key: cord-0830582-vmvmbvju
authors: Cantley, Richard L.; Hrycaj, Steven; Konopka, Kristine; Chan, May P.; Huang, Tao; Pantanowitz, Liron
title: Cytologic Findings in Effusions from Patients with SARS-CoV-2 Infection
date: 2021-01-22
journal: J Am Soc Cytopathol
DOI: 10.1016/j.jasc.2021.01.003
sha: cb0cae82039939e1543a7d78b592909714845112
doc_id: 830582
cord_uid: vmvmbvju

BACKGROUND Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is associated with “flu-like” upper respiratory tract symptoms and pneumonia. Body cavity effusions develop in a subset of patients with advanced disease. Although SARS-CoV-2 is known to be present in certain body fluids (e.g. blood) of COVID patients, it remains unclear if body cavity fluids are sites of infection. Our aim was to characterize the cytologic and clinical findings in COVID-19 patients with effusions. MATERIALS AND METHODS A record search for all cases of body cavity effusion cytology in SARS-CoV-2 positive patients from 3/1/2020 - 9/1/2020 was performed. Clinical history, fluid chemical analysis, cytologic findings, and patient outcomes were recorded. All cytology slides were reviewed. In situ hybridization (ISH) targeting SARS-CoV-2 spike protein transcript (V-nCoV2019-S) was performed on cell block material in all cases. RESULTS 17 effusion cytology cases were identified among 15 COVID patients, including 13 pleural, 2 pericardial, and 2 peritoneal. Most (13/15) patients were hospitalized for COVID complications. 8 patients died during hospitalization, 7 from COVID complications. All fluids were transudative by protein criteria. Lymphocytic or histiocytic inflammation predominated in 12/17 cases. 5 exhibited hemophagocytosis. No viral cytopathic changes or extra-medullary megakaryocytes were seen. Viral RNA was not detected in any case by ISH. CONCLUSION Body cavity effusion is an ominous finding in patients with advanced COVID-19 disease. Such effusions tend to be transudative with lymphohistiocytic inflammation, and commonly exhibit hemophagocytosis, an otherwise rare finding in effusion cytologies. No direct infection of cellular elements by SARS-CoV-2 was identified by ISH.

Severe acute respiratory syndrome coronavirus (SARS-CoV-2) is a novel RNA coronavirus that is the underlying cause of COVID Disease 2019 (COVID-19), which the World Health Organization declared a pandemic in March 2020. The symptoms of COVID-19 are variable and non-specific. 1 The majority of patients infected with SARS-CoV-2 are asymptomatic or develop mild "flu-like" symptoms such as cough, fever, and fatigue. However, a portion of patients (~10%) develop more severe respiratory disease, including interstitial pneumonia and acute respiratory distress syndrome (ARDS), as well as multiple organ dysfunction/failure. Severe cases of COVID have been linked to abundant release of pro-inflammatory cytokines, resulting in a so-called "cytokine storm" and hemophagocytic syndrome, a hyperactive autoinflammatory immune response which leads to tissue recruitment of T cells, neutrophils, and macrophages with resulting end-organ damage. 2 Chest radiographic findings in COVID-19 are typically non-specific. Common computer tomography (CT) findings include ground glass opacities, often with consolidation, typically in a bilateral and peripheral distribution. [3] [4] Body cavity effusions are relatively uncommon but are identifiable on CT imaging in a subset of COVID-19 patients, with pleural effusion present in 5.88% and pericardial effusion in 4.55% of cases in one meta-analysis. 4 Pleural effusions are more common in patients with severe disease. Acute perimyocarditis has been described in a small subset of COVID-19 patients, many of whom present with cardiac tamponade.

To date, literature on biopsy and/or cytology findings in living patients with COVID-19 have been scarce, and mostly limited to case studies and small case series. Pulmonary findings on biopsy have included diffuse alveolar damage (DAD), hyaline membranes, and interstitial inflammation composed predominantly of lymphocytes. [5] [6] [7] [8] Cytology studies in COVID-19 are particularly scarce. One recent report noted plasmacytosis in a bronchoalveolar lavage specimen. 9 Rare reports of pleural effusion specimens have generally noted reactive mesothelial cells and nonspecific mixed inflammation. 5 Autopsy studies of the lungs have generally shown DAD, often with superimposed bacterial bronchopneumonia. [7] [8] Proposed pneumocyte viral cytopathic change including hyperplasia, multinucleation, and intranuclear inclusion bodies have been reported. Systemic findings at autopsy have included hemophagocytosis, multiple thromboemboli, endotheliitis, and tissue megakaryocyte recruitment.

