stimulation with high density multiepitope protein and truncated orf protein were found higher than control group (p < . ); and these responses were observed higher with high density multiepitope protein than truncated orf protein in hev-recovered group (p ¼ . ) ( table i, fig. ). ifn-g elispot responses to high density multiepitope protein and truncated orf protein were found significantly higher in hev-recovered individuals than control group (p < . ). these responses to high density multiepitope protein were observed slightly higher than truncated orf protein in hevrecovered group, with no significant observation (p ¼ . ). table i and figure show the frequency of ifn-g-secreting cells following stimulation with high density multiepitope protein and truncated fig. . a: sds-page analysis of the expressed and purified high density multiepitope protein in e. coli. the high density multiepitope protein band is visualized at approximately kda. lane , uninduced control; lane , induction with mm iptg for hr; lane , induction with mm iptg for hr; lane , , flow-through; lane - , washing steps of the ni column; lane - , the first, the second, and the third purified eluates of high density multiepitope protein from the ni column; lane , prestained protein ladder (cinagen, tehran, iran). b: western blot analysis of the expressed and purified high density multiepitope protein in e. coli. the high density multiepitope protein band is visualized at approximately kda. lane , prestained protein ladder (cinagen); lane , flow-through; lane , wash; lane - , the first, the second, the third, and the fourth eluates. orf protein in hev-recovered individuals and control group. the levels of ifn-g and il- p responses to truncated orf protein and high density multiepitope in the recovered group were found significantly higher compared to control group (p < . ). however, the levels of these stimulated cytokines with high density multiepitope protein were found higher than truncated orf protein with no significant observation. the levels of specific il- for high density multiepitope protein and truncared orf protein were found moderate among the hev-recovered group, while the levels of specific il- for the both proteins were low (table i, fig. ). a better understanding of the hev-specific cellular immune responses may help us in designing and development of vaccine for the prevention and control of the hev infection. although some progress has been achieved in developing hev vaccine, but due to some limitations in current approach for vaccine preparation such as the duration of the response and cost effectiveness of vaccine, the attempt was made to develop an alternative hev vaccine [jimenez de oya et al., ] . the virus-specific t-helper cell immune responses have been shown to be essential for controlling and clearing viral infection by activating cytotoxic t cells and producing cytokines especially inf-g which can suppress viral replication. thus, viral proteins comprising helper t-lymphocyte (htl) epitopes may be useful as protective vaccines [aggarwal et al., ] . it has been shown that hev capsid protein has several immunogenic epitopes [gu et al., ] . shata et al. identified dominant and subdominant epitopes of hev capsid protein such as amino acids - , - , - , - , and - [shata et al., ] . aggarwal et al. mapped cd t-cell epitopes in hev orf protein, and found the immunodominant t-cell epitopes of capside protein are encompassed amino acids - , - , and - [aggarwal et al., ] . khudyakov et al. reported the antigenic regions in hev orf protein are encompassed amino acids - , - , and - [khudyakov et al., ] . selection of the epitopes is the most important part of the design of the epitope-based vaccines that can guarantee the effectiveness and extent of the desired immune responses. this study focused on the consereved epitopes with high-affinity binding to the different hla molecules to cover the majority of the human population, on this basis the multiepitope protein comprising these epitopes was constructed. this multiepitope design is a new strategy and may counter the limitations present in current vaccine against hev, but it requires further investigation. to date, no application of multiple hla-dr-restricted epitopes was reported to induse htl responses against hev infection. oany et al. designed a multiepitope vaccine candidate compromising b cell and t cell epitopes from spike protein of human coronavirus by in silico analysis. in their study, sequences of the spike proteins were collected from the uniprotkb database and the consereved epitopes with high-affinity binding to the different hla molecules were predicted using in silico tools to cover the majority of the world population. this computational design was a novel study to determine antigenic epitopes in spike protein and to design an epitope-based vaccine against human coronavirus [oany et al., ] . these type of vaccines were reported to have many advantages including the stability, lack of carcinogenic, large scale production with the least equipments, no need for cold chain storage, the ability to stimulate the immune system against a wide variety of strains and genotypes of a microorganism, and use in different races or high population coverage [elliott et al., ; livingston et al., ; sette and fikes, ; jackson et al., ] . meanwhile, some disadvantages like the creation of junctional epitopes in the sequential arrangement of the epitopes have limited using these constructs [livingston et al., ; depla et al., ] . junctional epitopes is created by the juxtaposition of two epitopes, and can suppress the immune responses to the main epitopes [livingston et al., ] . this problem was overcome through application of the spacer residues between the epitopes to improve appropriate epitope processing. furthermore, it has been reported that if the epitope is expressed with high density will enhance protective immunity [liu et al., ; lu et al., ] . lu et al. designed constructs consisting of various v epitope densities from hiv- gp , and found that high epitope density in one single protein molecule could enhance protective immunity [lu et al., ] . liu et al. reported that high epitope density from m protein of influenza virus enhanced specific humoral immunity against flu virus [liu et al., ] . vaccine development is dependent not only on selecting antigens and epitopes but also on defining the immune responses against target epitopes. by evaluating the antigenicity and immunogenicity of the high density multiepitope protein, it was indicated that the high density multiepitope protein stimulates slightly stronger t helper cell responses compared to truncated orf protein. it has been shown that during acute infection, virus-specific t helper cell responses can clear viral infection but lack of its activity leads to chronic infection [macdonald et al., ] . in this study, the hla typing was not carried out and the frequencies of various hla alleles among the participants were not determined. so it is not clear which type of hla alleles among the hev-recovered individuals had a low or highaffinity hla binding epitopes against the multiepitope protein. li et al. designed a multiepitope construct comprising six low-affinity hiv gag and pol ctl epitopes restricted to hla-a à and the hiv p antigen by bioinformatics analysis. they used cryptic epitope modification to enhance the immunogenicity of lowaffinity hla-a . -binding epitpes, and found the multiepitope can induce a strong cd þ t cell immune responses in mice [li et al., b] . jafarpour et al. designed and synthesized a multiepitope construct encoding conserved and immunogenic epitopes from hiv- tat, pol, gag, and env proteins by bioinformatics analyses as a new dna vaccine candidate. the designed multiepitope plasmid was able to induce proper immune responces in balb/c mice [jafarpour et al., ] . li et al. designed a multiepitope construct encoding t and b lymphocyte epitopes from latent membrane protein (lmp ) of epstein-barr virus (ebv) based on mammalian codon usage. the recombinant plasmid pcdna . þ/ ebv-lmp multiepitope was constructed and transfected into cos- cells. this multiepitope plasmid dna was able to stimulate strong cellular and humoral immunity in mice [li et al., a] . in the present study, high level of specific ifn-g and il- responses (t helper type cytokines) and low level of il- and very low level of il- responses (t helper type cytokines) against high density multiepitope protein and truncated orf protein were observed among the hev-recovered individuals. thus, both proteins can be considered as promising vaccine candidate against hev infection. hashish et al. designed a multiepitope fusion protein compromising two b cell and t cell epitopes from the e glycoprotein of bovine viral diarrhea virus (bvdv), and also k major subunit fanc and sta toxoid of enterotoxigenic escherichia coli (etec). in their study, fanc-sta-e gene construction was cloned into the expression vector pet and transformed into e. coli bl cells. fanc-sta-e fusion protein was well expressed by iptg-induction ( mm) at ˚c for hr and purified by ni-nta agarose using his-tag. this multiepitope fusion protein could develop neutralizing antibodies against etec and bvdv in vitro [hashish et al., ] . beside vaccine development, multiepitope technology can be used for diagnosis. several studies have been performed on multiepitopes proteins for development of diagnostic kit. the results of these studies showed that the use of multiepitope proteins can increase the sensitivity and specificity of the tests, since this kind of proteins has great efficiency to expose conserved and immunodominant epitopes [dipti et al., ; de souza et al., ] . de souza et al. designed a high density multiepitope protein containing three copies of four conserved epitopes from hepatitis b core protein (hbcag), and developed an elisa using this recombinant protein. the developed elisa was useful for hepatitis b diagnosis [de souza et al., ] . dipti et al. designed a novel recombinant multiepitope protein comprising six conserved and immunodominant epitopes from hepatitis c virus, and developed an anti-hcv diagnostic kit using this protein. the multiepitope protein was able to detect anti-hcv antibodies in human plasma with high degree of sensitivity and specificity [dipti et al., ] . although several microorganisms and cell lines are currently used for expression of foreign genes, but still e. coli are routinely used for expression of target proteins [vincentelli and romier, ] . fast cultivation, well-known genetics and high-level expression capabilities with high purity are some other advantages of e. coli expression system [fakruddin et al., ] . despite all these advantages, expression of a foreign gene in e. coli may be hampered by the presence of rare codons and high gc contents within the gene-coding region which result in decrease of the expression level or production of insoluble and nonfunctional proteins [mondal et al., ; elena et al., ] . in the present study, e. coli bl cell was used for expression of the high density multiepitope protein. the target genes were cloned in to the pet aþ plasmid and expressed by iptg-induction under control of strong bacteriophage t promoter and t rna polymerase of the e. coli bl cell. the iptg-induction is economically desirable method and increases protein production [mondal et al., ] . previous studies have reported high yield protein purification by ni þ chelate affinity chromatography using his-tag [glynou et al., ; farshadpour et al., ] . the application of affinity tags is simple, rapid, cost effective and also efficient for purification of proteins, and also has no effect on the structure, folding and function of the protein [glynou et al., ; carson et al., ] . in the present study, histag was used for the purification. the results of this study show that e. coli is a good system for expression of recombinant proteins with high yield. in conclusion, a high density multiepitope protein capable of induction of t helper type responses was successfully produced and evaluated in vitro. the results of this study provide much information for the design of hev multiepitope vaccine. however, further works will be needed to evaluate protective immune response of the high density multiepitope protein in vivo. t-cell epitope mapping of orf and orf proteins of human hepatitis e virus a rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the 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healthy adults: a large-scale, randomised, double-blind placebo-controlled key: cord- - jtfc authors: van nguyen, dung; van nguyen, cuong; bonsall, david; ngo, tue tri; carrique-mas, juan; pham, anh hong; bryant, juliet e.; thwaites, guy; baker, stephen; woolhouse, mark; simmonds, peter title: detection and characterization of homologues of human hepatitis viruses and pegiviruses in rodents and bats in vietnam date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: jtfc rodents and bats are now widely recognised as important sources of zoonotic virus infections in other mammals, including humans. numerous surveys have expanded our knowledge of diverse viruses in a range of rodent and bat species, including their origins, evolution, and range of hosts. in this study of pegivirus and human hepatitis-related viruses, liver and serum samples from vietnamese rodents and bats were examined by pcr and sequencing. nucleic acids homologous to human hepatitis b, c, e viruses were detected in liver samples of ( . %) of bats, ( . %), and ( %) of rodents, respectively. hepacivirus-like viruses were frequently detected ( . %) in the bamboo rat, rhizomys pruinosus, while pegivirus rna was only evident in ( . %) of rodent serum samples. complete or near-complete genome sequences of hbv, hev and pegivirus homologues closely resembled those previously reported from rodents and bats. however, complete coding region sequences of the rodent hepacivirus-like viruses substantially diverged from all of the currently classified variants and potentially represent a new species in the hepacivirus genus. of the viruses identified, their routes of transmission and potential to establish zoonoses remain to be determined. unlike many other communicable diseases, the burden of viral hepatitis has substantially increased over the last two decades to recently become the seventh leading cause of mortality worldwide. viral hepatitis now causes more deaths than tuberculosis, aids or malaria each year. hepatitis c virus (hcv) and hepatitis b virus (hbv) are responsible for > % ( % in ) of viral viruses , , of hepatitis-related mortality and disability. as such, these hepatitis viruses are the targets of efforts to combat viral hepatitis [ ] , including hbv vaccination, development of hcv vaccines, and highly effective drugs. in contrast, hepatitis e virus (hev) is endemic in many low-income countries [ ] but usually causes self-limiting hepatitis. infection with hev occasionally results in liver failure [ ] . hbv, hcv, and hev are members of virus families hepadnaviridae, flaviviridae, and hepeviridae, respectively. hbv has a partially double-stranded dna genome with overlapping open reading frames (orfs) [ ] , whereas hcv and hev have a single-stranded rna genome [ , ] . while the origins of these human viruses are unknown, rodents and bats are putative reservoir hosts because they host a diverse array of hepadnaviridae [ ] [ ] [ ] , hepeviridae [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , and genera pegivirus [ ] [ ] [ ] and hepacivirus [ , , ] of the flaviviridae family including homologues of the human hepatitis viruses under question. among these, it is of concern that a bat hepadnavirus may possess the ability to infect human liver cells [ ] . several factors may contribute to the risk of zoonotic rodent or bat virus transmission. rodent meat is a popular source of protein for human consumption in vietnam, particularly in the mekong delta, where rats (rattus spp. and bandicota indica) are commonly trapped and sold live in wet markets [ ] . the total annual consumption of rat meat in vietnam is estimated at - tonnes [ ] . hoary bamboo rats (rhizomys pruinosus) are additionally farmed for human consumption. moreover, bat faeces collected from bat caves or farms is used as natural fertilizer ("guano") in vietnam. as rodents and bats are reservoirs or carriers of a significant number of zoonotic pathogens [ ] and viruses with unknown zoonotic potential, there are health risks that are associated with exposure to these animals. however, a previous study [ ] showed none of the surveyed rat catchers or processors were aware of infection risks from contact with live rats. consequently, no precautions were taken for the handling of rodents. in the search for viral diversity and zoonotic viruses, novel paramyxovirus and coronavirus in vietnamese bats and rats were detected in a previous study [ ] . here, we report the detection pegivirus and human hepatitis-related viruses in these mammals. rodent and bat samples were collected within the vizions (vietnam initiative on zoonotic infections) framework [ ] for the screening of zoonotic microorganisms [ , [ ] [ ] [ ] . rodent samples. as it is important and essential to understand the risk associated with rodents, including those sold in the markets, a total of rats purchased from markets in five of twelve provinces in the mekong delta during - and farmed bamboo rats purchased from a market in dak lak in - were enrolled. in addition, trapped rats were also included. rat trapping was carried out in different locations (pig and poultry farms, rice fields, fruit groves, tropical forests, markets, slaughter-house) in the provinces of dong thap during march and dak lak in april , as previously described [ ] . serum and liver samples were collected post-mortem. species identification of rats was carried out on the basis of morphological characteristics and sequencing of a conserved housekeeping gene [ ] . all of the samples were stored in sterile tubes with rna later at − • c until nucleic acid extraction. special precautions were taken to avoid cross-contamination. bat samples. a total of bats were trapped at six sites in the provinces of dong nai (in cat tien national park) and quang ngai in may using mist nets and harp traps as described [ ] . trapped bats were speciated according to morphology [ ] , and liver samples were collected and stored as described above for rats. rna was manually extracted from rodent serum samples using qiaamp viral rna mini kit (qiagen, manchester, uk) and following instructions from the manufacturer. liver samples from bats and rodents were subjected to nucleic acid extraction using allprep dna/rna mini kit (qiagen, manchester, uk). briefly, about mg of liver per sample was first lysed and homogenised using tissuelyser (qiagen, manchester, uk). the lysate was applied to an allprep dna spin column for dna to bind onto the column. ethanol was added to the flow-through and rna and bound to the membrane when the sample was passed through an rneasy spin column. after washing steps, dna and rna was eluted separately in µl of nuclease-free water. extracted nucleic acid was used in screening for the targeted hepatitis viruses. in order to minimize contamination, pcr mastermix preparation, and the addition of templates were carried under separated laminar flow hoods and lab spaces. all of the surfaces, tubes, and equipment were uv irradiated between each pcr. reverse transcription using superscript iii reverse transcriptase (invitrogen, paisley, uk) was performed according to the manufacturer's instruction. synthesized cdna was screened for hepaciviruses and pegiviruses using a semi-nested pcr protocol with broad spectrum degenerate primers, which can detect all known hepaciviruses and pegiviruses. amplification conditions (using gotaq from promega, southampton, uk) included • c for min, and cycles of denaturation ( • c, s), annealing ( • c, s) and elongation ( • c, s). similarly, hev was screened using broadly reactive primers targeting viral rna-dependent rna polymerase as described in drexler et al., [ ] . dna extracted from liver samples was used for screening of hbv. degenerate primers targeting a highly conserved region of the polymerase gene of sequences from all known hbv hosts were designed for a nested pcr protocol using the above amplification conditions. primers for screening are listed in table s . the length of the sequenced screening fragments (excluding primer sequences) of homologues of hbv, hev, hcv, and pegivirus was , and nucleotides, respectively. for rodent hepacivirus, hev and pegivirus genomes, extracted rna from representative positive samples was subjected to deep sequencing using an illumina platform. libraries were prepared from total extracted rna using the nebnext ultra directional sequencing kit (new england biolabs, hitchin, uk) with omission of actinomycin d, then pooled and sequenced on a hiseq instrument (illumina, nr saffron walden, uk). quality control and trimming of reads were performed before genome mapping using clc genomics workbench (qiagen bioinformatics, redwood city, ca, usa) with the default affine gap cost parameters. the closest related virus genomes (genbank numbers kc , jx and kj for hepacivirus, hev and pegivirus, respectively) were used as templates for genome mapping. additional primers were designed using the obtained contigs for gap fillings. for bat hbv, primers were designed using sequences available from genbank and the obtained sequences from screening. these primers amplified amplicons, with overlapping regions as presented in table s . all of these nested or hemi-nested pcr protocols used superscript iii one-step rt-pcr system with platinum taq dna polymerase (invitrogen, paisley, uk) for rna viruses or q high-fidelity dna polymerase (new england biolabs, hitchin, uk) for hbv in the first round pcr, according to instructions from the manufacturers. q high-fidelity dna polymerase was also used in the second round pcr. two real-time pcr primer sets (table s ) in the untranslated region of bamboo rat hepaciviruses and the screening fragment of other rat hepaciviruses were designed using sequences available from this study. the targeted regions were amplified from positive samples and cloned into pgem-t easy vector (promega, southampton, uk) for in vitro transcription, as previously described [ ] . the obtained rna transcripts were used to generate standard curves of the real-time pcr assays for measurement of rodent hepacivirus rna titers using superscript iii reverse transcriptase (invitrogen, paisley, uk) and powerup sybr green master mix (thermo fisher scientific, northumberland, uk). sequences were imported into sse (simmonic sequence editor) [ ] for the alignment and calculation of sequence distance values from reference sequences of known viruses from which sequence identities were inferred. sequence distances instead of sequence identities in the regions used for classification of hepaciviruses and pegiviruses were presented to easily compare with the species p-distance thresholds set in the proposed update to the taxonomy of the genera hepacivirus and pegivirus [ ] . maximum-likelihood phylogenetic trees were reconstructed using the mega . software package [ ] with bootstrap resamples. the best-fitting model for each sequence dataset (as shown in figure captions) was first determined and used for phylogenetic reconstruction. sequences obtained in this study have been assigned the following genbank accession numbers mg -mg . nucleic acid that was extracted from liver samples of bats ( species; table s ) and rodents (six species) was screened for pegivirus and human hepatitis b, c, e viruses and their homologues ( table ) by nested and semi-nested pcr assays with degenerate primers. hepaciviruses were the most commonly detected ( . % of rodent samples, from three species), followed by hepatitis e related virus ( % of rodent samples, from four species) while hepatitis b related viral dna was only detectable in three bats ( species). most of the hepacivirus positive samples were from farmed hoary bamboo rats in dak lak province although the predominantly sampled rat species was rattus argentiventer. coinfection with hepacivirus and hev was observed in a sample from rattus losea. all liver samples from bats were negative for hepacivirus and hepatitis e related virus and no sample was positive for pegivirus. and other rats whose liver samples were screened for hepacivirus as above. hepacivirus rna was only detected in serum samples of bamboo rats with positive liver samples. pegivirus rna was detected in two samples from rattus tanezumi. two real-time pcr assays specific for bamboo rat hepaciviruses, and other hepaciviruses were used to determine viral rna concentration in the positive samples. the concentration of rna ( figure ) was high in both liver tissue (median, . × copies/gram; range, . × - . × ) and sera (median, . × copies/ml; range, . × - × ). viruses , , x of ( figure ) was high in both liver tissue (median, . × copies/gram; range, . × - . × ) and sera (median, . × copies/ml; range, . × - × ). amplicons derived from pcr screening experiments were all successfully sequenced and complete or near complete genome sequences were determined for representatives of the viruses by pcr or deep sequencing. after exclusion of primers, the obtained screening sequences from each targeted virus clustered in one or two clades of those with high identity and were most closely related to sequences previously reported from rodents or bats from the us [ , ] , china [ ] and vietnam [ ] . rodent hepacivirus sequences ( nucleotides) formed two well supported clades (figure a) . clade included all of sequences from rhizomys pruinosus which shared . - % pairwise nucleotide identity while three sequences (nucleotide identity of - %) from rattus losea and rattus argentiventer grouped in clade . these clades differed from each other by mean distances of . % and . % at nucleotide and amino acid levels, respectively. while the four vietnamese bamboo rat hepacivirus genomes were highly similar to each other (< % nucleotide and < % amino acid distances in the complete coding region (cds)), they were remarkably different from the closest sequence (kc ) with the corresponding distances of % and %, respectively ( table ). the amino acid p-distances of the obtained hepacivirus sequences and kc in the regions - and - (numbered relative to m ) were % and %, respectively. the newly identified hepaciviruses therefore could be provisionally classified as members of a new hepacivirus species (figure b ) [ ] . the other bamboo rat hepaciviruses in clade may also belong to this new virus species on the basis of the high sequence identity in this clade. similarly, although complete genome sequences were not determined for hepaciviruses in clade , they likely belong to species hepacivirus g due to their low amino acid p-distances ( . - . %) in the screening region to kj . the ' untranslated region sequences of these hepaciviruses contain two mir- seed sites (cacucc), which were located nucleotides from each other, as consistent with the suspected hepatotropism of the viruses. in contrast to host specificity of rodent hepaciviruses, the hev sequences ( nucleotides) from four rodent species were . - . % identical to each other and phylogenetically interspersed with each other and with reference sequences from other rat species (figure a) . the obtained complete genome of rat hev comprised of nucleotides excluding the poly a tail. its concatenated orf and orf differed by . % (table ) at amino acid level to the closest match (jx ) and these two sequences therefore belong to a same genotype in the species orthohepevirus c (figure b ), according to the latest proposal for classification of hepeviruses [ ] . amplicons derived from pcr screening experiments were all successfully sequenced and complete or near complete genome sequences were determined for representatives of the viruses by pcr or deep sequencing. after exclusion of primers, the obtained screening sequences from each targeted virus clustered in one or two clades of those with high identity and were most closely related to sequences previously reported from rodents or bats from the us [ , ] , china [ ] and vietnam [ ] . rodent hepacivirus sequences ( nucleotides) formed two well supported clades (figure a) . clade included all of sequences from rhizomys pruinosus which shared . - % pairwise nucleotide identity while three sequences (nucleotide identity of - %) from rattus losea and rattus argentiventer grouped in clade . these clades differed from each other by mean distances of . % and . % at nucleotide and amino acid levels, respectively. while the four vietnamese bamboo rat hepacivirus genomes were highly similar to each other (< % nucleotide and < % amino acid distances in the complete coding region (cds)), they were remarkably different from the closest sequence (kc ) with the corresponding distances of % and %, respectively ( table ). the amino acid p-distances of the obtained hepacivirus sequences and kc in the regions - and - (numbered relative to m ) were % and %, respectively. the newly identified hepaciviruses therefore could be provisionally classified as members of a new hepacivirus species (figure b ) [ ] . the other bamboo rat hepaciviruses in clade may also belong to this new virus species on the basis of the high sequence identity in this clade. similarly, although complete genome sequences were not determined for hepaciviruses in clade , they likely belong to species hepacivirus g due to their low amino acid p-distances ( . - . %) in the screening region to kj . the ' untranslated region sequences of these hepaciviruses contain two mir- seed sites (cacucc), which were located nucleotides from each other, as consistent with the suspected hepatotropism of the viruses. in contrast to host specificity of rodent hepaciviruses, the hev sequences ( nucleotides) from four rodent species were . - . % identical to each other and phylogenetically interspersed with each other and with reference sequences from other rat species (figure a) . the obtained complete genome of rat hev comprised of nucleotides excluding the poly a tail. its concatenated orf and orf differed by . % (table ) at amino acid level to the closest match (jx ) and these two sequences therefore belong to a same genotype in the species orthohepevirus c (figure b) , according to the latest proposal for classification of hepeviruses [ ] . the three hbv variants (from bat species hipposideros pomona and hipposideros larvatus) clustered with known bat hbv sequences (figure ). the two bat hbv complete genome sequences comprised and nucleotides. as with other hepadnaviruses, the bat hbv genomes have four open reading frames (orfs) encoding the surface (s), polymerase (p), core (c), and x proteins. a detailed comparison of the orfs of these viruses with their closest sequences is shown in table . bat consistently showed greatest sequence identity to ky in all orfs. in contrast, bat shared highest identity to ky in the p and s orfs, but was more similar to ky and ky in the orfs encoding for x and c, respectively. the two vietnamese pegivirus sequences from rattus tanezumi grouped with a sequence previously reported from rattus norvegicus (figure a ). the amino acid p-distance between the obtained rodent pegivirus sequence and kj in the region - (numbered relative to u ) was . % and the two viruses could be classified as members of species pegivirus j (figure b) , according in the update to the taxonomy of the pegivirus genus [ ] . the three hbv variants (from bat species hipposideros pomona and hipposideros larvatus) clustered with known bat hbv sequences (figure ) . the two bat hbv complete genome sequences comprised and nucleotides. as with other hepadnaviruses, the bat hbv genomes have four open reading frames (orfs) encoding the surface (s), polymerase (p), core (c), and x proteins. a detailed comparison of the orfs of these viruses with their closest sequences is shown in table . bat consistently showed greatest sequence identity to ky in all orfs. in contrast, bat shared highest identity to ky in the p and s orfs, but was more similar to ky and ky in the orfs encoding for x and c, respectively. the two vietnamese pegivirus sequences from rattus tanezumi grouped with a sequence previously reported from rattus norvegicus (figure a ). the amino acid p-distance between the obtained rodent pegivirus sequence and kj in the region - (numbered relative to u ) was . % and the two viruses could be classified as members of species pegivirus j (figure b) , according in the update to the taxonomy of the pegivirus genus [ ] . the present study reports the findings of pegivirus and human hepatitis-related viruses in vietnamese rodents and bats. the detection of hepacivirus, hev homologue and pegivirus in a number of rodent species, and hbv homologue in hipposideros larvatus indicates wider host ranges of these viruses. whereas, the identified rat hev and bat hbv showed relatively high sequence identity to previously characterized viruses infecting rattus rattus, rattus tanezumi and hipposideros pomona, the rodent pegivirus and hepacivirus showed substantial sequence distances to their closest sequences and represent new pegivirus variants and a new hepacivirus species. this highlights the incomplete genetic characterization of these viruses. thus far, only one complete genome and two complete coding sequences (including the one from this study) of rodent pegivirus are available on genbank. more highly divergent hepacivirus and pegivirus sequences are expected to be discovered from rodents in future studies. the absence of bat hev, hepacivirus, pegivirus, and low detection frequency of bat hbv are consistent with their low prevalence ( - %) reported in previous studies [ , , , ] . contrastingly, the prevalence of hepacivirus in the farmed bamboo rats was unprecedentedly high ( . %). the inbred nature of farmed bamboo rats that were investigated in this study may have contributed to susceptibility to infection and likelihood of persistence [ , ] . the existence of relatively homozygous individuals may play a key role in the maintenance of pathogens in a population [ ] . the high prevalence of hepacivirus in bamboo rats also indicates the need to reconsider transmission routes of hepaciviruses. among hepaciviruses, the transmission route of hcv has been relatively well studied, while those of other hepaciviruses are mostly speculative [ , ] . as a bloodborne virus, hcv is thought to be mainly transmitted through injections or blood transfusion. however, this does not explain how a range of divergent hcv strains have been maintained for centuries in some rural populations in central africa and southeast asia [ ] . equine hepacivirus has been shown to be transmittable via direct inoculation [ ] and via vertical transmission [ ] . rodent hepacivirus may utilize a similar transmission route as experimental intravenous injection of the supernatants of homogenised liver tissues from infected rats lead to viraemia [ ] . the high prevalence of hepacivirus in bamboo rats (in this study) and commensal rattus norvegicus ( . % in firth et al. [ ] ) indicates other more efficient transmission routes may exist such as via saliva the present study reports the findings of pegivirus and human hepatitis-related viruses in vietnamese rodents and bats. the detection of hepacivirus, hev homologue and pegivirus in a number of rodent species, and hbv homologue in hipposideros larvatus indicates wider host ranges of these viruses. whereas, the identified rat hev and bat hbv showed relatively high sequence identity to previously characterized viruses infecting rattus rattus, rattus tanezumi and hipposideros pomona, the rodent pegivirus and hepacivirus showed substantial sequence distances to their closest sequences and represent new pegivirus variants and a new hepacivirus species. this highlights the incomplete genetic characterization of these viruses. thus far, only one complete genome and two complete coding sequences (including the one from this study) of rodent pegivirus are available on genbank. more highly divergent hepacivirus and pegivirus sequences are expected to be discovered from rodents in future studies. the absence of bat hev, hepacivirus, pegivirus, and low detection frequency of bat hbv are consistent with their low prevalence ( - %) reported in previous studies [ , , , ] . contrastingly, the prevalence of hepacivirus in the farmed bamboo rats was unprecedentedly high ( . %). the inbred nature of farmed bamboo rats that were investigated in this study may have contributed to susceptibility to infection and likelihood of persistence [ , ] . the existence of relatively homozygous individuals may play a key role in the maintenance of pathogens in a population [ ] . the high prevalence of hepacivirus in bamboo rats also indicates the need to reconsider transmission routes of hepaciviruses. among hepaciviruses, the transmission route of hcv has been relatively well studied, while those of other hepaciviruses are mostly speculative [ , ] . as a bloodborne virus, hcv is thought to be mainly transmitted through injections or blood transfusion. however, this does not explain how a range of divergent hcv strains have been maintained for centuries in some rural populations in central africa and southeast asia [ ] . equine hepacivirus has been shown to be transmittable via direct inoculation [ ] and via vertical transmission [ ] . rodent hepacivirus may utilize a similar transmission route as experimental intravenous injection of the supernatants of homogenised liver tissues from infected rats lead to viraemia [ ] . the high prevalence of hepacivirus in bamboo rats (in this study) and commensal rattus norvegicus ( . % in firth et al. [ ] ) indicates other more efficient transmission routes may exist such as via saliva and bites, the zoonotic potential of the detected viruses is unknown and requires further investigation. while the identified rodent hepaciviruses appear host species specific, four rodent species were infected with highly similar hev homologues, which were phylogenetically interspersed, indicative of low host species specificity. this is a characteristic that may lead to their establishment and emergence in new hosts. understanding the receptor usage for cell entry of hev in rodents and other host species would potentially help predict the host range of the virus. furthermore, a surrogate assay with pseudotyped viruses carrying surface/envelope proteins of the identified viruses may be useful in assessing their potential to infect human liver cells. such an assay was used to show that a bat hbv could infect primary human hepatocytes [ ] . in summary, this study demonstrated the wide circulation of diverse pegivirus and human hepatitis-related viruses in new rodent and bat species. the presented findings form a framework for future investigations of human transmission risk, now that the rodent and bat species infected with these viruses have been identified and the human contact groups are better defined (e.g., bamboo rat farmers, rat catchers, rat sellers, and bat farmers). the transmission routes of the identified viruses are to be determined. the following are available online at http://www.mdpi.com/ - / / / / s . the global burden of viral hepatitis from to : findings from the global burden of disease study the global burden of hepatitis e virus genotypes and in hepatitis b: the virus and disease genetic organization and diversity of the hepatitis c virus hepatitis e virus: molecular virology, clinical features, diagnosis, transmission, epidemiology, and prevention bats carry pathogenic hepadnaviruses antigenically related to hepatitis b virus and capable of infecting human hepatocytes detection and genome characterization of four novel bat hepadnaviruses and a hepevirus in china hepatitis virus in long-fingered bats hepatitis e virus in rats hepatitis e virus genotype in wild rats, united states bats worldwide carry hepatitis e virus-related viruses that form a putative novel genus within the family hepeviridae complete genome sequence of a rat hepatitis e virus strain isolated in the united states hepatitis e virus in norway rats (rattus norvegicus) captured around a pig farm novel hepatitis e virus genotype in norway rats detection of a novel hepatitis e-like virus in faeces of wild rats using a nested broad-spectrum rt-pcr characterization of full genome of rat hepatitis e virus strain from vietnam identification of rodent homologs of hepatitis c virus and pegiviruses detection of zoonotic pathogens and characterization of novel viruses carried by commensal rattus norvegicus bats are a major natural reservoir for hepaciviruses and pegiviruses evidence for novel hepaciviruses in rodents rodents and risk in the mekong delta of vietnam: seroprevalence of selected zoonotic viruses in rodents and humans. vector-borne zoonotic dis market study of meat from field rats in the mekong delta aciar monograph rodent-borne diseases and their risks for public health detection of potentially novel paramyxovirus and coronavirus viral rna in bats and rats in the mekong delta region of southern viet nam the vietnam initiative on zoonotic infections (vizions): a strategic approach to studying emerging zoonotic infectious diseases bartonella species and trombiculid mites of rats from the mekong delta of vietnam. vector borne zoonotic dis how important are rats as vectors of leptospirosis in the mekong delta of vietnam? vector borne zoonotic dis a guide to the mammals of southeast asia large-scale screening and characterization of enteroviruses and kobuviruses infecting pigs in vietnam sse: a nucleotide and amino acid sequence analysis platform proposed update to the taxonomy of the genera hepacivirus and pegivirus within the flaviviridae family mega : molecular evolutionary genetics analysis version . for bigger datasets consensus proposals for classification of the family hepeviridae parasite-mediated selection against inbred soay sheep in a free-living, island population disease susceptibility in california sea lions consanguinity and susceptibility to infectious diseases in humans hepacivirus cross-species transmission and the origins of the hepatitis c virus viraemic frequencies and seroprevalence of non-primate hepacivirus and equine pegiviruses in horses and other mammalian species the virus whose family expanded experimental transmission of equine hepacivirus in horses as a model for hepatitis c virus vertical transmission of hepatitis c virus-like non-primate hepacivirus in horses mouse models of acute and chronic hepacivirus infection key: cord- -i w huke authors: xue, yong; sun, xiaohua; li, yinghui; liu, xin; dong, chen title: increased risk of hepatitis e virus infection in schizophrenia date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: i w huke until now, the risk of hev infection in schizophrenia was unknown. the present results showed that the seroprevalence of anti-hev igg and anti-hev igm in schizophrenia were significantly higher than that in healthy controls. anti-hev igg positivity increased with age and with the duration of disease in schizophrenia patients. moreover, schizophrenia patients with increased cd (+)/cd (+) t-cell ratios (> . ) had higher anti-hev igg detection rates than those with normal ratios ( . - . ). compared with the schizophrenia patients who tested anti-hev igg negative, the levels of interleukin- and interleukin- (th cytokines) were significantly higher, while the interleukin- (th cytokine) level was significantly lower, in those with anti-hev igg positivity. of five schizophrenia patients who were anti-hev igm positive, four had elevated cd (+)/cd (+) t-cell ratios. hev rna was isolated from one of these four patients and classified as genotype . anti-hev igm positivity was not detected among healthy controls. therefore, schizophrenia patients exhibited a higher risk of hev infection than controls. hepatitis e virus (hev) is a non-enveloped, single positive-strand rna virus in the family hepeviridae with a worldwide distribution [ ] . hepatitis e, caused by hev, is an important public-health problem in many developing countries and is the cause of sporadic acute hepatitis in developed countries. although the overall mortality of hepatitis e is less than % in the general population, it can reach up to % in pregnant women in the third trimester [ ] . hev infection has four documented routes of transmission: waterborne, foodborne, bloodborne and vertical transmission from mother to child [ , , , ] . epidemiological studies indicate that a higher prevalence of hev antibodies is observed in the elderly and people who live in rural areas with poor sanitation [ , ] . moreover, isolation of animal strains of hev and the existence of other animal species that tested seropositive for anti-hev igg and igm indicate that hepatitis e is a zoonotic disease [ , ] . schizophrenia is a complex mental disease that is highly heritable, and it involves interplay between genetic and environmental factors. immune system abnormalities have been reported in patients with schizophrenia, as evidenced by increased expression of proinflammatory cytokines, t-cell activation and high levels of autoantibodies [ ] . the alterations of circulating inflammatory cytokines, such as interleukin- (il- ), interleukin- (il- ) and interferon-c (ifn-c), reflecting the functions of t help type (th ), and interleukin- (il- ), interleukin- (il- ) and interleukin- (il- ), reflecting the functions of t help type (th ), were also observed in previous studies [ , ] . moreover, the previous results suggested there was an imbalance between th and th with a shift toward the th system in schizophrenia [ ] . epidemiological studies have already shown that the rates of hepatitis b and hepatitis c virus infection in patients with schizophrenia were five and times higher, respectively, than the estimated general population rates [ ] . however, the risk of hepatitis e virus infection in schizophrenia is still unknown. in the current study, we found that the detection rates for hev antibodies in schizophrenia patients were much higher than those in healthy controls. anti-hev igg positivity increased with age and with the duration of schizophrenia. moreover, schizophrenic patients with increased ratios of cd ? /cd ? t cells had higher anti-hev igg detection rates than those with normal ratios. compared with patients who tested anti-hev igg negative, the levels of il- and il- were significantly higher, while the il- level was significantly lower in those with anti-hev igg positivity. this study was carried out between august and march . two hundred sixty-nine individuals with schizophrenia (sz) were enrolled in the present study. the diagnosis of schizophrenia was based on criteria defined by the diagnostic and statistical manual of mental disorders (fourth edition, text revision) (dsm-iv tr). the mini international neuropsychiatric interview (mini) version . . dsm was used for screening patients for schizophrenia during data collection. all of the patients who were enrolled were inpatients and were diagnosed by the department of psychiatry, huai'an third hospital in jiangsu, china. moreover, all of the patients were free of acute infections and allergic reactions for at least two weeks prior to the collection of serum samples. two hundred sixty healthy controls (hc) were randomly recruited for age (± years) and gender matching from the annual health examination population ( , subjects) living in the same region. all of the healthy controls were found to be free of schizophrenia by screening using the mini test. moreover, the inclusion criteria for the healthy controls were the absence of any psychiatric and autoimmune disorders and the lack of a history of these disorders among immediate family members. additionally, healthy controls had to declare themselves free of acute infections and allergic reactions for at least two weeks prior to the collection of serum samples. this study conformed to the declaration of helsinki, and it was approved by the research ethics committee of the huaian third hospital, following established guidelines. all participants provided written informed consent after the study procedures were explained. anti-hev igg and igm were assayed using commercial immunoassay elisa kits (wantai biopharmaceutical, inc., beijing, china) as described previously [ ] . the sensitivity and specificity of the present assays were . % and . % for anti-hev igg detection, and . % and . % for anti-hev igm detection, respectively. in brief, plates precoated with recombinant hev orf proteins were incubated with test sera, as well as reactive and nonreactive controls at °c for min, washed five times and incubated with ll of human anti-igg-or anti-igm-horseradish peroxidase conjugate. after incubation at °c for min, the plates were washed and incubated at room temperature for min with enzyme substrate. absorbance was read at nm, and the cutoff value was determined according to the manufacturer's instructions. heparinized whole blood ( ll) was incubated with anti-cd (fitc), anti-cd (pe) and anti-cd (percp) and analyzed by four-color flow cytometry using a fluorescenceactivated cell sorter facscalibur (becton-dickinson, mountain view, ca) as described elsewhere [ ] . a total of , events were collected in the lymphocyte gate using morphological parameters. data were processed using cellquest pro software (becton-dickinson) and analyzed using the summit software. the cd ? t-cell, cd ? t-cell and cd ? t-cell counts were expressed as the proportion of all pbmcs. the levels of serum il- , il- , il- and ifn-c were assayed using commercially available elisa kits (yuanxiang biotech inc., shanghai, china). each cytokine assay was optimized, and a standard curve was established using standard procedures. in brief, plates were precoated with anti-il- , anti-il- , anti-il- or anti-ifn-c capture antibody. plates were washed and incubated with serial dilutions of standards and with test specimens for h at °c. plates were washed five times, incubated with appropriate biotinylated detection antibody for h at °c, and then washed and incubated for min with streptavidin-conjugated horseradish peroxidase. the reaction was developed with tetramethylbenzidine-h o substrate after the final wash. in all assays, the samples were analyzed in duplicate, and the case-control pairs were analyzed on the same plate. the lower limits of detection for il- and ifn-c were pg/ml, and for il- and il- , they were pg/ml, respectively. hev rna isolation, sequencing and analysis total rna from five anti-hev igm-positive samples as well as negative and positive controls was extracted from ll of serum using trizol-ls reagent (invitrogen, shanghai, china), reverse-transcribed and subjected to nested pcr to amplify the orf region as described previously [ ] . the limit of hev-rna detection in the present assay was copies/ml ( copies/reaction). after purification using a qiaquick gel extraction kit (qiagen, valencia, ca, usa), the pcr products were sent to invitrogen and sequenced using an abi genetic analyzer. genetic analysis was performed with mega version . (tempe, az, usa). an unpaired student's t-test was used to test for differences between sz and hc groups of continuous variables. differences in qualitative variables were compared using v or trend v tests. a two-sided p-value of . was set to denote statistical significance. all statistical analysis was performed using statistical product and service solutions . (spss; spss inc, chicago, usa). demographic and clinical characteristics for patients and controls are shown in table . there were no age or gender differences between schizophrenia patients and controls. increased levels of alanine aminotransferase (alt) and aspartate aminotransferase (ast) were detected in and schizophrenia patients, respectively. six patients had elevated levels of both alt and ast. in the hc group, only two and eight individuals had elevated levels of alt and ast, respectively. none of the controls had abnormal levels of both alt and ast. in the sz group, of ( . %) patients showed activation of t cells and increased ratios of cd ? /cd ? t cells ([ . ), while only eight people in hc group had increased ratios of cd ? /cd ? t cells (table ) . a significant difference was found between the sz group and the control. correlations between the detection rates, gender and duration of illness are summarized in table . ninety-four out of ( . %) in the sz group were positive for anti-hev igg, while out of people ( . %) in the hc group were positive (p \ . ). patients with a longer course of disease ([ years) tended to show higher rates of positivity than did patients with less than years of disease (p \ . ). the detection rates of hev igg antibody increased with age in the sz group but not in the healthy population. there was no significant difference between males and females in either the sz or the hc group. additionally, five cases, four males and one female, were hev igm antibody positive in the sz group, but no individuals were anti-hev igm positive in the hc group (p \ . ). the levels of serum il- , ifn-c, il- and il- were measured by elisa. significant differences were observed in serum il- , il- and il- levels between the schizophrenia patients with higher values of cd ? /cd ? t-cell ratios ([ . ) and those with normal values ( . ± . vs. . ± . for il- , . ± . vs. . ± . for il- and . ± . vs. . ± . for il- ), while no difference in serum ifn-c level was observed ( . ± . vs. . ± . for ifn-c). in comparison with schizophrenia patients who tested hev igg negative, hev-igg-positive patients had levels of serum il- and il- that were significantly higher, while the level of serum il- was significantly lower (p \ . , p \ . and p \ . , respectively) ( table ). there was no difference in the level of serum ifn-c between hev-igg-positive and negative patients. moreover, differences in cytokine levels were not observed within the hc group regardless of the hev igg status (data not shown). of five anti-hev-igm-positive samples, one sample with increased levels of alt and ast, and an elevated cd ? / cd ? t-cell ratio was positive for hev rna. the -nt orf sequence of the pcr product was sequenced and compared with those of known hev isolates. the isolate was . - . %, similar to the genotype isolates, but only . - . %, . % and . - . %, similar to the genotype , and hev strains, respectively. therefore, this hev isolate, named hev-huaiansz (genbank no. jx ) was classified as genotype , which was widely distributed in china. our study contributes to the sparse literature on hev seropositivity in schizophrenia patients. in previous studies, higher prevalence of antibodies to hbv nucleocapsid protein was seen in patients with psychiatric disorders [ ] . epidemiological studies also proved that patients with schizophrenia were significantly more likely to be infected with hcv than those without schizophrenia [ , ] . moreover, higher seroprevalence of antibodies to cytomegalovirus, herpes simplex virus, human immunodeficiency virus, influenza virus and coronaviruses were found in psychotic patients compared to the general population [ , , , , , , ] . therefore, it was reasonable for us to carry out the present study to evaluate the risk of hev infection in patients with schizophrenia. the results confirmed our hypothesis that anti-hev igg and igm detection rates in schizophrenia were significantly higher than in healthy controls. several factors could contribute to higher detection rates of hev antibody in schizophrenia patients. first, some symptoms of schizophrenia, in particular, the symptoms of cognitive impairment, may contribute directly to an inability to safeguard against the transmission of waterborne, foodborne or bloodborne infection [ ] . second, schizophrenia patients usually reside in settings such as psychiatric facilities, where they often forge social relationships with other higher-risk individuals [ , ] . third, financial instability and poverty are also associated with increased risks of hev infection [ ] . the clinical symptoms of hepatitis e are typical of selflimiting, acute viral hepatitis in general [ , ] . severe hepatitis e can occur in the elderly, pregnant women and people with chronic hepatitis b and chronic hepatitis c [ , , ] . chronic hepatitis e infection has also been documented in patients receiving immunosuppressive therapy following organ transplantation and those infected with hiv [ ] . the immune responses to hev involve antibody-mediated immunity, cell-mediated immunity, interferon and other host defenses [ , , , ] . data from cellular immune responses to hev suggest that the cd ? t-cell count is significantly higher in recovered hev-infected individuals than in acutely infected hev patients and the general population, but the levels of cd ? t-cells are similar in these groups [ , , ] . in schizophrenia, increased levels of cd ? t cells and higher cd ? /cd ? t-cell ratios were documented previously [ ] . in the present study, the detection rate of anti-hev igg in schizophrenia patients with increased cd ? /cd ? t-cell ratios ([ . ) was significantly higher than in patients with normal ratios ( . * . ). thus, further well-designed studies should be carried out to analyze the relationships between cd ? /cd ? t-cell levels and the increased risk of hev infection in schizophrenia patients. cellular immune responses to infections can be either of the th type, which is characterized by cytokines such as il- , ifn-c and il- , or the th type, which is characterized by cytokines such as il- , il- , il- and il- . in hepatitis b and c virus infections, the predominant immune response, which is th , is activated during liver trauma [ ] . according to pal et al. [ ] , ifn-c, a th cytokine, was lower in hev-infected pregnant women than in healthy people. furthermore, the production of il- , a th cytokine, was higher in hev-infected pregnant women than in controls [ ] . these results suggest that pregnant women with acute hev infection show a shift in the th / th balance towards a th response. this shift might play a role in the particularly severe clinical course and mortality of the hev infection during late pregnancy. it should be noted that schizophrenia might also be associated with an imbalance in th /th cytokines, and also with a shift toward the th system [ ] . in the present study, the th bias was observed in schizophrenia patients, as evidenced by the increased production of il- and il- and lower il- production. moreover, significant differences of the serum il- , il- and il- levels were observed among schizophrenia patients with and without hev igg antibodies. the major implication of this study is that there may be more potential risk factors in schizophrenia patients, which may be responsible for the increased transmission of hev. also, there are several limitations in our present study. first, the various drugs used in schizophrenia may affect the immune system, and virtually all our schizophrenia patients were on this type of medication. relationships between drugs and immune bias, and the effect of this relationship on the risk of hev infection in schizophrenia should be assessed in the future. second, we did not include inflammatory markers other than serum il- , il- , il- and ifn-c, and thus, future work should be carefully interpreted in the light of this. third, larger numbers of schizophrenia patients and controls as well as long-term follow-up will be required to further analyze the increased risk of hev infection in schizophrenia. taken together, schizophrenia patients exhibited higher risk of hev infection than controls in the present study. anti-hev igg seropositivity increased with age and with the duration of illness. moreover, schizophrenia patients with increased cd ? /cd ? t-cell ratios had higher anti-hev igg detection rates than those with normal ratios. the levels of il- and il- were significantly higher, while the il- level was significantly lower in schizophrenia patients with anti-hev igg positivity than in those who were antibody negative. in order to understand the questions behind the observed associations, further work is needed, with larger samples and longitudinal follow-ups. epidemiology of hepatitis e: current status the unique riverine ecology of hepatitis e virus transmission in south-east asia antibodies to measles in individuals with recent onset psychosis identification of genetic diversity of hepatitis e virus (hev) and determination of the seroprevalence of hev in eastern china the first experimental study on a candidate combined vaccine against hepatitis a and hepatitis e restricted enzooticity of hepatitis e virus genotypes to in the united states 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balance: an important indicator of efficacy for intra-arterial chemotherapy association of seropositivity for influenza and coronaviruses with history of mood disorders and suicide attempts immunological alterations in pregnant women with acute hepatitis e inflammatory cytokine alterations in schizophrenia: a systematic quantitative review hepatitis e: an emerging awareness of old disease prevalence of hiv, hepatitis b, and hepatitis c in people with severe mental illness a nationwide survey of hepatitis e virus (hev) infection in wild boars in japan: identification of boar hev strains of genotypes and and unrecognized genotypes coronavirus immunoreactivity in individuals with a recent onset of psychotic symptoms antibodies to cytomegalovirus and herpes simplex virus associated with cognitive function in schizophrenia inflammation in psychotic disorders: a populationbased study positive hepatitis e and epstein barr virus serology in a patient with jaundice after travel the two clinico-epidemiological forms of hepatitis e cytokine profiles, ctl response and t cell frequencies in the peripheral blood of acute patients and individuals recovered from hepatitis e infection efficacy and safety of a recombinant hepatitis e vaccine in healthy adults: a large-scale, randomised, double-blind placebocontrolled acknowledgments the authors thank dr. michael a. purdy, dr. maja kodani, dr. chong-gee teo and dr. saleem kamili for kindly reviewing the manuscript. we would also like to thank dr. chong shen, xianfeng cheng, yadong zhang and chunlin shi for their assistance in the present work. the authors declare no conflict of interest. key: cord- -wg vfh w authors: webb, glynn w.; kelly, sophie; dalton, harry r. title: hepatitis a and hepatitis e: clinical and epidemiological features, diagnosis, treatment, and prevention date: - - journal: clin microbiol newsl doi: . /j.clinmicnews. . . sha: doc_id: cord_uid: wg vfh w hepatitis a and e are both ancient diseases but have only been properly recognized as being caused by distinct pathogens in modern times. despite significantly different genomic structures, both viruses employ remarkably similar strategies to avoid host detection and increase environmental transmission. there are millions of cases of acute viral hepatitis due to hepatitis a virus (hav) and hepatitis e virus (hev) each year, resulting in tens of thousands of deaths. the presentations can be clinically indistinguishable, but each virus also has a range of less common but more specific phenotypes. the epidemiology of hav is complex, and is shifting in countries that are making improvements to public health and sanitation. hev presents a significant public health challenge in resource-limited settings but has historically been incorrectly regarded as having little clinical relevance in industrialized countries. descriptions of a clinical syndrome recognizable as hepatitis can be found in sumerian medical texts from the third millennium before the common era. approximately , years later, hippocrates recorded the features of "epidemic jaundice," including clinical descriptions suggestive of fulminant hepatitis. by the middle ages, the idea that jaundice might be transmissible had emerged; in the th century, pope zacharias had men with jaundice quarantined to control the spread of the disease, although he had no understanding of the exact etiology of the condition. large epidemics of jaundice, variously called "catarrhal jaundice," "infectious hepatitis," "epidemic hepatitis," or similar, were mainly associated with military campaigns and were a significant cause of morbidity and mortality among troops in the napoleonic wars, the american civil war, and both world wars. it was during the second world war that the first evidence of the viral etiology of epidemic jaundice, and the existence of distinct forms of the condition, emerged. a series of experiments in germany, the united kingdom, and the united states throughout the s used filtered materials from infected individuals to infect volunteers, or in some cases prisoners [ ] . by the end of the decade, these studies, along with epidemiological studies, had elucidated two subtypes of viral hepatitis, distinguished by the primary route of transmission and period of incubation: orally transmitted "infectious hepatitis," with a short incubation period, and parenterally transmitted "serum jaundice," with a prolonged incubation period. these were later termed hepatitis a and hepatitis b, respectively. it was the former that would be blamed for the large waterborne outbreaks of hepatitis that had plagued humankind since antiquity. vol. , no. november , www.cmnewsletter.com introduction by the end of the s, however, evidence of another pathogen causing epidemics of hepatitis started to emerge. work done during the preceding decade at the u.s. national institutes of health had already demonstrated that most cases of transfusion-associated hepatitis were due to neither hepatitis a virus (hav) nor hepatitis b virus (hbv). the cause of these cases of non-a, non-b hepatitis would eventually be identified as hepatitis c virus (hcv). an outbreak of hepatitis in kashmir, india, in provided the first evidence of another hepatitis virus. the epidemic was waterborne, and large, with around , cases and , deaths [ ] . the mode of transmission and clinical presentation were generally in keeping with hepatitis a; one key distinguishing feature was the excess morbidity and mortality seen among pregnant women. around the same time, another group was examining serum samples from three previous indian hepatitis outbreaks, including a large epidemic in delhi in [ ] . in all cases, serological testing of infected individuals found no evidence of infection with hav. further outbreaks of non-transfusion-associated non-a, non-b hepatitis were identified in other parts of asia, north africa, and the middle east [ ] . however, as no causative agent had been identified, it was impossible to determine if the same pathogen was involved in each outbreak. still, it was becoming increasingly clear that clinically apparent hepatitis a was actually very rare in developing countries [ ] . a few years later, a russian virologist named mikhail balayan was investigating an outbreak of non-a, non-b hepatitis among soviet troops stationed in tashkent, the capital of what is now uzbekistan. balayan wanted to take clinical samples back to moscow for further study, but that would have meant refrigerating the samples. as the infrastructure to do this was lacking, balayan instead ingested a pooled filtrate of samples from his patients [ ] . upon returning to moscow, he developed a case of acute hepatitis. electron microscopy of his stool identified viral particles that caused a hepatitis-like illness when experimentally inoculated into cynomolgus monkeys. crucially, these novel viral particles did not react with anti-hav igm. by the start of the s, the genome of the novel non-transfusion-associated non-a, non-b hepatitis virus had been sequenced, and it had been named hepatitis e virus (hev) [ ] . hav and hev can cause diseases that are clinically indistinguishable from each other, and despite being only distantly related, share some remarkable similarities in terms of the pathogenic strategies they employ. however, they are quite distinct in many other respects. this article reviews their similarities and differences, comparing the epidemiologies, clinical manifestations, diagnosis, treatment, and prevention of the two viruses. hav is a member of the family picornaviridae and was initially placed in the genus enterovirus. however, further study demonstrated that the virus was sufficiently different from other picornaviruses to be classified within its own genus, hepatovirus. hev was initially considered to be a member of the family calciviridae but was later reclassified as a member of the genus orthohepevirus, in the family hepeviridae. hav is one of nine species of hepatovirus and the only one known to infect humans. there are six genotypes of hav, three that infect humans and three affecting simians, but only one serotype [ ] . the remaining members of the genus infect a range of species, including bats, hedgehogs, shrews, and rodents. phylogenetic analysis suggests that the genus originated in small mammals and that human hav has a rodent origin [ ] , although a zoonotic reservoir no longer exists. the genus orthohepevirus contains four species (a to d). human disease is caused by orthohepevirus a, which has eight genotypes. the remaining three species are found in birds (hev-b), rodents and ferrets (hev-c), and bats (hev-d). two genotypes of orthohepevirus a are obligate human pathogens (hev and hev ), and two are endemic in a wide range of species and cause zoonotic infections in humans (hev and hev ). the remaining genotypes are primarily restricted to wild boar (hev and hev ) and camels (hev and hev ). however, human hev infection has been reported [ ] , and hev is capable of infecting primates [ ] . both hav and hev are non-enveloped icosahedral viruses. the lack of a lipid envelope offers both viruses a significant advantage in terms of their ability to spread in the environment, as demonstrated by the foodborne and waterborne outbreaks, which are synonymous with both hepatitis a and e [ , ] . this is because a stable, naked protein capsid offers the fragile rna genome significant protection against harsh environmental conditions. in comparison, the transmission of enveloped viruses tends to require at least close contact between individuals, if not the exchange of bodily fluids. this is because the viral envelope contains virus-encoded glycoproteins called peplomers, which mediate interactions with cell surface receptors. without these peplomers, the virion is unable to gain access to a host cell, so it is vulnerable to anything that would disrupt the lipid bilayer, such as drying, solvents, or detergents. within a host, however, enveloped viruses have several advantages over naked virions. the envelope facilitates crossing the plasma membrane, allowing new virions to leave the cell without the need for cell lysis, and also protects the virus from the immune response by hiding antigens from neutralizing antibodies [ ] . there is a significant body of evidence that suggests that although both hav and hev are non-enveloped viruses, they can also enjoy at least some of the benefits of enveloped viruses. hav and hev virions, which are shed in the stool, are naked protein capsids, ideally suited to their role of reaching new hosts across both time and distance in a potentially hostile environment [ ] . however, hav and hev, which are isolated from the serum of individuals suffering an acute infection, are wrapped in a hijacked layer of host cell membrane, similar to those found on classical enveloped viruses but distinguished by the lack of any virusencoded proteins at the surface [ ] . this allows circulating virions to avoid the immune response, as antigenic proteins are protected from neutralizing antibodies [ ] . however, the lack of peplomers raises questions as to how these quasi-enveloped virions achieve entrance into host cells [ ] . the world health organization estimates that there are . million cases of hepatitis a globally each year, resulting in approximately , deaths [ ] . in comparison, there are an estimated million hev infections each year, leading to . million symptomatic cases and around , deaths [ ] . these figures are concerned only with parts of the world where hev is endemic and, as such, are likely to represent a gross underestimate of the actual global disease burden [ ] . the primary route of transmission for hav is fecal-oral, primarily through direct person-to-person contact, but also via contaminated food or water. men who have sex with men are at increased risk of infection, as are any persons engaging in oral-anal sexual contact regardless of gender or sexual orientation [ ] . parenteral transmission via contaminated blood products has been described [ ] , and injecting drug users are at high risk [ ] , with increased prevalence positively correlated with low incomes [ ] . infected individuals shed virus in their stool for around weeks before becoming symptomatic and typically for a few days after but may continue to do so for several weeks. even with good standards of hygiene and sanitation facilities, the rate of infection in close contacts of cases is high, suggesting very efficient interpersonal transmission. the rate and pattern of hav transmission vary widely between different parts of the world, primarily determined by socioeconomic factors. in regions with smaller family sizes, better sanitation facilities, and greater access to clean drinking water, rates of infection are lower. counterintuitively, lower rates of transmission do not equate to less disease. in resource-poor areas of high endemicity, such as africa, parts of asia, and south america, infection with hav in early childhood is widespread. in most cases, young children are asymptomatic or experience a very mild illness, and hav infection typically confers lifelong immunity. conversely, in high-income regions of low endemicity, like north america, western europe, japan, and australia, exposure in childhood is rarer. as a result, a much smaller proportion of the adult population has anti-hav antibodies. if hav is introduced, significant outbreaks can result, particularly in high-risk groups, such as men who have sex with men [ ] , homeless people, and recreational drug users [ ] . these outbreaks primarily affect adolescents and adults, who are more susceptible to becoming seriously ill. over the past few decades, improvements in hygiene and sanitation in some lowand middle-income countries have reduced hav transmission and increased the average age at infection [ ] . this "epidemiological transition" shifts the epidemiological pattern closer to that seen in industrialized nations, producing a paradoxical increase in both morbidity and mortality associated with hepatitis a [ ] . the epidemiology of hav depends upon the genotype involved and, by extension, the geographical region under examination. as mentioned above, human hepatitis e is predominantly caused by four of the eight genotypes of orthohepevirus a. hev and hev are similar to hav in that they are endemic in lower-income countries [ ] , infect only humans, and are spread via the fecaloral route. however, unlike hav, infection with hev does not confer lifelong immunity. this results in both sporadic cases and periodic outbreaks, which occur periodically when anti-hev igg seroprevalence in the population drops below a critical threshold for herd immunity [ ] . hev and hev , in contrast, are zoonoses that infect a wide range of mammalian species; however, pigs constitute the primary viral reservoir [ ] . hev causes locally acquired infections in europe, north america, australasia, and japan [ ] . hev has historically been restricted to china and japan [ ] . transmission between pigs occurs via the fecal-oral route, and infection is typically apathogenic in the animals. consumption of infected meat is the primary vector for human infection, and hev has been identified in retail pork products at the point of sale [ ] . a range of other foods have also been implicated, with viruses isolated from shellfish, fruits, and vegetables [ ] ; this is likely due to pig slurry contaminating watercourses or being used as fertilizer. more than two-thirds of zoonotic hev infections are asymptomatic [ ] . of the remaining cases, only a small fraction are confirmed by serological or molecular testing; historically, most are either misdiagnosed or go unrecognized. in recent years, however, there has been a surge in reported cases. the number of laboratory-confirmed cases in europe increased by a factor of between and [ ] . in part, this likely reflects increased awareness of hev among clinicians, but in at least some countries, there has been an actual increase in incidence [ ] . reports from several different countries have described parenteral hev transmission via infected blood products [ ] . studies have also demonstrated viremia at the time of donation among healthy blood donors, with a wide variation in the rates of viremia in different countries, from : in india [ ] to : , in australia [ ] . the extent of the contribution that transfusion-associated hev infection makes to the global burden of disease is unclear, but it certainly presents less risk than the primary modes of transmission. however, patients who are at risk of chronic infection or more severe hepatitis are over-represented in the cohort of patients who are most likely to receive blood products. this includes transplant recipients, immunocompromised patients, and pregnant women. in response to this, a growing number of european countries now routinely screen blood donations for hev [ ] . in the last years, evidence has emerged of a new zoonotic source of hev infection. as mentioned above, hev-c affects rodents. genotype of hev-c (hev-c ) circulates in rats in europe, asia, and north america. a recent large prospective study in hong kong identified both acute and chronic hev-c infection in both immunocompetent and immunocompromised patients [ ] . extrahepatic manifestations were also described; one patient developed meningoencephalitis and died, with hev-c rna found in their cerebrospinal fluid (csf) [ ] . the presence of anti-hev-a antibodies does not appear to offer any protection, and molecular testing for hev-a does not detect hev-c due to sequence differences between the species. these findings suggest that hev-c could be prevalent around the world and yet be routinely missed, which would have important clinical implications and would pose a threat to the safety of blood products. clinical symptoms of hepatitis a typically occur following an incubation period of to days but can present as much as days after exposure (table ) [ ] . the clinical presentation ranges from asymptomatic to fulminant hepatitis [ ] . the disease course is typically more severe with increasing age; very young children often have no symptoms at all. the typical presentation involves two phases: a prodromal phase that lasts for to days and is characterized by malaise and myalgia, followed by an icteric phase [ ] . the icteric phase involves a mixed hepatic and cholestatic jaundice associated with anorexia, nausea, and fatigue. this stage lasts between and weeks. acute liver failure occurs in approximately . % of cases, although it is highly age dependent; in children and adults less than years of age, the case fatality rate is between . % and . %, while in adults over years of age, it is . % [ ] . co-existing chronic infection with hbv or hcv increases the chance of acute liver failure due to infection with hav [ ] . a subset of patients with hepatitis a may present atypically [ ] . up to % can develop cholestatic hepatitis, which includes a prolonged period of jaundice lasting weeks or more [ ] . the typical clinical course in these patients involves significant jaundice, pruritis, fever, weight loss, diarrhea, and malaise [ , ] . liver function tests (lfts) show a cholestatic picture, with marked elevation of bilirubin and alkaline phosphatase and a mild to moderate rise in alanine aminotransferase (alt). generally, patients with cholestatic hepatitis require only supportive treatment and go on to make a full recovery. a further % of patients experience a relapse of hepatitis a in the months following the primary infection [ , ] . following an apparent initial recovery, there is a biochemical relapse that may or may not be accompanied by clinical deterioration. alt levels can exceed , iu/dl, and patients generally remain anti-hav igm seropositive through the course of the illness [ ] . there are detectable levels of virus in the stool during relapses, so affected patients should be considered infectious. when symptoms recur, they are typically milder than the initial illness and of short duration, usually less than weeks [ ] ; however, it can take as long as a year for full biochemical resolution. it is not known what causes some patients to relapse, and no risk factors have been identified [ ] . multiple relapses can occur, but most patients go on to make a full recovery. most patients who develop clinically apparent hepatitis e experience an acute, self-limiting illness lasting to weeks [ ] . after an incubation period of between and weeks, patients develop features typical of hepatitis-jaundice, fatigue, fever, abdominal pain, nausea, and vomiting. in developing countries where hev and hev are predominant, young adults are the most commonly affected group. males are more likely to present clinically than women. overall mortality is between . % and % [ ] but is significantly higher in vulnerable groups, including children under years of age [ ] , individuals with pre-existing liver disease [ ] , and pregnant women (see below). in higher-resource settings, locally acquired cases are caused by hev and hev . there is significant heterogeneity in the clinical picture of acute infection in these areas; only a small minority of patients present with typical viral hepatitis as described above. despite this, hev is the most common cause of viral hepatitis in several european countries, including france, germany, and the united kingdom [ ] . as with hev and hev , genotypes and preferentially affect men, with a male-to-female ratio of around : , although they tend to be older, with a median age of [ ] . it this thought that this is a result of host factors rather than due to variations in exposure. pre-existing subclinical liver disease has been proposed as a potential risk factor, as both alcohol excess and diabetes, which are associated with hepatic steatosis and fibrosis, are over-represented among patients with acute hepatitis e [ ] . progression to liver failure is generally uncommon; however, a small number of cases have been reported, and a german study identified hev as the most likely precipitant of acute liver failure in % of a cohort of patients. individuals with pre-existing liver disease are at risk of decompensation or acute-on-chronic liver failure. however, hepatitis e is not as common a cause of decompensation in industrialized countries [ , ] . this likely represents differences in the pathogenicity of the genotypes, which are found in different regions. one of the most important public health challenges related to acute hepatitis e infection, which most commonly occurs in developing countries, is the excess morbidity and mortality seen among pregnant women (table ). in these parts of the world, where hev is the predominant genotype, around a quarter of pregnant women with acute hepatitis e die [ ] . this highest-risk period is the third trimester, with % of women infected during that stage of pregnancy developing fulminant hepatic failure [ ] . other than liver failure, the major causes of hev-related maternal death are obstetric complications, such as eclampsia or hemorrhage [ ] . there is also a significant risk to both the fetus and neonates. vertical transmission is common [ ] , and there is a substantial increase in the risk of intrauterine death, stillbirth, pre-term birth, and low birth weight [ ] . fetal mortality is approximately %, including those who die as a result of maternal death, and neonatal mortality is around % [ ] . the mechanisms that underlie the increased disease severity in pregnant women are not well understood. pathogenic differences between hev genotypes may play a part, as the rates high of mortality and morbidity are seen with hev and have not been described in the context of hev infection. both hev and hev are capable of infecting the decidua basalis (i.e., the uterine endometrium at the site of embryo implantation) and the placenta. however, hev replicates with greater efficiency in vitro in tissue explants and stromal cells from both tissues and is associated with increased tissue damage [ ] . hev also affects the secretion profile of the maternal-fetal interface, increasing the release of pro-inflammatory factors [ ] . both the viral load and serum inflammatory cytokine levels (tumor necrosis factor alpha, transforming growth factor β , interleukin and interferon gamma [ifn-γ]) are higher in pregnant women than in non-pregnant women infected with hev , and levels of the cytokines correlate positively with adverse pregnancy outcomes [ ] . other studies have shown that high levels of hev rna are also associated with worse outcomes in pregnancy [ ] . while hav does not cause chronic infection, persistent infection with hev has been documented. the majority of the literature on the subject describes solid organ transplant recipients, but chronic infection has been reported in other immunocompromised cohorts, as well, including in hiv-positive patients with cd + counts of < /mm , in individuals with hematological malignancies receiving chemotherapy and stem cell transplants, and in rheumatology patients treated with immunosuppressive drugs [ ] . in almost all cases, chronic infection involves hev or hev , but there has been a single report of chronic hev infection in a patient who had consumed camel meat and milk [ ] . chronic infection is defined as hev replication persisting for months or more [ ] . around two-thirds of solid organ transplant recipients who are exposed to hev develop a chronic infection [ ] . the risk is increased for those with immunosuppression and for those who are treated with tacrolimus. most chronic infections are asymptomatic, with only a mild or moderate rise in alt. some patients have normal lfts, and some remain seronegative for anti-hev antibodies despite active viral replication [ ] . without treatment, the development of fibrosis can occur, with a risk of rapid progression to cirrhosis, decompensation, and death [ ] . extrahepatic manifestations of hav infection are most commonly reported in patients who experience cholestatic hepatitis or relapsing hepatitis a. between and % of patients develop a rash and/or arthralgia, but a range of rarer complications have also been described. they include vasculitis, cryoglobulinemia, and thrombocytopenia [ , ] . a broad range of extrahepatic manifestations (table ) have been reported in the context of both acute and chronic hev infection, the most important of which are neurological and renal complications. the mechanisms that underlie these manifestations are not currently understood, but both immune-mediated processes and direct viral tropism have been suggested. neurological injury is the most frequently reported extrahepatic complication of hev infection, with around cases involving hev or hev currently described [ ] . in all cases, it is the neurological signs and symptoms that dominate the clinical picture. patients are typically anicteric and have either normal liver enzymes or at most mild to moderately deranged lfts. a wide variety of neurological illnesses have been reported in association with hev (table ), but the mostly strongly associated conditions are neuralgic amyotrophy (na), guillain-barré syndrome (gbs), and encephalitis/myelitis. na is an acute monophasic injury to the brachial plexus that results in pain, weakness, and sensory disturbance in the upper limbs. in a study of dutch and english patients with na, . % had evidence of acute hev infection at the onset of their neurological illness [ ] . hev-associated na has a characteristic clinical phenotype. the symptoms tend to be more severe, with more extensive bilateral damage to the brachial plexus than is seen in non-hev-associated cases [ ] . an international multi-center study in europe that prospectively tested patients with acute, non-traumatic neurological injury found evidence of hev infection in . % of the patients. there were three cases of na; all three had evidence of infection, and all three displayed the characteristic clinical phenotype of hev-associated na [ ] . gbs is an immune-mediated polyradiculopathy involving rapidly progressive muscle weakness, which can lead to respiratory failure. it is considered a post-infective condition, and campylobacter jejuni is the most common precipitant. the causative organism was not identified in over % of the cases [ ] . the earliest reports of hev-associated neurological illness involved gbs, following the observation that around a third of dutch gbs patients had lft derangement without an apparent cause [ ] . three case-control studies from the netherlands, bangladesh, and japan found evidence of recent hev infection at significantly higher rates among gbs patients than in control subjects [ ] . twelve cases of hev-associated encephalitis/myelitis have been reported in europe, asia, and the u.s. [ ] . five of them were chronically infected solid organ transplant recipients. five patients developed ataxia, which appears to be associated with worse outcomes; two of the ataxic patients died, and the remainder had a more significant long-term neurological deficit than those who did not have ataxia. six patients had hev rna in both blood and csf. in one case, the virus isolated from csf showed evidence of quasispecies compartmentalization, which may suggest the emergence of directly neurotropic strains [ ] . renal injury has been described in the context of both acute and chronic hev infection. renal biopsies of patients with hevassociated renal impairment show evidence of membranoproliferative glomerulonephritis (mpgn), cryoglobulinemia, and membranous glomerulonephritis [ ] . hev rna has also been isolated from the cryoprecipitate of an immunocompetent patient who presented with acute hev infection and mpgn [ ] . once viral clearance is achieved, renal function and proteinuria improve in most patients [ ] . the differential diagnosis of acute hepatitis is extensive. in addition to the five hepatitis viruses, there are several other viral causes, such as cytomegalovirus and epstein-barr virus. non-viral infectious causes include bacterial infections, such leptospirosis, rocky mountain spotted fever, and typhoid, or parasitic infection with liver flukes or roundworms. non-infectious causes include autoimmune hepatitis, systemic lupus erythematosus, drug-induced liver injury-iatrogenic or secondary to deliberate self-harmand toxins, most commonly alcohol. for several reasons, hev infection may not be considered a potential diagnosis. as it is still often incorrectly regarded as an "emerging" disease, many clinicians have limited knowledge of the disease. the heterogeneous presentation of hev infection, especially in developed countries, further compounds the problem. even when hev infection is considered, there are issues surrounding the availability and reliability of testing. in the u.s., there is no u.s. food, and drug administration-approved assay on the market. where they are available, there is significant variation in the sensitivity and specificity of tests [ ] , and the previous "gold standard" test has been shown to substantially underestimate seroprevalence in comparison to later-generation assays [ ] . in chronic hev infection, as previously described, patients are often seronegative for anti-hev antibodies, so molecular analysis must be used to detect hev rna directly (fig. a) . this type of analysis can be less useful in acute infection, as the period of viremia can be very narrow. however, virions are shed in the stool for a longer period. in contrast, highly sensitive and specific serological assays for anti-hav igm have been available for more than years (fig. b) [ ] . specific igm remains positive for a variable period of time following infection, disappearing from the sera of most patients within months but remaining for up to a year in some individuals [ ] . anti-hav igg is seropositive in both current and past infections, as it typically remains present in the serum for life. for both hepatitis a and acute hepatitis e, the majority of cases require no specific treatment. the minority of patients who develop fulminant hepatic failure should receive aggressive supportive therapy and be considered for liver transplantation. a small number of patients with severe, acute hepatitis e have been treated with ribavirin, producing a rapid resolution of liver enzyme derangement and viral clearance [ ] . it is, however, difficult to draw conclusions from these reports due to the lack of controls and range of different treatment protocols employed. in chronic infections in immunosuppressed transplant recipients, the first-line treatment is the reduction of immunosuppressive drugs, particularly those that target t cells. around % of patients clear the virus without any other intervention [ ] . for the remaining patients, ribavirin may be useful. the optimal duration of treatment is still to be fully determined, but the european association for the study of the liver guidelines suggest an initial -month course followed by a further -month course for those who fail to clear the virus after the first course [ ] . a large retrospective study suggested that around % of patients achieve a sustained virologic response following one course of treatment, rising to nearly % after a second [ ] . pegylated ifn-α can be considered for liver transplant recipients who cannot tolerate ribavirin or fail to respond but is generally contraindicated in patients who have received other organs as the risk of rejection is increased [ ] . in other immunosuppressed cohorts, successful treatment with ribavirin, ifn-α, or a combination of the two has been described [ ] . the main methods by which the spread of hav can be prevented are good hygiene practices, proper sanitation, case investigation and contact tracing during outbreaks, and active and passive immunoprophylaxis. thorough hand washing and careful food handling practices are of great importance. postexposure prophylaxis can be considered for close contacts of infected individuals, subject to local guidelines. this can consist of either vaccination or intramuscular immune globulin. the u.s. centers for disease control and prevention recommends vaccination for healthy people aged months to years; immune globulin for those aged over , although vaccination is acceptable; and immune globulin for children under months, immunocompromised individuals, patients with chronic liver disease, and anyone with contraindications for vaccination [ ] . screening for hav should be offered to at-risk individuals in sexual-health settings, mainly targeting men who have sex with men, intravenous drug users, and patients with hiv, hbv, or hcv disease [ ] . patients who test negative should be offered vaccination [ ] . in regions where hev and hev are endemic, the key prevention strategies surround improving sanitation facilities and access to clean, safe drinking water. in resource-rich settings, where zoonotic transmission is the major route of infection, proper preparation of food products is the primary preventive measure. meat products, in particular pork and game, should be thoroughly cooked [ ] . anyone with risk factors for developing a more severe illness, such as those with pre-existing liver disease or immunosuppressed individuals, should take particular care to avoid uncooked meat. people whose employment brings them into contact with pigs, boar, or deer and their products should take care to minimize direct contact and use appropriate protective equipment. an effective vaccine against hev, designated hev- , has been on the market in china for several years [ ] . the vaccine has been designed to offer long-term protection against all genotypes of hev but has yet to be licensed outside china. hav and hev share many similarities but are distinguishable from each other in many ways. they occupy similar ecological niches and have developed similar characteristics and strategies despite not being closely related. their status as quasi-enveloped viruses has not been seen in any other virus and likely represents an adaptation to take advantage of the secretory pathways that are accessible from hepatocytes. both viruses present public health challenges to both high-and low-income countries. the changing epidemiological patterns that hav displays in response to socioeconomic progress in developing countries require careful attention. interventions, such as universal childhood vaccination programs, must be properly implemented and timed correctly. if a vaccination program introduced to a low-endemicity region achieved inadequate coverage, it would likely exacerbate the epidemiological transition it was intended to ameliorate. our understanding of hev, particularly the zoonotic genotypes, remains inadequate, and the covid- pandemic has brought the risks presented by emerging zoonotic infections into sharp focus. there is mounting evidence that hev is significantly underdiagnosed, particularly in the context of its many extrahepatic manifestations. further attention is required to elucidate the actual burden of disease presented by hev and to better understand the risks presented by potential novel zoonoses, such and hev-c . hepatitis a: old and new discovery of hepatitis e: the epidemic non-a, non-b hepatitis years down the memory lane hepatitis e: an emerging awareness of an old disease evolutionary origins of hepatitis a virus in small mammals chronic infection with camelid hepatitis e virus in a liver transplant recipient who regularly consumes camel meat and milk genotype hepatitis e virus produced by a reverse genetics system has the potential for zoonotic infection new developments in an old disease naked viruses that aren't always naked: quasi-enveloped agents of acute hepatitis european association for the study of the liver. electronic address: easloffice@easloffice.eu; european association for the study of the liver. easl clinical practice guidelines on hepatitis e virus infection hepatitis a outbreak disproportionately affecting men who have sex with men (msm) in the european union and european economic area hepatitis a virus outbreaks associated with drug use and homelessness hepatitis a virus seroprevalence by age and world region increasing incidence of hepatitis a in korean adults transmission of hepatitis e virus in developing countries hepatitis e: an underestimated emerging threat high proportion of asymptomatic infections in an outbreak of hepatitis e associated with a spit-roasted piglet european centre for disease prevention and control. hepatitis e in the eu/eea hepatitis e virus (hev) in scotland: evidence of recent increase in viral circulation in humans hepatitis e virus infection may be transmitted through blood transfusions in an endemic area hepatitis e virus rna in australian blood donors: prevalence and risk assessment transmission of rat hepatitis e virus infection to humans in hong kong: a clinical and epidemiological analysis hepatitis a: clinical manifestations and management clinical course and management of acute hepatitis a infection in adults atypical clinical manifestations of hepatitis a prolonged intrahepatic cholestasis secondary to acute hepatitis a relapsing hepatitis a. review of cases and literature survey hepatitis e epidemic hepatitis e virus (hev) infection in patients with cirrhosis is associated with rapid decompensation and death hepatitis e virus: assessment of the epidemiological situation in humans in europe, / autochthonous hepatitis e in southwest england: natural history, complications and seasonal variation, and hepatitis e virus igg seroprevalence in blood donors, the elderly and patients with chronic liver disease host risk factors and autochthonous hepatitis e infection hepatitis e virus in patients with decompensated chronic liver disease: a prospective uk/french study hepatitis e infection in patients with severe acute alcoholic hepatitis hepatitis e during pregnancy: maternal and foetal case-fatality rates and adverse outcomes-a systematic review hepatitis e in pregnancy clinical course and duration of viremia in vertically transmitted hepatitis e virus (hev) infection in babies born to hev-infected mothers burden of hepatitis e virus infection in pregnancy and maternofoetal outcomes: a systematic review and meta-analysis clinical manifestations, pathogenesis and treatment of hepatitis e virus infections arthritis, vasculitis, and cryoglobulinemia associated with relapsing hepatitis a virus infection thrombocytopenia in hepatitis a-an atypical presentation hepatitis e virus and neurological injury neuralgic amyotrophy and hepatitis e virus infection clinical phenotype and outcome of hepatitis e virus-associated neuralgic amyotrophy hepatitis e virus infection and acute non-traumatic neurological injury: a prospective multicentre study the spectrum of antecedent infections in guillain-barré syndrome: a case-control study hepatitis e virus-induced neurological symptoms in a kidney-transplant patient with chronic hepatitis hepatitis e seroprevalence in europe: a meta-analysis two generations of "gold standards": the impact of a decade in hepatitis e virus testing innovation on population seroprevalence ribavirin for hepatitis e virus infection after organ transplantation: a large european retrospective multicenter study prevention of hepatitis a virus infection in the united states: recommendations of the advisory committee on immunization practices british association for sexual health and hiv. interim update of the bashh national guidelines for the management of the viral hepatitides key: cord- -ii xurmr authors: bachofen, claudia title: selected viruses detected on and in our food date: - - journal: curr clin microbiol rep doi: . /s - - - sha: doc_id: cord_uid: ii xurmr purpose of review: the purpose of this review is to provide an update on recent literature and findings concerning selected foodborne viruses. two groups of viruses were selected: (a) the most important viruses contaminating food, based on numbers of publications in the last years and (b) viruses infecting sources of food that might have an impact on human health. recent findings: important foodborne viruses such as norovirus, hepatitis a and rotavirus are usually “only” contaminating food and are detected on the surface of foodstuffs. however, they are threats to human public health and make up for the majority of cases. in contrast, the meaning of viruses born from within the food such as natural animal and plant viruses is still in many cases unknown. an exception is hepatitis e virus that is endemic in pigs, transmitted via pork meat and is recognised as an emerging zoonosis in industrialised countries. summary: even though the clinical meaning of “new” foodborne viruses, often detected by next generation sequencing, still needs clarification, the method has great potential to enhance surveillance and detection particularly in view of an increasingly globalised food trade. every form of live harbours its own range of viruses [ ] . no wonder, they are also present on and in our food, being it of animal or vegetable origin, and are consumed on a regular basis [ ] . considering this fact, only relatively few outbreaks or cases of disease due to foodborne viruses are reported-in contrast to bacterial infections [ • ]. this may be largely due to inability of these viruses to infect humans and/or inactivation during food processing. however, there are also several reasons that hamper recognition and reporting. foodborne viruses are highly diverse but the clinical signs they cause are usually not specific for the single virus and often rather general such as diarrhoea and malaise making diagnosis challenging and tedious. in addition, in case of human-to-human transmission after uptake of the virus, the initial foodborne origin may not be recognisable anymore if it is not extremely obvious. furthermore, lacking awareness of clinicians [ ] and a limitation of concerted surveillance programmes for foodborne viruses may contribute to underreporting [ ] . interestingly, the most important foodborne viruses regarding case numbers and economic impact are not really borne from within sources of food but are contaminating the surface of foodstuffs. examples are large outbreaks of hepatitis a from frozen berries or norovirus present on salad and vegetables [ •, ] . often, the people handling and processing food as well as contaminated water play a key role in transmitting viruses onto food [ ] . on the other hand, our foodstuffs originate from living organisms that carry their own set of viruses. hence, there is also a spectrum of viruses present within our food. the role of these viruses on our health is less studied and many viruses have only recently been detected by next generation sequencing (ngs). new diagnostic methods may improve detection of foodborne viruses dramatically, allowing for more efficient surveillance programmes and enhancing our knowledge on the importance of viruses in food [ •] . in contrast to traditional diagnostic methods, metagenomic approaches by ngs are untargeted and require no specific knowledge of the viral genome. for bacteria, conserved genetic markers such as s rrna allow for amplicon sequencing and subsequent taxonomic analysis resulting in an efficient representation of the bacterial abundance in a sample [ ] . since there are no conserved genetic markers present in different virus families, metagenomic analysis of viral populations is based on untargeted shotgun sequencing. basically, all nucleic acid present in a sample is sequenced concurrently and comparison of the resulting nucleotide or deduced protein sequence to existing databases allows identification of all viruses that are somewhat similar to existing database entries. thanks to ngs, the range of foodborne viruses detected on and in food is constantly increasing. however, a fully comprehensive overview is beyond the scope of this article. therefore, a selection had to be made. due to the lack of systematic surveillance on most foodborne viruses, it was not possible to use case numbers as selective parameter. therefore, the number of pubmed entries in the last years was taken as selective criterium (status on the th december ). the classics-viruses contaminating food (table ) norovirus of the articles matching to the search terms "foodborne" and "virus", comply with "foodborne" and "norovirus". undoubtedly, norovirus (nov) ranks top among the most important causes of acute gastroenteritis in humans worldwide. impressive annual numbers of up to million illnesses, , hospitalisation and , deaths that are accounted to nov in the usa alone show the importance of this virus for human public health [ •] . norovirus is a genus within the family of caliciviridae. virus particles are - nm in diameter and unenveloped which accounts for the high tenacity and resistance to disinfection. the genome is a single-stranded positive-sensed rna of . - . kb length and contains three open reading frames (orf) [ ] . propagation of nov in cell culture is notoriously difficult. successful cultivation requires for example the addition of enteric bacteria expressing strainsuitable histo-blood group antigen (hbga) [ ] or stem cellderived, nontransformed human intestinal enteroid monolayer cultures with addition of bile [ ] . due to these difficulties, the recently published iso standard method for nov in food is based on quantitative rt-pcr [ ] . beside of humans, several mammal species were shown to harbour noroviruses, such as pigs, cattle, rodents, dogs, cats, bats and even sea mammals such as harbour porpoises and sea lions [ ] . while nov are mostly species-specific, human noroviruses were detected in pigs and cattle in canada [ ] and some porcine nov isolates are genetically closely related to human nov [ ] . zoonotic transmission may hence be possible. however, due to the high contagiosity, the most important mode of transmission is directly from human-to-human or indirectly via the contaminated environment. particularly vulnerable are people on cruise ships, schools and other situations of dense clustering, even if hygiene standards are high. based on genotype profiles, only % of all nov outbreaks seem to be attributed to contaminated food [ ] . the most relevant source of food contamination with nov seems to be infected humans handling food rather than environmental contamination [ , ] . however, outbreaks due to contaminated fruit (e.g. raspberries and strawberries [ , ] ), leafy vegetables [ ] and particularly oysters [ ] show that the combination of faecally contaminated water and uncooked foods is particularly dangerous. thanks to molecular tracing of outbreaks and the acquired knowledge on sources and ways of transmission measures such as efficient disinfection and water treatment are increasingly taken to reduce the risk of foodborne nov infections [ ] . the second most frequently reported foodborne virus in pubmed is hepatitis a virus ( reports in the last years). however, in contrast to nov infection, hepatitis a (hav) is a vaccinable disease. hav is a member of the family picornaviridae and belongs to the genus hepatovirus. the virus particles come in two versions: naked, unenveloped icosahedral virions of nm diameter that are shed in faeces and pseudo-enveloped virions that are found in the blood of infected people and in cell cultures [ ] . the who estimates that hepatitis a virus (hav) accounted for million foodborne illnesses and . death in [ ••] . however, the prevalence and incidence vary significantly around the world. based on the level of anti-hav antibodies, endemicity is grouped into low (< %), intermediate ( - %) and high (> %). particularly high endemicity is observed in sub-saharan africa and india, while western europe, usa and australia have low levels [ ] . however, the disease burden in high endemicity regions is comparably low [ ] . this "paradox of hepatitis a risk" is due to the fact that in endemic areas children are infected at early age, e.g. in sub-saharan africa, % of the -year-olds are antibody positive [ ] but the infection in young children is often asymptomatic or atypical and triggers a robust immunity [ ] . in contrast, in regions with low endemicity, an increase in more severe clinical outbreaks can be observed due to a high degree of susceptible, non-vaccinated and older people. population movements and globalised markets play an important additional role [ ] . on the third place of the most frequently reported foodborne viruses is hepatitis e virus (hev) with publications. hev has gained a lot of attention in recent years. hepatitis e may clinically be indistinguishable from hepatitis a but hepatitis e virus (hev) is taxonomically unrelated to hav. hev is the most important member of the family hepeviridae and the genus orthohepevirus. interestingly, similar to hav, hepatitis e virus particles are unenveloped when excreted in faeces but pseudo-enveloped in blood and cell culture [ ] . hev viruses infecting humans belong to the species orthohepevirus a, whereas b, c and d infect birds, rodents and bats respectively [ ] . hepatitis e viruses of the orthohepevirus a species are further divided into the genotypes - . only the genotypes and are considered primary human pathogens that are transmitted by faecal-oral route. the genotypes and that are recognised as important zoonotic viruses are discussed in the second part of the review. the unenveloped viruses show high tenacity and can be detected in sewage and waste water [ ] . even though the main route of transmission is considered direct contact or faecal contamination of drinking water, hev- and strains may also be foodborne pathogens in developing countries with limited access to sanitation, hygiene and health systems. while the infections are usually self-limiting and often subclinical in immunocompetent people, these figures may rise to - % in pregnant women and immunocompromised individuals [ ] . insufficient immune response to the infection may lead to chronic disease and virus shedding [ ] . however, in western europe, infection with hev- or is usually considered an imported or travel disease [ ] . the fourth foodborne virus to be mentioned ( entries) is rotavirus. named after the wheel-like appearance they show on the electron microscope, rotaviruses (rv) are particularly dangerous for young animals and humans. the most important rv infecting mammals is the serogroup a. the who estimates that globally , children under years died in due to rotavirus infection; nearly half of them in india, nigeria, pakistan and the democratic republic of the congo [ ] . however, this figure was up to . in the year . a vaccine against rotavirus is on the market since and has significantly reduced the death rates due to rotavirus in children, e.g. by % in mexico [ ] . rotaviruses belongs to the family reoviridae and form an own genus. they are genetically highly diverse, forming up to eight serogroups [ ] . rva can be detected in many animal species and is an economically important pathogen in suckling pigs and calves but is usually species-specific. reports of zoonotic transmissions exist but the most important sources of human infection are humans [ , ] . foodborne transmission is mainly a problem in developing countries where also water contamination is frequent. in a recent surveillance study in columbia, % of analysed municipal drinking water samples were rvcontaminated [ ] . however, rv have also been detected in % of mussels in southern italy - [ ] . human adenoviruses (hadv), on the fifth place with reports, are often involved in gastroenteritis (ge) in children, where they are considered the second most frequent cause of ge after rotaviruses [ ] . however, they may also cause respiratory signs and eye infections. hadvs belong to the genus mastadenovirus within the family adenoviridae. to date, there are seven species of human adenoviruses known (human mastadenovirus a-g) (ictv th report ( )). the genus is non-segmented, linear double-stranded dna of - kb length and the approximately nm large virions are unenveloped, making them very resistant to environmental impacts [ ] . spread may be directly from person to person or via the faecal-oral route. contaminated recreational and irrigation water seems to be a frequent source of infection [ ] . contaminated water may also lead to contamination of food. for example, human adenoviruses were recently detected on fresh parsley using a metagenomic approach [ ] and are often contaminating shellfish [ ] . no zoonotic transmission has been reported as adenoviruses are usually speciesspecific [ ] . the last virus showing over ten pubmed entries related to "foodborne" in the last years is the human sapovirus ( publications) . belonging to the caliciviridae family (like nov) and forming an own genus, it is a small ( - nm) unenveloped rna virus. similar to nov, sapoviruses (sav) which were only detected in are highly resistant to unfavourable environmental conditions. currently, human and animal savs are classified into genogroups based on the vp protein [ ] . as hadvs, sapovirus outbreaks are often reported in kindergartens, nurseries and schools. the outbreaks are of sporadic nature and are reported world-wide but particularly many reports are from japan, where the virus was first studied and named after the city of origin of the first type strain: sapporo [ ] . transmission is mainly direct faecal-oral or via contaminated environmental surfaces. however, several reports show food as source of infection such as catered box lunches in a junior high school or food served at a wedding hall in japan [ ] . even though porcine sav are regularly detected in porcine faeces, transmission to humans and zoonotic outbreaks have not been reported to date [ ] . however, inter-genogroup recombination events of sav in pigs have been reported and highlight the high genetic diversity and evolution rate of sav [ ] . other potentially foodborne viruses that may cause disease in humans but have lower numbers of publications are human astroviruses (astroviridae), aichivirus/human kobuvirus (picornaviridae) and enteroviruses (picornaviridae) [ •] . astroviruses were even the most frequently detected viruses in mussels in southern italy [ ] . however, the clinical importance of these viruses is not completely clear, and literature is controversial. they show worldwide spread and are detected in faeces of healthy individuals and may hence be part of the normal flora of the gastrointestinal tract [ , ] . however, there are also reports of severe outbreaks of acute gastrointestinal disease, often in children under years [ , ] . enteroviruses in particular are frequently detected in environmental samples and waste water used for irrigation [ ] . it seems that most mammal species harbour their own astro-, entero-and kobuviruses [ , ] . while interspecies transmissions seem to occur [ , ] , their frequency and consequences for human health require further investigations. the "newbies"-viruses intrinsically present in food (table ) the vast majority of humans rely on plants as major source of food. metagenomic analysis of romaine and iceberg lettuce has shown the presence of wide range of plant and animal/ human viruses already on the field [ •]. it has also been shown that nov can be internalised by some plants such as lettuce and strawberries through the root system and can therefore be present inside the plant and fruits rather than just contaminating the surface [ , ] ; similar to the internalisation and retention of acid resistant viruses in water filtering molluscs [ ] . even though being internalised, nov, such as most animal and human viruses, is not able to infect or replicate in plant cells [ ] . however, there are numerous viruses specifically infecting plants. plant viruses are frequently detected in human and animal stool samples [ ] , but even though some virus families, like bunyaviridae, rhabdoviridae and reoviridae, contain plant-, animal-and human viruses, no plant virus is known to be a human pathogen [ ] . however, a report on the detection of antibodies against pepper mild mottle virus (pmmov) in patients with abdominal pains and virus positive stool samples points to interaction of the human immune system with this plant virus [ •] . furthermore, some plant viruses are known to infect and persist in insect-vectors and one of them, tomato spotted wilt virus, a member of the genus tospovirus of the bunyaviridae family, was even shown to replicate in human cell lines [ ] . due to the high genetic elasticity of their rna genome and the broad (plant-) host range, tospoviruses are indeed discussed to have the potential to cross the kingdom-barrier [ ] . in contrast to crossing the barrier between the plant and animal kingdom, it is far easier for viruses to cross species barriers. exposure to animal viruses, mainly of other mammals, represents therefore a greater risk for human health. since viruses are heat sensitive, raw or undercooked foodstuffs such as meat, milk and dairy products are the most likely sources for uptake of infectious animal viruses. only recently, the range of viruses intrinsically present in these products is being studied, mainly using ngs, and has revealed several "new" viruses. the most emerging zoonotic foodborne viruses in third world countries may well belong to the hepatitis e genotypes and . hev- and strains infect humans, but the reservoir is thought to be in several animal species, whereof the pig plays the most important role for foodborne transmission. hev- is distributed worldwide and represents the predominating genotype in europe while hev- is mainly reported from china, japan, india and indonesia [ •] but sporadic cases and virus detection have also occurred in italy [ ] , france [ ] and belgium [ ] . with seroprevalence rates of - % in many countries, hev seems to be endemic in piggeries and is also present in wild boar [ •] . in pigs, the virus causes only a subclinical disease with mild mostly microscopic lesions in the liver. virus is shed for - weeks in faeces and transmission between animals is via the faecal-oral route [ ] . the first foodborne transmission of hev was observed in japan where identical virus sequences were found in a diseased patient and in sika deer meat [ ] . additional reports, linking human cases to grilled wild boar meat in japan and pig meat in spain supported the initial finding. salines et al. ( ) [ ••] have summarised the reports of hev contaminations in meat products and found highest virus prevalence in pork liver pàtés ( % in canada and % in brazil) and drycured sausages containing pig liver such as the figatelli from corsica in france ( %). other sources of infection for humans are game meat (deer and probably rabbit) and food potentially contaminated by pig/wild boar faeces such as wild herbs [ ] . human-to-human transmission may occur but seems to be rare; however, blood transfusion has been reported as a transmission pathway [ ] . in humans, most infections seem to be subclinical; the most severe clinical sings are observed in older males and patients with pre-existing liver diseases or increased alcohol consumption [ , ] . a meta-analysis of studies on the hev seroprevalence in humans in europe from to showed a wide range from . to . %. contact to swine/wild animals had a significant influence as well as the type of assay used and the geographical area with highest mean values in france ( %) and lowest in italy ( . %) [ •] . the surveillance report of the european centre for disease control (ecdc) that includes data of eu/eea member states (accounting for > % of the total eu/eea population) provides data on confirmed cases of hev for - and shows an annual three-fold increase in the number of annual cases between and [ •] . however, hev- was already present in german wild boars as early as / [ ] and in india in [ ] as the analysis of archived samples has shown. therefore, it may well be that the disease and its zoonotic aspect has been largely overlooked for decades and acute cases regarded as hepatitis a. only recently, routine diagnostic tools are available and awareness in clinicians has grown. similar to havnet for hepatitis a, hevnet which was initiated aims at concerting and harmonising hev surveillance in europe [ •] and governs a hev sequencing repository for molecular tracing of outbreaks as well as a public hev online typing tool (http://www.rivm.nl/mpf/typingtool/ hev/). however, while research and surveillance of the disease in humans is increasing, many aspects of the virus life cycle and epidemiology in pigs are still unknown. due to the lack of clinical signs, hev has not been of veterinary importance so far. however, in order to fully understand and counteract the disease in humans, this gap needs to be closed. another animal product that may contain viruses is milk. while it is well known that several important bacterial diseases can be attained by consuming raw milk or dairy products such as brucellosis, tuberculosis and listeriosis, not so much is known about transmission of zoonotic viruses by milk. an exception is the confirmed transmission of tick-borne encephalitis virus (tbev) via goat milk leading to human infection [ , ] . a study in a high tbe risk region in poland showed . % of sheep milk positive for tbev by rt-pcr followed by goat milk ( . %) and cow milk ( . %) [ ] . importantly, even though tbev and other related tick-borne flaviviruses, being enveloped rna viruses, are not generally considered particularly resistant to environmental influences, they may survive low ph conditions such as present in the stomach. however, while langat virus, a close relative to tbev, survived conditions present during cheese production, it was completely inactivated by pasteurisation [ ] . raw milk cheese, particularly of sheep and goats may thus present a source of infection for the potentially lethal tbev and probably other tick-borne flaviviruses. while foodborne hev and tbev clearly represent a threat for human public health, the role of several other viruses of animal origin detected in food still needs to be assessed. zhang et al. ( ) [ ••] have used ngs to search for viruses present in beef, pork and chicken bought in supermarkets in san francisco. the muscle tissue they analysed was explicitly taken from the centre, not the surface of the meat pieces. interestingly, all viruses found were small, unenveloped dna viruses, mostly of the parvoviridae family. in pork, they found sequences of four different parvovirus species of three parvovirus genera. parvoviruses are among the smallest dna viruses (latin "parvus" = small) with a diameter of only - nm and are particularly resistant to disinfection and environmental conditions. they are frequently detected in faeces of diverse domesticated and wild animal species and humans, and may range from asymptomatic to highly virulent causing mainly enteric diseases [ ] . even though they are usually species-specific, interspecies transmission is occurring as in the case of canine parvovirus- that is thought to originate from cats [ ] . interestingly, porcine parvovirus was also detected in beef which may point to an interspecies transmission [ ••] . other dna viruses detected in beef and pork are torque teno viruses (ttv) of the anelloviridae family and members of the circoviridae family (e.g. porcine circovirus- in pork) [ ••] . for ttv alone, no clear association with disease, but a role in rendering other infections (e.g. by circoviruses) more virulent is discussed in pigs [ ] . however, newer studies have not observed a correlation of post-weaning multisystemic wasting syndrome (pmws) caused by porcine circovirus- and the presence of ttv [ ] . interestingly, human and animal ttvs are often detected in sera by ngs, even in healthy individuals [ , ] ; this may also be the reason why they are present in meat. porcine circovirus- (pcv- ) is endemic in pigs and associated with several clinical syndromes. it is also present in skeletal muscle tissue of infected pigs which was shown to be infectious for other pigs after oral uptake [ ] . no antibodies against pcv- were detected in humans so far but a certain risk for immunocompromised humans after receiving porcine xenotransplants is discussed [ ] . pcv- as well as parvovirus may also originate from live attenuated vaccines that are routinely used in piggeries [ ] . however, even though infectious pcv- was detected as a contaminant of a human rotavirus vaccine, no seroconversion was detected in the vaccinated children [ ] . in the analysed chicken, sequences matching to six different types of gyroviruses were detected [ ••] . they belong to the family of circoviridae but are structurally similar to anelloviridae and are small unenveloped viruses with a circular dna genome of about . kb. the most prevalent and world widely distributed representative of this genus is the chicken anaemia virus (cav) that infects and induces apoptosis in erythropoietic and lymphatic progenitor cells leading to anaemia and immunosuppression in young chicken. vaccines against this virus are routinely used in poultry farms [ ] . cav sequences have been detected in faeces of children and cats but due to the low copy numbers it is assumed that they are of dietary origin without infecting human cells [ ] . even though several gyroviruses with high similarity to cav were detected on the skin, in the blood and in faeces of immunocompromised humans, no antibody response against cav was observed [ ] . in contrast to gyrovirus, antibodies against an animal polyomavirus (bpyv) have been reported in humans. polyomaviridae are relatively small ( nm) unenveloped viruses with a circular, double-stranded dna genome of kb length and consist of the genera alpha-, beta-, gamma-, deltapolyomavirus. polyomaviruses have shown to persist in the organs of humans and many animal species, usually without causing overt clinical sings. however, in immunocompromised patients, an association with oncogenesis through transformation of infected cells has been proposed [ ] . since this transformation is due to integration of viral dna into the host genome-particularly in non-permissive infections-the relatively high seroprevalence against bovine polyomavirus (bpyv- ) in people handling cattle such as farmers, vets and abattoir workers was of some concern [ ] . based on epidemiological data, it was also speculated that bpyv in red meat might have an etiological role in the development of colorectal cancer in humans regularly consuming beef [ ] . furthermore, the presence of a novel bpyv- in beef was recently shown by metagenomic analysis [ ••] . however, while bpyv seems to be highly prevalent in cattle and may be transmitted to humans, no disease has yet been clearly attributable to the infection neither in cattle nor in man [ ] . another virus that is contradictorily discussed regarding its clinical meaning in humans after foodborne transmission is bovine leukaemia virus (blv). blv is endemic in cattle in many countries where it causes leukosis in - % of infected animals [ ] . its presence in meat and dairy products has been shown [ ] and buehring et al. ( ) [ ] found antibodies against blv capsid antigen in % of human serum samples using immunoblotting. there are also several reports claiming an association of breast or colon cancer with blv [ , ] . however, data from a recent study in china did not detect any blv genome or antibodies against blv in women with or without breast cancer even though the virus-and antibody prevalences of cows in the analysed regions were high [ ] . in addition, being a member of the retrovirus family, blv is highly susceptible to inactivation and it is unlikely to survive gastric conditions. however, more data are required to conclude on the foodborne zoonotic potential of blv. even viruses prone to inactivation by low gastric ph levels and proteases may cause foodborne infection if uptake occurs via a non-oral route. occupational groups like abattoir worker, vets and butchers may become infected thorough animal blood, body fluids and excretions via skin lesions, mucous membranes or by inhalation of aerosols [ •] . it is assumed that several important human viruses such as hiv, sars coronavirus and ebola were and still are crossing the species barrier not primarily through the consumption of meat but mainly during the butchering process of animals carrying the viruses such as primates and bats [ , ] . similarly, live poultry markets and culling of infected birds was shown to be a significant risk factor for human infection with h n influenza virus [ , ] . the classic foodborne pathogens such as nov, hav and rotavirus make up the majority of clinical cases in humans. these viruses are adapted to humans and are efficiently transmitted between humans, making it often difficult to untangle foodborne and direct human-to-human transmission. furthermore, in case of relatively "novel" foodborne viruses such as astroviridae or sapoviruses, it is often difficult to differentiate between "commensal" viruses, opportunistic pathogens and real threats for human public health. ngs has made it possible to non-specifically screen food for viruses and has shown that many intrinsic plant and animal viruses are also present in food products. the finding of several types of circo-and parvoviruses in beef, pork and poultry shows that also pure muscle tissue of apparently healthy animals harbours a surprising range of viruses [ ••] . the high tenacity of these small unenveloped dna viruses means that they can also be present in processed meat. this is supported by own observations, where ngs sequencing reads obtained from a dry cured pork sausage covered not only the whole genome of hev [ ] but also of porcine circo-and parvoviruses as well a plant virus (pepino mosaic virus) in high depth (unpublished data). it is not sure if these viruses remained infectious in the sausage. however, the hev sequence was identical to the virus isolated from a patient with acute hepatitis who consumed this type of sausage [ , ] . hence, new diagnostic tools have massively enhanced the possibility to detect foodborne viruses but subsequent virological, clinical and epidemiological studies are necessary to determine the importance of these findings for human health. however, increasing globalisation of the food trade facilitates introduction of new or "exotic" viruses and demands for broad range surveillance tool on an international scale. therefore, establishment of suitable protocols for non-specific screening of foods using ngs and endeavours towards concerted molecular tracing such as hav-and hevnet are essential tools for the control of foodborne viruses in the future. conflict of interest the author declares that she has no conflict of interest. human and animal rights and informed consent this article does not contain any studies with human or animal subjects performed by any of the authors. microbiology by numbers the presence of nuclear polyhedrosis viruses of trichoplusia ni on cabbage from the market shelf world health organization global estimates and regional 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transmission and spread of ebolavirus origins of hiv and the aids pandemic circulation of avian influenza h n in live bird markets in egypt avian influenza virus (h n ): a threat to human health complete genome sequences of two swiss hepatitis e virus isolates from human stool and raw pork sausage new subcluster of hev genotype strains linked to the first confrmed swiss case of foodborne hepatitis e infection key: cord- -uftc inx authors: nan title: abstract of th regional congress of the isbt date: - - journal: vox sang doi: . /vox. sha: doc_id: cord_uid: uftc inx nan in the fin de siecle was heavily concentrated in vienna. freud, boltzmann, schr€ odinger and mach might be the first names to find, whenever one cites austrian scientists. but more related to transfusion are the noble prize winners max perutz and karl landsteiner. landsteiner s fate illustrates the brain drain beginning in the early s escalating in with the "anschluss", which lead to the forced emigration of many scientists. a loss which was not regenerated in the post war years and was further aggravated by dubious and often undisclosed relations and scandals in the nazi-era. all together this leads to a severe loss of credibility and productivity of universities across decades. opening university access in the early s and intensive historical work-up of scandals transformed the austrian universities to open and effective scientific institutions driving innovation in the country. austria has achieved a great economic deal in recent decades, which was accelerated by the eu membership in . as a result of strong long-term economic performance, the country's gross domestic product (gdp) per capita is the eighth highest among oecd countries and fourth in the eu . levels of poverty and income inequality are both below the oecd average. investment in research and development (r&d) increased since the eu accession, when austria's r&d intensity (aggregate r&d expenditure as a percentage of gdp) was well below the oecd average and significantly far lower than switzerland -a country to which austria prefers comparison. the eu target of % r&d intensity was first met in and is the sixth highest among oecd countries and the second highest in the eu . austria showed the second highest increase in r&d intensity of all oecd countries, exceeded only by korea. the rapid expansion was matched by a similar increase in human resources and scientific output of universities. austrian science in quantum mechanics, quantum communication and information is world renown. vienna is a major biotech hub, as is linz in mathematics and mechatronics and graz in automotive and production technologies. austria has been a net resource recipient in the horizon and the preceding th framework programme. small and mediumsized enterprises show a high propensity to co-operate with universities and other research organisations and more and more included in scientific grant schemes. vienna is the largest student city in the german-speaking world and consistently ranks among the top cities in the world on quality-of-life indices. as austria possesses globally recognised cultural attractions ranging from famed salzburg festival to the vienna new year concert its inhabitants are not aware of the progress made in r&d and how thriving innovation is going on in their country. they still love to show their cultural heritage and events and impress the world with some kind of eternal sound of music. patients with refractory b-cell malignancies as non-hodgkin lymphomas (nhl) resistant to standard therapies have a dismal prognosis. the outcome is even poorer in patients relapsing after autologous stem cell transplantation. most of these patients do not qualify for an allogeneic hematopoietic cell transplantation (hct) due to refractory disease, lack of a suitable allogeneic donor, higher age or cumulative toxicity of previous chemotherapy. despite patients undergoing allogeneic hct normally profit from a graft-versus -lymphoma effect, overall survival in patients with nhl after hct remains short. a similar situation can be observed for patients with acute lymphoblastic leukemia (all). therefore novel treatment modalities are urgently needed. chimeric antigen receptor (car)-t cells, a new class of cellular immunotherapy involving ex vivo genetic modification of t cells to incorporate an engineered car have been used in clinical trials. in the majority of studies b-cell malignancies treated with cd targeting car-t cells have been analyzed. austria had the advantage to participate in two international trials in the past and is currently involved in further car-t studies. recently, results from cd directed car-t cell trials with an increased follow-up of patients led to fda (food and drug administration) and ema (european medicines agency) approval of tisagenlecleucel and axicabtagene ciloleucel. common adverse events (aes) include cytokine release syndrome and neurological toxicity, which may require admission to an intensive care unit, b cell aplasia and hemophagocytic lymphohistiocytosis. these aes are manageable when treated by an appropriately trained team following established algorithm. in this presentation, results of four large phase ii cd car-t cell trials for patients with nhl and all and focus on aes is summarized. preoperative anemia is a known risk factor for increased perioperative morbidity and mortality in patients undergoing major surgery. previous studies have not only shown higher in-hospital mortality, but also an increased hospital length of stay, greater postoperative admission rates to intensive care and prolonged use of mechanical ventilation and intensive care resources in patients with anemia compared to those with normal preoperative hemoglobin concentrations. about % of patients scheduled for major surgery suffer from preoperative anemia. this figure is even higher in patients requiring orthotopic liver transplantation, where up to % of all patients are diagnosed with anemia prior to surgery. transfusion of packed red blood cells (prbcs) is commonly used to correct anemic hemoglobin values. however, transfusion of prbcs has been associated with increased morbidity and mortality in patients undergoing cardiac, orthopedic, and abdominal surgery. additionally, transfusion of prbcs is associated with a greater incidence of postoperative acute kidney injury in patients undergoing orthotopic liver transplantation. as preoperative anemia might increase the perioperative use of prbcs, negative effects observed after prbc transfusions might even be augmented. data on the influence of preoperative anemia on morbidity and mortality after orthotopic liver transplantation are limited. thus, we retrospectively analyzed the association of preoperative anemia and mortality in adult patients undergoing orthotopic liver transplantation at our institution. in addition, we examined the influence of anemia on perioperative parameters such as transfusion requirements, surgical complications, early allograft dysfunction, acute kidney injury, and the need for renal replacement therapy. based on the results obtained in the retrospective analysis, an ongoing prospective randomized clinical trial was initiated. the two suspensive treatments in sickle cell disease (scd) are hydroxycarbamide, inducing the production of the functional hbf, normally repressed at birth, and red blood cell (rbc) transfusion, a critical component of scd management. however, rbc transfusion is not without risk. repeat exposure to allogeneic rbcs can result in the development of rbc alloantibodies which can make it difficult to find compatible rbcs for future transfusions. however, the main concern of alloimmunization is the development of hemolytic transfusion reaction, with in the most severe cases, hyperhemolysis, leading to multi organ failure and death in % of the cases. the prevention of this life threatening condition must be based on risk factors. however, although some risk factors, such as alloimmunization, have been identified, much of the mechanism underlying dhtr remains a mystery, particularly in severe cases presenting hyperhemolysis. here we will describe the current and future development to prevent and treat this severe syndrome in order to decrease exposure to transfusion in scd but also improve red blood cell quality, some new products are developed. oxidative damage is one of the parameter that could be diminished. some work is also ongoing to prevent filter blockage during leucodepletion of precious rbcs units from afro-caribbean donors carrying the sickle cell trait. finally, in countries with higher risks of transmission of infectious disease, treatment of red blood cell units against infectious agents can be discussed. the only current curative treatment of scd is hematopoietic stem cell transplantation (hcs). however, the occurrence, frequency, and effects of immune hematologic complications in hcs remain and will be discussed. finally, gene therapy is a real hope as a definitive curative treatment. clinical trials are ongoing in france and will be discussed as well as the remaining place of transfusion in this therapeutic. in the context of the chronic myeloid leukemia (cml), we have hypothesized that quiescent leukemic hematopoietic stem cells (hsc) compartment, escaping to the current tyrosine kinase inhibitors (tkis) treatment, in part associated in the molecular relapse, may be targeted by cart-cells immunotherapy. gene expression profiling studies have established that a cell surface biomarker il- rap is expressed by the leukemic but not by the normal cd + /cd -hsc. this talk will focus of the whole process of development of a cart-cells starting from recombinant il- rap protein mice immunization to produce a specific monoclonal antibody (mab), to the proof of concept demonstration, before moving into the clinic. we produced and selected a specific anti-il- rap mab (#a c clone, diaclone sa, besanc ßon, france). after molecular characterization of antigen-binding domain, nucleotide sequences were fused with rd generation t cell activation coding sequences and cloned as a single chain into a lentiviral backbone comprising a safety switch suicide gene icasp (inducible caspase ) and a monitoring/selection cell surface marker Δcd . we demonstrated in-vitro and in an in-vivo xenograft murine model that il- rap car t cells can be activated in the presence of il- rap+ cell lines or primary cml cells, secrete pro-inflammatory cytokines, degranulate and specifically killing them. we also demonstrated that multi-tkis treatment over a -year period does not affect transduction efficiency of cml patient t-cells by il- rap car vector and that autologous cart-cells are able to target il- rap+ leukemic primary hsc. "off-tumor-on target" toxicity prediction, by studying il- rap expression on a tissue macroarray comprising normal human tissues ( donors) , with #a c , detected various il- rap intensity staining in only few tissues. regarding the healthy hematopoietic system, #a c flow cytometry staining did not detect hematopoietic cells, except monocytes that express poorly il- rap. as expected, monocyte subpopulation is targeted by autologous il- rap cart cells (ratio e: t = : ), but at a lower level that il- rap cml cell line. in-vivo investigation of specific toxicities of autologous il- rap cart-cells against hsc and/or immune cells on a human-cd + cord blood cell engrafted/nog murine model, but also by an in-vitro cd + colony forming unit assay didn't reveal any significant toxicities in immunocompetent cell subpopulations, suggesting that healthy cd + hsc are not affected. finally, to overcome potential toxicity, functionality of the icasp / rimiducid â safety switch was demonstrated in-vitro but also in-vivo in a nsg tumor xenograft model, showing that, when activate, the system is able to eliminate more than % of cart-cells, after exposure to ap . in conclusion, based on cml model, we demonstrated that il- rap is an interesting target for cart-cell immunotherapy, with a limited "on target, off tumor" predictable toxicity. next step will be the up-scaling of the process in order to match with the use in human regarding also the regulatory requirements. this strategy may be applied, in the future, in other hematological malignancies. mortality ranges from to % for trauma victims with severe bleeding and is largely dependent on the transfusion therapy from which they can benefit. the nature of this therapy has an impact on prognosis with a halving of mortality when the plasma/prbc ratio is greater than ½ and a decrease of about % when the proportion of platelets transfused is close to that of whole blood. the speed with which such therapy is actually administered has a major impact as well with an increase in mortality of % for each minute of delay in making the entire therapy available. this can be explained mainly by the fact that the probability of death of these patients is greater within minutes of their admission to hospital with a median time to death of h after admission. to allow plasma, platelets and prbc to be made available in a timely manner, north american trauma centers have mandated that trauma centers have massive transfusion packs at the patient's bedside within min. to further simplify and speed up logistics from distribution to transfusion, several trauma centers now use whole blood stored at °c. this return of an "old" product is largely inspired by military experience where whole blood is mainly used "warm" immediately after collection with compelling evidence of its effectiveness. its return to civilian practice requires the ability to deleukocyte it while preserving platelets and to store it while maintaining their hemostatic functions. good quality data shows this is achievable and several clinical studies are planned to begin in the coming months. in france, the french blood establishment and the french army are cooperating to initiate the prospective randomized non-inferiority storhm trial (sang total pour la r eanimation des h emorragies massives) which will be comparing whole blood to separate blood components in an / / ratio in severely bleeding trauma patients. the primary endpoint will be a thromboelastographic parameter (maximum amplitude) assessed at the th hour after admission. secondary endpoints will include early and overall mortality, lactate clearance (reflection of the effectiveness of resuscitation) and h organ failure. this trial will be recruiting patients in french trauma centers and is planned to be initiated second half of . local/neighbours day: innovation in germany c- - mesenchymal stromal cells for regenerative therapy bm-msc / asc obtained from these protocols have been characterized in detail in preclinical evaluations. manufacturing licenses for msc and asc and a platelet-derived growth factor concentrate have been obtained and they have been explored in several clinical trials for treatment of bone defects (ortho-ct : eudract number: - - ; ortho-ct : eudract number: - - ; maxillo : eudract number: - - ) . we will summarize results of completed clinical trials which confirmed feasibility and safety of autologous msc /asc treatment and provided evidence for efficacy (gjerde et al, stem cell res. introduction: in vitro produced megakaryocytes (mks) may serve as source to produce platelets (plts) ex vivo or in vivo. we have established a strategy to differentiate mks from induced pluripotent stem cells (ipscs) in bioreactors. this study aimed at the large-scale production of mks using microcarriers to increase the mk yield and to characterize their phenotype and functionality after irradiation as a method to decrease possible safety concerns associated to the ipsc origin. methods: ipscs were cultured in an aggregate form in presence or absence of microcarriers using ml stirred flasks. cells were differentiated into mks using tpo, scf and il- in apel medium for a period of days. non-irradiated or irradiated ipsc-derived mks were analysed for polyploidy, phenotype and proplt production using flow cytometry and fluorescence microscopy. also, plt-production was investigated in vivo. non-irradiated or irradiated mks were transfused to nod/ scid/il- rcc-/-mice and blood was analyzed for human plts. results: differentiation of mks in presence of microcarriers resulted in an -fold increase of mks per ipsc in comparison to only aggregates. this resulted in mean of total mk harvest of . ae . in microcarrier-assisted bioreactors in comparison to . ae . mks collected from bioreactors containing only aggregates. interestingly, mks produced in microcarrier-assisted bioreactors showed higher proplt formation capacity than mks derived from only aggregates bioreactors. mk phenotype and dna content was comparable between mks derived from both types of bioreactors. irradiation of mks did not affect their phenotype and capability to form proplts or plts after transfusion into nod/scid/il- rcc -/mice. conclusion: microcarriers showed to significantly increase the yield of ipsc-derived mks in stirred bioreactors to clinically relevant numbers. this may facilitate the use of ipsc-derived mk for ex vivo production of plts, direct transfusion or for innovative mk-based regenerative therapies. although the rosetta stone, found by the troops of napoleon in egypt near the city of rosetta (rashid) contains only a small amount of text in three languages it was key in deciphering hieroglyphs. the rosetta mission tried to achieve something similar: by looking at a tiny body its goal was to decipher the origin of the solar system, planets including earth and life. after more than years the rosetta spacecraft softly crashlanded on comet churyumov-gerasimenko on september , . it has travelled billions of kilometers, just to study a small ( km diameter), black boulder named p/ churyumov-gerasimenko. the results of this mission now seem to fully justify the time and money spent in the last decades on this endeavor. where are we from? where are we going? are we alone in the universe? these are some of the big questions. in this talk i will show which answers we got from rosetta and comet chury. we follow the pathway of the material which makes up our solar system from a dark cloud to the solar nebula and finally to planets and life. i will show that indeed we are the result of stardust and that what happened here may happen elsewhere in the universe. cells, tissues and entire organs can collectively be seen as "living drugs". genetically unaltered cells are routinely used in clinical practice to treat diseases as diverse as anemia, bleeding disorders, leukemia and organ failure. ground-breaking advances in genetic and genome engineering technologies are propelling cell therapies to the frontline of medical research and practice. the hematopoietic system is particularly amenable to genetic engineering because specific cell types can be purified based on the expression of specific surface proteins and the ability to culture and expand cells ex vivo. the recent unprecedented clinical success of killer t cells reprogrammed by chimeric antigen receptors (cars) to attack cd expressing tumor cells demonstrates the power of immunotherapy with genetically engineered immune cells. however, given the rapid development of novel genome engineering and synthetic biology tools we are likely only at the beginning of a new era of engineered cellular therapies. i will present recent progress in immune cell reprogramming, gene correction, safety aspects and remaining challenges such as manufacturing. d- - cell free nucleic acids (cfna) circulate in the plasma of all individuals and are thought to be released by host and foreign cells into the circulation. after fractionation by centrifugation, cfnas can be extracted from the supernatant of whole blood samples or manufactured blood products. these dna or rna sequences can be of human, bacterial, viral or fungal origin. most of them are human double stranded dnas. research on cfnas is increasing, thanks to technological advancements in molecular biology. some of their results are already implemented in clinical practice in the areas of prenatal diagnosis, oncology and infectious diseases. the latter investigation focuses on the exploration of non-human cfnas, the field of metagenomics. high throughput sequencing associated with bioinformatics, the so-called new generation sequencing (ngs), has sped up the investigations of non-human cfnas. this tool provides the opportunity to classify cfnas into a human or non-human category, and then to identify them. it is thus possible to explore simultaneously the whole landscape of bacterial, viral and fungal populations. presently, ngs of human blood has already proven its feasibility and its value in identifying emerging viruses or investigating clinical cases of fever of unknown etiology. ngs of cfnas is also particularly effective in analyzing the different genotypes of a virus in case of a co-infection (e.g. hepatitis c virus). studying cfnas with the new molecular technologies is therefore of great importance in transfusion medicine, especially regarding security and clinical transfusion reactions. first, transfusion transmitted infections are the most feared adverse complications. second, febrile non-hemolytic transfusion reactions are also the most frequently reported adverse events in hemovigilance systems and their physiological mechanism -if only one-remains not clearly elucidated. investigating cfnas could thus improve our understanding and strategy aiming at reducing those two clinical adverse events. surveying comprehensively the composition of circulating infectious agents in a blood product by ngs technology could be very interesting for investigating a severe febrile transfusion reaction. moreover, when the costs of analysis will be reduced, it might be possible to screen prospectively and regularly the whole metagenomics of asymptomatic blood donors, in addition to the classical epidemiological surveillance. for instance, in a study testing a ngs method on manufactured fresh frozen plasmas, an astrovirus (mlb ) has been identified. finally, it is the responsibility of transfusion physicians implicated in the manufacturing of blood products to ensure that cfnas within a blood product do not have a clinical impact on the innate immunity of the recipients. according to recent research in vitro, cfnas purified from blood products can induce the transcription of inflammatory cytokines by mononuclear cells. as nonhuman cfna have an effect on toll-like receptors (tlr-linked inflammatory pathways), it would be also relevant to insure that donor's cfnas have no significant effect on the immune system of the recipient. in conclusion, cfnas are very diverse molecules contaminating blood products. technological progress makes now their investigation more available. besides being useful markers of infection in asymptomatic donors, their impact on the recipients' immunity should be further investigated. an active life and regular training is part of a healthy life style and for many this includes participation in endurance exercise competition at different levels. thus, it is highly relevant to know how a blood donation affects exercise performance and how close this can done to an endurance competition. endurance exercise performance is determined by many factors, but three of the primary are maximal oxygen uptake, the relative load that an individual can sustain over time and finally the efficiency of movement in the given discipline. over the years, a number of studies have sampled blood volumes ranging from - ml and applied different methodological approaches to measure maximal oxygen uptake over a recovery period ranging between - days. overall, the general finding is a reduction in blood haemoglobin and an attenuated maximal oxygen uptake as well endurance performance after blood donation. in normal to well-trained men maximal oxygen uptake and performance was normalized after two weeks in one study after a normal blood donation ( / ml), but remained attenuated after four weeks in another study, despite the change in blood haemoglobin concentration was similar and the design and methodology also similar in the two studies. in addition to maximal oxygen uptake the relative load that can be tolerated during exercise is probably also attenuated, through a decreased arterio-venous oxygen extraction, but the available data is very limited. the first part of this talk will highlight the major findings and discuss some of the methodological issues that complicate interpretation and conclusions. there are sex differences in circulating blood volume, haemoglobin concentration, haematocrit and hormone levels and thus it is entirely possible that there is a sex difference in the effect of blood donation on physical performance and the recovery after blood donation. in addition to the basic physiological sex differences, there is also a higher prevalence of iron deficiency in premenopausal women, physically active women and women donating blood. therefore, we studied the influence of a standard ml blood donation on maximal oxygen uptake and endurance performance and the subsequent recovery in physically active women. we observed that in iron sufficient women blood haemoglobin concentration and maximal oxygen uptake were back to baseline days after blood donation, but endurance performance was normalized already after days. the second part of this talk will discuss the sex differences in the effect of blood donation on maximal oxygen uptake and endurance performance. overall, the available data suggest that, with a careful conservative approach, - weeks are needed after a normal blood donation to be fully recovered to participate in endurance exercise competitions. more than one in ten attempts to donate blood result in a temporary deferral, due to concerns about the impact of the donation on the donor or recipient. there is well established evidence that temporary deferrals impact negatively on donors, with a large proportion of those deferred failing to return at the end of the deferral period. this presentation provides an overview of deferrals from the donor perspective, describing the likelihood of receiving a deferral for different donor subgroups. the impact of temporary deferrals on the future donation of donors, considering both short-term and longer-term donation patterns, will also be reviewed, outlining which donors are at highest risk of non-return following a deferral, and what is known about the accumulative impact of multiple deferrals on donors. several hypotheses have been proposed to account for the strong negative impact of deferrals on donor behaviour, and there is preliminary evidence of psychological factors, such as emotional reactions, predicting intention to return. research is also beginning to emerge on the effectiveness of tailored interventions to mitigate the impact of deferrals on donor behaviour. the evidence for these preventative interventions, and for strategies to reactive donors once they have lapsed post-deferral, will be reviewed. recommendations for blood centres will be made, as well as suggestions for future research to address continuing gaps in knowledge. in his influential study "the gift relationship" ( ) , richard titmuss coined the idea of voluntary, non-paid, blood donation being the gift of life for a fellow citizen. this metaphor has been powerful in mobilizing donors (busby ) . it conveys a direct relationship between blood donation and patients' vitality, as well as a difference between gains and costs. as the gift of life, blood donation is seen to symbolize pure altruism and promoting solidarity between strangers. but can we apply the metaphor as successfully into donating blood for research? we asked a group of finnish blood donors what they would think if the frc blood service invited them to give a blood sample and personal information for research. the blood donors were usually willing to contribute to research for the public benefit, because they saw great potential in science to create solutions to help patients in the future. however, based on our interview data and previous research, we suggest that the analogy between gift of life and donation for research did not work all the way. the metaphor fails to address donors' questions on new types of relationships, interests and risks related to the use of personal data for research. left unanswered these could discourage donating for research. hence, we argue that the gift of life metaphor is not applicable to donor recruitment at the research context. in this presentation we wish to look for a better metaphor for donation for research that blood services collecting research data could apply. academy day: transfusion challenges in patients with sickle cell disease a- - immunohaematological features of patients with sickle cell disease (lfa) should be considered as polymorphic antigens in the african population and these lfa are not present in most commercial panels. the situation is even more complicated when recipients lack high-frequency antigens, the most common ones being hr-, hr b -, sec-, u neg , u+ var , js(b-), (hy-), and jo(a-). finally, there is a high rh diversity among people of african descent. because they harbor variant alleles and/or partial rh antigens, they are at risk of developing alloantibodies. in this setting, screening for partial rh antigens makes sense. the figures illustrating this diversity vary with the approach used. one of them is to take into consideration rhd or rhce*ce variant alleles. in several american studies, their prevalence was estimated to be - % and - %, respectively. other teams take into consideration d, c and e partial antigens. their prevalence was estimated to be . - %, . - . %, . - . %, respectively, and the alloimmunization rates were . %, . - %, . %, respectively. as a result of these phenotype discrepancies, scd patients are more likely to be alloimmunized. an overall immunization rate of - % is commonly admitted in the general population. depending on the unit selection policy and/or the study design, the immunization rate in scd patients varies from % to %, the highest figures being established when an abo/rh -only matching policy is implemented. in a meta-analysis of publications, the overall alloimmunization rates were around %. alloimmunization is thought to be enhanced by an inflammatory state, which is often present in scd patients. they are more prone to develop a new alloantibody. using a stochastic modeling of alloimmunization, they have a % increased risk of producing additional antibodies versus % in the general population. autoantibodies have been identified as a risk factor of alloimmunization. as a result, scd patients often have complex mixtures of allo and autoantibodies. rh antibodies and those considered as irregular natural antibodies are present in a significant proportion. another characteristic of the antibodies in scd patients is their evanescence; up to % of alloantibodies become undetectable within a few years of their initial development. relatedly, about a third of dhtrs are reported to happen in patients with no previous history of immunization. in addition, a third of patients will not develop an antibody after a dhtr. identifying patients at risk of developing a dhtr is key to managing them properly. alloimmunization is a serious risk of red blood cell transfusion in patients with sickle cell disease (scd) and can result in severe (delayed) haemolytic transfusion reactions, exacerbation of clinical symptoms and life-threatening hyperhaemolysis. once alloimmunized, the presence of alloantibodies in the patients' blood further complicates pretransfusion testing and hampers the selection of compatible blood products. numerous studies have shown that scd patients have a relatively high risk of alloimmunization as compared to the 'general' population. this is not only explained by the large number of transfusions given but also by the increased exposure to foreign antigens as a result of differences in the antigen make-up of the scd recipients and the blood donor population. other factors involved in the immune response such as age at first transfusion, inflammatory state, hla typing are under investigation and are starting to unravel. because blood transfusion is still one of the main treatment modalities for scd and some patients have a life-long transfusion dependency it is important to minimize the alloimmunization rate. theoretically, complete matching for all relevant blood group antigens would prevent alloimmunization. this however, is only possible when all donors are comprehensively. matching strategies should be developed to minimize alloimmunization while balancing patients' need and donor availability and is cost effective. to develop a (preventive) matching strategy some factors need to be established; ) which antibody specificities are clinically relevant ) which antigens are most immunogenic ) what is the availability of specific antigen typings in the donor population ) how should recipients (and donors) be typed, phenotypically and/or genotypically and to what extent. the latter is especially important in scd patients since they are of african descent and the prevalence of genetic variations in this population is relatively high. rhd and rhce variants are common and can remain undetected when serological typing is used but can be discovered with high resolution molecular typing. patients with partial rh phenotypes are at risk for alloimmunization. apart from special rh phenotypes in individuals from african descent, the fy(a-b-) phenotype related to the gata-box mutation in the fyb allele and the u-or u var phenotype resulting from genetic variations in the mns alleles are also common. several studies have shown that in scd patients antibodies directed against rhd, rhe, rhc and k are most frequently found when unmatched transfusions are given. preventive matching for these antigens has proven successful in reducing alloimmunisation. extended matching for all rh antigens fy(a), jk(a) and jk(b) can further decrease the alloimmunisation rate. currently, different countries have preventive matching strategies in place for this vulnerable patient group. as genotyping is more and more available and within reach, optimal antigen typing approaches for patients and donors, combining serology and genetics are being developed. in this lecture several aspects of antigen typing approaches and preventive matching strategies that will most benefit scd patients of will be discussed. artificial intelligence has become a buzzword that will appear about anywhere in the media. we can forget that ai, or the subfield in this computer science field machine learning, has been around for over years. improvements in computing power, abundance of data, progress in computer science, and the arrival of affordable cloud solutions have now brought it to our daily lives. also in health care news about ai has become omnipresent. and some landmark papers have come out on algorithms outperforming (teams of) physicians in the diagnosis of all kinds of skin disease, eye disease from retina scans, and detect cancer in ct scans. however, little of these solutions have actually shown up in our clinical practice yet. in anaesthesia, we worked with the first algorithm to come to anaesthesia practice; hypotension during surgery is associated strongly with poor outcome like myocardial ischemia, surgical complications, renal failure and even mortality. we worked on a machine-learning trained algorithm that predicts hypotensive events using the arterial blood pressure curve up to - min before the actual event. to get fda and ce approval, however, mere mathematic validation is required. this can be achieved on retrospective datasets. in reality, we need more before we can use these algorithms to support our decision-making; after internal (retrospective) and external (prospective but passive use) validation steps, clinical (i.e. rct validation is needed. moreover, we will need to assess the economic impact too. ultimately this tool has now reached clinical practice and is starting to help us go from reactive to more proactive hemodynamic management. like this, we have started to work on machine-learning tools to predict the incidence of specific types of patients coming into a&e and predicting infections after surgery. we will discuss our approach, essentials to start with machine learning, practical learnings. we will also discuss a first project design to use machine learning in managing bleeding patients to get the best therapy advice for blood product use like plasma, fibrinogen et cetera. how can we start using this tool in unison with our existing tools to improve science and clinical practice in our respective (bio)medical fields? thrombocytopenia is a very common hematological abnormality found in newborns, especially in preterm neonates. two subgroups can be distinguished: early thrombocytopenia, occurring within the first h of life, and late thrombocytopenia, occurring after the first h of life. early thrombocytopenia is associated with intrauterine growth restriction, whereas late thrombocytopenia is caused mainly by sepsis and necrotizing enterocolitis. platelet transfusions are the hallmark of the treatment of neonatal thrombocytopenia. most of these transfusions are prophylactic, which means they are given in the absence of bleeding. however, the efficacy of these transfusions in preventing bleeding has never been proven. in addition, risks of platelet transfusion seem to be more pronounced in preterm neonates. because of lack of data, platelet transfusion guidelines differ widely between countries. in a recent randomized controlled trial (planet- /matisse study) among preterm infants with severe thrombocytopenia, we found that those randomly assigned to receive platelet transfusions at a platelet-count threshold of /l had a significantly higher rate of death or major bleeding within days after randomization than those who received platelet transfusions at a platelet-count threshold of /l. this presentation summarizes the current understanding of etiology and management of neonatal thrombocytopenia. transfusion-associated circulatory overload (taco) is a severe transfusion adverse reaction that is associated with increased mortality and morbidity. the incidence of taco in adults varies from % to %, but is probably underdiagnosed and underreported. the incidence in the pediatric population is undetermined. taco usually occurs in patients who receive a large volume of blood product over a short period of time. it is more common in patients with known risk factors such as cardiovascular disease, renal failure, and older or younger age (> years or < years). hospitalised patients and intensive care patients are also more at risk. the typical presentation of taco is respiratory distress (dyspnea, tachypnea) occurring within h of a blood transfusion. associated signs and symptoms are hypoxia, hypertension, tachycardia, positive fluid balance, high central venous pressure, and acute or worsening pulmonary edema on chest x-ray. echocardiography and measurement of brain natriuretic peptide (bnp) or its n-terminal prohormone (nt-probnp) is helpful for diagnosis. several definition criteria have been proposed for taco, but none are adapted for children, particularly critically-ill children who are more at risk. this is probably the main reason why taco is even more underdiagnosed and underreported in the pediatric population. in a recent study, we compared the incidence of taco in a pediatric intensive care unit using the international society of blood transfusion (isbt) criteria, with two different ways of defining abnormal values: ) using normal pediatric values published in the nelson textbook of pediatrics; and ) using patients as their own controls and comparing pre-and post-transfusion values with either a % or % difference threshold. we monitored for taco up to h post-transfusion. a total of patients were included. taco incidence varied from . % to %, depending on the definition used. with such wide variability, we conclude that a more operational definition of taco is needed in pediatrics, particularly for critically-ill children. differential diagnosis from other dyspnea-associated transfusion adverse reactions (e.g. transfusion-associated lung injury, anaphylaxis) is important because treatment differs, as do guidelines to the blood bank. treatment for taco is similar to that of any other cardiogenic pulmonary edema: oxygen, diuresis, ventilatory support. prevention is possible by avoiding unnecessary transfusions, transfusing only the necessary amount of blood product, avoiding rapid transfusions, and using diuretics. background: the risk and importance of transfusion-transmitted hepatitis e virus (tt-hev) infections by contaminated blood is currently a controversial discussed topic in transfusion medicine. in particular, the infectious dose is a not finally determined quantity. the different countries have chosen different approaches to deal with this pathogen. one central question is the need of individual nat screening (id) versus minipool nat screening (mp) approaches to identify all relevant viremias in blood donors. aims: comparison and evaluation of the available screening strategies in relation to the infectious dose to minimize the risk of tt-hev infections. methods: we systematically reviewed the presently known cases of tt-hev infections and available routine nat-screening assays. furthermore, blood donation screening strategies for hev ehev in effect in the european union were compared. we also describe our own experiences of hev screening utilising an id-nat-based donor screening algorithm compared to mp-nat in pools of samples. from november to january , a total of , blood donations were screened for the presence of hev rna using a mp-nat (in house, realstar hev rt-pcr kit) and an id-nat (cobas platform). results: the review of the literature revealed a significant variation regarding the infectious dose causing hepatitis e. in the systematic case review, all components with a viral load (vl) greater than . e+ iu caused infection (definitive infectious dose (difd) . the lowest infectious dose resulting in tt-hev infection observed in general was . e+ iu (minimal infectious dose (mifd). the infectious dose of the different blood products is mainly influenced by the remaining plasma content. our data comparing the two different hev screening algorithms revealed eight hev rna positive donations using a mp-nat (incidence : , ) , whereas hev rna positive donations were identified by id-nat (incidence : ); all id-nat only positive donations had vl < iu/ml. summary/conclusions: taken into account the current knowledge on the required mifd, the difd, and the analytical sensitivities of the screening methods, we extrapolated the detection probability of hev-rna positive blood donors using different test strategies (nat assay, id vs. minipool with different pool sizes). we also considered the amount of plasma in the different blood products and calculated the infectious doses needed to be detected. only id testing would be sufficient to detect the minimum vl in the donor to avoid tt-hev infections based on the currently known mifd, but a highly sensitive mp-nat should be adequate as a routine screening assay to identify high viremic donors and avoid tt-hev infections based on the difd. we have also determined that the incidence of hev infection was approximately % higher if id-nat was used. however, vl were below iu/ml and will most likely not result in tt-hev infection taken into account the currently known mifd or difd. the clinical relevance of and need of identification of these low level hev positive donors still require further investigation. in the last years several pathogen inactivation (pi) technologies have been developed to be applied to blood components. technologies for inactivating pathogens in plasma and platelets are available in the european union, and some others are currently under development. the first pi technology introduced in the market was for plasma, and was based on the addition of methylene blue and the illumination with light (theraflex mb-plasma, macopharma and gr ıfols). for platelets and plasma two technologies are licensed, one is based on the addition of amotosalen and the illumination with ultraviolet light (uv) (intercept â , cerus) and the other one combines the addition of riboflavin (vitamin b ) and the illumination with uv light (mirasol â , terumo bct). currently another technology for platelet inactivation, based on the illumination with uvc light and strong agitation is under development (theraflex, macopharma). for red blood cells one technology based on the addition of one molecule (amustaline, cerus) is being developed. the mechanism of action, and the spectrum and level of inactivation of pathogens varies among the different technologies. in addition, the number of studies with clinically relevant endpoints and the number of patients included in the studies is not homogeneous. there is published evidence for most of them that show that the treated blood components are safe and efficacious for the patients although, for treated platelet concentrates some decrease in the posttransfusion recovery and survival of the transfused platelets occur, with differences between the different technologies. however, cumulative experience on the use in routine, for some of the technologies for almost years, support the concept of the safety and efficacy of the blood components treated with pathogen inactivation technologies without a significant increase in utilization. the use of pathogen inactivation for blood components is not widespread. differences in epidemiology between countries, infectious risk perception, concerns about potential adverse effects associated with its use and economical considerations might explain the differences observed in its implementation. the history of the p and p k antigens is complicated and sometimes confusing because of several changes to the nomenclature. the association between the antigens and their genetic home has raised many questions as well as the longstanding enigma regarding the molecular mechanism underlying the common p and p phenotypes. the system (isbt no. ) currently includes three different antigens, p , p k and nor. the p antigen was discovered already in by landsteiner and levine while p k and nor were described in and , respectively. as for the abo system, naturally-occurring antibodies of igm and/or igg classes can be formed against the missing p /p k carbohydrate structures. anti-p is usually a weak and cold-reactive antibody very rarely implicated in hemolytic transfusion reaction (htr) or hemolytic disease of the fetus and newborn (hdfn). however, some antibodies against p have been reported to react at °c, bind complement and cause both immediate and delayed htrs. the p k antibodies can cause htr and anti-nor is regarded as a polyagglutinin with unknown clinical significance. a higher frequency of miscarriage is seen in women with the rare phenotypes p and p k /p k . the rbc of the fetus as well as of the newborn express low amounts of p , p and p k antigens but the placenta shows high expression and is consequently a possible target of the antibodies and the cause of the miscarriages. the p k and p antigens have wide tissue distributions and can act as host receptors for various pathogens and toxins. furthermore, altered expression of p k antigen has been described in several cancer forms. a longstanding question has been why individuals with p phenotype not only lack p k and p expression but also p . recently it was clarified that the same a galtencoded galactosyltransferase synthesizes both the p , p k and nor antigens and in addition the p and p phenotypes was confirmed to be caused by transcriptional regulation. transcription factors bind selectively to the p allele in the '-regulatory region of a galt, which enhance transcription of the gene. it has been debated whether the p k and p antigens exist on glycoproteins in the human rbc membrane or if glycolipids are the only membrane components carrying these epitopes. a recent publication shows that the p antigen can be detected on human rbc glycoproteins and thus glycosphingolipids can no longer be considered as the sole carriers of the antigens. the blood group system which started out with one antigen, p , has now gained two more members namely p k and nor. step by step the biochemical and genetic basis underlying the antigens expressed in this system has been revealed but still many questions remain to be solved. neither gata nor klf represent a blood group system but mutations in the genes encoding these transcription factors (tfs) have been shown to result in simultaneous altered expression of blood group antigens in certain rare blood group phenotypes. in particular, mutations in the klf gene are responsible for the dominantly inherited in(lu) phenotype, commonly referred to as lu(a-b-) because of the gross reduction in lutheran antigens expression. red cells from in(lu) individuals, though, have also weakened expression of other blood group antigens, like the high-incidence antigen anwj, the antigens of the indian blood group system (cd ) and p , among others. since the first description of klf variants associated with the in(lu) phenotype, many other variants of this gene have been reported with an impact on blood group antigen expression and they are listed on the klf table of blood group alleles. other than klf , a mutated gata gene has also been found associated with the x-linked form of the lutheran-mod phenotype and has likewise been registered in the gata allele table. besides the effect of tf variants on blood group antigen expression, there are transcription factor-binding site polymorphisms in regulatory regions of blood group genes, which also have an impact on the expression of the encoded antigens in red cells. the first example of such type of polymorphisms was described in , when the disruption of a gata motif in the ackr gene promoter was found to abolish erythroid gene expression in fy(a-b-) individuals of african descent. the impact of mutations affecting gata binding sites has also been described in some abo subgroups, like the am and bm phenotypes. a regulatory element with gata binding sites in the first intron of the abo gene has been found to be altered in individuals with these phenotypes, either by deletion or by a point mutation disrupting the gata motif. recent findings have also revealed that xga expression on red cells is dependent on gata binding to a control element located . kb upstream of the xg gene. a single nucleotide polymorphism (snp) within this region was shown to correlate very well with the expected distribution of the xga negative phenotype in different populations. further work has demonstrated that this g>c snp disrupts a gata binding site and consequently abolishes erythrocyte xg expression. overall, these investigations have allowed to elucidate the underlying genetic basis for xga expression and have made xga genotyping possible. similar to xga, the p antigen has been known for a long time to be determined by the a galt gene but the molecular basis underlying the common p /p phenotypes has remained elusive till recently. several cis-regulatory snps had been identified in non-coding sequences around exon a, which showed a very good correlation with p antigen expression. interestingly, potential binding sites for several hematopoietic tfs were identified in the same region. finally, recent investigations have demonstrated the role of the runx tf in the expression of p antigen, by selective binding to a regulatory site present in p but not in p alleles. to summarize, variation in blood group antigen expression may result from mutations or polymorphisms in the regulatory region of blood group genes. recent reports have unravelled the molecular mechanisms underlying the expression of p and xga blood group antigens, which involves tf binding to allele-specific regulatory elements. similar mechanisms may also regulate antigen expression in other blood group systems. c- - clinical immunology, copenhagen university hospital, copenhagen, denmark since the discovery of cell-free fetal dna (cffdna) in pregnant women's blood, the development of noninvasive prenatal testing (nipt) has provided new diagnostic applications in prenatal care. in transfusion medicine and clinical immunology, cffdna is extracted from maternal plasma to predict fetal blood groups with the purpose of ) guiding targeted rh prophylaxis in non-immunized rhd negative women and ) assessing the risk of hemolytic disease of the fetus and newborn (hdfn) in immunized women. i will give an overview of noninvasive prenatal testing of fetal blood groups. based on the literature, i will summarize the current experience with noninvasive prenatal testing of fetal rhd and other blood groups. for rhd negative pregnant women, routine clinical testing is available in several countries world-wide to assess the risk of hdfn in d immunized women, and routine testing to guide rh prophylaxis is now implemented as nationwide service in - european countries. noninvasive prenatal testing for fetal rhd is highly accurate with sensitivities of . %, as reported from clinical programs. in general, the sensitivity is challenged be low quantities of cffdna, especially in early pregnancy. the specificity is challenged by the polymorphic rh blood group system, where careful attention is needed to navigate among the many rhd variants. rhd variants may complicate cffdna analysis and interpretation of results, especially in populations with mixed ethnicities. despite these challenges, fetal rhd testing is very feasible when implemented with careful attention to these issues. for blood groups that are determined by snps, such as kel or rhc, the main challenge has been interference from the maternal dna when analyzing the fetal dna which has resulted in low accuracy and lower sensitivity, when using qpcr. with the application of more novel techniques such as next generation sequencing and droplet digital pcr, accurate noninvasive prediction of these fetal blood groups has been demonstrated. the success of predicting fetal rhd and its successful clinical implementation into national programs should encourage wide-spread use of cell-free dna based analysis. future work on noninvasive prenatal testing of fetal blood groups determined by snps may consolidate the application for cell-free dna testing for such targets, including human platelet antigens. at isbt, the newly formed cfdna subgroup of the red cell immunogenetics and blood group terminology working party will work to facilitate clinical applications, implementation and evaluation of cell-free dna testing. blood banks in most of the nordic countries all share a vein-to-vein approach which in short means that the collection of blood, the preparation of blood components, testing/release and storing is served by a single actor. on top of that recipient and donor blood grouping, crossmatching, delivery, registration of transfusion and of any complications is usually handled in a single blood banking information system (bbis). this means that blood banks in the nordics are traditionally operated by a single vendor. the needs for process control in a single vendor bbis, the present solutions, unsolved challenges and untapped possibilities of streamlining processes have been scrutinized with the intention to describe separate processes and to acquire best of breed or best of suite it-systems. the aim for integrations, rather than building an integrated it-system, to support the need for a vein-to-vein process is a precondition in the nordic countries. with multiple it-systems supporting isolated processes, we intend to facilitate development in these and furthermore increase the flexibility in the whole process. we set out to reveal any existing knowledge in the literature on it vendor strategies for blood banks, but we didn't succeed in identifying any relevant literature. however, a systematic literature study on vendor strategies when choosing health it was based on the prisma method, and identified studies, but only was eligible for full text review and met the inclusion criteria. even this broader literature study reveals very little evidence. two studies find single vendor strategies poor and conclude "best of suite" solutions to be optimal. one study was not able to correlate vendor strategies to the investigated productivity, but concludes that best of suite and best of breed strategies requires larger organizational changes than a single vendor strategy. in summary, the existing research is contradictory. this paper adds basic knowledge for breaking down the process control of blood banking in smaller processes. this adds the possibility for identifying best of breed or best of suite vendors, instead of relying on single vendor it solutions. furthermore it is a call for more research in the field of vendor selection strategies which this study didn't succeed in identifying. d- - applying drones to supply blood to remote areas: rwanda's experience biomedical services, rwanda-ministry of health-national blood services, kigali, rwanda background: in rwanda, blood transfusion services started in . during the genocide against the tutsis almost all the socioeconomic fabric of rwanda was destroyed as well as its health infrastructure. the healthcare system was suffering in its aftermath, and there were health inequalities between urban and rural areas, including access to blood for transfusion. from , the government started to rebuild all courses of life including the health system and the blood service in particular. the national center for blood transfusion (ncbt) was then mandated to provide safe, effective and adequate blood and blood components to all patients in need. this was pivotal in achieving health related mdgs , and . today, rwanda has an ambitious vision to put all million citizens within minutes of any essential medical product. while every second matters in emergency management, the use of drones was the perfect solution to many of the last mile challenges that have been traditionally difficult to overcome. it is impossible to forecast accurately down to the need of a single patient. the government has provided an easy solution by centralizing supply and providing on-demand, emergency medical deliveries by drone. doctors are now empowered to provide the quality care with all the supplies on hand, patients can now be treated close to home, and we eliminate waste from potential overstocking when health workers know that they have a quick and reliable source of supply. description: in , the government of rwanda started to operate the world's first national drone delivery program for blood and other lifesaving medical products. these drones can carry two to six units of blood at a time and deliver in - minutes depending on a hospital's location. the average duration was between - hours round trip with the vehicle system, before the use of drones. drones currently deliver blood to health facilities throughout the country and are set to reach % of transfusing health facilities outside kigali by the end of the year. within the first year, healthcare workers saved an average of . hours per delivery and a total of , hours of lost time on road pick up they could instead dedicate to patient care. by march , over , deliveries have been made, with % of those being emergency deliveries. a total of more than , blood units have been delivered. in february , zipline obtained the highest rating from the health facilities being served in a performance evaluation conducted by the national center for blood transfusion. when a doctor or medical staffer needs blood, they place an order through the hemovigilance order portal. they are then sent a confirmation message saying a drone is on its way. the drone flies to the health facility at up to km/h. when it is within five minutes of the destination, the medical staffer receives a notification. the drone then drops the package, attached to a parachute, into a special drop zone. conclusion: supply is not a developing country problem, it is a global issue. rwanda was just the first one to recognize the potential of this technology and decided to do something about it first and fast, to ensure access to universal access to all blood products. d- - scottish national blood transfusion service, edinburgh, united kingdom supporting the provision of viable transfusion services in remote/rural areas is more than just a geographical challenge. limited qualified blood bank resource; small throughput volumes; increased regulation are only three of the additional factors combining to threaten safe and sustainable transfusion service delivery. inventory management and out of hours service provision were identified as essential areas where it was thought that technology, in the form of a remotely controlled blood fridge, could provide a key element of the overall solution. a radio frequency identification (rfid) fridge racking system was installed in a standard blood bank fridge and connected to the laboratory information management system (lims) common to both the remote and central blood bank. the central blood bank was enabled to test patient samples from the remote laboratory, identify components located in the remote fridge suitable for the patient and allow correct component issue, even when qualified staff were unavailable in the remote laboratory. testing has concluded that installation of remote fridge management can play a major role in helping to maintain a remote inventory permitting patient compatible components to be issued. by sustaining transfusion services in remote communities we can avoid transportation of patients who require transfusion support to locations miles from home. antibodies to hna- b, fcgriiib and hna- have been reported, too. neonatal alloimmune neutropenia results from maternal antibodies transferred transplacentally to the fetus and is caused by all known hna-antibody specificities, i.e. hna- a, - b, - c, - d, hna- , hna- a, - b, hna- a, - b and hna- a specificity. hna and hla antibodies can induce mild febrile transfusion reactions and trali. since the introduction of the male only plasma strategy, in many countries the trali incidence decreased but it is still one of the most common causes of severe transfusion reactions. especially hna- a antibody containing plasma from female donors is responsible for severe or even fatal reactions. but also hna- a, - b, hna- and hla class i and class ii antibodies were reported. the latter activate monocytes to secrete soluble factors that act on the primed neutrophils in the narrow lung capillaries. laboratory testing: laboratory work-up requires the knowledge of the patient's clinical condition and the methods that are appropriate to detect the relevant antibodies. the classical granulocyte agglutination test (gat) in combination with the granulocyte indirect immunofluorescence test (gift) can detect nearly all relevant antibodies. hna- , - , - , - and hla class i antibodies are clearly detectable in the gift while hna- antibodies strongly agglutinate neutrophils in the gat. the monoclonal antibody-specific