Standardization of cytokine flow cytometry assays

Holden T. Maecker, Aline Rinfret, Patricia D'Souza, Janice Darden, Eva Roig, Claire Landry, Peter Hayes, Josephine Birungi, Omu Anzala, Miguel Garcia, Alexandre Harari, Ian Frank, Ruth Baydo, Megan Baker, Jennifer Holbrook, Janet Ottinger, Laurie Lamoreaux, C. Lorrie Epling, Elizabeth Sinclair, Maria A. SuniKara Punt, Sandra Calarota, Sophia El-Bahi, Gailet Alter, Hazel Maila, Ellen Kuta, Josephine Cox, Clive Gray, Marcus Altfeld, Nolwenn Nougarede, Jean Boyer, Lynda Tussey, Timothy Tobery, Barry Bredt, Mario Roederer, Richard Koup, Vernon C. Maino, Kent Weinhold, Giuseppe Pantaleo, Jill Gilmour, Helen Horton, Rafick P. Sekaly

Research output: Contribution to journalArticlepeer-review


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

Original languageEnglish
Article number13
JournalBMC Immunology
Publication statusPublished - Jun 24 2005

ASJC Scopus subject areas

  • Medicine(all)
  • Immunology


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