Quality assurance of intracellular cytokine staining assays: analysis of multiple rounds of proficiency testing.
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.
Jaimes
Maria C
MC
BD Biosciences, 2350 Qume Drive, San Jose, CA 95131, USA. maria_jaimes@bd.com
Maecker
Holden T
HT
Yan
Ming
M
Maino
Vernon C
VC
Hanley
Mary Beth
MB
Greer
Angela
A
Darden
Janice M
JM
D'Souza
M Patricia
MP
D015496:CD4-Positive T-Lymphocytes / Q000276:immunology*; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology*; D005434:Flow Cytometry / Q000379:methods* / Q000592:standards; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D007371:Interferon-gamma / Q000097:blood*; D007376:Interleukin-2 / Q000097:blood*; D007963:Leukocytes, Mononuclear / Q000276:immunology; D015588:Observer Variation; D010750:Phosphoproteins / Q000276:immunology; D029701:Saccharomyces cerevisiae Proteins / Q000276:immunology; D018709:Statistics, Nonparametric; D014409:Tumor Necrosis Factor-alpha / Q000097:blood*; D033921:Vesicular Transport Proteins / Q000276:immunology; D014763:Viral Matrix Proteins / Q000276:immunology
D005456:Fluorescent Dyes; D007376:Interleukin-2; D010750:Phosphoproteins; C492204:SFB2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014409:Tumor Necrosis Factor-alpha; D033921:Vesicular Transport Proteins; D014763:Viral Matrix Proteins; C065418:cytomegalovirus matrix protein 65kDa; D007371:Interferon-gamma