Daan Pieren

128 Chapter 5 Discovery Rate (FDR) of 0.10, and a -Log( p -value) of greater than 1.5 was used. p -values were calculated by Fisher’s Exact Test. Whole blood flow cytometry in COVID-19 FFX study Whole blood of participants in the FFX study, investigating the pandemic of SARS-CoV-2 in the Netherlands, was obtained from adults suffering from COVID-19 with a PCR-confirmed SARS-CoV-2 infection and that of asymptomatic SARS-CoV-2-PCR-negative household members. 100 µ L whole blood of each donor was labeled for surface markers at room temperature for 15 minutes with saturating concentrations of antibodies targeting: CD3, CD4, CD8, CD27, CD45, CD45RA, CD69, KIR2D, KIR3DL1, and NKG2A ( listed in Supplementary Table 4 ) in Brilliant Stain Buffer (BD Biosciences). Erythrocytes were lysed with 1mL lysing solution (BD Biosciences) for 15 minutes at room temperature. To fasten measurement of stained PBMC, cell suspensions were concentrated by centrifugation at 400 g for 5 minutes and resuspended in the 400 µ L remaining after removal of 700 µ L of fluid phase after centrifugation. Samples were measured on an LSRFortessa™ X-20 (BD Biosciences) and data were analyzed using FlowJo software (v10.6.1, TreeStar). Statistical analyses Statistical analysis was performed using GraphPad Prism version 8.4.1. The appropriate parametric or non-parametric tests were used based on the tested normality of distribution of the data. Two groups were compared by using Mann-Whitney U test (unpaired), or Wilcoxon Test (paired) with two- sided p -values. When comparing more than two groups, paired analyses were performed with either row-matched one-way ANOVA with Geisser-Greenhouse correction and Dunnett’s post-test, or with Friedman Test with Dunn’s post-test. Correlations between variables were analyzed using Spearman’s rank correlation coefficient ( r ). Linear regression analysis was performed to generate lines of best fit. Statistical significance was considered when p < 0.05, with * p <0.05, ** p <0.01, *** p <0.001, and **** p <0.0001. All data presented in bar graphs are depicted as mean ± s.d.