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Publication Details
AFRICAN RESEARCH NEXUS
SHINING A SPOTLIGHT ON AFRICAN RESEARCH
Standardization of cytokine flow cytometry assays
BMC Immunology, Volume 6, Article 13, Year 2005
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Description
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-ali quoted lyophilized reagents for stimulation and staining can provide further standardization to these assays. © 2005 Maecker et al; licensee BioMed Central Ltd.
Available Materials
https://efashare.b-cdn.net/share/pmc/articles/PMC1184077/bin/1471-2172-6-13-S1.pdf
https://efashare.b-cdn.net/share/pmc/articles/PMC1184077/bin/1471-2172-6-13-S2.pdf
https://efashare.b-cdn.net/share/pmc/articles/PMC1184077/bin/1471-2172-6-13-S3.pdf
https://efashare.b-cdn.net/share/pmc/articles/PMC1184077/bin/1471-2172-6-13-S4.pdf
Authors & Co-Authors
Maecker, Holden Terry
Unknown Affiliation
Rinfret, Aline
Unknown Affiliation
D'Souza, M. Patricia
Unknown Affiliation
Darden, Janice
Unknown Affiliation
Roig, Eva
Unknown Affiliation
Landry, Claire
Unknown Affiliation
Hayes, Peter John
Unknown Affiliation
Birungi, Josephine
Unknown Affiliation
Anzala, Aggrey Omu
Unknown Affiliation
Garcia, Miguel
Unknown Affiliation
Harari, Alexandre
Unknown Affiliation
Frank, Ian D.
Unknown Affiliation
Baydo, Ruth
Unknown Affiliation
Baker, Megan
Unknown Affiliation
Holbrook, Jennifer
Unknown Affiliation
Ottinger, Janet S.
Unknown Affiliation
Lamoreaux, Laurie
Unknown Affiliation
Epling, C. Lorrie
Unknown Affiliation
Sinclair, Elizabeth
Unknown Affiliation
Suni, Maria A.
Unknown Affiliation
Punt, Kara
Unknown Affiliation
Calarota, Sandra A.
Unknown Affiliation
El-Bahi, Sophia
Unknown Affiliation
Alter, Galit
Unknown Affiliation
Maila, Hazel T.
Unknown Affiliation
Kuta, Ellen G.
Unknown Affiliation
Cox, Josephine H.
Unknown Affiliation
Gray, Clive M.
Unknown Affiliation
Altfeld, Marcus A.
Unknown Affiliation
Nougarede, Nolwenn
Unknown Affiliation
Boyer, Jean
Unknown Affiliation
Tussey, Lynda
Unknown Affiliation
Tobery, Timothy
Unknown Affiliation
Bredt, Barry
Unknown Affiliation
Roederer, Mario
Unknown Affiliation
Koup, Richard A.
Unknown Affiliation
Maino, Vernon C.
Unknown Affiliation
Weinhold, Kent J.
Unknown Affiliation
Pantaleo, Giuseppe P.
Unknown Affiliation
Gilmour, Jill W.
Unknown Affiliation
Horton, Helen
Unknown Affiliation
Seḱaly, Rafick Pierre
Unknown Affiliation
Statistics
Citations: 228
Authors: 42
Affiliations: 18
Identifiers
Doi:
10.1186/1471-2172-6-13
ISSN:
14712172
e-ISSN:
14712172
Research Areas
Infectious Diseases
Study Approach
Quantitative