immobilization of granulocyte antigens (maiga) test detects all hnaantibodies except for hna- with high glycoprotein specificity and sensitivity but is time consuming and requires highly skilled personnel. for trali diagnostics laboratory testing is completed by methods like the indirect lymphocyte immunofluorescence test (lift) or elisa for hla class i antibodies and hla class ii specific elisas. since several years fluorescent bead based assays (luminex) enable faster and more automated hna antibody detection but to date not all specificities, especially hna- , can be reliably detected so that still the classical gift and gat have to complete the methodological spectrum. serological typing today is mostly reduced to the determination of hna- in the gift because the molecular reason for the hna- -null phenotype is not completely understood. establishing only one pcr-asp reaction for the main cd * a>t polymorphism would comprise the risk to miss other molecular causes. however, for all other hna allelotyping by pcr methods is the first choice. summary/conclusions: granulocyte serology still today is widely based on a variety of manual methods and will be reserved to specialized laboratories as it requires experienced laboratory staff and profound knowledge of granulocyte immunobiology. d- - norwegian national unit of platelet immunology, laboratory medicine, university hospital of north norway, tromso, norway maternal alloantibodies against antigens on human platelets can cause severe thrombocytopenia and bleeding in fetus or newborn, identified as fetal/neonatal alloimmune thrombocytopenia (fnait) . although most cases the thrombocytopenia is selfresolving within the two first weeks of life, some infants present bleeding symptoms and thus require platelet transfusion. a set of laboratory analyses are required to confirm the fnait diagnosis. in addition to guiding compatible platelets to the affected newborn, the correct diagnosis will be valuable to assess the risk of fnait in subsequent pregnancies. in addition, platelet alloantibodies may also complicate platelet transfusions by immune-mediated platelet refractoriness, and require proper identification of the patient's antibody specificities prior to selection of donor platelets. the algorithm for laboratory investigations include both serological and molecular assays, and depend on the objective and timing: whether there is an urgent need for platelet transfusion, follow-up of a pregnancy with known risk, or to do full-scale laboratory testing to confirm diagnosis. molecular genotyping should include all hpa systems relevant for the local population (in caucasians hpa- , - , - , - and - ), preferably with optional extended panels for systems for low frequency populations due to immigration/mobility and for less frequently seen alloantibodies (hpa- , - to - are most commonly included). serological testing of antibody binding to platelets is often initially tested by flow cytometry analysis (direct test and/or cross-match). however, the detection of platelet-specific antibodies is often complicated by the presence of anti-hla class i antibodies and thus require sensitive platelet glycoprotein-specific assays. serological testing for platelet-specific antibodies includes as a minimum panels of antigens on gpiib/iiia, gpib/ix, gpia/iia and cd and preferably additional targets for populations with asian/african origin. several methods are available; i.e. bead-based assays and elisa based methods. however, most reference laboratories perform variants of the monoclonal antibody immobilization of platelet antigens assay (maipa), as reported by the th international platelet immunology workshop of isbt ( ) . the investigations also include measurement of the anti-hpa- a by quantitative maipa if present, as this is reported to potentially predict the severity of fnait. for pregnancies with known risk of fnait, there are methods available to perform non-invasive prenatal typing from maternal plasma. the most feasible and so far appropriate for routine testing is fetal hpa- typing with quantitative pcr or by melting curve analysis. other sophisticated, yet resource-demanding techniques have also recently been reported -importantly also for typing of other hpa-systems. d- - molecular basis of hna- expression justus liebig university, institute for clinical immunology and transfusion medicine, giessen, germany human neutrophil antigen (hna- ) is a neutrophil-specific antigen located on gpi-anchored glycoprotein cd (also known as nb ). hna- is absent on the neutrophil surface of - % of the healthy individuals that divided the population to hna- positive and hna- null individuals. exposure of hna- null individuals to hna- positive neutrophils during pregnancy, after transfusion or transplantation, induces immunization against hna- and consequently the production of iso-antibodies. the hna- iso-antibodies are involved in the mechanism of neonatal alloimmune neutropenia (nain), transfusion-related acute lung injury (trali) and graft failure following bone marrow transplantation. presence of cd on a neutrophil surface of hna- positive individuals follows a bimodal expression that categorizes the circulating neutrophils to hna- positive and negative subsets. the cd gene contains exons encoding a protein of amino acids. the lack of hna- (in hna- null individuals) is associated with the presence of a missense mutation, cd *c. a>t in exon of cd gene inducing a premature stop codon in codon . this mutation alone or in combination with cd *c. delg has been introduced as the main reason for the absence of cd in hna- null individuals. a pseudogene (cd p ) highly homologous to exons - of cd gene is located downstream of the cd gene. conversion of exon of cd p into cd gene is responsible for the generation of cd *c. a>t missense mutation. in addition, the heterozygosity or homozygosity of cd *c. a>t is accounted for regulation of hna- negative and positive neutrophils subpopulations. genotyping has revealed the hna- null individuals, heterozygous for cd *c. a>t mutation without cd *c. delg, indicating the presence of a complementary mechanism regulating cd expression. newly in hna- null individuals and individuals with atypical cd expression a cd * g>a polymorphism in combination with cd * a>t is described. altogether these data indicate a complex compound mechanism(s) for regulation of cd expression on the neutrophil surface. this presentation will summarize recent findings on cd expression and highlights the potential genotyping methods for genetic assessment cd expression of donors and patients. blood products ordered, transfusion start and end times, whether patients experienced a reaction to the transfusion as well as vitals measurements taken before and during the transfusion, including temperature, oxygen level, blood pressure and heart rate. for the validation process, transfusion nursing notes were sampled and reviewers assessed the accuracy of the information regarding ) blood product ordered, ) whether the patient experienced a reaction, and ) the start and end times of the transfusion. for each of these fields across all sampled notes, the claritynlp tool reproduced these data points with percent accuracy. in addition, the tool supplied transfusion end times for numerous structured records that were missing this key data point. summary/conclusions: claritynlp can very efficiently digest a large number of transfusion nursing notes simultaneously and also does an excellent job of extracting the main characteristics of a transfusion, which can be used in partnership with structured data to produce a more accurate and more complete picture of patient transfusions. immunohematology and genomics, new york blood center, new york, united states antibody-based typing, with a positive result reflected in agglutination of the red cells (rbcs), has served the profession for nearly a century enabling safe and effective transfusion therapy. the power of rbc typing by serologic methods lies in the availability of standardized antibody reagents which target many of the specificities of significance for transfusion, and the ability to directly detect antigen expression on the rbcs. hemagglutination has historically been relatively inexpensive, particularly for abo and rhd as the most important blood groups in most populations. serologic rbc typing is reliable, requires no sophisticated equipment, is generally straightforward to perform, and is fast requiring less than h to results. hence, antibody-based testing has been considered the "gold standard" for blood group typing. with the age of genomics, dna-based genotyping is increasingly being used as an alternative to antibody-based methods. most antigens are associated with single nucleotide changes (snps) in the respective genes. genotyping has been validated by comparison with antibody-based typing and has been shown to be highly correlated. the power of genotyping of rbcs lies in the ability to test for antigens for which there are no serologic reagents, and to type numerous antigens in a single assay using automated dna-arrays. this increases accuracy and weak antigen expression can be revealed. fresh rbc samples are not required for dna extraction, and there is no interference from transfused rbcs or igg bound to the patient's rbcs. dnabased typing is economical in that it provides much more information, but testing requires special equipment, training, and -h turn around. what then is the best approach to use? will serologic typing be replaced by dnabased typing? indeed, genotyping will increasingly be used in the practice of transfusion medicine, especially with the growth of whole genome sequencing (wgs). however, because serologic typing for abo and rhd is fast and accurate and often relied on for sample identification, genotyping will not be the sole means for routine typing. genomic sequencing approaches will certainly reveal unrecognized changes and genetic variability in rbc membrane proteins, but not all variation will be immunogenic. a genetic polymorphism must be associated with antibody production to be considered a blood group antigen. the importance of an antibody, and antibody reactivity, then will continue to be the central defining principal in transfusion. as two sides of a coin, both are key to safe and effective transfusion therapy. since the mid- s, research in the molecular basis of structural and functional aspects of proteins carrying and producing the antigens has led to an upgraded and modern understanding of blood group variation. most commonly, single nucleotide polymorphism (snp) based basic molecular typing techniques were utilized to test new findings on a smaller subset of samples, and resulted in concordant sero-and genotyping results in general. however, quite commonly a small number of all samples delivered discrepant results, triggering consecutive rounds of analysis and resolution, finally resulting in a better knowledge with respect to the underlying blood group variation. such rounds of repetitions represented the synergistic incremental process of learning, learning for serologists and molecular biologists. inheritance of public, presence of high frequency, or low frequency, or partial antigens and notice of weakly expressed, or almost undetectable antigens marked the path of incremental learning and may best be exemplified by discoveries within the blood group systems abo, rhd and kell. naming for pheno-and genotypes coevolved alongside the permanent discovery of new antigens. at present, antigens and their antithetic counterparts (if tested and if existent), are more commonly reported independently, as exemplarily shown for the following kell phenotype consisting of three antithetic antigen-couples: kk, kp (a+b+), js(a+b+). alternatively, the same phenotype could be stated as: kel:- , , , , ( ), , . genotypes, on the other hand, rather mirror the actual biological background, e.g. display the two parental alleles (or "haplotypes") present in an individuals' sample. genotype of the above mentioned example would read: kel* . / . (italicized). in an idealized diagnostic environment and for most blood group systems encoded by proteins, every single blood group allele would be defined by its full genomic sequence, derived from one parental chromosome (including "some" 'and '-untranslated regions). thereby, every such "ideal allele" would fully declare presence or absence of all its public, low-and high-frequency antigens and possess its "ideal name". by trend, biallelic snps and their immediate relation to antithetic antigen couples might have distracted from the originally intended meaning of "blood group alleles", more recently. finally, genotypes only dependent on (ideal) allele names, and considering mendelian inheritance patterns (dominant, recessive), would allow for fully comprehensive phenotype predictions. more recently, blood group serology, e.g. the "second side of the coin", seems to gain momentum. since the advent of whole genome sequencing and access to many more than human genomes, it seems that dozens of new blood group alleles are discovered, almost on a daily basis. beside the challenge of naming this multitude of alleles, respective discoveries are frequently made in samples lacking any phenotypic blood group pre-values. clear procedures will be needed to address naming and analyzing the phenotypes resulting from previously unknown alleles. as a consequence, questions asked years ago have changed: today, molecular biologists looking at hundreds of newly discovered blood group alleles find themselves not being asked by serologists any more: "can you confirm my serology?", but instead, pose their question to the experts for the blood group phenotype: "can you confirm my genotype?" a-s - background: the screening of blood donors and returning travelers from active transmission areas have highlighted the importance of diagnosis of acute arboviral infections. in the context of co-infections and similar clinical signs in endemic zones, the differential diagnosis of arboviruses is essential to discriminate the causative agent. the detection of viral nucleic acids in serum or plasma provides a definitive diagnosis, however, in most instances, viremia is transient within less than two weeks after the onset of clinical illness. in addition, the cross reactivity due to the high degree of structural and sequence homology between zikv and other flaviviruses is a significant concern. the combination of molecular (identification of viral genomes) and immunological assays (detection of the immune response) is a key challenge to follow the natural history of these infections and to improve the patient management and the epidemiological surveillance. aims: in this context, we have developed an innovative platform based on agglutination of superparamagnetic nanoparticles (npmag) covalently grafted either with nucleic or proteic probes to face the continuing emergence of arboviruses. methods: dengue (denv) and zika (zikv) viruses are selected as models in this study. a pan-flavivirus rt-pcr is used for the molecular assay to amplify the viral genomes. then, biotinylated viral amplicons are captured specifically on complementary original polythiolated probes coated on npmag. for the immunological assay, npmag are grafted with viral ns proteins to capture anti-denv or anti-zikv antibodies potentially present in the plasma samples. both tests are performed in disposable cuvettes in a homogeneous format. a magnetic field generated by an electromagnet is applied to the reaction medium to align the npmag into chains to enhance the capture of the targets between two npmag. aggregates formed are detected when the field is turned off. the optical density is measured in real-time at nm during several cycles of magnetization / relaxation. results: in this study, molecular analytical performances were evaluated on human samples from blood donors with no history of infections as negative controls, on viral standards and on clinical samples. using viral references standards, we have observed sensitivities of - tcid /ml for zikv and denv (serotypes / / / ) after a detection phase of around min. the first results obtained on zikv (+) clinical samples previously tested by commercial real-time pcr (ct < , altona) showed an % correlation between the two detection methods. no false positive results or cross reactions were observed. concerning immunological assays, commercial human plasma from donors tested positive for denv or zikv antibodies were detected positive with our innovative approach in less than min (sampling + detection) instead of h with classical elisas. further assays on clinical samples are planned to confirm these preliminary results. summary/conclusions: this innovative strategy combining molecular and immunoassays on the same analytical platform offers new opportunities for rapid blood testing to improve the surveillance and the prevention of arboviral infections. background: zika virus (zikv) caused a dramatic epidemic in puerto rico (pr) during , with up to~ % of blood donors reactive for zikv rna in id-nat testing at the peak in june . aims: perform a serosurvey for anti-zikv igg using six panels of donor specimens each collected in march , at the beginning, peak and end of the epidemic, and from march and april . methods: we employed a commercially available zikv igg elisa antibody (ab) assay based on the zikv ns antigen from bio-techne to characterize zikv seroprevalence in the cross-sectional sample sets (anonymized with selected demographic information). results: pr donor samples collected in april were initially evaluated using the manufacturer supplied cut-off to confirm that the zikv ab results were largely negative ( positive, equivocal) despite the high dengue virus seroprevalence (> %) in pr that could potentially lead to false positive zikv ab results. we then used this dataset, together with known positives collected - months postdetection from zikv nat yield donors, to set a population-specific cut-off based on receiver operating characteristic (roc) curve analysis. this cut-off yielded sensitivity and specificity values of > %, and an area under the curve (auc) of . , demonstrating a highly accurate assay. we used this new cut-off to calculate final rates of seroreactivity in the additional sample sets ( samples) and estimate seasonal incidence. rates of reactivity, together with mean net od for only the reactives (shown in parentheses), were calculated for each sample set: background: sex hormone intake in blood donors occurs in three demographic groups: premenopausal women who take contraceptive drugs (progestins with or without estrogens), postmenopausal women who receive estrogen replacement therapies that may be combined with progestins, and testosterone therapies in men. we hypothesized that sex hormone therapies may modulate the quality of red blood cell (rbc) products via alterations of rbc function and predisposition to hemolysis during cold storage. aims: the objectives of this study were to evaluate the association between sex hormone intake and rbc measurements of hemolysis, and to examine possible mechanisms by which sex hormones interact with rbcs. methods: self-reported sex hormone intake and menstrual status were evaluated in , female blood donors from the national heart, lung and blood institute's rbc-omics study. the associations between hormone intake and donor scores of spontaneous storage, osmotic, or oxidative hemolysis were determined in all women and by menstrual state. the interactions between sex hormones and rbcs were determined by sex hormone (progesterone, b-estradiol, or testosterone) potency to inhibit calcium influx or hemolysis during incubations or cold storage. the calcium fluorophore, fluo- am, was used to define rbc calcium influx in response to treatments with sex hormones or drugs that modulate transient receptor potential cation (trpc) channel activity including hyp (a selective trpc activator). results: sex hormone intake by menstrual status was higher in premenopausal women ( . %) than in postmenopausal women ( . %). female hormone intake was significantly (all p < . ) associated with reduced storage hemolysis in all females ( . ae . % versus . ae . % in controls), enhanced susceptibility to oxidative hemolysis ( . ae . % versus . ae . % in controls), and reduced osmotic hemolysis in postmenopausal women ( . ae . % versus . ae . % in controls). in vitro, supraphysiological levels of progesterone ( or lmol/l), but not b-estradiol or testosterone, inhibited spontaneous or hyp -induced calcium influx into rbcs, and were associated with lower spontaneous hemolysis after day cold storage ( . ae . % versus . ae . %, progesterone lmol/l versus solvent control (dimethyl sulfoxide, . %), p < . ). co-incubations ( . h, °c) of rbcs in the presence of progesterone and a trpc activator (hyp , lmol/l) suggested that progesterone protected against hyp -induced hemolysis ( . ae . % and . ae . % versus . ae . %; hyp + progesterone at or lmol/l versus hyp alone, p < . by one-way anova). summary/conclusions: this study revealed that sex hormone intake in blood donors is capable of modulating rbc predisposition to hemolysis and led us to propose new mechanistic pathways by which progesterone regulates calcium influx and hemolysis in human rbcs. pre-and postmenopausal women respond differently to hormone intake and its effects on rbc responses to osmotic or oxidative stress. progesterone modulates calcium influx into rbcs via a mechanism that may involve interactions with membrane trpc channels, activation of which is associated with pre-hemolytic events such as senescence and eryptosis. a-s - international cooperation, swiss red cross, wabern, switzerland background: red cross and red crescent societies were playing an important role in setting up blood transfusion establishments in many low resource countries. by the mid- s, the red cross was active in the national blood programs in approximately % of countriesmostly in blood donor recruitment and education. today, major organizational developments in blood transfusion services were made in high income settings, where nearly % of all worldwide donations take place (home to only % of the population). who data shows that the median annual blood donation rate in high-income countries is . % of the population compared to . % in low-income countries. the factors for this low turnout are multilayered, but it is well-known that most resource poor settings suffer from a low rate of regular donors and challenges to set-up and financially sustain a national blood donor program. the red cross and red crescent (rc) societies assume a key role by reaching and retaining donors from the communities and contribute significantly to better safety and availability of blood. partnerships and international collaboration, such as the swiss red cross (src) program, are aiming to strengthen national structures to improve blood safety and to face today's epidemiological, demographical, and technological challenges. aims: the present work aims to review the role, the mandate and the impact of rc societies in improving blood safety through systematic "voluntary non-remunerated regular blood donor" (vnrbd) programming and international partnerships. methods: data and evidence is drawn from the src international cooperation projects over the last years, more specifically partnering with three rc societies, and the data from the global advisory panel (gap) of the ifrc including their global mapping. results: the promotion of vnrbd has been a specific objective in all src supported programs. through the engagement of the rc society, the training of volunteers and partnering with the health authorities, the projects significantly increased the blood donor rate by recruiting and retaining donors from the communities. for example, the rc societies increased total donations by % in lebanon; vnrbds by % annually in kirgizstan, and from practically zero to ' in south sudan. the importance of rc societies was also underlined in the published global mapping of gap, which showed that ( %) of them provide level a (full blood service), ( %) are level b (systematic blood donor recruitment) and ( %) are level c (vnrbd blood promotion) blood services. gap has also commenced a new three year vnrbd support program aimed at establishing tools and materials for national societies. summary/conclusions: the red cross / red crescent movement has a unique mandate and position in improving global blood safety at all levels; with its huge network of volunteers, even blood donors in remote communities can be reached and retained. rc societies in low resource settings with a level b or level c role should further capitalize on partnerships with local and international actors to leverage technical assistance and funding for their activities. background: it is essential to motivate and encourage the public to donate blood and be eager to help saving lives, in order to maintain safe and adequate national blood supply. aims: "technical assistance for recruitment of future blood donors (europeaid/ /d/ser/tr)" project aimed to avoid problems in supplying the safest blood to contribute to the improvement of public health by (i) increasing the knowledge of primary and secondary school students regarding blood donation, (ii) creating sensitivity in school principals and teachers regarding voluntary non-remunerated blood donation (vnrd), (iii) motivating family members of the students for blood donation and (iv) creating public awareness through media. methods: an effective coordination is established between ministry of health (moh), ministry of national education (mone) and turkish red crescent (trc). the existing curricula and textbooks of primary and secondary schools were reviewed and revised, and corresponding materials on the importance of blood donation were created. the human resources capacity of moh, mone and trc to support raising awareness on blood donation were developed. to raise public awareness on blood donation nationwide, education and recruitment campaigns on were organized in pilot schools. additionally, media and public relation campaigns on blood donation were organized throughout the country. results: ( ) existing curricula and textbooks relevant to promoting blood donation were reviewed, revised and reported to the board of education of mone. ( ) corresponding educational materials for students and teachers were developed and distributed. ( ) blood donation clubs were established in pilot schools. ( ) trainings were conducted for personnel of moh and trc on blood donation regarding their responsibilities. ( ) cascade trainings were conducted for personnel of transfusion centers and school principals in provinces. ( ) information seminars were delivered to . students and . teachers and family members of students during school campaigns. ( ) four animation films on blood donation were produced and broadcasted on the national tv channel (trt). ( ) three different computer games targeting different age groups were developed and uploaded to the web portal of the project and distributed to the pilot schools. ( ) media spots were produced and broadcasted . times in different tv and radio channels. ( ) billboard posters and brochures were prepared and distributed to provinces for raising public awareness. ( ) advertisements about the project and the importance of vnrd were displayed times on national and local newspapers, . times on online news, and broadcasted on national tv channels. ( ) during the campaigns, . units of whole blood were collected in pilot schools. ( ) visibility kits to recruit future blood donors are prepared and distributed throughout the project activities. ( ) awareness and knowledge level of students and their teachers/parents on the importance of blood donation are increased to . % and . % respectively, assessed through pretest and posttest. voluntary non-remunerated donation rate of national demand increased from . % to . in two years. summary/conclusions: training and campaign programmes successfully increased the knowledge on blood donation. to achieve national self-sufficient safe blood supply, efforts for recruitment should be continued. background: despite % of pakistan's population being under years, only % of blood supplies come from voluntary donors while remaining blood is collected from 'family replacement donors'. in pakistan the system has outsourced the mobilization of blood donors to the patient families. as a result many people reach out to their networks including on facebook to locate blood donors. there are thousands of posts each month in pakistan seeking blood donors on facebook. to facilitate needy families, the global social media giant facebook launched a special blood donation feature for pakistan in collaboration with sbtp, pakistan. the feature makes it easier for people to sign up to become blood donors and helps connect these voluntary donors with people and organizations in need of blood. similar features have been launched by facebook in india, bangladesh and brazil to address the problem of blood shortages in those countries. however, among these four countries pakistan has unique position because of the existence of a national counterpart, sbtp which can facilitate facebook in promoting its feature and provide the feedback on the impact of this innovative effort for continuous improvement of the feature. aims: to promote voluntary blood donations and blood safety in pakistan through facebook. methods: the facebook and sbtp teams launched a pilot to study the impact and effectiveness of the facebook blood donation feature as a tool of community engagement. a six months plan has been chalked out to measure the impact of this tool in five selected blood centres. a checklist called "p checklist" was shared with these blood centres to fulfill some basic requirements for an official blood bank page including a display picture, cover photo, contact information, directions, etc. regular skype meetings are held between the teams of sbtp, facebook (san francisco and singapore) and the blood centres to monitor the progress of the pilot and generate feedback. results: the facebook blood donation feature has recorded remarkable success with over one million signups within few months in pakistan. the blood centres participating in the pakistan study have experienced enhancement in the voluntary blood donations trend with - walk-in donors and an average of more than telephonic queries regarding voluntary blood donation per month in each center. the trend is gradually surging as the feature is being refined on the basis of feedback received. the pilot will end in june . the statistics generated since january are very encouraging and underscore the importance of social media in reaching out to the untapped potential blood donors. the study will be used to plan an effective nationwide strategy to increase donor mobilization, recruitment and retention. background: in our region, an increasing number of patients of african or asian origin with sickle cell disease (scd) or transfusion dependent thalassemia (tdt) require red blood cell (rbc) transfusions, and many have rbc alloantibodies. selecting optimally matched rbc units for these patients is essential for preventing not only acute hemolysis but also further alloimmunisation. beside antigen-matching for abo, rh d, c, c, e, e and k, patients with scd and tdt should ideally receive rbc units matched also for m, s, s, fya, fyb, jka and jkb (extended phenotype). this is the policy at our center, which currently provides rbc products to patients with hemoglobinopathies. because the vast majority of our blood donors are caucasians, the selection of matched rbc units for patients of different ethnic origin can be difficult. therefore, expanding the number of available african and asian blood donors is becoming increasingly necessary. aims: hereby the recruitment strategy of non-caucasian blood donors introduced at our center is described and the results obtained during six years are reported. methods: since . . , whenever a first-time blood donor of non-caucasian origin is registered, an alert is entered in the donor file to trigger the determination of the extended rbc phenotype along with routine testing. rbc antigen determination is performed in our laboratory with serologic methods. in selected cases (i.e. suspected rhd or rhce variant), samples are sent for molecular analysis (ssp pcr). rare rbc phenotypes relative to ethnicity are, among others, fy(a-b-), s-s-, lu(b-) and those with uncommon rh phenotypes. if a rare rbc phenotype is detected, a coded comment is entered in the donor data and the donor is listed in the national rare donor file. results: from . . until . . , an extended determination of rbc antigens was performed in subjects presenting for blood donation. twenty-nine rare donors ( %) were identified and included in the rare donor file: fy(a-b-), lu (a-b-), lu(b-), fy(a-b-) and s-, ccddee (r'r'). overall, these donors provided rbc units (range . to date, all donors are still active and are reserved for dedicated donations. the internal price of rbc antigen testing per donor is approximately . -chf, resulting in a total financial effort of around , .-chf in the time since the project was started. summary/conclusions: in our experience, a "passive" recruitment of non-caucasian blood donors based on ethnicity has an overall low efficiency from a logistic and financial point of view. moreover, african and asian blood donors may require investigations for hemoglobin variants and serology for malaria in addition to routine testing. nevertheless, a targeted determination of extended rbc antigen phenotype does allow the identification of persons with rare phenotypes. currently, measures for the active recruitment of potentially rare blood donors are being implemented at our center. after a pilot phase, a project for a nationwide recruitment strategy will be elaborated. a further goal is to build a national registry of patients with hemoglobinopathies requiring transfusions. blood transfusion is an essential treatment. transfusion safety consists of several components. although all are important, ion richer countries the order of priority is typically: .) avoidance of transfusion transmittable infections; .) quality of the blood product with a strong focus on component therapy; .) prevention of severe transfusion reactions; .) avoidance of clerical errors; .) sufficient availability of blood. the keynote of this lecture will be that the order of priorities on transfusion safety should probably be different in resource limited environments. .) sufficient availability of blood and proper utilisation; .) avoidance of transfusion transmittable infections; .) avoidance of clerical errors; .) prevention of severe transfusion reactions; .) quality of the blood product. most important, in regions with limited resources patients suffer from under-transfusion because not enough blood is available. all efforts should be made to reduce wastage of the available blood either by inappropriate storage, handling, or nonindicated transfusions. in addition, prevalence and number of pathogens transmittable by transfusion is much higher than in the richer parts of the world. this is aggravated by the fact that rejection of blood donors based on their history is problematic when the blood bank is empty. the aim to develop centralized national blood services with few sites manufacturing cost effective (low personnel costs) high-quality blood products, which are distributed to regional hospitals is not matching the reality of infrastructure, governmental support, and functionality. in healthcare systems with limited resources usually available personnel (hands) is not a problem, while reagents, equipment and even electricity are precious resources. the lecture will propose to focus on staff training and education, establishing local hospital-based transfusion services, which provide the blood products for the region, based on donor recruitment campaigns adjusted to the available technology, local culture, including replacement donor programs, with retention of safe donors as highest priority. fractionation of blood into components had been standard in transfusion medicine, but recently whole blood has experienced revival for patients with acute blood loss. given main transfusion indications such as postpartum hemorrhage, or severe trauma, in regions with limited infrastructure, whole blood might be the more appropriate product. most patients requiring transfusion in these regions are younger and volume overload by whole blood is not a major issue. in addition, frequent electricity failures do not allow prolonged storage of plasma at - °c (this is therefore mostly wasted), although this issue can be overcome by solar powered freezers. ideally whole blood should be pathogen inactivated for which two methods are currently available. to reduce frequencies of acute hemolytic transfusion reactions again education and training to minimize clerical errors in the transfusion process are most important. extended testing for other rhesus antigens and k beside abo and rh-d in transfusion dependent hemoglobinopathy patients may help to reduce delayed hemolytic transfusion reactions. currently a leukodepleted pathogen-inactivated whole blood product might mostly serve the needs for blood transfusion in regions with limited infrastructure . the developed world should invest research efforts to develop such a product available at affordable costs. background: in modern transfusion medicine, serological investigations for blood cell antigens are complemented by genotyping arrays and pcr assays. whilst these platforms are informative in the majority of reference investigations, they are limited in their ability to define nucleotide variants associated with rare antigens and unable to detect novel variants potentially affecting antigenicity. next-generation sequencing is increasingly being employed in reference settings, providing information that cannot be obtained through these methods. whole genome and whole exome sequencing have been successfully employed in many investigations of novel and rare antigens, however concerns remain regarding the collection of genomic data unrelated to reference investigations, and reporting clinically significant incidental findings. these concerns can be addressed through the use of targeted sequencing panels. we report on the design, testing and efficacy of a panel providing a comprehensive genotyping profile for red cell, platelet and neutrophil antigens in a single test. aims: design a customised targeted exome sequencing panel for red cell, platelet and neutrophil antigen genes, and benchmark the efficacy against a commercial medical sequencing panel (illumina trusight one -tso). -test the panel and in-house genotype prediction script on sequence outputs from samples with known red cell, platelet and neutrophil genotypes and phenotypes and determine whether predictions are concordant. methods: the panel was designed with probes covering exons of genes associated with red cell, platelet and neutrophil antigens. using illumina nextera rapid capture technology, samples were tested over five sequencing runs, on standard and micro chemistries, to determine optimal sample plexity per standard run, and the efficacy of smaller flow cells for lower throughput applications. an in-house python script was used to predict star-allele genotypes based on variants listed in isbt and embl databases. these predictions were compared to results from serology, snp array and previous tso data. results: coverage consistently averaged > , with % of target at a quality of q . optimal sample plexity for a standard run was determined to be samples, allowing for sufficient coverage of all clinically significant variants. for red cell samples with previous typing data (excluding rh structural variants), the script correctly predicted . % of snp based red cell genotypes. script predictions were % concordant for platelet genotypes, and four of five neutrophil antigen genotypes. hna genotypes defined by cd could not be reliably determined. the increased target coverage of the panel allowed for detection of a clinically significant heterozygous variant in scianna system, previously undetected by the tso panel due to extremely low coverage. additionally, a variant defining a potentially novel null allele was detected in the p pk system. summary/conclusions: the panel demonstrates considerably higher coverage, quality and throughput compared to the tso and allows for detection of variants previously overlooked due to low sequencing coverage. up to samples can be reliably sequenced in a single run. our script correctly predicts over % of snp based alleles; however, rh structural variants require further manual analysis. background: to ensure the safety of a transfusion it is critical to identify the blood type of both donor and recipient. serological methods for typing abo, rh and kel use monoclonal antibodies, however, typing reagents for rare blood groups are expensive, unavailable or unreliable. dna-based identification of human blood groups has been used to overcome these limitations and its application has reduced rates of alloimmunisation in chronically transfused patients. however, to date, the cost per sample has prevented the universal application of dna-based donor typing aims: to achieve universal adoption of dna based donor typing, the blood transfusion genomics consortium (bgc) set out to develop an affordable dna based platform, capable of typing all red cell antigens, hla class i and ii and human platelet antigens. methods: the uk biobank axiom array, previously used to type , uk citizens, was redesigned for donor typing using three approaches: i) mining transfusion medicine knowledge, e.g. isbt allele tables; ii) inclusion of loci associated with donor health; iii) extraction of all coding variants in relevant genes with a frequency > : , identified in large-scale sequencing data. samples from nhsbt and sanquin blood donors (n = , ) were used for performance assessment. red cell and platelet antigens for each donor were inferred from genotypes using the bloodtyper algorithm and concordance with clinical serological typing results assessed. results: concordance between genotypic and serological typing results was . % for , comparisons; of the discrepancies were serologically negative and genotypically positive for a given antigen (k/k, fy[a/b], lu[a/b]). in all cases dna variants known to modify or weaken antigen expression were detected, displaying the power of genotyping to detect variant 'weak-antigen' expression. across antigens for which serology was available, genotyping provided a . -fold increase in the number of typing results available per donor ( . vs . ). furthermore, genotyping provided data on an additional clinically relevant antigens, allowing identification of antigen-negative donors and blood group identification for which antibodies are not commercially available. the power of a genotyped panel of donors to support patient management was demonstrated by a retrospective analysis of clinical cases referred to nhsbt. from , patient referrals with > alloantibodies between and , unique alloantibody profiles were identified. we found that there was a . -fold greater likelihood of finding o negative compatible donors for these patients when using genotyping data from the , nhsbt donors. importantly, the number of alloantibody combinations for which no compatible antigen-negative donor could be identified fell from to , representing an additional patients that could be provided with directly compatible blood using the same donors. summary/conclusions: through the bgc efforts an affordable fully automated genotyping platform, including the processes for quality assurance and data analysis, has been developed. furthermore, we have demonstrated the real-world benefits more extensive donor characterisation can provide when selecting blood for patients with multiple antibodies. the results of this international collaboration provide opportunities to introduce fully-automated genotype-based donor typing in a safe and cost-efficient manner in blood supply organisations. c-s - biobank performs whole-genome sequencing (wgs) from individuals nation-wide. these data are suitable for allele frequency analysis to demonstrate gene expression and genetic profile in our population, also to estimate the significance of each antigen in transfusion practice. aims: we aim to provide and verify population-based blood group antigen profile using wgs and dna samples from taiwan biobank. methods: a near wgs and demographic data were analyzed. annotations of blood group antigen were performed according to variants from isbt allele tables, including transcription factors; variants for the lewis system were obtained from previous studies. annotations of blood group variants were verified by dna samples with targeted sequencing on illumina miseq, and specific variants were verified by dna samples with the commercial genotyping kit or sanger sequencing. allele frequencies from wgs analysis were compared with population serology data using two-proportion z test. results: population-wide blood group antigens were analyzed, revealed in-depth antigen expression profiles in all systems (except ch/rg). the antigen frequencies from wgs were similar compared with published serology data, except for the antigens and possible explanations listed as follow, ) m, n: insufficient sequencing reads, ) c, c: identical rhce exon with rhd exon for c allele, ) mur: insufficient read length/depth for gypa/gypb hybridization calling and individuals from high prevalence of mur antigen in aboriginal tribes were not enrolled. blood group antigen predictions and variants from wgs were accord to dna verification. furthermore, systems shown no genetic variations, predicting uniform antigen expression in our population, and we can manage transfusion with minimum concerns for antigen mismatch in these systems. moreover, we found weak and null alleles in our population for blood group systems that we had previously no knowledge of, such as lan, jr, and vel. these variants were helped to identify a patient with anti-jr a carrying homozygous jr a null alleles. summary/conclusions: taiwan biobank wgs is suitable for full blood group antigen profile determination with few adjustments required for specific antigens. the population antigen allele frequency provides valuable insight to antigen significance in transfusion practice, matching strategy for our patients, and estimation of the likelihood to obtain for specific antigen negative blood from mass population. also, the genetic variants revealed in this study can help us to locate rare donors, and to integrate variations into routine donor blood group screening to provide suitable blood at a low cost efficiently. background: providing adequate amounts of safe, appropriately matched blood to meet the demands of an expanding and aging patient population presents a challenging global problem. for these reasons, sustainable in vitro sources of red cells may offer a desirable alternative to reliance on donor blood. the first stable immortalized early adult erythroblast cell line, bel-a , has been shown to differentiate efficiently into mature, functional reticulocytes (trakarnsanga et al., nat commun. : , ) and consequently could provide a readily available tool for diagnostic use and proof of principle for future therapeutic use. aims: at ibgrl, next generation whole exome sequencing (wes) has been used previously to accurately predict blood group phenotype in a number of blood group systems, including abo, rh and mns (tilley & thornton, transfusion medicine (suppl. ) : , ). here we have used it to analyse and document bel-a blood group-related genotypes and predict blood group phenotypes. additional genes involved in cell-growth and enucleation were also analysed in order to further elucidate the characteristics of the bel-a cell-line. methods: bel-a cells (day ) were cultured in expansion medium and genomic dna (gdna) was isolated from cells on day . for wes, gdna libraries were prepared using nextera â rapid capture exome enrichment and sequenced on illumina â miseq. sequence alignments for genes encoding all known blood group systems and further genes encoding transcription factors and cell enucleation-associated proteins were visualised using integrative genomics viewer, whilst illumina â variant studio was used to identify observed mutations. mutations in coding regions were used to determine bel-a genotype and predicted phenotype. results: good coverage of most of the selected genes was achieved. alignment of homologous blood group genes including rhd/rhce, gypa/gypb and c a/c b was problematic and additional analysis of coverage of these genes was required for accurate interpretation. despite a number of polymorphisms observed across the tested genes, bel-a did not express any novel or rare blood group antigens. genotyping results predicted a common antigenic profile, in agreement with previous serological and genotyping results where available. although a number of missense single nucleotide variations were detected in analysed genes, including cr , cdan and tmx , these were common polymorphic variants and unlikely to be of any functional significance. summary/conclusions: wes was used to determine bel-a genotype in relation to blood group genes and selected genes encoding transcription factors and proteins associated with cell enucleation. wes allowed accurate prediction of blood group phenotypes, showing full concordance with available serological data (trakarnsanga et al, ) . a small number of mutations were identified which are of unknown significance and require further work to determine any potential phenotypic effects. this complete record of the bel-a blood group-related exome will enable reliable gene editing strategies for future diagnostic and therapeutic purposes. additionally, knowledge of the full cell line exome will allow analysis of any emerging genes of interest and provide better insight into the mechanisms of erythroid differentiation and enucleation. background: emerging evidence, especially in neonates, has shown potential harm associated with liberal platelet transfusion strategies. very little evidence exists regarding optimal platelet transfusion thresholds in critically ill children. randomized controlled trials may be difficult due to lack of equipoise from providers. if regional variation in practice exists, comparative effectiveness studies may be an alternative approach. aims: to describe regional variation in platelet transfusion practices in critically ill children. methods: secondary analysis of a prospective, observational study. subjects were grouped according to region (north america, europe, middle east, asia and oceania) and nation. transfusions were analyzed as prophylactic (given to prevent bleeding) or therapeutic (given to treat bleeding). the primary outcome was the total platelet count (tpc) prior to transfusion. sub-groups analyses were performed in children with an underlying oncologic diagnosis and those supported by extracorporeal life support (ecls). the dosing and processing of the platelet transfusions were analyzed as secondary outcomes. results: five hundred and forty-nine children from countries were enrolled ( % in north america, % in europe, % in oceania, % in asia, and % in the middle east). overall, the median (iqr) tpc prior to prophylactic transfusions (n = ) differed significantly on a regional basis (p = . ) and ranged from ( - ) x cells/l in the middle east to ( - ) x cells/l in asia. the median tpc prior to prophylactic transfusions did not significantly differ between countries (p = . ), nor did the tpc prior to therapeutic transfusions (n = ) differ on either a regional (p = . ) or national (p = . ) basis. for children supported by ecls (n = ), there were no regional (p = . ) or national (p = . ) differences for prophylactic transfusions. however, significant differences in the tpc prior to therapeutic transfusions were observed on both a regional (p = . ) and national ( . ) basis with the middle east, in particular israel, transfusing at the lowest median (iqr) tpc [ ( - ) x cells/l]. for children with an underlying oncologic diagnosis (n = ), no differences were seen in the tpc for prophylactic transfusions (n = ) on a regional (p = . ) or national (p = . ) basis. nor were differences seen in the tpc prior to therapeutic transfusions on a regional ( . ) or national (p = . ) basis. there was significant variability in the dosing of platelet transfusions on both a regional (p < . ) and national basis (p < . ). the median (iqr) dose based on volume ranged from . ( . - . ) ml/kg in north america to . ( . - . ) ml/kg in europe. the vast majority of transfusions were leukoreduced and irradiated but significant variation exists in storage duration on both a regional (p < . ) and national (p < . ) basis. summary/conclusions: regional and national variation exists in platelet transfusion practices among critically ill children, especially in those given therapeutic transfusions while supported by ecls. considering this variation, comparative effectiveness studies may be an appropriate approach to gain evidence to optimize platelet transfusion thresholds. background: the optimal threshold for prophylactic platelet (plts) transfusion in pediatric patients with cancer is still controversial and current clinical practice comes from studies on adults and on inpatient setting. the international guidelines (icmtg, ) recommend, for all age patients, a prophylactic platelet transfusion when plts count is ≤ /l and a platelet dose of . per square meter (sm) of body-surface area (bsa) in inpatient and . /sm in outpatient setting. aims: in january we started in our children's hospital a prospective protocol in order to evaluate the impact on bleeding risk of current clinical practice of prophylactic platelet transfusion in inpatients and outpatients onco-haematological patients. methods: bsa was calculated from age-standardized weight. inpatients received a dose per transfusion of . /sm and outpatients a dose per transfusion of . /sm. platelets were transfused when the count was ≤ /l or in presence of bleeding signs; pediatric aliquots were obtained from buffy coat derived pooled platelet concentrates or apheresis platelet concentrates, according disponibility. results: from january to december a total of platelet pediatric aliquots were transfused: ( . %) were obtained from apheresis platelet concentrates and ( . %) from buffy-coat-derived pooled platelet concentrates. the majority of platelets pediatric aliquots ( - . %) were transfused to onco-hematological patients undergoing hematopoietic stem cells transplant (hsct) or conventional chemotherapy. among them, aliquots were transfused in inpatient setting: ( %) in the hematology unit, ( . %) in the oncology unit and ( . %) in hsct unit. a total of ( . %) aliquots were transfused in outpatient setting: ( . %) to patients affected by hematological malignancies and ( %) to patients with solid tumors. five major bleeding events (who grade ≥ ) were observed during the study period and all of them occurred in hospitalized patients. two patients with solid neoplasm developed a who grade bleeding event. two patients with hematologic malignancies and a patient with neuroblastoma (n = , . %) developed intracranial bleeding (who grade ). the platelet count at the time of the event was /l, /l and /l, respectively. summary/conclusions: our results showed the efficacy, in onco-hematological pediatric patients, of a prophylactic platelet transfusion protocol based on international guidelines: a very low incidence of who grade bleeding has been observed in inpatients setting only ( . % vs % of plado trial, sj slichter, nejm, ) , while in outpatients setting the double platelet dose prevents the major bleeding event (who grade ≥ ) occurrence. background: the problem of blood-borne infections remains relevant in transfusion medicine. pathogen reduction technologies (prt) provide a preventive approach to a wide range of transfusion-transmitted infectious diseases. to date, prt widely used for platelet concentrates and blood plasma, however, the use of these technologies for the treatment of red blood cell-containing blood products undergo research. aims: the aim of our study was to evaluate the safety and efficacy of transfusions of pathogen-reduced (test group) red blood cell suspensions (rbcs) and compare these data with gamma-irradiated rbcs (control group). methods: the technology based on the combined action of riboflavin and ultraviolet (mirasol prt, terumo bct, belgium) was used to reduce pathogens in whole blood. subsequently, the rbcs of the test group were derived from pathogenreduced whole blood. the control rbcs were irradiated at the gammacell elite (best theratronics, canada) at a dose of gray. all rbcs were used for transfusion for days from the harvest day. pediatric patients with various oncological and hematological diseases were randomized to groups of members in each group. the test group of patients received transfusions of a pathogen-reduced rbcs; the control group received transfusions of a gamma-irradiated rbcs. the next day after transfusion were assessed hemoglobin and hematocrit increment, the level of potassium and haptoglobin in the patients' serum, the frequency and severity of transfusion reactions. - days after the transfusion, the direct antiglobulin test (dat) was performed and after - days the indirect antiglobulin test (iat) was performed. the interval to the next need for transfusion was also evaluated. results: the increase in hemoglobin and hematocrit (p = . ), as well as the concentration of potassium (p = . ) and haptoglobin (p = . ) in the patients' serum after the transfusion did not differ between groups. none of the patients in both groups had hyperkalemia after transfusion. in each group, two patients had febrile non-hemolytic transfusion reactions of comparable severity (p = ). all dat and iat tests were negative in both groups. the interval between transfusions were not significantly different between groups (p = . ). only in the test group was found the correlation between the increase in the hemoglobin and hematocrit values with the volume of transfusion, with the dose and the adjusted dose of hemoglobin obtained for the transfusion on body weight. and in this group was found inverse correlation between the hemoglobin and hematocrit increment with the level of hemolysis in the rbcs. summary/conclusions: we found that the clinical efficacy and safety of rbcs of the compared groups did not differ. there was no evidence of immune elimination and allo-sensitization caused by pathogen-reduced rbcs. according to our data, the spectrum of efficiency and safety indicators of pathogen-reduced rbcs is no worse than that of gamma-irradiated rbcs, provided that rbcs is used for days of storage. the founded correlation suggests that the efficiency of pathogen-reduced rbcs transfusions is more dependent on the characteristics of the rbcs. background: patient blood management (pbm) programs are expanding at an international level. a recent nationally representative study from united states observed pediatric age group as the only age group showing lack of objective evidence of pbm initiatives (goel et al, jama ) . aims: this study aims to identify trends in peri-operative blood utilization in children undergoing elective and non-elective surgeries over years duration from to . methods: using years data ( ) ( ) ( ) ( ) ( ) perioperative transfusions decreased steadily per year from . % in to . % ( % cumulative decline) in for children of all ages (or . ; % ci . - . ; p trend < . ). the cumulative change in elective procedures was . % versus . % decrease in urgent/emergent procedures (p trend < . ). summary/conclusions: in this large prospective registry study of > , children undergoing elective/non-elective surgeries, a statistically significant decrease in utilization of peri-operative rbc transfusions was seen across years from through with more significant decrease in urgent/emergent procedures than elective procedures while these findings need evaluation for non-surgical indications of transfusion, these results may provide first evidence of peri-operative pediatric patient blood management strategies being implemented to optimize transfusions in pediatric population. adverse events -tti, immune interactions and risk c-s - transfusion-transmitted infections (tti) are a long-standing and well recognized concern in medicine, which is tackled on the highest level to guarantee the safety of the transfusion procedure for all stake holders. these include the recipient patients, the donating volunteers, the health care workers involved, and their respective contact persons. accordingly, current national and international guidelines including expert societies and the who provide medical, technical, and legal frameworks, which are the basis for the standard operating procedures. nevertheless, there are important challenges, which render tti a "moving target", and reflect the dynamics in three main areas. first, a change in the type and number of recipient patients with past or ongoing immunomodulatory / immunodeficiency component (examples being hiv/aids, sot, allogenic hct, monoclonal antibody therapies, small molecule inhibitors). second, changing exposure to known agents in donors due to global travel, migration and displacement, as well as environmental/climate change. third, discovery and diagnostics of old and new agents with their known or presumed impact as tti. these aspects will require careful review of data and studies, and judicious discussion of the potential action such as selection versus close monitoring to keep tti rates as low as possible, to deliver maximal safety of patients and stakeholders. background: the implementation of nucleic acid testing (nat) and the development of sensitive and specific serologic assays to detect hbsag and anti-hbc antibodies significantly reduced the risk of hbv transfusion-transmission. the apparent redundant testing for two direct viral markers prompted debates on maintaining hbsag screening, particularly in low endemic countries where blood donations are screened for anti-hbc. however, frequencies of - % of hbsag-confirmed positive/nat negative donations have been reported depending on the sensitivity limit of the molecular assays used. the nature of this discrepancy between hbsag and dna remains largely unknown and it is essential to evaluate any potential negative impact on blood safety before considering removing hbsag testing. aims: the prevalence in blood donors and the molecular mechanisms responsible for a persistent undetectable or barely detectable level of viral replication in the presence of a sustained hbsag production were investigated in a collaborative study including five laboratories/blood centers in europe and south africa. discrepancy between viral dna and hbsag levels suggested the presence of mutations that may negatively affect hbv replication and/or infectious viral particle production. methods: donor samples from france, south africa, poland, and croatia were selected for having hbsag levels ≥ iu/ml and being id-nat (procleix-ultrio plus tm [ % lod: iu/ml]) non-reactive/non-repeatable reactive (nr/nrr) with undetectable viral load (vl) or < iu/ml (n = ) or nat repeat reactive (rr) with vl < iu/ml (n = ). french samples initially tested nat nr/nrr with procleix-ultrio (lod %: iu/ml) were retested with ultrio plus prior inclusion in the study. hbv dna load was quantified (cobas taqman hbv [loq: iu/ml]). hbv dna was purified from to ml of plasma after ultracentrifugation. the whole hbv genome, pre-s/s, precore/core and bcp regions were amplified and sequenced. results: following viral concentration, hbv dna presence was confirmed in % of all samples with undetectable or vl < iu/ml. hbv genotypes were a ( . %), a ( . %), a ( . %), b ( . %), c ( . %), d ( %), and e ( %). all samples were anti-hbc positive and % of ultrio-negative samples tested positive with ultrio plus. unusual - nt insertions/deletions identified in bcp regulatory elements (tata boxes, pginr, epsilon domain) suggest altered viral replication. amino acid substitutions (n = ) or deletions (n = ) at positions reported involved in nucleocapsid formation, particle envelopment and virion formation were observed in the core protein of samples. the replicative properties of the bcp and core variants are currently evaluated in vitro as a surrogate model for direct infectivity testing. preliminary results indicate that the variants tested so far have replicative capabilities similar to those of control viruses. analysis of pol, s, and hbx proteins is ongoing. summary/conclusions: these data confirmed the presence of extremely low level of circulating dna-containing viral particles in id-nat non-reactive or nonrepeated reactive blood donations with concomitant high hbsag levels and anti-hbc reactivity. despite the presence of mutations in the viral genomes potentially affecting virion production, preliminary data indicate that some of the viruses in plasma retain the ability to replicate in vitro and to constitute a potential infectious risk. c-s - background: in switzerland highly sensitive nucleic acid screening in an individual donation format for hepatitis b virus (hbv id-nat) and hepatitis b surface antigen (hbsag) detection is mandatorily performed (guidelines of swiss transfusion src, switzerland). since , hbv (hb) vaccination is recommended in switzerland for children and adolescents until the age of and for adults belonging to known risk groups. aims: to highlight that low anti-hbs titers several years following hbv vaccination still confer protection and enable the host immune system to clear hbv dna without development of serologic markers of disease. methods: a retrospective donor interview was conducted to complete information not covered by the questions included in the standard donor questionnaire. routine hbv serological donor screening was performed on a quadriga system (diasorin, former siemens) with the enzygnost hbsag assay (diasorin, former siemens). further hbv tests were performed on the abbott architect i analyser (hbsag neutralisation, hbeag, anti-hbc igg/igm, anti-hbc igm, anti-hbe and anti-hbs). routine id-nat screening for hiv/hcv/hbv was performed with the roche cobas mpx test on a roche cobas platform. hbv id-nat positive samples were confirmed with a quantitative hbv nat assay (abbott). background: hepatitis b core-related antigen (hbcrag) is a structural antigen of hbv, consisting in hbcag, hbeag and the p cr precore protein. quantitative hbcrag measurement is a sensitive marker of viral replication reflecting the cccdna content and persistence of disease. hbcrag positivity was found to be a significant risk factor of hbv reactivation in hbsag-, anti-hbc+, hbv dna-patients (occult hbv infection, obi) undergoing immunosuppressive therapy. aims: no data about hbcrag status in apparently healthy subject with obi are available. the aim of this study was to analyse this marker in our cohort of obi blood donors. methods: hbcrag was measured in blood donors confirmed to be carriers of obi (hbsag-, hbv dna+). of them, / ( . %) donors were anti-hbc positive, and ( . %) negative. donors had both anti-hbc and anti-hbe reactivities. a group of young blood donors vaccinated for hbv infection (hbsag-, hbv dna-, anti-hbc-), and patients with chronic hbv infection (hbsag+, hbv dna+) were used as negative and positive controls group, respectively. serum hbcrag was measured using a chemiluminescent enzyme immunoassay on the lumipulse g automated analyzer (fujirebio, tokyo, japan). the lower limit of detection (lod) of the quantitative assay is logu/ml and the lower limit of quantification (loq) is > logu/ml, due to nonlinearity results between and logu/ml. levels of hbcrag were tested in the three groups and analysed in comparison to the presence of anti-hbc and anti-hbe. statistical analysis was performed by the ibm statistics spss . . . results: all donors in the negative control group had undetectable hbcrag levels, whereas all patients in the positive control group have detectable hbcrag (mean value: . logu/ml, range . - . ), confirming that individuals without prior exposure to hbv would not have detectable hbcrag. hbcrag was detectable in / obi donors ( . %), with a mean value of . logu/ml (range . - . ). hbcrag could be measured only in obi donors ( . and . logu/ml), being below the loq of the test in the majority of obi ( / ). considering the presence of anti-hbc, hbcrag was detected in / ( . %) anti-hbc+ and in / ( %) anti-hbc-obi, with no significant difference in their mean levels ( . ae . vs . ae . ; p = . ). interestingly, the presence of anti-hbe ( / ) was independently associated with higher hbcrag levels ( . ae . vs . ae . ; p = . ). summary/conclusions: identification of donors with obi is critical to prevent the risk of hbv transfusion-transmission. being hbcrag associated with the cccdna content and replication, our results suggest that the presence of hbcrag, even if not quantifiable, could be useful marker to confirm the occult infection status, even in anti-hbc negative donors. the association between hbcrag, anti-hbc and anti-hbe could also be a useful marker to identify obi donors with a higher risk of hbv reactivation. c-s - hc group. human peripheral blood mononuclear cells (pbmcs) from blood donors were stimulated with hbv polypeptides pool in vitro. t cell proliferation assays (cfse) was used to detecting t cell proliferation, enzyme-linked immunospot assay (elispot) was used to detecting the frequency of hbv-specific ifn-c secreted t cells. spss . statistical analysis software was used for statistical analysis. the measurement data of normal distribution were tested by two independent samples t test; and the comparison between multiple groups was analyzed by one-way anova. mann-whitney u test was used for comparison between non-normal data sets. p < . was considered statistically significant. results: . proliferation characteristics of t cells. the proliferation of cd + t lymphocytes was mainly stimulated by specific hbv polypeptide pool, and the proliferation rates of obi group and chb group were significantly higher than those of hc group ( . %, . % vs. . %), with significant difference ( . % vs. . %, p = . , . % vs . %, p < . ). . the frequency of specific ifn-c secreted t cells. the response intensity of the obi group ( sfc/ pbmcs) and chb group ( sfc/ pbmcs) was higher than that of the hc group ( sfc/ pbmcs) under the stimulation of hbv polypeptide pool, and the positive rate of t cell response to the stimulation of hbv polypeptide pool was the highest in the obi group ( . %). summary/conclusions: both obi and chb had higher rates of hbv-specific t effector cell proliferation and ifn-c secretion than the healthy control group. compared with the chb group, obi group had a higher positive rate of t cell response, which may be one of the causes of host immunity resulting in obi. further studies on other immune factors are required. background: western blood transfusion practices are currently changing due to various drivers such as blood management policies, ongoing technological developments, and new therapeutic options. in the netherlands, as in many high-income countries, these have resulted in a diminishing trend of red blood cells. therefore, it is important for blood bank management to anticipate the future demand of blood products for the sake of medium and long term decision making. to support this decision making, we have employed scenario development, which is used in many other sectors (such as finance and transportation) and can also be applied to blood transfusion. building upon a prior literature review and semi-structured interviews of international experts, we gathered experts together for scenario sessions to assess the opportunities and threats for sanquin's medium-term ( - years) strategy using an online platform and face-to-face discussions. aims: to assess for opportunities, threats, and the organizational implications thereof for the medium-term future of sanquin, the dutch national blood bank. methods: twenty-one multidisciplinary experts in blood transfusion agreed to participate and were separated into two groups for half-day interactive sessions. using an iterative process through an online platform, experts brainstormed opportunities and threats for sanquin, which were categorized into themes. these themes were ranked according to importance and certainty, and through consensus, experts chose two themes with high impact and high uncertainty. for these chosen themes, specific actions for the blood bank were listed to mitigate and/or enhance the threat or opportunity. discussions were ample throughout. results: with regards to opportunities and threats for sanquin's medium term strategy, experts brainstormed many ideas and categorized them under themes: political context/ changing legislation, novel products and alternative applications, donors, international markets, commercialization, digitalization, change in perceptions, research, demand, and organizational structure. after ranking for importance and certainty, six themes were chosen: change in perceptions, international markets, political context (opportunities), demand (opportunities), research (vulnerabilities), and donor (vulnerabilities). for each of these themes experts provided specific actions for the organizations to mitigate threats or stimulate opportunities accordingly. these actions included increased transparency and improved communication with the (donor) public, lobbying in political spheres, increased activities in educational institutes and large funding organizations, and creating and collaborating on novel blood products on an international level, to name a few. summary/conclusions: these results show that mapping and assessing a blood bank's future using a multi-disciplinary group of experts is conducive as an effective means of collection a diverse range of opportunities and threats. this provides an opportunity for blood bank management to become proactive towards these potential opportunities and threats and possibly evolve future strategies for the organization. showed that iron-deficient female blood donors were more likely to have depressive symptoms than non-iron deficient female blood donors. among participants with depressive symptoms, females with low plasma ferritin levels had significantly increased odds for reporting a "feeling of lacking energy and strength" (or = . ; % ci: . - . ). as it is known that blood donors are at an increased risk of iron deficiency, it is important to determine whether those genetically predisposed to lower plasma ferritin levels have a higher risk of experiencing the tiredness/lack of energy symptom. aims: to investigate whether there is an association between polygenic risk scores (prss) based on plasma ferritin levels and the tiredness/lack of energy symptom in blood donors. methods: the dbds is an ongoing nationwide blood donor cohort, of which genome-wide genotype data are available for , participants. genotyping was performed using the infinium global screening array (illumina â ) and imputation was achieved based on a scandinavian reference genome. ferritin prss, based on an icelandic ferritin gwas (n = , ), were calculated for all dbds participants. , female donors were available for the analysis. data on depressive symptoms were obtained using the validated major depression inventory scale (mdi), a selfreport mood questionnaire, which assesses the presence of depressive symptoms. a donor was classified as "tired" if they responded "all the time" or "most of the time" to the question "how often do you feel that you lacked energy and strength?". logistic regression analysis was performed, adjusting for age. for generating the quantile plots, the participants were distributed evenly into six quantiles based on their prs, whereby quantile contained the donors with the lowest prss (genetically predisposed to lower ferritin levels) and was set as the reference quantile with or = from the age-adjusted regression analysis (tiredness~quantile). results: prss in females ranged between - . and . (mean . ). a total of , female donors were classified as "not tired" and ( . %) were classified as "tired". no significant difference in ferritin prs was found between "tired" and "not tired" female donors (tired mean prs: . ; not tired mean prs: . ). an age-adjusted logistic regression model found this to be insignificant (or: . , % ci: . - . ), p = . ). to visualise the lack of association, a quantile plot was created, separating the female donors into six equal quantiles based on their prs. no clear trend was observed; donors with the highest prss (in quantile ) had or = . (p = . ) of being tired when compared to those in quantile (or set as ). summary/conclusions: no significant association was found between the ferritin prss of female blood donors and the tiredness/lack of energy symptom. further studies are needed to understand the effect of blood donation versus genetic constitution on tiredness among female iron-deficient blood donors. background: antiretroviral therapy (art) is critical for the control of clinical progression of human immunodeficiency virus (hiv) infections. however, the outcome of art could be limited by drug resistance-associated mutations (drms), even lead to the transmission of drug-resistant hiv to treatment na€ ıve patients such as blood donors, which is a huge concern to art. drms surveillance in hiv infected groups is strongly recommended by world health organization. characteristics of genetic diversity and drms of hiv among blood donors may provide comprehensive data to monitor viral evolution and optimize art, play important roles in blood safety. aims: limited data concerning the epidemic of hiv- subtypes and drms of blood donors is available in china. this study is to investigate genetic characteristics and drms of hiv- infected blood donors. methods: from - , blood donations collected from blood centers, covering almost the whole of china, were confirmed as hiv- positive by national centers for clinical laboratories using abbott realtime hiv- assay or cobas taq-man hiv- test, version . . then hiv- gag ( bp, hxb : - ), pol genes ( bp, hxb : - ) (encoding the whole protease (pr) and a part of reverse transcriptase (rt)) was sequenced after viral rna extraction and amplification. hiv- subtype based on gag and pr-rt regions was determined by comprehensive analyses of los alamos hiv blast tool, rega hiv- subtyping tool, phylogenetic trees and online jphmm program. drms analysis was performed in the stanford hiv drug resistance database. results: among donations, gag and pr-rt regions of samples were sequenced successfully. the distribution of hiv- genotype was as follows: crf_ bc = ( . %), crf_ ae = ( . %), b = ( . %), crf_ bc = ( . %), crf _ b = ( . %), crf _ b = ( . %), crf _cpx = ( . %), crf _ b = ( . %), crf _ = ( . %), crf _bc = ( . %), urf_ = ( . %) and urf = ( . %). of hiv- isolates were identified to have drms. there were ( . %, / ) protease inhibitors (pi) accessory drms, pi major drms and ( . %, / ) non-nucleoside reverse transcriptase inhibitors (nnrti) drms. most of blood donors with drms were crf _ae and crf _bc ( . %, / ). of pi accessory drms were q e. the pi major drms included m l, m i and n s. n s could result in hlr to atazanavir (atv) and nfv, llr to indinavir (idv) and saquinavir (sqv). v d/e is main nnrti drm ( . %, / ). a combination of v d and k r among two samples acted synergistically to reduce efavirenz (efv) and nevirapine (nvp) susceptibility. furthermore, two blood donors with k n mutation in reverse transcriptase gene had high level-resistance to efv and nvp. summary/conclusions: overall, the most prevalent subtypes among blood donors in the study were crf _bc ( . %), crf _ae ( . %). besides, other rare crfs and several urf_ and urfs were also found in these hiv- isolates, which suggested the epidemic of hiv has been shifted from high risk populations into general populations, including blood donors in china. drms were observed in . % donors in the study, which may result in resistance to pis and nnrtis, especially the hiv- variants with n s mutation in pr gene and k n mutation in rt gene. in summary, our findings indicate that increasing diversity of hiv- in blood donors and remind us the necessity of timely genotypic drug resistance monitoring and molecular epidemiology surveillance of hiv- among blood donors. background: labeling of platelets is required to measure the recovery and survival of transfused platelets in vivo. currently a radioactive method is used to label platelets. however, its' application is limited, due to safety issues and the inability to isolate transfused platelets out of the circulation. biotin-labeling of platelets is an attractive non-radioactive option, however, no validated protocol to biotinylate platelets is currently available for clinical purposes. aims: the aim of this study is to develop a simple, standardized, reproducible method to label platelets with biotin as a non-radioactive alternative to trace transfused platelets in vivo. methods: six pooled buffy coats derived platelet concentrates (pcs) stored in % plasma were biotinylated at day and day of storage. to distinguish the effect of the processing steps from the effects of biotin incubation, 'sham' samples were processed. for the biotinylation procedure, ml of pcs was washed twice and incubated with mg/l biotin, dissolved in phosphate buffered saline-pas-e ( : ), for min. stability of the biotin labeled platelets after irradiation was tested. annexin v and cd p expression were assessed as measures of platelet activation. applicability of this method to other platelet products was assessed in three pooled pcs stored in % pas-e and three single donor apheresis pcs. results: the method was reproducible performed in a closed system. after biotinylation, . % ae . % of platelets were labeled. platelet counts, ph and 'swirling' were within the range accepted by the dutch blood bank for standard platelet products. the number of annexin v positive cells was not significantly altered by the biotinylation procedure in both fresh and stored platelets. in contrast, cd p expression was increased in biotinylated platelets . % iqr( . - . %) compared to the control samples . % iqr( . - . %) on day of storage. however, biotinylated platelets were not more activated compared to sham samples % iqr( . - . %). thus only the procedural steps led to increased cd p expression and not the biotin label itself. all samples showed maximal response to thrombin receptor-activating peptide. for platelets labeled at day , a similar pattern was observed. irradiation of biotin labeled platelets did not alter the stability of the biotin label nor cell quality. furthermore this method is also applicable to pooled pcs stored in pas-e and apheresis pcs, with similar patterns in annexin v and cd p expression. summary/conclusions: we developed a standardized and reproducible protocol according to good practice guidelines (gpg) standards, for biotin-labeling of platelets for clinical purposes. the procedural steps, which are similar to the steps used for production of hyperconcentrated platelet products, led to an increased cd p expression, but did not alter the annexin v expression. this method can be applied as non-radioactive alternative to trace and recover transfused platelets in vivo. blocking activity over the prototypic chs insulator in cell lines and substantially reducing genotoxicity in a c-retroviral vector-mediated carcinogenesis mouse model. in contrast to chs , these insulators are small-sized ( - bp vs . kb) and can be easily accommodated in gt vectors without detrimentally affecting vector titers. aims: we aimed to test whether a , one of the newly discovered cis, could reduce vector-mediated genotoxicity in the challenging context of sin-lvs, by insulating a therapeutic globin-vector. methods: we tested the genotoxicity effect in the il- -dependent d cells, which upon transduction with oncogenic vectors become il- -independent, leading to transformation. d cells were transduced with sin-lvs: the b-globin-ΤΝs . . -, the insulated b-globin-a -tns . . and the oncogenic sffv-gfp-vector. transduced cells were expanded in % il- and transduction efficiency was determined by vector copy number (vcn). transduced d cells were seeded in methylcellulose with % or - % il- to detect the il- -independent and potentially transformed clones. the il- -independent clones were further expanded in % il- and infused in partially myeloablated and il- -treated c h/hej mice. wbc analysis, blood smears and bone marrow(bm) cytospins were performed. results: the a insulator did not negatively affect vector titers (ΤΝs . . , a -tns . . , sffv-gfp: . , . , . x ^ iu/ml, respectively). d cells were successfully transduced with all vectors (%vcn positive colonies: - %) and expanded up to -fold. the a -insulator decreased the number of il- -independent colonies by - % over the uninsulated vectors. the uninsulated vector-transduced, il- -independent colonies, were greatly expanded in culture with % il- over the a -transduced colonies (sffv, ΤΝs . . , a -tns . . : , , fold change, respectively). il- independence as a transformation event was confirmed in vivo by the development of overt leukemia (hyperleukocytosis, splenomegaly, bm-and extramedullary site-infiltration) in mice transplanted with the il- -independent and expanded colonies. summary/conclusions: under forced oncogenic conditions, the a insulator effectively protected a therapeutic vector from vector-mediated genotoxicity. a may serve as a safety feature in the construction of globin-sin-lvs. background: novel rare nucleotide substitutions are frequently identified in rhd, the gene encoding the immunogenic d antigen of the clinically-relevant rh blood group system, resulting in d variant phenotype. so far, it has been commonly accepted that substitutions of amino acids located either in a transmembrane or intracellular domain of the rhd protein induce weak d phenotype, i.e. reduced d antigen density at the surface of red blood cells. recently we showed by functional analysis using a "minigene splicing assay" (msa) that a decrease in d antigen expression may be due also to alteration of cellular splicing. aims: here we pay attention to the general disruption of this mechanism and the related phenotypic consequences in novel and previously reported single-nucleotide variations in rhd. we then sought to characterize functionally by msa novel candidate splicing variants in rhd. then we extended the project by studying prospectively all single-nucleotide variations reported in rhd exons, in order to assess globally the correlation between in silico prediction and functional analysis and to gain insights into the reliability of bioinformatics tools in line with the available phenotypic and/or clinical data. methods: seventeen novel or uncharacterized rhd variations, including missense, synonymous and intronic substitutions, were selected for functional analysis by msa in human cell models. a second set, including missense variants reported in rhd exons and , was further analyzed. functional data were compared with an algorithm derived from the quepasa method and tools available in the alamut suite. a published d protein model was used to visualize the location of missense amino acid substitutions and to assess potentially their respective phenotypic consequence. results: a novel "universal" minigene was validated and used successfully to characterize eleven novel splicing variants. those variants include six intronic and four missense substitutions close to the consensus dinucleotide splice sites, as well as the c. c>t synonymous variation associated with a weak d phenotype, which creates a de novo splice site. very interestingly, c. g>t (gly val; d-negative) disrupts totally normal splicing, while c. g>c (gly ala; weak d) and c. g>a (gly asp; d-negative) only partially alter the mechanism. further visualization of amino acid changes in a d model suggests that gly asp, but not gly ala, dramatically impair rhd protein structure/folding. subsequently the global analysis of mutations in rhd exons and by msa showed that inclusion of whole exon sequence in the mature transcript is significantly reduced in / ( . %) variants, which correlates well with the quepasa-like prediction (sensibility = . , specificity = . ). additionally, while normal exon inclusion is affected by c. c>g (weak d type ), the associated leu val substitution does not seem to be deleterious to the protein. summary/conclusions: on the basis of our functional data, this work shows that splicing disruption in the presence of rhd variants is a common and general mechanism that may act independently or synergistically with alteration of protein structure through amino acid substitutions, resulting in a weak d phenotype. it also illustrates the potency of combining functional tests and in silico tools towards the phenotypic/clinical interpretation of rare variants. background/aims: monetary and non-monetary incentives may support blood services in recruiting blood donors but have also been criticized for violating ethical principles and threatening blood safety by attracting donors with a high risk for infectious diseases. although incentives for blood donors have been discussed extensively over the past decades, empirical research on this topic remains limited. the aim of this study was to describe attitudes towards incentives for blood donors in europe and show donor return rates of compensated and non-compensated blood donors in south-west germany. methods: first, we present results of a secondary analysis of the eurobarometer, a nationally representative survey in all member states of the european union. in , participants were asked to evaluate eight potential incentives for blood donations as acceptable or unacceptable. these incentives were refreshments (e.g. coffee), physical check-ups (e.g. blood pressure), free (testing) laboratory parameters, free medical treatment, complimentary items (e.g. first aid kits), monetary travel reimbursements, additional cash reimbursements, and release from work. second, we conducted a retrospective analysis of donor return patterns of . compensated and . non-compensated donors who started donating blood at mobile and fixed donation sites. compensated donors received either eur as a regular reimbursement for their expenses (at a fixed donation site), in accordance with the german transfusion law, or a singular free entrance for an amusement park (at a mobile donation site). these compensated donors were compared with noncompensated donors who started either at a fixed or mobile donation site. chisquare statistics were used to test for differences in regular donor status after , , and months between compensated and non-compensated first-time donors. results: among german participants of the eurobarometer, physical check-ups ( . %), refreshments ( . %) and free (testing) laboratory parameters ( . %) showed the highest acceptance as an incentive for blood donors. travel reimbursements and free medical treatment were rated as acceptable by . % and . %, respectively. the lowest acceptance was for release from work ( . %), complementary items ( . %) and additional cash reimbursement ( . %). interestingly, the acceptance of potential incentives varies considerably across europe. in south-west germany, donor return of first-time donors differed significantly by type of compensation. among compensated first-time donors, who received eur as a monetary reimbursement, the proportion of regular donors after months ( . %) was significantly higher than among comparable non-compensated donors ( . %). however, a non-monetary compensation (free entrance) did not increase donor return rates. conclusion: the eurobarometer survey indicates that in most european countries monetary incentives are only accepted by a small minority. refreshments, checkups, free (testing) laboratory parameters and free medical treatment were most popular as incentives for blood donors. however, results of our four non-randomized donor samples from south-west germany suggest that monetary compensation may increase the likelihood of donors returning to fixed donation sites. regular monetary reward may therefore help to recruit regular donors especially in urban settings. incidentally, non-monetary compensation by a free entrance, however, may not affect donor return. background: previous research showed that whole blood (wb) donors that are temporarily deferred on-site are at higher risk of lapsing, yet very little studies have focused on differentiating the effects that different deferral reasons (e.g., travel, hemoglobin [hb]) may have on donor lapse. in addition, donor experience (i.e., firsttime or repeat donor) has also previously been found to affect donor lapse, yet novice ( - prior donations) and reactivated donors (returning after years of not donating) may respond differently. finally, it is currently unclear how and why different deferral reasons and donor experience interact in influencing donor lapse. aims: our aims were to understand ) how deferral reasons and donor experience jointly affect donor lapse, and ) why donors may lapse after temporary deferral. methods: a mixed methods approach was used. first, we used sanquin's donor database for a quantitative analysis of return behavior of all dutch wb donors between and (n = , ). the first wb donation for each donor was identified as the target donation. lapse was defined as non-return within a followup period of two years after the target donation. target donations included % new donors, % novice donors, % experienced donors, and % reactivated donors. deferral reasons included travel, hb, medical short-term (< days duration), medical long-term (> days duration), and miscellaneous. next, we interviewed temporarily deferred donors to understand the deferral process from their perspective. semi-structured interviews were used to understand how these donors cognitively and emotionally experienced on-site temporary deferral. we analyzed the interviews (using the framework approach, cf. hillgrove et al., bmc public health, ) to identify key topics and underlying themes. results: of target donations, % were deferred, mostly for travel ( %), medical short-term ( %), and hb ( %). survival and time-to-events methods showed that the different deferral reasons and donor experience levels differentially impacted donor return or lapse. importantly, experience and deferral interacted in influencing return (rate). for instance, deferred new donors were more likely to lapse than eligible or experienced donors (ors < . , p's< . ). even though deferral also affected return of experienced donors, this effect was smaller or even non-existent for certain deferral reasons (e.g., travel-and hb-related deferrals). qualitative results showed that almost all donors experienced temporary deferral as disappointing, particularly when it was unexpected (e.g., first-time deferral). not all donors (fully) understood the aims of deferral or how to prevent on-site deferral. donor beliefs about why deferral would lead to lapse were related to recurring deferrals, (mistakenly) interpreting deferral as permanent, or feeling all the effort did not pay off. summary/conclusions: reasons for temporary deferral differently impact risks of donor lapse at different levels of donor experience. for new donors all reasons for deferral are related to higher risks of lapse, whereas some reasons for deferral seem not to affect lapse among more experienced donors. unexpected or recurring deferrals may explain why donors lapse after temporary deferral. blood banks may tackle disappointment after deferral by explicitly showing that the donor is still valued, for instance by using personalized communication or offering an alternative good deed. background: blood donors experience a temporary reduction in their hemoglobin (hb) value after whole blood donation. in the netherlands, the hb value is measured before each donation, and a too low hb value (cut-off values: . mmol/l ( g/l) for men and . mmol/l ( g/l) for women) leads to a deferral for donation, in order to prevent iron deficiency and anaemia. the minimum interval between two donations is internationally set at weeks, but over time donors exhibit iron deficiency so that blood donors are temporarily deferred from donation each year. in the us % - % of deferrals are due to low hb, especially in women (editorial, transfusion, ) . due to the recovery process after each donation and the unobserved heterogeneity of donors, advanced statistical methods are needed to model the longitudinal data of hb values of blood donors. aims: to estimate the shape and duration of the recovery process of hb until the hb value has returned to its pre-donation level, to assess whether one can distinguish between donors with fast and/or slow recovery of their hb level and to predict future hb values. methods: the study is based on data of the donor insight study, which was a prospective cohort study performed by sanquin in the netherlands from to . we employed three statistical models for the hb value: (i) a mixed-effects models, (ii) a latent-class mixed effects model, and (iii) a latent-class mixed-effects transition model. in each model, a flexible function was used to model the recovery process after donation. the latent classes identify groups of donors with fast or slow recovery times, and donors whose recovery time increases with the number of donations. the transition effect accounts for possible state dependence in the observed data. all models were estimated in a bayesian way, using data of a sample of new entrant donors ( males and females). prior information from the clinical literature (boulton, vox sanguinis ) about the recovery process three days after blood donation was incorporated into the analysis since these values were not identified in the observed data. results: the results show that the latent-class mixed-effects transition model fits the data best. we also found that the recovery process shows a concave process (initially fast followed by slower recovery). the estimated recovery time is much longer than the current minimum interval of days between donations. namely, depending on the subgroup that the donor belonged to, males showed a recovery time of to days, while the estimated recovery time for females varies between to days. these results suggest that an increase of this interval may be warranted. summary/conclusions: the analysis shows the usefulness of the sophisticated statistical models that make use of historical information to model complex processes in time, in this case the hb trajectory over time across repeated donations. in addition, our results suggest a (much) longer time lag between subsequent donations to avoid anemia. background: complications of blood donation are known to reduce donors' return for future donation. the episode study (experience success in donation) showed that water drinking shortly before donation had an effect of % reduction of selfreported vasovagal reactions (vvr) in younger novice whole blood donors (wiersum-osselton, transfusion, ) . aims: in this study we analysed the return for a subsequent donation of the donors participating in the episode study. this was a predefined secondary outcome of the episode study. methods: the episode study was conducted in young (< years) whole blood donors making their first, second, third or fourth donation in geographically selected collection centres. the study interventions were: ml water drink, ml water drink or squeezing a ball (placebo intervention) during the wait after the screening interview and before phlebotomy, and a control group without intervention. participating donors were sent an online questionnaire about their experience within a week following their donation attempt. in the netherlands donors are usually invited for blood donation in accordance with hospitals' needs; the aim is to invite eligible donors at least once a year. donors were included in the return analysis if they had received at least one invitation within days after the index donation and we analysed their return for a donation attempt within days. associations with the interventions and donors' donation status, gender and reported symptoms at their index donation were analysed by calculating return percentage of eligible donors and by binomial logistic regression. results: out of the episode participants who had received an invitation, ( . %) returned within the study period. there was no difference in donor return between the two water groups. the likelihood of return was significantly increased in both water and placebo intervention donors compared to the questionnaire group (or . , % ci . - . and . , . - . respectively). return was slightly lower in women (or . , ) and lower in first-time donors (or . , . - . ) than after a nd - th donation. a staff-recorded or self-reported vvr at the index donation reduced donor return (or . , % ci . - . and or . , . - . respectively). other symptoms following donation were also associated with a lower return percentage. summary/conclusions: in this cohort of younger new and novice blood donors, . % returned for a subsequent donation. a vvr (either staff-recorded or selfreported) reduced donor return. donors who received a study intervention, either water or placebo, were more likely to return, whether or not they had suffered a vvr. it is conceivable that the mere fact of study participation could also have increased donor return, even in de questionnaire group; this will be examined in the total population of target group donors. background: the contribution of older blood donors to the blood supply is substantial. in australia, donors aged > years contributed % of all donations made in . however, with ageing, the general health status of older donors changes relatively faster, thus progressively affecting their ability to donate. an indepth understanding of the relationship between older donors' health status, future donation patterns, and risk of iron-deficiency could be of a great value to inform the blood service to predict the number of future donations, and manage the risk of iron-deficiency. aims: to understand the relationship between self-reported health, blood donation patterns, and the management of identified iron-deficiency in older blood donors. methods: we linked the sax institute's and up study baseline data collected between and to the blood service donation records, inpatient records, and medicare records*. the data-linkage was conducted by centre for health record linkage. using these linked data, we examined the relationship between health, donation patterns, and iron-deficiency and its management. results: we followed up , active whole blood donors for , eligible person-years (average age at recruitment . years, . % female, average follow up . years per-person). after adjusting for the effect of age, sex, body-mass index, education, non-english language spoken at home, country of birth, smoking, physical activity, regular use of multivitamins, alcohol consumption at enrolment, and total number of whole blood donations in the years prior to enrolment, participants with better self-reported health at recruitment showed significantly higher rates of donation. excellent, very good, good, and fair/poor health status donors made ( % ci - ), ( - ), ( - ), and ( - ) donations per person-years, respectively. iron-deficiency was identified in . % of donors in the study (n = , % ci . - . ) . sixty percent of those with iron deficiency (n = , , % ci . - . ) visited their general practitioner (gp) within days of the identification of irondeficiency, and . % ( % ci . - . ) of those visiting gp underwent further iron status examination and monitoring. after adjusting for several potential confounders including the total number of donations made during the follow-up period, excellent self-reported health status was independently associated with lower risk of iron-deficiency (p for trend = . ). summary/conclusions: information on self-reported health status can be an effective indicator to estimate the future donation yield of an older blood donor panel, and risk of developing iron-deficiency. donors with better self-reported health had a higher number of future whole blood donations and a lower risk of iron-deficiency. donors referred to gps for management of their iron status utilised the health services as expected, however there is an opportunity to improve their contact with their gps. * medicare records was provided by australian government department of human services. anaemia is a major public health issue, affecting % of the population worldwide according to the world health organization. iron deficiency is responsible for approximately half of all cases globally, with other causes including anaemia of chronic disease, other nutritional deficiencies, haemoglobinopathies, renal impairment, malignancy and bone marrow disease. in the elderly, where anaemia is even more common, the cause is frequently multifactorial. anaemia is associated with increased mortality, decreased cognitive and physical function, depressive symptoms and fatigue, particularly in older adults. poor outcomes have also been reported in anaemic patients with underlying comorbidities such as cardiac and renal disease, and cancer. within a hospital setting, anaemia is highly prevalent. preoperative anaemia, affecting up to % of patients, is associated with poor clinical outcomes including higher in-hospital mortality, longer length of stay and higher icu admission rates. anaemia management requires a proactive and multi-faceted approach, typically involving a multi-disciplinary team in which the transfusion practitioner plays a vital role. this includes screening of high-risk patients and pre-admission clinics to identify and manage patients at high risk of peri-operative anaemia. implementation of patient blood management (pbm) guideline recommendations has been shown to be effective to prevent and optimally manage anaemia within the community and hospital settings. the transfusion practitioner has key roles in the coordination, monitoring and auditing of pbm programs. active patient involvement and engagement of all members of the multidisciplinary team, including primary care clinicians, are also key to enhance the success of such programs. tp - the role of the transfusion practitioner in anaemia assessment and management: processes, tips and resources for creating background: patient blood management (pbm) is an evidenced based integrated multi-disciplinary approach aimed to improve clinical outcomes by effectively managing and conserving the patient's own blood, thus reducing unnecessary exposure to transfusion. pbm has the patient as the central focus with the aim being to improve their outcomes and include them in the process. pbm includes three pillars: ) optimising the patient's own blood, ) minimising blood loss and ) optimising a patient's physiological tolerance of anaemia. delayed assessment/management of anaemia contributes to increased health costs and unnecessary blood transfusions, and transfusion has been recognised to be associated with increase morbidity and mortality. the term transfusion practitioner (tp) includes those known as transfusion nurses, transfusion safety officers, haemovigilance officers, or patient blood management (pbm) coordinators. a key aspect of the role is driving and influencing clinical blood management activities to help align practice to internationally recognised guidelines and standards, including pbm. aim: to demonstrate the tp role in anaemia assessment & management and discuss strategies, processes, tips and resources for creating organisational and cultural change to implement pbm. context: literature outlines the importance of a multidisciplinary team to implement pbm related changes, and tps play a fundamental role within these teams to support 'buy in'. tps are seen as enablers, pulling resources together, engaging with those involved, providing education and facilitating change. they are often the ones to conduct audits, collating data and evaluating outcomes. approaches to implement pbm should be tailored to suit individual organisations. the authors will outline different approaches, highlighting where the tp can support or lead activities. one approach to anaemia assessment is to undertake an audit, examples of available tools will be shown. with this data, the tp along with the pbm team can explore options for corrective action. these could include interventions such as developing a pathway where all or a specific group of patients are assessed and or treated either at a preoperative clinic, or with their local general practitioner; through to more complicated strategies such as establishing anaemia clinics. the skills of the tp are a valuable asset to analyse clinical specialties/patient mix who should be targeted to achieve best outcomes, they know the organisation and as such are well placed to help develop a process/concept that will suit, and they can provide education and support to promote and embed these practices. conclusion: appropriate assessment and management of anaemia requires a multidisciplinary approach. the tp plays an active and crucial role in this team. examples of processes, tips and resources to support change and embed a pbm culture across the clinical spectrum will be shared. d-s - department of hematology and central hematology laboratory, inselspital bern, bern, switzerland immune haemolytic anaemia (iha) is characterized by an increased breakdown of red blood cells (rbcs) due to allo-and/or autoantibodies directed to rbc antigens with or without complement activation. clinical and laboratory signs of haemolysis in concert with the presence of a positive direct antiglobulin test characterize iha. alloantibodies formed during pregnancy and/or after prior transfusions may cause acute or delayed haemolytic transfusion reaction after transfusion of a rbc product incompatible with the specificity of the alloantibody. autoantibodies to rbcs reduce the survival of endogenous and hamper the recovery of donor rbcs after transfusion. lymphoproliferative disease, autoimmune disease, infection or drugs often cause autoantibodies to rbc, but frequently no obvious cause can be identified. besides the antigen specificity, the isotype critically determines the biological activity of rbc antibodies in vivo. the isotype defines the affinity to fc-gamma receptors on cells of the reticuloendothelial system as well as the capacity to activate the classical pathway of complement, igm being the most effective. antibody-mediated complement activation results in the opsonisation of rbc with c bc/c d with subsequent complement receptor-mediated removal by phagocytes (extravascular haemolysis). occasionally, complement activation proceeds via the activation of c to the formation and insertion of the membrane attack complex resulting in intravascular haemolysis. there is growing evidence that the innate immune system plays an important role in the pathogenesis of iha. the process of complement-mediated haemolysis results in systemic inflammation, which contributes to morbidity and mortality of patients suffering from iha. complement activation results in the release of anaphylatoxins, which are strongly vasoactive and mediate chemotaxis, inflammation and formation of radical oxygen species. release of cell-free haemoglobin and cell-free haeme upon haemolysis induces endothelial cell activation, no-depletion, cytotoxicity, ros formation and neutrophil activation. natural plasma scavengers, such as haptoglobin and hemopexin complex with their target molecules, cell-free haemoglobin and haeme, with subsequent removal of the complexes via cd and cd -mediated phagocytosis. although being positive acute phase proteins due to consumption the plasma scavengers become exhausted during chronic haemolysis thereby failing to prevent the adverse biological effects of cell-free haemoglobin and haeme in the circulation. inducible haeme oxygenase- (ho- ) is an efficient cellular scavenger by breaking down haeme into biliverdin with subsequent formation of bilirubin, co and ferrous iron with subsequent oxidation to ferric iron and storage by the ferritin h chain. ho- has an established role in the systemic protection from systemic inflammation induced by haemolytic and non-haemolytic diseases. the lecture will emphasise the role of innate immunity with a special focus on different plasma-and cellular systems involved in the pathogenesis of systemic inflammation in patients suffering from iha. d-s - understanding erythrocyte clearance c roussel, p amireault, p ndour and p buffet research and teaching, institut national de la transfusion sanguine, paris, france the clearance of erythrocytes is essential in physiology, disease and transfusion. elimination of erythrocytes altered because of senescence or pathological processes is expected to protect the microcirculation from obstruction by adhesive or rigid erythrocytes. it also contributes to the harmful consequences of anemia and hemolysis in hereditary and acquired red blood cells diseases as well as in conditions associated with auto-or allo-immunization. immunobiology has explored in great details antibody-mediated clearance of erythrocytes but conventional approaches may not be fully operational to explain delayed hemolytic transfusion reactions. some important clearance processes are independent from the recognition of molecules or antigens on the erythrocyte surface. increased erythrocyte stiffness triggers their clearance in hereditary spherocytosis, malaria and possibly also in the context of autoimmune anemia. knowns and unknowns on the mechanisms and sites of erythrocyte clearance will be presented based on a critical review of old and recent contributions. d-s - cardiovascular and endocrine-metabolic diseases and aging, istituto superiore di sanit a, rome, italy existing literature indicates that red blood cells (rbcs), beyond gas transport, exert a complex role in human physiology, being involved in many functions essential to maintain ion, metabolic and immunological homeostasis. rbcs display an immunomodulatory activity on adaptive immune cells by promoting t cell growth and survival and inhibiting activation-induced cell death. the balance between cell death and survival controls t cell homeostasis and anomalies in this balance account for diseases linked to excessive or faulty t cell growth. rbcs are able to modulate innate immunity by binding endogenous molecules such as chemokines and mitochondria-derived dna, as well as external agents such as pathogens. rbcs can also directly modulate innate immune cell activation or tolerance by controlling the maturation of the circulating pro-inflammatory subset of dendritic cells (dcs). these cells are potent inducers of primary antigen-specific t cell responses, produce tnf-a when stimulated by lps and are the principal il- p -producing cells among leukocytes. the pro-inflammatory capacity of circulating dcs is controlled by rbcs that are able to inhibit their maturation and il- production. in diseases characterized by local th inflammatory response such as psoriasis vulgaris and rheumatoid arthritis, pro-inflammatory dcs play a role in the induction and perpetuation of inflammation. collectively, literature data indicate that rbcs exert important modulatory functions that may result in immune activation or quiescence, depending on the environmental conditions. when rbcs encounter a microenvironment characterized by an intense production of ros, the rbc defenses get overwhelmed or are unable to counteract the new pro-oxidant status and become themselves a source of ros, which cause the generation of senescent signals on rbcs. the major feature of oxidized rbcs is the clustering and/or the breakdown of band . other features are the complexation of hb with spectrin, the loss of glycophorin a, the externalization of phosphatidylserine and the reduction of the "marker of self" integrin-associated protein cd . a similar senescence phenotype has been documented in rbcs during the storage period. oxidized, senescent or stored rbcs, due to surface antigen modification and to the release of pro-inflammatory molecules, fail to control immune cell homeostasis thus contributing to the perpetuation of inflammation and to the pathogenesis of immune-mediated diseases associated to oxidative stress, such as autoimmune diseases and atherosclerosis. our research group demonstrated that rbcs from patients with carotid atherosclerosis presented a senescent phenotype similar to that acquired by rbcs from healthy subjects following to in vitro oxidation. oxidized erythrocytes fail to control t lymphocytes apoptosis and lipopolysaccharide-induced monocyte-derived dc maturation, thus representing dangerous signals for adaptive and innate immunity and contributing to the pathogenesis of atherosclerosis. in conclusion, the crosstalk between rbcs and the immune system represents a mechanism to maintain immunological homeostasis. however, in high oxidative stress conditions, that can take place during a prolonged storage period or in particular diseases, rbcs can acquire a pro-oxidant behaviour and lose their functional and homeostatic features. by interfering in immune system homeostasis, rbcs become a potential tool that can be manipulated to improve or reverse pathological situations characterized by anomalies in the control of adaptive and innate immunity. transfusion therapy remains an important treatment modality for patients with sickle cell disease (scd). transfusions are given to lower the percentage of circulating sickle rbcs, and to decrease blood viscosity and have been shown in clinical trials to reduce the risk of stroke by %. however, many indications for transfusion in scd remain controversial partly due to insufficient randomized clinical trials data and in part because of our limited understanding of the complex pathologic networks leading to diverse disease complications in scd despite the common single mutation. similarly, we have incomplete mechanistic understanding of why chronic transfusion protocols must be continued for those indications supported by clinical data. the beneficial effects of transfusion therapy in scd need to also be weighed against potential transfusion risks including alloimmunization associated with lifethreatening delayed transfusion reactions, increased iron stores associated with increased oxidative stress and exposure to infectious agents. we believe that a deeper understanding of the benefits as well as harmful effects of transfusions is crucial to optimize our current transfusion therapy protocols in scd. this knowledge may provide highly needed guidance, which is currently lacking, for expansion or limiting existing indications for chronic transfusions in scd. d-s - treatment of thalassaemia department of pediatric hematology, ege university, faculty of medicine, bornova/ izmir, turkey thalassaemia is a devastating blood disease with a significant worldwide burden. annually, , children are born with a major thalassemia. life-time rbcc transfusions and iron chelation remain standard of care treatment in thalassaemia. transfusion therapy still account for significant iron overload related morbidity and mortality despite chelation therapy which is associated with poor adherence, safety concerns and varied efficacy. higher risk for transfusion transmitted infections (ttis) exists for thalassemia patients whose transfusion exposure sustains lifelong. although, the risk of transmission for traditional viruses is exceedingly rare in the modern era, emerging infectious diseases continue to be recognized as potential threats to transfusion safety. the inadequacy of blood safety points to the necessity for an additional layer of security for the blood supply in the developing world. pathogen reduction technologies for rbcc may imply a proactive, more generalized approach against new and re-emerging pathogens in the developed world and may be an ultimate safeguard for transfusion safety in the developing countries. rbc alloimmunization may become a major challenge in thalassaemia management. prevention is the key reducing the burden of alloimmunization. while the recommendation is to transfuse thalassaemic patients with c/c,e/e,kell compatible blood, it is not universally practiced. extended molecular rbc typing may be an appropriate adjunctive test in addition to serological typing before embarking on transfusion therapy. if a complete rbc antigen profile has not yet been performed in an alloimmunized patient, genotyping is the only option for accurate detection of rbc antigens that may guide the antibody identification. allogeneic stem cell transplantation (a-sct) is the only available curative therapy in children with hla matched sibling which is available to approximately % patients. in the absence of msd, mud transplant with high compatibility criteria has still limited experience. mismatch related, cord blood and haploidentical donor scts are considered experimental. a-sct carries a substantial risk of saes and mortality, both increasing with recipient age and disease severity. dfs is % in paediatric and % in adults. gene therapy for correction of the a-globin chain imbalance overcomes the problems of donor availability and immunologic complications associated with a-sct. multicenter clinical studies on gene addition therapy by using self-inactivating lentiviral vector are currently underway. recently, gene editing by either gene disruption or gene correction emerged as a potential alternative to gene addition therapy in beta-thalassaemia. a new era of novel therapeutics is unfolding in thalassemia management. several targets have been identified that can improve alpha/beta chains imbalance, ineffective erythropoiesis, or iron dysregulation and a number of those now have agents in preclinical and clinical development. hydroxyurea may improve globin chain imbalance and be beneficial for reducing or omitting transfusion requirement in selected group of patients. ruxolitinib has shown the limited effect on pretransfusion haemoglobin and reduction in transfusion needs, but allowed steady decrease in spleen volume that may serve for avoiding splenectomy in beta thalassaemia. luspatercept may restore normal erythroid differentiation and improves anaemia and hepcidin mimetics or tmprss inhibitors may modulates ineffective erythropoiesis by iron restriction and improves anaemia and organ iron loading. background: thalassaemia major (particularly b-type) and sickle cell disease (scd) are the commonest clinically important haemoglobinopathies, representing major sources of morbidity. recommended therapy is regular transfusion of safe, good quality blood, and monitoring of related complications. thalassaemia international federation (tif) guidelines, in place since , include strategies for precautionary measures and use of scientific progress in detection, inactivation and elimination of transfusion transmissible pathogens. antigen-matching strategies to avoid alloimmunization against rbc antigens and other measures including haemovigilance are key components for safe blood, alongside voluntary, non-remunerated blood donation and laboratory quality assurance programmes. aims: we present the contribution of tif and the greek experience in ensuring safety and availability of blood for thalassaemia patients applying internationally accepted standards and recommendations. methods: tif -a non-profit, patient-driven organization with national thalassemia associations in countries -promotes national control programmes for prevention and management contributing to the achievement of final cure. the main working methods are provision of education, expert support, networking, communications and projects to support improvements in the quality of health, social and other care. in greece, technical standards for blood donor selection and testing are applied in compliance with directive / /ec as well as haemovigilance programmes and traceability procedures for recording adverse reactions and events associated with the transfusion of rbcs (directive / /ec). pre-transfusion and transfusion measures recommended by the council of europe are applied. in particular, measures for transfusion of "the right blood at the right time for the right patient", leucodepletion, rbc washing and accurate cross-matching and antigen and antibody screening for an extended matching policy are practised. fresh (up to days old) rbcs are used. molecular testing for abo and rh d is performed in cases with blood group discrepancies. haemovigilance in greece covers % of total blood supply. data on ttis in , patients with thalassaemia and scd-thalassaemia in - are analysed. results: tti prevalence in thalassaemia syndromes was: hbv . % (occult type . %), hcv %, hiv . %, htlv . %, wnv . % and hev %. most frequent adverse reactions in - were allergic (incidence : ), non-haemolytic febrile reactions : , , "other" : , , alloimmunisation : , , taco : , , tad : , , tt-hev : , . hyperhaemolysis was diagnosed in two scd patients, delayed haemolytic transfusion reaction in one thalassaemia intermedia patient. trends in - show reduced incidence of alloimmunisation against rbcs. rates of allergic and pyrexial ars remained stable. no major abo incompatibility case was reported and no fatal transfusion reaction of transfusion has been recorded. summary/conclusions: blood safety in transfusion has significantly improved in high and upper-middle income but unfortunately not in lower and low income countries. blood shortages and lack of stringent protective measures for thalassaemia patients is the reality for many developing countries. tif focuses particular attention on the provision of support and the promotion of initiatives promoting the safety and adequacy of blooda key component of the lifelong management of patients with transfusion-dependent thalassaemia. background: b thalassemia is the most common group of hereditary hemoglobinopathy diseases. affected people with major thalassemia are dependent on regular blood transfusion which leads to iron overload. hepcidin is a peptide and an important regulator of iron homeostasis. expression of this hormone is influenced by polymorphisms within the hepcidin gene, hamp. aims: this study aimed to analyze the association of three polymorphisms in promoter of hamp, rs , rs , and rs with iron overload in major b thalassemia patients who do not respond to iron chelating therapy. materials and methods: a total of samples from major b thalassemia patients were collected. genomic dna was extracted and sequenced for snps rs , rs , and rs . statistical analysis was performed on ibm*spss* statistic using independent t test and fisher test. results: our analysis revealed statistically significantly difference between the level of cardiac iron concentration and c.- a>g variant (p = . ). for rs statistical analysis was on the edge of significant relationship between minor allele and serum ferritin (p = . ). all samples were homozygous for allele t of rs . summary/conclusions: different factors affect iron overload in thalassemia. our findings and others emphasize the role of hepcidin polymorphism as a key component in iron homeostasis. ten to twenty years ago, countries in south eastern africa faced the peak of the devasting hiv/aids epidemic leading to an up to years drop in general life expectancy. with the burden of hiv/aids falling mainly on the economically active population of young and medium-aged adults, the epidemic endangered social and economic stability in nations most heavily affected. today, despite aids still being a major cause of death in south eastern africa, the epidemic has become an example of public health gains that can be achieved through programmatic, evidencebased approaches that are endorsed by globally aligned policy and funding strategies. based on his work from lesotho, where one out of four among adults is infected by hiv, niklaus labhardt will take the auditors through the history of hiv programs in south eastern africa and show how innovative, pragmatic and evidence based implementation brought the region to a stage where the goal to end the aids epidemic by might be in reach. background: in france, the deferral for men who have sex with men (msm) was reduced from permanent to months in july . since this change has not impacted the residual risk (rr) of undetected hiv among blood donations, the ministry of health is considering a greater access of blood donation to msm. two scenarios have been studied: s . deferral of msm during the months preceding the donation; s . deferral of msm who have had more than one sexual partner in the months preceding the donation, similarly to all other blood donors in france. aims: to assess the impact of these two scenarios on the hiv rr estimated over the period july -december which is the baseline rr with the current month deferral for msm. methods: baseline hiv rr was calculated with the classical incidence-window-period method, where hiv incidence was derived from a detuned assay (eia-ri) detecting recent infections (≤ days) since all hiv- antibodies positive blood donations are tested with this test. the assessment of the impact of both scenarios on the baseline hiv rr was based on (i) data obtained from surveys among msm in the general population and in blood donors (compliance survey), to estimate the number of additional msm who would give blood in each scenario, and on (ii) hiv incidence estimate among these additional donors. this incidence was estimated: for s , from msm blood donors with the current deferral policy ( months) and for s , from monogamous msm of the general population. results: from july to december , / ( %) hiv- positive blood donors tested with the eia-ri were identified as recently infected, allowing to estimate the baseline hiv rr at . in million donations [ % ci: . - . ], or in , , donations. for s , the number of additional msm donors was estimated at and the number of additional hiv positive donations at . , resulting in an hiv rr of . in million donations [ % ci: . - . ] or in , , donations. for s , the number of additional msm donors was estimated at , and the number of additional hiv positive donations at . , resulting in an hiv rr of . in million donations [ % ci: . - . ] or in , , donations. sensitivity analysis shows that if both the number of msm and the hiv incidence were multiplied by . , the risk would be in , , donations for s , and in , , for s . summary/conclusions: for both scenarios, the hiv rr remains very low. for s ( -month deferral), the risk is identical to the baseline rr and is very robust to variations in the model parameters. for s (no more than one sexual partner, months), the risk is . higher than the point estimate of the baseline rr and sensitivity analysis shows that this estimate is less robust than for s , since the risk could be times higher than the baseline rr. for both scenarios, there was a modest increase in eligible msm donating. d-s - background: recruiting safe blood donors amongst the largest hiv-positive population in the world is a major challenge for south african blood transfusion services. south african donor deferral criteria and deferral periods for perceived high risk activities have evolved over time, but current risk factors for infection have not been formally assessed. in addition, most studies have reported risk factors for prevalent hiv infection whereas risk behaviours for incident infection are more informative as donations with these infections could occur during the window periods of available screening assays. aims: to identify the demographic and behavioural risk factors associated with incident hiv infection among blood donors in south africa. methods: we conducted a case-control study with incident hiv-infected blood donors compared to infectious marker negative controls. incident hiv cases and controls seronegative for hiv, hepatitis b and c viruses and syphilis were accrued from a donor pool covering of provinces in south africa. controls were frequency matched at a : ratio to cases on race, age and geography. incident hivinfections were hiv rna positive by individual donation nucleic acid amplification testing (id-nat; procleix, grifols) but antibody (ab) negative (prism, abbott) as well as those rna+/ab+ donors with recently-acquired hiv based on limiting antigen avidity (lag) assay results with normalized optical density values of < . . eligible cases and controls completed a confidential audio computer assisted structured interview (acasi) on motivations for blood donation and behavioural factors, including behaviours in the months before donation. frequencies and measures of statistical association for risk behaviours comparing cases and controls are reported after adjusting for multiple comparisons. results: from november to january , we enrolled incident hiv cases and controls; ( . %) cases and ( %) controls were ≤ years old. there were significantly more female cases ( . %) than female controls ( . %) (p < . ). significant hiv risk factors (all p < . ) reported within the -months before donation included: having a primary sex partner who is male; reporting increasing numbers of male sexual partners for both females and males; frequency of vaginal sex; frequency of vaginal sex without condoms; use of methods to clean, dry, or tighten one's anus before sex; and having visited a traditional healer for medical care. lack of medical aid (private health insurance) and reports of injury or accident with blood loss were also associated with an incident hiv infection. summary/conclusions: our study has identified a set of novel, putative risk factors for incident hiv infection among south african blood donors while confirming a number of previously known sexual risk behaviours. not having private health insurance and being injured may be markers of socio-economic context that place individuals at higher risk rather than behaviours that directly increase hiv transmission risk. the detection of risk behaviours by acasi in donors who passed predonation questionnaires and interviews suggests that acasi has the potential to improve risk behaviour identification. background: in france from to , among male blood donors (mbds) found hiv- positive at blood donation screening, % did not disclose any risk factor for hiv infection during post-donation interviews, while % reported having sex with men (msm), and % and % reported heterosexual sex (hts) and other risk factors, respectively. aims: in order to gain new insights into the risk factors for hiv- infection in mbds, we performed an hiv- genetic network analysis, including hiv- positive mbds and patients included in the french primary hiv infection anrs co primo cohort (pc). methods: mbds, who donated blood between and , and pcs, included between and , were studied. epidemiological data were collected by the french blood service (efs) upon blood donation or post-donation interviews for mbds, and upon inclusion for cps. viral strains were sequenced and genotyped in pol gene, and a recent infection assay was performed to date infection in mbds (recent: < months). a partial transmission network was computed based on tamura-nei nucleotidic distance (threshold for hiv- s/t b = . %; for non-b s/ t = . %) and assortative mixing was evaluated for mbds epidemiological data, including risk factors for hiv infection (msm, hts, others and unknown). selfreported data were then compared to assortativity-enhanced data. results: hiv- strains from mbds and pcs were linked into clusters including at least one mbd. primo-only clusters were excluded from the analysis. compared to mbds who did not cluster, those found linked to the network were younger ( vs. year-old; p < . ) and were more likely to have a recent infection ( % vs. %; p = . ). assortative mixing indexes showed that paired individuals were more likely to live in the same area (p < . ) and to have the same risk factor for hiv infection (p < . ) compared to a random distribution. imputing msm risk factor to non-msm individuals paired with msm changed the distribution of risk factors as follows: msm: % vs. %, hts: % vs. %, other: % vs. % and unknown: vs. %. summary/conclusions: after validating the assortativity of risk factors between paired individuals, and imputing msm risk factor to individuals self-reported as non-msm (including those with no identified risk factor), up to % ( / ) of mbds could be reclassified as msm. this is a worst-case scenario, as the network analysis does not exclude the possibility of one or several persons between two paired individuals (missing link). altogether, these results could help reevaluate the hiv residual risk linked to msm mbds, especially in the frame of the evolution of blood donor deferral criteria. background: although most individuals remain asymptomatic, htlv infection can lead to adult t-cell leukaemia/lymphoma (atll) and htlv- associated myelopathy (ham). the serious nature of these diseases, evidence of transmission via non-leucodepleted blood, and concern about a high prevalence among donors originating from endemic areas led to the uk blood services introducing universal blood donation screening in . monitoring through routine surveillance commenced and htlvinfected donors were invited to participate in the htlv national register cohort study to assess disease progression. these data together with evidence from lookback to previously untested donations and cost-effectiveness analysis were reviewed by an expert working group in and . aims: to describe the epidemiology of htlv among uk blood donors and evidence of disease progression from long term follow up of asymptomatic donors. methods: uk blood donations screened, and infected donors identified are reported to a national surveillance scheme. these donors are contacted, their results explained and information about clinical history and possible sources of infection are collected. where appropriate, htlv-infected donors are consented to the register, with participants completing a baseline questionnaire about their health, flagged in registries for cancer or death, and followed up about every - years. results: in the uk - , htlv-infected donors were identified. prevalence among new donors was steady around / donations. prevalence among repeat donors peaked in ( . / donations), with most in previously untested. from to , prevalence of . per , donations (average of one positive/year) was recorded. in , prevalence among new donors increased to . / , donations ( positives), with increased numbers associated with asian ethnicity and coinciding with an increase in collections from bame groups. overall, most were women ( / , %), uk-born ( / ; %) and htlv- infections ( / ; %). mean age was years. almost all positive donations were from previously untested donors ( / ), with seroconversion within a year of previous donation confirmed for only of the previously tested donors. typically, infections were associated with endemic countries (including caribbean region, west africa, iran, india and japan), acquired through breast feeding or from their heterosexual partner originating from these countries. interestingly, three were thought to have been infected through self-flagellation. a total of htlv-positive asymptomatic blood donors have already been recruited to the htlv national register, and during over -person years follow-up, none had developed atll or ham. summary/conclusions: over years of testing, few seroconverters were identified, suggesting very little ongoing transmission among uk blood donors. the lack of disease among the cohort study was also reassuring, although it is likely too early to detect associated symptoms of a slow progressing disease. recruitment to this unique dataset continues, also outside of the blood donation setting. as a result of these surveillance data, evidence from lookback, and cost-effective analysis, in nhsbt ceased to test donations from previously tested donors unless the donation was being used to manufacture a non-leucodepleted component. finland lies in northern europe between the °and °n latitude. the length of the country is km and width km. by surface area it is the fifth largest country in eu. the population of the country is . million resulting in the lowest population density in eu ( . inhabitants/km ). the whole country is inhabited, although most of the population is packed in the south. the climate of finland is influenced mainly by its latitude, but the warm waters of the gulf stream and the north atlantic drift current also play a role. due to finland's northern location, winter is the longest season. the southern portions of the country are snow-covered about three or four months of the year, and the northern regions for about seven months. long distances, low population density and the extreme climate give logistical challenges. it is estimated that these logistical costs can be as much as - % of gdp in finland. the finnish red cross blood service (frc bs) has been the nationwide blood service provider in finland since . frc bs collects annually about whole blood units of which % are collected in fixed sites and % in mobile sessions around the country. central activities (donor recruitment, medical support, production, testing, supply chain management, digital services and administration) are located in helsinki. management of transfusion is highly dependent on the logistical arrangements from blood donation sites to the central facilities and from the central inventory to the hospitals. the logistics is outsourced to three major partners all of whom have their roots in nationwide public transportation and logistics services. posti ltd is a state owned company having its roots in the national postal and telecom office. today it is the leading postal and logistics service company having the widest network coverage in finland. blood units collected at different fixed sites and mobile sessions are transported overnight by posti ltd to the frc bs central facilities by am on the day following the blood donation. posti ltd is also used for the regular deliveries of blood products to the hospitals. the other important partner is matkahuolto ltd, which was founded in the s to maintain bus stations and to serve as a common marketing company for the bus and coach services in finland. it maintains a nation-wide package delivery system based on the scheduled bus route network. matkahuolto ltd is used to transport donor testing samples from the donation sites to the central laboratory. by this arrangement it is possible to obtain most of the donor samples to the laboratory around midnight, which significantly speeds up the completion of laboratory results. the third logistics partner is jetpak finland ltd, which operates the air freight for the national flight company finnair. blood transfusion services can be managed centrally in a large sparsely populated country in a manner that is of high quality, safe and cost effective. however, the supply chain has to be planned carefully. background: elearning is a divisive topic. it is often criticised as an inferior form of education while simultaneously being promoted as a means to provide education to large numbers of people in a consistent, cost-effective manner. bloodsafe elearning australia (bea) is a government-funded blood transfusion education program that commenced in and provides courses in clinical transfusion practice and patient blood management (pbm) including: -clinical transfusion practice ( courses) -pbm: general ( courses) -pbm: medical ( courses) -pbm: acute care and surgical ( courses) -pbm: obstetrics and maternity ( courses) -pbm: neonates and paediatrics ( courses) aims: to determine the engagement, outcomes and impact of learning of bloodsafe elearning australia courses. methods: a retrospective analysis of user registrations, course completion records, course evaluation data and red cell usage in australia to determine learner demographics, and the impact on acquisition of knowledge and application to clinical practice. results: in the period from july to january : - , people registered as learners - , , courses were completed -these learners came from countries, with , ( . %) of them from outside of australia. analysis by profession shows that: - . % are nurses and/or midwives - . % are medical - . % are laboratory, anaesthetic technicians or other. analysis of user evaluation data (n = , ) from april to january shows that these courses have a positive impact, with . % of respondents stating they gained additional knowledge, . % able to make changes to clinical practice, and . % reporting that these changes will improve patient safety and outcomes. analysis of international participants shows greater benefits with . % gaining knowledge, . % able to change their clinical practice and . % believing this will improve patient outcomes. analysis of red cell usage in australia shows that since there has been a . % reduction in red cells issued. this has been achieved through a number of pbm activities including development of guidelines, research and audits, education, waste reduction strategies, and promotional campaigns. bloodsafe elearning australia courses on pbm were released in and are one part of this pbm activity, and it is notable that these courses have the widest reach as they are undertaken by a large proportion of doctors, nurses and midwives in australia who are not directly involved with the blood sector. stakeholder feedback shows that the program provides credible, consistent education that is cost-effective, reduces duplication, is 'best-practice' elearning, is readily accessible, and allows institutions to focus on the development of practical transfusion skills. summary/conclusions: this analysis shows that elearning is a well-accepted, wellutilised form of education for healthcare workers to learn about clinical transfusion practice and patient blood management, and learners gain knowledge that can change their clinical practice and improve patient outcomes. it is also likely that these courses have contributed to better utilisation of a scarce, freely-donated resource. this approach has global reach and availability, and is a cost-effective model for improving transfusion practice in the developing world by providing education for millions of healthcare workers. d-s - prospective platelet auditing: analysis of trainee compliance with guidelines pathology, columbia university, new york, united states background: apheresis platelets are a component product with high cost and limited supply. furthermore, there is a potential for severe transfusion reactions associated with this product such as transfusion related lung injury (trali), and sepsis due to bacterial contamination. therefore, transfusion guideline compliance is closely monitored by many centers. this quality assurance analysis describes our experience with prospective platelet auditing performed by physicians in pathology residency training. aims: this study aims to evaluate the ability of physicians in training to perform prospective auditing and compare policy compliance for different levels of experience. methods: this is a quality assurance analysis of a prospective platelet audit program for a -month period (january -december ). the blood bank paged the on call physician any time an order was placed for a patient with a platelet count of > , /ll, ≥ doses of platelets with no interim repeat count, or an unknown platelet count. audit records created by physician trainees in their first post graduate year (pgy ) were compared to subsequent years (pgy > ). information collected included the total number of doses requiring approval, number of products approved, training year for the approving physician, and transfusion indication. cost analyses assumed $ for a dose of platelets. descriptive statistics and comparative analysis using a pearson's chi-square were used with a difference of p < . considered statistically significant. results: there were platelet doses requiring approval with ( %) routed to the pgy group and ( %) to the pgy > group. there were ( %) ordered doses that were in compliance with hospital transfusion policy and ( %) that were not in compliance with hospital policy. of the appropriately ordered doses, the pgy group declined release of necessitating the clinical team to insist upon release without approval, and there were zero such instances in the pgy > group. when paged by the blood bank, pgy physicians approved product release not in compliance with policy for / ( %) doses while pgy > physicians approved not indicated products for / ( %) of doses (p < . ). products not indicated by hospital policy were held from release by pgy physicians for / ( %) doses and / ( %) doses by pgy > physicians (p < . ). the ordered doses not in compliance with hospital policy had an estimated cost of $ , . of this cost, there was a calculated $ , savings of products not released due to prospective auditing. there was an additional potential savings of $ , for products not indicated but released ($ , from the pgy and $ , from the pgy > group). summary/conclusions: despite a higher number of requests being routed to the more senior pgy > group, there were a disproportionately higher number of out of compliance platelet orders being released by the pgy group in addition to withholding needed products on several occasions. potential mitigation strategies for this could include a closer level of oversight for pgy physicians, and the potential monetary savings could justify a hiring a dedicated patient blood management team or quality assurance manager to monitor compliance and provide feedback to clinicians. d-s - what can we learn from how adverse events are detected? norwegian directorate of health, oslo, norway background: the primary aim of reporting systems, such as haemovigilance systems, should be learning and improvement and to identify risk areas, not simply counting errors. to understand and learn from adverse events the description of how, where and why they occur, and how they are detected, is important. to support our understanding, we use a predetermined classification that is required for reporting to eu, supplemented by classification suggested by ihn, who and ourselves. in we started asking the blood establishments what steps they would take to prevent recurrence of the event, and we added a simple classification to tell how the adverse event had been detected. aims: this study aims to analyze how different types of adverse events reported to the haemovigilance system were detected, whether the current quality management systems used in norwegian blood establishments had effective barriers and whether new barriers should be considered. methods: adverse events reported to the norwegian haemovigilance system in and were analyzed with focus on how the adverse event had been detected. in all cases classification had been performed by the reporter of adverse events and were confirmed, or reclassified if necessary, by the haemovigilance team before analysis. for analysis based on classification we used powerbi (microsoft). results: a total of adverse events were reported from norwegian blood establishments. all had been classified according to how the adverse event had been detected. twenty ( . %) adverse events were detected because of alarms or warnings from it-systems or equipment. routine checks by blood establishment staff detected ( . %) events and formal internal or external reviews detected one event. seven ( . %) events were detected because the donor became ill shortly after donation, but the illness was not caused by the donation. sixty-four percent of events were detected in a way that did not fit our present classification and hence were classified as "other". twelve out of wrong blood in tube were detected by an alarm from the it-system or routine check, as were six of events related to blood ordering, two of seven errors in testing, six of events where incorrect blood had been transfused, and eight of events related to donor selection. in reports human error was listed as the cause of the event and of these were detected by alarms or routine checks. summary/conclusions: detection of adverse events by alarms or routine checks are highly efficient when the blood establishment has historic data to check against, as exemplified with wrong blood in tube or a patient require irradiated blood components. when no historical data exists or when the quality management systems do not require routine checks, events are usually detected by chance. further analysis is needed to see if and where the quality management systems should be improved. the wide variety of adverse events can make it difficult to select which area to prioritize in the improvement work. results: the hb measurements from the finger prick were on average . g/l ( . %) higher than from the venous blood samples. the range of the difference was - -+ g/l. these results were used in order to add novel information to determine the measuring uncertainty of hb measurement in frcbs. in . % ( / ) of the donors in this study the venous hemoglobin measurements were below the cut-off point of donor eligibility. in those measurements the difference of the finger prick and venous hemoglobin measurement was at most + g/l. % of the hemoglobin results from the finger prick were in the range ae g/l compared to the venous hemoglobin results. % of the results from the finger prick were between ae g/l (the precision of the device) compared to venous hemoglobin results. in cases the difference between finger prick and venous measurements was outside standard deviations from the mean i.e. . % from the bottom (n = ) or top (n = ) of distribution. systematic errors were seen in some nurse's results both towards too low or too high hb result in the finger prick measurement and some nurses had random errors in both directions. the batch of cuvettes, donors' age, gender or the time of sampling were not detected to have an impact on the difference between finger prick and venous hb measurements in this study. summary/conclusions: the results of the poc measurements compared to the cell counter were in agreement with published data and with manufacturers' information on the device. the practical skill test is a workable way to develop competence and operations to measure the hemoglobin from the finger prick. it offers an opportunity to give personal feedback to nurses concerning their personal performance in the use of the current hb measurement technic. it also provided data on the accuracy of the poc method in the everyday donor selection process. background: whole blood donation has frequently been related to iron deficiency. a blood donor loses per donation about % (men) to % (menstruating women) of iron stores. to replenish the iron lost by blood donation in a donation interval of days, a donor needs to absorb . mg iron per day. this amount exceeds the reported maximal amount of absorbed iron of - mg/day, eventually leading to iron deficiency, with consequences such as donor deferral and possibly iron deficiency-related symptoms (decreased physical endurance, fatigue, pica, restless legs syndrome, and cognitive functions). since hb levels do not reflect donors' true iron status, measuring ferritin is a better way to detect low iron stores in whole blood donors. studies from usa and denmark showed that on the introduction of ferritin measurement with either extension of donation intervals or iron supplementation in case of low iron stores, deferral percentages for low hb declined in both male and female donors. aims: to gain more insight in iron status of whole blood donors during their donor career, how this affects donor health and which measures may prevent low iron stores in donors. methods: in the netherlands, sanquin blood bank is currently implementing a policy with ferritin-guided donation intervals. in brief, ferritin levels are measured in all new donors and in repeat donors every th donation or in case of an hb below the deferral threshold. donation intervals are extended if ferritin levels are < lg/ l, or ≥ and ≤ lg/l (for and months respectively). we anticipate that routine ferritin measurement will ultimately result in a lower prevalence of iron deficiency, less hb deferrals and improved donor retention. this will be further evaluated in a stepped wedge cluster-randomized trial 'find'em', which may also identify subgroups of donors prone to develop (symptoms of) iron deficiency. in addition, implementing ferritin screening may lead to a decreased donor availability. for this purpose, we modeled the impact of the implementation of our ferritin deferral policy on donor availability over time, which provides insight for both the expected size of the impact of the ferritin deferral policy and the time and rate at which this impact is expected to occur. this allows the blood bank to timely plan actions to counterbalance possible donor shortage and ensure an adequate blood supply. lastly, iron supplementation can be an alternative measure instead of donation deferral. as the used and recommended dosage of iron supplementation varies widely across blood services, sanquin is planning to start a new study in whole blood donors to gain evidence on the dosage and frequency of iron supplementation and its effect on ferritin and hemoglobin levels and donor health. results: the before-mentioned studies are ongoing and results will be expected from onwards. summary/conclusions: iron deficiency is a frequent side effect of whole blood donation. to prevent iron deficiency and its consequences, like donation deferral and health issues, more evidence-based insight in iron management of whole blood donors is being generated. d-s - superdonors -genetic risk profile and risk of low hemoglobin deferral background: no reliable method exists for stratifying new blood donors into those who can maintain sufficient hemoglobin (hb) levels and those who will be deferred because of a low hb (< . mmol/l [< . g/dl] for women and < . mmol/l [< . g/dl] for men). polygenic risk scores (prss) have shown great promise in predicting complex disease risk. prss could also prove useful for identification of donors genetically predisposed to low hb levels, and, thus, to an increased risk of deferral. aims: the objective of the study was to evaluate the association between prs (modelled to predict hb level as a quantitative trait) and risk of deferral as a binary outcome. methods: the danish blood donor study (dbds) is an ongoing nationwide blood donor cohort since with more than , participants. extensive genotyping has been performed on approximately , dbds participants using the infinium global screening array (illumina â ) and extended by use of imputing based on the pan-scandinavian reference genome. based on hb and genetic data on more than , icelandic individuals (an independent discovery cohort), we constructed different weighted prss for individuals from dbds. information on the donors' whole blood donations following inclusion into dbds unto end of was obtained from a nationwide donation database, scandat. the best predictor of hb among the nine prss was chosen and used in all subsequent analyses. we performed multilevel mixed-effects linear regression analysis with hb as outcome, and prs as factorized explanatory variable with cutoffs at , , , , , , , and th percentiles, respectively. moreover, the models had a two-level clustering on donor id and donation site and an id-specific random intercept; and further adjusted for: sex(binary), age(continuous), year of donation(factorized), and time since last donation (continuous). lastly, risk of deferral was evaluated in random effects logit models with similar covariables and clustering structure. results: mean number of donations per donor after dbds inclusion was . donations. generally, we observed a statistically significant positive association between prs(hb) and current hb levels. compared with donors in the - prs percentile group, donors below the th percentile had lower (- . hb mmol/l ( % ci: - . ; - . )) and donors above the th percentile higher (+ . hb mmol/l ( % ci: . ; . ) hb levels. in the random effects logit models we observed a marked increase in deferral risk with decreasing prs percentile strata. with the - prs percentile stratum as reference, donors below the th percentile and donors above the th percentile had odds ratios of deferral of or = . ( % ci: . ; . ) and or = . ( % ci: . ; . ), respectively. summary/conclusions: we found a statistically significant positive association between prs(hb) and hb levels and a markedly increased risk of deferral with decreasing prs(hb). from a scientific point of view, it is unsurprising that a genetic score for hb from an independent cohort is associated with hb in another cohort. however, from a practical perspective, prss may be the first step in a personalized donation approach to donors and their risk of deferral. background: individually calibrated inter-donation intervals for repeat blood donors have the potential to minimize the risk of iron related adverse outcomes (e.g., hemoglobin deferral or collecting a donation from a donor with low or absent iron stores) without unduly impacting the donated blood supply. machine learning has shown promise for personalized clinical risk assessment. aims: our aim is to use machine learning to develop donor-specific, personalized inter-donation intervals that minimize the risk of adverse outcomes while maintaining or improving the adequacy of the donated blood supply. methods: using a public use dataset from the reds-ii donor iron status evaluation (rise) study (cable, transfusion, ) we defined donor profiles with physiological measures including hemoglobin, ferritin and soluble transferrin receptor along with questionnaire responses regarding diet, reproductive health indicators, and demographics. we used these profiles ( features, , donations from , repeat donors) and the time until the next donation attempt to predict iron-related outcomes of the next donation attempt. possible outcomes were no adverse outcome, hemoglobin deferral, low-iron donation (ferritin < ng/ml for women and < ng/ml for men), or absent-iron donation (ferritin < ng/ml for men and women). we trained multiple machine learning models on , of the donations and selected the model with the best performance (lowest cross-entropy loss in cross validation). we assessed the best model's performance on a hold-out test set of donations, which were not used to train or select the model. we then used our model to generate risk estimates for these test donors as a function of days since their last donation, which varied from days to days. to show individual variation, we generated graphical representations of individual donors' risk over time. results: ferritin, log ferritin, body iron, and time since last donation were most useful for predicting iron-related adverse outcomes at the next donation attempt. the estimated risk of adverse outcomes at the next donation attempt varied considerably across donors. as expected, the risk of adverse outcomes days after the last donation was lower than the risk days after the last donation for most donors (risk of hemoglobin deferral decreased for % of donors; risk for low-iron donation decreased for %; and risk for absent-iron donation decreased for %). summary/conclusions: the risk of iron-related adverse outcomes as a function of time since last donation varies considerably between donors. machine learning models trained on relevant donor profiles can effectively estimate how an individual's risk will change over time. individual risk estimates could allow blood centers to protect highrisk repeat donors while continuing to allow more frequent collections from low-risk donors. further study is needed to ensure this approach works well for donor classes that are not well-represented by the rise dataset, to assess risk prediction outside of the physiological measures collected in the rise study, and to determine the viability of assigning an optimal inter-donation interval to a first-time donor using this approach. background: iron depletion is common among repeat blood donors, who contribute a large proportion of the blood supply in many countries. exogenous iron from multivitamins with iron or iron-only supplements helps prevent donation-induced iron depletion, but whether dietary iron protects against iron depletion in repeat donors has not been rigorously evaluated. available data from the reds-ii rise study in the us (cable, transfusion, ) and from the danish blood donor study (rigas, transfusion, ) suggest minor impact of dietary iron consumption on blood donor iron status in multivariable regression models. both studies, however, analyzed food items singly, such as beef or fish, rather than in aggregate, so precision was limited. aims: to evaluate whether a composite measure of dietary heme iron consumption, weighted for frequency and iron content, was associated with incident iron depletion among repeat blood donors. methods: a re-analysis of the rise cohort was undertaken to test the hypothesis that reported levels of animal protein consumption was associated with lower risk for incident iron depletion among repeat blood donors. the six blood centers participating in rise enrolled first-time and frequent donors for - month follow-up of donation frequency and iron status. a brief checklist of food categories was administered at baseline to assess frequency of consumption of several categories of animal protein that are rich in heme iron, the biochemical form of iron most readily absorbed. an iron composite score (ics) weighted for frequency and heme iron content was derived and subjects were grouped into tertiles (thirds) of ics. iron status was assayed at enrollment and study completion and at roughly one-third of donation visits in between. modified poisson regression with generalized estimating equations was used to generate risk ratios controlling for donation frequency and other covariates. results: of enrolled donors, were iron replete at baseline and completed the food checklist. the median value of the ics for each tertile (lowest to highest) was . , . , and . mg of heme iron weekly. these values are equivalent to approximately , , and servings of beef per week, or alternately twice as many servings of chicken or pork. across follow-up visits with iron outcomes assayed, almost % of donor visits were associated with intermediate iron depletion (serum ferritin < ng/ml) and . % with complete depletion of iron stores, representing serum ferritin < ng/ml. after controlling for demographic factors and donation frequency, the lowest tertile of ics was associated with a greater than fold higher risk for complete iron depletion during all follow-up visits (rr . , % ci . , . , compared to the highest tertile). summary/conclusions: in this longitudinal evaluation of dietary iron and iron status, blood donors with low intake of heme iron had an elevated risk for developing advanced iron depletion. these results suggest that blood centers should continue to recommend iron-rich diets to repeat blood donors. background: blood donors lose approximately mg of iron with every blood donation. as a result, frequent blood donors are at risk of iron deficiency and low hemoglobin (hb) levels, which may affect their health and eligibility to donate. lifestyle behaviors such as dietary iron intake and physical activity, may influence iron stores and thereby hb levels. gaining insight into associations between lifestyle behaviors and hb levels is valuable for blood supply organizations, as lifestyle behaviors can potentially be considered to prevent hb deferrals. examining the mediating role of ferritin, a measure reflecting iron stores, in these associations will help to gain insight into whether iron stores could be the limiting or enabling factor that links lifestyle behaviors to hb recovery after donation. aims: to investigate associations between lifestyle behaviors (dietary heme and non-heme iron intake and physical activity) and hb levels, and whether ferritin mediates these associations. methods: donor insight-iii (dis-iii) is a dutch cohort study of blood and plasma donors and included , donors. participants who were pregnant, had hemochromatosis, used iron supplements/medication, got a hysterectomy or bilateral oophorectomy were excluded (n = ). hb levels were measured in edta whole blood samples using a hematology analyzer (xt- , sysmex, japan) and ferritin was measured in plasma from lithium heparin tubes (architect ci , abbott laboratories, u.s.a.). dietary heme and non-heme iron intake (grams/day) were assessed using a food frequency questionnaire adapted to measure iron intake. moderate-tovigorous physical activity (mvpa, minutes/day) was assessed using the international physical activity questionnaire (ipaq)-short form. results: in total, , ( , female) participants were included. donors with higher intakes of heme iron had significantly higher hb levels (regression coefficient (b) ( % confidence interval ( % ci)) in men and women respectively: . ( . to . ) and . ( . to . ) mmol/l), independent of age, smoking, menstruation, number of donations in previous two years, donation interval, sedentary behavior, the other lifestyle variable (i.e. (non-)heme iron intake or mvpa), and initial hb level. non-heme iron intake was negatively associated with hb levels (- . (- . to - . ) and - . (- . to - . ) mmol/l for men and women respectively). ferritin mediated associations between dietary iron intake and hb levels (indirect effect in men and women respectively: . ( . to . ) and . ( . to . ) lg/l for heme and - . (- . to . ) and - . (- . to - . ) for non-heme). more mvpa was negatively associated with hb levels in men only (- . (- . to - . )), which was not mediated by ferritin. summary/conclusions: in conclusion, higher heme and lower non-heme iron consumption are associated with higher hb levels in donors via higher ferritin levels, indicating that donors with high heme iron consumption may be more capable of maintaining iron stores to recover hb levels after blood donation. more mvpa was associated with lower hb levels, although effect sizes were small, independent of ferritin. taking a donor's lifestyle behaviors into account may be useful in preventing low hb levels in blood donors. immune thrombocytopenia (itp) is still diagnosed by exclusion of other causes for thrombocytopenia. sensitive and specific detection of platelet autoantibodies may support the clinical diagnosis and prevent misdiagnosis of itp. for example, the direct monoclonal antibody immobilization of platelet antigens (maipa) assay, performed with in vivo sensitized patient platelets, offers platelet glycoprotein specific autoantibody detection with high accuracy. a drawback is that low platelet counts demand a large blood sample to have sufficient patient platelets available for analysis. circulating platelet autoantibodies are more difficult to detect by maipa; and may demand more sensitive detection platforms, such as those using surface plasmon resonance. in general, the presence of anti-gpiib/iiia, anti-gpib/ix and anti-gpv platelet autoantibodies is investigated. all these antibody specificities have been found in patients with itp. in itp, platelet autoantibody-mediated destruction via the spleen has been proposed; but also other mechanisms leading to low platelet counts in itp may play a role. inhibition of megakaryocytopoiesis by autoantibodies or by t cells has been suggested. in mice, gpib-directed antibodies induce loss of platelet-sugar epitopes, inducing hepatocyte-medicated platelet destruction. platelet autoantibodies can cause complement activation, which may contribute to platelet autoantibodymediated destruction. interestingly, we recently found that lack of detectable platelet autoantibodies is correlated with non-responsiveness to rituximab (cd moab) treatment in itp patients. in children with newly diagnosed and often transient itp, platelet autoantibodies of igg class or not often found, but of igm class are present for short duration. in conclusion, testing for platelet autoantibody characteristics and their pathologic effect may be helpful in establishing the diagnosis of itp and in choosing the best individualized therapy for itp patients. a-s - thrombopoietin receptor agonist (tpo-ra) treatment raises platelet counts and induces immunomodulation in immune thrombocytopenia (itp) jw semple , r aslam , e speck , j rebetz and r kapur lund university, lund, sweden st. michael's hospital, toronto, canada background: itp is an autoimmune bleeding disorder in which autoantibodies and/ or autoreactive t cells target the destruction of platelets and megakaryocytes in the spleen and bone marrow. several therapeutic options e.g. corticosteroids, intravenous immunoglobulins (ivig), rituximab and splenectomy are available for patients but inadequate efficacy, side effects and/or expense can make them undesirable. for the last years, tpo-ra e.g. romiplostim and eltrombopag have made a substantial contribution to the treatment of itp patient's refractory to first-line treatments. of interest, approximately % of patients that are tapered from tpo-ra therapy show a sustained response (e.g. a stable higher platelet count than before treatment). the mechanism of how tpo-ra induce these sustained responses is unknown. aims: to analyze the efficacy and immunomodulatory properties of a murine tpo-ra (amp , amgen) in a well-established murine model of itp that demonstrates both antibody-and t cell-mediated thrombocytopenia (chow l et al., blood ) . methods: platelet glycoprotein (gp) iiia (cd ) knockout (ko) mice were immunized with cd + platelets and itp was initiated by the transfer of their splenocytes into mice with severe combined immunodeficiency (scid). the scid mice were treated with either placebo or tpo-ra weekly and platelet counts and serum anti-platelet antibodies were measured weekly. results: in an initial pilot dose escalation study, control na€ ıve scid mice treated with a single subcutaneous bolus of different concentrations of murine tpo-ra ( , and ug/kg) had significantly higher platelet counts by h post infusion. in addition, compared with untreated mice, bone marrow histology revealed significantly increased numbers of megakaryocytes. maximal platelet count increases were observed with the highest tpo-ra dose and this dose was chosen to treat scid mice suffering from itp. when scid mice were treated with weekly injections of tpo-ra, platelet counts began to increase after weeks and were fully rescued to control levels after weeks post splenocyte transfer. of interest, compared with non-treated itp mice, serum igg anti-platelet antibody production in the tpo-treated mice was significantly reduced starting from two weeks post splenocyte infusion. summary/conclusions: these results suggest that murine tpo-ra is not only an efficacious therapy for murine itp but also induces immunomodulation indicative of immunosuppression. thus, this model may be able to elucidate the mechanism of how tpo-ra's induced immunosuppression in patients with itp. background: desialylation, the loss of sialic acid content on platelets (plts) glycoproteins (gps) was recently identified to contribute in immune thrombocytopenia (itp). however, the potential impact of autoantibodies (aabs) on megakaryocyte sialylation remains unclear. aims: to investigate the effect of itp aabs on plts and megakaryocytes (mks) sialylation and the subsequent impact on plt survival. methods: aabs from well-characterised itp patients induced gp-modifications were tested using a lectin binding assay. after incubation of mks or plts with itp or control sera, glycan changes were analysed by flow cytometry (fc). to investigate the impact of desialylation on plts life-span, the nod/scid mouse model was used. results: itp sera were investigated in this study. ( %) sera induced a significant increase in rca signal on plt surface compared to control sera from healthy donors (rca-mean fold increase (rca-fi): . , range: . - . , p = . ). in addition, ( %) sera caused higher ecl binding to test plts (ecl-fi: . , range: . - . , p = . ). injection of desialylating aabs resulted in accelerated clearance of human plts from the circulation of the nod/scid mice which was significantly reduced by a specific neuraminidase inhibitor that prevents background: autoimmune hemolytic anemia (aiha) is a rare autoimmune disease characterised by hemolysis associated with the presence of immunoglobulins (igg, igm, or iga) and/or components of complement system on red blood cells (rbcs), which is usually demonstrated by a positive direct antiglobulin test (dat). depending on the presence of an underlying disorder, aiha can be subdivided into primary and secondary and, by the temperature at which autoantibodies bind optimally to rbcs, into warm antibody aiha (waiha), mixed aiha (including both warm igg and cold igm antibodies), cold agglutinin disease (cad), paroxysmal cold hemoglobinuria (pch) and dat negative aiha. a frequent finding in immunohematology is the presence autoantibodies on rbcs without clinical symptoms of hemolysis that may later develop. aims: the aim of this study was to analyse serologic findings and transfusion support in patients with aiha and also to analyse dat positive patients without clinical symptoms. methods: we included data for all adult patients with aiha and dat positive patients without clinical symptoms diagnosed and/or treated at the university hospital centre (uhc) zagreb, croatia in the period between and . the diagnosis of aiha was defined by anemia with features of hemolysis (elevated bilirubin and/ or elevated lactate dehydrogenase and/or low haptoglobin level) and a positive dat. results: the data from patients ( % women) meeting the inclusion criteria was analysed. the mean age at the time of aiha was years (range - years). the mean hg level at diagnosis was . g/l. dat results were positive mostly with igg+c d ( %) or igg ( %). most patients had warm aiha ( %). other types of aiha diagnosed were mixed aiha ( %), cad ( %), pch ( . %) and dat negative aiha ( . %). in cases alloantibodies were detected with autoantibodies in the patient's plasma. patients were treated with corticosteroids as st line therapy and some with intravenous immunoglobulins (ivig). in severe or refractory patients rituximab and/or splenectomy was applied. a total of % of patients were transfused at a mean hemoglobin level of . g/l. during this period we detected dat positive patients without clinical symptoms. summary/conclusions: most patients from our study were diagnosed with warm type of aiha, followed by mixed type aiha and cad. on the other hand, pch and dat negative aiha were very rare, which is in concordance with relevant literature. most patients were transfused despite therapy used, which is not desirable in patients with aiha and should be better controlled, especially in moderate cases of anemias, where this is rarely necessary. a significant number of patients that were dat positive without clinical symptoms may later develop aiha and should be closely monitored. background: autoimmune haemolytic anaemia (aiha) is a decompensated acquired haemolysis caused by the host's immune system acting against its own red cell antigens. aiha is a rare disorder and although british society of haematology (bsh) guidelines for diagnosis and treatment were published in february , there is little evidence for clinical practice in the united kingdom. aims: to investigate the approach to diagnosis, investigation and management of patients with aiha in english nhs trusts. methods: a survey of diagnostic and management practice was designed, piloted and disseminated to clinical transfusion leads in all english acute nhs trusts from november to march . completion was by a consultant haematologist treating patients with aiha but a response that represented a departmental consensus was encouraged. results: responses represented % ( / ) of english acute trusts. median number of adults with aiha diagnosed annually was - . in the preceding years, % ( / ) recalled at least one patient who had died due to aiha. although % ( / ) undertook a bone marrow biopsy in all patients, % required additional features, mainly: neoplasia, age over or being treatment-refractory. for patients with suspected drug-induced immune haemolysis, % ( / ) would not organise confirmatory tests, either because it was considered unnecessary ( / ), or because clinicians were unsure how to access tests ( / ). when determining aiha subtype, % ( / ) indicated there were no circumstances in which they would undertake cold antibody testing (antibody titre and/or thermal amplitude), with considering this unnecessary and unsure how to access tests. in clinical scenarios of patients with aiha and dat positive to c d ae igg ae cold associated symptoms, up to % ( / ) of respondents would not test for cold antibodies. for first line treatment of primary warm aiha, mean duration of prednisolone mg/kg given before judging the patient refractory and reducing the dose was . weeks (sd . , range - weeks). second line treatment of choice was rituximab for % ( / ) of respondents and splenectomy for %. intravenous immunoglobulin and splenectomy were the most cited rescue therapies. for primary cold haemagglutinin disease (chad), first line treatment was rituximab-based for % ( / ) but single agent steroid for %. we also explored the potential for future audit and research. % ( / ) of respondents were able to identify patients with aiha who previously required transfusion. % ( / ) of respondents would consider supporting a registry of patients with aiha requiring transfusion. the key questions that respondents thought a registry should address were: morbidity and mortality, treatment response, and differences in the diagnosis and treatment of aiha subtypes. there was uncertainty over access to cold and drug-induced antibody tests and clinicians do not always conduct bshrecommended cold antibody tests for aiha with c d positive dat. initial treatment of primary warm aiha and chad broadly matched bsh guidelines although % ( / ) would continue prednisolone at mg/kg beyond the recommended days before starting a taper, with greater toxicity risk. summary/conclusions: the findings support the need for a range of research initiatives, including creation of an aiha registry. preoperative anemia is common and is associated with adverse outcomes in the peri-operative period. preoperative anemia also increases the risk of allogeneic blood transfusions, which may lead to increased perioperative mortality, increased hospital length of stay and infections. diagnosis and treatment of anemia is one of the tenets of patient blood management (pbm), along with reduction in unnecessary transfusions and diagnostic phlebotomy, as well as use of hemostatic agents to reduce bleeding among many others. effective pbm is multi-disciplinary, multi-modal, timely, individualized and patient-centered. early referral to pbm and multi-modal pbm interventions are associated with greater improvement in pre-operative hemoglobin. pbm has been shown to reduce transfusions and cost, while system-wide, multi-modal programs may also be associated with improvement in mortality. using examples from our local research and practice, i will discuss three aspects of pbm. iron and erythropoiesis stimulating agents (esa) are effective, safe and used extensively in management of pre-operative anemia. previous studies have questioned whether esa leads to increased risk of thrombosis, however, recent systematic reviews do not support these concerns. another pbm approach is to reduce bleeding during surgery by using hemostatic agents such as tranexamic acid (txa). txa reduces transfusion requirements in knee and hip arthroplasty, and is safe, widely available and relatively cheap. txa is effective in both anemic and non-anemic patients, making it an attractive universal pbm strategy. finally, recommendations and evidence-based guidelines on pbm exist, including the most recent international guidelines developed by the pbm international consensus conference. however, knowledge translation in pbm has been a problem and a number of barriers to its implementation have been identified. these include perceived or actual lack of expertise, time, and resources, as well as lack of physician and patient engagement. one way to address patient engagement is education through character driven animation and we are currently trying this approach. a-s - low vs . high hemoglobin trigger for transfusion in vascular surgery (tv): a randomized clinical feasibility trial (the tv trial) background: current guidelines advocate to limit red-cell transfusion during surgery, but the feasibility and safety of such strategy remains unclear as the majority of evidence is based on postoperative stable patients. aims: we assessed the effects of a protocol aiming to restrict red-cell transfusion during elective vascular surgery. methods: fifty-eight patients scheduled for lower limb-bypass or open surgery of abdominal aortic aneurysm were randomized to a low-trigger (hemoglobin < . g/ dl, mmol/l) vs. high-trigger (hemoglobin < . g/dl, mmol/l) for red-cell transfusion throughout hospitalization. intraoperative change in cerebral-and muscle tissue oxygenation was assessed by near-infrared spectroscopy. we used a nationwide registry to collect data on death and major cardiovascular events, which encompassed ( ) severe adverse transfusion reaction, ( ) acute myocardial infarction, ( ) stroke, ( ) new-onset renal replacement therapy, ( ) vascular reoperation, and ( ) amputation of the lower limb. results: the primary outcome, mean hemoglobin within days of surgery, was significantly lower in the low-trigger group: . g/dl vs. . g/dl in the hightrigger group (mean difference . g/dl; p = . , longitudinal analysis) as were units of red-cells transfused ( background: controlled non-hemato-oncological studies have consistently demonstrated a single-unit red blood cell (rbc) transfusion policy as well as a stringent hemoglobin (hb) rbc transfusion threshold to be safe and reduce blood product utilization. yet, it is unclear whether these conclusions also apply to the hemato-oncological patient population. aims: to quantify reduction of rbc blood product utilization by the introduction of a restrictive single-unit hb-triggered rbc transfusion policy among the inpatient hemato-oncological population. methods: under the liberal transfusion protocol, applied up till november , , standard double-unit rbc transfusion was indicated with a hb threshold ≤ . g/dl and/or anemia-related symptoms. following this date, the restrictive transfusion protocol was introduced involving a lowering of threshold to . g/dl and single-unit transfusion. for patients with an asa-score of ii-iii and iv, a hb threshold of respectively ≤ . g/dl and ≤ . g/dl applied. we evaluated rbc blood product utilization over a month period starting december , (liberal protocol) and december , (restrictive protocol) in all hemato-oncological patients admitted for chemotherapeutic treatment including hematopoietic stem cell transplantation (hsct) with an expected duration of neutropenia of ≥ days. analysis of categorical and continuous data was performed using the chi-square and mann-whitney test, respectively. results: during both observational periods, patients were admitted who in total received therapy cycles, including acute myeloid leukemia (aml) induction cycles and autologous hscts. distribution of indications of admittance, median age, duration of hospitalization and duration of neutropenia did not differ between both periods. during the restrictive period, in / ( . %) of transfusions the assigned hb trigger was adhered to. the percentage of single-unit transfusion episodes increased from / ( . %) to / ( . %) with the introduction of the restrictive protocol. overall, rbc blood product utilization per admittance did not reduce under the restrictive protocol (cumulative number of transfused rbc units . (interquartile range (iqr) . - . ) during the liberal versus . (iqr . - . ) during the restrictive period (p = . )). however, rbc blood product utilization per neutropenic day demonstrated a trend towards reduction: . (iqr . - . ) versus . (iqr . - . ) units per day during the liberal versus restrictive period, respectively (p = . ). this reduction was mainly attributed to autologous hscts during which rbc blood product utilization decreased from . (iqr . - . ) to . (iqr . - . ) units (p = . ), corresponding to a reduction from . (iqr . - . ) to . (iqr . - . ) (p = . ) units per neutropenic day. moreover, / ( . %) patients during the liberal versus / ( . %) during the restrictive period did not require rbc transfusion during admittance. consequently, stringent hb thresholds as compared to single-unit transfusions seem to more strongly impact rbc blood product utilization. summary/conclusions: a hb-triggered single-unit transfusion policy results in a strong reduction of rbc blood product utilization in the setting of autologous hsct. no utilization reduction was observed among other hemato-oncological inpatient populations receiving intensive chemotherapy. further improvement of protocol adherence rates could potentially increase the benefit of this blood saving strategy. a-s - assessment of hb content of packed red cells (prbc): is it time to label each unit with hb content? r jain , n marwaha and s sachdev transfusion medicine, aiims, new delhi transfusion medicine, pgimer, chandigarh, india background: in the current era of evidence based medicine and individualized care of patients, rbc transfusion continues to be administered on the basis of conventional wisdom and the notion of an average benefit per unit. the existing blood transfusion practice based on the "number of units transfused" ignores the fact that the total hb varies markedly among the individual rbc units. aims: the present study was aimed at estimating the hb content in packed red cell unit prepared by three different protocols from ml and ml whole blood collection in three types of blood donors: replacement blood donor (rd), first time voluntary donor (ftvd) and regular voluntary blood donor (rtvd). methods: a total of prospective blood donors were included in this study. three hundred whole blood collections were performed in each of the three groups of donors (rd, ftvd, and rtvd). within each group collections were done in double ml, triple ml and quadruple ml blood bags respectively. a pre-donation venous sample was drawn from sample collection pouch for analysis in hematology analyzer as reference method for hb concentration of donor. the hb content of packed red cell units were estimated after collection of representative sample from the blood unit. volume of prbc unit was estimated by the formula of weight of blood in prbc divided by specific gravity. the hb content in unit was estimated by the formula: hb content in unit = hb value of the prbc unit (g/dl) volume of prbc unit (dl). results: in this study the hb concentration (g/dl) was comparable among three types of blood donors except that rtvd had lower hb values when compared to rd (p = . ). hemoglobin concentration of prbc ranged from . - . g/dl; mean hb was . ae . g/dl. net hb content of prbc bag was lower in prbc prepared from rd as compared to ftvd (p = . ) and rtvd (p = . ). the hb content of prbc units prepared from ml collection ranged from . - . g and from ml collection ranged from . - . g. we observed a wide range of net hb content in the prbc units and the correlation coefficient showed the strongest association of net hb content of the prbc unit with the overall volume of prbc (r = . , p = . ).higher volume prbcs have more hb content. volume of prbc bags in the study ranged from ml to ml (including both and ml collections). summary/conclusions: the present study shows that labelling hb content of the prbc unit help in better inventory management for patients. the hb content may help in decision making for release of units for paediatric/low weight versus adults/ higher weight patients. adopting a policy of optimizing dosage of rbc transfusion could have the potential to significantly improve rbc utilization and decrease patient exposure to allogenic blood. this would help further in the clinical transfusion practices based on evidence. a-s - nv more, p desai, s rajadhyaksha, a navkudkar and n deshpande transfusion medicine, tata memorial centre, homi bhabha national institute, mumbai, india background: red blood cell (rbc) transfusion is an important medical therapy benefiting the patient in a wide spectrum of clinical setting. critically ill intensive care unit patients in particular, as well as medical and hemato-oncology patients, are among the largest group of the user of rbc. periodic review of blood components usage is essential to assess the blood utilization pattern in any hospital or health care set up. our institute is a bedded tertiary care oncology centre with approximately , to , rbc transfusions annually. these transfusions are required in various stages of patient treatment like chemotherapy, radiotherapy, surgical and palliative care and there are established guidelines by the institute to be followed by clinicians. aims: to study clinical practices of rbc transfusions based on indications and to evaluate appropriateness of rbc utilization practices at the institute. methods: this was a prospective observational study, started after approval from institutional ethics committee. total of rbc transfusion events in adult patients over a period of four months were included and analyzed as per institutional guidelines for their appropriateness. details of transfusion events in form of pre transfusion hemoglobin, indication of transfusion, type of request, number of unit requested and issued, time of issue, site of transfusion and adverse reactions, etc were obtained from department of transfusion medicine records. overall statistical analysis was descriptive using spss software. chi-square test in cross tables was applied to see the relationship between different variables and considered significant if p-value was < . . results: total rbc transfusion events for patients were analyzed. there were ( %) events in patients of medical oncology and ( %) in patients of surgical oncology. maximum transfusions were received by patients in age group of to years ( %). total % of transfusion events were appropriate as per institutional guidelines. all transfusions administered in operation theatre were found to be appropriate with p value < . . inappropriateness was more %( / ) and significant in daycare setup (p < . ). anemia was the most common indication of rbc transfusion observed in % of events ( / ). total % rbc transfusions were given as planned and % as urgent transfusions. most common adverse transfusion event observed was allergic reaction in . % of total transfusion reactions. summary/conclusions: clinical practice of rbc transfusions in our hospital was largely found to be appropriate and rational with adherence to institutional guidelines. blood utilization audits should be conducted regularly by transfusion services and results should be discussed with clinician for ensuring judicious use of the scarce resource. the concept of transfusion safety officer (tso) can be introduced for better coordination between clinicians and blood transfusion services to improve practices. a-s - paul-ehrlich-institut, langen, germany on a global scale, blood services are quite diverse in regard to aspects like organisational structure, regulatory background, donor populations, donation rates or pathogen epidemiology. the world health organization (who) recognizes blood and blood products as essential medicines and provides guidance to member states for various aspects like blood regulation, best practices in blood collection and transfusion, or screening parameters. more recently a who guideline on residual risk of transfusion associated infections has been established which may facilitate decision-making for the most appropriate screening algorithms. it emphasizes the need for regional evaluation of screening assays and regulatory control of blood-associated ivds. background: babesia, a protozoan parasite that infects red blood cells, is a leading infectious cause of mortality in u.s. transfusion recipients. babesia is usually transmitted through the bite of an infected tick but may be transfusion transmitted (tt) or transmitted from mother to child during pregnancy. babesiosis is a world-wide disease; the ticks that carry babesia have a global