Standardization of cytokine flow cytometry assays

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