key: cord-319013-oytqcifa
authors: Focosi, Daniele; Anderson, Arthur O.; Tang, Julian W.; Tuccori, Marco
title: Convalescent Plasma Therapy for COVID-19: State of the Art
date: 2020-08-12
journal: Clin Microbiol Rev
DOI: 10.1128/cmr.00072-20
sha: 
doc_id: 319013
cord_uid: oytqcifa

Convalescent plasma (CP) therapy has been used since the early 1900s to treat emerging infectious diseases; its efficacy was later associated with the evidence that polyclonal neutralizing antibodies can reduce the duration of viremia. Recent large outbreaks of viral diseases for which effective antivirals or vaccines are still lacking has renewed the interest in CP as a life-saving treatment. The ongoing COVID-19 pandemic has led to the scaling up of CP therapy to unprecedented levels. Compared with historical usage, pathogen reduction technologies have now added an extra layer of safety to the use of CP, and new manufacturing approaches are being explored. This review summarizes historical settings of application, with a focus on betacoronaviruses, and surveys current approaches for donor selection and CP collection, pooling technologies, pathogen inactivation systems, and banking of CP. We additionally list the ongoing registered clinical trials for CP throughout the world and discuss the trial results published thus far.

dengue virus, and Zika virus) (2) , chikungunya virus (3) , influenza viruses A [e.g., A(H1N1) and A(H5N1)] (4), Ebola virus (EBOV) (5) , and respiratory betacoronaviruses (SARS-CoV and Middle East respiratory syndrome-CoV [MERS-CoV]), which could put us in situations very similar to the situation with the current pandemic and which require the development of specific intervention protocols.

While vaccination strategy is undoubtedly a viable goal, development of a vaccine requires a time frame not compatible with an emergency situation. It is also a prophylactic approach that has no use in the therapeutic setting. On the other hand, the use of antivirals is valuable for the therapeutic setting (6, 7) . For the limited number of antiviral agents currently available, unless provided free of charge to developing countries, financial cost is an issue. Additionally, manufacturing is hard to scale up in short time frames.

In situations in which the new pathogen is able to induce an immune response with the production of neutralizing antibodies, passive transfusion of convalescent blood products (CBPs), in particular, convalescent plasma (CP), has proven to be a winning and logistically feasible therapeutic strategy (8) . CBPs can be manufactured by collecting whole blood or apheresis plasma from a convalescent donor. This approach has been used since 1900 (9) , and previous experiences have been reported elsewhere (10) .

The main accepted mechanism of action for CBP therapy is clearance of viremia, which typically happens 10 to 14 days after infection (11) . So CBP has been typically administered after the appearance of early symptoms to maximize efficacy. Convalescent whole blood (CWB), in addition to antibodies, provides control of hemorrhagic events, as in Ebola virus disease, if transfusion occurs within 24 h to maintain viable platelets and clotting factors. Nevertheless, CP best fits settings where only antibodies are required.

In this review, we have described current technologies for CP collection, manufacturing, pathogen inactivation, and banking of CP. Then we have summarized historical settings of CBP application, with a specific focus on applications for COVID-19 and other future pandemics. Several articles included in this review are available as preprints which have not yet passed peer review, as indicated in the reference section.

Convalescent donor testing for neutralizing antibodies is mandatory in upstream donor selection. Donor selection is generally based on neutralizing antibody titer, as assessed with a plaque reduction neutralization test (PRNT) (12) , which requires a viable isolate, replication-competent cell lines, and skilled personnel. Since PRNT takes time to be set up and requires expensive facilities, in resource-poor settings or in time-sensitive scenarios, collection based on a retrospective PRNT or, alternatively, on an enzymelinked immunosorbent assay (ELISA) targeting the recombinant receptor binding domains (RBDs) of the viral antireceptor has often been implemented; under these circumstances, studies have suggested that ELISA ratios/indexes have good correlations with PRNT titers; e.g., the Euroimmun ELISA IgG score detected 60% of samples with PRNT titers of Ͼ1:100, with 100% specificity using a signal/cutoff reactivity index of 9.1 (13) . The current understanding of neutralization suggests that the virus-blocking effect is related to the amount of antibodies against different epitopes coating the virion, whose stoichiometry is in turn affected by antibody concentration and affinity.

The donor should preferably live in the same area as the intended recipient(s) to allow consideration of mutations of the target viral antigens. SARS-CoV-2 S protein has already mutated after a few months of viral circulation (14) , with one mutation outside the receptor-binding motif (23403A¡G single nucleotide polymorphism, corresponding to a D614G amino acid change) currently defining a dominant clade (15) characterized by reduced S1 shedding and increased infectivity (16) . Nevertheless, it should be considered that preferring indigenous donors could represent a drawback in areas with epidemics of other infectious diseases (e.g., malaria).

Three approaches are theoretically available to recruit CP donors, with each having pros and cons. The least cost-effective approach is screening the general regular blood donor population for the presence of anti-SARS-CoV-2 antibodies. In areas of endemicity, such a strategy provides many fit donors with the additional benefit of seroprevalence study in the general population (80% of cases being asymptomatic) but requires a large budget.

Alternatively, recruitment of hospital-discharged patients is highly cost-effective (patients can be easily tested before discharge and tracked), but patients who have required hospitalization are highly likely to be elderly with comorbidities and, hence, unfit to donate.

The intermediate approach, whenever allowed by privacy regulations, is making calls to positive cases under home-based quarantine to solicit donations; given the large numbers of such cases, some of them are likely to be regular donors, and home-based convalescence suggests that they are fit enough to donate. Nevertheless, lessons from MERS (17) and preliminary evidence with COVID-19 (18) (19) (20) suggest that patients with mild symptoms may develop low-titer antibodies, making antibody titration even more important in the population-wide and home-based approaches. Plasma samples collected an average of 30 days after the onset of symptoms had undetectable half-maximal neutralizing titers in 18% of donors (21) .