distribution. babesiosis has been reported throughout europe and in canada, korea, india, and japan. prospective testing of blood donations in endemic areas of the u.s. revealed . % of donors were positive for babesia dna or antibodies (moritz, nejm, ) aims: -to report results of ongoing babesia clinical trial -to explain significance of babesia as a tt infection methods: in cobas â babesia for use on the cobas â / systems, is a qualitative polymerase chain reaction nucleic acid amplification test, developed to detect in whole blood (wb) donor samples the babesia species that cause human disease: b. microti, b. duncani, b. divergens, and b. venatorum. testing began in october under a u.s. fda-approved investigational new drug application. wb was collected into a proprietary medium that lysed red blood cells and stabilized babesia rna and dna. donations were collected in states with high, low, and no babesia endemicity and screened as individual blood donor (idt) samples. reactive index donations were retested in simulated minipools of (mp ), plus idt replicates with cobas â babesia. reactive index donations were also tested with validated alternate babesia nat and for b. microti igm and igg antibodies. donors with reactive results were invited to enroll in a follow-up study to test for additional evidence of infection. results: to date, , valid donations have been screened with cobas â babesia, and ( . %) were reactive. of ( %) initially-reactive donations were confirmed to be positive for babesia with a positive alternate nat or serology result. of ( %) confirmed-positive donations was collected in a state with low babesia endemicity (pennsylvania), and ( %) was collected in a state where babesia is not considered endemic (iowa). of ( %) confirmed positive donations were collected in states with high endemicity. of ( %) confirmed babesia-positive donations were detected in late fall or winter. all ( %) confirmed babesia-positive donations were reactive in mp . serology results are available for of confirmed-positive donations: at index, of confirmed babesia-positive donations were only igg-positive, while none were only igm-positive; were positive for both igg and igm. of the confirmed-babesia positive donations were negative for both igg and igm antibodies. cobas â babesia showed an overall specificity of . % ( , / , ; % exact ci: . %> %). summary/conclusions: the cobas â babesia test successfully identified babesiapositive donations, including confirmed-positive donations with no igm or igg reactivity. donations were collected in states considered low-or non-endemic for babesia. confirmed-positive donations were collected outside of the summer babesia season, when most clinical cases occur. screening with cobas â babesia continues in several laboratories. cobas â babesia is not fda licensed or available commercially. background: babesiosis in humans is caused by the erythrocytic protozoan parasite, babesia microti which is transmitted by tick bites, but is also transfusion transmitted. although frequently asymptomatic or presenting with flu-like symptoms in a normal host, if immunocompromised infection can lead to severe complications and death. b. microti is endemic in the north eastern/upper midwest united states where partial testing of donations has been implemented. in canada, a study of~ , donors did not identify any b. microti antibody-positive samples, suggesting low risk at that time, but risk should be monitored. aims: to evaluate the prevalence of b. microti-positive donations in potentially atrisk areas in canada. methods: between july and november , , blood donor samples were selected from sites near the us border. minipools were tested for b. microti nucleic acid by transcription mediated amplification (tma) using the procleix â babesia assay on the panther â system with individual testing on reactive pools. reactive donations were also tested by b. microti-specific: american red cross (arc) igg immunofluorescence assay [ifa] and imugen ifa/pcr. a subset of , tma-negative samples, primarily from the province of manitoba and eastwards to nova scotia, were tested for b. microti antibody using the arc ifa and if positive, the imugen ifa/pcr. donor age, sex, donation status, residential location and collection site location were recorded. donors who tested reactive/positive were informed, deferred and asked about risk factors (possible tick exposure and travel within canada, the usa and elsewhere, history of symptoms) and a follow-up sample was requested for supplemental testing (tma, arc ifa). reactive donations were removed from inventory. results: the , donor samples were proportional to collections in target geographic regions. age group, sex and donation status were also similar to the donor base in the collection areas. one sample from winnipeg, manitoba was tma reactive and antibody positive on supplementary testing. the donor did not remember symptoms or spending time in wooded areas. he visited the city of fargo, north dakota, usa. the subset of , samples tested for antibody were also proportional to collections in the targeted areas. four antibody-positive samples were identified from mid-september to october , all in south western ontario near lake erie. none were tma reactive. three were interviewed and none remembered any symptoms, any likely tick exposure, or relevant travel within canada or the usa. summary/conclusions: this is the largest b. microti prevalence study in canada. the results indicate very low prevalence with only tma-confirmed-positive donation of , tested. the donor was from the only region in canada where one autochthonous human case has been reported and active tick surveillance identified b. microti positive tick populations. seropositive donations in south western ontario may suggest low prevalence in that region, but interpretation is less certain due to lack of corroborating supplementary results or case history. given the close proximity to the us border, forgotten us travel should not be ruled out. a-s - background: the protozoan parasite toxoplasma gondii is prevalent in animals and humans worldwide. wild and domestic felids are the definitive hosts, and homoeothermic animals serve as the intermediate ones. after primary infection, the parasite persists lifelong within latent tissue cysts. transmission is by ingestion of undercooked or raw meat infected with cysts, by ingestion of food or water contaminated with oocysts, or transplacentally. however, it can also be acquired by blood transfusion and organ transplantation. toxoplasmosis can be a severe disease in immunosuppressed people and neonates whose mothers have acquired primary infection during pregnancy. aims: there is no information about the specific epidemiology of t. gondii infection in blood donors in portugal. therefore, we sought to determine the seroprevalence of t. gondii and associated risk factors in the population of blood donors in portugal. methods: between september and july , blood donors who attended the portuguese blood and transplantation institute blood banks located in oporto, coimbra and lisbon, and also at regional blood collection meetings, were invited to participate in the study. a written informed consent was obtained and a questionnaire about socio-demographic and behavioural variables was answered. sera were assessed for igg antibodies to t. gondii by a modified agglutination test (mat) commercial kit (toxo-screen da â biom erieux, lyon, france). results: of the blood donors (mean age . ae . ; range - years old), . % were positive for antibodies to t. gondii. when questioned about toxoplasmosis, almost half the blood donors did not have any knowledge about the disease. the centre of portugal had the highest seroprevalence ( . %) followed by the north ( . %) and the south ( . %). blood donors living in rural areas had a significantly higher seroprevalence (p = . ) than those living in urban areas. seroprevalence increased with age, with the highest seroprevalence ( . %) found in the age group of - years old (multiple logistic regression [mlr]: or = . ; ci: . - . ; p < . ), and decreased with educational level (p < . ). engaging in soil-related activities (gardening or agriculture) was significantly related to t. gondii seropositivity (p = . ). regarding water consumption, untreated sources (even though including mineral and tap water) was confirmed as a risk factor (mlr: or = . ; ci: . - . ; p = . ). other behavioural and eating characteristics, including cats in the household, eating raw or undercooked meat, processed pork products, or not washing raw fruit and vegetables before eating, were not associated with t. gondii infection. summary/conclusions: the risk of t. gondii transmission through blood transfusion is low, and serologic testing of antibodies, with exclusion of blood donors, appears not to be feasible. immunosuppressed individuals, organ transplant patients and pregnant women, should receive t. gondii antibody-negative blood components for transfusion. this study explored the epidemiology of t. gondii in portugal thus providing useful information on the seroprevalence and potential risk factors for t. gondii transmission. information regarding toxoplasmosis and its prevention could be promoted by medical and public health authorities among blood donors, and also the general population, when addressing policies, and designing screening programs, for monitoring and controlling infection and disease in portugal. a-s - who is syphilis testing excluding? c reynolds , c pearson , k davison and s brailsford nhsbt/phe epidemiology unit, nhs blood and transplant nhsbt/phe epidemiology unit, public health england, london, united kingdom background: screening for treponemal antibodies to detect syphilis in blood donors has been in place in england since the s. there have been no reported syphilis transfusion transmissions in england since records began in part due to sensitivity of the organism to cold storage. since we have specific tests in place for other sexually transmitted infections such as hiv and hepatitis b virus (hbv), the utility of syphilis screening is often questioned. however, it may be a useful proxy for higher risk behaviours particularly following shortening of deferrals for higher risk sexual behaviours from to months in november and against a background of increasing infectious syphilis in the general population. aims: here we describe the epidemiology of recently-acquired syphilis in blood donors in england compared with hiv and acute hbv infection between and . methods: monthly donation testing results are collected from the nhs blood and transplant (nhsbt) screening centres and reference laboratory. the demographics, possible sources of infection, and compliance to donor selection in confirmed positive donors are collected by proforma at post-test discussion with the nhsbt clinical team. recent syphilis is classified as igm positive and/or recent history including a negative donation within months for regular donors. results: between and there were recent syphilis cases, hiv and acute hbv infections identified by donation screening. recent syphilis rates per , donations increased from . to . whereas hiv decreased from . to . with less than positive donations in . acute hbv rates rose slightly from . to . in . males outweighed females accounting for . %, . % and . % of cases of recent syphilis, hiv and acute hbv respectively. nearly a quarter of cases of recent syphilis and hiv were seen in donors below years old. of the male donors with recent syphilis, . % reported sex between men and women (sbmw), . % sex between men (sbm) and . % did not report a risk. this contrasted with hiv where . % of male donors reported sbm, just . % not reporting a risk. overall , and males with recent syphilis, hiv or acute hbv respectively were non-compliant to the sbm deferral in place at the time of donation. in , donors with recent syphilis aged - years (median years) were excluded from the donor pool, including non-compliant to the sbm deferral. there were fewer than hiv cases identified in , all over years old, all compliant, reporting sbmw. of the hbv acute cases in , were male, all but one in the and over age-group. summary/conclusions: over the year period demographics of recent syphilis cases appeared similar to hiv with highest rates in young males, albeit lower proportions reporting sbm. following the switch to a month deferral, hiv case detection continued at low level, while syphilis screening continued to exclude higher numbers spanning all age-groups, potentially at risk of other sexually transmitted infections, including non-compliant donors. background: globally, an estimated million blood donations are given annually. in the blood service we are obliged to monitor donor health and ensure that blood donation is safe. in recent years, large-scale blood donor cohort studies in several countries have increased our knowledge on health effects of blood donation. health concerns relate both to immediate side effects like fainting and to possible long-term health issues related to repeated blood or plasma donation. the studies have provided us with data that can now help us introduce an evidence-based individualised donor care -a parallel to personalised medicine. individualised donor care in the management of iron depletion: studies have shown that a large percentage of our frequent whole blood donors, especially young women, are iron depleted. iron depletion is a strong predictor of deferral for low haemoglobin but has also been associated with e.g. restless legs syndrome and lower birth weight in children of frequent donors. the risk of iron deficiency can be mitigated by ferritin-guided prolongation of interdonation intervals or by iron supplementation. prolongation of interdonation intervals can challenge our inventories. iron supplementation, on the other hand, may give gastrointestinal side effects and other effects have been proposed as well, e.g. the masking of malignant disease and increased iron availability with subsequent risk of infection. in a large study we found that iron supplementation is not associated with increased risk of infection. what is the optimal balance between iron supplementation and prolongation of interdonation intervals? a growing number of blood services have implemented various flavours of iron management regimens generating more results. moreover, genetic studies in e.g. the uk, us, holland, and denmark can help us to find donors at high risk of iron depletion or low haemoglobin. we can use all these data in a big data approach in the pursuit of an individualised risk assessment model. other risks for blood donors: the presentation will also cover other risks associated with donation. new studies identify predictors of fainting after blood donation and also new interventions to prevent fainting. the global demand for plasma derived medicinal products has increased severalfold the last years. plasma donors are bled up to times per year in the us. very little is known about the health effects of frequent plasma donation. we know that immunoglobulin levels decrease with frequent donation but how does this affect health? summary/conclusions: the precautionary principle mitigates risk through early intervention prior to evidence. we tolerate next to no risk of transfusion-transmitted infectious diseases. the health of the blood donors, however, has not been protected similarly. we owe to our whole blood and plasma donors to investigate health effects of blood donation and ensure their safety. while the first attempts may not be perfect, we now have the tools to construct models for individualised donor care. background: in , the isbt, aabb, ihn and eba jointly issued the standard for surveillance of complications related to blood donation which categorized donor adverse events (dae) into categories ( subcategories) defined by specific criteria. severity and imputability were briefly described but were optional. subsequent validation of these categories demonstrated consistency in categorizing reactions, but wide variation in assignment of severity. in , with international input, the aabb donor biovigilance committee developed a severity grading tool (sgt) using a recognized medical adverse event grading system in which neutral grades replace subjective terms (mild, moderate, severe). aims: a large us blood collection establishment (bce) applied the draft sgt to assess its use in real cases of dae. methods: we performed retrospective analysis of all allogeneic and apheresis needle-in donations between / / to / / . severity grading was assigned based on criteria defined by the sgt. database review of dae was performed, and each event was assigned a grade based on the type of outside medical care (omc), and on specific key search terms. search terms for omc included emergency room, emergency medical response, urgent care, healthcare professional, and hospital admission. additional specific key search terms included fracture, concussion, laceration, dental injury, surgery, and hospitalization. since duration and activities of daily living (adl) limitations were not captured in our dae database, cases in our dae claims' database were reviewed. case files of events classified as grade or higher were individually evaluated by a physician for grading accuracy. results: in , , needle-in collections, , dae were graded for severity. the majority ( , , . %) were vasovagal reactions (vvr), followed by , apheresis-related ( . %), , needle-related ( . %) and allergic ( . %) events. the majority of dae were grade accounting for . % of all dae, followed by grade ( . %), and grade ( . %). there were grade and no grade dae. among the vvr, . %, . %, . % and . % were grade , , , and respectively. grade vvrs included concussions, fractures, dental injury, and pre-faint and fainting events requiring hospitalization for work-up. two grade vvrs involved falls resulting in intracranial hemorrhage requiring immediate medical intervention. for allergic and apheresis dae, there were only and grade reactions respectively, and no grade or events. needle-related dae included . % grade , . % grade , . % grade , and no grade events. of the six grade needle-related dae, were nerve irritations lasting > months, and were dvts requiring hospitalization. summary/conclusions: the sgt provided consistent assignment of severity for the majority of dae, based on outside medical care and specific key search terms. assignment of severity based on impact on activities of daily living or on duration of injury/condition requires tracking over time making such assignments more difficult; modification of our dae tracking database and claims database to capture adl and duration should improve severity assignment for such cases. background: the international haemovigilance network (ihn) has collected aggregate data on complications of whole blood and apheresis donations from member national haemovigilance systems (hvs) since . aims: we analysed the data collected in - in order to learn from the data and consider future improvement of data collection. methods: national hvs entered annual data on donor complications in the passwordprotected "istare" (international surveillance of transfusion adverse reactions and events) online database. from the donor complication spreadsheet allowed entry of separate data for whole blood donation (wbd) and apheresis, but also provided an option for entering data for all donation types. annual numbers of whole blood and apheresis donations were also collected. the harmonised international standard definitions were implemented in . reactions were captured according to severity level (mild, moderate, severe) but without distinction between donor sex or first time vs repeat donation. extracted data were used to calculate national and aggregate donor complication rates (generally per donations). results: twenty-four hvs provided figures for donations and donor complications for one or more years (median years per country was , iqr - ). the total number of country years (cy) was , covering million donations. the overall complication rate was . / donations and the median country rate was . complications/ donations (iqr . - . ). rates were generally consistent within a hvs from year to year but showed considerable variation between hvs; this was also the case for reactions classed as severe. not all countries differentiated between mild and moderate reactions and some reported all reactions under a single severity level. vasovagal reactions were the most commonly reported complication: overall . / donations, median country rate . / donations (iqr . - . ). rare and apheresis-related types of complications such as generalized allergic reaction ( . per , , cy), and major blood vessel injury (category available since ; overall . per , , cy) were only reported occasionally. eighteen of the hvs provided separate data for complications of whole blood and apheresis donations in one or more years (total cy, . million wbd and . million aphereses, total million donations). for these hvs the median rate of vasovagal reactions was . / wbd (iqr . - . ) and . / apheresis procedures ( . - . ) . reported haematoma rates were higher for apheresis than for wbd: the median per hvs was . / wbd (iqr . - . ) vs . / aphereses ( . - . ); rates of arm pain and/or nerve injury (not separated in - ) also tended to be higher: median . / with wbd, iqr . - . , vs . / with apheresis, . - . . summary/conclusions: international reporting allows hvs to study rates of blood donation complications, to distinguish between wbd and apheresis complication rates and capture information about very rare events. variability of reporting and of severity assessment between countries impairs the feasibility of comparisons between hvs. work is needed to improve harmonisation of classification of donation complications and severity assessment for data comparison and research. background: to prevent iron related hb loss, screening with ferritin testing was implemented in stockholm county (approx. registered blood donors) during a two-year roll-out. iron supplementation is offered to blood donors but has not prevented hb deferrals resulting in control visits per year. ferritin testing is hypothesized to increase iron compliance. aims: implementation of ferritin testing for surveillance of iron levels for the entire blood donor population with specific attention to new donors, women returning after pregnancy, donors with low hb and at return visit after low hb. yearly testing of plasma and platelet donors. methods: ferritin testing, following a staff education program, was implemented for applicant donors, donors with low hb, women after pregnancy, apheresis donors, followed by screening of registered blood donors per donation site. after initial screening, donors will be tested at each th (women) or th (men) donation, and with yearly testing of young adult blood donors below years. six nurses were educated to process ferritin and blood count results. donors with aberrant ferritin were contacted by letter. results: establishment of cut-off levels and algorithms for ferritin testing and iron treatment was evidence based but met practical limitations such as number of analyses and results that could be processed per week, limitations in liss set-up, blood demand contra preferred cut-offs, iron supplementation compliance. for applicant donors, hb testing show that % of female and % of male applicants cannot be registered because of low hb ( and mg/l respectively). adding ferritin testing, a preferred cut-off level of lg/ml (male reference level), would result in additionally % female and . % male applicant donor loss. as this would threat the blood demand, cut-off was set to lg/ml for women, above the female reference lg/ml, with an acceptable % loss of female applicant donors. for registered blood donors, mg of extra iron tablets were offered at low ferritin ( - lg/ml). this was sometimes combined with prolonged intervals and often repeated before ferritin was restored above lg/ml. donors with ferritin below lg/ml (in . % applicant donors, . % registered donors) or above lg/ml ( . % applicant donors, . % registered donors) were deferred and recommended to see their physician. for hb deferral, the interval was prolonged from to months, irrespective of ferritin levels. this, together with iron supplementation, resulted in an increase from % to % approved hb at return. the team of nurses processing ferritin and blood count results ( ½ nurse fulltime weekdays) reacted to approximately donor results daily, representing % of test results. summary/conclusions: many female donor applicants have suboptimal ferritin levels although they meet required hb for donation. iron treatment was added to retain donors with low ferritin as only prolonged intervals may danger the blood supply. for implementation of ferritin testing, it is necessary to have a well-functioning and agile organization to create and apply algorithms for testing, extension of intervals and iron treatment. background: since november a new donor screening regime is introduced in the netherlands where serum ferritin levels in whole blood donors are measured periodically to further control potential iron deficiency in donors. donor deferral thresholds are set at and ng/ml, and donors are deferred for six and twelve months respectively if ferritin levels are below these values. as limited information is available on ferritin recovery in whole-blood donors, the policy is introduced in parts such that adaptations to the implementation may be considered based on intermediate results and the impact of the measure on donor well-being can be evaluated. aims: to assess the effect of donor deferral on donor ferritin levels. methods: ferritin levels are measured in new donors and at every fifth donation in repeat donors. donors with ferritin levels below the indicated thresholds are deferred and ferritin is re-evaluated at their return for donation after six or twelve months. the policy allows estimating long term trends in ferritin levels post donation in repeat donors. as ferritin levels are measured in all new donors a reference distribution of ferritin levels in healthy individuals is obtained as well. results: among repeat donors % ( % of , male donors, and % of , female donors) have ferritin levels below ng/ml and are deferred for their next donation. furthermore, the distributions of ferritin levels in repeat male and female donors are similar and each has an average ferritin level of ng/ml. in contrast, we found that only % of new female donors (n = , ) and . % of new male donors (n = , ) have a ferritin levels below ng/ml. the average ferritin level in new donors was ng/ml for males and ng/ml for females. comparing the ferritin levels in new and repeat donors, a reduction in average ferritin levels between . and . was observed in female donors and between . and . in male donors. both ratios increased with donor age. at the end of december donors with low ferritin levels returned for donation after six or twelve months deferral. repeat ferritin measurements show that on average the ferritin levels in female donors increased by ng/ml per year whereas average ferritin levels in male donors increased by ng/ml per year. summary/conclusions: in line with earlier findings in literature our results show that repeat donations substantially reduce ferritin levels in repeat donors. these range from . to . in female and from . to . in male donors, who generally have higher ferritin levels. deferral of donors with low ferritin levels seems to be effective in increasing ferritin levels in donors, however, further monitoring of follow-up in repeat donors is warranted to see whether the proposed scheme allows for sufficient donor recovery over time. there are~ different rare diseases and the genes for half have been identified. approximately . million uk citizens experience premature ill-health because of a rare disease. a conclusive diagnosis is generally not reached and on average the diagnostic odyssey lasts . years. the main aims of the , genomes project are to reduce the diagnostic delay by embedding whole genome sequencing (wgs) to accredited standards in the care path of patients with undiagnosed rare diseases. the project started in and dna samples from , nhs patients and their close relatives have been analysed by wgs. here we review the results from the nihr bioresource pilot study for the , genomes project comprising phenotype and genotype data from , individuals recruited at hospitals using approved eligibility criteria for rare disease domains. we determined the population structure including ethnicity and relatedness estimation, high level phenotypes collected using human phenotype ontology (hpo) terms and quality control and summary metrics for samples and variants. the sequence resource contains over million unique variants in the , genetically independent samples, with % of variants previously unobserved in other large scale publicly available genome datasets. we summarise the curation of gene lists and pertinent findings in , unique diagnostic-grade genes for the domains. over , reports assigning pathogenic or likely pathogenic causal variants have been issued with diagnostic yields varying between domains from . % to %, while the proportion of novel causal variants ranged between % and %. we show the power of the bayesian association test, bevimed, to recapitulate decades of clinical genetics discoveries and by identifying > novel genes and novel diseasecausing variants in the non-coding space of the genome. we show how typing data for all red cell, hpa and hla class antigens can be extracted from wgs data. we mined the data from the , genomes project and similar sequence resources to re-version the probe content of the uk biobank axiom array. we genotyped donors from england and the netherlands with this new array and observed a . % concordance when comparing , blood centredetermined antigen typing results with genotype-determined ones. for the red cell and hpa antigens that were available for , donors, the array typing provided a . -fold increase in typing results per donor ( . vs . ) and rare donors were identified. using the genotyping data we identified . times more compatible units among this cohort of donors when blood demand was modelled using referral data from , english patients with more than three red cell alloantibodies. in conclusion the , genomes project has shown the feasibility of using wgs across a universal healthcare system to deliver a diagnosis for patients with rare diseases. based on these results the nhs has commissioned the analysis of another , dna samples from patients with cancer and rare disease. with analysis of dna by wgs and arrays becoming part of routine clinical care, blood services must develop competencies to extract transfusion and transplant relevant information from clinical-grade genotyping data. next-generation sequencing (ngs) enables the sequencing of thousands of genes, the exomes, and even entire genomes by single experiments at a reasonable price. there have also been advances in cytometry: use of antibodies with different fluorescence tags enables simultaneous monitoring of the expression of dozens of antigens. however, immunological methods cannot detect every variant discovered by ngs. genome sequencing reveals not only the exome but also the regulatory elements of transcription/translation, such as promoters and enhancers. rna sequencing determines which genes and spliced transcripts are expressed. it is amazing to realize how much this novel technology has been contributing to the better understanding of various biological phenomena. since the initial cloning of the human blood group a transferase cdnas in the early s, we have been studying the abo genes, a and b glycosyltransferases, and a and b oligosaccharide antigens. various scientific disciplines including genetics, immunohematology, biochemistry, enzymology, and glycobiology have been applied to their study. we have made several important scientific contributions. we demonstrated the central dogma of abo: the a and b alleles at the abo genetic locus encode a and b transferases, which synthesize a and b antigens, respectively. we elucidated the allelic basis of the abo system. we found amino acid substitutions between a and b transferases and inactivating mutations in o alleles. we became the first who succeeded in the abo genotyping, discriminating the aa and ao genotypes, as well as the bb and bo, which was impossible by the immunological approach. we have taken a simple experimental strategy: preparation of eukaryotic expression constructs of a/b transferases and their derivatives, dna transfection to human hela cells or their sublines, and immunological detection of the a/b antigen and/or biochemical examination of the enzymatic activity. we used this to show that the codons and are crucial in determining the sugar specificities of galnac/galactose of a/b transferases. we also identified mutations in several subgroup alleles causing restricted substrate use and diminished transferase activity. we also showed that cis-ab and b(a) alleles specifying the expression of both a and b antigens by single alleles encode a-b transferase chimeras. since then, other scientists have characterized more than abo alleles. recent human genome sequencings have identified many more single nucleotide polymorphism variations. the genome sequences of many species are also available. taking advantage of those sequences and associated information, we have expanded our research to include evolutionarily related a , -gal(nac) transferases and their genes and scaled it up from the genetic to genomic level. in this talk, i would like to present the followings. : our elucidation of the molecular genetic basis of the abo blood group system (as requested by the organizer); : identification of novel abo alleles by others; : more snp data from genome sequences and potential problems for abo genotyping; : findings obtained from analysis of abo genes from other species; bacteria, vertebrates, to primates; : a , -gal(nac) transferases and their genes and the crosstalk between a transferase and forssman glycolipid synthase (fs); and : the potential causes of generation of abo polymorphism and of species variations of the gbgt gene specifying the fors polymorphism. in recent years, there has been a concerted effort to improve our understanding of the quality and effectiveness of transfused blood components. the expanding use of large datasets built from electronic health records allows the investigation of potential benefits or adverse outcomes associated with transfusion therapy. together with data collected on blood donors and components, these datasets permit an evaluation of the effect of donor and blood component factors on transfusion recipient outcomes. large linked donor-component recipient datasets provide the power to study exposures relevant to transfusion efficacy and safety, many of which may not otherwise be amenable to study for practicality or sample size reasons. analysis of these large blood banking-transfusion medicine datasets allow for characterization of the populations under study and provide an evidence base for future clinical studies. knowledge generated from linked analyses has the potential to change the way donors are selected and how components are processed, stored and allocated. however, unrecognized confounding and biased statistical methods continue to be limitations in the study of transfusion exposures and patient outcomes. results of observational studies of blood donor demographics, storage age, and transfusion practice have been conflicting. this review will summarize statistical and methodological challenges in the analysis of linked blood donor, component, and transfusion recipient outcomes. c-s - a large deletion spanning xg xg and gyg gyg constitutes a genetic basis of the xg null phenotype, underlying anti-xg a production background: the xg blood group system comprises the homologous antigens xg a and cd . the cd gene resides within pseudoautosomal region on the short arms of the sex chromosomes and thus mimics autosomal inheritance. xg, on the other hand, is x-linked and straddles the pseudoautosomal boundary; a truncated pseudogene composed of only the first exons remains on the y chromosome and therefore males carry a sole full-length copy of xg. this phenomenon manifests as asymmetric frequencies of the xg(a+) phenotype between the sexes: roughly % of women and % of men are xg(aÀ). also, whilst xg a immunization is rare, the vast majority of all anti-xg a makers reported are men. recently, we reported that the rs c variant disrupts a gata motif between xg and cd . this abolishes erythroid xg a expression and causes the common xg(a-) red cell phenotype. however, rare individuals who produce anti-xg a cannot be accounted for by this finding. we hypothesized that a structural defect in the xg coding region causes the true xg null phenotype underlying anti-xg a production. aims: we undertook to determine a genetic explanation for anti-xg a production. methods: genomic dna (gdna) was extracted from two whole blood samples and cell-free dna (cfdna) from archived plasma samples from donors producing anti-xg a ; one cfdna sample was from a female donor and the rest from males. polymerase chain reaction (pcr) experiments, sanger sequencing, and database searches were performed to identify and confirm the deletion. aliquots of gdna from four males reported to carry a similar deletion in the genomes project were also tested. results: in one gdna sample, exon-specific pcr identified a deletion involving part of xg and the downstream gene gyg . database searches indicated that the most likely deletion was the infrequent genomic structural variant esv reported in the genomes project. further analyses with a short ( bp) and a long ( bp) pcr amplicon across the suspected breakpoint determined that this deletion was approximately kb and corresponded well with esv . this finding was confirmed in the second gdna sample. given the rarity of anti-xg a producers, we decided to test for the same deletion in cfdna extracted from old archived plasma samples. of the cfdna samples, poor quality in four samples prevented amplification even from control reactions and one was contaminated with bacterial dna. in the remaining nine samples, eight could be amplified for the deletion-specific -bp short amplicon while one was negative for the deletion. sanger sequencing of the amplicons revealed a heterogeneous repetitive dna element, ltr b, hinting at a previously-reported recombination event. this deletion was not detected in the samples from the genomes project which reiterates the previously identified deficiency in data interpretation and reporting for deletions. summary/conclusions: a large deletion disrupting the xg and gyg genes accounts for the xg null phenotype underlying the majority ( of ) of anti-xg a makers. one sample remained unexplained, indicating further heterogeneity to be explored. our data help to explain why anti-xg a production is rare and has primarily been reported in men. background: s and s antigens encoded by gypb differ by one nucleotide (nt), c. c>t, p.thr met. two different genetic backgrounds are associated with silencing of s antigen and a u+ w phenotype. these include the nt change c. c>t (p.thr met) causing partial exon skipping and designated gybp* n. (gypb*ny) and c. + g>t, an intron change causing complete skipping of exon , designated gypb* n. (gypb*p ). aims: samples from three individuals, a previously transfused african american sickle cell patient (p ), a blood donor of unknown ethnicity (p ), and an african american patient (p ) (lapadat r. aabb abstract) were investigated for discrepant serologic and molecular results when determining s and s phenotype. methods: standard methods were used for rbc typing with licensed s and s reagents and rbcs from donor p were also tested with monoclonal and polyclonal anti-s and anti-s. dna was isolated from wbcs and hea precisetype performed on p and p . p was also tested by gypb*s/s as-pcr, exon pcr-rflp for c. + g>t and as-pcr for c. c>t. p was tested for gypb*s/s and c. c>t and c. + g>t changes by a real-time pcr-fluorogenic ' nuclease taqman chemistry. for all, gypb exons - were amplified and sanger sequenced and aligned to consensus using clustal x. results: rbcs of all three probands typed s-and strongly s+ while dna testing indicated c. t/c (gypb*s/s). assay for the two common gypb*s silenced alleles, c. c>t and c. + g>t, indicated all three samples had both silencing mutations previously reported to be independently associated with a sÀu+ w phenotype. hea precisetype could not interpret this novel allele combination and indicated gypb*s as pv (possible variant). samples were confirmed to be heterozygous for c. c/t, c. c/t and c. + g/t by exon specific sequencing and as-pcr, pcr-rflp and real-time pcr. by long range sequencing of gypb, all three were heterozygous c. t/g and c. a/g (p. leu/trp), c. a/t (p. thr/ser), c. a/g and c. g/t (p. glu/gly), c. c/t (s/s), c. g/t (p. val/leu), c. c/t (p. thr/met), and c. + g/t. all samples were also c. g/g (p. ser) and heterozygous for several previously recognized silent changes in exon , c. t/c, c. t/c and c. a/g. summary/conclusions: we report a novel silenced gypb*s allele that can confound gypb genotyping interpretation. the allele was found in three probands associated with a sÀs+ phenotype. in these samples, two changes previously reported to be inherited independently and both associated with silencing of s antigen are carried on the same allele. dna-based testing could not rule out that c. t or c. + t are separate and that gypb*s was also silenced. robust s+ rbc typing indicates both changes are on gypb*s. gene sequencing confirms the c. + t change is on a gypb* n. [gyp*he(ny)] background. c. c>t (rs ) and c. + g>t (rs ) have a frequency of . respectively . in the african population (exac). although we identified samples, the frequency of this novel allele is unknown. background: the lutheran blood group system currently consists of antigens. these antigens are of low immunogenicity and may cause mild-to-moderate transfusion reactions and hemolytic disease of the fetus and newborn. the activation of lu-glycoprotein/bcam on red blood cells (rbcs) and its interaction with laminin- a is thought to play a role in vaso-occlusion in sickle cell disease and other hematological disorders. the two glycoprotein isoforms lu-glycoprotein and bcam are encoded by the bcam gene which consists of exons located on chromosome q . . a number of rare lutheran phenotypes have been previously recorded in israel, including lu:- , observed among iranian jews, lu:- in one thalassemia patient and one case of lu:- . in this report, a previously transfused pregnant arab patient with b-thalassemia intermedia was investigated because she presented with an antibody to an unknown high frequency antigen (hfa), potentially related to the lutheran system. aims: to characterize a novel lutheran antigen through serological and molecular investigation of a patient with a lutheran related antibody. methods: initially, the red cell phenotype and the presence of a lutheran related antibody in the serum of the patient were detected by standard serological techniques, utilizing enzyme treated and chemically modified cells and rare cells and sera from the nbgrl collection. further serological investigations were carried out using standard iat (liss tube and bio-rad gel) technique. plasma inhibition studies were performed using soluble recombinant lu protein (srlu). eluates were prepared using acid elution method (gamma elu-kit ii). genomic dna was isolated from whole blood and all exons of the bcam gene were amplified by pcr and directly sequenced by sanger sequencing. the impact of the identified mutation on lutheran glycoprotein structure was studied by molecular dynamics calculations. results: the patient's plasma reacted with all cells tested, except for three examples of in(lu) cells and cells treated with -aminoethylisothiouronium bromide, trypsin and a-chymotrypsin. inhibition studies with srlu protein showed complete inhibition of the antibody, thereby confirming the antibody to be directed toward an epitope on the lu-glycoprotein. in addition, testing of inhibited plasma revealed the presence of underlying anti-e and anti-fy a . an eluate was prepared to isolate the patient's lu-related antibody and this eluate was found to be incompatible with examples of lu:- , lu:- , lu:- , lu:- , lu:- , lu:- , and lu:- cells, whereas in(lu) were compatible. results of serological typing of the patient's cells, for lu system hfas, could not be conclusively determined due to the patient having been recently transfused. however, results suggested (through absence of mixed field reactivity) the patient's cells to be lu: - , , , , ,- , . bcam sequence analysis confirmed the patient to be lu* , lu* and revealed a novel homozygous mutation c. a>c in exon , encoding p.lys gln in the lutheran glycoprotein. summary/conclusions: a novel homozygous mutation c. a>c (p.lys gln) in exon of bcam was identified in a patient with an antibody to a lutheran hfa. serological and genetic evidence presented here indicates discovery of a novel antigen of the lutheran blood group system, which we propose to name lura. background: lutheran glycoprotein and basal cell adhesion molecule antigen b-cam are two isoforms of a type i membrane glycoprotein residing on red cell surfaces. both isoforms are adhesion molecules with the main function of laminin binding, and both carry antigens of the lutheran blood group system (lu). the system currently comprises antigens, all encoded by mutations in the alternatively spliced single gene bcam located on chromosome . currently, isbt lists high incidence antigens in the system. aims: we report a case study of an individual with an unidentified alloantibody to high incidence antigen present in her plasma. samples from the patient and her family were investigated. we provide here serological and molecular evidence for a novel high incidence antigen of the lutheran blood group system. methods: serological investigations were performed by standard iat (liss tube and bio-rad gel) technique. plasma inhibition studies were completed with soluble recombinant lu (srlu) protein. genomic dna was isolated from whole blood of the patient and her family members; all the exons of the bcam gene were amplified by pcr and analysed by direct sanger sequencing. the impact of the identified mutation on lutheran glycoprotein structure was studied by molecular dynamics calculations. results: presence of a lu-related antibody in the patient's plasma was confirmed, reacting moderate strength by liss iat with untreated and papain treated cells. cells from the patient's mother, father and two siblings were all incompatible with her plasma, though weaker than panel cells, reflecting dosage. only in(lu) cells were compatible with patient's plasma. the antibody was successfully inhibited with srlu protein, thereby confirming the epitope recognised by the antibody resides on the lutheran glycoprotein. the patient's cells were found to be lu: - , , , , , , , , . bcam sequencing revealed a novel homozygous mutation c. g>a in exon , encoding p.val met in the lu glycoprotein. the c. g>a change appears to be an extremely rare mutation, listed in gnomad database with a frequency of . - and with no known homozygous examples. homology model of the novel lutheran glycoprotein was subjected to all-atom molecular dynamics calculations to analyse potential conformational changes. summary/conclusions: we report serological and genetic evidence for a novel antigen of the lutheran system, which we propose to name lunu. the evidence will be submitted to the isbt red cell immunogenetics and blood group terminology working party for consideration for allocation of antigen status. the absence of this high incidence antigen arises from a rare single amino acid change p.val met in the lutheran glycoprotein and the presence of anti-lunu in the patients' plasma was presumed to have been made in response to previous pregnancy. on native, papain-treated (diagast) and trypsin-treated (sigma) rbcs. genomic dna was extracted from peripheral blood cells by an automated method, amplified by sema a exon-specific primers and sequenced. results: the proband was a -year-old female patient of moroccan origin, group a, d+c+e-c+e+, k-, without transfusion history. she was hospitalized at weeks gestation for a blighted ovum requiring a manual vacuum aspiration, with a significant hemorrhage risk. a rbc antibody screening was performed by a first laboratory. the antibody reacted + by iat on all native reagent rbcs, with negative autocontrols, but was nonreactive on papain-and trypsin-treated cells. an anti-ge was initially suspected, due to the pattern of reactivity and ethnic background. new blood samples were referred to our national immunohematology reference laboratory. the antibody showed the same profile. anti-ge and anti-ch could be ruled out. the serum was nonreactive with two jmh:- and positive with two jmh:- samples. the patient was found to be jmh positive. in addition, a soluble recombinant jmh protein (jmh imusyn/inno-train) fully abolished the reactivity of the panagglutinating antibody. the antibody was an igg . overall, these results were consistent with a probable jmh variant and prompted us to perform sema a sequencing. three nucleotide changes were found, in homozygous state: a rare nonsynonymous change in exon , c. g>a (p.asp asn, rs , maf < . , sift score = ); a common synonymous change in exon , c. a>g (p.gln gln, rs , maf = . ); a rare non-synonymous change in exon , c. g>a (p.arg his, rs , maf < . , sift score = . ). the analysis of surface accessibility of asp and arg using the d structure of sema a (rcsb pdb- nvq https://www.rcsb.org/structure/ nvq) showed that only arg was predicted to be an exposed-epitope. interestingly, all other reported jmh variant phenotypes correspond to an arginine substitution. of note, we retrospectively found another individual of algerian ancestry (pregnant woman) with a pan-agglutinating igg antibody showing a similar pattern of reactivity, and with the same three changes in sema a. we unfortunately could not perform a cross-compatibility testing with the proband (no material left and unsuccessful contact). summary/conclusions: serological and molecular studies allowed us to provide evidence for a novel high-prevalence antigen in the jmh blood group system, very likely encoded by the p.arg his substitution in sema a. we propose to provisionally assign the name jmh for this antigen. interestingly, our two unrelated jmh:- individuals were from north african ancestry. background: the abo system was discovered almost years ago and the underlying structures later elucidated as carbohydrates carried by glycoproteins and glycolipids. the terminal trisaccharides galnaca (fuca )gal and gala (fuca )gal constitute the clinically important a and b epitopes, respectively. clausen et al. (pnas, ) showed that the a antigen could be extended to a repetitive glycolipid a epitope, galnaca (fuca )galb galnaca (fuca )galb glcnac-r. however, extended forms of b antigen have not been described. we encountered two related situations with unexplained serological reactivity. firstly, enzyme-conversion to group o treatment of group b (b-eco) red blood cells (rbcs) with a -specific gh family exogalactosidase (bzyme) abolishes b antigens as detected by hemagglutination and flow cytometry with all monoclonal anti-b tested. despite this, % of group o plasmas have been reported to give positive crossmatch results with b-eco rbcs. secondly, plasmas from ab and b individuals of the globoside-deficient p k phenotype contain anti-p and anti-px but react stronger with bpp-rbc than with app/opp-rbc. based on these findings, we hypothesized the presence of a bzyme-resistant, b-related glycolipid. aims: to identify the molecular basis of the enigmatic serological observations outlined above. methods: plasma and eluates from an a b individual with the p k phenotype were investigated by hemagglutination and flow cytometry, as were eluates from b p k and o plasma. rbc membrane glycolipids were extracted from two batches of pooled, expired group b-rbc units (frozen -litre reference preparation and confirmatory preparation from freshly collected units). native or enzyme-treated glycolipid fractions were analysed by liquid chromatography electrospray ionizationmass spectrometry (lc-esi/ms) and immunostaining of thin layer chromatography (tlc) plates. antigen expression in the h+bÀ human erythroleukemia (hel) cell line was analysed by flow cytometry following overexpression of selected glycosyltransferases. results: anti-p-depleted eluates made from a b p k plasma contained anti-px and antibodies of unknown specificity that reacted stronger with native or papaintreated bpp-rbcs compared to app/opp-rbcs. anti-px was removed by adsorption onto opp-rbcs but reactivity (here designated anti-extb) remained against b/bpp/b-eco rbcs. lc-esi/ms of glycolipid fractions from group b units revealed an unknown hexnac-hex-(fuc-)hex- hexnac-hex- hex heptasaccharide. upon b-nacetylhexosaminidase treatment of this candidate structure, a group b type hexasaccharide was produced, demonstrating that the terminal hexnac of the hexnac-gala (fuca )galb glcnacb galb glc heptasaccharide was b-linked. since the discovery of the anti-platelet effects of aspirin platelets have been a major therapeutic target for pharmaceutical companies and also a very profitable target. however, the effectiveness of aspirin has also been a challenge as it is an inexpensive drug and any new agent needs to show clear benefit over aspirin. furthermore the risk of bleeding from anti-platelet agents, especially cerebral bleeds, has also presented challenges. in the 's orally active gpiib/iiia antagonists were considered to be the 'super aspirin' but clinical trials showed increased mortality and ultimately this class of drugs was relegated to iv use only in high-risk patients. gpib/ix/v antagonists were also a promising drug target but no agent made it to market. the real breakthrough was the discovery of the p y antagonist clopidogrel which, in conjunction with aspirin, proved to be very effective at preventing thrombotic events and as a result it became the biggest selling drug in the world at the time. with clopidogrel now offpatent the combination of aspirin and clopidogrel is a formidable challenge to any new agent both in efficacy terms and pharmacoeonomic terms. so is there a future for new anti-platelet agents? with the growing awareness of the role of platelets in inflammation and an understanding of how the immune activation of platelets differs from the classical haemostatic activation of platelets it is now possible to develop novel anti-platelet agents that target inflammation without compromising haemostasis. it is here that we should look for the next generation of anti-platelet agent. c-s - university hospitals of geneva, geneva, switzerland platelet function defects, either congenital or acquired, are associated with increased bleeding risk, particularly in a perioperative setting. the use of platelet function assays is therefore tempting in order to tailor transfusion and limit platelet transfusion to those bleeding patients with impaired platelet function, as assessed by those assays. however, the current guidelines provide only weak recommendations supporting the routine use of these assays. indeed, there are numerous platelet function assays on the market that differ in their method of evaluation of platelet function and agreement between their results is at best moderate. the threshold values beyond which procedure-associated bleeding risk becomes worrisome is not standardized. moreover, observational studies addressing the predictive value of platelet function testing in perioperative or spontaneous bleeding are not consistent. finally, management trials with randomized patients assessing the benefit of platelet function testing are scarce. more recent data identified selected situations where platelet function testing may be useful though. i will review the different platelet function assays as well as selected clinical studies addressing the impact of platelet function testing to improve bleeding and transfusion-related outcomes. the latest recommendation will be addressed too. background: platelet refractoriness complicates the provision of platelet transfusions in management of thrombocytopenia in oncology patients. platelet refractoriness poses challenge due to alloimmunization to hla and human platelet antigens and is associated with adverse clinical outcomes. aims: a prospective study was undertaken to analyse result of platelet compatibility with post-transfusion platelet count increment and to ascertain presence of platelet antibodies as causative factor in platelet refractory oncology patients. pulmonary complication after blood transfusion is the leading cause of transfusionrelated morbidity and mortality, with an incidence reported between . - % of all transfused patients. the most important transfusion related pulmonary complications are transfusion associated circulatory overload (taco), transfusion related acute lung injury (trali) and transfusion associated dyspnea (tad). in this presentation the recent changes in the international definitions will be presented and discussed. furthermore, insights in the different underlying pathophysiologic mechanisms will be highlighted. in the past decades only for trali prevention strategies have successfully been designed and implemented. currently no evidencebased treatment strategy is available for any of these life-threatening syndromes. insight in the pathogenesis of pulmonary complications after transfusion should pave the way for future prevention and treatment studies. the issue of the impact of iron overload / toxicity on the hematopoietic stem transplantation (hct) outcome has been firstly addressed in the field of transfusion dependent thalassemia. today the concept has been extended to other diseases characterized by periods of variable duration of transfusion dependence such as myelodysplastic syndrome (mds) and myeloproliferative diseases. patients requiring regular blood transfusions certainly develop iron overload leading to tissues and organ damage. iron burden before transplant significantly impacts outcome and long-life posttransplant. it is well known that iron overload is deleterious for organs such as liver, heart and endocrine glands and it has been postulated could also increases the risk of infections and severe graft versus host disease early after hct. recent preclinical data has shown how increased production of reactive oxygen species (ros) resulting under iron overload condition, could impair the stem cells clonality capacity, proliferation and maturation. also, microenvironment cells could be affected through this mechanism. for this reason, iron overload is becoming an important issue also in the engraftment period early post-transplant. high baseline ferritin levels before hct have been shown to negatively influence clinical outcome, but nowadays, ferritin is considered a steady and not biologically active form of iron, while free iron forms as non -transferrin bound iron (ntbi) and labile plasma iron (lpi) are considered the main trigger of cell damage more representative of the dynamic tissue damage. the scientific community is moving the iron disease from a "bulky" disease, such as classically in thalassemia (based on quantitative iron parameters as ferritin, red blood cell transfusion number, mri) to a "toxic" disease (based on active and dynamic biological markers as ntbi/lpi). at this time in all the studies published on hct setting, only the correlation between direct or indirect estimates of iron overload (mainly serum ferritin) and outcome parameters has been explored, while the duration of exposure to toxic iron species has not been taken into account. the first study that explored the lpi role in relationship with outcome was published by wermke and colleagues in malignancies. they investigated the predictive value of both stored (mri-derived liver iron content) and non-transferrin-bound-iron, defined as enhanced labile plasma iron (elpi) on post-transplantation outcomes in patients with acute myeloid leukemia or mds. their prospective, observational all-ive study showed that patients who had raised elpi concentration at baseline, also had significantly increased incidence of non-relapse mortality at day ( %) compared with those who had normal elpi at baseline ( %) (p = . ). reinterpreting transplant predictive factors in the light of the current advances in understanding iron homeostasis further supports the concept that the key to successful transplantation is regular and life-long chelation therapy to consistently suppress tissue reactive iron species and prevent tissue damage in the years before hct. in transfusion medicine, the role of donor sex was long considered to be limited to the increased risk of trali observed after transfusions from female donors. this risk has been shown to be limited to female donors with a history of pregnancy and to plasma rich products (i.e. excluding red blood cell products, typically containing < ml plasma). until, in , we found that sex-mismatched red blood cell transfusions were associated with increased recipient mortality. since then, several other studies have confirmed these findings, but some studies also did not find an association. all of these studies relied on the analyses of routinely collected health care data, which was not primarily intended to be used for research. as a result, analyses are complex and often difficult to properly appraise based on published descriptions. therefore, the discussion about possible reasons for these discordant findings has largely focused on the methodological approaches of the different studies. other potential explanations include differences in donor or patient populations, production methods, or storage time of blood products. the different potential explanations are expected to be associated with different underlying biological mechanisms. therefore, further delineating which donor, patient, and product characteristics modify the observed association could provide more insight into the underlying mechanism. in , we observed that only transfusions from female donors with previous pregnancies were associated with increased mortality and only in male recipients under years. this leads us to postulate that pregnancy induced long term changes in the female immune system are transferred during red cell transfusion, with negative consequences for young male recipients. the low amount of plasma present in red cell products further lead us to assume a cellular component, like passenger leukocytes, to be involved. it has been shown that micro-chimerism of passenger leukocytes can persist for decades after transfusion, even of leuko-reduced blood products, suggesting long term immune-modulation could play a role. we hypothesized that passenger leukocytes would die during storage of blood products and the negative effect of ever-pregnant female donors, on the survival of young male red cell recipients, would therefore be attenuated by increased storage time. however, our data seem to indicate the opposite. the risk of death was increased over three-fold for young male recipients of old (> days storage) red cells from ever-pregnant donors, compared to for young male recipients of fresh (< days storage) red cells from ever-pregnant donors ( -year cumulative incidence of death . % versus . %). the negative control group (i.e. young male recipients of red cells from male donors) showed a much weaker association of mortality with storage time (i.e. . % versus . %). these findings seem to falsify our hypothesis that mortality could be caused by passenger leukocytes, establishing long term immune-modulatory effects. another potential mechanism that has been suggested could be the presence of cellfree dna in transfused blood products. this cell-free dna increases during storage. however, more research is needed both to establish if cell-free dna can also be linked to previously pregnant blood donors and by which mechanism it could negatively affect young male transfusion recipients. clinical trials (cts), the gems in clinical research for generating robust evidence in medicine and public health, are costly and complicated undertakings. in resource limited setting like sub-saharan africa (ssa) where the health systems are sub-optimal and where capacity for research is limited, the conducting of cts can be a daunting challenge. the challenges of undertaking cts in rls may be categorized based on the occurrence of the bottleneck(s) in relation to the ethics and regulatory approval process: pre-approval: protocol development: in order to develop a context-specific protocol which is subsequently subjected to an ethics and regulatory approval process, investigators need to review and ensure that the protocol is pragmatic and feasible with respect to implementation. this results into a time-consuming reiterative process of reality-checking the protocol. site selection: in light of the limited research infrastructure, investigators in rls and their developed world partners spend considerable time reviewing and selecting suitable sites for participation in the anticipated protocol for the cts. suitable sites are usually very few and with competing on-going studies. approval: institutional review board (irb) approval: the irb approval process can be quite lengthy ( - months) with considerable unpredictability in the periods between the initial and subsequent irb reviews. national regulatory approval: the requirements by national regulators are unusually innumerable with limited flexibility to accommodate specific cts. post-approval: the key post-approval challenges for cts implementation in rls are attaining appropriate participant enrolment and maintaining high retention rates. specifically, for participant enrollment, the challenge may be unforeseen competing cts targeting the same participant pool or community perspectives that may discourage participants from getting screened for the cts. retention may also be a challenge particularly where participants view enrollment as a chance to access healthcare services may therefore not have any incentive to keep in a study after the initial study visits. in conclusion, cts are complex undertakings wherever they are conducted but are doubly challenging in rls like sub-saharan africa. the bottlenecks at the preapproval, approval and post-approval stages are considerable. nevertheless, it is rewarding to perform ctus in rls given that the data generated therein is highly valued by national regulators and may hasten the registration process for medical products. background: interest in an appropriate and effective whole blood (wb) pathogen reduction technology (prt) is growing, especially in sub-saharan africa where the residual risk of transfusion-transmitted infections (ttis) remains unacceptably high and wb is still frequently used. cerus corporation, manufacturer of the intercept tm blood system, and swiss transfusion src are collaborating on a clinical development program to adapt intercept prt using amustaline (s- ) and glutathione (gsh) for red blood cells (rbcs) into an appropriate prt for wb in resource-limited settings in africa. treatment with amustaline/gsh has been shown to inactivate a broad spectrum of transfusion-transmissible pathogens in rbcs. studies with amustaline/gsh in wb have shown effectiveness against a duck hepatitis b virus (> . log reduction) and plasmodium falciparum (> . log reduction), with future studies planned. a wb prt system with amustaline/gsh also has the potential benefit of minimal electricity requirements. aims: to describe the safety and clinical objectives for a phase clinical trial using the amustaline/gsh prt system for wb in africa, and describe research and development efforts to adapt the intercept prt system for rbcs into a robust and appropriate wb system for settings with high burdens of tti and limited resources. methods: the protocol for a phase clinical trial using pathogen-reduced wb treated with amustaline/gsh in an african country is presented, as are current research and development activities related to the development of a prt system for wb. results: in the planned phase clinical trial in africa, clinically stable patients with anemia who require wb transfusion will be randomized into two study arms at a large medical center in a sub-saharan african country. enrolled patients will receive one unit of non-leucocyte-reduced wb treated with amustaline/gsh, or a unit of untreated control wb or rbcs. the primary safety endpoint will be the incidence of high-imputability transfusion reactions (swissmedic ≥grade ) within the first hours of transfusion. data will also be collected on all adverse events and transfusion reactions (all grades) and the development of treatment-emergent antibodies to pathogen-reduced wb or auto-antibodies within (ae ) days of the study transfusion. clinical efficacy will be characterized by hemoglobin increment hours after transfusion adjusted to hemoglobin dose and body weight. summary/conclusions: a prt system for wb is being developed based on the intercept prt for rbcs that is in advanced development in europe and the united states. intercept-treated rbcs have met efficacy and safety endpoints in phase clinical trials. the amustaline/gsh prt system used to treat intercept rbcs has demonstrated effective inactivation against a broad spectrum of agents that may result in ttis. a phase clinical trial using an adapted prt system for wb in africa is the first step in a clinical development program that includes additional pathogen inactivation efficacy studies and improvements to the wb prt implementation process. together, these developments and evaluations represent progress toward a realistic and appropriate prt for wb in africa and other resource-limited settings. background: in australia, demand for plasma-derived products has increased dramatically, and there is a need to increase plasma collections. first-time donor retention, including the rate at which first-time donors return, is a pressing issue. a quick return is optimal as this increases the overall plasma yield and is associated with long-term retention. however, we lack evidence of effective interventions to encourage first-time donors, particularly those donating plasma, to return and to establish a higher frequency donation routine. working from schultz's ( ) framework, this intervention study was based upon insights from interviews with first-time plasmapheresis donors. participants identified barriers such as time and lack of knowledge about plasmapheresis. facilitators included being able to help more people and to donate more frequently than allowed with whole blood. participants generally favoured donating at a frequency of every weeks. aims: the aim of this study was to test the effectiveness of three intervention conditions compared with the business-as-usual (bau) procedure on the proportion of donors returning to donate plasma and the number of plasma donations. we report on the data from months post-donation. methods: donors were randomly assigned to one of four study conditions. in conditions and , donors received an email one day after their initial donation. in the first condition, donors received the bau 'thankyou' email. donors in the second condition received an alternative email with content derived from the interview study. donors in the remaining conditions received either the bau email (condition ) or the revised email (condition ) coupled with a telephone call. the phone call was scripted to provide additional information about plasma, including how often plasma can be donated, a suggestion to donate every weeks, and a prompt to forward-book appointments. results: the final sample (n = ) comprised women ( %) and men ( %) aged - (mean = ). after two months . % of donors returned to donate plasma at least once. after controlling for gender, age, and blood group, donors in each of the intervention conditions were more likely to return to donate plasma than were donors in the bau condition. the greatest effect was found between donors randomized to condition (revised email + phone call), or = . , ci = . - . , and bau. donors assigned to the two telephone conditions (condition and ) donated plasma at a higher frequency than bau. summary/conclusions: this study tested the effectiveness of interventions designed to encourage first-time plasma donors to return to donate plasma and to establish a routine of donation. early indicators suggest that the evidence-based email and phone call elements are more effective than bau in bringing donors back to donate plasma, and the revised email combined with a phone call had the greatest positive effect on short-term plasma yield. background: healthy individuals with hereditary hemochromatosis (hh defined as hyperferritinemia and homozygous p.c y mutation), but also carriers of other hfe mutations (p.c y/p.h d or homozygous h d) with elevated serum ferritin (sf) are accepted as blood donors, if allowed by local regulations and if eligibility is fulfilled. generally, blood components are released for transfusion at normal sf levels (< ng/ml in females, < ng/ml in males). aims: prospective, two-center, randomized study comparing the efficacy and tolerability of double-erythrocyte apheresis ( rbcaph) and whole blood phlebotomy (wbph) for iron depletion in asymptomatic subjects with hh or hyperferritinemia and other hfe mutations in the setting of routine blood donation. methods: eligibility criteria included age ≥ - years, total blood volume ≥ l, bmi < kg/m , hb ≥ g/l, elevated sf levels and no end organ damage due to iron overload. rbcaph ( ml rbc) were scheduled every days and wbph ( ml) every days until sf was < ng/ml. a complete blood count and sf were measured at baseline, at every visit and at follow up weeks after completion of the study. adverse events were systematically recorded. the treatment effect was tested by poisson regression, with gender, hfe mutation, bmi and baseline sf as covariates. results: subjects ( females; mean age years) were randomized to wbph (n = ; female) or rbcaph (n = ; females). hfe mutations were p.c / p.c y in subjects, p.c y/p.h d in , and p.h d/p.h d in . at baseline, mean hb was g/l (sd . ) and median sf was ng/ml (iqr - ng/ml). procedures (wbph n = , rbcaph n = ) were completed; were interrupted (local hematoma, insufficient flow); ( wbph, rbcaph) were postponed because of low hb and for non medical reasons. there were drop-outs in the wbph arm due to depression and poor compliance, respectively. anemia (hb < g/l in males, < g/l in females) occurred after visits in wbph subjects and after visits in rbcaph subjects. fatigue was reported after phlebotomies and aphereses. only participants ( %) completed the study per protocol. blood components ( rbc concentrates and plasma units) for transfusion were obtained. overall, a median of . wbph (iqr . - . ) was needed to reach sf < ng/ml, corresponding to . times of rbcaph (median . , iqr . - . ) (p = . ). analyzing separately p.c /p.c y and p.c y/p.h d carriers, the relation wbph to rbcaph was . and . , respectively. treatment arm and hfe mutation were the covariates with significant effect on the primary endpoint (p = . and . , respectively). summary/conclusions: rbcaph is more efficient than wbph for iron depletion in healthy subjects with hh or other hfe mutations and moderate hyperferritinemia. intensive treatment schedules, generally recommended for hh, are difficult to keep because of hb drop and compliance. less intensive treatment in asymptomatic individuals with hh and their inclusion in blood donation would avoid negative effects on quality of life and benefit blood collection centers in the long term. background: serum ferritin (sf) measurements in whole blood (wb) donors demonstrated that female sex and intensity of donation are major risk factors for iron deficiency. approximately ml red blood cells (rbc) and - mg iron are lost with wb donation. double unit rbc ( rbc) collections of ml (ca. ml less than the rbc amount of two wb donations) lead to a loss of about mg iron. in switzerland, the maximal allowed donation frequency for male donors is once every months for rbc and once every months for wb donation. aims: to describe and compare the course of hemoglobin (hb) and sf in male subjects donating wb and rbc at our institution. methods: we included wb and rbc donors (n = ) who donated with the maximal allowed donation frequency over months between and , yielding , wb and , rbc donations. we excluded subjects with hyperferritinemia and known hfe mutations. hb limits were g/l for wb and g/l for rbc donation. with rbc apheresis ml rbc were collected. sf was measured on a predonation serum sample; hb was determined from finger prick samples. the donors received no iron substitution. we used generalized estimating equation models for hb and sf trajectories. results: mean age at the first blood donation was (wb) and years ( rbc), respectively. at the first donation, mean hb was g/l (sd ) in wb and g/l (sd ) in rbc donors; mean sf was (sd ) and lg/l (sd ), respectively. on average, hb and sf were higher in rbc donors ( . g/l and lg/l, respectively; p < . ). there were subjects with sf < lg/l in wb and in rbc group, and with sf < lg/l (but > lg/l) and , respectively. in rbc donors, between the first and the last donation, mean hb declined from g/l to g/l (p < . ) and mean sf from lg/l to lg/l (ns). in wb donors, mean hb dropped from g/l to g/l (p < . ) and sf from lg/l to lg/l (p < . ). similar results were found when adjusting for age and season. hb values dropped from baseline until the th donation for wb donors and until the th donation for rbc donors with an upward trend thereafter. in both groups, no hb value below the limits of blood donation and no anemia were observed. sf reached a nadir at the th donation in both wb and rbc donors ( lg/l and lg/l) and increased thereafter in rbc donors. in wb donors, sf followed a parabolic trend that peaked at the th donation, and then declined until the last donation. summary/conclusions: the maximal allowed blood donation frequency for wb and rbc male donors in switzerland is not only protective for the development of anemia, but also for deferral of blood donors because of low hb. this was observed even in subjects with low sf at baseline. background: granulocyte concentrate transfusion is a potentially lifesaving option for patients without functional neutrophils. however, recent studies have failed to demonstrate the anticipated clinical effectiveness of this procedure. granulocyte concentrates are manufactured using sedimentation agents to separate granulocytes from red blood cells and enhance granulocyte collection efficiency. high-molecularweight hydroxyethyl starch (hes) is most commonly used for this. however, authorities recently restricted the use of hes due to its unfavorable risk-benefit-profile. modified fluid gelatin (mfg) is an already used alternative sedimentation agent. as the granulocyte product contains these substances, any impact of the sedimentation agent on granulocyte function may affect the clinical effectiveness of granulocyte transfusion. aims: we tested the hypothesis that mfg is not inferior to hes in terms of the functionality and viability of granulocytes. methods: granulocytes from ten healthy donors were isolated, aliquoted and incubated in parallel for hours with either % (control), . %, % or % mfg (gelafundin %, b. braun melsungen ag) or hes (hespan %/ / . , b. braun medical inc.), respectively, and granulocyte migration, chemotaxis, reactive oxygen species (ros) production, neutrophil extracellular trap formation (netosis), antigen expression of cd b, cd l and cd b, and viability were subsequently investigated in vitro. testing was performed using live cell imaging of the cells embedded into a collagen i matrix for parallel testing of migration, ros production and netosis. in addition, flow cytometric (facs) analysis was utilized for surface marker expression, viability and respiratory burst measurement. results: granulocyte migration decreased in a dose-dependent manner in response to hes and mfg. relative to the controls, all three concentrations of hes lowered migration distances (p < . respectively), whereas only the higher concentrations ( % and %) of mfg showed lower relative migration distances (p < . respectively). track straightness was reduced with both sedimentation agents at % and % to the same extent (p < . respectively). hes resulted in lower cd b expression (p = . ) and higher cd l expression (p = . ) compared to the controls, whereas the differences for cd b did not reach statistical significance. mfg did not affect the expression of any investigated surface antigen mediating endothelial adhesion and transmigration in comparison to the controls. no significant differences in the timing of ros production or netosis, or in neutrophil viability or respiratory burst were observed. summary/conclusions: these results indicate that mfg is not inferior to hes in terms of granulocyte phenotype and function in vitro when used at equal concentrations, and that potential impairment of granulocyte function can occur with hes. background: plateletpheresis donation leads to a well-known transient decrease of donor's platelets. the question of long-term effects raised with the development of regular donations by some donors in order to satisfy a growing demand. a seminal work (lazarus, transfusion, ) stated that there is a sustained thrombopenia in frequent plateletpheresis donors, correlated with the total number of donations. aims: french regulation authorizes up to plateletpheresis donations per year, with a minimum weeks interval between them. we tried to evaluate the risk of sustained thrombopenia under these conditions. methods: we retrieved all plateletpheresis donations occurring between / / and / / from the french civilian blood donors' base and then selected a cohort of donors with at least donations during that period. in order to minimize measurement errors, platelet counts analysed were means of three consecutive donations, i.e. measures for each donor. results: the cohort includes , donors ( women and , men). mean platelet counts fluctuate between . and . platelets/ml. analysis of variance does not show any statistically significant difference (f = . ), even taking donor's sex or age in consideration. there is no difference if we consider the total duration of the donations, either. donors with the lowest first counts show a significant rise in subsequent measures and donors with the highest counts show a decrease trend, exhibiting a classical regression toward the mean. summary/conclusions: plateletpheresis french regulation does not seem to be at risk of sustained donor thrombopenia. this conclusion is in agreement with recent literature data. the primary biological role of the human leukocyte antigen (hla) system is the regulation of the immune response to foreign antigens. because of this role, hla genes and molecules have an important role in transplantation, etiology of many autoimmune, non-autoimmune and infection diseases, but also in transfusion medicine. an increasing probability of an hla non-compatible blood products, tissues or organs exists due to the extremely high polymorphism of hla genes, with more than , described alleles to date, and their different frequency distribution in various worldwide populations. the hla system, originally discovered as a result of a transfusion reaction in the s, can cause detrimental immune reactions in transfusion therapy. hla antibodies present in the patient are responsible for some of these reactions, while in other cases hla antibodies or hla reactive cells present in the transfused product are accountable for the immunoreactivity. hla antibodies form as a result of exposure to foreign hla antigens during pregnancy, transplantations and blood transfusions and can cause platelet immune refractoriness, febrile transfusion reaction, transfusion-related acute lung injury, and transfusion associated graft versus host disease. in order to avoid or reduce the development of these transfusion-related events, hla antibody negative or compatible products should be used. almost all existing methods presently used for molecular typing of hla polymorphisms are based on polymerase chain reaction, but with different resolution levels (low resolution -two digits or high resolution -four digits). in addition to providing a more precise detection of polymorphisms at hla classical loci (e.g. hla-a, -b, -c, -drb , -dqb ), molecular methods can also determine polymorphisms at hla loci which previously could not be typed by serology (e.g. hla-drb , -drb , -drb , -dqa , -dpa ). the most commonly used method for the detection of hla antibodies was until recently complement-dependent cytotoxicity (cdc) technique, but it is increasingly being replaced by a more sensitive, solid phase based method (luminex technology). in conclusion, an accurate and precise determination of both hla gene polymorphism and hla antibodies presence is essential for the safe and efficient administration of transfusion products. background: in only a minority of pregnancies complicated with anti-hpa a antibodies serious fetal/neonatal disease develops. the difficulty in predicting which mothers should be treated with ivig hampers implementation of fnait screening. we found that fc-core fucosylation and galactosylation are highly variable in anti-hpa a igg, and that these glycan features strongly affect binding to fccriiia receptor. the level of fc-core fucosylation of anti-hpa a alloantibodies was found to correlate with platelet count and outcome of the newborn, suggesting that antibodyspecific fucosylation might serve as a biomarker in fnait screening. however, at present the fc-glycosylation pattern can only be determined by complicated methods involving purification of the antigen-specific igg, and analyzing trypticly released -igg-derived-glycopeptides by tandem liquid chromatography-mass-spectrometry (ms) techniques. these methods, although powerful, are not yet suited for high throughput clinical screening. aims: our aim was to provide a simplified method to quantify the biological activity of anti-hpa- a antibodies, and possibly other alloantibodies against blood cells. methods: here we explored if cellular surface plasmon resonance (spr) imaging can replace ms, resulting in less complicated handling of patient sera and donorantigen-bearing cells. the strength of the binding of platelets to fccr on spr sensor was monitored under flow. the spr sensor was equipped with both wt fccriiia (sensitive to fc-glycosylation status) and mutant fccriiia-n a (insensitive to fcglycosylation status). in addition, the biosensor was prepared with anti-platelet cd (c ) and anti-igg to calibrate the number of injected platelet as well as to quantify igg-opsonization. the quality of the anti-hpa a glycosylation was monitored as the ratio of the binding of opsonized platelets to the wt and the mutant n a-fccriiia. platelets opsonized with recombinant glycoengineered anti-platelet antibodies with different levels of fc-fucosylation were used as standards. for validation, plasma samples with anti-hpa a antibodies, already analyzed by mass spectrometry and with known clinical outcome were tested (sonneveld, bjh, ) . results: we found that the ratio between the binding to the wt fccriiia and to the mutant n a-fccriiia correlated with the level of fucosylation of the hpa a antibodies, as measured by mass-spectrometry (r = À . ; p < . ). overall, a similar predictive value for disease severity was obtained as we previously reported for this retrospective cohort. in addition, quantitative information on antibody concentration can also be extracted using the fccriiia-n a receptor as sensor on the chip, while anti-igg gave aspecific signals, presumably because it recognized cytophilic platelet-fccriia-bound antibodies as well. summary/conclusions: in conclusion, the combined use of wt and mutant fccriiia in a label free spr assay provides both quantitative and qualitative information of platelet bound anti-hpa a antibodies, which circumvents the need for purification of specific antibodies and laborious mass spectrometric analysis. this approach might be generally applicable to determine the biological activity of cell bound antibodies not only for anti-hpa a in fnait, but also for anti-rhd alloantibodies in hdfn or anti-platelet antibodies in itp. background: immunization against the human platelet hpa- a alloantigen is the most common cause of severe fetal and neonatal alloimmune thrombocytopenia (fnait) in otherwise healthy term newborns. the screening for hpa- a antigen in pregnant women is an important tool for identification of pregnant women at risk of having a fetus/neonate with fnait. any targeted intervention depends on efficient screening methods as well as sensitive and specific methods for detection of anti-hpa- a. within the framework of the polish-norwegian project (prevfnait) we have performed hpa- a screening program in poland. aims: our aim was to assess the frequency anti-hpa- a antibody detection and the clinical outcome of newborns identified through the study. women who joined the program due to the fnait in the previous child or in the current newborn are not analyzed in this study. methods: hpa- a screening of pregnant women in - gestational weeks was performed by facs phenotyping or rq-pcr genotyping at ihtm in warsaw. hpa- a negative/hpa- b/ b women were tested for hla drb * : and for anti-hpa- a antibodies by maipa (followed up at week - , , , - and weeks after delivery). if anti-hpa- a were detected, quantitative maipa was performed. all hpa- a negative women were contacted for information concerning the newborn. if the baby had thrombocytopenia and anti-hpa- a were not detected by maipa, the look back samples were tested retrospectively by paklx test (immucor). results: hpa- a negative women were identified ( . %). anti-hpa- a was antibodies were detected by maipa in women (two delivered tweens). in addition, anti-hpa- a antibodies were later detected by paklx in further women who delivered baby with severe thrombocytopenia and/or ich. total number of immunized mothers was ( . %). they delivered babies; were boys. three women were treated by ivig: two by and injections since th and th gw respectively. the anti-hpa- a concentration in the st one was . ; . ; . iu/ml in , , gw respectively and in the nd < . iu/ml in all examined samples. the decision on treatment was based on the low plt count~ g/l in the fetus in cordocentesis. their newborns (one delivered tweens) were healthy. the rd treated woman entered the program in gw (anti-hpa- a concentration was high . iu/ml). she obtained one injection of ivig. her baby was born with mild thrombocytopenia with no ich. severe fnait occurred in / newborns: in with anti-hpa- a detected in paklx only and in with antibody concentration in maipa - st : . / . / . at / / th gw respectively; nd : . / . at / th gw respectively. ich was observed in all of them; plt count was < x in four, / in one. summary/conclusions: / the severe thrombocytopenia due to anti-hpa- a alloimmunisation in our prospective study occurred in / pregnancies / the paklx could improve anti-hpa detection in the screening program and should be considered as an additional diagnostic test, if maipa result is negative / the hpa- a alloimmunisation frequency is higher in pregnancies with male than female fetus. background: foeto-maternal platelet alloimmunization (fmpai) is mainly characterized by foetal and / or neonatal thrombocytopenia (fnait), sometimes revealed by intracranial hemorrhage (ich) or even by foetal death in utero (fdiu). the experience of the pnil milwaukee (usa) reported in that the diagnosis of alloimmunization was carried in only % of neonatal thrombocytopenia cases with a clinical symptomatology highly suggestive of an alloimmune etiology. aims: the aim of this two-year study was i) to determine the frequency of platelet incompatibilities in fnait, ich and fdiu and ii) to evaluate the frequency of detectable platelet alloantibodies (alloab) and their specificity in cases of incompatibility. methods: platelet genotyping was performed by hpa beadchip genotyping kit (bioarray solutions, immucor, warren, nj). serology investigation was carried out by different methods: complete maipa kit (apdia bvba, turnhout, belgium), pack lxtm assay (immucor gti diagnostics, waukesha, wi) and « in house » maipa. all and data were collected using the laboratory information management system. results: patient files were analyzed. no incompatibility is demonstrated in hpa- to - , - and - systems in . % (n = ). hpa- and / or and / or incompatibilities were found in cases ( . %), hpa- and / or in cases ( %). platelet alloimmunization was globally confirmed in only . % of the cases. platelet alloabs were identified regardless of clinical manifestations: anti-hpa- a ( . %), anti-hpa- b ( . %), anti-cd ( . %), anti-hpa- a and anti-hpa- b ( . % respectively) and anti-hpa- b and anti-cd ( . % respectively). alloabs were found in the context of neonatal thrombocytopenia, in ich and in fdiu, and in a follow-up of pregnancy. even if no anti-hpa- alloab could be identified, the incompatibility in this system was highly associated with fnait, ich and fdiu (n = , n = and n = on cases). summary/conclusions: this study strongly confirmed the known immunogenicity of some hpa systems and highlighted overall the severity of hpa- and hpa- incompatibilities. the definite diagnosis of fmpai is difficult to make due to the present technical difficulties in the detection of antibodies against the hpa- and hpa- systems. however, our results suggest that special attention should be paid to the management of pregnancies with these incompatibilities due to the frequency of severe foetal/neonatal adverse events. background: fetal and neonatal alloimmune thrombocytopenia (fnait) is a potentially life threatening disease caused by maternal alloantibody formation against fetal human platelet antigens (hpas), of which anti-hpa- a is accountable for the fast majority of the cases. population-based screening for fnait has been topic of debate for over decades. logistically as well as financially, the major challenge of such a screening is the typing of pregnant women to recognize the % hpa- a negative women. at present, hpa- a typing is mostly done by genotyping. for costeffective implementation of anti-hpa- a screening there is need for a high-throughput, quick and low-cost phenotyping assay. aims: the aim was to develop a high-throughput, quick and low-cost phenotyping assay in order to identify hpa- a negative pregnant women. methods: an automated sandwich elisa was developed to perform hpa- a phenotyping using a murine monoclonal anti-gpiiia as coating antibody and horseradishperoxidase-conjugated recombinant igg anti-hpa- a as detecting antibody. to ensure the applicability for high-throughput testing in a potential screening setting, ll of the uppermost plasma of - days-old stored edta anticoagulated blood tubes was used, without first swirling or spinning them. in two phases, samples of pregnant women were tested and compared to an allelic discrimination polymerase chain reaction assay as golden standard. in the first phase, samples from unselected consecutive pregnant women were tested. the second phase was part of a prospective screening study in pregnancy and confirmatory genotyping was restricted to samples with an arbitrary set od < . in the hpa- a elisa. the developed elisa was optimized to require no additional handling (swirling or spinning) of stored tubes. during phase i, consecutive samples were tested. in phase ii, the hpa- a elisa was performed in another , consecutive samples, with confirmatory q-pcr in , . the two phases combined, samples from in total , hpa- a negative and hpa- a positive pregnant women were genotyped. the assay reached a % sensitivity with a cut-off od between . and . , leading to a specificity of . %. summary/conclusions: a quick, low-cost and reliable assay for hpa- a phenotyping was developed that can be used in a population-based screening setting to select samples that has to be tested for the presence of anti-hpa a antibodies. because plasma from non-mixed or spinned tubes of three to six day-old samples can be used, this assay is applicable to settings with suboptimal conditions. background: cytomegalovirus (cmv) sero-prevalence in ireland is lower than that which is reported in many other european countries. a study of pregnant women in found that . % of irish women were cmv seropositive in comparison to % from western europe and % eastern europe and % from africa. an internal study carried out by the irish blood transfusion service (ibts) in indicated the rate of cmv seropositivity in irish blood donors was . %. therefore a significant proportion of the irish donor and recipient population are susceptible to primary cmv. this is of particular concern for patients for certain at-risk groups such as very-low birthweight cmv seronegative neonates, cmv seronegative patients undergoing transplantation and other cmv seronegative immunocompromised patients. this results in a demand for the provision of cmv sero-negative blood components. in the ibts evaluated the abbott alinity s cmv igg assay as a replacement for the cmv mastazyme eia (total ab eia). aims: to assess the performance of the abbott alinity s cmv igg screening assay in comparison to the cmv mastazyme eia (total ab eia). methods: diagnostic sensitivity was determined by testing confirmed cmv igg positive donors from an external laboratory. sensitivity was assessed using three seroconversion panels (n = ). analytical sensitivity was calculated using linear regression analysis of the who first international standard for anti-cmv igg. diagnostic specificity was determined by testing donors. further evaluation of discordant results was carried out using the architect anti-cmv igg and igm assays and vidas anti-cmv igg and igm assays. results: the diagnostic sensitivity of the alinity s anti-cmv igg assay was determined to be %. the seroconversion sensitivity reported out of samples reactive. the analytical sensitivity of the alinity s cmv igg assay was determined to be . iu/ml. the validation reported discordant results from donor samples tested with both the alinity s cmv igg assay and the current mastazyme total assay. discordant results were observed (alinity s anti-cmv igg positive/mastazyme total negative). further testing of these samples classified discordant results as positive, as negative and as indeterminate. discordant results were observed (alinity s anti-cmv igg negative/mastazyme total positive). further testing classified these samples as negative. overall the diagnostic specificity was determined to be . %. summary/conclusions: both the seroconversion and analytical sensitivities are comparable between the alinity s cmv igg assay, the cmv mastazyme total ab assay, the architect cmv igg assay and the vidas igg assay. the slight variations can be attributed to the individual assay cut-off definitions, which can vary greatly between cmv assays. it must be noted that the determination of the diagnostic specificity ( . %) does not include indeterminate discordant results. further testing will be carried out to try to characterize all discordant samples in collaboration with abbott. this evaluation did not identify any donors with isolated confirmed cmv igm antibodies in a pool of donors. based on this evaluation the abbott alinity s cmv igg assay is a suitable replacement to the mastazyme total ab assay for blood donor screening. background: africa has a unique set of challenges regarding safe blood transfusion. two of the largest contributing factors are: ) the most common disease states in sub-saharan africa (ssa) require large amounts of blood as lifesaving interventions e.g. malaria, ) the highest burden of infectious diseases transmissible through transfusion (tapko, toure, & sambo, ) is found in ssa. this has often led to the binary donor base that exists in ssa, consisting of voluntary non-remunerated blood donors (vnbd) and family or replacement donors (frd) as transfusion centres are unable to supply the demand when relying only on vnbd. voluntary non-remunerated donors are the safest blood donors as they have no incentive (other than altruistic motives) and are not under social pressure to donate, both factors that may induce individuals knowing or suspecting themselves to be infected with a blood-borne agent to donate blood. nucleic acid testing (nat) in conjunction with serological testing is the gold standard for testing, however, the vast distances and high temperatures of africa makes transport of traditional plasma samples a logistical challenge. many publications evaluating the stability, suitability, and ease of use of dried blood spots (dbs) for nat have been published. generally, results have been shown to be comparable to traditional plasma samples. dbs is being used successfully in the early infant diagnosis (eid) programs for hiv by means of pcr testing, especially in africa. aims: . to demonstrate that dbs and/or dried plasma spot (dps) testing is suitable for blood donor screening and can make nat testing more widely available in africa . to determine the diagnostic sensitivity and specificity of testing dps and dbs samples, in comparison to testing of plasma samples. methods: negative new donor samples and confirmed positive donor samples, as defined by routine blood safety screening done at western cape blood service, were screened using a dried blood spot kit. after routine testing was completed, one dbs sample and one dps sample for each blood donor were prepared and analysed with the ultrio elite assay on the panther analyser. summary/conclusions: dbs/dps can be used as a sample for screening blood donors as the invalid rate was . %, and only found on dbs samples. logistically dbs/dps is well suited for the resource-poor countries as samples are: -easy to obtain (fingerpick samples could be used.) -transport is simplified as samples will not leak or haemolyse due to high temperatures. -samples can be stored at room temperature dbs/dps demonstrated acceptable specificity. the ultrio elite performed well with regards to hiv and hcv sensitivity. sensitivity with regard to hbv was not as high but this could be due to very low and erratic viral loads. background: sanquin blood supply is responsible for the blood transfusion services in the netherlands. at the national screening laboratory sanquin (nss) annually more than . blood and plasma donations are tested, on average . samples per day. for more than years, infection serology testing was performed using the prism (abbott diagnostics), but since mid of july , serological testing for the hbsag, hiv ag/ab, anti-hcv and anti-hbc is done with abbott's alinity s system. aims: to compare the numbers of initially and repeatedly reactive results of whole blood and plasma donation samples and the rate of non-specific results leading to deferral of donations and donors for prism and alinity s assays using data from months before and months after implementation of the alinity s systems at nss. methods: initial and repeat reactive rate of the assays run by either prism (hbsag, hiv o plus, hcv) or alinity s (hbsag, hiv ag/ab combo, anti-hcv,) were calculated for january to june (prism) and august to december (alinity s). due to the lack of a true confirmatory method for anti-hbc, we only compared the rate of repeatedly reactive results for prism hbc and alinity s anti-hbc. results: the rate of repeat reactive results for prism (p) and alinity s (a) assays were as follows: ) hbsag p . % ( / . ) versus a . % ( / . ); ) hiv p . % ( / . ) versus a . % ( / . ); ) anti-hcv p . % ( / . ) versus a . % ( / . ). the rate of anti-hbc reactive samples was not significantly different between prism ( . %) and alinity s ( . %). over the study period, the rate of initially reactive samples for the three main screening assays (hbsag, hiv, hcv) was also comparable between alinity s ( . %) and prism assays ( . %), mainly attributable to a rather high number of initially reactive alinity s hiv ag/ab results. this was due to initial issues with blood collection tubes that were resolved. as a result in december, the rate of initially reactive samples decreased to . %, which was significantly lower than for the three prism assays ( . %). summary/conclusions: the introduction of the alinity s assays lead to a decrease of the average repeat reactive test results (hbsag, hiv, hcv) by . % as compared to the prism, mainly due to a lower false reactive rate of the alinity s anti-hcv assay. this will be further investigated for first time and multiple time donors. with the implementation of the alinity s at sanquin we aimed to improve not only the operational efficiency but also to further minimize unjustified disapproval of donors. these first data show that the low initial and repeat reactive rates of the alinity s assays indeed have a positive impact on unnecessary deferrals of donations and donors. background: in blood banks, testing all blood donations for markers of infectious diseases plays an important role in maintaining the safety of blood transfusions. mandatory serological testing in switzerland is performed for anti-hcv, hiv ag/ab, hbsag and syphilis. highly specific and sensitive tests with corresponding automation are essential for this purpose. aims: a comparative study was carried out to evaluate the usability of the newly launched alinity s system (abbott) and the specificity of the infectious disease parameters hbsag, anti-hcv, hiv combo and syphilis (abbott) with the currently used elisa methods on the quadriga befree system (all diasorin, formerly siemens healthcare diagnostics). methods: the study took place at the interregional blood transfusion service in berne, switzerland. the specificity of the parameters was studied on , blood donor sera from both first time and repeat donors. the samples were tested first on the quadriga be free system with enzygnost hbsag . , enzygnost anti-hcv . , and enzygnost hiv integral assays and on the pk with the newbio-pk tpha assay (newmarket biomedical). all samples were retested on the same day with hbsag, anti-hcv, hiv combo and syphilis on the alinity s. initial reactive samples were repeated in duplicate. discriminatory tests were carried out for repeatedly reactive samples using alternative screening tests and neutralisation (for hbsag) on an abbott architect i system and immunoblots (hiv-, hcv-, syphilis-inno-lia, fujirebio). for all samples, results from our routine individual donation nucleic acid testing (hcv, hiv, hbv, roche cobas system) were available. results: based on the results from testing , blood donations, the observed specificities of alinity s assays (a) and enzygnost assays ( summary/conclusions: the alinity s system was easy to use by the operators after a very short introductory training and provides good operational efficiency such as high throughput even when selective testing for samples is needed. the observed specificity of abbott alinity s versus siemens enzygnost assays is comparable in a blood donor screening setting. unfortunately, we were not able to analyse statistically the specificity data due to the insufficient number of donor samples tested in parallel. it is worth mentioning that around % of the samples included in the study derived from repeat donors who had been previously tested with the enzygnost assays but were "first time donors" for the alinity s assays. all four assays from both systems exhibit a very good specificity and are highly suitable and practicable for routine blood donor screening. background: effective screening for transfusion-transmissible infections is essential to ensure safe blood transfusions. the world health organization recommends mandatory serological testing of blood donations for human immunodeficiency virus (hiv), hepatitis b (hbv)/c (hcv), and syphilis. due to increasing demands on clinical laboratories, there is a need for reliable and accurate automated blood screening tests. the fully automated cobas e analyser can be used with elecsys â infectious disease parameters to screen donor blood samples. aims: to compare the performance of elecsys â infectious disease parameters on the cobas e analyser (roche diagnostics) with other commercially available assays for routine first-time blood donor screening. methods: we provide results from etablissement franc ßais du sang (montpellier), a blood bank which participated in a large, multicentre study of the cobas e analyser. the following infectious disease marker assays were compared: hiv, elecsys â hiv duo versus prism hiv o plus; hcv, elecsys â anti-hcv ii versus prism hcv; hbv surface antigen (hbsag), elecsys â hbsag ii versus prism hbsag; hbv core antigen antibodies (anti-hbc), elecsys â anti-hbc ii versus prism hbcore; syphilis, elecsys â syphilis versus newbio pk tpha assay. specificity was tested using residual fresh serum samples from unselected first-time blood donors, and calculated according to assay package inserts and site-specific cutoffs. samples were tested using comparator assays, then retested the same day using elecsys â assays. initially reactive samples were repeated in duplicate; confirmatory tests were conducted on repeatedly reactive samples. confirmatory tests: hiv, nucleic acid testing (nat), architect hiv ag/ab and inno-lia â hiv i/ii score assays; hcv, nat, archi-tect hcv and inno-lia â hcv score assays; hbsag, nat, architect hbsag and elecsys â /prism hbsag confirmatory assays; anti-hbc, nat, hbsag, anti-hbs, and architect anti-hbc assays; syphilis, architect syphilis tp and inno-lia â syphilis score assays. sensitivity was tested using preselected, anonymised, positive, citrate-phosphate-dextrose-plasma samples (plasmatec laboratory products) and compared with archived data for comparator assays. sensitivity was calculated according to the final nat result. results: across all infectious disease markers, specificity to detect repeatedly reactive samples using elecsys â versus comparator assays was similar ( . - . % versus . - . %; n ≥ ). in specificity analyses, there were discrepant results for hiv testing, for hcv, two for hbsag, eight for anti-hbc, and five for syphilis. sensitivity of the elecsys â hiv duo assay ( . %; % ci . - . ) was higher than the prism hiv o plus assay ( . %; % ci . - . ), but the difference was not statistically significant. sensitivities of elecsys â and comparator assays were the same for hcv ( . %; % ci . - . ), hbsag ( . %; % ci . - . ), anti-hbc ( . %; % ci . - . ), and syphilis ( . %; % ci . - . ); three hcv and six anti-hbc samples were classified negative/ indeterminate and excluded from the analyses. in sensitivity analyses, there were two discrepant results for hiv testing, three for hcv, and five for anti-hbc. summary/conclusions: elecsys â infectious disease parameters on the cobas e analyser demonstrate high specificity/sensitivity for screening first-time blood donor samples, with similar clinical performance to other commercially available assays. background: individual plasma and serum specimens from whole blood or plasmapheresis donors are tested for absence of infectious agents by serological assays prior to use for transfusion or production of blood derived therapeutics. the department of plasma analytics (pa), takeda (austria), and haema ag, grifols (germany), both labs with high throughput and a high level of automation, were seeking for alternatives to replace their current serological test systems (abbott prism next). aims: to allow a direct comparison of the two final candidate analyzers alinity s (abbott) and cobas e (roche diagnostics gmbh), a side by side evaluation was carried out by the pa and haema with support from abbott and roche (provision of instruments and reagents). the aim was to compare assay specificities as well as handling and performance of the instruments. the outcome should be used to better understand potential specificity differences and practical handling aspects (throughput, etc.) of a next generation serological analyzer. methods: the two candidate instruments were installed in the pa. from march to june , close to , aliquots from routine preselected repeat donors, provided by haema, were run on both study instruments in parallel. plasma samples were tested for hbs antigen (ag), hcv antibody (ab), hiv ag/ab, and partially for syphilis ab. serum samples were additionally tested on hbc ab. samples with repeat reactive results ("rr", two reactive results out of three tests) not confirmed by confirmatory tests were counted as false reactive. the necessary sample size was calculated based on a one-sided comparison of proportions with the aim to detect potential specificity differences (a = %) in the size of those specified by the manufacturers' instructions. two different lots were tested for the three main assays. results: out of , plasma and , serum samples, test results representing individual donations were found rr on one or both instruments. two samples were confirmed positive ( hbsag, hcv), two others were indeterminate. the sample containing low level antibodies against hcv was pcr negative and only detected by the roche system. the percentage of false reactive results for the five assays on the two systems were (alinity s/e ): hbs ag: . / . % in a total of / samples tested; hcv ab: . / . % in / , p < %; hiv ag/ab: . / . % in / , p < %; syphilis ab: . / . % in / ; hbc: / % in / . no significant difference was found between the calculated specificities in our study and the manufacturers' data. a potential influence of sample matrix and kit lots was assessed. a trend towards more false reactive results in serum vs plasma was found for nearly all assays. no clear-cut statistical difference was seen between lots. summary/conclusions: the study results are in line with the manufacturers' specificity data, showing that the alinity s hcv ab and hiv ag/ab assay show a slightly higher specificity in a population of plasma and serum samples from repeat donors prescreened by prism. a possible influence on the test specificity by the sample matrix was detected but needs further investigation. the possibility to edit complex genomes in a targeted fashion has not only revolutionized basic research but biotechnological and therapeutic applications as well. with the rapid development of genome editing tools, in particular zinc-finger nucleases (zfns), transcription activator-like effector nucleases (talens), and the crispr-cas system, a wide range of therapeutic options have beenand will bedeveloped at an unprecedented speed. therapeutic genome editing in hematopoietic cells enable new interventions in the blood and immune system, including novel approaches to treat immunological disorders, infectious diseases, and cancer. we have developed gmp-compliant protocols to manufacture gene edited cd + hematopoietic stem and precursor cells (hspcs) as well as chimeric antigen receptor (car) t cells, with the final goal to provide novel cell therapies for patients suffering from primary immunodeficiencies, chronic infection with human immunodeficiency virus type (hiv- ), and some tumor entities. despite great success in improving their specificity, engineered designer nucleases can induce genotoxic side effects by introducing mutations or chromosomal aberrations. we have established novel genome-wide assays that enable us to detect chromosomal aberrations induced not only by off-target activity but also by on-target activity, such as micro-aberrations and translocations, with unparalleled sensitivity. in toto, our developed protocols allow us to achieve genome editing in hematopoietic cells with high efficiency and to assess the genotoxic risk associated with the expression of crispr-cas nucleases and talens in clinically relevant human cells, so forming the basis for planned phase i/ii clinical studies. adoptive t cell therapy (act) has proven a potent means to treat blood-borne tumors and solid tumors. adoptive cell therapies include t cells that are genetically engineered with tumor specific t cell receptors (tcrs), or with chimeric antigen receptors (cars). in addition, tumor infiltrating cells (tils) can be isolated from tumor lesions, which are then expanded and reprogrammed in vitro prior to transfusion into the patient. the anti-tumoral efficacy of act products depends on several parameters, including the capacity of cd + t cells to produce cytokines, chemokines and granzymes, a feature that is critical for effective anti-tumoral responses. here i will discuss our efforts to develop and improve act products for future clinical use. i will present pre-clinical work on developing til therapy for non-small cell lung cancers. in addition, i will show that human cd + t cells can be divided into different subsets, and that only one of those subsets is highly cytotoxic. this finding may help improve the quality of genetically engineered t cell products, like tcr and car t cell products. background: the baltic states -estonia, latvia and lithuania have a lot in common. we are located side by side, share the baltic sea as a gate to the west, and more importantly, a common history. we were members of the ussr and suffered years of soviet occupation. we held hands in a km long human . . .chain" across the three states to express our mutual support, and later on, even joined the european union on the very same day -june st , . the three differ a bit in size, population and more in the languages spoken in each one, but that does not explain why the path towards voluntary unpaid donation varies as it does. aim: the aim is to describe the journey towards voluntary non-remunerated blood donation in the baltic states after regaining independence from the soviet union. methods: the information was collected from published and unpublished memories, annual reports and written interviews with latvian and lithuanian colleagues. results: in soviet times, all orders came from moscow and quality control was conducted from the capital city of latvia, riga. donors were mostly paid and given an extra vacation day. big factories were the best places to collect blood and people were queuing to donate. in , the soviet union fell apart and the baltic states suddenly got the freedom and responsibility to decide. in estonia the first edition of "guidelines for the preparation, use and quality assurance of blood components" was taken as guidance in . a lot of advice came from finnish colleagues. in , it was decided to move towards non-paid voluntary donations. the process took years. the first couple of years were economically difficult for the reborn state, as money had less value than food. instead of cash, donors were given rapeseed oil, sugar and pasta, for example. as the situation improved, food items were replaced by small symbolic gifts that carry sentimental value. it has been this way for more than years by now. in lithuania, the process started later, the first program for developing a framework for voluntary non-remunerated donations being carried out in - . it resulted in % of the donations being unpaid. the second program initiated in is still ongoing, aiming towards % non-remunerated donations by . by the end of , they had reached . %. in the beginning, the main obstacle was a private blood center creating unfair market conditions. in latvia, monetary compensation for blood donations still exists, but the younger generation has been encouraged to donate blood for free and some results can already be seen. summary/conclusions: a common starting point does not guarantee the same results, at least not at the exact same time. examining the circumstances leading to the different outcomes could benefit countries yet to start moving towards non-remunerated donations as well as those considering the opposite. haemoglobin (hb) was as expected significantly different between women and men (meanaesem: . ae . vs . ae . g/dl; p < . ). percentage of females with low hb < . g/dl were . %, . %, . %, . % and . %, percentage of males with hb < . g/dl were . %, . %, . %, . % and . % for the age groups - respectively. ferritin values were higher in males compared to females (median; th - th %>tile: ; - vs ; - lg/l; p < . ) and in older age groups compared to younger age groups (median; range in age groups - in females: ; - , ; - , ; - , ; - , ; - and in males: ; - , ; - , ; - , ; - , ; - respectively) . percentage of females with ferritin ≤ lg/l were . %, . %, . %, . % and . %, while percentage of males with ferritin ≤ lg/l were . %, . %, . %, . % and . % for the age groups - respectively. white blood cell counts (wbc) were slightly higher in females compared to males (meanaesem: . ae . vs . ae . ; p < . ). percentage of females with wbc > x /l were . %, . %, . %, . % and . %, while percentage of males with wbc > x /l were . %, . %, . %, . % and . % for the age groups - respectively. none had wbc < x /l. platelet counts (plt) were higher in females compared to males (meanaesem: ae . vs ae . ; p < . ).percentage of females with plt < x /l were . %, . %, . %, . % and . %, while percentage of males with plt < x /l were . %, . %, . %, . % and . % for the age groups - respectively. among the low plt counts most were caused by edta-dependent pseudothrombocytopenia. extreme deviations from normality were seldom and referred to gps for further investigations. summary/conclusions: first time donors are young with % younger than years of age and the female/male ratio was / . of the first time donors with data on ferritin available, % had low ferritin (≤ lg/l). the typical male first time donors neither had low hb nor low ferritin, even with a significantly lower ferritin in younger donors. in female first time donors the prevalence of low hb ( %< . g/dl) and low iron stores ( %≤ lg/l) is high. in all, while all first time donors are highly appreciated, campaigns could target the male population to even out the gender imbalance. blood centers must be aware of the higher prevalence of low iron stores in the youngest donors. background: the aim of assessing suitability of prospective blood donors is protection of their health and the safety of transfused patients. selection process is not always effective in obtaining all relevant information from blood donors in a timely manner. for several reasons, some risks remain undetected or they are disclosed at a future donation(s). therefore, recording and management of post-donation information (pdi) are of great importance for improvement of transfusion safety, donor counselling and education as well as overall improvement of the selection process. aims: the aim of the study was to present results of pdi management at croatian institute of transfusion medicine (citm) and the effect of education activities on their trends. methods: we have analyzed reports on pdi recorded in two-year period ( - ), according to the types of information obtained, age and sex of blood donors, total number of their donations preceding pdi, and the time of receiving the information. the effect of an information leaflet on pdi launched in november was assessed by comparing results in two study years. results: a total of pdi were recorded: in ( / donations) and in ( / donations) with the following distribution: nonsexual risk as tattoo and piercing ( . %), surgical procedures ( . %), travel history ( . %), infections/ contact ( . %), other medical reasons ( . %), endoscopy/invasive diagnostic procedures ( . %), malignancy ( . %), autoimmune diseases ( . %) and sexual risks ( . %). majority ( . %) were late pdi, revealed on the future donation(s): . % on the first next donation, . % on the second and . % after more than subsequent donations. the mean age of blood donors associated with pdi was ae years (median years), while the mean age of all donors in / was years (median years). of all pdi, . % were related to male donors ( % in total pool of citm donors). using chi-square test there were no significant difference between female and male donors in total pdi frequency and in their distribution to early and late pdi (p > . ). the median number of all donations preceding pdi was for female donors and for male donors. implementation of education leaflet for blood donors resulted in . % reduction of pdi in compared with (p > . ). the effect is more pronounced (p < . ) when comparing second and first half of (- . %). reduction is observed in all types of pdi with the exception of infections/contact (because they are mostly early pdi) and malignant diseases. the share of early pdi increased from . % in to . % in , which may suggest better awareness of blood donors on the importance to inform blood bank on changes in their health status. summary/conclusions: our study points to the importance of systematic recording and management of pdi, including education of blood donors about the need of providing all relevant facts related to their health and the safety of donated blood in a timely manner. we are planning further improvements by providing information on this topic on posters and screens on donation sites. background: currently, the transfer of data between organizations and/or computer systems is very limited, and where present is typically proprietary. in the absence of a standardized reference format individual organizations and vendors attempting to integrate disparate databases must develop unique solutions. aggregation of information from multiple sources is complex and costly, constituting a significant barrier to effective analysis of data to improve practice and inform policy. aims: to standardize the definitions and facilitate integration of key data items used in blood donation and transfusion. we report here on an initial effort to map internationally harmonized critical steps in the blood collection/donation process in order to test the approach. methods: through a collaborative process of serial conference calls and correspondence, an informal multi-national consortium of experts across the transfusion industry are attempting to create a vocabulary with sufficiently precise definitions to be usable by automated systems and that can be the foundation of a blood collection/transfusion medicine common data model (cdm), using the following steps: -define the scope of activity to be addressed and segment into key processes. -identify the set of data elements in each segment that are common to all systems. -review and consider existing standards and definitions for each data element. -develop draft definitions for each data element. -release draft to public domain for critical review and refinement with long-term goal of gaining widespread endorsement. results: a standardized approach to blood donation was mapped through identification of common pathways and core mappable data elements. denominator data associated with donor characteristics and blood collection was selected as the first segment to address. a dictionary (or vocabulary) of common terms has been created and will be presented for international comment. summary/conclusions: developing an international consensus on the core elements and their definitions across the transfusion chain is critical for data integration and automation efforts. the expected benefits of this endeavor include that it allows the establishment of algorithms to automate reporting and thus reduce hands-on staff time; reduces time and resources needed to integrate new databases; allows systems to continue to use existing concepts and definitions internally while also providing data output in a standardized format; supports the ability to consistently analyse, interpret and present information regardless of the data source; establishes data definitions against which new systems can be developed; helps to improve comparability of results by providing a common data model for researchers and policy makers; improves confidence in data integrity and reliability of the derived information as a © the authors vox sanguinis © international society of blood transfusion vox sanguinis ( ) (suppl. ), - basis for rational decision making; and reduces data gathering effort and cost thus improving opportunities for more efficient/complex data analysis. standardizing the transfusion medicine dataset is the first step in achieving the automation of data transfer and analysis needed globally to drive patient safety, research innovation, and best business practices. further steps must address the precise methods of data exchange, identification of responsible entities for maintenance and further development, and engagement of computer system developers. red blood cell (rbc) alloantibodies develop in a subset of individuals following exposure to non-self rbcs through transfusion, pregnancy, or other activities; these antibodies can lead to difficulty locating compatible rbcs, acute or delayed hemolytic transfusion reactions, or hemolytic disease of the newborn. alloimmunization is underestimated due in part to antibody evanescence, the random nature of posttransfusion antibody screens, fragmented medical care, and the lack of widespread antibody registries. factors that influence who will develop detectable alloantibodies are not well understood. transfusion burden is one risk factor for alloimmunization, though many highly transfused individuals never form alloantibodies despite exposure to many rbc units (and many non-self abo blood group antigens). individuals with sickle cell disease (scd) and myelodysplastic syndrome (mds) are more likely to form rbc alloantibodies than most other patient populations. individuals with rheumatologic and other forms of autoimmunity, though not chronically transfused, are also at higher than average risk of forming rbc alloantibodies. inflammation, in a broad sense, is one common thread among these diagnoses associated with high prevalence rates of rbc alloimmunization. reductionist murine models support some types of inflammation (including viral-like stimuli) around the time of rbc exposure as being associated with an increased likelihood of alloantibody formation. strategies other than transfusion avoidance or extended antigen matching beyond abo/ rh would be beneficial to prevent new rbc alloantibody formation, especially in patients at highest risk. background: the unique genetic makeup of the omani population makes them rich in the genetic blood disorder. % of omani populations are Àa/Àa gene carriers, % Àa/aa, and % of the population are aa/aa. around % of omani nationals carry the gene for hbs, and - % carry the gene for b-thalassaemia. recent statistics show that there are around patients with thalassaemia major and with scd in oman. the other rbc abnormality that is common in oman is g pd deficiency which is found in % of males and % of females. omanis are known to have the highest frequency of a thalassaemia and g pd reported so far in any race. although blood transfusion is one of the supporting treatments of scd, it can cause some serious complications for the patients. alloimmunization of red blood cells is one of the consequences of blood transfusion. alloimmunisation of the rbcs can cause haemolytic transfusion reactions and may trigger hyperhaemolysis, in which transfused and patient's own rbcs are destroyed. alloantibodies can cause delay in the process of transfusion, it can be costly and time consuming. high number of patients developing alloantibodies may indicate a major difference in the patient and donor population. it may also indicate lack of a controlled, generalised sickle patients management policy. in oman the decision of transfusing scd patient is left to physicians attending the patient. aims: this study is aimed to highlight the increasing number of alloimmunised sickle cell patients. in the royal hospital we get new cases of sickle patient with alloantibodies each year. the acknowledgement of these cases may help in is assessing the current practice of transfusing scd patients, or will help to define the donor and patient population difference. methods: patients were recruited in the royal hospital for this study. edta blood samples were taken for antibody screening test and in the positive cases antibody was identified, all tests done by capture technique using immucor neo machine. results: of the scd patients, % of the patients were male and % female, mean age was years, in the range of - years. % of the scd cases were positive for the alloantibodies, % were female and % were male, the age range was from - years. % of the positive were scd, % s trait and % were s/ bthal. most of the patients developed one antibody, however cases of multiples antibodies were also detected. % of the patients were with single alloantibody, % of them with two antibodies, % with three antibodies, % with four antibodies and % with five antibodies. the majority of the cases were igg against rh antigens anti-e is being the majority %, followed by anti-d %, anti-k %, anti-c %, anti-c %, anti-jk a %, anti-jk b %, anti-fy a %, anti-e %, anti-s %, antis %, anti-kp a . %, anti-fy b . % and igm being %. summary/conclusions: rbc alloimmunisation rate is high in oman majority of the patient affected are female. interestingly sickle trait patients were also transfused and % of them developed alloantibodies. the practice of transfusing rh and kell matching blood unit is implemented four years ago and still high alloimmunization percentage is achieved. background: in ghana, routine pre-transfusion investigations for patients with sickle cell disease (scd) involve only abo-d typing and immediate spin crossmatch, without screening for irregular rbc antibodies aims: determine the prevalence and specificities of and risk factors for rbc alloantibodies in multi-transfused patients with scd methods: in , a cross-sectional study in multi-transfused patients with scd, from two tertiary hospitals in ghana was performed. participants' data on demography, transfusion and medical history were recorded. antibody screening and identification tests were done at sanquin, the netherlands, with standard serology using liss as enhancer and with papain treated rbc panel cells ('enzyme only'). characterization of rhd genes was done by multiplex ligase amplification assay. logistic regression was used to determine the association of patient characteristics, i.e. sex, age at enrollment (continuous), age at first transfusion (categorized as ≤ , - , - and ≥ ), previous pregnancy, number of transfused units ( , - and - and > ), and years after last transfusion (< , - , - , > y) with presence of alloantibodies results: patients ( males and females, median age years, range . - ) were included. the median number of transfusions was (range - ). the median years after last transfusion was (range weeks- . years). in patients, anti-rbc antibodies were detected. in of them the antibodies were weakly reactive with enzyme treated cells only or pan-reactive, possibly some of them representing autoantibodies or antibodies against high frequency antigens. in seven patients enzyme-only anti-le a was demonstrated, likely naturally occurring antibodies. thus, in at least patients ( . %) alloimmunization was demonstrated or suspected; in patients the alloantibodies were 'enzyme only'. besides, the alloantibodies of known specificity ( anti-d, anti-d+c, anti-e, anti-c, anti-e, anti-k, anti-s, anti-le a , anti-go a ), three antibodies reactive only with fy(a-b-) cells and two antibodies of yet unidentified specificity were detected. in six d-patients ( had been pregnant) anti-d (together with anti-c in two patients) was found. in three out of four d+ patients with anti-d, an rhd variant gene was demonstrated ( dau-alleles and diii type or diva- ). logistic regression revealed that none of the risk factors analysed was associated with the presence of antibodies in the patients. immunobiology -red cell alloimmunity fifty-eight patients, had experienced an adverse reaction during or shortly after transfusion ( patients had dark urine). adverse reactions were associated with the number of units received (or . ( % ci, . - . ; p = . ), but not with the presence of antibodies (p > . ) summary/conclusions: in at least % of multi-transfused patients with scd alloimmunization could be demonstrated, mainly ( %) directed against rh antigens. the enzyme only reactivity, coupled with absence of antibodies in seven of patients with probable haemolytic reaction and known evanescence of especially non rh antibodies suggest possible low titre and disappearance of some clinically relevant antibodies. given the high immunization rate together with the high frequency of adverse transfusion reactions, pre-transfusion screening for rbc antibodies should be considered for patients with scd. background: rh blood group system and mainly antigen d is one of the most immunogenic, diverse and clinically important protein-based blood group. antibody anti-d may induce hemolytic transfusion reactions and hemolytic disease of the fetus and newborn. anti-d prophylaxes become ineffective if an anti-d immunization has occurred. approximately % of the d+ population carries rhd alleles associated with reduced d antigen expression. qualitative variants, in which some epitopes are lacking and can produce anti-d antibody, are usually termed partial d. by contrast, d weak is commonly defined as a quantitative variant that have all d epitopes and should not make anti-d. del is a very weak form of d antigen and cannot be detected by routine serological tests. because some of del individuals have already developed an anti-d antibody whereas others did not this group contains both qualitative and quantitative changes. aims: investigation was prompted by finding discrepant results in typing of d antigen in a pregnant woman / rd pregnancy, st delivery, abortions in st trimester/. routine serological techniques detected d negativity and the presence of antibody allo-anti-d in clinically significant titre. the non-invasive testing of d status of the foetus from maternal peripheral blood was indicated, but this was not applicable due to presence of the rhd gene in the woman's dna sample isolated from buccal swab. our aim was to investigate the discrepancy and determine the underlying rhd genotype. methods: blood samples, dna from peripheral blood and buccal swab of the pregnant woman were investigated. routine blood grouping and antibody testing were performed by column agglutination. two anti-d sera (id-diaclon anti-d igg (cell line esd ) by biorad and anti-d duo igm+igg, clone: th + ms by immucor) were used for adsorption/elution test for identification of del phenotype. initial rhd genotyping was performed by rt-pcr (exons , , ) with the dna from buccal swab; further resolution was performed using pcr-ssp (fluogene; inno-train diagnostik gmbh); sequencing was performed by sanger analysis (inno-train diagnostik gmbh). results: genotype was identified as rhd positive by ce-certified pcr-ssp kits (fluogene). sanger sequencing of rhd from exon to revealed presence of a nucleotide deletion in position c. dela, which is specific for allele rhd* el. . this nucleotide change results in the amino acid change p.val leufs* causing the del phenotype. presence of antigen d was proved by adsorption/elution technique. titre of the anti-d was rising during the pregnancy to the level two weeks before the delivery. the newborn was delivered by s.c. without a sign of hemolytic disease. blood grouping of the newborn revealed blood group a, d negative, dat negative, testing for del was not performed. summary/conclusions: the case reported here shows that females with rhd* el. allele are able develop strong anti-d immunization, so this type of del phenotype belongs to the "partial del subgroup". presence of variant rhd gene in mother disabling antenatal fetal genotyping from maternal blood by current methods requires a more attentive approach to care for such pregnancies. supported by mh cz-dro uhkt and rvo-vfn . a-s - ea scharberg , s rothenberger , a st€ urtzel , n gillhuber , s seyboth , e richter , g rink and p bugert institute for transfusion medicine and immunohematology, drk-bsd ba-w€ u-he, baden-baden institute for transfusion medicine and immunology, heidelberg university, medical faculty mannheim, mannheim, germany background: rb a (di ) is a low prevalence antigen of the diego blood group system. it has been found in few families only. the clinical significance of anti-rb a is unknown so far. the slc a *c. c>t (p.pro leu; isbt allele name: di* . ) allele is the molecular basis of the rb a antigen. in the gnomad database this gene variant was found in only one of , sequenced genomes (allele frequency: . ). aims: to prove the frequency of the allele in our population and gain an rb a positive donor we performed a molecular screening for di in , blood donors. after our antibody screening test accidentally contained an rb a positive test cell we found out that anti-rb a is a very common antibody specificity. the frequency of the antibody in patients and blood donors was proved. methods: for the molecular screening of the blood donors we developed a pcr-ssp method. the antibody screening test in , patients and in blood donors was performed in the gel technique (biorad ahg id-cards) using a cell screening panel (drk-bsd src) including an rb a positive test cell. positive reactions with the rb a positive cell were confirmed by an additional rb a positive test cell of different source. additional antibodies were excluded or identified in the same method using an antibody identification panel (drk-bsd irc). results: the molecular screening for the di* . allele in , blood donors revealed no single positive individual. within the first weeks of usage of our antibody screening test which accidentally contained the rb a positive test cell patients with anti-rb a were found. it was . % of , patients tested in laboratories in different parts of germany. some laboratories stopped using the rb a positive lot to avoid expensive and time consuming identification and conformation tests. in of randomly tested blood donors ( . %) anti-anti-rb a was also present. summary/conclusions: despite the very low frequency of the di* . allele, anti-rb a is a very frequent unexpected antibody in patients and blood donors in germany. it is obviously naturally occurring and is even more frequent than anti-wr a and anti-vw we found in previous studies in around % of patients and donors. a-s - national blood center, ministry of health and sports, yangon, myanmar hemovigilance which detects every event not only for patient' reactions and donor's complications but also incidents and near misses definitely improve quality of blood transfusion services especially for those situations where implementation of all the standards in one time is not possible. healthcare system in myanmar is still in the stage of requiring priority for clinical professions and has limited resources for supportive roles. supportive services including transfusion service are still not a center of interest from prioritization of health care system. blood transfusion service has been practiced in myanmar since . real essence of transfusion service is hidden behind laboratory practice and transfusion is regarded as part of laboratory investigation. hospital laboratories take care of testing of blood donated by replacement donors. this kind of transfusion services under laboratory umbrella is still being practiced in myanmar except national blood center (nbc) which was established in in accordance with blood and blood product law. this law was formulated cohesively with who strategies of blood safety. in , who global data-based study sent questionnaires for assessment of safety status of transfusion service. nbc noticed that there was no data which can support corrective actions for safety. from that time onward, active retrospective review of existing data and introduction of records, prospective finding of process errors and any events from hospital blood banks were recorded and taking into actions at local level. cost of every unit of blood is supported by government. in , national blood and blood product committee was established. the steering committee is working hard to get cooperation from every service by aiming to prevent those undesirable events before establishment of national level policy, standards and guidelines for sustainable service quality. in conclusion, by using essence of hemovigilance as a tool, quality of transfusion service can be improved step by step to fulfil the gap in spite of limited resources. the system started in local, extends to regional level by getting agreement of importance from hemovigilance results and is finally approaching to national level endorsement. background: erroneous transfusion of abo-incompatible(aboi) blood almost always reflects a preventable breakdown in transfusion protocols and standard operating procedures and can have disastrous consequences, with significant morbidity and mortality. these incidents need to be investigated in a systematic manner to identify system vulnerabilities to mitigate risks and improve patient safety. since , reporters to shot have been asked to score( - ) the extent to which the cause of incidents can be attributed to key factors: staff, environmental, organisational and government/regulatory which helps recognise the key factors identified whilst investigating these incidents. aims: to understand why unintentional transfusion of aboi blood components continue to happen despite standard procedures and national guidance available. methods: retrospective analysis of unintentional transfusion of aboi blood components reported to shot between - (inclusive) was done to identify common themes and recognise areas of improvement. information provided using the shot human factors investigation tool (hfit) between - was reviewed to understand more about why the errors occurred. results: sixty-seven unintentional aboi transfusions were reported between - ; majority ( / , . %) were red cell transfusions but aboi plasma ( / ) and platelet transfusions ( / ) were also seen. most errors occurred in the clinical area ( / , %), and could have been detected at point of administration. in ( %) cases, the error could not have be detected at the point of administration with a primary laboratory error in / ( %) incidents. reviewing data from hfit for cases in - ( aboi cases), the total score for staff culpability was , compared to a total score of for all the other three organisational and system factors. this disparity is most obvious for the aboi red cell cases, all of which scored the maximum for staff culpability, i.e. / compared to / as the combined total score given to the other factors. in the preceding years ( to ), there were no hf scores available; however, the emphasis on staff-related culpability is demonstrated by cases that included an outcome of the local case review and ( . %) mentioned staff-related retraining or disciplinary procedures. the risk of haemolysis and serious harm is more likely with aboi red cells than with other components with / ( %) that resulted in death, / ( %) major morbidity and / ( %) no or minor adverse reaction. of these cases, one resulted in conviction for manslaughter and at least two staff dismissals. summary/conclusions: transfusion never events continue to occur, and it is evident that investigations into such incidents focus mainly on staff failings and do not consistently identify system wide changes that need to be incorporated to address prevalent issues. national recommendations and a safety alert to 'use a bedside checklist' immediately prior to administration were issued between - to support prevention of such errors but never events continue to persist. current approach is ineffective because it often leads to apportioning blame, rather than understanding the often-complicated and multidimensional factors contributing to the error. this must be replaced by a holistic approach which addresses local work pressures and embraces advances in automated technology like electronic prescribing and barcode scanning. of the confirmed trars, n = were possibly related to treatment, n = trars were probable, and n = were definitely related to treatment; n = trars were grade , n = were grade , and none were grade . in recipients of conventional wb, there were n = ( . %) ars, n = ( . %) fnhtrs, n = ( . %) taco, n = trali, and n = ( . %) unclassified transfusion reactions. of the confirmed trars, n = were possibly related to treatment, n = trar was probable, and n = were definitely related to treatment; n = trars were grade , n = was grade and n = was grade . there were mirasoltreated wb transfusions in pregnant women and trars ( . %), both grade and probably related. there were transfusions of mirasol-treated wb and transfusions of conventional wb in patients < years old resulting in n = ( . %) trars in recipients of mirasol-treated wb and n = ( . %) in recipients of conventional wb. summary/conclusions: timely data reporting of trars and expanding the hv infrastructure has helped to improve the hv system in ghana. of wb transfusions in routine use in ghana, there were . % trars in recipients of mirasol-treated wb and . % in recipients of conventional wb. additionally, mirasol-treated wb was safely transfused in pregnant women and pediatric patients. haematology, monash health, melbourne, australia background: transfusion-associated graft-versus-host disease (ta-gvhd) is rare and usually fatal. it can be prevented by provision of irradiated blood products to at-risk individuals, such as those receiving nucleoside analogues, alemtuzumab, bendamustine or with hodgkin lymphoma (hl). duration of risk is uncertain, so ensuring these individuals correctly receive lifelong irradiated blood components, as currently recommended by anzsbt and bsh guidelines, is challenging. in australia, platelets are routinely irradiated, but red blood cells (rbc) are not. aims: to determine whether patients receiving fludarabine, cladribine, bendamustine, alemtuzumab, or dacarbazine (for hl), appropriately received irradiated rbcs. secondary outcomes included rates of ta-gvhd after unintended exposure to non-irradiated components, factors influencing correct issue of irradiated rbcs such as transfusion management plans, and provision of adequate clinical information on blood requests. methods: we performed a retrospective audit to identify patients receiving therapies indicating risk for ta-gvhd using pharmacy dispensing records from january to october at monash health, a multi-campus university hospital in melbourne, australia. diagnosis, treatment dates, group and hold (g&h) requests, rbc transfusions, and follow-up information were sourced from laboratory and medical records. results: we identified patients who received fludarabine (n = , %), bendamustine (n = , %), cladribine (n = , %), dacarbazine for hl (n = , %) and alemtuzumab (n = , %). the median age of patients was years (range - ) and ( %) were male. median follow-up was months (range - ). post-exposure, patients ( %) received transfusions with % correctly receiving irradiated rbcs. the remaining , all from haematology/oncology, received a total of unirradiated rbcs. in patients, this was rectified on subsequent transfusions. there were no cases of ta-gvhd at median follow-up of . months (range - ) from first rbc transfusion. after medication administration, patients had g&h requests after a median of months (range - ). only % of requests had sufficient clinical information to prompt irradiation, such as hl or medication details, and only % asked for irradiated components. preventive strategies have now been employed. transfusion management plans for haematology patients were implemented in march . for audited patients, these were written from days prior to days after medication exposure. two were written following inadvertent unirradiated rbc transfusion. patients identified in this audit will have a laboratory flag generated and prospectively, pharmacy dispensing records will be sent to blood bank to identify at-risk patients. our hospital is transitioning to electronic medical records (emr). an alert will be generated in emr when ordering transfusions if there has been exposure to these medications. however, clinical awareness and documentation remain vital. additional measures include patient education, alert cards, and ongoing collaboration with medical staff to encourage transfusion planning. summary/conclusions: recognition of patients at risk for ta-gvhd remains low, even among haematology units. we are making progress on ensuring provision of lifelong irradiated blood components in patients exposed to nucleoside analogues or alemtuzumab, as well as hl patients. implementation of an emr and additional strategies in this domain is important to prevent ta-gvhd. background: blood transfusion is considered an essential element in the management of patients globally. it might be risky and transfusion related adverse reactions may occur with the less adherence of transfusion policies. standard guidelines regarding the screening of blood for infectious disease, genuine need of transfusion and abo compatibility are followed and monitored drastically. however, patient assessment during transfusion especially at patient bedside and post transfusion is also equally important. aims: we are a newly established hospital and are working towards the best possible management of patients. in this regard to minimize the transfusion errors and to highlight if any lacking being practiced during transfusion, we conducted this study to observe the compliance rate of documentation of transfusion form by the healthcare staff and also to observe the compliance of line of action taken in case of occurrence of transfusion reactions. methods: this was a observational study conducted at nibd and bmt, pechs campus from february to february . ethical approval was obtained prior to the study. transfusion form for each transfusion was filled. the form provided information on documentation of blood product receiver name, employee identity number, date and time of receiving blood product, patient name, medical record number on units, on patient's wrist band and on transfusion form. abo compatibility on the unit and on form, medical record number from wrist band, name and employee identity number of two healthcare staff started transfusion, transfusion start and completion time. time, temperature, blood pressure, pulse and initials of staff at the time of order, onset, after minutes and at the completion of transfusion were also included. transfusion reaction form was also filled by the healthcare staff. data was analyzed by using spss version . . results: a total of transfusions forms were analyzed. over all compliance rate was %. out of , ( %) forms were available in source notes and of , ( %) were partially and completely filled. higher compliance was seen in the initial months of hospital establishment than later months (p-value = . ). highest non compliance was seen in documentation of initials of duty doctors on transfusion form at the completion of transfusion( %) and highest compliance was seen in documentation of name by healthcare nursing staff at the start of transfusion( %). a total of ( . %) adverse events were reported from red blood cells and platelets. mean time of start of symptoms was hours and minutes for red blood cells and for platelets it was hour and minutes. transfusion was instantly stopped as the symptoms appeared with no delay of time and actions were taken to resolve the reactions. time of appearance of symptoms and time of start of medication were documented and error free. all blood bags were returned to the blood bank and discarded after hours as per the policy of hospital. summary/conclusions: the study was conducted to highlight the scarce practices that are being implemented by healthcare staff in context of documentation and reporting of transfusion reactions at our hospital. stringent actions should be taken for the adherence of compliance by healthcare staff to avoid morbidities and mortalities. we believe that it will also be helpful to provide baseline information in the process of preparation of a national guidelines and protocol on blood transfusion procedures. a-s - as buser, a holbro and l infanti regional blood transfusion service, swiss red cross, basel, basel, switzerland to make blood supply safer, pathogen inactivation (pi) technologies have been developed. they are based on photochemical (amotosalen/uva or riboflavin/ uv) or uv-c light treatment to reduce potential pathogens in blood components. this gain of safety might however be offset by "off target" effects of these technologies. in virtually all clinical platelet transfusion trials, it has been demonstrated that post transfusion increments with pi platelet (plt) components are lower as compared to conventional components, indicating different biological behaviour such as survival/ clearance of nontreated and treated plt. published studies have also suggested shorter survival of platelets in vivo in animal studies. additionally, data of the rates of alloimmunization and refractoriness after transfusion of pi platelets are show discrepant results. animal studies suggest a reduction of the rates of alloimmunization when transfusing (leukoreduced) pi plt as compare to conventional plt. in the clinical setting, published data, including very recent reports, showed different rates of hla class i and ii alloimmunization with the two currently available photochemical based pi technologies. while pi of plt components surely benefit patients regarding pathogen safety, the impact of potential off target effects possibly impairing efficacy of pi plt transfusions need more investigation. background: brucellosis is an endemic disease and still a major health problem in saudi arabia. ministry of health in saudi arabia listed brucellosis as a notifiable disease due to its endemicity. in the last ten years, the incidence has decreased significantly to approximately cases per , but is still higher than that in developed countries. human-to human transmission is extremely rare including breast feeding, transplacental, sexually and blood transfusion. five cases of brucellosis through blood transfusion have been reported in the literature. brucella transmission through blood transfusion is likely underreporting due to the long incubation time of - weeks (range, days to months),vagueness of clinical presentation and lack of hemovigilance systems in endemic areas. (allohsct) and ( . %) autologous (autohsct) hsct patients, with mean corrected count increments (cci) of . , . and . , respectively. mean cci decreased in a linear fashion between day ≤ and day pcs ( . , . and . at ≤ days; . , . and . at days, respectively), although the number of pc transfused on day to autohsct patients was small (n = ). background: nipah virus (niv) is a paramyxovirus (genus henipavirus) that emerged in the late s in malaysia and has since been identified as the cause of sporadic outbreaks of severe febrile disease in bangladesh and india. niv infection is frequently associated with severe respiratory or neurological disease in infected humans with transmission to humans through inhalation, contact or consumption of niv contaminated foods. nipah virus (niv) belongs to the list of pathogens identified by the who to have the potential for a global pandemic. aims: this study aimed to investigate the efficacy of the theraflex uv-platelets system to inactivate niv in platelet concentrates (pcs). the theraflex uv-platelets system (macopharma) uses uvc light without the need of any additional photoactive compound. methods: plasma reduced pcs from bcs ( % plasma in additive solution ssp+) were spiked with virus suspension ( % v/v). pcs (n = , ml) were then uvcirradiated on the macotronic uv machine (macopharma) and samples were taken after spiking (load and hold sample) and after illumination with different light doses ( . , . , . and . (standard) j/cm )). the titre of the niv (malaysia) was determined as tissue culture infective dose (tcid ) by endpoint titration in microtitre plate assays on vero cells (atcc â crl- tm ). the results of the infectivity assay demonstrated that uvc irradiation dosedependently inactivated niv. after spiking a niv titer of . (bag no. ) and . (bag no. ) log tcid /ml was received in the pcs. at a uvc dose of . j/cm and higher niv was inactivated down to the detection limit of the system ( . log tcid / ml), resulting in log reduction factors of ≥ . (bag no. ) and ≥ . (bag no. ). summary/conclusions: our results demonstrate that the theraflex uv-platelets procedure is an effective technology to inactivate niv in contaminated pcs. vs. ae . e platelets/unit, p < . ), whereas the platelet content of apheresis pc did not change ( ae . vs. , ae . , p = . ). summary/conclusions: pathogen reduction resulted in the transfusion of older pc on average, but without altering the number of pc ordered or the use of pc per patient. pathogen reduction has improved pc stock management without an increase in platelet demand, despite lower platelet content of buffy coat pc after pr implementation. donors and donation -donor adherence -are we doing the right thing? the transfusion procedure is the last step in a multi-process supply chain. the task of matching supply with demand requires donor managers to consider average consumption rates on a weekly or monthly basis, but to also have insight into variability in order distribution and possible attribute (blood groups) requirements. since hospitals and blood banks are usually not deeply interwoven and often only ex-post data is available, forecasting methods should be implemented. a thorough analysis of order pattern to set weekly target inventories and safety levels is required to close the information gap. a collection plan needs to identify possible bottlenecks which can be prevented through the planning of inter-shipping, changes in message urgency and building of reserve donor pools. constant analysis of collection and mobilization kpis allows donor managers to implement the rolling-wave planning approach and continually adapt to changing requirements, unexpected events and overall systematic variability. the variability happens on the demand side, as order quantities and their attributes, such as blood group distribution, are subject to change. however, also the supply is subject to significant variation, as donor response rates, attrition, deferrals and overall availability of donors are not constant. the data was collected with the face-to-face interview method right after the donation. first-time donors has attended to the study in regional blood centres in cities in turkey. the survey included items in accordance with the standard tpb predictors of attitude, self-efficacy, and intention. self-identity, anticipated regret, donation anxiety, paraphernalia anxiety, personal moral norm, descriptive norm, satisfaction, motivation also assessed for the first-time donors. the relation between the predictors and intention confirmed with correlation analyses. the predictors' distribution analysed by multiple linear regression. a number of goodness-of-fit indices were calculated and examined for each tested models (ibm, amos spss). the results of goodnessof-fit tests for proposed model provided a better fit to the data than these models (cmin/df = ). moreover, this result indicated that the fit between the proposed model and the data could be improved with further modifications with the inclusion of paths between motivation and attitude, self-identity and intention. moreover, inclusion the paths between donation anxiety and intention and between self-efficacy and attitude, on contrary to recent analyses suggesting opposite paths. evaluation of goodness-of-fit tests showed good result for revised model with a value of cmin/df = . , close to perfect fit. the revised model revealed that attitude was the strongest positive direct predictor of intention followed by personal moral norm, self-identity, motivation and anticipated regret (path coefficients: . , . . . , . , and . , respectively). donation anxiety was the negative direct predictor of intention (- . ). satisfaction was the strongest positive indirect predictor of intention via attitude and followed by self-efficacy ( . and . ). paraphernalia anxiety was the negative indirect predictor of intention (- . ). descriptive norm did not show any significance. our model accounted for . % of the variance in intention. summary/conclusions: these findings suggest several potential avenues for enhancing donor retention. the results obtained with this study provide important data from the standpoint of donor retention, which should be, implemented in the future strategies of turkish red crescent. background: transpose-transfusion and transplantation: protection and selection of donors, is a european consortium project, including partners from countries, reviewing donor selection and protection policies for substances of human origin (soho).one of the main issues in the current donor selection system, which transpose aims to tackle, is that for many, if not most criteria, is not evidence based. the transpose consortium therefore tries to re-assess selection criteria, revised them where needed and provide recommendations as evidence-based as possible. transpose additionally adds to the current european directorate for the quality of medicines & healthcare (edqm) guidelines by emphasizing donor safety. aims: the aim is to compare existing donor eligibility criteria throughout europe, and to compile a list of risks to consider, with evidence-or consensus-based deferral criteria to provide more uniform donor screening criteria. methods: there are three horizontal work-packages (wps); wp coordination, wp dissemination, and wp evaluation of the project, and four technical ones with specific deliverables and milestones to be regularly produced: -wp inventory of donor selection & protection practices; -wp development of risk-based guidelines for donor selection and protection; -wp development of a standard donor health questionnaire (dhq); -wp training course/workshop on the use of the guiding principles, guidelines and the dhq. the transpose project launched in september and will complete in spring . wp has completed its work in october, wp will complete its work in june , and wp and wp have recently commenced. results: with the use of the deliverables created by wp , we have created an indepth inventory of current practices in donor selection and protection, including overview of similarities and differences across european countries and across soho types. there is an agreement amongst experts that existing guidelines are often based on the precautionary principle rather than on risk assessment. consequently, in the development of wp 's guidelines for donor selection and protection, we now make an effort to also emphasize donor safety, in a more evidence-based way via the use of risk-based assessments. this will result in a standardized dhq with a common trunk and more in -depth questions per soho. summary/conclusions: the impact of the outcomes of transpose will be threefold. first, outcomes are expected to be of help in revising donor selection and protection related eu directives. second, the set of guiding principles and donor selection & protection guidelines will facilitate eu member states to take a next step in implementing donor selection and protection policies in a consistent and clear-cut way to the benefit of both donors and recipients of soho. third, a standard donor health questionnaire with carefully guided local/regional/national adjustments will become available per soho which can be used widely and will consequently enable comparisons of the prevalence of certain risks and risky behaviours throughout europe. background: transpose-transfusion and transplantation protection and selection of donors is a european consortium project, including partners from countries, that reviews donor selection and protection policies for blood, plasma, tissues, assisted reproductive technology (art) and stem cells (together soho). donor selection criteria (dsc) in europe are based on eu-directives, guidelines and countries' own additional criteria. literature shows that particular criteria are outdated or not risk-based, often leading to unnecessary donor deferral or an underestimation of risks for donors. aims: to ) provide a comprehensive inventory of current systems for selection and protection of donors and donations, ) critically review them and ) recommend an over-arching donor health questionnaire (dhq) including all necessary criteria currently used by different eu-member states (eu-ms). methods: in-depth semi-structured interviews with key stakeholders in blood collection were conducted to identify main topics for improvement in the current dsc. these formed the basis for a survey sent to professionals from collection institutions of all soho to get feedback on current systems from as many eu-ms organisations as possible. questionnaires were sent to a total of experts ( blood; plasma; tissues; stem cells; art) and ( %) completed questionnaires were received. where information was lacking, additional experts were asked to recommend upon dsc. results: for blood and plasma donation four main areas of concern in dsc were identified: risk-based selection, adaptability, flexibility and consistency. the stakeholders agreed that dsc are often outdated and lack evidence, hence leading to unnecessary deferral of donors and underestimated risks for donors. they suggested to base dsc on group risk-assessment (risk-based selection) and on conducting more research to achieve standardized risk perceptions and evidence-based deferrals, either for safety of recipient or donors. criteria could be made more detailed to fit specific groups to defer less donors (adaptability). furthermore, implementing criteria was considered easy, but abolish criteria when not regarded as a risk anymore seems almost impossible (flexibility). additionally, deferral periods are perceived too long, seen as both negative, i.e. jeopardizing donor return intention and positive, i.e. no risk for safety (consistency). changing legislation into guidance was an often-mentioned suggestion to improve dsc. specific feedback on plasma donations revealed that many whole blood topics are not applicable to plasma-only donors, e.g. parasite infections such as malaria (no deferral needed); travel history (no deferral needed), and recent bacterial and viral infections (deferral periods currently too long). a clear need for more research on plasma collection-related issues was identified. summary/conclusions: dsc are perceived redundant on a substantial number of aspects by most stakeholders. besides achieving the goal of save and sufficient soho for patients, many regulations could be improved to diminish deferrals and decrease donor risks. transpose will add to reviewing, improving and harmonising these regulations and criteria. furthermore, transpose will provide suggestions to improve directives and guidelines and a dhq, focusing on both donor health protection and safety of donations, but also removing deferral criteria that are not relevant (anymore), and offer a future research agenda to make dsc more evidence-based. background: transpose -transfusion and transplantation: protection and selection of donors, is a european commission co-funded project with participation of stakeholders from both not-for-profit and private blood collecting organizations as well as researchers and officials. the project aims to create new evidencebased donor selection criteria as well as guiding principles for risk assessment of threats to the safety of all substances of human origin (soho) except solid organs. as part of this, an inventory of current donation-related risks was performed, including an investigation of both type and number of adverse events reported. aims: we here aim to present an overview of reported adverse events in plasma and whole blood donation in europe and to compare this to the anticipated risks rated by transpose stakeholders. methods: national or local data on adverse reactions from the years - , both serious and mild, in whole blood and plasma donors was collected from the relevant stakeholders (eighteen and nineteen respectively). stakeholders were also asked to grade the most important anticipated donor risks according to severity, level of evidence and prevalence. we then compared the relevant risk categories as evaluated by the stakeholders with the categories of the provided data, as well as the heterogeneity of category numbers. results: thirteen stakeholders provided data on adverse events during whole blood donation in a given year, including in total thirty-three different categories of adverse events, ranging from only one unspecified reaction to seventeen different categories, with an average of nine categories per stakeholder. the most frequently used categories were hematoma (included by %), arterial puncture ( %) and nerve damage ( %). vasovagal reactions were also frequently included ( %); however, this was being done variably as vasovagal reactions unspecified, and acute and/or delayed vasovagal reactions. only one stakeholder reported iron deficiency. for plasma donation, seven stakeholders provided data on adverse events. a total of twenty-seven different categories were reported, ranging from one to seventeen per stakeholder, with an average of nine. the most frequently reported adverse events were hematoma ( %), citrate reactions ( %) and arm pains and nerve damage (both %, respectively). anticipated risks in blood donation were rated by nine stakeholders rating iron deficiency, vasovagal reactions and hematomas the greatest risks to donors. for plasmapheresis, six stakeholders rated vasovagal reactions, hematomas and citrate reactions as highest risk. summary/conclusions: as shown, categories used to describe adverse events in blood donation vary tremendously across europe, with some countries only being able to provide total numbers of adverse events without further specification. furthermore, there is a gap between perceived high donor risks and reported adverse reaction categories in donor vigilance for whole blood, as reports on iron deficiency are virtually absent despite being considered the most significant risk. our findings show the need for international collaboration on creating an international standardized donor vigilance system, to gather more insight into donor risks to protect the health of donors. plenary session -a glimpse of the future pl- - modern transplantation medicine has made significant progress within the last decades due to a better immunological understanding of rejection and advances in immunosuppression. however, the severe side effects of long-term, typically lifelong, immunosuppression and the shortage of donor organs remain the major restrictions in transplantation. the idea behind all research to improve transplant outcome has always been the modification of the recipient's immune system to ideally induce a specific tolerance towards the donor's graft. in fact, the immunological blindness of the recipient towards the donor's graft is achieved by a general reduction of the immune system's competence and represents a major burden for transplant patients. the idea of invisible organs is an entirely different approach to solve the problem: instead of inducing an immunological blindness of the recipient's immune system an immunological invisibility of the donor's organ is created. this is achieved by genetically engineering the transplant to eliminate the organ's immunogenicity defined by the gene products of the major histocompatibility complex (mhc) and minor histocompatibility antigens. in addition to manipulating the expression of mhc genes required for immune recognition, immune cloaking strategies are used to evade immune rejection. these approaches take advantage of creating an immunosuppressive environment and expressing immune suppressive molecules by immunomodulatory transgenes. mhc engineering and immune cloaking in an entire organ is achieved during ex vivo perfusion by lentiviral transduction of gene expression modifiers and transgenes to induce a permanent immunological invisibility of the organ. importantly, mhc engineering also prevents the presentation of minor histocompatibility antigens, which usually are not possible to match between donor and recipient, but which trigger potent immune responses and graft rejection. eliminating the targets of cellular and humoral rejection as well as creating an allograft-specific immune environment through immune cloaking camouflages the organ and equips it with a powerful set of defense weaponry. immune-engineering of transplants achieved during the inevitable ex vivo period of the allograft after explantation without the need to accept off-target effects allows keeping the recipient's immune system fully functional and capable to combat infections and cancer. in pre-clinical in vivo studies from rodents to minipigs a clear survival advantage of ex vivo engineered transplants could be demonstrated. this approach has the potential of eliminating the burden of organ rejection and immunosuppression, thereby sustainably increasing transplant survival, organ availability and quality of life. gene editing for sickle cell disease: re-expression of the fetal c-globin genes (hbg / ) could be a universal strategy to ameliorate the severe b-globin disorders sickle cell disease (scd) and b-thalassemia by induction of fetal hemoglobin (hbf, a c ). we have previously identified bcl a erythroid enhancer sequences, marked by hbf-associated common genetic variants, that are required for repression of hbf in adult-stage erythroid cells but dispensable in non-erythroid cells. recently we have optimized conditions for selection-free on-target crispr-cas editing in human hscs as a nearly complete reaction without detectable genotoxicity or deleterious impact on stem cell function. we demonstrate that cas :sgrna ribonucleoprotein (rnp) mediated cleavage at core sequences of the + bcl a erythroid enhancer results in highly penetrant disruption of gata binding motif, reduction of bcl a expression, and induction of fetal c-globin. erythroid progeny of edited engrafting scd hscs express therapeutic levels of hbf and resist sickling, while those from b-thalassemia patients show restored globin chain balance. moreover we find that hscs preferentially undergo nonhomologous as compared to microhomology mediated end-joining repair. nhej-based bcl a enhancer editing approaching complete allelic disruption in hscs appears to be a feasible therapeutic strategy to produce durable hbf induction. in this presentation, i will compare and contrast bcl a enhancer editing to other autologous curative gene therapy and gene editing approaches at various stages of clinical and pre-clinical evaluation. oxygen is vital for life. without oxygen death is assured for aerobic organisms. although everybody knows this fact a lot of medical acts forget to take care of it, leading to a lot of potential troubles. indeed, during cell respiration the glucose oxidation by oxygen gives carbon dioxide, water and energy. this energy also called atp is necessary for cellular metabolism and consequently for life. we have identified an extracellular hemoglobin coming from a marine worm, called arenicola marina, which is able to deliver oxygen to this animal living in the intertidal areas on the atlantic coast in france between the north sea and biarritz. this molecule called m was developed in the medical device named hemo life â . we have showed that this product was very efficient to protect organs before transplantation. a multi centers clinical trial performed under the supervision of pr. le meur from the chu of brest, on patients waiting kidney grafts showed a delay graft function reduced roughly by three between the two kidneys harvested on the same donor with and without hemo life â and grafted on recipients. in , a world first was realized in france by the pr. lantieri to georges pompidou hospital in paris, france. indeed, it was the first time that a patient received a second graft face. this surgery was realized with hemo life â and showed a very nice result according the pr lantieri, the anastomosis were very easy and no edema was observed. furthermore, we have developed dressing incorporating m making a product called hemhealing â . preclinical data on diabetic mice showed an increase of healing process. hemoxycarrier â , a therapeutical oxygen carrier, is also in progress of development in order to address ischemic diseases such as the sickle cells disease, myocardiac infraction and stroke. this universal oxygen carrier without blood typing, which is the ancestor of our red blood cell containing hemoglobin showed that it is able to deliver oxygen at different biological levels, cellular, tissues and organs and could address a multitude of medical applications. background: main goal of transfusion is saving life and/or improve the health status of human by "safe blood" which needs regular, voluntary, unpaid blood donors. donor recruitment is being more sensitive and challenging part of the blood supply system in actual global socio-economic conditions. achievement to enough voluntary non-remunerated blood donation (vnrbd) can be established by an efficient donor recruitment. efficiency of the donor recruitment has still close relation with blood donor recruiter although there are so many new tools. occupational specifications, rights and responsibilities of blood donor recruiter have wide range differences between countries which cannot be explained completely by the specific conditions of each country. also, a concrete document which has an international consensus was not existing on this subject. turkish blood foundation (tbf) has been organizing an international workshop since ; anatolian blood days (abd). "who is a blood donor recruiter?" was the topic of abd-vii at - march . aims: main aim of the workshop was to check and evaluate the existing systems of the participant countries. than create a model for clearly defining occupational specifications, responsibilities and rights of blood donor recruiter. methods: experts from countries participated in the workshop. those countries are albania, algeria, bosnia-herzegovina, estonia, france, germany, hungary, india, kazakhstan, lithuania, macedonia, montenegro, oman, portugal, qatar, romania, russia, saudi arabia, serbia, slovenia, sri lanka, tajikistan, turkey, uganda, uzbekistan. these countries reflect almost all religious, ethnical, social, cultural and economic situations of the world. a questionnaire which was analyzing existing systems at participant countries sent before the workshop. after country presentations different discussion groups were organized. below listed topics were announced at final declaration. results: donor recruiter: . should have university degree preferably in marketing and business administration field. . should have a certificate and/or professional experience in public relation . should have efficient skill in conversation, sociability, independence, self-confidence, reliability, resilience and conscientiousness as well as to work in a team . should get a special training which includes not only social topics such as public relation, marketing, etc. and medical topics related bb&tm before practicing alone as a donor recruiter . should be a permanent staff . should have basic salary and performance bonus might be given . is eligible to monitor and modify mobile team working period at blood drive . should participate the mobile blood drive which he/she has organized . should participate the group who will create promotional materials for national blood service . number at each blood establishment should be defined based on annual blood collection such as staffs for , whole blood collection annually in germany. summary/conclusions: in conclusion; both donor recruitment and retention are not easy tasks to undergo while public are aging, and birth rates are decreasing all around the world. dedicated blood donor recruiter whose occupational specifications, rights and responsibilities are clearly defined will be the corner stone of the success for providing enough safe blood for transfusion. ct smit sibinga and j emmanuel background: africa is a large continent with independent states and a total population of , , , (february ) . healthcare policies and strategies are developed through who's advocacy, guidance, and support from hq in geneva and the who regional offices; eastern mediterranean regional office (emro) supporting arabic speaking countries and the african regional office (afro) responsible for sub-saharan countries. population distribution is approximately . % urban. there are a large number of different local dialects and languages spoken. the main languages spoken are english, french, portuguese, spanish and arabic. countries are mainly classified by undp as being of low and medium human development index the africa society for blood transfusion (afsbt) has members in most countries, advocates for the development of sustainable and effective blood services, and has developed a stepwise level accreditation program. in emro held a consensus meeting developing a "strategic framework for blood safety and availability for - " with a set of priority interventions focusing on leadership and governance, cooperation and collaboration, provision of safe blood and blood products, appropriate clinical use of blood, and quality system management. in all member states of the african union (au) countries, in abuja, nigeria, pledged that national budget for health should be at least % of the national fiscal budget. in ministers of health of who member states endorsed that blood and blood products be included in the essential medicines list; these endorsements and who's universal health coverage (uhc), have yet to be fully implemented. aims: to analyze (gap-analysis) to what extend countries in africa implement the world health assembly resolution wha . on availability, safety and quality of blood products, which urges governments to ensure safe, accessible, affordable and available supplies of blood and components from voluntary non-remunerated blood donors, which meet clinical transfusion requirements and achieve national self-sufficiency, following who guidelines and recommendations. methods: to provide an overview of the current status of the blood supply in africa strengths and weaknesses, data from who's global status report on blood safety and availability were analyzed and used. the study has been descriptive and explorative. results: the report identified a number of areas requiring attention; principle amongst these were -inadequate funding; -lack of governance and leadership; -ineffective public education on blood donation; -absence of capacity building for clinicians on rational use of blood; -lack of haemovigilance and implementation of quality management systems; -the need for regulatory or oversight mechanisms. summary/conclusions: national authorities should address areas requiring attention if progress towards ensuring a sustainable safe and sufficient supply of blood products is to be achieved. key is the commitment and support of national governments, which should implement resolutions and recommendations agreed by ministers of health at wha and african union. background: the core function of the blood donation testing (bdt) laboratory is to screen every unit of blood collected from a donor for blood group type and infectious disease markers to ensure safety of the national blood supply prior to transfusion. the lab operates daily on two work shifts, comprising of staff on the morning (am) shift (from : to : ) and staff on the afternoon (pm) shift (from : to : ) on weekdays and staff on the am shift and staff on the pm shift on weekends. bdt lab has a staff strength of to be rostered for the work shifts. each staff is on a five-day work week and has to work pm shift and am shift per month on average. the higher number of pm shift leads to staff feedback that they do not get sufficient time in the evenings for family and social or leisure activities. a lean six sigma project was initiated to review the work rostering to improve the work-life balance of the staff. aims: the project aims to reduce the number of staff working on the pm shift without affecting the downstream processes and continues to meet the timely release of blood supply to the hospitals. methods: lean six sigma tools were used to study the bdt lab workflow process and to identify factors that contributes to the higher number of pm shifts that the staff has to take on. data on the turn-around time and the man-effort required for each screening tests performed was analyzed. a survey was also conducted to understand the preference of the staff on the acceptable number of pm shifts per month. results: the main contributing factor for more staff required to perform the pm shift is due to majority of the daily donation samples being received only in the evening. as this factor is beyond the control of the bdt lab, redeploying work from the pm shift to the am shift was eventually selected as the solution to reduce the number of staff needed for the pm shift. the screening test that was shifted was determined based on the test system that has the shortest turn-around-time and is able to allow continuous release of results. at the same time, most of the staff must be trained for that test system. a trial on the new roster involving staff on am shift and staff on pm shift was conducted. the total number of pm shift per month was reduced from to using the re-defined process. the % reduction translates to fewer number of pm shifts that the staff has to undertake and was able to meet the staff's expectation. summary/conclusions: with the adoption of the new process workflow, bdt lab was able to reduce the number of pm shifts that the staff needs to be rostered using evidence based process improvement method. most importantly, the lab has a satisfied team of staff with better work-life balance. background: preparedness of blood transfusion services for emergencies and crisis situations is an important issue concerned with patient and transfusion safety. aims: having an experience of delay in blood component supply in an emergency situation due to partial interruption of hospital information system (his), it is aimed to create a crisis kit and constitute an alternative work flow for emergency in crisis situations. methods: it is stipulated that the failure of his which is normally conducts all process for transfusion would be disabled in a disaster or crisis situation. a brain storm was made on possible challenges associated with disability of his during transfusion emergencies. according to the scenarios a kit was developed for the process management of transfusion emergencies. results: a flow chart was designed in proper with transfusion emergency definitions of who and instructions were written to explain the flow chart. all forms categorized with different colour codes are designed to fill with handwriting. the kit consists of flow chart and instructions, analysis request forms (blue coloured), blood component request forms (pink coloured), proceeding forms (green coloured), pens and blood sample tubes with edta were put into a plastic folder labelled as transfusion emergency disaster & crisis (tedc) kit. additionally, the kit is placed in a sealed clear plastic bag and delivered to all inpatient and intensive care units of pediatrics and pediatric surgery. a training programme concerned with transfusion emergency situations and usage of the tedc kit was developed for health care workers involved in blood transfusion process. pre and post-assessment tests were developed for the evaluation of effectiveness of the training programme. summary/conclusions: it's challenging to improve the response capacity of blood transfusion services during emergencies and crisis situations. abstract withdrawn. background: india is a developing country having licensed blood banks majority have manual documentation which causes inaccuracies and errors in blood bank activities. monitoring is a herculean task. computerisation is the need of the hour but this goal involves many hurdles and challenges aims: the aim of this study is to discuss the challenges faced during computerisation of blood bank activities and the remedial solutions for it methods: department of transfusion medicine, king george medical university, lucknow is one of the biggest blood bank of the country with annual collection of , blood units. two years ago, the blood bank worked on totally manual system. computerisation involved challenges associated with hardware and software installation and personnel training. hardware was installed in two phases. initially hp system but later shifted to apple imac due to frequent breakdowns. with hp server. software installation (easy software) involved erratic internet connectivity hence changed to lan. customisation involved radical changes according to our needs. at times we had to change our way of working to suit the software. biometrics linking, medical registration, cash id generation, donation, serological crossmatch, automated blood grouping, labelling, chemiluminescence & nat testing, blood component preparation and camps were all included with challenges at every level. remedial actions were taken from small to big. training of the staff was the most essential part of the implementation of computerisation who initially showed considerable resistance and at time faking ignorance due to apprehension that their mistakes will be highlighted and they will be penalised for it. it was a herculean task in creating their password protected identity and enforcing them to use it. gradually the staff realised that computerisation made their task easier as it cut down on paper work and repetition and also prevented serious mistakes from happening. hard copies at certain essential areas were still maintained to continue work in the event of major breakdown of computer results: computerisation aided us in regulating the movement of the donor which at times was repetitive due to manual entry. transfer of data ensured a safe supply and the mistakes could be retraced very easily. implementation which included installation, training and enforcement took a period of months. after overcoming all the challenges we minimised hard copies to registers and started taking printouts of the other necessary details. the turnover time for the employees due to computerisation decreased by %. waiting time for attendant decreased by %. traceability of all the units became %.supervision of the activities being carried out was % accurate. identification of the donors was easy due to biometrics which included thumb impression and iris scanning. the decision making time for donors decreased by % thus making the system more efficient. summary/conclusions: manual to computerisation involves involvement from source to supply and it is essential to anticipate the challenges and be prepared for solutions in order to make its implementation successful p- ct smit sibinga , y abdella and f konings iqm consulting, zuidhorn, netherlands who eastern mediterranean office, cairo, egypt background: who defined essential medicines (ems) as medicinal products that satisfy health-care needs of the majority of a population. they should be available at all times, in adequate amounts, in appropriate dosage forms, with assured quality and affordability. in blood and blood products (whole blood, red cells, platelets, plasma, and plasma-derived products) were added to the who model list of ems. appropriate and effective regulatory framework (legislations, regulations, etc.) and a functioning regulatory authority (ra) is crucial for management of blood products as ems. however, particularly in the less developed world, these prerequisites have barely been implemented. aims: to analyze and advise on existing legislation and regulations. existing legislative instruments of the member states of who eastern mediterranean region (emr) were collected and analysed for relevance and appropriateness for preparation and use of blood and blood products as well as use of associated substances and relevant medical devices. a literature search was done on matching combinations of regulatory system, regulatory framework, legislation, regulation, with production and use of blood and blood products, which resulted in almost exclusively references with respect to national and international legislation. benchmark: who recommendations (aide m emoires) and eu directives. methods: existing legislative instruments of the member states of who eastern mediterranean region (emr) were collected and analysed for relevance and appropriateness for preparation and use of blood and blood products as well as use of associated substances and relevant medical devices. a literature search was done on matching combinations of regulatory system, regulatory framework, legislation, regulation, with production and use of blood and blood products, which resulted in almost exclusively references with respect to national and international legislation. benchmark: who recommendations (aide m emoires) and eu directives. results: various formal legislative documents of only / countries are put in force by governments [ (egypt) till (pakistan -sindh)]. most are detailed descriptions of ra, operational establishments, and specific requirements. however, none of these legislations complies with who and eu recommended formats and contents, and will not support effective regulatory oversight to promote and enhance quality, safety and availability of these ems. summary/conclusions: government should provide effective leadership and governance in developing a national blood system (nbs, fully integrated into the national health-care system. essential functions of a nbs include an appropriate regulatory framework with legislations, regulations and other non-legislative instruments administered by a ra. these documents should spell out principles and cadres, standard setting, and organization of the blood system to ensure an adequate supply of blood and blood products and safe clinical transfusion for which a model was designed. the structure of nbs will depend on organization and level of development of the health-care system. however, all critical activities within nbs should be coordinated nationally to promote uniform standards, economies-of-scale, consistency in staff competency, quality and safety of these ems, and best transfusion practices. key is formulation of an appropriate regulatory framework administered by a ra responsible for regulating the vein-to-vein chain in the preparation and use of these ems. background: the capacity of blood transfusion service to provide adequate supplies of blood components is the issue of concern for health providers worldwide; longer term observations of trends in this respect are therefore of crucial value. aims: the study aim was analysis of some basic activities of the polish blood transfusion service in - including organizational changes, numbers of donors, donations and blood components as well as activities directed at increasing their safety. methods: retrospective analysis of data supplied by the regional blood transfusion centers (btcs). results: in the discussed period, blood and blood components were collected in regional btcs and local collection sites as well as during mobile collections. although the number of local collection sites decreased from in to in in favor of mobile collections, which increased from , to , , the former is still the number one location for donating blood. on average . % of all donations were performed in local collection sites. the total number of blood donors both at the beginning and the end of the discussed period was similar ( , in and , in ); over % of all donors were non-remunerated. however, the number of first-time donors dropped significantly (from , in to , in ). the total number of donations increased from , , in to , , in ; most frequent were whole blood collections (from , , in to , , in ) . some blood components (mostly plasma and platelet concentrates) were also collected by apheresis. most frequently prepared blood components were red blood cell concentrates -rbcs ( , - , , units per year), fresh frozen plasma -ffp ( , , - , , units) and platelet concentrates -pcs ( , - , units, with significant increasing tendency). additional processing methods such as leukocyte depletion and irradiation were more frequently applied to pcs ( - . % in respective years irradiated, . - . % leukocyte-depleted), than to rbcs ( . - . % irradiated, . - . % leukocyte-depleted). in , the pathogen reduction technologies in plasma and the pcs were implemented. up to date however the use of these technologies is limited in most btcs. in approximately . % of pcs and % ffp units issued for transfusion were subjected to pathogen reduction technologies. summary/conclusions: our study data may contribute to the assessment of some long-term tendencies observed in polish blood transfusion service and may serve practical-benchmarking. this in turn may prove beneficial to the transfusion community as a whole. background: in poland % of hospitals depend on blood for the treatment of patients; over . mln units of blood components are annually transfused. it is therefore purposeful to expand the knowledge on factors impacting on blood transfusion service (bts). the institute of hematology and transfusion medicine (ihtm) as competent authority is responsible for collection of data related to the activity of all polish blood transfusion centers (bcts). this data is exploited to a much lesser degree than the recently available statistical methods and data processing tools would allow. moreover, survey of research in the field of public health indicates a negligible share of issues related to bts. it seemed therefore necessary to "fill in the gap" with true assessment of performance of the polish bcts for improvement of bts activity. st stage of our investigation refers to collection, merging of data from different sources, their unification and preparation (big data) for further analysis to be performed using multidimensional statistical analysis and data mining methods. aims: assessment of the activity of the polish btcs over the year-period in two stages. goals at st stage: . data digitalization; scanning of paper documents. . development of a uniform template for collecting digital data from various sources. . standardization, unification and quality improvement of available data: filling in missing data, elimination of errors, duplicate records etc, that may distort the outcome of analyzes. . selection of data for analysis. methods: digitalization and big data methods for processing various types of data: a) stored in paper form ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) , b) digital stored in two file types (.doc and .xls, for the years - and - , respectively). for each data-type, a separate excel file model was created. the models were then merged into one analytical table with data processing methods. results: . pages of paper documents were scanned. . models developed for data from different sources: a. paper-data were rewritten and ascribed to its model; outcome - tables, columns, , rows. b. .doc and .xls. filesdata were ascribed to other models; outcome - tables, columns, rows. . the models were merged into analytical table to create a mb database (comparable to approx. min of music). . the data was subjected to standardization, unification and quality improvement: filling in missing data, elimination of errors and duplicate records that may distort the results of analyzes. . selection of data for analysis at nd stage. summary/conclusions: the st stage provided a set of selected data for analysis in the nd stage which will rely on multidimensional statistical analysis and data mining methods. the outcome of such analysis will contribute to optimal realization of objectives: a) gaining in-depth knowledge about the fundamental phenomena that shape polish bts, b) identification of potential changes bcts, c) development of overall guidelines for change management. aimed to touch untouched or less touched topics of bb&tm. so far workshops were organized and each year around countries were participated from almost all religious, ethnical, social, cultural and economic situations of the world. . supported realization of major changes in bb&tm in turkey; a convincing medical individuals and agencies mainly moh to give the deserved consideration to bb&tm b encouraging the recognition and establishment of national blood program c issuing a new blood act and numerous necessary bylaws, etc. d creating an appropriate standard donor questionnaire form e changing blood transfusion practice from % whole blood (at ) to %. f changing donated blood screening criteria; while anti-hcv screening became obligatory malaria, screening cancelled g preparing national guidelines h promoting haemovigilance nurse post i promoting patient blood management . around , blood bankers attended national courses, , attended national congresses, , attended nationwide symposiums. summary/conclusions: bbtst can be accepted as a sample how a scientific nongovernmental organization may give a very positive impact on developing and progressing bb&tm activities with close collaboration moh and other related organizations abstract withdrawn. background: globally there is growing investment in information technology (it) in business. this similar trend has been observed in blood establishment computerized systems (becs). the it investment can be high hence it decisions need to be properly informed. the africa society for blood transfusion (afsbt) encourages the use of its in african blood services as this optimize quality blood services, thereby improving patient's outcomes. afsbt established in an it working group (afsbt itwg) with the support of the swiss red cross (src) to spearhead the it standards among member blood services. a number of priority it thematic areas were identified. these includes it governance which focuses on creation (strategic alignment) and preservation (risk management) of business value. there is absence of published literature on how a structured it governance framework can be implemented in a resource constrained setting. a review of the national blood service zimbabwe (nbsz) it governance was done based on published it governance framework. aims: to explore how a structured it governance can be developed, implemented and monitored in a resource constrained setting. methods: a published mit-cisr framework which has six components was used to assess the strength, gaps and opportunities of the it governance. results: nbsz has been implementing an evolving structured it governance system. in terms of service strategy and organization there is a well-established it function which is reflected in the nbsz strategic plans. this ensures it annual budgetary support, which averages . % of the total budget. the it governance arrangements are such that decision rights are assigned to different it staff (executives, it specialist, and users). a range of it solutions have been embedded within the nbsz operations such as becs, financial, donor mobile application, social media, temperature monitoring, and human resources. the business performance goals are defined and are congruent across the various business units. it organization and desirable behaviors are documented in the ict policies and procedures and were needed remedial actions are available through the code of conduct. the it metrics are included within the nbsz monitoring and evaluation system which use a four colored traffic lighting reporting system. it was noted that the it accountabilities are undesirably tilted to the it specialist only hence some ict projects tend to have delayed deliverables. the it governance mechanisms are supported with tools such as service level agreements and established communication approaches. simple excel based solutions are used to track critical performance metrics such as on the interactive blood supply management status, which averages . % ( ) based on a -day projected stocking and supply levels. nbsz need to properly document the return on investments on all these ict initiatives, which is estimated ( / ) to be at . % of annual savings. summary/conclusions: blood services in resource constrained settings can implement a properly structured it governance and this will ensure maximum return on it investments. the nbsz approach will be shared and further developed in the afsbt itwg to support other blood services in improving their it governance. haematology/blood transfusion, alfred health, melbourne, australia background: in october , an integrated electronic medical record (emr) was implemented at an australian metropolitan multi-campus heath service using cerner millennium tm , aiming to achieve himss (healthcare information and management systems society) level . prior to implementation, large numbers of blood specimens were collected from patients unnecessarily and sent to pathology without a test request attached (no blood test requested -ntr). these specimens required additional processing in the laboratory. electronic specimen collection using cerner specimen collect tm allowed streamlining of specimen processing by eliminating paper requests. as part of the new workflow, individual specimen labels are printed with the specified blood test and correct tube type. this helps prevent the practice of collecting additional specimens due to uncertainty of the collection requirements. aims: • to quantify the expected reduction in ntr specimens following introduction of electronic specimen collection, and outline the benefits • to determine the impact on collection errors and wrong blood in tube (wbit) events methods: data was obtained directly from cerner millennium tm using a ccl (cerner command language) query which is run monthly by pathology it staff. this data includes all specimens registered for the month with indication of rejected specimens, wbit & ntr samples. 