Open Access Highly Accessed Methodology article

Standardization of cytokine flow cytometry assays

Holden T Maecker1*, Aline Rinfret2, Patricia D'Souza3, Janice Darden3, Eva Roig2, Claire Landry2, Peter Hayes4, Josephine Birungi5, Omu Anzala6, Miguel Garcia7, Alexandre Harari7, Ian Frank8, Ruth Baydo8, Megan Baker9, Jennifer Holbrook9, Janet Ottinger9, Laurie Lamoreaux10, C Lorrie Epling11, Elizabeth Sinclair11, Maria A Suni1, Kara Punt12, Sandra Calarota13, Sophia El-Bahi14, Gailet Alter15, Hazel Maila16, Ellen Kuta17, Josephine Cox17, Clive Gray16, Marcus Altfeld15, Nolwenn Nougarede14, Jean Boyer13, Lynda Tussey12, Timothy Tobery12, Barry Bredt11, Mario Roederer10, Richard Koup10, Vernon C Maino1, Kent Weinhold9, Giuseppe Pantaleo7, Jill Gilmour4, Helen Horton8 and Rafick P Sekaly2

Author Affiliations

1 BD Biosciences, San Jose, USA

2 Université de Montreal and CANVAC, the Canadian Network for Vaccines and Immunotherapeutics, Montreal, Canada

3 National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, USA

4 Chelsea and Westminster Hospital and IAVI, London, UK

5 Uganda Virus Research Institute and IAVI, Entebbe, Uganda

6 Kenya AIDS Vaccine Initiative (KAVI), University of Nairobi, Kenya

7 Centre Hospitalier Universitaire Vaudois and EUROVAC, Lausanne, Switzerland

8 University of Washington and HVTN, Fred Hutchinson Cancer Research Center, Seattle, USA

9 Duke University Medical Center and HVTN, Durham, USA

10 Vaccine Research Center, National Institutes of Health, Bethesda, USA

11 University of California, San Francisco, USA

12 Merck and Co., West Point, USA

13 University of Pennsylvania, Philadelphia, USA

14 Sanofi Pasteur, Lyon, France

15 Massachusetts General Hospital, Boston, USA

16 National Institute for Communicable Diseases, Johannesburg, South Africa

17 Henry Jackson Foundation, Rockville, USA

For all author emails, please log on.

BMC Immunology 2005, 6:13  doi:10.1186/1471-2172-6-13

Published: 24 June 2005

Abstract

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.