Under emergency settings, it has often happened that donors are not screened for high-titer neutralizing antibodies or that low-titer donations are collected; nevertheless, as soon as the urgent requests are satisfied and a buffer stock has been created, repeat donations should preferably focus on donors with high titers (22) .

As recently suggested, plasmapheresis could additionally benefit the convalescent COVID-19 donor by reducing the prothrombotic state via the citrate-based anticoagulants administered during donation and by removal of high-molecular-weight viscous components (23) .

In addition to interventional trials, in the United States several trials have been initiated to create registries (e.g., ClinicalTrials.gov registration no. NCT04359602) or collect plasma with titers of Ͼ1:64 from immune donors for banking purposes, without immediate reinfusion (e.g., trial NCT04360278, NCT04344977, or NCT04344015). These approaches should be encouraged to better face the next waves of the COVID-19 pandemic.

CP should be collected by apheresis in order to ensure larger volumes than available with whole-blood donations and more frequent donations and to avoid causing unnecessary anemia in the convalescent donor. Double filtration plasmapheresis (DFPP) using fractionation filter 2A20 is under investigation as an approach to increase IgG yield by 3 to 4 times ( Table 1 , trial NCT04346589 in Italy); since DFPP-derived plasma is not an ordinary blood component but, rather, a discard product, additional regulations could apply in different countries. A very exploratory approach is under investigation in a Chinese trial collecting immunoglobulins from convalescent donors by immunoadsorption (trial NCT04264858), which could be an alternative to plasma fractionation.

Although neither the U.S. Food and Drug Administration (FDA) (24) nor the European Center for Disease Control (ECDC) is recommending pathogen reduction technologies (PRT) for CP (25) , several national authorities consider that, under emergency settings, donor screening and conventional viral nucleic acid testing (NAT) (i.e., HIV, hepatitis C virus [HCV] , and hepatitis B virus [HBV] NAT) would not be enough to ensure CP safety (12) . Under this scenario, additional virological testing and PRT approximately double the final cost of the therapeutic dose. Several technologies for PRT have been approved and are currently marketed.

Solvent/detergent (S/D)-filtered plasma provides quick inactivation of Ͼ4 logs of most enveloped viruses; although the technology was developed and is widely used for large plasma pools, small-scale reduction has been reported. The technology relies on several steps: addition of 1% tri(n-butyl) phosphate-1% Triton X-45, elimination of solvent and detergent via oil extraction and filtration, and finally sterile filtration (26) . Filtration across 75-to 35-nm-pore-size hollow fibers could remove large viruses (such as betacoronaviruses) while preserving IgG (27), but this has not been implemented yet.

In recent years photoinactivation in the presence of a photosensitizer has become the standard for single-unit inactivation; approved technologies include combinations of methylene blue and visible light (28) (Theraflex), amotosalen (S-59) and UV A (29) (Intercept), and riboflavin and UV B (30) (Mirasol). These methods do not affect immunoglobulin activity.

Fatty acids are also an option. In 2002 it was reported that caprylic acid (31) and octanoic acid (32) were as effective as S/D at inactivating enveloped viruses.

Heat treatment of plasma has been used in the past (33, 34) but comes with a risk of aggregation of immunoglobulins (35, 36) . Figure 1 represents how CP and intravenous immunoglobulin (IVIg) can be obtained under modern fractionation procedures. As per CP collection, two approaches can be pursued. Large-pool products. Pharmaceutical-grade facilities typically pool 100 to 2,500 donors to manufacture S/D-inactivated plasma. IVIgs are similarly prepared from pools of 2,000 to 4,000 liters of plasma (or 100 to 1,000 liters in the case of hyperimmune IVIg) (37, 38) . Such volumes can hardly be obtained from CP donors, and timely creation of dedicated CP production chains pose difficult good manufacturing practice (GMP) issues within plasma vendor plants (38) .

MPFS into immunoglobulins. In order to be economically sustainable, contract (private-run) fractionation typically requires well over 10,000 liters of plasma per year, and domestic (state-owned) fractionation typically requires over 100,000 to 200,000 liters per year in addition to starting up a fractionation facility. An "on-the-bench" minipool fractionation scale (MPFS) process (5 to 10 liters of plasma, i.e., approximately 20 recovered plasma units) using disposable devices and based on caprylic acid precipitation has been under development in Egypt since 2003 and has proved effective at purifying coagulation factors (39) and immunoglobulins (6-fold enrichment) (40) . The same disposable bag system has also been combined with S/D reduction (26) .

CP can be either frozen or transfused as a fresh product. Aliquots of 200 to 300 ml can be easily achieved from a single unit using modern PRT kits. Banking CP at temperatures below Ϫ25˚C (according to European Directorate for the Quality of Medicines [EDQM] or FDA guidelines for ordinary plasma for clinical use [41] ) is encouraged in order to produce CP as an off-the-shelf, ready-to-use product. Most regulatory systems require that CP be tracked informatically as a blood component different from ordinary plasma for clinical use. The final validation label should report that the donor has tested negative by PCR for the convalescent disorder and additional microbiological tests and should describe the inactivation method. A single cycle of freezing and thawing does not significantly affect the quantity or function of immunoglobulins (42) . Given that COVID-19 AB blood group recipients can receive CP units only from scarce matched blood group AB donors, to increase the pool of compatible units several authors have recommended titration of anti-A and anti-B isoagglutinins and transfusion of low-titer (Ͻ1:32) non-ABO-compatible CP units (i.e., O, A, and B) to AB recipients (22, 43) .

