key: cord-0710794-2rjtzcwn authors: Santoro, Angela; Angelico, Giuseppe; Mastrosimini, Maria Gaia; Inzani, Frediano; Cianfrini, Federica; Straccia, Patrizia; Arciuolo, Damiano; D'Alessandris, Nicoletta; Scaglione, Giulia; Spadola, Saveria; Rossi, Esther Diana; Zannoni, Gian Franco title: Diagnostic impact of safety protocols for processing peritoneal washing specimens during global pandemic of Coronavirus disease 2019 : a comparative study from 195 cytological samples date: 2021-08-19 journal: Cytopathology DOI: 10.1111/cyt.13053 sha: 6edd5f7144146832ae139f618e91c65ad88b1e2c doc_id: 710794 cord_uid: 2rjtzcwn BACKGROUND: Global pandemic of Coronavirus disease 2019 represented a major concern for the health services worldwide, also determining major changes in cytopathology practice. AIM: We aimed to establish the diagnostic performance of cytologic evaluation with the new safety protocol to process cytological samples compared to the standard one. We also aimed to assess how cytological diagnoses and sampling were impacted during the pandemic period compared to the pandemic‐free period in 2019. MATERIALS & METHODS: Peritoneal washings cytology samples received during COVID‐19 emergency in Italy (March 11, 2020 – January 11, 2021) were compared to the samples received during the same time frame in year 2019. RESULTS: 195 specimens were analyzed in the present study. We did not observe noticeable differences in cytological diagnoses during the pandemic period compared to the pre‐pandemic period in 2019. In fact, during pandemic and pre‐pandemic periods, we observed respectively 0 vs 0 non‐diagnostic (ND), 1 vs 7 atypia of uncertain significance (AUS), 3 vs 0 suspicious (SFM) diagnoses, 52 vs 86 negative for malignancy (NFM) and 4 vs 42 malignancies (MAL). CONCLUSION: A consistent reduction in number of cytological samples has been observed during the COVID‐19 period. Finally, our institutional safety protocol for processing cytological samples did not affect the diagnostic reliability of peritoneal washing samples. This article is protected by copyright. All rights reserved Intraperitoneal spread of neoplastic cells is a well-established phenomenon occurring in several advanced-stage epithelial neoplasms, which may result in serosal involvement with or without concomitant effusion. 1 Therefore, peritoneal washings (PW) in patients with abdomino-pelvic neoplasms, represent an important additional value that can be easily processed for cytologic examination. Neoplastic cells in PW indicate intraperitoneal diffusion of the neoplasm beyond the originary tumor site; this phenomenon often correlates with a worst prognosis and aggressive biological behaviour. 1, 2 The prognostic significance of PW cytology is well documented in gynecological tumors, gastric, pancreatic, and oesophageal tumours. [3] [4] [5] [6] [7] Recently, the worldwide cytopathologists'community introduced a 5-tier international system for reporting serous fluid cytology (TIS) using specific diagnostic categories defined by well-defined criteria and risk of malignancy (ROM): unsatisfactory (ND), benign (NFM), atypia of uncertain significance (AUS), suspicious (SFM), malignant (MAL). 8 In this regard, a recent paper on cytology of serous fluids demonstrated the good diagnostic performance of TIS system in terms of specificity and predictive value, confirming its useful role in the cytological diagnosis of effusions. 9 The global spread of Coronavirus disease 2019 (COVID-19) determined major changes in public health services, also affecting cytopathology practice. 10 In fact, according to the World Health Organization guidelines, all cytological samples must be processed and handled as they carry an infectious potential. 11 There are only two large institutional experiences reported in cytopathology practice during the pandemic period. 12, 13 Both studies reported a significant reduction in the number of the cytological procedures; at the same time, given the prioritization of oncological conditions, an increase in malignant diagnoses was recorded. Recent international survey studies have investigated the major changes related to biosafety procedures affecting cytopathology services worldwide following the spread of Coronavirus disease. 12 The global pandemic determined a significant reduction in the number of cytological procedures performed; however, an increase of malignant diagnoses rate was recorded. 12, 13 In Italy, a novel method for decontamination of cytological materials processed by liquid-based cytology (LBC) has been proposed and utilized to protect the laboratory personnel from infectious risk. 14-16 This article is protected by copyright. All rights reserved In the present study we aimed to ensure the diagnostic performances of cytological evaluation with the new safety protocol to process cytological samples compared to the standard one. Moreover, we also assessed how cytological diagnoses and sampling were impacted during the pandemic period compared to the pandemic-free period in 2019. Data regarding PW were collected during the ten months before and after the March 11, 2020, the watershed between the standard and the new protocol. From March 11, 2020 all cytological samples processed in our institution were considered as potentially infectious. Serous fluids from PW, were processed by specialized technicians wearing protective equipments inside a dedicated biosafe hood. Glass slides were then placed into a 70% alcohol fixative solution and 99% ethanol was added to the solution for decontamination purposes. 