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J Immunol Methods. 2011 Jan 5;363(2):143-57. doi: 10.1016/j.jim.2010.08.004. Epub 2010 Aug 19.

Quality assurance of intracellular cytokine staining assays: analysis of multiple rounds of proficiency testing.

Author information

1
BD Biosciences, 2350 Qume Drive, San Jose, CA 95131, USA. maria_jaimes@bd.com

Abstract

When evaluating candidate prophylactic HIV and cancer vaccines, intracellular cytokine staining (ICS) assays that measure the frequency and magnitude of antigen-specific T-cell subsets are one tool to monitor immunogen performance and make product advancement decisions. To assess the inter-laboratory assay variation among multiple laboratories testing vaccine candidates, the NIH/NIAID/DAIDS in collaboration with BD Biosciences implemented an ICS Quality Assurance Program (QAP). Seven rounds of testing have been conducted in which 16 laboratories worldwide participated. In each round, IFN-γ, IL-2 and/or TNF-α responses in CD4+ and CD8+ T-cells to CEF or CMV pp65 peptide mixes were tested using cryopreserved peripheral blood mononuclear cells (PBMC) from CMV seropositive donors. We found that for responses measured above 0.2%, inter-laboratory %CVs were, on average, 35%. No differences in inter-laboratory variation were observed if a 4-color antibody cocktail or a 7-color combination was used. Moreover, the data allowed identification of important sources of variability for flow cytometry-based assays, including: number of collected events, gating strategy and instrument setup and performance. As a consequence, in this multi-site study we were able to define pass and fail criteria for ICS assays, which will be adopted in the subsequent rounds of testing and could be easily extrapolated to QAP for other flow cytometry-based assays.

PMID:
20727897
PMCID:
PMC3003767
DOI:
10.1016/j.jim.2010.08.004
[Indexed for MEDLINE]
Free PMC Article

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