SARS-specific neutralizing antibodies usually persist for 2 years (44), and a decline in prevalence and titers occurs in the third year (45) . Convalescent anti-SARS immunoglobulins were manufactured on a small scale (8, 46) . Three infected health care workers with SARS progression despite the best supportive care (BSC) survived after transfusion with 500 ml of CP; viral load dropped to zero at 1 day after transfusion (47). Soo et al. reported in a retrospective nonrandomized trial that treatment with CP (titer of Ͼ1:160) in 19 patients was associated with a shorter hospital stay and lower mortality than continuing treatment with high-dose methylprednisolone (48) . Amotosalen photochemical inactivation of apheresis platelet concentrates demonstrated a Ͼ6.2 log 10 mean reduction of SARS-CoV (49) . Theraflex reduces infectivity of SARS-CoV in plasma (50) . Heating at 60°C for 15 to 30 min reduces SARS-CoV from plasma without cells (51) , while maintaining 60°C for 10 h is required for plasma products (52) . In addition, SARS-CoV was found to be sensitive to S/D (51, 53) .

Antibody responses to MERS persist for less than 1 year, and the magnitude correlates with the duration of viral RNA shedding in sputum (but not with viral load). Patients with mild disease have very low antibody titers, making CP collection challenging in MERS convalescents (54) . A study reported that only 2.7% (12 out of 443) exposed cases tested positive by ELISA, and only 75% of them had reactive microneutralization assay titers (17) . CP with a PRNT titer of Ն1:80 provides clinical benefit in MERS (55) . A case of transfusion-related acute lung injury (TRALI) following CP transfusion in a patient with MERS was reported (56, 57) . MERS-CoV load in plasma was reduced by Theraflex (58), Intercept (59), Mirasol (60) , and heating at 56°C for 25 min (61); in all cases, passaging of inactivated plasma in replication-competent cells showed no viral replication.

As soon as the COVID-19 pandemic appeared (62, 63) , several authors suggested CP as a potential therapeutic agent (64, 65) . Of interest, the most critically ill patients show prolonged viremia (strongly correlated with serum interleukin-6 [IL-6] levels) (10), which makes feasible therapeutic intervention with antiviral agents and immunoglobulins even at late stages. Viral shedding in survivors can last as long as 37 days (62), mandating SARS-CoV-2 RNA screening in CP donors. Serum IgM and IgA antibodies appear in COVID-19 patients as early as 5 days after symptom onset (66), while IgG can be detected at day 14 (67) . IgGs are generally detected after 20 days (68, 69) . Severely ill female patients generate IgG earlier and at higher titers (70, 71) ; the greatest part of the neutralizing antibody response has been shown to be associated with the IgG 1 and IgG 3 subclasses (72, 73) . Duration of anti-SARS-CoV-2 antibodies in plasma is currently unknown; while the overall antibody responses for other betacoronaviruses typically declines after 6 to 12 months (74), SARS-specific neutralizing antibodies usually persist for 2 years (44) . So, in the vast majority of countries, a suitable donor could donate 600 ml of plasma (equivalent to 3 therapeutic doses under most current trials) every 14 days for a minimum of 6 months. Up to 7 plasma donations have been proven not to decrease antibody titers in convalescent donors (18) . In contrast to SARS and MERS patients, most COVID-19 patients exhibit few or no symptoms and do not require hospitalization; this could suggest that the majority of convalescent donors are best sought in the general population although specific studies on antibody titers in mildly symptomatic patients suggest low titers (18) (19) (20) .

SARS-CoV-2 is reduced by Ͼ3.4 logs by Mirasol (75) (and likely by other PRTs); nevertheless, SARS-CoV-2 viral RNA (vRNA) is detectable at low viral loads in a minority of serum samples collected in acute infection but is not associated with infectious SARS-CoV-2 (76) . Intercept treatment has been proven not to reduce SARS-CoV-2 neutralizing antibody titers (77) .

The main contraindications to CP therapy are allergy to plasma protein or sodium citrate, selective IgA deficiency (Ͻ70 mg/dl in patients 4 years old or older), leading to anaphylaxis from IgA-containing CP (78) , or treatment with immunoglobulins in the last 30 days (because of a risk of developing serum sickness). As in many other trial settings, concurrent viral or bacterial infections, thrombosis, poor compliance, short life expectancy (e.g., multiple-organ failure), and pregnancy or breastfeeding are also contraindications (79) .

In an early case series from China, five patients under mechanical ventilation (4 of 5 with no preexisting medical conditions) received transfusions of CP with an ELISA IgG titer of Ͼ1:1,000 and a PRNT titer of Ͼ40 at days 10 to 22 after admission. Four patients recovered from acute respiratory disease syndrome (ARDS), and three were weaned from mechanical ventilation within 2 weeks of treatment, with the remaining patients being stable (80) .

Another Chinese pilot study (ChiCTR2000030046) of 10 critically ill patients showed that one dose of 200 ml of CP with a neutralizing antibody titer of Ͼ1:640 resulted in an undetectable viral load in 7 patients, with radiological and clinical improvement (81) .

A third series of 6 cases with COVID-19 pneumonia in Wuhan showed that a single 200-ml dose of CP (with titers of anti-S antibodies determined by chemiluminescent immunoassay [CLIA] only) administered at a late stage led to viral clearance in 2 patients and radiological resolution in 5 patients (82) . Pei et al. reported successful treatment of 2 out of 3 patients with 200-to 500-ml doses of CP (83) . Recovery from mechanical ventilation was also reported by Zhang et al. in a single patient after antibodies in CP were titrated with an anti-N protein ELISA (84) . No improvement in mortality despite viral clearance was reported in a retrospective observational study recruiting 6 late-stage, critically ill patients treated with gold-immunochromatographytitrated CP, compared to results in 13 untreated controls (85) . One case of recovery in a centenarian patient who received 2 CP units (S-RBD-specific IgG titer of Ͼ1:640) was also reported (86) .

