key: cord-0801975-pixroj1k
authors: Wang, Zhao; He, Yu; Shu, Huaqing; Wang, Ping; Xing, Hui; Zeng, Xiaoqian; Tang, Zhaoming
title: High Fluorescent Lymphocytes Are Increased in COVID‐19 Patients
date: 2020-05-20
journal: Br J Haematol
DOI: 10.1111/bjh.16867
sha: 072e21962df98e35eed7a824d654bc1db3b9a206
doc_id: 801975
cord_uid: pixroj1k

Since the outbreak of SARS‐CoV‐2 in December 2019, the etiology of Coronavirus Disease 2019 (COVID‐19) has spread to most of the world within months. The understanding of pathomechanism of SARS‐CoV‐2 is extremely limited.

blood lymphocytes are decreased in COVID-19 patients (Guan et al., 2020) . As to lymphocyte subgroups, CD8+ T cells are decreased during the process of the COVID-19 disease while the percentage of exhausted CD 8+ T cells (NKG2A+ T cells) are increased (Zheng et al., 2020a) . The mechanism of lymphopenia in COVID-19 patients is that lymphocytes, especially T cells, are attracted into the infection sites or killed by the coronavirus. These were described in detail in a recent review (Tay et al., 2020) . B cells are the source of antibodies to SARS-CoV-2 and part of lymphocytes, but the levels of circulating B cells especially activated B cells or plasma cells are unclear. One study found that B cells were decreased substantially in severe COVID-19 patients (Zheng et al., 2020b) , while another study suggested the level was not changed .

High Fluorescent Lymphocytes (HFL) in blood are cells related with activated B cells or plasma cells (van Mirre et al., 2011) . HFL can be easily counted by an automated hematology analyzer as one of the parameters of complete blood count (van Mirre et al., 2011) . In this study, we conducted retrospective analyses of HFL counts of 111 COVID-19 patients. HFL levels of COVID-19 patients were compared with that of healthy individuals. To our knowledge, this was the first study to count and compare the numbers of high fluorescent lymphocytes in different severity of COVID-19

patients. This study may provide insight into understanding the interaction of B cells with SARS-CoV-2 and a clue to monitoring disease severity in COVID-19 patients.

We retrospectively analyzed complete blood count results of COVID-19 cases admitted to Wuhan Union Hospital, China, during January 29 th to March 8 th 2020. The diagnosis of COVID-19 was based on the Guideline provided by the National Health Commission of China (National Health Commission of the People's Republic of China, 2020). COVID-19 cases were classified into mild or severe subgroups according to the Guideline. Severe cases were defined as having any of the following features: 1. respiratory rates ≥ 30 per min; 2. Oxygen saturation at rest ≤ 93%; 3. PaO2/FIO2 ≤ 300; 4. pulmonary imaging shows that patients' lesions increase more than 50% within 24 to 48 hours. Cases other than severe condition were classified into mild group. Complete blood count results including high fluorescent lymphocyte count were performed with Sysmex XE-5000 automated cytometry (Sysmex, Kobe, Japan). Healthy controls were enrolled before SARS-CoV-2 outbreak in the late November 2019. Healthy controls were from regular physical examination groups. Subjects with fever, cough, diarrhea symptoms were excluded. Subjects with any lung, heart, liver, kidney and other infectious diseases were excluded in this study. All the results of complete blood count including high fluorescent lymphocyte count were retrospectively retrieved from information-processing unit (IPU) of Sysmex XE-5000 automated cytometry. The study was approved by the Ethics Committee of Wuhan Union Hospital. Non-normal data were described as median with interquartile range. The Kruskal-Wallis test and Dunn's multiple comparisons test were used to compare variables among groups. All statistical analyses were performed by Graphpad Prism version 8.0 (Graphpad Software, California, USA). P <0.05 was considered as statistically significant.

There were 111 COVID-19 patients enrolled into this study (Table 1. ). All were from the epicenter of Wuhan city at the beginning of the COVID-19 pandemic. Forty-six (41.4%) were male. The median age of overall COVID-19 patients was 48.6 years (24 years to 89 years). Nineteen patients (17.1%) were classified into the severe group and five (4.5%) of them died. Patients in the severe group were older than patients in the mild group (median age: 66 y vs 42 y, P < 0.001). There was no significant difference in gender and age between COVID-19 patients and healthy controls.

