Phenotypic Characterization of Lymphocytes in COVID-19

To detect the phenotypic characteristics of the lymphocytes (CD4⁺ and CD8⁺ T-cells, and B-cells and NK cells), samples of ethylenediaminetetraacetic acid anticoagulated peripheral blood (2 mL) were collected from COVID-19 patients before initial treatment and a second sample was collected after 12 days of treatment. Measurements were performed as previously described [6 (link)]. Briefly, CD4⁺ and CD8⁺ T-cell, CD19⁺ B-cell, and CD16⁺ CD56⁺ NK-cell staining was performed with the following antibodies: Peridin chlorophyll protein-conjugated anti-human CD3 mAb (BD Biosciences, California, USA), allophycocyanin (APC)-conjugated anti-human CD4 mAb (BD Biosciences), APC/Cy7-conjugated anti-human CD8 mAb (Biolegend, USA), APC-conjugated anti-human CD19 mAb (BD Biosciences), APC-conjugated anti-human CD16, and Brilliant™ Violet 510 (BV-510)-conjugated anti-human CD56 mAb (Biolegend). The gate strategy of CD4⁺ T-cells, CD8⁺ T-cells, B-cells, and NK cells was executed as CD3⁺CD4⁺, CD3⁺CD8⁺, CD3⁻CD19⁺, and CD3⁻CD16⁺/CD56⁺, respectively, and the cells were analyzed using multiple-color flow cytometry on a BD FACS Canto II flow cytometry system (BD Biosciences).