Many more case reports and small case series are accumulating in the literature; successful treatment was reported in 3 cases with ARDS and mechanical ventilation using two 250-ml CP doses (titrated with ELISA only) in South Korea (43, 87) , in 2 cases from Iraq (88), in 8 out 10 severe cases from Mexico (89) , in 20 out of 26 severe cases from Turkey (90), in a kidney transplant recipient from China (91) , in a case with severe aplastic anemia in Poland (92) , in a case with X-linked agammaglobulinemia in Spain (93) , and in 1 patient with marginal-zone lymphoma treated with bendamustine and rituximab in the United Kingdom (94) . Centers in the United States reported successful treatment with CP in 18 out of 20 patients in a series (95) , in 27 out of 31 patients with severe to life-threatening disease in another series (96) , in one case with myelodysplastic syndrome (97) , in a critically ill obstetric patient (in combination with remdesivir) (98) , and in an allogeneic stem cell transplant recipient (99) .

In a single-arm phase II trial (NCT04321421 [100] ) run in Lombardy, 49 patients with moderate to severe disease were treated with up to 3 units of PRT-treated CP (250 to 300 ml/48 h) having neutralizing antibody titers of Ն1:160 in 96% of cases. Importantly, the viral inoculum was 50 50% tissue culture infective doses (TCID 50 ) instead of the usual 100 TCID 50 . Seven-day mortality was 6% versus 16% in a historical cohort. One case of TRALI was reported (101) .

In a large case series from Wuhan, 138 patients were transfused with 200 to 1,200 ml of CP at a median of 45 days after symptom onset and experienced a 50% lower intensive care unit (ICU) admission rate and mortality than the group treated with best supportive care. Responders had higher lymphocyte counts, lower neutrophil counts, and lower lactate dehydrogenase (LDH), type B natriuretic peptide (BNP), urea nitrogen, procalcitonin, glucose, and C-reactive protein (CRP) levels. Complete data on neutralizing antibody titers in COVID-19 convalescent plasma (CCP) units were not available, but responders tended to have received CP units with higher antibody levels (102) .

In the first retrospective, randomized controlled trial published to date, 39 patients in New York with severe COVID-19 were transfused with 2 units of ABO-type matched CP with anti-Spike antibody titers of Ն1:320 (measured by a two-step Spike proteindirected ELISA). CP recipients were more likely than control patients to not increase their supplemental oxygen requirements by posttransfusion day 14 (odds ratio [OR], 0.86), but survival improved only for nonintubated patients (hazard ratio [HR], 0.19) (103) .

Another prospective, multicenter randomized controlled trial from China (ChiCTR2000029757) enrolled 103 patients with severe to life-threatening COVID-19. The study was underpowered because of earlier than expected (200 cases) termination. CP (9 to 13 ml/kg from donors with S-RBD IgG titer of Ն1:640) was associated with a negative SARS-CoV-2 PCR test at 72 h in 87.2% of the CP group versus 37.5% of the BSC group, but clinical improvement at 28 days was statistically different only in patients with severe, but not in life-threatening, disease (104) . Table 1 lists the other ongoing CP trials in COVID-19 patients collected from different web portals. The United States has developed a specific platform for facilitating clinical trials (https://ccpp19.org/), while the International Society of Blood Transfusion created a resource library (https://isbtweb.org/coronaoutbreak/covid-19 -convalescent-plasma-document-library/). At the same time, in the United States an expanded-access program (EAP) has been approved by the FDA and coordinated by Mayo Clinic and has led to treatment of more than 30,000 patients as of 8 July 2020 (https://www.uscovidplasma.org). A preliminary report on the first 20,000 patients (66% from intensive care units) confirms safety (Ͻ1% severe adverse events and 14.9% mortality at 14 days) and suggests a benefit compared to results with historical cohorts, especially if CP is administered before mechanical ventilation (105, 106) ; donor titers were not disclosed, and evidently some donations were not titrated before reinfusion. Largely similar data have been reported from a 25-patient case series from Houston, Texas, where CP has been used as an emerging investigational new drug (eIND) (107).

Typically, 1 or 2 doses of 200 ml are administered (if 2 doses are used, they are administered at least 12 h apart), with infusion rates of 100 to 200 ml/h. The cumulative dose should be targeted according to body weight and antibody titer (22) .

Several authors have suggested plasma exchange with CP (i.e., high-volume therapeutic plasmapheresis followed by CP transfusion) rather than CP transfusion alone in order to clear proinflammatory cytokines from the bloodstream (108, 109) , and several successful case reports deploying nonconvalescent plasma have been reported (110) (111) (112) . One randomized controlled trial (NCT04374539) is ongoing in patients with severe COVID-19, but unfortunately no trial to date is testing plasmapheresis followed by CP.

Unfortunately, most trials in westernized countries (in contrast to ones ongoing in China) have no control arm, which will impair efficacy interpretation. When present, the control arm consists of the best supportive care alone (typically oxygen and hydroxychloroquine at 400 mg twice a day [b.i.d.] for 10 days) or combined with intravenous placebo or standard (nonconvalescent) plasma (eventually of pharmaceutical grade). Since other plasma components (e.g., aspecific immunoglobulins or isoagglutinins; see below) could contribute to clinical benefit, the latter approach is ideal for dissecting the specific contribution of neutralizing antibodies although concerns could be raised by the prothrombotic nature of COVID-19 pathology (see Side Benefits from CP in COVID-19, below). Even using a placebo control in late-stage patients (refractory to former lines) could pose some ethical concerns because it denies treatment opportunities to an unresponsive disease. Future trials should investigate combined antiviral and CP therapies.