'rejected specimens' includes incomplete specimen and/or request certification, unlabelled specimens/requests and mismatched specimens. further information about wbit events was collated from riskman reports and staff interviews. results: data from the months prior to emr implementation was compared with months post. ntr numbers reduced from /month to /month ( % reduction), freeing up more storage space in fridges. rejected specimens due to inadequate patient request labelling reduced from a mean of /mth to /mth. wbit numbers have increased slightly: before having median (range - ), after with median (range - ). although it was hoped that wbit incidence may reduce with the new emr, of the post implementation wbits involved electronic specimen collection. departure from planned protocols involving a lack of working printers, causing staff to print patient labels away from the patient's bedside, as well as multiple patient labels printing on individual printers appear to be a main cause of the emr wbits. summary/conclusions: emr implementation has led to a reduction in ntr, and rejected specimens due to inadequate request labelling, as well as increased storage space in laboratory refrigerators. associated benefits include: • decreased financial costs of the wasted equipment • decreased staff time collecting and processing unusable specimens • decreased environmental impact of manufacture and disposal of unused specimens • decreased potential of iatrogenic anaemia work in preventing the occurrence of further wbits is ongoing, by ensuring that label printers are in working order, are in plentiful supply and easily accessible to staff; and also ensuring positive patient identification and blood collection by the patient's bedside remains a priority. jm mustaffa , k teo , s tsai , p heng , r sagun and m wong laboratory medicine khoo teck puat hospital, singapore, singapore background: khoo teck puat hospital (ktph) is a -bed general and acute care hospital, opened in , serving more than , people living in the northern sector of singapore. the blood bank of ktph department of laboratory medicine provides specimen testing and blood transfusion services for ktph as well as the neighbouring yishun community hospital (ych), one of the largest community hospitals in singapore providing intermediate care for recuperating patients including rehabilitative services. the process of ordering transfusion-related test requests in both hospitals is through printed forms. aims: in line with the hospital directive to move towards electronic patient management, the ktph blood bank intended to implement an electronic type and screen (e-t&s) system. the goal of the project is to ensure zero patient identification errors and maintain full traceability and accountability for the blood collection process in all transfusion-related testing. another aim of the system is to reduce repeated venepunctures when specimens are rejected due to missing essential patient information on the printed forms by implementing mandatory fields in the e-t&s form. methods: the e-t&s was implemented in phases. phase : an online version of the printed form was signed electronically by the ordering doctor and a witness within the electronic medical record system, sunrise clinical manager (scm) system with the doctor counter-checking by signing on the specimen label to ensure correct patient identification. phase : the ordering doctor is not required to sign on the specimen label since fingerprint biometrics are required for the electronic signin. phase : elimination of the witness step for blood collection. specimen collection and rejection data from to was analysed. specimen rejection rate was presented as percentage of rejected specimens (mislabelled, unlabelled and clerical errors) over total specimen count for each month. results: between january and march , before the implementation of the e-t&s phase , the average rejection rate for blood bank specimens was . % and . % for identification and clerical errors respectively. during phases and of implementation, rejection rate increased due to unfamiliarity to the new work processes. by february , with the implementation of the final phase of the e-t&s system the specimen rejection rate was . % and . % for identification and clerical errors respectively. rejected specimens were mostly from the few locations that had to use paper requisition due to workflow or infrastructure limitations. summary/conclusions: the e-t&s system was implemented successfully in ktph. full traceability and accountability of the blood collection process was maintained with the fully electronic system. the adoption of electronic documentation has also reduced the number of preventable repeated venepunctures that were due to incomplete order information on the printed forms. future developments in technology and full implementation of e-t&s system in all hospital locations may make zero patient identification error achievable and ensure transfusion safety in all patients in the near future. background: blood component administration represents a critical phase due to the possible occurrence of errors during the different steps from the identification of the patient to the infusion of the product. error occurrence can be reduced by the implementation of validated information systems. we tested the scweb â system at the bedside in a transfusion outpatient clinic. aims: the aim of the study is to validate a system designed to assist and to control blood administration step by step using electronic devices to ensure traceability and documentation of the process methods: the scweb â system is based on it monitored checklists which guide the personnel to follow the procedure, according to best practices; the system must initially be activated by the operator which is recognized by an auto-signing system based on bluetooth low energy which avoids the operator having to identify himself/herself beforehand. appropriate privacy protection is provided. thereafter the system takes up the task to give instructions and to verify the adherence, by asking an active confirmation of the proper fulfillment of the activities; a continuous registration and documentation is made by the system. standards and specifications for each step of the procedure have been configured on scweb â system to track in detail operator and patient identification, presence of informed consent to transfusion, blood pressure, pulse and temperature recording, vein access, verification of the blood unit. an alarm has been set after min, to ensure the control of patient's conditions. for each step, an active confirmation of the action is required and nurse and doctor direct involvement must be actively confirmed on the device by both operators. the system has been tested at the bedside on patients admitted to the outpatient clinic for red cell concentrate transfusion; compliance of the personnel and organizational impact has been recorded. results: the system required a very short training: ease of scweb â system allows its implementation without negative impact on organization of transfusion outpatient clinic and without difficulties by operators (nurses and doctors), who appreciated the help given by the it check system. the registration of the electronic check list offered a reliable tool for the traceability of the transfusion procedure, also granting a paperless and timely available documentation of the entire process through a registration in electronic format of all the operator's action in every single phase of the transfusion process. when prescribed, confirmation of the checklist was only possible in the presence and with the active confirmation of two operators (doctor and nurse). summary/conclusions: the scweb â system is useful as a barrier against the mismatch of transfusion (preventive measure), as a traceability and documentation measure and as a tool for training of personnel in blood transfusion administration; it avoids paper registration during the transfusion process, due to the timely registration of the activities performed by operators recognized by the system thanks to the bluetooth low energy auto-signing device. the scweb â system will be connected to the transfusion data management system, to monitor all the process from the arrival of the unit from the blood bank. background: he blood banks aims at reducing cost and increasing customer satisfaction by providing quality in service. the quality in service can be attained by streamlining the processes and restructuring the supply chain of the organization by implementing it tools. aims: aim is to understand the complex flow of information and processes within the supply chain of the blood bank. the requirement of such a study is a part of the integrated erp modeling for the integrated functioning of a blood bank. methods: he approach used to understand and map the sequence of processes, and the work responsibilities of each process and the operational decisions involved at each step is process mapping and data flow diagrams for front end system modeling and analysis. the processes are mapped and represented in a schematic diagram. dfd (data flow diagram) are constructed for representing the system. a context diagram is also constructed for understanding the entities interacting with the system. the emr systems aim at replacing (or supporting) the paper based medical records. the whole model of the system is divided into two parts-front end and back end. the front end design and analysis is done using epc (event-driven process chains), resource views, data flow diagram for data view. reporting was on donor selection, finance and collection of blood bag, blood collection process, component preparation, blood testing and blood distribution results: process mapping using event driven process chain generated a whole view of the processes involved. the resource view gave an organizational structure and the personnel involved. the data view using context diagram and data flow diagram gives a flow of data and amount of data involved. this framework can be used for business process reengineering for the blood banks by conducting a time study and removing non value added activities. data view helps analyze redundant data in each process. it also helps in staff training and orientation within the department. summary/conclusions: a systematic overview presented in this paper facilitates in removal of non value added processes, duplication of data, bottlenecks, reduction of cycle time and thereby improving service quality in blood banks. background: the transfusion of blood components, one of the most prevalent interventions in clinical practice is a major expenditure item in healthcare services which tend to increase in recent years. aims: it is intended to investigate the impact of transfusion associated costs to hospital costs in pediatric intensive care unit (picu) patients. methods: during a year period (january -december ) patients, females and males receiving transfusion with blood components along the stay in picu were included in the study. transfusion associated costs and total costs for healthcare services for children treated in picu was collected by using hospital information system (his). statistical analysis of data was performed by spss software (version . , spss inc., chicago, il, usa). mann-whitney u test and kruskal-wallis test was performed for comparison of independent categoric variables and numeric data; chi-square analysis was performed for comparison of two numeric variables and spearman correlation analysis was performed for associations. results: the median age of patients was . months (interquantile range-iqr ). the median length of stay was days (iqr ). in total blood components were transfused in which of red blood cell concentrates, apheresis platelet concentrates, granulocyte concentrates, fresh frozen plasmas, and cryoprecipitate and whole blood. the ratios of transfusion associated expenditures to hospital costs were categorized in intervals of percentages as < %, - %, - % and > %. most of the patients ( . %) were ranked in the lowest interval. the medians for hospital cost and transfusion associated cost were . euros (iqr = . ) and . euros (iqr = . ), respectively. a significant strong positive correlation between numbers of transfusions and hospitalization cost of picu was detected (r: . , p < . ). while it was found a significant weak positive correlation between transfusion associated cost and hospital cost (r: . , p = . ) there was also a significant weak positive correlation between the age and transfusion associated cost (p = . , r: . ). a significant difference was found between the patients with and without hematological malignancies (p < . ) for transfusion associated cost. the reason why pediatric dosages are mostly prefer is that the hospital provides healthcare for only children and splitting of the blood components was common in the hospital. but unexpectedly a significant increase on the transfusion associated costs which is related to split blood components was detected (p < . ). summary/conclusions: studies on the economics of blood transfusion have been conducted mostly in patients who require chronic or multiple transfusions. picus, specialized facilities that provide care for patients with severe life-threatening diseases are major departments often necessitate multiple transfusions. there are many variables to evaluate the impact of transfusion associated cost to hospital cost in picu patients, but the major factors are underlying conditions, admitting diagnoses and transfusion strategies. although there are unexpected data in our study demonstrated the increasing impact on transfusion associated cost originated from blood components split for pediatric usage no significant relationship was determined to explain this situation. further studies on the economics of blood transfusions have to be carried out to clarify the variables of transfusion associated costs. background: approximately . % of the transfused blood component is packed red cell (prc). over ordering of prc unit is a common practice and excessive pretransfusion testing was being wasteful of resources and have adverse consequences on cost. high crossmatch to transfusion (c/t) ratio as quality indicator of blood bank implies that crossmatches were performed unnecessarily. aims: the aims of this study were to evaluate the cost effectiveness of strategies for limiting the number of pretransfusion testing of ordering prc. methods: all prc units who ordered from dr. hasan sadikin hospital from january to december were collected in this retrospective study. number of ordering prc unit, completed pretransfusion testing of ordering prc units, and prc units that were transfused were recorded. restrictive pretransfusion testing strategies were done based on the hemoglobin level and diagnosis as transfusion indication criteria. cost effectiveness was measured by multiplying the unit cost of pretransfusion testing and number of prc unit. results: out of total , ordered prc unit, , ( . %) were subjected to pretransfusion testing and . % ( , ) of ordering prc unit which are pretransfusion testing were transfused. this means that . % ( , ) of ordering prc unit were not subjected to pretransfusion test. this showed savings of , , , rupiah. c/t ratio was . which demonstrate a good ordering pattern. however, . % ( , ) of completed pretransfusion testing of ordering prc unit were not transfused, leading to blood bank loss of , , , rupiah. summary/conclusions: strategies for limiting the number of pretransfusion testing on the good c/t ratio was still associated with saving cost effective background: blood is a precious resource for saving patient lives. the purpose of blood and blood component therapy is to provide suitable and safe blood products to achieve best clinical outcomes. nurses have an important role in ensuring safe blood transfusion. it is crucial for nurses to have sufficient knowledge about blood donation and collection, storage, component preparation, possible adverse effects of blood transfusion and necessary management and care. aims: the aim of this study was to assess the impact of an educational intervention on knowledge and awareness of nurses regarding blood transfusion services and practices. methods: the baseline study to assess the knowledge and awareness regarding blood transfusion services and practices of the nurses posted at various areas of the hospital including wards, operation theatres and critical care areas was carried out at our institute hospital which is a tertiary care teaching centre. the nurses were then sensitized and educated regarding blood transfusion services and practices during their day to day activities by referring them to the blood transfusion guidelines of the institute. subsequently, a self-developed questionnaire which was used for the baseline assessment of knowledge and awareness of the nurses was again used to reassess them. a total of questions were included in the questionnaire pertaining to: general awareness (two questions), blood donation (two questions), testing and blood component preparation related (two questions), storage of blood/blood components (two questions) and pre-transfusion checks and bed side transfusion practices (eleven questions). fifty nurses each were included for both the baseline as well as post-sensitization assessment. for different category of questions, the correct response rates were compared with those obtained in the baseline study using mann-whitney test. the entire study duration was spread over a period of three months (december, to february, . results: the overall mean percentage of 'correct' responses for questions in the baseline study was . %, whereas post sensitization it was . %. the mean percentage increase in general awareness related questions was . %, . % for storage of blood/blood components related questions, . % for pre-transfusion checks and bedside transfusion practices related questions, . % for testing and blood component preparation related questions and . % for blood donation related questions. the percentage increase in correct response was found to be statically significant for each of the five categories of questions. the overall mean percentage increase in correct response rate was also statistically significant (p < . ). summary/conclusions: this study revealed that after sensitization and educational intervention there was a significant improvement in the knowledge and awareness of nurses regarding the blood transfusion services and practices. abstract withdrawn. background: tact, introduced in the uk in to support managers, provides resource-saving, continual, 'real-time' monitoring of knowledge-based competency of staff in transfusion laboratories. tact is available online / , complementing existing practical competency schemes and external quality assessment. multiple variations on a standard pre-transfusion testing scenario are generated using constrained randomisation; logic rules for automatic assessment of sample acceptance, abo/d, antibody screen and identification (as/id), and component issue are based on bsh guidance. during , tact was offered internationally to transfusion laboratory managers to trial, and saw uptake in five countries. the core tact programme, based upon uk guidelines, is under review for programming conversion, to be customisable for the international community. aims: to assess the feasibility of tact programming conversion to meet the requirements of country-specific pre-transfusion testing guidelines, and to direct future programming in line with feedback from international users. methods: guidelines from / international users were obtained and translated where necessary. these were compared against the core assessment elements of current tact programming. international users were approached for their feedback on the current version of tact, as it compared to their local policies and practices. results: the following criteria were cross-referenced: specification of transfusion request forms, sample label acceptance criteria, reagents used for abo/d and as/id, resolution of grouping anomalies, alloantibody confirmation/exclusion, and selection criteria of blood components for transfusion-dependent patients and women of child-bearing potential. apparent differences included:-australia:--selection of red cells for patients with immune anti-d. greece:--inclusion of the name of the patient's father on the transfusion request. italy:--testing of all new patients with an anti-a,b reagent and two different monoclonal anti-d reagents. international users in the same three countries supplied feedback. this included suggestions for:-greater complexity of cases presented, provision of patient history, inclusion of follow-on tests e.g. phenotyping and cells for antibody confirmation/exclusion, broader range of reaction strength grading, and official professional cpd credits. the following differences were noted:-nomenclature used, the format and content of the request form, use of english abbreviations of patient clinical details, and the availability, provision and specification of blood components. summary/conclusions: this analysis has shown very few instances where the current tact iteration differs from the guidelines reviewed, and that it is feasible to expand the use of tact on a more international basis. the current iteration of tact has been developed to represent an abbreviated scope of pre-transfusion testing practices, which can be applied to laboratory practice outside of the uk without difficulty. further work is required to enable international users to configure tact such that the system represents all laboratory practice on an international basis. aims: these courses provide education to clinicians on patient blood management and safe transfusion in neonatal and paediatric settings in order to improve patient outcomes and increase awareness of the national patient blood management guidelines. this analysis aimed to investigate the uptake, practical use, and perceived value of the courses by learners. methods: a retrospective analysis of course completion statistics and course evaluation data. results: there have been , paediatric and neonatal courses completed from march to february with . % of learners being nurses and/or midwives. analysis of course evaluation data (n = ) showed that these courses: -provide knowledge ( . %) -improve patient safety and outcomes ( . %) -result in change to clinical practice ( . %) -are relevant to clinical practice ( . %) -are easy to use ( . %) -are readily accessible ( . %). examples that learners provided of how they can apply this learning to their clinical practice include: -"[i am now] more aware of special requirements for neonatal blood transfusion" -"[i] feel more confident especially when talking with parents" -"[i will now be] checking the patient's blood results and will speak up for unnecessary blood sampling" -"[it's good that] when there is ambiguity in clinical practice [this is] very well shown by explanation from experts in the field" -"we don't do a lot of transfusions [and this is] a reminder that transfusions are not always the first answer to the baby's clinical picture". summary/conclusions: analysis of course and user evaluation data demonstrates that these courses are being used by nurses and doctors working in the neonatal and paediatric settings and that they provide knowledge of pbm that can be applied to clinical practice, thereby contributing to improved patient care. background: blood transfusion is a high-risk clinical activity that must be mastered both theoretically and practically in order to guarantee the required result without any incident or complications. the mastery of transfusion knowledge among nurses represents a very important link in the transfusion chain. the objective of this work is to compare the theoretical and practical knowledge of transfusion among two groups of nurses divided according to their seniority. aims: this is a cross-sectional descriptive study conducted over a period of month [ st april- th april] . we selected two groups of care staff: the st group consists of students at the end of their training at the higher institute of nursing sciences. the nd is made up of nurses working in university hospitals of tunis, currently practicing blood transfusion. the evaluation's tool used was a questionnaire of simple or multiple choice questions, were related to theoretical knowledge of labile blood products and to transfusion practice. ten questions were considered "life-threatening" if their answers were false. a comparative study was made between the two groups. methods: this is a cross-sectional descriptive study conducted over a period of month [ st april- th april] . we selected two groups of care staff: the st group consists of students at the end of their training at the higher institute of nursing sciences. the nd is made up of nurses working in university hospitals of tunis, currently practicing blood transfusion. the evaluation's tool used was a questionnaire of simple or multiple choice questions, were related to theoretical knowledge of labile blood products and to transfusion practice. ten questions were considered "life-threatening" if their answers were false. a comparative study was made between the two groups. results: the participation rate in the survey was %. the nd group participants had an average seniority of years . more than half of them ( %) had seniority of less than years. only % had more than years of experience. the rate of correct answers for all items combined was . % for students versus . % for practicing nurses. the theoretical knowledge part was more mastered in the st group than that of practicing nurses ( . % vs . % of correct answers). on the other hand, the control of the transfusion act was better in nd group ( % vs . %). the overall "dangerous" response rate was % for students and . % for practicing nurses. false practical knowledge was more common in group ( . % vs. . %). summary/conclusions: the theoretical as well as the practical knowledge remains not well mastered by the care staff. our study highlighted the best theoretical mastery for young students and practical for practicing nurses. this could be explained by the freshness of knowledge in the first group and the daily practice in the second group. background: the european commission (ec) directive / /ec on blood donor selection criteria is years old. in the meantime, knowledge on risks related to blood donor selection has progressed and challenged several obligatory rules. transpose -transfusion and transplantation: protection and selection of donors, is a european commission co-funded project with participation of more than stakeholders from both not-for-profit and private organizations providing substances of human origin (soho). the project aims to provide evidence-based donor selection criteria and guiding principles for risk assessment of threats to the safety of soho. as part of this work, an inventory of current blood donor selection criteria in europe and an evaluation of the evidence behind current practice was performed by experts working on this project. aims: to identify the gap between the ec directive / /ec on whole blood donor selection criteria and a current evaluation of the clinical relevance of the criteria based on scientific literature by a panel of european experts within the transpose project. methods: in , we performed an inventory of blood donor and transfusion recipient risks in participating european countries. project members were asked to provide the existing donor selection criteria related to these risks and to carry out a risk-based evaluation for each of them. the evaluation was based on the available scientific literature and on a risk assessment template based on the abo risk-based decision-making framework, developed by transpose. all risks with divergent assessments within the panel were resolved through discussion; in all cases an expert consensus was established. subsequently we compared the results with the content of the ec directive / /ec for every risk, thereby identifying discrepancies and missing items in the directive. results: the panel identified risks considered to be significant, distributed between donors and recipients. for / ( %) of them the expert evaluation deviated from the content of the ec directive, or the ec directive provided no information about the decision making. in particular, a discrepancy was observed for / criteria concerning general health and medication, / for transfusion transmissible infections, / for high-risk behaviour and travel, and / for other diseases. summary/conclusions: our results highlight a significant gap between the whole blood donor selection criteria stated in the ec directive / /ec and the scientific evaluation performed by a panel of transpose participating experts. this gap includes both new risks not addressed in the ec directive and addressed risks that are however evaluated differently. this involves both blood donor and transfusion recipient safety, and various medical and epidemiological topics covering several aspects of the blood donation criteria. we strongly recommend a change in the european legislation, allowing a procedure to guarantee that blood donor selection criteria are updated regularly within the framework of the european institutions, to keep aligned with scientific progress, epidemiology and changes in medical practice, in order to enable an updated risk-and evidence-based framework for donor selection criteria. the risk-assessment method elaborated in the transpose project is a valuable instrument for this purpose. background: the brazilian health regulatory agency -anvisa has developed the method for assessment of potential risk in hemotherapy services (marpsh) which is based on the data collected during the inspections of blood services carried out by regulatory authorities. using marpsh any blood service can be classified in one of possible potential risk categories: high, medium-high, medium, medium-low and low risk. each category represents a different potential risk level, according to the proportion of compliance with the established regulatory requirements. marpsh has been used since , showing a trend of risk reduction on blood services evaluated all over the country. aims: this work aims to describe the utilization of marpsh as a tool for an integrated risk management model. also, it shows the main results obtained after years of data monitoring and coordination of regulatory actions and policies by anvisa, targeting quality and safety of blood products. methods: the utilization of marpsh follows a network risk management model since the inspections are carried out by decentralized organs in all states and some municipalities. the inspectors fulfill a standardized inspection guide containing the regulatory requirements, where each item is associated with a level of risk, varying from i to iii as the risk increases. at the end of the inspection, after a statistical calculation, the service is categorized. this classification gives an estimate of its quality profile, guiding the adoption of suitable measures for risk management by local authorities and services. these data are send to the states (if realized by municipalities) and to anvisa that perform consolidation in a national level. either states or anvisa use data to coordinate risk management measures in a broader spectrum. data are continuously monitored by anvisa as part of its strategical panel of indicators. anvisa follows up specially blood services in high and medium-high risk with the aim of helping or complementing local authorities' actions. additionally, anvisa periodically sends this information to the brazilian ministry of health and local governmental organs from brazilian national blood system that also support actions to improve quality in their blood services networks. results: since , when the assessment covered blood services, marpsh reached blood services in ( % of the blood services registered) what corresponded to almost % of the inspection cover in this year. over this period ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) it is possible to notice a dramatic decreasing in trend for proportion of blood services classified as high and medium-high risk, varying from % to %. summary/conclusions: marpsh generates data necessary to the categorization of blood services into five levels of potential risk. as a result, the regulatory actions are applied by local organs with the purpose of reducing or eliminating risks involved in the production and use of blood components. data have shown a significant risk reduction over years of marpsh's utilization. additionally, monitoring of this data at a national level has been permitting appropriate planning and prioritization of integrated strategies directed to risk management and strengthening of blood services in brazil. background: sub-saharan africa has the highest need of blood transfusion in the world, mainly for childbearing women and children suffering from malaria. meeting basic quality and operational requirements to provide patients with safe blood remains a challenge in these settings. aims: the aim was to identify and prioritize potential hazards for patients in blood bank practices in the democratic republic of the congo (drc). we focused on two subsets: (i) sensibilisation and selection of donors, and (ii) qualification and production of blood products, using failure mode effect analysis (fmea). methods: two risk analysis workshops were organized at the national blood transfusion centre in kinshasa, the democratic republic of the congo. in both workshops, a multidisciplinary team was invited to represent different hospitals and profiles in transfusion management in drc: quality coordinators (n = ), training coordinator (n = ), medical doctor for donor selection (n = ), hemovigilance officer (n = ), laboratory technicians performing donor sampling, blood qualification and production (n = ), biomedical scientists (n = ), microbiologist (n = ), clinical biologist (n = ), nurse (n = ). the principle of fmea was applied, which implies identification of possible hazards (failures) in a process flow, followed by scoring each hazard according to their impact, probability, detection and feasibility. in the both workshops, participants were guided by an external facilitator who guaranteed common understanding of the methodology. main focus of the risk analysis was potential harm to the transfused patient in the process of (i) sensibilisation and selection of donors, and (ii) qualification and production of blood products. all ideas were written on coloured cards and mapped on a chart according to their impact, probability, detection and feasibility score. hazards were ranked according to their final risk score by multiplying these four scores. results: in the process of sensibilisation and selection of donors, the three hazards with the highest final score for impact, probability, detection and feasibility were: (i) a paid donor is recruited after sensibilisation by family members of the patient, (ii) donor selection staff approves a non-eligible donor for blood donation because of personal/financial motivation, (iii) blood donors are not correctly informed about blood donation during sensibilisation by family members of patients. in the flow of qualification of blood products, highest scores were made for: (i) no double check for validation and sorting of qualified blood products, (ii) stock-out of reagents, (iii) no check for match between registered test result and tested blood tube. regarding production of blood products, the top three consisted of: (i) transport time between blood collection and processing is > h, (ii) storage of qualified and quarantined blood products in the same fridge (some sites only), (iii) power cuts. summary/conclusions: the risk analysis resulted in three prioritized hazards in the process of donor selection/sensibilisation and blood product qualification/production. some are very specific to the sub-saharan african setting and have been described before (power cut, family and paid donors, stock rupture,. . .). an action plan needs to be put in place to reduce their final risk score. the risk analysis needs to be continued for the remaining blood transfusion flows. background: . million of germany's population, so just under a quarter of residents, have a migration background. the majority of these has roots in regions where the population has a distribution pattern of blood group and hla-antigens that differs considerably from the predominant one in the german population. sufficient supply of these individuals with red blood cell (rbc) and platelet concentrates (tc) will continue to be a major challenge in the future, as blood donors with compatible blood group antigens are dramatically underrepresented in the local donor pools. many migrants suffer from severe hematological disorders such as b-thalassemia or sickle cell disease and will not only need compatible blood transfusions, but an allogeneic stem cell transplantation in the foreseeable future. as healthy family donors often are not available, at present suitable stem cell donors with a similar genetic background can only be found in international donor registries. aims: this project was initiated to recruit new donors with a migration background for blood donation and to increase the number of blood stem cell donors among this group. methods: serological extended blood group phenotyping was performed by automated gel card technique (fa. grifols, erytra) and included ab , rh (ccdee), kk, fy (ab), jk(ab), lu(ab), m, n, s, s. hla typing for hla-a, -b, -c, -dr, -dq, and -dp was performed by next generation sequencing. allele frequencies were analysed using genepop version . ; the rare and very rare alleles were defined according to the allele frequency database (www.allelefrequencies.net) rbc genotyping using next generation sequencing is currently being established and will include additional antigens with the most frequent distribution pattern differences between migrant and resident populations according to literature. results: so far, more than blood donors with a migration background have been recruited for a blood donation in this project. amongst this group, over blood donors from more than non-european countries enrolled as potential stem cell donors. an initial evaluation of the data revealed a very similar distribution of blood groups compared to the current blood donor population in north rhine-westphalia. of migrant donors, ten fy(a-b-) donors were identified, which corresponds to a percentage of . %. amongst hla-typed potential stem cell donors, we found ( . %) with rare and very rare alleles. summary/conclusions: blood donors with rare blood group and hla phenotypes (e.g. null types such as fy(a-b-)), are in demand for adequate medical care of people with a migration background. the technological development of blood group determination by next generation sequencing will significantly improve the supply for all blood transfusion recipients in germany. this project is funded by the european development fund - (erdf) and the european union. background: mortality due to uncontrolled haemorrhage following trauma is the most important cause of potentially preventable deaths. trauma care systems in low and middle income countries like india, are still in developing phase. also, the role of blood component therapy in improving patient outcomes has been mostly derived from combat settings. application of these protocols in an urban setup has not been well established and marked variation in practice exists. hence this study aims to identify the key components of transfusion practices to optimize the transfusion protocol in trauma settings. aims: to study the current transfusion practices in severely injured trauma patients, admitted to the red area/resuscitation bay after initial triage in ed methods: this prospective observational study was conducted over a period of year starting from june to may at the department of transfusion medicine in collaboration with emergency department at jpnatc, aiims, new delhi. the study included severely injured patients (iss ≥ ) that were admitted within h to the red area/resuscitation bay after triage. data collected included the demographics, injury, laboratory and transfusion details for these patients results: during the study period patients ( . % males) were enrolled. mean iss scores was . . mean time to hospital admission after injury was : (iqr . - : ) hours. mean time to first rbc transfusion following admission was : (iqr : - : ) hours. approximately . % ( ) patients were in shock (sbp < mm hg &/or pulse rate > /min). whereas, ( . %) patients were coagulopathic (pt ≥ . times of normal). during initial h of admission, these patients were transfused with ( . %) rbc, ( . %) ffp, ( . %) rdp and ( . %) cryoprecipitate of total blood components utilized for these patients. massive transfusion (defined as transfusion of ≥ units/ h) was given to ( . %) patients. summary/conclusions: significant quantity of blood components were required during initial resuscitation in severely injured patients. pre-hospital transfusion can significantly reduce the time to transfusion. further studies are needed to assess utility of pre hospital transfusion in severely injured patients. background: allogenic stem cell transplantation recipients are known to be the main consumers of platelet concentrates (pc). the geneva university hospital is one of the three allogeneic hematopoietic stem cell transplantation (hsct) centers in switzerland. since the blood center is also part of the hospital, data of pc consumption are easily available. as needs rose steadily since several years, with an average increase of % per year, pc supply is a serious concern for our center. aims: in this study we tried to evaluate if any pre-transplant indicator could help to forecast the number of pc needed after an allogeneic hematopoietic stem cell transplantation. methods: this observational retrospective study was conducted in geneva hospital on patients suffering from various inherited or acquired disorders of the hematopoietic system who were treated by hsct in . pc consumption was examined from january to december . the five indicators were: gender, stem cell source (bone marrow (bm) vs peripheral blood stem cell (pbsc)), donor type (hla matched ( - / ) vs haploidentical), conditioning regimen (standard vs reduced intensity), and cmv serology of the recipient. results: data for a total of patients aged from to years were analyzed; ( %) were male and ( %) female; ( %) were cmv-negative and ( %) were cmv-positive. out of a total of transplants, ( . %) were haploidentical and ( . %) hla-matched. according to the stem cell source, bm was transplanted in cases ( . %), and pbsc in cases ( . %). two patients also received a cd + stem cell boost. our analysis showed that, with a mean follow-up of days, the number of pc transfused to our patients treated by hsct ranged from to units, with an average of and a median of , illustrating a high variability. the results indicated that gender, stem cell source (bm vs pbsc), conditioning regimen (standard vs reduced intensity), and cmv serology of the recipient do not have any statistical impact on platelet consumption. however, we observed a tendency of an increased need for platelet transfusion when patients were cmv positive. our results also showed a statistically significant (p = . ) higher number of pc transfused for patients treated with a haploidentical ( ) versus hla-matched ( ) transplant. summary/conclusions: this study points out the high variability of platelet consumption after hsct, which limits the forecast of platelet production needed to support allogeneic hsct recipients. a larger cohort would be required to confirm a potentially higher platelet consumption in cmv positive patients, and to consolidate our results showing a higher pc consumption for patients treated with haploidentical transplant. abstract withdrawn. background: historically at our institution, a minimum of four red blood cell (rbc) units were crossmatched for all cardiac surgery cases regardless of surgical case-type or patient characteristics. two rbc units were packed in validated blood product coolers and brought to the operating room (or); the balance of crossmatched units remained in the blood bank. a retrospective review revealed that very few rbcs were transfused ( : % ( / ), : % ( / )). moreover, approximately products were wasted each month as a direct result of this practice. thus, we recognized an opportunity to improve inventory management in terms of personnel activities and blood component utilization. aims: the goal of this study was to reduce advance preparation of coolers in cardiac surgery cases without compromising patient care and safety. we limited our intervention to those patients who were eligible for electronic crossmatch. we maintained the aforementioned historical practice for those patients with history of and/ or those who currently demonstrated clinically significant red blood cell alloantibodies. methods: a multidisciplinary group consisting of representatives from the blood bank, cardiac surgery, cardiac nursing, cardiac anesthesia and surgery quality department was assembled in october to determine whether a modification of practice was reasonable and safe. group members evaluated site specific society of thoracic surgery (sts) cardiac surgical data between july and december to establish intraoperative red cell transfusion rates classified by type and urgency of surgery. the group's main goal was to discontinue preparation of default coolers for patients eligible for electronic crossmatch who were scheduled for all types of non-emergency cardiac surgery cases in which ≤ % of historical cases required at least one red cell transfusion. additionally, team members simulated the multiple protocols by which red blood cells could be prepared and delivered to the or and estimated the time for each scenario. results: review of sts data showed that the following cases met the criteria of ≤ %: elective primary coronary artery bypass graft (cabg), urgent primary cabg, elective mitral valve repairs, and elective aortic valve replacements. simulation showed that, in patients eligible for electronic crossmatch, preparation from receipt of order to completion of unit packing for delivery took . min using the pneumatic tube system (maximum of units per tube) and . min using delivery of a cooler using a human courier. summary/conclusions: based on the simulation results, and with consensus agreement from the multidisciplinary group, default cooler preparation for elective primary cabg, urgent primary cabg, elective mvr, and elective avr was discontinued in december . one year following implementation of the change in policy rbc units were issued to the or (a % reduction); % ( ) were transfused, compared to % in . wastage rates decreased from products a month to per month on average. summary/conclusions: the most obvious drawback of pabd is the higher cost in running the program in comparison with collection of allogeneic blood in the areas of additional patient attention and clerical input in labeling, separate storage and so on. in this audit, % of the autologous blood components were not transfused into the intended recipients and wasted; in this context, the pabd program could not be considered as a cost-effective approach in protecting blood safety. background: the national blood service zimbabwe (nbsz)'s blood supply management status (bsms) is an integral process of ensuring the availability of a safe and sufficient blood supply provision. nbsz introduced a new daily blood bank statement with improved metrics from may . the new analytics approach focuses on three interactive components of the blood bank statement; the available stock, quarantine stock (as per the desired -days stocks level), and the demand versus supply. it is imperative to have a closely monitored blood supply chain because blood has limited shelf life with uncertainties in both supply and demand. the 'blood-for-free' proclamation by the government of zimbabwe in july set more pressure on the blood demand. these metric-based analytics seek to assess if the nbsz's improved blood bank statement is a realistic model for the bsms. aims: to assess the use of the interactive metrics in monitoring the blood supply management status. methods: a prospective cross-sectional study was conducted. a total of daily blood bank statements which were submitted between may and december from each of the five branches were analyzed. the bsms which is calculated as the average of the three interactive measures of quarantine stock, available stock and demand versus supply was determined. sub-analysis of branches was done to determine individual branch performance. analysis by month was done to assess seasonal variations. findings and recommendations were shared among key stakeholders to validate the bsms methodology. results: overall the quarantine stock average was . % (sd +/- . ), the available stock was . %: (sd +/- . ) and the demand versus supply was at . % (sd +/- . ).the overall bsms was . %; (sd +/- . ) for the study period. gweru and masvingo nearly supplied all the demanded blood with . %, overall bsms of . % and . %, overall bsms of . % respectively. bulawayo supplied . % of the blood demanded with an overall bsms of . %. mutare supplied . % with a bsms of . % and harare . % and a bsms of . %. there were monthly variations but the service could supply above % of the blood demand. in the month of may the service met . % of the demand and a bsms of . %. in november and december it supplied . %, bsms of . % and . %, bsms . % respectively. august also had a below average supply of %, bsms - . %. june, october and september recorded above the average values; . %, bsms of . % and . %, with a bsms of . % respectively. summary/conclusions: the overall bsms performance was satisfactory and it was noted that branches capacitated according to demand. the new interactive analytics approach is appropriate for showing the blood bank status and assessing the performance of the branches. this new approach has optimized the decision-making process in blood supply management. the metrics are tracked using excel based model hence this approach is suitable for resource constrained settings with limited ict infrastructure . st vincent's hospital melbourne (svhm), a tertiary hospital supporting medicine, surgery and non-major trauma emergency and itu services implemented a mtp in . subsequent mtp reassessment has led to implementation of regular multi-disciplinary review of all mts to identify areas for improvement in transfusion and other aspects of support for critically bleeding patients. aims: to implement a systematic service-wide stakeholder review of mt events at svhm aiming to identify deficiencies and implement improvements in mt management. methods: a multi-disciplinary mt review team was established as a subcommittee of the hospital transfusion committee (tc) to update the organisational mtp in and subsequently continued to meet quarterly as the mt review subcommittee (mtrs) of the tc, systematically reviewing all aspects of mts at svhm. instances where or more red cell units are transfused in < h are identified from the laboratory information system and reviewed by the mtrs which includes representatives from accident and emergency, intensive care, operating suite (os) and transfusion laboratory staff; the head of the patient's treating unit is also invited to contribute. reviews include: demographics, clinical details, comorbidities, time from patient arrival to pre-transfusion specimen collection/receipt, time from blood request to release/transfusion, regularity of full blood examination (fbe)/coagulation (coag) testing, timing of blood component transfusion, total component provision/ratios, component waste, patient outcome, and communication between various clinical areas and also the laboratory. a discussion summary with actions/ recommendations is provided to the tc and some cases referred to the hospital mortality/clinical review committee. results: cases reviewed: from treating units including cardiothoracic surgery ( ) hepatobiliary/gastrointestinal/colorectal surgery ( ), vascular surgery ( ), neurosurgery ( ), orthopaedic surgery ( ), endocrine ( ) and "other" (encompassing general surgery, urology, general medicine and oncology - ). areas for monitoring/improvement identified: transfusion documentation, regularity of fbe/coag specimen submission, reducing time between patient arrival and specimen collection, reducing specimen transport time, interfacing point of care bloodgas analysers to the central pathology result management system as well as component management/waste reduction and the introduction of viscoelastometry assessment in the os. of reviewed cases involved the transfusion of emergency uncrossmatched o rhd negative red cell units. the appropriateness of the use of this precious resource is also reviewed by the mtrs. summary/conclusions: the svhm mtrs meets regularly to review mt events and formalise multidisciplinary collaboration in identifying possible improvements to support these often critically ill patients. matters highlighted include communication issues, delays in specimen delivery and blood component waste minimisation. areas for further work include minimising delay between mt events and review, and formalisation of key performance indicators for mts. background: the use of radio frequency identification (rfid) technology to manage the blood supply chain is recognized as a major enhancement to the operations of blood banks and hospital transfusion services. to facilitate optimal blood supply management, it is crucial to guarantee the integrity of rfid tags throughout the transfusion chain. since rfid tags can be affixed to blood products very early in the process, these tags undergo the same process-steps as the blood products themselves (e.g. centrifugation, label printing, shock-freezing and irradiation). aims: the goal of this study was to validate the mechanical and functional resistance of biolog-id rfid tags through different blood related processes: centrifugation, label printing, shock-freezing, intensive reading at À °c, and irradiation. biolog-id tags are passive hf ( . mhz) tags. they are compliant with is , iso - and follow the guidelines for the use of rfid technology in transfusion medicine (vox sanguinis, ). methods: biolog-id tags were evaluated using a series of rfid encoding and reading tests. before each of the processing steps, each tag was encoded with donation number, site id, product code, blood group and expiry date. the data was encoded using the isbt format. the different processing steps and conditions tested were: -centrifugation: quintuple whole blood bags, filled with ml water. centrifugation at , rpm for min. tags processed, tags per kit affixed at different positions. -shock-freezing at À °c: shock-freezer (angelantoni, sf ), units processed, reading immediately after removal from shock freezer. water, tags irradiated at gy and tags at gy results: all biolog-id tags were encoded and read with a % success rate in all series of tests. summary/conclusions: biolog-id rfid tags can be encoded and read through common processes used throughout the blood transfusion chain. their mechanical and functional integrity is not affected by centrifugation, shock-freezing, intensive reading at À °c, printing, eto sterilization and irradiation. background: the provisioning of compatible red blood cells by international cooperation is presented. the units were meant for an -year old female, with homozygous sickle cell disease (scd) and multiple complications. patients' blood group was a positive with anti-c, -e, -wr a and an antibody to a high prevalence antigen in the rh system, anti-hr b possibly combined with anti-hr b (rh ). the antibody was not reactive with rh null , -d-or hr b negative cells. the donor center put out an international request for group a or o, rh null or -d-units lacking wr a and possibly k, fy a , jk a , wr a , do a and s (the latter antigens for prophylactic matching). the patient sample had been genotyped for rhd and rhce using mlpa and sanger sequencing and the patient was found to carry rhd* /rhd* n. and rhce*cevs. / rhce*cevs. . aims: the request was sent to the american rare donor program (ardp). the ardp working with the american red cross national molecular laboratory, used the rh genotype information to identify donors carrying the same or similar rh variant alleles using the rh allele matching approach described previously (keller et al. transfusion ( s): a). methods: a recent blood sample was used to confirm anti-hr b ; no anti-hr b was detected. the patient rhd and rhce alleles were used to build punnett squares for both genes with donors carrying the same and similar alleles that would be predicted to be compatible. tier donors are those predicted to carry the same combination of rhd and rhce alleles as the patient. tier donors are those predicted to be homozygous for one of the allele combinations carried by the patient. tier donors are those predicted to carry alleles similar (but not identical) to those carried by the patient, with similar predicted phenotype. the database of donors in the ardp carrying rh variant alleles was queried against the alleles in the patient-specific punnett square. results: donors of group a or o and matched for rh alleles were identified as follows: tier , tier and tier donors. after the clinical team agreed to drop one or more of the prophylactic antigen matches, one tier unit lacking s and jk a was identified at the american red cross. while the request was being processed, the patient experienced a sickle cell crisis, red cell aplasia and recurrent aiha and her hemoglobin level dropped from to . g/dl. at that time, she was transfused the only compatible units available - of the rare -dphenotype and her hb increased to . g/dl and eventually to g/dl. the tier rh allele matched unit was shipped to amsterdam where it was frozen, and reserved for the transfusion care of this patient. summary/conclusions: this case illustrates how rh allele matched blood can be found for a highly rh alloimmunized patient, and can avoid use of the exquisitely rare -d-or rh null blood. background: blood transfusion has been a complicated and high-risky clinical procedure. any error could cause serious injuries to patients. to better assure the procedure safety. aims: we enhanced and built a blood transfusion database platform and develop inventory management strategies to better guarantee the patient transfusion safety. methods: we designed six new features of the platform ( ) assuring the patient identification with barcode techniques; ( ) designing a structured order entry; ( ) proactively reminding the physicians with patient's previous blood transfusion reaction with related precautions including the use of leukoreduction filter; ( ) automatically reminding physicians the happening of reaction and suggesting relevant test; ( ) building a complete traceability log system; and ( ) supporting data analysis. the blood transfusion safety team includes medical technologists, nurses, physicians, system analysts, and blood transporter and the whole process is electronic management. the new blood transfusion platform integrated the workflow, reduced the incidence of abnormal blood samples collected ( % after implementation, p < . ), reduced the time of call for medical technologists with blood component preparation and improved the achievement rate of emergency -min blood crossmatch ( . % after implementation, p < . ). the barcode correctly identified patients and monitored the entire transfusion process to reduce the error rate of blood component supply ( % after implementation, p < . ). summary/conclusions: after the transdisciplinary team approach with e-monitoring and a better design of clinical decision support module with barcode technology, blood transfusion database platform improve the blood supply efficiency and assure blood transfusion safety. background: in the modern world, terrorist acts are characterized by a multiplicity of combined injuries to a large number of victims. qualified medical care is urgently required for a large number of patients in one locality at the same time. it leads to increase in emergency demand for blood components, mostly red blood cells. the desire to donate blood to the victims is a natural manifestation of society's solidarity in response to tragic events. however, donor activity and patient needs do not always correlate. aims: to analyze the donor activity during the terrorist attacks. methods: a retrospective analysis of donation activity in periods of terrorist attacks in moscow ( moscow ( - . the average daily blood donations' number (dbdn) before ta compared with the number of donations in day after ta and with the dbdn during days after ta. also the number of delivered rbc units (d-rbcu) daily before ta and daily in days after were compared. results: in - , terrible ta occurred in moscow: people died and more than were injured. with the explosion in subway in / people died, were injured. the number of d-rbcus increased by % on ta-day, and by % during next days. dbdn in the st day after ta increased , times, and in the next days - , times. second explosion in subway in / resulted in died, injured. the number of d-rbcus increased by % on ta-day, and by % during days. dbdn in the st day after ta increased , times, and in the next days - , times. in (explosion on market) resulted in died, injured. d-rbcus delivery increased by % on ta-day, and by % during days. dbdn in the st day increased , times, but decreased to , times during the next week. with subway explosion in people died, were injured. the number of d-rbcus increased by % on ta-day, and by % during days. dbdn in the st day after ta increased , times, and in the next days - , times. with the explosion in airport in people died, were injured. rbcus delivery increased by % on ta-day, and by % during next days. dbdn in the st day after ta increased , times, and in the next days - , times. summary/conclusions: an increase in donor activity is observed already the next day after ta and usually lasts for days, but does not correlate with the number of victims. the rbcs' delivery from blood bank increases in all cases on the day of the ta. therefore, the guarantee for patients is the maintenance of rbcs' stock, including cryopreserved ones. it is also necessary to promptly send excess of red blood cells harvested at the peak of activity to the cryobank. background: rh system is the major blood group system besides abo system. even after proper blood grouping and cross matching there is a possibility of alloimmunisation in recipients against the rh or minor blood group antigens like kell, mnss, duffy etc. in medical colleges which cannot bear the financial burden of complete phenotyping of patient and donor, implementation of rh & kell phenotypes match blood transfusion can play a major role in preventing alloimmunisation and adverse events in multitransfusion patients aims: to evaluate the efficacy of rh & kell phenotyping as a cost effective measure instead of extended phenotyping in multitransfused patients methods: study was carried out in the department of transfusion medicine, one of the biggest blood bank of the country with annual collection of , blood units. patients of thalassemia, aplastic anemia and leukemia were taken who required multiple transfusions. complete phenotyping was done initially of all the patients before transfusion. patients were taken as control and the other were taken as cases. blood units of healthy donors were chosen ( were males and were females). in all the donor units, identification of rh & kell phenotyping was done by the antigen antibody agglutination test by the erythrocyte magnetize technology on fully automated immunohaematology analyzer qwalys. these blood units were transfused to patients who had been selected as cases. in the control group, patients were transfused blood units which were not phenotyped for rh & kell but gel crossmatching was done. follow-up was done on these patients for transfusion reactions and at the end of six months they were evaluated for any alloimmunisation. results: at the end of months, no reactions were reported in cases receiving rh & kell phenotype blood and no alloimmunisation was seen on repeat phenotyping. the control group on the other hand reported reactions in cases ( . %) and phenotype at the end of three months showed alloimmunisation with 'e' antibody. the phenotypic frequencies of rh & kell blood groups in the population were comparable with other published studies. amongst the rh antigens (e) was the most common ( . %) followed by d ( . %), c ( . %), c ( . %) and e ( . %). thus 'e' was the most common and e was the least common of all the rh types. background: the prevalence of a particular blood group has an uneven distribution in different geographic areas and is largely determined by the national composition of the population. moscow is one of the largest city of europe with population of . million. the understanding of prevalence of red blood cells antigens (rbc-ag) among the population has great importance for blood banking planning. aims: to determine frequency and distribution patterns of transfusion-significant rbc-ag among donors in the moscow region. methods: the results of immunohematological studies on ab , rhesus and kell systems were analyzed retrospectively in blood donors for years ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) in moscow. data collection and processing was carried out using the regional information system for transfusiology. rbc-ag detection (ab , rh, kell) systems was performed using microplate method (automatic immunohematological analyzer "galileo neo" (immucor, inc., usa)) and "ih- " (bio-rad laboratories, usa) with diagnostic cards. results: the most frequent blood group is a (ii) . %, (i) blood group . %, b (iii) . %, ab (iv) . % (n = ). rh(d+) was established as positive in the presence of antigen d and as rh(d-) negative in its absence. donors with weak variants of antigen d (du) were determined as rh (d+) positive. the ratio of rh (d+) and rh (d-) was . % and . %, respectively. donor's phenotype detection was routinely conducted from the year, therefore the number of donors was . the most common phenotype among donors ccdee ( . %), the second in frequency ccdee ( . %), the third in frequency rhesus negative phenotype ccddee ( . %) in the studied population. the ccdee and ccdee phenotypes were . % and . %, respectively. the most rare are ccdee ( . %), ccdee ( . %), ccddee ( . %). other options: ccddee, ccdee, ccdee, ccddee, ccddee, ccdee, ccddee were detected in single cases and amounted to a total of . % (n = ). cw antigen was tested in donors and was detected in . %. cw is most commonly found in donors with ccddee phenotypes ( . %), ccdee ( . %) and ccdee ( . %), with other variants of the data phenotype, the antigen was detected in . % of the examined (n = ). antigen k was detected in . % of donors, in . % of this antigen is absent (n = ). summary/conclusions: the study of transfusion-relevant antigens distribution in population is necessary for building of effective and flexible model for blood service managing. a differentiated approach in choosing a strategy to form a long-term bank for storing blood components, taking into account the frequency of various antigen variants, contributes to improving the quality, accessibility and safety of medical care. of dislocated division of our blood establishment in orthopaedic hospital valdoltra (obv) in the number of outdated units at the hospital side dropped considerably. results: since when the issued number of red blood cell units (rbc) was the amount of issued units rose to in and then dropped more or less steadily to in . in this period the hospitals' programmes rose for % in all areas. number of donated units declined from in to in . after reorganization in the number of outdated units fell from % of stocked units to . %. after setting a dislocated unit of ctdiz on obv location the number of discarded rbc fell from to in . for transfusion specialist who is constantly in contact with the clinician in the hospital the most important day routine is when the stock availability is displayed. it happens times a day; at a.m. when the previews' day collection is released and another three times a day when the updates occur. the central base is led in ljubljana (the capital) and all centres are able to control and order the stock for the blood banking. blood wastage remained low and the traceability of the blood usage in south-western region remains high ( %). though it is not supported by an informational system the traceability form the blood bank to the patient is done on paper. this issue demands a big effort by the staff in blood bank and in hospitals. summary/conclusions: reorganization enabled better stock utilization and traceability of issued units. sometimes it is impossible to predict the peak demand of rbc especially during the summer season when the population of the area doubles and car accidents as well. transfusion specialist's effort in assuring the optimal blood stock represents the crucial daily routine. blood bank, grande international hospital, kathmandu, nepal background: voluntary non-remunerated blood donor consists of % blood donor's population in nepal. therefore demographic about the distribution of blood donors according to the age group is important to achieve % voluntary nonremunerated blood donors in nepal. aims: to explore the demographic distribution of the blood donor in different age group in the kathmandu nepal. methods: this is retrospective study conducted at nepal red cross society central blood transfusion service. data from january to january were collected from donor management software. the data includes socio demographic data. data has been process with spss version - results: during years study period, total of , blood donation happened from both mobile blood collection and in-house blood collection. out of , collection, ( . %) are from - age group; ( . %) are from - age group ( . %) are from - age group; ( . %) are from group - age group; ( . %) are from age group - and ( . %) from age group above respectively. summary/conclusions: the distribution of abo blood group varies regionally and from one population to another. in kathmandu, nepal - years age group is the most common age group encountered donating blood. the data generated in the present study and several other studies of different geographical region of india will be useful to health planners and future health challenges in the region. background: the information system on hemovigilance sihevi-ins©, coordinated by the national health institute was available in to all blood banks in the country. this software allows to centralize and record the identification data of a donor, its infectious and immunohematological tests, as well as the fractionation and final destination of each blood component obtained from a donor. aims: to describe the abo and rhd typing discrepancies in blood donors found in the blood group variables registered by each blood bank to sihevi-ins©. methods: retrospective analysis of the information registered by of the blood banks authorized nationwide between january and december . results: sihevi-ins© received information of , accepted donors, % of them with more than one donation in the same blood bank in a period of months. a total of abo or rhd discrepancies were identified in people, who made donations in blood banks (estimated risk: one discrepancy per , accepted donors). five of the blood banks implicated in these discrepancies are hospital-based (annual average collection of , ae , units, representing . % of the national collect). the remaining blood banks are distributors (average collection: , ae , units per year, representing % of the national collect). % of blood group typing discrepancies (n = ) were related to the abo group. the most common discrepancy was between a typing group and ab typing group ( %) . in % of the cases, the same blood bank initially registered in the same donor, an o blood type donation and later an a blood type (n = ) or b type (n = ). rhd typing discrepancies account for % (n = ) of the total. additionally, in three donors, a simultaneous discrepancy between abo and rhd typing was detected in the same blood bank. the results could be due to: a) failure in the warning mechanism before the release of the blood component; b) errors in typing the information of the donor registered in the system or c) failures in the identification of the donors at the time of selection. the above shows risk in the process of control of blood components release, which can impact patient safety unless abo and rhd typing blood groups are systematically verified before transfusion. summary/conclusions: despite blood banks have a verification and validation process through software to release blood components, flaws were detected. although sihevi-ins© is not a software to validate the information before the release of blood components, it was through this program that abo and rhd typing discrepancies were identified in donors who attended the same blood bank multiple times. this finding implies increasing the controls that should be used in each blood bank, to avoid lose traceability of the processes and to put at risk the life of the recipients. blood donor testing department, blood transfusion institute of nis, nis, serbia background: the ability to automate blood grouping and antibody detection procedures is a requirement for blood donor testing laboratories. mistakes in the sample identification and testing procedures could be prevented by testing on automated immuno-hematology systems. irregular antibody screening and abo/rhd grouping of blood donors are tests performed routinely in blood transfusion institute of nis. neo iris (immucor, usa) is a fully automated instrument for the abo and rh d grouping using microplate hemagglutination technique and antibody screening and identification using solid phase red cell adherence (sprca). aims: evaluation of the automated neo iris system for abo and d grouping and irregular antibody screening of blood donors in blood transfusion institute of nis. methods: during the evaluation period a total of edta-anticoagulated samples for abo and d forward and reverse grouping using microplate anti-s, anti-s, anti-jka and anti-k. in one case ih- failed to identify anti-c antibody in very low titer in sample with anti c+d antibody presence. in two samples ( . %) false-positive result were observed both on ih- system and neo iris and in two cases ( . %)only on neo iris due to nonspecific reasons. summary/conclusions: abo/rhd grouping results obtained on neo iris system, using microplate method, have a good correlation with results on ih- system as our routine column agglutination method. for antibody screening and identification neo iris showed high sensitivity for detection of clinically significant antibodies which is important step for increasing blood transfusion safety. background: continuing improvement of laboratory quality to provide accuracy test results for precise diagnosis and treatment is the mission of advanced laboratory. immunogenetic testing for histocompatibility including human leukocyte antigen (hla) typing, hla antibody detection and cytotoxicity test is critical for diagnosis and evaluation of transplantation and prognosis monitoring. in order to improve the quality of experiment competency, an external quality assurance schemes with review and education per year program was established and performed during the period from to in taiwan. aims: the proficiency testing (pt) held semiannually from to were reviewed to investigate the outcome of competency improvement of laboratories participated in the program. methods: the test items in the exercises were classified into groups, hla genotyping (including pharmacogenetics hla typing), cytotoxicity test, hla and platelet antibody. the methods of hla genotyping include ssp (sequence specific primer), sso (sequence specific oligonucleotide), sbt (sequence based typing) and either ssp+sso or ssp+sbt were used, the methods of hla antibody including elisa, flow cytometry and luminex were used and the methods of platelet antibody including sprca and elisa were used. there are four shipments of exercise materials in two years and each shipment include two positive and one negative samples for antibody detection, two each of whole blood and serum for cytotoxicity of t and b cell and three whole blood for hla genotyping. aims: this study aims to survey transfusion related laboratory tests for the quality improvement of hospital's blood bank management. methods: we analyzed survey results of kinds of routine work categories of blood banks that were registered on korean association of external quality assessment service. blood bank worker voluntarily replied this electronic survey. the categories were as follows: . characteristics of institution . the equipment of blood bank . the kinds of tube in blood bank . the present kinds of blood bank tests . abo and rh type tests . the cross-match tests . the irregular antibody tests . hemovigilance system . other blood bank tests . massive transfusion protocol . quality control issues we analyzed and compared each category data according to considering characteristics of hospitals. results: there were consensus and some differences of current blood bank tests. we presents the result of a pilot survey. especially the cross-match tests were divided by saline phase method added with irregular antibody tests or completion of rd step anti-human globulin phase according to institutional environment. automated typing machines or automated irregular antibody test devices were more increased in large-scale hospitals than small-scale hospitals. different kinds of tubes were used such as edta tube for abo and rh typing, plain tube for cross-match test. the retention segments of rbc were reserved for minimum days. most blood bank were registered and regularly listed up transfusion events to korean hemovigilance system for safety transfusion. also, a lot of institution have none or underdeveloped massive transfusion protocol. more specific survey results will be analyzed in further poster presentation. summary/conclusions: this survey will show the current status of transfusion related blood bank test. this institutional blood bank comparison will be helpful to assess the currency of individual blood bank environments. abstract withdrawn. background: we know that quality management is a continuous process, involving implementation, maintenance and improvement. aims: our purpose is to show our experience in implementing the quality management system in the whole institution and our first steps in achieving the jacie accreditation in the stem cell collection facility in order to provide our patients and donors the best possible care. methods: the institute for transfusion medicine of the republic of macedonia (itm) is the main institution in charge of blood transfusion service (bts) in the whole country, which is the national unified system. the stem cell collection facility is a part of the itm. this facility is operational since year with collections of stem cells ( in patients and collections in sibling donors) till now. we are obtaining the implementation and maintenance of qms through the establishing of the iso standardization for the whole institution (itm), as well as of implementing jacie standards in the stem cell collection facility. the two of our colleagues became the jacie inspectors and the standard operating procedures (sops) were developed, followed by regular meetings, trainings and self-evaluation of the personnel. we asked for the orientation visit from the independent jacie inspector in order to come one step closer to the jacie accreditation and to improve our overall qms. results: the institute for transfusion medicine of rm was a part of the ipa project "strengthening the blood supply system". this project aimed to ultimately bring the blood transfusion service to european union standards allowing the exchange of blood components and all other types of collaboration with other european union countries in future. the project put the basis for unification of blood transfusion standards and operating procedures in the whole country as well as set up essential education of blood transfusion personnel. although a lot of strengths were found during the orientation visit from jacie inspector, there are still a lot of areas for improvement. our strengths are motivated team and supportive institutional leadership including macedonian ministry of health. areas for improvement are: labeling of cellular therapy products and lack of laboratory for quality control. there is a national regulatory framework in place and who and world bank initiatives in macedonia which support quality in health care and accreditation. summary/conclusions: our institution has in plan to implement isbt standards for labeling of cellular therapy products and to establish a laboratory for quality control of cellular therapy, as well as to meet all the requirements to become jacie accredited facility. working by standards, following the rules and regular self-evaluations will help us to maintain the strong quality management system. every institution will benefit from a quality management system that brings you into line with international standards. ensuring the quality of our services and products is essential to keep safe and strong blood transfusion service. background: implementation of robust quality assurance program is key to high performing blood establishments. quality control and quality assurance systems together constitute the key quality systems and are parts of quality management. effective and efficient quality control policies not only provide guidance that help to increase the reliability of results but also maintains the laboratory's consistence performance overtime. aims: therefore, we established a set of qc limit using historical data which can timely identify unexpected variation in the testing systems and trigger a review of test processes in blood screening laboratories as part of quality assurance system. methods: last two consecutive years (jan, to dec, ) qc data from archi-tect i sr (abbott laboratories, chicago) was extracted using abbottlink for philippines red cross tower national blood center total of data points ( data points in & data points in ) were obtained for different qc levels for four serological blood screening assays (hiv combo, hbsag, anti-hcv, syphilis). the data was sorted for each assay/lot and qc level combination by year. qc limits were calculated using simple mean, standard deviation (sd) and coefficient of variation (cv%) and were validated and compared with manufacturer's recommendation. results: all the six positive quality control levels cv% ( . - . ) were within manufacturer's precision recommendation (within lab precision hbsag ≤ %, anti-hcv ≤ %, syphilis ≤ %, hiv ≤ %) in . five out of six positive quality control levels cv% ( . - . ) showed within manufacturer's precision recommendation (within lab precision hbsag ≤ %, anti-hcv ≤ %, syphilis ≤ %, hiv ≤ %) in except syphilis tp positive control ( . %). all four negative quality control levels showed the sd values within . - . in and . - . in respectively. summary/conclusions: excellent qc performance was observed in philippines red cross tower national blood center blood screening laboratory based on historical data and evidence-based laboratory qc limit for blood screening assays were established using historical data which takes into account total variation expected in a test system and offers a more robust and meaningful mechanism for setting control limits, for the first time. background: quality indicators (qi) in transfusion medicine (tm) are 'critically important aspects of transfusion medicine practice that are measured and utilized to gain insight for continuous quality improvement, into the degree to which the tm is capable of providing quality tm care, products or services for the aspect of practice measured following comparison of the measurement against acceptable local or international reference thresholds, benchmarks, standards, or practice guidelines'. the critical control point (ccp) selected for this study is 'administration techniques and monitoring of key elements'. this has been selected since the clinical fraternity plays a larger role in ensuring quality services in administration of blood components. there was a need to follow up compliance to standard protocol for bedside transfusion practices hence was decided to study the same with four selected quality indicators and introduce corrective measures if necessary. aims: . to assess the existing transfusion practices in the institute with specific quality indicators . to introduce corrective reforms to improve the existing practice . to assess the transfusion practices after interventions using the same quality indicators methods: to assess the existing transfusion practices in our centre, transfusions were prospectively followed up with a structured checklist. the quality indicators used were (i) verification of blood components prior to transfusion (ii)initiation of transfusion within min of release from the blood bank (iii) close observation of transfusions for the first min (iv)completion of transfusion within the right time frame for each component. as a corrective measure, a transfusion monitoring format was designed which was distributed in every ward and the nursing officers were informed to monitor and document transfusions using that. in addition, the blood bank staff was made to call up the wards and ensure that the transfusions of every component had been initiated within min of issue. transfusion practices were once again monitored by following up transfusions using the same quality indicators. results: there was significant difference in all the four variables between the two phases. . % transfusions were verified in phase i while . % were verified in phase ii (p < . ). . % transfusions were started within half an hour of issue while in the second phase, it rose to . % (p < . ). . % transfusions were observed in the first min in phase i and . % were observed in the second phase (p < . ). in phase i, . % transfusions were completed within right time while the same in phase ii was . % (p < . ). summary/conclusions: we recommend the following as quality indicators for bedside transfusion practices: background: antibody titration consists in performing antibody detection with selected red cells of different sample dilutions. the titer is reported as the reciprocal of the highest dilution that induces macroscopic agglutination. the usual applications of titration are prenatal studies and complex antibodies identification. some publications have demonstrated that more variation in antibody titer and titration score are noted upon repeat testing of the same sample when testing was performed in tubes as compared to repeat testing in gel. aims: to evaluate the efficacy of automated antibody titration versus manual method by using gel microcolumn technology. methods: edta-anticoagulated whole blood donors' and plasma frozen samples containing a known irregular (rh, kidd, duffy, mns, etc.) and regular (a & b) antibodies were selected. the titers of samples were determined in parallel by using grifols analyzers (erytra and erytra eflexis) and compared versus grifols gel manual method by using grifols gel microcolumn technology and grifols red blood cell reagents. sixty of these also processed in parallel in erytra and erytra eflexis analyzers for comparison. for the precision study, of these samples were tested in the automated systems for times ( datapoints for each analyzer) on different testing days. the hands-on (manual intervention) average time required to complete a titration was measured ( expert technicians) in different sample workload ( and samples testing). these results were compared with the same number of independent titrations performed in grifols analyzers. for the walk-away time, different sample workload ( and samples testing) were assessed in manual method ( expert technicians) and compared to timings obtained when reproduced in grifols analyzers. results provided by analyzers were reviewed and compared to manual method. results: titer obtained by erytra or erytra eflexis was equivalent to the titer obtained manually (differences ≤ titer: % ≤ . titer). the results proved that both instruments were equivalent in performing titration (differences ≤ titer; % ≤ . titer). the precision results showed no difference between titers obtained through the % of the runs performed with the grifols analyzers (differences ≤ titer: % ≤ . titer). the manual hands-on in automated system was reduced in a % compared to manual method for sample. when the number of samples was increased ( samples), the difference in hands-on in was even more reduced ( %). in addition, the walk-away was % higher in automated system compared to manual method. furthermore, when the number of samples was increased ( samples), the walk-away difference was increased even more ( %). finally, automated system software demonstrated to increase the standardization of the test as all samples, results and reagents traceability were automatically managed. summary/conclusions: grifols gel system including erytra and erytra eflexis analyzers provided a scalable and efficient solution to perform standardized titrations in the immunohematology lab. the study proved that using grifols gel system, titrations can be run in an automated reliable way (less than one-fold differences versus manual gel), thus reducing at least % the hands-on, increasing at least % the walk-away, rising the standardization and automating all testing traceability. , and fourth case of use (results) scenarios, tasks were considered "very easy" by %> % of users and "easy" and by - % of users; %> % of the users considered "sufficient" the design to ease the interaction; and %> % of users never founding any situation of not knowing how to proceed. for the fifth case of use (user roles), % of users considered tasks "very easy" or "easy"; % of users considered "sufficient" the design to ease the interaction; and % of users never found any situation of not knowing how to proceed. for the sixth case of use (maintenance plan), % of users considered tasks considered "very easy" or "easy"; % of users never found any situation of not knowing how to proceed; and % of users considered the maintenance plan similar or better than other instruments. reliability analysis ( background: quality control procedures in blood group serology for reagents, techniques, personnel working and automated equipment are essential for the accuracy of the laboratory results. the observation of high number of uninterpreted results during blood donor grouping was a motive for investigation and possible targeting the problem. aims: to identify blood group interpretation problems by analyzing the testing results obtained with the commercial quality control samples routinely used during blood grouping. methods: a microplate (mp) system for performing abo and rhd, as well as rh phenotype and kell blood group determination with two automated analyzers techno ( and ) and correspondent two mp-readers lyra ( and ) using maestro software from diamed is currently in use for blood donor typing. three types of mp are being used such as: a, b, ab, dvi-, dvi+, ctl/a , b profile for first time donors, then a, b, d ctl for repeat donors and finally, the c, c, e, e, k, ctl profile. the accuracy and safety of the blood grouping results is ensured by using the diamed q.c. system which consists of + tubes of whole blood and tubes containing serum with known specific antibodies. we analyzed and compared the interpretation of the q.c. whole blood samples' results from both of the analyzers after a new optic camera was installed on the techno /lyra system. ward to ward. methods: a prospective observational pilot study was done for around prbc unit issues which were followed in real time for understanding the tat within blood bank & from ward to ward. as per the definitions, the areas where the times are documented perfectly are understood and considered for calculations. based on the conclusions of pilot study a monitoring form has been designed and utilised to monitor the tat within bb & wtw. the data is analysed monthly and an avg tat for bb & wtw is calculated. the common causes of delay in providing the blood components were analysed and strengthened to both reduce & control the tat. results: in pilot study, total wtw tat averaged to min, with highest time taken min, where there were additional processings like leukodepletion, irradiation, saline washing of red cells and holding the transfusion. lowest wtw tat was found to be min where there was a prior information for crossmatch. after the surveillance form has been started, the average time taken for wtw tat came down to min, maximum being min (jan ), the areas where delay happened were identified as internal courier delays, technician delays, billing & other logistics delay. the concerned staff are put on regular training to maintain the tat. summary/conclusions: although ethically all the staff work for providing better care for patients, there will be few areas that delay the life supporting blood transfusion. monitoring using tat surveillance forms help in avoiding the delays and hence provide better & timely transfusion support. blood donation -blood donor recruitment p- hematological and physiological characteristics of regular blood donors with beta-thalassemia traits background: according to recent evidence, the physiological variability observed in the hematological characteristics of regular blood donors (linked -in certain caseswith genetic factors or the donor's lifestyle) may affect red blood cell (rbc) storage lesion. beta-thalassemia heterozygous (b-thal-het) blood donors represent a group of particular interest because of a) the high frequency of thalassemia mutations in specific geographical areas b) the physiology of the b-thal-het rbcs, which predisposes towards more effective management of storage-associated stress. aims: the goal of the present study was the comparative examination of the hematological and rbc physiological features of regular blood donors with or without beta-thalassemia traits before blood processing for transfusion purposes. methods: healthy blood donors of greek origin ( - years old), who met the blood donation criteria were recruited in this study. plasma/serum (uric acid, electrolytes, extracellular hemoglobin, antioxidant capacity), cellular (rbc indices) and biological parameters (corpuscular fragility, proteasomal activity etc) were measured. the results were statistically analyzed and topologically represented in biological networks for both donor groups (+/-b-thal-het). significance was accepted at p < . . results: b-thal-het represented % of the donor cohort. no differences in lifestyle (smoking, alcohol consumption, physical exercise) were observed between the two groups. nevertheless, regardless of sex and sex-dependent parameters (e.g. hct, hb concentration), b-thal-het demonstrated: a) reduced hct, mcv and mch ( % p = . , % p = . and % p = . , respectively) and b) increased rbc count ( %, p = . ) compared to the average donors. moreover, mpv platelet index was found slightly elevated (p = . ) and serum total protein concentration slightly reduced (p = . ) in the same group. a trend for higher plasma antioxidant capacity (p = . ) was evident in the group of b-thal-het, in addition to statistically significant lower levels of osmotic fragility (by %, p = . ) and hemolysis (by %, p = . ) compared to controls. finally, analysis of the three proteasome-associated enzymatic activities (n = per group) in the rbc cytosol and the membrane, revealed similar levels in the two groups (p > . ). the b-thalhet and control biological networks showed insignificant variations in respect to the amount of connections and their hub profiles. however, differences were observed regarding the number or type of connections, or even their topology in the network, in the cluster of lipids (triglycerides, ldl etc), nitric oxide, clusterin, carbonylated plasma proteins and rbc osmotic fragility (correlated with the concentration of electrolytes selectively in b-thal-het donors) between the two groups. summary/conclusions: b-thal-het who meet the criteria for blood donation are a non-negligible sub-group of the total donor population in greece. they exhibit several similarities to the general cohort, but differ in fine characteristics of rbc physiology, including resistance to hemolysis and extracellular antioxidant capacity. the differential network profile of hematological and redox parameters may be important in respect to the subsequent blood processing and storage of b-thal-het erythrocytes for transfusion purposes. background: blood service in poland is based on voluntary and non-remunerated donations. regional blood donor centre in poznan as well as other regional centres (total of ) are the only entities authorized to collect, process, store and distribute blood and its components to hospitals in the region of their activity but they are also responsible to provide sufficient amounts of blood and its components. regional blood donor centre in poznan is one of the largest blood centers in poland with the total number of donations exceeding , per year. in the recent years we have observed a growing popularity of tattoos among various age groups as well as among people registering to donate blood (first time and repeat donors) hence, it is critical to introduce suitable measures to ensure the safety of blood and its components. aims: the aim was to analyse the correlation between the increasing number of donors deferred from donating blood due to having tattoos made and the number of recorded confirmed hcv infections and the effect it may have on the safety of blood and its components. methods: the analysis was made using the data for the years - obtained from the computer system 'blood bank' which is in operation in regional blood centre in poznan, poland. we have analysed the total number of deferrals of donors due to recently acquired tattoo and the total number of recorded confirmed hepatitis c infections. we must note that the category of temporary deferrals due to tattoos is a broad one: it includes so called regular 'artistic' tattoos, permanent make-up procedures as well as medical tattoos. results: we have recorded a significant increase in number of deferrals due to tattoos from in to in (+ %). in the group of male donors this trend remained rather stable with a slight decrease: from in to in (À . %). in the group of female donors the growth was more prominent: from in to in (+ %). in terms of the recorded confirmed hcv infections a downward trend can be observed: from in to in (À . %). in the group of male donors from in to in (À %), in the group of female donors from in to in (À %). summary/conclusions: as we can conclude from the analysis the applied policy of temporary deferrals of donors with recently acquired tattoos (in the last months) proves to be a reliable method of increasing the safety of blood and its components. nevertheless, the current conduct of the qualification of the donors which requires a month deferral following the new tattoo must be complemented by various and numerous educational activities regarding the means of hcv transmission (and other bloodborne viruses such as hbv, hiv) and ways of protection from possible infections. special emphasis must be put on the group of female donors as the growth of deferrals was more prominent among them. at the same time it is vital to ensure for constant availability for all donors of well designed, concise educational materials (hard copies on the premises, articles, infographics, downloadables etc. on the website). background: a temporary deferral has a negative impact on donor retention, with many donors failing to return at the end of their deferral period. anecdotal evidence collected by the australian red cross blood service suggested that many donors do not know when they are eligible to return to donate, suggesting that a reminder message may be effective at promoting donor return once the deferral has ended. aims: the aim of this study is to evaluate the effectiveness of a reminder message on the return rates of deferred donors at the end of their deferral period. this reminder message notified donors that their deferral period was ending and encouraged them to make an appointment to donate. this study also aimed to determine the most effective time to send the message, message content, and mode of communication (sms vs email) in optimising donor retention post deferral. methods: three separate randomised controlled trials were conducted to answer these questions. data on donors' attempted return behaviour and subsequent deferrals, appointments and donations made one month after the deferral end date were collected and analysed. results: overall, . % of donors who received a reminder message attempted to return compared to . % of donors in the control group (p < . ). looking at each time point, donors who received the message week before their deferral ended were % more likely to attempt to return compared to the control group (p < . ). the week prior reminder message was particularly effective with males, with . % attempting to return to donate, compared with . % of females (p < . ). there were no significant differences in the return rates of donors who received the recipient versus non-recipient focused message, or donors who received the message via email or sms. summary/conclusions: a reminder message sent to deferred donors at the end of their deferral period is a simple, cost-effective way to promote donor retention, providing clear information regarding the date on which the donors can return to donate as well as a prompt to make an appointment background: our challenge is to provide % voluntary donation for safe blood, thus taking into account the current history of family donation, promotion of blood donation, level of awareness and voluntary donations from various institutions, the opinion of interviewees will give us a clearer idea of what we want to achieve and what needs to be improved in the future. aims: . provide % voluntary donation for safe blood. . establishing a special department within the national blood transfusion center responsible for marketing and promotion of voluntary blood donation. methods: this study was conducted as a combination of qualitative and quantitative methods. the study was a combination and identification of existing data, direct interviews with persons of different age groups, preparation and dissemination of questionnaires and analytical processing of the collected information. the study questionnaire with questions in total was divided into sections out of which questions on blood practices were answered by all interviewees. people answered questions on the blood transfusion service. questions on blood knowledge were answered by people. questions on the knowledge of the blood transfusion were answered by people, questions on blood donation were answered by people and questions on the communication channels were answered by people. results: out of interviewees, % have never donated and did not intend to donate, due to the fact that most of them were afraid of needles and infections, while the smallest part didn't donate blood because it was not allowed by the religion, % did not donate, but expressed the readiness to donate in the future, % have donated voluntarily only once, % were family donors, % regular volunteer donors, and % have donated voluntarily several times and did not want to donate anymore. from those who have donated, % have donated for one of their relatives, % have donated for thalassemic children, % have donated to benefit free check-up and % have donated because it was valuable for their health. the question as to whether they would voluntarily donate again, % have answered yes, % no and % were still not sure. this means that donation of those who have donated once did not leave a positive impression, did not increase the desire to repeat the donation once again, rather it has restrained or made it unsafe for them to repeat donation. among the causes mentioned by the interviewees were bad conditions in the donation facilities, staff behavior, inadequate treatment, they did not feel good after donation and had hematoma at the venipuncture. summary/conclusions: based on the results obtained from the study, the national blood transfusion center needs the establishment of a genuine promotion department where there is a need for a transfusion doctor who should be an active part of it. the national blood transfusion center should build up and implement a rigorous retention policy for voluntary blood donors, as the study found out that around % of donors who have donated once would like to donate again. their attraction through a donor retention policy will surely lead to self-sufficiency with safe blood. the safe blood is a public good and for this reason it is the duty of all state instances, the media and non-governmental organizations to give their support in the promotion of voluntary blood donation. background: smoking, unhealthy diet, sedentary behavior and inability to maintain adequate exercise have significant consequences for several chronic disorders, including obesity. a balanced and equilibrate nutrition may prevent the negative consequences associated to the status of obesity. in italy, overweight and obesity is increasing with adults of overweight and of obese in with a higher frequency in the south. blood centers can play a public health role in obesity surveillance and interventions. aims: since the quality of life, self-reported by the patient, related to health and adequate quali-quantitative nutrition, are becoming necessary and relevant in the field of nutrition, we conducted a demographic study to evaluate the health status of the blood donors by monitoring the nutritional habits and lifestyle. methods: a descriptive cross-sectional face-to-face questionnaire was developed. it included a item dietary assessment, reporting semi-quantitative food frequency, dietary behavior and questions on self-rated health status. normal weight was established with bmi < kg/m , overweight with a bmi ≥ and < kg/m , and obesity with bmi ≥ kg/m . obesity prevalence was standardized by sex. donors were repeat blood donors, who had made at least donations in the last years, and were eligible to donate. results: of the blood donors enrolled between july and january , were regular repeat donors, did not wish or chose not to respond at survey for several reasons (i.e. lack of time or privacy) and accepted, of which were deferred from blood donation and were excluded from the analysis. among the included participants . % (n = ) were male, age ranged from - years with a mean age of . ae . sd and . % (n = ) were female age ranged from - years with a mean age of . ae . sd. data showed that donors followed mainly a mediterranean diet and had more awareness to lifestyle, women more than men, in comparison with general population. the prevalence of overweight was found . % in men and . % in women. our survey showed that . % of the participants evaluated their health as "good", without gender difference (men, . % vs women, . %). besides, . % reported their health as "very good". summary/conclusions: overweight and obesity are common among regular blood donors and it is more frequent in men than women. our preliminary data showed that women have a better knowledge of the nutritional properties of food and consequently adopt a more balanced and proper diet. furthermore, it is clear that they are aware about the relationship between lifestyle and health putting into practice their information. unfortunately, the survey structure, of observational nature, does not make it possible to establish whether women are more alert to health to participate more in donation programs or if, on the contrary, the status of regular donor could help the improvement of knowledge and healthy lifestyle. background: donor recruitment pose an ongoing challenge to blood banks worldwide. one approach to improve the effectiveness of donor recruitment is to target influencing factors. a yearly league is conducted at the sultan qaboos university (squ) to encourage university students and faculty to donate blood. during this, the colleges are evaluated based on different measures including the number of donors recruited from each college and the efforts made by the students in increasing the awareness of blood donation in the colleges and in the society via different means including the utilization of the social media. the whole competition is organized and ran by an independent group of students. aims: this study aims at studying the impact of the yearly squ college competition on the perception of blood donation among squ students. methods: a comprehensive anonymous voluntary survey was developed and used to assess perception of students aged - attending squ and other universities (non-squ) over a two years' period. analysis was performed using ibm spss statistics . . categorized variables were presented in numbers with percentages and associations between the groups were analyzed using chi-square test. a p-value of < . was considered statistically significant. results: a total of students were surveyed ( squ, non-squ). there was no statistical difference between squ and non-squ students with regard to past history of blood donation and the number of donations made. when comparing between both cohorts, % of the squ and % of non-squ students reported the university as the main source for information (p < . ), while % of squ and % of non-squ students reported that the social media was the main source respectively (p = . ). there was no statistical difference between male and female donors on their perception of level of self-knowledge on blood donation (p = . ). about % of the youth agreed that blood donation is one of the duties toward the community. squ students reported higher rates of respond to specific requests for blood donation ( . % vs . %, p < . ). squ students reported greater influence of peers ( % vs . %, p < . ), personal knowledge ( % vs . %, p = . ) and personal experience ( . % vs %, p = . ) when compared to non-squ students. they also reported more feeling of commitment to the society ( . % vs %, p < . ). squ students reported lower influence of parents ( % vs %, p = . ), lower rates of fear from needles ( % vs %, p < . ) and lower rates of fear from blood ( % vs %, p < . ). there was no difference between male and female genders in any of the discouraging factors. summary/conclusions: these results highlighted the positive impact and important rule of the youth in the promoting blood donations among themselves through this yearly college competition; in recruiting blood donors and in the dissemination of the knowledge of blood donation. distinct promotion strategies should be adopted to increased first time and repeated blood donation among the youth. we advocate for similar initiatives in encouraging blood donation and disseminate knowledge among individuals in the community. dubai blood donation center, dubai health authority, dubai, united arab emirates background: dubai is multicultural city in united arab emirates. only about % of the population consists of uae nationals with the rest comprising expatriates from various countries all over the world. approximately % of the expatriate population (and % of the emirate's total population) are asian, chiefly indian ( %) and pakistani ( %). dubai blood donation centre is the only centre providing blood donation services in dubai. arabic is the national and official language and english is used as a second language. in order to have good quality screening, it is important that blood donors understand the educational material and questionnaire properly. aims: dubai blood donation centre receives donors (nationals, residents and gcc card holders) from various countries. the aim of this study is to analyze the multinational profile of donors and to find out the need to add any third language to meet the customer needs and expectations. methods: a cross-sectional study of blood donors was conducted in dubai blood donation centre in . the donors were asked about their country of origin, languages which they can read & understand and about the preferred mode of communication. results: a total of donors were surveyed and asked about the languages which they can read and understand and responses were obtained. the most common languages which can be read and understood by blood donors in dbdc are english (n = ; %), arabic (n = ; . %), hindi (n = ; . %) and malayalam (n = ; . %). the donors come from different countries, most common ( . %) donors are indian and ( . %) are from uae. it was found that % donors can read and understand only one language. majority ( . %) donors can read and understand either of the official languages arabic or english. however, ( . %) donors can't read and understand these two official languages, the other common languages being hindi and malayalam. the donors were asked about the preferred mode of communication, responses were obtained. the most common mode of communication were sms and telephone ( % together). summary/conclusions: based on the above findings, it can be concluded that the blood donor profile in our centre is multinational which is a unique and almost similar to the population profile of dubai. as . % donors can't read and understand arabic and english, so it has been decided that the educational material and questionnaire need to be prepared in one more language. hindi has been decided as the third language in the centre and donor questionnaire and educational materials in hindi will also be made available to the donors. further,the donors will be communicated through sms for routine messaging and disease notification while telephonic calls will be done only when the blood is urgently required. background: metabolic disorders (metds), including hypertension, dyslipidemia, hyperglycemia, and central obesity, are tightly associated with cardiovascular diseases and type diabetes mellitus. due to the sedentary lifestyle and increased consumption of high-calorie diet in modern society, metds have become serious health problems worldwide. to have a better understanding and possible improvement on blood donors' health condition, we conducted a survey of the prevalence of metds among blood donors in a blood donation station located in the hsinchu science park in taiwan. participants with metds will be provided with health education materials about metabolic risk reduction, in order to prevent the development of future complications. aims: the aims of this study were to determine the prevalence of metabolic disorders among blood donors, and to calculate how much money would be paid to identify a case of hyperglycemia, hyperlipidemia, or undiagnosed diabetes. methods: this study was approved by the institutional review board of taiwan blood services foundation (tbsf). the body weight, body height, waist circumference (wc) and blood pressure (bp) of participants were measured. blood samples were obtained to determine the values of hemoglobin a c (hba c) background: the law on blood donation supports the development of the blood service and guarantees the protection of the donor's rights and the maintenance of health during blood donation in the russian federation. national criteria for donor selection for blood donation are used in the activities of blood service establishments and are aimed at ensuring the blood products safety. the study of the characteristics of blood donors allows to predict the development of blood service and to plan the volume of blood products for transfusion and plasma fractionation. aims: the aim of this work was to study the characteristics of whole blood and apheresis donors in the blood service in the russian federation. methods: indicators of activity in the blood service establishments in the russian federation in sectoral statistical observations over the period - and the calculation of indices characterizing the whole blood and apheresis donors were analyzed. data are presented according to the administrative division of russian federation into federal districts (fd). results: the proportion of whole blood donors was . %, plasmapheresis donors - . %, blood cell apheresis, including plateletapheresis, donors - . %. for the period - , the percentage of repeated and regular whole blood and apheresis donors increased from . % to . %. the percentage of first-time donors ranged from . % to . %. the largest proportion of plasmapheresis donors was observed in the volga fd ( . %). about . % of the total plasma was collected by apheresis from donors. the percentage of plateletapheresis donors increased from . % to . %. the largest percentage of plateletapheresis donors was observed in the central fd ( . %), where a significant part of medical centers of cardiac surgery, hematology and bone marrow transplantation are located. the proportion of platelet concentrate collected by apheresis increased to . % in . actions to recruit young donors for blood donation and its components were regularly carried out in all federal districts. summary/conclusions: in the russian federation, the structure of donation is characterized by an increase in the proportion of plateletapheresis donors, stabilization of the percentage of plasmapheresis donors and an increase in the proportion of repeated and regular whole blood and apheresis donors. there are significant regional variations of donor's characteristics in the federal districts. background: shortage of blood supply despite continuous blood donation campaigns especially during local festive seasons has been a major issue in our country. thus, our faculty initiated blood donation drives in collaboration with national blood centre in order meet the demand for the blood requirements. however, the pre-donation deferral rate was relatively high among our young blood donors leading to loss of valuable blood units. understanding the causes of donor deferral provides direction on strategies for young donor recruitment and retention of future blood donation. aims: the aim of this study is to evaluate the young donor deferral pattern and to identify factors which could help in minimizing the preventable deferrals. methods: this is a retrospective study of voluntary young blood donors age between to years old recruited during mobile blood donation in faculty of medicine, universiti teknologi mara, malaysia. the study was conducted between january to december . the data were retrieved from the official reports of each mobile blood donation. results: a total of young blood donors had attended mobile blood donation during the study period. the overall pre-donation deferral rate is . %. the main causes of deferral are low haemoglobin (hb) level ( . %) followed by low blood pressure ( . %), upper respiratory tract infection ( . %) and sleep less than h ( . %). summary/conclusions: low haemoglobin and low blood pressure are the two common reasons for blood donation deferral among our young blood donors. in our study a significant proportion of deferrals are due to sleep less than h whereby this could be prevented if the donors are aware of the donor selection criteria. strategies to mitigate preventable deferrals and improve blood donor retention particularly young blood donors as source of motivation for future blood donation are urged to avoid additional stress on the blood supply. background: in the modern world, donating blood has become a humane manner for saving of patients life. but there are barriers to blood donation which are designed to ensure both donor and blood recipients' safety. anemia is one of the most common health problems in the world. based on the who estimation, nearly a quarter of the world's population are suffering from anemia, its prevalence varies among the populations and age groups. the prevalence of anemia among men is . % and in non-pregnant women is . %. aims: the aim of this study was to determine the status of hemoglobin in volunteer blood donors referring to fars province blood transfusion service and to determine the demographic status of them during the last two years. our study included blood donors for all blood donors during the last two years. methods: the study is descriptive cross-sectional and our sampling was non-random and simple sampling method. all parameters related to the donors, including age, sex and type of donation were investigated and analyzed in spss software. results: the total number of referrals for blood donation was . repeated blood donors was . % of total population and had the highest number of referrals, followed by first and lapsed donors with . % and % respectively. in terms of gender distribution, . % were female and . % were male. the highest rate of hemoglobin level less than . g/dl was found in first-time donors with . % and the lowest prevalence was observed in lapsed donors, followed by repeated donors with . %. . % of the repeated blood donors have hemoglobin higher than . . there was a significant difference between blood donation type and hemoglobin level. summary/conclusions: according to our findings, low hemoglobin levels are more common among first-time and female donors, and this requires a special training among these groups. because the high share of first time donors in blood supply and the positive impact of female donors on the blood safety, corrective action for that groups is recommended. finnish blood donor biobank j partanen, t wahlfors, m arvas, j clancy, k l€ ahteenm€ aki, e palokangas and n nikiforow background: the increasing need for large, well-characterized cohorts of healthy individuals for modern biomedical research, such as genomics or phenomics studies typically including tens or even hundreds of thousands of subjects, has posed the possibility of using blood services as an option for collecting samples and related data. the possibility to re-contact blood donors for repeated sampling or asking for additional data has further increased interest in collecting large biobanks from blood donors. there is also a need to study more thoroughly the effects of blood donation on donor health. aims: the first-phase goal is to recruit , blood donors with broad biobank consent for the finngen (https://www.finngen.fi/) project, a large publicprivate effort aiming to collect genome and health-related registry data of % ( , ) of the finnish population. ( . %), dental examination ( . %) and medication history ( . %). permanent deferral namely, risk factor involving transfusion transmitted infections and chronic disease were ( . %) and ( . %) respectively. the prime cause of permanent deferral was risk factor involving transfusion transmitted infections while the temporary deferral was bed side hypertension. gender wise, the leading cause of donor deferral in male was bed side hypertension and anaemia was the major cause in female. summary/conclusions: the findings of the survey aid to evaluate the significant causes of blood donor deferral. this study suggests that the restrictive criteria can be used for blood donor selection. this will in turn increase the blood supply of tertiary care hospital. background: donor selection is the first step towards safe blood but retaining blood donors is also very important for the blood supply. donor questionnaire and the medical interview should provide optimal doctor deferral. aims: to evaluate deferral rate in blood donors in order to identify the main reasons and to target eventual corrective activities. methods: we analysed the data concerning blood donors who were registered in the period of three years ( - ). we used data from the information system e-delphyn. background: iron deficiency (id) in blood donors is an underestimated issue in many countries and may cause symptoms to blood donors even without anemia. id prevention is mainly based on the prevention of anemia in whole blood donors, which is done by deferring donors whose haemoglobin level is under defined threshold ( g/l in women, g/l in men in france). efs (french blood establishment) studies has observed that the rate of deferral for anemia is significantly higher in women than in men, either in french west indies ( . % versus . %) or in continental france ( . % and . %). assessing the prevalence of id is of great interest since strategies to counteract it must deal with donor health and self-supply. however, data on id are missing in france. aims: to estimate the prevalence of id in french whole blood donors and to identify risk factors associated with id. methods: this non-interventional, cross-sectional, multicenter study is performed in blood donors of efs and ctsa (blood center of the french military health service). all whole blood donors who met selection criteria are potentially included. donors coming for bloodletting and donors who refuse to participate to the study are excluded. no additional sample is taken for the study, ferritin is tested after blood qualification on surplus amount. samples are selected at random within all the geographical areas and all mobile blood drives and blood centers between march and march , . results: this study ferridon has been approved by ethical research committee. nine thousand ( ) whole blood donors will be included in efs centers in continental france. to have information on donors of afro-caribbean origin and comoros origin, donations should be included in the french west indies and in reunion island. additionally, whole blood donors will be included in ctsa centers. in this study, id is defined by ferritin lower than ng/ml and iron overload is defined by ferritin higher than ng/ml. all donors with iron deficiency or overload will received a letter advising to consult their general practitioner. weights will be calibrated on age, sex and geographical area so the sample will be representative of the french whole blood donors. estimation of id prevalence will take into account the weights and logistic regression model will be used to analyze risk factors associated with id. data will be analyzed during april and may to get result at the end of may. summary/conclusions: ferridon will be the first study on id in the french blood donors. considering the french health care system and diet, it will be interesting to compare those results to results from other countries. mostly this study will allow to consider various strategies dealing both with donor safety and self-supply. background: in portugal, with an aging population of around million people, only . % are blood donors. the country has a national center of blood supply and some central hospitals with a blood donation center. despite the growing practice of the excellent concepts of patient blood management, it is imperious to attract new donors. this need has been our inspiration to use new approaches towards people, in a constant work of promotion. aims: reach the majority of our local population using radio and telecommunication as well as social networks in an attempt to raise the number of new blood donors in a central hospital of the north of portugal. methods: active communication with the population of our reference area, via the social networks facebook tm and instagram tm , through educational digital posters and messenger service to answer any kind of questions. establish contact with radio and television stations as well as with the mayor of the city, journalists, schools, town hall deputies and celebrities, through email and telephone calls. design posters, flyers and public advertising to distribute in the city. results: through the social networks it has been possible to reach a population of dozens of thousands in our city, in a daily basis. the national and world donor days were celebrated with success, in our health facility, with city mayor and journalists, and also in three television stations with national broadcast, reaching millions of people. celebrities (sport, television, music, stand-up comedy, journalists and a magician) have accepted our challenge through videos or donating blood, appealing to blood donation and sponsoring our cause. these projects and continuous availability to innovate have given our hospital a self-sufficiency of % in , instead of % in , which implied receiving less blood unities from the national center of blood supply. our most recent project involves high schools, in an attempt to educate our next generation of donors, with meetings in the town hall with deputies and district school delegates. summary/conclusions: the aging population and the low percentage of blood donors are an important issue concerning public health. nevertheless, the good will and continuous advertising and educative work towards the population, appealing to the ethical and civil responsibility since young ages have shown to improve our capacity of response as a central hospital, increasing the auto-sufficiency of blood unities and the interest of younger donors. it is of the utmost importance to understand that this is a continuous and a hard work of the professional team of our hospital, involving countless calls, emails and hours to obtain some positive response, in an endless job. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the mean interval between donations is shorter for former regular donors ( . months, p < . ) whereas donors with an interval of to months are more likely to be regular (aor ; % ci . - ). summary/conclusions: at the provincial blood transfusion centre of bukavu, the percentage of regular donors is low and there is a substantial loss of former regular donors. some factors identified to be linked to fidelity are unique to our study: female gender and a longer interdonation interval. other factors that are similar to those found elsewhere have a particular significance in our donor population which consists mainly of young people and people without income. efforts must be undertaken to ensure a supply from voluntary donations; recruitment strategies and target groups must be refined. future qualitative studies are needed to explain the various associated factors and better understand the motivations of regular and non-regular donors to improve donor retention. results: in kazakhstan, the proportion of donors is higher, especially primary. the number of blood and especially plasma donations is higher, which can be explained by the presence of several albumin and immunoglobulin production sites. increased evidence of blood transfusion rules, the development of a patient's blood management in combination with an increase in the quality of blood components cause a reduction in clinical need for red blood cells and plasma for transfusion. at the same time, the need for platelets is growing. it is difficult to assess the correctness of comparing the amount of banked whole blood. it is equally difficult to compare the number of received and distributed donor red blood cells and plasma: in russia they are measured in liters, and in kazakhstan in doses. with a certain degree of conditionality platelet extraction can be compared. in russia, they are counted in equivalent doses isolated from a dose of whole blood (at least cells per dose), and in kazakhstanin therapeutic doses (at least cells per dose). in , the estimated consumption of platelets in kazakhstan exceeded the russian indicator by . %. % of platelets in kazakhstan and . % in russia are harvested by the apheresis method. inactivation of pathogens is performed in % of platelets in kazakhstan and in . % in russia. pathogen inactivation with amotosalen allows us to abandon the examination of donors for cytomegalovirus and irradiation of platelet concentrate. the main modern trend in the use of cryoprecipitate is using it as a source of fibrinogen, a blood coagulation factor that is first depleted in coagulopathy associated with injury and massive bleeding. its production is growing in both countries, endowment in kazakhstan in exceeded the russian indicator by . %. a significant plasma percentage in both countries does not pass quarantine due to repeated non-appearance of the donor and is subject to destruction. inactivation of pathogens is performed in % of plasma in kazakhstan and in . % in russia. despite the instruction and selection of donors, laboratory screening of infection markers remains effective: russia more often identifies hiv and viral hepatitis c from potential donors, and viral hepatitis b and syphilis are detected in kazakhstan. . % of donors in kazakhstan are exempted due to the results of multiplex screening of nucleic acids of three hemotransmissive viruses. summary/conclusions: the blood services of russia and kazakhstan perform tasks to provide medical organizations with effective blood components. in conditions of decreasing demand for red blood cells and plasma, it is advisable to focus on the efficiency of resource use and improving the quality of blood components produced. blood collection including apheresis p- finnish red cross blood service, helsinki, finland background: skin disinfectant must effectively reduce microbes from the arm of the donor. as a result of poor disinfecting microbes may be transferred from skin via venepuncture to the collected blood and contaminate the blood components. aims: to ensure the efficiency of the skin disinfectant used for donor arm disinfecting by validation. the validation has two criteria which the post-disinfection samples must achieve: . no bacteria growth near the puncture spot (result cfu) in ≥ % of the samples. . total amount of bacteria on average is at most cfu/ cm . at most % of samples are allowed to have - cfu/ cm . methods: microbiological samples were taken with contact plates from voluntary persons' elbow folds before and after skin disinfection. the disinfecting was performed according to normal procedure by five nurses altogether with ethanol based disinfectant used in blood donation. on the sample plates an x was marked and this was directed to the puncture spot pointed by nurse. post-disinfection sample was taken at the moment the skin would be punctured with needle. results: the amount of bacteria varied from to above cfu/ cm in the pre-disinfection samples. disinfection reduced bacteria very well; the critical puncture spot was totally clean ( cfu/ cm ) in . % of the samples and . % of the samples had or only cfu/ cm . the average number of bacteria after disinfection was , cfu/ cm and the maximum number was cfu/ cm . most of the remaining bacteria were single colonies at the edges of the plates. summary/conclusions: both main criteria are fulfilled. the sub criteria of the second main criteria is also full filled if the not so critical colonies at the edges of the plates are not taken into account. the skin disinfectant in question is shown to be effective and can still be used in blood donation paying attention to thorough procedure performance according to the instructions and sufficient drying time of the disinfectant. background: research questions involving blood donation and recipient data often require advanced statistical methodologies. while such methodologies may appear in other medical research areas, specific tailor-made statistical tools and approaches are required for the analysis of blood-related data. these toolkits, which often require collaboration, are not always readily available to blood services, especially so in resource-limited settings. an international network of statisticians, epidemiologists and clinical researchers has been established for this purpose, which started with an invited session at the meetings of the international biometric society. aims: to exchange ideas, experience and knowledge to further improve the quality of blood sector research. methods: currently our network covers four major blood services and members from five different countries. the network has monthly conference calls about past and current research topics. we wish to extend this network further, and establish a subcommittee on statistical and epidemiological methodology with regular face-toface meetings at an international organization such as isbt. results: the monthly meetings have already demonstrated that the members share common problems and interests. for example, we are discussing techniques to analyze data with repeated measurements, e.g. eligibility haemoglobin tests, ways to assess the healthy donor effect, e.g. determining appropriate controls groups, and predictive models of blood supply and demand, e.g. stochastic processes and queuing models. the network also aims to organize training sessions in methodology either on site and/or by developing web lectures. summary/conclusions: an international network on statistical methodology for the analysis of blood donation and recipient data will improve the quality of research in the field of transfusion medicine research. expanding the network to include countries and blood services in research limited settings needs to be actively pursued. background: blood donor hemoglobin concentration (hb) is commonly measured from a skin-prick sample at the donation site, and low hb is the most common reason for temporary donor deferral. while a proportion of the deferrals do reflect true low hb, the skin-prick sample is prone to preanalytical error and variation resulting in false deferrals. aims: we assessed the applicability of a venous blood sample for second-line hb screening in blood donors failing the initial skin-prick test. methods: initial hb was measured from a skin-prick sample with the hemocue hb + (hemocue ab) point-of-care (poc) device. donors with hb < g/l for females or < g/l for males or with a decrease > g/l from latest donation were included in the study. in the study group, a venous blood sample was collected for hb measurement with the poc device at the donation site. donation eligibility was based on this hb result. venous hb was also determined with a hematology analyzer (sysmex xn, sysmex co.). the blood service's current workflow served as the control group: two more skin-prick samples were collected and the donor's final hb and donation eligibility assessed with an algorithm based on all three skin-prick hb results. results: in the study (n = ) and control (n = ) groups, the proportion of male donors ( % and %) and the mean initial skin-prick hb ( g/l and g/l) were similar. significantly less donors were deferred from donation in the study group ( %) than in the control group ( %; chi-square test p = . ). the mean difference in venous hb with the poc device versus the hematology analyzer was À g/l (range À to + g/l). two donors were incorrectly accepted based on venous sample poc result; however, in both, hb measured with the hematology analyzer was only g/l below the limit of donation eligibility ( g/l for a female and g/l for a male). interestingly, a further donors ( % of all deferred in the study group) would have been eligible for donation based on the hematology analyzer result. summary/conclusions: utilizing a venous blood sample for second-line screening of donors failing the initial skin-prick hb test significantly decreased low hb deferrals without compromising donor health. blood donors' and blood service nurses' reactions to the new workflow have been favorable. we conclude that valuable donations can be recovered and donor satisfaction increased by implementing a second-line hb screening model utilizing venous sample analysis at the donation site. background: there is a paucity of literature on haemoglobin (hb) reference values for adults above years of age. this age group has been reported to use up to % the blood supply. some studies report a decline of mean hb with age, but others have found no change with age. conflicting findings of hb levels in the healthy elderly population may be associated with challenges in accessing data from healthy older adults, small sample sizes, selection bias and recent health population data. to donate blood, each individual is assessed as 'healthy' and must meet the minimum hb criteria. however, the hb criteria across countries vary and many blood collection services have an upper age limit for donors. as many populations around world are aging, restricting the upper age limit for blood donation may potentially affect the size of the donor pool and consequently the nation's blood supply. aims: to explore the hb levels of healthy older adults, through a multi-centre retrospective observational study of blood donors aged years or older. methods: over a one-year period, hb values were collected from blood donors aged ≥ years from blood centres of four countries. the estimated proportion of blood donors aged ≥ years old for each country was . % in south korea (sk); . % in hong kong (hk), < . % indonesia (indo) and % in japan (jap). the minimum hb criteria varied between each country and ranged from . - . g/dl for women and . - . g/dl for men. hb levels were determined using point of care testing (hemocue, compolab, hemcontrol) or the xe- d sysmex dependant on the country of origin. statistical analysis of the mean, standard deviation and cumulative distribution of hb were determined by gender and age. background: medication usage is assessed to determine donor eligibility from the perspective of both recipient and donor safety. different time frames since last taken apply to different medications. assessment of medication use varies by jurisdiction, but most european centres use multiple questions. these often include a general question about recent medication use whereas the usa does not. at canadian blood services there are medication questions on the donor history questionnaire (dhq), including any medication use in the last days, vaccination and specific medications over different time frames (high teratogenicity medications). the name of each medication taken and reason for use are documented by staff at each donation attempt. assessment of the frequency with which this process occurs is the first step in improving efficiency of this aspect of donor screening. aims: to determine the percentages donors answering yes to medication questions by demographic variables. methods: all whole blood donors who completed the dhq (full length or abbreviated) in were included in the analysis. donors' answers to each of the medication questions were extracted from the national epidemiology donor database, as well as sex and age. the number and percentage of donation attempts in which a donor answered yes to each medication question were calculated. donors who answered yes to any medication question were sorted by sex and by age group, the totals and percentages calculated. results: there were , donation attempts with a completed dhq. overall, % of donors answered yes to medications in the last days, % to vaccination, and less than . % to others ( % any). slightly more were female ( vs %) of those who answered yes to any medication question, as well as by individual question. the percentage of donors answering yes to any medication question increased progressively in each age group from % of - year olds to % aged + (p < . for trend). summary/conclusions: more than one third of all donation attempts answer yes to a medication question and require further questioning and documentation. this is more common in older donors and follows a similar trend to general population medication use. comparison of ways of assessing medication use in different countries may help identify effective but more efficient approaches. in addition, the contribution to donor and recipient safety of assessing all medications should be assessed. blood center experience with trima accel and tomes software j schreier , a davison , j gambarte , y l opez , c calonge and e herranz terumo bct, lakewood, united states centro de transfusi on de la comunidad de madrid, madrid, spain background: in the madrid community, more than apheresis platelet collections were completed in , of which almost were completed in the blood transfusion center and the remainder in several hospitals in the region. trima accel was implemented to meet the productivity needs of the blood transfusion center while improving the donation experience. tomes (terumo operational medical equipment software), which enables bidirectional communication with trima accel devices, was used to connect and centrally manage all trima accel devices with automated data capture and reporting. aims: the aim of this study was to evaluate operational improvements using trima accel with tomes compared to trima accel version . methods: this was a retrospective study analyzing apheresis procedures on trima accel version during the control period from january to september compared to apheresis procedures on trima accel during the test period from september to december . this was not a paired study. operator interventions, and completed procedure rate comparisons, were analyzed using a -proportions test, whereas donor demographic data were analyzed using a -sample t-test. results: trima accel was used to collect single and double platelet products stored in platelet additive solution. operators selected either a single (target platelet yield = . or . ) or double (target platelet yield = . ) platelet donation based on desired procedure time not the maximum number of products that could have been collected per donor. no statistically significant differences were observed for donors in the test arm compared to donors in the control arm for total blood volume (control = ml, test = ml, p = . ), hematocrit (control = . %, test = . %, p = . ), or platelet pre-count (control = / ll, test = /ll, p = . ). females represented % of donors in the control arm compared to % of donors in the test arm. platelet split rate (platelet products per procedure) increased from . with trima accel version to . with trima accel ; procedure time decreased from . min to . min for single collections and from . min to . min for double collections with trima accel (these differences were not statistically significant). the percentage of procedures that completed with no operator interventions due to access alerts increased from . % to . % (p < . ) and the rate of completed apheresis procedures increased from . % to . % (p = . ) with trima accel . manual transcription of data during the procedure was discontinued with the implementation of trima accel with tomes. tomes captured procedural data and operator steps with barcode scanning and tracking of configured events. this information was transferred to tomes post procedure and printed as a final report. summary/conclusions: trima accel significantly decreased operator interventions, and automated data capture with tomes eliminated manual transcription of data. both outcomes freed operators to complete other tasks and focus on donor well-being. background: the european committee (partial agreement) on blood transfusion (cd-p-ts) of the council of europe (coe) has appointed a working group (wg) to focus on issues with plasma supply management (psm). the task of the wg is, among others, to collect and analyze data in order to fill knowledge gaps concerning donor safety in plasmapheresis. in doing so, the working group will gather evidence base data to support the upcoming revision of the th edition of coe's "guide to the preparation, use and quality assurance of blood components", the blood guide. an international survey was conducted sept-dec , distributed to blood establishments (bes) by the cd-p-ts representatives to coe's member and observer states. the questionnaire included sections covering collection practices (volume and frequency), management of red cell loss, donor panel demographics and data on donor adverse events. aims: the aim of this study was to investigate whether collection practices following the recommendations published in the blood guide for maximal collection volumes and number of donations per year were indeed associated with higher levels of donor safety and improved donor base sustainability. methods: from the total of respondents, bes collected plasma for fractionation (pff) by apheresis and the study had a dataset covering , , plasma donations in the latest fiscal year (lfy). the parameter used as marker of donor safety was the rate of immediate vasovagal reactions with loss of consciousness (vvr with loc) per , plasma collections. the parameter used as marker of donor base sustainability was the retention rate of donors, ie % donors active in the previous year returning to make a donation in the lfy. results: in the blood guide, the collection volume per apheresis is limited to % of the estimated total blood volume but maximally ml, including anticoagulant. respondents had differing practices and scale of collection program be were aligned or lower, and be had higher collection volumes. altogether reported the immediate vvr with loc rate, which mainly was lower than / collections. there was a small trend towards reduced rate with larger collection volumes than allowed by the current blood guide. saline compensation during or after collection did not affect the rate of vvr with loc. no correlation was observed between the annual donor retention rate and the rate of vvr with loc or saline compensation practices, as reported by respondents. the retention rate banded in the range of %> % (mean = %, min = %, interquartile range = %, max = %). the association between maximum allowed yearly plasma collection ( l) appears to be reasonably constant and showed no clear association with the donor retention rate. summary/conclusions: restricting the maximum collection limit according to the current blood guide was not associated with either lower vvr with loc or with higher donor retention rate. this study supports reassessment of current blood guide s limits for collection volume of maximum of ml per donation and l per year per donor. methods: serum ferritin concentrations were established from sera stored at À °c from repetitive platelet donors between and , using architect â ferritin assay chemiluminescent microparticle immunoassay (cmia). the hematimetric parameters were evaluated in a total blood sample using the celldyn â . sixteen samples were obtained from women (age: . ae . years, range: - ) and samples from men (age: . ae . years, range - ), corresponding to . % and . % of the total female and male repetitive donors of platelets by apheresis using trima accel â terumo-bct and amicus tm fresenius-kabi. the difference in the concentration of serum ferritin between the last and first donation was established, as well as the change in the predonation platelet count between the last and first event. results: in the study population, . % of women and % of men performed repetitive donations of platelets by apheresis with an interval of less than three months. the change in ferritin concentration was evaluated according to the interval between donations. in women ferritin delta was À . ae . ng/ml when the donations had an interval less than three months, vs . ae . ng/ml when the time between donations was higher (p = . ). in men the change in the ferritin levels was À . ae . ng/ml with donation times less than three months vs © the authors vox sanguinis © international society of blood transfusion vox sanguinis ( ) (suppl. ), - À . ae . ng/ml with prolonged donation times (p = . ). in women, the change in platelet count was À ae . /ul, when the donations had an interval less than three months vs À ae . /ul when the time between donations was greater (p = . ). in men, the delta of the platelet count was À ae . /ul in donation times less than three months vs À ae . with higher donation times (p = . ). no correlation was found between the concentrations of serum ferritin and the platelet count (r = . , q = . for males, and r = . , q = . for females). summary/conclusions: the data obtained suggest that repetitive donation of platelets by apheresis with intervals between donations of less than three months, significantly reduce serum ferritin concentrations in women and men, although normal levels were maintained in both groups. there was no correlation with platelet count. therefore, it is proposed to develop prospective studies to establish the minimum time interval safety for platelet apheresis donor procedures. background: the demand for platelets concentrates is increasing continuously and becomes a challenge for the blood establishments. apheresis platelet collections may be a solution for this challenge. improving apheresis collection efficiency while maintaining blood donor safety is an important goal for the service du sang of the belgian red cross. aims: our establishment evaluated the improvements of the trima accel automated blood collection system version (ta ) by comparing its routine performance with that of the previous software version . (ta ). methods: prospective, multi-site, controlled, non-randomized trial. apheresis collections were performed in three sfs sites: liege, mons and namur using the two trima software versions ta and ta sequentially. data were collected from december to april on ta and from june to july on ta . simple and double doses of platelets (respectively . , . and . , . ) were collected in platelet additive solution (ssp+, macopharma) with concurrent plasma from the same cohort of donors in accordance to donor's eligibility and preferences. in order to maintain the same final platelets content in platelets concentrates, the trima accel's tool yield scaling factor (ysf) was subsequently adjusted from . (ta ) to (ta ). platelet yield, duration of procedure, number of alarms requiring operators' interventions were recorded and evaluated. donor's hypocalcemia was avoided by giving preventively oral or intravenous calcium which was documented by the operators. results: five hundred ninety ( ) collections with ta and with ta were recorded, with % and % complete procedures respectively. mean duration of procedures was min on ta against min on ta , p < . . the mean alerts number per procedure on ta was . against . on ta , p < . whereas the maximum alerts number per procedure was and respectively. on ta , % procedures did not require operator's intervention against % on ta ,. with ta the inlet flowrate was automatically adjusted in . % procedures. the inlet flowrate was increased in response to access pressure in . % of procedures, for % of the procedures the inlet flowrate was decreased and for . % of the procedures the inlet flowrate was increase and decreased on the same procedure by the ta autoflow system. summary/conclusions: ta with its autoflow function improves apheresis donors experience while decreasing operator' interventions through a significant reduction of access draw alerts. as expected from the trima accel platform, post-donation safety remains high. a weak increase in procedure duration was observed for the same platelet yield which may be resolved with further adjustments. background: trima accel system is an apheresis platform relying on continuous flow centrifugation to collect from a donor platelets, plasma or rbcs based on donor qualification. the latest software version -trima accel (ta ) introduced the autoflow feature which allows for automated flow rate adjustments. moreover, ta leverages the mobilization capacity of the spleen increasing potential platelet productivity while maintaining high post-donation safety standards characteristic of trima accel. aims: the objective of this evaluation was to assess the impact of ta software by retrospective comparison of procedure data and potential for increased productivity with those of trima accel version (ta ) in the same cohort of platelet donors. methods: eight hundred twenty one procedures, started on ta from th january to th october were compared to procedures, started on ta from th october to st december . procedural data from the trima devices were captured using the cadence system (terumo bct, lakewood co). parameters investigated were the number of machine access pressure alerts per procedure, the potential for higher platelet yield collections and the actual collected yields within the same cohort of platelet donors. results: both donor populations (ta vs. ta respectively) were comparable and were characterized by: tbv - vs. ml; platelet count pre-procedure - ³/ll vs. ³/ll; hematocrit pre-procedure - % vs. %. gender distribution was % female with ta vs. % with ta . venous access pressure alerts were significantly improved by ta with an average of . alerts per procedure as compared to . with ta , i.e. % decrease. this decrease went down to % if only male procedures were analyzed. the maximum number of pressure alerts went down by % from alerts in one particular run in the ta cohort to alerts in one ta procedure. procedure time for single platelet products was reduced from to min and for double platelet products from to min (ta and ta respectively). donor qualification possible was % of procedures yielding single products and % of procedures yielding double products with ta . the percentage of procedures qualifying for doubles increased to % with ta . in terms of split rates, i.e. how many platelet doses could be produced per apheresis collection, potential split rates increased from . to . from ta to ta , respectively. in fact, the observed split rate rose modestly from . to . , as shorter procedures were generally selected according to donors' preferences. summary/conclusions: in comparable donor populations, implementation of ta decreased the number of access pressure alerts significantly compared to previous trima versions. the average procedure duration was also found to be slightly reduced. implementation of ta has the potential to increase productivity significantly. the observed modest actual rise in split rate suggested that factors related to donor and inventory management will determine at which extent the potential of the new software will be used. donor compared experience on trima accel to trima accel version august to october or trima accel during the test period from november to january . this was not a paired study. donors completed the survey while recovering from the apheresis procedure in the cantina. results from the paper survey were transcribed into excel for analysis. results: donors completed the survey during the control period whereas donors completed the survey during the test period. the mean number of previous donations for the control period was . (min max ) and for the test period was . (min max ). there were no first time donors during the control period and first time donors during the test period. % of donors rated their overall donation experience as good on trima accel compared to % on trima accel version . zero ( ) donor rated their experience on either trima accel device as poor. % of donors who responded to the question said they would donate on trima accel again. summary/conclusions: no significant difference was observed in donor experience between trima accel version and trima accel as both versions receive high marks. background: the trend on growth of query for donor platelet concentrates is observed in russia for past few years. as reported by edqm in , a higher number of the platelets was consumed compare to by . %. patients' with hematological malignancies treatment requires platelet concentrates transfusions during chemotherapy, immunotherapy and hematopoietic stem cells transplantation. according to the data collected in national research center for hematology (nrch) in , ( . %) of the , patients, treated within facility, received platelet transfusions as transfusion therapy. the total number transfused units is , , which is higher (by . %) comparing to . platelet concentrates production can be performed either by apheresis process or by pooling individual units recovered from the whole blood. taking into account that the nrch produces blood units for its own needs, the pooling is not suitable method for production because its implementation doesn't cover require for platelets and overproduces rbcs. that is why platelet concentrates in nrch are obtained by apheresis only. in summary, the growth on requirement for platelet concentrates and their safeness explains the need for a comparative study for effectiveness of platelet production using various apheresis systems. aims: the aim of the study is to compare effectiveness of platelet concentrate production using mcs + (haemonetics), upp and trima accel (terumo bct) version . protocols. methods: the data for protocols of platelet donations performed in were analyzed: on trima accel and on msc + . all donors were voluntary and non-paid donors with previous experience of blood donations. the choice of the platelet collection device was random; analysis of the main characteristics of donors did not reveal any significant differences between the groups. the median age of the donor was years old, height - cm, weight - kg, platelet count before donation - /l, hematocrit - %. detailed data are presented in table . student's t-test for unrelated sets was used for statistical analysis of the data. a value p of less than . was considered as significant. results: the data obtained showed significant difference (p < . ) between average number of platelets collected on trima accel ( . ae . /l) and on mcs + ( . ae . /l). while cost of consumables are comparable, trima accel demonstrated . % higher efficiency. procedure duration also was comparable and averaged within min for both devices. detailed data are presented in table . it is crucial to mention that proportion of trima accel's donations was significantly increased in nrch during and reached , % in total ( - . %). a flexible usage of trima accel's consumables for different procedures (regular platelet collection and collection in pas) allowed to change the pas/regular platelets collection ratio from . % up to . %. summary/conclusions: obtained results proved the effectiveness of the trima accel's use for platelets concentrates production. it allowed to increase the average count of platelets obtained for one procedure by . % compared to mcs + while the cost of consumables and procedure duration are comparable. the donor's comfort during procedure did not affect either. in long terms increasing of number of platelets collected is reducing the cost of platelet concentrates production. abstract withdrawn. background: apheresis collected platelet concentrate is preferable in terms of reducing the risks of adverse reactions in platelet transfusion when compared to random donor platelet concentrates. aims: the aim of our study is to present our experience in collection donation of single donor platelets with apheresis. methods: this is a retrospective study performed in the institute for transfusion medicine from till . all donors were fully informed on the donation procedure and signed an informed consent for donation. the optimal platelet count that we want to achieve was ≥ . equal to random donor platelet doses. minimum preapheresis platelet count in donors requested to start the apheresis collection was . /ll. platelet collection was performed using flow cell separators haemonetics mcs+ and trima accel. acid citrate dextrose formula a was used for anticoagulation. median precollection platelet count of donors was . /ll, with range from . /ll to . /ll. male were % of the donors and females were %. the single procedure usually took - min. the median platelet count collected was . , range - . . the median processed blood volume was ml and median used acd-a was ml. mean total volume of collected product was ml. the adverse effects included vein perforation and the numbness of the extremities as reaction of acd-a (hypocalcemia), which occur rarely and was very mild. summary/conclusions: the collected platelet count was more than the wanted optimum platelet count. the number of apheresis donors is increasing and we are working on expanding our voluntary platelet donors registry and increasing the number of typed donors in the registry. background: to determine value of hemoglobin in blood donors, there are some tools or methods used, such as: cyanmethemoglobin method that can detect of hemoglobin quantitative and methods cupric sulfate solutions (cuso ) can detect of hemoglobin qualitative. according to who (world health organization) to determine the level of hemoglobin in blood donors enough used cuso solutions with specific weight (y) . can to detect value of hemoglobin above or same with . gr/dl. but, cuso solution specific weight . can not to detect and elimination value of hemoglobin above gr/dl or polycythemia sick. because it, central blood transfusion unit (utdp) as the central of blood service in indonesia to manufacture cupric sulfate solution (cuso ) with a specific weight (y) to detect value of hemoglobin below gr/ dl and determine value of hemoglobin above gr/dl. because it, do the testing the accuracy of the solution cupric sulfate in detecting and eliminating donor with value of hemoglobin above gr/dl with the test of samples. aims: to determine accuracy and effectiveness by blood donors unit in indonesia red cross to use cuso solution with specific weight . in to detect and elimination value of hemoglobin donors above gr/dl. methods: used the method cyanmethemoglobin and cuso (y) . determination value of hemoglobin donor. test results were analyzed with spss software version using nonparametric analysis wilcoxon test. results: this research testing the accuracy and effectiveness of using a cuso solution with specific weight (y): . in detecting and eliminating hemoglobin value donors above gr/dl. from data processing using spss with the wilcoxon test p value . . summary/conclusions: it was found that the cuso solution (y): can detect hemoglobins value above gr/dl and more effective in checking the hemoglobin in blood donors. it can be seen from the data processing with spss version with the wilcoxon test p value < . . it is important to monitor the precise course by which repeated blood donation affects hb and the probability of low hb deferral. zinc protoporphyrin (zpp) is a functional indicator of body iron levels and is hypothesized to predict hb levels among blood donors. advanced statistical methods are necessary to properly analyze the longitudinal associations between zpp and hb in data with repeated donations per donor. aims: to determine whether predictions of future hb levels using current hb levels can be improved by taking zpp levels into account, and to illustrate the use of statistical models for repeated measurements of blood donors. methods: we used data from the zpp and iron in the netherlands cohort (zinc) study. we identified previous zpp levels (log-transformed) as the main predictor and adjusted for previous hb level, age, day and time of donation, donation history, bmi, blood volume and blood pressure. we used linear mixed models, which take into account missing data in the outcome and associations between repeated measurements, to investigate the longitudinal association between previous zpp and current hb levels. the longitudinal analysis with linear mixed models was contrasted with a simpler analysis based on the area under the receiver-operating-characteristic (roc) curve for the probability of low hb deferral. results: in total, whole blood donors ( , whole-blood donations) were included in the zinc study, % being female donors. previous zpp showed a statistically significant association (p < . ) with hb levels in females, but the size of the association was quite small (regression coefficient, b = À . , % confidence interval À . to À . ). the same was true for males, but the size of the association was even smaller. blood volume and age for women were significant secondary predictor variables; blood volume, age and donation interval for men. by comparison, the roc analyses showed relatively larger, but less statistically significant predictive effects of zpp on hb. summary/conclusions: zpp is a statistically significant predictor of hb levels, but the size of the effect after adjustment for previous hb and other variables is small. the results cast doubt on whether zpp is an effective predictive marker for hb level and low hb deferral, and suggest that zpp should not be included in prediction models for hb levels. by properly adjusting for associations between repeated measurements and by using all available data, longitudinal models provide less biased and more precise estimates than simpler cross-sectional analyses. background: finnish red cross blood service (frcbs) is a national blood service and is responsible for all blood collection and component production in finland. the highest age for blood donors was years until the end of year and since beginning of donors between and years have been able to donate blood. blood donation after the age is possible if the donor has donated within the last months. the upper age limit was raised up based on adverse event data from frcbs donors up to years and on published data from other blood establishments. aims: the aim of this study is to find out if the new policy from with upper age limit of years is safe. therefore donor adverse event data was analyzed in order to evaluate if the blood donors older than years have more adverse events compared to other donors. the most common donor adverse event for donors over years, haematoma, was registered times ( . %). in the other age groups haematoma was registered times ( . %) and the difference between the oldest age group compared to all other donors was statistically not significant (chi , p = . ). vvrs with loc were registered times ( . %), vvrs without loc times ( . ), and the total number of all daes was ( . %) in the age group years or older. the respective numbers in the other age group were: , . %; , . %; ( . %). the number of vvrs with and without loc, and total number of all daes in the age group years and older was smaller than in the other groups and the difference between these groups was statistical significant (chi , p < . ). summary/conclusions: donors over the age of years have less donor adverse events than other age groups. decision to raise the age limit from to seems proven to be right as the older age group has even less donor adverse events than other donors. background: deep vein thrombosis (dvt) of the donor's phlebotomy arm is a rare, but serious complication of blood donation that needs to be recognised and managed appropriately in a timely manner. post donation dvt will be classed as a 'serious adverse events of donation' (saeds)these are events that either result in donor death, hospitalisation, intervention or significant symptoms persisting for more than one-year post donation. aims: to review cases of dvt post donation reported in uk in - years and identify any common themes for improving practice methods: all data relating to saeds from the four uk blood services reported to shot in the last years ( - inclusive) were reviewed to look for reports of dvt post donation results: a total of saeds were reported in uk from approximately . million donations (whole blood and apheresis) collected during this period. three cases of upper limb dvt were reported during this time accounting for % of the saeds reported and rate of dvt of in . million donations collected. -case : a regular male whole blood donor in his early s reported worsening arm pain days following blood donation, had a painful venepuncture and a small bruise at site of donation. he was diagnosed to have an upper limb dvt extending to the subclavian and brachiocephalic vein and started on oral anticoagulants. no other contributory factor was obvious -case : a female donor in her mid- s gave her sixth whole blood donation without event. days after donation, she developed worsening arm pain in the donation arm and was diagnosed with an upper limb dvt and commenced oral anticoagulation. there was no other identifiable risk factor for the thrombosis -case : a female donor in her s developed pain, swelling, redness and itchiness in her donation arm and chest wall two days after donation. she also described prominent veins on the affected side compared to her other arm. she contacted the transfusion service one week after donation; by this time she was also breathless on minimal exertion. she was admitted to hospital and commenced on anticoagulant therapy. a diagnosis of dvt and associated pulmonary embolus was confirmed. the donor's only risk factor for thrombosis was use of the oral contraceptive pill. summary/conclusions: rare complications of blood donation, like dvt, can occur. superficial venous thrombosis may occasionally progress into the deeper veins of the donor's arm but dvt can also occur without signs and symptoms of superficial thrombosis. none of our patients had any overt evidence of superficial thrombosis. one patient in our series reported using oral contraceptive pill. no other risk factors for thrombosis was forthcoming. transfusion services should encourage donors to make early contact with the blood service if they experience arm complications so that they can be investigated and managed in a timely manner. staff dealing with such donors must recognise the possibility of this rare complication, explore other additional contributory factors and initiate prompt and appropriate management. background: voluntary blood donation is widely considered to be safe with very minimum chance of adverse reaction, which may occur during or after the end of phlebotomy procedure. aims: to find the adverse blood donor reaction among voluntary blood donors in tertiary care hospital in kathmandu methods: this is a prospective study done among voluntary blood donors at grande international hospital, kathmandu, nepal from february to march . the outlines of reported and communicated adverse donor reaction were also collected after the blood donation from voluntary blood donors in different locations including outdoor and in-house blood donation drive results: in the present study , whole blood donors were included, during the period of years, ( . %) adverse donor reactions were reported. majority ( . %) of adverse donor reactions were mild in nature such as, sweating; ( . %), light headedness; ( . %), nausea and vomiting; ( . ), allergy and bruises; ( . %), sore arm; ( . %) and hematoma; ( . %) while ( . %) were severe adverse reactions similarly, anaphylaxis; ( . %), loss of consciousness; ( . %) and convulsive syncope; ( . %). markers of the adverse donor reaction were age, sex, pulse, weight, blood pressure and donation status. age and first time status were related with significantly higher risk of adverse reaction with - years old at higher risk compared to - years old. first time donors were at higher risk compared to repeated volunteer donors. summary/conclusions: the results of the study are helpful to identify and understand the complication of adverse donor reactions though the incidence of reactions in the blood donors is lower than in other studies. donor age and donation status were strong possibilities of complications. background: blood donors with pollen-induced allergy and asthma must often refrain from donation in pollen season despite medication, because of symptom severity or similarity to airways infection. extracts of the medicinal mushroom agaricus blazei murill (abm) given orally have been found to reduce ige anti-ovalbumin levels and ameliorate the skewed th /th cytokine balance in mice sensitized to ovalbumin (takimoto, immunopharm immunotox, ; ellertsen & hetland, clin mol allergy, ). aims: the objective was now to examine whether supplementation with the abmbased extract that we used in the mouse model for allergy, could alleviate allergy and asthma in blood donors by reducing specific ige levels and basophil sensitization. methods: sixty donors at oslo blood bank with self-reported birch pollen allergy and/or asthma were recruited and randomized in a double-blinded, placebo-controlled study with oral supplementation for weeks before the birch pollen season with the abm-based extract andosan tm (immunopharma, oslo, norway). this is a water extract of the bacidomycetes mushrooms abm ( %), hericeum erinaceus and grifola frondosa. the participants filled in questionnaires for allergic conjunctivitis & rhinitis, asthma and medication. serum ige (immunocap â , immunodiagnostics, sweden) and bet v -induced basophil activation in whole blood determined by cd expression in a flow cytometer (flow cast â , b€ uhlmann lab ag, switzerland), were analyzed before and after the pollen season. (trial record: nct , clinical.trials.gov). results: there was significant reduction in allover allergy-related ailments and types of allergy medication used in the abm extract compared with placebo group during the pollen season and no side effects. also, abm treated asthmatics had fewer symptoms and used less medication than controls. in the abm group, serum levels of specific ige anti-bet v and anti-t , were significantly reduced during the pollen season as compared with levels in the placebo group. whereas the maximal allergen concentrations needed for eliciting basophil activation before the season changed significantly to lower concentrations (i.e. enhanced sensitization) after the season in the placebo group, these concentrations remained similar in the group given the mushroom extract. summary/conclusions: oral pre-seasonal supplementation with an abm-based extract for months reduced general allergy ailments, asthma symptoms and medication in blood donors with birch pollen-induced allergy and asthma during the pollen season. this was due to reduced specific ige levels and basophils rendered less sensitive to allergen activation. the study suggests that supplementation with abm mushroom extract can have prophylactic effect on aeroallergen-induced allergy and asthma in blood donors. it may therefore reduce such ailments in affected blood donors and impact blood donations in the pollen season. results: dhv started in / / . the data presented in this abstract is till / / . data is collected from total of blood donors ( . % male donors and . % female donors). repeat donors accounted for . % against . % of first time donors. of the total number of donor adverse events recorded, . % ( ) reported for male donors and . % ( ) for female donors when the donor adverse events stratified age-wise, the highest incidence reported in age group - years (male . % and female %). among age group - years, (male . % and female . %), whereas in age group - years, (male . % and female . %) data analysis of total reported and registered donor adverse events, are categorized as hyperventilation ( ), sweating ( ), dizziness (pre-syncopal ), loss of consciousness ( ), vomiting ( ), convulsions ( ), hematomas with re-bleed ( ), nerve irritation ( ) and off-site reactions ( ). many donors showed multiple forms of reactions. summary/conclusions: evaluation of donor side effects helps to improve donation process and donor compliance. most frequently recorded reaction remains dizziness (pre-syncopal). our donor vigilance data show reactions occurred more frequently in younger age, female and first time donors. repeat donation and age are predictors for low rates of adverse events. participation in dhv implies an effort to improve donor care and safety infrastructure and a desire for national and international comparisons to determine best practices and also to look into effectiveness of risk reduction strategies and follow-up trends. pre-donation hydration was implemented as an interventional tool to test the effects of hydration on pre-syncopal reactions to blood donation, specifically targeting those at highest risk such as female, first-time, high school donors. the results are awaited. background: descriptions of deferral categories and a knowledge in the percentage of deferrals in each category are of value in formulating recruitment and retention strategies. this can also help in planning more efficient recruitment strategies and thereby assist in reducing the shortage in blood supply. aims: the aim of the study is to categorize all donors who were deferred during medical checkup and find out the donor deferral rate in dubai blood donation center from january st to december st and also to find out whether there is any yearly or seasonal trend in any of the categories of deferral criteria's which can aid in forecasting and managing donor pool. methods: a retrospective study of donors deferred during last three years from january st to december st was done in dubai blood donation centre. the donors deferred during pre-donation medical check-up were categorized into categories including low hemoglobin, high and low bp, intake of antibiotic, fever and flu, taking other medications, travel history etc. the deferrals were analyzed monthly and yearly and then were compiled to find any yearly trend or seasonal trend in the donor deferral rate in any of the categories. the data were analyzed using the spss software and a p value of < . was considered significant. the assessment of donor suitability is in accordance with aabb standards and is consistently applied in every blood donation setting on each occasion of donation to all blood donors. results: during this study, , donors were registered from january st to december st and , ( . %) donors were deferred. the common reasons of deferral were low hb, high bp, travel history, intake of antibiotics and cough/flu symptoms. there was a significant decrease in deferral rate from . % ( / ) in to . % ( / )in and further to . % ( / ) in (p < . ). the specific deferral rate due to low hb also significantly (p decreased during these three years ( / in , / in , / in ), though no change was seen in the deferral due to other reasons. the reduction in the rate of deferral due to low hemoglobin may-be linked to the change in the staff performing the hemoglobin testing in dbdc (nurses instead of phlebotomist were assigned to perform hb estimation of donors). there was a seasonal variation in the deferral rate in all the three years-lowest in june ( / ) and then increasing with a peak in october ( / ) and plateauing till january. this pattern of deferral corroborated with the rate of deferral due to flu/fever and cough and antibiotics with an average of / in june and increasing to / in october (p < . ). summary/conclusions: staff competency is pertinent in accurately deferring donors. there is also a significant seasonal pattern in flu/fever and intake of antibiotics deferral rate that is reflected in the total donor deferral pattern. seasonal variation of specific category of donor deferral should be taken into account for donor recruitment and retention efforts. background: west nile virus (wnv) is a mosquito transmissible flavivirus. it has been shown (vogels thesis) that the common mosquito in the netherlands can transmit wnv in laboratory circumstances but presently does not