Nine-color flow cytometry for accurate measurement of T cell subsets and cytokine responses. Part II: Panel performance across different instrument platforms.
Cellular immune responses elicited by vaccination are complex and require polychromatic analysis to accurately characterize the phenotype and function of rare, responding cells. Technical challenges and a lack of instrument standardization between research sites have limited the application of polychromatic cytometry in multicenter clinical trials. Two previously developed six-color T cell subset immunophenotyping reagent panels deliberately designed to accommodate three additional low frequency functional measurements were compared for their reproducibility of staining across three different flow cytometers. We repeatedly measured similar T cell subset frequencies between the two reagent panels and across the three different cytometers. Spectral overlap reduced sensitivity in two of the three open measurement channels (PE [IL-2] and APC [IFN gamma]) for one reagent combination, particularly in subsets with low cytokine expression. There was no significant interassay variation for measurements across instrument platforms. Careful panel design will identify reagent combinations that minimize spectral spillover into channels reserved for cytokine measurement and comparable results can be achieved using different cytometers, however, it is important to establish standardized quality control procedures for each instrument to minimize variation between cytometers.
McLaughlin
Bridget E
BE
University of California Davis, Internal Medicine, Infectious Diseases, Davis, CA, USA.
Baumgarth
Nicole
N
Bigos
Martin
M
Roederer
Mario
M
De Rosa
Stephen C
SC
Altman
John D
JD
Nixon
Douglas F
DF
Ottinger
Janet
J
Li
Judy
J
Beckett
Laurel
L
Shacklett
Barbara L
BL
Evans
Thomas G
TG
Asmuth
David M
DM
D000911:Antibodies, Monoclonal; D000945:Antigens, Differentiation, T-Lymphocyte / Q000378:metabolism; D016207:Cytokines / Q000032:analysis*; D005434:Flow Cytometry / Q000295:instrumentation / Q000379:methods* / Q000592:standards; D005456:Fluorescent Dyes; D006801:Humans; D016130:Immunophenotyping / Q000295:instrumentation / Q000379:methods* / Q000592:standards; D007202:Indicators and Reagents; D011786:Quality Control; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013194:Staining and Labeling; D016176:T-Lymphocyte Subsets / Q000166:cytology* / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000945:Antigens, Differentiation, T-Lymphocyte; D016207:Cytokines; D005456:Fluorescent Dyes; D007202:Indicators and Reagents