Notably, several plasma manufacturers are attempting to develop SARS-CoV-2specific hyperimmune sera (e.g., Takeda's TAK-888 merged with Biotest, BPL, LFB, Octapharma, and CSL Behring into the Convalescent Plasma Coalition [113] ; Kedrion and Kamada have joint ventures [81] ).

CP is considered an experimental therapy, and, as such, phase 3 randomized controlled trials should be encouraged. Despite this recommendation, in emergency settings phase 2 trials are usually started, hampering efficacy analysis. Response in published trials is generally measured clinically (PaO 2 /FiO 2 ratio) or radiologically according to target organs. Nevertheless, surrogate endpoints can include anti-SARS-CoV-2 antibody titer or absolute lymphocyte count increases in recipients, as well as decreases in recipients' SARS-CoV-2 viral load or IL-6 levels. Whenever quantitative PCR is not available, cycle threshold (C T ) value increases in qualitative PCR after transfusion could be a proxy for reduced viral load.

The first concern is transfusion-transmitted infection (TTI (114) or by some individuals. In addition, there is a now a recognized risk of hepatitis E the within the U.K. blood donor population (115) , most likely due to the consumption of poorly cooked pork products (116, 117) , for which screening has only relatively recently been initiated (71) . Although this does not preclude such SARS-CoV-2 convalescent plasma/serum from being used therapeutically within the United Kingdom, these other risks should be considered during larger clinical trials or with compassionate use in individual patients. Respiratory betacoronaviruses produce only a mild and transient viremia. With SARS-CoV, limited replication in lymphocytes (118) leads to significant risk only for recipients of blood products with high concentrations of donor lymphocytes (peripheral blood stem cells, bone marrow, granulocyte concentrates, etc.). Preliminary reports have shown that SARS-CoV-2 viremia persists only in critically ill patients (10) .

The second concern is TRALI, which can be life-threatening in patients who are already suffering from ALI. Male donors are usually preferred in order to avoid the risk of transfusing anti-HLA/HNA/HPA antibodies from parous women. In the case of COVID-19, where female patients have been shown to have higher IgG levels, this could be detrimental, and anti-HLA/HNA/HPA antibody screening could be implemented.

Antibody-dependent enhancement (ADE) is also a theoretical concern related to passive or active antibodies (targeting S protein domains other than the RBD) facilitating IgG-coated virion entry into macrophages via Fc␥ receptors and/or complement receptors (119, 120) , leading to activation of the RNA sensing Toll-like receptors (TLR) 3, 7, and 8 and finally to elevated production of tumor necrosis factor (TNF) and IL-6 (a so-called cytokine storm). ELISAs discriminating the difference between total and RBD-binding antibodies could be useful to inspect the occurrence of ADE. Genetic polymorphisms (e.g., Fc␥RIIa [121] ) can also contribute to ADE. To date, potential evidence supporting a role for ADE in COVID-19 include the following: (i) the correlation between disease severity and total anti-SARS-CoV-2 antibody levels (70, (122) (123) (124) , including neutralizing antibodies (125, 126) ; (ii) the low prevalence of symptoms in COVID-19 patients younger than 20 (who have likely not been primed by infection with the other common cross-reacting coronavirus 229E or OC43 or anyway have low-affinity anti-coronavirus IgG [127, 128] ); (iii) the occurrence, in SARS, of ADE at low antibody titers in vitro (129) and correlation in patients of high IgG titers and early seroconversion with disease severity (130) . Overall, these findings raise concerns for usage of low-titer CP units (131) . Other evidence is the high level of afucosylated IgG against S protein, facilitating FcR binding, that is produced in the most severely ill patients (132, 133) .

A last, COVID-19-specific, concern is worsening of the underlying coagulopathy (134) from clotting factors in transfused plasma (not only CP but also nonconvalescent plasma in control arms); since this has not been reported to date, it remains a theoretical concern.

Obviously, patients with humoral immune deficiencies can benefit from polyclonal antibodies contained in CP, and patients with hemorrhagic diathesis can benefit from clotting factors.

Plasma is also likely to contain antibodies against other common betacoronaviruses associated with the common cold, which have been shown to cross-react with SARS-CoV-2 antigens in intravenous immunoglobulin (IVIg) preparations (135) , likely stemming from recent infection with another human betacoronavirus (128) . Accordingly, IVIg led to clinical and radiological recovery in 3 Chinese patients with severe COVID-19 (136) , and the same team is now leading a randomized controlled trial (NCT04261426).

After demonstration that blood group O health care workers were less likely to become infected with SARS-CoV (137), a research group proved that anti-A blood group natural isoagglutinins (which can also be found in CP plasma from blood group O and B donors) inhibit SARS-CoV entry into competent cells (138) . Such binding could opsonize virions and induce complement-mediated neutralization (139) . Since SARS-CoV-2 uses the same receptor as SARS-CoV, anti-A isoagglutinins are expected to have similar effects against SARS-CoV-2 (140); accordingly, clusters of glycosylation sites exist proximal to the receptor-binding motif of the S protein from both SARS-CoV (141) and SARS-CoV-2 (142) . Several publications showed that the odds ratio for acquiring COVID-19 is higher in blood group A than in blood group O (143) (144) (145) (146) (147) , and one showed the ABO gene polymorphism to be the most significant at predicting severity of COVID-19 (147) . COVID-19 has more severe clinical presentations and outcomes in the elderly and in males; intriguingly, elderly males are known to experience reductions in isoagglutinin titers (148, 149) . Although alternative explanations exist (150, 151) , studies are hence ongoing to evaluate correlations between isoagglutinin titers and outcomes in blood group O and B patients (152) . If the correlations are confirmed, while preserving ABO match compatibility, blood group O and B donors for CP in COVID-19 could be preferred, and their anti-A isoagglutinin titers should be tested.