All the 111 patients had lymphocyte count results for analyses. Twenty-four patients had no HFL count results, the remaining 87 were enrolled for HFL analyses. Lymphocyte counts were significantly lower in mild and severe COVID-19 patients compared with healthy controls (1400 x 10 6 /L and 820 x 10 6 /L vs 2100 x 10 6 /L, P < 0.0001). Lymphocyte counts in the severe group demonstrated a lower trend as compared to that in the mild group (P = 0.080 , Fig 1. ).

HFL counts were significantly higher in mild and severe COVID-19 patients compared with healthy controls (11.8 x 10 6 /L and 20.4 x 10 6 /L vs 0.0 x 10 6 /L, P < 0.0001). The HFL counts tended to be higher in severe COVID-19 patients than in mild patients (Fig 1.) . To explore the relative change

This article is protected by copyright. All rights reserved between HFL and lymphocytes in individuals, the ratio of HFL/lymphocyte was calculated. The changing trends of HFL/lymphocyte were similar to HFL. Two patients (2/5) with the highest HFL/lymphocyte ratio died.

Since the beginning of the SARS-CoV-2 outbreak, the epidemiological and clinical features of COVID-19 have been reported (Guan et al., 2020; Huang et al., 2020) . Consistent with the reports, this present study showed that middle-aged people are more susceptible to SARS-Cov-2, senior people are more likely to develop a more severe condition. The fatality rate in this study was 4.5% which fell between 1.4% (Guan et al., 2020) and 15% reported by two other studies. One characteristic that was different from other reports was that female occupied the main part of the overall patients.

In this study, we found the circulating lymphocytes were decreased in COVID-19 patients. Severe patients tended to has fewer circulating lymphocytes than mild patients. Our data further demonstrated the decreasing trend of lymphocyte in COVID-19 patients (Guan et al., 2020; . Contrary to the overall decreasing trend of lymphocyte, we found that HFL were increased in COVID-19 patients. Our result concurred with a recent case report that found plasmacytoid lymphocytes in blood film of COVID-19 patients (Gérard et al., 2020) . Study found that the levels of antibodies specific to SARS-Cov-2 were higher in severe patients . We found HFL tended to be higher as COVID-19 patients got worse. This is reasonable since HFL is related with the level of circulating plasma cells (van Mirre et al., 2011) .

In conclusion, the SARS-CoV-2 infection induced lymphopenia but increased HFL. The HFL level might be correlated with disease severity of COVID-19 patients. Given that HFL can be conveniently counted by a hematology analyzer, it might be a useful parameter for clinical monitoring and mechanism studies of COVID-19. Studies based on larger samples are warranted to confirm our finding. 

Clinical and immunologic features in severe and moderate Coronavirus Disease

National Health Commission of the People's Republic of China. (2020) New coronavirus pneumonia prevention and control program

SARS-CoV-2: A new aetiology for atypical lymphocytes

Clinical Characteristics of Coronavirus Disease 2019 in China

2020) Coronavirus infections and immune responses

The trinity of COVID-19: immunity, inflammation and intervention

Sensitivity and specificity of the high fluorescent lymphocyte count-gate on the Sysmex XE-5000 hematology analyzer for detection of peripheral plasma cells

Antibody responses to SARS-CoV-2

Functional exhaustion of antiviral lymphocytes in COVID-19 patients

Study of the lymphocyte change between COVID-19 and non-COVID-19 pneumonia cases suggesting other factors besides uncontrolled inflammation contributed to multi-organ injury. medRxiv

Accepted Article Characteristics Males, n (%)

We appreciate all the colleagues who worked in the front line or as medical logistics support staffs during the SARS-CoV-2 outbreak.

ZW and ZT conceived the study and wrote the paper. YH and HS analyzed the data and wrote the paper. PW, HX and XZ collected the data. All authors contributed to critical revision and final approval of the manuscript to be published.

This article is protected by copyright. All rights reservedThe authors do not have any conflict of interest to declare.