key: cord-0861805-e0176dxk authors: Löhr, Phillip; Schiele, Stefan; Arndt, Tim Tobias; Grützner, Stefanie; Claus, Rainer; Römmele, Christoph; Müller, Gernot; Schmid, Christoph; Dennehy, Kevin M.; Rank, Andreas title: Impact of age and gender on lymphocyte subset counts in patients with COVID‐19 date: 2021-06-23 journal: Cytometry A DOI: 10.1002/cyto.a.24470 sha: 146e627e5983e657b05d3d0c64cefe6435e2ba6e doc_id: 861805 cord_uid: e0176dxk In symptomatic patients with acute Coronavirus disease 2019 (COVID‐19), lymphocytopenia is one of the most prominent laboratory findings. However, to date age and gender have not been considered in assessment of COVID‐19‐related cell count alterations. In this study, the impact of COVID‐19 as well as age and gender on a large variety of lymphocyte subsets was analyzed in 33 COVID‐19 patients and compared with cell counts in 50 healthy humans. We confirm that cell counts of total lymphocytes, B, NK, cytotoxic and helper T cells are reduced in patients with severe COVID‐19, and this tendency was observed in patients with moderate COVID‐19. Decreased cell counts were also found in all subsets of these cell types, except for CD4+ and CD8+ effector memory RA+ (EMRA) and terminal effector CD8+ cells. In multivariate analysis however, we show that in addition to COVID‐19, there is an age‐dependent reduction of total, central memory (CM), and early CD8+ cell subsets, as well as naïve, CM, and regulatory CD4+ cell subsets. Remarkably, reduced naïve CD8+ cell counts could be attributed to age alone, and not to COVID‐19. By contrast, decreases in other subsets could be largely attributed to COVID‐19, and only partly to age. In addition to COVID‐19, male gender was a major factor influencing lower counts of CD3+ and CD4+ lymphocyte numbers. Our study confirms that cell counts of lymphocytes and their subsets are reduced in patients with COVID‐19, but that age and gender must be considered when interpreting the altered cell counts. the respiratory system is thought to be mediated by the virus itself and/or an overstimulated immune system. 5 COVID-19 is associated with various lab abnormalities of coagulation, inflammatory biomarkers, and biochemical as well as hematologic values dependent on the severity of COVID-19. 6 The most striking finding in the blood count of patients with COVID-19 is lymphocytopenia that correlates with the severity of disease. 7, 8 However, interpretation of virusinduced alterations of lymphocyte counts should also be considered in the light of age, given that COVID-19 patients mostly belong to the middle-aged and old population. A large basic effect of aging is seen on counts of cytotoxic and helper T cells. During the human lifetime, the proportion of naïve T cells within both CD4+ and CD8+ cells decreases dramatically from 45% and 43% to 20% and 10%, respectively. 9 In contrast, the proportion of central memory and effector memory cells within cytotoxic and helper T lymphocytes increases with age. Furthermore, also gender has an impact on lymphocyte counts. In general, counts of total CD3+ T lymphocytes as well as CD8+ cytotoxic T cells and especially CD4+ T helper cells are higher in females compared to males. 10 Thus, the question arises whether the widely reported alterations in counts of lymphocytes and their subsets in patients with COVID-19 are indeed primarily due to coronavirus infection, or whether gender and age might have a significant impact. While lymphocytopenia in COVID-19 patients is well known, the influence of age and gender has hardly been considered so far. The aim of this study was to analyze the influence of age and gender on the total number of lymphocytes, as well as on B-, T-and NK-cell subsets during COVID-19. Study participants with COVID-19 were recruited for this prospective study at the University Medical Hospital, Augsburg, between April and October 2020. Patients were included immediately after clinical admission and confirmation of a positive qPCR test for SARS-CoV-2 from oropharyngeal swab. The time period between onset of COVID-19 symptoms and study inclusion was restricted to a maximum of 28 days. Exclusion criteria were malignancy, immune deficiency, autoimmune disorder, chronic infection disease, and pregnancy. Every participant was classified according to interim guidance of the World Health Organization (WHO) as mild infection (uncomplicated upper respiratory tract infection), pneumonia (without need for supplemental oxygen), severe pneumonia (with need for supplemental oxygen), acute respiratory distress syndrome (ARDS), sepsis, or septic shock. 