13, 15 Cytological materials were prepared by ThinPrep method (Liquid Based Cytology -LBC). In detail, all LBC specimens processed in our laboratory followed a strict protocol to reduce the risk of contamination (Table 1): 1. Sample collection in a 70% ethyl alcohol solution. 2. Centrifugation: 600g for 10 minutes or 1200g for 5 minutes. Cytological specimens before March 11, 2020 were processed with our institutional standard liquid-based cytology protocol 16,17 in detail, cytological samples were fixed in methanol based buffered preservative solution and processed using ThinPrep (Hologic Inc) ( Table 1) In both pandemic and pandemic-free groups, the demographic details and the patient's history were recorded. This article is protected by copyright. All rights reserved PW samples were classified into five categories according to the well-defined criteria and risk of malignancy (ROM) for serous fluids proposed in 2019 as TIS (International reporting system for serous fluid cytology): unsatisfactory (ND), negative (NFM), atypia of uncertain significance (AUS), suspicious (SFM), malignant (MAL). 8 Cytological procedures performed prior and before the turning point (March 11, 2020) were compared, as well as the percentage of all five categories by the pandemic protocol and the prepandemic standard procedure. Furthermore, 48 and 13 PW patients, from the pre-pandemic and pandemic periods, respectively, had corresponding peritoneal biopsies. Continuous data were reported as median and ranges. Categorical data were reported as counts and percentages. The variations between the two groups about the percentage of all five cytological categories and the concordance with the peritoneal biopsies were evaluated using the Person's chi squared (χ 2 ) or Fisher's exact test. P values lower than 0.05 were considered statistically significant. (Table 2 ). Concerning the rate of benign (40% vs 32%) and malignant conditions (60% vs 57%) justifying the need to perform PW cytology, for both reference periods, we did not observe significant differences. Gynecological tumors represented half of the neoplastic conditions under both groups (30% vs 29%). Data were summarized in Table 3 ). Clinical conditions determining PW cytological exams during pandemic period were ascites (n = 41), including 24 due to chronic liver disease, inflammation and heart failure (40%), and 17 associated to malignancy, 9 of which related to a clinical-instrumental suspect of carcinosis. This article is protected by copyright. All rights reserved During the COVD-19 period there was 73% reduction of peritoneal biopsies performed compared to the pre-pandemic period (13 against 48). Within pre-COVID-19 period, 97% of MAL serous fluids had biopsy-proven peritoneal involvement (n=32). Two AUS PWs were unsolved also on biopsies because of inadequate bioptic sampling; both cases showed low cellularity and artefactual changes that altered the morphology of malignant cells, invalidating also the immunohistochemical results. The primary tumor was a gastric poorly cohesive carcinoma in one case and an unknown primary site in the other case. Over the course of the COVID-19 period, 100% of MAL PWs had biopsy-proven peritoneal involvement (n=4). However, 8 out of 52 NFM PWs underwent peritoneal biopsy, and 38% resulted positive on histology (n=3). By comparing the two groups, statistically significant differences were not observed. Examples on cytohistological comparison between peritoneal washing specimens and corresponding histological samples are illustrated in Figure 3 . Finally, regarding the COVID-19 status of the laboratory staff, none of the staff got COVID-19 during processing the fluids due to the precaution steps taken. The COVID-19 emergency drastically changed laboratory organization and cytopathology practice. 10 The potential presence of Coronavirus-19 in cytological samples, imposes strict biosafety protocols, according to the recent World Health Organization laboratory biosafety guidelines. 11 Liquid-based cytology appears as a still safer technical opportunity, considering that according to this procedure the cytological specimen is directly collected in the fixative, able to inactivate the virus, and processed in a closed system. Moreover, the adoption of TIS diagnostic criteria for reporting serous fluid cytology represented an important step to standardize the cytological procedures, the laboratory handling and the reporting terminology. 8 The results from our study indicate that, from 195 PW specimens analysed according to TIS terminology, significant differences in diagnostic cytological categories during the pandemic and pre-pandemic period were not observed. However, a relative decrease of 24% (31% vs 7%) was recorded in the malignant category. The cases in benign category increased of 23% (64% vs 87%), however this was not statistically significant (P-value 0.079). This difference between malignant and benign diagnoses observed can be explained by: 1) a reduction in the number of cytological procedures performed during the pandemic period; 2) the fact that our hospital is considered a tertiary referral center mainly focused in oncological patients. This article is protected by copyright. All rights reserved However, we have also observed an intriguing result: despite the recommended prioritization of oncological patients during COVID-19 