lead to effective transmission. however, the number of outbreaks of wnv is increasing and moving from the eastern and southern european borders towards the traditionally more colder western and northern parts of europe. in order to prevent wnv transmission to blood transfusion recipients, dutch donors that travelled to regions with wnv risk are deferred for a period of days for whole blood, platelet donations and quarantine plasma in order to exclude potentially infected asymptomatic donors. aims: to assess numbers of dutch donors who are deferred for travelling to wnv risk areas within europe, and the return after onsite and offsite deferrals of donors. methods: data from to on donation attempts and deferral were retrieved from eprogesa, the blood bank information system. onsite deferral is defined as a donation that was attempted in the deferral period or in the days prior to deferral, all other deferrals are considered as offsite. a generalized estimating equation model was used to assess the association between onsite versus offsite wnv risk deferrals in - and subsequent return rates within two years (after which a donor is inactive according to domaine). results: in - , , donation attempts led to onsite deferral for wnv risk; % at whole blood donation attempts, % at new donor examinations. in total , offsite deferrals could not be traced directly to a donation, but based on the next donation more than % were probably whole blood donors. the number of deferrals peaks each year during august, the major holiday period in the netherlands, and increased from in august to in august . this increase is probably caused by the expansion of wnv risk regions. the return rate of wnv deferred whole blood donors is slightly lower than for donors who are not deferred ( % versus %); for wnv deferred new donors the return rate is % (versus % for no deferral). thus wnv deferral resulted in approximately - extra lapsing donors during these years. however wnv deferred donors, that are older (odds ratio (or) . ; % confidence interval ( % ci) . - . ), of male sex (or . ; % ci . - . ) and whole blood donors as opposed to new donors (or . ; % ci . - . ) were more likely to return to donate. there was no difference in return rate by offsite and onsite deferral. summary/conclusions: travel-related wnv deferrals are increasing with expanding risk regions, especially in the holiday season where the availability of donors is already low. although the numbers of donors who are permanently lost after wnv deferral are limited, the increasing numbers of lost donations make it important to consider alternatives to donor deferral such as wnv nat testing. background: low haemoglobin due to iron deficiency is increasingly recognized as a serious problem in many blood centers. donor education, iron supplementation, ferritin monitoring, and lengthening of inter-donation interval are currently the main mitigation measures. however, a number of factors in particular donor knowledge could impact their success. locally, iron supplementation programme was implemented since with target group of donors who have given blood within the last six months. aims: here we look at an online donor survey to gain insight on their view of the programme and knowledge. methods: donors with successful blood donation in the past six months would be given days of one tablet of iron supplementation ( mg elemental iron) since . an electronic questionnaire was sent to blood donors in to assess their view on the programme and knowledge which focused on iron store and absorption, compliance and any side effects occurred. results: donors (male to female was : . ) replied to the questionnaire. of them, received iron supplementation (male to female was : . ). most of the respondents ( %) had one or more donations in the preceding months. of the donors received iron tablets, only ( %) took all; ( %) took more than % but not all; ( %) took some but less than % and ( %) did not take any. gastrointestinal upset was reported in ( %) donors and constipation seen in ( %) among those who took at least some of the iron supplementation. most respondents answered correctly to the questions on the knowledge on iron store and absorption. when comparing those with better compliance (took more than %) to those who did not (took less than %), significantly more donors in the former knew vitamin c could enhance iron absorption (p < . ). on the other hand, no difference was seen when they were asked if ) iron can only be absorbed from meat; ) tea and coffee consumed during meal can enhance iron absorption; ) everyone can take iron supplementation on their own; and ) iron store in male is always more than female. summary/conclusions: the results suggested that there is definitely more room to enhance the blood donors' knowledge on iron store and absorption in order to improve the effectiveness of iron supplementation programme. besides, the side effects reported by the donors could be an important limiting factor that better alternatives should be explored and considered. background: vasovagal reactions (vvr) are a well-established deterrent to donor return. however, the correspondence between vvr experience and donor lapse is not perfect. in australia, for example, vvrs only reduce two-year return rates by % for whole blood donors and % for plasma donors. the elements of a vvr and the donor's interpretation of this event that protect against or encourage lapse have not yet been identified. aims: in this study we explored the views of donors on donating following a vvr, with a particular interest in their emotional reaction to the vvr, their understanding of what caused the reaction, and their intentions to return. methods: semi-structured telephone interviews were conducted with whole blood and plasma donors who had a recent vvr experience. data were analysed using the framework approach. results: donors are generally motivated to give blood to help others and to positively impact on those in their communities. they anticipate feeling good after their donation but in contrast, for many, a vvr leaves them feeling anxious, embarrassed, and disappointed. for donors, the experience of a vvr negatively influences their perceived ability to donate successfully, and many fear it will happen again. however, this effect appears minimised among donors who at least partially attributed their reaction to their own behaviour, such as poor hydration. for donors already juggling multiple demands, a vvr may tip the balance with donating becoming too much of an effort and perceived risk. however, donors appeared more confident to return if they felt supported by staff or if they could donate with family or friends. summary/conclusions: this study provides valuable insight into the vvr experience, which will aid in the improvement of donor safety and retention. the findings highlight the need to improve communication at the time of and following a vvr, to educate donors on how to reduce their vvr risk, and to intervene to help donors maintain their perceived ability to give blood in order to maximise retention following a vvr. background: frequent blood donation depletes the iron stores of blood donors. iron depletion might have negative effects on the health of the general population, but its effect on the blood donor population is not well known. aims: to investigate the iron status of finnish blood donor population and how it relates to donor health, the finnish red cross blood service set up the findonor , study in . we investigated whether there were changes in donors' selfrated health and if these possible changes could be associated with differences in iron biomarkers (ferritin and soluble transferrin receptor -stfr) or hemoglobin levels during the first study visit. methods: participants were recruited in three donation sites in the capital region of finland between may and december . participants filled out an electronic questionnaire about their health and lifestyle at the donation site during their enrollment visit. participants were asked by letter to fill out the same questionnaire electronically during the summer . we included the participants ( men and premenopausal and postmenopausal women) who completed both health questionnaires. to evaluate self-rated health we used the well-validated single question: "how would you rate your health in general?". participants were able to evaluate their health status on a five-point scale: excellent, very good, good, moderate, and poor. iron biomarkers and venous hemoglobin were measured from blood samples collected at the first study visit. we first computed the odds-ratios of reporting poorer health depending on demographic group. we then compared iron biomarker and hemoglobin levels between donors who reported improved, similar or poorer health rating. results: donors who rated their health in the first questionnaire as moderate (n = ), good (n = ), very good (n = ) or excellent (n = ) health tended to report improved ( %), similar ( %), similar ( %) or poorer ( %) health ratings respectively in the second questionnaire. pre-menopausal women reported their health poorer in the second questionnaire compared to the first questionnaire more often than post-menopausal women (pre-menopausal %, post-menopausal women %), or = . % ci . - . ). there were no differences between other groups. there were no significant differences in iron biomarkers levels (ferritin and stfr) or hemoglobin levels between donors whose health ratings were improved, similar or poorer. summary/conclusions: in this cohort, pre-menopausal women rated their health poorer at the end than at the beginning of the study more often than post-menopausal women. no association was found between changes in self-rated health and iron levels (ferritin, stfr) or hemoglobin levels. further studies about the factors relating to blood donors' self-rated health need to be carried out. background: in recent years, the blood donation business has made great achievements, but it still cannot avoid the occurrence of adverse reactions to blood donation which not only brings certain obstacles to the blood donation work, but also affects the enthusiasm of blood donors. aims: to understood the causes and other relevant factors of adverse reaction among blood donors, the information of blood donors at dai autonomous prefecture of xishuangbanna were analyzed in . methods: the data of volunteers from january to december were analyzed. the causes of adverse reactions were classified, and the incidence of adverse reactions was compared in terms of gender, frequency, age and blood type of blood donors. results: there were blood donors in , ( . %) of whom had adverse reactions and causes were induced, among which mental stress was the most common factor that accounted for . % ( cases). there was no significant difference in the incidence of adverse reactions between men and female (p > . ). from the frequency of blood donation, the incidence in the first donor was significantly higher than that in the second donor (p < . ). when it comes to age, the incidence was different and the - age group was the highest ( . %). among different blood group donations, there was no significant difference (p > . ). summary/conclusions: adverse reactions of blood donation is closely related to the psychological state and age of the blood donors. the staff of the blood center should further optimize the service, strengthen the communication and publicize the knowledge of blood donation. the ultimate goal is to increase the blood donation rate on the basis of reducing adverse reactions. background: blood loss due to repeated blood donation can lead to iron deficiency or anemia, but currently there is no management plan for the prevention of iron deficiency in korean blood donors. female and male donors are required to wait at least weeks between blood donations in korea, which is the shortest period among all northeast asian countries. female and male donors are allowed to donate whole blood up to five times per year and platelets up to times per year (if spaced more than days apart for the latter) due to the chronic blood supply shortage. these facts induce concern about the impact of blood donations on the donors' iron status. aims: this study aimed to evaluate the effect of oral iron supplementation in repeat donors based solely on donation history. methods: the high-risk group included male donors with ≥ whole blood donations or plasmapheresis or plateletpheresis donations, and female donors with ≥ whole blood donations or component donations, both within the previous year. the control group consisted of first-time or reactivated (ft-ra) donors who had no history of blood donation in the past years. the hemoglobin (hb) level, ferritin level, total iron binding capacity (tibc), transferrin saturation, and soluble transferrin receptor (stfr) of repeat donors at high risk for iron deficiency were compared to those of ft-ra donors. iron deficient erythropoiesis (ide) is defined as present if the log of the ratio of soluble transferrin receptor to ferritin was ≥ . . the repeat donors took iron supplements for weeks and the same tests were repeated after and weeks to evaluate their effects and the side effect and compliance was assessed. results: a total of male and female repeat donors were recruited, and each male and female ft-ra donors were recruited to the control group. after week iron supplementation, among male donors, the prevalence of: low hb level (< . g/dl) decreased from . % to . %; low ferritin level (< . ng/ml) decreased from . % to . %; high tibc level (> lg/dl) decreased from . % to . %; low transferrin saturation (< . %) decreased from . % to . %; and ide (stfr/ferritin ≥ . ) decreased from . % to . %. among female donors, the percentage of: low hb level (< . g/dl) decreased from . % to . %; low ferritin level (< . ng/ml) decreased from . % to . %; high tibc level (> lg/dl) decreased from . % to . %; low transferrin saturation level (< . %) decreased from . % to . %; and ide (stfr/ferritin ≥ . ) decreased from . % to . %. in total, male ( . %) and female ( . %) blood donors reported undesirable side effects related to iron supplementation. a total of male ( . %) and female ( . %) blood donors were administered iron supplementations for days. participants ( . %) answered that they were willing to take a complimentary iron supplementation. summary/conclusions: ferritin level, considered a reliable indicator of iron status, increased and ide decreased significantly after iron supplementation in female donor group, but not in male donor group, compared to the ferritin levels and ide of control donors. iron supplementation in repeat donors at a high risk of iron deficiency was shown to reduce their risk of iron deficiency or anemia irrespective of gender; however, -week oral iron supplement was not enough to restore iron storage level in the male donor group. background: c-reactive protein (crp) is an acute-phase protein and a non-specific maker of inflammation and tissue damage produced by the liver. several prospective epidemiologic studies have demonstrated that high-sensitivity c-reactive protein (hs-crp) is a predictor of future coronary events among apparently healthy men and women, hs-crp level greater than mg/l has been independently associated with a % excess risk in incident of coronary heart disease (chd) as compared with levels less than mg/l. frequent blood donation has been associated with a lower incidence of coronary artery disease (cad); however, there is a dearth of information on serum levels of crp in the nigerian donor population. aims: to investigate whether regular blood donation is associated with lower serum hs-crp level in nigerian blood donors. methods: a descriptive cross-sectional study carried out to measure serum levels of high sensitive c-reactive protein (hs-crp) and ferritin among blood donors attending the donors' clinic in lagos university teaching hospital (luth). subjects who did not meet criteria for blood donation were excluded. additional data on sociodemographic characteristics was collected using interviewer-administered questionnaire. serum ferritin was analysed using chemiluminescent microparticle immunoassay performed on the abbott architect ci (abbott laboratories, abbott park, il, usa). serum concentration of hscrp was estimated by immunoturbidimetry method using analytical kits from erba diagnostics mannheim gmbh in semi-autoanalyzer (xl , erba mannheim). data was analysed using stata version (stata corp) statistical software. results: in total of blood donors, ( . %) were males and ( . %) were females, the mean age was . ae . years. two hundred and thirty four ( . %) were first time donors and ( . %) were regular donors, serum levels of hs-crp was slightly higher in regular donors compared to first time donors ( . ae . vs . ae . mg/l, p = . ) though the difference was not significant. serum levels of ferritin was significantly higher in first time donors compared to regular donors ( . ae . vs . ae . ng/ml, p = . ). interestingly, levels of serum hs-crp were significantly higher in male than female population ( . ae . vs . ae . mg/l, p < . ) and smokers than non-smokers ( . ae . mg/l vs . ae . mg/l, p = . ). correlation analysis showed no correlation between serum hscrp and serum ferritin levels in both categories of donors while there was a weak positive correlation between hs-crp levels and white blood cells among the first time donors. summary/conclusions: this present study did not reveal any decrease in baseline levels of serum hs-crp with regular blood donation; smoking status and gender were however associated with an increase in baseline hscrp. this finding suggests that hs-crp level might not be a useful marker of future coronary events in healthy blood donors in nigeria. background: because the blood donation removes mg of iron from the donor, iron deficiency, frequently occurs in regular blood donors leading at a long term to the anemia. aims: to determine the effect of blood donations on ferritin levels in regular blood donors. methods: all prospective donors have been submitted to a physical examination and a health history assessment intended to ensure that the prospective donor is in a good general health and eligible to donate blood. the acceptance criteria are: • hemoglobin > or = . g/dl for male and > or = . g/dl for female • inter donation interval = days • donations/year for male and /year for female all eligible donors and deferred donors for all reasons except for low hemoglobin who accepted to enroll in this study and signed a consent. in addition to the medical exam, two samples have been collected one for cbc and another for ferritin. donation history, sex, age and weight have been documented. results: first time and regular donors accepted to enroll in this study. only female donors ( . %) participated to this study. . % of the participants were first time donor. % of male and % of female frequent donors are iron deficient out of male blood donors were iron deficient ( %) with serum ferritin < ng/ml. . % were repeat donors. out of female donors were iron deficient ( . %) with serum ferritin < ng/ ml, all were repeat donors. . % of repeat donors were iron deficient / of the deplete donors were first time donors summary/conclusions: frequent blood donors have higher prevalence of iron deficiency than first time donors. female donors have a slightly higher prevalence of iron deficiency than male donors. prevalence of iron deficiency in abu dhabi donor population is lower than the published data. changes need to be done on: increase inter donation interval or restrict the total number of allowable donations in a -month period for whole blood and red cells modifying donor hemoglobin requirements testing for serum ferritin iron supplementation donor education abstract withdrawn. background: haemovigilance procedures aim to guarantee not only the safety of the recipients of blood and its components but the safety of the donors as well. every adverse reaction that occurs during the donation of blood or its components can potentially be a threat to the health of the donor which can subsequently lead to the decision of the donor to resign from donating blood. aims: the aim is to analyse the type and the frequency of occurrence of adverse reactions among the donors donating blood or its components independently of the method of the donation. methods: we have analysed the number of collected donations and the number of adverse reactions in the years - in the group donors of aged - . we have specified following adverse reactions: vasovagal response without fainting, vasovagal response with fainting, vascular reactions (bruises) and other (e.g. allergic reaction to the anticoagulant, loss of blood pressure due to hypovolemia). the analysis was made using data obtained from computer system blood bank which is in operation in blood center in pozna n, poland. results: in years - the total number of adverse reactions among the donors was recorded which is . % of the total number of collected donations. % of the adverse reactions occurred in the group of donors aged - . vasovagal response without fainting was the most common adverse reaction in the total number of reactions and totalled . % of all adverse reactions. in the group of donors ages - it totalled % of all adverse reactions. the second most common type of adverse reactions was vasovagal syncope that totalled . %, in the analysed group of donors . %. vascular reactions (bruises) totalled . % of all adverse reaction, in the analysed group . %. the remaining adverse reactions totalled . %. summary/conclusions: . vasovagal reactions (with and without fainting) were proved to be most common adverse reactions in the group of donors aged - i.e. in the groups of donors just starting to donate blood. it seems reasonable to continue with further research into the reasons for the occurrence of this psychosomatic reactions. . it seems beneficial to provide constant educational activities of young donors regarding the preparation for the process of donation of blood and its components (proper nourishment, hydration as well as planning the time for scheduled donation long enough for a safe and pleasant procedure. . it seems beneficial to provide constant training for the medical staff involved in the process of donation regarding active observation of donors, proper conduct in the situation when the adverse reactions occur during the blood donation, ways to minimize the fear of donors, effective communication with the donors (explaining the process of blood donation, proper behaviour after the donation e.g. avoiding physical exercise or straining the arm). blood products -blood processing, storage and release background: the accumulation of microvesicles (mvs) in rbc concentrates during storage may be responsible for clinical symptoms such as inflammation, coagulation, and immunization. aims: our aims was to determine whether any of cd molecules responsible for important functions are present on the microvesicles, and if their expression level is dependent on the storage period of rbc units. additionally, by using cytometric analysis and phagocytosis visualization in a confocal microscope, we examined the interactions of donor monocytes with erythrocyte microvesicles, depending on their time of storage. methods: erythrocyte microvesicles were isolated from "fresh" ( nd day) and "old" ( nd day) stored rbc units. qualitative and quantitative cytometric analysis of these membrane structures was performed using the annexin v-fitc, anti-cd a-pe antibody, and calibrated beads. the microvesicles were also visualized under a confocal microscope. the expression of the molecules cd a, cd , cd , cd , cd , and of phosphatidylserine was analysed using flow cytometry. measurements of microvesicle phagocytosis by human monocytes were carried out using a flow cytometer and a confocal microscope. results: the analysis of the microvesicles with calibration beads allowed us to identify these structures with a diameter of about . lm in the "fresh" and "old" blood samples. we observed a statistically significant increase in the number of microvesicles in the "old" units ( ae mvs per ll), as compared to the microvesicles in the "fresh" ( ae mvs per ll). at day , the microvesicles had elevated expression levels of cd and reduced expression levels of phosphatidylserine. significant changes were also observed in the case of cd and cd molecules. the expression of these molecules of vesicles isolated from "fresh" rbcs was lower than in the case of -day vesicles. the phagocytosis index was significantly higher ( . %) for the microvesicles isolated from -day stored rbcs than for microvesicles from the - background: platelet concentrates (pcs) are conventionally stored at room temperature with a limited shelf-life of - days. alternative storage methods, such as cold storage and cryopreservation are attractive options due to the potential for extended storage, reduced bacterial growth and improved hemostatic function. cryopreservation of human pcs has been associated with formation of more microparticles and elevated procoagulant activity compared to liquid-stored (room temperature-and cold-stored) pcs. microparticles are submicron plasma membrane particles that have been postulated as potential mediators of adverse transfusion outcomes. similarities in the size and storage-related changes up to days suggest that sheep may be a suitable model in which to investigate the effects of pc transfusion. previous research has established that room temperature stored sheep pcs contain fewer microparticles than human pcs. however, nothing is known of the effect of other storage conditions. aims: this study aimed to determine whether cold storage and cryopreservation contribute to variation in concentration and size of sheep platelet derived microparticles compared to conventionally stored sheep pcs. methods: sheep buffy coat derived pcs in % plasma/ % ssp+ were prepared with minor modifications to standard procedures for preparation of human pcs. sheep pcs were split into units (n = of each) on day and stored either at room temperature (rt; - °c with agitation) for days, cold stored for days ( - °c no agitation) or cryopreserved (À °c with the addition of - % dimethyl sulfoxide) for - days and sampled post-thaw. platelet supernatant, prepared by double centrifugation, was stored at À °c. the mean size and concentration of microparticles were measured using nanosight ns nanoparticle tracking analysis system (malvern instrument). results are mean ae standard deviation. storage associated changes overtime were determined using a one-way analysis of variance with bonferroni's post-test. paired t-tests were applied to determine the effect of cryopreservation. a p-value of < . was considered significant. results: at day , sheep pcs had a microparticle concentration of . ae . microparticles/ml with a mean size of . ae . nm. storage duration at rt sheep pcs was not associated with significant changes to microparticle concentration or size. cryopreservation of sheep pcs significantly increased the concentration ( . ae . microparticles/ ml; p = . ) and the mean size ( . ae . nm; p = . ) of microparticles post-thaw. the mean size and concentration of microparticles in the cold-stored pcs at day was comparable to room temperature pcs stored for days ( . ae . nm vs. . ae . nm; p = . and . ae . microparticles/ml vs. . ae . microparticles/ml; p = . respectively). summary/conclusions: cold storage of sheep pcs did not impact formation of microparticles over the days storage period; however, cryopreservation increased microparticle concentration and the size post-thaw. further investigation is required to determine whether these findings are influence hemostatic function. a pre-clinical sheep model of cold-stored and cryopreserved pc transfusions can facilitate mechanistic studies and complement clinical trials. background: during storage, the properties of rbc in storage solution change ("storage lesion"). for instance, ph, atp and , -dpg concentrations decrease upon prolonged storage. these changes can affect oxygen delivery by the cells. the capacity to deliver oxygen is defined as p : the oxygen tension (po ) at which % of the hemoglobin is saturated with o . an oxygen dissociation curve (odc) represents the non-linear relationship between saturated hemoglobin and po . this relationship is dependent on temperature, ph, pco and , -dpg. due to changes in these factors, the curve will shift along the x-axis. in whole blood, p is at a po of about mm hg. not much is known about p of rbcs in storage solution, and the changes during storage. aims: to determine the oxygen dissociation of rbcs stored in standard red cell additive solution sagm and in pagggm (an experimental red cell additive solution, transfusion. ; : - ). methods: rbcs were prepared in sagm (n = ) or pagggm (n = ). pagggm is designed to better maintain both atp and , -dpg during storage. rbcs were stored at - °c and sampled on day , and for (internal) ph, atp, , -dpg and p . p was determined by hemox analyzer (tcs scientific corp.). the principle of the hemox is based on the measurement of spectrophotometric properties of hemoglobin at different oxygen pressure. rbc samples were brought from oxygen-rich environment to oxygen-poor environment ( %) using n gas. p was determined from the obtained odc. results: the whole storage period, ph i of pagggm-rbcs was higher compared to sagm-rbcs. , -dpg content of sagm-rbcs decreased during storage and was below the detection limit after day . , -dpg content of the pagggm-rbcs increased the first days of storage and slowly decreased from day on. at day , pagggm-rbcs still contained . -dpg ( . lmol/g hb). p values decreased during storage from mmhg at day to mmhg at day for sagm-rbc and from mmhg to mmhg for pagggm-rbc. p values of pagggm-rbcs were higher during the entire storage period. summary/conclusions: during storage, the p decreased in all rbcs. the p was higher for the pagggm-rbcs during the whole storage period. the higher p in pagggm-rbcs seems to correlate with the higher , -dpg content in these cells. background: in belgium % of the platelets are pathogen inactivated (pi) and legislation requires a minimum platelet content of . per platelet concentrates (pc). therefore routine pools are produced with buffy-coats (bc). facing increased demand of pc and stable to slightly declining red blood cells (rbc) demand, production of whole blood (wb) derived platelets must be adapted to switch flexibly from to bc per pool. this dual pooling strategy should allow alignment between wb collection forecast, pc inventory, pc demand and pc production. aims: first develop a pooling procedure with bc and ml platelets additive solution (pas) instead of ml for bc, without changing the settings of our wb separators and platelets separators. maintain a content of ≥ . platelets with a ratio plasma/pas between to % required for pi. after validation, deploy a dual pooling strategy ( or bc/pool). methods: wb is collected with top and bottom kit (composelect; fresenius kabi) and separated (macopress; macopharma) to produce ml bc with % haematocrit (htc) and > % platelets recovery with average platelets content of . random bc are pooled with ml or bc are pooled with ml of pas-e, platelets are then extracted on tacsi pl (terumo bct) and pc are treated for pi (intercept blood system; cerus). each pc is sampled and platelet content is determined (abx pentra xl ; horiba). results: during the study bc were processed into pools ( ( . %) with bc and ( . %) with bc). before tacsi separation, bc mixture with pas-e had volumes of ae ml ( bc) and ae ml ( bc) with respectively htc of ae % and ae %. the plasma/ pas ratio was ae % in both cases. tacsi separation was performed with one same program for both types of pools. after pi, platelets content of the pools was . ae . with bc and . ae . with bc (average ae standard deviation). pools below the limit of < . were / ( . %) with bc and / ( . %) with bc. the platelets concentrations ( /ll) were ae ( bc) and ae ( bc). platelets recovery was % ae for bc and % ae for bc. summary/conclusions: bc could theoretically produce pools of bc or pools of bc. this means a maximum potential gain of + % pc. in practice during shortage periods we switched from to bc when dictated by the actual inventory levels and hospital needs. the advantage of this dual pooling strategy was a gain in production capacity to cover these shortage periods ( pc, + %). the disadvantage of pooling randomly bc is that pools contained less than . platelets per pool potentially limiting their usage to low weight or paediatric patients. a preselection of the bc based on platelet count could optimize the bc pooling procedure. background: apheresis-derived platelet concentrates (apcs) is a standard medical therapy indispensable to contrast bleeding or hemorrhage. however, bacterial infection caused by storage at room temperature (rt) still remains the major drawback. recently, we showed that cold-stored apcs are associated with better plt functionality but with accelerated clearance (haematologica , pmid: ). cold-induced apoptosis was identified as a potential mechanism of the shorter plt survival aims: to investigate the protective effect of apoptotic inhibitors during cold storage of apcs methods: apcs were collected and stored at rt and °c in the presence or in the absence of caspase- inhibitor. the phosphatidylserine exposure and the mitochondrial membrane potential (mmp) (tetramethylrhodamine ethyl ester perchlorate [tmre ] staining) were measured using flow cytometry. the protein expression was quantified by western blot results: a higher expression of the apoptotic marker phosphatidylserine was detected in cold-stored apc compared to rt (% apoptotic events meanaesem: ae % vs. ae % p = . ). to verify if the apoptotic signal, observed with phosphatidylserine, specifically involved the intrinsic pathway, the mmp was analyzed as a marker of alive cells. interestingly, after cold storage a decrease of the mmp was observed compared to rt indicating the activation of the intrinsic pathway (mean fluorescence intensity tmre meanaesem: . ae . vs. . ae . , p = . ). accordingly, a decrease of the procaspase- level after cold storage was detected by western blot analysis. however, when plts were stored in the presence of caspase- inhibitor a significant rescue of the cold-stored cells viability was observed (tmre staining: % alive cells meanaesem: ae % vs. ae %, caspase inhibitor vs. ionomycin, p = . ). this indicates that the activation of the apoptotic pathway, induced during cold storage, can be prevented using caspase inhibitor summary/conclusions: our results show that the reduction of cold-stored plt viability can be prevented by a specific caspase inhibitor. consequently, cold storage, associated with a better plt functionality, may become an efficient strategy for apc storage in combination with apoptotic inhibitors background: gamma-irradiation is used to treat red blood cell (rbc) concentrates (rccs) for patients who are immunosuppressed. this treatment is known to damage rbcs and to increase storage lesions. one of the causes of the storage lesions is the presence of oxygen. several studies have shown, based on different strategies to reduce o , a reduction of storage lesions related to metabolism, protein modifications and cell morphology. aims: the present research work investigated the effect of gamma-irradiation on rccs stored under normal condition and hypoxia/hypocapnia. methods: saturation of o (so )-and abo-matched rccs from whole blood donations, leukoreduced and prepared in paggsm (macopharma, france) were pooled and split in two identical rccs within h post-donation. one bag (treated) was submitted to oxygen and carbon dioxide adsorption (oxygen reduction bag, hemanext, usa) for h on an orbital shaker ( rpm) at °cae and then transferred to a storage bag impermeable to gas. the other one (control) was left as it is. the two bags were then stored at °c. a g-irradiation treatment ( gy, gammacell elan, theratronics) was applied at day or and the rccs (expiry dates at day or day , respectively) were stored until day . hematological parameters, glycolytic metabolites, extracellular potassium level, antioxidant power, morphology and deformability were measured. results: starting so values were of . %ae . (n = ) in control and of . %ae . (n = ) in treated bags, and reached . %ae . and . %ae . at day , respectively. as expected, an increase in glycolysis rate was observed during deoxygenation without any influence from the irradiation. potassium levels were identical in treated and control, and reached around mm at expiry with an irradiation-dependent kinetic release. antioxidant power and deformability were identical in both conditions. no difference in hemolysis was observed after irradiation on day and the values stayed equivalent through end of storage (at day , hemolysis (control) = . %ae . , hemolysis (treated) = . %ae . , p-value > . ). when irradiated at day , hemolysis was lower (p-value = . ) in treated rccs at the end of storage (day , . %ae . ) compared to control ( . %ae . ). seven days post-irradiation, two-third of the control rccs were above the limit of . % whereas all the treated rccs remain below the limit. quantification of microvesicles and morphological analysis confirmed these data. summary/conclusions: the storage under hypoxia has a beneficial effect on rbc storage thanks to a decrease in o content and to an improvement of metabolism. this benefit provided equivalent storage when rccs were irradiated at day and was an advantage when irradiated at day . importantly, the results show that combining irradiation with hypoxia/hypocapnia retained the improved hemolysis profile of o depleted rbc. in summary, the reduction of o level in rccs enables a better storage of rcc when a late irradiation is applied. background: in vitro blood circuit machines require a constant monitoring of blood flow rate which have to be maintained at a constant value. also, measuring the hematocrit of flowing blood in such machines is essential for performing real-time diagnostics. recently, acoustophoresis has emerged has a promising blood separation technology capable of replacing centrifugation for the preparation of platelet concentrate. to avoid damaging blood cells, the technique is used without infusing pumps thus increasing the need of flow monitoring. however, acoustophoresis chips performs at low flow rates, outside the range of available commercial flow meters. in addition, hematocrit measurement is of a particular interest for acoustophoresis since it is a direct indicator of the separation efficiency. aims: in this study, we present a straightforward doppler ultrasound system designed for measuring blood flow rate and hematocrit in an acoustophoresis chip [bohec et al, platelets, ] . we show that the stability of the in vitro environment can be used to obtain high level of accuracy of the doppler method using a basic and low-cost experimental set-up. this improvement allows a precise measurement of flow rates as low as . ml/min in sub-millimeter tubing. furthermore we evaluate the capability of the system to measure hematocrit of human blood samples coming from different donors. methods: the experimental set-up was constituted of an ultrasonic continuous wave doppler probe mounted on a d printed support. the accuracy of flow rate measurements between . ml/min and . ml/min was evaluated as well as the optimal measurement time. for different blood bags, the relationship linking the total energy of doppler signals and hematocrit was derived. hematocrit in a range under % was estimated from doppler signals for each blood bag. results: the system is able to acquire exploitable doppler signals for the whole flow rate and hematocrit range. flow rate estimation from the signals shows a high accuracy with a mean measurement error under % for a measurement time of s. the mean error is still under % for a measurement time of . s. hematocrit estimation from doppler signals shows a good linear correlation with reference measurements for bags , and . hematocrit estimation for bag diverges from reference for values above %. summary/conclusions: the proposed doppler ultrasound system is capable of measuring low blood flow rate in narrow medical tubing with a high accuracy. it is particularly suited for an acoustophoresis device but the versatility of the system makes it easily applicable to any in vitro blood circuit. we furthermore demonstrated that the system can be used for measuring hematocrit under % without additional developments. this finds interesting applications in blood sorting technologies but also demonstrates that doppler ultrasound is a potential simple and low cost method for measuring hematocrit of flowing blood in vitro. background: hereditary hemochromatosis (hh) is the most common genetic disorder in populations of northern european descent manifesting with high levels of storage iron (ferritin) in blood and tissues. the standard treatment is serial therapeutic phlebotomy to decrease iron overload. the collected blood is frequently discarded but some blood banks allow "healthy" hh patients to donate blood for patient use. red cell concentrates from hh donors have been reported safe for transfusion, but little or no data is available on platelet concentrates from hh donors, including the potential contribution of surplus iron to the "platelet storage lesion". aims: the aim of this study was to compare platelet quality, activation and aggregation over seven-day storage in platelet-rich plasma from patients with newly diagnosed hh and from healthy controls. methods: whole blood ( ml) was drawn into compoflow blood bags containing cpd and sag-m from healthy controls and newly diagnosed hh patients. platelet-rich plasma (prp) was prepared from whole blood and split into four compo-flex bags each containing ml prp (range - platelets/l). platelet quality tests were performed on days , , , and of storage. platelet aggregation was tested using a chrono-log aggregometer and four agonists (adp, arachidonic acid, collagen, and epinephrine). platelet expression of cd , cd b, and cd p was measured with flow cytometry while ph and metabolites were measured with a blood gas analyzer. scd l and scd p in the supernatant were quantified using enzyme-linked immunosorbent assays. results: both hh and control groups included males and females. the mean age was significantly lower in the control group, years ( - years), than in the hh group, . years ( - years) (p = . ) while ferritin levels were significantly higher in hh patients (median . , range - ) than in controls (median . ng/ml, range . - ng/ml) (p < . ). in the hh group, each had the c y/c y and c y/h d genotypes. results of prp quality control tests were comparable between the two study groups over seven days of storage (p < . ) with the exception of glucose (higher in hh patients on all time points, p < . ). platelet aggregation and the expression of activation markers (cd p and cd b) on platelets and in the supernatant (scd p and cd l) were comparable between hh and control prp units over all seven days of storage. the analysis revealed comparable and expected alterations in metabolic and platelet activation markers over seven-day storage in both groups. ph increased, glucose decreased, and lactate increased over time while cd b expression decreased and cd p increased. platelet aggregation responses decreased during storage but to a varying degree depending on the agonist, however, the decrease was comparable in cases and controls. summary/conclusions: these results suggest that high iron stores in hh do not adversely affect the quality of platelet units produced from hh patients. furthermore, the data also suggest that blood from hh patients, including platelets, can be donated for patient use. background: platelets are often shipped over long distances from collection centres to blood processing centres and subsequently to hospitals. platelet agitation facilitates oxygen transfer, thus promoting aerobic metabolism, and maintaining platelet ph. during shipment, platelets cannot be agitated continuously, which may promote anaerobic metabolism. previous studies have examined the effects of prolonged periods without agitation on apheresis platelets collected in plasma, but not platelets in platelet additive solution (pas). it is therefore important to determine whether platelet quality and function are maintained during prolonged transport or hold time in a shipper. aims: the aim of this study was to evaluate the effects of prolonged storage without agitation on the in vitro quality of apheresis platelets in pas. methods: triple dose apheresis platelets (n = ) were collected using a trima accel platform in % plasma/ % pas (ssp+). after resting for h, platelets were split equally into three components, packed into a shipper and transported immediately to the blood centre. upon arrival, one of the platelet components was removed (< h; t ), and the others remained within the shipper, without agitation. the second component was removed at h post-collection (t ), and the third was removed at . h post-collection (rounded up to h; t ). platelets were tested on day , and post-collection and in vitro quality and function were monitored. data were analysed using a two-way repeated measures anova, where a p-value of < . was considered significant. results: platelets held without agitation for h consumed significantly more glucose than those removed at h or immediately upon arrival (p < . ), even on day post-collection. this was accompanied by increased lactate production (p < . ), indicating increased anaerobic glycolysis. consequently, the ph was significantly lower in t platelets (p < . ), and on average it was . ph units lower than in platelets held in the shipper for h or less. however, the ph remained above . in all components. mean platelet volume was also reduced in t platelets (p < . ), suggesting acceleration of the platelet storage lesion. phosphatidylserine exposure, surface expression of cd p and microparticle generation were significantly higher in the t platelets throughout the storage period (all p < . ), suggesting platelet activation. release of scd p was also increased in t platelets (p = . ), whereas extended storage in a shipper did not affect release of rantes (p = . ). adp-induced activation of glycoprotein iib/iiia, measured by pac- binding, was decreased in t platelets (p < . ), indicating reduced platelet responsiveness to agonist stimulation. additionally aggregation in response to collagen (p = . ) and adp (p = . ) were significantly lower in t platelets, suggesting a decrement in platelet function after prolonged storage without agitation. summary/conclusions: significant in vitro changes were observed in platelets held without agitation for h. these results suggest that the length of time that platelets are held in a shipper should be minimised where possible. background: the shelf-life for platelet products has been restricted to days. this very limited window of time is intended to sustain the quality of platelet and to reduce the risk of bacterial growth. we have recently demonstrated that in suitable platelet bags, the platelet product quality remains high after days of storage. this was proved by examining in vitro, the quality parameters of platelets such as platelet concentration, glucose, ldh, and ph (alexopoulos k. et al., haema, , ) . our new target is to extend this research in extra days of storage. we also want to determine if there is any bacterial development in this period. aims: the goal is to investigate the capability of storage period for platelet units, from to days. methods: in this study, platelets were collected from normal blood donors in the blood bank department of general hospital of patras "agios andreas". a total of ae ml of whole blood was drawn into triple cpd/sag-m top-top bags blood container systems, lmb technologie (gmbh). the platelet concentrates were prepared by platelet rich plasma (prp) method and then they were placed in a platelet incubator with agitator (helmer pc ). samples were drawn aseptically with a needless access coupler (cair-lgl) on days , , and . platelet count was done by ceeldyn ruby (abbott all data shown are reported as mean ae standard deviation (sd). the swirling effect remained positive (+) during the seven days storage period. the bacterial screening was found negative. summary/conclusions: platelet concentration in the bag remained constant between day and day , maintaining platelet yield. the decrease in glucose and increase in lactate, along with the decreased ph, show that the platelets remain metabolically active between days and of storage. the ph remained well within the acceptable range. no bacterial contamination was reported. thus, we conclude that platelet concentrates in these specific bags may be used with an extended shelf life of days. further studies are needed with other platelet bags to confirm our hypothesis. abstract withdrawn. aims: we introduce rt-dc as a fast, robust and unbiased quality control tool for pc, rcc and hpsc. utilizing the interdependency between cell deformation and the molecular state of the cytoskeleton, we demonstrate that rt-dc is capable to assess the quality of blood products. methods: by rt-dc we assessed: i) platelets after storage at °c or room temperature (rt) over days for apheresis pcs in addition to standard in vitro platelet function assays; ii). red blood cells before and after gamma irradiation in addition to hemolysis; and iii) hpsc after cryopreservation with % or % dmso in addition to cell count, and in vitro viability. in addition we compared the regeneration time of patients' platelets and leukocytes after transplantation of hpsc products containing either % or % dmso. results: for pcs standard quality assurance tests did not show a major difference between °c and room temperature storage while rt-dc showed a highly significant difference between both start conditions (day - , p < . and day , p < . ). for red cells, we found by rt-dc no impact of gamma irradiation with gy over the entire storage period of days assessing different rcc. for hpsc, rt-dc showed that cryopreservation in liquid nitrogen resulted in a significant increase in deformation ( . for % dmso versus . for the control without dmso; p < . ). however, this did not differ to high extent whether % or % dmso were used for cryopreservation ( . and . , respectively; p < . ). hpsc viability was lower after cryopreservation using % dmso in comparison to using % dmso. overall, blood cell regeneration is comparable between % and % dmso. summary/conclusions: studying platelet and red blood cell concentrates as well as hematopoietic stem cells under different, clinically relevant, storage conditions our results demonstrate that intrinsic material properties reveal insights into cell function and allow to predict cellular state in a robust way and using small sample volumes. in order to offer more flexibility to the production process, the storage of bcs overnight ( h) has been validated in our blood center. aims: the aim of the study was to assess the platelet quality in platelet concentrates derived from overnight stored buffy coats. methods: whole blood collected at day was separated into plasma, bc and red cell concentrates either at day or at day . bcs were then stored until the pooling step at °c without agitation and pcs were prepared at day by pooling isogroup bcs. seven " h-pcs" were prepared from bcs stored for h (whole blood separation at day ) and six " min-pcs" from bcs stored for min (whole blood separation at day ). standard quality control measurements were performed during the process and the storage. in addition, the quality of the platelets into the prepared pcs was assessed throughout the period of storage by measuring the hypotonic shock response (hsr) and by measuring by flow cytometry the proportions of platelets in apoptosis (marked with annexin v), of functional platelets (marked with cd ) and of activated platelets (marked with cd the changes observed during the -h storage period appear to be limited and compatible with a further pr process using a photochemical treatment (amotosalen and uva) with intercept. summary/conclusions: leukocyte-depleted "double dose" buffy coat platelets with a high platelet content and ready for pathogen reduction can be obtained with the ipp pooling and leukodepletion set developed by kansuk. a storage period of h before applying the photochemical treatment is feasible without significantly altering the biological quality of platelets. methods: dd-bc-pc were prepared with bc and ml of pas (intersol, fresenius kabi (germany) are sterile docked to the octopus harness and combined into a ml pooling bag. the pool is centrifuged and the pc supernatant expressed through a bioflex cs leukodepletion filter into a temporary platelet storage container. the obtained dd-bc-pc were tested within h of preparation and after storage for h in the platelet storage container for volume, platelet content, residual leukocytes (wbc), plasma ratio and biological parameters, ph, po , pco , glucose, lactate, mpv, ldh, p-selectin and swirling. results: the platelet content of dd-bc-pc (n = ) was on average . ae . . in a volume of ae ml. the mean of plasma ratio was % [min: . max: . ]. all pc contain < . wbc [min: <lq; max: . ]. po decreased from ae mmhg after separation ( h) to ae mmhg after h, ph (+ °c) and pco was stable during the same time period. glucose decreased from . ae . mmol/l ( h) to . ae . mmol/l ( h) while lactate increased from . ae . mmol/l ( h) to . ae . mmol/l ( h). the vpm was stable with . ae . at h and h. ldh increased from . ae . u/l at h and . ae . u/l at h as p-selectin . ae . ng/ml at h and . ae . ng/ ml. all units have maintained swirling. the changes observed during the -h storage period appear to be limited and compatible with a further pr process using a photochemical treatment with intercept. summary/conclusions: leukocyte-depleted "double dose" buffy coat platelets with a high platelet content and ready for pathogen reduction can be obtained with the platelet pooling and leukodepletion set developed by fresenius. a storage period of h before applying the photochemical treatment is feasible without significantly altering the biological quality of platelets. background: the irish blood transfusion service (ibts) is the statutory body with the responsibility for ireland's national blood supply. in the ibts collected , whole blood units and performed , platelet apheresis procedures. blood is collected in three fixed and six mobile sites and is returned overnight in temperature controlled vans to a single processing site. in september , the ibts implemented the tacsi automated system for the preparation of buffy-coat derived platelets as an alternative to the orbisac system. aims: the aim of this study is to compare operational aspects and quality data of buffy-coat derived platelets prepared by the tacsi (t-pcs) and orbisac (o-pcs) automated systems. methods: qc data of t-pcs and o-pcs from years to were analyzed. in , the ibts prepared , orbisac platelet pools of which , were qc tested. in , orbisac and , tacsi platelet pools were prepared and tested. in , tacsi platelet pools were prepared of which were qc tested. qc data comprised pc volume, platelet content ( /unit), residual leucocyte content ( /unit) and ph at expiry. feedback from the processing laboratory operators was collected comparing operational aspects of preparation of t-pcs and o-pcs. results: from the , o-pcs tested in and , mean pc volume was ae ml. initially the t-pcs mean volume was ae ml but more recently the volumes were increased by addition of a larger volume of additive solution to ae ml. platelet content ( /unit) was significantly improved in t-pcs ( ae ) compared to o-pcs ( ae ) [p < . ] as well as residual leucocyte content ( /unit) which was lower in t-pcs ( . ae . ) compared to o-pcs ( . ae . ) [p < . ]. ph at expiry was lower in t-pcs ( . ae . ) compared to -pcs ( . ae . ) but remained within the quality requirements. from an operational perspective with two tacsi devices, five operators produce approximately t-pcs per hour compared to a lower throughput of o-pcs per hour with five orbisac devices. the tacsi system is more compatible with batch processing which aligns with our staffing arrangements in routine production and has resulted in greater productivity since its introduction. with tacsi implementation, we reverted to manual pooling of buffy coats and new staff had to undergo training to achieve optimal platelet recovery. once staff were trained during validation, we observed a % increase in platelet recovery. processing laboratory staff indicated that tacsi devices were easier and simpler to maintain in comparison to orbisac, but highlighted that some improvements to tacsi system boxes could be made to make the system more robust. summary/conclusions: pcs prepared with tacsi and orbisac automated systems both met national and european quality requirements, however we observed a significant improvement in quality markers of pcs prepared with tacsi. processing staff reported easier maintenance and processing with tacsi compared to orbisac. throughput is higher with tacsi and there is greater additional capacity to increase production in the future if required. a perez aliaga , f puente , a aranda , j domingo , l call en and a bah banco de sangre y tejidos de arag on, aragon, spain terumo bct europe n.v., zaventem, belgium background: the blood bank and tissues of arag on (bsta) is responsible for the collection, processing and distribution of blood in the region of aragon in spain. with five fixed and five mobile sites, the bsta collects and processes , whole blood units per year. to improve quality of processed units and working conditions of personnel, the bsta decided to implement the reveos automated system. validation studies were carried out in november and routine use started in july . in parallel, the bsta initiated pathogen inactivation of platelet concentrates (pc) in november to improve transfusion safety. in november , the mirasol prt system was implemented due to simplicity of this platform. aims: the aim of this abstract is to (i) describe the results of the validation studies carried out during reveos implementation (ii) present routine use data of products processed with reveos and (iii) provide information on mirasol implementation. methods: in , whole blood units were processed using reveos. quality parameters for red cell concentrates (rcc), such as: hemoglobin (hb), hematocrit (hct) and volume; plasma: volume and residual platelet content; and pcs: volume and yield, were analyzed. a control group consisting of units processed with a semiautomated platform was used as comparison. a survey was sent to the collection and processing staff for feedback on operational aspects of the use of reveos. from to , qc data of rccs (n = ), plasma (n = ) and pcs (n = ) processed with reveos were collected. number of mirasol treated pcs and hemovigilance data from to were collected. results: volume, hb and hct of rccs processed with reveos were significantly higher compared to control rccs (volume: ae ml vs ae ml; hb: . ae . g/ unit vs . ae . g/unit; hct: . ae . % vs . ae . %). control plasma units had higher volume ( ae ml) and lower residual platelet content ( ae /l) compared to reveos plasma units (volume: ae ml; residual platelet content ae /l). for pcs, volume was higher for units processed with reveos compared to control ( ae ml vs ae ), but no statistical difference was observed in platelet content (reveos: ae /unit; control: ae . /unit). we observed either an increase or stabilization in qc parameters of rccs, plasma and pcs during the years routine use of reveos. for example, percentage of rccs units hemolyzed dropped from % to % between and and platelet content in pcs increased from /unit (first six months in routine) to /unit. plasma volume increased from ml during validation to ml in . survey results from staff highlighted the user friendliness of the reveos device. number of mirasol treated pcs increased from % in to . % in with no pathogen transmission nor serious adverse events reported following transfusion of these products. summary/conclusions: with implementation of two user-friendly systems, the reveos whole blood automation system and mirasol prt system, we observed an increased standardization of our processes enabling us to provide higher quality and safer blood products to hospitals and the patients they serve. background: white blood cells (wbc) may be regarded as contaminants in blood components and potentially reduce transfusion safety. there is currently enough evidence showing that wbc reduction through pre-storage filtration reduces the incidence of three major complications of allogeneic blood transfusions: febrile nonhemolytic transfusion reactions (fnhtr), refractoriness to random-donor platelet transfusions and transmission of cmv. leukodepletion is now mandatory in canada and several european countries. aims: the aim of this study was to evaluate leukoreduction performance and the quality of the resulting red blood cell concentrate (rbc) produced with new quadruple system top & bottom imuflex crc with an integrated red blood cells filter which was used in whole blood fractionation under routine conditions of san paolo asl blood bank methods: twenty-seven whole blood donations ( ml) were collected with using quadruple blood bags top & bottom system with an integrated rbc filter (imuflex crc, terumo bct). centrifugation and processing (using t-ace ii + -terumo bct) of whole blood units followed the blood bank sop to obtain rbcs, plasma and buffy coat. rbcs were diluted with sagm ( ml) and then filtered by gravity in average - h after blood collection to obtain leukoreduced rbc. average temperature during filtration was °c. samples were collected before and after leukoreduction for calculation of hemoglobin loss through the filtration process. filtration time was recorded. final hb, hematocrit (hct), residual platelets and white blood cells were determined after filtration using standard laboratory methods (blood cell counter) results: wb donations (n = ) were performed. rbc characteristics post-filtration and separation were as follows: volume ( . ae . ml), hematocrit ( . ae . %) hemoglobin ( . ae . g/dl) and hemoglobin per unit ( . ae . g); residual platelets ( . ae . x ³/ll); residual wbcs ( À . ³/ll). the mean filtration time was ae min and no filter blocks were observed summary/conclusions: the usability and performance of the new quadruple system top & bottom imuflex â crc with integrated rbc filter was evaluated. all quality parameters complied with italian and european guidelines and requirements. in conclusion, the set was found to be a reliable and efficient collection and separation system that performs consistently background: novel devices for automated whole blood processing (reveos tm terumo bct) have been installed in regional center for blood donation and blood treatment in katowice to modernize and automate production of blood components. this system allows to separation of four whole blood units into four buffy coat depleted red blood cell concentrates (rbc), four units of plasma and four interim platelet concentrate (ipu) units in one automatic cycle. an ipu is the starting blood component for the production of pooled, leucoreduced platelet concentrates (lpc). residual leukocytes (wbc) are obtained as a byproduct and discarded. the use of this system shortens production time because it combines otherwise two separate steps: centrifugation and blood separation in press. aims: to evaluate the quality of the blood components obtained with the newly developed reveos nlr kits, which are a whole blood collection kits containing no leucocyte reduction filter for the rbcs. methods: blood [wb] was collected into nlr reveos set (terumo bct), and processed between and h from collection using the c protocol. quality control was performed on rbc, plasma and ipu. rbc were tested for hemoglobin (hb) content, hematocrit (ht), residual white blood cells (wbc), volume and hemolysis level on the last day ( nd day) of storage. the content of wbc and platelet count (plt), protein, factor viii on the day of production and after one month of storage were determined in plasma. for ipu, platelet and leucocyte counts, volume and average platelet yield index (pyi) were displayed at the device after processing was recorded. furthermore, a comparison between fully automated and semi-automated whole blood processing was done, by comparing the amount of time necessary to fractionate whole blood units using the two different processes. background: direct, single step automatic blood separation devices (reveos tm terumo bct) was installed in regional center in katowice in . the system allows for full automation of whole blood fractionation. one of produced blood components is an interim platelet unit (ipu), containing most of the platelets and limited of white blood cells (wbcs). it is then pooled through a filter to form a therapeutic unit of leucoreduced platelet concentrate (lpc). a rough estimate of the platelet yield in the ipu, the so-called platelet yield index (pyi) is calculated and displayed by the device after the end of the separation phase. it is a useful tool to optimize selection of ipu for pools to produce lpc. aims: to determine the quality of polled lpc obtained on the basis of ipu from whole blood processed up to h after collection and the usefulness of this method in daily practice. methods: ipus used for production of lpc were obtained using fully automated whole blood processing single step system -reveos tm (terumo bct). from the beginning of the application of the method, therapeutic units of lpcs were produced after overnight hold: pools of ipus and of ipus were made using platelet pooling set (terumo bct), using ml ssp+ solution (macopharma). results: therapeutic units of lpc were analyzed. the mean volume of lpc was ae ml, platelet content was . ae . x , and white blood cells (wbc) count was . ae . x , % all lpcs had wbc below . ph measurements were made on the fifth day of storage, the ph was determined in units of lpc and was . ae . . summary/conclusions: the use of interim platelet units in daily practice increases the possibility of producing high quality blood components for patients and is a great alternative to the laborintensive manual methods of lpc's production. background: the development of cold-stored platelets has been hampered by the rapid removal of blood after transfusion by apoptosis and desialylation due to platelet storage lesions caused by refrigeration. previous studies have shown that antioxidants inhibited the activation and desialylation of cold-stored platelets for days and also inhibited rapid elimination after transfusion in animal models. aims: it was tried whether in long-term ( days or longer) storage conditions, antioxidants showed similar effects inhibiting the activation and desialylation of cold-stored platelets. methods: platelet-rich plasma (about ml) were obtained from a healthy blood donors of type o blood group and divided into a ml platelet storage bag manufactured by the manufacturer, and the conditions including room temperature and refrigerated condition and various kinds of antioxidants were prepared, (ph, glucose, lactic acid) and activation index (cd p, gpiba (cd b), annexin v), degree of reactive oxygen species and sialidase activity on the th, th and th day, and phagocytosis using the hepg cell line and platelet recovery after in vivo transfusion using scid mice were compared and compared. results: cold-stored platelets showed autoaggregation on the nd day of storage, and apparent aggregation on the fifth day of visual observation but not in the antioxidanttreated group. as expected, the ph, glucose and lactate levels of the cold platelets were maintained and the platelets at room temperature changed significantly. nac (n-acetylcysteine) -treated platelets were less active on day and sialidase activity was maintained lower than that of control group on day . the degree of phagocytosis by the hepg cell line remained at the same level during the storage period, and the in vivo recovery rate in the animal model was superior to that of the antioxidanttreated cold platelets for h and h after transfusion. summary/conclusions: cold platelets could be developed as clinically effective agents for up to two weeks if appropriate preservatives were added, and antioxidants, especially n-acetylcysteine, were effective. background: the reveos c procedure produces the following three blood components for transfusion: plasma, rbcs, and a single interim platelet unit (ipu). whole blood is collected into the reveos blood bag set and held at room temperature a minimum of h before processing on the device. after processing on the reveos device, the plasma product is ready to freeze. the rbc product is ready to combine with sag-m additive solution and leukoreduce using the integrated leukoreduction filter. the ipus are rested and agitated for the recommended time. for example, when whole blood is processed within h of collection, the ipu is agitated overnight before pooling. because the ipu is made from one unit of whole blood, approximately to ipus are pooled together and leukoreduced by filtration prior to storage in a platelet storage bag. this leukoreduced platelet pool ( units) can be supplied to the demand of hospitals like a platelet apheresis product. aims: to assess the usability of the reveos automated blood processing system to collect units of whole blood and to process the units on the reveos device, producing blood components that meet product quality specifications. methods: this study will evaluate approximately units of whole blood using the reveos system and approximately units of pooled platelet products. a total of units of whole blood (wb) have been collected into the reveos blood bag set and then held at room temperature without the use of an active cooling system (or temperature control system), such as cooling plates, or in this case, ice packs. of the units, units have been processed on the reveos device using the fresh procedure (within h of collection). nine units have been processed using the overnight wbhold procedure. all units have processed successfully without alarms, resulting in three components (rbcs, plasma, platelets). we also evaluated four platelet pools created from eighteen interim platelet units (ipus). one ipu has been discarded due to a -min whole blood collection time. the leukocyte count on all component using the automatic sysmex-xn cell blood counter. results: the red blood cells meet the criteria for hematocrit, hemoglobin and residual wbc counts are less than million per unit. the plasma units meet the criteria for displayed volume accuracy compared to measured volume and also for factor viii levels. the average residual platelets in plasma levels meet the criteria. the platelet pools meet the acceptance criteria for platelet yield and volume. limited data (n = ) suggests that this criteria will be easily met, provided that wb hold conditions are suitable for platelets. summary/conclusions: final results indicate that products produced from the reveos system meet or exceed product quality expectations. staff report that the reveos system is very easy to use. abstract withdrawn. background: thorough manual mixing of overnight-held whole blood (wb) units prior to centrifugation to separate red cell (rc) from platelet-rich-plasma (prp) is currently practised at the hong kong red cross blood transfusion service (hkrcbts). the mixing step is crucial to maximise platelet yield but laborious, and may cause elbow and wrist injuries in operators. a laboratory shaker, reax (heidolph instruments, schwabach, germany), originally designed for mixing solvents, was modified to allow mixing source wb units by the supplier. to enhance the well-being of the operators in the context of occupation health and safety (oh&s) management, feasibility of the application of such blood mixer to replace the manual mixing steps in blood component preparation was explored. aims: to evaluate the in vitro quality control (qc) parameters of the blood components prepared with the use of the modified mixer. methods: fifty-eight wb collections, including units in ml quadruple bags ( q) with/without integrated leucofilter and units in ml quadruple bags ( q), were processed in accordance with hkrcbts' prevailing component processing protocols except using the motor-driven mixer in lieu of manual mixing. wb collections were divided into groups, mixed at revolutions per minute (rpm) for min (n = ) and min (n = ), then processed into rc, prp platelet and plasma components. in vitro qc assessments including haematocrit (hct), haemoglobulin (hb) and haemolysis at expiry for rc; absolute platelet count and ph for platelet concentrates (plt); volume and residual platelet count for plasma units were evaluated and compared. results: all components derived from both q and q processed with the blood mixer fulfilled the qc requirements stipulated by aabb standards and council of europe (coe) guidelines. there were no significant differences for all the qc parameters in rc and plasma components produced when mixed for or min. summary/conclusions: the overall performance of blood components processed with the motor-driven mixer met the acceptance criteria at the hkrcbts in accordance with the aabb and coe standards. higher platelet yield was observed with prolonged mixing for min. however, prolonged mixing will much delay routine component processing. further evaluation on intermediate mixing time such as , or min may help determine an optimal mixing duration to maximise platelet yield without compromising processing workflow. to conclude, using the modified blood mixer to replace laborious manual mixing in components preparation is promising to improve the oh&s for blood component preparation operators. background: leukocyte reduction is widely used to reduce the risk of non-hemolytic febrile transfusion reactions and alloimmunization by multiple blood transfusions. around and after year , universal leukocyte reduction was adopted in many developed countries. there are mainly two types of filter, one to filter whole blood and the other to filter red cell concentrate (rcc) used during pre-storage blood processing. aims: in this study, we conducted the filtration tests with sepacell tm rs for rcc in comparison with sepacell tm r-s and sepacell tm pure rc widely used in the world. background: the cardarelli hospital blood bank processes over , whole blood units yearly. in order to constantly cope with the huge demand for platelets from oncohematology and intensive care departments, a progressive review of the processing protocols for platelet concentrates (pc) from buffy-coat (bc) pools has recently been implemented to reduce the number of units assembled, from (routine in italy) to . the -bc pc units have slightly lower volume and plasma content, factors that further limit the transfusion risk. in addition, the -bc assembly allows to facilitate the production of the pc units obtained from the same blood type, in order to minimize both the adverse reactions related to the abo/rh incompatibility and the amount of bcs not used. finally, the described innovation also impacts on the workload, with reduction of assembly, centrifugation and separation time. aims: the aim of this work is to define an optimized protocol for the preparation of pc units from -bc pools, so that the platelet yield is always higher than the minimum value required by current regulatory ( . cells/unit). background: blood is a basic component for human life and hence blood transfusion is an integral intervention in the treatment of patients. the need of blood transfusion is increasing with decreasing blood supply due to limited blood donations. conversely wastage of blood products is also a topic for discussion. to ensure the availability of blood products when they are needed, wastage of blood products should be controlled and minimized. aims: the study was designed to investigate the quantity and reasons of wastage of blood products at our hospital. methods: this was an observational study based on the statistical data available from blood bank department of nibd and bmt, pechs campus, karachi, pakistan. the study period was from february to february . study was approved from institutional review board. for all the blood products wastage and reasons of wastage were evaluated. frequencies were calculated by using spss version . results: a total of blood products were available at our institute including each of platelets, pack red cells and fresh frozen plasma(ffp). the overall wastage rate was . %. pack red cells and platelets were fully consumed yet shortage of supply was observed. however, highest wastage was observed in ffp in our blood bank i.e. ( %). results of investigating the common causes of blood product wastage at the hospital revealed that it was highly associated with common causes, including the expiry of unused products ( %) followed by broken bags ( %). summary/conclusions: this study showed over all diminutive wastage rate. ffp wastage was highest among all the blood products. wastage of blood products is a genuine issue in a hospital setup, strategies and plan of action should be discussed and implemented including enhanced collaboration with other centres to outsource the blood products or even donate at times, education and training of staff for better clinical use of blood products and ensure the availability when and where they are needed most. background: in the early s, the blood service of the republic of kazakhstan functioned along the lines of the soviet period: donation of blood was approached to the place of its use (carried out at hospitals), screening of donor blood was carried out at one stage (enzyme immunoassay) on analyzers of semi-automatic type, there was no mechanism calculating the cost of blood components, the activity of blood centers was funded at the rate of liter of whole blood. donation was not popular in society, household fears of infection were common when donated. aims: research objective is to study the main stages of reforming of blood services of the republic of kazakhstan (rok). the research methods are based on the system analysis of the condition of the rok's blood services based on the indicators of production activity for the period of - . results: in order to effectively reform the blood services, the following steps were taken. the blood supply was centralized and optimized. blood collection at hospitals was terminated, more than blood collection subjects were closed. the result was a more efficient use of technical, human and financial resources, as well as ensuring the high quality of the blood components produced. today, there are blood centers in the republicone for each of the administrative territorial units of the republic. the screening of donor blood for transfusion infection markers has been transferred into a two-step principleimmunological analysis and nat testing. a fully automated analytical equipment of a closed type is used for screening, unified for all blood centers of the republic. unification of the equipment choice allowed to provide centralized service maintenance of complex equipment and its uninterrupted operation. since , the national reference laboratory has been operating in the blood service, which conducts an external assessment of the quality of laboratory research in blood centers, as well as research in controversial and complex cases. introduced pathogen inactivation of platelet concentrates in % of cases of platelets. erythrocytes are used only in the weighing solution and only after leukoreduction. the use of resource-saving blood harvesting technologies, as well as methods to further enhance the infectious and immunological safety of components, are expanding annually. the foundations for the development of applied scientific research in the field of transfusion medicine were laid, and an own school of transfusiologists was created. as a result of reforming the blood services, the mechanisms of reimbursement of blood services costs from the budget of the republic have been changed, uniform tariffs have been defined by type of blood components for all blood components suppliers in the country. summary/conclusions: the transformations made it possible to create a blood service in kazakhstan that corresponds to the best international practice and ensures the quality, efficacy and safety of transfusion therapy. further development of the blood service is aimed at deepening reforms, studying and developing cellular technologies, new properties of plasma blood components, and improving the clinical practice of using donor blood components. background: reconstituted platelet concentrate (rpc) is a component that contains platelets (thrombocytes) resuspended in plasma that is blood group compatible with the recipient. it is used when transfusion of platelet concentrate is necessary but the fresh platelet concentrates of the specific blood group are not available. group rh-(negative) platelets resuspended in the ab group plasma are of universal use as such components can be used for all recipients independently of their blood group. in the area of activity of regional blood center in poznan rpc is used most often for patients after stem cell transplant incompatible with their blood group. aims: the aim of the analysis is to evaluate the loss in the number of platelets in the reconstituted platelet concentrates in order to determine their quality and usage. methods: we used for the analysis units of pooled platelet concentrate derived from buffy coats and units of pooled platelet concentrate derived from the apheresis. for the reconstitution group ab plasma was used. we investigated the number of platelets prior to and after the reconstitution. following measures were calculated: number of platelets in pooled platelet concentrates derived from buffy coats, number of platelets in pooled platelet concentrates derived from apheresis; average values and the standard deviation as well as the loss in number of platelets due to the reconstitution. the number of platelets was calculated with impedance method using horiba pentra xl analyser. . pooled platelet concentrates derived from buffy coats the number of platelets before the reconstitution: . /unit ae . the number of platelets after the reconstitution: . /unit ae . the loss in the number of platelets: . % . platelet concentrates derived from apheresis the number of platelets before the reconstitution: . /unit ae . the number of platelets after the reconstitution: . /unit ae . the loss in the number of platelets: . % summary/conclusions: . reconstitution results in the loss of . % of platelets in comparison to the initial value of pooled platelet concentrate derived from buffy coats. . the average number of platelets after the reconstitution is . /unit, which fulfils the quality requirements. . reconstitution results in the loss of . % of platelets in comparison to the initial value of pooled platelet concentrate derived from apheresis. . the average number of platelets after the reconstitution is . /unit, which slightly deviates from the quality requirements. . the results that we obtained show that the process of reconstitution always results in the loss of number of platelets and that the quality of platelet concentrates that have not been reprocessed is always higher. because of that we recommend that the reconstitution should only be performed in special cases such as for patients after stem cell transplant incompatible with their blood group and that hospitals should be supplied with platelet concentrates derived from buffy coats and apheresis. methods: pf was extracted from human pcs by using immunoaffinity chromatography and specific anti-pf antibody on the column. hereafter, pf was purified with concentration tubes having kda molecular weight cut-off and dialyzed via dialysis tubing with . kda cut-off. pf concentration was measured by bradford method. specific enzyme-linked immunosorbent assay (elisa) and dot blot analysis were used to determine the pf specificity. molecular weight of obtained pf was determined by polyacrylamide gel electrophoresis (page). results: our data confirmed clearly that separated pf had kda molecular weight which is corresponding with a tetramer pf . in addition, elisa showed that the pf qualified true antigenicity. dot blot analysis helped us to assure the specificity of pf . summary/conclusions: we used and optimized a homemade protocol to isolate and purify the pf . nevertheless, there was no any scientific report among the databases that utilized the similar purification method. recombinant pf is more feasible and ready-to-use, but its price may discourage the researchers to study the biology of pf . unused human pcs and specific anti-pf antibody can considered as an alternative to separate the pf particularly in labs with the shortage of resources. background: whole blood is increasingly used in civilian hospitals as an alternative to blood components in trauma resuscitation with massive bleeding. however, the hemostatic effect of whole blood depends on the initial platelet content and decreases rapidly within the first weeks of storage at °c. consequently the problem arises of how to provide wb to trauma centers without losing valuable low titer group o units if the units are not used within the expiry date of use. aims: to assess the in-vitro quality of red cell concentrates (rcc) produced from days cold stored leucodepleted whole blood units filtered with a platelet-sparing whole blood filtration filter in order to save the rcc once the wb units has reached the expiry date. methods: units of whole blood were leucodepleted within h using a platelet sparing filter (imuflex wb-sp/terumo bct) and stored for days at ae °c. on the th day of storage, units are centrifuged and the rcc is supplemented with sag-m additive solution for an additional storage of days at ae °c. in vitro parameters such as hemoglobin, hematocrit, residual leucocytes, platelet content, hemolysis, glucose, lactate, atp, . dpg were tested during storage. results: the imuflex wb-sp provided satisfying leucodepletion of the wb units ( . ae . . e /u)) while maintaining % of the initial platelet content ( ae . e /u). mean volume ( ae ml), hemoglobin content ( ae g/u) and hematocrit ( ae %) was within conformance range. after days of storage, % of the initial platelet content at collection is maintained and mean hemolysis is . ae . %. rcc processing at day was uneventful, without loss of hemoglobin or noticeable hemolysis. mean volume ( ae ml), hematocrit ( ae %), hemolysis ( . ae . %) were in conformance with mandatory standards at day . after days of storage (i.e. day after collection), rcc quality parameters were maintained with however an increase in hemolysis ( . ae . %) which was above usual values routinely observed with rcc produced from day processed units. rcc out of were above the maximum limit of . % at day after collection such increased hemolysis was more pronounced at day after collection ( . ae . %) with a proportion of rcc with a non-conforming hemolysis ratio above the accepted threshold ( % of the tested units). summary/conclusions: rcc units can be readily obtained from leucodepleted wb units with spared platelet contents after days of cold storage. in vitro quality attributes of such rcc are within conforming ranges for hemoglobin, hematocrit, volume and hemolysis. hemolysis increases more rapidly than in standard rcc, thus limiting the possibility of use within days after collection. additional studies are being initiated to reduce hemolysis during storage. g hetland , , f mahmood , l nissen-meyer and i nentwich immunology and transfusion medicine, oslo university hospital immunology, university of oslo, oslo immunology and transfusion medicine, akershus university hospital, lørenskog, norway background: allergen-specific ige in donors' plasma can be transferred from blood donors to recipients via plasma-containing products and consequently sensitize recipient's effector cells bearing receptors for ige (mast cells, basophils, thrombocytes etc.). these cells can then degranulate after exposure to allergen. this can cause allergy symptoms of various degrees (johansson, allergy, ) . food allergen-specific ige antibodies, although causing mild or no symptoms in blood donors, can be of clinical importance to plasma recipients due to varying sensitization and activation grade of recipients' effector cells. aims: the aims of the study were ) to assess sensitization profiles against an array of allergens in randomly selected blood donors and ) to identify donors with high concentrations of food-specific ige antibodies that could bear risk of clinically important sensitization of plasma product recipients. methods: we tested serum samples from blood donors randomly selected on one donation day outside the pollen season. we used ll serum per sample in a multiplex ige line-blot enzymoimmunoassay for analytes (euroline atopy screen, euroimmun, germany), which comprised food allergen extracts and single allergens, inhalant allergen extracts, insect venom extracts and one carbohydrate allergen ccd. this high-throughput method enables scanning of serum samples for ige against allergens in less than h. results are expressed as east (enzymoimmunoassay) classes: class (no sensitization), - (weak sensitization), (moderate sensitization), - (strong and very strong sensitization). results: east class, allergen, number of samples. food allergens: class none detected. class , sesame seed: . class , apple ; hazelnut . class , apple ; almond ; hazelnut ; sesame seed ; soybean ; cow milk alpha-lactalbumin . class : donors weakly sensitized to one or more allergens as kiwi, apple, almond, hazelnut, sesame, soybean, wheat, mutton, beef, cow milk beta-lactoglobulin and alpha-lactalbumin. we did not find any donors sensitized to apricot, peanut, rice, rye, yeast, cow milk casein and carbohydrate allergen ccd. insect venoms: class , and none, class , wasp venom ; honey bee . lower classes ( - ) not reported in this abstract. summary/conclusions: we found considerable sensitization to inhalant allergens but such sensitization probably represents low risk of clinical allergies due to passive transfer of antibodies. sensitization to insect venoms was negligible. we disclosed only one blood donor ( %) with high ige to sesame seed with a potential to transfer clinically relevant ige antibodies via plasma products. the need for a broad screening of donors for ige sensitization remains still questionable. background: the platelet storage lesion (psl) is one of the key factors that limit the platelet shelf-life to days. the mechanisms mediating the psl are not well understood, but it is becoming increasingly evident that platelets from some donors do not store as well as others. similarly, assessment of many in vitro quality parameters has been used to predict transfusion outcomes, with varying degrees of success. donor attributes such as age, and body mass index (bmi) may contribute to these differences. aims: to characterise the variability in the quality and function of apheresis platelet donations and to examine associations between donor attributes and platelet characteristics. methods: apheresis platelets (n = ) were collected, stored at °c with agitation, and tested at day , day and post-collection. vwf and fibrinogen binding (indicators of platelet adhesion; ae ristocetin stimulation), phosphatidylserine (ps) exposure (annexin v binding) and microparticles (cd + /annexin-v + ) were measured by flow cytometry. platelet aggregation was initiated with collagen, thrombin or arachidonic acid. thromboxane a (txa ) was measured by elisa. the proportion of procoagulant platelets (% coated platelets) was assessed by fibrinogen binding following collagen/thrombin stimulation. platelet-attached glycans were measured using flow cytometry and lectins ricinus communis agglutinin- (rca- ; detecting galactose-residues) and wheat germ agglutinin (wga; detecting sialic acid and nacetyl-d-glucosamine, glcnac-residues) ae stimulation with ristocetin. donation time, donor age, blood pressure (bp) and bmi were recorded. linear regression was performed using graphpad prism software. results: for many of the parameters measured, there was high inter-donor variability. platelets from subsets of donors showed up to -fold higher vwf and fibrinogen binding, formation of coated platelets and microparticle numbers. binding of lectins was also highly variable between the donors, indicating variability in sugar cleavage. following ristocetin stimulation, binding of all lectins increased, and again there was high variability in the responses, with up to -fold higher binding in platelets from a subset of donors. of particular interest, aggregation in response to arachidonic acid was observed in only % of platelet donations tested; although platelets from non-responders aggregated in response to the collagen and thrombin. txa was also higher in the arachidonic acid responders (p = . ). there were few associations between platelet parameters and donor attributes, and these associations were typically weak. ph at day was weakly associated with donor age (p = . , r = . ) but not bmi (p = . , r < . ), with a trend towards lower ph in younger donors. summary/conclusions: these data indicate that apheresis platelet products are highly variable. whilst donor characteristics may have some influence on the quality of the donated platelet product, there is a high level of inherent inter-donor biological variability. a much larger sample size is required to confirm these findings, and determine whether they have clinical implications. background: the frequency of beta thalassemia mutations, and thus, of beta thalassemia heterozygotes (b-thal-het) is particularly high in certain geographical regions. it has been reported that a significant proportion of donors (occasionally more than %) are b-thal-het that meet the criteria for blood donation (hb > . g/ dl). red blood cells (rbcs) of b-thal-het donors are characterized, in vivo, by particular geometry and redox status. despite sporadic indications that the rbc storage lesion may be milder in b-thal-het, targeted research on this donor group is still missing. aims: the aim of this study was to investigate whether b-thal-het rbcs storage at blood banks leads to a distinctive hemolytic, physiological and redox profile, thus, making b-thal-het a unique blood donor group. methods: blood samples from healthy non-smoker donors ( b-thal-het carriers and controls) were analyzed before and after preparation and storage of leukoreduced packed rbc units in cpd/sagm at various time intervals. susceptibility in hemolysis (in the presence/absence of oxidative, mechanic and osmotic stimuli), redox status (lipid peroxidation, reactive oxygen species (ros) accumulation, antioxidant capacity), intracellular ca + and proteasomal activities were determined. for statistical analysis, significance was accepted at p < . . samples from the red cell units were collected aseptically, processed (dual centrifugation at , g for min) and stored at À °c. processed samples were thawed, and then analysed using the nanosight ns nanoparticle tracking analysis system (malvern instruments). samples from all time-points from each unit were analysed on the same day. data were analysed by one-way anova with bonferroni's multiple comparisons test. results: at d , red cell units contained an average of . ae . mvs/ ml. the mean size of these mvs was . ae . nm and the mode size was . ae . nm. the concentration of mvs increased gradually throughout storage (p = . ), reaching a maximum at d of . ae . mvs/ml. both the mean (p < . ) and mode (p < . ) size of the mvs increased during storage; however, this size increase primarily occurred in the first week of storage (d vs. d : p < . for both mean and mode). by d , mean and mode size of mvs was . ae . nm and . ae . nm summary/conclusions: nanoparticle tracking analysis demonstrated the presence of mvs smaller than nm in red cell units. both the concentration and size of mvs present in red cell units increased during the days of routine storage. the concentration of these mvs was approximately -fold higher than we had previously detected using flow cytometry (aung, pathology, ) indicating the advantages of more sensitive techniques in characterisation of mvs. background: the lack of availability of sterile saline in a format suitable for use in blood centers for manual washing has led to an urgent need for blood services to consider alternative methods. for operational flexibility it would be desirable to be able to produce a washed rbc unit that had a shelf life longer than h. aims: the aim of this study was to validate the manual method for washing rbcs using sagm solution both as wash and storage solution and to ascertain whether an extended storage period for washed rbcs may be feasible. methods: six day old leukocyte depleted red blood cells (ld-rbc) and six day old ld-rbcs were manually washed and stored in sagm, and half of the units were pre-stored irradiated ( gy). a volume of ml wash solution (sagm) was added to the ld-rbss by sterile connection. after mixing the units were centrifuged for . min at g at °c (hettich roto silenta rs) before removing the supernatant using compomat g extractor. wash procedure was repeated twice using ml sagm solution, and after removal of the last supernatant, ml of sagm solution was added. all units were immediately measured for volume, haematocrit, albumin, iga, potassium, haemolysis, haemoglobin, ph, glucose and lactate and tested again after h, days and days storage at ae °c. results: all washed ld-rbcs met european specification for haematocrit ( . - . ) and all but one for hb content (≥ g/unit). hemolysis increased during storage. the rate of hemolysis in irradiated ld-rbcs was greater over time than in nonirradiated units. all units, both irradiated and nonirradiated, met european specification for hemolysis (less than . %) days after washing. after wash, potassium levels were low and then increased during storage; increase was greater in irradiated than nonirradiated units. potassium concentration days after washing and irradiation did not exceed those levels found at the end of shelf life (day ) of standard ld-rbcs. ph decreased during storage due to the metabolic activity of red blood cells converting glucose to lactate. the ph level of the supernatant depends on the age of the unit and not on the irradiation. the glucose concentration of the supernatant after washing is high due to sagm solution. the concentration of glucose decreased and lactate increased due to the metabolic activity of red blood cells. there is currently no specification in europe or finland for iga in washed rbcs. aabb and american red cross rare donor program stipulate that level of iga should be less than . mg/dl ( . mg/l). our iga method s lower limit for detection is . mg/l and all results were below this level. total albumin were well below finnish specification (< mg/unit). background: room temperature has been the standard storage condition for platelets since the s, when it was shown that this improved in vivo survival compared to when stored at °c. however, storage at room temperature has several disadvantages, including risk for bacterial contamination and short outdating. recently, the interest in cold-stored platelets increased, especially for patients with a hemostatic need. using extensive analysis techniques, we evaluated the in vitro quality of cold stored platelets in additive solution. aims: investigation of the in vitro quality of platelets stored at - °c in pas-e. methods: three experiments were performed, in which two platelet concentrates, prepared from buffy coats and ml of pas-e (pcs) were pooled and split in equal pcs. pcs were stored for days at - °c, one of each pair with agitation on a flatbed shaker and the other without agitation. various parameters were analyzed to study the in vitro quality during storage and compared to routine room temperature storage. results: during cold storage, the swirling phenomenon disappeared within one day. due to the lower temperature, metabolism of the platelets was lower as compared to room temperature storage. the metabolic conditions were acceptable with ph d -d : . - . with platelet count /u and glucose still at mm at least until days of storage. platelet activation maintained acceptable levels with cd p expression < %, while ps exposure increased rapidly; > % after days of storage. aggregation tests showed functional platelets until days of storage. agitation during storage had no effect on any of the tested parameters. summary/conclusions: during storage of platelets at - °c, the hematological parameters and ph met routine requirements, while swirling phenomenon disappeared already at the first day. the functionality of the platelets did not decrease during cold storage, indicating that the swirling phenomenon is not a good surrogate marker under these conditions. the strong increase of ps exposure might be involved in the observed short survival of cold-stored platelets. platelet concentrates stored at - °c are potentially suitable as a hemostatic agent for patients with a bleeding in need of platelets, but more studies are needed. aims: the goal of the study is the reinforcement of platelet reserves for case of emergency events and increasing their availability for treatment, preferentially in patients with massive bleeding. methods: we performed a comparative study with cpp and fp in vitro. buffy coatderived pooled leukoreduced platelets rhd negative were frozen in - % dmso and stored at À °c for months. cpp were thawed at °c, then reconstituted in platelet additive solution ssp+ and compared to fp. we measured these parameters: platelet content, platelet concentration, platelet loss during preparation process, coagulation properties, volume, ph, dmso concentration, titres of anti-a and anti-b antibodies. results: the average platelet loss after the process of freezing and reconstitution was %. the amount of platelets and platelet concentration in unit was lower in cpp compared to fp, but high enough (amount /unit, concentration . /unit). both types of plts (either pcc or fp) maintained an acceptable ph during storage. swirl was on value in fp and on value in cpp. the average plasma content in fp was % compare to . % in cpp after reconstitution. measured titres of igm anti-a and anti-b antibodies were very low ( - : ). cpp had faster clot initiation (rotem clotting time (ct) in cpp . s, fp . s). cpp contributes to a sufficient clot (rotem maximum clot firmness (mcf) in cpp . mm, fp . mm). summary/conclusions: our results shows, that cpp have higher procoagulation activity and simultaneously lower clot firmness. thawing and reconstitution of platelets are easy and fast processes if platelet additive solution is used. this method helps to increase the availability of platelets in emergency medicine. low plasma content in cpp enables their use as washed platelet product in specific groups of patients. methods: after donation, the whole blood was stored in room temperature overnight before separating next morning by reveos â system. seven abo compatible ipus, each with a target volume of ml, were selected and then they were connected to the pooling set provided by terumo bct. prior to the pooling of ipus, ml of additive solution (t-pas+ provided by terumo bct) was added and distributed evenly between the ipu bags. the pooling set was then kept h on bench in room temperature followed by h on agitator at ae °c. after filtration, the pool might be manually adjusted if its volume exceeded the maximum of ml to meet the requirements by the intercept tm blood system. the final products were two pathogen-reduced platelet units with a shelf life of days. results: during validation of the method, pathogen-reduced platelet units were controlled, in addition to the platelet count, for ph, glucose, po , pco and lactate on day , and of storage. the platelet count was . ae per unit on day . the ph value was . on day , . on day , and . on day . the glucose concentration decreased from . to . and . mmol/l on day , and , respectively. the mean po level was . , . and . kpa while the mean pco was . , . and . kpa and the lactate concentration was . , . and . mmol/l on day , and , respectively. since routine implementation of the method in april , regular quality controls showed an average of platelet count of . ae (n = ) with a volume of ae ml per unit. summary/conclusions: the validation of the method and the following two years of experience in routine shows that the pooling of ipus processed in reveos â system meet the requirements needed for intercept tm ds processing set for pathogen reduction of platelets. the results from the quality controls of the final platelet units were in accordance with the local and eu guidelines. methods: data was analyzed from published and unpublished clinical studies that performed both primary and secondary testing of platelets using the bta system. the studies included apheresis and whole blood derived buffy coat platelets and tested - ml sample volume per culture bottle. the studies classified results based on aabb bulletin - definitions with some modifications. the following assumptions were made including: • data was summarized as total number of positive tests, observed by the total number of tests performed on each day post collection; • it was assumed that one test was performed per platelet unit; • all units eligible for secondary testing were negative by the primary test the data needed to demonstrate a benefit for the use of the bta d systems for detecting contamination that was not revealed by previous bacterial testing as well as clinical specificity. results: a total of , platelet units from the studies where secondary testing of platelets was performed were analyzed. platelets were tested on day , , or ≥ , and represented . %, %, and % of the units tested, respectively. true positives were detected in platelet units representing . % of the total platelets tested. the majority were reported from platelets tested on day ≥ with a total of . data showed the bta d system used for secondary testing detects the most prevalent contaminates reported, staphylococcus spp., in ≤ h with the majority detected in ≤ h after incubation, allowing for interdiction of the units prior to transfusion. instrument specificity was reported in of the studies for platelets tested at days and ≥ days with a total false positive rate of . % (range of - . %). instrument sensitivity when used for secondary testing could not be determined since subculture of negative bottles is not performed during routine use. during previous validation testing of lrap and lrwbpc, , culture bottles were confirmed true negatives by subculture. summary/conclusions: data from the studies that tested platelets at to days post collection provided evidence that the bta d with bpa & bpn detects contaminants missed in previously tested platelet units. the data supports that the bact/alert d system is an effective safety measure for secondary testing of platelet products to extend platelet dating beyond day and up to day when testing is performed using the test parameters described in the bpa and bpn bottle ifus and according to the fda draft guidance. background: magnetic nanoparticles have recently shown great potential in nonradioactive labeling of platelets. platelet labeling efficiency is enhanced when particles are conjugated with proteins like human serum albumin (hsa). however, the optimal hsa density coated on particles and the uptake mechanism of single particles in platelets remain unclear. aims: we characterize the interaction between single particles and platelets and determine the optimal hsa amount required to coat particles. methods: ferucarbotran iron oxide nanoparticles were coated with hsa in different amounts ( . - mg/ml) and we confirmed successful hsa coating by addition of a crosslinking hsa antibody (dynamic light scattering). we labeled platelets from pooled platelet concentrates with mm ferucarbotran coated nanoparticles and analyzed labeled platelets for iron content (atomic absorption spectroscopy) and particle localization (transmission electron microscopy). single-molecule force spectroscopy was used to determine binding forces of nanoparticles to platelet compartments. we applied hsa-particles via linkers of different length (i.e. short~ nm, medium nm and long~ nm) on the cantilever tip and let them interact with a platelet provided on a collagen surface. after interaction we determined the rupture force required for platelet retrieval. results: the iron content per platelet reached a maximum at . - . mg/ml hsa coated particles with . ae . and . ae . pg/platelet, respectively. however, the . mg/ml hsa coating resulted in~ -fold higher binding affinity to platelets than particles coated with . mg/ml hsa. depending on peg length between tip and particle, particles interacted differently with platelets as shown by one, two or three force distributions of , , and pn, which correspond up to three different binding pathways, respectively. the results indicate that a particle can interact with three targets including platelet membrane, open canalicular system, and platelet granules. summary/conclusions: our results reveal mechanism of platelet-particle interaction on a single particle level and provide an optimal hsa concentration coated on particles to gain maximal platelet labeling efficiency. labelling of platelets by magnetic nanoparticles may substitute radioactive labeling. results: the activation/lesions on total platelets and small and medium-sized platelets platelet population was detected on storage day , by the increased expression of cd . the percentage of cd -positive cells among the population of large platelets did not change during storage. on the day , increased expression of cd b and cd p was detected, but only on large platelets. small and medium-sized platelets had increased cd p expression only on day . the expression of cd a on total platelets increased significantly on day , and stayed unchanged until day . the same pattern of cd a expression was detected for small and medium-sized platelets, whereas on large platelets the expression continued to increase until the end of storage. a decreased percentage of cd -positive cells was detected among the total platelets and populations of medium-sized and large platelets. the storage induced externalization of phosphatidylserine on total platelets or on any of the platelet populations was not detected. the levels of tgf-b and p-selectin in the pc-bc supernatants were unchanged during the -day storage period. increased annexin and pf concentrations were detected on day . the concentration of b-tg increased on day of storage, and continued to rise until the end of storage. summary/conclusions: the evaluation of expression of activation markers on different platelet populations could be used as an additional analysis in quality control of platelet concentrates, and in the assessment of novel approaches to platelet concentrate manipulation i.e. for testing new additive solutions, cryoconservation protocols, and cryoprotectants. background: the primary goal of autologous blood transfusion is to reduce the risks related to allogeneic blood transfusion, including transfusion-associated graft-versus-host disease (ta-gvhd). although downward trends in rates of autologous blood transfusion have been reported in europe and the americas, it still plays a role in eliminating risks related to allogeneic blood transfusion in japan, especially ta-gvhd. since february , the transfusion service in our hospital has managed autologous blood conservation techniques and helped to prevent mistransfusion by employing a bar code-based electronic identification system. aims: the objective of this study was to determine the use of types of autologous blood components in a single institution over an approximately -year period. methods: between february and december , we retrospectively analyzed autologous blood transfusion, including perioperative autologous cell salvage (pacs), pre-operative autologous blood donation (pad), and acute normovolemic hemodilution (anh). we investigated the use of autologous blood components and the rate of complying with electronic pre-transfusion check at the bedside in the operating rooms. we also determined the adverse reactions to autologous blood transfusion, which were categorized according to the definitions proposed by the international society of blood transfusion (isbt) working party on haemovigilance. results: a total of , patients ( % of whom received operations) received blood transfusion, of which , ( %) received autologous blood transfusion alone, , ( %) both autologous and allogeneic blood transfusions, and , ( %) allogeneic blood transfusion alone. the rate of autologous blood transfusion among patients who received blood transfusion was %. pacs units were transfused to , patients ( %), pad units to , patients ( %), and anh units to patients ( %), and multiple blood conservation techniques were used for one patient. the overall compliance rate with electronic pre-transfusion check at the bedside in autologous blood components was . %. adverse reactions were observed only with pad transfusion and not pacs nor anh. the number and rate of adverse reactions to pad transfusion were and . %, respectively, of which the most common was febrile non-hemolytic transfusion reaction at ( %), followed by allergic reactions at ( %), and hypotensive transfusion reaction at ( %). the severity of adverse reactions to pad transfusion was grade (non-severe) in all cases, and no serious adverse reactions were observed. among pad units, the rate of adverse reactions to whole blood pad units was . %, that to frozen pad units was . %, and that to autologous ffp units was . %. summary/conclusions: our observations indicate that the rate of autologous blood transfusion among patients who received blood transfusion was %, when all types of autologous blood conservation techniques were included. to accurately determine the rate of autologous blood transfusion in a hospital, it may be better for the transfusion service to manage all types of autologous blood conservation techniques. they are now clinically available as a blood product. the residual plasma level of this product, which is prepared using the automated cell processor acp (haemonetics corp.), is approximately %. recently, a retrospective multicenter study was reported that this product was effective and safety for prevention of recurrent or severe transfusion reactions. plt products are generally stored with continuous agitation to maintain their quality. the interrupted agitation of plt suspended in additive solution (plasma carryover: - %) for up to h was previously found to have only a slight impact on in vitro plt properties. however, in some small hospitals with no agitator, if the initiation of transfusion is delayed by an emergent change in a patient's condition, the interruption of agitation may be prolonged. aims: the aim of this study was to evaluate the effects the interruption of agitation for h (shelf life of wpc in japan) on the in vitro qualities of plt. methods: leukoreduced apheresis platelet concentrates in % plasma were washed on day one after blood collection using the automated closed-system cell processor acp (n = ). wpc, which were rested h after preparing, were divided equally into control and test units using polyolefin containers on day one. control units were agitated from days one to seven. test units were stored without agitation from days one to three, and agitation was subsequently performed until day seven. both units were stored at - °c. in vitro plt quality was examined on days one, three, four and seven. results: the plt concentration of prepared wpc was . ae . ( /l) and the volumes of the control and test units after the division were ae and ae (ml). the ph values of the test units on day three were lower than those of the control units; however, the ph of both units were maintained at higher than . during the seven-day storage period. swirling was well maintained and no clumping was visible in both units during storage period. no significant differences were observed in plts concentrates, mpv, hsr, aggregation response. the pco , po , bicarbonate, glucose and lactate mean values of test units were slightly lower or higher than those of the control units on days three or four. the levels of cd p expression were significantly higher in the test units than in the control units on days three ( . % ae . vs . ae . , p < . ); however, this difference decreased in a time-dependent manner after agitation resumed. the levels of cd b expression of test units were relatively lower than those of the control units until day seven, but no significant difference between the two units. background: monitoring residual white blood cells (rwbcs) is a requirement for quality monitoring (qm) the production of leucocyte depleted blood components. although flow cytometry is widely used for monitoring rwbc, there are no widely accepted methods to accurately and consistently measure rrbcs in blood components. sysmex have developed a novel algorithm, termed the blood bank (bb) mode for their xn-series of haematology analysers which is specifically designed to quantitate the levels of rwbcs and rrbcs in blood components. aims: we have previously assessed the linearity, accuracy and reproducibility of the bb mode on spiked samples in an r+d lab. we sought to further assess the performance of the bb software in a routine, high throughput blood component manufacturing department. methods: units of plasma, platelets (pcs) and red cell concentrates (rccs) were produced according to standard uk specifications within nhs blood and transplant (nhsbt). qm of residual cells was tested using the bb mode whilst rwbc was additionally analysed by flow cytometry using bd leucocount kit. results: during a -month field trial over , data points were collected representing all types of manufactured component. for some pcs, bb mode results from some sample tubes that did not contain edta gave very high rwbc values, indicating a potential large number of ld failures. the results were significantly different from those obtained from pcs using edta samples (p < . ) which did not show the same high values. for rccs or plasma, the range of results from plain and edta tubes were not significantly different (p ≥ . ). the analyses of ld platelet and plasma concentrates by either bb mode or flow cytometry both show more than > % of ld components have less than rwbc/unit. for ld failures (n = ) there was a good correlation (r = . ) between flow cytometry and bb mode measurements. spiking studies suggested that the limits of detection and quantitation of rrbc were around and rrbc/ll respectively. residual red cell counts from manufactured components showed a wide variation in their numbers between units. as expected platelet production methods also showed a significant difference (p < . ) in rrbc contamination, with lower levels in apheresis platelets (median = rbc/ll, n = ) compared to those produced from buffy coats (median = rbc/ll, n = ) in our hands, although the time taken to analyse samples is similar for flow cytometry and bb mode, considerable time can be saved on manual handling and the processing of samples for flow cytometry (approximately - h for - samples). summary/conclusions: we have been able to embed the sysmex bb mode into a routine production environment and confirm that its performance in spiked samples is mirrored in routine use. for platelets, sample collection in edta is essential. the bb mode offers an opportunity to reduce operator time compared to flow cytometry whilst gaining additional information on rrbc. abstract withdrawn. results: in our experiment, the typical size of a spectrin matrix section (l) was to nm (without oxidation). the heights (h) of dips were to nm. due to oxidation, the junctional complexes between spectrin and membrane proteins can rupture. only % to % of the spectrin surface has the same structure as in the control group. the values of l and h vary significantly depending on the intensity and time of exposure. we observed significant changes in the spectrin matrix after exposure to uv radiation in a model experiment. the local topological defects in the membrane arise from the action of oxidizing agents on the red blood cells. the mechanism of their appearance is connected mainly with the distortion of the spectrin matrix. as a result of oxidation processes, the spectrin molecules can be damaged. there is a transformation of tetramers to dimers. additionally, it can be easily seen with the afm, that spectrin network structure was essentially destroyed. most parts of the spectrin matrix have damaged structures with mesh breaks and dips after uv irradiation. also the results of network distortions in response to temperature changes were obtained. there are presented preliminary results of spectrin matrix change during long-term storage of prbc. summary/conclusions: atomic force microscopy in direct biophysics experiment allows to observe and to quantitatively measure the disturbances in the spectrin matrix nanostructure in response to oxidation processes in rbcs. these studies are important for the fundamental research of interactions of rbcs on the molecular level during redox processes and the consequences to their structure and function on the cellular level. this is important for the advancement of transfusion medicine, intensive care medicine, and molecular and radiation biology. methods: blood samples were taken from donors during a prophylactic examination and collected with edta-filled microvettes (sarstedt ag and co., germany). all experiments were carried out in accordance with the institute guidelines and regulations. the polylysine-coated glass was used to perform the sedimentation method for formation of native rbc smears. it is important that any fixatives weren't used. the stiffness of rbc membranes was studied in native rbcs (control) and native cells after the application of modifiers: glutaraldehyde, hemin, zn + (heavy metal ions). local stiffness was studied by atomic force spectroscopy (afs) (ntegra prima, (nt-mdt, russian federation). results: experimental kinetic curves i(z) were measured. nonlinear fitting method was used to determine the young's modulus. the experimental dependences of membrane bending were approximated by the hertz model to a depth up to nm. the young's modulus e = ae kpa for control rbc. it was shown that some natural oxidants (hemin), membrane fixatives (glutaraldehyde) modifiers, heavy metal ions (zn + ) significantly increased the absolute value of the young's modulus of rbc membranes up - times. the biophysical parameter hertz depth (h hz ) was determined for each curve. under the influence of modifiers the hertz depth h hz was changed from nm to nm. there are presented preliminary results during long-term storage of blood. summary/conclusions: the blood rheology is determined by rbc deformability, associated with membrane stiffness. the young's modulus can be used as a quantitative criterion to estimate the membrane state of a native cell. the results of the work can be used in clinical practice, in assessment of the quality of donor blood during storage before transfusion, in biophysical studies of rbc state. abstract withdrawn. immunohemotherapy, centro hospitalar universit ario são joão, epe, porto, portugal background: transfusion of blood and blood components is an essential resource in modern medicine. a proper use of human blood, being an irreplaceable resource, is necessary in order to achieve minimal wastage. blood wastage may occur for a number of reasons, like expiry date, haemolysis, seroreactivity or low volume. monitoring wastage of blood product during collection, testing and processing of blood is used as a quality indicator. aims: to determine the annual rate of discarded blood components due to expiry date in a portuguese university hospital blood bank (bb) from january to december , in order to implement appropriate measures to minimize the number of discarded blood to a reasonable rate. methods: we retrospectively analysed the rates of blood components discarded after meeting their expiry date of a portuguese university hospital blood bank from january st to december st . results: a total of , whole blood units and , apheresis platelets were collected during the study period. of the , blood components (packed red cells, whole blood pooled platelets and apheresis platelets) prepared during the study period, a total of , ( . %) blood components were discarded, of those . % due to expiry date. the rate of discarded packed red cells, according to this component production, decreased considerably over the years, in was . %, in was . % and . % in . similar tendency was shown in the pooled platelets for consecutive years with . % ( ) and . % ( ), but with an increase in ( . %). the rate of apheresis platelets had a more variable behaviour from to with rates of . %, . %, and . % respectively. summary/conclusions: blood transfusion is an essential part of patient care. for this reason, the implementation of a quality system and continuous evaluation of all activities of the bb can help to achieve maximum quantity and quality of safe blood. we identified that date expiry was the main reason of discarded blood components, although there was a significant decrease in the rate of discarded packed red cells over these three years. properly implemented blood transfusion policies, donor screening and training staff as well as implementation of automation also helps to improve the process, reducing the discarding rates of blood and blood component. background: storage performance of platelets (plt) is associated with age of the donor. the risk for plt with poor storage performance, characterized by high lactate production and rapid acidification of a plt concentrate (pc), shows a positive correlation with age. we wished to explore whether high lactate production was associated with donor health issues. aims: to investigate high lactate production by stored platelets in relationship to donor health. methods: single-donor pc were collected by apheresis or prepared from buffy coat and donors were evaluated who could be marked as 'rapid acidifiers'. in total, apheresis pcs and pcs from whole blood were included in four studies. information about donor health was obtained either from the blood bank information system or using questionnaires. in some donors, the lipid profile was measured from plasma, and the diabetes marker hba c from red cells. triglyceride levels > . mmol/l and hba c levels > mmol/mol were defined as high. results: twenty two percent ( / ) of the donors were marked as 'rapid acidifiers' and % ( / ) of these donors had health issues. 'rapid acidifiers' were of age , - (median, range) years. three groups of donors can be distinguished: a) donors affected by metabolic syndrome, prediabetes and type diabetes, indicated by high cholesterol and/or triglycerides, high hba c and/or the use of medication to treat diabetes. b) donors affected by vascular diseases who reported or used medication to treat high blood pressure. c) "other" donors who used other medication to treat various other conditions. the remaining 'rapid acidifiers' ( %) did not have high triglyceride or hba c levels and did not report health issues. summary/conclusions: pcs with rapid acidification by high lactate production are mainly collected from older donors with health issues. we postulate that high lactate production by stored plts is associated with health issues, and we will combine detailed donor information (health and behavior) with in vitro quality for a significant number of donations. background: the development of applied biotechnologies requires a search and creation of new methods of cells' functional completeness analysis. the instrumental assessment of platelets quality for the selection of the most effective donors, quality assessment of platelet concentrates for short and long-term storage and for the selection of platelets for cryopreservation is in demand in blood service. assessment of platelets morphofunctional status is possible using morphological studies of various platelet granules fractions (makarov, med. alfavit, ). among the biologically complete platelets there is a special population of cells, the so-called granule-rich platelets (grp). these cells contain the largest number of cytoplasmic granules (more than visually distinct granules). it is established that grp have increased viability and functional activity. earlier we found a correlation between the grp level in blood plasma and shift of the redox potential value in blood plasma after cryodestruction of platelets (tsivadze et al., doklady physical chemistry, ). it was suggested that the shift of the redox potential may be partly due to the release of the low molecular weight antioxidants contained in functionally complete platelets outside the cell. in turn, the concentration of low molecular weight antioxidants can be estimated using the cyclic voltammetry. aims: the aim of the study was to estimate of cyclic voltammetry method possibilities for quality assessment of platelets. methods: the functionality of platelets was examined in platelet concentrate (pc) obtained by apheresis ( . ae . /ll). voltammetric analysis in pc before and after the platelets cryodestruction was carried out on platinum electrode in the potential range from À mv to + mv using a potentiostat ipc pro l and saturated ag/agcl electrode as reference. for the morphofunctional analysis platelets were vitally stained with fluorochrome stains trypaflavin and acridine orange. microscopic examination of platelets was carried out using confocal microscope nikon eclipse i. the following parameters were evaluated: concentration and percentage of platelets with granules and concentration and percentage of grp. results: voltammetric studies in pc show that there are two oxidation peaks of low molecular weight antioxidants on voltammogram at potentials + mv and + mv. analysis of pc before and after the cells cryodestruction showed that changes in the height of oxidation peaks occur, indicating an increase of antioxidant content in blood plasma. at the same time a correlation between the changes in the height of oxidation peaks and the grp content in the sample was found. in samples with reduced initial grp content (less than %) after the cells cryodestruction significant changes in the height of oxidation peaks were not observed, regardless of the total number of cells in the pc. summary/conclusions: in conclusion, voltammetric analysis allows to indirectly estimate the population of functionally active platelets that in combination with other methods of analysis can serve to assess the quality of platelet products. background: determination of hemoglobin derivatives in blood is one of the most important studies in clinical laboratory diagnostics, especially during the storage of donor blood and its transfusion. concentration of hemoglobin derivatives can be changed during redox process. aims: to show the possibility of using non-linear fitting method to calculate concentrations of hemoglobin derivatives during reduction-oxidation processes. methods: for this we performed model biophysical experiment, in vitro. blood samples were collected into edta microvettes from healthy donors (sarstedt ag and co., germany) during prophylactic examinations. all the donors gave their consent to participate in the study. a suspension of erythrocytes was prepared in pbs buffer with ph . . we used ultraviolet (uv) irradiation of blood or nano as oxidizing agent. the drug cytoflavin (stpf "polisan", russian federation) was used as an antioxidant. in our study we used digital spectrophotometer (unico , usa) to measure the absorption and scattering of light ( . nm step). the method of nonlinear fitting was used to find the concentrations of hemoglobin derivatives. the empirical spectrum d l (k) exp was approximated by the theoretical curve d l (k l ) theor , which fits the experimental curve in the best way. under approximation the light absorption by different hemoglobin derivatives was considered in model. simultaneously effects of rayleigh light scattering on structures with size d<< k (coefficient s) and light scattering on particles with size d≥ k (coefficient k) were taken into account: d l (k l ) theor = e hbo ,l c hbo l+ e hb,l c hb l+ e methb,l c methb l+ e hbno,l c hbno l+ e methbno -,l c methbno -l+ e methbno,l c methbno l+k+s/k l ( ), where e h,l is the molar absorption coefficient for each hb h derivative at given wavelengths k l , c h is the concentration of the derivatives hb h , l is the thickness of the solution layer, d l (k l ) is the optical density of the substance, k and s are the parameters of the model. results: we determined the concentrations of hemoglobin derivatives without any additional chemicals in blood. there were measured experimental spectra for different agents action on blood. it was shown that concentration of methb increased after uv irradiation and nano action (up to %). there were calculated c h for each hb h derivative. it was established that theoretical curves coincide with experimental data with good accuracy (r = . ). incubation of rbcs with cytoflavin leads to reduction of methb to hbo . summary/conclusions: the determination of hemoglobin derivative concentrations by the method of nonlinear fitting (without adding special chemical agents to blood) can be used for measurement of carboxyhemoglobin in blood during toxic state of organism. also it is important for assessment of rbcs quality before blood transfusion. background: the use of in line leukoreduction filters have been highly expanded in iranian blood transfusion centers within the last decade in order to provide sufficient leukoreduced blood fractions from healthy safe frequent blood donations to be supplied to the leukocyte sensitive patients. leukoflex lcr , the dominant brand of such filters procured by iranian blood transfusion organization, is the most updated generation of the filters used around the world. aims: in this study, it is tried to recover the trapped leukocytes from this novel filter by different buffering systems and having optimized the elution mode, the cell differential of the viable recovered white blood cells were determined by flow cytometry. methods: having passed the routine virological tests, eight leukoflex lcr leukoreduction filters freshly used in tehran blood transfusion center were daily collected and each were back flushed by a self-designed mechanical system (a peristaltic pump, a triple junction with regulator part and an air pump) using various conditions and additives for pbs buffer at different phs in order to find the highest recovery yield for leukocytes. the optimized elute was characterized by flow cytometry for subcellular profile to be determined. results: it was illustrated that a system consisting of pbs (without cacl and mgcl ) in ph . containing mm edta and %(w/w) dextran without additive amounts of triton x was the most optimized buffering system for lcr filter back flushing. total cell content was also determined as . * granulocytes, . * lymphocytes and . * monocytes using auto hemoanalysis and flow cytometric methods. summary/conclusions: in addition to partly compensating of the overhead expenses inflicted by application of leukoreduction filters on healthcare system, the results will assist blood organization system to be more classified in rational profile design, future cell therapy strategies and exceptional blood management. also, the recovered cells could be of significance in stem cell science, cellular interaction studies as well as novel molecular developments in drug discovery. vox sanguinis ( ) results: three lines of strategy are in place to pursue self-sufficiency of the largest number of pdmps. first strategy line: maximizing the yield of driving proteins, represented by immunoglobulins (ig) and albumin; this was assured by csl behring with a yield of . g ig ( % intravenous -privigenand % subcutaneous -hizentra) and g albumin (alburex) per kg plasma fractionated, corresponding to . g ig and . . g albumin; based on present demand, this represents % and % self-sufficiency, respectively, for naip regions. second strategy line: ensuring other products from plasma fractionation; the fractionator granted . g fibrinogen (riastap) and . . iu vwf/fviii (haemate p) per year, which corresponds to the present demand of naip regions for both products, but it is under the full potential of plasma, thus providing a high margin of safety in case of increased demand (now the case of fibrinogen). third strategy line: exchanging cryoprecipitate, fibrinogen and vwf with italian regions whose plasma is fractionated by other companies to obtain prothrombin complex concentrates (pcc -kedcom) and antithrombin (atked) as to satisfy naip regions demand; this strategy allowed a supply of . . iu at and . . iu pcc, capable of ensuring self-sufficiency for naip regions until . summary/conclusions: in italy, differentiation of plasma contract manufacturing among companies with different portfolios allowed naip regions to obtain a significant contribution to self-sufficiency from vnrd plasma for a variety of pdmps by different and complementary strategies consisting in maximizing the yield and the portfolio of proteins from the fractionator and exchanging products among regions for other pdmps at high demand but not included in the portfolio of a single fractionator. plasma check system: a valuable tool for plasma freezing validation and monitoring. background: the validation of plasma freezing processes may result problematic in the monitoring/control of critical process parameters (cpp). in in italy , litres of plasma were produced and frozen. aims: in order to assist plasma freezing validation and cpp monitoring, of the italian bes performing plasma freezing utilize the plasma check system (pcs), a system able to record, store and certify the temperature (t) detected at the core of "surrogate" bags during the entire freezing session, consistently with gmp requirements. pcs is patented and commercialized by expertmed srl, verona, italy (http://www.expertmed.it). methods: pcs consists of parts: a) "surrogate" bags (check-bags) of and ml corresponding to the average standard volumes of the real products, containing a fluid validated to simulate the thermal behaviour of plasma; b) a mobile probe (cryo-med) positionable at the core of the check-bags; c) a dedicated software (memo-track). plasma freezing session data are tracked via barcode/rfid and can be consulted by the pcs that associates blast freezer code, operator code, cryo-med and check-bag. data on plasma freezing are stored in a shared folder and transferred to the be information system. the pcs can also be used to check and monitor the out-of-storage variations of core t of frozen plasma unit, i.e. during labelling and packaging procedures, thus allowing to establish optimal timeframes and operations and suitably validate these procedures. in the period - , at the pievesestina be of emilia romagna region , plasma units were frozen so as to allow complete freezing within to a temperature below À °c, in , freezing sessions, using the pcs both for process validation, change control and for the systematic monitoring of core t at each freezing session. furthermore, at the bologna be tests on the out-of-storage conditions of plasma units were carried out to revalidate the procedures of labelling and packaging. results: out of , freezing sessions carried out at the pievesestina be, ( . %) were detected to fail to reach À °c at the core of the check-bags within . of the latter, in most cases ( %) a technical error in the activation of the cryo-med was identified. in addition, the pcs was systematically utilized for periodical revalidation of the freezing procedures. the tests performed at the bologna be to validate the out-of-storage procedures of frozen plasma labelling and packaging allowed to modify the operating procedures in place so as to establish optimal timeframes and operations. this prompted corrective actions regarding: i) number of units to be taken out of storage sites at each labelling session (< units), ii) labelling time (< ), iii) optimal storage t (À °c vs. À °c), iv) optimal time between two openings of storage sites (> ). summary/conclusions: the pcs is a valuable system for plasma freezing validation and monitoring, as well as to perform monitoring and control of the whole pathway of frozen plasma in the be. it is a technologically advanced, easy-to-use and costeffective tool that can efficiently replace other traditional methods commonly used for the above-mentioned purposes. assessment of blood group matching quality using six sigma metrics background: six-sigma metrics provides a general methodology to evaluate a process performance on a sigma scale. implementation of six-sigma for quality assurance can benefit the health care sectors. one of the most important health care sectors is blood transfusion service. for that reason, maintaining a high quality in blood transfusion service is required. pathogen in activated plasma is one of the main products that are provided by the blood transfusion service. the process of producing pathogen inactivated plasma involves blood group matching step. the quality of this blood group matching is extremely significant for the delivery of plasma that satisfies the recipient need. aims: the aim of this study is to assess the quality of blood group matching of pooled plasma units using six sigma metrics, and to clarify the potential implementation of six sigma metrics as a quality management tool. methods: this retrospective study was conducted in the component preparation lab of kuwait central blood bank. the twelve months (january -december ) data of pooled fresh frozen plasma units were recruited and examined. the data was separated to data without double check ( months) and data with double check ( months). data statistics and analysis were conducted by the use of six sigma metrics. results: in a sample size of from the first six months a mismatch was found which equals dpmo and . sigma metric. and in a sample size of from the second six months a mismatch was found which equals dpmo and . sigma metric. out of the whole pooled units were found to be mismatched. some of which were found to be discarded as abo discrepancy, broken, or expired. other was still available in the system, while the rest of the mismatched units were issued. summary/conclusions: using the six sigma principle the study presents a successful assessment of blood group matching quality. as a . sigma metrics obtained from the first months, were shifted to a sigma metrics of . in the second months, after the addition of a double-checking step to the blood group matching of pooled plasma process. the implementation of these metrics in our laboratory quality management has been shown to be very beneficial. in which six-sigma metrics were able to clarify the reduction in blood group matching errors. although six-sigma benefits in major quality improvements and helps to reach an error free laboratory services, yet it presents a new challenge to laboratory practitioners. currently, the hemophilia a patients treated with factor viii concentrated as the first line of therapy but it is more expensive and the supply is not sufficient so for now they have not used factor viii concentrate as prophylaxis therapy. for some cases, hemophilia patients in indonesia depend on subsidy from the world federation of hemophilia. the first handicapped concentrated case is just for therapy not for prophylaxis. big blood centers in indonesia produce routinely fresh frozen plasma (ffp) and cryoprecipitate-anti hemophilic factor (ahf) as replacement therapy for hemophilia a, but its content and safety of factor viii from ml ffp need to be improved. nowadays, there is an available kit for producing minipool cryoprecipitate (mc) that has better safety and quality but it is available as liquid products, stored in very strict and specific temperature (À °c). prophylaxis therapy for hemophilia patients needs a stable product, easy to use and convenient treatment for patients. aims: to analyze the content and safety of f viii with minipool cryoprecipitate (mc) and lyophilized mc for home therapy. methods: we produced mc; mc as the control, mc were lyophilized with excipient and mc without excipient. we analyzed the number of factor viii, the safety, and stability. we count the erythrocyte, leukocyte and platelet residual in mc using flow cytometry. we also measure the ph, osmolality, solubility to learn its stability after storage at days at room temperature ( - °c) and blood bank refrigerator temperature ( - °c) at central blood transfusion services (cbts). results: we found the content f viii with excipient is higher ( . iu/ml) than without excipient ( . iu/ml) and the storage at blood bank refrigerator ( - °c) is better than at room temperature ( - °c) . in both group, there were no residual cells and bacterial found in mc. no significant difference in the ph, osmolality and solubility in both groups. summary/conclusions: the lyophilized mc with excipient stored at blood bank temperature ( - °c) is better than room temperature. this experiment will be continued to know its stability in extended storage time. background: peptic ulcer disease (pud) is a multifactorial and complex disease, and it affects a wide range of people in the world. however, a perfect therapy for pud has not yet been available at present. therefore, we provided a novel therapeutic approach for pud patients and observed its effect in this study. aims: we provided a novel therapeutic approach for pud patients and observed its effect in this study. methods: in this randomized controlled trial, pud patients residing in chongqing were enrolled from to . they were randomly assigned to two groups: (a) a control group used only rabeprazole, and (b) a platelet-rich plasma (prp) group that received a combined therapy of autologous platelet-rich plasma (aprp) and rabeprazole. the aggregation rate of aprp was measured via aggregation remote analyzer module. the therapeutic effect was assessed via the ulcer size and the symptom score. all data were recorded and analyzed statistically using spss. results: a total of patients were included ( patients as control group) and ( patients as prp group) in the analysis. we found that the aggregation rate of aprp is not affected in ph . after treatment with pepsin. our results showed that there were no significant differences between the prp group and control group before the treatment, and there was also no significant difference in healing time between the two groups in different variables. however, regression analysis revealed that the healing time was . d less in the prp group than in the control group, and the patients with higher symptom scores in the initial examination need more time to heal in treatment. summary/conclusions: this study showed an encouraging preliminary result that aprp has a positive result in the peptic ulcer patients, and it seems to be a better choice for refractory pud patients. despite the further follow-up studies are needed to determine the duration of efficacy of aprp, the approach will be helpful for improving the pud treatment in clinical. background: the croatian institute of transfusion medicine (citm) collects, produces and distributes blood components in an area of . million habitants. annually, it collects about , whole blood and , apheresis donations. platelet concentrates (pcs) are more inclined to bacterial contamination due to storage conditions that favor bacterial replication. the citm decided to evaluate the mirasol pathogen reduction technology (prt) system as it offers the possibility to work with a non-toxic, non-mutagenic compound that upon uv illumination induce nucleic acid damage, reducing the risk of septic transfusion. aims: the study objective was to evaluate quality of pcs treated with the mirasol prt system for platelets and stored in tpas+ for days at °c on a platelet shaker. methods: pcs were produced according to the citm's s.o.p., either through pooling of bc with tpas+, "wbd", or through apheresis collection using two devices: the fresenius amicus, "ad" and haemonetics mcs+ system, "mcd". pcs were stored in % plasma and % pas and mcd were subsequently evaluated also in % of plasma and % pas. identical pcs were produced with a pool-split protocol to be prt-treated or serve as untreated control. pcs were treated with the mirasol system according to manufacturer's instructions. qc parameters, such as yield, ph and swirl were measured at days , and . bacteria sterility test was performed at day for a sample of all treated platelets. protein content of pcs produced routinely at the citm was determined to assess accuracy of plasma carry-over calculations for all processed pcs. results: mirasol-treated wbd (n = ) and ad pcs (n = ) stored in % plasma showed at day an average ph ≥ . ; swirl ≥ . and yield = . . their untreated counterparts showed average values for ph ≥ : , swirl ≥ . and yield . - . . mcd stored in % plasma (n = ) that underwent prt showed at day average values for ph = . , swirl = . and yield = . . control mcd showed average values for ph = . , swirl = . and yield = . . mcd stored in % plasma (n = ) that underwent prt showed average values for ph = . ., swirl = and yield = . . their untreated counterparts had average ph = . , swirl = . and yield = . . total protein content in pcs derived from wbd (n = ), ad (n = ) and mcd (n = ) was g/l, g/l and g/l, respectively. while the coefficient of variation of wbd and ad ranged from % to %, plasma products respectively, the one of mcd reached %. all prt-pcs were negative for bacterial growth at day . summary/conclusions: mirasol treated wbd and ad produced according to citm current s.o.p. were quite similar to untreated controls at expiry, on day and passed the requirements of the eu guidelines ( th edition). quality of mcd units met eu criteria at day ; swirl decreased significantly at day which might be explained by the variability in plasma content of mcs+ -derived platelets, challenging the accurate calculation of illumination index for the mirasol treatment. all mirasol treated pcs showed minimal platelet loss at the end of storage. as the implementation of pr had to be cost-neutral it could only be implemented for~ % of the annual produced buffy coat platelet concentrates (bcp) (~ . bcp/year) and required a change in the bcp production method. the primary aim of the implementation was to offer increased blood safety to our most vulnerable patients. the secondary aim was to ensure that we built-up enough routine experience with pr to enable us to quickly ramp-up the production of pr-bcp to % if there were an outbreak of an emergent pathogen in the madrid region. aims: to verify if we could produce~ % pr-bcp without increasing the overall production cost (opc) for bcp. also evaluate the impact of pr on overall scrap rates of bcp, outdate rates and usage of other safety measures. methods: we compared opc for bcp between the pre-pr period ( ) . this cost was offset by substituting a semi-automated production method for bcp, which was used in to produce . % of bcp-units. a manual double dose buffy coat production method (dd-bcp) in combination with pr enabled us to reduce the bcp-disposables cost by . %. despite the moves from a semi-automated to a manual production method the overall scrap rates during production decreased in by . %. the extension of max. storage time from to days for % of the bcp-units that were pr resulted in decreasing our overall outdating rates by % (versus ). this reduction in outdating rates reduced our opc in by . %. in we gamma-irradiated . % fewer bcp-units, but this had only a minimal impact on the opc. summary/conclusions: results of this study confirmed that we reached our initial objectives of producing~ % pr-bcp without increasing the overall production cost (opc) of bcp. it enabled us to offer increased blood safety to the most vulnerable patients. we built-up enough routine experience with pr so we could quickly rampup the production of pr-bcp to % if there were an outbreak of an emergent pathogen in the madrid region. background: irradiation of red cell units is undertaken to prevent transfusion-associated-graft-versus-host-disease (ta-gvhd) in immuno-compromised patients. while irradiators using radioactive c-ray sources are primarily found in blood establishments, they require regular recalibration and supplementary safety measures. xirradiation has been shown to have similar biological effectiveness to c-irradiation and does not require a radioactive source. there is international interest in moving away from gamma sources to reduce vulnerability to terrorism. although damaging, impacts of irradiation on red cells are well recognised. only a limited number of studies have compared red cell component quality following cand x-irradiation for both standard volume red cell concentrates (rcc) and neonatal red cell splits (rcs). aims: to compare the in vitro quality of rcc and rcs when subjected to cor xirradiation on day of storage then stored for a further days. rcs were also irradiated on day of storage as that is most common practice in nhs blood and transplant (nhsbt). methods: four rcc were pooled and split into arms on day of storage, with units in each arm. all units received an irradiation dose of . - . gy. two arms remained as standard volume rcc and were either cor x-irradiated on day of storage. the other two arms were both split into rcs on day of storage before being irradiated on day (early arm) or day (late arm) of storage. for each replicate in these arms, splits were c-irradiated and splits x-irradiated. all arms were tested a day prior to irradiation and , and days post-irradiation for red cell quality parameters: haemolysis, intracellular atp and , dpg, supernatant potassium, glucose and lactate, ph and red cell microvesicle release. the rcc arms were sampled over storage; while for the rcs arms, split was tested on each testing day post-irradiation. a -way anova was used to detect statistical differences over storage between cand x-irradiation for the same components. results: all components produced were within nhsbt specification for volume, haemoglobin and haematocrit. there were no significant differences in red cell in vitro quality parameters studied over storage between cand x-irradiated units, for standard volume arms or neonatal arms and whether rcs were irradiated early or late in storage. moreover, all arms were within haemolysis specification for the end of storage (> % of units with < . % haemolysis) and % of units had atp levels above the recommended minimum for acceptable post-transfusion survival ( . lmol/ghb). both haemolysis and potassium levels at the end of storage for the standard c-irradiated rcc were comparable to our laboratory's historic data for the same component. summary/conclusions: in summary, the storage quality of rcc and rcs post-xirradiation did not differ from c-irradiation in this study, providing reassurance that either method could be used in routine manufacturing. a pajares herraiz , c coello de portugal , m morales , f solano , c perez parrillas , a rodriguez hidalgo , t diaz rueda and m flores direccion, regional transfusion center toledo-guadalajara transfusion service, toledo hospital complex, toledo transfusion service, general university hospital of guadalajara, guadalajara transfusion service, hospital nuestra señora del prado de talavera de la reina, talavera regional transfusion center, regional transfusion center toledo-guadalajara, toledo, spain background: the regional transfusion center of toledo-guadalajara (rtc) manages the collection, processing and distribution of blood components for the hemotherapy area of castilla la mancha (spain) that serves general hospitals (hospital complex of toledo (hct), university general hospital of guadalajara (ughg) and hospital nuestra señora del prado de talavera (nspt)) and the needs of , inhabitants. by also managing the hct transfusion service, it facilitates the handling of stocks. since , rtc has initiated pathogen inactivation (pi) for a part of its platelet components(pc) with the intercept blood system (cerus) using a photochemical treatment with amotosalen and ultraviolet-a. this system allows the inactivation of a broad panel of pathogens and leukocytes, extending the shelf-life of the cp from to days. this affects the expiry and discards of this blood component, allows a better management of the inventory and has an influence on production costs. aims: the objective was to evaluate the influence of pi in the production of cp at rtc and the expiry in the hemotherapy area during the last years divided into four periods ( results: pc were predominantly obtained from whole blood collections with % of bc platelets/ % of apheresis platelets. % of the available bc were used in production for period a and % for periods b, c and d. after wastes of approximately . %, the distribution of pc was stable for the periods studied. pc were distributed for period a, pc for b, pc for c and pc for d. the % of pi platelets with -day shelf life available in the hospitals was limited to % during period a. it was then increased to . %, . % and % for periods b, c and d respectively. the percentage of wastes was stable at . - . % but the discards due to expiry went down from . % (period a) to stabilize at . % in periods b and c and . % in period d. in the general hospitals the expiry went down from % to . %(hct), . % to . % (ughg) and . % to . %(nspt) respectively. summary/conclusions: greater control of pc stocks through historical analysis and consumption projection, together with it tools and the use of pi pc with -day shelf life allowed reducing discards for expiry from . % to . % in the last period analyzed at rtc and the major hospitals of the hemotherapy area. this has a great value in cost-reduction and improves inventory management and the efficiency of the processes. background: blood centers are faced with many challenges including availability of concentrate platelets as well as ensuring highest quality of the product. overcoming the shortage of platelet apheresis by using pooled platelet derived from whole blood units separated using automated standardized system, which can assist blood banks to meet the increase demand in platelets. the pathogen inactivation (pi) technology can improve the quality of the product by mitigating the risk of transfusion-transmitted diseases (ttd) and residual white cells, resulting in minimizing non -hemolytic transfusion reactions. however, the pathogen inactivation treatment must not impact the platelet quality and functionality significantly, as well as the patient safety. aims: evaluate the quality of pooled platelets derived from whole blood (five interim platelet units), separated using reveos automated blood processing system (terumo bct), pooled in % donor plasma and pathogen inactivated by amotosalen/uva technology. methods: five interim platelet units (ipus) produced with reveos device (terumo bct) from single whole blood donations, were pooled with a platelet pooling set (terumo bct) and leucodepleted with a lrf-xl filter (haemonetics). thirty pools have been included in this study, the units were treated using a large volume cerus intercept processing set for platelets according to the manufacturer's instructions and stored until day . the swirling was determined by visual inspection. the volume and yield content were assessed preinactivation and after treatment by pathogen inactivation with a cell counter (dxh- , beckman coulter), rbc contamination was also measured preinactivation with a cell counter (beckman coulter), bacterial contamination was assessed by automated blood culture with a bact/ alert system (biomerieux). the ph of the platelet units was assessed with a phmeter (jenway), and residual amotosalen levels were assessed by an hplc assay. results: the impact of amotosalen/uva pathogen-inactivated pool platelet products quality were assessed. the pre and post-inactivation of the units showed a swirling score of - . the average volume per unit of the pre-inactivation was ml ( - ml) and post inactivation was ml ( - ml), with average volume loss during inactivation was ml ( - ml), corresponding to % ( - %). the average platelet yield per unit pre-inactivation was . ( . - . ) and post inactivation . ( . - . ) with an average platelet loss of % ( - %) . the average rbc contamination per unit ( . - . rbc/ml). the culture tests were negative, the average ph at day was . ( . - . ), average ph at day / was . ( . - . ). the average residual amotosalen concentration post treatment was . lm ( . - . lm). summary/conclusions: the quality of pathogen-inactivated pool platelets tested, met the criteria set by aabb guidelines. the volume and platelet loss were in acceptable range, in alignment with previously published data. a residual amotosalen concentration below lm is considered safe and acceptable by french and german authorities. the evaluated data support the reasonable assurance of quality and effectiveness of the device when used in accordance with indication for use. background: the implementation of a pathogen inactivation process (pi) allows the redesign of processes to obtaining safe blood components by reducing the need for additional testing for pathogens detection, minimizing the residual risks (such as the infectious window period for those pathogens that are detected as usual), eliminates the need for selective tests (eg cytomegalovirus serology test) and complements gamma irradiation given its ability to inactivate white blood cells. in addition, the routine implementation of pi reduces the incidence of bacterial infection in recipients of blood components and allows blood services to proactively protect the blood supply against future emerging infections. aims: to verify the functional integrity and viability of platelet concentrates after being inactivated of any pathogenic agent, to be used as safe and functional components for transfusions. methods: a total of independent platelet concentrates were studied. platelets are donated through a process called plateletpheresis according to the established norms, after the process, platelet concentrates were submitted to pi on the intercept blood system tm platform with uv-a illuminator; an immediate sampling of each donation of platelet concentrates was carried out taking a sample of ml pre-inactivation and another sample post-inactivation ( h after pi). the platelet viability of each sample was evaluated by demonstrating the cd p expression marker by flow cytometry. once processed, platelet concentrates were released as safe components for donation. compiled the experimental data of the platelet count with platelet activation marker with respect to the total platelet, a comparative, nonparametric test of wilcoxon was carried out between two measurements (pre vs post) and the platelet viability after pi was determined. results: a total of independent platelet concentrates were studied, where the average percentage of pre-inactivated platelets with expression of the cd p marker, was %, while the percentage of functional platelets post inactivation was %, this result only shows that the functionality of the platelets is not being altered after the inactivation process. the wilcoxon test confirms that there is no significant difference between platelet activity pre-and post-inactivation, with a % confidence level. summary/conclusions: the process of photochemical treatment with amotosalen hydrochloride and long-wavelength ultraviolet light (uva) applied to platelet concentrates provides functional products without alterations in platelet function to be transfused. background: treatment of platelet concentrates (pcs) with pathogen reduction technologies is widely implemented in blood establishments to reduce the risk of bacterial contamination and to face the presence of new emerging agents in blood components. aims: the reduction of antioxidant power (aop) could be a quality control test to prove the complete viro-inactivation treatment. this evaluation has the goal to study the feasibility of the method from "abonnenc et al., transfusion, " in another blood service, assessing the aop of platelet units treated by intercept technology. methods: the aop is expressed in edel value, one edel being equivalent to lmol/l ascorbic acid. repeatability, intermediate precision and accuracy were determined. linearity was evaluated using the linear regression and the calculation of pearson's coefficient (r²). limit of quantification (loq) was determined by measuring aop using nacl samples to define the background. roc curves were used to determine a threshold to discriminate pcs before and after treatment. a distinction was realized between men and women and between apheresis (a) pc and buffy coats (bc) pcs. a one-year evaluation was assessed on pcs before and after treatment on the routine production. results: the coefficient of variation for the repeatability was less than %. for the intermediate precision, the coefficient of variation was less than %, but for the pcs after treatment, this result rose up to %. the r² value for the linearity was . %. the detection limit corresponded to a result of edel and the loq (equal to xsd) is edel. concerning roc curves, the men apcs threshold was . edel compared to women apcs with . edel. the threshold for bcpc was edel. all of these results had % of specificity. below this threshold, intercept treatment was considered to be executed. about the one-year experience on routine pcs production, apcs ( women and men) and bcpcs were tested. all of the bcpcs and women apcs were under the threshold after treatment. concerning men apcs, . % of the pcs after treatment were not under the threshold. summary/conclusions: the device validation was satisfied. for the one-year evaluation and concerning men group apcs, the threshold found by abonnenc et al. was edel. our study showed a threshold with % specificity and % sensitivity at . edel which is much lower. specificity was favored compared to sensitivity but the analysis should be revised to adapt the threshold to get higher sensitivity. this can lead to reduce the non-conformity and allows measuring the aop only after treatment. for women, our threshold was found at . edel compared to . edel for abonnenc et al. concerning sex in apcs, results were statistically lower in women group than men group before and after treatment. and for bcpcs, the two populations (before and after treatment) were very distinguishable and our threshold ( edel) was lower than abonnenc threshold which was at . edel. in conclusion, edel threshold enables the segregation and depends on the preparation process adapted in each blood service. aims: this study has the goal of measuring antioxidant power (aop) level in plasma units treated by mb technology. the aim is to use such a test as a quality control assay for documenting the execution of pathogen inactivation treatments during the preparation of plasma units. methods: aop measurements were performed using a potentiostat electrochemical analyzer. a -ll volume of sample is deposited over the electrodes on a single-use microship. the aop is expressed in edel value, one edel being equivalent to lmol/l ascorbic acid and reflects the redox status of the plasma units. different protocols were established to understand the role of mb, the illumination and the filtration on the aop variation measure: ) complete treatment, ) plasmas with mb without illumination, ) plasmas without mb with illumination and ) plasmas without mb without illumination. ten dosages on men donor samples, except for protocol where n = , were realized during the viro-inactivation process, t corresponds to a dosage of plasmas before treatment, t the plasma after the mb dry tablet passage, t is the time after illumination and t corresponds to the final product (after filtration). results: in each protocol with mb, an increase was observed after addition of mb before illumination. after illumination, the edel values decreased for about less than %, which was expected because of the degradation of mb in its photoproducts during the illumination. in the series and , the illumination seemed to have an effect by itself, with or without mb because the aop increased. the final filtration has the goal to eliminate the residual mb and its photoproducts. after this step, the aop values fell down. the series was a confirmation of the efficacy of the filter to remove the mb as shown by the decreased aop in t ( ae edel at t and ae edel at t ). however, in the absence of mb (series and ), the results at t and t were not statistically different. summary/conclusions: the filtration decreases the aop rate, except when there was no mb. the results of non-complete viro-inactivation treatment allow concluding that the measure of aop rate may not indicate that the treatment was completed or not since significant differences before and after treatments were found in the non-complete treatment series. vox sanguinis ( ) background: the intercept blood system (ibs), a photochemical treatment with amotosalen and uva, is used to inactivate pathogens and leukocytes in plasma. the intercept tm plasma processing set (cerus bv, netherlands) was modified to incorporate plastic containers in non-pvc materials sourced from alternate suppliers and connecting parts and accessories in non-dehp pvc formulations, making the system dehp-free. the final storage container was modified with a higher contact surface with plasma to limit the thawing time. proportion of units with a fibrinogen concentration ≥ . g/l was % (> % required). mean recovery fviii fibrinogen after ibs treatment and frozen storage were % and %, respectively. residual platelets were < /l, leucocytes < /l and red blood cells < /l. all units had a protein content > g/l. residual amotosalen was below lm in all post-cad samples. the concentration of tat complexes was slightly reduced after treatment and frozen storage. concentrations of c a and c a were significantly reduced with the cad treatment. the plasma thawing time in a water bath at °c was consistently short ( - min). summary/conclusions: pathogen inactivated plasma units (ffp-a-ibs and ffp-wb-ibs) prepared with dehp free intercept processing sets retained in vitro characteristics which meet the quality standards for therapeutic plasma. the process did not activate coagulation or complement. reducing ffp thawing time from routine - to - min is an important benefit for emergency use. background: plasma coagulation factor concentrations usually differ for individual donors, therefore pooling of whole-blood derived plasma units moderates high or low coagulation factor concentrations and ensures transfusion of more standardized blood components. moreover, pooling contributes to dilution of reactive antibodies and may reduce the risk of non-hemolytic transfusion reactions and trali. additionally pathogen inactivation reduces the risk of transfusion-transmitted infections, and non-hemolytic transfusion reactions as well as gvhd through inactivation of residual lymphocytes. aims: assessment of the impact of plasma pooling and pathogen inactivation on the standardization of blood components and plasma quality. methods: the study included experiments. for each experiment male-donor, abo-compatible whole-blood derived plasma units (≥ ml) were collected from different donors and pooled using the donopack optipool plasma pooling set (cerus europe b.v.). each of the -unit pools were split into equal minipools which were subsequently treated with the in intercept blood system (cerus europe b.v.). then, each minipool was split into (≥ ml) therapeutic units. samples were collected before and after pooling as well as after inactivation to assess the coagulation factor content (fviii, fix, fibrinogen, vwf antigen using elisa) and coagulation time (aptt, pt). the study-analysis included samples from five pools from single plasma units respectively ( background: biotin (bio) is an alternative to radioactive red blood cell (rbc) tracers which allows one to concurrently track in vivo multiple cell populations labeled at different bio densities. in american clinical trials, multi-labeled biorbc have been transfused in man to assess their survival (mock et al, transfusion, ) . in these studies, the different biorbc populations were monitored by ex vivo flow cytometry analysis using streptavidin. so far, the biotinylation reagents biosulfonhs was not complying with good manufacturing practices (gmp). moreover biorbc, with bio ≥ lg/ml, have induced immunization of the recipient, in rare cases (schmidt et al, transfusion, ) . this represents an obstacle regarding the regulatory european authorities. aims: the aim of this study is to describe a procedure of biotinylation of rbc intended for clinical trials while refining the levels of bio ≤ lg/ml. methods: sterile status is met throughout the process. rbc are taken from standard rbc concentrates and treated with biosulfonhs of gmp-grade ( to lg/ml) recently commercialized. washing buffer is of injectable-grade. biotinylation efficacy is controlled by flow cytometry with streptavidin conjugated to different fluorochromes: phycoerythrin (pe) or brilliant violet (bv ). results: labeling with biosulfonhs of gmp-grade or non gmp-grade is comparable and populations of rbc could be easily distinguished between themselves and from unlabeled blood cells. biosulfonhs (lg/ml): (mfi . ), (gmp mfi ; non gmp mfi . ), (gmp mfi ; non gmp mfi ), (gmp mfi ; non gmp mfi ). streptavidin-bv brighter than streptavidin-pe is a promising tool because it amplifies by . the signal of fluorescence and allows a good differentiation of the populations of rbc treated with only , , and lg/ml biosulfonhs. summary/conclusions: this preliminary study explores the feasibility of multilabeled biorbc production for clinical trials. the benefits of this approach are to overcome the need for non-radioactive tracers, to follow simultaneously various populations of rbc and consequently to limit the number of volunteers, and to reduce the risk of immunization using bio ≤ lg/ml. background: rejuvenation is aiming to revert ageing-related disease development. heterochronic parabiosis studies revealed eotaxin in young and old murine blood as a regulator of brain aging and neurogenesis. umbilical cord blood (ucb)-borne factors including tissue inhibitor of metalloproteinases (timp ) and neonatal immune cells also contributed to rejuvenation in animal models. human platelet lysate (hpl) is commonly used by us and others for highly efficient cell propagation in vitro (burnouf et al., biomaterials, ) . published data indicate only limited differences between adult and ucb-derived hpl, partly questioning enigmatic rejuvenation effects. aims: to verify candidate regenerative factors in neonatal blood products we compared protein contents of neonatal and adult plasma and platelets, respectively. methods: heparinized ucb samples (n = ) were centrifuged within h to collect neonatal platelet rich plasma. aliquots from apheresis platelet concentrates (n = ) were used as adult counterpart. platelet concentration was adjusted to - / l. plasma supernatants and platelets were obtained by centrifugation and platelet pellets were re-suspended in saline. after two freeze/thaw cycles at À °c/ °c for platelet lysis (npl; apl) the platelet fragments were removed by centrifugation. the protein content was analyzed with a proteome profiler tm array. nine samples of each group were pooled to avoid individual donor variations. a threshold of , au spot density was defined as cut-off. data were analyzed by graphpad prism using two-way anova. results: semi-quantitative evaluation of analytes per array revealed significant differences. in plasma samples and platelets and analytes were detected above cut-off, respectively. in neonatal plasma we found more highly prevalent proteins (> , au spot density) compared to adult plasma ( / vs. / ). thirteen proteins were significantly elevated in neonatal plasma including growth/differentiation factor (gdf ), platelet derived growth factor aa (pdgf-aa) and serpin e (p < . ). more highly prevalent proteins were detected in npl ( / ) compared to apl ( / ), and proteins were significantly elevated including vascular cell adhesion molecule- (vcam- ), platelet factor (pf /cxcl ), epidermal growth factor and lipocalin- (p < . ). in adult samples only proteins were significantly higher in plasma and three proteins in apl compared to the neonatal groups (p < . to p < . ). summary/conclusions: we detected significant differences in regenerative growth factor and cytokine contents of neonatal and adult plasma and platelet samples, respectively. additional experiments are underway to further characterize their impact in distinct functional readouts. background: the production and storage conditions of platelet (pl) products intended for transfusion are constantly evolving and need sometimes in vivo evaluations in clinical trials to ascertain whether the platelets have retained their ability to survive in the circulation. this requires that the transfused platelets can be distinguished from the recipient's circulating platelets. labeling of platelets with biotin (bio) affords to track in vivo and concurrently, multiple cell populations covered with various biotin densities as already described for red blood cells (mock, transfusion, ) . surprisingly, there is only one study describing the transfusion of human biopl (stohlawetz, transfusion, ) . so far, the biotinylation reagent bio-sulfonhs was not complying with good manufacturing practices (gmp), which represents an obstacle regarding the regulatory authorities. aims: the aims of this study are ) to describe a procedure to label injectable human platelets with densities of biotin, ) to evaluate the impact of biotinylation on platelet functions, ) to track human biopl in the circulation of the mouse. methods: platelets are taken from standard platelets concentrates and treated with . and lg/ml biosulfonhs of gmp-grade, recently commercialized. main platelet functions are assessed in vitro. human biopl survival is evaluated in immunodeficient nsg-mice treated with liposome-clodronate to eliminate macrophages and to prevent rejection. circulating human biopl are detected ex vivo by flow cytometry with streptavidin phycoerythrin. results: using trap ( lm), p-selectin externalization reveals a normal capacity of secretion for all biopl. gpiba and gpiibiiia expression is not affected by the biotinylation process. biopl have the ability to aggregate: using arachidonic acid ( mm), amplitude of aggregation is . ae . % (bio ); . ae . % (bio . lg/ ml); . ae . % (bio lg/ml). using collagen ( . lg/ml), amplitude of aggregation is . ae . % (bio ); . ae . % (bio . lg/ml) . ae . % (bio lg/ml). the biopl populations could be easily distinguished between themselves and from unlabeled blood cells in the mouse circulation during more than h. after h, the mean fluorescence intensities are . ae . for unlabeled circulating mouse platelets, . ae . and . ae . for circulating human biopl covered respectively with . and lg/ml biotin. summary/conclusions: this labeling approach should be helpful to evaluate new platelet products in vivo and represents an alternative to radioactive tracers. it allows to follow simultaneously different platelet populations and consequently limits the number of volunteers in clinical trials. background: severe ocular surface diseases, dry eye syndrome, persistent and recurrent corneal epithelial defects and diabetic or neurotrophic keratopathy are mainly successfully cured by standard treatment protocols. however, not rarely does refractory to these usual treatments appear, especially with serious forms of disease. in military medical academy, autologous serum eye drops -auto seds and autologous platelet lysate -apl eye drops have been being applied in the treatment of ophthalmological patients in these categories, who were previously resistant to standard therapy. aims: to show the achieved results of therapeutic use of autologous blood products (auto seds and apl) in the treatment of ophthalmological patients who previously had not responded to conventional therapysingle center experience. methods: auto seds are prepared by taking autologous blood into tubes (bd vacutainer, cat, ml) and apl in tubes with anticoagulants (greiner bio-one, acd-a, ml). control on tti of every patient and sterility of every series has been conducted. before and after the treatment, subjective ocular discomfort (ocular surface disease index -osdi), objective parameters of the tear film (schirmer's test, rose bengal, tear breakup time -tbut) and measuring of epithelialization zone were analyzed. apl, obtained from platelet-rich plasma which had been frozen, unfrozen and diluted with nacl solution, up to %. auto seds were administered in the form of % eye drops. results: auto seds have been applied to ophthalmological patients ( men and women), previously resistant to standard therapy. in total treatments were performed (each lasted days). for successful curing, one or two treatments per patient, in average, were applied. apl has been used multiple times to one patient with sj€ ogren syndrome and severe multiple tropical corneal changes. all ophthalmological patients had subjective improvements (the average pre and post treatment osdi scores were . and . respectively). also, objective progress was present in % of all patients (p < . ). summary/conclusions: the use of auto seds and apl in the treatment of ophthalmological patients, previously resistant to standard therapy, is in constant increase, because of its simplicity and low expenses. apl has turned out to be better than auto seds for patients with severe trophic changes, because apl contains larger amounts of the nerve growth factor, tgf-b, vegf and platelet derived growth factor. however, a larger number of clinical cases is needed for future conclusions. background: whole blood (wb) has recently regained favor in treatment of massively bleeding patients in military and civilian settings. platelets (plts) are a vital component in clot formation. as a component of wb, it is critical that they maintain functionality throughout storage. red blood cells (rbcs) stored in hypoxic/ hypocapnic conditions preserve high level of , -dpg while reducing storage lesions stemming from oxidative stress. , on the other hand, effects of steady hypoxia (pco ~ - mmhg) on plts contained in leukoreduced wb is poorly characterized. aims: examine the effects of hypoxic conditions on plt function and microvesicle (mv) formation in wb stored hypoxically (h) and conventionally (c) for -week storage at - °c. methods: units of wb were collected at mayo clinic rochester blood donor center from normal healthy volunteers into ml cp d. wb was leukoreduced using plt-sparing filter (terumo wb-s) then split into control (c) and hypoxic (h). h-wb was processed by the oxygen-reduction bag (hemanext, lexington ma) and unit was stored in o -free bag. ml of wb were collected from each unit at day , weeks , , . plt counts, agonists (thrombin receptor agonist peptide (trap), adenosine diphosphate (adp) and collagen stimulated platelets aggregation, nonactivated and agonists activated plt surface expression of phosphatidylserine (ps, annexin-v binding), p-selectin, fibrinogen receptor (pac- binding), and microvesicles (mv) were measured by coulter counter and digital flow cytometer. paired student t-tests were used to analyzed differences in degradation rates; significance: p < . . results: h-plt counts declined to~ % by the o -reduction process, while similar decline was observed after week in c, and thereafter remained steady. plt activation (ps) increased over time (h >> c after processing; c increasing more rapidly during storage). p-selectin increased over time (h < c), while pac- showed large increase after week, then remained steady (h << c). plt activation by trap or adp declined modestly over weeks (~ %) while h-plt showed additional~ % reduction for all time points. collagen activation for c-plt increased after week ( %) and gradually increased to % after weeks (~ % reduction with h compared to c). plt-derived mv (cd and cd /annexin v) increased~ -fold over storage time; day mv levers were significantly higher for h, but subsequent increase rates were similar or lower. total number of plt-derived mv (cd a) in wb supernatant increased -fold after weeks for c, while h suppressed increase to -fold. (majority of the trends described above showed significant differences between h and c.) summary/conclusions: plts were activated over -week period when stored at - °c in leukoreduced wb, accompanied by a modest loss of agonist-induced activation. oxygen reduction treatment initially activated h-plts, while subsequent increase in activation rates were suppressed compared to c-plts. wb plts retained activatability, and hypoxic condition showed only modest further reduction on the activatability. hypoxic wb may provide higher quality wb for trauma patients if the levels of initial plt activation can improved during oxygen reduction procedure. methods: after informed consent, eligible patients were randomized to either first receive autologous followed by allogeneic seds or first receive allogeneic followed by autologous seds. each sed treatment phase was one month, separated by one month of patient's standard treatment (wash out period) between sed treatment phases. the patients each donated ml whole blood from which the autologous seds were prepared. allogeneic seds were prepared from blood from never-transfused male donors with blood group ab. all serum was diluted : by adding saline, and aliquoted in an eye drop dispensing system (meise, schalksm€ uhle, germany). at each visit, the osdi was determined using a validated questionnaire, with higher scores reflecting poorer outcomes. the results were analyzed intention-to-treat, and a random effects linear mixed model for cross-over design was used. results: in total, patients were enrolled, of whom were excluded because they failed the autologous blood donation. background: the following blood components for non-transfusional use (bcntu) are produced in our transfusional center (tc): ) allogeneic platelet gel (pg), derived from buffy-coats (bc) and human cord blood platelet gel (cbpg); ) autologous serum eye drops (sed). the creation of both types of platelet gel started in but only in we confirmed the process for daily production: these blood components are used to treat pediatric patients with epidermolysis bullosa. the sed, produced from , is dedicated to treat patients with dry eye syndrome. aims: production and storage bcntu. methods: the whole process production of bcntu is traced on the transfusional informatic system (emonet-insielmercato), under the same conditions of another blood transfusional components. the process takes place in closed circuit using the laminar flow hood. ) pg production starts from the bc resuspended in plasma that are not used for daily platelet concentrates, instead the cbpg is produced using cord blood units that are not used for hematopoietic transplant. both have a platelet concentration between - /ll and negative blood cultures, required by the italian law; the units are frozen at À °c and last -year. pg and cbpg must be activated with calcium gluconate or batroxobin to be used. ) the ophthalmologist's patients, with dry eye syndrome, donate ml of autologous blood; the serum is separated and after the dilution with a balanced saline solution ( %) are divided in boxes containing single-dose vials each: they are stored at À °c and they last one year. negative blood culture was evaluated. results : background: candida albicans is the most common pathogen detected in fungal infections. aims: in this study, we aimed to evaluate the in vitro antifungal activity of volunteerderived platelet rich plasma (prp) against c. albicans atcc strain and the possible effects of certain chemokines, kinocidins that might play a role in this activity. methods: prp from nine volunteers were derived by using magellan prp â kit. % calcium gluconate was used to obtain autologous thrombin. c. albicans isolates with a final yeast concentration of cfu/ml and cfu/ml were inoculated on sabouraud dextrose agar at the st , nd , th , th and th hours of incubation to reveal the antifungal activity of autologous thrombin-activated prp. the colonies were counted after - h of incubation at °c. chemokines and kinocidins (platelet factor- , interleukin- and thymosin-b ) were also measured simultaneously by elisa method. results: compared with the pbs-control group, the prp- group showed that the antifungal activity was still going on at the th hour. the difference in colony production between the two groups at th hour was statistically significant (p < . ). it was observed that the antifungal activity continued at the th hour, decreased at the th hour in the group prp- group. although the same amount of prp was used and the same amount of chemokine and kinocidins were released in both groups, the concentration of c. albicans was considered to be important in the detection of more effective prp- group. although there was an increase in il- levels by hours in the two prp groups by elisa method, no antifungal effect was detected against c. albicans. it was observed that decrease in tmsb values results from the antifungal activity on the advancing hours in the prp groups. whereas pf- did not act an antifungal activity on prp- and prp- . summary/conclusions: even in our study group where the highest platelet counts were obtained at the lowest concentration, c. albicans reproduction could not completely eliminated as mentioned in the literature. repeated doses of prp applications, such as drugs used in patients, may have longer duration of action and even complete repression of reproductive outcomes. background: generally, blood is available in developed countries for transfusion. sometimes, transfused or previously pregnant patients form alloantibodies to red cell antigens and rarely, to antigens of high prevalence. this case focuses on a twoyear-old girl, of pakistani descent, diagnosed with neuroblastoma stage iv with anti-in b and -e. although the publications indicate that % of the pakistani, indian or iranian populations are in(b-), it was discovered that this blood type is exceedingly rare. an international search was required to ensure blood product availability for chemotherapy and autologous hematopoietic progenitor cell transplants aims: illustrate the response of the public to a powerful story of a child needing rare blood for treatment and international collaboration for provision of very rare units. methods case report: a two-year-old patient's sample was referred for antibody identification. the patient had received four transfusions ( ml of red cells) in the preceding -day period. hgb level fluctuations were consistent with decreased transfused red cell survival. following the last transfusion of ml, the hemoglobin decreased from . to . . anti-in b , and a ficin-only reactive anti-e was identified in the serum and anti-in b in the eluate. the monocyte monolayer assay predicted the anti-in b to be clinically significant ( % reactivity). transfusion of antigen neg units once obtained, resulted in a stable transfusion response. although it was expected that in(b-) blood would be more easily sourced, only two donors in the usa were in (b-) e-. as - units of blood were requested for the post-transplant period, a national and international search was initiated, as was a robust media appeal to donors resulting in many donors for an intense domestic screening effort in the usa. the search of the who international rare donor panel by the international blood group reference laboratory revealed three known in(b-) e-donors; two british and one australian. they were contacted, recruited, collected and shipped to the usa with the work of the american rare donor program (ardp) staff and the isbt working party on rare donors (isbt wprd) members in each of the countries. results: the intense media coverage of oneblood (the florida blood center collaborating on treatment with the hospital) included online news outlets (youtube, facebook) resulted in over , responses from national and international potential donors to be tested for in b . isbt wprd members were sent the web information of potential donors identified in their countries by the ardp. over , samples from blood centers and associated laboratories tested with anti-in b by oneblood. two new in(b-) donors were discovered ( . %); but both typed e+, thus were not a match for the child. summary/conclusions: this intense media coverage and the overwhelming donor response was unprecedented in our experience. the coordination and cooperation among the numerous blood centers reflect the deep dedication of the blood banking community to the well-being of special patients in need. this case illustrates the response potential that a powerful story and a medical appeal for exquisitely rare blood utilizing social media and other online news outlets can generate. background: blood platelet units are generally stored in blood banks for - days, afterwards they are discarded. prepared infusible platelet membrane (ipm) from fresh or outdated human platelets correct the prolonged bleeding times in thrombocytopenic animals such as rabbits. infusible platelet membrane (ipm) as a platelet substitute may be the most feasible approach to reach the target market. our previous experiments have shown that ipm has a hemostatic efficacy to shorten bleeding time without any adverse effects in rabbits. aims: abnormal toxicity is the european pharmacopoeia standard for assessment of biological products which the test material is administered to the mice. in this study, abnormal toxicity of ipm was evaluated in experimental animal model such as mice to assure the safety of ipm without any evidence of serious toxicity. methods: in this experimental study, infusible platelet membrane (ipm) was prepared from outdated platelet concentrates. platelet concentrates were pooled, disrupted by freeze-thaw procedure, pasteurized for h to inactivate the possible viral or bacterial contaminants with a sodium caprylate stabilizer, formulated by sucrose and human serum albumin and finally lyophilized. at first, the test for sterility is carried out under aseptic conditions for ipm vials and then we injected . ml of ipm ( mg/kg) intravenously between to seconds into each health mice, weighing - grams. these tests were performed according to eu pharmacopeia monographs. results: in the sterility test no evidence of microbial growth in our product is found. the abnormal toxicity test will be passed if none of animals die during h after injection. if more than one animal dies, the preparation fails the test. if one of the animals just dies, the test is repeated. in our experiment all five mice were alive after h of ipm injection. summary/conclusions: in this research the results showed that ipm as a platelet substitute is free of abnormal toxicity with adequate safety and it may be used in human clinical trial studies as a feasible approach to develop a platelet substitute in the future. however, further studies are required to confirm the different aspects of its safety as well. the success of such investigations may affect patients' care in transfusion medicine in the future. a substantial number of infants, especially premature infants, are unable to receive adequate amounts of their mothers' milk for a variety of reasons. the world health organization recommends that infants, especially preterm and ill infants are fed with quality-controlled donor milk if they cannot be fed with their own mother s milk. due to the possible transmission of the human immunodeficiency virus many human milk banks closed in the s, therefore the availability of donor milk has decreased. aims: we analyzed the processing of donor milk and the required laboratory tests to establish a human milk bank within our blood donation service in cooperation with the department of neonatology at the frankfurt university hospital. methods: based on the recommendations for promoting human milk banks in germany, austria, and switzerland (efcni) we evaluated the manufacturing steps and