15353-j-veluchamy

2 Harmonized NK FACS panels to study NK cell phenotype and function | 53 not have a significant effect on the NK cell phenotype in both non-activated and cytokine activated PBMC samples. On the other hand, the decrease in the NK cell marker CD16 was more prominent in A431 stimulated cryopreserved NK cells. Importantly however, in the ADCC condition (NK+A431+CET), the decrease in CD16 expression levels was comparable between fresh and cryopreserved NK cells. Our study also indicated that there is no need for IL-2 and IL-15 cytokine stimulation preceding in vitro NK cytotoxicity experiments in this setting, as the influence of these cytokines was restricted to increase in CD25 expression on NK cells, with significantly higher CD25 levels in A431 stimulated fresh NK cells, but did not translate into significantly higher degranulation. In general, NK cell cytotoxicity assays are performed using fresh specimens, whereas our results here support the feasibility to use cryopreserved NK cells for this type of assays. The use of cryopreserved PBMC further limits the drawbacks involved in real time analysis of fresh samples over different time points, thus reducing instrument variability and allows generating more reproducible data sets in longitudinal analysis. Multicenter trials should aim for harmonized panels and reproducible data generation, but have faced challenges in this respect due to variability in flow cytometers and antibody- fluorochrome conjugates resulting in inconsistent data sets 47-49 . With our panel design, uniform gating strategy and implementation of standardized procedures, we were able to obtain reproducible data in three different centers, thereby overcoming inter-laboratory variability issues. Further, differences related to instrument set-up or operators, did not significantly influence the data set. The unique ability to obtain highly reproducible data between three flow cytometers independent of operators and machines, offers an ideal opportunity to use these FACS panels in multicenter trials for monitoring of clinical specimens. The generation of comparable results between different centers could further help in designing cost effective clinical trials by reducing shipment costs effectively. Material & Methods PBMC isolation and activation Whole blood samples were collected from healthy donors after obtaining informed consent in accordance with the ‘‘Code for Proper Use of Human Tissues’’ as formulated by the Dutch Federation of Medical Scientific Organizations (www.fmwv.nl ) 50 . PBMC were isolated from whole blood using Lymphoprep™ (STEMCELL Technologies, Vancouver, Canada) density gradient centrifugation. PBMC were incubated at a concentration of 5 x 10 6 /ml in 6 well plates in Roswell Park Memorial Institute (RPMI) medium (Sigma Aldrich, Zwijndrecht, The Netherlands) containing 2% human serum albumin (HSA) overnight at 37°C, 95% humidity, 5% CO2 atmosphere. PBMC viability, numbers and NK cell content were counted