%0 Journal Article
%T Standardization of cytokine flow cytometry assays
%A Holden T Maecker
%A Aline Rinfret
%A Patricia D'Souza
%A Janice Darden
%A Eva Roig
%A Claire Landry
%A Peter Hayes
%A Josephine Birungi
%A Omu Anzala
%A Miguel Garcia
%A Alexandre Harari
%A Ian Frank
%A Ruth Baydo
%A Megan Baker
%A Jennifer Holbrook
%A Janet Ottinger
%A Laurie Lamoreaux
%A C Lorrie Epling
%A Elizabeth Sinclair
%A Maria A Suni
%A Kara Punt
%A Sandra Calarota
%A Sophia El-Bahi
%A Gailet Alter
%A Hazel Maila
%A Ellen Kuta
%A Josephine Cox
%A Clive Gray
%A Marcus Altfeld
%A Nolwenn Nougarede
%J BMC Immunology
%D 2005
%I BioMed Central
%R 10.1186/1471-2172-6-13
%X 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¨C44% 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¨C20%, depending upon the experiment. The inter-lab C.V. was lowest (18¨C24%) for samples with a mean of >0.5% IFN¦Ã + T cells, and highest (57¨C82%) for samples with a mean of <0.1% IFN¦Ã + cells.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.Enzyme-linked immunospot (ELISPOT) and cytokine flow cytometry (CFC) (or more specifically, intracellular cytokine staining (ICS)) are popular methods for single-cell analysis of antigen-specific T cell cytokine production. T cell production of IFN¦Ã, and increasingly also IL-2, is taken as a measure of vaccine immunogenicity in experimental vaccine trials. Of
%U http://www.biomedcentral.com/1471-2172/6/13