Standardization of cytokine flow cytometry assays
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Date
2005-06Author
Maecker, Holden T
Rinfret, Aline
D'Souza, Patricia
Darden, Janice
Roig, Eva
Landry, Claire
Hayes, Peter
Birungi, Josephine
Anzala, Omu
Garcia, Miguel
Harari, Alexandre
Frank, Ian
Baydo, Ruth
Baker, Megan
Holbrook, Jennifer
Ottinger, Janet
Lamoreaux, Laurie
Epling, C Lorrie
Sinclair, Elizabeth
Suni, Maria A
Punt, Kara
Calarota, Sandra
El-Bahi, Sophia
Type
ArticleLanguage
enMetadata
Show full item recordAbstract
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-aliquoted lyophilized
reagents for stimulation and staining can provide further standardization to these assays.
Citation
BMC Immunology 2005, 6:13Collections
- Faculty of Health Sciences (FHS) [10377]