title: Standardized and flexible eight colour flow cytometry panels harmonized between different laboratories to study human NK cell phenotype and function.
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.
authors.affiliation: Glycostem Therapeutics, Oss, The Netherlands.|Department of Medical Oncology, VU University Medical Cancer Center Amsterdam, De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands.
authors.forename: John P
authors.initials: JP
authors.lastname: Veluchamy
authors.affiliation: Miltenyi Biotec GmbH, Bergisch Gladbach, Germany.
authors.forename: María
authors.initials: M
authors.lastname: Delso-Vallejo
authors.affiliation: Glycostem Therapeutics, Oss, The Netherlands.
authors.forename: Nina
authors.initials: N
authors.lastname: Kok
authors.affiliation: Glycostem Therapeutics, Oss, The Netherlands.
authors.forename: Fenna
authors.initials: F
authors.lastname: Bohme
authors.affiliation: Department of Haematology and Medical Oncology, University Hospital of Wuerzburg, Wuerzburg, Germany.|Department of Haematology and Medical Oncology, Sozialstiftung Bamberg, Bamberg, Germany.
authors.forename: Ruth
authors.initials: R
authors.lastname: Seggewiss-Bernhardt
authors.affiliation: Department of Medical Oncology, VU University Medical Cancer Center Amsterdam, De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands.
authors.forename: Hans J
authors.initials: HJ
authors.lastname: van der Vliet
authors.affiliation: Department of Medical Oncology, VU University Medical Cancer Center Amsterdam, De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands.
authors.forename: Tanja D
authors.initials: TD
authors.lastname: de Gruijl
authors.affiliation: Miltenyi Biotec GmbH, Bergisch Gladbach, Germany.
authors.forename: Volker
authors.initials: V
authors.lastname: Huppert
authors.affiliation: Glycostem Therapeutics, Oss, The Netherlands.
authors.forename: Jan
authors.initials: J
authors.lastname: Spanholtz
mesh_terms: D000941:Antigens / Q000276:immunology; D015415:Biomarkers / Q000032:analysis; D045744:Cell Line, Tumor; D003116:Color; D005260:Female; D005434:Flow Cytometry / Q000379:methods* / Q000592:standards*; D006801:Humans; D007694:Killer Cells, Natural / Q000166:cytology / Q000276:immunology* / Q000378:metabolism; D007753:Laboratories / Q000592:standards*; D007963:Leukocytes, Mononuclear / Q000166:cytology / Q000276:immunology / Q000378:metabolism; D010641:Phenotype; D055607:Receptors, Natural Killer Cell / Q000276:immunology; D012015:Reference Standards; D015203:Reproducibility of Results
chemical_list: D000941:Antigens; D015415:Biomarkers; D055607:Receptors, Natural Killer Cell