the quality controls require to establish a human milk bank. background: for patients suffering from severe ocular surface disorders treatment with blood derived serum eye drops (sed) is a highly effective therapy. autologous sed, prepared from the patient's own blood, is used preferably. for this approach we have more than years of experience. if auto-sed cannot be manufactured due to medical reasons allogeneic sed present an alternative. since years, the allogeneic approach is well established in our center. aims: retrospectively evaluation of our experience with allo-sed. methods: in germany manufacturing of allo-sed is only possible as an "individual healing attempt". for each patient experienced regular ab -identical male donors without blood borne disease, who never received blood products and not taking any kind of medication are selected. additionally, donors must pass a questionnaire excluding any form of dry eye syndrome. allo-sed are manufactured directed for each individual patient according to the process for auto-sed in a closed system. patient files of our serum eye drops donors were screened for patients receiving an allogeneic treatment. data concerning indication for allo-sed, contraindication for phlebotomy, problems with donor selection and manufacturing, as well as serological and microbiological testing results were obtained. clinical results were evaluated © the authors vox sanguinis © international society of blood transfusion vox sanguinis ( ) (suppl. ), - by ocular surface disease index (osdi) and patient's questionnaire, asking for subjective benefit, symptom reduction, possible side effects, consumption and comparison with artificial eye drops or, if applicable, with auto-sed. furthermore patients are undergoing regular ophthalmologic examination within a special consultation for dry eye syndrome at our hospital. results: patients were identified receiving allogeneic sed, patients had been treated autologous previously. in total, allogeneic sed have been produced times since june . indications were ocular gvhd (n = . %), neurotrophic keratopathy (n = . %), mucous membrane pemphigoid (n = . %), sj€ ogren syndrome (n = . %) and secondary keratoconjunctivitis sicca by virtue of chemotherapy, meige syndrome, rosacea, morbus bruton (n = . %). contraindications for autologous donation were underlying disease (n = . %), poor venous access (n = . %), low haemoglobin (n = . %), low body weight (n = . %), very young age (n = . %), circulatory disturbances (n = . %) and lack of response to auto-sed (n = . %). some patients presented more than one contraindication. manufacturing problems were: lipemic donor plasma (n = . %), high donor haemoglobin (n = . %) and unspecific positive serological findings (anti-hbs n = . %). microbiological testing was sterile every time. as side effects one case of allergic reaction, suspected as serum protein allergy, appeared. clinical outcome can be considered equivalent to ased. subjectively, all patients benefited from the therapy and reported an alleviation of their symptoms. for some indications (highly active gvhd) allo-sed might even be the better option. summary/conclusions: considering our previous experience, allo-sed seem to be a safe and equally effective alternative to auto-sed for patients unable to donate blood. in case of urgent indication, timely supply can sometimes be difficult. to overcome this disadvantage licensing allo-sed as a new blood product with the possibility of production and storage in advance would be a desirable goal. in addition supply would become even safer by preparing allo-sed according to a quarantine principle like ffp. abstract withdrawn. background: vernal keratoconjunctivitis is a chronic, recurrent bilateral inflammation of the outer ocular layer. mostly affected are children and young people and the condition is more common in boys. the disease presents with eye pruritus (itching eye), photophobia (sensitivity to bright light), excessive tearing and foreign eye syndrome. severe cases manifest with diffusion of overgrown papillae usually of the upper eyelid, bursting of the connective tissue barriers and appearance of giant papillae that press on the cornea. corneal ulceration is a severe complication of vernal keratoconjunctivitis that may induce scarring, corneal neovascularization and occasionally perforation. treatment of keratoconjunctivitis mainly relies on steroids, mast cell stabilizers, antihistamines, immunosuppressive drugs (cyclosporine), artificial tears, contact lensdressing, cryotherapy and surgical papillae removal. we present the case of a year-old girl with corneal ulceration who was applied artificial tears after traditional methods of treatment proved unsuccessful. aims: the aim was to share our experience on artificial tears therapy applied in ophthalmic disorders. methods: autologous blood ( ml) was collected into disposable, sterile transfer bags used for routine blood component preparation (no anticoagulant) and incubated for h at °c. the clot was then removed by centrifugation and the serum containing erythrocytes was press extracted. centrifugation was applied again to obtain serum free of cellular components. the serum was then divided into . ml segments (capsules)and the artificial tears applied to the left eye daily. results: ulcer healing was reported after weeks of therapy with artificial tears. the dosage was reduced to daily. no recurrence of corneal ulceration was observed after subsequent weeks. summary/conclusions: artificial tears are a safe and effective therapy for ophthalmic disorders in children. background: arv non-disclosure among hiv-positive donors who tested hiv antibody (ab) positive but rna negative (ab+/rna-), so-called false elite controllers, was previously described by our group in south africa, with > % of ab+/rnadonations since testing arv positive. the extent of undisclosed arv use at time of donation represents a significant risk to blood safety in a country with a growing treated hiv population. aims: to establish the prevalence of arv non-disclosure among four subgroups of hiv-positive donors in south africa along with demographic correlates of non-disclosure. methods: south african blood donors are screened by a self-administered questionnaire, which includes questions on current hiv status and arv use, followed by a semi-structured personal interview. specimens for hiv, hepatitis b and c testing are collected at time of donation. based on id-nat (procleix, grifols) and antibody (prism, abbott; western blot) testing, hiv-positive blood donations were classified as acute (ab-/rna+), recent (ab+/rna+, limiting antigen avidity [lag] odn ≤ . ), longstanding (ab+/rna+, lag odn > . ) and potential elite controller (ab+/rna-) cases. stored plasma from these donations were tested for four arv drugs using qualitative liquid chromatography-tandem mass spectrometry (detection limit . lg/ml). chi-square tests were used to assess associations of hiv case type, gender, ethnicity, age, donor type, and donor clinic (fixed, mobile) type with arv non-disclosure. results: during , donors tested hiv-positive of whom had samples available that were tested for arvs. the overall prevalence of undisclosed arv use was . % (n = ) with efavirenz most frequently detected ( ), followed by lopinavir ( ) and nevirapine ( ) . potential elite controller cases had the highest proportion of detectable arv ( / ; %) (p < . ) followed by longstanding ( / ; . %) and recent ( / ; . %) infections. none of acute hiv cases tested positive for arvs. there were no associations between arv use and gender or ethnicity. however, older ( to years) hiv-positive donors ( / ; . %) were significantly more likely to test positive for arv than younger ( to years) donors ( / ; . %) (p < . ). arv use was more frequent among first time ( / ; . %) than in lapsed ( / ; . %) or repeat ( / ; . %) donors (p < . ). donors at mobile clinics had significantly higher arv non-disclosure than donors at fixed sites ( . % vs . %; p = . ). summary/conclusions: the . % prevalence of undisclosed infection and arv use among hiv-positive south african blood donors is alarming. higher rates of nondisclosure among first-time donors was expected, but non-disclosure among repeat and lapsed donors suggests failure in donor education and assessment. the . % prevalence among concordant ab+/rna+ cases may suggest sub-optimal viral suppression. lack of detection of arvs in acute cases should be qualified because the samples were not tested for tenofovir, the most common drug used in pre-exposure prophylaxis. donor motivation for non-disclosure of known hiv infection and arv use needs further investigation, since early arv initiation or infection while on prep could lead to low ab and rna levels, failure to detect hiv-infected donations and transfusion-transmission of hiv. blood bank, rotary blood bank, new delhi, india background: voluntary blood donation ensures safe blood transfusion. careful blood donor selection is of importance to provide safe blood to patients, although new methodologies have also been adopted by blood centers for blood safety and to minimize risk of transmitting infections through blood transfusion. the quality and the availability of blood components depend on the willingness to donate and reliability of the information given by the donors about their own health, including risk behaviour. blood donor history questionnaire is designed to evaluate donor's history in accordance with the guidelines laid down by the fda. donors, once deferred by the blood bank, will be less motivated to return for donation if he is not counseled effectively. it is important to reduce the number of deferrals by good donor comprehension and the centre should have a mechanism to recall temporarily deferred donors aims: the aim of the study is to analyse donor history and test results of those who donated blood with past history of jaundice. based on their history which suggested the type of viral infection they had, these donors were accepted or deferred. data was collected from voluntary blood donors who were screened for blood donation in the year . methods: in this study, donor history was analysed with reference to history of jaundice. jaundice in donors after the age of yrs, history of surgery, blood transfusion, body tattoos and acupuncture treatment within past one year of donation, history of multiple sex partners and related history and intravenous drug abuse history was taken into consideration. donors who revealed past history of jaundice were asked in detail about their illness and recovery. blood was donated by donors from whom the history of jaundice was elicited and it was understood that the type of virus which caused jaundice was not hepatitis b or c. those who could not give the correct history or were not sure of the cause of hepatitis, those individuals were deferred. aims: to assess the performance of this follow-up program in terms of donor participations, successful confirmed positivity rates, and potential reentry rates. methods: eligible donors were tested for hbsag, hcvab, hivab/ag, and tpab with two eias for each marker. samples reactive with at least one assay were tested further with electro-chemiluminescence assay (eca) and reactive samples were considered repeated reactive (rr). tpab reactive donations were re-tested with particle agglutination assay (tppa). samples eca or tppa non-reactive were considered non-repeatable reactive (nrr background: the blood donation service in suhl processes more than . samples annually from whole blood and apheresis donations, testing on average around samples per day. for the last years, serology screening was performed on the architect instruments (abbott) (arc), but will be changed to the alinity s system (aly) by middle of . although the design of the aly assays is based on those of the arc assays, we undertook a thorough evaluation of the four mandatory screening assays detecting hbsag, hiv ag/ab, anti-hcv and anti-hbc. aims: to validate the mandatory screening assays on the new aly system in our lab in terms of sensitivity and specificity, also including samples with known falsereactive results. determine the rate of false reactive results for hbsag, anti-hcv and anti-hiv that may lead to deferrals of donations and donors. methods: for sensitivity, we used known positive samples confirmed by immunoblot or nat. known unspecific positive samples for arc not confirmed by immunoblot or nat were testes for aly also. close to . unselected samples (edta plasma) from routine blood and apheresis donors were tested in parallel on both systems, arc and aly to determine the rate of initial and repeat reactive results. results: all known confirmed positive samples were identical detected by aly. samples with known unspecific reactive results were retested by aly with the following results: / anti-hcv, / hiv ag/ab and / hbsag were found reactive by aly to. one donation from an acute hiv infection in the early seroconversion period was detected by both methods in routine testing. there are no reactive results for aly not already known for arc. the specificity for the screening assays on aly versus arc assays were as follows: ) hbsag aly . % ( % ( / % ( ) vs arc . % ( % ( / ; ) hiv aly and arc . % ( / ); ) anti-hcv aly . % ( % ( / % ( ) vs arc . % ( % ( / . the number of anti-hbc reactive samples did not differ between aly and arc. summary/conclusions: while the switch to the new system is mainly driven by operational efficiency, obviously, the high specificity of the alinity s assay will reduce unnecessary deferrals of donations and donors. abstract withdrawn. background: blood donor selection is the cornerstone for blood transfusion safety, designed to safeguard the health of both donors and recipients. donor safety is targeted by reducing the risk of complications associated with blood donation and transfusion safety by reducing the risk of transfusion-transmitted infections (tti) and other preventable transfusion reactions. there is always a compromise on blood donor safety as well as blood safety during outdoor mega blood donation drives due to various reasons, mainly due to more number of donations within a stipulated time. aims: to compare the blood donor selection patterns between in house blood donations and donations at mega blood donation drives and its influence on donor safety and blood safety in a tertiary care hospital in india. methods: a retro prospective study was done to audit and compare blood donor safety and blood safety over a period of years from january to december . blood donor safety was analyzed by two indicators: donor health questionnaire (dhq) monitoring and blood donor reaction rates and blood safety through tti positivity rates. ( ) during mega blood donation drive. summary/conclusions: a good donor selection is a lengthy process which involves pre-donation information and advice: this is usually provided in a leaflet, especially about transfusion-transmitted infections (and the associated risk factors) and the potential risks of donation, filling of dhqs by the donor himself, donor interview: conducted by a qualified medical specialist trained in donor selection process and health assessment at the end of the interview to declare if the donor is eligible to give blood or deferred temporarily or permanently. it was observed that seroprevalence rates, number of donor reactions and incompletely filled dhqs were more among blood donations at mega blood donation drives when compared to blood donations during in house collections. this is mainly due huge number of blood donations with in a stipulated time where there is limited time spent on proper donor selection. stringent implementation of who strategy: "safe donor safe blood" is the only way for blood donor and transfusion safety. background: safety of blood transfusion is a great concern especially in crisis countries and during humanitarian emergencies. transfusion transmitted infections (ttis) are one of the major health problem in yemen that are associated with blood transfusion complications. aims: the aim of this study is to determine the prevalence of ttis among blood donors at national blood transfusion and researcher center (nbtrc this contributed to an additional reactivity of . %, thereby total reactivity being . %. % ( / ) of these were hcv reactive & % ( / ) for hbv. the nat yield was in and the viral loads of nat reactives ranged from - x iu/ml for hcv & all the hbv yields had an extremely viral load of < iu/ml. / nat reactive showed sero-conversion after - months with follow-up eclia screening, and of these were hcv reactive and hbv reactives. summary/conclusions: incidence rate indicate that the current risk of transfusion transmitted viral infections attributable to blood donation is relatively high in our country. parallel use of both serology and nat screening of donated blood in countries that have high seroprevalence can improve the blood safety. at our centre, by using best in class serology and nat technologies, we were would add an extra layer of safety to blood supply by interdicting samples from donor with recent infections. abstract withdrawn. abstract withdrawn. ( / , ) . the both hiv-rna and hcv-rna detected donors by nat were identified in the window period. summary/conclusions: in this study, we found that nat could detect infected cases with hbv-dna, hiv-rna and hcv-rna which were forgotten by serological methods therefore, nat is a sensitive screening method to detect low viral load and shorten the window period of the virus infection to ensure the safety of blood transfusions. service du sang, croix rouge de belgique, namur, belgium background: due to enhancement of kits specificity and machines throughput, roche elecsys â technology is a potential partner for blood donations screening laboratories. aims: the aim of the study was to assess the performance of the elecsys serology assays on a cobas e equipment for clinical specificity, analytical sensitivity and reproducibility. background: deceased donors are the primary source of organs and tissues for transplantation but the risk of infectious complications in the recipient is high and is the main cause of morbidity and mortality after transplantation. to minimize the risk of infections by organ or tissue transplantation, donors should be tested for anti-hiv- / , hbsag, anti-hbc, anti-hcv, and syphilis. further laboratory tests may be required depending on the history of the donor and on the tissue properties. certain grafts can be donated after circulatory death of the donor; however, the absence of the heartbeat may change dramatically the blood composition by e.g., haemolysis and proteolysis. this may have an impact on test performance and lead to false results. therefore, an assay validation is needed for testing of cadaveric samples. aims: a validation study was performed to demonstrate the suitability of elecsys hbsag ii, anti-hbc ii, anti-hcv ii, hiv combi pt, hiv duo, syphilis, htlv-i/ii, and chagas for the use in cadaveric samples from non-heart beating donors. methods: as the basis for validation, we followed the recommendations of the paul-ehrlich-institut (pei) "proposal for the validation of anti-hiv- / or hiv ag/ ab combination assays, anti-hcv assays, hbsag and anti-hbc assays for use with cadaveric samples". comparison of spiked samples from living donors and cadaveric donors was used to demonstrate accuracy. to determine precision, two cadaveric specimens were tested in several replicates. acceptance criteria were implemented according to the pei recommendations. results: results were found to be within specifications requested by the pei recommendations for all tested assays summary/conclusions: the evaluated results support the extension of the use of these assays with cadaveric specimens. background: in developed countries, blood donors are routinely screened for a range of blood borne viruses (hiv, hbv, hcv and htlv) using highly sensitive screening tests. this has dramatically improved the safety of blood supply. however, transmission by transfusion of unknown or unsuspected viruses remains a continuing threat. this is particularly relevant considering that a significant proportion of transfused patients are immunocompromised and more frequently subjected to fatal outcomes. in developed countries, blood donors are routinely screened for a range of blood borne viruses (hiv, hbv, hcv and htlv) using highly sensitive screening tests. this has dramatically improved the safety of blood supply. however, transmission by transfusion of unknown or unsuspected viruses remains a continuing threat. this is particularly relevant considering that a significant proportion of transfused patients are immunocompromised and more frequently subjected to fatal outcomes. aims: in this context, metagenomic analyses of viral content in blood donations collected in geographical zones recognized as "hotspot" for viral emergence represents a suitable approach without any a priori for the identification of a potential emerging viral risk that may compromise blood safety. methods: in the framework of a viral discovery program founded by the french national agency for medicine security (ansm in french), more than plasma samples collected in sub-saharan africa countries ( ) ( ) and the amazon region of brazil ( ) have already been analysed by metagenomics. results: although no viral sequence could be described as novel (i.e. new species or even a new genus), we unexpectedly identified a feline bocavirus in two donors from mauritania. a large diversity of known viruses that are not part of the regularly monitored agents were also observed, among which anelloviruses, hpgv- (formerly known as gbv-c), papillomaviruses, herpes viruses, parvovirus b , chikungunya virus, enterovirus, and various small circular viruses (circo-, cycloand gemycircularviruses). while no significative differences was observed in the higher classification of detected virus (above families/genera) between africa and brazil, we observed variations at the sequence level allowing better resolution of the genetic diversity for several viruses (for example characterization of hpgv- genotypes). summary/conclusions: overall, the absence of novel viruses in blood samples collected across countries of two distant continents is reassuring regarding threats emergence. however, continuous monitoring of prospective blood banks should be continued. summary/conclusions: after the high peak observed in during the first period, this study shows that the decrease in the seroprevalence of viral markers is continuous over the next five years. the second period is marked by an irregular evolution of seroprevalence but with lower levels than the first period. the recruitment of new donors allows a quantitative increase in donations. however, improving the quality of blood products essential condition of transfusion safety is achieved through retention of recruited blood donors. background: in blood screening laboratories, samples may be transferred between automated serological and molecular instruments, and the potential for sample contamination is a serious risk to the integrity of nucleic acid testing (nat) results. the sensitive limit of detection (lod) for hiv and hcv nat assays combined with the high viral titers encountered in specimens from patients with acute infections presents a challenge for maintaining the sample integrity of negative specimens. at additional cost per test, this risk can be reduced with single-use filter pipette tips. aims: we evaluate the efficacy of applying induction heated washes to a non-disposable pipettor on serology instruments-alinity s, alinity i, and architect i sr (abbott diagnostics)-to preserve the integrity of samples transferred to a downstream molecular instrument, the m realtime (abbott molecular diagnostics), which amplifies viral nucleic acid targets exponentially. methods: in this application of induction heating, the metallic pipettor warms under its own resistance to coil-induced electrical currents. by sweeping the pipettor through an induction coil, temperatures on the pipettor are elevated throughout its length. single donor high viral titer hiv genotypes a ( . log iu/ml), b ( . log iu/ml), c ( . log iu/ml), crf ( . log iu/ml), crf ( . log iu/ml), and urf ( . log iu/ml), as well as single donor high viral titer hcv genotypes a ( . log iu/ml), b ( . log iu/ml), a ( . log iu/ml), ( . log iu/ml), q ( . log iu/ ml), and t ( . log iu/ml) were used as potential sources of contamination; these genotypes account for the majority of hiv and hcv infections worldwide. on serology instruments, one high viral titer hiv or hcv specimen and three consecutive susceptible negative samples (hiv/hcv rna negative human plasma, abbott molecular diagnostics) were tested on an hiv ag/ab combo or anti-hcv immunoassay (abbott diagnostics), and this schema was repeated four times per positive specimen. induction heated washes occurred between all samples processed on the serology instruments. the first susceptible negative from each testing block, with approximately ml of residual sample volume, was then tested using the . ml abbott realtime hiv assay (lod copies/ml) or . ml abbott realtime hcv assay (lod iu/ml) and an hcv ag immunoassay (lod . fmol/l; abbott diagnostics). study acceptance criteria required that any susceptible negative sample had no detectable level of hiv or hcv rna. results: all first susceptible negative samples (n = per platform per virus schema) run on alinity s, alinity i, and architect i sr using induction heated washes after a high viral titer hiv specimen or hcv specimen were hiv ag/ab combo nonreactive (< . s/co) and reported no detectable level of the hiv rna target, or were anti-hcv nonreactive (< . s/co) and reported no detectable level of the hcv rna or core antigen targets. summary/conclusions: while precautions should continue to be taken for samples run on molecular instruments, the integrity of samples originally tested on the alinity s, alinity i, and architect i sr was preserved for downstream molecular testing through the use of induction heated washes. aims: increasing the safety of blood and blood products -motivating the blood donors to be regular donors methods: national reporting system showed the high prevalence of ttis among first blood donors in compares with the regular donors. in per . . donations, % % of confirmed positive hiv, % of hcv, and % of hbv cases has been reported among first blood donors. in the end of a national program named "pre-donation screening tests "has been developed and has been implemented in high prevalence provinces in whole country. based on this program, all first blood donors who accept in donation sites, if after donor selection process are eligible to donate blood, they refer to give just a blood sample for screening ttis tests. after months, the invitation letters and smss send to the donors who have negative results for all screening ttis tests, and they can be eligible to donate blood after another donor selection process. in , about . % of all donations have been rejected because of at least one of hiv, hcv, or hbv confirmed positive results, while this reject rate in was . %, which shows a significant decreasing the ttis prevalence among blood donation from to . the prevalence of hiv, hcv, and hbv among donations has been decreased significantly in compared with the . prevalence of hiv among donations reduce from . % in to . % in , for hcv and hbv the same results have been experienced, respectively from . % and . % in reduce to . % and . % in . it seems this applied study could effectively scale up the safety of national blood supplies. in addition this intervention could support iranian blood transfusion service to increase the proportion of regular blood donors from . % in to . % in . it means that with increasing the regular blood donor population sizes, the safety of iranian blood and blood products will be more and more scaled up. summary/conclusions: evidence based reports show there is a high rate of prevalence of transfusion transmitted infections (ttis) among first blood donors. so an effective intervention which can reduce the risk of unsafe first blood donation can effectively increase the safety of blood and blood products. pre donation screening tests program in iran can support the national program to decrease the rate of ttis among blood donations from . % in to . % in . abstract withdrawn. abstract withdrawn. background: despite the universal application of viral inactivation and elimination technologies during the preparation of plasma-derived products, the exclusion of infectious donations before any other procedure remains the first essential step as well as the major determinant for the safety of untreated labile blood products. current selection and screening techniques have reduced the risk of viral transmission to very low levels, but there is still a very low but quantifiable risk of transmission through donations beyond routine detection, particularly during the " seroconversion window". "of an infection in a blood donor that is to say during the period when the recently infected donor has not yet developed a serological response. the level of residual risk, which must be as low as possible, is mainly conditioned by the rates of the infections concerned (hiv and hepatitis b virus (hbv) and c (hcv)) to blood donors. summary/conclusions: the evolution of serologic markers is generally satisfactory with continued regression, which has improved particularly for hiv. on the other hand, hepatitis b is still a concern because of its still high rate among new donors. it is desirable to initiate a regular donor vaccination program to protect against hepatitis b. background: blood centres require high throughput assays with a high level of reproducibility to assure consistent results and minimize unnecessary retesting of samples and deferral of donors. in addition, continued economic pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. aims: evaluate the reproducibility of hcvab, havab igm and havab igg essays using abbott alinity s when compared to architect i sr. determine repeatability of these tests in alinity s essays. methods: during months a study was conducted where several samples (minimum samples per test) were randomly sorted and tested using alinity s and architect i sr, results were compared using ibm spss statistics â . in order to evaluate repeatability, at least samples with different reactive degrees (high, intermediate and low) per test were repeated, using alinity s, an average of times per sample and it was determined the percentage of coefficient of variation (%cv). results: a total of samples were tested ( for hcvab, for havab igm and for havab igg) using alinity s and architect i sr, and it was ensured that there were no statistically significant differences between results (p > . ). using samples and a total of essays we found the %cv hcvab ranged from to . %. samples were tested for havab igm in a total of essays and the %cv ranged from to . %. havab igg was tested in samples during essays and the %cv ranged from to . %. summary/conclusions: the new automated equipment alinity s system demonstrated no statistically difference when compared with architect i sr and repeatability was ensured. this demonstrates the precision of results generated by this fully automated blood screening analyzer, which helps assure consistent results for the testing and retesting of blood specimens for hcvab, havab igm and havab igg. background: blood centres require high throughput assays with a high level of reproducibility to assure consistent results and minimize unnecessary retesting of samples and deferral of donors. in addition, continued economic pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. aims: evaluate the reproducibility of hbsag, hbsab, hbcab, hbeag and hbeab essays using abbott alinity s when compared to architect i sr. determine repeatability of these tests in alinity s essays. methods: during months a study was conducted where several samples (minimum samples per test) were randomly sorted and tested using alinity s and architect i sr, results were compared using ibm spss statistics â . in order to evaluate repeatability, at least samples with different reactive degrees (high, intermediate and low) per test were repeated, using alinity s, an average of times per sample and it was determined the percentage of coefficient of variation (%cv). results: a total of samples were tested ( for hbsag, for hbsab, for hbcab, for hbeag and for hbeab) using alinity s and architect i sr, and it was ensured that there were no statistically significant differences between results (p > . ). using samples and a total of essays we found the %cv hbsag ranged from - . %. samples were tested for hbsab in a total of essays and the % cv ranged from - . %. hbcab was tested in samples during essays and the %cv ranged from - . %. using samples and a total of essays we found the %cv hbeag ranged from . - . %. samples were tested for hbeab in a total of essays and the %cv ranged from - . %. summary/conclusions: the new automated equipment alinity s system demonstrated no statistically difference when compared with architect i sr and repeatability was ensured. this demonstrates the precision of results generated by this fully automated blood screening analyzer, which helps assure consistent results for the testing and retesting of blood specimens for hbsag, hbsab, hbcab, hbeag and hbeab. background: blood centres require high throughput assays with a high level of reproducibility to assure consistent results and minimize unnecessary retesting of samples and deferral of donors. in addition, continued economic pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. aims: evaluate the reproducibility of hivag/ab, syphilis and htlv i/ii essays using abbott alinity s when compared to architect i sr. determine repeatability of these tests in alinity s essays. methods: during months a study was conducted where several samples (minimum samples per test) were randomly sorted and tested using alinity s and architect i sr, results were compared using ibm spss statistics â . in order to evaluate repeatability, at least samples with different reactive degrees (high, intermediate and low) per test were repeated, using alinity s, an average of times per sample and it was determined the percentage of coefficient of variation (%cv). results: a total of samples were tested ( for hivag/ab, for syphilis and for htlv i/ii) using alinity s and architect i sr, and it was ensured that there were no statistically significant differences between results (p > . ). using samples and a total of essays we found the %cv hivag/ab ranged from to . %. samples were tested for syphilis in a total of essays and the %cv ranged from to . %. htlv i/ii was tested in samples during essays and the %cv ranged from . to . %. summary/conclusions: the new automated equipment alinity s system demonstrated no statistically difference when compared with architect i sr and repeatability was ensured. this demonstrates the precision of results generated by this fully automated blood screening analyzer, which helps assure consistent results for the testing and retesting of blood specimens for hivag/ab, syphilis and htlv i/ii. , human immunodeficiency (hiv) and hepatitis c (hcv) viruses' infection in blood donors were . %, . % and . % respectively. consecutive positive results for hbv were . % ( / ), for hcv were . % ( / ) and nil for hiv. there was no sample carry over in this. out of consecutive reactive donors were donated for same patients and were related with infected patient which were statistically significant (p < . ). summary/conclusions: among all tti reactive donors . % ( / ) were consecutive reactive. the reason for the same may be process related like sample carry over or reagent carry over or donor related. donor related reasons may be, one of the close relative is reactive for virus and that is transmitted to other family members. in our study reactive donors either had close contacts with persons with history of infective disease or were their first degree family relatives. these findings were found statistically significant (p < . ). this study recommends that in analysis of consecutive positive results in elisa along with looking for procedure/sample error, there is also a need to take retrospective history of donors for close contact with infected patient. background: screening for transfusion-transmitted infections (ttis) is critical in ensuring safety of blood products. transmission of infections through transfusion remains a major source of viral hepatitis especially hbv and hcv. the effectiveness of rapid immunochromatographic test (ict) devices for screening of blood is a concern and needs validation through advanced methods like chemiluminescence immunoassay (clia) and polymerase chain reaction (pcr). aims: the current study was conducted to evaluate the performance and screening effectiveness of commercially available rapid screening kits in comparison with clia and pcr. methods: this single centre, cross sectional study was conducted at the department of blood transfusion services, shaheed zulfiqar ali bhutto medical university, islamabad, from january -june . a total of ten commercially available ict devices and one clia kit (liaisonr xl) were tested for their sensitivity, specificity, positive predictive value (ppv), negative predictive value (npv) and accuracy using positive and negative samples each for hbv and hcv respectively, in comparison with the values determined by pcr. the ict kits included hightop, rightsign, wondfo, accu-chek, fastep, abon, immumed, insta-answer, biocheck and ctk. results: the sensitivities and specificities of ict kits for hbsag were % and % (hightop), % and % (rightsign), % and % (wondfo), % and % (accu-chek), % and % (fastep), % and % (abon), % and % (immumed), % and % (insta-answer), % and % (biocheck) and % and % (ctk) respectively. similarly the sensitivities and specificities of different ict kits for hcv were % and % (hightop), % and % (rightsign), % and % (wondfo), % and % (accu check), % and % (fastep), % and % (abon), % and % (immu-med), % and % (insta answer), % and % (biochek) and % and % (ctk) respectively. the sensitivity and specificity of diasorin liaison murex assay for both hbv and hcv were found to be %, when compared with pcr. the ppv, npv and accuracy were determined accordingly. summary/conclusions: rapid testing ict devices for both hbv and hcv available in pakistan were found to have a variable degree of sensitivity and specificity, when compared with pcr. comparatively expensive but quality methods are more reliable as compared to rapid devices. the data generated will help policy makers to prepare future plan of action and introduce the concept of quality control in blood centres. the analysis has shown that the population of blood donors also included people infected with syphilis. in reference to the number of the tested samples this number is quite significant. the analysis proved the increase in the number of syphilis infections among the blood donors which is consistent with the general trend in the population. summary/conclusions: we have proved that testing blood donors for the treponema pallidum infection increases the safety of recipients of blood and its components and that obligatory testing of donors is fully justified. , and , the use of third or fourth generation serological assays is mandatory for screening of blood donor units for hbv and hcv infection before transfusion. routinely, blood banks in india screen the units by the elisa testing. nat is not very common due to cost constraints. aims: the aim of this study is to determine the frequency and load of hbv dna and hcv rna in hbs and hcv reactive blood donors respectively, and hence it was intended to contribute to determining whether routine hbs and hcv screening of blood donors, using elisa method alone, provides any concrete benefits with regard to hbv and hcv risk reduction or whether the implementation of nat will be of great benefit to low-resource countries like india, which has high prevalence of hbv and hcv. abstract withdrawn. , donors were routinely tested for hbv dna by using cobastaqscreen mpx- and mpx- (roche) or procleix ultrio and ultrio plus id (grifols) assays. obi was confirmed by repeat dna testing and by performing additional serological and molecular investigations on index and follow-up samples. anti-hbs concentrations were determined and anti-hbc antibodies were tested with three distinct commercial clia assays (anti-hbc elecsys roche, architect anti-hbc ii abbott, and hiscl anti-hbc assay sysmex). hbv pre-s/s, precore/core and bcp regions were pcramplified after viral particle concentration and viral amplicons were sequenced. results: hbsag-/dna+ donors ( : , ) including confirmed obi were identified ( : , prevalence). among obi donors, ( . %) tested anti-hbc+/anti-hbs-, ( . %) were anti-hbc+/anti-hbs+, ( . %) were anti-hbc-/anti-hbs+, and anti-hbc-/anti-hbs-primary obi ( . %). anti-hbc-/anti-hbs+ obi donors were significantly younger (mean: years [range: - years]) than those with anti-hbc+/anti-hbs+ (mean: years [range: - years]) and anti-hbc+/anti-hbs-(median: years [range: - years]) profiles (p < . ). hbv vaccination was documented for ( %) of these donors and was reported in one donor but without definitive evidence. extremely low hbv dna loads (range: < - iu/ml) were transiently detected in seven donors during follow up. genotypes identified were genotype b (n = / ) and genotype c (n = / ). preliminary analysis of core protein (n = ) and bcp (n = ) sequences showed no particular genetic feature that could be associated with altered antigenicity or core gene expression. follow-up was available for / anti-hbc-/anti-hbs+ donors ( - samples/donor; range: - months). anti-hbc remained undetectable with all clia assays in these donors except one. low transiently detectable levels of hbv dna were observed overtime with anti-hbs levels fluctuating between and , iu/l. no significant difference in hla-a, -b (except hla-b* more frequently detected in anti-hbc negative obi), and -drb *. summary/conclusions: overall, the . % prevalence of anti-hbs-only in hbv dna positive obi carriers ( : , of total donors) in dalian blood donors confirmed previous reports from south east asia. this phenomenon was not related to core antigenic variations but was significantly associated with younger age of carriers. a particular route and natural history of the infection may be considered. the hypothesis of acute-phase vaccine breakthrough was ruled out in / donors by the over months stability of this serological profile. breakthrough in immunized donors may still be suspected. further studies are needed to evaluate the potential infectivity of anti-hbs-only/hbv dna+ obi carriers, and to characterize the potential viral and immunological mechanisms responsible for this unusual hbv infection profile. confirmatory laboratory, hungarian national blood transfusion service (hnbts), budapest, hungary background: vaccination against hepatitis b virus (hbv) is an effective tool to avoid the infection. in hungary, population born after is considered to be immunized, because inoculation has been mandatory for children as campaign vaccination since . hbv vaccine is strongly recommended for healthcare workers, moreover trips to endemic countries and awareness of individuals could also be reasons of vaccination in immunologically na€ ıve age-groups. since the hbv vaccine contains surface antigen, a recent inoculation can cause reactivity of hbsag screening assays and positivity of confirmatory tests for several days resulting in deferral of donors from blood donation. the former regulation of hnbts, which was valid until december , , allowed the re-entry of donors whose immunization records and negative hbv confirmation of the second blood samples proved that the previous vaccination had resulted in the hbsag confirmed positivity. aims: the aim of this study was to strengthen that vaccination against hbv before blood donation had resulted in the reactivity of hbsag screening and confirmatory assays between and . background: in brazil, the introduction of nucleic acid tests (nat) for hbv-dna detection in the routine screening at public blood banks is relatively recent. at fundac ßão pr o-sangue/hemocentro de são paulo (fps-sp), about , blood donors are submitted to serological screening tests (hbv, hcv, hiv- / , chagas disease, syphilis and htlv- / ) and nat for hiv, hcv and hbv per year. approximately % of the blood donations are discarded due to some reactive result; of these, the hbv discard rate was . % in . aims: our study aims to determine the potential infectious cases among samples that had one or more hbv-reactive screening results (anti-hbc, hbsag and mp-nat-hbv) and verify the different categories of hbv infection (acute, chronic, occult hepatitis b infection (obi) and immunological window). furthermore, to characterize the distribution of hbv genotypes, drug resistance and escape mutations and analyze the risk factors. methods: we carried out a cross-sectional study of roughly , donations from may to december . hbv antibodies and antigen screening were performed using cmia kits architect â -abbott/hbsag and architect â -abbott/anti-hbc. nat screening was performed in minipools (mp) of six samples using kit nat hiv/hcv/ hbv -bio-manguinhos (sensitivity % lod iu/ml for hbv). reagent samples (n = ) that presented one or more hbv-reactive screening results (anti-hbc, hbsag and/or mp-nat-hbv), were submitted to individual nucleic acid extraction and "in house" quantitative real-time pcr-hbv (id-pcr-hbv) targeting the hbsag region (sensitivity - ui/ml). the hbv genotypes and mutation analyses were determined by direct sequencing of the hbv pol-gene/surface-gene and use of the online analysis tool geno pheno [hbv] . . socio-demographic and epidemiological data were also analyzed. financial support: fapesp / - . results: among the hbv reactive samples, were reactive for anti-hbc only ( . %), for hbsag only ( . %) and were reactive for both markers and hbv dna ( . %). routine testing and id-pcr-hbv identified ( . %) samples of active infections that had all hbv reactive/positive tests results. no hbv dnayield samples or hbsagyield or obi were observed. viral loads for active infections samples ranged from . e+ to . e+ cop/ml (median, . e+ cop/ml). hbv sub-genotypes a , a , c , d and f were found in %, %, %, %, and % of the donors, respectively. no reverse transcriptase inhibitor-resistant variants were detected. escape mutations in small hbsag protein shb region were detected in % ( / ), with the following main substitutions c ( x), r, n and g. the mean age of donors with active hbv infection was years, mostly donors were males ( %), mixed ( %) or white ( %) and had concluded high school ( %). summary/conclusions: discard rate due to isolated anti-hbc is high but no obi was found in the blood donor population studied. in addition, no case of immunological window for hbv or hbsagyield was detected. there was a predominance of subgenotype a and mutations associated with escape were found in % of hbv-dnapositive samples. continuous research and surveillance about hbv prevalence among blood donors are needed to maintain and evenly increase blood safety in brazil. background: screening for anti-hbcore antibodies in blood donors is considered to contribute significantly to blood safety, since it reveals donors with occult or probable occult hepatitis b, with variable results in molecular screening, due to very low viral load. however, universal anti-hbcore testing in blood donors, might exclude a considerable number of blood donors in countries with high hbv prevalence and even in countries with low to moderate prevalence, like greece. aims: the aim was to investigate the percentage of blood donors with natural hbv infection (confirmed positive anti-hbcore) or hbv immunization due to vaccination (anti-hbs+ only, due to vaccination) and predict the impact of generalized anti-hbcore testing. methods: during the period - november , all blood donors were asked to give their consensus for additional screening for hepatitis b anti-hbcore and anti-hbs antibodies, besides the obligatory serological and molecular screening, the samples of few donors who disagreed, were not examined. all samples with repeated positive anti-hbcore results, were further examined for anti-hbcore igm and anti-hbe antibodies. furthermore, a new donor sample was requested, to confirm reactivity. the serology results were recorded in an excel spreadsheet. additional data, including age, sex, nationality, number of previous blood donations, abo blood group, family history of hbv infection, hbv vaccination, were also recorded and statistically evaluated. donors were informed of the positive results. results: a total of edta samples were tested using the architect anti-hbcore, anti-hbs, nti-hbcore-m and anti-hbe assays (chemiluminescent microparticle immunoassay (cmia). repeated anti-hbcore(+) occurred in ( . %) samples, among which ( %) were also anti-hbe(+), while anti-hbs was found > m iu/ml in ( . %), between and miu/ml in ( . %), and < miu/ml in ( %). among anti-hbcore positive donors, / were foreigners ( . %) and were greeks, while foreigners consisted , % ( / ) of donors examined. so, anti-hbcore was found positive in , % of foreigners ( / , all from countries with high prevalence for hepatitis b infection) and in , % of greeks ( / ). in total, ( . %) samples had anti-hbs > miu/ml (considered seroprotective for the donor). summary/conclusions: almost half of our blood donors ( . %) were immunized, by vaccination and ( . %) by natural infection. the incidence of natural infection was significantly higher in foreigners ( . % versus , %). if not all anti-hbcore+ donors, % with anti-hbs < iu/ml, might be potentially infectious, especially for immunocompromised patients. if we choose to screen all blood donors for anti-hbcore and reject those with positive results, regardless of the anti-hbs levels, we would probably lose a significant number of donors and jeopardize blood sufficiency. alternatively, we could reject only those with anti-hbs < or < , or choose to selectively screen pre-donation blood donors from countries with high prevalence of hbv infection. following this pilot study, the prevalence of immunization against hbv in large numbers of blood donors from various parts of greece, must be investigated, in order to decide whether to introduce such screening. aims: the aim of this study was to perform phylogenetic analysis of the donor samples with hcv found in the neighbouring villages to determine the nature of transmission. methods: altogether, approximately million blood donor samples were screened with anti-hcv immunoassay (architect anti-hcv, abbott gmbh, wiesbaden, germany) and reactive results were confirmed with anti-hcv line-immunoassays (inno-lia hcv score, fujirebio europe, gent, belgium). based on lia positivity, in samples an association of hcv infection was supposed, because the residence places of donors were in three neighbouring villages situated less than km to each other. pcr was positive in samples. from these samples, hcv sequencing and phylogenetic analysis were performed. fourteen hcv infected samples of general population and of ivd users were also included into the study. results: phylogenetic analysis detected genetic relationship among the hcv virus sequences in donor samples. the most abundant was the a subtype, and it formed two different groups on the phylogenetic tree. according to their genetic distance, a more distant mutual ancestor could be supposed. two samples with b subtype originated from the same village, and their difference was only nucleotides. three hcv from the ivd user control group showed close genetic relationship with the viruses detected in the donor samples. summary/conclusions: based on our phylogenetic analysis, hcv transmission in blood donors could be the consequence of the ivd use and the origin might be related to or primary human sources. during and , a significant increase in the hcv seroprevalence among the ivd users was observed, which was approximately threefold in the rural areas of hungary. our recent findings highlight the importance of the proper donor selection, which can identify the typical signs of the ivd use. moreover, enhancing awareness of blood donors with education is a further significant issue in order to reduce the risk of transfusion transmitted infections. abstract withdrawn. background: in china, the residual risk of transfusion-transmitted hcv has been declining since screening of blood donors for anti-hcv and/or hcv nat from . however, many high sensitivity reagent, using to test blood donors' samples, lead to false-positive results and donors loss. aims: this study intended to establish a donor reentry procedure for hcv screening reactive donors in china. methods: from march to december , there were blood donor samples which were screened reactive or belonged to "grey zone" by elisa and/or reactive by nucleic acid test(nat) at the local blood centers were collected from chinese blood centers. all these samples were sent to institute of blood transfusion (ibt) national reference laboratory where anti-hcv and hcv individual nucleic acid test (id-nat) were conducted. if the results were reactive for anti-hcv, then the samples were tested with a recombinant immunoblot assay (riba). results: based on this study, of donors in the study who could be classified into two categories for hcv status: ( . %) true positive and ( . %) false positive. a total of of donors lost to follow-up, their hcv status cannot be determined with certainty. based on these data, a reentry procedure for hcv screening reactive donors was proposed. summary/conclusions: based on our proposed donor reentry procedure for hcv screening reactive donors, a majority of screening false-positive donors ( . %) can re-entry safely. abstract withdrawn. background: providing safe blood for transfusion in sub-saharan africa (ssa) is a particular challenge due to a combination of factors; limited resources and infrastructure, suboptimal diagnostics and a high prevalence of the major transfusion-transmissible infections (ttis). average seroprevalence data estimates from the ugandan and kenyan blood transfusion services (bts) for hepatitis c (hcv) currently stand at . % and . % respectively. between january and december , in mbale (eastern uganda) the hcv prevalence amongst blood donors was an alarming . %. with no provision or funds for confirmatory testing, the bts are unable to confirm or refute a diagnosis of active hcv. this results in large quantities of blood wastage, unnecessary anxiety in potential donors and high donor deferral rates limiting the donor pool. aims: we aim to determine the true prevalence of active hcv infection amongst seropositive donors in bts in uganda and kenya. in addition, we aim to compare the performance of locally used serodiagnostics and best available alternative tests and to examine the feasibility of cost-effective additional or alternative tests to help provide accurate results on the infectious status of blood. methods: hcv seropositive blood samples from bts study sites (kampala, mbale, mombasa) will be re-tested using the local serology screening test (abbott architect anti-hcv), an alternative who pre-qualified rapid antibody test (sd bioline) and a confirmatory test (hcv core antigen test). where there is discrepancy in the results or need for clarification, samples will be tested on the cepheid xpert platform by reverse-transcriptase pcr to obtain a quantitative rna result. s/co (specimen to cut-off) values for false positive samples (by screening serology) will be analysed and presented. pre-analytical factors (centrifugation speed, haemolysis check, time delay between collection and testing) will be controlled for and documented. results: pilot data from re-testing quarantined hcv seropositive donor blood (mbale bts) in uganda demonstrated that / seropositive blood ( %) was rna pcr negative. in december , / ( %) of seropositive samples (by screening anti-hcv serology) in kampala bts had s/co values between . - . ( . is the cut-off indicating a positive sample). data from the re-testing of seropositive samples as true representation of active hcv will be demonstrated and s/co values for the study period concomitantly with a retrospective analysis of january to december . preanalytical factors, cost analysis comparisons of the diagnostic platforms coupled with costs of the donor deferral process in false positive cases will be presented. summary/conclusions: for the bts in ssa there are significant resource and financial implications, as repeat testing and donor deferral counselling is required. evaluating and introducing new and appropriate diagnostics and algorithms in the screening of hcv is crucial in improving the supply of safe blood transfusion services in east africa. background: in november , the blood services of england, scotland and wales reduced donor deferral to three-months for commercial sex workers and individuals with higher risk sexual partners, including sex between men. the change was recommended after a detailed review by an external expert committee (sabto) which recommended that a shortened deferral of months would allow detection of recently acquired infection and maintain residual risk (rr) at a tolerable level. recommendations were accepted by government but with a government commitment to explore a more individualised approach. aims: to assess the impact of a -month deferral on blood safety in terms of epidemiology of infections in blood donors and compliance with donor selection criteria, and to explore evidence required to develop a more individualised approach to donor selection policy methods: routine uk blood donation surveillance data for - ( : preliminary) were reviewed. annual prevalence and incidence of hbv and hiv infection were estimated, with a poisson regression models to test for trends. incidence was calculated from donors seroconverting within -months, and/or microbiological and clinical evidence of recent infection. for donors positive in , compliance with the -month deferral was determined. uk hemovigilance data were scrutinised for evidence of transfusion transmitted infections (tti) associated with newly eligible donors. results: from to among new donors, annual hiv prevalence decreased significantly by an average of . % each year (p = . ) to . / , donations in ; no significant trend was observed for hbv. annual hiv incidence among repeat donors also decreased significantly by an average of . % each year (p = . ) to . / , -person years (pyrs) in (based on seroconverters). there was no significant trend in hbv incidence over the study period, however between and incidence increased from . / , pyrs to . / , /pyrs (based on and seroconverters respectively). with the information available to date, none of the seroconverting donors were non-compliant, and there was no reported confirmed ttis associated with the policy change. summary/conclusions: hiv prevalence and incidence has continued to decline. hbv incidence in repeat donors increased in although initial analysis suggests this is not associated with the policy change. monitoring continues, and residual risks will be re-estimated as data post-change accumulate. these data are reassuring, and therefore it is appropriate to scope the evidence for, and feasibility of, a more individualised approach to selection policy. a multidisciplinary steering group has been convened including representation from patient and stakeholder groups. gaps in knowledge are being defined, and a package of work is in development under the project of fair (for the assessment of individualised risk), using the abo rdf for guidance. background: permanent deferral of men who have sex with men (msm), established in the s, primarily to minimise the risk of hiv transfusion-transmitted infections is increasingly challenged. accordingly, blood services in many countries have changed to time-based deferral. in canada, a -year deferral was implemented in , reduced to -months in ; a -month deferral is now being considered. aims: to estimate the risk of undetected hiv among screened blood donations under a -month deferral since last sex between men. methods: the applied model combines features of previously published english and canadian models to estimate hiv risk under a -month deferral. three scenarios varying hiv incidence, prevalence and non-compliance under a -month deferral were modelled. assumed constants were the hiv nucleic acid window period, testing procedure error rate and assay sensitivity. model inputs were incidence under the current -month deferral, calculated as hiv positive donors with a previous negative within months divided by number of person years, numbers of hiv positive donations, hiv positive msm, hiv msm incident cases and newly eligible msm donors (from donor surveillance and compliance surveys). the risk with a -month deferral was estimated for three scenarios, one determined "most likely", where msm donor non-compliance, hiv incidence and hiv positive donations do not change and msm newly eligible to donate are estimated from compliance surveys. this scenario is based on data from two sequential policy changes in canada. an "optimistic" scenario where non-compliance halves and a "pessimistic" scenario where msm hiv incidence, hiv positive donations, non-compliance and new msm donors double were also used. the median hiv residual risk was used as the final estimate. the uncertainty in this estimate was assessed with the . th and . th percentiles over the simulation ( % ci). results: incidence, per , donations, was estimated to be . , . and . for the "most likely" "optimistic" and "pessimistic" scenarios, respectively. for the month deferral "most likely" scenario, hiv residual risk was predicted to be in . million donations ( % ci: in , million to in . million). for the "optimistic" scenario, hiv residual risk was estimated to be in . million donations ( % ci: in , million to in . million). finally, for the "pessimistic" scenario, hiv residual risk was estimated to be in . million donations ( % ci: in , million to in . million). with these residual risk estimates, based on the number of donations in canada, one hiv infectious donation would be in inventory every years for the "most likely" scenario, every years for the "optimistic" scenario and every years for the "pessimistic" scenario. summary/conclusions: the risks of hiv entering the blood supply in canada for a -month msm deferral are predicted to be very low for all modelled scenarios, including a "pessimistic" doubling of hiv incidence post change. background: safety of blood and blood products is a major concern in pakistan. the prevalence of transfusion transmitted infections among multi-transfused thalassaemia patients is high (above %). the hiv epidemic in pakistan is following the asian epidemic model where after establishment among the high risk groups, its transmission to general public is rapid. fear, stigma and ignorance have contributed heavily to hiv transmission in pakistan. the hiv detection among blood donors is on the rise and reports occur in media repeatedly. aims: to investigate the possible transmission of hiv through blood transfusion in punjab, pakistan and to highlight the steps being taken to reduce further transmission of infections methods: in september , a report of hiv transmission through blood transfusion was reported in the media where a mother and her newborn acquired hiv after blood transfusion from a hiv positive donor (confirmed later). the case was referred to and investigated by the punjab blood transfusion authority (pbta). the pbta team took blood samples of both recipients (mother and her newborn) and the blood donor who was a family relative. the samples were tested by highly sensitive chemiluminescence immunoassay (clia). the clia results confirmed the presence of hiv in both recipients and the blood donor. due to maternal hiv antibodies transfer through the placenta, the infection status of the newborn was not re-confirmed as he died within two weeks. the donor informed that he had donated times in the past few years. the pbta was able to trace only one earlier donation three months ago. the recipient (a female) was found, tested by clia and was found to be hiv positive. all these cases occurred in unlicensed private blood banks that were screening for hiv on rapid manual devices. the blood banks were sealed by the authority and infected cases were registered by the provincial aids control programme and are being treated. summary/conclusions: the main reasons for hiv spread through blood transfusion is the use of sub-standard rapid screening devices which are not evaluated and validated at a national level. in addition, the existing system relies on the family/replacement donors. the national safe blood transfusion programme, is implementing blood safety system reforms as recommended by who. under the reform agenda, the blood transfusion authorities have been made functional and grant licenses to only those blood banks with proper systems to ensure quality and safety of blood products. the programme is developing a national system for the evaluation, selection and validation of all assays used for screening of blood in close coordination with the drug regulatory authority of pakistan. to promote the culture of voluntary blood donations, the programme has taken concrete steps initiating with the formulation of a national blood donor policy, interaction with celebrities, celebration of world blood donor day and more recently the launch of blood donation feature through 'facebook'. the promotion of voluntary blood donation concept along with regulation of blood sector will reduce the risk of hiv transmission through blood transfusions in pakistan. mianyang blood center, mianyang urumqi blood center, urumqi, china rti international, rockville national heart, lung, and blood institute, bethesda stanford university, stanford, united states background: the incidence of hiv infections has increased substantially over the past decade in china, especially among young people, who represent nearly half of the chinese blood donor population. this upward trend in hiv infections underscores the importance of monitoring hiv prevalence and incidence in chinese blood donors. aims: to estimate hiv prevalence and incidence rate (ir) among chinese blood donors using blood donation data from five geographically-disperse blood centers in - participating in the recipient epidemiology and donor evaluation study-iii (reds-iii) china program. methods: western blot confirmatory testing was done on samples of blood donations reactive for hiv- / on one or both rounds of routine elisa tests or positive by nucleic acid amplification testing (nat). multiple imputation was used for samples with missing confirmatory test results. hiv prevalence was calculated among first-time donors. to estimate hiv ir in first-time donors, single-well lag-avidity eia testing was conducted with first-time hiv recent (incident) infections defined as being infected within approximately days based on avidity of hiv antibodies. a novel model was derived to estimate hiv ir among infrequent repeat donors who had provided only one donation in the - estimation interval. to derive an overall hiv ir for repeat donors, this estimate was combined with the classical-model ir estimated for repeat donors who had given at least donations in the estimation interval. multivariable logistic regression model was used to examine factors associated with hiv infection. results: a total of , , whole blood and apheresis platelet donations with postdonation screening results were collected at the five blood centers between and , including , donations from first-time donors and , donations from repeat donors. hiv prevalence among first-time donors was . per , donors ( % ci, . - . ). hiv ir was estimated to be . per , person-years ( % ci, . - . ) among first-time donors and . per , person-years ( % ci, . - . ) among repeat donors. hiv prevalence and ir varied across regions with an increasing trend observed at some blood centers. among first-time donors, being male, older than years, minority ethnicity, less than college education, and certain occupations (commercial services, factory workers, retired, unemployed, or self-employed) were associated with positive hiv confirmatory testing results. summary/conclusions: although hiv prevalence and incidence remain low among chinese blood donors, it is important to monitor hiv epidemiology in blood donors on a continuous basis, especially among populations and regions of higher risk. further donor screening and education strategies need to be developed and evaluated to reduce these risks. the ir methods used in this study for first time donors as well as repeat donors who donate very infrequently is readily applicable to other countries who have similar donation patterns. background: in thailand, the national blood centre is responsible for blood donation service which includes follow-up and blood donor counseling in order to indicate the infection status, especially hiv-positive blood donors. currently, although the epidemic of hiv infection in thailand is in decline, the hiv-positive cases still have been found in blood donors screening. thus, monitoring of hiv infection status in blood donors and post-blood donor counseling are important for providing the hiv-positive infected donors lead to access the hiv treatment immediately. aims: to study the hiv follow-up cases on serological testing over years for assessment of the hiv infection in thai blood donors. the retrospective analysis of hiv follow-up cases on serological testing (cmia, ics and western blot) was conducted during to at thai national blood centre. results: a total of , , voluntary blood donations over years, the repeated reactive results on hiv serological screening were , ( . %) cases and only half of these hiv reactive donors returned to follow-up testing for ascertaining their hiv status. for hiv follow-up process, the hiv reactive screening donors must be followed for months and tested by using the different three principles of hiv serological testing. a total of , hiv reactive results were separated to three patterns including hiv positive results, inconclusive results and negative results which the number of each group was , ( . %) cases, ( . %) cases and , ( . %) cases respectively. out of , hiv positive results, we found that , ( . %) cases were positive with all hiv serological testing for the first-time follow-up and ( . %) cases were tested and become to positive results after follow-up more than one time. in cases of inconclusive results, ( . %) cases were reactive only or testing(s) which these donors did not return to confirm again leading to temporarily deferred donors in blood donor system. in addition, ( . %) cases of inconclusive results could not conclude the hiv result although they were repeated several times. for the last pattern, , negative results cases showed ( . %) cases were negative results after follow-up over months while ( . %) cases were inconclusive results before changing to negative results which almost cases of this group were reentry as blood donor after deferral period is over. summary/conclusions: the number of repeated reactive results on hiv screening was constant over years of which returned to follow-up only half of hiv reactive donors leading to accumulation of temporarily deferred donors in blood donation system. hiv follow-up positive cases were informed and counseled immediately then referring to anonymous clinic for treatment. the problem and challenges of hiv follow-up were inconclusive results that were unclear and some of these did not return to retest lead to loss of re-entry donor who might be changed to negative result afterward. hence, the effective counseling and follow-up system need to be taken urgently to encourage the temporarily deferral donors returned to retest for reducing stigma of deferred donors in hiv follow-up cases. . we only analyzed the information that had non-reactive results for infectious markers reported by blood banks to sihevi-ins©, because they represent a risk for blood recipients. results: when loading the information of sivigila in sihevi-ins©, donors were found ( % men); of these people donations were obtained ( % whole blood). donors ( %) had a reactive result for hiv being subsequently reported in sivigila. in addition, five of them were reactive simultaneously for hbv in blood banks and took on average ae days to be reported in sivigila. donors ( . %) had an hiv reactive result notified by sivigila and subsequently they were reactive in blood banks. this behavior may suggest an attempt to spread the disease. donors ( % men) despite being initially reported in the sivigila database, presented a non-reactive result in a blood bank for hiv; one of them was reactive for syphilis and hbv and only one for hbv. this pattern may suggest false positive or negative results in one of the two databases analyzed. fourteen donors had negative test in blood banks for hiv and in a range of up to months they were reactive by sivigila ( % of them donate whole blood). this conduct may suggest that accepted donations were in a window period and therefore warrant further investigation. considering that two blood components could be obtained on average from each donor, a potential risk is estimated for recipients. summary/conclusions: the donors reported first in the blood banks through sihevi-ins© and later in sivigila allow to estimate an adequate orientation to the health services. the information from general epidemiological surveillance programs could improve the selection of donors and transfusion safety. background: it is assumed that bacterial contamination of blood products most often takes place during the donation process. the number of bacteria at this time point is estimated to be around - cfu per bag. little is known about the growth behavior of different bacteria species in whole blood (wb) units during storage and the distribution of bacteria to the different blood products. aims: aim of the current study was to determine the growth of different bacteria species in contaminated wb units and to study the distribution of the bacteria to the different blood components. methods: whole blood (n = - per species) was inoculated with approximately cfu of different bacteria species (escherichia coli, klebsiella pneumoniae, pseudomonas fluorescens, staphylococcus aureus, staphylococcus epidermidis, streptococcus dysgalactiae, streptococcus pyogenes) and stored for to h at room temperature before centrifugation and separation into red blood cells (rbc), buffy coat (bc) and plasma. bcs from spiked wb were each pooled with random bcs to prepare plasmareduced platelet concentrates (pc). samples were taken from wb after storage and from the blood products (rbc, bc, plasma and pc) right after preparation, and the bacterial titer was determined. sterility of pcs was tested by bact/alert after seven days of storage. results: bacterial growth in wb varied remarkably between donations and bacteria species. the highest titers in wb were detected for the streptococcus species, whereas staphylococcus aureus, staphylococcus epidermidis, escherichia coli and pseudomonas fluorescens did not multiply. bacteria preferably accumulated in the bcs during separation, reaching titers of up to . cfu/ml in bcs and up to . cfu/ml in the corresponding pcs right after preparation. in total, out of pcs tested positive for bacteria at the end of storage. the results were dependent on the species used: e.g., / pcs tested positive after spiking with streptococcus pyogenes, while only / pcs tested positive after spiking with escherichia coli. bacterial contamination of rbc and plasma units was much less frequent and associated with higher bacterial titers in the parental wb units. summary/conclusions: the growth and distribution of bacteria during processing of wb into the different blood products is species-dependent and remarkably varies between donations. results: both patients were male ( yo and yo) with a history of acute myelogenous leukemia status-post haploidentical stem cell transplant. the patients were thrombocytopenic and underwent simultaneous transfusion of irradiated, non-pr, day platelets stored in platelet additive solution, from a single apheresis collection. the blood supplier's primary pre-release bacterial cultures were negative, and the on-site point of release secondary safety measure pan genera detection (pgd) testing was negative for both gram positive (gp) and gram negative (gn) organisms. both apheresis units also passed visual inspection prior to release from the blood bank. during transfusion, both patients displayed signs of septic transfusion reaction including rigors, fever, hypoxemia, tachypnea, tachycardia, and hypotension. transfusion reaction evaluations were initiated, and both patients were admitted to the medical intensive care unit and started on broad-spectrum antibiotics. gram stain of one platelet unit demonstrated gram negative rods (gnr) and gram positive cocci (gpc) in clusters, and the second platelet unit demonstrated gnr only. repeat secondary safety measure pgd testing of both units was negative for both gp and gn organisms. direct bacterial cultures of both platelet units grew both gnr and gpc identified as a. baumanii and s. saprophyticus after h of incubation. colonies on the initial bacterial plates were too numerous to count (tntc), and subsequent re-plating of the platelet units showed: unit : a. baumanii tntc and s. saprophyticus with . cfu/ml unit : a. baumanii . cfu/ml and s. saprophyticus . cfu/ml blood cultures collected from both patients became positive within h with gnr on gram stain, and both blood cultures ultimately grew both a. baumanii and s. saprophyticus. the primary pre-release cultures at the blood supplier remained negative. after days on antibiotics and pressors, both patients stabilized and were discharged home. the blood donor was interviewed, and he was well. no cultures were collected. summary/conclusions: this case documents failure of both primary pre-release bacterial testing and secondary on-site point of release pgd testing to identify two pathogenic organisms. a. baumanii and s. saprophyticus are unusual causes of septic transfusion reactions, and it is possible that these organisms have different limits of detection in the pgd assay compared to other organisms. rapid attention to clinical signs during transfusion and prompt initiation of antibiotics is critical for the management of septic transfusion reactions. we are currently evaluating ways to further reduce septic transfusion reactions, including increasing the utilization of pathogen reduced platelets. background: transfusion-associated infections due to the transmission of bacteria still represents a major risk in developed countries nowadays. despite the refrigerated storage of red blood cells (rbc), fatal reactions of patients receiving contaminated rbc units are repeatedly reported. in order to further increase the safety of blood transfusions, new strategies and measures have to be developed. in this context, transfusion-relevant bacteria reference strains can serve as a valuable tool for the validation, comparison and interpretation of these new developments. aims: conducting a collaborative study to establish the first repository for red blood cell transfusion-relevant bacteria reference strains. methods: six bacterial strains (serratia liquefaciens, serratia marcescens, pseudomonas fluorescens, listeria monocytogenes, yersinia enterocolitica a- and yersinia enterocolitica a- ) were distributed from the paul-ehrlich-institut to laboratories in countries for enumeration, identification and growth measurement in a -day interval for a total of days after low-count spiking of rbc bags ( - colony-forming units (cfu)/rbc bag). results: except for s. marcescens, all other strains showed good-to-excellent growth in rbc. s. liquefaciens, p. fluorescens, y. enterocolitica a- and y. enterocolitica a- achieved > cfu/ml at day and cfu/ml at day . growth of l. monocytogenes was lower reaching a maximum concentration of > cfu/ml at day . in out of laboratories, s. marcescens showed no growth at all. summary/conclusions: five of the six tested strains showed robust growth in rbc independent of donor variability and are promising candidates to be adopted as official rbc transfusion-relevant reference strains by the world health organization. background: the samplok sampling kit (ssk), itl biomedical, is used by nhs blood and transplant (nhsbt) for sampling of platelet components (pc) for bacterial screening using the bact/alert d system. inoculation of bact/alert bottles is performed immediately after sampling. validation of delayed inoculation, with retention of the sample within the ssk devices, would allow a contingency for transport to other screening sites in the event of an incident that prevented testing at the sampling site. ssk maintain a closed system for sampling of pc and are compatible with standard blood collection bags. a graduated chamber ( or ml) ensures that only the required sample volume is collected and an integrated needle allows inoculation into bact/alert bottles. aims: the national bacteriology laboratory, nhsbt, evaluated the impact of prolonged storage of pc samples in ssk devices with regard to bacterial viability and detection. methods: four reference species were assessed: staphylococcus aureus (atcc ), streptococcus agalactiae (atcc ), escherichia coli (atcc ), clostridium perfringens (atcc ). a pool and split method was used with apheresis pc suspended in plasma. units were screened using bact/alert d prior to spiking to prove the absence of contamination. pc were spiked with a single species (range - . cfu/ml) and tested on bact/alert with a ml inoculation into anaerobic and aerobic bottles (positive control). enumeration was performed to confirm the bacterial concentration. each unit was sampled using two ml ssk, which were held for a period of h at - °c. the process was repeated with a -h period. once elapsed, ml of each ssk was inoculated into an aerobic and anaerobic bact/alert bottle, one ssk per atmosphere per species and the remaining sample was enumerated. all bottles were incubated on the bact/ alert system for a maximum of days ( ae . °c) and subcultured if positive. results: positive controls had detectable growth by bact/alert, excluding aerobic bottles with c. perfringens. this was expected as it is an anaerobic organism. after the storage periods, all bottles had detectable growth by bact/alert. s. aureus showed an increase of . -log after h ( . to . cfu/ml) and . -log after h ( . cfu/ml to . cfu/ml). s. agalactiae increased by . -log after h ( . cfu/ml to . cfu/ml) and . -log after h ( . cfu/ml to . cfu/ml). c perfringens increased by . -log after h ( . cfu/ml to . cfu/ml) and . -log after h ( . cfu/ml and . cfu/ml). for e. coli, the concentration after h was reduced by . -log ( . cfu/ml and . cfu/ml), however this was possibly a result of inherent counting errors. at h, an increase in growth by . -log ( . to . cfu/ml) was obtained. summary/conclusions: storage of pc samples in ssk devices for up to h does not have a negative effect on bacterial viability and detection using the bact/alert d system. background: the intercept tm blood system for platelets efficiently inactivates pathogens and leukocytes in platelet concentrates (pc). the system utilizes amotosalen and uva light and is available for the treatment of apheresis and whole blood (wb) derived platelets (mostly buffy coat pools) in europe in plasma or platelet additive solution (pas), and the treatment of apheresis platelets in the us (trima tm in % plasma or amicus tm for % intersol pas/ % plasma). acinetobacter baumanii and staphylococcus saprophyticus strains were isolated from a saline flush taken h after successful and complete transfusion of an apheresis intercept-treated pc in % pas/ % plasma, from a patient with a suspected septic reaction that occurred h post transfusion. bacterial isolates, and a sample of a gram stain-negative and culture-negative sister split pc were submitted for evaluation. we report here the in vitro inactivation of the fast growing, gram negative bacterium a. baumanii and slower growing gram positive s. saprophyticus. the sister unit was assessed for amotosalen break down products as an indication of successful inter-cept treatment. aims: the objective of the study was to evaluate bacterial inactivation of a. baumanii and s. saprophyticus in apheresis platelets, assessed immediately after treatment and with re-culture at the end of a day shelf-life. methods: a double apheresis pc collected in % pas/ % plasma was split into three equal components, yielding approximately ml of platelets/pc. a. baumanii and s. saprophyticus were grown in lb broth and each pc unit was inoculated with either bacterial strain or the combination of both strains, each at~ log colony forming units/ml (cfu/ml). after inoculation, the contaminated units were treated in small volume (sv) intercept kits. samples were taken: pre and post-inactivation treatment, and at , and days of storage. the samples were analyzed by plating on lb plates ( ll- ml). residual amotosalen levels were assessed by hplc. results: initial bacterial titers were . - . cfu/ml. following the inactivation treatment, no viable bacteria were detected by plate culture. no bacteria were detected after , and days of storage, corresponding to > . log inactivation of both bacterial strains. performance of the intercept treatment process was confirmed in the sister pc unit as evidenced by levels of amotosalen and its byproducts characteristic of intercept treatment, as well as by review of the process documentation. summary/conclusions: amotosalen/uva effectively inactivated a. baumanii and s. saprophyticus individually and together below the limit of detection after days storage. no bacteria in the sister pc by gram stain and culture, and the presence of amotosalen byproducts suggested that the pc collection involved in the septic reaction was sterile at the time of intercept treatment and was successfully illuminated. the possibility of only one-of-two split pc being contaminated due to biofilm formation is minimized in the intercept system which decants platelets into virgin storage bags at the end of treatment. contamination of the source pc container likely occurred after intercept treatment, possibly at the time of spiking for transfusion. background: studies in sub-saharan africa have documented bacterial contamination of blood products for transfusion varying between , %> , %, up to times higher than in the north. published data from central africa are lacking. aims: the aim of this study was to determine the proportion of blood products contaminated with bacteria in three hospitals in the democratic republic of the congo (drc). to assure aseptic sampling, we used a closed system of sampling bags. in addition to the presence of contamination, we assessed semi-quantitative colony counts. methods: from july to december , a total of blood products were sampled, of which in hôpital provincial g en eral de r ef erence, kinshasa (hpgrk), in hôpital p ediatrique kalembe lembe, kinshasa (hpkll) and in hôpital saint-luc, kisantu (hslk). after compatibilization of blood products, ml of blood was transferred from the primary blood bag to an attached sampling bag. sampling bags were sealed off, stored in the fridge and transported once daily to the bacteriology laboratory. using the adapter connected to the sampling bag, ml of blood was inoculated in a blood culture (bactalertpf, biom erieux) and incubated at °c for days. cultures were checked daily for signs of growth. in addition, ml of blood was equally distributed on the cled and macconkey agar-coated sides of a dipslide (meus s.r.l.). dipslides were incubated h for semi-quantitative culture, expressed as colony-formingunits (cfu) per ml. in case of blood culture growth, bacteria were identified and a second blood culture was inoculated to exclude contamination during processing. bacteria grown on semi-quantitative culture were also identified. results: a total of . % ( / ) of whole blood and red cell concentrates were contaminated with bacteria. in hpgrk, . % ( / ) of blood products were contaminated, in hpkll . % ( / ) and in hslk . % ( / ) . the proportion of contaminated blood products was significantly higher in hpgrk compared to hslk (p = . ). there was no significant difference between the other sites (p = . and p = . ). majority of isolated bacterial species were coagulase-negative staphylococcus spp./micrococcus spp. ( . %) and bacillus spp. ( . %). the remaining % of bacterial isolates were identified as non-fermentative gram-negative rods, klebsiella pneumoniae, staphylococcus aureus, mould, listeria spp., corynebacterium spp./coryneform bacteria. the concentration of all isolated bacteria was lower than ³ cfu/ml, except for one coagulase-negative staphylococcus spp. found in hpgrk at ³ cfu/ml. summary/conclusions: to our knowledge, we were the first to reach a sample size of more than blood products for bacterial culture and the first to use a closed system of sampling bags in sub-saharan africa, which guarantees aseptic sampling of blood cultures. this might explain the low bacterial contamination rate ( . %) of blood products in three hospitals in drc compared to previous studies in other sub-saharan african countries. moreover, bacterial concentrations in the contaminated blood products were low (< ³ cfu/ml). the different proportions of contamination between study sites suggest that different environments and practices play a role in the risk for bacterial contamination. background: although the screening of the treponema pallidum (tp) is mandatory in blood donations, its necessity is controversial, because there have been no transmissions by blood products documented in the developed countries in the last few decades. aims: based on laboratory markers, active and early tp infected donors (aeid) were determined. the demographics of aeid and the frequency measures of cases were compared with that of early infected syphilis cases (syc) notified from the to -year-old general population registered at the nphc. methods: altogether, , to -year-old donors were screened with anti-tp immunoassay (architect syphilis tp, abbott, wiesbaden, germany) between and . reactive results were confirmed with immunoblots (viramed biotech ag, planegg, germany), which discriminated both specific anti-tp (igg, igm) and non-specific vdrl antibodies in five dilutions. meeting the criteria of anti-tp igg positivity with a vdrl titer of ≥ : and anti-tp igm positivity, donors were considered as aeid. they were stratified by age, gender and residence regions. the proportion of aeid (paeid) and syc (psyc) were calculated in first time (ft), and repeat tested (rt) donors and in the to -year-old general population, respectively, in each year studied. the period prevalence (pp) of aeid and syc was estimated in the populations at risk , across - . statistics: weighted proportions and one-way anova with tukey post-hoc test and z score test were applied. statistical significance was defined as p < . . results: anti-tp reactivity was confirmed in blood donors. aeid was proved in cases with ft and rt donors. in that period, syc were notified. both in aeid and syc, the age group of - years with approximately % and % of individuals was the dominant. the proportion of men was % and % (p = . ) in the aeid and syc, respectively. paeid estimated in ft donors was significantly higher ( . %; . %; . %, p < . ) than that of rt donors ( . %; . %; . %) and the proportion of syc ( . %; . %; . %) in the general population. pp of aeid showed a roughly homogenous distribution in the regions ( . %- . %), however, pp of syc had a significant ( . %; p < . ) central hungary dominance in relation to the other regions ( . %- . %). comparing the pp of aeid to syc, central hungary indicated a significant difference ( . % vs. . %, p < . ), however, other regions showed no substantial differences. summary/conclusions: donors with anti-tp confirmed positivity are referred to the healthcare system. based on the laboratory markers tested, aeid could be separated and their demographical characteristics are pretty similar to that of syc notified from the general hungarian population. the proportion of early and active infection in ft donors is significantly higher than that of rt donors and the proportion of syc in the general population. given the considerable number of tp infection in background: quality assurance and safety of hematopoietic stem cells (hsc) with emphasis on prevention of bacterial and fungal contamination are the prerequisites for any transplantation procedure. bacterial contamination is of particular significance as it occurs relatively more frequently and bacteria are gradually gaining more antimicrobial resistance. aims: the aim was to determine the incidence rate of bacterial and fungal contamination during processing of transplantation material at the institute of hematology and transfusion medicine (ihtm) taking into account the hsc sourceperipheral blood (pbsc), bone marrow (bm) or cord blood (cb). methods: analysis involved both autologous and allogenic components collected at ihtm and other hospitals and dedicated for ihtm patients. in all, the analysis comprised donations, including bm ( . %), pbsc ( . %) and cb ( . %) donations processed in our laboratory in the years - . bm was collected in operating theatre-conditions, pbsc with cell separators -cs- (baxter), cobe spectra (gambro) and trima accel (terumo bct) and cb was acquired from umbilical vein at obstetrics and gynaecology wards. aerobic and anaerobic bacteria contamination was determined at various incubation temperatures (room temperature and °c) using a variety of culture media. pbsc and bm were tested using samples with trypcase-soy broth (tsb-t) and with schaedler + vit k (biomerieux). cb was tested using bactec peds plus/f and bactec lytic/ /anaerobic/f (becton-dickinson). results: in the - period contaminated products were found: pbsc ( . % of all tested pbsc units) and cb ( . % of all tested cb units). no infected bm products were determined. the overall percentage of contaminated products was estimated at , %. in , three ( ) products were found contaminated with staphylococcus epidermidis; all came from one patient with central venous catheter and were collected on consecutive days. other products were contaminated mostly with staphylococcus epidermidis ( . %). detailed results to be presented on the poster. summary/conclusions: according to ihtm policy no contaminated product is admitted to clinical use. the outcome of our study identifies processing experience of the staff as the main indicator of product quality. important is also proper assessment of donor health and condition of the injection site as products are usually collected from central venous catheter. the closed system is an additional safeguard against contamination during processing. the sample collecting procedure should help to avoid false positive results. background: syphilis is considered a global public health problem. the world health organization (who) estimates that there are annually around million new cases of syphilis in the world, more than % occurring in developing countries. despite significant decrease in syphilis transfusion transmission. the recent increase in worldwide incidence associated with the risk of transmission through platelet concentrates (cp), which are stored at room temperature, have called attention to the potential residual risk of syphilis transmission by transfusion. between and we observed in our institution a significant increase of % in positivity of syphilis among blood donors from . % in to . % in and . % in (p < . ). aims: to determine the prevalence of active syphilis in blood donors and characterize the serological profile of syphilis positive donors. methods: each positive sample in a treponemic chemiluminescence assay (cmia, abbott architect) performed during blood donor screening in was submitted to a treponemic elisa anti-treponema pallidum igm (euroimmun) and a non-treponemic test (antigen-omega diagnostics). samples with positive results for one or two of these tests (indicating recent syphilis) were submitted to a real-time pcr for syphilis. the inno-lia syphilis-fujirebio immunoblot test was also performed for samples that presented a positive result for elisa-igm alone. financial support: fapesp / - . results: among , samples screened in , ( . %) presented a positive result for cmia -syphilis. of these, ( . %) were included in the study. a total of samples ( %) showed vdrl+/igm+; ( %) vdrl+/igmand ( . %) vdrl -/elisa igm+. the inno-lia syphilis test was performed as a confirmatory test in ( . %) samples that presented positive results for elisa igm and vdrl negative with ( . %) positive results, ( . %) undetermined and ( . %) negative. none of the samples showed the presence of treponema dna by real-time pcr. the prevalence was . %, the incidence was . % in the year , and the incidence in relation to the total positivity was . %. both, prevalence and incidence were higher in men, white, not married, aging - years and high school educational level. we observed a % a-hbc seroprevalence in the elisa igm-syphilis positive samples and a prevalence of . % htlv coinfection. summary/conclusions: we observed a significant increase in prevalence of syphilis in ( . %) with an incidence of . % of the total of cases initially positive in the cmia test. according to our data, we could identify a risk of syphilis transfusion transmission in blood banks that exclusively use the vdrl for donor screening, once we found ( . %) cases with negative vdrl and elisa igm and inno-lia positive. continuous monitoring of the profile of donors infected with syphilis at this time of reemergence of the disease is useful and important not just for blood banks, as it reflects the epidemiological situation of disease in community, and can contribute to the definition of health policies. background: transfusion related sepsis is a serious concern limiting platelet storage time to days at room temperature. while most units are screened for bacterial contamination when collected, bacterial monitoring methods can take up to days to detect contamination. thus, cold storage of platelets represents an attractive alternative for improving platelet safety. in this study, we assessed bacterial growth in platelets stored either at room-temperature (rt; °c) or refrigerated (cs; °c). aims: the aims of this study were to ) assess the effect of storage temperature on platelet function and bacterial growth in "contaminated" platelet units, and ) identify factors contributing to bacterial growth during rt storage. methods: apheresis platelets in plasma (plt) were obtained from healthy donors using the terumo trima accel automated blood collection system (terumo bct). fresh plasma (fp) was collected similarly. aliquots of plt or fp were transferred to ph safe minibags (blood cell storage, inc) and "contaminated" with acinetobacter baumannii, escherichia coli, pseudomonas aeruginosa, staphylococcus aureus, staphylococcus epidermidis, or pbs (uninfected control). minibags stored at rt were agitated using an orbital shaker set to rpm while cs aliquots were stored under static conditions. bacterial growth was monitored daily through dilution plating. lactate levels were assessed by istat (abbott) cg + test cartridges. plasma glucose levels were assessed using blood glucose testing strips (germaine laboratories). platelet activation and aggregation were assessed on days , , , and by flow cytometry and multiplate platelet aggregometry, respectively. results: bacterial growth progressed rapidly over the first - days post-collection in all plt aliquots stored at rt except those challenged with s. epidermidis. significant growth of s. epidermidis was not detected until day . bacterial numbers remained unchanged in refrigerated aliquots through day . rt storage resulted in significantly (p < . ) decreased platelet aggregation over time which was exacerbated by bacterial challenge. plt function was largely preserved with refrigeration regardless of challenge. bacterial growth was significantly reduced, or at least delayed, in fp. fp challenged with gram-negative pathogens exhibited a significant (p < . ) delay in bacterial growth at day . while growth of e. coli and p. aeruginosa recovered by day , growth of a. baumannii was significantly (p < . ) inhibited throughout. fp challenged with gram-positive pathogens exhibited significant (p < . ) reduction in bacterial growth relative to plt aliquots. bacterial growth correlated with plt lactate production. lactate levels in plts challenged with e. coli showed diminished significantly after day , indicative of lactate utilization. with exception of fp challenged with s. aureus, bacterial growth was restored in fp supplemented with lactic acid in all challenge groups. summary/conclusions: refrigeration preserved platelet function while both inhibiting bacterial growth and lactate production. conversely, the opposite was observed with rt storage. these data demonstrate that bacterial growth can be controlled through refrigeration without loss of function and rt storage may potentiate growth of certain bacterial strains through accelerated plt metabolism. background: bacterial contamination of peripheral blood progenitor cell (pbpc) for transfusion has been the cause of serious sepsis and life-threatening infections. however, a standard procedure or choice of test sample(s) has not been established to screen pbpc products for microbial contamination, because these products are not large enough to facilitate inoculation of the recommended volume for the automated blood culture systems. samples taken from by-product plasma and low volume pbpc product were cultured in routine sterility test. aims: to evaluate the residual risk of microbial contamination in pbpc products for transplantation, we cultured sufficient post-thaw inoculation volumes from pbpc products which were discarded for various reasons in our blood center. methods: in routine sterility test, a -ml sample of by-product plasma collected with pbpc product was inoculated into bact/alert bpa and bpn culture bottle ( ml each) within h after collection. the bottles were then placed in the bact/ alert system and incubated for at least days or when a positive reaction was indicated by the automated liquid-media culture system. moreover, a -ml postthaw sample would be cultured before transplantation performed. in the residual risk investigation, discarded pbpc products were thawed, and then a -ml and a -ml aliquot were taken and cultured with the same method. all positive bottles were subcultured for bacterial isolation and identification. results: in september and march , after maintaining in liquid nitrogen for to years, pbpc products collected from patients, which was preserved in a volume between and ml, were discarded. all of these products had been cultured negative in routine sterility tests with plasma samples. these final products were thawed and cultured with both the -ml and the -ml aliquot. one of these pbpc products had the positive culture result with the -ml retested samples. nevertheless, the same pbpc product had the negative result with the -ml post-thaw pbpc sample and the -ml by-product plasma sample. propionibacterium acnes was isolated from the bpn positive bottle. summary/conclusions: the residual risk of microbial contamination in pbpc postthaw products still exist after routine sterility test with the plasma sample and the -ml volume of pbpc sample. the bacterium isolated from pbpc product was normal skin flora bacterium. an optimal screening method of pbpc products merits further study to increase the safety of the blood supply. background: hospital hygiene tools that serve as a proxy for assessment of microbial contamination are increasingly used. they include adenosine triphosphate (atp) bioluminescence and air particle counting. however, their use for microbial monitoring of blood banks remains underexplored. they could be of particular interest in a sub-saharan african setting (temperatures, dust) to circumvent bacterial culture and provide direct results usable for monitoring over time. aims: the aim of this study was (i) to quantify environmental bacteria in the air and on surfaces that are regularly in contact with blood products, and (ii) to evaluate atp bioluminescence techniques and particle counts as a predictor for bacterial contamination, in three blood banks in the democratic republic of the congo (drc). methods: samples were taken in three blood banks in the democratic republic of the congo: hôpital p ediatrique de kalembelembe (hpkll) ( surfaces, air), hôpital provincial g en eral de r ef erence (hpgrk) ( surfaces, air) and the national blood transfusion centre (cnts) ( surfaces, air). surfaces that are regularly in contact with blood products were selected (sealer, fridge, donor chair,. . ..). regular surfaces were sampled using rodac contact plates ( . cm²) containing cled and macconkey agar, irregular surfaces using swabs (nrsii, medicalwire). atp was measured on the same surface (pd , kikkoman), expressed as relative light units (rlu) per cm². air was sampled by active sampling ( liter; spinair, iul) on cled and macconkey medium. in parallel, particles > . lm and > lm were counted using a particle counter ( , liter; metone a). culture media were incubated for h at °c before counting colony forming units (cfu). results: for regular surfaces, the median (range) viable bacterial count was ( - ) cfu/rodac, ( - ) cfu/rodac, ( - ) cfu/rodac for hpkll, cnts and hpgrk, respectively. at hpkll, highest viable counts were found in the sink (plain growth) and cool boxes ( and cfu/rodac). in cnts the blood processing bench, the donor chair arm support and washing basin showed the highest counts (plain growth). whereas in hpgrk, most bacteria were found in a fridge (plain growth), blood bag trolley (plain growth) and manual separator ( cfu/ rodac). gram-negative bacilli were isolated from water basins and sink in cnts and hpkll, but also surfaces close to donor chairs at hpgrk. the median (range) of atp per cm² was . ( - . ) rlu at hpkll, ) rlu at cnts and . ( . - . ) at hpgrk. atp results and total viable count were not correlated (n = , p = . ). median (range) bacterial count in the air was ( - ) cfu/ l for all sites together. there was no correlation found between total bacterial count and particles > . lm or > lm (r = . and r = . respectively; p < . ; n = ) summary/conclusions: total viable bacterial count of surfaces varies over blood bank sites. according to our results, atp and particle counts did not correlate with bacterial counts on surfaces and in the air, respectively. bacterial isolates from blood bank environments in drc need to be identified and seasonal variations need to be evaluated. background: the risk of transfusion-transmitted hepatitis e virus (tt-hev) infections in line with the question of the necessity of hev-nat screening of blood products is currently subject to an ongoing debate on the importance of timely introduction of hev screening of blood donors and the impact of blood safety. different countries have chosen different regulatory approaches. just recently, the german federal authorities have introduced mandatory testing of all therapeutic blood products beginning from january st . however, we already decided to voluntarily test all our blood products since january . aims: in this study, we present our results of a % screening of therapeutic blood products for hev rna including four years of active surveillance of hepatitis e infection among blood donors in germany. methods: from january to december , a total of , allogenic blood donations from , individual german blood donors were screened in a minipool format of samples of for the presence of hev rna (realstar hev rt-pcr kit, altona diagnostic technologies (adt), hamburg, germany). nucleic acids were extracted from . ml plasma using the chemagen msm-i extractor (viral k, perkin elmer chemagen gmbh). the % lod of the assay was determined to . iu/ml ( iu/ml per single donation). the presence of hev-specific igm and igg antibodies was determined using the anti-hev igm/igg elisa (euroimmun, luebeck). hev rna concentrations were quantified using the first who international standard for hepatitis e virus rna for nat-based assays. all hev rna positive donors were deferred from donation for months. follow-up samples were tested for the presence of hev rna and hev-specific antibodies. genotyping was performed by sequencing of the hypervariable region (hvr) and orf . results: in total, hev rna positive donors were identified. of these, hev rna-positive donors, were nat-only positive donations ( . %, negative for anti-hev igm and anti-hev igg), three donors had a positive igm titer ( . %), donors showed reactive igm and igg titers ( . %), donors already had isolated igg titers ( . %). median values of viral loads were approximately twice as high in index donations that were antibody negative. merely donors showed elevated alt levels ( . %), mostly within a double increase within the reference range ( . %), only . % of donors had even further elevated alt levels. significantly higher alt values were found in donors with a viral load > , iu/ml compared to the group with viral loads between and iu/ml. available follow-up samples confirmed igg seroconversion for all donors, however we also observed long-term igm positivity in some donors. genotyping revealed genotype in all cases. the month-dependent incidence ranges from : to : , blood donations with a peak in june and july. summary/conclusions: the high number of identified hev rna positive donors emphasizes the need for hev-nat screening to increase the safety of blood products. this study further confirmed that hev infection is common in german blood donors. background: zika virus (zikv) is a mosquito-borne virus that has caused outbreaks in central and south america in february , and has threatened the safety of blood transfusion globally. there is a high risk of zikv transmission by whole blood and blood components transfusion. it was reported that, zikv rna in infected patients plasma can only be detected within to weeks. however, in whole blood, zikv rna might present positive up to day after the symptoms appear in some patients, even if the clinical symptoms disappeared with zikv rna negative in plasma. this phenomenon suggested that the presence of zika is associated with red blood cells (rbcs). moreover, another report showed that viral load in whole blood of type a west nile virus (wnv) patients was higher than type o, implying that the binding of virus to rbcs may be related to blood group glycoprotein. both of zikv and wnv are member of the flavivirus genus. the study is intended to explore whether zikv have the same adherence mechanism to rbcs as wnv. aims: to investigate the distribution of zikv in blood components and adherence of zikv to different blood types of rbcs in whole blood. methods: five units for each blood type of a, b, o and ab whole blood were randomly selected. each unit of ml whole blood was divided into two half-unit. zikv was added to one half-unit in a certain proportion, and incubated at °c for days. each component of whole blood was collected for viral load detection. in the other half-unit,rbcs were suspended in the same type pools of plasma with equal volume after the plasma removed from the whole blood after centrifugation. zikv was added with the same certain proportion, and then incubated at °c for days. the whole blood samples and the upper plasma by centrifugation were collected detected for zikv rna. meanwhile, rbcs were washed and resuspended with normal saline followed by viral load detection. results: zika rna of these samples which extracted from whole blood, rbcs, and plasma were determined in a quantitative reverse transcription pcr, and viral rna of each component was all up to copies/ml. although, zikv rna loads did not show significant difference in distribution between rbcs and their corresponding plasma components, zikv rna quantification were significantly higher than those in plasma (p < . ) in type o rbcs and lower than those in plasma (p < . ) in type ab rbcs. summary/conclusions: in our study, we detected high viral rna loads in rbcs. it was demonstrated that zikv adheres to erythrocyte in whole blood, and the blood type may have influence on the adherence. background: hong kong is not endemic for dengue virus (denv) with most of the documented cases being imported. the presence of sufficient number of mosquito vectors, aedes albopictus, in the territory has led to two self-limiting indigenous outbreaks affecting residents from to . during august to september , another outbreak of confirmed cases of autochthonous dengue fever were reported to the department of health, linked to two epidemiological clusters, one in lion rock park near wong tai sin (wts) district ( cases) and the other in cheung chau, an outlying island ( cases). aims: we assessed the risk of dengue transmission from blood donors during the outbreak using a simplified version of the probabilistic model developed by biggerstaff and petersen (b-p) and the european up-front risk assessment tool (eufrat) model (oei, transfusion, ) . methods: patient demographic and general population data were obtained from the centre for health protection and the department of census and statistics of the hong kong government for the number of -to -year-old patients in the outbreak and residents of the same age range in hong kong and wts district as at mid- respectively ( - years old being the eligible age range for first time donation). to apply the b-p model, we estimated denv incidence among donors in hong kong territory and in wts with confirmed denv infection during august to september after correction for clinical:subclinical infections ratio, the mean length of asymptomatic viraemia and the probability of collecting blood from asymptomatic donors as described previously (seed, transfusion, ). to estimate the risk using eufrat model, outbreak and blood donation variables were entered into eufrat's web-based interface (https://eufrattool.ecdc.europa.eu/), which provided automatic calculation of risk-related output parameters. results: while using the b-p model, the estimated risk of collecting a denv viraemic donation was one in , ( , - , , ) territory-wide for the -day study period but increased to one in , ( , - , ) in wts. similarly while applying the eufrat model, the risk of encountering a viraemic donor was in , ( , - , ) territory-wide and in , ( , - , ) in wts during the same period. the eufrat also predicted a territory-wide issue of . unit of denv-contaminated labile blood component during the outbreak period. summary/conclusions: like many mosquito-borne infections such as denv, the risk is characteristically localised and varies geographically and seasonally during outbreaks. the average predicted risk of collecting a denv-viraemic donation territory-wide is low at in , during the outbreak based on the b-p model, which was generally considered as tolerable. however, the risk increased by folds when blood donations were collected from wts residents, who had higher chances of paying visits to lion rock park in close proximity. it was then justifiable to institute risk mitigation policies such as geographically-based deferral and/or fresh component restriction, enhanced post-donation reporting, etc. to protect against blood safety. background: hev is a developing threat to blood safety following the reporting of several cases of transfusion transmission hev (tt-hev). transfusion-related hev infection has been reported in several countries but its true frequency is probably underestimated because it is often asymptomatic and testing of blood donors is infrequent. pakistan is classified as a highly endemic region; with sporadic cases of hev occurring throughout the year, mainly affecting the adult population. to the best of our knowledge, no studies have been reported from pakistan on the epidemiology of hev in blood donors. aims: to assess the epidemiology of the hev specific antibodies and serum alt levels in blood donors of capital twin cities of pakistan. methods: this cross sectional study was conducted from july to december at three blood banks in the capital twin cities (rawalpindi and islamabad) of pakistan. the blood donors were equally distributed across the three blood banks. only donors who tested negative for hiv, hbv and hcv were included in the study. serum alt levels were analyzed by using automated clinical chemistry analyzer (selctra pro m) using merck kits. all samples were tested for hev-specific antibodies (igm and igg) by using enzyme linked immunosorbent assay (elisa) kits (adaltis, italy). statistical analyses were performed using spss software version . (ibm). results: in our study population there were ( . %) males and ( . %) females. the mean age of recruited blood donors was . (sd ae . ), with a range of - . younger donors were more common with a frequency of - year olds of ( . %). we found an overall hev igg prevalence of . % and an hev igm prevalence of . %. there were ( . %) blood donors who were positive for both igg and igm antibodies. our results revealed that the hev specific antibodies (igg, igm) prevalence increased with age. the mean value of serum alt was . (sd ae . ) with a range of - iu/l. the serum alt levels were elevated (> iu/l) in ( . %) blood donors. there was significant correlation (p=< . ) between serum alt level and hev specific antibodies for igg and igm. summary/conclusions: this study shows that a significant proportion of blood donors at our blood centers have been infected with hev and may be able to cause tt-hev. as we have not yet measured hev rna, we have used igm antibodies as a proxy for donors who have active infection. hev is generally asymptomatic, so it is debatable whether mandatory hev screening in blood donors should be required. results of this pilot study show that there is a need to conduct a larger study at national level with highly sensitive assays before making screening for hev mandatory in pakistan. background: hepatitis e virus (hev) is a zoonotic virus. who estimates that there are million hev infections, million acute hev cases and thousands hevrelated deaths worldwide each year. in recent years, the prevalence of hev in european and american countries has increased significantly. the survey results show that the positive rate of hev igg in blood donors is respectively . % in new zealand, . % in britain, . % in denmark, % in the united states and . % in the netherlands. hev has become a global public health concern. in addition to the food route of infection, several cases have been reported that hev can be transmitted via blood products. aims: to investigate the prevalence of hepatitis e virus (hev) infection among voluntary blood donors and potential impact on blood safety in guangzhou china. methods: blood samples from blood donors were collected from april to april at the guangzhou blood center and were tested for anti-hev igg antibody (hev igg), anti-hev igm antibody (hev igm) and hev antigen (hev ag)by enzyme linked immunosorbent assay (elisa). hev rna detection was performed on hev antigen positive samples by rt-pcr. the association of age, gender, ethnicity, occupation and alt with hev igg and igm were analyzed by chi-square test. multivariate logistic regression analysis was applied to identify the independent risk factors of hev infection. results: the positive rates of hev igg, igm and hev ag were . % ( / ), . % ( / ) and . % ( / ), respectively. no positive hev rna was detected. age and ethnicity were independent risk factors for hev igg and hev igm. the rate of hev antibody increased significantly with age (igg or = . , p < . ; igm or = . , p < . ). donors who were zhuang minority ( . %, . %) showed higher anti-hev than those who were han ethnicity ( . %, . %), and the difference was statistically significant (igg or = . , p = . ; igm or = . , p < . ). in addition, we found that occupation was an independent risk factor for hev infection, where students showed the lowest anti-hev rate. summary/conclusions: the results indicate that the positive rate of hev antibody among blood donors in guangzhou is high, and the infection status differs in different populations. our study provides basic data for the estimation of risk of transfusion-transmitted hev. background: human cytomegalovirus (hcmv) belongs to the viral family of herpesviridae. it is an enveloped double-stranded dna virus, widely distributed in the human population ( - % seropositive subjects worldwide) and cause of severe disease in immunocompromised patients and upon infection of the foetus. in normally healthy subjects, hcmv persists lifelong without clinical manifestation undergoing alternating phases of active viral replication and latency. since hcmv can be readily detected in blood, as free virus as well as associated to neutrophils and monocytes, hcmv transmission is a complication of blood transfusion. even though leukoreduction of blood products has been shown to significantly reduce the risk of hcmv transmission, higher inactivation standards may be required for high-risk, immunocompromised groups of patients. aims: in this study, murine macrophages infected with murine cytomegalovirus (mcmv) were used as a model to study the inactivation cell-associated cmv in human plasma using the theraflex mb-plasma system (macopharma). methods: mcmv expressing the green fluorescent protein was used to infect murine macrophages. infected macrophages were harvested h after infection, washed and used for spiking of plasma. plasma units (n = , ml) were spiked with infected cell suspension ( % v/v) and treated with the theraflex mb-plasma system according to the manufacturer's protocol using the macotronic-b illumination device (macopharma). samples were taken after spiking (load and hold sample), after illumination with different light doses ( after addition of mb, , , and [standard] j/cm ) and after blueflex filtration. mcmv titers were determined by endpoint titration and large volume plating on murine fibroblasts. infectious virus, which expressed gfp in infected cells, was detected using a fluorescence microscope. results: the results of infectivity assay showed that the treatment of human plasma by the theraflex mb-plasma system inactivated cell-associated mcmv in a dosedependent manner. after spiking with mcmv infected macrophages a mcmv titer of . (bag no. ) and . (bag no. ) log tcid /ml was achieved in the plasma units. in hold samples, a mcmv titer of . (bag no. and bag no. ) log tcid /ml was determined. the illumination step of the theraflex mb-plasma treatment procedure efficiently inactivated mcmv. already three-fourths of the standard light dose decreased infectivity of cell associated and remaining cell-free mcmv to infectivity levels below the limit of detection (≥ . log). further investigations would be needed to evaluate the log reduction capacity of the blueflex filtration step for cell-associated mcmv. summary/conclusions: the results with the murine model virus suggest that the theraflex mb-plasma system is an effective technology to inactivate cell-associated cmv in human plasma units. background: the use of pathogen inactivation (pi) technologies for platelet concentrates and plasma is slowly but steadily increasing. methods for treatment of red blood cells (rbcs), the most commonly used blood component, are still under development. aims: a novel approach for pi in rbc units employing uvc light was developed. methods: pi treatment was applied to full-scale rbc units after leukodepletion. the pi capacity of the uvc-based method was evaluated by bacteria and virus infectivity assays. a panel of in vitro assays to measure quality, metabolism, functional, morphologic, and blood group serology variables was applied to a pool-and-split approach in which pathogen-reduced rbcs were investigated in comparison to untreated rbcs. results: uvc treatment caused dose-dependent inactivation of bacteria and enveloped and non-enveloped viruses in rbc units. at a full dose, the mean log reduction factors ranged from . (bacillus cereus) to . (serratia liquefaciens) for the tested bacteria, and from . (emcv) to ≥ . (vsv) for the tested viruses. uvc treatment did not alter rbc blood group antigen expression. quality of uvc-treated rbcs was maintained during storage, e.g. hemolysis in uvc-treated and untreated rbcs were well below . % until day of storage. summary/conclusions: the data obtained until now show that uvc irradiation is a potential new method for pi in rbcs and justify further development of this process. background: histo-blood abh antigens are the mayor allogeneic antigens in human and they are widely distributed in almost all tissues. the expression of a- , -fucosyltransferase (fuct ), encoded by fut gene, determines the secretor status of an individual. about % of caucasian population have a functional copy of fut (secretor gene) expressing abh blood group soluble antigens in organic fluids such as saliva and seminal plasma. this individuals are known as "secretors". soluble abh blood group antigens have been associated with several metabolic and infectious diseases as well as reproductive failures. the incidence of infertility related of both male and female factors continues to rise despite many advances in reproductive technologies. it is well known that abo antigens are expressed on sperm membrane and in seminal fluid of secretors as well as abo antibodies are present in cervical mucus. in previous studies we observed significant loss in progressive motility of spermatozoa of non-secretors compared to secretor ones caused by specific cervical mucus antibodies in abo-incompatible couples. in addition, sperm cells are haploid cells, so that a heterozygous individual has two sperm subpopulations, each expressing the corresponding allele. the specific antibody of cervical mucus will attack only its complementary sperm. aims: to evaluate the prevalence of secretor character in men belonging to fertile and infertile couples in order to investigate a possible association with reproductive success. methods: samples of semen, from infertile men and from fertile controls were studied. comprehensive infertility evaluation was performed in all patients according to who criteria. secretor phenotype was evaluated in seminal plasma by inhibition of hemagglutination assay using saline erythrocyte suspensions, monoclonal antibodies anti-a, anti-b and lectin from ulex europaeus (anti-h). to distinguish between abo genes, genomic dna was extracted by an enzymatic digestion method. pcr was designed with two sets of oligonucleotides that allow to amplificate two different regions of the transferases without use of restriction enzymes. by comparison of bands of the pcr products, the individual genotype was determine. cervical mucus antibodies of their female partners were titrated with the corresponding red blood cells. results: results were analysed in both groups. in infertile couples with abo incompatibility, the frequency of non-secretor phenotype of male partners ( . %) were significantly higher than those from fertile couples ( . %) (p < . ) the results obtained by pcr in sperm cells correlated % with red cells phenotypes. summary/conclusions: incidence of infertility continues to increase. several factors have a negative impact on men's reproductive health. immunological implications are now being studied and considered as a cause of failure in sperm-egg interaction, even among normal gametes. secretor phenotype in male partners could help reproductive success by blocking cervical abo antibodies. furthermore, if the male is heterozygous, cervical mucus antibodies will only affect the corresponding sperm. we propose to evaluate abh antigen expression on sperm membrane and seminal plasma as well as abo antibodies in cervical mucus to contribute to the diagnosis and treatment of human infertility. background: the h blood group contains one antigen, the h antigen, which is present on virtually all red blood cells (rbc) and is the acceptor substrate of both a and b gene-specified glycosyltransferases. in blood group o the h antigen remains unmodified and therefore its rbcs show the highest and the rbcs of blood type ab the least amounts of h antigen. individuals with the so called bombay phenotype carry homozygous h null alleles (h | h) and do not produce any h antigen. the para-bombay phenotype retains some h antigen on rbcs either induced by a weakly active (h+ w | h+ w ) or completely silenced fut gene (h | h), mandatory linked with an active fut gene. aims: in this study, we aimed to develop an adapted flow cytometric method to quantify the relative amount of h substance present on rbcs in order to distinguish different abo phenotypes in routine diagnostics as well as to capture rare h-deficient phenotypes. methods: analyses were performed on a flow cytometer (facs canto ii, bd biosciences, ch) and measured with identical instrument settings. list mode data were evaluated and visualised using bd facsdiva software. rbcs were incubated with increasing concentrations of monoclonal anti-h antibodies (bric -pe and a : mixture of bric -pe/bric , ibgrl, uk). after rinsing the cells with pbs, micro-aggregates were mechanically dissolved. rbcs from blood donors with different abo phenotypes (o ( ), a ( ), a ( ), b ( ), a b ( ), a b ( )) and patients with genetically confirmed bombay and para-bombay phenotype were assessed. results: saturation of h antigen binding sites on type o rbcs was achieved only upon use of a : antibody mixture (bric -pe/bric ) covering approx. half of the h-binding sites by unconjugated bric . in contrast, non-o type rbcs reached saturation of h-binding sites using pure bric -pe. rbcs coated with bric -pe at saturation revealed a distinct pattern of mfi (mean fluorescence intensity) depending on the abo phenotype. in addition, mfis of rbcs upon staining with bric -pe did discriminate bombay-and para-bombay type rbcs, respectively. summary/conclusions: adapted flow cytometry is able to distinguish variant expressions of rbcs h antigen. thus, our flow cytometric method may complement traditional serological and genetic analyses in routine abo phenotype cases or, more intriguing, when the bombay or para-bombay phenotype is suspected. it will be of interest to further prove this method by investigating additional rare h-deficient phenotype cases. s chen , , x xu , , x hong , , k ma , , j he , , j chen , and f zhu , blood centre of zhejiang province zhejiang provincial key laboratory of blood safety research, hangzhou, china background: weakened a and b antigen expression results in abo typing discrepancies. h gene controls the development of h substance from which a and b antigens develop. depressed a and b antigen expression and strengthened h antigen expression are always simultaneously observed in abo subgroups. there are other possibilities for weak antigen expression of abo system such as leukemic change and pregnancy. it is undiscovered whether abnormal expressions of a, b and h antigen stand for abo subgroups in hemopathic patients. aims: the aim of this study is to explore the role of enhanced reactions with anti-h in direction to abo subgroups of hemopathic patients. methods: samples from blood donors and hemopathic patients with nonconcordant abo typing by serological tests were collected after consent information. the agglutination strength of these rbcs with anti-h reagent was recorded. enhanced reactions were determined by comparison with the results from normal abo groups. the genomic dnas of samples were extracted and genotyped for abo system. this work was sponsored by the medical science research foundation of zhejiang province ( rc ). results: samples in blood donors showed increased expression of h antigen, of which were identified as abo subgroups. there were enhanced reactions in hemopathic patients. however, were finally confirmed as normal abo genotypes. no statistical significance ( . % vs . %, p > . ) in the frequency of strengthened h antigen expressions was observed between donors and hemopathic patients. the total number of subgroups is and respectively in blood donors and hemopathic patients. extremely significant statistical differences ( . % vs . %, p < . ) existed in the frequency of subgroups with enhanced h antigen, which meant the possibility of subgroups in hemopathic patients samples was less. summary/conclusions: the expression of h antigen is comparably enhanced in subgroups and hemopathies. but most of hemopathic patients with strengthened h antigen expression present normal abo genotypes. as a result, the enhanced reaction with anti-h is necessary but not sufficient for serological identification of abo subgroups in hemopathic patients. background: although the use of automated blood bank analyzer with the advantages of speed and efficiency has recently increased, the abo discrepancies in automated blood bank analyzer have caused the reporting delays of the results and increase of the task. aims: we analyzed the causes of abo discrepancies in automated blood bank analyzer and suggested a solution strategy based on the causes. methods: from november to january , cases ( . %) of abo discrepancies among , abo blood type tests performed using the -min reaction mode of ih- in chonbuk national university hospital blood bank were identified. we compared the test results of -min mode with results of immediate mode using different red cell reagents, and analyzed the causes of discrepancies by performing additional tests such as microscopy, auto-control, antibody screening and identification, anti-a and abo genotyping. results: in the immediate reaction using different red cell reagents, cases ( . %) of discrepancies disappeared and cases ( . %) remained discrepancies. all abo discrepancies observed in the -min reaction were due to serum side causes, and one case ( . %) was due to both of serum and red cells side cause. nonspecific response ( cases, . %), cold antibody ( cases, . %), rouleaux formation ( cases, . %), cis-ab ( cases, . %), and abo subtype ( case, . %) were analyzed as causes of discrepancies. one discrepancy due to cis-ab was disappeared in the immediate reaction using different red cell reagents, abo subtype was changed to totally different blood group, a. on the other hand, in cases of the discrepancy corrected by the immediate reaction using different red cell reagents, the intensity of the positive results still observed in immediate reaction was not different from the -min reaction. summary/conclusions: ih- , an automated blood bank analyzer, was considered useful for automation of abo blood typing, and some observable abo discrepancies are expected to be mostly addressed by reexamining with immediate reaction mode using different red cell reagents. abstract withdrawn. background: abo blood group antigens mainly expressed on red blood cells, but along with that they also present on many organs and tissues like epithelia, platelets, vascular endothelia and neurons etc. the importance of abo antigens extends beyond transfusion medicine by association with various systemic diseases like cardiovascular diseases, gastric diseases, cancers, infectious diseases etc has been proven previously. previous researchers also tried to find out the involvement of abo antigens in neurological diseases like alzheimer's disease, parkinson diseases etc. but association with neurological tumours is less explored. aims: this study aimed to analyse the association of abo blood group antigens with neurological tumours. methods: a retrospective study in a tertiary care institute in india analysed the years data from jan to dec . the carcinoma patient's admitted in neurosurgical department during study period were included in our study. their diagnosis and abo blood groups were collected from hospital information system. data were analysed into microsoft excel and spss (version ). results: during study period a total of patients with neurological tumours were admitted in our hospital. the blood group frequency of these patients were . %, . %, . %, . % for a, b, o and ab respectively. the common neurological tumours found in our study were glioma ( . %) followed by pituitary adenoma ( . %), meningioma ( . %), schwannoma ( . %), cavernoma ( . %), neuroma ( . %) and space occupying lesions ( . %). the prevalence of abo antigens was almost similar in all neurological tumours except in neuroma. neuroma was found in . % o group patients as compared to other blood groups which was found statistically significant (p < . ). summary/conclusions: in this study we tried to analyse the association of neurological tumours with abo blood groups antigens. we found there is no association of neurological tumours with abo blood groups because the prevalence on abo group in general population is almost similar in patient with neurological tumours except neuroma. neuroma group of tumours like neurofibroma, neuroblastoma, nerve tumours etc. were more common in o group of patients while in our population frequency of b blood group antigen ( . %) is more common as compared to o blood group( . %). background: rhd and rhce represent homologous genes in head-to-head position on chromosome (chr , p . ). they encode for the proteins rhd resp. rhce which compose together with rhesus associated glycoprotein (rhag), band and ankyrin the ankyrin complex (ac) linking the red blood cell (rbc) membrane to aspectrin of rbc cytoskeleton (s.e. lux, blood, ). cooperatively, the proteins of ac are important for maturation and physiologic properties of rbcs. many proteins of the rbc membrane express blood group antigens on their extracellular surface and are therefore of concern in transfusion medicine. cepellini et al. described weakened hemagglutination reactions of rhd+ rbcs in the presence of an rhc+ antigen (cepellini et al, pnas, ) . we attempted to further elucidate the expression of rhd/rhag proteins in various rhce/rhce pheno-/genotypes using a sophisticated flow cytometry approach. aims: in this study, we investigated a flow cytometric method for measurement of the antigen-density of various rhce-phenotypes. methods: analysis was performed on a flow cytometer (facscanto ii, becton dickinson (bd)) using bd facsdiva software and identical instrument settings for all samples. optimized number of rbcs was incubated with saturating concentration of pe-conjugated anti-rhd antibodies brad- /brad- /fog- (ibgrl, bristol, uk). debris was excluded by rbc gating in fsc/ssc plot. quantibrite-pe beats (bd) were applied according to manufacturer's instruction to quantify the relative expression of rhd epitopes. in addition a representative number of samples from common phenotypes were assessed for expression of rhag using bric- pe (ibgrl). results: a total of samples from healthy blood donors with serologically defined rhcde phenotypes were included into this study (rr( ), r r( ), r r ( ), r r( ), r r( ), r r ( ), r r ( )). variant expression of rhd by different rhce phenotypes using brad- -pe was shown. rhd was weakly expressed in presence of rhc antigen (cepellini effect). effect of rhd gene dose on rhd protein expression is mitigated by rhc/c genotypes. when only samples with molecularly confirmed phenotypes were assessed, the rhdce genotype predicts consistently the strength of rhd protein expression. outlier samples ( ) were retrospectively genotyped and revealed rhdce genotypes as expected from the strength of rhd expression falsifying serological rhcde phenotypes. in contrast, rhe/e polymorphic site does not correlate with rhd expression. in addition, rhag protein is equally present across all rhcde phenotypes. similar results were obtained by using alternative anti-d antibodies such as brad- -pe and fog- -pe, although different antibody's avidity precludes quantitative comparison of antigen expression on rbcs. summary/conclusions: sophisticated facs methods reveal different expression of rhd on rbcs according to rhce/rhce phenotype/genotype. rhc/c polymorphic sites (c. g>c, c. a>g, c. a>g of exon , exon resp. and intron ) are in linkage with rhd expression, confirming the observation by cepellini et al. in contrast, rhe/e (c. c>g, exon ) is not in linkage with rhd expression. based on epigenomic signature it is conceivable that altered transcription factor binding sites (tbs) of rhd mirrored by homologous rhc/c may cause variant rhd expression. rhe/e snp mirroring the homologous sequence of rhd in exon is not recognised as tbs. in addition, although ac comprises all three rh proteins (rhd, rhce, rhag), their transcriptional regulations seem to be distinct. red cell reference laboratory, australian red cross blood service, perth, australia background: the rh antigen was first described when an antisera thought to contain a potent anti-c did not react with all c+ cells. these non-reacting c+ cells were classified as c+, rh:- , and the antibody specificity anti-rh . most polyclonal anti-c contain anti-c and anti-rh . previous studies have shown of monoclonal anti-c reagents are actually anti-rh . these reagents will not detect the c antigen where the red cells are rh:- . aims: the australian red cross blood service investigated a phenotype discrepancy in a blood donor. the donor's historic phenotype c+ (r r) was inconsistent with the current donation phenotype c-(r r ). we aimed to investigate the cause of the discrepancy so the donor could be assigned the correct phenotype, identify the root cause of the discrepancy and implement any corrective actions. methods: the donor's red cells were phenotyped with all available anti-c reagents as per the manufacturers product insert across both manual and automated testing platforms. following variable results and weaker reactions with some reagents, dna was extracted from the edta sample and was genotyped using immucor bioarray tm hea precise and rhce beadchip tm . targeted dna sequencing of rhd and rhce was also performed using the trusight tm one sequencing panel. a review of the historical phenotype results, including the testing platform and reagents used at the time was also performed. results: on the current sample the donor's red cells gave a + reaction by tube with bio-rad seraclone â ( ) [clone ms ] and immulab epiclone tm [clone anti-c reagents. the sample tested negative on the beckman coulter pk using beckman coulter anti-c [clone ] blood grouping reagent and tested positive ( ) reaction on the immucor neo using immuclone â ( ) anti-c [clone . immucor bioarray tm hea precise beadchip tm predicted a c+ phenotype and no variants were detected with the bioarray tm rhce beadchip tm . the trusight tm one sequencing panel genotyped the donor as rhd* /* n. and rhce* . /* with a predicted phenotype of c+, c+ w , d+, e-, e+, rh: (locr+), rh:- . a review of the donor's historical records indicated the donor tested as c+ on the pk , which at the time was being used with an in-house bromelain preparation (sigma-aldrich) and diagast olymp pheno anti-c reagent [clone ms ]. summary/conclusions: results indicated the phenotype discrepancy was caused by the c+ rh:- variant associated with the rhce* . allele. reagents containing clones ms- and ms correctly phenotyped the donor as c+, with the manual tube reagents showing a weaker reaction which may alert the operator to a possible variant which is important in the patient setting. the beckman coulter pk and associated anti-c [clone ] failed to detect the c antigen. this reagent appears not to detect the c antigen where it is associated with the rh:- phenotype, which is in contrast to the previous report by faas et al, transfusion, where it was demonstrated that clone reacted with c+ rh:- bromelain treated red cells. abstract withdrawn. background: although serological rhd typing has always been challenging due to variation of techniques and variable sensitivity of anti-d reagents, most individuals are unequivocally typed as either rhd positive or rhd negative. however, variants of d (weak d and partial d phenotypes) may present typing difficulties. individuals with partial d (missing epitopes of the d antigen) must be typed as rhd negative as blood receivers, but as rhd positive, as blood donors. aims: the aim of our study was to evaluate the algorithm used since at ahepa university blood center, to resolve rhd typing problems among first time donors. methods: since automatic analyzers may type variants of rhd as rhd+, our practice is to routinely perform two different typing methods in first time donors: an automated microplate method on the neo analyzer (immucor) and the slide test, using a potent reagent (anti-d blend-immunodiagnostika). in case of negative, weak, slow or mixed-field reaction, further testing with an automated microplate weak d method [immucor-modified indirect antiglobulin (anti-igg) test] follows. the next step of the protocol consists of testing with the commercial id-partial rhd typing kit (bio-rad) comprising a panel of monoclonal anti-d reagents, in an indirect coombs gel test assay. the patterns obtained with this kit can distinguish between d weak and partial d and can also differentiate between categories ii, iv, v, vi, vii dfr, dbt and dhar. the last step of our algorithm consists of molecular testing (immucor bioarray rhce and rhd beadchip assays) at the hellenic national blood transfusion center, in case of remaining uncertainty. results: we applied the above algorithm in samples: a) by using the partial d kit, samples were typed: four samples were characterized as "partial d" ( dfr, diii) and as "weak d". four of the weak d samples (all from women of reproductive age) were confirmed by molecular typing ("weak d type " three samples, "weak d type . or . " one sample). b) the nine ( ) remaining samples that showed atypical serological pattern, were sent for molecular testing, which characterized samples as "weak d type ", one sample as "weak d type " and another as "weak d type ". results are pending for samples. summary/conclusions: in our experience some partial rhds may be mistyped as rhd+ if the technologist does not inspect the pattern of the reactions and only takes into account the assignment by the automatic analyzer as d+ or d-. by use of our algorithm, serological characterization was sufficient to distinguish between weak d and partial d in , % of cases. molecular typing was necessary in the rest. the integration of molecular techniques improves the quality and accuracy of d typing of blood donors. if applied to patients, it also allows administration of d positive blood without compromising safety to those carrying prevalent weak d types that have not been reported to produce anti-d. furthermore, it permits withholding rhig in case of pregnant women carrying such weak d types. background: rhd antigen is one of the most clinically significant blood group antigens. except d positive and d negative phenotypes, there are over rhd variants, which represent as serologic weak d phenotypes (swd). patients with certain swd can make anti-d alloantibodies. by serology testing it is not possible to clearly distinguish among different swd. in croatian institute of transfusion medicine (citm) patients and pregnant females with swd are mostly reported as rhd negative and generally did not refer for confirmation, because molecular testing was not part of the algorithm. that remains the risk of shortages of rhd negative blood and overuse of anti-d immunoprophylaxis for pregnant females. according to uk guidelines patients with swd who are likely to require chronic transfusion support and females ≤ years are treated as d negative and refer for confirmation of d type. people who are rhd genotyped as weak d type , or are not susceptible for rhd alloimmunisation. one study showed that in croatian population the most frequent variants are weak d type , and . aims: the aim of this study is to estimate the prevalence of swd in patients and pregnant females and to find out serologic and molecular characteristics of swd referred for confirmation. methods: from / / to / / we analysed . samples of patients and pregnant females. rhd typing was performed by anti-d igm monoclonal reagents in direct agglutination micromethod on tango (bs , bs ) (biorad, dreieich, germany), swing maestro [lmh / (ldm ) + - and th- + rum- + ldm ] and ih- [lmh / (ldm ) + - ] (id-card, biorad, cressier, switzerland). cut-off value for tango was determined as ++ and for gel microtyping as +++. the samples with results below the cut-off were reported and treated as rhd negative, all except those which gave discrepant results at current testing or with historical data. these were sent to rhd genotyping for confirmation. dna extraction was done by qiaamp blood mini kit (qiagen, hilden, germany) and rhd genotyping by pcr-ssp kits ready geneweak d and ready genecde (inno-train, kronberg im taunus, germany). results: from . samples ( , %) were swd. / ( %) were referred to rhd genotyping. / ( %) samples were weak d type , or , while / ( %) were weak d type and partial d variants vii and va. serologic reactions with monoclonal igm anti-d reagents showed different pattern for weak d types , and . clearly negative serologic reactions were given in / samples with bs and bs , in / samples with lmh / (ldm ) + - and in / samples with th- + rum- + ldm . summary/conclusions: the prevalence of swd in this study is rather low ( , %). after rhd genotyping % of referred samples were finally reported as d positive. serologic determination of d variants is inconsistent and only rhd genotyping can resolve rhd status in swd. to define the permanent rhd status of swd female of childbearing potential and patients who are likely to be chronically transfused we will introduce rhd genotyping in the new algorithm. background: among all blood group systems, the antigens of the abo system are by far the most clinically significant. comes second in importance is the antigens of the rh system, which comprise d, c, e, c, and e antigens. another clinically relevant antigen is the k of the kell blood group system, which is known to be involved in both htr and hdfn. the distribution of the major blood group antigens, such as rh, and kell, is well-studied among populations of developing countries. in contrary, a relatively few studies have addressed their frequencies in saudi arabian population this is also the case in jazan province, where only two published studies have analysed the prevalence of abo and d antigens, while the frequency of other clinically important antigens, such as rh and kell antigens, is yet to be explored. aims: to determine the frequency of the following clinically relevant blood group antigens; rh(d, c, e, c, e) and k among saudi blood donors in king fahd central hospital in jazan province. methods: a retrospective, cross-sectional study was carried out in the blood bank of king fahd central hospital in jazan province. the red cell phenotyping records for blood donation of randomly selected saudi donors, who donated blood between january and june , were reviewed to identify the prevalence for the following antigens: d, c, e, c, e and k. the hospital blood bank routinely performs rh/k phenotyping for all blood donation using either bio-rad or ortho diagnostic column agglutination technology (cat) platforms. phenotype frequencies were expressed as percentages. results: this study included a total of saudi voluntary as well as family replacement blood donors. the d antigen was found to be positive in . %, while k antigen was positive in . %. among other studied rh antigens, e was the most common ( . %) followed by c ( . %), c ( . %) and e( . %). dce/dce ( . %) and dce/dce ( . %) were the most common phenotypes amongst d-positive and dnegative donors, respectively. surprisingly, dce/dce phenotype was significantly prevalent ( . %) with almost times higher frequency compared that reported in caucasians ( . %). the rare phenotype dce/dce was found in donors ( . %), while dce/dce and dce/dce phenotypes were found in only one donor each. summary/conclusions: this study is the first to determine the frequency of rh and k antigens in saudi blood donors in jazan province. determination of the frequency of these clinically significant antigens in our geographical area will facilitate the selection of antigen-matched red cell units for transfusion in recipients with multiple alloantibodies. it will also help in the management of blood donation processes and planning the estimated need of blood stock of different blood group phenotypes to meet the patient's needs. abstract withdrawn. background: the gerbich (ge) blood group system includes several high-frequency antigens located on glycophorin c and d. with only few reports published on the clinical significance of antibodies directed against these antigens, it is unclear whether blood transfusions have to be antigen negative in the presence of an anti-ge antibody. the monocyte monolayer assay (mma) is an in-vitro method used to estimate the clinical significance of alloantibodies. aims: to illustrate the role of the monocyte monolayer assay (mma) in the transfusion management of a patient with an anti-ge alloantibody. methods: the clinical and transfusion history was retrospectively retrieved from the patient's medical records. serological investigations were performed by indirect antihuman globulin test. papain and trypsin treated cells were also used. the clinical significance of the antibody was assessed by mma. genomic dna was isolated from whole blood and the samples were further characterized by pcr. results: a -year-old male patient with lung cancer without previous transfusions was admitted ( / ) for surgery. his hemoglobin was . g/l. an anti-ge antibody was detected and it was decided to transfuse ge-positive packed red blood cells (prbcs). however, no blood transfusion was needed. in july , the patient was admitted for colon cancer surgery with a hemoglobin of , g/dl. the anti-ge alloantibody was still detectable and a ssp-pcr revealed the genotype ge* .- . an mma performed on the pre-transfusion sample revealed a monocyte index (mi) of . % and the antibody was considered not to be clinically relevant. the mi was interpreted as following: - % not significant; - % inconclusive; > % clinical significant. however, due to the clinical background of the patient it was decided to transfuse ge-negative prbcs, which were obtained from etablissement francais du sang (efs), paris, france. two days after surgery, the patient received units of ge:- ,- prbcs without any transfusion reaction. one and a half year later ( / ), peritoneal carcinomatosis, as a complication of colon cancer, was diagnosed. the patient's hemoglobin was g/l and he had a passage disorder, symptoms of deterioration and an adynamia. based on the mma results from july indicating no clinical significance of the antibody, it was decided to transfuse ge-positive prbcs. in the following days the patient received a total of units of gepositive prbcs no immediate or delayed transfusion reaction were observed following these transfusions. two further mma's, performed on samples drawn on december th and th ( days after transfusion of a total of three and two days after two further prbcs respectively), showed a mi of . % und % respectively and the anti-ge antibody was considered still not to be clinically significant. summary/conclusions: we report the case of a patient with an anti-ge antibody transfused with ge-positive prbc. as ge-negative prbc are not available in switzerland and not easy to obtain internationally the mma can help in the decision on how to transfuse. in this case, the clinical course confirmed the mma-based prediction. transfusions of ge-positive prbcs were tolerated without signs or symptoms of immediate or delayed transfusion reactions. background: abo grouping discrepancies occur when the results of forward grouping are not corroborative to those of the reverse grouping. these may be due to weak subgroups of a and b, missing or weak abo antibodies or red cell alloantibodies. determination of correct abo blood group of a donor is essential for preventing abo incompatible transfusions and to avoid hemolytic transfusion reactions in the recipient. aims: to determine the frequency of abo discrepancies and their resolution to correctly identify the blood group of the donors. we also determined the frequency of 'weak d' positivity in rhd negative donors. methods: this was a retrospective study on donor samples collected from st april, to th september, (two and a half years). for discrepant samples, the abo and rhd grouping was repeated using tube technique using commercial antisera {anti-a, anti-b, anti-ab and anti-d (igm), anti-d blend (igm+igg), anti-h and anti-a lectins}. adsorption-elution testing was done for detecting weak subgroups of a and b. antibody screen ( -cell) and identification ( -cell) was done by gel technique (bio-rad, switzerland). 'weak d' testing in rhd negative donors was also performed by gel technique. antibody titration was done using tube technique. the donor details including name, age and the registration/unit number of the donation were also checked for all the discrepancies to avoid repetition while data analysis. results: we detected ( . %) abo discrepancies out of the total donor samples tested during the study period. out of these, ( . %) were rhd positive. the most common cause of abo discrepancies was weak anti-b antibody ( / ; . %), followed by weak anti-a antibody and weak subgroups of a ( / each; . % each) and weak subgroups of b ( / ; . %). the remaining . % ( / ) discrepancies were due to agglutination with o cells in reverse grouping. the overall frequency of weak subgroups of a and b collectively was . % ( background: detection of unexpected red blood cell (rbc) antibodies before transfusion is critical for prevention of hemolytic transfusion reaction. ideally, unexpected rbc antibody detection is carried out within days after receiving a patient's sample. however, in some cases, retests could be performed after more than days for evaluation of any transfusion reaction, quality control or research. therefore, it is necessary to determine the stability of antibodies after refrigeration or freezing for a certain period of time. aims: we carried out antibody identification test with fresh, refrigerated and frozen samples using automated analyzer ih- and manual tube methods to evaluate the stability of antibodies after storage and compare the results between the two methods methods: antibody identification tests were performed using ih- (bio-rad, cressier fr, switzerland) and manual tube methods. fifty samples showing positive results in antibody screening test by both methods were divided into three and tested immediately, week after storage at °c and month after storage at À °c. the specificities and reactivities of antibodies at each storage state were recorded and compared between the two methods. results: specificities of antibodies identified were concordant between ih- and manual tube methods irrespective of the storage state. the results were as follows: anti-e/e+c, ; anti-le a , ; anti-di a , ; anti-c+e, ; anti-m, ; anti-d, ; anti-c, ; anti-k, ; anti-jk a , : anti-xg a , ; unidentified antibody, ; autoantibody, cases. with regard to the changes in reactivity owing to storage, ( %) samples (anti-e+c, ; anti-m, ; anti-di a , ; anti-d, ; anti-c+e, ; anti-le a , ; anti-c, : autoantibody, ; unidentified antibody, ) showed identical reactivities after week and month storage by both ih- and tube methods. however, ( %) samples, comprising unidentified antibodies, anti-le a , anti-c+e, anti-e, anti-e+c, and autoantibody, showed decreased reactivities after storage in both methods. three samples, comprising anti-di a , anti-e+c and anti-k antibodies, showed increased reactivities after storage. one sample with anti-jk a showed increased reactivity only after month storage, while one sample with anti-xg a showed decreased reactivity only after month storage. higher reactivities were observed in all samples detected using the ih- analyzer than manual tube methods (p < . , wilcoxon rank sum test). summary/conclusions: the specificities of unexpected antibodies detected by ih- and tube methods were the same in all storage states; however, reactivities were higher in ih- than in the tube method. twenty-six ( %) of samples showed identical reactivities after week refrigeration and month freezing. nineteen ( %) samples showed decreased reactivities after storage; however, ( / , %) of them were nonspecific antibodies, unable to identify using commercial id panels. therefore, it is suggested that retests for evaluation of transfusion reaction, quality control or research could be reliably performed after more than days, if stored appropriately in refrigerated or frozen states. abstract withdrawn. t gleich-nagel , d huber-marcantonio , n rufer , g canellini and c niederhauser unit of transfusion medicine, interregional blood transfusion src, lausanne laboratory diagnostics, interregional blood transfusion src, bern, switzerland background: a positive direct antiglobulin test (dat) is mainly found in patients with warm/cold autoantibodies or alloantibodies directed against transfused erythrocytes. the identification of antibodies fixed on red cells is important for the clinician, allowing the further evaluation of a patient's clinical situation including their current medication. in immunohematology the elution of a positive dat remains a tedious and expensive procedure. the blood transfusion service src (bts src) has derived a flow chart that indicates in which situation an elution of dat positive samples should be performed. in order to follow the bts src guidelines, it is mandatory to obtain additional data related to the patient's condition, such as haemolytic parameters and recent transfusion history. currently, our laboratory is not always able to apply the recommended flowchart, since information is often unavailable. aims: here, we performed a comparative study between the algorithm provided by bts src and our in-house strategy, which is based on the qualitative changes of a positive dat, without the need for additional patient and biological information. methods: details of dat positivity and the patient's transfusion history was taken from the software eprogesa (mak-system) and analysed in excel. we analysed a total of ' dats and evaluated them for their positivity, whether an elution was performed or whether antibodies were detectable in the eluate. furthermore, we performed an additional analysis on those samples, that were derived from recently transfused patients (< days). results: a positive dat was found for igg and c d in out of ' ( . %) samples, a level similar to previous reports of positive dats for hospitalized patients. among these positive samples, ( %) were eluted because of a qualitative change in their positivity according to our in-house algorithm. identification of warm autoantibodies or alloantibodies occurred in only . % ( / ) of the cases. from the patients transfused within the last days and having a positive dat, ( %) were eluted according to our in-house algorithm. the same samples would have been analysed if the swiss transfusion guidelines had been applied. however, this comparative study reveals a significant discrepancy in regards to overall sample numbers that should have been eluted according to the two algorithms ( versus samples). this is mainly due to the fact that the swiss transfusion based algorithm does not recommend an elution of positive dats from patients who did not receive a transfusion within the last -days, except if there is a significant clinical suspicion (e.g. haemolysis). summary/conclusions: this comparative study indicates that our elution-based algorithm was performed on all clinically relevant samples as recommended by the bts src guidelines. qualitative changes in the dat positivity represent our main parameter for selecting those samples to eluate. besides ensuring that no clinically relevant samples were missed, this strategy also led to a large number of unnecessary elution analyses. in conclusion, a significant reduction in the laboratory workload and economical savings arises if the relevant clinical information and patients history is known prior to laboratory analysis. background: novel anti-cd monoclonal antibodies, such as daratumumab (dara) and isatuximab, used in treatment of multiple myeloma, interfere with routine blood bank serologic tests. as part of the strategies to manage these patients, it is recommended to perform extended phenotyping to provide matched units (aabb association bulletin # - ). many investigations have focused on the interference with iat for the screening and identification of underlying alloantibodies and how to overcome them, but less has been published on the potential interference with extended phenotyping techniques. aims: the purpose of this study is to compare different technologies to type the most important antigens in myeloma patients before and during the treatment with therapeutic anti-cd antibodies. vox sanguinis ( ) (suppl. ), - methods: edta-anticoagulated whole blood samples coming from patients in different stages of treatment with daratumumab and with isatuximab have been typed in parallel with dg gel microcolumn (grifols) and mdmulticard technology (grifols). the results are also compared with genotyping results obtained with id core xt (grifols). direct coombs, autocontrol and antibody screening has also been performed as complementary tests. results: the study provides that four patients had positive dat and/or ac before therapeutic cd antibodies treatment. in these cases, of negative antigens (fy / jk and/or s) turn to positive in gel technology but mdmulticard showed % agreement with genotype id core xt results. focusing in the data obtained during the treatment, negative antigens were type as positive in gel technology ( % of the tests). mdmulticard agreed with genotype in % of the analyzed antigens. as complementary data, of patient-treated samples had dat or ac positive and showed panagglutination. summary/conclusions: the results demonstrated that mdmulticard is an effective method to type cd -directed cytolytic antibodies treated samples in addition to dat and or autocontrol positive samples. background: antibody titration is a semi-quantitative method to estimate the strength and concentration of antibodies present in plasma or serum sample. titration methodology should be validated together with clinical data to evaluate the relevance of the titer value in each application. the titer of an antibody depends on different parameters: the antibody concentration in the sample, the density of the corresponding antigen expressed on the red blood cells used, the affinity constant of the antibody-antigen and other parameters regarding the technique used (e.g. gel cards or tube test). gel cards technology reduces the intra and inter-laboratory variation in titration studies comparing with the tube technique. aims: to evaluate the suitability of dg gel coombs, dg gel anti-igg and dg gel neutral (grifols) for titrations using two sample volumes ll and ll. methods: twenty frozen plasma samples containing unexpected antibodies from different specificities (anti-jk a , -fy a , -k, -d, -e and -c) were titrated in dg gel coombs and dg gel anti-igg cards and donor fresh plasmas with natural occurring antibodies (anti-a and -b) were titrated in dg gel coombs and dg gel neutral (saline technique). the titer of the antibodies was determined by testing two-fold dilutions of the plasma with selected red blood cells depending on the antibody tested. plasma samples were diluted in dg gel sol. selected red blood cells serascan diana, serigrup diana or donor blood were added into the card ( ll at . %). further, sample dilutions were dispensed into the card ( ll or ll). subsequently, cards were incubated min, °c (coombs technique) and min, - °c (saline technique), centrifuged in dg spin and the results read. agglutination intensity was graded visually according to the instructions for use of dg gel cards. the reciprocal of the highest plasma dilution that gives macroscopic agglutination was interpreted as the titer. results: titers obtained with dg gel coombs and anti-igg (n = titrations, titer ranged - ) were compared for each sample with unexpected antibodies. no differences were found between gel cards types (differences were ≤ . titer in the % of the cases). differences between dg gel coombs and neutral (saline technique) (n = titrations, titer ranged - ) were observed when anti-a and -b antibodies were titrated using the same sample. the titer was similar or higher in coombs in comparison to the saline technique. coombs titers may be a mix of igm antibodies reacting at °c and igg antibodies. differences were > titer in % of the comparisons and ≤ titer in the rest of the cases ( %). comparing sample volumes of ll and ll in all cards (n = titrations), higher titers were observed using ll, as expected. differences were titer in the % of the comparisons, < titer in % and > titer in the % of the cases. background: autoimmune haemolytic anemias (aiha) are characterized by production of antibodies directed against red blood cells and destruction by the mononuclear phagocytic system or complement system. aiha observed in paediatrics is usually self-limiting and often precipitated by viral infections. in some, the condition is secondary to autoimmune diseases, drugs, infections or underlying primary immune deficiencies. appropriate immuno hematological evaluation to characterise the underlying autoantibody can help identify the type of aiha to aid in diagnosis & treatment of these cases. aims: retrospective analysis of immune-hematological evaluation, treatment and outcome of aiha in paediatrics. methods: patients aged - years, diagnosed with aiha, between april -december ( months) were included in this analysis. aiha was defined as positive direct coombs' test (dct) with anemia associated with corroborative evidence of haemolysis in the form of raised indirect hyperbilirubinemia, raised ldh, raised reticulocyte counts or red cell agglutination on peripheral smear. further monospecific dct and evaluation for the specificity of autoantibody was done for all patients using biorad gel cards and panel cells. steroids were given as first line in all; second line agents included cyclosporine and rituximab. red cell transfusion was given in those with severe anemia with cardiac decompensation. results: patients were diagnosed during the study period with autoimmune haemolytic anemia. haemoglobin at presentation ranged from . to grams/dl. the initial presentation was severe anemia in children and mild-moderate anemia with thrombocytopenia (evan's syndrome) in . the trigger for haemolysis was infection in children. rheumatological evaluation was performed for children out of whom were diagnosed as evolving lupus. primary immune deficiency evaluation was advised for and one child was diagnosed as suffering from combined immunodeficiency. dat was positive in out of aiha patients as one of the infant had dat negative iga mediated aiha secondary to viral infection. two out of dat positive cases had igg & c d positivity on monoclonal dat testing whereas rest had only igg coating the red cells. dat titration was more than : in patients; where only of these patients had both igg and igg coating and rest had only igg . alloantibody screen was negative in all. specificity of autoantibody was found only in one case, which was against rh blood group antigen (anti e). all patients received prednisolone as the primary treatment. three children attained remission following a - weeks of steroids. in those who were steroid dependent, cyclosporine was used as the second line agent in and rituximab was used in . out of these children children are in sustained remission and off any medication, whereas the rest are on low dose steroids with cyclosporine. summary/conclusions: aiha is not an uncommon problem in children and can vary in its clinical severity. the proper diagnosis and management involves efficient immuno-hematological evaluation, as detailed characterization of the autoantibody coating the red cell is very important in guiding the clinician for management and prognosis. abstract withdrawn. background: drug-induced immune hemolytic anemia (diiha) is rare and has only been described once with dexchlorpheniramine (polaramine tm), an antihistaminic agent widely used in the treatment of a variety of allergic reactions. we report a case of diiha complicated with acute renal failure associated with antibodies to dexchlorpheniramine. a -year-old woman with no history of transfusion, was treated semimonthly with a combination of chemotherapy and targeted therapy for metastatic colorectal adenocarcinoma. her chemotherapy regimen consisted of oxaliplatin and -fu with leucovorin rescue (folfox). panitumumab (monoclonal antibody anti-egfr) was used as targeted therapy. premedication with dexchlorpheniramine iv was systematically given at the beginning of each cycle of treatment to reduce the risk of perfusion reactions mainly associated with panitumumab. the patient developed chills and febrile agranulocytosis during the first and second infusion respectively. the third infusion was not performed due to pyrexia, chills, general discomfort experienced by the patient at the beginning of chemotherapy. probabilistic antibiotherapy was administered and the patient recovered rapidly. during the next infusion (day ), following premedication with dexchlorpheniramine, a more "impressive" reaction including all the above mentioned symptoms occured along with back pain and dark colored urine. the infusion was halted and no chemotherapy was delivered. bacterial infection at the implantable port was first thought to be the cause of this adverse event but was not confirmed. additional laboratory findings revealed biological signs of inflammation associated with iha and acute renal failure. the patient was treated with hemodialysis (day ), two units of rbcs (day ) and was discharged one week later in stable condition. dexchlorpheniramine was then suspected and samples collected on day were sent for a diiha laboratory workup. aims: the aim of this study was to support a clinical diagnosis of diiha. methods: laboratory workup included direct and indirect antiglobulin tests (dat and iat). drug antibodies investigation was performed by incubating patient's serum and eluate from patient's rbcs in the presence of drug against normal donor rbcs that had not been previously treated with the drug (i.e., by the so-called "immune complex" method). control tests were performed in parallel. drug was diluted in pbs and tested at and mg/ml. results: dat was positive (anti-igg + , anti-c d + ) and no unexpected rbcs antibodies were detected by iat in patient's serum and eluate without the in vitro addition of the drug. an antibody directed against untreated (titer ) and enzymetreated (titer ) normal donor rbcs was demonstrated only in patient's serum in the presence of the drug tested at mg/ml by the gel method. the pool of normal sera did not react in the presence of the drug. summary/conclusions: the multi-drug treated patient described in this study was demonstrated to have dexchlorpheniramine dependent antibody detected by the "immune complex" method. the key to the diagnosis was the observation of positive dat with negative eluate tests which prompted a reexamination of the medications administered in temporal relationship with the hemolytic event. although rare, this case report should alert physicians to the need to investigate the possibility of dexchlorpheniramine induced hemolytic anemia in any patient who develop unexpected anemia after hematologic or oncologic procedures p- singapore, singapore, singapore background: daratumumab is a monoclonal antibody against cd used in the treatment of multiple myeloma and has been known to bind to cd on rbc's and interfere with indirect antiglobulin based serologic tests such as red cell antibody screens and crossmatch compatibility testing. in order to negate the interference of daratumumab, our reference laboratory follows the daratumumab protocol recommended by the aabb which uses dithiothreitol (dtt) treated reagent red cells in red cell antibody screening and identification test in patients known to have received daratumumab. aims: the objective of this study is to determine the impact of daratumumab in the turnaround time (tat) for red cell antibody screening and identification. methods: a retrospective review of the tat for red cell antibody screening and identification samples of patients known to be treated with daratumumab from october to december was performed. turnaround time is defined as the time the sample is received up the time the results were reported. the tat for routine red cell antibody screen and identifications were also reviewed during the same period and was compared with the tat of samples from patients treated with daratumumab. results: a total of patients on daratumumab had samples sent to our reference laboratory for red cell antibody screen and identification during the study period. information on daratumumab treatment was not provided to the reference lab prior to the start of testing in of the patients while the use daratumumab was mentioned in the serology request form of the other patients. the median tat for red cell antibody screen and identification is min (range: - ) if information on daratumumab was provided prior to start of testing and min (range: - ) if information was not provided prior to testing. the median tat for routine testing is min (range: - ). using wilcoxon rank-sum test, turn-around time for antibody screening and identification for daratumumab treated patients was observed as statistically not significant when compared to routine samples (p value . ). however, tat for serologic tests requests with appropriate medical history compared to the testing requests without relevant information was also observed to be significantly difference (p value . ). summary/conclusions: there is no significant impact in the tat of red cell antibody screen and identification in patients known to receive daratumumab as compared to routine testing. however, there is a significant difference in the tat if information on daratumumab treatment is not provided prior to testing. this highlights the importance of providing the relevant medication information in the request form in order to prevent delays in testing and provision of blood to patients on daratumumab, which can result in improved organizational efficiency and have positive impact on cost and resource savings. background: daratumumab, an anti-cd monoclonal antibody, has been shown to be highly efficacious in the treatment of multiple myeloma (mm). cd is a glycoprotein highly expressed on plasma cells and, to a less extend, on the surface of red blood cells (rbc). when bound to cd on rbc, daratumumab interferes with the pretransfusion tests, with positive antibody screening and crossmatch. anti-cd interference is an important challenge as many mm patients will require blood transfusions during their treatment. dithiothreitol is a reducing reagent with multiple applications in blood bank testing. treatment of rbc with dithiothreitol irreversibly removes cell surface cd tertiary structure, avoiding the binding and testing interference by the anti-cd daratumumab. aims: to demonstrate the efficacy, safety and celerity of the protocol between the blood bank (bb) and haemato-oncology of our institutions, using just the crossmatch. methods: a retrospective research was used for the evaluation of the results obtained from the implemented protocol. this comprehends a previous contact by haemato-oncology that leads to a study of the patient before the beginning of daratumumab treatment, and consists in: abo/rhd grouping; rh and kell phenotyping, and other clinically significant antigens; antibody screening; and direct antiglobulin test. genotyping may be required for some patients who received previous blood transfusions. before the beginning of the therapy, a blood sample of the patient is sent to the bb to perform laboratory tests and frozen after. this frozen sample is used for crossmatching in patients that already started therapy, did not have a blood transfusion in between, and have a positive antibody screening and/or crossmatch. in further transfusions, in case of positive tests, the dithiothreitol-treated donor rbc is applied. the donor rbc antigens are always selected accordingly to patients negative clinically significant antigens, when transfusional support is needed. the laboratory tests are executed in gel column agglutination technique. results: since , patients were studied, from which were transfused with blood units, according to the protocol. there were no immunizations or adverse reactions to transfusion registered within the transfused patients, neither delay on the availability of blood units. patient blood sample collected and frozen prior to the beginning of the treatment, has shown to be a good strategy by reducing significantly the waiting time for the blood unit in the first transfusion. summary/conclusions: this protocol, which defines the communication among the involved professionals, has shown to be a secure and effective way of reducing interferences caused by daratumumab. it ensures the previous study of the patients and their transfusion with rbc respecting the patients negative clinically significant antigens. if not adopted, the mitigation measures described in this protocol, delays in the availability of the rbc requested and alloimmunizations, may and will possibly occur. a good communication between the bb and the haemato-oncology is crucial for a good time management when a transfusion is requested for these patients. three methods were used to resolve this dara interference. reagent rbc's were treated with dtt, which know to denature cd and then tested with patient plasma. allo-adsorption study was performed using a certain ratio of red cells to plasma. in addition, a selection of phenotyped cord cells were used as an antibody screening panel. results: dtt treatment of reagent red cells was successful at eliminating dara interference and allowing for the presence of underlying antibodies to be identified. in this case, underlying antibodies were not detected by using reagent dtt treated red cells or phenotyped cord cells. adsorption technique was ineffective at elimination the reactivity. summary/conclusions: dara is the first commercial fda-approved therapeutic monoclonal antibody used in treating multiple myeloma patients. • since cd is weakly expressed on normal red blood cells, dara attachment to red blood cells can interfere with pre-transfusion iat testing. • dtt treatment of reagent red blood cells and cord cells can abolish the interference of dara to test for the presence of underlying alloantibodies. • to prevent delays in issuing red blood cell units to patients, hospitals should send patient samples to be tested before receiving dara treatment to ensure that clinically significant alloantibodies are not being masked. background: antibody screening (as)is considered superior to antihuman globulin (ahg) cross match during pretransfusion compatibility testing. in spite of knowing the utility and superiority of as, it has not been adopted uniformly in india. therefore, scarce data is available from this subcontinent in terms of optimisation of red cell antibody detection during pretransfusion testing in form of "type and screen" aims: the main objective was to study the benefits of performing simultaneous antibody screening along with the blood grouping during the first hospital visit to the hospital. other objectives were to study the prevalence of clinically significant antibody among the indian population and to follow up the patients who were transfused antibody screen negative but cross match incompatible blood. we also studied some other relevant quality indicators related to efficiency of blood transfusion services methods: this prospective study was carried out at a tertiary healthcare centre in india between july and dec ( months). the study protocol was submitted to institutional review board and permission was granted. blood grouping and as were done during patients' first hospital visit, which we called "type and screen". when the patient got admitted to the hospital and required blood transfusion, a blood request form was generated by the user and sent to blood bank. depending upon the results of antibody detection, further course of action was chosen. if patient was found to have no antibody, immediate spin test (ist) cross match compatible blood was issued and transfused. in such cases the procedure of ahg crossmatch testing was continued even after issue of blood. cases where ahg cross match test was found negative no further follow-up of the patient was done whereas when ahg cross match was found positive, patients were followed after the transfusion results: a total of patients were "type and screened". majority were from hemato-oncology, bmt, liver transplant, paediatric cardiac surgery, and medical icu units. clinically significant allo-antibody was detected in patients and autoantibody was detected in patients. alloantibody was detected mainly against rh and kell blood group systems. in diagnosed aiha cases, majority were in the form of warm aiha ( %) and % of aiha cases were having hidden single or multiple alloantibody. significantly higher proportion of patients in as positive group required blood transfusion than as negative group ( % vs %, p < . ). in both the groups, in planned cases, most of the time blood was issued immediately within the defined turnaround time except in where either transfusion was delayed or surgery was postponed. it happened only in trauma or surgical bleed cases. expiry of blood was decreased significantly due to no usage of blood ( . % vs. %, p < . ). during the period of study we obtained cases where the ist cross match was compatible but the ahg cross match was incompatible. during follow up none of the cases demonstrated any sign of hemolysis summary/conclusions: in developing countries like us, optimization of as during pretransfusion testing increases operational efficiency and significantly decreases the expiry of blood. results: during the period when absc was performed on pk , , donation samples were tested and , ( . %) were found absc positive. antibodies to red cells were identified in donations out of , ( . %) absc positive samples and in the rest, no irregular antibodies were detected. the prevalence rate for atypical antibody was . %. the top most frequent antibody specificities were: nonspecific cold antibodies ( . %), anti-e ( . %), anti-mi a ( . %), anti-m ( . %) and anti-le a ( . %). a total of , donations were screened for atypical antibodies by ih- and , ( . %) were screened positive. among these, anti-red cell antibodies were identified in , samples ( . %), which was significantly higher than those identified in pk screened positive samples (p < . ). the prevalence rate for atypical antibody as screened positive by ih- and with confirmed red cell specificities was . %, which was also significantly higher (p < . ). the top most frequent antibody specificities were: anti-mi a ( . %), anti-m ( . %), anti-le a ( . %), anti-e ( . %) and non-specific cold antibodies ( . %). anti-fy b was detected in cases, which would be missed detection by enzyme treated reagent cells on pk system. summary/conclusions: the performance of the ih- system using a -cell screening panel including one cell with mi(a+) expression and column agglutination technology with iat phase was superior in comparison with that of pk in the context of higher sensitivity in detecting more true positive results and higher specificity in detecting more true negative and less false positive results. this has translated into the advantages of reduction in workload of reference laboratory in performing less antibody identifications in those false positive samples as well as enhanced transfusion safety by removing more irregular red cell antibody positive plasma-containing components from the issuable inventory, which may potentially lead to haemolytic transfusion reactions. the prevalence of irregular red cell antibodies of . % in healthy blood donors in hong kong reflects more the true statistical figure. background: chronic red blood cell (rbc) transfusion is the upfront therapy for thalassaemia patients, however this therapy is featured by several adverse events including rbc alloimmunization. phenotype matched products transfusion policy can prevent alloantibody formation, but it makes routine transfusion more difficult for both the donor center and the transfusion service. a recent systematic review (franchini et al, blood transfus ) reported a rbcalloimmunization prevalence of . %, with a higher incidence against rh and kell systems in thalassemia intermedia patients. aims: the aim of our retrospective study is to evaluate the rbc alloimmunization prevalence in thalassemia patients transfused in a single center over a years period with limited phenotype matched rbc (rh and kell system antigens) units. methods: from to thalassaemia patients, with a minimum follow up of year and transfused with more than > rbc units, were included in our study. patients were studied for: blood group and rh / k phenotype determination, direct antiglobulin test (dat), irregular antibodies research (abirr). cross-match and detection of alloantibodies were performed using the indirect antiglobulin test by the column agglutination method. six-monthly dat and antibody screening were performed using the indirect antiglobulin test and enzymatic papain-treated rbc test. results: overall patients ( females, males) were included in our retrospective analysis: patients were affected by thalassaemia major and by thalassaemia intermedia. rbc alloimmunization prevalence was . % ( patients): patients were found to be positive for rbc alloantibodies, four with alloantibodies and autoantibodies. eleven alloantibodies were detected ( anti-h, anti-cw, anti-e, kpa, anti-jka, anti-jkb, anti-m and anti-lua). in out of alloimmunized patients we found an anti-e antibody reactive in enzymatic papain-treated rbc test only, in the third alloimmunized patient anti-kpa and anti-lua antibodies were detected, while in the remaining patients, in which auto and alloantibodies were detected, a severe autoimmune hemolytic anaemia (aea) requiring therapy was diagnosed. in these cases the appearance of alloantibodies is concomitant with the presence of autoantibodies. among the patients positive for alloantibodies, were affected by major thalassemia and one by intermedia thalassaemia summary/conclusions: in our experience a limited phenotype matched rbc transfusion policy showed a rbc alloimmunization prevalence similar to literature data: . % vs . %; we didn't find higher alloimmunization prevalence in thalassemia intermedia patients may be due to the low patients number. we believe that introduction, in our department, of an extended-phenotype matched transfusion, including antigens of the main group systems (fy, jk, mns) and the main rare antigens (cw, kp, lu), could reduce the risk of red blood cell alloimmunization in thalassemia patients. abstract withdrawn. background: undoubtedly, preventing alloimmunization has an advantage over overcoming its consequences. however, the high cost of technical and organizational aspects of preventive measures requires their scientific substantiation confirmed by clinical and laboratory data. selection of donors of the rhesus (d, c) and kell (k) antigens for the red blood cell transfusions to hematological patients has been regulated in the russian federation since . recipients with the phenotype c+c-transfuse red blood cell only with the same antigenic combination. for transfusions red blood cell obtained from k-negative donors are used. the compatibility of the donor and recipient with the antigens c, e, e, c w (rhesus system) and k (kell system) is additionally taken into account from april . that is, transfuse red blood cell that do not contain antigens in the phenotype that are not in the recipient's phenotype. aims: to evaluate the efficiency of red blood cell donor selection using antigens of rhesus (c, c, e, e, c w ) and kell (k, k) systems for the prevention of the recipient alloimmunization. methods: immunohaematological studies using equipment and reagents of biorad (usa) were performed in patients of the hematology clinic. non-hodgkin lymphoma was diagnosed in patients, acute leukemia in , multiple myeloma in , chronic lymphatic leukemia in , chronic myeloid leukemia in , aplastic anemia in , hemophilia in , myelodysplastic syndrome in , and other hematological diseases in . the frequency of detection of antibodies to antigen c ( . % vs . %) and to antigen e ( . % vs . %) decreased four times. the frequency of detection of antibodies to the c w antigen has not changed significantly ( . % vs . %, respectively). selection of antigens c (rhesus) and k (kell) has been carried out in the clinic since , therefore the immunization index for these antigens remained unchanged and amounted to . % vs . % for anti-c antibodies; . % vs . %for anti-k antibodies. alloantibodies to the antigens e (rhesus) and k (kell) were not detected for the entire observation period. summary/conclusions: research verified the effectiveness of alloimmunization prevention of recipients by selecting red blood cell for antigens c, c, e of the rhesus system and k (kell). the study concluded that selection of red blood cells for the antigens c w , e (rhesus) and k (kell) does not affect the level of alloimmunization of patients and is not clinically justified. background: in the russian federation, there is an order according to which patients requiring multiple transfusions, who are at high risk of immunological complications are to typed for red blood cell antigens: abo, d, c, c, e, e, cw, k, k. selection of erythrocyte-containing blood components is carried out taking into account the donorrecipient compatibility according to all the listed antigens. aims: analysis of results of immunological evaluation of patients of hematological clinic. methods: the study included first time patients of hematology clinic in - . typing of antigens of abo, rhesus, kell systems, screening and identification of antibodies were carried out using equipment and reagents from bio rad (usa). results: interpretation of results of immunohematological screening was complicated in ( . %) patients. the total number of complex cases was . the double population of red blood cell was most often determined in antigens of the rhesus system ( . % of the total number of patients) as a result of previous transfusion therapy. of those, chimera for the antigen e was detected in cases ( . % of patients with the chimera for rhesus and kell antigens), cin ( . %), sin ( . %), e - ( . %), cw - ( . %), k - ( . %). in such cases, donor red blood cells were chosen not carrying chimeric antigen for transfusions, in the presence of chimeras in both paired antigensred blood cell transfusion with the cc phenotype and / ee. chimera for abo antigens was detected in . % of the examined individuals. the discrepancy between the direct and reverse blood grouping of the abo system in patients ( . %) was due to a decrease in the production of antibodies - cases and the appearance of extra agglutinins - case. autoantibodies were detected in . % of all patients, including . % of patients, when they caused panagglutination phenomenon. upon detection of autoantibodies that complicate the individual selection of donors, transfused red blood cells that are compatible with antigens of abo, rhesus, kell, duffy, kidd, mns systems. alloantibodies were detected in . % of patients, including specific anti-din ( . %), anti-dcin ( . %), anti-kin ( . %); antibodies of unidentified specificityin ( . %), polyspecificin ( . %). summary/conclusions: the complexity of interpreting immuno-hematological tests in hematological patients is due to intensive transfusion therapy, changes in red blood cell antigens and appearance of nonspecific antibodies due to underlying disease. red blood cell for transfusion in these patients should be selected taking into account the expanded red blood cell antigen profile. abstract withdrawn. background: blood transfusion is an essential part of therapy for many