key: cord-0942332-we1w2cxp authors: Berg, Ronan M.G.; Ronit, Andreas; Rørvig, Sara B.; Plovsing, Ronni R. title: Atypical lymphocytes in bronchoalveolar lavage fluid from patients with COVID-19 ARDS date: 2020-10-08 journal: Pathol Res Pract DOI: 10.1016/j.prp.2020.153242 sha: 9e5eb91b700859e25eb53011e416c65629c0ffa4 doc_id: 942332 cord_uid: we1w2cxp nan Atypical lymphocytes have recently been reported in peripheral blood smears from patients with COVID-19. Here, we report results of cytomorphological analyses of the bronchoalveolar lavage fluid in four patients with COVID-19 ARDS in which we consistently found high fractions of similar atypical lymphocytes. Immunohistochemical staining revealed that they expressed CD3 but not CD138, thus likely representing T cells rather than plasma cells. Further studies are required to determine whether these cells are pathognomonic for COVID-19, and whether the cells previously observed in peripheral blood smears represent a 'spill over' from the highly inflamed lungs. Keywords: Acute respiratory distress syndrome; SARS-CoV2; T cell We read with interest the recent paper by El Jamal and coworkers published in Pathology - [1] which reported that conspicuous atypical plasmacytoid lymphocytes are present in peripheral blood smears from patients with coronavirus disease 2019 (COVID-19), which is in line with other recent studies [2, 3] . Indeed, many of the patients in these previous reports were critically ill and required intensive care therapy, and in one of the studies, similar cells were noted bronchoalveolar lavage fluid (BALF) [2] . The exact nature and role of these atypical lymphocytes in the pathophysiology of COVID-19 nevertheless remains a terra incognita. In a recent study [4] , we investigated the compartmental cellular and humoral immuno-phenotype in blood and BALF of patients with COVID-19 acute respiratory distress syndrome (ARDS). The study encompassed four mechanically ventilated patients admitted to the ICU with moderate to severe ARDS (for patient characteristics, see Table 1 ), and was approved by the Regional Ethics Committee of Copenhagen (H-20023159), the Knowledge Centre for Data Review of Copenhagen (P-2020-399) and registered at clinicaltrials.gov (NCT04354584), and oral and written informed proxy consent was obtained from the next of kin as well as the patient's general practitioner. They patients were all were included <72 hours after intubation, and none were treated with corticosteroids, antivirals or immunomodulatory drugs off-protocol. In terms of lymphocyte counts and function, we found notable changes in T cells, including a reduced thymic output with a depleted and hyperactivated T cell population, of which the latter was more prominent in the lungs than in blood [4] . Here, we report additional cytomorphological analyses of BALF from this study. Five cytospins each containing one to three drops of BALF were made from each of the four patients. Two cytospins were stained with May-Grünwald-Giemsa (MGG), and three were left for immuno-histochemical staining for CD3 and CD138, which are surface markers used to identify T cells and plasma cells, respectively. MGG revealed the presence of the same atypical plasmacytoid lymphocytes previously described in blood smears in the BALF of all four patients ( Figure 1A) . Hence, the atypical lymphocytes contained a basophilic cytoplasm with a perinuclear clearing and in some cells small intracytoplasmic vacuoles were observed. The nucleus was enlarged with an oval to irregularly shape and contained clumped chromatin and prominent nucleoli. Immunohistochemical subtype characterisation was somewhat difficult due to the relatively sparse amounts of cell in the cell-block and the cells were not all well conserved. However, they did express CD3 ( Figure 1B) while only relatively few were found weakly positive for CD138 ( Figure 1C) . Thus, this cell population likely represents highly reactive virocyte-like T cells rather than plasma cells. The variation in the count of this cell type in each sample was significant ranging from only a few cells to 20-30% of the leukocytes, but was nonetheless was a consistent finding in all four patients. Similar but not identical cell-types have previously been described in case reports of influenza A/H1N1 pneumonia complicated by ARDS [5] , but atypical lymphocytes with this morphology is not typical of other know pneumonia-causing viral diseases [1, 2] , and has not, to the best of our knowledge, previously been described in BALF of any other disease. Potentially, this cytomorphological pattern may thus be pathognomonic for COVID-19. hyperactivated T-cell population in the lungs are important to the progression of lung parenchymal damage in COVID-19, or if they alternatively merely comprise an epiphenomenon to the severe pulmonary hyperinflammation, which is often coined a 'cytokine storm' [6] . In any event, we speculate that the relatively high occurrence of these cells in the lungs compared to that of blood J o u r n a l P r e -p r o o f (<10% of lymphocytes [2] ) reflects that their terminal differentiation occurs in the lungs, and that the atypical lymphocytes reported in peripheral blood mainly reflects a 'spill-over' from the alveolar compartment. Future studies should perform an in-depth characterisation of the virocytelike lymphocytes by additional immuno-histochemical analysis and cell sorting, as well as functional studies, so that it may be determined if the circulating atypical lymphocytes are indeed derived from the lungs, whether they are pathognomonic for COVID-19, and how they potentially contribute to the severity and progression of disease. Atypical lymphocyte morphology in SARS-CoV-2 infection Atypical lymphocytes in peripheral blood of patients with COVID-19 SARS-CoV-2: a new aetiology for atypical lymphocytes Compartmental immunophenotyping and cytomorphology in COVID-19 ARDS: a case series Influenza A/H1N1 severe pneumonia: Novel morphocytological findings in bronchoalveolar lavage COVID-19: immunopathology and its implications for therapy We thank all the COVID-19 patients and their families for their participation. We also thank Anna Louise Sørensen, Unni Nielsen and the staff at the Department of Pathology for their outstanding technical laboratory assistance. We also thank the clinical staff and nurses at the Department of Anaesthesiology and Intensive Care for their dedicated contribution.