CP manufacturing should be considered among the first responses during a pandemic while antivirals and vaccines are tested. Despite huge competition from trials employing small molecules, multicenter randomized controlled trials should be encouraged in order to establish efficacy and provide hints about the most effective schedule (timing and dose).

Coronavirus disease 2019 -COVID-19

Zika virus

Chikungunya: epidemiology, pathogenesis, clinical features, management, and prevention

Broadly protective strategies against influenza viruses: universal vaccines and therapeutics

Therapeutic strategies to target the Ebola virus life cycle

That escalated quickly: remdesivir's place in therapy for COVID-19

Exploring pharmacological approaches for managing cytokine storm associated with pneumonia and acute respiratory distress syndrome in COVID-19 patients

The effectiveness of convalescent plasma and hyperimmune immunoglobulin for the treatment of severe acute respiratory infections of viral etiology: a systematic review and exploratory meta-analysis

The true historical origin of convalescent plasma therapy

Detectable serum SARS-CoV-2 viral load (RNAaemia) is closely correlated with drastically elevated interleukin 6 (IL-6) level in critically ill COVID-19 patients

Use of convalescent plasma therapy in SARS patients in Hong Kong

Operational protocol for donation of anti-COVID-19 convalescent plasma in Italy

Convalescent plasma therapy for the treatment of patients with COVID-19: assessment of methods available for antibody detection and their correlation with neutralising antibody levels

Controlling the SARS-CoV-2 outbreak, insights from large scale whole genome sequences generated across the world

Emergence of drift variants that may affect COVID-19 vaccine development and antibody treatment

The D614G mutation in the SARS-CoV-2 spike protein reduces S1 shedding and increases infectivity

Feasibility of using convalescent plasma immunotherapy for MERS-CoV infection, Saudi Arabia

Relationship between anti-Spike protein antibody titers and SARS-CoV-2 in vitro virus neutralization in convalescent plasma

Sex, age, and hospitalization drive antibody responses in a COVID-19 convalescent plasma donor population

Clinical predictors of donor antibody titer and correlation with recipient antibody response in a COVID-19 convalescent plasma clinical trial

Convergent antibody responses to SARS-CoV-2 infection in convalescent individuals

COVID-19 convalescent plasma: phase 2

Convalescent plasma, an apheresis research project targeting and motivating the fully recovered COVID 19 patients: a rousing message of clinical benefit to both donors and recipients alike

Recommendations for investigational COVID-19 convalescent plasma

An EU programme of COVID-19 convalescent plasma collection and transfusion: guidance on collection, testing, processing, storage, distribution and monitored use

Solvent-detergent filtered (S/D-F) fresh frozen plasma and cryoprecipitate minipools prepared in a newly designed integral disposable processing bag system

Nanofiltration of single plasma donations: feasibility study

Practical limitations of convalescent plasma collection: a case scenario in pandemic preparation for influenza A (H1N1) infection

Photochemical treatment of plasma with amotosalen and longwavelength ultraviolet light inactivates pathogens while retaining coagulation function

Characterization of plasma protein activity in riboflavin and UV light-treated fresh frozen plasma during 2 years of storage at Ϫ30°C

Enveloped virus inactivation by caprylate: a robust alternative to solvent-detergent treatment in plasma derived intermediates

Inactivation of lipid enveloped viruses by octanoic acid treatment of immunoglobulin solution

Virucidal heat-treatment of single plasma units: a potential approach for developing countries

Passive transfer of scrub typhus plasma to patients with AIDS: a descriptive clinical study

Passive immunotherapy in AIDS: a randomized trial of serial human immunodeficiency virus-positive transfusions of plasma rich in p24 antibodies versus transfusions of seronegative plasma

Passive immunotherapy in AIDS: a double-blind randomized study based on transfusions of plasma rich in anti-human immunodeficiency virus 1 antibodies vs. transfusions of seronegative plasma

Intravenous immunoglobulin G: trends in production methods, quality control and quality assurance

Annex 4. Recommendations for the collection, quality control and regulation of human plasma for fractionation

New methods of plasma fractionation -a presentation of the "minipool" fractionation procedure developed in Egypt

Minipool caprylic acid fractionation of plasma using disposable equipment: a practical method to enhance immunoglobulin supply in developing countries

Guide to the preparation, use and quality assurance of blood components, 19th ed. European Directorate for the Quality of Medicines and Health Care

Effect of multiple freeze-thaw cycles on detection of measles, mumps, and rubella virus antibodies

Convalescent plasma therapy in coronavirus disease 2019: a case report and suggestions to overcome obstacles

Two-year prospective study of the humoral immune response of patients with severe acute respiratory syndrome

Duration of antibody responses after severe acute respiratory syndrome

Purification of severe acute respiratory syndrome hyperimmune globulins for intravenous injection from convalescent plasma

Experience of using convalescent plasma for severe acute respiratory syndrome among healthcare workers in a Taiwan hospital

Retrospective comparison of convalescent plasma with continuing high-dose methylprednisolone treatment in SARS patients

Amotosalen photochemical inactivation of severe acute respiratory syndrome coronavirus in human platelet concentrates

Inactivation of three emerging virusessevere acute respiratory syndrome coronavirus, Crimean-Congo haemorrhagic fever virus and Nipah virus-in platelet concentrates by ultraviolet C light and in plasma by methylene blue plus visible light

