15353-j-veluchamy

2 Harmonized NK FACS panels to study NK cell phenotype and function | 43 Standardized FACS panels and protocols overcome variability between flow cytometers and centers The initial selection of antibodies to design NK cell FACS panels, handling of samples and development of a common gating strategy, was used to harmonize data acquisition and analysis between the participating centers. To verify that use of the individual instrument settings and optimized protocols would lead to comparable data sets, experiments were performed in three different centers with different flow cytometers using the same reference PBMC samples (n=6). First, single-stain antigen expression levels, acquired from each center, were compared for the NK cell phenotype and function panel (Figure 2). Data from all FCS files, collected from individual experiments, were analysed using the harmonized analysis protocol based on a fixed gating strategy. Using a predefined gating strategy, average frequencies (technical replicates divided by 2) for each parameter were compared between the three flow cytometers (BD FACSCanto TM II, BD LSRFortessa TM or MACSQuant ® Analyzer X) at three different sites using a non-parametric Kruskal-Wallis test. With exception of CD14 VioBlue in BD LSRFortessa TM (Reference sample III, **p-value=0.001) and CD19 VioBlue in MACSQuant® Analyzer X (Reference sample III, *p-value=0.03), the rest of the measured parameters was comparable across different centers (Figure 2 and Table 4). Specifically looking at the variability of more sensitive antigens used for discrimination of NK phenotype, no differences were detected for expression of NKG2A, NKG2C, NKG2D, KIR2D (Figure 3A) between the three flow cytometers (p value range 0.09- 0.13). In order to detect the NK function-associated antigens, PBMC were exposed to target cells (A431) for 4hr and expression of CD107a, CD25, NKp44 and CD16 levels were analysed (Figure 3B). Standardized FACS panels, sample handling, individual acquisition protocols for the three different flow cytometers, as well as harmonized gating strategies for the analysis of the same reference PBMC samples, resulted in a dataset without inter-center variability. These findings thus support the use of these protocols and NK cell FACS panels for use in the monitoring of multi-center trials. Figure 2: Comparison of FACS panels antibody fluorochrome intensities between three flow cytometers All antibodies in the NK cell phenotype and function panel were tested for their fluorochrome intensities using the same staining and acquisition protocols on three different flow cytometers. Single stains from back bone antigens were evaluated for their antigen expression levels. PBMC were gated on NK cells for detecting NK receptors and NK functional antigens in the drop-in channels. One representative set of histograms (n=3) is shown for each marker tested at three different centers using three different flow cytometers. Antigen expression is expressed as percentage of positive cells for back bone antibodies in figure A and for drop-ins in figure B.