{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,23]],"date-time":"2026-10-23T06:07:39Z","timestamp":1792735659873,"version":"4.5.0"},"reference-count":34,"publisher":"Springer Science and Business Media LLC","issue":"1","license":[{"start":{"date-parts":[[2005,6,24]],"date-time":"2005-06-24T00:00:00Z","timestamp":1119571200000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/2.0\/"},{"start":{"date-parts":[[2005,6,24]],"date-time":"2005-06-24T00:00:00Z","timestamp":1119571200000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/2.0\/"}],"content-domain":{"domain":["link.springer.com"],"crossmark-restriction":false},"short-container-title":["BMC Immunol"],"abstract":"<jats:title>Abstract<\/jats:title><jats:sec>\n                        <jats:title>Background<\/jats:title>\n                        <jats:p>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).<\/jats:p>\n                     <\/jats:sec><jats:sec>\n                        <jats:title>Results<\/jats:title>\n                        <jats:p>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<jats:sup>+<\/jats:sup>cytokine<jats:sup>+<\/jats:sup> cells and CD8<jats:sup>+<\/jats:sup>cytokine<jats:sup>+<\/jats:sup> cells) were determined by each site. Raw data were also sent to a central site for batch analysis with a dynamic gating template.<\/jats:p>\n                        <jats:p>Mean inter-laboratory coefficient of variation (C.V.) ranged from 17\u201344% 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\u201320%, depending upon the experiment. The inter-lab C.V. was lowest (18\u201324%) for samples with a mean of &gt;0.5% IFN\u03b3 + T cells, and highest (57\u201382%) for samples with a mean of &lt;0.1% IFN\u03b3 + cells.<\/jats:p>\n                     <\/jats:sec><jats:sec>\n                        <jats:title>Conclusion<\/jats:title>\n                        <jats:p>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.<\/jats:p>\n                     <\/jats:sec>","DOI":"10.1186\/1471-2172-6-13","type":"journal-article","created":{"date-parts":[[2005,6,24]],"date-time":"2005-06-24T18:13:37Z","timestamp":1119636817000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":211,"title":["Standardization of cytokine flow cytometry assays"],"prefix":"10.1186","volume":"6","author":[{"given":"Holden 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