key: cord-0737781-lczz8rv3 authors: Boulanger, Mary; Molina, Emily; Wang, Kunbo; Kickler, Thomas; Xu, Yanxun; Garibaldi, Brian T. title: Peripheral Plasma Cells Associated with Mortality Benefit in Severe COVID-19: A Marker of Disease Resolution date: 2021-03-31 journal: Am J Med DOI: 10.1016/j.amjmed.2021.01.040 sha: c46d28d689bee6e1ab62076a123d2d0d0ae8c562 doc_id: 737781 cord_uid: lczz8rv3 BACKGROUND: Cytokines seen in severe COVID-19 are associated with proliferation, differentiation, and survival of plasma cells. Plasma cells are not routinely found in peripheral blood, though may produce virus-neutralizing antibodies in COVID-19 later in the course of an infection. METHODS: Using the Johns Hopkins COVID-19 Precision Medicine Analytics Platform Registry, we identified hospitalized adult patients with confirmed SARS-CoV-2 infection and stratified by presence of plasma cells and WHO disease severity. To identify plasma cells, we employed a sensitive flow cytometric screening method for highly fluorescent lymphocytes and confirmed these microscopically. Cox regression models were used to evaluate time to death and time to clinical improvement by the presence of plasma cells in patients with severe disease. RESULTS: Of 2,301 hospitalized patients with confirmed infection, 371 had plasma cells identified. Patients with plasma cells were more likely to have severe disease, though 86.6% developed plasma cells after onset of severe disease. In patients with severe disease, after adjusting for age, sex, BMI, race and other covariates associated with disease severity, patients with plasma cells had a reduced hazard of death (aHR: 0.57; 95%CI: 0.38 to 0.87; p-value: 0.008). There was no significant association with the presence of plasma cells and time to clinical improvement. CONCLUSIONS: Patients with severe disease who have detectable plasma cells in the peripheral blood have improved mortality despite adjusting for known covariates associated with disease severity in COVID-19. Further investigation is warranted to understand the role of plasma cells in the immune response to COVID-19. Since the emergence of SARS-CoV-2 in Wuhan, China in December, 2020, a number of laboratory findings have been associated with severe disease and death including lymphopenia and elevated levels of inflammatory markers. 1 These observations suggest a viral-induced systemic hyper-inflammatory state driving lung injury and acute respiratory distress syndrome (ARDS). 2, 3 However, the key mediators driving this response are still under investigation. Importantly, the immune response to COVID-19 varies over time in a given patient. 4 Initially, innate immune cells, including macrophages and neutrophils are prominent, driving high levels of pro-inflammatory cytokines to target the virus. Within several days of infection, the adaptive immune response, including T-and B-lymphocytes, begins to activate. Dysregulation of the innate response may be a major contributor to pathologic inflammation and morbidity. 5 Elevated levels of IL-6, IL-2R, IL-10 and TNF-alpha correlate with increased COVID-19 disease severity. 2, 3, 6 Interestingly, these same cytokines are linked to the proliferation, differentiation, and survival of plasma cells. 7, 8 Immunologically, plasma cells are responsible for synthesizing antibodies and have been identified as possibly producing virus-neutralizing antibodies in COVID-19. 9,10 Not only have plasma cells been noted in bronchoalveolar lavage (BAL) fluid 11 and gross lung pathology 12 in COVID-19, but plasmablast expansion and extrafollicular B cell activation has also been described in patients who are critically ill. 13 Interestingly, plasmablast frequency in patients with severe COVID-19 did not correlate with age, days since symptom onset, comorbidities, or levels of receptor binding domain specific IgM or IgG. 13 In a study of eleven patients, B-cell populations did not correlate with cytokine levels. 14 Increasing our understanding of cytokines and immune cells throughout the clinical course of COVID-19 is critical, as this could lead to potential therapeutic insights for both targeting dysregulated as well as promoting "protective" immune responses. 15 This is the first multi-center retrospective study to describe hospitalized patients with confirmed SARS-CoV-2 infection with plasma cells on complete blood count with differential by disease severity and mortality. By using a blood cell counter widely available in clinical laboratories we have a sensitive tool to detect an important mediator in the disease process of COVID-19 that may also be useful as a marker of disease resolution. Using the Johns Hopkins COVID-19 Precision Medicine Analytics Platform Registry (JH-CROWN), we identified 2,540 patients with confirmed SARS-CoV-2 infection by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) who were hospitalized at our 5hospital health system between March 3 and August 29, 2020. We excluded patients who were discharged or deceased within 24-hours from admission. Patients with a past medical history of of multiple myeloma or plasma cell leukemia were excluded from analysis. Corp., Kobe, Japan) to measure the immune status of patents with COVID-19. These analyzers can differentiate leukocyte subsets by flow cytometry based forward scatter, side scatter and RNA content. Antibody-synthesizing lymphoplasmacytoid B cells and plasma cells have high fluorescence intensity above that of normal lymphocytes and can be detected using this method. 