Evaluation of inactivation methods for severe acute respiratory syndrome coronavirus in noncellular blood products

Heat sensitivity of a SARS-associated coronavirus introduced into plasma products

SARS-coronavirus (SARS-CoV) and the safety of a solvent/detergent (S/D) treated immunoglobulin preparation

MERS-CoV antibody responses 1 year after symptom onset, South Korea

Challenges of convalescent plasma infusion therapy in Middle East respiratory coronavirus infection: a single centre experience

Possible transfusion-related acute lung injury following convalescent plasma transfusion in a patient with Middle East respiratory syndrome

Feasibility, safety, clinical, and laboratory effects of convalescent plasma therapy for patients with Middle East respiratory syndrome coronavirus infection: a study protocol

Inactivation of Ebola virus and Middle East respiratory syndrome coronavirus in platelet concentrates and plasma by ultraviolet C light and methylene blue plus visible light, respectively

Inactivation of Middle East respiratory syndrome-coronavirus in human plasma using amotosalen and ultraviolet A light

Inactivation of Middle East respiratory syndrome coronavirus (MERS-CoV) in plasma products using a riboflavin-based and ultraviolet light-based photochemical treatment

Heat inactivation of the Middle East respiratory syndrome coronavirus

Clinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan, China: a retrospective cohort study

China Novel Coronavirus Investigating and Research Team. 2020. A novel coronavirus from patients with pneumonia in China

The convalescent sera option for containing COVID-19

Convalescent plasma as a potential therapy for COVID-19

IgA dominates the early neutralizing antibody response to SARS-CoV-2

Antibody responses to SARS-CoV-2 in COVID-19 patients: the perspective application of Convalescent Plasma Therapy for COVID-19 Clinical Microbiology Reviews serological tests in clinical practice

Antibody responses to SARS-CoV-2 in patients with COVID-19

Antibody responses to SARS-CoV-2 in patients of novel coronavirus disease 2019

Guidelines from the expert advisory committee on the Safety of Blood, Tissues and Organs (SaBTO) on measures to protect patients from acquiring hepatitis E virus via transfusion or transplantation

Rapid generation of neutralizing antibody responses in COVID-19 patients

A serological assay to detect SARS-CoV-2 seroconversion in humans

Serological responses in patients with severe acute respiratory syndrome coronavirus infection and cross-reactivity with human coronaviruses 229E, OC43, and NL63

Inactivation of severe acute respiratory syndrome coronavirus 2 in plasma and platelet products using a riboflavin and ultraviolet light-based photochemical treatment

SARS-CoV-2 RNA detected in blood samples from patients with COVID-19 is not associated with infectious virus

Stability and neutralising capacity of SARS-CoV-2-specific antibodies in convalescent plasma

IgA anaphylactic transfusion reactions

Convalescent plasma to treat coronavirus disease 2019 (COVID-19): considerations for clinical trial design

Treatment of 5 critically ill patients with COVID-19 with convalescent plasma

Effectiveness of convalescent plasma therapy in severe COVID-19 patients

Treatment with convalescent plasma for COVID-19 patients in Wuhan

Convalescent plasma to Treat COVID-19: Chinese strategy and experiences

Anti-SARS-CoV-2 virus antibody levels in convalescent plasma of six donors who have recovered from COVID-19

Effect of convalescent plasma therapy on viral shedding and survival in COVID-19 patients

Successful treatment of a centenarian with coronavirus disease 2019 (COVID-19) using convalescent plasma

Use of convalescent plasma therapy in two COVID-19 patients with acute respiratory distress syndrome in Korea

A severe refractory COVID-19 patient responding to convalescent plasma; a case series

Infusion of convalescent plasma is associated with clinical improvement in critically ill patients with COVID-19: a pilot study

Life-saving effect of convalescent plasma treatment in covid-19 disease: clinical trial from eastern Anatolia

Convalescent plasma therapy: helpful treatment of COVID-19 in a kidney transplant recipient presenting with serve clinical manifestation and complex complications

First case of convalescent plasma transfusion in a child with COVID-19-associated severe aplastic anemia

Rapid recovery of a SARS-CoV-2 infected X-linked agammaglobulinemia patient after infusion of COVID-19 convalescent plasma

Convalescent plasma for persisting Covid-19 following therapeutic lymphocyte depletion: a report of rapid recovery

Use of convalescent plasma in hospitalized patients with Covid-19 -case series

Hospitalized COVID-19 patients treated with convalescent plasma in a mid-size city in the midwest

Convalescent (immune) plasma treatment in a myelodysplastic COVID-19 patient with disseminated tuberculosis

The use of convalescent plasma therapy and remdesivir in the successful management of a critically ill obstetric patient with novel coronavirus 2019 infection: a case report

COVID-19 in allogeneic stem cell transplant: high falsenegative probability and role of CRISPR and convalescent plasma

Plasma from donors recovered from the new coronavirus 2019 as therapy for critical patients with COVID-19 (COVID-19 plasma study): a multicentre study protocol

Mortality reduction in 46 severe Covid-19 patients treated with hyperimmune plasma. A proof of concept single arm multicenter interventional trial

Improved clinical symptoms and mortality on severe/critical COVID-19 patients utilizing convalescent plasma transfusion

Convalescent plasma treatment of severe COVID-19: a matched control study

Effect of convalescent plasma therapy on time to clinical improvement in patients with severe and life-threatening COVID-19: a randomized clinical trial

Early safety indicators of COVID-19 convalescent plasma in 5,000 patients

Safety update: COVID-19 convalescent plasma in 20,000 hospitalized patients

COVID-19) patients with convalescent plasma

Get rid of the bad first: therapeutic plasma exchange with convalescent plasma for severe COVID-19

The successful use of therapeutic plasma exchange for severe COVID-19 acute respiratory distress syndrome with multiple organ failure

Efficacy of therapeutic plasma exchange in severe COVID-19 patients

Successful treatment of plasma exchange followed by intravenous immunoglobulin in a critically ill patient with 2019 novel coronavirus infection

Potential effect of blood purification therapy in reducing cytokine storm as a late complication of critically ill COVID-19

Global Vaccine Business Unit on the latest on the coronavirus and Takeda

Is it time to rethink UK restrictions on blood donation?

