15353-j-veluchamy

2 Harmonized NK FACS panels to study NK cell phenotype and function | 37 Introduction Flow cytometry serves as a powerful analytical platform for rapid measurement, characterization and functional analysis of individual cells within heterogenic cell populations 1 . The ability to simultaneously detect multiple parameters in different cell types, promoted fluorescent activated cell sorting (FACS) analysis as a crucial tool to study the complexity of the immune system 2 . Recent advances in flow cytometry instruments and reagents have increased the possibilities for development of more complex multi-colour FACS panels, resulting in their extended use in research and clinical studies 3 . Multi-colour FACS panels facilitate a deeper understanding of the biology, distribution and interaction of different immune cell types, offering valuable information to more accurately diagnose, monitor and treat various immunological disorders and malignancies 4,5 . There is an ever- increasing number of multi-center clinical trials studying cellular therapy approaches. Thus, immune monitoring of patients should be eased using harmonized multi-colour FACS panels to yield reliable and reproducible data. However, despite the routine use of multi- colour FACS panels in such trials, limitations of implementing standardized methodologies and data analysis protocols have led to a high degree of variation, severely limiting data interpretation from different centers 6,7 . Extensive work done by several groups has identified the main issues that need to be carefully considered when developing multi-colour flow cytometry panels for harmonized use 8-10 , which involve sample type, sample handling, panel design, selection of reagents, instrument set-up, and data analysis. They have also created a series of guidelines recommended to harmonize those processes. Briefly, the design of optimal multi-colour FACS panels requires careful selection of the most appropriate fluorochrome-conjugated antibodies to identify and characterize rare cell populations 11 . Prior to sample acquisition, it is crucial to optimize instrument settings, involving fine-tuning of the light scatters and photomultiplier tube (PMT) voltages for each detector, followed by accurate compensation for spectral overlap of all fluorochromes used. Furthermore, standard operating procedures (SOPs) for sample preparation, staining, acquisition, gating strategy and data analysis methods are essential to reduce data variability of multi-center FACS monitoring. Most of the available multi-colour FACS panels for immune subset analysis are designed for general characterization of major leukocyte populations 2,3,12 . There is an obvious need for similarly standardized and harmonized multi-colour FACS panels for specific subsets such as for instance natural killer (NK) cells. In particular, their increased use in cellular therapy approaches, as they are perceived as a safer option for targeted anti-cancer therapy than T cells 13 , calls for the development of NK specific polychromatic FACS panels. NK cells are innate lymphocytes mediating cytotoxic responses against virally infected or tumour cells. The vast majority of peripheral blood NK cells are CD56+CD16+ effector cells