patients. although life-saving for many patients, blood transfusion is not without risk. the main goal of blood transfusion services is that transfused blood should be compatible with the patient. the clinical and serologic evaluation, which allows for the transfusion of the most compatible (or "least incompatible") blood, requires a joint effort between the clinician and the transfusion medicine physician. aims: root cause analysis of incompatible cross matches in patients. methods: in this prospective study, total of , , crossmatches were performed over period of last four & half years, out of which units were found incompatible by column agglutination method-cat in polyspecific (anti-igg+ c d) gel media. a root cause analysis protocol was formulated to resolve incompatibility to ensure safe transfusion. results: on the evaluation of , , crossmatches, only units were found to be incompatible ( . %). the major cause for incompatibility found in patients was aiha-( . %). other causes of incompatibility were infections ( . %), multiple transfusions ( . %), trauma ( . %), evan's syndrome ( . %), rh negative mother ( . %), sca ( . %) & incompatibility due to dat positive packed red cells ( . %).the most common antibody found were anti-'c', anti-'s' & anti-'m'. summary/conclusions: the rca protocol involves a thorough evaluation of the patient's clinical condition and underlying pathology to identify the cause. a logical stepwise approach will enable provision of safe transfusion to the patient. background: antibodies to high-frequency antigens (hfas) are a transfusion hazard, as compatible blood is often very difficult to obtain. other clinically significant alloantibodies represent an additional transfusion risk. in patients treated with allogeneic bone marrow transplantation (bmt) recipient red cell alloantibodies may cause acute or delayed haemolysis of donor red blood cells (rbc) and contribute to morbidity and mortality. aims: the aim is to present the case of a patient with myelodysplastic syndrome (mds), multiple "common" alloantibodies and an additional alloantibody to a highfrequency antigen, treated with allogeneic bmt. methods: a forty-one-year-old caucasian patient with mds (raeb- ) was admitted to our hospital in january for unrelated allogeneic bmt. she previously received myeloablative conditioning therapy according to the flu / bu / atg protocol ( days of mg iv. fludarabine, days of busulfan mg iv, days of mg iv. antithymocyte globulin). the indirect antiglobulin test (iat), done in august and december of , was negative. according to anamnestic data, the patient had two pregnancies. she received red cell transfusions during childbirth and platelets in december . results: the patient's blood group was o rhd positive, iat positive. the donor blood group was a rhd positive, iat negative. phenotype of the recipient's rbcs, as well as the donor rbcs, was also determined. anti-e and -c w were found in the patient's plasma, but an additional alloantibody was suspected. the autocontrol was negative. column agglutination technology (cat) and tube technology were used to identify rbc antibodies. plasma was tested with pheno-matched rbcs, papain-and . m dithiothreitol-treated rbcs, as well as cord and autologous rbcs. adsorption and elution tests were done, excluding other "usual" clinically significant alloantibodies, and the patient received three incompatible (xm in iat, cat) yt(a+), e-, c w -, k-red cell units. the sample was urgently sent for an antibody investigation at the international blood group reference laboratory (bristol, uk). in the reference laboratory, anti-e, -c w and an alloantibody to a high-frequency antigen were confirmed, whose specificity was determined to be anti-yt a . anti-jk b was also suspected and later confirmed. before the patient was discharged from the hospital, she received eight more red cell units (yt(a+), e-, c w -, jk(b-)), during which she was serologically closely monitored. summary/conclusions: the results of the antibody investigation in this case study indicate the presence of multiple alloantibodies in a patient who has previously received immunosuppressive myeloablative conditioning therapy. in addition to the "common" alloantibodies (anti-e, -c w , -jk b ), an alloantibody to a high-frequency antigen (anti-yt a ) was detected in the patient. this patient was transfused with incompatible red cell units (yt(a+)) in an emergency, with no ill effects. although anti-yt a is rarely a clinically significant antibody, according to literature, it can cause immediate haemolytic transfusion reaction. additional risk were "common" clinically significant alloantibodies, especially anti-jk b , which was in this case extremely difficult to detect and had further complicated the selection of blood. background: the identification of an antibody against a high-incidence antigen always introduces a challenge due to the difficulty in finding compatible units of red blood cells (rbcs). in patients needing surgery it is important to minimize their transfusional needs by implementing patient blood management programs (pbm). tests that predict the clinical significance of antibodies, such as monocyte monolayer assay (mma) are also useful in guiding clinical decisions. kell blood group system contains highly immunogenic antigens. antibodies against these antigens are immunoglobulin g, and can cause severe hemolytic transfusion reactions and fetal anemia. results: case report we report the case of a -year-old female, with non-hodgkin lymphoma, chronic anemia and scoliosis with severe neurological compromise, proposed for lumbar spinal stabilization surgery. she had a total hip replacement surgery in , with unknown transfusion history. her obstetric history was g p a . the patient had no history of thromboembolic or hemorrhagic events. during pre-transfusional tests, she was typed as a rr and had a positive antibody screening test. the identification studies were suggestive of an antibody against a highincidence antigen, so the surgery was delayed until clarification of these results. she was also referred to a pbm appointment where her hemoglobin was improved from . g/dl to . g/dl by administration of ferric carboxymaltose iv and darbepoetin sc. the patient was phenotyped as kp(a+b-) with anti-kpb, an antibody against a highincidence antigen (> % prevalence worldwide). it is a rare antibody with variable reactivity, causing from none to moderate/delayed transfusion reactions. to access the clinical significance of this antibody, a mma was performed, resulting in a reactivity of . %, suggesting no clinical relevance, however it could be altered after transfusion of kpb+ blood. in order to find compatible rbc's, several family members were phenotyped, however they were all positive to the kpb antigen. in portugal there were no rr kp(b-) blood donors, as it is extremely rare, so we searched in the international rare donor panel (irdp) and two donors were found in spain. two units of compatible rbc's were requested prior to the surgery, which was performed successfully four months later without transfusional support. summary/conclusions: anti-kpb is a rare antibody that in some cases can cause hemolysis of the transfused kp(b+) red blood cells. the combination of kp(b-) and o rr, an extremely rare phenotype, presented a challenge in finding compatible rbcs. this case illustrates not only the complex transfusional and logistic problems that an antibody against a high-incidence antigen can pose, but also the importance of an efficient pbm programme to mitigate the transfusional needs in these patients. background: blood transfusion is an integral part of the supportive care for patients with sickle cell disease (scd). allo-immunization is a recognized complication to red blood cells transfusion (rbc) in those patients. this may result in difficulties in providing compatible blood, and may be associated with the risk of acute of delayed hemolytic transfusion reactions. aims: to describe transfusion management in a patient with scd who has multiple alloantibodies with difficulty in obtaining compatible blood, in order to highlight the importance and clinical consequences of this complication and suggest a possible management approach methods: an -years-old female patient with scd presented to our hospital with hemoglobin level of g/dl secondary to acute splenic sequestration. she had a history of multiple previous admissions and many previous rbc transfusions. blood grouping and pre-transfusion compatibility testing were performed in addition to phenotyping of the patient's red cells. screening was done using column agglutination technique by automated machine (ortho; usa) and antibody identification was performed manually using commercial cells identification panel. phenotyping for the patient was done using haemagglutination technique with mono-specific anti sera (bio-rad; switzerland). results: the patient was of group o rhd (positive). antibody screening was positive and antibody identification revealed probable anti-e and anti-fya with possible development of anti-k allo-antibodies, in addition to recent development of autoantibodies; giving pan-positive reactivity with the identification panels. phenotyping of the patient's rbcs was found to be r r and k-negative. other masked allo-antibodies of undefined specificities were suspected and no compatible blood was found. the clinical condition warranted a blood transfusion, so least incompatible phenotypically matched rbc unit was released. the patient developed acute hemolytic transfusion reaction with drop of the hb level to . g/dl. despite screening hundreds of rbc units, no compatible units were identified, and no transfusion was given. the patient was managed conservatively using hydration, analgesics, hydroxyurea, erythropoietin, intravenous immune globulin (ivig), steroids, and rituximab. hb level increased to g/dl in weeks, and the patient was discharged from the hospital. the sample of the patient was sent to a reference lab (institut fur klinische chemie und laboratoriumsmedizin-regensburg -germany) for further investigations, clarifications and advice for compatible transfusion in case of need. the report of the reference lab revealed the development of additional anti-m and anti-s with confirmation of the presence of anti-fya, anti-k and warm auto-antibodies. phenotyping of rbcs was confirmed by molecular diagnostic testing done in the reference lab; as r r, k-neg. summary/conclusions: finding compatible blood may be extremely difficult in patients with scd who develop multiple alloantibodies. it is therefore essential to perform an initial extended red cell phenotyping for the patients at diagnosis and to have on shelf ready phenotyped blood units for issuing to the patients, to minimize allo-immunization. transfusion may occasionally be avoided in allo-immunized patients, utilizing alternative options of treatment and reducing the risk of serious complications such as hemolytic transfusion reactions. background: red blood cell (rbc) antigens that are present on less than % of most populations are known as low incidence antigens and those present on more than % are known has high incidence antigens. the mns blood group system consists of antigens carried on glycophorin a (gpa), glycophorin b (gpb) or on hybrids of these glycophorins. there are low incidence and high incidence antigens in the mns blood group system. an individual that is homozygous for gp.mur will be negative for the high incidence jenu (mns ) antigen. anti-jenu was first described in a thai patient with thalassemia where only compatible units were found following screening of units. the jenu negative phenotype is a rare phenotype with an estimated frequency of . %. a male patient with a history of previous transfusion presented with an anti-e and a weak auto antibody with no apparent specificity. a donor unit selected for cross match (group o rhd positive, c+e-c-e+, k-) was incompatible with a reaction grade of + by column agglutination technology. the patient's sample and donor unit were referred to the red cell reference laboratory for investigation for a possible antibody to a low incidence antigen. aims: we aim to characterize the phenotype of the incompatible donor unit. methods: standard serological procedures were used to identify the antibody specificities in the patient's sample. blood group phenotyping of the patient and donor was performed by standard serological procedures. genotyping and zygosity testing was performed using polymerase chain reaction (pcr) high-resolution melting (hrm) assay. gp.mur is a gp(b-a-b) hybrid glycophorin resulting from a gene conversion event between gypa and gypb . this disruption to gpb impacts s expression. the donor was negative with anti-s moab (albaclone), positive with anti-s polyclonal (immulab) and negative with anti-s monoclonal antibody (immulab). this s and s phenotype was consistent with the previously reported examples of gp.mur homozygote jenu negative individuals. molecular testing was consistent with serology supporting gp.mur homozygosity and jenu negative phenotype. summary/conclusions: this donor has been added to our rare donor panel and their red cell donations are cryopreserved for future use in our rare donor frozen inventory. there is limited anti-jenu antiserum available to confirm the jenu negative phenotype. we currently rely on the serological profile of red cells presenting with the gp.mur phenotype, s negative and the discrepant s phenotyping to identify jenu negative donors. this case has highlighted the importance of following up unexplained serological incompatibilities. the development of a monoclonal antibody directed against jenu antigen would provide an opportunity to screen for suitable donors for this rare phenotype. background: molecules expressed on tumor cells are a target of interest for drug development by the use of monoclonal antibodies or blocking proteins. however these drugs have the potential to interfere in pretransfusion testing when the target molecule such as cd is also expressed on red blood cells (rbcs). recently, many drugs targeting cd have been developed but appropriate mitigation strategies and approach to selecting rbcs for safe transfusion is still an obstacle. aims: we describe a case of delayed hemolytic transfusion reaction (dhtr) by anti-jk a in a patient treated previously with cd targeted high affinity sirpa fusion protein alx . methods: a -year-old woman diagnosed with nasal cavity squamous cell carcinoma was enrolled in an alx clinical trial. her blood type was group ab, rhd positive, and the antibody screening test was negative for the past months. she had no previous transfusion history during the past two years. after two infusions of alx , two units of apheresis platelets were requested for transfusion. the blood bank noticed that the antibody screening was positive and further investigation was proceeded. results: antibody screening showed trace positivity in both panel cells (i & ii) at room temperature (rt) and °c albumin phase, and + at anti-human globulin (ahg) phase by tube method. the auto control was negative at rt and °c albumin phase, but + at ahg phase. antibody screening ( cells) and identification ( cells) all showed + at ahg phase using gel cards. direct antiglobulin test was + for anti-igg and + for anti-c d using gel cards. two units of rbcs were requested for transfusion after hemoglobin decrease to . g/dl. rbc genotyping was unavailable at the moment. as her previous antibody screening was negative (anti-jk a not detectable), e-, c-, fy b -rbcs were given as a second best option, considering the phenotype distribution of major blood groups in the korean population. the hemoglobin level was well sustained between . - . g/dl but it decreased again to . g/dl twenty days after rbc transfusion. further laboratory investigation was consistent with a dhtr. the patient was no longer being given alx , and antibody screening and dat decreased to - + reactivity. we presumed that antigen typing results would be reliable after chloroquine dissociation and cell washing using antisera that did not require ahg for testing. serologic phenotyping showed that the patient's cells were c+, e+, c+, e+, jk a -, jk b +, fy a +, fy b -, s-, s+, m+, n + . antibody identification using papainized panel cells revealed anti-jk a antibody. we concluded that the dhtr was due to anti-jk a , and jk a -, fy b -, s-rbcs were issued for further transfusion requests. the patient's hemoglobin level recovered to . g/ dl. the patient's genotype was later identified to be the same as serologic typing. summary/conclusions: communication with the physician and blood bank to perform adequate pretransfusion testing before administration of drugs targeted to cd is important to achieve safe transfusion for patients. serologic phenotyping using antisera which do not require ahg for testing can be used as a second option when genotyping is unavailable in a timely manner. background: transfusion is still a key treatment for sickle cell disease (scd) patients. as a result, these patients are much more exposed to transfusions' risks, the most feared one being a delayed hemolytic transfusion reaction (dhtr). we investigated a female scd pediatric patient with no known antibody, who was referred to us for a suspicion of two dhtrs. three transfusion episodes were reported (a total of four units collected from four donors). for the last transfusion, a premedication with rituximab was done. the patient was planned to undergo a bone marrow transplant with her brother as her donor. aims: to describe the molecular and serological workups needed to investigate a dhtr in a scd patient. methods: antibody identification and crossmatches were performed by iat gel testing with red blood cells/panels, which were used raw, papain-treated and trypsintreated. rbcs' phenotypes were determined by conventional techniques. semi-quantitative phenotypes were conducted by serial dilutions with a monoclonal anti-jk a (ms /seraclone â ). dna was extracted using the magna pure compact instrument (roche). sequencing of jk exons - was carried out by in-house techniques. results: the antibody identification showed a very weak anti-jk a , which was only reactive on papain-treated rbcs. autologous control was also only positive in this technique. dat and the eluate were negative. as the patient had recently been transfused (less than four months earlier), on this first sample we were neither able to perform autologous adsorptions, nor verify her jk a /jk b phenotypes. in order to rule out the imputability of an anti-lfa in the dhtr outcome, crossmatches with her donors' rbcs were undertaken. three out of the four donors were tested. apart from the anti-jk a reactivity, none of them was reactive. because the patient had previously been phenotyped as jk(a+b+), her jk gene was sequenced. her genotype was determined as jk* ( a, a, a, g)/jk* . to confirm this jk a variant allele, a family study was conducted. all her siblings were found to harbor the same genotype. her mother's and father's genotypes were jk* ( a, a, a, g)/ jk* and jk* /jk* , respectively. subsequently, autologous adsorptions were performed, which proved the anti-jk a to be an autoantibody. considering the weakness of this antibody, internal controls were used, in order to evaluate a possible dilution effect of this technique. finally, serial dilutions with the anti-jk a reagent showed a weakened jk a expression encoded by the jk* ( a, a, a, g) variant allele. this finding is consistent with the fact that the crossmatches between the proband's serum and her brother's rbcs were weaker than those performed with (jka+b+) rbcs. summary/conclusions: about a third of dhtrs are reported to happen in patients with no previous history of immunization. performing sensitive serological techniques in order to identify antibodies is necessary to select the most appropriate units. molecular work and extra serological testing can be useful to determine whether an antibody is an allo or autoantibody. even though in this case the anti-jk a was the only antibody identified, because it was proven to be an autoantibody, it is difficult to conclude if it was the cause of the dhtr. nevertheless, jk(a-b+) blood was issued, and no adverse events have been reported. luckily, the patient's bone marrow donor harbors the same variant allele. background: according to the aabb, a pre-transfusion sample must be obtained within days of transfusion if a patient has been transfused or pregnant in the preceding months. despite this safeguard, high risk patients (i.e. those recently transfused with a history of pregnancy or transfusion) may develop antibody during this day window. to avoid issuing incompatible red blood cells (rbcs) to these patients, a new antibody screen (abs) sample should be drawn and tested shortly before anticipated transfusion. aims: we report a case of a y/o man who presented to the ed (hospital day , hd ) with a post-fall intracranial hemorrhage and multiple fractures. anti-e and anti-jka were identified after admission on a new specimen prior to current specimen expiration (< days). methods: specimen # (s ) was sent on hd for type & abs (t&s) and crossmatch (xm) of rbcs. abs and immediate spin xm were negative; there was no patient history. by hd , he had negative t&s specimens (hd : s ; hd : s & ; hd : s ) and had been transfused rbcs (hd : ; hd : ) via electronic xm (exm). at hr on hd , rbcs were requested and could have been issued via exm since s was not expiring until midnight. however, given recent transfusions, bb staff first called the patient's nurse to review history. patient was uncommunicative, but had scars suggesting past trauma or surgery. s was requested and received at hr. results: s showed anti-e and anti-jk a in plasma and eluate. his hemoglobin/hematocrit (h/h) decreased from . ( . - . g/dl)/ . ( . - . %) on hd to . / . on hd . during this period, he underwent several surgeries without unexpected bleeding, documented jaundice or dark urine. two e-jk(a-) rbcs were transfused on hd , which he tolerated well with an increase of hemoglobin from . g/dl to . g/ dl. he did well post transfusion with stable h/h between . / . . to . / . . he was discharged on hd . repeat abs on s was negative. of the rbcs transfused before s , one was e+ and four jk(a+). the family reported that he was injured years prior and had been admitted to hospitals, but was unaware of transfusion. hospital # (h ) reported admissions years ago ( rbcs transfused) and years ago; all abs were negative. h admission was years ago with positive abs and inconclusive workup. h admission years ago showed negative abs. summary/conclusions: the patient developed a significant antibody response in less than days from the specimen collection, likely a secondary immune response to sensitization from a transfusion years earlier. a new specimen was requested prior to transfusion even though the existing sample (which was abs negative) had not expired. this approach identified new antibodies, preventing transfusion of incompatible rbcs, and a potentially serious hemolytic transfusion reaction. this case suggests that for high-risk patients, abs more frequently than every days may be beneficial. it is important to increase clinicians' and laboratorians' awareness of this issue. background: red cells with partial d antigen have historically been classified as such, based on the fact that the red cells type as d positive, but individuals make anti-d antibody when exposed to conventional d antigen. a definitive confirmation of the variant of d antigen is obtained after the rh d genotyping. aims: to present a case study of the patient's alloimmunisation with the present d partial antigen type dnb, most likely on previously received transfusions. methods: the patient's pretransfusion testing included the determination of the abo blood group and rhd type (id card diaclon abo/d dv+, dv-, reverse grouping, monoclonal antibodies, gel method), antiglobulin crossmatch, additional phenotyping (gel and tube methods), antibody screening, identification of the specificity of irregular anti-erythrocyte antibodies by commercially available red cell panels (id dia-panel bio rad gel method, panocell immucor, tube method) through an indirect antiglobulin test (iat) and enzymes. after routine rhd typing we continued further characterisation of the rhd antigen by serologic assay (bio-rad id-partial rhd typing),and finally by rhd antigen molecular genotyping (fluogene method on fluo vista machine). results: our patient is a year old woman with a diagnosis of tu mammae who was preparing for total mastectomy surgery. she had a history of blood transfusions twenty years ago, and she also had two births. the blood group typing was: o, ccdee, k-, fy (a-b +), jk (a+b +), ss, mn, le (a+b -). the agglutination reactions that we tested with anti d serums were strong ( +). the compatibility test with rhd positive donated blood units was positive. the presence of anti-d and anti-fya antibodies in the serum of the patient was determined. we prepared one compatible blood unit, rhd negative and fya negative, for a surgery. interpretation of the id-partial rh d typing set indicated that this is a diii category of d partial antigen. a sample of blood of our patient was sent to the blood transfusion institute of serbia, where molecular typing of d antigen was performed and the presence of partial form of antigen d, dnb type, was found. summary/conclusions: rhd positive patients or donors with anti-d antibody presents in their serum should be tested for d genotyping. the recommendation for further transfusions of our patient with dnb d partial and her alloimmunisation is to prepare d negative, fya negative erythrocytic blood components, and as a possible blood donor it would be labeled as rh d positive. background: the jr blood group system consists of jr a (jr ), a high frequency antigen expressed by the abcg gene. the individuals with jr(a-) phenotype are mainly found in the japanese population and may develop anti-jr a when stimulated by blood transfusion or pregnancy. anti-jr a is a dangerous antibody for pregnancy, but also could cause mild or moderate neonatal jaundice. aims: to conduct the antibody specificity identification of the high frequency antibody in a pregnant woman with history of pregnancy but no transfusion. methods: abo, rhd and some special blood group antigens were identified by tube method in saline. antibody screening and blood group specific antibody identification were performed by indirect antiglobulin test (iat) in gel column. the reagent cells treated with trypsin, chymotrypsin and papain, were used to test the antiserum to obtain the characteristic of antibody reaction. the antibody titer in the patient's serum was detected. dna sample was extracted and exons and adjacent intronic sequence of the abcg gene were sequenced. the sample of one family member was collected for testing. results: the blood groups of the patient were b, rhd(+), lu b (+) and kp b (+). the negative reaction of the serum reacted with all reagent cells were tested in saline, but positive ( +) in iat test, while the self-control was negative. the antiserums reacted strongly ( + in iat test in gel card) with the papain-treated cells, but kept the same reaction strength ( +) with trypsin-and chymotrypsin-treated cells, which indicated the possible existence of anti-jr a . the titer of igg antibody in serum was . in cross matching test, the red blood cell of the patient's brother with the same abo and rhd blood group with the patient was successfully matched with the serum of the patient. the sequencing analysis of the abcg gene in the patient and her brother revealed one homozygous nonsense mutation in exon (c. c>t, p.gln x). after the delivery of the pregnant women, no pathological jaundice was seen in the newborn. summary/conclusions: in the condition of the anti-jr a reagent was unavailable for the identification of jr a antigen in the patient, having an indication with anti-jr a by serological test, the alternative genotyping method was used. the most common silencing jr allele reported in asian population, especially in japanese population, was identified to indicate jr(a-) phenotype. immunohemotherapy, centro hospitalar vila nova de gaia/espinho, vila nova de gaia, portugal background: if the investigation of irregular/unexpected antibodies reveals a pattern in which all or most screen and panel cells are positive, with reactions in the same phase and with the same strength, along with a negative autocontrol, an irregular antibody to a high-prevalence antigen may be suspected. high-prevalence antigens are those that are present in almost all individuals ( % or more). fortunately, because these antigens do occur so frequently, it is not common to find a patient with an antibody to one of them. however, when it happens, it may become a troubling situation. aims: clinical case report of panagglutination in assessment of irregular antibodies. methods: collection of clinical data in scl ınico â and sibas â applications. results: woman, years old, o rhd+, previously transfused with red blood cells concentrates in , was proposed to a correction surgery of a periprosthetic hip fracture. pretransfusion serologic tests were requested and irregular antibodies were detected ( + in all the screening cells). in order to identify the specificity of the antibody, a panel of cells was tested; the result was considered inconclusive, due to positive reactions ( +) with all test cells in liss/coombs and atypical positivity with dragging in all cells in enzymatic environment. autocontrol and direct antiglobulin test were negative. it was decided to send two blood samples to the reference laboratory for a more complete immunohematological study. compatibilization of red blood cells to this patient was also requested. during the waiting period, haematopoiesis was optimized. although the patient did not present anaemia at admission, the analytical study revealed iron deficiency; therefore, supplementation with intravenous iron was performed. the reference laboratory also obtained a panreactive panel ( + with all cells) in liss/coombs and weak positivity in papain. after allo-adsorption, the search for irregular antibodies was negative. an anti-yt a , apparently without clinical significance (negative igg and igg ) was then identified. transfusion was not needed either during or after the surgery, with a good recovery of the haemoglobin value in the postoperative period. summary/conclusions: yt a , which belongs to cartwright system, is a high-prevalence antigen in all populations. anti-yt a , an igg antibody stimulated by pregnancy or transfusion, is not as uncommon as we may think, which suggests that it is reasonably immunogenic. these antibodies are not generally considered clinically significant, but there are reported cases of acute and delayed haemolytic transfusion reactions in which anti-yt a has been implicated. therefore, although the described pattern of panagglutination in assessment of irregular antibodies may suggest the presence of an alloantibody directed against a highfrequency antigen, it is very important to confirm that hypothesis, recurring to a reference laboratory if necessary, to identify the antibody and to determine its clinical relevance. even if the identified antibody is associated with rare haemolytic transfusion reactions, it is crucial to optimize haematopoiesis when it is not an emergent procedure, in order to minimize transfusion and its associated risks. both for emergent and elective procedures, the creation of a national database of patients with already identified irregular antibodies would facilitate the administration of red cells concentrates without the implicated antigen. aims: to investigate the frequency and explore the genomic characterization of jk (a-b-) phenotype in blood donors in harbin, china. methods: all samples were screened for jk(a-b-) phenotype using a direct urea lysis test. and the results were confirmed with by iat using anti-jka and anti-jkb with a standard tube test. additionally, polymerase chain reaction amplification and sequence analysis of the jk gene were performed. results: from blood samples, four donors with jk (a-b-) were selected, at a frequency of . %. among these four samples available for sequencing jk gene, a total of two genotypes were discovered: heterozygote of ivs - g>a combining with heterozygote of g>a (gly glu) and heterozygote of g>a (gly glu) combining with heterozygote of c>t(thr met). summary/conclusions: the frequency of jk(a-b-) phenotype in blood donors in harbin area was lower than the reported data from the populations in other areas of china and in finland, but higher than that in japan. ivs - g>a, g>a and c>t were common mutations in the before reports, while g>a was reported first time. in addition, it is an effective measure which establish the jk(a-b-) phenotype donors in this region, to solve the blood transfusion problem in patients with anti-jk . background: blood types, indicating the type of blood group antigen expressed in the red blood cells, is determined by the type of allele at the blood group gene locus. therefore, when allele frequency of each blood group gene is determined, it is possible to predict the frequency of a specific blood type donor with a homozygous allele. it is also possible to estimate the proportion of donors within a particular blood type through combination of specific alleles. and because the ratio of blood group allele differs between ethnicity and race, this can be used as basic data for population genetics and anthropology. therefore, we present a study that examined the allele frequencies of blood group systems in the korean population through blood group genotyping. aims: the purpose of this study is to determine the frequencies of blood group alleles in the korean population, to predict the proportion of homozygous donors, and to obtain the basic data of population genetics. methods: , blood donors from age to were recruited at korean red cross blood centers located nationwide. acquired samples were examined by blood group genotyping methods using the rbc genotyping system id core xt (progenika biopharma). for each donor, genotypes of blood group systems, excluding abo and rhd, were identified. calculation of the frequencies of blood group alleles in the korean population was done. results: we conducted molecular genotyping of the rhce, kell, kidd, duffy, mnss, diego, dombrock, colton, cartwright, and lutheran blood group systems. the allele frequencies of these blood group systems in the korean population were estimated as follows. -rhce*ce . %, rhce*ce . %, rhce*ce . %, rhce*ce . % -kel*k_kpb_jsb allele % -jk*a allele . %, jk*b allele . %, jk*b_null allele . % -fy*a allele . %, fy*b allele . % -gypa*m allele . %, gypa*n allele . % -gypb*s allele . %, gypb*s allele . %, gypb*mur allele . % -di*a allele . %, di*b allele . % -do*a allele . %, do*b allele . % -co*a allele % -yt*a allele % -lu*b allele % summary/conclusions: the significance of this study is accumulation of data on the allele frequencies of blood group genes through highly accurate genotyping method in the east asia region. this enables the prediction of the proportion of donors with a combination of specific blood group alleles in the korean population, which accounts for a decent percentage of the population in this region. background: in donors from arabian countries only little is known about blood groups other than abo and rhesus. during the last years increased migration to central europe has put a focus on the question how to guarantee blood supply for patients from these countries, particularly because hemoglobinopathies with the need of regular blood support are more frequent in patients from that region. aims: blood group allele frequencies should be determined in individuals from syria, other arabian countries, and iran by molecular typing. methods: as most blood groups are defined by single nucleotide polymorphisms (snps) we introduced a maldi-tof ms assay to detect alleles encoding blood groups including kk, fy (a/b), fy null , c w , jk(a/b), jo(a+/a-), lu(a/b), lu ( / ), ss, do (a/b), co(a/b), in(a/b), js(a/b), kp(a/b), rhce*c. c>g, and rhce*c. c>g. additional blood groups and polymorphisms like yt(a/b), s-s-u-, vel null , co null and rhce*c. g>t were tested by pcr-ssp. a total of probands including individuals from syria, from iran, from the arabian peninsula and from northern african countries were included. results: % of the donors were homozygous for the fy null (fy*- t>c, fy* n. ) mutation, . % carried the heterozygous mutation. . % of the syrian probands were heterozygous for the do* c>t mutation (both, do*jo and do*jo ; jo(a+/ a-)) that is virtually unknown in caucasian donors. . % of the syrian donors heterozygously carried the kel* . allele coding for js(a) (phenotype js(a+/ b+)) that is very rare in caucasians. however, no homozygous kel* . carriers were identified. . % of the syrian and . % of all donors were negative for yt*a, which is definitely more frequent than in europeans. one donor from northern africa homozygously carried the gypb*c. c>g, intron + g mutation, inducing the s-u+ w phenotype. . % of all and . % of northern african donors were heterozygous for the rhce*c. c>g substitution, . % of the syrian donors carried rhce*c. c>g (heterozygously) and . % of all donors were heterozygous for rhce* g>t. heterozygosity for vel deficiency (vel*- ) was detected in individuals ( %; of them from syria) whereas only one syrian donor carried the homozygous mutation. none of the donors carried the aqp *c. delg (co* n. ) mutation that induces the co null phenotype. summary/conclusions: the study provides a first overview on a number of different blood group alleles in blood donors from arabian countries. some blood group alleles that largely are lacking in europeans but had been described in african individuals are present in arabian populations at a somewhat lower frequency. in single cases it could be challenging to provide immunized arabian patients with compatible blood. methods: three unrelated individuals ( blood donors and one pregnant woman) of polish origin who were typed as ab group with a very weak a antigen and normal b antigen expression were subjected to extended abo typing. in one case family studies were performed (blood samples from donor's mother, father and sister). sequencing analysis of this donor dna was performed three times (from two blood samples and buccal swab). serologic investigations were performed with standard methods: /gel cards diaclon id abo/d (anti-a: clone a , anti-b: clone g / , anti-a,b: clone es , es + birma + es ; bio-rad) and diaclon id abd-confirmation for donors (anti-a: clone m / = la- ; bio-rad); /tube techniques with: anti-a (birma ; a- h , a s.pa m , c. d ), anti-b (lb- , b- f , c. a ). genotyping was determined by rbc fluogene abo basic kit (inno-train, germany) and by sequencing of + . -kb site of abo gene to cover sequences ranging from the end of intron to utr of the abo gene. additionally sequence of exon of the abo gene was analyzed. results: abo typing showed normal b and a very weak a antigens on rbcs of all three individuals ( blood donors and one pregnant woman). the a antigen was detected by tube technique only using anti-a clones: birma ( + to +), a- h ( + to +) and c. d (weak+ to +); negative reaction of a antigen typing by gel cards was observed. the sera of all individuals contained anti-a antibodies. commercial pcr-ssp kit revealed three heterozygous a/b genotypes (absence of delc typical for abo*a alleles). in all these individuals abo sequencing of . -kb fragment confirmed the heterozygous genotype with polymorphisms characteristic for abo*b. allele ( a>g; c>g; c>t; g>a; c>a; g>c; g>a) and detected a novel abo*a allele sequence with duplication-based insertion of bp after position (abo*a c.dup _ ; gcaggacgtgtccatgcgccg). as a consequence, the online protein translation predicts an in-frame duplication of seven amino acids after codon (p.dup_ _ qdvsmrr), with synonymous change of the repeated codon (cgc>cgg) and (cgg>cgc) but both coding arginine (r). inheritance of abo*a c.dup _ allele was confirmed by family studies of one donor: his father and sister had a/b genotype associated with normal a and b antigens expression; his mother had normal a antigen expression. she carried abo*a . allele and the same abo*a c.dup _ allele as a son. summary/conclusions: a novel a weak allele at the abo gene detected in three unrelated polish individuals is an in-frame insertion of seven amino acids to the wild-type glycosyltransferase a. the stability of the encoded protein may be affected causing the weak a phenotype. the inheritance of this mutation was confirmed in the family studies. background: since the cloning in of cdna corresponding to mrna transcribed at the blood group abo locus, polymorphisms and phenotype-genotype correlations have been reported by many investigators. although many subgroups have been explained at the genetic level, unresolved samples are still encountered in clinical practice. we report here the result of an abo investigation of a sample from a swedish blood donor that showed a very weak agglutination of rbcs with anti-a in routine forward typing. aims: to elucidate the genetic basis of the apparent weak a subgroup. methods: routine abo genotyping by pcr-asp and pcr-rflp including pcrbased analysis of the upstream cbf/nf-y-binding enhancer region was carried out. further genetic analysis was performed by dna sequencing of abo exons - (including base pairs of the adjacent introns) and the proximal promoter. flow cytometric testing of rbcs was performed with monoclonal anti-a, anti-b and anti-h. results: the weak agglutination of erythrocytes with anti-a was accompanied by the expected lack of anti-a and anti-a in plasma. abo genotyping gave the genotype abo*a . /o . usually consistent with normal expression of a antigen. enhancer analysis resulted in an amplification product corresponding to the expected single cbf/nf-y binding motif. flow cytometric testing of the sample showed a antigen expression with an almost chimeric pattern where the majority of the cells (approximately %) expressed the a antigen at a very low level, marginally distinguishable from the group o control. the remaining approximately % of the cells displayed an a antigen level ranging from normal to very weak. genomic abo sequencing showed an abo*a . -like allele except for a novel mutation located in intron , c. + g. the o allele had an additional snp, c. g>a, consistent with the abo*o . allele variant summary/conclusions: a previously unreported variant, c. + a>g, likely effecting the -donor splice site of intron was found in an a weak sample. this type of mutations is expected to decrease mrna stability and/or cause skipping of the preceding exon in the mrna. however, small amounts of full-length enzyme might still be made, being able to give rise to the weak a antigen expression seen in this individual. interestingly, this mutation is very similar to the genetic variant underlying the weak a subgroup a finn . in this case, however, the c. + a>g mutation is located in the -end of intron and is predicted to cause partial skipping of exon . strikingly, the a finn phenotype also results in a pseudochimeric pattern by flow cytometry but with only approximately % positively staining erythrocytes. due to the well documented lack of a-allele-derived mrna in peripheral blood, further transcript studies could not be undertaken. further studies are needed to investigate the exact mechanisms underlying the pseudochimeric pattern observed by flow cytometry in these two interesting genotypes/phenotypes abstract withdrawn. background: abo is the clinically most relevant blood group system in transfusion and transplantation medicine. abo genotyping is potentially useful in clarifying serologic blood grouping discrepancies. this scenario includes inherited subgroups alleles, temporary acquired variant abo phenotypes in disease or pregnancy, and chimerism due to exchange of progenitor cells early in fetal life or after blood progenitor cell transplantation. aims: to investigate the molecular basis for abo discrepancies detected in clinical samples, including donors and patients, sent to our reference laboratory during the past years. methods: if routine abo grouping showed weak agglutination or forward vs reverse typing discrepancy, further abo typing studies were performed manually. adsorption-elution tests were also performed in some cases with polyclonal anti-a and anti-b to confirm whether a or b antigens were weakly expressed on the rbcs membrane. a pcr approach using sequence specific primers for a , b, o and o alleles was used for initial genotype determination. the full abo coding region was analysed as previously described in selected samples for which abo discrepancy was still unexplained. allele specific fragments spanning exon , intron and exon were amplified using a forward primer targeting the g nucleotide (to exclude o alleles amplification) in combination with either b, a or a generic reverse primer. analysis was carried out by sanger sequencing. results: a total of samples with suspected inherited abo subgroup alleles were selected for further molecular studies by sequence analysis. a subgroup alleles: in out of samples with suspected a subgroup alleles, the c. insg insertion was detected corresponding to the abo*ael. allele. the abo*aw . - variant, a hybrid a -o v allele, was found in cases. in case we found the c. g>c change, previously reported associated with weak a antigen expression. finally, a novel c. c>g change was detected in an a allele. b(a) or cis-ab suspected alleles: the abo*b(a) variant carrying the c. a>g change was found in of samples with bo genotype but a weak antigen expression. in the remaining cases, a consensus b allele was detected, thus pointing to a potential chimerism as the cause of the results observed in abo grouping. finally, we have identified an abo*b . allele carrying the nucleotidic change c. a>g in the context of an abo phenotype vs genotype discrepancy. summary/conclusions: the sanger sequencing approach applied in this study have proved to be informative and helpful to determine the molecular basis of abo grouping discrepancies with suspected inherited subgroups. we found mutations, within exon of the abo gene, in out of samples, including novel alleles. chimerism was suspected in cases of a antigen expression in samples with b o genetic background carrying an apparent normal b allele. we are evaluating at the moment a deep sequencing approach by next generation sequencing to determine the presence of a small amount of a minor allele in the presence of a large surplus of the other two alleles. background: recently, the multiple pregnancy rate has been increasing due to advances in artificial fertilization including in vitro fertilization-embryo transfer. most dizygotic twins have dichorionic placenta, but % of them share the placenta. monochorionic dizygotic twins can have blood chimerism, leading to double rbc populations in routine abo serologic typing. recently, more sensitive and objective column agglutination tests with automated systems are being widely used. therefore, blood chimerisms in dizygotic twins can be detected more easily by routine abo blood typing. aims: we report congenital blood chimerism in monochorionic dizygotic twins of triplets, found incidentally during abo serological testing and confirmed by abo genotyping and str marker analysis. methods: a -year-old male (one of triplets) was admitted to the hospital for medical checkup. he did not have any history of transfusion or bone marrow transplantation. routine abo blood grouping test was performed using automated blood bank system ih- ; however, it showed abo discrepancy. the red blood cells showed double cell populations in a gel column with anti-a and anti-b. we carried out abo genotyping both from the blood and from a buccal swab. for the further evaluation, we performed abo serologic testing, abo genotyping, and str marker analysis in his family members. results: among the triplets, blood chimerism was demonstrated in the patient and his brother. they both showed a b phenotypes in the serologic test and tri-allelic abo genotypes in the blood, a /b /o . however, in buccal swabs, the patient showed a /o and his brother showed b /o . other members of the family (father, mother, and dizygotic sister) had regular abo blood types in the serologic test. we performed str analysis in the triplets and parents. eleven loci (d s , d s , d s , csf po, th , d s , d s , d s , d s , d s , and fga) revealed more than one additional allele in the blood sample, apart from those in the buccal swabs. str marker analysis showed that his brother too had blood chimerism. summary/conclusions: we found blood chimerism in monochorionic dizygotic twins of triplets during routine abo blood typing, and this was confirmed by str analysis. as the application of assisted reproductive technology increases, the incidence of blood chimerism will also increase. blood chimerism can often create confusion during abo serologic typing and microchimerism can be overlooked in routine methods. therefore, it is helpful to use an automated blood bank system to improve sensitivity and blood chimerism should be considered if abo blood grouping reveals double populations. background: expression of abo transferase genes can be affected by genetic variants located within the coding sequence, at splice junctions, in the proximal promoter and in the intron enhancer. here we describe five new alleles with singlenucleotide substitutions found in samples with discrepant or unusual abo serology. aims: to resolve serological discrepancies or unusual serological findings in the abo blood group system by molecular methods, in particular by sanger sequencing. methods: forward and reverse abo phenotyping was performed by the gel or tube methods. genomic dna extracted from whole blood was pcr amplified to cover the entire abo coding sequence, splice junctions, proximal promoter and intron enhancer. amplification products were sanger sequenced directly or after cloning in a bacterial host. results: case # is a patient with an unclear abo phenotype: forward type b, reverse type ab. sequencing of genomic dna and cloned abo exon detected variant c. - t>g in heterozygosity on an otherwise common a allele, and in trans an abo*b. allele. case # is a caucasian donor with an abo discrepancy: forward type aweak/o, reverse type a. sequencing also detected variant c. - t>g in heterozygosity on an a background, and in trans an abo*o. . allele. given that this variant is located near the intron splice acceptor site, abo* - g transcripts are postulated to undergo altered splicing, leading to an aweak phenotype. case # is a prenatal sickle-cell disease patient with an abo discrepancy: forward type aweak, reverse type a. dna sequencing detected variants c. c>t (pro leu) and c. t>a (tyr asn), both in heterozygosity on an otherwise common a allele, with an abo*o. . allele in trans. thus, the data establish an association of allele abo* t, a with an aweak-like phenotype. case # is a donor with an abo typing discrepancy: forward type o, reverse type a. sequencing detected variant c. c>g (pro arg) in heterozygosity on an a background, and in trans an abo*o. allele. an interpretation of the data is that variant c. c>g weakens the activity of the a transferase, with allele abo*a ( g) encoding the aweak-like phenotype detected by serology. case # is a year-old patient with an abo discrepancy: forward type o, reverse type ab. sequencing of genomic dna and cloned abo exon detected variant c. g>c (gly ala) in heterozygosity on an a background, and in trans an abo*o. . allele. the serology and molecular results suggest that allele abo*a ( c) encodes a cisab weak phenotype. case # is a caucasian donor with an abo typing discrepancy: forward type o with a weak agglutination with anti-ab, reverse type o. dna sequencing detected variants c. g>a (glu lys) and c. g>a (asp asn), both in heterozygosity, in trans, and on a backgrounds. variant c. g>a by itself constitutes allele abo*a . . the phenotype encoded by abo* a is uncertain. summary/conclusions: molecular characterization of abo alleles can help in their future identification and discrepancy resolution. background: expression of abo transferase genes can be affected by genetic variants located within the coding sequence, at splice junctions, in the proximal promoter and in the intron enhancer. here we describe five new alleles with singlenucleotide substitutions found in samples with discrepant or unusual abo serology. aims: to resolve serological discrepancies or unusual serological findings in the abo blood group system by molecular methods, in particular by sanger sequencing. methods: forward and reverse abo phenotyping was performed by the gel or tube methods. genomic dna extracted from whole blood was pcr amplified to cover the entire abo coding sequence, splice junctions, proximal promoter and intron enhancer. amplification products were sanger sequenced directly or after cloning in a bacterial plasmid vector. results: case # is a year-old pregnant female with an abo typing discrepancy: forward type o, reverse type a. pcr-rflp predicted abo*a /abo*o . sequencing detected variant c. insg (val gly>fs ter) in heterozygosity on an otherwise common a allele, and in trans an abo*o. . allele. it is unclear how the early truncation of the a transferase encoded by allele abo* insg still allows for some residual enzyme activity, as suggested by the reverse a type. case # is a recently-transfused year-old black patient with an unresolved abo type. sequencing detected variant c. a>g (silent) in homozygosity and variant c. c>t (ala val) in heterozygosity, both on an o background, with an abo*b. allele in trans. although variants c. a>g and c. c>t are likely of no consequence to the abo phenotype of this patient, they are reported here as components of a new abo*o ( g, t) allele. case # is a year-old prenatal female with a rhd typing discrepancy. failure to yield an abo genotype on blood-chip (progenika), a genotyping microarray that interrogates polymorphic positions in rhd and abo, prompted dna sequencing. sequencing of genomic dna and cloned abo exon detected variant c. c>t (arg cys) in heterozygosity on an abo*b allele background, and in trans an abo*o. . allele. the phenotype encoded by allele abo*b( t) is predicted to be b, as evidenced by forward typing on immucor neo and reverse manual typing. case # is a prenatal black patient with an abo typing discrepancy: forward type o in gel, a + mixed field (mf) in tube. reverse type on a cells + in gel, / + in tube. sequencing of genomic dna and cloned pcr products covering exons - detected variant c. g>c (asp his) in heterozygosity, and in trans an abo*o. . allele. case # is the newborn baby of case # , with a forward type a + mf in gel, a + mf in tube. sequencing of the baby's dna detected variant c. g>c (asp his) in heterozygosity, and in trans an abo*b. allele. from these results it is inferred that the phenotype encoded by allele abo* c is a -like. case # is an year-old hispanic donor with an abo typing discrepancy: forward type a, reverse type o. sequencing of genomic dna and abo exons - and - detected variant c. c>t (gln ter) in heterozygosity, and in trans an abo*o. . allele. the truncation of the a transferase at such a relatively late position is consistent with the retention of some enzyme activity, explaining the forward a type encoded by allele abo* t. summary/conclusions: molecular characterization of abo alleles can help in their future identification and discrepancy resolution. background: expression of abo transferase genes can be affected by genetic variants located within the coding sequence, at splice junctions, in the proximal promoter and in the intron enhancer. variants reported to date in the intron enhancer include large deletions, small deletions and single-nucleotide substitutions. here we describe four new alleles with single-nucleotide substitutions found in samples with discrepant or unusual abo serology. aims: to resolve serological discrepancies or unusual serological findings in the abo blood group system by molecular methods, in particular by sanger sequencing. methods: forward and reverse abo phenotyping was performed by the gel or tube methods. adsorption-elution by the heat elution method and testing for h and a substances in saliva were performed by following the procedures in the aabb technical manual. genomic dna extracted from whole blood was pcr amplified to cover the entire abo coding sequence, splice junctions, proximal promoter and intron enhancer. amplification products were sanger sequenced directly or after cloning in a bacterial plasmid vector. background: inactive alleles of the fut could be decreased or aborted the activity of the fucosyltransferase, which results in to form the bombay or para-bombay phenotype with weak or no h antigen expression on erythrocytes. now many para-bombay individuals have been found in the chinese population. according to names for h blood group alleles v . of red cell immunogenetics and blood group terminology working group of the isbt, fut alleles were identified for bombay or para-bombay phenotype around the world. aims: the study was explored the distribution of fut alleles for the chinese individuals with para-bombay phenotype. methods: the samples were come from the blood donors or the patients. the a, b, h antigens were determined using conventional serological method according to the manufacture's instruction. the sequences of the full coding region for fut was amplified, then amplicon was purified with enzymes digestion and used as template for sequencing bidirectionally. all nucleotide sequences obtained were analyzed and compared with standard fut sequence. results: nineteen chinese individuals with para-bombay phenotype were identified. ten of them were the donors and nine individuals were come from the hospital. the rbcs had a very weak agglutination reaction with anti-h in the most of the individuals. fut homozygous mutations were found in the individuals and fut heterozygous changes were existed in individuals after bidirectionally sequencing. . %, . %, . %, . %, . %, . %, . %, . % respectively in the individuals with para-bombay phenotype. according to our previously reports, the fucosyltransferase activity of fut * n. (c. _ delag), fut * w. (c. c>t) and fut * w. (c. c>t) were abolished in vitro assay, while fut mrna levels of them had no effect compared with wild type. summary/conclusions: the fut mutations in the para-bombay individuals were various. the most common fut allele in the chinese individuals with para-bombay phenotype was fut * n. (c. _ delag). background: the regulatory mechanism of the abo gene is complicated and has been investigated extensively.variation in a antigen expression was recognized very early in the twentieth century and the a blood group was divided into a and a . later the a blood group was subdivided further based on characteristic reactivity with human polyclonal antisera, i.e., strength of reactivity and presence of mixed field agglutination; presence of anti-a , and whether a or h blood group substance was present in the saliva of secretor subjects. mutations critical for abo blood group phenotypes have predominantly been found in exons and of the abo gene, both of which encode the catalytic domain of abo glycosyltransferase. in our case report we show how mutation ranging from single nucleotide in the intron enhancer element can alter the efficacy of enzyme and alter antigen expression. aims: this study aims to investigate the molecular basis of discrepant results of abo forward/reverse typing in blood donor. methods: the abo typing was performed using tube technique and column agglutination tests (bio-rad, grifols). standard tests were completed with adsorption-elution study using o plasma as a source of anti-a, and with saliva testing for presence of a and h substances. we performed quality control for these methods. abo group genotyping was performed using pcr with sequence-specific primer by commercial kit (abo-variant; bag healthcare, lich, germany). pcr-amplified exons and intron enhancer were subject to bi-directional dna sequence analysis using standard sanger dideoxy chemistry. seqscape software (abi) was used to analyze sequence data by comparing the obtained sequence to a reference sequence from ncbi. results: standard serological forward tests identified blood group o, however, only anti-b iso-agglutinins were present. anti-a in adsorption-elution study was successfully adsorbed and eluted from the investigated cells. a and h substances were detected in saliva. abo genotyping using pcr-ssp indicated genotype o v/a . dna sequence analysis showed result abo*a ( + a), abo*o. . . the specimen was revealed as an a subgroup, probably a m with an unusual genetic variant in the intron region of the abo gene, the enhancer of the gene expression. summary/conclusions: we report the first case of abo*a ( + a), the mutation located in the enhancer region of gene expression in allele a, that causes discrepant results not only in abo forward/reverse typing but also in molecular blood grouping tests. based on our serological findings, this subgroup is considered as a m . background: a chimera is a single organism composed of cells with distinct phenotypes and/or genotypes. several different types of chimeras are described: artificial, twin and dispermic. the artificial chimerism can be seen following hematopoietic stem cell transplantation, or more transiently following blood transfusion. the second type may also be inherited most commonly through blood exchange in utero between twins. dispermic chimerism is induced by the fertilization of two maternal eggs with two spermatozoa and their fusion into one body. this one is also called tetra-gametic chimerism. in transfusion medicine, chimeras are often detected when mixed field reactivity is observed in abo/d typing or, less commonly, when phenotyping for other blood group antigens. aims: this investigation was prompted by finding a double population of erythrocytes in a surgery patient with no transfusion history. our aim was to investigate the chimera and determine the underlying abo genotype of this patient. methods: routine blood grouping was performed by column agglutination. separation of the double cell populations was performed by differential agglutination with igm anti-d (immuclone, anti-d fast igm, clone: d - , immucor). initial abo genotyping was performed by pcr-ssp (fluogene; inno-train diagnostik gmbh); further resolution was performed using in-house pcr-asp and pcr-rflp methods. next generation sequencing (monotype abo; omixon using illumina sequencing platform) and sanger sequencing analysis were also performed. identification of reference alleles was investigated by fragment analysis of short tandem repeats (str) polymorphisms. results: double population was found in column agglutination in tests with anti-a and anti-ab, and subsequently when typing for d and c antigens, with approximately % of o d+c+ cells. the patient's genotype was identified as abo*o. /*a by ce-certified pcr-ssp kit (fluogene). routine pcr-asp and pcr-rflp could not resolve the patient's genotype possible abo*a /*o genotype was detected by pcr-rflp, but the pcr-asp analysis gave an apparent abo*a homozygote result. sanger sequencing of abo exons and also gave anomalous reactions: no abo*a allele was detected. homozygosity for c. delg was observed as well as heterozygosity for c c/a. this result therefore suggests the patient's genotype is abo*o. /*o. . . next generation sequencing (omixon) revealed the same result. however, when pcr amplification of the cbf/nf-y enhancer vntr -region was performed, possible heterozygosity was observed, i.e. a weak band representing a single copy, and one representing copies of the enhancer region were present. presence of a single copy of the -bp cbf/nf-y enhancer vntr region is unique background: del is a very weak form of d antigen with low density expression of d antigen on the surface of red blood cell, which is generally typed as d-blood group as couldn't form agglutination in routine rhd blood group testing and could only be detected by the non-routine adsorption-elution test. in the east asian and southeast asian population, - % of the individuals with serologically apparent d-phenotype are not these with truly d-phenotype, but del phenotype, which is very rare in caucasian and black ethic groups. and the rhd* el. (rhd* a) is most prevalent (> %) in del people in these regions, so the del carried this allele was commonly known as "asia type" del. in previous studies, no alloanti-d was observed in a large cohort of chinese "asia type" del pregnant women with d+ fetus to indicate no occurrence of alloanti-d immunization against d+ red cell in "asia type" del individuals. aims: to conduct genotyping analysis in the chinese patients having serologically apparent d-phenotype simultaneous with alloanti-d to confirm the existence of the "asia type" individuals to produce alloanti-d or not. methods: from to , the blood sample of the patients or pregnant women identified with alloanti-d in our reference lab were collected. d antigen was confirmed again using the blend anti-d reagent (clone th- /ms- , igm/igg) by tube method in saline and indirect antiglobulin test (iat) in gel card. the zygosity of rhd gene was detected by hybrid rhesus box pcr with psti digestion. for the samples with d or dd genotypes obtained by rhd zygosity analysis, multiplex ligation-dependent probe amplification (mlpa) genotyping was conducted for rhd genotyping analysis. results: a total of serologically apparent d-chinese patients (female, n = ; male, n = ) with alloanti-d were identified. different titers of alloanti-d from : to : (≤ : , n = ; > : , n = ) were detected including few cases with mixed antibodies (anti-d mixed with anti-c, n = ; anti-d mixed anti-e, n = ). serological rhd typing confirmed the serologically apparent d-phenotype. rhd* n. / n. (homozygous rhd gene deletion) genotype was identified in majority of them ( / , . %) by rhd zygosity analysis, while rhd* n. / n. genotype (n = ) and rhd* n. / n. genotype (n = ) carried the rhd non-functional hybrid alleles were detected by mlpa. summary/conclusions: compared with the distribution of average % frequency of "asia type" del in serologically apparent dpopulation in guangzhou of china, no one case of "asia type" del was identified in the cohort of serologically apparent d-patients with alloanti-d in this study. this also provides evidence to confirm no occurrence of alloanti-d immunization in "asia type" del individuals. aims: a serologically rhd-negative donor was found to be rhd-positive in the routine rhd screen. to solve the discrepancy between serology and molecular screen, the sample was sequenced on dna and rna level. methods: phenotyping on id/iat-cards (bio-rad) was done using commercial anti-d antibodies. the adsorption-elution analysis was performed using an in-house pool of polyclonal anti-d antibodies. furthermore an antibody d-screen was performed (diagast). for rhd genotyping rh-type and partial d-type assays (bag health care) were carried out. the sample was further characterized by exon sequencing including flanking intronic regions. rna was extracted from whole blood, reverse transcribed and the cdna sequenced. for amplification and sequencing, both published (gassner, transfusion, ; legler, trans. med., ; richard, transfusion, ) and in-house primers were used. results: repeated phenotyping of the sample with commercial as well as, in-house anti-d antibodies confirmed the rhd negativity. in addition, the adsorption-elution analysis showed a negative result. however, genotyping, using commercially available kits, yielded a rhd positive result and no variants were detected. to investigate this discrepancy, all rhd exons were sequenced. the sequencing data revealed the mutation c. + delt in the splice donor site of exon . to confirm the effect of the splice site mutation on transcription, rna from a fresh whole blood sample was analysed. as a positive control, gypb was amplified and sequenced from the same cdna. wild-type gypb (mns ) was found. with rhd specific primers, no product could be amplified. summary/conclusions: we present a serologically rhd negative case, that was identified as rhd positive by standard commercial genotyping kits. sequencing revealed the new splice site mutation c. + delt. rna sequencing yielded no detectable product. the donor was classified as rhd negative. this case of a discrepant result between serology and genetics shows the importance of a profound and highly sophisticated genetic investigation of conflicting laboratory results. j stettler, s lejon crottet, h hustinx, c von arx, f still, j graber, c niederhauser and c henny interregional blood transfusion src berne ltd., berne, switzerland background: one of the most immunogenic and clinically significant blood group antigens in transfusion medicine is the rhd antigen. variant rhd phenotypes with weakened or absent antigen expression pose a challenge for rhd status assignment in blood donors. to ensure patient safety, it is necessary to fully characterize these variants at the molecular level. aims: samples from two donors were investigated in our laboratory due to discrepancy in rhd typing. methods: rh blood group phenotyping was done by standard serological column agglutination testing (id-system, biorad). further rhd characterization was performed by an anti-d antibody panel containing monoclonal antibodies (d-screen, diagast) and an adsorption-elution test using an in-house pool of polyclonal anti-d antibodies. molecular investigation was initially performed by ssp-pcr detecting common rhd variants (rbc-ready gene cde inno-train; rh-type bag health care). rhd sequencing was done on either dna or rna using published and inhouse primers for amplification and sequencing. results: by tube testing, the rbcs of donor were predicted to be rh:- ,- ,- , , . however, all ten exons of the rhd gene could be detected by routine genotyping. sequencing of rhd revealed a homozygous mutation c>g at position which is the second last nucleotide of exon and thus might have an influence on exon splicing. by cdna analysis a transcript with a correctly spliced exon was identified. the mutation c. c>g leads to the amino acid substitution t r located in the twelfth transmembrane domain of rhd using the model of flegel (transfus apher sci., ) as reference. adsorption-elution testing using a pool of polyclonal anti-d showed a weak positive reaction, re-classifying the donor as rhd positive. this novel allele, rhd* g, could thus be categorized as a del allele. serological results displayed an almost normal rhd antigen expression for donor . further serological determination of the rhd antigen with different antisera, however, showed a reaction pattern typically observed with a weak d variant. with commercial available kits no rhd variant could be detected. rhd sequencing revealed a novel homozygous mutation c. g>c in exon . this mutation causes a p.a p exchange in the sixth membrane-spanning domain of rhd. based on serological data, the donor is rhd positive and in case of transfusion the patient would be treated as rhd negative. summary/conclusions: here we report two novel rhd missense mutations c. c>g and c. g>c harbouring an amino acid substitution within a transmembrane segment. the c. c>g variation displayed an unusual low rhd antigen reactivity and would have been mistyped as rhd negative without extensive genotypic testing. molecular analysis of variant c. c>g suggests that the t r exchange causes a del phenotype rather than a miss splicing event. this was also confirmed by adsorption-elution testing. interestingly, variant c. g>c could only be detected due to comprehensive serological and genetically investigation. background: the rh blood group system is highly polymorphic and one of the most clinically relevant systems in transfusion. actually d antigen is of critical importance due to its involvement in hemolytic transfusion reaction and hemolytic disease of the fetus and newborn. rhd gene variants are common in africans and mostly related to partial d phenotype. aims: rhd gene sequence was investigated in two african brazilian samples. we further attempted to take advantage of combining the molecular data and the available in silico tools for the functional interpretation of the variations, in order to get insights into the clinical phenotype that may be predicted a priori from genotyping. methods: sample #id is a d-negative donor self-declared as african descent. sample #id is a patient with sickle cell disease (scd) typed as d-positive with anti-d in his serum. serologic d typing was determined by manual gel test and by microplate in an automated instrument. sample #id was also submitted to adsorption/elution test. after genomic dna extraction, all ten rhd exons and flanking intronic regions from sample #id were pcr-amplified with rhd-specific primers and analyzed by sanger sequencing. sample #id was investigated by next-generation sequencing on the miniseq platform (illumina) by using a previously published, custom (selected blood group genes) ampliseq panel. a reported three-dimensional ( d) structural model of the rhd-rhd-rhag heterotrimer was used to visualize the position of variations and predict their putative functional/clinical effect. results: in sample #id , a single nucleotide missense change, i.e. c. c>g in exon , was identified. this transversion is thought to replace a threonine by an arginine residue at amino acid position (p.thr arg) of the rhd protein. analysis in the d model clearly suggests a dramatic impact of the p.thr arg substitution occurring in a functionally-critical, conserved motif in terms of interhelix interaction, which is supposed to be highly deleterious to the stability of the protein, and potentially impairs totally its expression at the red blood cell plasma membrane. this predicted functional effect is definitely in accordance with the d-negative phenotype reported in sample #id . in sample #id , the single c. a>g transition was found in exon leading to a threonine-to-alanine substitution at amino acid position (p.thr ala). amino acid is located in rhd protein extracellular loop , and is thus thought to alter d antigen structure, resulting in a partial d phenotype. this hypothesis is in accordance with anti-d found in the serum of sample #id . summary/conclusions: for the past years, due to the advent of next-generation sequencing and the subsequent identification of numerous rare variants, bioinformatics prediction and modelling tools have evolved and currently help physicians in diagnostics, clinical management and genetic counselling. we took advantage of some of those in silico methods to predict retrospectively the effect of two novel variant rhd alleles, including one d-negative and one partial d alleles. although phenotype and clinical symptoms remain definitely the standard determinants to assess the effect of genetic variations, use of those approaches may soon become valuable for guiding subsequent investigations in immunohaematology. abstract withdrawn. alleles of the weak d type and diva cluster. in africans, the most frequent were typically associated with alleles of the weak d type (including dol and rhdpsi), diva and dau clusters with f v occurring in > % of alleles; in addition the key mutations of weak d type and dii and two inactivating mutations (c. _ inst and c. delg) not reported in rhb were among the first polymorphisms. in east asians, rhd( g>a) at . % was most frequent, followed by dfv, weak d type , dbo- , key mutations of diva and weak d type cluster as well as rhce-like substitutions and the mutations of weak d type , type , type , rhd(a v), dvl- , weak d and rhd(n s). weak d type and rhd(t r) were frequent in south asia but not elsewhere. summary/conclusions: data from tgp and gnomad add relevantly to the knowledge on rhd alleles; tgd discloses linked intron polymorphisms, gnomad frequency data not biased by the likelihood of serologic detection. current typing strategies usually start with serology later complemented by molecular typing. in the future, molecular methods will gain importance and frequent alleles currently not distinguished from "standard rhd" may need a rational transfusion strategy. in this respect, the high frequency of weak d type and type in europeans was surprising, might warrant confirmation by alternative methods and should trigger discussion on rational transfusion strategies for these alleles. consistent with an r haplotype and probable dc-. two siblings that were abo compatible including the dc-sibling were incompatible at iat phase. reactivity could be completely adsorbed from the serum using r r , r r , and rr rbcs indicating the antibody is probably a single specificity. the donor returned in and to continue autologous donations. the aim of this case study was to examine the genetic framework of the rhd and rhce genes and to characterize the rh epitope recognized by the antibody. aims: the donor returned in and to continue autologous donations. the aim of this case study was to examine the genetic framework of the rhd and rhce genes and to characterize the rh epitope recognized by the antibody. methods: serologic testing was performed by manual tube testing using ahg in the indirect antiglobulin phase. rbc phenotyping was performed by standard tube hemagglutination testing from edta anticoagulated blood. rhd and rhce exons were sequenced using genomic dna and standard sanger dideoxy method with the bigdye terminator v . cycle sequencing kit. sequence data was aligned to rhd_ng_ . . rhd zygosity was performed using pcr-rflp with mspi. background: according to recent findings in molecular immuno-hematology, rhd genotyping is strongly indicated in rhc+ and rhe+ donors classified in routine as d-negative. among these, one could find a non-negligible share of entirely new genetic alterations or even del alleles, which are often not identifiable with routine serological methods due to the low number of antigenic sites. aims: the present study reports the genotyping data of rhd on rhc+ and rhe+ caucasian donors classified serologically as d-negative, all enrolled by a single transfusion center in italy methods: rhd serological typing was carried out in microplate direct agglutination tests (iris, immucor) by using different anti-d igm clones (clone , dvi+: ldm +esd m; clone , dvi-: rum- , th ) and different anti-d igg clones (clone : ms ; clone : d e ). all donors with d-negative results (n = , divided into subjects with rhc+, with rhe+ and with both rhc+ and rhe+) were addressed to genotype analysis with rhd beadchip molecular test (immucor), pcr-ssp (bagene, inno-train) and/or rbc-fluogene (inno-train). the discrepant results between serology (d-neg) and molecular biology (wild-type or full-length rhd gene) were further investigated by bi-directional sequencing of the rhd coding regions. results: one-hundred donors have been analyzed retrospectively, as part of a pilot study. following the data obtained in this first phase, the analysis methods described above have been implemented in routine, allowing to include further donors, studied prospectively. in . % of donors (n = ), the molecular analyses showed the complete deletion of the rhd locus, while in cases ( . %) a genetic status was found with "non-deleted" rhd. over all, bi-directional sequencing on these donors revealed the presence of negative and weak-d variants. the list of rhd alleles we have identified at the molecular level is as follows: rhd* n. ( cases), rhd* n. ( ), rhd* n. ( ), rhd* n. ( ) , rhd* n. ( ), rhd* el. ( ), rhd* el. ( ), rhd* el. ( ), rhd* w. ( ) . moreover we found a donor with a lack of signal encompassing exons - of the rhd sequence (bioarray rhd beadchip), while additional cases are currently under investigation. summary/conclusions: our study confirms that a non-negligible number of caucasian subjects, classified serologically as d-negative, present rhd gene alterations that differ from the common total deletion. in line with the literature data, we also found a frequency of about in cases ( subjects out of ), in which a donor re-classification as d-positive (weak d type) was necessary. hence, a wider use of molecular typing methods is desirable in order to achieve the correct genetic characterization and the appropriate phenotypic classification of "apparently" d-negative donors. background: without evidence of abnormal serological d antigen expression there will be no quest for weak d, partial d or d variant on the red blood cells. according to our blood donor registry we found that out of serologically typed donors, . % were d+, . % d-and . % weak d. aims: to compare different weak serological reactions of the d antigen to the rhd genotyping. methods: molecular rhd typing using isolated dna and rbc-ready gene cde and rbc-ready gene d weak kits was performed in blood donors, who were serologically typed as weak d using monoclonal blended igm/igg and dvi-and dvi+ anti-d reagents by slide and microplate (mp) technique respectfully, as well as by the antiglobulin test (iat) in gel and with the set of monoclonal partial d typing reagents (biorad). in addition, rhccee phenotyping and genotyping was also performed. results: all of the donors with serologically weak reactions were confirmed to be weak d variants by genotyping except one donor whose iat was false positive due to rbc autoantibodies. the frequency of d variant genotypes was as follows: % weak d type , % weak d type , one donor was typed as weak d type and another one as weak d type . these weak d types were associated with different degrees of serologically determined weakness ranging from negative to weak positive reactions concerning slide and mp. all of them gave positive reaction ranging from + to + with iat, except for the weak d type with the score of < + which gave negative reaction by slide and mp and inconclusive result with the set of monoclonal anti-d reagents for partial d typing. the percentage of donors, who, at serological typing were only found to be d positive in the iat was %. one of the weak d type donors was negative with dvi-and positive with dvi+ reagent in the mp. the additional rh phenotype (genotype) was ccee in all of the donors except in the one who was genotyped as weak d type , as well as in the d negative donor, being ccee. summary/conclusions: further rhd genotyping is required to estimate the actual frequency of d variants in our blood donors. in practice, current serological methods are sufficient to detect almost all variant d phenotypes. there is a consensus that routine molecular d antigen screening in d negative donors in order to detect del variant when ddccee phenotyped red blood cell transfusion is practiced in all d negative patients does not seem to be cost-effective. background: rh null or rh mod -the so-called rh-deficiency phenotypes-are characterized by a null or severely reduced rh antigen expression (including d, c/c and e/ e), respectively. molecular genetic studies showed that these phenotypes are transmitted in an autosomal recessive manner. rh null phenotype originates from two different molecular events giving rise to the amorph type and the regulator type. the former is caused by homozygosity for silent genes at rhd and rhce loci, caused by inactivating mutations in rhce and deletion of rhd. on the other hand, the regulator rh null type as well as the rh mod phenotype are attributed to mutations in rhag gene when in homozygous state or when in heterozygosity with another rhag allele containing an inactivating mutation. a functional rhag is essential both for the correct rh complex assembly and rh antigen expression in the erythrocyte membrane. aims: the aim of this study was to investigate the molecular genetic basis of an argentinean proband with no detectable d, c, c, e and e antigens by standard serological techniques. methods: blood samples were collected from the proband, her parents and sister. the proband was a year-old young woman with parameters of hemolytic anemia: low hemoglobin level ( g/dl), reticulocytosis ( %), hyperbilirubinemia, increased ldh and marked spherocytosis. the d, c, c, e and e status was determined by standard serologic hemagglutination techniques using specific monoclonal antibodies. genomic dna was isolated using a modified salting-out method. dna samples were initially screened for the presence of intron and the untranslated region of the rhd gene using pcr strategies. rhc/c, and rhe/e alleles were studied by allele-specific pcrs to determine the rhce genotype. rhd zygosity was analyzed by pcr-rflp. rhd exon polymorphisms were studied by rhd exon scanning procedure based on pcr-ssp. rhag gene was investigated by exon-specific pcr amplification and sanger sequencing. results: no d, c, c, e and e antigens were detected in the proband's erythrocytes. the father and sister rh phenotype was: d+, c+, c+, e+, e+ whereas the mother rh phenotype was: d+, c+, c-, e-, e+. rh genotyping confirmed the rh phenotypes for all family members except for the proposita who genotyped rhd+, rhc+ and rhe+. all samples showed an homozygous status for the rhd gene and all rhd exons were detected by exon scanning. sequencing analysis revealed an homozygous c. c>t mutation in rhag exon in the proband whereas the rest of the family showed an heterozygous state in the same nucleotide position. the c. c>t mutation is responsible for the p.ser phe amino acid substitution predicted to be in the th rhag glycoprotein transmembrane segment. summary/conclusions: this study described the molecular background responsible for an rh-deficiency phenotype in an argentinean proband. we identified the novel missense mutation c. c>t in the rhag gene which results in the ser to phe single amino acid substitution that shows to be critical for rh antigen complex assembly within the erythrocyte membrane. further studies are being performed in order to determine whether the proband is rh null or rh mod . background: rh blood group system is the most immunogenetic blood group system and blood donor typing should account for all expressing antigens in order to prevent anti-d alloimmunization. aims: the objective of this prospective study was to investigate rhd alleles among blood donors who typed d-by serologic methods and positive for c and/or e. for this reason we developed an easy-to-perform dna-based screening method for the detection of rhd gene and positive samples were further characterized by two commercial pcr-ssp kits. methods: of individual blood donors within a month period, ( . %) typed as d-with standardized immunohematologic methods including the indirect antiglobulin test (iat). residual edta-anticoagulated blood samples were used to isolate genomic dna using the qiaamp dna blood kit (qiagen, germany) from out of ( . %) c/e+ and serologically d-donors. all dna samples were tested individually for the presence of rhd-specific dna sequences in the rhd promoter, intron , exon and exon by a multiplex pcr-ssp method. the reaction was conducted in a final volume of ll with primers that were applied as described by f. wagner et al. (bmc genetics, ) except antisense primer for exon and the two primers amplifying an hgh gene fragment as internal control, designed by our laboratory. pcr products were visualized by electrophoresis on a % agarose gel with ethidium bromide staining. in case of a positive reaction the sample was analyzed by pcr-ssp d weak and pcr-ssp cde (inno-train, germany). results: out of d-individuals analyzed, were ddccee, ddccee, ddccee and one had a ddccee phenotype. molecular analysis showed that ( . %) were negative for all four rhd dna regions. among the other samples, all of ddccee phenotype, three were found to be positive for rhd promoter, intron , exon and exon , three for rhd promoter and exon , and two for exon alone. further genotyping revealed five hybrid rhd-ce-d alleles [ rhd-ce( - )-d and rhd-ce( - )-d], one allele represented the del(m i) genotype, while the remaining two samples did not show an allele that could be determined with the pcr-ssp kits. summary/conclusions: serotyping is the standard method to assign transfusion strategies but it is not always capable to correctly define all samples that show weak reactions in d. a rhd genotyping strategy is needed to confirm d-blood donors and thus to avoid anti-d immunizations. for these reasons we suggest the implementation of an easy and possible cost-effective method. background: more than weak d types have been described to date. transfusion recipients with weak d type , , or are not at risk for forming allo anti-d when exposed to conventional rh d-positive rbcs. molecular analysis of weak d offers a more reliable basis than serotyping to determine the prevalence of weak d types and optimal d transfusion strategies. background: the d antigen, which consists of a mosaic of epitopes, is determined in all the blood donors and patients. most people are either rhd-positive or rhdnegative, but there is a certain number of people who have a variation of the d antigen, which are called weak d, partial d and del phenotypes. aims: the objective is to use molecular methods to determine whether blood donors in republika srpska (with whom a serological weak d antigen has been detected) really have the weak d antigen. in addition, determine whether blood donors, who have been determined as persons who are rhd-negative, with the phenotypes c and/ or e, who have the rhd gene and d antigen on the erythrocyte membrane, so weak that it could not be determined by serological techniques. methods: blood samples were used from regular blood donors, who have been determined as persons with a weaker d antigen, as rhd-negative or as c and/or e positive (based on the agglutination strength) using serological techniques, the test tube method, the microplate method and the gel method. gp.mur was also modelled and shown to closely resemble the tertiary structure of glycophorin a. the predicted structure is anti-parallel b sheets arranged in a "b barrel" also referred to as an ob-like-fold. the regions in which blood group antigens were identified in the predicted stable dimeric structure. summary/conclusions: ob-like-fold structures typically to bind oligonucleotides or oligosaccharides and are associated with cold shock proteins. further modelling is in progress to predict the structure of gpa/gpb heterodimers as a basis for understanding the presentation of blood group antigens. of interest, this finding is consistent with a previous report showing that this gpa binds to carbohydrates. this model serves as a foundation for future work regarding the properties of gpa, which includes identifying locations of specific interactions between gpa and other rbc surface proteins such as gpb and band , as well as identifying structural features of antigenic regions on gpa. . even though no significant differences were found among the groups studied, haplotypes containing the mcc b and sl polymorphisms were identified in d samples but were not found in tb and l groups. summary/conclusions: this preliminary data obtained suggests that cr polymorphisms and haplotypes, especially those containing mcc b and sl snps, could be involved in the disease pathogenesis of tuberculosis and leprosy. the entrance of mycobacteria into macrophages is mediated by complement receptors that facilitate their uptake by host cells so the combined haplotypes could be enhancing parasite phagocytosis and inflammation. further studies are being carried out to establish whether cr polymorphisms are risk or protective factors and whether other genetic variations in this receptor are also involved. abstract withdrawn. background: the dombrock blood group system consists of two antithetical antigens, do a and do b , and three high-prevalence antigens, gregory (gy a ), holley (hy), and joseph (jo a ). the rare do null or gy(a-) phenotype lacks all dombrock antigens, and the do null alleles vary with both do* and do* backgrounds. here we report the molecular basis of a novel do null allele in a gy(a-) brazilian patient with anti-gy a . aims: case presentation: an alloantibody to a high-prevalence antigen was detected in the serum of a year old woman from the northeast brazil with a history of pregnancies but no history of previous transfusion. she required transfusion because of a schedule for total thyroidectomy surgery due to a large compressive nodular goiter. the antibody did not react with the autologous rbcs but reacted by the indirect antiglobulin test in liss with all panel rbcs and other rbc samples tested except with the gy(a-) phenotype. the corresponding antigen was resistant to treatment with papain but sensitive to dtt and trypsin. these results suggested that the antibody recognized an antigen in the dombrock blood group system. the purpose of this study was to identify the antibody specificity and to determine the molecular basis of the phenotype detected. methods: the red cells phenotype and the presence of the dombrock related antibody in the serum were detected by standard hemagglutination techniques. rbcs and antibodies were from our in-house collection of rare samples. genomic dna was prepared from peripheral blood of the patient. dombrock genotyping was performed by id-core xt platform (grifols, spain). the exons of the do gene were amplified by pcr and directly sequenced. experimental immunohematology and diagnostic immunohematology diagnostic immunohematology experimental immunohematology, sanquin, amsterdam, netherlands background: typing of blood group antigens is essential to prevent transfusion reactions or haemolytic disease of the foetus and newborn. to date, the isbt recognises blood group antigens. most antigens ( ) belong to one of the blood group systems. since the genetic basis of these systems is known, genotyping of these antigens is possible. the molecular background of antigens is unknown and can only be determined serologically. one of these antigens is sd a (sid), first reported in .~ % of the population carry sd a on erythrocytes, but this frequency might be higher since identification is difficult due to variability in expression. in % of individuals sd a is present in urine. cells with a high expression of sd a (cad/sda++) are used for detection of antibodies. recently, a -cells antibody detection panel of bio-rad contained a sda++ cell and many individuals with anti-sd a were detected. the b galnt gene has been implicated in the synthesis of sd a . we collected individuals with and without anti-sd a to elucidate the genetic background of the antigen. aims: elucidation of the genetic basis responsible for loss of the sd a antigen on red blood cells. methods: routine diagnostics to identify antibodies in patients was performed using a bio-rad -cells panel, containing donor with high expression of sd a . additionally, pregnant women were screened for anti-sd a . dna of eight samples with anti-sd a and eight samples without anti-sd a was isolated for further analysis. sanger sequencing was performed on b galtnt exon - . results: sequencing of b galtnt revealed two homozygous mutations which are present in all eight individuals with anti-sd a , but not present in controls. the remaining two controls are heterozygous for these mutations. the first mutation within exon , c. t>c (enst . , rs ) changes a cysteine to arginine at position of the protein. the second mutation in exon c. a>g (rs ) does not change an amino acid. both snps have a maf of . and therefore we expect that . % of the population is homozygous for the minor allele. genotyping of a large population of pregnant women and the serological detection of anti-sd a in women with a homozygous mutation is in progress. summary/conclusions: the high frequency antigen sd a has not been linked to a blood group system because the molecular basis for loss of the antigen has not been elucidated. the b galtnt gene has been associated with sd a synthesis and therefore we analysed this gene for mutations in individuals with antibodies against sd a . a single homozygous mutation within exon causing an amino acid change was found in all individuals with anti-sd a , and no individuals without antibodies were homozygous for this snp. from population studies we expected~ % sd a -negatives, but either this frequency is an overestimation because of difficulties to detect low expressed antigens or mutations in other genes are interfering with sd a synthesis. a larger study of individuals with homozygous mutations in b galnt and linkage to sd a -negativity and presence of antibodies will be performed before sd a can be assigned to a new blood group system. abstract withdrawn. abstract withdrawn. background: erythrocyte duffy blood group antigen can scavenge chemokines in whole blood. duffy blood group gene consists of two major alleles: fy*a and fy*b. however, little is known regarding the association of duffy blood group polymorphisms with the red blood cell (rbc) chemokine scavenging. aims: the aim of this study was to determine the association of duffy blood group polymorphism with the rbc chemokine scavenging. methods: the duffy blood group were genotyped by ˊ-nuclease assay in healthy chinese han individuals, while erythrocyte chemokine scavenging function and duffy antigen expression from the same samples were measured using erythrocyte chemokine binding assays and quantitative flow cytometry respectively. results: rbc chemokine scavenging of cxcl was significantly lower in the individuals with the fy*a/fy*a genotype compared to those with fy*a/fy*b genotype (p = . ). similar result was also observed in rbc chemokine scavenging of ccl (p = . ). the expression of duffy antigen on rbc surface in the individuals with the fy*a/fy*a genotype was significantly higher compared to those with fy*a/ fy*b genotype (p = . ). summary/conclusions: duffy blood group polymorphism is associated with the differential rbc chemokine scavenging. it is probable that a change in duffy antigen structure caused by duffy blood group polymorphism is responsible for the differential rbc chemokine scavenging. background: individuals with p-phenotype can develop a naturally occurring anti-pp pk and has clinical significance, causing hemolytic transfusion reactions or hemolytic disease of the fetus and newborn. finding and procuring blood units of pphenotype is a challenge because of its rarity throughout the world. therefore, acute normovolemic hemodilution (anh) can be an on hand tool in the perioperative successful management of patient with rare blood group. however, this approach has not been commonly used aims: n/a. methods: n/a. results: a -year-old korean woman was referred to samsung medical center for surgical management for gallbladder malignancy. her blood type was group a, d-positive. the patient had no known history of transfusion. however, antibody screening and identification test using the column agglutination method (bio-rad, cressier, switzerland) showed panagglutination with negative reactions to autologous red blood cells, indicating the presence of alloantibodies to high frequency antigens. the specimen obtained from the patient was sent to the central laboratory of the swiss red cross (bern, switzerland) and confirmed as anti-pp pk. at first, the transfusion team of our hospital recommended the surgical team to postpone the surgery. however, anh was planned because postponing surgery was not preferred and the patient's preoperative hemoglobin was . g/dl. ml of blood was withdrawn through a radial arterial catheter in two ml blood bags containing citrate-phosphate-dextrose-a solution after anesthetic induction. equal volume of % hydroxyethyl starch solution was infused during the procedure. the patient underwent radical cholecystectomy and liver wedge resection with lymph node dissection, and two units of autologous blood were returned to the patient during surgery. she was then discharged h later with a hemoglobin level of . g/dl. later, the family study was performed with the standard serologic method using the proband's plasma containing anti-pp pk and sequencing of the a galt gene, which were conducted according to the protocols by koda et al.(transfusion. ) . the proband and her brother were homozygous for c. dupc, indicating a rare p phenotype. summary/conclusions: we experienced that autologous blood transfusions via anh is an alternative to allogenic rbc blood transfusion in patients who have no blood available because of high alloimmunization antibodies against rare blood groups. " and the third sample as "gypb*s_gyp*[ a], gypb*s_null(ivs + t)" with a predicted phenotype: s-s+ mi a + and s+s-mi a +, respectively. the gypa specific primers used for discrepancy resolution detected the nucleotide substitution, gyp.c. c>a, in gypa-b-a hybrid associated to gp.hut allele, thus confirming the id core xt result. the expression of mi a for one of these samples was confirmed using non-commercial anti-sera. hence, these three samples were not gp.mur but gp.hut phenotype. both alleles codify for the expression of mi a antigen since it is expressed on several hybrids between the usual forms of glycophorin a and b. two of these three gp.hut samples are african-american donors. gp.hut was reported in white people with a frequency about . % and in thais with . %. these three gp.hut cases found by id core xt in this study point to a higher frequency of this glycophorin variant and also to the presence in african american population. summary/conclusions: id core xt was able to detect two glycophorin phenotypes, gp.mur and gp.hut, which codify for the expression of mi a antigen. standard molecular methods should be implemented in pre-transfusion testing and obstetrical care routine to detect the most clinically relevant glycophorin variants in mns system. background: serf(+) is a high prevalence antigen in the cromer blood group system, which is encoded by a crom* allele. the lack of the serf antigen, serf(À) on red cells is caused by a single nucleotide polymorphism, c. c>t in exon of the decay-accelerating factor, daf gene. alloanti-serf has been found in thai pregnant woman with serf(À) and a serf(À) individual was found among thai blood donors. anti-serf is not a marketed product; hence, a molecular technique has to be implemented to genotype for the crom* allele among blood donors. aims: this study aimed to identify the crom* allele among thai blood donors leading to predicted serf(+) and serf(À) phenotypes. methods: dna samples obtained from , central thai blood donors were genotyped for serf allele detection using in-house pcr with sequence-specific primer (pcr-ssp) and confirmed by dna sequencing. results: the allele frequencies of crom* (+) and crom* (À) among , central thais were . ( , / , ) and . ( / , ), respectively. the homozygous of crom* (À/À) alleles was not found in this study. additionally, the pcr-ssp technique was validated by dna sequencing using randomly chosen samples together with heterozygous crom* (+/À) samples and the results were in agreement. summary/conclusions: our results confirm a high frequency of the crom* (+) allele in the thai population and their frequencies were similar to those formerly reported among thai blood donors. this study would be beneficial to predict the serf antigen from genotyping results due to unavailability of commercial antiserum. background: there is increasing interest in the use of molecular methods for predicting abo grouping. though nextgen and sanger sequencing have both been used to predict abo type, predicting abo type from buccal swab-derived dna and from deceased donors benefits from a quick and reliable method. besides a pcr-rflp that has been used by many labs for more than years, there is a commercially-available research use only (ruo) kit, and both interrogate nucleotides associated with o , o , a and b with a representing the ancestral allele. aims: the aim of this report is to compare two low-resolution polymerase chain reaction (pcr)-based methods, for investigation of samples submitted to a reference molecular immunohematology laboratory for abo typing discrepancies. fifty-six peripheral blood samples were tested, from patients and from blood donors. methods: genomic dna was isolated from peripheral blood mononuclear cells. background: del is the weakest known d positive phenotype in the rh blood group system and detectable only by adsorption and elution tests. the rhd g>a change is an important marker for del phenotype in east asians. a rapid and efficient pcr method for rhd gene g>a genotyping is useful in east asian countries. aims: the aim of this study was to develop a method for rhd g>a genotyping by using single-tube pcr with melting temperature(t m )-shift primers. methods: two allele-specific primer for rhd g>a and a common primer were designed and synthesized. two gc-rich tails of different lengths were attached to ends of the allele-specific pcr primers. single-tube pcr with t m -shift primers was carried out with the three primers. after pcr, melting curve analysis was performed. rhd g>a could be genotyped by differences of the t m s of the pcr products. all of genotyping results were compared with those obtained from conventional pcr-ssp. for the discordant results, rhd exon sequencing was performed to determine rhd g>a genotype. results: a total of samples were genotyped for rhd g>a by pcr with t mshift primers. samples were typed as a+/g-, samples were typed as a-/g+, samples were typed as a+/g+ and samples were typed as a-/g-. two samples typed as a+/g+ by pcr-ssp but a+/g-by pcr with t m -shift primers were confirmed as a+/g-by rhd exon sequencing. summary/conclusions: the single-tube pcr with t m -shift primers for rhd g>a genotyping is simple, rapid, accurate, and it is superior to conventional pcr-ssp. abstract withdrawn. background: the rh blood group system has numerous variant alleles, which may affect rh antigen expression, including rhd-rhce (d-ce) hybrid genes. these variant alleles are frequently found in people of african descent, and typically result in either d-negative (d-) phenotype, or partial d antigen expression, including silencing of high-frequency antigens and/or expression of low-frequency antigens. patients carrying those alleles are particularly at risk of alloimmunization, suggesting that their identification is important in diagnostics. quantitative multiplex polymerase chain reaction (pcr) of short fluorescent fragments (qmpsf) has proven successful for genotyping those dna samples carrying d-ce hybrid genes by assessing both qualitatively and quantitatively rhd and rhce gene exons. aims: the aim of this project was to genotype both rh genes in a cohort of brazilian patients with sickle cell disease (scd), which are known to be of african descent, by using the qmpsf approach and report hybrid gene variability in this population. methods: one-hundred fifteen dna samples were selected for the study and analyzed prospectively by the rhd-qmpsf and rhce-qmpsf approaches to investigate the copy number of all exons in both rh genes. genotypes were further confirmed or investigated by sanger sequencing and conventional pcr-rflp assays. results: in the dna samples, ( . %) exhibited a "wild-type" profile by qmpsf analysis. hybrid genes involving exon , which is functionally not relevant as reported before, was found in samples, including and samples carrying respectively rhd-ce( )-d and rhce-d( )-ce (two homozygous each). except two samples that require additional studies ( . %), rhd zygosity was resolved successfully: (n = rhd gene copies; . %), ( ; . %) and ( ; . %). clinically relevant, i.e. partial d, genotypes were identified in four hemizygous samples ( / , . %) carrying rhd*dau , rhd*dv. , a rhd*diiia-like allele, and a novel rhd*d-ce( :g h-y s-n i)-d allele, as confirmed by sequencing. other hybrid alleles, such as rhd* n. and rhd*diiic, were also found in trans with a normal rhd* allele. in rhce, c/c genotype could be resolved. the rhce*ce (rhce*ce ( c)-d( )-ce) allele, which is commonly cis-associated with rhd*Ψ, was observed in four samples. however the clinically relevant polymorphisms in variant rhce alleles, such as those involved in cemo, cear, ceag, and ceti, were mostly identified by other standard methods. summary/conclusions: although most of the brazilian patients with scd investigated in this study did not carry rhd-rhce hybrid genes, qmpsf analysis has been shown to be an efficient tool in the whole genotyping process to investigate rh gene variation. as previously reported, it has been conclusive for characterization of rhd zygosity and identification of rare, as well as novel, variant alleles. additionally, our results show a large diversity of hybrid genes among the brazilian patients with scd. therefore, we suggest that qmpsf may be used as a complementary screening approach for assessing rh genotype in selected patients and donors. = ) vs. non-bleeding (n = ) patients. platelet, pmp and cp phenotype and function were evaluated by flow cytometry: activation and granule release were examined by antibodies against granulphysin (cd ), p-selectin (cd p), activated gpiib/iiia (pac- ) and phosphatidylserine (ps) (lactadherin) unstimulated and adp, trap or collagen stimulated. coated platelets were identified as a highly granulated independent cell population appearing following collagen stimulation, gated on side scatter and gpiba (cd b). normal healthy reference levels were available. results: the platelet count in bleeding ( /l) and non-bleeding ( /l) patients was comparable (p = , ). bleeding patients had a higher bat score compared to non-bleeding patients ( vs. , p < , ). the proportion of cps was normal in all patients. however, in non-bleeding patients the proportion of ps+cps and per cell ps expression (mfi) ( , % and , mfi) were higher, compared to bleeding patients ( , % and , mfi, both p < , ), and the proportion of ps+cps correlated negatively with bat score (r = , , p < , ). cd + cp was higher in non-bleeding ( , % and , mfi) compared to both bleeding patients ( , % and , mfi) and significantly higher than the reference level ( , % and , mfi, both p < , ). finally, the proportion of ps+pmps was normal in bleeding patients, but their pmps expressed higher than reference ps per cell, both unstimulated and for all agonist ( , mfi unstimulated vs , mfi reference, p < , ). summary/conclusions: patients with it exhibited different bleeding tendency despite comparable thrombocytopenia. in non-bleeding patients the proportion and per cell level of ps+ were higher, indicating that generation of cps with high ps expression is a critical factor determining bleeding phenotype. the finding of high pmp ps per cell level in bleeding patients could represent an inadequate compensation for lack of cp function, indicating that procoagulant pmps may be less important than cps for thrombocytopenic bleeding. quantification and characterization of cps may be a useful tool for future assessment of bleeding risk as well as a therapeutic target in it and other conditions with bleeding diathesis and/or thrombocytopenia. more studies investigating this field are warranted. background: alloantibodies against human platelet antigens (hpas) and human leukocyte antigen (hla) are implicated in several immune-mediated platelet disorders. detection of these antibodies is crucial in the diagnosis and management of these disorders. aims: to establish a method detecting hpa- , hpa- , hpa- , hpa- and hla antibodies using luminex bead technology. methods: monoclonal antibodies specific for platelet glycoproteins and hla class i molecules were separately coupled to the luminex microbeads. positive anti-hpa- a, anti-hpa- b, anti-hpa- a, anti-hpa- a samples were used to validate the specificities of the luminex assay. the anti-hpa- a, anti-hpa- a standard samples were used to evaluate the sensitivities of the luminex assay by serial dilutions (from neat to / ). results: samples collected from patients or isbt platelet workshop were tested by the luminex assay. the results showed that luminex assay could detect antibodies against hpa- a, hpa- b, hpa- a, or hpa- a successfully from the known samples. the sensitivities of the luminex assay detecting anti-hpa- a, and anti-hpa- a were : and : , respectively, using the standard samples. no cross-reactivity was observed in the samples containing multi-platelet antibodies, or mixture antibodies against hpa and hla. the results of samples with platelet disorders were agreement with those of monoclonal antibody immobilization of platelet antigens (maipa) assay. summary/conclusions: luminex beads coupled with monoclonal antibodies could be successfully used to detect hpa and hla antibodies with high sensitivity. background: platelet transfusion is important in clinical treatment. the expression of abo antigen on platelet surface is differential, so it is usually need to ensure the consistency of the abo antigen in clinical transfusion. but in many cases, it is difficult to find the platelets that the abo blood type matched between the recipient and donor, and abo-incompatible platelet infusion is required in these cases. to data, the expression of abo antigens on platelets in normal blood group individuals is rarely reported in chinese population. aims: to understand the differential expression of abo antigen on platelet surface in population of zhejiang province, china. methods: total of individuals with normal abo groups ( group a, group b and group ab individuals, and group o as negative control of abo antigens on platelets) were analyzed. the expression of abo antigens on platelets was determined by flow cytometry using monoclonal antibodies: fluorescein isothiocyanate (fitc)-conjugated mouse antihuman blood group a and pe-conjugated murine igg anti-b antibody ( pe bgrl ). flow cytometric parameters were statistically analyzed by the mann-whitney test or the kruskal-wallis test to observe the difference in two or more groups using graphpad software v . . the correlation and regression analysis between a and b antigen in the platelets and rbcs were also performed by the software. population studies were reported as the mean and standard deviation (sd), and p values less than . were considered statistically significant. results: according to mfi values of abo antigens expression on platelets, the samples were divided into three groups: low expression (le), high expression(he) and moderate expression (me) according to the background mfi observed in group o samples. it was found that about . % of the individuals had a weak expression of abo antigen on the platelet surface in zhejiang province. there was a significant difference in the intensity of antigen expression between these three different groups of the same blood group. for each blood group, there was a positive correlation between the intensity of abo antigen expressed on the platelet membrane and red blood cells of the individuals. results: cases were found with antibody positive. among them, cases ( %) were only anti-hla-i positive, cases ( %) were only anti-hpa positive, cases ( %) were both anti-hla-i and anti-hpa positive. cases were found without anti-hla-i or anti-hpa. among the cases with anti-hpa positive, the distributions of anti-gpiib/iiia, anti-gpia/iia, anti-gpib/ix, anti-gpiv were . %, . %, %, . %, respectively., hla antibody positive rate in the female patients was higher than that in the male and hpa antibody positive rate in the female was lower than that in male, but there was no significance difference between them (p > . ). summary/conclusions: in ptr patients, the platelet antibody was mainly hla-i antibody combined with hpa antibody. background: human neutrophil antigens (hna) are polymorphic structures located on surface membrane of human neutrophils. alloantibodies against hna are implicated in a number of clinical conditions, including immune-mediated neutropenia and transfusion reactions. genotyping for human neutrophil antigen (hna) systems is an important in the diagnosis of disorders involving alloimmunization to hna. aims: the aim of this study was to investigate the hna allele frequencies among blood donors and hematological patients undergoing blood transfusions and to estimate possible hna incompatibilities and risk of hna alloimmunization. methods: a total of blood donors and hematological patients from the north-west region of the russian federation were recruited. dna samples were obtained and typed for hna- , - , - and - systems using polymerase chain reactions with sequence-specific primers (pcr-ssp). specific primers for hna were designed and the polymerase chain reaction amplification conditions were optimized. the v test was used to test for the hardy-weinberg equilibrium for the hna systems. the probabilities of the incompatibility and the potential risk for alloimmunization against different hna systems after random transfusions were estimated based on the hna allele and genotype frequencies. results: in blood donors, the frequencies for the fcgr b* (hna- a), fcgr b* (hna- bd), and fcgr b* (hna- bc) alleles were . , . and . ; for the slc a * (hna- a) and slc a * (hna- b) alleles, . and . ; for the itgam* (hna- a) and itgam* (hna- b) alleles, . and . ; for the itgal* (hna- a) and itgal* (hna- b) alleles, . and . , respectively. in hematological patients, the gene frequencies for hna- a/ bd/bc, - a/ b, - a/ b, and - a/ b were . / . / . , . / . , . / . , and . / . , respectively. no statistic significant difference between genotypes in these groups was observed. since the allele frequencies of hna - , - - for hematological patients and donors did not have statistically significant differences, possible hna incompatibilities and risk of hna alloimmunization were estimated based on the obtained data on the allele and genotype frequencies of hna in a group that combines donors and hematological patients (n = ). the predicted risk of hna- , - , - , - incompatibilities in this cohort were . %, . %, %, and . %, respectively. the possible risk of hna- a, - bd, and - bc alloimmunization were . , . , and . , respectively; of hna- a and - b alloimmunization, . and . ; of hna- a and - b alloimmunization, . and . ; of hna- a and - b alloimmunization, . and . , respectively. summary/conclusions: the information about hna gene frequencies can be used not only in blood services for detection and identification of hna alloantibodies in donors and assessment of alloimmunization risk but also for anthropological studies. background: non-invasive fetal rhd genotyping is performed using circulating cell-free fetal dna from maternal plasma sample and real-time polymerase chain reaction. this antenatal routine dna test is used to target rh-ig administration to prevent hemolytic disease of the newborn. aims: the aim of this study is to characterize maternal rhd variants responsible for indeterminate results during fetal rhd genotyping due to early amplification of at least one of the exons ( , or ) of the rhd gene. methods: samples were tested from / / to / / using free dna fetal kit â rhd. samples ( , %) yielded a premature signal for one or more exons of the rhd gene. after extraction of maternal cellular dna, the maternal rhd was characterized using rhd beadchip assay (immucor/bioarray). rhdiiia-ce( - )-d summary/conclusions: greater diversity is observed in the caucasian population rather than in the afro-caribbean. % of the identified variants are rhd negative alleles including alleles leading to partial rh antigen expression. unexpected alleles are found such as weak d type , , or . these data underline the benefits of maternal rhd genotyping when abnormal early signals are detected during noninvasive fetal rhd genotyping. background: a considerable number of rhd alleles responsible for weak d phenotypes have been identified. serologic determination of these phenotypes is often doubtful and makes genetic analysis of rhd gene highly desirable in transfusion recipients and pregnant women. dna-based methods are useful for enhancing immunohematology typing in doubtful d phenotypes at pregnant women. aims: determination of the rhd gene in a cohort of pregnant women with doubtful d phenotypes. methods: determination of the rhd phenotyping was performed with microagglutination technique biorad and ortho diagnostic simultaneously. rhd genotyping was performed on cases with d typing serological discrepancies with ready-to-use inno-train rbc-ready gene cde and rbc-ready gene d weak test kits based on polymerase chain reaction with sequence-specific priming (pcr-ssp) to unclear serologic findings. results: molecular analyses showed of ( %) pregnant women were rhd*weak d type and not at risk for anti-d. rhd*weak d type were typed in cases ( %) and case was rhd*weak partial . and potentially at risk for being alloimmunized producing anti-d allo-antibodies. summary/conclusions: appropriate classification of rhd phenotypes is recommended for correct indication of rhig in pregnant women. however, the serologic differences between rhd-negative and rhd-positive pregnant women is a real problem for unnecessary application of rhig prophylaxis in pregnant women with d variants. conclusion: antenatal rhig prophylaxis is useful in rhd negative pregnant women. with genotyping we found that % of serological doubtful rhd negative women was d variants that not produce anti d antibodies. in that cases those rhig prophylaxis was unnecessary and harmful as a product of human origin. on other hand there is a save up of a stock of rhig which is any way in deficit. is it time to think about cost benefit of rhig prophylaxis and genotyping in pregnant women. background: in may , uk neqas (btlp) created an external quality assessment (eqa) sample designed to mimic a feto-maternal haemorrhage (fmh) bleed of ml. all material used passed pre-acceptance serological testing; samples were dispatched to participants in countries. post-dispatch testing by flow cytometry (fc) using an anti-d marker showed a bleed volume of . ml so an investigation was initiated. aims: to determine the cause of the unexpectedly low bleed volume and what lessons could be learnt. methods: production methodology and results of pre-acceptance testing were reviewed. fc testing was repeated, plots examined, and the fmh scientific advisory group consulted for advice. further fc testing was performed at wbs using alternative markers, and the material used was investigated at ibgrl. participant results were examined to determine if the sample should be withdrawn from scoring. a questionnaire on how results were managed was sent to the participants using fc with an anti-d marker. results: a material production methodology review showed no obvious cause of the erroneous in-house result. review of pre-acceptance testing images showed no issues, further d-typing of the cord showed + reactions vs. two reagents by tube, cf. + with two different reagents by column agglutination technology. repeat fc testing using the anti-d marker gave similar results; however, closer examination of the plots showed a left shift in the positive peak, indicating reduced fluorochrome binding, possibly due to reduced d antigen density on the cord cells. further fc testing at wbs demonstrated a marked reduction in fluorescence intensity with an anti-d marker. further investigation using an anti-hbf marker showed a bleed volume of . ml, indicating the correct proportion of cord material had been used during sample production. additional serology at ibgrl on the cord material showed reactions which were weaker than the control with / anti-d reagents. overall, the investigation supported the hypothesis that the cord material was d variant. a review of results submitted by participants mirrored the fc investigation and the sample was withdrawn from scoring, as the fc median result is used to calculate scores and the d variant cord was clearly affecting testing with an anti-d marker. the questionnaire showed that all respondents examine fc plots and the gating used, but not all act on them before reporting results, and not all have a back-up plan for anti-d ig dosing in a similar situation. later sequencing of the d gene revealed the cord donor to be dvii which can have a lower than normal d antigen density. summary/conclusions: the use of a d variant cord in an eqa sample was not planned, but allowed uk neqas to highlight some important learning points: -thorough examination of fc plots is essential to avoid underestimation of fmh; a controlled procedure should be in place if modification of gates is required -access to cord/neonatal blood to allow serological investigation may be useful in a similar clinical situation -it is important to have a back-up plan for issuing anti-d ig in the event of an uninterpretable fmh result background: allo-antibodies against fetal blood group and platelet antigens produced by antigen-negative pregnant women can cause hemolytic disease of fetus and newborn (hdfn) and fetal and neonatal alloimmune thrombocytopenia (fnait). prediction of the fetus antigen status in immunized women is important for making decisions concerning further management of pregnancy. nipt is widely used for determination of fetal blood groups but determination of proper specificity in the real-time amplification of a single nucleotide polymorphism (snp), such as k or hpa- a, requires modified protocols. droplet digital pcr (ddpcr) permits detection of low-grade fetal chimerism in maternal plasma dna with higher specificity using allelic discrimination pcr protocols. aims: to establish ddpcr protocols for non-invasive prenatal diagnostics (nipd) of clinically important blood group antigens. methods: dna was isolated from plasma samples of pregnant women and donors with known genotypes (easymag, biomerieux). allelic discrimination protocols for determination of k/k (n = ), s/s (n = ), hpa- a (n = ), hpa- (n = ), hpa- (n = ), hpa- (n = ) genotypes were performed using ddpcr method with droplet digital tm (biorad). the results of allelic discrimination performed using ddpcr were concordant with the already known phenotype/genotype of donors and pregnant women. ddpcr enabled the detection of - , reads for total dna from plasma in tested samples. all fetal results were in agreement with antigen positive genotype of the neonates and the fetal chimerism was from , % to , % (one case was for advanced pregnancy - week of gestation). in / tested samples false positive results were detected at the level of or unspecific reads. summary/conclusions: the implementation of allelic discrimination protocols for ddpcr allowed detection of fetal-maternal incompatibility in k/k, s/s and hpa- a, - a/b, - a/b, - a/b antigens encoded by snp. background: in france, for pregnancies complicated by anti-d (rh ) and anti-c (rh ) allo-immunization, the tests currently used to quantitate maternal antibodies are tube method titration and continuous flow analysis determination of the antibodies concentration. recently, an automated assay was developed using the column agglutination technology on the ih- system (bio-rad â). aims: we wanted to evaluate the score, calculated from the agglutination profile of the antibodies on the ih- system, as a quantitative data to appreciate the level of maternal antibodies. methods: titers from samples containing anti-d and containing anti-c have been established using the semi-automated tube method performed since decades in our lab and the fully automated gel method on the ih- system. scores were calculated manually in both cases. antibodies concentrations were also determined for all samples by continuous flow analysis on our auto-analyzer device (evolution iii ams alliance). we looked for a possible correlation between anti-d and anti-c scores and the corresponding concentrations using the spearman correlation test. results: anti-d tube and gel scores were significantly correlated with the anti-d concentration values (p < . , r = . and p < . , r = . respectively). anti-c scores were also significantly correlated with anti-c concentration values (p < . ) but gel scores have a better correlation coefficient than tube scores (r = . versus . ). it was easier to extrapolate gel score thresholds than tube score thresholds from the autoanalyzer values, with the aim of triggering fetal monitoring by ultrasounds and measurements of the peak systolic velocity in the middle cerebral artery only for risk pregnancies. the determined gel score thresholds were and , corresponding respectively to ui/ml ( uchp/ml) of anti-d and . ui/ml ( uchp/ml) of anti-c. conclusions: calculating the score from the hemagglutination profile displayed by the ih- system provides added values compared to the sole reading of the titer. for anti-c immunization, gel scores are more discriminant than tube ones and better correlated to the concentration values established by continuous flow analysis. the proposed score thresholds to trigger fetal antenatal monitoring need, however, to be confirmed on more samples and to be clinically documented. background: hdnf is due to maternal igg alloantibodies directed against fetal antigens that cross the placenta during pregnancy, causing hemolysis in the fetus, anemia that can lead to edema, ascites, hydrops and, in some cases, death. the diagnosis and management of hdnf is based on maternal screening, and middle cerebral artery (mca) doppler monitoring. in severe hdnf intrauterine blood transfusions (iuts) and or exchange transfusion (et) after birth are necessary to correct anemia, to prevent and treat fetal hydrops. aims: we report eight years of experience in our immunohematology reference laboratory (irl) to highlight the importance of red cell antibody detection as a fundamental parameter to identify pregnancies with high fetal risk and to drive a correct treatment. methods: we report laboratory data from pregnant women with a positive indirect antiglobulin test (iat) referred to our irl from january to december . we performed antibody screening and identification by indirect antiglobulin test (iat) in microcolumn method with biovue system (ortho-clinical diagnostics, raritan, usa), and the title of antibodies in iat by tube method without additive. follow-up tests were also performed in the presence of significant red cell antibodies in order to check antibody title and begin clinical monitoring. threshold values were ≥ : for anti kell antibodies and ≥ : for other specificities. results: out of women, ( . %) displayed clinically significant antibodies, ( . %) clinically insignificant antibodies and ( %) natural antibodies of different specificities. among women with clinically significant antibodies the most frequent was anti-d ( . %) also in combination with other rh antibodies ( . %), while anti-k accounted for %, anti-e for % and antibodies against high-incidence antigens for . %. anti-m and anti-le a antibodies were also found ( . % and % respectively) but they were not clinically significant. among women with clinically relevant antibodies, showed a critic antibody title and they underwent gynecological and obstetric monitoring. fetuses resulted affected by hdfn, displaying anti-d in cases and anti-kell in . fetuses with severe hdfn (anti-d in and anti-kell in ) required iuts, were treated with et, received red blood cells units at birth. summary/conclusions: the mother screening program led to important improvements in the outcomes of hdfn. the identification of women with clinically significant antibodies allowed an appropriate monitoring program and therapy. background: the hemolytic disease of the fetus and newborn (hdfn) is a severe disease, resulting from maternal erythrocyte alloantibodies directed against fetal erythrocytes. alloimmunization in pregnant women has been found to range from , % to , % worldwide. there are over erythrocyte surface antigens, of which more than have been reported to be associated with hdfn. although anti-rhesus d was once the major etiology of hdfn, the universal introduction of antenatal and postpartum rh immunoglobulin has resulted in a marked decrease in the prevalence of alloimmunization to the rhd antigen in pregnancy. consequently, alloantibodies other than anti-d emerged as an important cause of severe hdnf, in particular anti-k and anti-c. however, there are other antigens that have also been found to be associated with hdfn. aims: retrospective identification of erythrocyte antibodies in pregnant women in hospital de braga in and . methods: this study was planned to assess the prevalence of erythrocyte antibodies responsible for alloimmunization, excluding abo-immunizations, in pregnant women attending the antenatal clinics of hospital braga during years, from january to december . in this study, we retrospectively evaluated the erythrocyte antibody screening results of pregnant women. women with positive erythrocyte antibody screening also underwent identification with gel card system following the manufacturer's instructions (diamed â ). the outcomes of infants, whose mother's indirect antiglobulin tests were found to be positive, were examined. direct antiglobulin tests, jaundice and phototherapy history, transfusion and mortality of the newborns were recorded. results: during the study period, pregnant women were attended in hospital de braga. the laboratory registered positive erythrocyte antibody screening tests. the prevalence of positive erythrocyte antibody screening was , %. anti-d was the most common antibody found ( , %). anti-d prophylaxis given during pregnancy was responsible for of cases and maternal antibody titer levels did not exceed among these cases. the prevalence of non-rhd immunization was %. anti-e ( , %) was the most frequent alloantibody other than anti-d followed by anti-m ( , %) and anti-c ( , %). multiple maternal antibodies were found in pregnant women. four women had types of alloantibodies: anti-c and anti-e; anti-c and anti-d; anti-k and anti-cw; anti-e and a non-identified antibody. one pregnant had types of alloantibodies: anti-d, anti-c and anti-e. of all cases of newborns whose mothers had a positive antibody screen tests, icterus occurred in % of them and phototherapy was given in %. summary/conclusions: the prevalence of positive erythrocyte antibody screening in hospital de braga was , %. the erythrocyte antibody screening showed that anti-d was the most common antibody found ( , %) in most of the cases because of anti-d prophylaxis. the prevalence of non-rhd immunization was %. the other most frequent alloantibodies were anti-e ( , %), anti-m ( , %) and anti-c ( , %). an increasing prevalence of non-anti-d alloimmunization was found and there are currently no preventive strategies. in contrast to rhd alloimmunization, the main risk factor for non-anti-d alloimmunization is a previous transfusion therapy. thus, it is important to minimize the exposure of women to incompatible erythrocyte antigens through unnecessary transfusions when possible. background: the mns blood group system is one of the most complex blood group systems. although alloanti-m is a common antibody observed in pregnant women and could also be found in the serum of individuals who have not been exposed to m positive erythrocytes, it is rarely clinically significant and has been regarded as an unimportant antibody to cause hemolytic disease of the fetus and newborn (hdfn), especially in caucasian and black ethnic groups, for a long time. however, an increasing number of cases of severe hdfn resulting in fetal hydrops and recurrent abortion caused by alloanti-m have been reported mainly in the asian population, especially in the japanese and chinese populations. aims: to summarize the characters of serological testing in preterm twins newborns suffered with severe hdfn. methods: the blood sample of two newborns with severe hdfn and the mother, who had the history with three hydrops fetus, were collected. abo, rhd, rhce, and mn blood group typing of the twins newborn and their mother were performed in saline with tube or gel card. direct agglutination test (dat), elution test, antibody specificity identification and antibody titer detection were conducted by iat method in gel card. results: o, rhd(+), and ccee blood groups were identified both in the mother and the twins newborn. background: in france, since may , the legislation does not promote anymore the use of the reference tube method for titration of anti-red blood cells antibodies. this opened the way to the use of newly developed automated anti-red blood cells antibodies quantitation by column agglutination technology. aims: we wanted to assess the performance of titration and scoring by the id-gel test on the ih- system (bio-radâ) and to compare it with the performance established for the reference tube method, used in our lab since decades. another objective of the study was to determine titer thresholds for the gel method, to trigger fetal monitoring by ultrasounds and measurements of the peak systolic velocity in the middle cerebral artery. methods: an home-made internal quality control (iqc) prepared and calibrated using the international anti-d standard ( / ) was used to determine the intraassay and interassay imprecisions, regarding the score and the titer results. patients samples for testing were chosen during the -months assay period, regarding the specificity of the antibodies and the tube titer in order to cover a wide range of have lower values. the highest differences (more than to dilutions higher) were seen for antibodies directed against rh system antigens. among the other specificities, anti-k (kel ) and anti-m (mns ) antibodies show the most samples with equal or lower titers compared to the tube method. conclusions: automated anti-red blood cell antibodies titration by column agglutination technology on ih- system shows better intra and interassay cvs compared to the tube method. it is explained by the fully automated process that includes the reading step. titer results are almost always higher with the gel technology. thus, it seems possible to safely extrapolate the titer thresholds defined for anti-red blood cells antibodies by the tube method to the gel method. however, based on future clinical studies and fetal/neonatal outcomes, it would probably be necessary to increase these thresholds, at least for anti-rh antibodies, in order to avoid heavy, expensive, stressful and useless monitoring of some pregnancies. results: the first case was a -day-old female infant, yellowish skin developed the next day after birth. her capillary bilirubin level was mg/dl, the evidence favored neonatal hyperbilirubinemia and the clinical manifestation revealed hemolysis symptoms. her laboratory findings showed elevated reticulocytes ( . %), ldh ( iu/l) and g pd ( . u/ghb); dat (+/-), iat (-), anemia (hb . g/dl, hct %), and blood smear showed anisocytosis, spherocytes, and polychromatic rbc. her mother blood typed o, d positive, while her blood type was b, d positive and anti-b was found from her elution rbcs ( + ). due to rarely severe anemia with abo incompatibility, maternal plasma was analysed for abo igg antibodies and showed high antibody a and b titre with : and : . the female infant received one unit washed-prbcs for anemia and intensive phototherapy for hyperbilirubinemia. her clinical condition improved significantly, hb rose to . , bilirubin level was within normal range, she was discharged. another -days-old male infant was our second case. on the third day after birth, yellowish skin discoloration developed and bilirubin level was mg/dl. two days later, his transcutaneous bilirubin (tcb) measurement data was high and laboratory findings also showed raised reticulocytes ( . %), dat (+/À), iat (À), hb . background: anti-indian b is a rare alloantibody against the high frequency antigen in b . individuals with the in: ,- phenotype (in(a+b-)) are observed with a frequency of < . % in the indian population and have not been described in caucasians. the majority of anti-in b antibodies have been reported in individuals without previous transfusions, indicating the possibility of a naturally occurring antibody. anti-in b is considered clinically significant and haemolytic reactions after in b -incompatible transfusions have been reported. haemolytic disease of the foetus and newborn (hdfn) due to anti-in b has not been described. however, a positive direct antihuman globulin test (dat) may be observed. aims: to describe the challenges of managing a pregnancy and childbirth of a woman with an anti-in b . methods: serological investigations were performed by iat (tube and column agglutination). papain and trypsin treated cells were also utilised. soluble recombinant in blood group proteins (in-rbgp) (inno-train, germany) were used in neutralization tests. the clinical significance of the anti-in b antibody was determined by monocyte monolayer assay (mma). genomic dna was isolated from whole blood and the samples were further characterized by pcr amplification and sanger sequencing of exon of cd . results: in a -year-old pregnant (para ) woman of indian origin without previous transfusions, an alloantibody of the specificity anti-in b with a titer of : was detected by iat (negative with papain-treated cells) at gestational week (gw) and . the mma, performed in duplicate on samples taken at these dates, showed a mi of . %/ . % and . %/ . % respectively. the mi was interpreted as follows: - % not relevant; - % inconclusive; > % clinical significant. the patient's parents were typed heterozygous, in: , whereas her husband was homozygous, in:- , . due to the husbands phenotype, the fetus was predicted to be in b positive. doppler flow measurement of the peak systolic velocity in the middle cerebral artery of the foetus was normal. delivery took place at gw without increased bleeding. the neonate presented no clinical manifestation of hdfn. neither the mother nor the baby required blood transfusions. summary/conclusions: we report the case of a pregnant woman of indian origin with an anti-in b alloantibody. the first mma, performed in gw , was inconclusive whereas the second mma, performed in gw , indicated that the antibody was clinically significant. if the mi-increase is only due to the pregnancy or has also a clinical significance, cannot be stated. in b negative blood components were not available and the patient's relatives were all in b positive. therefore, measures to avoid transfusions, including optimised peripartal management of haemostasis, was of utmost importance. with only few cases published, the risk of hdfn could not be excluded with certainty. an intrauterine investigation by doppler was performed to exclude relevant anaemia of the fetus. no transfusion was needed at delivery as there were no haemorrhagic complications. the neonate presented no clinical signs of hdfn. background: hemolytic disease of the fetus and newborn (hdfn) is a disease which if untreatedcan cause perinatal mortality and morbidity with a substantial risk for long-term sequela. in albania we lack of studies in this field. aims: the aim of this study is to determine the predictive value and the reliability of the "critical titre" during the evaluation of red cells alloantibodies ability to cause the hemolytic disease of fetus and newborn. methods: we conducted a descriptive, cross-sectional study. the data were collected in the university hospital for obstetrics and gynecology in albania. in the study were included immunized pregnant woman for anti-d antibodies and their newborns which were affected from the hemolytic disease of fetus and newborn. the data belong to the period and . results: the "critical titre" in our study was , meaning that this was the minimal value of the titre antibodies that could cause hemolytic disease of fetus and newborn. our study concluded that only newborns were born without the hemolytic disease of fetus and newborn and the titre values were less than . moderate hemolytic disease of fetus and newborn were caused between the titre values - . the summary/conclusions: the titre values of the mothers are a predictive option of the high risk of giving birth to a child with the hemolytic disease of fetus and newborn. it is recommended that in this cases the mother should be followed with doppler ultrasonography to measure the blood flow of the middle cerebral artery. also the doctors should recommend in pregnant women with positive coombs test not only the identification of the anti-d antibodies but also the identification of the other antibodies such as anti-e, anti-c, anti-k. background: rhd-negative pregnant women with allo-anti-d are at risk of having a fetus affected by haemolytic disease of the fetus and newborn (hdfn) where the fetus is rhd-positive. the rhd allele is highly polymorphic and many rhd variants give rise to an array of partial d phenotypes. the clinical significance for many partial d phenotypes is not well-established. rhd genotyping by non-invasive prenatal testing (nipt) to assess the fetal rhd status determines whether the fetus is at risk for hdfn. nipt tests also include strategies for detecting maternal rhd variants to provide for accurate reporting. however, the presence of a paternal rhd variant, while having the potential to confound nipt interpretation, is often not recognised. we report a "trio" family study triggered by a request for nipt for an rhd-negative pregnant mother, weeks gestation, who presented with allo-anti-d and anti-jk a antibody. subsequent paternal and fetal rhd genotyping was conducted and revealed a novel variant rhd allele. aims: we aim to characterise the paternal rhd allele and review clinical case features. methods: rh phenotyping was performed by standard serological procedures. nipt tested for fetal rhd exons , and . rhd genotyping on whole blood/cord blood dna was performed on the immucor bioarray rhd beadchip kit which predicts a rhd phenotypic variant of best fit. dna sequencing was performed using the illumina trusight one sequencing panel. copy number variation (cnv) analysis was used to assess the rhd exon structure and zygosity. results: the paternal red cells typed as group o rhd+c-c+e-e+, (ror). nipt genotyping detected fetal rhd signals for all exons, predicting rhd-positive. no maternal rhd sequences were detected consistent with homozygosity for the rhd deleted haplotype. for both paternal and cord genomic dna (gdna), beadchip genotyping predicted a rhd variant "diiia/cehar". furthermore, signal drop out was observed at nucleotide positions (c. , c. , c. ) located in rhd exon suggesting exon was either deleted or rhce-replaced. paternal and cord gdna sequencing detected out of snps (c. g>t, c. c>t, c. a>c, c. c>g) associated with diiia phenotype plus additional snps (c. g>a, c. g>c) on the rhd gene. both were rhd hemizygote by cnv analysis. no rhce variants were detected. clinical case features: the maternal anti-d quantitation increased from . iu/ml ( weeks gestation) to iu/ml ( weeks gestation). the fetus required intrauterine transfusions during the pregnancy to manage the hdfn. summary/conclusions: both father and fetus carry an rhd allele that does not align with alleles encoding diii phenotypes. this putative novel rhd variant allele comprises snps associated with diiia and with a possible exon deletion/rhcereplaced. a similar allele was reported in literature, although based on sequence analysis only, with no phenotype data. the variant allele here encodes rhd-positive phenotype and we predict that there may be a loss of d-epitopes. notwithstanding, the clinical presentation shows that maternal anti-d against this rhd phenotype (presumed partial) is associated with a severe hdfn and that such rhd blood group phenotypes are of clinical significance for alloimmunised pregnancies. abstract withdrawn. background: cd is a glycosylphosphatidylinositol (gpi)-anchored protein with apparent molecular mass of kda. in addition to being expressed on human plts, cd is expressed on activated t-cells, endothelial cells, cd + hematopoietic stem cells as well as on progenitor cells. in the chinese population, the calculated allele frequencies of hpa- a and - b are . and . , respectively. based on these data, the risk of alloimmunization against hpa- alloantibodies due to incompatible plt transfusion or pregnancy is expected to occur in relatively high frequency. however, until today there is no report of hpa- alloimmunization in the chinese population. in this study, we analyzed sera from hydrop fetus cases by maipa technique and icfa. aims: to detect the anti-hpa b alloantibodies by maipa and icfa. methods: a -year-old mother, gravida /para . the mother in the first pregnancy was diagnosed hydrop fetus at pregnancy weeks by ultrasound. in the second pregnancy, fetal hydrops was observed by ultrasound at pregnancy weeks. the mother's irregular antibody test was negative. the maternal platelet specific antibodies and hla antibodies were negative. blood routine and morphological examination of fetal umbilical cord blood showed that plt count dropped to . /l, wbc count dropped to . /l, including neutrophil %, lymphocyte %, mononuclear %, eosinophil %, basophil %, red blood cells were normal, hb was g/l. screening for hla and plt-specific antibodies was performed using a elisa-based plt antibody kit (pakplus, gti diagnostics) as recommended by the manufacturer. plt antibodies were detected by icfa and maipa.hpa genotyping was detected by cpr-ssp. results: the fetus's genotype was hpa- a/a, - a/a, - a/a, - a/a. - a/a, a/a, a/a, a/b, naka (+) and the maternal was hpa- a/a, - a/b, - a/a, - a/a. - a/a, a/a, a/ a, a/a, naka (+). the paternal genotype was hpa- a/a, a/b, a/a, a/a, a/a, a/a, a/a, a/b, naka (+), which was the only incompatible antigen compared with the maternal hpa. samples were tested using the fresh plt panels consisting of hpa- aa and - bb homozygous donors. the reactivity of the negative control and the mother's sera with the plts from hpa- a/a (donors ), hpa- a/b (donors ) and hpa- b/b (donors ) donors by maipa. the mother's serum showed no reactivity against a/a plts, weak positive reactivity against a/b plts (od values . ), but strong reactivity against b/b plts (od values . ).this finding could be confirmed by one of the reference plt laboratories (japanese red cross kanto-koshinetsu block blood center, japan) using freshly isolated plts from hpa- genotyped donors (anti-hpa- b average value . ). summary/conclusions: in this study, we found anti-hpa- b in a case of fnait (patient hpa- aa, blood group o) using the maipa technique. we were able to detect the presence of hpa- b alloantibody in one case of nait. background: fetal and neonatal alloimmune thrombocytopenia (fnait) occurs in : live births in caucasians. serological and molecular human platelet antigens (hpa) genotyping tests are performed to investigate and conclude to fnait diagnosis. however, in few cases and particularly in case of suspicion of private platelet antigen, some specialized analyzes must be performed in the laboratory (lab). these analyzes can range from sanger or ngs sequencing to platelet serology with transfected cells. aims: the aim of our study was to explore where the frontier between research and care takes place in the field of platelet immunology through the prism of the fnait investigations carried out by the platelet immunology laboratories. methods: a two-part electronic survey have been sent to foreign platelet immunology experts (pie) from platelet immunobiology working party (piwp) members and espgi board members (n = ). the first part focused on the lab practices and regulatory environment regarding to accreditation, contact with patient, informed consent and patient results. the second part stressed on the investigations performed to discover new platelet antigen and more precisely on the perceived status of these analyzes ( background: haemolytic disease of the fetus and newborn (hdfn) can occur when maternal red cell antibodies, directed against red cell antigens present on the fetal red blood cells, cross the placenta and enter the fetal circulation. in a "traditionally" conceived pregnancy, when hdfn occurs, it is as a result of maternal antibodies directed against fetal red cell antigens in the heterozygous state, whereby the antigen is inherited from the father only. with the advent of donor oocyte (do) in-vitro fertilisation (ivf), the addition of a third person into the reproductive equation allows for the possibility of a more severe form of hdfn when fetal red cell antigens are present in the homozygous state (one copy from father and one copy from donor) and maternal antibodies are directed against these. antigens expressed in the homozygous state will have more antigens sites per red blood cell and therefore are at an increased risk of red cell destruction from the maternal derived cognate antibodies. aims: to raise awareness of increased severity risk of hdfn in donor oocyte conceived pregnancies. methods: we describe two unusual cases of hdfn in our institution of two women whose pregnancy was induced using a donor oocyte and their offspring requiring transfusion support in the postnatal period to treat hdfn. results: the first is a case previously reported (doyle, quigley, fitzgerald et. al. transfusion medicine, ) of protracted hdfn due to anti-c, managed with phototherapy initially, then intervention with red cell top-up transfusion at weeks post-delivery. the second is an unusual case of severe abo hdfn requiring exchange transfusion therapy (pre-publication). summary/conclusions: given the increased number of pregnancies conceived using do we recommend that antenatal guidelines are reviewed to create awareness regarding the potential increased risk of hdfn in do pregnancies complicated by allo-immunisation. critically, antenatal testing guidelines should highlight that the predicted outcomes associated with quantitation/titres can only be used when do has not been used to obtain the pregnancy. it is also essential that clinicians inform the blood transfusion laboratory when do has been used. abstract withdrawn. %) are deceased due to organ rejection, and / patients ( %) are deceased due to disease not related to rejection. summary/conclusions: the use of therapeutic plasma exchange for the treatment of antibody mediated rejection in solid organ transplant is safe and effective when used along with other treatment modalities. further studies will help determine whether it can be reproduced in larger cohorts and whether it is more effective in certain organs. background: extracorporeal photopheresis (ecp) is an important cellular therapy for the treatment of several (auto-)immune diseases such as graft-versus-host disease. the international standard for the ex vivo treatment of the leukapheresis product is the application of -methoxypsoralen ( -mop) and irradiation with uv-a light. however, the addition of -mop to the illumination bag is associated with a potential risk of contamination. aims: the basic principle of the ecp is the induction of apoptosis in the leukocytes. our aim was to find an alternative for the conventional apoptosis induction without the need of external substance application. the objective of the study was the investigation of the apoptosis levels and kinetics in leukocytes after treatment with -mop+uv-a compared to uv-c treatment without additional -mop. methods: we used an in vitro h cell culture approach with human mononuclear cells from healthy blood donors. untreated control cells were compared with , lg/ ml -mop plus j/cm uv-a treated cells and j/cm (effective dose) uv-c treated cells. apoptosis in several leukocyte sub-populations was detected daily with annexin v and -aad flow cytometry standings. results: the apoptosis analysis of cd cd t-helper cells, cd cd cytotoxic tcells, cd b-cells, cd monocytes, cd neg cd nk-cells and cd cd nkt cells revealed no statistical differences in almost all of these cell types after treatment with -mop/uv-a or uv-c light. the apoptosis kinetic as well as the final apoptosis after h were similar in both treatment groups. summary/conclusions: the addition of -mop to the photopheresis irradiation bag is a risk for potential infections. the main effect of the -mop/uv-a treatment is most probably the induction of apoptosis in the leukocytes. here, we provide information that this induction of apoptosis can also be achieved with uv-c irradiation without the need of -mop addition. the apoptosis patterns in most leukocyte subpopulations are very similar after treatment with uv-c compared with -mop/uv-a treatment. future in vivo studies are needed to prove the therapeutic effect of uv-c treated cells in the ecp setting. abstract withdrawn. background: therapeutic plasma exchange (tpe) is performed to remove the implicating substances from the plasma causing the disease. a periodic appraisal of tpe data is important to get insight into the procedural related effects and toxicities and overall outcome in order to have a guided future approach. aims: the purpose of this study is to observe the overall profile and outcome of the patients receiving the tpe in the medicine intensive care unit (micu) of a tertiary care hospital in south india. methods: a record based audit was conducted for the all the patients who were admitted to our tertiary care hospital of south india with bedded micu and received tpe therapy between june, and december . all the tpe procedures were performed using haemonetics multicomponents system (mcs) + ln apheresis system based on intermittent flow centrifugation. we audited our tpe for: number of treatments, clinical indications, treatments prescribed and administered, any procedural or patient complications, and adherence to current best practice recommendations. results: sixty nine patients had undergone tpe procedures. among them, thirty were female patients ( %). the median age ( - ) years. guillain-barre syndrome (gbs) was the most common indication ( %) followed by cases of thrombotic thrombocytopenic purpura, diffuse alveolar hemorrhage, myasthenia gravis, autoimmune encephalitis and hypertriglyceridemia respectively. the tpe regimens received by patients in this icu were not always prescribed in accordance with current best practice recommendations. there were ( %) episodes of patient related complications during the tpe treatments. in ( %) procedures, technical error in the machine was encountered. summary/conclusions: the findings of this audit have identified differences between the current prescription recommendations for tpe and those applied. the infrequency of the therapy and the different indications may present a challenge for medicine intensive care clinicians to provide best care in all cases. background: microangiopathic hemolytic anaemia (maha) encompasses a spectrum of disorders characterised by widely disseminated thrombosis in small blood vessels resulting in formation of schistocytes and concomitant thrombocytopenia. plasma exchange (pe) needs to be considered as empirical and urgent life saving therapy in these disorders irrespective of waiting for specific testing like adamts levels in thrombotic thrombocytopenic purpura (ttp) or complement levels or factor h antibodies in atypical hemolytic uremic syndrome (ahus). aims: to assess the efficacy and safety of plasma exchange in patients diagnosed as having microangiopathic hemolytic anaemias. methods: a retrospective analysis of all pe procedures performed in patients diagnosed as having maha was done over a period of years ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . procedures were done on apheretic device (cobe spectra, terumo bct, lakewood co. usa). patients' pre and post procedural hematological and renal parameters were analyzed by applying paired t test. adverse event if any was recorded. results: pe was performed in patients with diagnosis of maha ( -ahus, -ttp, each of post stem cell transplantation drug induced thrombotic microangiopathy (tma), post thyroidectomy tma and post-partum tma). the mean age of patient was . ae . years with m:f as . : . number of procedures per patient varied from to . post pe recovery was observed within - days with statistically significant increase in mean platelet count from . ae . to . ae . /l (p = . ) and significant decline in mean lactate dehydrogenase level from . ae . to . ae . lkat/l (p = . ). there was also significant decline in mean percentage of schistocytes in peripheral smear from . ae . % to . ae . % (p = . ). the mean serum urea changed from . ae . to . ae . mmol/l and creatinine from . ae . to . ae . lmol/l (p = . and . respectively) with significant increase in urine output from . ae . to . ae . ml/kg/h (p = . ). adverse events were observed in patients ( %), allergic reaction to replacement fluid (n = ) being the commonest followed by hypotension (n = ), rigors and chills (n = ). overall survival rate at months was %. summary/conclusions: pe had proven its safety and usefulness as life-saving first line treatment modality in maha. prompt and aggressive treatment helps in achieving early and complete remission in these patients. background: neuromyelitis optica (nmo) also known as devic's disease or devic's syndrome is a rare demyelinating disease of the central nervous system that most often results in selective involvement of the optic nerves (optic neuritis) and spinal cord (myelitis)and has female preponderance. neuromyelitis optica (nmo) attacks are poorly controlled by steroids and evolve in stepwise neurological impairments. assuming the strong humoral response underlying nmo attacks, therapeutic plasma exchange is an appropriate technique in severe nmo attacks. aims: to study the effect of tpe in neuromyelitis optica. methods: a year old female in the medicine department, civil hospital, ahmedabad admitted with chief complains of weakness and numbness in the arms and legs, blurred vision, reduced sensation, difficulty in controlling bladder and bowels, uncontrollable vomiting and hiccups since - days in the medicine department, civil hospital, ahmedabad. attacks were treated with short courses of high doses of intravenous corticosteroid -methylprednisolone intravenous. but there was no clinical improvement. results: clinician advised for the trial of tpe in this patient. the procedure was performed by automated device with continuous flow centrifuge machine fresenius kabi-com.tec using double lumen femoral catheter. after obtaining informed consent from the relative of the patient, cycles of tpe were performed on daily basis. after cycles, both subjective and objective clinical response to tpe was estimated by three different sources (the patient, a transfusion medicine physician, and the treating neurologist). [ ] for motor performance, patient was assessed on a disability scale ( = healthy; = minor symptoms; = able to walk meters without support; = able to walk meters with support; = confined to bed or wheelchair; = requiring assisted ventilation; = dead).patient's motor performance was increased to scale (upper limb) and (lower limb) from scale , deep tendon reflexes were normal. visual function began to improve week after the treatment. visual acuity was / after weeks. summary/conclusions: assuming the strong humoral response underlying nmo attacks, therapeutic plasma exchange is an appropriate technique in severe nmo attacks. this suggests that tpe is beneficial in nmo patients during acute attack if there is no response to corticosteroid treatment. background: babesiosis is a tick borne infectious disease caused by the protozoa babesia. while most infections with babesia are asymptomatic, some patients present with a symptomatic infections and rarely this can be a severe life threatening illness. treatment is primarily with antibiotics but red cell exchange (rce) has been used in the more severe cases which are characterized by high grade parasitemia, evidence of severe hemolysis and or multi-organ failure involving the kidney, lung or liver. a threshold parasite level of % has arbitrarily been applied as an indication for rce, however, this threshold is not evidence based. aims: to report on patients with babesiosis and high grade parasitemia who were treated with antibiotics only without rce methods: data were collected from july to july . a case was defined as a patient diagnosed with babesiosis for whom rce was requested on the basis of a parasitemia of > % but on clinical evaluation it was considered that rce could be withheld and the patient monitored awaiting response to antibiotics. results: three cases of severe babesiosis in which the use of rce was requested on the basis of a parasite level of greater than %, but was not performed. the rce was deferred on account of the good clinical state of the patient and the absence of renal failure. levels of parasite at diagnosis were . %, % and %. all patients were followed daily until discharge. two of these patients had been splenectomized and each received a single unit of red blood cells during the hospitalization. the third patient had a long history of refractory lymphoma and was pancytopenic requiring multiple transfusions during the years before the diagnosis of babesiosis. she had transfusion transmitted babesiosis from a red blood cell transfused days prior to the diagnosis. all three patients responded well to antibiotics and were discharged between - days with undetectable parasites. summary/conclusions: this small case series suggests that requests for rce solely on the basis of an arbitrary level of parasitemia should be questioned and the clinical state and evidence of organ failure considered in the decision to perform rce. abstract withdrawn. chronic transfusion program (ctp) remains the gold standard therapy for stroke prevention and for patients with a severe disease who have inadequate response to hydroxyurea treatment. aims: to evaluate the safety, efficacy and cost between scd patients on ctp that underwent both aet and partial manual exchange transfusion (pmet) procedures. methods: retrospective observational cohort study of patients with scd on ctp that have switched between pmet and aet. this study was carried out from / / to / / in a hospital in portugal. data on patient history, haematological values, duration of the procedure, intervals between them, adverse events as well as the cost of material and working hours were collected and compared between both procedures. results: a total of patients met the inclusion criteria described. however, patient was excluded from our study because of the lack of attendance to the ctp. during the study, we recorded exchange procedures ( pmet and aet), both on peripheral venous access. from all those procedures the major concern was the poor venous access, which was the reason why patients had returned to pmet. no major complication or alloimmunization was observed. the indications for ctp were cerebral vasculopathy (n = ), stroke (n = ) and recurrent vaso-occlusive crisis with multiorgan failure (n = ). for both procedures, target values were to obtain a pre-exchange hbs level ≤ % for stroke and cerebral vasculopathy and ≤ - % for other indications. the median hbs level before pmet was , % ( , - , ) and , % ( , ) before aet. we documented a higher hbs level prior to the next procedure in , % of patients (n = ). despite that all patients remained stable without any major scd related event. both procedures were well tolerated and iron overload was well controlled (median ferritin level pmet: , vs. aet: , ng/ml). the duration of the exchange procedure was longer and the intervals between procedures were shorter with pmet (median pmet: vs. aet: min and pmet: vs. aet: weeks, respectively). annual rbc requirements per procedure were superior (median vs. units) and the overall costs related with aet were , times higher - . , € and . , € aet and pmet, respectively (estimated cost per session aet: , € and pmet: , €). summary/conclusions: our study shows, that the hbs level before both procedures, performed during the same interval, was similar. we verified that pmet has a comparable efficacy with aet in terms of preventing the development or progression of chronic complications and that the cost per procedure is significantly higher with aet. however, in a clinical situation where it is important to rapidly reduce the hbs level, and/or where the control of the target hbs is stricter so that the patients are clinically controlled without an increase in hospital visits, aet is preferred. we conclude that aet is more effective in the rapid reduction of hbs and ferritin levels, as well as being less time consuming. despite this, for the reasons described above, it is more cost-effective to maintain both aet and pmet procedures. background: erythrocytapheresis/red blood cell (rbc) exchange, involves removing of a large number of rbcs from the patient and returning the patient's plasma and platelets along with compatible allogenic donor rbcs. typical indication for rbc exchange is sickle cell disease and its related complications. however, one of the miscellaneous indications of rbc exchange is for the patients of methemoglobinemia who are refractory to treatment by methylene blue. acquired methemoglobinemia is more common than any genetic causes. acquired methemoglobinemia is caused by toxins that oxidize heme iron, notably nitrate and nitrite-containing compounds. for patients failing to respond to standard treatment with methylene blue or in whom its use is contraindicated; hyperbaric oxygen or rbc exchange is indicated aims: case reports on use of rbc exchange in methemoglobinemia are few and indications are based on anecdotal reports. methods: exchange was performed on the cell separator machine, com tec by fresenius kabi. results: we report a case of acquired methemoglobinemia where patient was admitted with peripheral capillary oxygen saturation (spo ) of % on air. the patient did not show improvement in spo level with effective emergency treatment of methylene blue. since, the patient was refractory to treatment with methylene blue, the decision was made by clinician to proceed with rbc exchange. the patient improved significantly after two cycles of one rbc volume automated rbc exchange, and was discharged with spo of % on air. summary/conclusions: automated rbc exchange can be used in patients of acquired methemoglobinemia successfully when methylene blue is ineffective, and may be superior to manual one. background: therapeutic plasma exchange (tpe) is known to disturb the ph and electrolyte status. patients with compromised liver functions may be at a higher risk of electrolyte imbalance due to metabolic abnormalities. aims: the aim of this study was to analyze the variation in ph, ionized calcium, sodium, potassium, and bicarbonate in liver disease patients undergoing tpe. methods: patients with liver disease undergoing tpe during the period from july to august were included in the study. data on patient demographics, details of the tpe procedure, blood gas analysis report and adverse effects of tpe (if any) were collected and analyzed. results: one hundred and seven procedures were done during the study period; of these ( %) were done on the mcs plus (haemonetics corporation) and rest ( %) were done on the spectra optia (terumo bct). the percentage change in ionized calcium, sodium, and potassium due to the procedure was found to be statistically significant (p = . ). the systolic (p = . ) and diastolic ( . ) blood pressure also changed significantly with the procedure. the predictors for the change in ionized calcium were found to be pre-procedure ionized calcium (p < . ), the age of the patient (p < . ) and the pre-procedure ph (p = . ). procedurerelated complications occurred during procedures of which complications ( . %) were categorized as features of hypocalcemia. no association was found between hypocalcemic manifestations and pre-procedure calcium, change in calcium, age or gender of the patient. summary/conclusions: the tpe procedure in liver disease patients causes remarkable changes in ionized calcium, sodium, potassium and bicarbonate ions. the decrease in ionized calcium during the procedure is predicted by pre-procedure ionized calcium levels, ph and age of the patient. monitoring of these parameters and appropriate corrective measures are imperative to patient safety. background: therapeutic plasma exchange (tpe) in pediatric age group is technically demanding because of low blood volume, difficult venous access and poor cooperation of the patient during the procedure. we here present our experience of tpe in pediatric patients from our centre. aims: to assess the challenges during tpe in pediatric patients and formulate appropriate strategies. methods: we did retrospective analysis of all tpe procedures performed in pediatric patients over a period of years ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . tpe procedures were done on two different apheretic devices (cs plus, fenwal usa and cobe spectra, terumo bct lakewood, colorado) daily or on alternate days depending on clinical condition of the patient. for all procedures, kit was primed with compatible packed red cells. adverse events during the procedure were noted and analyzed. results: a total of tpe (range - /patient with mean of . procedures) were performed for pediatric patients with different indications like atypical hus (category i as per american society for apheresis (asfa) in total patients, neuromyelitis optica (category ii) in patients, rapid proliferative glomerulonephritis (category i), c glomerulopathy in patients each and one patient of infective hemophagocytosis. the average age of patient population was . yrs ( . - years) . the male:female ratio was : with an average weight of . kgs. adverse events were observed during ( . %) procedures. most commonly observed adverse events were allergic reaction to replacement fluid ( . %) followed by hypotension ( . %), line occlusion ( . %), vasovagal, endotracheal tube blockage and symptomatic hypocalcemia was observed in one procedure each ( . %).there was no corelation observed between physical parameters of patient with adverse events. all adverse events were managed as per departmental standard operating procedures (sops) and procedures were completed successfully except in one where the procedure was abandoned. no mortality was observed during the procedures. background: the hemoglobin (hb) content of packed red blood cell (prbc) units is heterogenous. the patient's blood volume is also variable which can be calculated based on the weight, height and body surface area (bsa) of the patient. the efficacy of a transfusion episode can be assessed if the hb content of prbc is known and the patient's post-transfusion hb increment is determined. aims: this prospective study was performed to compare the efficacy of the transfusion of prbcs based on hb content versus the standard transfusion practice in thalassemia major patients. we also determined the correlation between hb increment and the hb content of prbc units transfused. methods: a total of registered thalassemia major patients of our institute were included in the study after excluding the patients who had allo-or auto-antibodies. the study was approved by the institute ethics committee. the enrolled patients were randomly divided into two groups: group i (n = )they received abo/rhd identical prbcs suspended in additive solution (saline, adenine, glucose, mannitol: sagm-prbcs) after determining its hb content (units with hb content ≥ g); and group ii (n = )they received randomly selected abo/rhd identical sagm-prbcs. the hb estimation of the randomly selected units in group ii was blinded. following tests were done on pre-transfusion sample: hb estimation using the hematology analyzer (orion , ocean medical technologies, india), blood grouping using tube technique, anti-human globulin (ahg) crossmatch and direct antiglobulin test (dat) using gel technique (biorad, switzerland), antibody screening (abs) using a fully automated immunohematology analyzer (neo, immucor, usa). on the posttransfusion sample collected h after transfusion, hb estimation and dat were performed. results: there was no significant difference among the patient characteristics of the two groups. the mean hb content of the sagm-prbc units was significantly higher (p = . ) in group i (mean ae standard deviation: . ae . g; range: . - . g) than group ii ( . ae . g; range: . - . g). the mean hb increment in group i patients ( . ae . g/dl) was significantly higher (p = . ) than the group ii patients ( . ae . g/dl). in both the groups i and ii, there was a significant negative correlation between hb increment and weight (p = . in groups i and ii), age (p = . for group i; p = . for group ii), body surface area (bsa) (p = . for group i; p = . for group ii) and blood volume (p = . for group i; p = . in group ii). in both the groups i and ii, there was a significant positive correlation between hb increment and hb dose adjusted for bsa and the hb dose adjusted for blood volume (p = . in both groups i and ii for both the parameters). summary/conclusions: the efficacy of transfusion is more when patients are transfused with sagm-prbcs having hb content of g or more as compared to those who are transfused with randomly selected units. for optimal hb increment in thalassemia major patients, the transfusion strategy should be based on the hb content of the sagm-prbcs. background: in male transfusion recipients under years of age, receiving red blood cells (rbcs) from an ever-pregnant blood donor has been associated with increased mortality, compared to receiving a product from a male donor. although it has been suggested that older units of rbcs could be associated with increased mortality, there are significant methodological challenges in these studies. other studies indicated the freshest units of rbcs could be associated with increased mortality among transfusion recipients. we hypothesize both the association between ever-pregnant donors, and fresh units, with mortality could be caused by passenger leukocytes in the transfused rbc units, which decay during storage. aims: to quantify modification of the effect of ever-pregnant donors on mortality in young male rbc transfusion recipients, by storage time. methods: data on transfusion recipients receiving their first-ever rbc transfusion in one of six major dutch hospitals between / / and / / was collected. for the current study, male transfusion recipients under years receiving only transfusions from one donor sex exposure category were selected and followup was censored at three years after transfusion. differences in storage time between groups were estimated by linear regression, adjusted for total number of transfusions, patient age, blood group, transfusion year and month. in a single-unit, single-transfusion cohort, cumulative mortality was estimated separately for patients receiving transfusions from ever-pregnant or male donors and for 'fresh' (< days storage) or 'old' (> up to days storage) rbcs. results: for recipients of only blood from male donors, the storage time of the freshest unit was . day shorter when comparing the patients who died, to , patients who survived (ci: À . to . ). for recipients of only blood from ever-pregnant donors, the storage time of the freshest unit was . day longer when comparing the patients who died, to patients who survived (ci: À . to . ). in the single-transfusion cohort, , patients received a fresh rbc transfusion from a male donor, of whom died; patients received a fresh transfusion from an ever-pregnant female donor, of whom died. patients received an old transfusion from a male donor, of whom died; patients received an old transfusion from an ever-pregnant female, of whom died. the -years cumulative incidence of death among young male recipients was . % (confidence interval (ci): . % to . %) after a fresh transfusion from a male donor and . % (ci: . % to . %) after a fresh transfusion from an ever-pregnant female donor. the -years cumulative incidence of death was . % (ci: . % to . %) after an old transfusion from a male donor and . % (ci: . % to . %) after an old transfusion from an everpregnant female donor. summary/conclusions: prolonged storage of rbcs from ever-pregnant donors was not associated with decreased mortality at years. contrary to our expectations, our results indicate older units may potentiate the effect of ever-pregnant donors. however, due to limited sample size the observed differences were not statistically significant. background: according to the literature review, there was limited impact of premedication (antipyretics, antihistamines and steroids) before transfusion on the prevention of adverse transfusion reactions (atrs). however, the necessity of premedication remains controversial. the premedication before transfusion is still a common clinical practice in pacific-asian countries, along with the premedication rate ranging from to %. in our previous investigation, we found that premedication rate was . % in the outpatients in , which was much higher than the reported rate in asia. aims: to investigate the incidence of atrs and decrease premedication rate without increasing the rate of atrs via education and evidence-based clinical practice. methods: the incidence of atrs from april to december, was retrospectively surveyed. evidence-based clinical practice was initiated since january, . clinical data of the outpatients receiving transfusion therapy were requested and analyzed from january to september, . the incidences of atrs and premedication rates in and were compared using chi-square test. a p value less than . was statistically significant. besides, feedback of the incidence of atrs and premedication rate was given quarterly to the clinicians during the investigation. results: from april, to september, , a total of , blood units were transfused in the outpatients with , transfusion events. of these, cases of atrs, including febrile nonhemolytic transfusion reactions (fnhtr) and minor allergic reactions were reported. the overall premedication rate in the outpatients was . % in , and was significantly decreased to . % in (p < . ). it was reported that the incidences of atrs in and were . % and . % per unit, respectively. there was no remarkable difference between the incidence of atrs in and (p = . ). summary/conclusions: via education and evidence-based clinical practice, we successfully reduced premedication rate without increasing the rate of atrs in the outpatients. furthermore, introduction of computerized provider order entry (cpoe) and clinical decision support system (cdss) could be considered and be expected to prevent unnecessary premedication before transfusion, increasing the compliance with optimized transfusion strategies in the future. methods: a retrospective analysis was done over a period of one year to evaluate clinical efficacy of granulocyte transfusions in hemato-oncology patients with febrile neutropenia. mobilization of granulocyte donors was done as per standard protocol, which included subcutaneous injection of granulocyte colony stimulating factor (g-csf) - lg/kg and tablet dexamethasone mg, - h prior to granulocyte harvest by apheresis. all granulocyte products were gamma irradiated before transfusion. patient parameters like white blood count (wbc), absolute neutrophil count (anc), hemoglobin and platelet count were recorded pre-and post-granulocyte transfusion. infection related mortality (irm) within days of granulocyte transfusion was also recorded. results: minimum adequate granulocyte yield of per unit was fulfilled in % of granulocyte harvests. clinical indications for granulocyte transfusions were fever, an absolute neutrophil count (anc) < /ll, evidence of bacterial and/or fungal infections (i.e. clinical signs of infection, positive cultures and radiological evidence) and unresponsiveness to appropriate antimicrobial therapy for at least h. effects of clinical, microbiological and granulocyte transfusion related variables on infection-related mortality were investigated. the post transfusion anc (within h) increased significantly (median value: /ll) as compared to baseline levels (median value: /ll) (p < . ). infection related mortality was observed in only % ( out of ) of patients. patients became afebrile within - days and culture negative within - days after granulocyte transfusion. for analysis purpose granulocyte transfusion episodes were grouped according to doses of granulocyte transfusions, based on european guidelines (standard dose: . - . cells/kg and high dose: > . cells/kg background: hsa's blood services group (bsg) is singapore's national blood service. in , we conducted our pilot national pbm audit to promote pbm practices. it was agreed that the audit would be performed annually with incorporation of a new indicator to continue promotion of pbm and sharing of good practices. aims: to provide an update on the second national pbm audit for . results are compared to the pilot audit and summarized below. methods: we collected data on performance indicators from acute public care hospitals for weeks each in march and august (the pilot audit covered weeks in ). the performance indicators were: ). percentage compliance to documentation of red blood cell transfusion indications ). percentage of patients screened for pre-operative anaemia, to days before surgery ). peri-operative transfusion rates ( days before to days after surgery) for commonly performed surgeries: coronary artery bypass graft surgery (cabg), total knee replacement (tkr), total hip replacement (thr), nephrectomy, colectomy and hysterectomy. the first two indicators assess pbm efforts and were measured in the pilot audit. indicator ) was added to the second audit to assess impact of pbm practices on transfusion in surgical patients. it was an appropriate time to incorporate this indicator as the hospitals would have been familiar with pbm since its introduction in . results and recommendations were shared with the senior management and hospital transfusion committees of the participating hospitals. results: for indicator ), hospitals had a compliance of - %, the remaining had a compliance of - %. all hospitals incorporated electronic blood ordering but the usage was not compulsory in some. hospitals which mandated electronic ordering performed better as doctors could only order blood products after entering the transfusion indication. we saw compliance increase from % in to % in a hospital that had newly mandated electronic ordering. for indicator ), results ranged from % to %. hospitals made notable improvements when compared to , achieving % and % respectively. they had implemented pre-operative workflows screening all elective surgical cases for anaemia at least weeks before surgery. one hospital also started an outpatient intravenous iron service which reduced pre-operative anaemia rates. for indicator ), mean number of transfused units for each surgery ranged from . to . units per patient, lowest being thr and highest being cabg. this suggests that some transfusions were potentially avoidable with more robust pbm practices. the rate of perioperative transfusions was highest for cabg at % and lowest for tkr at %. summary/conclusions: the annual national pbm audit increases pbm awareness, allowing hospitals to share and learn good practices and implement measurable improvements. based on this audit, a recommendation to mandate electronic ordering of blood products to improve adherence to red cell transfusion indications and implementing pre-operative workflows with consideration for intravenous iron support was made. this audit was more representative than the pilot, with a longer duration of data collection and incorporation of indicator ) showing impact of pbm practices. background: autoimmune haemolytic anaemia (aiha) is a decompensated acquired haemolysis caused by the host's immune system acting against its own red cell antigens. aiha is a rare disorder and although british society of haematology (bsh) guidelines for diagnosis and treatment were published in february , there is little evidence for clinical practice in the united kingdom. aims: to investigate the approach to the diagnosis, investigation and management of patients with autoimmune haemolytic anaemia (aiha) in english nhs trusts. methods: we designed and distributed a survey to the clinical transfusion leads at all english nhs trusts between november and march . the survey requested information on detailed, simulated clinical scenarios. the first simulated scenario described a young patient with active aiha months after an allogeneic stem cell transplant, who has received multiple transfusions in the last weeks and is hypotensive, tachycardic, with a falling haemoglobin (hb), currently g/l. the second scenario describes a young man with a new diagnosis of warm aiha who has an initial hb of g/l and returns to clinic at a -week interval with symptoms of fatigue. he is actively haemolysing and commenced on mg/kg prednisolone. results: there was a % ( / ) response rate by trusts. faced with a - h delay for allo-adsorption studies, % ( / ) of respondents would instead transfuse acutely with abo, rh and k matched red cells negative for any previously detected alloantibodies, % ( / ) would transfuse with o rh d negative red cells and % ( / ) would wait for completion of allo-adsorption studies before transfusing. in this first scenario, a quarter of respondents appeared to delay a potentially lifesaving blood transfusion. british society of haematology guidelines recommend that when anaemia is life-threatening in the time required for full compatibility testing, abo, rh and k matched red cells should be transfused. in the serious hazards of transfusion (shot) report, the most serious and fatal of cases of preventable delayed transfusion was a patient with aiha who died untransfused with an hb of g/l, while awaiting alloadsorption studies. a key shot message was that if clinical harm to patients from withholding blood outweighs safety concerns over a possible delayed haemolytic transfusion reaction, emergency blood is essential and should be offered. the second scenario also identified considerable variation in transfusion practice. it can take several weeks for patients with aiha to respond to prednisolone so a transfusion threshold < g/l after an hb fall of at least g/l in the previous weeks is perhaps overly conservative. summary/conclusions: the overall findings support a need for studies to explore barriers to uptake of guidelines, and to identify areas for further audit and research to guide safe and appropriate transfusion practice in aiha. background: balance between supply and demand of o d negative red cells remains a challenge for almost every blood service. with this re-audit, we wanted to collect objective and comprehensive information regarding usage of o d negative red cells supplied by nhs blood and transplant (nhsbt) to private and nhs hospitals in england. aims: the aim was to understand hospital practices, actual needs and possible avoidable usage of o d negative red cells. where possible, comparisons were made with two previous audits ( ) ( ) ( ) . methods: participating hospitals were asked to determine the fate of all group o d negative red cells they received between th and th may excluding substitutions and complete an organisational survey regarding activities, policies and stockholding practices with respect to o d negative blood. participating hospitals were asked to provide (if available) the prevalence (as a percentage) of o d negative patients in their population. this information, in conjunctions with hospital activities, will be used to estimate appropriate o d negative stockholding levels. background: o rhd-negative (neg) red blood cells (rbcs) are a precious resource, are often in short supply and transfusion of these units in emergency settings carries the potential risk of transfusion-related adverse outcomes such as haemolytic reaction due to minor blood group incompatibility. as such, their use should be closely monitored within health services. most recent australian guidelines ( ) for their use in emergency settings include pre-menopausal females of unknown blood group (mandatory indication) or while the blood group is being established; use should be limited to or less units where possible before a switch to group-specific rbcs (acceptable indication). aims: audit of use of emergency uncrossmatched o rhd-neg rbcs against national guidelines in our institution (an australian tertiary metropolitan public hospital providing acute medical and surgical, emergency and critical care services). methods: use of emergency uncrossmatched o rhd-neg rbcs units over a six-year period was retrospectively reviewed. we collected information about rbcs transfused and discarded, adverse outcomes, patient characteristics, clinical indications and whether use met national guidelines or could have been avoided. results: episodes of emergency uncrossmatched o rhd-neg rbcs were identified, encompassing transfusion of rbc units to patients and the discard of rbcs (due to incorrect transport). of the episodes, episodes ( %) involved an eventual switch to group-specific rbcs (range of emergency units, - units). the main requester was the emergency department ( %). the most common clinical indication for transfusion was acute gastrointestinal bleeding ( %). of the episodes, episodes ( %) did not meet the guidelines for emergency use because > units of emergency uncrossmatched o rhd-neg rbcs were issued. episodes ( %) were flagged as potentially inappropriate as the patients were clinically stable according to documentation in the medical records. episodes ( %) were identified as potentially preventable due to delay in pre-transfusion sample collection (defined as > h elapsed between patient arrival and group and screen sample collection) in the setting of acute bleeding ( %), receipt of an unsuitable pretransfusion sample requiring sample recollection ( %), delay in pre-transfusion sample processing ( %), no valid pre-transfusion sample being available at the time of the bleeding episode despite having a planned elective procedure or being an inpatient with recent clinical bleeding ( %). only one patient was investigated for potential transfusion-related adverse outcome ( %) which was thought likely due to concurrent sepsis. summary/conclusions: over six years, episodes utilising emergency uncrossmatched o rhd-neg rbcs were identified with rbcs issued and rbcs discarded. a significant proportion of episodes ( %) were potentially avoidable if there had been a valid pre-transfusion sample available in the transfusion laboratory at the time of the episode. efforts to minimise use of this precious resource are ongoing, and include feedback to clinical units regarding importance of valid pretransfusion samples prior to applicable invasive procedures and in bleeding patients, ongoing education to medical and nursing staff, and continuing audit of use of this blood component in the hospital haemovigilance programme. abstract withdrawn. abstract withdrawn. background: platelet transfusions are often given prophylactically to thrombocytopenic hematology patients. to which extent platelet function improves after transfusion, and how this improvement correlates with an increase in platelet count, is not well studied. flow cytometry has been used to evaluate platelet function after transfusion in a few studies and can be performed even at low platelet counts. rotational thromboelastometry (rotem) represents a more physiological measure of platelet function in whole blood that has not been extensively used in transfusion settings. we used these methods to investigate if platelet transfusion improves platelet function in hematology patients and if improvement correlates with increased platelet counts. aims: the aim was to evaluate the relationship between response to platelet transfusion, measured as corrected count increments (cci), and platelet function in thrombocytopenic patients with hematological disorders. methods: blood samples (sodium-citrate anticoagulated) were collected from unselected hematology patients receiving prophylactic platelet transfusions, after informed consent had been obtained. samples were taken at three time-points: within h before transfusion, h after and - h after transfusion (via a central venous catheter or a subcutaneous venous port). for each time-point, platelet response to adenosine diphosphate (adp) and thrombin receptor-activating peptide (trap- ) was assessed by flow cytometry by measuring p-selectin and pac- expression on single platelets. rotem analysis was also performed on all samples, using intem and extem reagents. results: an interim analysis was performed after inclusion of patients. the mean platelet count before transfusion was /l (range - /l). h cci was /l and - h cci was /l, but response was highly variable. pselectin expression after stimulation with adp and trap was significantly higher at h after and - h after transfusion compared to before transfusion (p < . ). pac- expression after stimulation with adp was significantly higher at - h after transfusion (p < . ), but not at h after transfusion. in rotem, clot amplitude at and min (a and a ) as well as maximum clot firmness (mcf) improved after transfusion (p < . ). a significant correlation between absolute platelet count and p-selectin expression after trap and adp stimulation was found (r s = . and . respectively, p < . ). absolute platelet count was also significantly correlated with mcf (r s = . , p < . ), where % of patients with a platelet count of more than /l reached mcf values within the reference interval. summary/conclusions: platelet function generally improves after transfusion and was in our patient population correlated to the absolute platelet count, but was also seen at the single platelet level in flow cytometry. a post transfusion platelet count of more than /l might be sufficient to significantly improve coagulation in heavily thrombocytopenic patients, but larger studies are needed to confirm this conclusion. abstract withdrawn. abstract withdrawn. background: sickle cell disease (scd) is a genetic disorder that is frequently referred to as a hypercoagulable state. hydroxyurea (hu) is known to decrease the frequency of vaso-occlusive complications and need for blood transfusions in severely affected individuals. although cross-sectional studies show that treatment with hu is associated with decreased coagulation activation, there are no prospective studies evaluating the effect of hu on coagulation activation. aims: to assess the effect of hu on markers of fibrinolysis (d-dimer) and endothelial activation (soluble vascular cell adhesion molecule- [soluble vcam- ]) in patients with scd in their non-crisis, "steady state." methods: patients, at least years of age, with documented hbss or hbsb-thalassemia, eligible for treatment with hu were studied in this prospective, observational study. laboratory investigations were obtained at baseline, prior to commencement of therapy with hu, with repeat evaluations at three and six months of therapy. non-parametric test was applied to observe the association between hu therapy and the biomarkers of interest. results: twenty-five patients with scd (hbss: , hbsb thalassemia: ) were enrolled (females: [ %]), with a median age of years (iqr: ). following months of hu, median values for wbc count ( . /l vs. . /l, p = . ) and d-dimer ( . ng/ml vs. . ng/ml, p = . ) were significantly lower than baseline values, while the mean corpuscular volume ( . fl vs. . fl, p = . ) was significantly higher than the baseline value. no significant differences from baseline were observed in the median values for hemoglobin ( . g/ dl vs. . g/dl, p = . ), platelet count ( /l vs. . /l, p = . ), lactate dehydrogenase ( u/l vs. . u/l, p = . ) or soluble vcam- ( . ng/ ml vs. . ng/ml, p = . ) following months of hu therapy. summary/conclusions: this exploratory study confirms that treatment with hu is associated with decreased coagulation activation in patients with scd, although no effect on endothelial activation was observed. by decreasing coagulation activation, hu may decrease the risk of thrombotic complications in scd. abstract withdrawn. abstract withdrawn. transfusion medicine, apollo gleneagles hospitals, kolkata, india background: reduction of immune responsiveness through blood transfusion has been documented by previous authors. breast cancer is considered as one of the commonest cancer globally and the second main cause of death in females transfusion of allogeneic blood in breast cancer surgery is variable and differences of transfusion incidence have been observed in the literature. where the maximum surgical blood ordering schedule (msbos) dictates cross matching and reservation of blood before surgery, factors deciding their utilization are varied and numerous. our hospital protocol guides that every patient planned for elective breast cancer surgery should routinely have a blood sample sent for reservation of one unit of compatible packed red blood cell (prbc) in the blood bank. aims: in this prospective study we aimed to audit the blood utilization in patients undergoing elective breast surgery and thereby optimize the blood ordering schedule, economic burden and loss of clinical resources. methods: the study included confirmed breast cancer patients planned for elective breast surgeries from january to december . patient and disease details like age, stage, tnm status, estrogen receptor (er) and progesterone receptor (pr) status, human epidermal growth factor receptor (her - ) expression, triple negative breast cancer (tnbc) status, reproductive and treatment status were documented. patients were divided into younger group [≤ years] and older group (> years). before surgery blood samples for compatibility testing were sent to blood bank for blood reservation. details of test, blood issue and blood transfusion were documented in the blood bank. approximate loss of time in minutes and wastage of resources in terms of money (inr) in the blood bank were noted. all results were calculated as mean ae sd and a 'p' value of < . was considered statistically significant. results: of the total patients most underwent wide local excision of the breast and modified radical mastectomy. a total of patients received units of blood and blood components in all categories of surgeries. only were younger women (≤ years) with mean age of years. non-transfused patients were significantly more than transfused ones (p < . ). frequency of blood transfusion was more in young patients ( . %). seven ( . %) of the total stage iv patients received blood transfusions. frequency of blood transfusion was more in patients undergoing surgery after chemotherapy ( . %). a significant loss of time and loss of revenue was observed. summary/conclusions: we conclude that routine compatibility test is not justified for all patients undergoing breast surgery. a more targeted approach is needed to reduce blood demand and associated cost to patient and blood transfusion services. background: blood transfusion guidelines are not only essential for the optimal use of blood products, but also help reduce transfusion-related adverse reactions and improve patient outcomes. the korean national transfusion guidelines were developed in and fully revised in by the korean centers for disease control and prevention and the korean society of blood transfusion. in our hospital, which is a -bed university hospital, a transfusion-indication data-entry program based on the national transfusion guidelines was developed in . it was applied to the electronic medical record system and all transfusion orders, except emergencies, have been performed through this program since then. aims: we planned to record and analyze the reasons for transfusion in order to monitor blood product usage and provide feedback to clinicians. furthermore, we intended to contribute to patient safety through the appropriate use of blood products. methods: we classified transfusion-indications by the blood product requested and created a pop-up window listing these indications, which would appear at each regular transfusion order. indications for transfusion with each blood product were as follows: red blood cells (rbcs)acute blood loss, chronic disease (sub-classified as hb ≤ g/dl, cardiovascular disease, cerebrovascular disease, peripheral vascular disease, respiratory disease, age ≥ years, age ≤ months, chemotherapy), surgery/ procedure, transplantation and 'other'; platelets (plts)present bleeding, bleeding prevention (sub-classified as hematologic disease, solid tumor, peripheral blood stem cell transplantation, disseminated intravascular coagulopathy, infant), surgery/procedure, massive transfusion and 'other'; fresh frozen plasma (ffp)bleeding in coagulopathy, bleeding prevention in coagulopathy, massive transfusion, plasma exchange and 'other'. transfusion indications entered into the data-entry program from sep to feb were analyzed. results: the number of transfusion-indications analyzed was for rbcs, for plts and for ffps. the most common indications for transfusion were chronic disease for rbcs ( / , . %), bleeding prevention for plts ( / , . %) and 'other' for ffp ( / , . %). 