Standardization of cytokine flow cytometry assays

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dc.contributor.authorMaecker, Holden T.
dc.contributor.authorRinfret, Aline
dc.contributor.authorD'Souza, Patricia
dc.contributor.authorDarden, Janice
dc.contributor.authorRoig, Eva
dc.contributor.authorLandry, Claire
dc.contributor.authorHayes, Peter
dc.contributor.authorBirungi, Josephine
dc.contributor.authorAnzala, Omu
dc.contributor.authorGarcia, Miguel
dc.contributor.authorHarari, Alexandre
dc.contributor.authorFrank, Ian
dc.contributor.authorBaydo, Ruth
dc.contributor.authorBaker, Megan
dc.contributor.authorHolbrook, Jennifer
dc.contributor.authorOttinger, Janet
dc.contributor.authorLamoreaux, Laurie
dc.contributor.authorEpling, C. Lorrie
dc.contributor.authorSinclair, Elizabeth
dc.contributor.authorSuni, Maria A.
dc.contributor.authorPunt, Kara
dc.contributor.authorCalarota, Sandra
dc.contributor.authorEl-Bahi, Sophia
dc.contributor.authorAlter, Gailet
dc.contributor.authorMaila, Hazel
dc.contributor.authorKuta, Ellen
dc.contributor.authorCox, Josephine
dc.contributor.authorGray, Clive
dc.contributor.authorAltfeld, Marcus
dc.contributor.authorNougarede, Nolwenn
dc.contributor.authorBoyer, Jean
dc.contributor.authorTussey, Lynda
dc.contributor.authorTobery, Timothy
dc.contributor.authorBredt, Barry
dc.contributor.authorRoederer, Mario
dc.contributor.authorKoup, Richard
dc.contributor.authorMaino, Vernon C.
dc.contributor.authorWeinhold, Kent
dc.contributor.authorPantaleo, Giuseppe
dc.contributor.authorGilmour, Jill
dc.contributor.authorHorton, Helen
dc.contributor.authorSekaly, Rafick P.
dc.date.accessioned2018-07-26T09:33:27Z
dc.date.available2018-07-26T09:33:27Z
dc.date.issued2005-06-24
dc.descriptionICS 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.en_US
dc.description.abstractBackground: 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.en_US
dc.identifier.citation: 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., 2005. Standardization of cytokine flow Cytometry assays.en_US
dc.identifier.issn14712172
dc.identifier.urihttps://hdl.handle.net/20.500.11951/312
dc.language.isoenen_US
dc.publisherBioMed Central Ltd.en_US
dc.subjectCytokine flow cytometryen_US
dc.subjectIntracellular cytokine stainingen_US
dc.titleStandardization of cytokine flow cytometry assaysen_US
dc.typeArticleen_US
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The final, definitive version of this paper has been published in the BMC Immunology, Vol.6, Issue 13, June/2005. https://doi.org/10.1186/1471-2172-6-13; published by BioMed Central Ltd. All rights reserved.
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