Nine-color flow cytometry for accurate measurement of T cell subsets and cytokine responses. Part II: Panel performance across different instrument platforms.

TitleNine-color flow cytometry for accurate measurement of T cell subsets and cytokine responses. Part II: Panel performance across different instrument platforms.
Publication TypeJournal Article
Year of Publication2008
AuthorsMcLaughlin BE, Baumgarth N, Bigos M, Roederer M, De Rosa SC, Altman JD, Nixon DF, Ottinger J, Li J, Beckett L, Shacklett BL, Evans TG, Asmuth DM
JournalCytometry A
Volume73
Issue5
Pagination411-20
Date Published2008 May
ISSN1552-4930
KeywordsAntibodies, Monoclonal, Antigens, Differentiation, T-Lymphocyte, Cytokines, Flow Cytometry, Fluorescent Dyes, Humans, Immunophenotyping, Indicators and Reagents, Quality Control, Reproducibility of Results, Sensitivity and Specificity, Staining and Labeling, T-Lymphocyte Subsets
Abstract

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.

DOI10.1002/cyto.a.20556
Alternate JournalCytometry A
PubMed ID18383309
Grant ListAI051999 / AI / NIAID NIH HHS / United States
C06RR-12088-01 / RR / NCRR NIH HHS / United States
R01 AI051999-03 / AI / NIAID NIH HHS / United States