Standardization of cytokine flow cytometry assaysReport as inadecuate

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BMC Immunology

, 6:13

First Online: 24 June 2005Received: 04 December 2004Accepted: 24 June 2005


BackgroundCytokine 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.

ResultsThree 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 CD4cytokine cells and CD8cytokine 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.

ConclusionICS 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.

Electronic supplementary materialThe online version of this article doi:10.1186-1471-2172-6-13 contains supplementary material, which is available to authorized users.

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Author: Holden T Maecker - Aline Rinfret - Patricia D-Souza - Janice Darden - Eva Roig - Claire Landry - Peter Hayes - Josephine 


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