16, 17 To confirm that these cells were plasma cells, blood samples that were flagged for high fluorescent lymphocytes had blood smears prepared and stained with Wright's stain. The slides were scanned with a Cellavision scanner and results reviewed by a hematology technician and hematologist, who were both blinded concerning clinical information. We stratified patients by disease severity using the WHO ordinal disease scale. 18 We performed Cox regression analyses to evaluate time to death and time to clinical improvement in severely ill patients (i.e. those requiring high-flow nasal cannula, non-invasive ventilation, mechanical ventilation, ECMO or with multisystem organ failure) with and without plasma cells after adjusting for age, sex, BMI, race as well as other covariates known to be associated with severe disease as described elsewhere. 19, 20 Other covariates included SpO2/FiO2 ratio, respiratory rate, blood pressure, heart rate, temperate, white blood cell count, absolute lymphocyte count, hemoglobin, albumin, glomerular filtration rate, alanine transaminase, D-dimer, ferritin, CRP, Charlson Comorbidity Index, and DNR/DNI status. 19, 20 Cox regression analyses included all events that occurred up to 28 days after the first day of severe disease. Time to clinical improvement was determined by a 2-point decrease in WHO score or discharge from the hospital. 18 Statistical analyses were conducted using R software. 21 Our study suggests that patients with severe COVID-19 who have circulating plasma cells were less likely to die. Plasma cells appeared after progression to severe disease in most patients, and thus will not serve as an early marker for severe disease. However, the association between plasma cells in severe COVID-19 and the increased likelihood of survival suggests that plasma cells may play a crucial role in the immune response to COVID-19. Plasma cells likely secrete neutralizing antibodies in COVID-19. 9,10 Therefore, investigating the titer of COVID-19 antibodies in recovered patients who had circulating plasma cells would give insight into whether these cells correlate with a more robust or prolonged antibody response. 22 If patients with circulating plasma cells have more neutralizing antibodies, this could serve as a marker to identify potential donors for convalescent plasma. As transfusion of convalescent plasma with higher anti-SARS-CoV-2 IgG antibody levels is associated with a lower risk of death, it is critical to identify donors who may have higher antibody titers. 23 Interestingly, in patients with dengue fever, another syndrome associated with a positive-stranded RNA virus, plasmablast response is similarly associated with disease severity regardless of viral load; however, there was no correlation between neutralizing antibody titer and plasmablast response. 24 This suggests that immunomodulatory factors promoting viral clearance and immunity are multifactorial. Although our study is the first multi-center retrospective study following hospitalized patients with COVID-19 with confirmed plasma cells in the peripheral blood there are several limitations. While patients had CBCs monitored regularly during their hospital admission, the frequency of the CBC with differential was provider dependent. Our study suggests an association between the presence of plasma cells and the resolution of severe disease but we are unable to assess for a causal link with our current data set. Continued research on circulating plasma cells in patients with COVID-19 can better elucidate the immunologic response to SARS-CoV-2 and help identify factors associated with recovery from severe disease. The data utilized were part of the JHCROWN: The COVID PMAP Registry, which is based on the contribution of many patients and clinicians and funded by Hopkins in Health, the Johns Hopkins Precision Medicine Program. Response (to BTG, KW, and YX). The funders were not in involved in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication. 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Accessed at www.r-project.org/ on 17 The Power of Antibody-Based Surveillance Convalescent Plasma Antibody Levels and the Risk of Death from Covid-19 Association between magnitude of the virus-specific plasmablast response and disease severity in dengue patients Figure 1: Survival probability by presence of peripheral plasma cell in patients with severe COVID-19 Figure 2: Time to clinical improvement by presence of plasma cells in patients with severe COVID-19