COVID-19: Lymphocyte Subpopulations Monitoring in Critically Ill Patients

Background: The alteration of lymphocyte subpopulations can help to predict the severity and the prognosis of severe Coronavirus disease 2019 (COVID-19). Our goal was to describe the kinetics of lymphocyte subsets, and their impact on the severity and mortality in critically ill COVID-19 patients. Methods: We collected demographic data, comorbidities, clinical signs on admission, laboratory findings on admission then a follow-up during hospitalization. Lymphocyte subsets including CD3+ T cells, CD4+ T cells, CD8+ T cells, B cells, and natural killer (NK) cells were counted by flow cytometer. Results: On admission, we observed lymphopenia in 57% of cases, decreased CD3+ T cells in 76% of cases, decreased CD4+ T cells in 81% of cases, decreased CD8+ T cells in 62% of cases, decreased B cells in 52% of cases, and decreased natural killer (NK) cells in 33% of cases. After treatment, decreased CD3+ T cells, decreased CD4+ T cells, decreased CD8+ T cells, and decreased natural killer cells were predictor factors of mortality, in the univariable analysis. Conclusion: CD3+ T cells, CD4+ T cells, CD8+ T cells, and natural killer cells were predictor factors of severity, ICU mortality, and also a useful tool for predicting disease progression.


Introduction
In December 2019, a new disease due to SARS-CoV-2 infection appeared in China. A few weeks later, it spreads and becomes a pandemic; about 6,931,000 cases have been infected globally with 400,857 (5.8%) deaths, as of June 8, 2020 [1]. The pathophysiology of this infection remains not fully clarified. Although, it has been observed that SARS-Cov-2 is responsible for a reactive inflammatory storm as a result of an exaggerated host immune system response, with deleterious effects on multiple organs; firstly, in the most of the cases, it affects pulmonary tract, then, the others systems including cardiovascular, gastrointestinal, neurological, hematopoietic and immune system [2] [3] [4].

Methods
For this prospective single-center study, we included all adult patients with con- Critically ill patients were defined as admitted in the ICU because they required mechanical ventilation or more than eight liters per minute of oxygen to maintain pulse oxygen saturation (SpO 2 ) > 90% or had a respiratory rate of more than 40 breaths per minute.
We collected demographic data, clinical signs, laboratory findings on admission then a follow-up during hospitalization (lymphocytes, D-dimer, ferritin, lactate dehydrogenase (LDH), C-reactive protein, procalcitonin, PaO 2 :FiO 2 ), chest CT scan if available, outcomes, time from onset of the first symptom to ICU admission, Charlson Comorbidity Index [18] and sequential organ failure assess-

ment (SOFA) scores [19]. CD3+ T cells, CD4+ T cells, CD8+ T cells, B cells, and
natural killer (NK) cells were counted by flow cytometer. All tests were performed at the discretion of the treating physician.
We expressed continuous variables as medians and interquartile (IQR) ranges or means (standard deviations (SD)), as appropriate. Categorical variables were described using percentages and compared using the χ 2 test, although Fisher exact test was used when the data were sparse. We performed a univariable analysis to evaluate the risk factors of mortality. The analysis was processed by SPSS 10.0 for Windows (SPSS, Chicago, IL, USA). A p-value of less than 0.05 was considered statistically significant.
Informed consent was waived due to the emergency of the disease, and researchers analyzed only anonymized data. All research was conducted following the national guidelines and regulations.

Results
Of 1618 COVID-19 patients hospitalized in our university center, 55 patients (3.4%) were admitted to the ICU. We list the basic, clinical characteristics, biological and radiological findings in Table 1. The mean age was 59 (16.5) years; 74.5% were men. Among all the patients, 84% had chronic medical conditions. The frequent symptoms were dyspnoea (85%), and cough (80%). The median length from the onset of symptoms to ICU admission was 7 (6 -8) days. The median SOFA score on admission was 5 (4 -17). On admission, lymphopenia was common (76%) with a median of 980/mm 3 , the median LDH was 560 IU/L, the median D-dimer was 2975 mg/L, the median ferritin was 1135 ng/mL. The chest CT scan showed bilateral ground-glass opacification > 50% in 74% of cases.
Among the 55 patients, we analyzed the lymphocyte subsets from 21 patients.

Discussion
In our population, after treatment, decreased CD3+ T cells, decreased CD4+ T cells, decreased CD8+ T cells, and decreased natural killer cells were predictor factors of mortality; further, a large proportion of patients recovered lymphocyte subpopulations in survivor cases. At the best of our knowledge, this is the first report of the monitoring lymphocyte subsets in a Moroccan cohort of critically ill COVID-19 patients.
These findings were in line with the publishing data. Wang et al. [20] declared the count alteration of total lymphocytes, CD4+ T cells, CD8+ T cells, B cells, and natural killer cells in COVID-19 patients; as well, the CD8+ cells was an independent marker of severity and efficacy of treatment. And accordingly to Sun et al. [21] who observed that total T lymphocytes, CD4+ T lymphocytes, CD8+ T lymphocytes, and NK cells decreased in un-discharged/died group at two weeks after treatment, compared with the discharged group. Additionally, CD4+ T cells and CD8+ T cells could help to evaluate the disease evolvement [22]. Besides, a recent meta-analysis concluded that lymphopenia was related to worsened outcomes [23].
This study has some limitations. The first concern was that our study was a single-centered study with only 55 severe patients, of whom only 21 patients who had lymphocytes subset counts. Secondly, we collected lymphocyte subset counts on admission in the intensive care unit, but some patients were initially hospitalized in a general ward for some days. Thirdly, the interpretation of our findings might be limited by the sample size. However, our ICU was the referral center in the region, thus we consider our study population is representative of cases diagnosed and treated in our region. Further studies through the country still needed.

CD3+ T cells, CD4+ T cells, CD8+ T cells, and natural killer cells were predictor
factors of severity and ICU mortality. As well, they were a useful tool for predicting disease progression. Other larger sample studies are needed to validate risk factors and the lymphocyte threshold.

Ethical Considerations
Informed consent was waived due to the emergency of the disease, and researchers analyzed only anonymized data. All research was conducted following the national guidelines and regulations.