Dataset Information

Standardization of cytokine flow cytometry assays.

ABSTRACT:

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% IFNgamma + T cells, and highest (57-82%) for samples with a mean of < 0.1% IFNgamma + 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.

SUBMITTER: Maecker HT

PROVIDER: S-EPMC1184077 | biostudies-literature | 2005 Jun

REPOSITORIES: biostudies-literature

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Publications

Standardization of cytokine flow cytometry assays.

Maecker Holden T HT Rinfret Aline A D'Souza Patricia P Darden Janice J Roig Eva E Landry Claire C Hayes Peter P Birungi Josephine J Anzala Omu O Garcia Miguel M Harari Alexandre A Frank Ian I Baydo Ruth R Baker Megan M Holbrook Jennifer J Ottinger Janet J Lamoreaux Laurie L Epling C Lorrie CL Sinclair Elizabeth E Suni Maria A MA Punt Kara K Calarota Sandra S El-Bahi Sophia S Alter Gailet G Maila Hazel H Kuta Ellen E Cox Josephine J Gray Clive C Altfeld Marcus M Nougarede Nolwenn N Boyer Jean J Tussey Lynda L Tobery Timothy T Bredt Barry B Roederer Mario M Koup Richard R Maino Vernon C VC Weinhold Kent K Pantaleo Giuseppe G Gilmour Jill J Horton Helen H Sekaly Rafick P RP

BMC immunology 20050624

<h4>Background</h4>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 ...[more]

PMID: 15978127

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Dataset Information

Standardization of cytokine flow cytometry assays.

Background

Results

Conclusion

Publications

Standardization of cytokine flow cytometry assays.

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