11 Patients suffering from severe pneumonia, ARDS, sepsis, or septic shock were categorized as "severe" COVID-19 in our study, while all other causes were defined as "moderate" COVID-19. The study was performed in accordance with the revised declaration of Helsinki and approved by the internal ethics committee (internal review board No. 2020-12). Signed informed consent was obtained from all participants allowing analysis of all laboratory data presented in this manuscript. Additionally, 50 healthy adult blood donors from the blood bank at University Medical Hospital, Augsburg, served as control group. Control samples were collected, measured, and analyzed during the pre-COVID-19 era excluding infection with SARS-CoV-2 as described below. Table S2 . The gating strategy is shown in Figure S1 as already described by our research group. 12 Table 2 . Age had an impact on total lymphocyte count and most subsets in entire cohort of COVID-19 patients and healthy controls, as listed in Table 3 . Median numbers of total lymphocytes were reduced by 12.5% per 10 years (/10y; Figure 2A ). This aging effect could be observed also for CD3+ cells (À14.1%/10y), CD8+ cells (À19.1%/10y; Figure 2B ) and some of their subsets (naïve (À38.8%/10y; Figure 2C ), memory (À19.6%/10y), CM (À21.1%/10y), EM (À18.3%/10y), early (À29.5%/10y; Figure 2D ), intermediate (À14.5%/10y), and exhausted CD8+ cells (À9.6%/10y) as well as for CD4+ cells (À14.8%/10y) and some of their subsets (naïve (À21.5%/10y), memory (À14.2%/10y), CM (À16.0%/10y), EM (À13.0%/10y), regulatory (À12.5%/10y), and Il2R+ CD4+ cells (À18.2%/10y)). Aging had also a negative influence on NK cells (À13.6% /10y) and their subsets (CD56+ CD16+ (À13.4%/10y), CD56dim CD16bright (À17.8%/10y), CD56bright CD16dim (À19.0%/10y)), as well as on B cells (À14.4%/10y) and their subsets (naïve (À23.4%/10y), memory non-class switch (À24.0%/10y), memory class switch (À10.8%/10y), transitional (À79.3%/10y)). Gender influenced the ratio of CD4/CD8 lymphocyte counts in univariate analysis. Female participants had a higher median ratio compared to male in study participants and healthy controls (2.89 (quartile: 1.85-3.94) vs. 2.06 (1.44-3.10); p = 0.024). The impact of moderate/severe COVID-19, gender and increasing age Table S1 ). Total lymphocyte count was decreased in moderate (cB: 0.640, p = 0.008) and severe (cB: 0.427, p < 0.001) COVID-19 patients compared to healthy controls, and this decrease was independent of age and gender. was observed on CD56bright CD16dim (cB: 0.902, p = 0.008) and CD56dim CD16bright (cB: 0.873, p = 0.018) NK cells. In this prospective study, we analyzed lymphocyte subsets of 33 elderly patients with different degrees of COVID-19 severity. In univariate analysis we found lower counts of total lymphocytes and all their subsets, with the exception of CD4+ and CD8+ EMRA cells as well as late and terminal effector T helper cells, in patients with severe COVID-19. In patients affected by moderate COVID-19, a similar trend was found for most analyzed cell populations, although this did not always reach statistical significance. well known. [13] [14] [15] Usually, the human immune system reacts by inducing proliferation of lymphocytes during the acute phase of viral infections including cytomegalovirus, 16 Epstein Barr virus, 17 and also T A B L E 3 Median cell counts of lymphocytes and their subsets of total cohort including COVID-19 patients and healthy controls (n = 83), deviation of cell counts dependent on age over lifetime, and p value A novel coronavirus from patients with pneumonia in China Clinical characteristics of 138 hospitalized patients with 2019 novel coronavirusinfected pneumonia in Wuhan Postmortem examination of patients with COVID-19 Clinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan, China: a retrospective cohort study Immunology of COVID-19: current state of the science Hematologic, biochemical and immune biomarker abnormalities associated with severe illness and mortality in coronavirus disease 2019 (COVID-19): a meta-analysis Clinical and immunological features of severe and moderate coronavirus disease 2019 Lymphocyte subset counts in COVID-19 patients: a meta-analysis Age and gender leucocytes variances and references values generated using the standardized ONE-study protocol Values for alphabeta and gammadelta T-lymphocytes and CD4+, CD8+, and CD56+ subsets in healthy adult subjects: assessment by age and gender Clinical management of severe acute respiratory infection when COVID-19 is suspected. 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The involvement of natural killer cells in the pathogenesis of severe acute respiratory syndrome Impact of age and gender on lymphocyte The authors declare that there is no conflict of interest. https://orcid.org/0000-0003-4939-279XRainer Claus https://orcid.org/0000-0003-2617-8766