Browsing by Author "Maecker, Holden T."

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    Standardization of cytokine flow cytometry assays
    (BioMed Central Ltd., 2005-06-24) Maecker, Holden T.; Rinfret, Aline; D'Souza, Patricia; Darden, Janice; Roig, Eva; Landry, Claire; Hayes, Peter; Birungi, Josephine; Anzala, Omu; Garcia, Miguel; Harari, Alexandre; Frank, Ian; Baydo, Ruth; Baker, Megan; Holbrook, Jennifer; Ottinger, Janet; Lamoreaux, Laurie; Epling, C. Lorrie; Sinclair, Elizabeth; Suni, Maria A.; Punt, Kara; Calarota, Sandra; El-Bahi, Sophia; Alter, Gailet; Maila, Hazel; Kuta, Ellen; Cox, Josephine; Gray, Clive; Altfeld, Marcus; Nougarede, Nolwenn; Boyer, Jean; Tussey, Lynda; Tobery, Timothy; Bredt, Barry; Roederer, Mario; Koup, Richard; Maino, Vernon C.; Weinhold, Kent; Pantaleo, Giuseppe; Gilmour, Jill; Horton, Helen; Sekaly, Rafick P.
    Background: Cytokine flow cytometry (CFC) or intracellular cytokine staining (ICS) can quantitate antigen-specific T cell responses in settings such as experimental vaccination. Standardization of ICS among laboratories performing vaccine studies would provide a common platform by which to compare the immunogenicity of different vaccine candidates across multiple international organizations conducting clinical trials. As such, a study was carried out among several laboratories involved in HIV clinical trials, to define the inter-lab precision of ICS using various sample types, and using a common protocol for each experiment (see additional files online). Results: Three sample types (activated, fixed, and frozen whole blood; fresh whole blood; and cryopreserved PBMC) were shipped to various sites, where ICS assays using cytomegalovirus (CMV) pp65 peptide mix or control antigens were performed in parallel in 96-well plates. For one experiment, antigens and antibody cocktails were lyophilised into 96-well plates to simplify and standardize the assay setup. Results (CD4+cytokine+ cells and CD8+cytokine+ cells) were determined by each site. Raw data were also sent to a central site for batch analysis with a dynamic gating template. Mean inter-laboratory coefficient of variation (C.V.) ranged from 17–44% depending upon the sample type and analysis method. Cryopreserved peripheral blood mononuclear cells (PBMC) yielded lower inter-lab C.V.'s than whole blood. Centralized analysis (using a dynamic gating template) reduced the inter-lab C.V. by 5–20%, depending upon the experiment. The inter-lab C.V. was lowest (18–24%) for samples with a mean of >0.5% IFNγ + T cells, and highest (57–82%) for samples with a mean of <0.1% IFNγ + cells. Conclusion: ICS assays can be performed by multiple laboratories using a common protocol with good inter-laboratory precision, which improves as the frequency of responding cells increases. Cryopreserved PBMC may yield slightly more consistent results than shipped whole blood. Analysis, particularly gating, is a significant source of variability, and can be reduced by centralized analysis and/or use of a standardized dynamic gating template. Use of pre-aliquoted lyophilized reagents for stimulation and staining can provide further standardization to these assays.