SARS-CoV-2 RNA has been detected in a number of bodily fluids including bronchoalveolar fluid, sputum, feces, blood, and urine. 10 In addition, recent single case studies have identified viral RNA in pleural and pericardial fluid, respectively, by reverse transcriptase polymerase chain reaction (RT-PCR). [11] [12] At this time, it is not clear whether body cavities such as the pleura or pericardium may serve as reservoirs for ongoing or repeat COVID-19 disease, or whether SARS-CoV-2 infection may be present within any of the cellular elements of effusion specimens.

To the best our knowledge, no studies have examined the specific findings in patients with COVID-19 and serous cavity effusions. The goals of our study are therefore to determine the pertinent clinical and pathologic findings in patients with COVID infection and body cavity effusions, and to determine if effusion fluids may serve as viral reservoirs. To that end, we 

The study was performed under approval from the University of Michigan Institutional Review

Board. An ongoing search of the University of Michigan Anatomic Pathology database (SoftPathologyDx) was performed to identify cytologic cases of pleural, pericardial, or peritoneal fluids in patients who also had a positive COVID-19 nasopharyngeal PCR test from March 2020 to September 2020.

A total of 17 body cavity effusion specimens in 15 patients were identified. All cytology slides including Thinprep™ (Papanicolaou stained), smears (Diff Quik stained), and sectioned cell block slides (H&E stained) were retrieved from the archives along with cell block tissue cassettes. The electronic medical record was accessed to determine patient demographics (including age and gender), past medical history, history of present illness, volume of fluid collected, hospital course, and patient outcomes. In all cases, data from any concurrent fluid chemical analyses were recorded, including color and appearance and levels of protein, pH, and glucose. Also collected were results from any associated microbiology culture findings.

Finally, erythrocyte and leukocyte counts and 100 cell count differentials in these effusion samples were noted. 

We identified 15 patients who did fit study inclusion parameters, among whom a total of 17 effusion cytology specimens were collected (Table 1 ). There were 11 (73%) patients that had unilateral pleural effusions sampled, including 2 (13%) patients with two samples collected each from the same site. Two patients had pericardial effusions sampled and two patients had peritoneal fluid sampling (1 for ascites, 1 peritoneal washing). Patients were 32-81 years old, with 9 male and 6 female patients. Four (27%) patients had a known diagnosis of malignancy (1 gastric carcinoma, 1 hepatocellular carcinoma, 1 chronic lymphocytic leukemia, and 1 follicular thyroid carcinoma). Most (13/15) patients had respiratory tract symptoms ranging from dyspnea and cough to acute hypoxic respiratory failure. Of the remaining two patients, one presented with abdominal pain and bloating while another was found to have SARS-CoV-2 incidentally on pre-surgical workup.

J o u r n a l P r e -p r o o f There were 8/15 (53%) patients who died during hospitalization, including seven (47%) from COVID-related illness. There were 6/11 (55%) patients with pleural effusion sampling who died during the course of hospitalization, including five of COVID-related illness and one from gastrointestinaI bleeding secondary to diverticular disease. One of the two patients with pericardial effusion died, as a result of cardiac tamponade secondary to COVID-19. Both patients with peritoneal fluid sampling are alive and were discharged.

Fluid chemical analysis was performed at the time of collection in most cases (Tables 2-3) .