'hb ≤ g/dl' was the most frequent sub-indication of chronic disease ( / , . %), and hematologic disease was the most frequent sub-indication of bleeding prevention ( / , . %). many clinicians entered transfusion indication as 'other': rbcs ( / , . %), plts ( / , . %) and ffp ( / , . %). however, the free-text supplied by the clinician when 'other' was selected, often corresponded to an indication already categorized in the transfusion-indication data-entry program; . % of rbcs and % of plts. of the indications entered as 'other' in ffp, . % were surgery/procedure-related. summary/conclusions: in our hospital, the release of blood products has been dependent on the data-entry of transfusion indications (except in emergencies) since sep . transfusions of rbcs and plts were most common for chronic disease and bleeding prevention, respectively, but many cases entered as 'other' could have been categorized as existing indications in our data-entry program. therefore, we conclude that additional training is needed for clinicians regarding the determination of transfusion-indications and correct use of the transfusion-indication dataentry program, in order to use blood products more appropriately. methods: this was a prospective cohort designed study. subjects were children aged - years with indication of platelet transfusions in sardjito hospital yogyakarta indonesia. the patient samples were collected before and h post-transfusion, the expression of cd p on platelet was determined by flow cytometry method. results: there were subjects who were divided into two groups. fifty-one subjects received non-leukodepleted pcs and the other fifty-one transfused by pre-storage leukodepleted pcs. the mean of pre-transfusion platelet cd p for nonleukodepleted and leukodepleted groups were . % and . %, and the mean increase of post-transfusion platelet cd p for non-leukodepleted was . % and the mean decrease of leukodepleted groups was . %. it was shown the increase of post-transfusion platelet cd p for non-leukodepleted group, and it was significantly (p < . ) higher than in the leukodepleted groups. summary/conclusions: there was an increase of post-transfusion platelet cd p expression in patients received non-leukodepleted, but a decrease in leukodepleted pc transfusions. background: preoperative anaemia is a common finding in patients undergoing surgery and often neglected in our country. aims: the objective of this study was to evaluate hb(values and the identification of cardiac patients who entered operation with anaemia. and also to study the correlation between hb values and the number of rbc (red blood cell) transfused unit methods: this is a retrospective, descriptive and analytical study. the data for this study was collected from the files in the statistic's service at qsut (university hospital center "mother teresa"). the object of our study were the files of patients hospitalized in the period january -may in the cardiac surgery ward, which were subjected to cardiac surgery. from the files were collected data on age, gender, primary diagnosis, accompanying diseases. we also collected hb, rbc, htc (hematocrit), mcv (mean corpuscular volume), mch (mean corpuscular hemoglobin), mchc (mean corpuscular hemoglobin concentration). from the transfusion service at qsut and from the files were pulled out the transfused patients and the number of transfused units. results: based on the who definition for anemia (females < g/dl and males < g/dl), from the patients included in the study, ( %) were anaemic. from males in the study, ( %) of them were anaemic based on hb lab values, whereas from women in the study anaemic were found to be ( %) of them. from the anaemic patients in the study, ( . %) of them with mild anaemia, ( . %) with moderate anaemia and ( . %) with severe anaemia. in the total of anaemic female . % are under , while . % are over/or years old. in the total of anaemic males, % are under , while % are over/or years old. it is noticed that most of them are with normochromic normocytic . %, normocytic hypochromic anaemia . %, hyperchromic microcytic anaemia . %, macrocytic normochromic anaemia and macrocytic hypochromic anaemia respectively . % and microcytic normochromic anaemia . %. the average value of preoperative hb decreased from . g/dl before surgery to . g/dl after surgery, so there is a decrease of approximately . g/dl of hb value. in our patients, % ( ) were transfused and the remaining % ( ) were not transfused. from transfused patients ( %) patients were anaemic. the correlation between the values of hb, rbc, htc and the number of transfusions shows that with the decrease of these values the number of transfused units increases. summary/conclusions: the diagnose of anaemia is underestimated before surgical intervention in our country and investigation of hb low values do not take the proper importance to find probable cause and correct it before surgical intervention. the lower the hb values, the greater the chance to be transfused and the number of rbc transfused units. failure to correct hb values before surgery results in unnecessary transfusions for the patients or which could have been avoided, eliminating also the risk of transfusion complications. background: alloimmunization after red blood cell transfusion is affected by various factors. it is known that the incidence of alloimmunization increases in certain diseases. extended red blood cell matching can be used to prevent the development of alloimmunization in diseases which the rate of alloimmunization is increased. in asia, extended red blood cell matching is not actively implemented. aims: we tried to investigate whether there is a difference in the disease categories between unexpected red blood cell antibody positive and negative groups. methods: from january, to december, , the diseases of the patients who had undergone unexpected red blood cell antibody identification test at dong-a university hospital was examined through medical records. from january to december , the diagnosis was made on patients who had two or more unexpected antibody screening tests. we analyzed the frequency difference of disease category between two groups. results: a total of patients were performed with unexpected antibody identification tests. of patients who underwent more than screening tests, ( . %) were positive. were consistently unexpected antibody negative. the patients with solid tumors (n = , . %) and those with hematologic diseases (n = , . %) had a higher incidence in unexpected antibody positive group. the patients with myeloid malignancy had a significantly higher frequency than lymphoid malignancy (p = . ). the frequency of patients with liver cirrhosis was significantly higher in the unexpected antibody positive group ( / , . %) than in the negative group ( / , . %) (p = . ). the incidence of non-hodgkin lymphoma was significantly higher in the unexpected antibody negative group ( / , . %) than in the positive group ( / , . %) (p = . ). summary/conclusions: there was a difference in the distribution of diseases between unexpected antibody positive group and negative group. the patients with liver cirrhosis were more frequent in unexpected antibody positive group, suggesting that extended red blood cell matching would be considered. background: in hematological patients with multiple platelet transfusions (pc) often develop immune response to human leukocyte associated antigens (hla-i) and human platelet-specific associated antigens (hpa). besides, platelet associated immunoglobulins (paig) and complement components (pac) are found on platelet. this leads to increased platelet destruction and development of refractoriness to transfusions of donors' platelets. transfusion therapy using an individual selection of platelets and plasmapheresis, contribute, in the majority of cases, to the realization of efficient transfusion by pc. but, in difficult cases, there is a need to use intravenous immunoglobulin, which may promote the efficient transfusion of pc. aims: evaluate the algorithms of using the complex therapy of refractoriness to transfusions of donors' platelets with additional application of intravenous immunoglobulin (ivig). methods: in there were three female patients in the clinics of the centre for observation, age between and years (me = ) with the ineffectiveness of complex therapy for overcoming refractoriness to transfusions of donors' platelets due to selection and plasmapheresis. the diagnoses were as follows: aplastic anaemia (aa)- , acute myeloid leukemia (aml)- . individual selection of platelets was carried out by the adhesion method on the solid phase (immucor "galileo neo"). paig and pac / were evaluated by the method of flow cytometry (bd facscanto ii) by the method of double staining with cd a. the density of fixed paig, pac was © the authors vox sanguinis © international society of blood transfusion vox sanguinis ( ) (suppl. ), - evaluated by the median fluorescence intensity (mfi). the two patients with aa received ivig-igg therapy in the standard dose , g/kg per day, for days. one patient with aml received ivig-iggam therapy in the standard dose , ml/kg/day for . results: under pressure of the complex therapy with the use of ivig in the standard dose there was are decrease in mfi over time in the case of two patients: aa- mfi-paigg reduced from to ; while the patient with aml: paiga reduced from to , and paigm from to , pac from to , pac from to . the patient with aa- over time, regardless of the treatment, there was an increase of mfi, but the effect of pc transfusions was achieved under pressure of complex therapy. under pressure of complex therapy all the patients also reduced the frequency of reaction of alloantibodies when resorting to an individual selection and increasing the frequency of compatible couples "donor-recipient". summary/conclusions: delivery of complex therapy and the additional application of ivig enables an adequate transfusion therapy of pc, neutralize hemorrhagic syndrome and continue the treatment of the main disease. detection and monitoring of paig/pac during the development of refractoriness to transfusions of donors' platelets are additional markers for prescription of ivig therapy. anaesthesia, tan tong seng hospital, singapore, singapore background: blood transfusion is quite prevalent in paediatric cardiac surgical procedures. we hypothesized that the routine use of rotational thromboelastography (rotem) to guide transfusion decisions would reduce the overall proportion of patients receiving transfusions in paediatric cardiac surgery aims: the aim of the study was to find if the use of blood and blood products in pediatric cardiac surgical cases in a single centre is affected due to rotem. methods: sixty paediatric cardiac surgical patients undergoing cpb were included in this study. thirty patients (study group) were prospectively included and compared with thirty procedure and age-matched control patients (control group). in the study group, rotem, performed during cpb guided intraoperative transfusions. perioperative transfusions of blood and blood products, postoperative blood loss and hemoglobin levels were compared between the two groups. results: the patients in the control group received fewer transfusions of packed cells ( % vs %) and fresh frozen plasma ( % vs % p<o.oo ). more patients in the study group received platelets ( % vs %) and cryoprecipitate ( % vs %). the postoperative blood loss was lower in study group compared to the control group. the postoperative hemoglobin did not differ between the two groups. summary/conclusions: the results suggest that the routine use of rotem in paediatric cardiac surgery to guide transfusions is associated with reduced proportions of patients receiving transfusions and is evidence based practice. abstract withdrawn. abstract withdrawn. abstract withdrawn. background: mycoplasma pneumonia (mp) infection has often been implicated in respiratory tract infections. although this agent has also been associated with other non-respiratory diseases, hemolytic anemia is the most common hematologic complication of mp infection. episodes of sudden onset of severe anemia are rarely seen in beta thalassemia. we report a -years-old female with beta thalassemia major who was diagnosed to have severe anemia secondary to autoimmune hemolytic anemia induced from mp infection aims: to report a case of patient with beta thalassemia major who was diagnosed to have severe anemia secondary to autoimmune hemolytic anemia induced from mp infection and how it was managed with minimal transfusion. methods: a -year-old female case of beta thalassemia major who developed autoimmune hemolytic anemia following mycoplasma infection. the patient is on monthly blood transfusion since the age of years at dubai thalassemia center. she was admitted to the hospital with days history of dizziness, severe low back and joint pain and her travel history indicated a four-week visit to pakistan. initial investigations showed a hemoglobin level of . g/dl the direct and indirect coombs test were positive. patient was transfused with two units leuko-depleted compatible prbc (o, rh +, negative for e and kell antigens.) and discharged next day. three weeks later, the patient was still with lower limbs pain and the hemoglobin was . g/dl with a positive dat with both igg+ and c + , patient was not transfused as it was difficult to find matching blood; therefore she was given one week follow up. four days later, patient reported to the center with fever, generalized body pain and fatigue. she had significant drop in hemoglobin . g/dl, mcv . , platelets count , crp . , retic count . , blood culture no growth, total bilirubin . , malaria parasite smear was negative, urine culture and microscopy were normal. mycoplasma pneumonia antibody showed positive with igg . and igm . , she was started on azithromycin for days and received units prbc least incompatible leuko-depleted (o, rh +, negative for e and kell antigens.). after which her hemoglobin increased to . g/dl. she did not show any improvement in her condition over the following weeks and repeat mycoplasma pneumonia antibody showed an increased titer, which denoted resistance to azithromycin; hence, we started her with second line of treatment levofloxacin for days. screening for collagen disease were all negative apart from high esr of mm/hr. patient remained afebrile and transfused with least incompatible available blood, post transfusion hb was . g/dl. three weeks later, during her routine follow up, she was doing well with esr dropping to mm/hr and hemoglobin . g/dl, which indicated gradual settling of hemolysis, although the last dat remained positive. subsequent visits showed hb drop of . g/dl/week which is acceptable for transfusion dependant thalassemia. results: with conservative management patient condition improved. summary/conclusions: hemolytic anemia associated with mycoplasma pneumonia is usually self-limited and resolves spontaneously after mycoplasma infection elimination. packed red cell transfusion in transfusion dependent patient may be troublesome and can aggravate the condition; therefore it should be utilized and limited to significant symptomatic anaemia background: early plasma transfusion in women with severe postpartum hemorrhage is often instituted to prevent and treat coagulopathy, but whether maternal outcomes improve with this treatment is unclear. aims: to quantify the association of plasma transfusion within the first min after diagnosing persistent postpartum hemorrhage and adverse maternal outcome. methods: we performed a retrospective cohort study of women with persistent postpartum hemorrhage, defined as postpartum hemorrhage refractory to first-line measures to control bleeding. women were enrolled from january , to january , in dutch hospitals. in a time-dependent propensity score-matched analysis, women with transfusion of fresh frozen plasma within min following the diagnosis of persistent postpartum hemorrhage were matched to women without plasma transfusion or plasma transfusion at a later time point during hemorrhage. plasma transfusion was not guided by coagulation tests. the main outcome measure was adverse maternal outcome, defined as a composite of mortality, hysterectomy and arterial embolization to control bleeding. we performed sensitivity analyses with initiation of plasma transfusion within and within min following diagnosis persistent postpartum hemorrhage, compared with other plasma transfusion strategies. results results: the cohort consisted of women with persistent postpartum hemorrhage, with uterine atony as cause of hemorrhage in women ( . %). in this cohort, seven maternal deaths ( . %), hysterectomies ( . %) and arterial embolizations ( . %) were observed. plasma transfusion within min in women was matched on time-dependent propensity scores with women with no or later plasma transfusion. rate of adverse maternal outcome was similar between women with and without early plasma transfusion, . % versus . %, adjusted odds ratio . ( % confidence interval . - . ). results of sensitivity analyses were comparable to the primary results. summary/conclusions: among women with persistent postpartum hemorrhage, initiation of plasma transfusion within min following this diagnosis was not associated with improved maternal outcomes, compared with no or later plasma transfusion. whether other transfusion strategies or identification of women with high risk of coagulopathy will improve maternal outcomes remains to be studied. background: preoperative evaluation of increased risk for perioperative bleeding is essential for cardiac surgery patients. platelet aggregation, as a laboratory test for assessment of platelet function, is very efficient for optimal antiplatelet treatment and also for assessment of the increased risk of postoperative bleeding and perioperative application of allogenic red blood cell transfusions. aims: the aim of this study was to establish whether preoperative platelet function testing for the presence of residual effect of acetylsalicylic acid (asa) on platelet aggregation can be correlated with an increased risk for intraoperative bleeding during cardiac interventions. methods: a retrospective study included patients who underwent surgical procedures (cabg ii and cabg iii) at the department of cardiovascular surgery, clinical center in ni s in period november -july . patients were previously taken asa at a dose of mg per day, but therapy was discontinued days before surgery. platelet aggregation was measured on the day of surgery using impedance aggregometer multiplate (multiplate platelet function analyzer, roche), using trap and aspi tests. patients were divided into two groups: control group (cg), which included patients with preserved function of platelets (aspi - au*min), and examined group (eg), which included patients with inhibited platelet aggregation in aspi test (< au* min background: hemophilia b is an x-chromosome-linked inherited bleeding disorder resulting from reduced levels or an absence of factor ix clinically represented by recurrent prolonged bleeding. mutations can be inherited or acquired de novo. de novo mutations arise in about one-third of patients with haemophilia. the disease affects primarily males, but approximately % of carrier females have factor ix clotting activity lower than % and are at risk for bleeding. aims: case report of a hemophilia b carrier. methods: the clinical history of the patient was thoroughly studied by analyzing medical records and analytical results using the software available in the health institution and paper records. results: year old woman, with no history of abnormal haemorrhage, healthy, up to when, after a dystocic delivery, presents with subcutaneous and ocular haemorrhage and dispersed bruises. a basic coagulation study is performed and a level of % fix is found. genetic analysis reveals a mutation in fix gene and she is diagnosed as a carrier of hemophilia b. except for a son, no other direct family members carry the mutation. in , after total hysterectomy with bilateral adnexectomy due to an invasive carcinoma in the uterine cervix, the patient suffers a haemorrhagic shock with multiple transfusion support and around six months later undergoes an ureteronephrectomy also with the need for post-surgery transfusion support. the patient is referred to the immunohemotherapy department where she has regular appointments as an outpatient since . in , she requires surgery for the removal of a thyroglossal duct cyst, which is performed under prophylactic treatment with iu recombinant fix (rfix) daily, for four days, and up to h after discharge maintaining basal levels of fix of % and with no major complications reported. in , the patient is admitted to the er in haemodynamic shock and a history of black stools and syncope. at admittance, she receives a bolus of iu of rfix and requires severe transfusion support due to active bleeding. she is diagnosed with colonic angiodysplasia and undergoes surgery. daily doses of iu rfix are administered from day (d ) and up to d maintaining fix levels around - %. the patient is operated at d under the cover of iu of rfix, twice per day, and up to d after surgery reaching fix levels of around - %. at d the dose was diminished to iu of rfix daily and at d she is discharged with no rfix replacement therapy necessary in ambulatory. since then, no complications or new occurrences have been reported. the patient is under prophylactic administration of rfix prior to minor invasive procedures (nodule biopsy, dental extractions and colonoscopies). summary/conclusions: hemophilia b carriers usually do not require any major health care for their daily routine or invasive procedures. however, in the case reported, basal fix levels conditioned any minor intervention to be performed. thus, this carrier must be considered as a moderate hemophilia b patient and mandatory prophylaxis with rfix must be performed prior to any invasive procedure. background: blood transfusion is a life saving procedure but transfusion associated complications are equally important that need to be addressed. with limited resources of blood donations and screening, excessive transfusions must be avoided to prevent risk and economic burden on patients. on the other hand discussion on appropriate use of blood product is a matter of debate in medical literature. it is recommended to restrict the liberal use of transfusion. aims: in this regard we aimed to observe the frequency of genuine and non genuine transfusion at our hospital and also to compare the outcomes of liberal and restrictive transfusions. methods: it was a cross sectional study carried out at nibd and bmt, pechs campus, karachi, pakistan, from february to january . the study was approved from institutional review board. pack red cells (prc) transfusion in case of < g/dl hemoglobin (hb) with cardiac history and < g/dl hb with symptomatic anemia was considered genuine. platelets transfusion in case of sepsis and count < /l, sepsis with bleeding and < /l, patients on active chemotherapy without bleeding but platelet count < /l and in patients presented with bleeding was considered genuine. patients who were presented with bleeding, deranged coagulation profile and with sepsis + bleeding considered genuine for fresh frozen plasma (ffp) transfusion. spss version . was used for descriptive and inferential statistics. results: a total of transfusions were evaluated for genuine and non genuine transfusion. male predominance was high in our data ( %). out of , ( %), ( %) and ( %) prcs, platelets and ffp were transfused respectively. out of platelets, ( %) were platelet apheresis. / ( %) prcs and / ( %) platelets were non genuinely transfused as per the criteria described in methods. however, ffp and platelet apheresis were genuinely transfused. in non genuine group of platelet transfusion, all patients received more than unit platelet and the increment of platelet count post transfusion was found statistically non significant (p = . ). thirty six ( %) of transfusions were done with more than units of prcs in non genuine group. the mean hb level of patients received unit and more than units prcs was statistically same (p = . ) and it was found out that there was same increment in hb in both the groups post transfusion. (p = . ). summary/conclusions: we concluded that although blood transfusion is an integral procedure for saving life yet correct use of blood products is necessary in the era where blood donations are scarce and blood transfusion has its potential associated hazards. the approach towards the appropriate use of blood products will not only minimize the risk of transmission of infectious diseases but also will reduce the economic burden. longitudinal studies on large sample size are needed to validate this study. uncertain. an ovine model enables comparisons of; (a) invasive and non-invasive methods to measure oxygen delivery and utilisation at a tissue level, and of (b) the efficacy of various resuscitation fluids and transfusion protocols in treating haemorrhagic shock. aims: to develop an ovine model of haemorrhagic shock. methods: sheep (n = ) were anaesthetised, ventilated and instrumented. instrumentation included blood flow, perfusion, and microdialysis probes placed in the brain, kidney, liver and skeletal muscle. haemodynamic, tissue oxygenation, tissue flow and respiratory data were recorded continuously by data capture software. non-invasive assessments, including brain and muscle oxygen saturation by nirs, and visualisation of sub-lingual microvascular perfusion, as well as arterial blood gas measurements and plasma/serum/urine collections were made at specific timepoints. after a baseline period ( h) sheep were bled~ % of their estimated blood volume (estimated at ml/kg) to a mean arterial blood pressure (map) of - mmhg. sheep were then resuscitated with either plasmalyte â (up to ml/kg/ hr; n = ) or transfused with sheep red cells (n = ) to achieve a map > mmhg. sheep were euthanised h after resuscitation. data are presented as mean ae standard deviation. results: sheep were haemorrhaged an average of . ae . ml blood which combined with iatrogenic blood loss (~ ml) corresponded to an average . ae . % blood loss. two out of the four sheep met clinical criteria for haemorrhagic shock (map = - mmhg, lactate > mm, svo < %). across all four sheep the nadir map averaged . ae . mmhg, lactate peaked at . ae mmol/ l, and nadir svo was . ae . %. all sheep survived to the end of the experimental protocol. summary/conclusions: these data demonstrate the successful induction of haemorrhagic shock in an ovine model. further experiments are planned to improve the protocol and to achieve % incidence of haemorrhagic shock, and then to compare invasive and non-invasive measures of oxygen delivery and utilisation as well as the efficacy of different resuscitation fluids and red cell transfusion. adverse events, including trali p- bilirubin were recorded within the -day period. the clinical parameters were compared against the reaction strength of the antibody reactions. the automated strength was measured by solid phase. the manual testing consisted of a -min incubation using liss and adding monospecific igg. the dat was performed manually by adding poly-specific igg and then testing with monospecific igg and c d. the rh group and non-rh group had and cases performed manually, and results were + or weaker further indicating the manual strength did not correlate with the clinical hemolysis. likewise, in / ( %) the dat was negative, and did not show any correlation with clinical hemolysis. however, when ldh and bilirubin were measured, the two parameters increased as the automated strength of the antibodies increased. summary/conclusions: most of the dshtr investigation was not associated with overt accelerated red cell destruction. a strong correlation was observed only between the automated immunohematology testing results and other laboratory markers of hemolysis. in our experience, the direct antiglobulin test and manual strength showed no correlation. background: numerous transfused patients present severe, sometimes critical clinical conditions. the occurrence of adverse transfusion reactions (atr) may induce deterioration in the clinical condition with a worsened clinical course and a lifethreatening or fatal outcome as is the case with nervous system impairment. in france, in , out of , notified atrs, ( . %) and ( . %) were life-threatening and death respectively. aims: our aim was to evaluate the notified atrs with neurological signs that occurred in transfused patients over a period of six years and six months in hospitals in the auvergne rhône alpes area. the study included patients with reported atrs in hospitals in this area from january st to june th . each atr was registered in the national haemovigilance database system. two signs observed at the time of the atr were analyzed: unconsciousness and convulsions. stroke was excluded. the type of atr, its severity, the blood product involved and its imputability were studied. results: during the period under study, , atr were reported, of which included unconsciousness and/or convulsions ( . %). of these patients, were females ( . %) and males ( . %). unconsciousness alone was frequently observed ( reports, . %). convulsions were notified in reports ( . %) and were associated with unconsciousness in of them. the diagnosis of seizure, with no other clinical signs, was established in cases ( . %). unconsciousness and/or convulsions were present in allergic reactions ( . %), cases of transfusion-associated circulating overload ( . %), cases of suspected transfusion-transmitted bacterial infections and hypertensive reactions. in allergic atrs, unconsciousness was notified in cases and unconsciousness associated with convulsions in one. twelve atrs were severe ( . %), were life-threatening ( . %) and in cases, they resulted in the death of the recipient ( . %). of the allergic atrs, were severe and life-threatening. red blood cell concentrate was involved in atrs ( . %) and platelet concentrate in ( . %), including cases with apheresis platelet concentrate and cases with pooled platelet concentrate. fresh frozen plasma was involved in atrs ( . %). nevertheless, the imputability of the blood product was excluded or unlikely in atrs ( . %). in the suspected transfusion-transmitted bacterial infections, the imputability of the transfusion was ultimately excluded after a negative result was obtained in the bacterial culture of the blood product. the imputability of the blood product was probable or possible in and atrs respectively, but was certain in only atrs. summary/conclusions: unconsciousness and/or convulsions were rarely observed in atrs notified in transfused patients. nevertheless, the presence of these signs highlights the seriousness of the atr ( ars, . %). lastly, the imputability of the blood product was often excluded or unlikely. in the multivariate cox model for the effect of lpi on overall survival, adjusted for age and ipss-r category, elevated lpi levels were associated with inferior overall survival (hr . , % ci . - . , p = . ). this effect was most pronounced in the td-rs subgroup (hr . , % ci . - . , p < . ). similarly, elevated lpi levels were associated with inferior pfs (hr . , % ci . - . , p < . ) for the whole study population and the td-rs subgroup (hr . , % ci . - . , p < . ). in total patients received iron chelation during the sample collection period ( patients deferasirox, patients desferrioxamine). lpi levels were normal in out of the samples collected during deferasirox treatment and in out of samples collected during desferrioxamine treatment. summary/conclusions: transfusion dependency is associated with the presence of toxic iron species and inferior overall and progression-free survival in lower-risk mds patients. in td-rs patients the effects were most pronounced indicating ineffective erythropoiesis leading to additional iron toxicity. background: post-transfusion immunomodulation has been reported to contribute to poor patient outcomes. clinically relevant transfusion models are needed to improve our understanding of underlying mechanisms. sheep transfusion models are of increasing importance in blood transfusion research as they provide several advantages over small animals, including their size, anatomy, physiology and similar blood volume compared to human. a current limitation of sheep transfusion models is the lack of characterisation of the sheep immune system. understanding the sheep immunology is necessary to advance sheep transfusion models, identify mechanisms that contribute to post-transfusion immunomodulation and facilitate the translation of findings into clinical settings. aims: to characterise the sheep leukocyte inflammatory responses to in vitro lipopolysaccharide (lps) challenge in edta and heparinized whole blood. methods: edta and heparinized sheep whole blood (n = of each) was cultured with rpmi media ( °c, % co ) alone or with the addition of lps ( - lg/ml; derived from escherichia coli : b ). the inflammatory response was assessed after h (h), h, h, h, h and h. supernatant was harvested at each time point and stored at À °c. inflammatory cytokine/chemokine production was determined using sheep specific in-house elisa (il- b, il- , il- and il- ). twoway analysis of variance with bonferroni's post-test was used to measure the effect of incubation time and concentration compared to no lps matched samples. results: when edta was used as an anticoagulant, addition of lps resulted in production of sheep il- b and il- but not il- or il- . il- b production was significantly increased following stimulation of lg/ml lps for h (p = . ) and declined following h incubation. release of il- was significant h post-lps stimulation with lg/ml (p = . ) and reached a maximum at h. the use of heparinized blood resulted in a different immune profile as all inflammatory markers tested were detected following stimulation with much lower concentrations of lps ( lg/ml), although the incubation times differed. il- b was significantly increased following h incubation (p = . ), with increasing levels observed up to h post-lps stimulation. il- production was evident from h and reached significance at h post-lps stimulation (p = . ). il- was significantly increased following stimulation of lg/ml lps for hr (p = . ) with lower concentrations of lps resulting in il- production at h (p = . ). release of il- was significant after h of lg/ml lps stimulation (p = . ), with lower concentrations of lps resulting in il- production at h (p = . ). in heparinized whole blood an lps concentration-dependent effect was evident for all cytokines. summary/conclusions: using a time-and concentration-approach our findings indicate that sheep are more tolerant and have a delayed response to lps stimulation compared to previous research using similar human in vitro whole blood culture models. in addition, data suggest that sheep have greater immune responses using heparin as anticoagulant for the collection of blood samples. improving our understanding of sheep immunology and development of relevant sheep transfusion models will provide a bridge between sheep models of transfusion and clinical settings. . rhdig inappropriately administered (unnecessary exposure) (n = , %) administered to: -rhd positive woman (n = ) -rhd negative mother with rhd negative neonate (n = ) -woman with immune anti d (n = ) -administered in error (instead of other ig) (n = ) rhdig delayed/omitted/wrong dose (risk of sensitisation to the d antigen) (n = , %) -omitted (n = ) -delayed (n = ) -inadequate dose (n = ) administration without correct patient identification (n = , %) storage & handling (n = , %) failure to check the maternal and neonatal blood groups prior to administration was identified as a source of error. misinterpretation of blood results also led to women receiving product inappropriately. e.g. reading a negative antibody screen as the mother being rhd negative. patient identification was raised as an issue. rhdig is often stored in satellite blood fridges for easy access. collection from these areas did not always require confirmation against patient identifiers and there was no register of women who received product or link to the batch number to ensure traceability. two incidents involved the administration of rhdig when the prescription for other immunoglobulin products was not clear, leading to a child and a baby receiving rhdig instead of the intended immunoglobulin. summary/conclusions: these incidents indicate problems with the processes of appropriate identification of women who need rhdig, the use and interpretation of pathology tests and requirements for prescription and administration. these resulted in omitted and inappropriate doses of rhdig. blood matters has made a number of recommendations regarding rhdig administration: -all health professionals involved in rhdig administration should be appropriately trained in the use of rhdig -confirmation of the maternal rhd status is essential prior to prescription or administration -positive patient identification must be used prior to administration of rhdig -health services should consider regular auditing to identify areas for improvement relating to rhdig blood matters continues to work with maternity care providers to improve practice. centro comunitario de sangre y tejidos de asturias, oviedo agencia gallega de sangre, organos y tejidos, galicia banco de sangre y tejidos de cantabria, cantabria banco de sangre de la rioja, la rioja banco de sangre y tejidos de navarra, navarra banco de sangre y tejidos de arag on, aragon fundaci on de hemoterapia y hemodonaci on de castilla y le on, castilla y leon fundaci on banco de sangre y tejidos de las islas baleares, islas baleares, spain terumo bct europe nv, zaventem, belgium background: hemovigilance, a long-term monitoring process made mandatory by national and supranational regulations, begins with a systematic whole blood or blood component collection and ends with an examining period after transfusion of blood components into the patients. in spain, organized in autonomous regions, the hemovigilance system is structured in three levels: ( ) the local level comprised of transfusion centers and hospital based transfusion services that monitor and collect all transfusion related adverse events (ae) and level them up to ( ) the regional hemovigilance coordinator, who communicates all the region's data to the ( ) spanish ministry of health which issues an annual report and corresponds with european institutions. to ensure safer blood supply, pathogen reduction technology (prt) was approved and implementation started in spain in . the mirasol prt system for platelets and plasma was introduced in and is currently being used in of the spanish regions. aims: to monitor the safety of the system, a passive hemovigilance study on mirasol treated products was initiated in the region of asturias and collaboration was extended to other regions (baleares, galicia, la rioja, cantabria, navarra, castilla y leon and aragon). methods: collected data included allergic and febrile reactions, trali and all other adverse event observed. severity of the event and level on imputability of the transfusion were also assessed using the who grading scale. hemovigilance data of mirasol treated products (platelets or m-pc and plasma or m-p) are included from to as blood centers started to apply the technology in routine. results: increase adoption of the mirasol system is observed between , when , mirasol treated blood products were issued to hospitals and with , mirasol products issued. due to low number of transfusions of mirasol-treated blood components in and , notification rates began to be analyzed in , showing ae rates of . %, similar to reports at the national level. stable transfusion reaction rates were observed with m-pc (around . ). rate of ae after transfusion of m-p is fluctuant between . and . . this fluctuation could be due to the inconsistent numbers of m-p transfused from one year to the other. most of transfusion reactions (around %) were of grade i severity and grade ii level of imputability. allergic reactions accounted for most of the adverse events, with g&i > reactions in and of respectively . and . no bacterial nor viral transfusion transmission was recorded on mirasol products during the study period ( ) ( ) ( ) ( ) ( ) ( ) . at the national level, nine cases of bacterial transfusion transmission (with g&i > ) were reported. these transmissions were probably due to transfusion of non-pathogen reduced products. summary/conclusions: the observed notification rate of ae is similar to the national rate but allergic reactions with g&i > is inferior with mirasol treated products. also, we found no reports of transfusion transmission infections nor cases of transfusion associated graft-vs-host disease, demonstrating safety of mirasol treated products. were attributed to human error ( %) with the lowest frequency in equipment failure ( %), compared to % and %, respectively, in the following three years. root cause analysis demonstrated failures in the quality management system including failures in administration, inadequate staffing for blood collection as well as in distribution and processing, and failures arising from institutional constraints and system failures in hospital management. high numbers of "other" aes ( %) in distribution and whole blood collection call for further investigation to indicate measures necessary for prevention and correction. errors related to incorrect blood component transfused (ibct) in - were in , , blood units ( / , ) issued for transfusion. these resulted in serious reactions ( %) ( fatal, life-threatening) . another ( %) were related with ibct that did not cause a reaction. near misses (component not transfused) were ( %) summary/conclusions: our data demonstrate increasing compliance with reporting requirements. questions about the initial factors for deviations in certain activities specifying failures in equipment and materials due to system as well as human errors, highlight the need for further specifications beyond "other" and "human error". background: the weakest link in the transfusion chain currently is the handling of blood components after their issue and the bedside blood administration practices. aims: to evaluate compliance with standard procedures for bedside blood transfusion practices by analysis of the "transfusion feedback forms" in a tertiary care multi-specialty hospital setting. methods: during the study period of months, the transfusion feedback forms received from various clinical areas of the hospital were studied with special reference to the transfusion times. the data was categorized based on the patient's location as well as the time of transfusion, whether done in routine or emergency hours. results: , blood components were issued during the study period, while transfusion feedback forms for , components ( . %) were received in the transfusion medicine department. delay in starting the transfusion (more than min after issue) was observed in transfusion events ( . %). the component transfusion time exceeded the permissible limits for component ( . %).the overall total permissible time for completion of components exceeded permissible limit in ( . %) of transfusion events. the pediatric ward ( . %), icu and ot complex ( . %) were found to be the most non-compliant delay in transfusion, transfusion time and total transfusion time. amongst the delayed transfusions after issue, ( . %) were during the routine hours i.e. between am to pm and ( . %) were in the non routine hours i.e. between pm to am. summary/conclusions: the audit of bedside blood transfusion practices has given us a good insight into various areas of noncompliances as well as the predominant locations in the hospital where the practices need to strengthened further. focused training program on safe blood administration practices for all staff involved in handling and transfusion of blood components is now planned to combat this issue. background: the international surveillance of transfusion adverse reactions (ars) and events (aes) (istare) of the international haemovigilance network (ihn) collects aggregate data from member national haemovigilance systems (hvs) in order to estimate the morbidity and mortality of blood transfusion in a holistic approach. the ultimate goal is to contribute to improving the safety of transfusion by close monitoring throughout the chain "from vein to vein". aims: we analyse recent istare data on suspected transfusion transmitted infections (sttis) for - in comparison to previous years of surveillance, [ ] [ ] [ ] [ ] [ ] [ ] [ ] methods: annual aggregate data from ihn member hvs on transfusion associated bacterial, viral and parasitic infections collected online in istare are analyzed by incidence in blood components (bcs) issued for transfusion, by severity and imputability as well as by blood component. ars with definite, probable or possible imputability were included in the analysis. trend analysis is performed to allow comparisons and to collect information on established and newly emerging infectious threats of blood transfusion. results: for - sets of annual aggregated data from countries covering , , bcs issued were analyzed. all ars totalled , and infectious ars amounted to ( . %). the overall incidence of the infectious ars was . / , units of bcs issued. bacterial infections were the most frequent ( , %), next viral ( , . %) and then parasitic ( , . %). serious were % and there were fatalities ( . %, incidence . / , ). nine deaths were attributed to sepsis and the other two were associated with non-malarial parasitic pathogens. one geotrichum clavatum fungal infection associated with apheresis platelets was reported as a free text comment. this very rarely recognized fungal pathogen caused a very severe infection in a patient but the route of transmission is inconclusive. the viral sttis included hbv ( %), hcv ( %) and hiv ( . %). the recorded as "other" ( . %) including cases of hev, one case of parvovirus b , one cmv and one ebv. no case of tt-malaria was reported. other stt-pi were (two fatal). the prevailing bcs were in general rbcs followed by platelets. comparison with corresponding data for - shows a consistent overall incidence in total sttis ( . vs . / , ). however, considerable differences were seen in separate categories, such as bacterial infections (significantly increased rate in - , p < . ) and an almost doubled rate of parasitic infections (p < . ). compared to the earlier period, there were many fewer hbv infections ( vs ) and many more hev. a similar reduction in the rate of hcv and hiv was observed in - in comparison with previous years. this may be explained by the fact that nat testing for hcv/hiv/hbv has been implemented in many countries in the last decade. summary/conclusions: the infectious risk of transfusion overall remains very low. the rate of bacterial cases has increased and among other viral sttis the frequency of hev is increasing. the mortality of transfusion due to sttis is lower than in the previous period of surveillance. abstract withdrawn. background: one of the main aspects of haemovigilance system in hospitals is following of adverse events and reactions related to blood transfusions. aims: it was intended to analyse the adverse reactions related to transfusion of blood components in pediatric patients. methods: over a four year period (january -december ), the haemovigilance records of all patients receiving blood transfusions procedures were reviewed and transfusion reactions were analysed. statistical analysis of data was performed by spss software (version . , spss inc., chicago, il, usa). majority of blood components were provided by regional blood center organized by national red crescent society. but granulocytes collected by apheresis after donor mobilization and reconstituted whole blood for exchange transfusions were prepared in the transfusion center of the hospital. results: the median age of patients who developed transfusion reactions was months (interquantile range-iqr ). the median for the numbers of individual transfusions in children in a year was (iqr ). the median for the numbers of blood components individually transfused to patients was (iqr ). patients, anaphylactic transfusion reactions in patients and transfusion-related lung injury (trali) in a patient. the overall incidence of transfusion reactions was estimated at a rate of . per units. summary/conclusions: it was reported that adverse effects related to blood transfusion, especially allergic reactions and fnhtrs are common in pediatric patients than adults. in a multinational study concerned about the transfusion reactions related with red cell concentrates, allergic transfusion reactions and fnhtrs were reported at a rate of and in units and in units, respectively. while the incidence of transfusion reactions in children was found . % in a study from the u.s.a., the overall incidence of transfusion reactions in our study which was estimated at a rate of . per units represents a lower rate. hospital gran canaria dr. negr ın, gran canaria hospital general universitario, ciudad real, spain hospital nostra senior de meritxell, andorra, andorra banco sangre y tejidos, santander banc de sang i teixits, barcelona, spain fundaci on hematol ogica colombia, bogot a, colombia centro regional de transfusi on de almer ıa, almeria complejo hospitalario de navarra, pamplona fundaci on banc de sang i teixits illes balears, palma de mallorca hospital de cabueñes, gij on, spain background: root analysis cause is defined as the cause of an error that, if it is treated, eliminates the repetition of the error aims: describe types of human and latent errors detected by a work group in the root analysis cause of transfusion incidents, analyze the concordance between the individual responses of the members and propose recommendations in order to improve transfusional safety. methods: in fifteen participants (nurses and hematologists dedicated to transfusion and component preparation) studied some incidents of administration of nonirradiated components and tried to approach the root causes by applying the classification of errors in mers-tm transfusion medicine. they transferred the answers to a questionnaire (simple or chain error, initial process affected, human and latent errors and measures derived from the analysis to correct the errors). the communication was made by mail and by the spanish transfusion society web forum, which contained the consultation documents. data and percentages are exposed for each type of error and the answers of the participants are tabulated. results: cases corresponded to patients. two patients of years of age diagnosed of acute myeloblastic leukemia (case and ) and chronic lymphatic leukemia (case ). in one case, the hematologist of the transfusion service canceled an irradiation prescription; in another, a patient with fever was transfused in the emergency room without the irradiation requirement and it was later discovered that he had received a transplant of hemopoietic progenitors month earlier; in the last case, neither the requesting doctor nor the laboratory technician nor the following doctor (prescriber) detected the alerts located in their respective computer applications. in all cases, the story was judged as sufficient for analysis. the majority of reviewers ( %) diagnosed a chain of errors. there was agreement of % with respect to the initial process affected. the initial error was communication ( %), monitoring ( %) and compliance ( %), in cases , , and . - human errors were detected per case (average: . , . and . errors respectively) and - latent errors per case (average: . , . and . , respectively). the latent errors most punctuated were: failures in the quality of the protocols ( %), in the transfer of important knowledge ( %), in the available technology ( %) and in the information to the patient ( %). all the participants contributed feasible measures of improvement according to root causes: ) improve the quality and drafting of work procedures and their compliance, including procedures of effective communication between professionals, ) train staff in knowledge important for safety, ) communicate with computer application providers to improve the effectiveness and visibility of the alerts and ) involve the patient with essential information to ensure transfusion safety. the measures were processed later as recommendations. summary/conclusions: the root analysis shows agreement between participants and allows the elaboration of useful recommendations to increase patient safety. this strategy can contribute to the comprehensive prevention of errors. background: in transfusion-associated circulatory overload (taco), pulmonary oedema develops primarily due to volume excess. data from the uk haemovigilance scheme, serious hazards of transfusion (shot) suggest that either the incidence of taco, or the recognition and reporting of taco, has increased over time. from to , reports of taco increased from to ; deaths from to , major morbidity from to . known risk factors include pre-existing cardiac and/or renal dysfunction, low body weight, extremes of age (eg, < years, > years), concomitant fluid administration, positive fluid balance, peripheral oedema and hypoalbuminemia. in a small subset of cases reported to shot, taco developed following transfusion for severe anaemia in the absence of other risk factors. this may be an under-recognised independent risk-factor. aims: to raise awareness of severe anaemia as an under-recognised risk factor for taco and is potentially life-threatening transfusion. methods: cases of taco submitted to shot over the last years were reviewed to identify cases where transfusion for severe anaemia was a key identifiable patient risk factor. results: the following are illustrative cases: -case : a patient in their s weighing kg was prescribed six units of red cells for iron deficiency anaemia after being admitted with hb g/l. the patient had no risk factors for taco except for profound anaemia. during transfusion of the fifth unit the patient became dyspnoeic, hypoxic and hypertensive. the patient recovered after diuretic therapy and had a post-transfusion hb level of g/l. -case : a patient in their s presented with a -week history of weakness and dizziness and had felt unwell for months. the hb was g/l, ferritin lg/l and ecg showed cardiac ischaemia. two units of red cells were transfused. after the second unit oxygen saturations fell despite supplemental oxygen, post-transfusion hb of g/l. a third unit was transfused over min and the hypoxia worsened with dyspnoea and crackles on chest auscultation. the chest x-ray showed an enlarged cardiac silhouette and pulmonary congestion. the patient improved with diuretics. -case : a patient in their s with severe megaloblastic anaemia, hb g/l and peripheral oedema developed taco after transfusion of units and recovered with diuretic therapy. summary/conclusions: chronic and acute anaemia are associated with compensatory cardiac changes irrespective of the aetiology of anaemia. this is further compounded by the underlying cause of anaemia particularly haematinic deficiencies (iron/b deficiency) that independently affect myocardial function. hyperdynamic circulation related to anaemia increases the load on the heart, causing myocardial ischaemia and hypoxia and if the anaemia is not corrected, eventually leads to heart failure. clinicians need to make an accurate diagnosis and avoid excessive transfusion in patients with severe anaemia with or without other additional risk factors. patients with chronic iron/folate/b deficiency without haemodynamic instability should be given the appropriate haematinic replacement. haematinic deficiency responds rapidly to appropriate vitamin/mineral. blood transfusions are to be given only when clinically indicated and even then, only the minimum volume needed for symptomatic relief transfused with consideration for diuretic therapy. methods: legal forms for reporting transfusion reactions were used in the retrospective analysis, which were adjusted by the department of quality assurance and quality control in the electronic form and distributed to clinics using blood components. clinicians were trained to report transfusion reactions through the hospital's transfusion board and through the "service for improvement of the quality and safety of health services" at the clinical center of sarajevo. analysis of the reported reactions in the institute include immunohematological and microbiological examination based on which the guidelines for further treatment with blood components are being made. users of registered services are obliged to report since . results: total of , different blood components were applied in the period between - . department for quality assurance and control has received serious adverse reactions, serious adverse event, reports of transfusion reactions, of which ( %) were inadequately filled, in the same period. from the above, ( . %) were transfusion reactions to erythrocyte blood components which were applied, ( . %) to platelet components and ( . %) were transfusion reactions after the application of fresh frozen plasma. the analysis has shown that the most frequent were febrile non-haemolysis reactions ( or . %), followed by allergic reactions ( or . %). two transfusion reactions ( . %) were characterized as circulation overload. summary/conclusions: the frequency of serious adverse reactions and events was . % ( of , ) and . % were reported transfusion reactions ( of , ). with the establishment of the hospital transfusion board and with the increase of collaboration with the clinical center, significant progress has been made. it is necessary to increase awareness among clinicians in regards to the safe transfusion practice. reporting transfusion reactions should be a mandatory procedure, a path to the proper selection of blood components, monitoring adverse reactions, and for us, transfusiologist, guide to the safest, most efficient blood components. j garc ıa-gala, e martinez-revuelta, a caro-g omez, c castañ on-fern andez and i fern andez-rodriguez hospital universitario central de asturias, oviedo, spain background: elderly patients are the main group of transfusion recipients in our country. given their comorbidities are a risk group for the development complications related to transfusion. aims: analyze the incidence of adverse effects related to transfusion in the elderly population and to assess what factors may influence its appearance methods: transfusions were reviewed in patients > years old. the variables analyzed were: sex, age, diagnosis/reason for transfusion, pre-transfusion hemoglobin (hb), number of transfused units, infusion rate and transfusion side effects, as well as the measures used to prevent or treat the transfusions. effects of circulatory overload results: a total of patients were analyzed ( women, men), with a median age of years ( - ). in total, ch were transfused. patients received ch, patients ch, patients received ch. patients were transfused at two different times. the median hb prior to transfusion was . g/dl. in the patients who received ch was . g/dl, those who received ch: g/dl and those who received ch: . g/dl. the infusion time could be estimated in % of the patients. in those who received ch was . min; . min in those who received ch and . min in those who received ch. patients ( % of the total) suffered some type of adverse effect related to the transfusion. in patients there was an increase in posterior ta, in an increase in hr, in an episode of hypotension and in another one episode of acute respiratory failure. % of those who had an adverse effect were older than years. patients with aht after transfusion, % received ch and the remainder ch. among their background, % had a history of ischemic heart disease. % also had a positive balance. the average previous bp was / mmhg and the subsequent one was / mmhg. % of patients did not receive diuretic treatment. in the case of the patient with acute respiratory failure was in oligoanuria, with positive balances. ch was transfused in total. she was treated with oxygen therapy and with intensification of the diuretic treatment, recovering later. summary/conclusions: -patients > years have a higher risk of suffering some type of adverse effect related to transfusion because they have pre-existing risk factors such as ischemic heart disease or heart failure. -we see that the risk of suffering some type of adverse effect in the elderly population is greater when we transfuse ch than ch. -we have appreciated that in those patients receiving ch, the infusion rate is higher. -the study highlights the lack of methods to prevent the development of circulatory overload. background: iron deficiency anemia is the commonest cause of anemia worldwide. weakness, fatigue, reduced physical activity and difficulty in concentration are the symptoms which are associated with its deficiency. the forefront treatment available is oral iron replacement therapy which is convenient, cost effective and has substantial outcome. another option is intravenous (i/v) iron when oral is not tolerable. despite of potential transfusion associated hazards and limited availability of blood due to shortage of voluntary blood donations, it is insisted by the patients prior to iron therapy. aims: the aim of conducting this study was to observe the impact of administration of oral iron, i/v iron and transfusion on hemoglobin levels in patients presented with iron deficiency anemia. methods: this was an observational study carried out at nibd and bmt, pechs campus, karachi, pakistan from february to december . the study was approved by the institutional review board. diagnosed ida patients presented at our hospital were recruited for analysis who were given oral iron, i/v iron and transfusion for the correction of anemia. informed consent was taken from the participants. descriptive and inferential statistics was applied by using spss version . . results: a total of ida patients were analyzed in which ( %) were females and ( %) were males. the most common symptom in females and males was fatigue followed by body aches in females ( %) and pallor in males ( %). menorrhagia was present in ( %) of females of reproductive age. surgical history was present in ( %) of females while there was no surgical history in males. mean hemoglobin, mch and mcv of females at baseline was . ae . , . ae . , and ae . while in males it was . ae . , ae . and . ae . respectively. sixty two ( %) females were advised oral and i/v iron and ( %) received transfusion. however, in males ( %) received transfusion and ( %) were advised oral and i/v iron therapy. it was observed that the increment of hemoglobin after oral/iv iron at average of months follow up in males and females was same as that the transfusion (p > . ). summary/conclusions: in our society where blood donations are scarce especially voluntary blood donations that are considered to be the safest type of blood donation. we would like to draw attention towards the alternatives to correct anemia such as oral and i/v iron replacement therapy as our results revealed that there was no difference in the increment of hemoglobin between the two groups. we need to educate our society especially the older age adults and young women who are more vulnerable of getting ida to opt oral and i/v iron therapy. it will be cost effective, convenient and also has less risk than transfusion. cellular therapies -stem cell and tissue banking, including cord blood background: the differentiation of megakaryocytes plays an important role in the production of platelets. however, the underlying mechanisms regulating megakaryocytes differentiation have rarely been studied. aims: to identify candidate genes involved in megakaryocytes differentiation and investigate the potential regulatory mechanisms of megakaryocytes differentiation from human cord blood hematopoietic stem cells in vitro. methods: cb-derived cd + cells were isolated using density gradient centrifugation and magnetic activated cell sorting (macs). cultures were stimulated with only recombinant human tpo ( ng/ml). after , and days, the mk fraction was selected by immunomagnetic sorting from the non-mk fraction using an anti-cd a monoclonal antibody. rna-seq-derived gene expression data was performed on uncultured samples (day ), cultured but unselected samples (day ), and cultured, selected samples (day , and ) by using the next-generation sequencing (ngs) platform, and rq-pcr technology was used to verify the expression of transcription factors. results: the comparison of the transcriptome profiles among the five stages showed that a massive gene expression change occurred in megakaryocytes differentiation. a total of genes were detected, of which showed up-regulation and down-regulation. among these genes, differentially expressed genes (degs) (fold change ≥ ; false discovery rate < . ) were selected were further validated with rq-pcr, including gabre, cdhr , wasf , pkhd l , thbs , pf v , lrrc and lgals . the rq-pcr result indicated that the mrna expression level increased with the prolongation of culture time. however, pf v mrna expression level was highest at day , lgals was highest at day . summary/conclusions: conclusion: our study identified a series of genes that may participate in the regulation of megakaryocytes differentiation. these results should serve as an important resource revealing the molecular basis of megakaryocytes differentiation and thrombocytopoiesis. preoperative anemia and blood transfusion requirement during hip and knee surgery rambam health care campus, haifa, israel background: blood transfusion (bt) is independently associated with increased morbidity, mortality and hospitalization length across different patient populations. due to bt-related risks, the concept of "patient blood management" (pbm) has been introduced to clinical practice. the three pbm pillars are: optimizing red cell mass, minimizing blood loss and optimizing physiological reserve. bt indications during orthopedic surgery include excessive bleeding or hemodynamic instability and not the hemoglobin (hb) level. in most clinical scenarios, a restrictive transfusion threshold (hb level: - g/dl) appears to be non-inferior to the liberal transfusion strategy in terms of blood use, morbidity and mortality. similar results are observed in highrisk patients after hip surgery. we hypothesize that preoperative anemia may lead to blood product overuse and its complications. aims: evaluating potential correlation between preoperative anemia and bt requirement during hip or knee surgery. methods: we reviewed medical files of patients who underwent hip or knee replacement surgery at rambam between - . patients with hb level measurement within days pre-surgery were included. receiving > blood unit was considered a surgery complication and such patients were excluded. patient demographic and clinical data, including comorbidities, surgery type, length of hospital stay, were collected. we created a synthetic data cohort using mdclone healthcare data sandbox, an environment enabling fast data extraction and producing synthetic data for analysis that does not require irb approval. results: during the evaluated period, patients underwent hip or knee surgery; were excluded from the analysis due to receiving > blood unit. hb measurement within days pre-surgery was available for patients. hip or knee surgery was performed in ( %) and ( %) patients, respectively. women comprised % (n = ) of patients who underwent hip surgery. in the hip-surgery group, . % of patients required bt, with this need being slightly higher among women ( . % vs. . %; p-value=ns). only ( %) patients were transfused during knee surgery and this cohort was not further analyzed. patients receiving bt had a significantly lower mean hb level than those who didn't require it ( . g/dl versus . g/dl for women and . g/dl vs. . g/dl for men; p-value < . ). hospitalization was longer in transfused patients compared to non-transfused ones (mean . vs. . days, p-value = . ) and in patients with a low hb level (female < , male < . ) than in those with a high hb level, irrespective of receiving bt (p-values < . ). patients with at least one of the following diagnoses: diabetes, renal failure, ischemic heart disease, were significantly more likely to have a lower preoperative hb level (p-value < . ). no other factors (e.g., patient's weight, rdw value or blood pressure) were predictive of transfusion need. the probability of a need for blood unit was . in the hb g/dl group and . in hb g/ dl group ( %>reduction). summary/conclusions: anemia presence before elective hip surgery is a risk factor for bt requirement and longer hospitalization. diagnosis and management of anemia using timely pre-surgery consultations may minimize intraoperative bt, particularly in women and patients with comorbidities. real-patient data and prospective trials are warranted. abstract withdrawn. abstract withdrawn. background: cd , known as platelet glycoprotein iv, belongs to type b scavenger receptor and is related to the pathogenesis of many diseases. type i cd deficiency was cd not expressed on platelets and monocytes. individuals with type i deficiency can produce homologous antibodies and cause related immune diseases. in recent years, it has been reported that cd deficient individuals cause fetal immune thrombocytopenia with fetal edema syndrome in asia. cd is not expressed in mature rbc, but exists in hematopoietic stem (progenitor) cells. anemia is an important cause of edema. in view of the phenomena of clinical and animal experiments, cd + hematopoietic stem (progenitor) cells were cultured in vitro to observe the effect of anti-cd monoclonal antibody on cd + hematopoietic stem (progenitor) cells. aims: to investigate the effect of anti-cd monoclonal antibody on proliferation and differentiation of human cd + hematopoietic stem (progenitor) cells in vitro. methods: choose healthy full-term maternal women without various obstetric complications, take cord blood ml. after density gradient centrifugation of ficoll cell separation solution, cd + hematopoietic stem (progenitor) cells were sorted by flow cytometry and cultured for - generations. mtt was used to examine the effect of anti-cd monoclonal antibody on the growth of hematopoietic stem (progenitor) cells. flow cytometry analysis was used to detect the apoptosis and cell cycle of cd + hematopoietic stem (progenitor) cells. the effect of anti-cd monoclonal antibody on the formation of cfu-e/bfu-e in hematopoietic stem (progenitor) cells was analysis by cfu-e/bfu-e account after - days culture. results: after umbilical cord blood was isolated by ficoll to obtain mononuclear cells, the hematopoietic stem (progenitor) cells of cd + were sorted by flow cytometry, and about . % of cd + hematopoietic stem (progenitor) cells were isolated. different concentrations of anti-cd monoclonal antibody and hematopoietic stem (progenitor) cells were cultured in vitro. the od value of value ( . ae . ) of anti-cd monoclonal antibody group ( mg/ml) was decreased than normal group ( . ae . ) (p < . ), and the od value ( . ae . ) was significantly decreased at the cd monoclonal antibody concentration of mg/ml (p < . ). there was no significant difference between the hematopoietic stem (progenitor) cells culture group and the igg control group (p > . ). in the annexin v flow detection, the apoptotic rate of anti-cd monoclonal antibody group ( mg/ml) was statistically increased than the normal group (p < . ). anti-cd monoclonal antibody significantly induced hematopoietic stem (progenitor) cells to undergo s phase cell reduction, g phase cells increased, and g /s phase cell arrest occurred. the number of cfu-e/bfu-e clones in the normal group was ae , the number of cfu-e/bfu-e clones in the control group was ae , and the number of cfu-e/bfu-e clones in the anti-cd monoclonal antibody culture group was ae . the number of colonies formed by hematopoietic stem (progenitor) cells in the anti-cd monoclonal antibody culture group was significantly lower than that of the other groups (p < . ). summary/conclusions: anti-cd monoclonal antibody can reduce the proliferation of human cd + hematopoietic stem (progenitor) cells and reduce the ability of erythroid differentiation. background: recently the new modern collection techniques were introduced in the apheresis procedures. cobe spectra system was replaced with spectra optia, and it was necessary to verify the efficiency of spectra optia in pbpc collections. aims: the aim of the study was to evaluate and optimize the new cmnc protocol spectra optia v. (terumo) for pbpc collections in patients with haemato-oncological malignant diseases. methods: the results of autologous pbpc collections were evaluated in: (a) well mobilized patients with precollection cd + cells concentration in blood higher than /ll, (b) from only the first collections, which were performed either by the use cmnc spectra optia v. or cobe spectra v. , v. , terumo (c) collections were performed in the standard and large volume leukapheresis regimen, lvl. engraftment data in transplanted patients were assessed. results: standard collections were performed in patients. the yield of cd + cells was high, and no significant differences were found between the numbers of cd + cells prepared from spectra optia , ( , - ) and cobe spectra , ( , ) /kg b. w. (a = , ; pval , ). the dependence of cd + cell yield on the precollection concentration of cd + cells in blood can be considered as linear with high correlation coefficients in cmnc spectra optia r = , , and cobe spectra r = , . lvl collections were performed in of patients, and there were no significant differences between the numbers of cd + cells prepared by cmnc spectra optia , ( - , ) and cobe spectra , ( , - ) /kg b.w. (a = , ; pval , ). the relations between the precollection cd + cells concentration in blood and the numbers of cd + cells from collections can also be considered as linear with the correlation coefficients in cmnc spectra optia r = , , and cobe spectra r = , , respectively. in lvl, the median platelet loss was significantly lower in cmnc spectra optia ( %) than in cobe spectra ( %). a group of patients was transplanted by means of pbpc prepared in the standard regimen. median time in the neutrophil reconstitution was in cmnc spectra optia as well as cobe spectra days, while in platelets from cmnc days, and from cobe spectra days, respectively. the number of patients obtained pbpc from lvl. the median time in neutrophils and platelets reconstitution was in cmnc spectra optia as well as cobe spectra the same, and corresponded with and days, respectively. summary/conclusions: cmnc protocol spectra optia is a modern, efficient and the safe system, which can be used for both standard and lvl procedures. in well mobilized patients the sufficient dose of cd + cells for transplantation could be prepared from one standard or one lvl procedure. no serious adverse reaction have been observed. background: peripheral hematopoietic stem cells are collected from patients/donors after mobilization with g-scf. the aim of the collection is a fixed number of cd + cells/kg. this number depends on the pre-apheresis cd + number, the blood processed and the collection efficiency of the procedure. the aim should be to collect all the requested cells in day, whenever possible. this is in order to reduce the dose of g-csf given to donor/patient and the resources used in the collection centre. the only parameter that can be adjusted is the volume of blood processed, if this is increased, the likelihood of collecting the requested amount of cells is increased, but only if the pre-apheresis cd number is high enough. therefore, you need to know, when it is feasible to increase the volume and thereby increasing the time of the procedure with the intention to collect all the requested cells in day. it can also show if it is possible to reduce the volume of blood processed, thereby reducing the time of the procedure. aims: to develop an easy tool to calculate the volume of blood processed in order to collect the requested cells in day. methods: the mean collection efficiency (ce) was calculated. ce is calculated as cd + cells collected/(pre-apheresis cd + number*processed volume)* %. based on the mean ce, an excel sheet was generated to calculate the volume of blood that should be processed in order to collect all the requested cells. the excel sheet is designed so the user enters the pre-apheresis cd + number, patient weight and the requested number of cd + cells. the ce is fixed according to the mean ce calculated. the result is the volume of blood processed in order to collect the requested yield. based on that result, the apheresis machine will provide time for the procedure, thereby it is possible to evaluate if the collection can be finished in day or not, e.g. by increasing the volume of blood processed. spectra optia â was used for all collections, cmnc for allogeneic donors and mnc for autologous patients. results: mean ce = % (n = ). a ce of % was chosen as the cut-off for the cd calculation tool. using the cd calculation tool: allogeneic donors (n = ): mean ce = %, mean blood volume processed = . tbv, mean time: min, donors were finished in day collection ( %) autologous patients (n = ): mean ce = %, mean blood volume processed = . tbv, mean time = min, patients were finished in day ( %). the calculation failed in only case ( . %). in this case the volume of blood processed was reduced according to the calculation, but because of unexpected low ce, the requested number of cells was not achieved. summary/conclusions: the cd calculation tool based on an excel sheet has shown to be simple and easy to use in order to personalize the stem cell collection. immunotherapy products: blood product, pharmaceutical, or a new category all together? from till . all donors were hla typed and matched; they were fully informed on the donation procedure and signed an informed consent for donation. minimum dose required to ensure successful and sustained engraftment was /kg cd + cells and /kg mono-nucleated cells (mnc). pbsc harvesting was performed with continuous flow cell separator baxter c , cobe spectra and spectra optia using conventional-volume apheresis processing the - . total blood volumes per apheresis. a femoral catheter was used for harvesting and acid citrate dextrose formula a is used for anticoagulation. recombinant human granulocyte colony-stimulating factor (g-csf) is used to mobilize pbpc for collection. harvesting of pbsc is usually performed after to days of g-csf subcutaneous administration at a dose of lg/kg body weight. results: all the donors were siblings of the patients treated at the university hematology hospital. there were apheresis procedures performed in healthy sibling donors. there were males and females, aged - . one to two apheresis procedures were required to collect adequate graft. the single procedure usually took - h and the volume of collected stem cells was - ml. the needed number of mnc and cd + cells was successfully collected by . apheresis. there were abo incompatible donors. procedures for mobilization and collection of pbpc from healthy donors are generally well tolerated. the only adverse effects of the apheresis procedure were bone pain as reaction of g-csf and numbness of the extremities as reaction of acd-a (hypocalcemia), which occur rarely and were very mild. the collected pbsc were used in allogeneic stem cell transplantation in patients with: acute myeloid leukemia - patients ( . %), acute lymphoblastic leukemia - patients ( . %), chronic myeloid leukemia - patients ( . %), myeloproliferative disorders - patients ( . %), myelofibrosis - patients ( . %), severe aplastic anemia - patient ( . %), non-hodgkin lymphoma - patient ( . %), multiple myeloma - patient ( . %), chronic lymphoblastic leukemia - patients ( . ), hodgkin disease - patient ( . %). summary/conclusions: the apheresis collection of pbsc in healthy donors is an effective and safe procedure. we are developing our national stem cell donors registry as a part of bone marrow donors worldwide. in that way we hope we will help widen the world network of stem cell donors and enlarge the possibility for each patient to find the right match. background: leukocyte-removing filters for blood are being used widely as a universal leukocyte reduction policy is being adopted progressively throughout the world. filtration is one of the most effective methods for preventing various adverse transfusion effects caused by leukocytes included in blood components, such as febrile reaction, alloimmunization, and transmission of leukotropic viruses. aims: the goal was to evaluate whether the new domestic blood filter finecell, developed by kolon industries, gumi, korea, is appropriately suited to the international standards. and to reveal its efficacy and safety in the settlement of leukocyte reduction system in korea. methods: thirty-two units of packed red blood cells obtained from ml whole blood collected from healthy donors were used. this was done by analyzing the filtration time, residual leukocyte count, rbc recovery, and hemolysis rate during a storage period of days after leukoreduction. results: the standards commonly used for the evaluation of leukocyte-removing filters are set by the food and drug administration of the usa and the council of europe. the results of our study satisfied these international standards. summary/conclusions: the newly developed domestic leukoreduction filter was, thus, found to be efficient and will contribute to the improvement of the quality of isolated blood components used in korea. faculty of science, humanities and education, technical university of liberec, liberec, czech republic background: as polymeric fibrous scaffold fabrication techniques strive to create structures that more closely replicate tentative extracellular matrix form and function, the need for increased scaffold bioactivity becomes more pronounced. the fibrous structure made from biocompatible and nontoxic polymers ensures mechanical stability, however cell proliferation requires further stimulation. platelet-rich plasma, which has been shown to contain over bioactive molecules, has the potential to deliver a combination of growth factors (gfs) and cytokines capable of stimulating cellular activity. aims: the presented work deals with the preparation of nanofibrous materials with platelet growth factors incorporated into the internal fiber structure. polyvinyl alcohol (pva) was used for the preparation a material providing a progressive release of native gfs without need of subsequent crosslinking. methods: materials were prepared from pva (mw , , % of hydrolysis) using electrospinning technology (nanospider tm ws u). platelet lysate (pl) was prepared from thrombocyte rich solution (obtained from regional hospital in liberec, the concentration of - x plt/ml, freeze-thaw method with subsequent centrifugation). nanofibers were electrospun from % pva solution using water: ethanol ( : ) solvent system. materials with proteins were electrospun from solution containing % of pva and % of pl. morphological analysis was performed by scanning electron microscopy. protein release was monitored using spectrophotometry (bradford method) and chromatography. results: the prepared fibrous materials consisted of random oriented end-less fiber with smooth surface with minimal defects in structure. the morphology of materials was not altered by the addition of proteins. the average fiber diameter was: ae nm for pristine pva fibers and ae nm for pva with incorporated proteins (pva/pl). pva/pl layers contain - mg of protein per gram of pva. % of the proteins are released during the first day (burst release) followed by a gradual release of up to weeks. summary/conclusions: nanofibrous pva-based nanofiber materials were prepared with native growth factors. the process used for the preparation of solutions and subsequent spinning does not affect the activity of the incorporated proteins, which are being gradually released. therefore, we believe that the developed material has great potential for use in tissue engineering e.g. to promote healing of chronic wounds. acknowledgements: supported by the czech health research council, project no nv - - . background: human a-defensins are small cationic peptides with antimicrobial and anticancer activity. up till now, six a-defensins have been described in humans. they include the human neutrophil peptides (hnp) , and which present in large amounts in neutrophil azurophilic granules and differed from each other only in the first amino acid. a fourth defensin, termed hnp- , comprises less than % of the total defensins in neutrophils and has a distinct sequence from hnp - . the other two, human defensin and , are synthesized mostly by intestinal paneth cells. neutrophil defensins (hnp - ) are . kda peptides that are characterized by three disulfide bridges. the pattern of disulfide bonds in the mature forms is crucial for the functional properties. due to this structural feature, synthesis of defensins using the chemical and recombinant approach presents quite a challenge. moreover, purification from the natural source can be very difficult because the large number of neutrophils is needed to obtain a sufficient amount of protein. in blood banks, leukocyte reduction filters are used to remove leukocytes from blood components in order to prevent adverse transfusion reactions. leukofilters contain high numbers of normal human cells and discard after use. aims: the aim of this study was to purify a-defensins from neutrophils trapped in leukocyte reduction filters. methods: blood bags from healthy blood donors were collected after written consent. all donors were screened for infectious diseases (hbv, hcv and hiv) and negative samples were included in the study. blood bags were filtered at °c by leukoflex lst- filters. the cells were extracted from the filters by back-flushing with cold phosphate buffered saline (pbs), ph . , without mgcl and cacl , containing mm edta and . % sucrose. the pbs was homogenized with the filter content and then collected in a sterile tube. neutrophils were separated from mononuclear cells by ficoll. isolated neutrophils resuspended in pbs x at a concentration of cells/ml. for degranulation, cells were stimulated with nm of formylmethionyl-leucyl-phenylalanine (fmlp) for min followed by stimulation with lm of cytochalasin b for min. supernatant was collected by centrifugation at g for min. supernatant was incubated with mouse monoclonal antibody to hnp - and purification of hnp - was performed by lmacs protein g microbeads system. the presence of protein was confirmed by western blot. results: the presence of the . kda band was confirmed by western blot, which corresponded to the size of the a-defensins. summary/conclusions: the development of defensins as therapeutic products requires access to a steady supply of neutrophils. our results indicated that lst- filters are economical source for purifying a-defensins. anatomical sciences, abadan school of medical sciences, abadan, iran background: epigenetic reprogramming of terminally differentiated cell can modify somatic cells to a pluripotential state. there are several approaches that induce pluripotency in somatic cells. exposure the cells with the embryonic stem cell extract is an easy way, and some investigations were done on fibroblast cell line. however, its efficiency was low aims: the purpose of this study was to increase the number of reprogrammed granulosa cell as a full differentiated cell into pluripotential state methods: the human granulosa cells were cultured in the medium containing -aza-deoxycytidine and trichostatin a. then, the cells were exposed to mouse escs extract and co-cultured with mouse embryonic fibroblast in the presence of leukemia inhibitory factor (lif). alkaline phosphatase test and also immunohistochemistry staining for oct , sox and nanog were performed after and h and week results: the results indicated that after h the granulosa cells were revealed a round and expanded morphology. the cells in all groups except in negative control, were showed alkaline phosphatase activity. the cells that were cultured in medium containing -aza-deoxycytidine and trichostatin a and exposed to the extract had the most numbers of alkaline phosphatase positive cells. immunocytochemistry showed the granulosa cells that were cultured in medium containing -aza-deoxycytidine and trichostatin a with extract expressed oct with weak intensity after h. no expression of oct , sox and nanog were observed in other groups at the same time. after h, oct , sox and nanog were over expressed in the cells that were treated with -aza-deoxycytidine, trichostatin a and extract. furthermore, there was high expression of oct in the granulosa cells that were cultured in medium containing dmso and exposed to the extract. after one week, the expression of oct and sox in the granulosa cells that were cultured in medium containing dmso and exposed to the extract was continued while its expression ceased in the other groups. the expression of nanog were ceased in all groups after one week summary/conclusions: present study revealed that the inhibitors of the methyl transferase ( -aza-deoxycytidine) and histone deacetylase (trichostatin a) could delete the epigenetic markers and improved cells reprogramming by administration of the extract abstract withdrawn. abstract withdrawn. abstract withdrawn. background: mesenchymal stem cells (mscs) are adherent spindle shape cells expressing different surface markers. they show special criteria including, paracrine effects, differentiation to several tissue cells, migration, immunomodulatory and regenerative potentialities. mscs are isolated from different sources and employed as therapeutic tools to treat several diseases and injuries. however, some of mscs properties including secretion of growth factors and migration ability are controversial especially during remission or in presence of tumor. interestingly, msc-derived compartment could be used as practical tools in term of diagnosis, follow up, management and monitoring of disease instead of intact mscs. exosomes are kind of extracellular vesicles (evs) characterized via their size and releasing mechanism. usually they defined as less than nm in diameter vesicles. they secreted from different cells and are also found in urine, blood, breast milk, cerebrospinal fluid and other body fluids. exosomes contain genetic material including dna, mrnas, micrornas (mirnas) and other biomolecules. mirnas are single stranded non-coding rnas transcribed from dna. immature mirnas are subjected to two known cleavages to modify to mature mirna that involve to either mrna degradation and gene expression process or cell-cell interaction and communication via secretion as the part of exosomes. aims: this study was aimed to discuss some aspects of exosomal micrornas derived from mscs in progression, diagnosis and treatment of some diseases. methods: different scientific data bases including pubmed, google scholar and scopus were used to find and review related articles. results: evs play important role either in intercellular communication related to pathological and physiological situation or intracellular communication, angiogenesis, immune system modulation and metastasis progression. mirnas could regulate expression of multiple mrnas then they play important role in different biological processes and contribute cell-cell interaction as well as influence in the progression of different disease. exosomal mirna-derived mscs are involved in cancer procession, tumor growth, angiogenesis and metastasis. they are used as diagnosis and therapeutic tools to treat different diseases such as renal failure, liver fibrosis, myocardial infarction. summary/conclusions: due to controversial aspect of using of intact mscs especially during remission or in presence of tumor, msc-derived exosome could be used as practical tools in term of diagnosis, follow up, management and monitoring of disease instead of intact mscs. aims: the aim of study try to use sybr green i based real-time pcr to identify homozygous, heterozygous, gene deletion or wild type for rhd exon , , and a. methods: for this study, we used real-time pcr with high resolution melting curve mode, and matrix mix containing sybr-green i were used for sequence specific primers of g>a and rhd exon , , . samples with rhd gene deletion homozygous/heterozygous, g>a heterozygous with rhd gene deletion and normal rhd, normal rhd homozygous/heterozygous and rhd -rhce( - )-rhd homozygous/heterozygous were enrolled in our study. all samples were screened using rhd exon genotyping, sanger sequencing and rhesus box analysis. concentration and mass of dna samples were in alleles of normal rhd/rhd gene deletion. the tm ratio of rhd exon ( °c) to internal control ( °c) were . in alleles of normal rhd/rhd -rhce( - )-rhd , . - . in alleles of rhd gene deletion/normal rhd, . - . in alleles of normal rhd and < . alleles of rhd gene deletion. the tm ratio of rhd exon ( °c) to internal control ( °c) were . in alleles of normal rhd/rhd -rhce( - )-rhd , . - . in alleles of rhd gene deletion/normal rhd, . - . in alleles of normal rhd and < . alleles of rhd gene deletion. the tm ratio of rhd exon ( °c) to internal control ( °c) were . in alleles of normal rhd/rhd -rhce( - )-rhd , . in alleles of rhd gene deletion/rhd -rhce( - )-rhd . - . in alleles of rhd gene deletion/normal rhd, . - . in alleles of normal rhd and < . alleles of rhd gene deletion. summary/conclusions: using the tm ratio of sequence specific primers to internal control is an effective way to detect the rhd gene deletion or rhd weak d types , and not detected") were tested with a method based on next generation sequencing (ngs) using the illumina miseq platform to detect a possible rhd variant not interrogated by id rhd xt. results: in total dna samples were tested in pools. fifteen ( ) pools ( samples) gave rhd deletion genotype and seventy two ( ) pools ( samples) resulted to the presence of rhd gene. the positive pools were also analyzed individually. the genotype results obtained were: rhd exon no amplification ( ), rhd exon and the genotype results obtained with id rhd xt (in pools and individually) were concordant with the results provided by the centers. hence, % accuracy was obtained using id rhd xt with dna pooled samples. the results of rh ngs for the samples with inconclusive results by id rhd xt showed rhd variants previously described: sample rhd* - inst (del), sample rhd*ivs + g (del), samples rhd*weak d type (partial d), sample rhd*weak d type (weak d), sample rhd*weak d type (weak d) and not described: sample rhd*del - (unknown) summary/conclusions: id rhd xt is a high accurate tool for genotyping the most common rhd alleles associated to weak d and d negative phenotype in up to pooled dna samples. use of rhd genotyping improve rhd typing in blood donations variant rhd alleles generate qualitative/quantitative alteration in serological expression of d antigen such as weak and partial rhd phenotypes which are clinically important in transfusion setting. population studies have shown varied distribution of the variant d alleles in caucasians, africans, east asians and indians. many countries have developed their own population-specific strategy for identifying d variants. our previous study in indian population showed absence of weak d type , , and which are commonly found in caucasians d variant individuals. instead, a novel population-specific pattern i.e.~ -kilobase duplication event, including exon , was predominantly identified in . % d variant samples. functional analyses showed that this genetic variation results in the expression of several transcripts, including a wild-type product. commercial genotyping assays available, mainly detect common d variants found in caucasians and africans, thus limiting its usefulness in india. hence, based on our findings we have designed a multiplex pcr assay specific for indian population that can be easily implemented at the laboratory level for genotyping variant rhd. aims: to characterize rhd variants using "indian-specific, rhd genotyping assay". methods: seventy samples referred to our laboratory for molecular characterization of rhd variants were included in this study. all rhd variant samples were serologically typed for results: out of the rhd variants included in this study, samples ( %) showed presence of indian specific allele i.e. exon duplication. seventeen rhd variants samples showed presence of both exon and . qmpsf analysis of these samples excluded involvement of rhd-rhce-rhd hybrids. sixty of the seventy d variant individual had r r genotype this assay thus can be used routinely in indian laboratories to identify and characterize rhd variants. - non-invasive fetal kel genotypes from allo-immunized anti-kel women were done ( positive confirmed fetuses, undetermined, positive non-confirmed, negative non-confirmed and negative confirmed). - non-invasive fetal rhc genotypes from allo-immunized anti-rh women were done non-invasive fetal rhe genotypes from allo-immunized anti-rh women were done ( positive foetuses, undetermined for , % of the allo-immunized women, the pregnancy was compatible and no specific antenatal monitoring was necessary summary/conclusions: non-invasive fetal red blood cell genotype is a powerful tool to diagnose a feto-maternal red blood cells incompatibility and allows to legitimize a costly and heavy specific antenatal monitoring s purchla-szepioła , m krzemienowska , m pelc-kłopotowska , m jurkowska , m debska , m uhrynowska and e brojer the test developed by ihtm has been offered to clinicians and pregnant women since but it is not covered by the health care system. rhd nipt is not informative for mothers with rhd variant. in such cases further analysis of the molecular background is offered to exclude from immunoprophylaxis the women with weak rhd type , and . aims: summary of a -year period of routine rhd nipt available at ihtm. methods: cffdna isolated using easymag, biomerieux from plasma of pregnant women determined with standard serology as rhd-negative (in - week of gestation) was examined for the presence of exons and of rhd and ccr by realtime pcr using lc ii (roche). maternal dna from whole blood was tested for identification of rhd variant using rbc fluogene rbc-dweak/variant (inno-train, germany) or the home-made protocol. results: in cases the rhd gene was not detected in cffdna and the administration of rhig was not recommended. in seven cases ct-values for rhd and ccr indicated a maternal d variant (d ct ccr -rhd > ); the genetic follow-up of six of them identified: rhd* w. in cases, rhd* w. and rhd* . in cases the rhd nipd results indicated that a fetus is rhd positive and rhig administration was recommended it was recommended in the remaining % of mothers. in . % cases with maternal d variant rhd nipt was not possible. however, in / such cases the test is unnecessary because follow up analysis revealed maternal rhd variant of the weak d type and in switzerland extended antigen-matching for duffy, kidd and mnsbesides rhesus and kell -is recommended for sickle cell disease (scd) patients. the ethnic diversity of red blood cell (rbc) antigen polymorphism engender that these patients are often transfused with rbcs from donors of african origin. this strategy, however, increases the likelihood of being exposed to certain low-prevalence antigens, such as rh (d w ), as these are almost exclusive to african populations. rh is encoded by several types of rhd*dv as well as by dau- . anti-rh is associated with delayed hemolytic transfusion reactions (htr) aims: here we report a specific low-prevalence antibody newly formed by the same patient, meanwhile gravida , para , causing positive crossmatches with the rbcs of two of the four selected donors. subsequently, advanced serologic and genetic workup and close international collaboration enabled optimal patient care. methods: standard serological methods were used for antibody specification (biorad, cressier, ch and in-house). crossmatches were carried out by indirect antiglobulin test at °c. molecular typing of donors' and parental blood group antigens was performed by further serological analysis (institute national de transfusion sanguine, paris) revealed an anti-rh in addition to anti-fy , anti-e and anti-jk a . genotyping of the two donors causing positive crossmatches presented heterozygosity for rhd* . which encodes rh . the newborn's phenotype was a r r k-, fy(a-b+) and most likely rh -and jk (a+b+), considering both maternal and paternal (a r r, k-k+, fy(a-b+), jk(a+b-), rh -) predicted phenotypes. the neonatal serum contained maternal anti-a , anti-rh and anti-e. the direct antiglobulin test was positive but elution only showed nonspecific reactions with papain-treated cells. latter might have been caused by anti-fy during her present pregnancy we were able to demonstrate that two positive crossmatches of two former compatible donors were caused by a new alloantibody against a low-prevalence antigen, namely anti-rh , derived from several rh + rbc transfusions during the previous pregnancy. despite this challenging blood supply we were able to support the patient with a total of ten antigen compatible and crossmatch negative rbc units from french and swiss donors until delivery with increasing age, the relative number of women in the study population raise from % in the patients younger than years to % in the patients older than years. our study showed that cardiovascular diseases were the commonest indications for warfarin use in older patients with %. only , % achieved target therapeutic range while the risk of thromboembolism and the subsequent need for proper anticoagulant therapy increases sharply with age, the bleeding risk rises as well. older patients are more sensitive to any given dose of warfarin and need a significantly lower total weekly dose. a well-informed patient provides one of the best defenses against bleeding complications. recent data demonstrate doacs advantages over warfarin, especially for older population: more predictable dosing, fewer drug interactions and reduced risk of intracranial bleeding although vast majority of fh cases are caused by mutations in ldl-r gene %- % patients do not harbor genetic cause in the known loci. patients with homozygous/severe heterozygous fh are unresponsive (ldlc above mg/dl with diet and drug therapy) and require additional extracorporeal therapy to reduce ldlc concentrations to prevent the development/progression of cad. ldl apheresis techniques remove apolipoprotein b-containing lipoproteins from blood and include heparin-induced extracorporeal ldl precipitation(help), immunoadsorption, dextran-sulfate adsorption methods: a y iraqi male visited cardiac-opd. ct coronary angiogram showed cad-dvd. he had multiple tendinous xanthomas and xanthelasmas. family history was significant for death of elder brother from coronary event at y, a sister with similar profile age y and one sister apparently normal. he was taking medical treatment for dyslipoproteinemia (ecosprin mg od, ticagrelor mg bd, rosuvastatin mg od). despite dietary and medical treatment his dyslipoproteinemia was refractory. therefore cascade-filtration was planned with evaflux a plasma fractionator. one procedure of cascade plasmapheresis was done on com.tec apheresis system (fresenius kabi, germany) separating patient's plasma as the first step and passing it through a pore sized based filter column a (evaflux, kawasumi, japan) as the second step. a total of . x plasma volume( ml) was processed. the patient was given continuous calcium infusion. the flow rate of ml/min was maintained immunoglobulins) were not assessed. summary/conclusions: the procedure successfully met the requirement of reduction of cholesterol by %. the patient became responsive to the medical treatment. follow up of the patient up to a year has been uneventful with no additional procedure requirement actions included development of major haemorrhage protocols with improved communication and required instances of delayed transfusion to be reported to the uk national haemovigilance scheme (serious hazards of transfusion, shot) methods: delayed transfusion data have been collected from . hospitals identify incidents and report them via an online database. mh may also result in avoidable or overtransfusion. reports are analysed and collated data published in the shot annual reports in july each year. results: the total number of reports of delayed transfusion has increased with time: , , in the last years. delayed transfusion in relation to mh was reported for cases - contributing to death in patients %) miscommunication was noted between clinical different teams, between emergency departments, porters and the transfusion laboratory, failure of bleeps, failure to communicate the urgency, failure to confirm the patient location. failures to follow mhp correctly occurred in / ( . %): incorrect activation including failed alerts to porters, wrong contact telephone details and wrong components in the mhp packs most transfusions included red blood cells (median, units); % of women were transfused with fresh frozen plasma (median, units) and % with platelets. mean pre-and post-transfusion hemoglobin levels were . g/dl and . g/dl, respectively, representing an increment of . g/dl per rbc unit transfused ( . g/dl in soweto and . g/dl in durban). indications for transfusion included obstetric hemorrhage ( %), chronic anemia ( %), surgery or anesthesia ( %), other ( %) and not specified ( %). transfusion for chronic anemia (vs. hemorrhage) was associated with gestation ≥ weeks (odds ratio = . , % confidence interval . - . ). surgical blood loss was a common indication in trimester ( %) that declined to % then % in trimesters and . summary/conclusions: hemorrhagic complications accompanying spontaneous abortions and ectopic pregnancies in the first and second trimesters were the most common reasons for antenatal transfusion surveillance and analysis of blood transfusion reactions represents inseparable part of hemovigilance. aims: summarization of data on reported cases of transfusion reactions. methods: analysis of serious undesirable reactions to blood products administration in the czech republic (cr) during period - . results: there were evaluated , of blood products administrations in , patients in the cr during defined three years period. announced , ( , %) transfusion reactions including severe transfusion reactions ( adjudged with grade ). the most frequent types of severe transfusion reactions: anaphylaxis , trali x, taco x, hcv x, hbc x, bacterial infection x, delayed hemolysis x. transfusion reactions incidence according to administered bp: red blood cells products: , administrations, transfusion reactions (fnhtr x, allergy x, circulatory overload x, anaphylaxis x, trali x, hbv x, hcv x) platelets: , thrombocyte administrations, including transfusion reactions (allergy x, fnhtr x, anaphylaxis x, circulatory overload x, delayed immune hemolysis x, acute circulatory overload x. granulocytes: administrations, transfusion reactions plasma: , administrations, reactions reported (allergy , fnhr , circulatory overload , anaphylaxis x, trali x, hbv x, ards x. summary/conclusions: conclusions: comprehensive analysis and data processing help to appropriate prospective setting of blood products (bp) production and hemotherapy. concrete outputs from processed data triggered undermentioned changes in many departments in the cr: . plasma for clinical uses from male blood donors, . prestorage of leucocyte reduced blood products, . production of platelets in additive solutions, . implementation of pcr testing method for blood donors screening. background: skae's basic activities include epidemiological surveillance of all adverse events (aes) associated with deviations in the collecting, testing, processing, storage and distribution of blood and blood components that may affect quality and safety near misses" and "uneventful transfusion errors" are collected to identify preventable causes. incorrect blood component transfused (ibct) events are reported following ihn instructions. results: a total of they were mainly associated with deviation in processing ( . %) and attributed to equipment failure and materials ( %) whole blood collection, materials and distribution, as a result of product defect, equipment failure, human error and other. trend analysis showed a significantly increasing (p < . ) annual rate of total aes by % ( % confidence interval - ) ) % fibrinogen-depleted phpl or ( ) % fibrinogen-depleted phpl plus heparin. internalization of fluoresceinamine-labeled heparin in stcs was investigated by flow cytometry and immunocytochemistry. all stromal cells were subjected to whole genome expression analysis (affymetrix human gene . st array) and data were analyzed using r/bioconductor and panther analysis tools. confirmative qrt-pcr was performed and protein levels of selected pathways were analyzed by a bead-based western blot system (digiwest â ). immunophenotyping, in vitro differentiation, longterm proliferation and colony forming units (cfu) assays were done for all cell types. results: in vitro exposure of heparin induced differential internalization and lysosomal accumulation in stromal cells, as well as regulation of distinct gene sets, both in a tissue-source dependent manner. affected signaling cascades were mainly involved in proliferation, cell adhesion, apoptosis, inflammation and angiogenesis. the influence of heparin on protein expression and phosphorylation of four pathways (wnt, pdgf, notch and tgfbeta) was further analyzed, revealing most alterations in bm-stcs. independent of origin and medium composition, flow cytometry analysis revealed the characteristic fibroblastoid phenotype profile (cd +/ +/ + and cd -/ -/ -/ -/hla-dr-). in addition a comparable osteogenic and adipogenic differentiation capacity was found summary/conclusions: internalization of heparin in lysosomes by stromal cells, differential gene and protein expression and phosphorylation changes were observed in a tissue-source dependent manner. however, stromal cell characteristics as immunophenotype pattern, long-term proliferation, clonogenicity and in vitro differentiation were unaffected, putatively by post-translational protein modifications. in this respect, application of porcine heparin is compatible with efficient manufacturing of stromal cell based medicinal products abo incompatibility may have no effect on the clinical outcome after allogeneic hematopoietic stem cell transplantation. however, it carries additional risks of hemolytic reactions, delayed red blood cell (rbc) engraftment and incidence of graft-versus patients were categorized according to abo compatible and mismatched groups; these were further sub-categorized into major, minor and bidirectional. direct coombs test (dct) was performed when hemolysis was suspected in the post-transplantation period along with serum lactate dehydrogenase (ldh) %) were male and ( . %) female. mean age of abo matched and mismatched groups were ( . ae . ) years. most common indications for transplant included beta thalassemia major ( . %), aplastic anemia in ( . ) and pure red cell aplasia ( . %). source of stem cell was bone marrow in and peripheral blood patients abo matched while abo mismatched group comprised of ( . %) patients with further subdivision into major (n = ), minor (n = ) and bidirectional in the post transplantation period, packed red blood cell and platelets were transfused in matched group (n = ) and (n = ) comparably(n = ) and (n = ) in mismatched group. primary and secondary graft failure in matched group was . % and . % patients while in mismatched group graft failure was observed in ( . %) patients respectively. positive dct in abo matched group in ( . %) patient, whereas ( . %) patients with major and minor abo mismatch group with raised ldh levels and deranged lfts were found. episodes of acute and chronic gvhd in abo compatible and incompatible groups were insignificant. overall survival in abo summary/conclusions: these results indicate that abo incompatibility does not seem to influence outcome of the patients undergoing allogeneic hematopoietic stem cell transplantation. careful monitoring of patients can help detect problems early and treat them efficiently, thus, reducing the number of life threatening events a picascia , c sabia , f cavalca , g nicoletti and c napoli in our routine work with one lambda sab class ii reagents, we observed non-specific reactivity with some beads bearing dr and dr in patients without sensitizing events. this pattern was not confirmed by testing same sera with screening-and pra-beads suggesting non-specific reactions. aims: here, we sought to determine if fetal bovine serum (fbs) treatment would be effective in reducing/eliminating non-specific reactivity. methods: we tested sera pre-treated with fbs from non-sensitized patients that showed the dr /dr pattern. in particular, ll of fbs was added to ll of patient serum; incubated for min at °c; centrifuged and subsequently tested in the sab assay. as controls, we treated sera from patients with documented dsa including dr /dr and patients without hla antibodies. results: dr /dr non-specific reactivity was eliminated or significantly reduced after fbs treatment. we found that patients with dr and dr dsa had no change in mfi values and additional reactivity was not observed in negative fbs treated sera transfusion medicine, national blood transfusion centre transfusion medicine, national blood transfusion center transfusion medicine, national blood transfusion centre, tirana, albania background: abo blood group, has been associated with many diseases, although the explanation for abo's blood group association and some illnesses is still unclear. aims: to find the distribution of cases by blood groups in patients with malignant pathology compared to donors in order to assess the presence of the abo blood group as an epidemiological indicator to identify populations exposed to different malignant pathologies methods: we conducted a case-control study. abo blood group and diagnosis of all patients have been studied. the control sample was collected from , healthy donors from which group a ( , %), group o ( , %), b ( , %) and group ab ( , %) resulted. the study was conducted in patients who have been transfused and submitted a request to determine the blood group at the blood bank at qsut during the period - results: among the patients, when all malignant pathologies were taken together, the highest frequency was seen in blood group a ( . %), followed by ( . %), b ( . %) and ab ( . %). group a frequency was higher and o was lower compared to controls. a high incidence of blood group a is seen in: pancreatic cancer a ( %), in gastric cancer a ( %), colorectal cancer a ( , %), breast cancer a ( %), cervical cancer a ( %) and ovarian cancer a ( %) versus a ( . %) in the control group. a high incidence of blood b is seen in multiple myeloma b ( %) and cervical cancer b ( %) versus b ( . %) in the control group. blood group ab has a high incidence in malignant lymphoma ab ( %) versus ( . %) in the control group summary/conclusions: it appears that individuals with blood groups a, b and ab are more at risk of developing malignant pathologies and individuals with blood group o are more protected. background: the high homology and opposite orientation of rh genes promote many rearrangements between them and generate a large number of rhd and rhce variants which can be inherited together. several studies have shown that those rh variants in patients with scd represent an additional risk for alloimmunization and delayed hemolytic transfusion reactions (dhtrs), but little clinical or biological evidence related to alloimmunization and dhtr are presented for all the rh variant alleles. it is well established that transfusion recipients with the most common weak d types , and , are not at risk for forming alloanti-d when exposed to conventional rhd-positive rbcs. aims: we report here a case of a -year-old patient typed as weak d type , receiving d+ rbc units who presented anti-d in his plasma detected three weeks after the last transfusion. methods: rhd beadchip (immucor, nj, usa), was performed to identify the rhd variant allele associated with the weak expression of d. rhce genotyping was performed by laboratory developed tests. sequencing of rhd, rhce and rhag were performed to determine if there were other mutations that could explain the production of alloanti-d. serologic testing was by standard hemagglutination methods. the clinical significance of the antibody was evaluated by monocyte monolayer assay (mma). results: serological analysis showed a negative dat and the presence of anti-d in plasma ( + by gel). anti-lw was ruled out. rhd genotyping revealed the patient was rhd*weak d type . rhce genotyping predicted the d+c+c+e-e+ phenotype. sequencing of rhd, rhce and rhag found no additional changes and confirmed the presence of rhd*weak d type . mma showed the anti-d was clinically significant (> %). summary/conclusions: we report the production of alloanti-d in a scd patient with rhd*weak d type allele. weak d type patients are not considered to be at risk for allo anti-d but our results show that there are exceptions and that these anti-d can be associated with clinically significant rbc destruction. background: the mns blood group system is located on glycophorin a (gpa), glycophorin b (gpb) and hybrid glycophorins on the surface of the red blood cell (rbc). these glycoproteins are involved in complex structures interacting with other rbc surface proteins including the band /diego protein. the glycophorins are heavily glycosylated and contains multiple clinically significant blood group antigens. it has proved difficult to model the gpa extracellular structure due to its heavy glycosylation, and lack of homology with existing modelled proteins. aims: to develop an in silico model of gpa as a basis for improved predictions of structure function relationships methods: prediction of secondary structure and disorder: . . predictprotein (https://predictprotein.org); . . spider (http://sparks-lab.org/server/spider /); . . dsc (discrimination of protein secondary structure class): using an in-house implementation; . . jpred (http://www.compbio.dundee.ac.uk/jpred /); . . raptorx (http://raptorx.uchicago.edu). prediction of secondary structure: . . robetta (http://robetta.bakerlab.org/submit. jsp); . . falcon (http://protein.ict.ac.cn/treethreader/); . . itasser (https://zha nglab.ccmb.med.umich.edu/i-tasser/) threading methods to evaluate the quality of protein structures: . . verify d (http://servicesn.mbi.ucla.edu/verify d/); . . prosa (https://prosa.services.came.sb g.ac.at/prosa.php) protein-protein docking: . . gramm-x protein-protein docking web server (http://vakser.compbio.ku.edu/resources/gramm/grammx/); . . gramm (http://va kser.compbio.ku.edu/main/resources_gramm . .php) results: using in silico modelling we derived a stable tertiary glycosylated structure for gpa both as an individual protein and a homodimer. the hybrid glycophorin background: non-invasive prenatal testing of fetal antigen using cell-free fetal (cff) dna from maternal plasma of immunized women is widely implemented into clinical routine but the sensitivity and specificity of the method, especially for genotyping antigens encoded by single nucleotide polymorphisms such as k antigen, is limited by low proportion of cffdna in maternal plasma dna. according to literature reports detection of circulating tumour (ct)dna can be improved by selection of short ctdna fragments using automated electrophoresis methods. aims: the aim was to assess the feasibility for enrichment of cffdna fraction in maternal plasma dna by size selection using the pippin prep gel selection system. methods: plasma dna isolated using easymag (biomerieux) from rhd negative and k-negative pregnant women (n = ) carrying fetuses with known genotype was loaded into % agarose gel casette ( % df marker q , sage bioscience) and size selection of fraction was performed on a blue pippin tm (sage bioscience) with the elution from min to h min of electrophoresis. results for real-time pcr detection of fetal rhd, kel* and ccr (as a marker of total plasma dna) in dna fraction after gel selection were compared to results obtained from non-processed plasma dna. results: the total dna level (measured by ccr ) was significantly lower in dna samples tested after gel selection (from . to . geq/pcr) versus the level obtained from non-processed plasma dna (from to geq/pcr). the results for fetal fraction (measured by rhd) from dna samples of rhd-negative pregnant women carrying rhd positive fetus tested after gel selection were from , to . geq/pcr versus . - . geq/pcr for non-processed plasma dna. results for kel* detection in plasma dna from k-negative pregnant women carrying k-negative fetus were kel* -negative in dna samples tested after gel selection comparing to nonprocessed dna samples were false kel* positive amplification was observed. however, kel* detection in plasma dna from two k-negative pregnant women carrying k-positive fetus gave false kel* -negative results in dna samples tested after gel selection comparing to non-processed dna samples were kel* positive genotype was obtained. the total dna level in samples from k-negative women was from . to . geq ccr /pcr after gel selection versus from to geq ccr / pcr in non-processed dna samples. summary/conclusions: using the pippin prep gel selection system increases the proportion of cffdna fraction in total plasma dna by retaining long maternal dna fragments in the gel cassettes, but the protocol of gel separation dilutes the separated material decreasing the concentration of fetal dna and leading to false negative results of nipt. anti-rh quantification assay using ih- (bio-rad â ): promising results for monitoring rh:- pregnant women j beaud, h delaby, c toly-ndour, a mailloux and s huguet-jacquot centre national de r ef erence en h emobiologie p erinatale (cnrhp), hôpital saint-antoine, paris, francebackground: the generalization of immunoprophylaxis by anti-rh immunoglobulins since complicates the interpretation of the anti-red blood cell antibodies screening during pregnancy. to distinguish an alloantibody from a passive one, many laboratories in france use anti-rh microtitration. it is a column agglutination technology using red blood cells rh: , - , - , , (r r) . it permits to quantify low levels of anti-rh in comparison to a range of an anti-rh standard. performed since at the cnrhp and automated on evo clinical base tecan in (dilutions and distribution), anti-rh microtitration is well adapted to rh prophylaxis. aims: the aim of this study was to evaluate this technique on the ih- system from bio-rad â . methods: on ih- , the reactivity of the bio-rad â reagents was compared with the cnrhp reagents (red blood cells r r, anti-rh standard). the performances of the method were evaluated using three internal quality control (icq) ( cnrhp home-made at and ng/ml and bio-rad â at ng/ml) on papainized r r (plc) and native r r (nlc). a comparison of results from patient sera ranging from . to ng/ml was done between ih- and evo clinical base tecan. results: the results of the qci are similar between the different reagents used. there is no significant difference between the types of red blood cells except for the limit of detection: . ng/ml in plc - ng/ml in nlc. for the qci, the intra and interassay imprecision based on the dilution degree show coefficients of variation between and %, similar to those found with the evo clinical base. the correlation with the cnrhp technique performed on samples in plc and samples in nlc was satisfactory (deming plc: r = . y = . x + . -nlc: r = . y = . x- . ). summary/conclusions: the anti-rh microtitration on the ih- offers similar performances to the method conducted at the cnrhp. the ih- allows automated reading of gel cards. however, it does not have a calculation or interpretation algorithm and does not directly give the concentration of anti-rh . this final part remains manual and requires trained staff. background: haemolytic disease of the foetus and newborn (hdfn) due to maternal-foetal incompatibility has been perfectly framed for decades from the etiologic, pathogenetic and therapeutic point of view. the anti-d alloantibody is most frequently responsible for the most serious form of hdfn due to rhd incompatibility (rhdi hdfn). although immunoprophylaxis (ip) has reduced the number of cases of rhdi hdfn, this disease continues to occur and red blood cell alloimmunization still remains the most common cause of foetal anaemia. hdfn due to abo incompatibility (ab i hdfn) is currently the most common neonatal haemolytic disease in the western world. however, only in about . - % of cases haemolytic disease demands transfusion support. aims: analysis hdfn from to . methods: the hdfn's transfusional support is: intrauterine transfusion (iut) in the antenatal period; exchange transfusion (et) for severe hyperbilirubinaemia and neonatal transfusion of small volumes red cells for the newborn's late anaemia in the postnatal period. our policy for iut, for et and for the neonatal transfusion requires the use of a concentrated blood cells (ec) preferably group rh negative (cde/cde) or negative for any red cell antigens if the mother has antibodies, fresh (< days), preferably cmv safe. for iut, the ec must be compatible with mother's plasma, must have hematocrit + %, and irradiated. the unit for et must be compatible with the newborn's plasma, whit hematocrit % - % and irradiated. the ec used in post-natal transfusions is usually divided into rates of ml, hematocrit ae %. results: in last years, we calculated neonates with hdfn ( males and females): with rhdi hdfn, with ab i hdfn and with hdfn due to incompatibility for other red blood cell antigens. we have produced iut: for our hospitalized patients and for patients located in other hospitals. of these patients, who received iut, were immunized: showed anti-d antibody and antibodies different from anti-a and anti-b. , of the infants with rhdi hdfn, were transfused in utero. neonates on ( . %) have performed et: with ab i hdfn and with rhdi hdfn; the latter had also been transfused in utero. neonates on were transfused after birth: with rhdi hdfn, with ab i hdfn and with hdfn due to incompatibility for other antigens. summary/conclusions: our case studies reflect the literature data. neonates with rhdi hdfn are the most numerous ( . % of the total) and are those who have requested the highest blood supply both in the antenatal period ( . %) that postnatal ( . % performs et, . % performs postnatal transfusions). neonates with aboi hdfn are . %: nobody has received iut, only one has been subjected to et, and % has transfused after birth. patients with hdfn due to other antigens are %, have undergone iut . % and were transfused after birth . %. background: according to british guidelines on neonatal transfusion, since we shared with neonatologists a transfusion protocol for preterm babies. patients are anemic premature newborns with a gestational age ≤ weeks and/or a birthweight lower than g, until months of age. aims: reduce the incidence of iatrogenic anemia. methods: pre-transfusion tests were based on ab direct test, rh phenotype, direct and indirect antiglobulin test (dat, iat). a second blood sample was required for ab /rh confirmation. blood transfusions were performed with negative kell negative ( cde/cde/kk) cmv negative irradiated erythrocyte concentrates (ec) of less than days. ec were subdivided in ml aliquots with a hematocrit of ae %. according to the definition of "small volume transfusions", our protocol established that further four transfusions had to be delivered free of testing. after the fifth ec transfusion the supplementary release of ec was provided of type and screen (t&s) test with h of validity. serological investigation and full compatibility testing were applied in the presence of a iat and/or dat positivity and in the case of mother alloimmunization. results: from october to the end of , premature newborns received ec transfusions within their first months of life. in % of cases (n = ), transfusion requirement was comprised within the 'small volume transfusions'. another % of cases (n = ), requiring further ec administration, was requested of a blood sample for t&s determination and % (n = ) for a cross-match test. in . % of newborns (n = ), being transfused within the " small volume transfusions", blood requirement of ec was fulfilled by the initial blood test ( blood samples). . % of newborns (n = ) received more than transfusions ( - ; median = ) accounting for ec released and for this group blood samples were required. summary/conclusions: with the exception of babies requiring crossmatch test, blood tests were performed to sustain infants transfused with units. the alternative option of omitting crossmatch test (otherwise suggested by italian directives), allowed a reduction of % of blood drawn without any adverse effect or incident reported. due to the relevance of anemia in premature babies, we suggest the application of this transfusion policy in all newborns in the first months of life. background: glucose- -phosphate dehydrogenase deficiency (g pdd) is a sexlinked enzymopathy which is usually asymptomatic unless individuals are exposed to oxidative stress agents. the g pd genotype is the most common g pd genotype in sub saharan africa (ssa). some studies have linked blood from g pdd donors to poor outcome of a transfusion. however, there are no genetic screening programmes for blood donors in the region hence the contribution of g pdd to the donor pool in the ssa setting had not been described.aims: this study aimed to describe the prevalence of g pdd genotype among donors in two regions in uganda. it also described the effect of g pdd and the coinheritance of haemoglobin s and a-thalassaemia on the haematological quality of blood. methods: , blood samples from donor packs were utilized in a transfusion trial conducted in uganda, were genotyped for g pd , haemoglobin s and a-thalassaemia. haemoglobin and haematocrit measurements for the donor units (packs) at the time of transfusion were used to describe the effect of g pd and co-inheritance with a-thalassaemia (n = , ) and haemoglobin s (n = , ) on the haematological quality of blood packs. a subset of donor blood packs was utilized to determine the sensitivity and specificity of the carestarttm rapid diagnostic kit (rdt) for g pdd. results: based on g pd genotyping, . % (n = ) of the blood samples used in the trial were deficient for g pd enzyme while . % (n = ) were heterozygous. significant lower hemoglobin values were observed in red cell concentrates (p = . ) and whole blood (p = . ) donations of heterozygous g pd genotype. co-inheritance of g pdd and a-thalassaemia resulted in significantly lower haemoglobin levels. the carestarttm rdt test was . % sensitive and . % specific for detecting donor blood packs with g pdd. summary/conclusions: the prevalence of g pdd among ugandan blood donors was similar to that in the general population. the heterozygous genotype resulted in lower haemoglobin concentration of the blood units. the use of carestarttm rdt for screening of stored blood units was not as efficient in this study hence further testing for the determination of g pdd needs to be done on fresh samples from donors. transfusion medicine, jaypee hospital, noida, india background: during last two decade there has been a continuous remarkable improvement in desensitization therapy in high risk hla sensitized kidney recipients. in india there has been a tremendous increase in the number of kidney transplantations in patients having anti-hla antibodies (hla sensitized) with excellent success rate. aims: in present study, we are describing successful role of desensitization in hla sensitized patients having preformed donor-specific hla antibody (dsa). methods: all patients were preconditioned with combined modality of a standard dose of rituximab, therapeutic plasma exchange (tpe) and low dose ivig. tpe was started using com. tec (fresenius kabi, germany) after days of administration of rituximab. complement dependent cytotoxicity cross match (cdc-xm), luminex cross match with donor lysates (lm-xm, immucor inc., ga, usa) and flow cytometry cross match (fc-xm, bd facs canto ii).) was done in all cases. if any of the three tests was positive, single antigen bead assay (sab) was performed. desensitization therapy was given in all cases where dsa was detected. pretransplant tpe procedures were done until dsa (mfi < ) and cdc-xm became negative. cdcxm labeled positive at ≥ %. t-cell fcmx was considered positive above mfi and b-cell fcmx was considered positive above mfi. lmxm was considered positive above mfi. sab was performed using lifecodes single antigen (lsa) class i and class ii kits (immucor, usa). if the specificity of anti-hla antibodies was against donor hla antigen(s) it was called as donor specific antibody (dsa). results: present study demonstrated the diagnostic and clinical superiority of adding fc-xm and lm-xm in pretransplant compatibility testing algorithm over cdc-xm. cdc-xm alone was not able to detect anti-hla antibody in patients ( . %). among the three pretransplant compatibility tests, fcxm demonstrated highest sensitivity. among the cases initially screened showed dsa positivity in sab. desensitization was done in those cases only. in our study, sab was positive for class ii alone in ( %) while in remaining ( %) cases it was positive for both class and class ii. the number of pre transplant tpe procedures required was . ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . the mean number of post-transplant tpe sessions required was . (range, - ). during pretransplant and post transplant tpe procedures, five ( . %) patients presented with allergic or hypotensive reactions which were managed conservatively. most of the patients were discharged after seven days of hospital stay whereas patients who required post-transplant tpe were discharged after a relatively longer hospital stay (mean- . , median- days). after three months, protocol biopsy was done in those cases only where post transplant tpe was required. protocol biopsy showed normal findings. in present study, the mean duration of follow up was approx months with the longest duration of follow up of months. summary/conclusions: in a country like india where there is a huge gap in the demand and supply of kidney and a large no. of patients waiting for a suitable organ, transplant across hla barrier could a good doable option. thorough pretransplant compatibility and tpe are essential tools to make these transplants program successful background: most transfusion-dependent chronically anemic patients are managed by simple red cell transfusions. however, some patients are not able to tolerate the additional volume associated with simple transfusions and are at a high risk of developing transfusion associated circulatory overload, if transfused with multiple red cell units. plasma-to red cell exchange (prx) is a modified procedure wherein an apheresis machine is used to remove patient's plasma, while simultaneously replacing with donor rbcs. this procedure allows for a rapid euvolemic transfusion of rbcs to patients that are severely anemic and intolerant to excess fluid volume. others as well as our group have previously described this procedure. we now summarize our institutions nearly seven years of experience performing this procedure on a routine basis. aims: to document patient experience with prx. methods: we performed a retrospective chart review of all patients that underwent prx at our institution in the last seven years. our protocol for prx has evolved during this period. currently, we perform the procedures using spectra optia (terumo bct, lakewood, co) machine using the plasma-exchange program and tubing set. if the patient's pre-procedure hematocrit (hct) is < %, we custom prime the tubing set with % albumin. the number of red cell units transfused to the patient depends on their pre-procedure hematocrit and is individualized to the patients. results: we have treated four patients with prx procedure. patient # is a -year-old transfusion-dependent male with beta-thalassemia major. the patient had experienced multiple congestive heart failure exacerbations secondary to simple transfusions and we consequently performed prx procedure, every weeks, starting in . the patient has completed procedures with - units of washed red cells transfused to achieve a target hct goal of to %. he tolerated all procedures without any volume overload issues. he continues on this transfusion regimen. patient # was a -year-old female who had symptomatic anemia secondary to sickle cell disease (hb ss complicated by end-stage renal disease (esrd). she had progressively become intolerant to simple transfusions, including an episode of severe dyspnea, which required intubation. she underwent prx procedures with - units of washed red cells. patient tolerated the procedures without any significant complications. however, during a different surgical procedure, she experienced cardiac arrest and subsequently passed away. patient # is a -year-old transfusion-dependent male with severe anemia secondary to sickle cell anemia (hb ss). he was intolerant to excess fluid because of esrd and congestive heart failure. he has undergone prx procedures with - red cell units transfused to achieve a hct goal of %. he tolerated all procedures without any volume overload issues. he continues on this transfusion regimen. patient # is a -year-old male with a sickle cell disorder (hb ss) complicated by esrd, heart failure and chronic hypoxemic respiratory failure. the patient has undergone two prx procedures with - red cell units. other than an episode of non-bloody emesis that was symptomatically treated, he tolerated both procedures. he continues to be managed on this regimen. however, the patient remains noncompliant with treatment. summary/conclusions: prx is a safe and efficient method to transfuse multiple red cell units to volume-intolerant anemic patients. background: transplanted organ failure due to antibody mediated rejection in abo-compatible organs is a serious complication with a bad prognosis. the goal treatment in these cases encompasses the following strategies: adjustment of the immunosuppressive medications, ivig infusion, antibody removal by therapeutic plasma exchange, and/or the use of target-specific monoclonal antibody medications to lymphocytes, plasma cells, and/or complement. the american society for apheresis has assigned a category i to the use of therapeutic plasma exchange for the treatment of abo-compatible antibody mediated rejection in kidney, but a category iii to all other abo compatible organs: liver, lung, and heart. at our institution, a standardized approach for the use of therapeutic plasma exchange as a supportive intervention for abo-compatible immune mediated rejection, regardless of the organ type, has been in place since . aims: a retrospective review was performed to evaluate our patient outcomes using therapeutic plasma exchange for the treatment of antibody mediated allograft rejection in abo-compatible solid organ transplantation. methods: patients used for the retrospective review were selected from an existing therapeutic apheresis list. the therapeutic plasma exchange protocol consists of: adjustment of the immunosuppressive medications, ivig infusion, antibody removal by therapeutic plasma exchange, and/or the use of target-specific monoclonal antibody medications to lymphocytes, plasma cells, and/or complement. it is performed as follows: one plasma volume exchange is performed on days , , , , , along with one or more of the above strategies followed by an ivig infusion. cases with allograft rejection in which plasmapheresis was not used were excluded. and t devos aims: this study aimed to explore the possible causes of the decreased transfusion rate for all adult cardiac surgery patients. methods: data were collected from adult cardiac surgery patients during the mentioned time frame and were extracted from electronic patient files and a database of the department of cardiac surgery. a set of variables was defined as possible confounders by a panel of experts. after discussion, global variables (age, gender, duration of surgery, use of cpb (cardio-pulmonary bypass), american society of anesthesiologists (asa) risk score, type of surgery, urgency, attending cardiac surgeon and attending anesthesiologist) and cpb-related variables (administration of cardioplegia yes/no (cpg), duration of cpb, circulatory arrest, hypothermia, duration of aortic cross-clamp, baseline hemoglobin and cpb-priming volume) were retained. negative binomial models for counts were used for data analysis. all analyses were performed with spss. results: patients were extracted from databases and further analyzed. the mean age of this group was , years (sd +/- , years) and . % of them were male. the mean duration of surgery was min (sd +/- , min). the decrease of perioperative rbc transfusion rate over four years was statistically significant (p < . ). in , the mean use was , units per operation (sd +/- , ), which changed to , units (sd +/- , ) in . three variables (urgency, attending cardiac surgeon, attending anesthesiologist) changed significantly over years and were used in a multivariable model as confounders together with rbc transfusions and year. even after adjustment for these factors, the decrease in rbc transfusion rate was still statistically significant (p < . ). in the specific group of patients undergoing cardiac surgery with cpb (n = ), the use of rbc was also significantly reduced (p < . ). in , the mean use was , units per operation (sd +/- , ) and this changed to , units (sd +/- , ) in . after correction for the cpb variables that notably changed over the years (cpg, priming volume and hypothermia) and the three previously defined confounders (urgency, attending cardiac surgeon and attending anesthesiologist) the reduction of rbc transfusions over years still remained statistically significant (p < . ). summary/conclusions: our study shows evidence for a decreased rbc transfusion rate in adult patients undergoing cardiac surgery between and . this tendency was also seen in the subgroup of patients undergoing surgery with cpb. possible explanations of the decrease are implementation of various established parts of patient blood management. however, a unique reason could not be identified in this study. background: growing worldwide demand for immunoglobulin products such as intravenous immunoglobulin (ivig) and subcutaneous immunoglobulin (scig) is driving plasma collection. patients with primary immunodeficiency (pid) or secondary immunodeficiency due to haematological malignancy or its treatment (sid) rely on these products to maintain therapeutic serum igg levels to minimise recurrent infection. efficacy of immunoglobulin replacement therapy (irt) in pid is well established but information on sid is limited. the different aetiologies of hypogammaglobulinaemia between pid and sid raised the question of whether sid patients on irt experience similar clinical and quality of life (qol) benefits as reported in pid patients. aims: to assess whether sid patients experience similar clinical and qol benefits while on irt as pid patients. methods: following ethics approval, data on dosage, serum igg trough levels and infection (bacterial, viral and fungal requiring treatment such as antibiotics) was collected from adult pid and adult sid patients from medical records and pathology reports, for their last months of ivig and their first months of scig. the starting and maintenance dose was . g/kg/month for ivig, transitioning immediately to . g/kg/week for scig without a washout period. a study specific questionnaire was developed to gather data on patient perceived side effects, treatment satisfaction and impact of irt on social/family life, work/study and their overall qol. paired t-test was used for parametric data and the wilcoxon signed-rank test for non-parametric data. results: sid patients were significantly older with a mean age of . years versus . years in pid patients (p = . ). a mean of three training session was required to reach competency in scig administration in both cohorts. there was a trend of reduced side effects on scig for pid and sid patients compared to ivig, with a significant reduction of headaches in the pid cohort (p = . ). the majority of patients experienced infusion site reactions, which were predominantly perceived as manageable. % of infections were respiratory tract infections. pid patients had slightly higher mean serum igg trough levels with scig ( . g/l) compared to ivig ( . g/l), and fewer infections on scig than ivig (mean annual infection rate of . vs . respectively). sid patients had higher mean serum igg trough levels on scig ( . g/l) than ivig ( . g/l) (p = . ) but experienced more infections while on scig versus ivig (mean annual infection rate of . vs . respectively). the number of hospitalisation due to infection decreased in both cohorts with scig. pid patients perceived that switching from ivig to scig improved their health and qol. in contrast sid patients perceived no improvements in health and qol. summary/conclusions: data from this pilot study suggests that the clinical and qol impact of irt in sid patients is different to that of pid patients. to support evidence based irt management and effective use of this limited and expensive blood product in sid, larger studies which account for different stages of malignancy and associated treatment regimes are required. background: there is an increasing platelet transfusion for treatment and prophylaxis of bleeding in patients with hematologic disorders and malignancies. because of limited resources, leukoreduced platelet concentrates is not yet implemented in most indonesian hospitals. in vitro platelet activation may cause morphology, functional, and ultrastructure changes. those changes will reduce the platelet viability, in vivo functions, and clinical efficacy. high platelet cd p expression is the cause of faster platelet destruction in the reticuloendothelial systems. post-transfusion in vivo hemostatic efficacy can be determined by the measurement of corrected count increment (cci), recovery, and platelet cd p expression. aims: to analyze the increase of platelet cd p expression in patients of non-leukodepleted compared to pre-storage leukodepleted pc transfusion.background: haemorrhage is a leading cause of preventable death not only in the military trauma care, but also for civilian population suffering accidents or bleeding injuries in regions with low population density where health services should reach people in remote areas. resuscitation using blood products and limited infusion of normal saline improves survival for critically bleeding patients. nowadays there are hems programs (helicopter emergency medical system) carrying blood products around the world. the hems in castilla-la mancha, with physician and nurse, is the first out-of-hospital emergency service in spain that provides packed red blood cells (prbc) transfusion where the accidents happen without delaying the transport to the proper hospital for definitive treatment. this program has been developed between the blood center of ciudad real and the hems team ('gigante ', emergency service castilla-la mancha). the goal of the designed protocol was to preserve the properties of the product to be transfused in out-of-hospital environment by hems teams. aims: to describe the process for out-of-hospital prbc transfusion in hems of ciudad-real. the protocol for out-of-hospital blood transfusion was developed according to criteria of medical indications and security, monitoring, and tracking of the product. methods: data for the observational retrospective study were collected from june to august . the medical helicopter (ec t ) was provided with two prbc o rh(d) negative. the shock index was selected for the indication for transfusion according to the literature revised and as a simple rate to obtain out-of-hospital data. to achieve the feasibility and preservation of the prbc a prospective monitoring of volume was established, haematocrit, haemoglobin, leucocytes, coulter, hemolysis and microbiological culture. blood center established two groups: the case group for the prbc kept in the hems base and helicopter and the control group for the units remaining in the blood center with standardized blood conservation. for both groups, control and comparison of immediately obtained hematologic analyses, and days after collection, were performed. the statistical analysis used spss . version (significance p < , ). results: prbc samples were evaluated, , % ( ) from case group and , % ( ) from control group. analyses were tested day and day after collection. haemolysis was not observed. all cultures were negative. although significant differences were found between the parameter in the value of before-after in the value of the hematocrit, leukocytes and coulter, there are no differences between the prbc that flew and those conserved in the transfusion-service. all results comply with current legislation and blood transfusion standards. there have been administered prbc transfusion to patients during out-of-hospital advanced medical assistance. no post-transfusion reactions have been registered. prbc units have a -day rotation to allow the use of the units in the hospital after achieving their optimal status. summary/conclusions: the out-of-hospital transfusion protocol designed to transport blood (prbc) in the helicopter for hems has demonstrated to keep the standard conditions and properties of the product to be considered useful in the treatment for critically bleeding patients in the out-of-hospital. background: early and adequate treatment of major bleeding is important for survival and a good outcome. blood and plasma are given increasingly early including before hospital admission in trauma based on successes reported from combat experience. in the national patient safety agency issued a rapid response report requiring national health service hospitals in england to take actions to improve provision of blood in an emergency including provision of major haemorrhage protocols (mhp) and drills. the national reporting and learning scheme had identified reports of deaths and instances of harm due to delay over a -year period. aims: the aim of the study was to monitor the acid-base status of the patient by means of abg and to administer the blood component therapy based on teg results. methods: this study was a prospective observational study of adult patients over a period of months. serial monitoring of the abg in the intra-operative period was done. teg guided resuscitation was performed in all cases. results: the abg analysis of all patients showed decrease in the ph, increase in pco , decrease in serum bicarbonate level and elevation in negative base excess. these components of metabolic acidosis can be attributed to massive transfusion. increased lactate, an independent parameter, which reflects poor tissue perfusion or shock and predicts need for massive transfusion was observed in all patients. all the cases showed a decrease in ionized calcium levels which could be a result of citrate related toxicity. increased glucose was observed in all patients which may be due to increase in the catecholamine release as a response to haemorrhagic shock. electrolyte correction was given depending on results of the abg analysis wherever appropriate. two out of cases showed an increase in r time indicating deficient coagulation factors, which was corrected with fresh frozen plasma (ffp). three cases showed elevation in k time indicating deficient fibrinogen levels, which was corrected by ffp. fresh frozen plasma was also given in cases, which showed decrease in the alpha angle, indicating deficient fibrinogen, and cryoprecipitate was given in cases. platelets were transfused in patients showing a decrease in the maximum amplitude (ma), which indicates deficient platelets. summary/conclusions: teg as poc testing is an important tool in appropriate blood component therapy in massive transfusions. serial monitoring of abg helps in monitoring acid-base status of the patient and therefore is a guide in the correcting electrolyte level in patients undergoing massive transfusion. background: massive blood loss is encountered in various situations like trauma, major surgeries, gastrointestinal bleeds and obstetric haemorrhages. haemorrhage is an important cause of mortality and morbidity in massively bleeding patients. early recognition of haemorrhage and intervention is essential for survival. massive transfusion (mt) of blood is required to replenish blood losses and is a lifesaving treatment in these patients. a variety of haemostasis and pathophysiological changes occur during massive haemorrhage and massive transfusion. all of these changes contribute to the vicious cycle of progressive coagulopathy due to the 'lethal triad' of refractory coagulopathy, progressive hypothermia and persistent metabolic acidosis. aims: the aims of the study included understanding management of cases of massive blood transfusion in surgical patients, impact of mt of blood components on patient outcome, evaluating post-operative complications of massive transfusion and the development of institutional massive transfusion protocol (mtp).methods: this prospective observational study commenced after institutional ethics committee (iec) approval. it comprised of adult surgical oncology patients who received massive transfusions and was conducted for a period of months. every case of a massive transfusion was studied under the following headings ( ) patient's details ( ) patients base-line laboratory tests ( ) resuscitation with transfusion ( ) intra-operative laboratory tests ( ) thromboelastography (teg) ( ) post-operative complications ( ) duration of stay in the hospital ( ) day mortality rate. results: complete blood count, serum electrolytes, arterial blood gases, coagulation profile and teg were used to monitor transfusion therapy in the intraoperative period. intraoperative laboratory parameters of patients showed dilutional coagulopathy, metabolic acidosis, hypocalcaemia, hypomagnesaemia, hyperkalaemia and hypokalaemia, increased lactates and glucose. electrolyte correction was done based on the derangement. the derangements were on a decreasing trend in the postoperative period and returned to baseline level by nd or rd post-operative day with no requirement of correction in the post-operative period. the post-operative outcomes were evaluated in terms of the surgical site infection (ssi) as per the centers for disease control (cdc) criteria, surgical complications as per modified clavien-dindo classification and respiratory complications. a total of ( . %) patients had ssi, ( %) had surgical complications and ( %) patients had respiratory complications. the length of the stay in the hospital was longer for patients who had postoperative complications. despite complications, owing to excellent peri-operative care, ( %) patients were discharged alive. summary/conclusions: surgeries associated with massive transfusion are at an increased risk of ssi as well as morbidity and mortality. complications associated with rapid transfusions of blood, acute haemorrhage and associated risk of the surgery lead to a prolonged icu stay and increased length of stay in the hospital. a well-developed massive transfusion protocol optimizing the ratio and dose of the blood component therapy results in excellent patient outcome with minimal postoperative morbidity and mortality. background: despite the introduction of new oral anticoagulants (dabigatran, rivaroxaban, apixaban), vitamin k antagonists (vka), such as warfarin and acenocoumarol are still the most widely used oral anticoagulants for the treatment of non-valvular atrial fibrillation (nvaf). the use of vka must be regularly and often laboratory controlled in order to ensure the adequacy of therapy and to avoid subdosing or drug overdose. the most commonly used test for the control of oral anticoagulant therapy is the prothrombin time (pt), expressed in inr system, which provides an internationally standardized monitoring of the treatment. time in therapeutic range (ttr) represents a measure of the quality of the anticoagulant effect of vka and estimates a percentage of time a patient's inr is within the desired therapeutic. aims: the aim of this study was to evaluate of the effectiveness of vka therapy in patients with nvaf and to identify factors affecting the anticoagulation efficacy. methods: a retrospective study was conducted on a population of outpatients with nvaf, treated with vka and followed in blood transfusion institute of ni s from january to december . laboratory control of inr was done from capillary blood of patients on thrombotrack solo (axis shield, norway) and thrombostat (behnk elektronik, germany). targeted ae . %, but . % of patients had a ttr less than %. patients were at high risk of thrombosis in . % of time (inr < . ) and high risk of bleeding in . % of time (inr > . ). the most significant independent factors affecting the quality of vka therapy are gender, arterial hypertension, diabetes mellitus and the use of amiodarone and antiplatelet drugs (aspirin, clopidogrel). summary/conclusions: the ttr is undoubtedly useful indicator of the effectiveness of vka treatment. the most important predictors of poorer efficacy of vka therapy are arterial hypertension, diabetes mellitus, patients' gender and the use of amiodarone and antiplatelet (aspirin, clopidogrel) drugs. to improve the quality of vka therapy, education of patient and better collaboration with them, as well as a successful teamwork with clinicians are also imperative. background: an estimated . million deaths per year result from haemorrhagic blood loss. at a cellular level, haemorrhagic shock develops when oxygen delivery is insufficient to meet oxygen requirements to maintain aerobic metabolism. successful resuscitation prevents further oxygen debt and repays the prior oxygen debt. this includes the administration of fluids and blood components (e.g. plasma, red cells and platelets). measurement of oxygen delivery and utilisation at a tissue level requires invasive monitoring not possible clinically, meaning that surrogate markers such as lactate and venous oxygen saturation (svo ) are used instead. new technologies such as incident dark field imaging and near-infrared spectroscopy may offer a non-invasive alternative; however their utility in haemorrhagic shock remains background: transfusion-induced red cell alloimmunization is still a major challenge in transfusion practice. besides logistic problems for the transfusion laboratory, it may compromise available blood supply, and when undetected and unanticipated, it may risk haemolytic transfusion reactions. knowledge about risk factors can help to optimize preventive matching strategies. severe renal failure and subsequent renal replacement therapy influence the immune system and could therefore modulate the risk of alloantibody formation against foreign red cell antigens subsequent to transfusion. aims: this study aims to quantify the association between renal failure, according to its degree and its treatment with renal replacement modalities, and transfusioninduced red cell alloantibody formation. methods: we performed a multicenter case-control study within a source population of patients receiving their first and subsequent red cell transfusion between and in the netherlands (risk factors for alloimmunization after red cell transfusion, r-fact study). using a conditional multivariate logistic regression, we compared first-time transfusion-induced red cell alloantibody formers (n = ) with two similarly exposed non-alloimmunized control recipients (n = ) during a five-week alloimmunization risk period. degree of renal function was categorized as: 'no renal failure' i.e. glomerular filtration rate (gfr) > ml/min/ . m , 'moderate renal failure' i.e. gfr ≥ - ml/min/ . m during a continuous period of minimally seven days, 'severe renal failure' i.e. gfr < ml/min/ . m and/or use of any type of renal replacement therapy during at least one day of the alloimmunization risk period. odds ratios were interpreted and presented as relative risks (rr). adjusted rrs were conditioned on the matching variables and identified confounders. results: no renal failure was observed among ( . %) cases versus ( . %) controls; moderate renal failure among ( . %) cases versus ( . %) controls; and severe renal failure among ( . %) cases versus ( . %) controls. among the latter, cases and controls underwent renal replacement therapy. moderate renal failure and severe renal failure without renal replacement therapy were not significantly associated with red cell alloimmunization (adjusted rr . , % ci . - . and adjusted rr . , % ci . - . , respectively). however, patients undergoing renal replacement therapy had a two-fold lower alloimmunization risk (adjusted rr . , % ci . - . ) as compared to transfusion recipients without renal failure, unrelated to type and duration of renal replacement therapy. summary/conclusions: these findings suggest that patients undergoing renal replacement therapy have strongly diminished red cell alloimmunization risks. further research should confirm these results and elucidate the underlying pathophysiological protective mechanism. background: the ability of allogeneic hematopoietic stem cell transplantation(allo-hsct) to prevent relapse depends partly on donor natural killer (nk) cell alloreactivity. nk effector function depends on specific killer-cell immunoglobulin-like receptors (kir) and hla interactions. thus, it is important to identify optimal combinations of kir-hla genotypes in donors and recipients that could improve hematopoietic transplantation outcome. aims: to obtain the optimal combinations of inhibitory kir and its ligand between donor and recipient which is helpful for the guidance of selecting donors and recipients in hsct. methods: the pcr-sbt method was used for kir dl , kir dl /kir dl , kir dl , kir dl and hla-a, -b, -c, -drb , -dqb genotyping. pairs of hla / identical donor/recipients matching samples were retrospectively analyzed. three different models of kir were established. there were kir-kir gene model, kirligand model and haploid model. in kir-ligand model, patients were divided into three groups: c /c homozygote group ( cases), c /c heterozygote group ( cases) and c /c homozygote group ( cases). according to the expression of dl , cases were dl positive and cases were dl negative. there were cases of bw /bw , cases of bw /bw and cases of bw /bw in the dl positive samples. according to the expression of a /a , they were divided into three groups: a /a negative group ( cases), a /a heterozygous group ( cases) and a / a homozygote ( cases). according to kir genotyping, kir haploidentical group ( cases) and kir haploid mismatched group ( cases) were divided. the clinical data on neutrophil and platelet remodeling time, gvhd and os of cases were statistically analyzed by the mann-whitney test or the kruskal-wallis test using graph-pad software v . . results: there was no significant difference in the time of neutrophil and platelet remodeling, the incidence of agvhd and the survival time after transplantation in the kir genotype model. in haplotype model, there was no significant difference in neutrophil and platelet remodeling time and survival time after transplantation. the incidence of agvhd was low when the kir haploid mismatched and kir dl was positive. it was conducive to neutrophil and platelet remodeling when bw /bw and a /a was heterozygosity. summary/conclusions: it is important to establish the three different models of kir genotypes, haplotypes and receptor-ligand mismatches for analyzing the effect on the prognosis of allo-hsct. kir-ligand model plays an important role in hla unre-background: transfusion of platelet concentrates (pcs) has been associated with adverse outcomes including transfusion-related acute lung injury (trali). the underlying mechanism of trali has been related to the accumulation of immunomodulatory mediators (e.g. lipids, cytokines/chemokines) present in pcs. current room temperature storage limits the shelf-life of conventional pcs to - days. alternative storage conditions, including cryopreservation, offers extended storage and a solution for blood banking logistics. cryopreservation of human pcs has been associated with higher concentrations of immunomodulatory mediators compared to room temperature stored pcs and it has been suggested that cryopreserved pcs may be immunomodulatory. to investigate the effects of cryopreserved pcs, a transfusion sheep model would be a beneficial approach. aims: to characterize immunomodulatory mediators in supernatants of sheep conventional and cryopreserved pc and to investigate whether storage duration and cryopreservation impacts the accumulation of these mediators. methods: buffy coat pooled sheep pcs (n = ), prepared in % plasma/ % ssp+ with minor modifications to standard human procedures, were stored room temperature (rt) for days (d) and sampled on d , d and d . cryopreserved sheep pcs (n = ), prepared by the addition of - % dimethyl sulfoxide, were stored at À °c and sampled pre-freeze and post-thaw. supernatant was prepared at each time point with double centrifugation and stored at À °c. concentrations of pro-inflammatory cytokines (interleukin (il)- , il- b, il- a), anti-inflammatory cytokine il- and chemokines (il- , monokine induced by gamma interferon (mig) and interferongamma induced protein (ip)- ) were measured using sheep specific in-house and commercial enzyme linked immunoassays (elisa). levels of non-polar lipid mediators, such as arachidonic acid (aa), -hete and -hete were assessed in the stored sheep pc-and cryo-pc supernatant using commercial elisa. results shown as mean ae standard deviation. the effect of storage was determined at p < . using paired t-test. results: in rt stored sheep pc supernatant il- , il- b, il- a, il- , il- , mig, ip- , -hete and -hete were detected at d , d and d . storage duration significantly increased accumulation of ip- at d ( . ae . pg/ml compared to . ae . pg/ml, p = . ) and further increased at d , and il- at d ( ae . pg/ml compared to ae . pg/ml, p = . ). cryopreserved sheep pc supernatant pre-freeze and post-thaw contained equivalent or higher concentrations of il- , il- b, il- a, il- , il- , mig, ip- , -hete and -hete than rt stored d pcs. however, cryopreservation did not impact levels of any of the platelet derived mediators. summary/conclusions: several platelet-derived cytokines/chemokines, including high concentration of il- with neutrophil priming activity, and non-polar lipids were found in stored sheep pc supernatant. these immunomodulatory mediators may contribute to adverse outcomes associated with pc transfusion. storage at rt, but not cryopreservation was associated with increased accumulation of immunomodulatory mediators in sheep pcs. most importantly, similar to human pcs, sheep cryopreserved pcs contained at least if not higher concentrations of majority of cytokines as pcs stored at rt, therefore making sheep a suitable model in which to investigate immunomodulatory effects of cryopreserved pc transfusion. background: transfusion, despite being a lifesaving therapy, has been associated with adverse transfusion outcomes. transfusion related acute lung injury (trali) remains one of the leading causes of transfusion-related mortality. accumulation of immunomodulatory mediators (e.g. lipids, cytokines/chemokines) present in blood products, including packed red blood cells (prbcs), have been implicated with the development of non-antibody mediated trali. however, how specific mediators contribute to the underlying mechanism is yet to be defined. during routine storage of human prbcs fewer than cytokines/chemokines and several biologically active lipids have been identified. a sheep model of trali has successfully been developed using human prbc supernatant, however transfusing sheep prbc has not been investigated. to support the use of sheep prbc in the trali model and to better understand the precise mechanism, characterization of the potential mediators in sheep prbc is required. aims: to characterize immunomodulatory mediators in sheep prbc supernatants and to investigate whether storage duration impacts the accumulation of these mediators. methods: sheep prbcs (n = ), prepared according to standard human procedures with minor modifications, were stored ( - °c, days (d) ) and sampled at d and d . supernatant was prepared by double centrifugation and stored at À °c. concentrations of pro-inflammatory cytokines (interleukin (il)- , il- b, il- a), antiinflammatory cytokine il- and chemokines (il- , monokine induced by gamma interferon (mig) and interferon-gamma induced protein (ip)- ) were measured using sheep specific in-house and commercial enzyme linked immunoassays (elisa). levels of potential non-polar lipid mediators (arachidonic acid (aa), -hydroxyeicosatetraenoic acid (hete) and -hete) were assessed in the sheep prbc supernatant using commercial elisa. paired t-test was used to compare fresh and stored prbc supernatant (p < . ). results are mean ae standard deviation. results: at day , aa ( , ae , pg/ml), -hete ( . ae . pg/ml), -hete ( . ae . pg/ml) and il- b ( . ae . pg/ml) were detectable in sheep prbcs supernatant. at day , storage duration significantly increased concentrations of aa ( , ae , pg/ml, p = . ) and -hete ( . ae . pg/ml, p = . ) in sheep prbcs supernatant. summary/conclusions: similar to reported findings of human prbcs, the predominant type of immunomodulatory mediators present in sheep prbcs were non-polar lipids. the concentration of these non-polar lipids increased during storage. these immunomodulatory mediators may contribute greatly to adverse outcomes associated with prbc transfusions. further investigation is required to determine whether stored sheep prbcs supernatant induce immunomodulation in sheep in vitro and in vivo transfusion models. background: dshtr incidence is reported as in , transfusions, presenting days to months after the transfusion. the published data addressing the correlation between the strength of the antibodies detected after a dshtr has taken place and the corresponding clinical symptoms as measured by laboratory parameters that assess the presence of hemolysis is limited. aims: the aim of this study is to evaluate the correlation between the results of the dat, automated and manual antibody reactivity strength with the corresponding clinical parameters of hemoglobin, lactate dehydrogenase (ldh), bilirubin, and haptoglobin. methods: a dshtr is defined as discovering a new antibody within days of a transfusion. for all positive antibody screens, a work-up is initiated consisting of identification panels, dats, antigen typing of the red cells transfused, and eluates at the discretion of the transfusion medicine physician. additional laboratory testing for hemolysis is requested when indicated. a retrospective review was conducted of patients who were identified as having a dshtr. levels of hemoglobin, ldh, and transfusion safety background: rhd immunoglobulin (rhdig) has been available for years in australia. since its introduction for routine antenatal and postpartum prophylaxis, alloimmunisation has decreased from % to . %, reducing the number of australian deaths from haemolytic disease of the newborn over a hundred-fold, to approximately . deaths per . blood matters serious transfusion incident reporting (stir) system has been collecting transfusion incidents and adverse events across four australian jurisdictions since . since january , rhdig administration errors have been reported. aims: to understand incidents relating to the administration of rhdig and increase safety and awareness of risks. methods: health services registered with stir (n = ) were notified of the inclusion of reporting rhdig incidents. when an incident is identified, the reporter sends an online notification to stir, prompting the appropriate investigation form to be sent for completion. the completed incident data are reviewed and validated by an expert group. data is de-identified and collated for reporting. results: during the period january -december , reports were received; reports were validated, with reports excluded (reactions rather than administration errors). reports were categorised as below: background: following the nice transfusion guidelines, recommending offering iron before and after surgery to patients with iron-deficiency anaemia (ida), we worked collaboratively with the anaesthetic and pre-operative team to implement a clear and robust anaemia pathway for pre-operative haemoglobin (hb) optimisation. oral iron was started, where appropriate, and our anaemia pro-forma was sent for review in a virtual clinic to assess eligibility for intravenous iron. we performed a retrospective evaluation of the patients who received iv iron during the anaemia pathway. aims: the aim of this retrospective evaluation was to look at the cohort of patients who had received iv iron in and assess the effect of iv iron on haemoglobin levels for different defined groups. methods: we classified patients, as described in munting and klein, , depending on their iron parameters as having either: -idaserum ferritin < mg/l -chronic inflammation with idaserum ferritin - mg/l with transferrin % of < %/crp > mg/l -anaemia of chronic inflammationserum ferritin > with transferrin % of < %/crp > mg/l patients were considered eligible for iv iron if the following criteria were met: . an inadequate response to oral iron, or were unable to tolerate oral iron or the interval between diagnosis and surgery was short . the anaemia pro-forma was completed . hb was ≤ g/l . they were classified as either having ida or chronic inflammation with ida or anaemia of chronic inflammation hb was measured prior and on average, days following the iv iron infusion. we excluded patients who had their post iv iron follow up blood tests done after surgery. results: this retrospective evaluation included patients. patients were classified as having ida and patients classified as having chronic inflammation with ida. those classified with ida had a mean hb of g/l ( - ), a mean mcv of . fl and a mean serum ferritin of lg/l. those with chronic inflammation with ida had a mean hb of g/l ( - ), a mean mcv of . and a mean serum ferritin of lg/l. follow-up hb was measured on average twenty days post iv iron infusion in both groups. the average hb post iv iron infusion in the ida group was g/l ( - ) with an average increment of g/l and in the group with chronic inflammation with ida the average post iv iron hb was g/l ( - ) with an average increment of g/l. summary/conclusions: in conclusion the group with ida had, on average, a lower starting hb that the group with chronic inflammation with ida and the average increment in hb days post iv iron infusion was greater in the group with ida. however, the group with chronic inflammation with ida cases also responded to iv iron and therefore we strongly consider the use of iv iron in both groups. further studies to evaluate the ongoing effect of iv iron would help assess whether the same level of increment seen with ida can also been seen for the group with chronic inflammation with ida over a longer period and how long the increment was sustained. background: the expansion of personalized genomic medicine has led to the development of targeted therapeutic approaches for patients. one example is sipuleucel-t, an autologous cellular immunotherapy product used to treat prostate cancer manufactured from the patient's white blood cells. this study describes our experience with incorporating autologous cellular immunotherapy products into the workflow of the blood bank. the policies supporting the workflow are outlined and compliance with them is assessed. aims: this study aims to evaluate the process and method used to dispense and track the infusion of sipuleucel-t. methods: this is a retrospective analysis of the dispensation and administration of sipuleucel-t from january -august , which was handled exclusively by the blood bank. standard operating procedures and hospital policies were reviewed and compliance with these policies evaluated. included were patients who had the sipuleucel-t product dispensed and administered. information collected included the total number of products dispensed, patient age, adverse reactions/incidents, premedication, and patient outcome. descriptive statistics were used for data analysis. results: there were products dispensed to patients. the recipients were male patients diagnosed with prostate cancer with a mean age of years. there were doses (a complete course) administered to / ( %) recipients and a partial course ( - doses) administered to / ( %) recipients, for a total of products. the blood bank workflow treated sipuleucel-t as a derivative in the computer system, listing the manufacturer (dendreon corporation) as the supplier. health care providers were instructed to follow the nursing policy for the administration of blood products and derivatives for the infusion of sipuleucel-t. this policy required documentation of the infusion in a transfusion nursing note and reporting adverse events to the blood bank as transfusion reactions. there were no adverse events reported to the blood bank, yet there were adverse events described in provider notes; of them necessitating transfer to the emergency department, and requiring hospital admission. of the infusions, infusions were documented in a chemotherapy note rather than a transfusion note ( %), and ( %) were documented as both a transfusion and a chemotherapy administration. there were additional deviations from the blood product administration policy: cases where the consent check was not performed, case where the product was infused with ringer's lactate rather than normal saline, and cases where the -person -way check erroneously indicated the product was irradiated. summary/conclusions: this study describes one approach to managing cellular therapy products as an extension to existing blood products dispensed by a blood bank. the findings demonstrate noncompliance with hospital policy with this new product as evidenced by failure to report adverse events and failure to follow hospital practices regarding administration. although sipuleucel-t is a product manufactured from an autologous blood product donation, the administration and perceptions of this product may be more similar to a pharmaceutical. as the field of immunotherapies derived from blood product donations continues to expand, these products may necessitate an entirely new approach to ensure proper management. abstract withdrawn. (ref ) . while the mnc procedure is fully automated, cmnc requires frequent interface checks to ensure the collection of the correct cell layer. at the rambam health care campus, a tertiary care center, solely the mnc procedure had been employed till , at which point, the cmnc has been introduced for the use in patients with a white blood cell (wbc) count of ≥ , /ll on the collection day. aims: the current study aimed to compare various parameters of peripheral blood stem cell (pbsc) collection, using the cmnc protocol in allogeneic donors and patients undergoing autologous stem cell (autosc) transplantation. additionally, data on autosc collection using mnc (n = ) and cmnc (n = ) procedures were compared. methods: data were retrospectively obtained from pbsc collection reports in consecutive cmnc procedures, including autologous and allogeneic donors. the following comparisons were made: cmnc results of allogeneic versus autologous donors, a sub-analysis of cmnc results for autologous donors with a pb cd + count ≥ /ll versus allogeneic donors as well as mnc versus cmnc results in autologous donors. the collection efficiency- (ce- ) was defined as the total cd + amount in the collection bag divided by the amount of cd + cells in the pb processed by the collection apparatus. results: in the cmnc, the following parameters significantly differed between autologous and allogeneic donors: mean collection time ( ae and ae min, respectively; p = . ), the total blood volume processed ( . ae . and . ae . , respectively; p = . ) and the final volume in the collection bag ( ae and ae ml; p = . ). the mean ce- in autologous versus allogeneic donors was ae and ae , respectively (p = . ). using cmnc, the collection was effective in % of allogeneic and % of autologous donors. in autologous donors, a significantly lower collection bag volume ( ae and ae , respectively; p < . ) and increased total wbc in the collection bag ( ae versus ae , respectively; p < . ) were obtained using cmnc compared to mnc protocol. thirteen patients were treated with plerixafor due to a low pb cd + count following g-csf therapy; of them achieved a cd count ≥ and their collection was considered effective. summary/conclusions: the cmnc protocol is highly effective in terms of the cell yield in both allogeneic and autologous donors with a pb cd + count ≥ /ll. significantly superior collection results are obtained in allogeneic donors versus autologous ones. cmnc provides a significantly higher wbc and a lower final collection volume than mnc. similar total cd + cell counts are obtained with both methods. . tbv processed ranged from - . tbv with mean of . , average was . tbv for females and . tbv for males mean pre-apheresis cd + count was . cells/ll (range . - . ). mean postapheresis cd + count was . cells/ll ( . - . ). mean cd + cells x / kg recipients body weight was . (range: . - . ). our target yield was ≥ cd + cells/kg body weight of the recipient and in only / ( %) cases, the yield was < . / ( . %) procedures were lvl and / ( . %) were svl. summary/conclusions: most of our pbsc were done for haematological indications ( . %) and the target dose was cells/ll in single leukapheresis. in cases ( %), target yield was achieved, only cases had < but > yield. in our study donors < years have shown to mobilize better than the older children. hematocrit (hct) and weight showed correlation with cd + cell yield but they cannot be taken absolute predictors. wbc count cannot be taken as a predictor for cd yield as high wbc count did not convert into high cd yield or vice versa. high prepheresis cd + count gave higher postpheresis cd + count. large volume leukapheresis (lvl), > tbv gave higher yield as compared to standard volume leukapheresis (svl). blood volume processed related to prepheresis cd + count and/or the weight difference between the donor and recipient. other parameters like hematocrit, wbc count, age etc did not show correlation to the volume processed. in our study, younger age and prepheresis cd + count were found as the most relevant predictors for stem cell yield. background: allogeneic hematopoietic stem cell transplantation is an established therapy for many hematologic disorders. since the discoveries of the potential of peripheral blood stem cells (pbsc) in the hematopoietic reconstitution mid s and early s pbsc gradually replaced bone marrow as the preferred source of stem cells. the introduction of hematopoietic cytokines that can mobilize large number of progenitors into circulation accelerated pbsc usage. aims: the aim of our study is to present our year experience with apheresis collecting of pbsc in donors. methods: this is a retrospective study performed in the institute for transfusion medicine of republic of macedonia and university hematology hospital for period background: obtaining unambiguous results of hla typing plays an important role in the transplantation of hematopoietic stem cells. appropriate selection of alleles in the level of hla between recipients and unrelated bone marrow donors reduces the risk of transplant rejection and graft-versus-host disease. new generation technology ensures the highest possible resolution and obtaining unambiguous genotyping results due to the high complexity of the hla system. currently, this is the selection method for obtaining hla test results at the high resolution level. aims: the aim of this study was to determine hla loci (hla-a, -b, -c, drb / / / , dqb , dpb , dpa , dqa ) in potential bone marrow donors from poland. the research included , potential bone marrow donors registered between and . a novelty of this paper was that the amplification of all hla loci was performed by using multiplex pcr primers in a single tube. that solution completely eliminated the need to pool amplicons. methods: the typing of the hla loci (hla-a, -b, -c, drb / / / , dqb , dpb , dpa , dqa ) of potential bone marrow donors was made by using the alltype tm ngs -loci amplification kit (one lambda). genomic dna was isolated from peripheral blood of , donors. hla genotypes were determined according to the manufacturer's protocol on the miseq illumina platform. the obtained sequencing data was evaluated by using the typestream tm visual ngs analysis software. results: the ngs method allowed to obtaining unambiguous results of genotyping of potential bone marrow donors, and also provided the identification of rare alleles, such as: c* : , c* : , c* : , c* : , drb * : , c* : , b* : , c* : , dqb * : , drb * : , drb * : . summary/conclusions: . new generation sequencing technology (ngs), which is based on pcr, ensures the highest possible resolution. . the ngs method allows to obtain more accurate sequencing results compared to the conventional methods. . the research has confirmed the superiority of the ngs method over conventional methods in obtaining unambiguous hla genotyping results at the high resolution level. background: the accurate results of hla typing are significant for ensuring the success rate of hematopoietic stem cell transplantation. currently, hla typing is mainly based on sanger sequencing, which has a high proportion of ambiguous combination results indicating potential errors for hla typing. it is necessary for finding a more accurate typing method to reduce the risk. next-generation sequencing (ngs) method could provide clonal sequencing of single molecules, which has been used for hla genotyping and improved the scope and precision of hla study. aims: to establish a full-length precision sequencing platform for hla-i gene (hla-a, -b, -c) based on ngs technology and be evaluated by classical sangersequencing method, which can effectively improve the accuracy of hla typing for donor and recipient in hematopoietic stem cell transplantation. methods: hla-i (hla-a, -b, -c) gene-specific primers were screened, and the amplification parameters were optimized to obtain full-length sequences of hla-i gene under the same condition. the sample library for the amplicon was prepared with transngs tn dna library prep kit and the sequencing step was carried out with illumina miseq platform according to the manufacturer' protocol. all the sequencing data in fastq format were analyzed by typestream visual software version . . (one lambda inc.)with the default setting. cord blood samples were collected for hla typing with the mentioned above next-generation sequencing method in our study. in parallel, all the sample were also tested with the sanger sequencing method according to the previous study in our laboratory. results: samples were successfully tested with two methods and the coincidence rate between two sequencing methods was %. with the next-generation sequencing method, the probability of ambiguous results among samples in our study is . %( / ) for hla-a, . % ( / ) for hla-b and % ( / )for hla-c. however, the probability of ambiguous results with the sanger sequencing method is . % for hla-a, . % for hla-b, % for hla-c. summary/conclusions: the full-length precision sequencing platform for hla-i gene (hla-a, -b, -c) based on ngs technology was established, which could greatly reduce the probability of ambiguous results and effectively improve the accuracy of existing hla typing techniques.