Hepatitis E virus in blood components: a prevalence and transmission study in southeast England

Pork products associated with human infection caused by an emerging phylotype of hepatitis E virus in England and Wales

Hepatitis E risks: pigs or blood-that is the question

SARS-coronavirus replication in human peripheral monocytes/macrophages

Epitopes required for antibody-dependent enhancement of Ebola virus infection

Antibodydependent enhancement of Ebola virus infection

Influence of Fc␥RIIA and MBL polymorphisms on severe acute respiratory syndrome

Immune phenotyping based on neutrophil-tolymphocyte ratio and IgG predicts disease severity and outcome for patients with COVID-19

Viral kinetics and antibody responses in patients with COVID-19

Global profiling of SARS-CoV-2 specific IgG/IgM responses of convalescents using a proteome microarray

Neutralizing antibodies responses to SARS-CoV-2 in COVID-19 inpatients and convalescent patients

Neutralizing antibody responses to SARS-CoV-2 in a COVID-19 recovered patient cohort and their implications

First infection by all four non-severe acute respiratory syndrome human coronaviruses takes place during childhood

Pre-existing and de novo humoral immunity to SARS-CoV-2 in humans

Antibody-dependent SARS coronavirus infection is mediated by antibodies against spike proteins

Anti-SARS-CoV IgG response in relation to disease severity of severe acute respiratory syndrome

The potential danger of suboptimal antibody responses in COVID-19

Symptomatic SARS-CoV-2 infections display specific IgG Fc structures

Afucosylated immunoglobulin G responses are a hallmark of enveloped virus infections and show an exacerbated phenotype in COVID-19

Autopsy findings and venous thromboembolism in patients with COVID-19: a prospective cohort study

Currently available intravenous immunoglobulin (Gamunex © -C and Flebogamma © DIF) contains antibodies reacting against SARS-CoV-2 antigens

High-dose intravenous immunoglobulin as a therapeutic option for deteriorating patients with coronavirus disease 2019

ABO blood group and susceptibility to severe acute respiratory syndrome

Inhibition of the interaction between the SARS-CoV Spike protein and its cellular receptor by anti-histo-blood group antibodies

HIV-1 incorporates ABO histo-blood group antigens that sensitize virions to complementmediated inactivation

Harnessing the natural anti-glycan immune response to limit the transmission of enveloped viruses such as SARS-CoV-2

Specific asparagine-linked glycosylation sites are critical for DC-SIGN-and L-SIGN-mediated severe acute respiratory syndrome coronavirus entry

Structural, glycosylation and antigenic variation between 2019 novel coronavirus (2019-nCoV) and SARS coronavirus (SARS-CoV)

Relationship between the ABO blood group and the COVID-19 susceptibility

Association between ABO blood groups and risk of SARS-CoV-2 pneumonia

Association between ABO blood groups and clinical outcome of coronavirus disease 2019: evidence from two cohorts

Testing the association between blood type and COVID-19 infection, intubation, and death

Genomewide association study of severe COVID-19 with respiratory failure

Antibody titers in group O platelet donors

Titers of ABO antibodies in group O blood donors

ABO blood group predisposes to COVID-19 severity and cardiovascular diseases

C3 and ACE1 polymorphisms are more important confounders in the spread and outcome of COVID-19 in comparison with ABO polymorphism

Anti-A isohemagglutinin titers and SARS-CoV2 neutralization: implications for children and convalescent plasma selection

Ebola virus convalescent blood products: where we are now and where we may need to go

Focosi is a hematologist employed as resident transfusion physician at the largest blood bank in Italy since 2009. He has been a transplant immunologist and immunogeneticist, quality assurance manager, and production manager. He has received awards from the European Federation of Immunogenetics, the European Society of Organ Transplantation, and the Italian Society of Hematology. He has a Ph.D. degree in Clinical and Fundamental Virology, and a master's degree in Clinical Trials. He has authored 124 articles indexed in PubMed, for a global h-index of 24, on topics ranging from emerging viral infections to new markers of immune competence. Julian W. Tang is a hospital consultant and medical virologist, with special interests in the diagnosis, treatment, epidemiology, and infection control of influenza and respiratory viruses, congenital viral infections, HIV, and blood-borne viruses. He also has a Ph.D. in zoology. He has formerly been associate professor at the University of Alberta and assistant professor at the Chinese University of Hong Kong.Marco Tuccori is a clinical pharmacologist with special interest in pharmacovigilance and pharmacoepidemiology. He also has a Ph.D. in pharmacology and medical physiology. He is currently pharmacovigilance manager at the Unit of Adverse Drug Reactions Monitoring of the University Hospital of Pisa and coordinator of the Tuscan Regional Centre of Pharmacovigilance. He collaborates with the Agenzia Italiana del Farmaco (AIFA) as a member of the Working Group for Signal Detection Analysis on Drugs and Vaccines. He was a member of the Advisory Board (formerly Executive Committee) of the International Society of Pharmacovigilance (ISoP) from 2012 to 2019. He is the author of about 90 articles in peer-reviewed scientific journals and four chapters of books.