Protein levels were consistently low in these effusion specimens (range <0.8 -5.9 g/L). The pH ranged from 6.89 -8.15, and glucose from 19 -150 mg/dL. Leukocyte count in these specimens ranged from 75 -6676 cells/mL. A differential 100 cell count was performed in 14/17 cases. In Cytology reports and slides were re-reviewed by two cytopathologists. All 17 specimens were negative for malignancy, confirmed on re-review. Lymphohistiocytic inflammation predominated in 12/17 cases (5 lymphohistiocytic predominant, 4 histiocytic predominant, 3 lymphocytic predominant) ( Table 4 ) (Figures 1-2) . The remaining five cases included four with mixed acute and chronic inflammation and one with predominantly acute inflammation. Notably, hemophagocytosis was present in five (29%) cases including erythrophagocytosis in three In all cases, ISH for SARS-CoV-2 was negative in all cellular elements, including mesothelial cells and inflammatory cells ( Figure 4 ). All ISH slides were reviewed by two cytopathologists (R.C. and L.P.). Appropriate positive controls were observed.

The signs and symptoms of COVID-19 disease are wide-ranging and may be non-specific.

Though most patients present with relatively mild "flu-like" illness, approximately 10% of patients can develop more severe disease marked by lower respiratory tract involvement and/or systemic symptoms. Although only a minority of patients with SARS-CoV-2 develop body cavity effusions overall, a significant portion of patients with severe disease will develop pleural J o u r n a l P r e -p r o o f effusions and/or pericardial effusion. One recent study found that while pleural effusion is rare early in the COVID-19 disease phase (2.5%), it is a relatively common occurrence in patients with more advanced-phase disease (22.7%). 13 However, to date, the pathologic features of body cavity effusions in COVID-19 patients have not been well described.

In our study, the presence of a pleural effusion was an ominous finding, as 7/13 (54%) patients with a pleural effusion died of COVID-19 during the course of their hospitalization. This is in line with the high mortality associated with pleural effusions in the setting of acute Middle East Respiratory Syndrome, caused by a related coronavirus (MERS-CoV). 14 Only two cases were identified with pericardial effusion in our study, both of whom had severe systemic and cardiac disease. One patient died of cardiac tamponade secondary to COVID-19, while the other patient has recovered from COVID-associated myocarditis with ongoing chest pain and dyspnea. The two cases of peritoneal fluid were from patients with known or suspected neoplasms, one with a history of pancreatic carcinoma and another with an adnexal cyst which proved to be a mature cystic teratoma on resection. Both patients are alive and without COVID-19 disease.

Fluid analysis showed that, in all COVID-19 cases, the effusions were transudative in nature (<30 g/L protein), including in both cases with aerobic bacterial growth. The latter two cases did show reduced glucose. The quantity of inflammatory cell involvement varied widely (75-6,676 cells/mL), and in most cases lymphohistiocytic inflammation predominated. The cytologic findings were overall non-specific in nature. Mesothelial cellularity ranged from scanty and nonreactive to hypercellular and reactive. Microscopic re-examination confirmed that lymphocytic and histiocytic inflammation predominated in most cases, with only one case showing a predominance of neutrophils. Megakaryocytes, the presence of which has been described in biopsy and autopsy tissue from patients with COVID-19, were not detected in any case. Also absent were viral cytopathic effects.

The presence of hemophagocytosis in a subset of effusions is noteworthy. It has been hypothesized that severe cases of COVID-19 disease are linked to a "cytokine storm" with an associated hyperactive immune response and hemophagocytic lymphohistiocytosis. 15 Hemophagocytosis is a common histologic finding in thoracic lymph nodes, liver, spleen, and bone marrow, and it is frequently seen at autopsy in COVID-19 patients who had clinical features of hemophagocytic syndrome such as high fever, hyperferritinemia, and cytopenias. [15] [16] There were five (29%) cases in our study that did show hemophagocytosis, being present in both pleural effusions from one patient who underwent repeat sampling. In three cases the only hemophagocytosis identified was of erythrocytes, while two cases showed hemophagocytosis of both erythrocytes and leukocytes. Interestingly, one of the two cases exhibiting phagocytosis of leukocytes was from an asymptomatic patient with an ovarian cyst who was found to be SARS-CoV-2 positive on pre-surgical workup and who did not develop symptomatic disease. Notably, four patients (5, 7, 8, and 12) had clinical and laboratory findings consistent with "cytokine storm," including increased serum ferritin, cytopenias, and vascular accidents, but no hemophagocytosis was identified in any of their cytology samples. In total, 2 out of 4 patients with hemophagocytosis present in effusion cytology material died of COVID-related illness while 2 are alive without evidence of disease. Thus, while hemophagocytosis is a common effusion cytology finding in patients with COVID-19, in our study it was a non-specific finding which did not correlate with severity of COVID-related disease.

