Standardized and flexible eight colour flow cytometry panels harmonized between different laboratories to study human NK cell phenotype and function

• Published in: Scientific reports 2017-03, Vol.7 (1), p.43873-43873, Article 43873
• Main Authors: Veluchamy, John P., Delso-Vallejo, María, Kok, Nina, Bohme, Fenna, Seggewiss-Bernhardt, Ruth, van der Vliet, Hans J., de Gruijl, Tanja D., Huppert, Volker, Spanholtz, Jan
• Format: Article
• Language: English
• Subjects: 13/1; 13/21; 13/56; 692/308/575; 692/4028/67/580; 96/106; Antigens; Antigens - immunology; Biomarkers - analysis; CD14 antigen; CD16 antigen; CD19 antigen; CD25 antigen; CD3 antigen; CD45 antigen; CD56 antigen; Cell activation; Cell Line, Tumor; Clinical trials; Color; Cryopreservation; Data processing; Female; Flow cytometry; Flow Cytometry - methods; Flow Cytometry - standards; Freezing; Genotype & phenotype; Humanities and Social Sciences; Humans; Immunotherapy; Killer Cells, Natural - cytology; Killer Cells, Natural - immunology; Killer Cells, Natural - metabolism; Laboratories; Laboratories - standards; Leukocytes (mononuclear); Leukocytes, Mononuclear - cytology; Leukocytes, Mononuclear - immunology; Leukocytes, Mononuclear - metabolism; multidisciplinary; Natural killer cells; NKG2 antigen; Peripheral blood mononuclear cells; Phenotype; Potassium channels (inwardly-rectifying); Receptors, Natural Killer Cell - immunology; Reference Standards; Reproducibility of Results; Science; T cell receptors; Thawing
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/srep43873

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.