Standardization of cytokine flow cytometry assays

摘要: 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 different candidates across multiple international organizations conducting clinical trials. As such, study was carried out several involved HIV trials, define inter-lab precision using various sample types, and protocol for each experiment (see additional files online). Three types (activated, fixed, frozen whole blood; fresh cryopreserved PBMC) were shipped sites, where assays cytomegalovirus (CMV) pp65 peptide mix control antigens performed parallel 96-well plates. For one experiment, antibody cocktails lyophilised into plates simplify standardize assay setup. Results (CD4+cytokine+ cells CD8+cytokine+ cells) determined site. Raw data also sent central site batch analysis with dynamic gating template. Mean inter-laboratory coefficient variation (C.V.) ranged from 17–44% depending upon type method. Cryopreserved peripheral blood mononuclear (PBMC) yielded lower C.V.'s than blood. Centralized (using template) reduced C.V. 5–20%, experiment. The lowest (18–24%) samples mean >0.5% IFNγ + cells, highest (57–82%) <0.1% cells. be good precision, improves frequency responding increases. PBMC may yield slightly more consistent results Analysis, particularly gating, is significant source variability, centralized and/or use standardized Use pre-aliquoted lyophilized reagents stimulation further standardization these assays.