Data analysis and statistical evaluation
Flow cytometry data were analysed using FlowJo version 10.6.2 (BD
Biosciences). Gating was performed according to single stains and
fluorescent-minus-one controls. The corresponding gating strategy is
presented in Figure 2. Lymphocyte identification and doublet exclusion
were performed by forward-scatter (FSC) versus sideward parameters (SSC)
and FSC/SSC-height (H) versus FSC/SSC-area (A) plotting, respectively.
Cells positive for DAPI (dead cells), CD14 (mainly monocytes), or CD3 (T
cells) were excluded from further analyses. CD19 expressing cells were
further characterized for IgD, CD27, and CD38 expression. Frequencies of
SARS-CoV-2-protein specific B cells in recovered COVID-19 patients and
healthy donors were compared using two-way repeated measurements ANOVA
and Sidak’s multiple comparisons test. Patient and healthy donor age was
compared using unpaired two-tailed t-test, and gender was compared using
two-tailed Fisher’s exact test. Correlations were calculated using
Spearman’s rank coefficient. Statistical analysis and graphical
representation were realized with GraphPad Prism v7 and R v3.6.2.