It has not been established whether hemophagocytosis in effusion cytologies is more common Angiotensin-converting enzyme 2 (ACE2), the primary receptor of SARS-CoV-2 cellular entry, is expressed on multiple human tissue types, notably epithelium of the oral, nasal, and respiratory tracts, gastrointestinal epithelium, and endothelial cells. 17 However, it is not typically expressed on inflammatory cells such as B and T lymphocytes and macrophages, and the potential expression of ACE2 in human mesothelial cells has not been well established.

ISH for SARS-CoV-2 was negative in all cases in our series, as no viral RNA was detected in either mesothelial or inflammatory elements within serous cavity samples. As noted previously, case reports have detected SARS-CoV-2 RNA by RT-PCR in effusion cytology specimens.

However, it is not clear that such effusions harbor infectious SARS-CoV-2 viral particles or infected cellular elements. One limitation of this study is that RT-PCR was unavailable to be performed in our samples. Further, the effusions in our series were uniformly transudative by protein criteria (<30 g/L). Transudative effusions occur due to imbalances of hydrostatic and J o u r n a l P r e -p r o o f osmotic pressure such as congestive heart failure, cirrhosis, and pulmonary edema, whereas exudative effusions occur in settings of direct tissue damage. 18 The transudative nature of the fluids and negative findings by ISH in our study suggests that while body cavity effusions were common in these advanced COVID-19 cases, they were likely secondary in nature to acute cardiopulmonary dysfunction in COVID-19 rather than direct viral-induced tissue damage.

In summary, the presence of pleural or pericardial effusion requiring clinical intervention was an ominous finding among COVID-19 patients at our institution, with 62% (8) of patients in this small series dying, including 54% (7) from COVID-related illness. The effusions in these patients were transudative in nature in all cases. Inflammation in these effusion cytology specimens was predominantly histiocytic and/or lymphocytic in most cases. Hemophagocytosis, typically a rare finding in effusion cytology specimens, is not an uncommon finding in body cavity effusion specimens from COVID-19 patients, present in 29% (5/17) of cases compared to 0% (0/16) of pleural effusions from non-COVID-19 patients in our study. 

COVID-19 diagnosis and management: a comprehensive review

Could hemophagocytic lymphohistiocytosis be the core issue of severe COVID-19 cases

COVID-19): A Systematic Review of Imaging Findings in 919 Patients

Coronavirus Disease 2019 (COVID-19) CT Findings: A Systematic Review and Meta-analysis

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and coronavirus disease 19 (COVID-19) -anatomic pathology perspective on current knowledge

Pathological findings of COVID-19 associated with acute respiratory distress syndrome

Late histopathologic characteristics of critically ill COVID-19 patients: Different phenotypes without evidence of invasive aspergillosis, a case series

Pulmonary pathology and COVID-19: lessons from autopsy. The experience of European Pulmonary Pathologists

Bronchoalveolar Lavage Specimen of the First Patient Requiring Extracorporeal Membrane Oxygenation for SARS-CoV-2 in Europe

Detection of SARS-CoV-2 in Different Types of Clinical Specimens

First Detection of SARS-CoV-2 by Real-Time Reverse Transcriptase-Polymerase Chain Reaction Assay in Pleural Fluid

SARS-CoV-2 detection in the pericardial fluid of a patient with cardiac tamponade

CT Features of Coronavirus Disease

Acute Middle East Respiratory Syndrome Coronavirus: Temporal Lung Changes Observed on the Chest Radiographs of 55 Patients

Rodríguez-Morales AJ. A review of the main histopathological findings in coronavirus disease 2019. Hum Pathol

Epub ahead of print

SARS-CoV-2 Infection-Associated Hemophagocytic Lymphohistiocytosis

Tissue distribution of ACE2 protein, the functional receptor for SARS coronavirus. A first step in understanding SARS pathogenesis

Diagnostic approach to pleural effusion in adults