pandemic, the number of malignant cases was not superior to the pre-lockdown period. This result seems to be quite different from other studies [11] [12] , that not only reported a significant reduction in the number of the cytological procedures but at the same time also described an increase in malignant diagnoses. We have to precise that all analysed samples come from: 1) patients with suspicion for neoplastic/malignant disease; 2) patients affected by malignant neoplasms, already treated in our Hospital. In these latter cases the PW would represent a stadiative procedure, a prognostic tool or also a component of follow-up programmes in the patient clinical management. In this perspective, despite the decrease of overall malignancy rate, the prioritization of neoplastic patients has been ensured. Furthermore, an important finding emerged from our study is represented by the diagnostic performance of our institutional ethanol-based safety protocol for processing specimens. The latter protocol have caused only very limited changes mainly concerning cellular morphology and nuclear details. By this modified technique, we have observed a slight increase of the fibrin amount in the background, probably due to the rapid fixation of the hemorrhagic material in a large volume of ethanol. Moreover, a decrease in cellularity with smaller and more scattered cells in comparison with the pre-pandemic method of preparation was also noted. However, based on our routinary experience in the pre-COVID period with the standard methanolbased protocol, we did not observe significant differences in cytoarchitectural features as well as diagnostic categories. These results are in line with a previous study reporting the cytological experience with this novel ethanol-based protocol. 9 In fact, ND, AUS and SFM diagnoses were respectively 0, 7, 0 during COVID 19 period and 0,1,2 in 2019, before pandemic spread. Finally, during the COVD-19, similarly to the reduction of cytological procedures, there was also 73% reduction of peritoneal performed biopsies compared to the pre-COVID-19 period. Moreover, within both periods, by the different adopted procedures, we can confirm a good correlation between MAL cytological diagnosis and biopsy-proven peritoneal involvement histology, in this way highlighting TIS role in confirming the definitive diagnosis of malignancy. We reported our institutional experience in processing and evaluation of cytological specimens during COVID-19 period where a consistent reduction in number of performed procedures has This article is protected by copyright. All rights reserved been observed. Despite high-risk, oncological patients have been prioritized, the overall malignancy rate did not increase. Moreover, given the risks associated with the methanol-based procedure, our results showed that the new described laboratory adaptations to COVID-19 emergency provided safe procedures for sanitary operators, without compromising diagnostic conclusions and making our experience useful also for other pathological labs dealing with pandemic. Finally, the implementation of TIS in diagnostic report has provided good results in term of diagnostic performance and adequate clinical decision-making. The present research received no fundings. No conflict of interest declared. The data that support the findings of this study are available from the corresponding author upon reasonable request. Table 1 . Slide preparation methods in pre-pandemic and pandemic period Table 2 . Demographic details of patients in pre-COVID-19 and COVID-19 period. Diagnostic Principles and Clinical Correlates Practical Pathology of Serous Membranes Accepted Article This article is protected by copyright. All rights reserved Peritoneal washing cytology. Uses and diagnostic criteria in gynecologic neoplasms The role of cytology in endometrial cancer: Diagnostic and clinical considerations from peritoneal/pelvic washings. Is it still a heated debate? Cancer Cytopathol Evaluation of pelvic washing specimens in patients with endometrial cancer: Cytomorphological features, diagnostic agreement, and pathologist experience The Morphological Analysis of Cells in the Peritoneal Washing Fluids of Patients with Gastric Cancer Predicting positive peritoneal cytology in pancreatic cancer The International System for Reporting Serous Fluid Cytopathology (TIS) -Diagnostic categories and clinical management Cytohistological correlation in serous effusions using the newly proposed International System for Reporting Serous Fluid Cytopathology: Experience of an oncological center Defining the epidemiology of Covid-19 -Studies needed Laboratory biosafety guidance related to coronavirus disease 2019 (COVID-19). Interim guidance Global impact of the COVID-19 pandemic on cytopathology practice: Results from an international survey of laboratories in 23 countries Impact of ongoing COVID-19 pandemic on cytology: An institutional experience Biosafety procedures for handling intraoperative surgical samples during COVID-19 pandemic: an Italian pathology laboratory experience Description of a new biosafe procedure for cytological specimens from patients with COVID-19 processed by liquid-based preparations Fix the collected sample in the haemolytic and methanol-based, buffered,preservative solution Cytolyt™ Resuspend the cell pellet Evaluate the cell pellet Add an appropriate amount of the specimen (depending on the size of the cell pellet) to the PreservCyt solution vial