Abstract
Advancements in multi-colour fluorescence activated cell sorting (FACS) panel warrant harmonized procedures to obtain comparable data between various laboratories. The intensifying clinical exploration of Natural Killer (NK) cell-based immunotherapy demands standardized and harmonized NK cell FACS panels and acquisition protocols. Eight colour FACS panels were designed to study human NK cell phenotype and function within peripheral blood mononuclear cells (PBMC). The panels were designed around fixed backbone markers and channels, covering antigens for non-NK lineage exclusion (CD3, TCRγδ, CD19, CD14, SYTOX® Blue) and NK cell selection (CD45, CD56, CD16), complemented with variable drop-in markers/channels to study NK cell phenotype (NKG2A, NKG2C, NKG2D and KIR2D) or NK cell function and activation (CD25, NKp44 and CD107a). Harmonized FACS set-up and data analysis for three different flow cytometers has been established, leading to highly comparable and reproducible data sets using the same PBMC reference samples (n = 6). Further studies of NK cells in fresh or cryopreserved PBMC samples (n = 12) confirmed that freezing and thawing of PBMC samples did not significantly affect NK phenotype or function. In conclusion, our data demonstrate that cryopreserved PBMC samples analysed by standardized FACS panels and harmonized analysis protocols will generate highly reliable data sets for multi-center clinical trials under validated conditions.
Highlights
Flow cytometry serves as a powerful analytical platform for rapid measurement, characterization and functional analysis of individual cells within heterogenic cell populations[1]
To harmonize multicolour flow cytometry analysis for studying natural killer (NK) cell phenotype and function, three independent research centers using different flow cytometers equipped with compatible laser and detector/filter settings (Table 1) tested comparability and reproducibility of obtained data sets between centers
We aimed to develop harmonized fluorescent activated cell sorting (FACS) panels for the phenotypic and functional assessment of NK cells
Summary
Flow cytometry serves as a powerful analytical platform for rapid measurement, characterization and functional analysis of individual cells within heterogenic cell populations[1]. 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[8,9,10], which involve sample type, sample handling, panel design, selection of reagents, instrument set-up, and data analysis. They have created a series of guidelines recommended to harmonize those processes. 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
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