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.