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

¹BD Biosciences, San Jose, USA

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

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

⁴Chelsea and Westminster Hospital and IAVI, London, UK

⁵Uganda Virus Research Institute and IAVI, Entebbe, Uganda

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

⁷Centre Hospitalier Universitaire Vaudois and EUROVAC, Lausanne, Switzerland

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

⁹Duke University Medical Center and HVTN, Durham, USA

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

¹¹University of California, San Francisco, USA

¹²Merck and Co., West Point, USA

¹³University of Pennsylvania, Philadelphia, USA

¹⁴Sanofi Pasteur, Lyon, France

¹⁵Massachusetts General Hospital, Boston, USA

¹⁶National Institute for Communicable Diseases, Johannesburg, South Africa

¹⁷Henry Jackson Foundation, Rockville, USA

author email corresponding author email

BMC Immunology 2005, 6:13doi: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.