PATIENTS and METHODS
Patients:  A total of 103 patients infected with the new Coronavirus-2019, who applied to Istanbul Memorial Şişli Hospital and Marmara University Pendik Training and Research Hospital with various symptoms and signs from sore throat to shortness of breath were included in the study [52 female, 51 male with the mean age of 53,9 ± 15 (25-88)]. Covid-19 RT-PCR positivity was the inclusion criterion in the study (tested by Covid-19 kit, Bioeksen R&D Istanbul, Turkey). However, a group of patients was diagnosed by computed tomography (CT) with the ground glass appearance in their lungs. Thus, 76.7% of our patient group was PCR and CT positive; 16.5% of the patients were only PCR positive, outpatient group; and 6.8% were only CT positive. 52.4% of our patient group also had chronic diseases such as diabetes, hypertension, coronary artery disease, and renal insufficiency. Mean hospitalization time was 8 ±8.5 days (range:1-51).
The patients were clinically classified; a group of patients with mild CT findings without lymphopenia (percentage>20%) were added to the outpatient group and classified as ”mild group” (35%); patients who had more common CT involvement, but with oxygen saturation above 90% were classified as “moderate group” (40.8%); in addition to widespread lung involvement, patients with oxygen saturation below 90% at room air were grouped as “severe group” (24.3%) and their immunological features were examined accordingly. According to the instructions of our Ministry of Health for patient treatments; That Hydroxychloroquine alone or hydroxychloroquine and azithromycin combination in mild patients; favipiravir in addition to the combination of hydroxychloroquine azithromycin in moderate patients; additional favipiravir and/or tocilizumab and/or convalescent plasma treatments in addition to the hydroxychloroquine azithromycin combination in severe patients have been used.
The study was approved by both Ministry of Health, Scientific Research Platform (no:2020-04-30_11_31) and Marmara University Ethics Committee (no:08.05.2020/09.2020.541). Informed consent was obtained from the participants before the study.
Controls:  In the comparison of immunological parameters with healthy controls, raw flow cytometric data from healthy controls that have been tested before for the laboratory routine norm studies in Ekşioğlu-Demiralp’s Laboratory and samples that have been used as controls to the diseases in the theses instructed by Ekşioğlu-Demiralp were used. Age and gender matched 100 historical controls were included the study.
Isolation of White Blood Cells and Immunophenotyping:  Peripheral blood White Blood Cells (WBC) of all participants were isolated from their hemogram tubes with EDTA taken for their routine tests by using erythrocytes lysing solution (155 mM NH4Cl; 10 mM KHCO3; 0,1mM EDTA; pH:7.3). The following fluorochrome labelled monoclonal antibodies (mAb) and isotype-matched controls were used for two-three color phenotypic analysis: anti-IgG1, anti-IgG2a, anti-CD45, anti-CD3; anti-CD4; anti-CD8; anti-CD16; anti-CD56; anti-CD19; anti-CD20, anti-HLA-DR; anti-CD10; anti-CD25; anti-CD28, anti-CD69, (Becton&Dickinson Inc, San Jose, CA, USA). Cells were acquired and analyzed using CellQuest software on a FACSCalibur flow cytometer (Becton Dickinson Inc, San Jose, CA, USA). Lymphocytes, monocytes, and neutrophils were gated according to their forward and side scatter characteristics and their specific CD markers. Populations were evaluated as percentage. The absolute number of subsets was calculated in accordance with the absolute number of lymphocytes for each individual sample. Multiple gating strategies were applied when required. Mean fluorescence intensities (MFIs) of CD10 and CD16 antigens on neutrophils and MFIs of CD16 and HLA-DR on monocytes were also evaluated as an indication of activation/exhaustion criterion.
Oxidative Burst and Phagocytosis:  White blood cells from 13 patients in moderate clinical course and 5 healthy controls were isolated from their heparinized peripheral blood samples and simultaneously tested for oxidative burst and phagocytosis functions of their neutrophils and monocytes by applying instructions of a standard kit (PhagoBurst Test, Becton Dickinson Inc, San Jose, CA, USA). Percentage results were evaluated by using the test’s standard values and, also by comparing to controls. Moreover, the mean fluorescence intensity (MFI) of each PMA-stimulated sample was divided into that of its non-stimulated sample. Thus, the folds increases in DHR123 fluorescence following PMA stimulation were calculated for neutrophils and monocytes and compared between patients with COVID-19 and the controls.
Apoptosis and Cell Death:  White blood cells from 13 patients in moderate clinical course and 5 healthy controls were isolated from their heparinized peripheral blood samples. A cell-permeable fluorogenic caspase-3 substrate, Phi-Philux.G1D2 (OncoImmunin Inc, Kensington, MD), and propidium iodide (PI) were used to evaluate early, late apoptosis and cell death. Briefly, cells (1x105/ml) were resuspended in phosphate-buffered saline (PBS) containing 5 % fetal bovine serum (FBS) and one tube was stimulated for apoptosis using 100 ng/ml of PMA at 370C for 1 hour, while other was incubated at the same temperature without stimulation, as a control. Then 9 mM Phi-Philux.G1D2 was added to the tubes and samples were incubated for an additional one hour at the same conditions. Following the incubation, PI (20mg/ml in PBS) was added to evaluate cell death simultaneously. Then the cells were immediately acquired by flow cytometry (FACSCalibur, Becton Dickinson Inc, San Jose, CA, USA) using CellQuest software, where at least 40,000 cells were acquired. PhiPhilux stained only cells were evaluated as early apoptosis or activation; PhiPhilux+PI stained cells as late apoptosis; and PI stained only cells as cell death. All were assessed in lymphocyte, monocyte, and neutrophil gates. Initial MFI values of PhiPhilux fluorescence in unstimulated samples that demonstrate the baseline, spontaneous activation status of the cells were measured and compared to the controls.
Statistical analysis:  The statistical software package SPSS version 25.0 was used for all statistical analyses. Values were presented as percentages and absolute numbers ± standard deviation of the mean. Statistical significance of differences between the groups was calculated using with an analysis of variance (ANOVA) and followed by Bonferonni test as post-hoc. The significance of differences in pre-after measurements was calculated using the Wilcoxon signed ranks test. Mann-Whitney U test was used for non-parametric comparisons in small groups. Values of p<0.05 were regarded as significant.