Standardization of cytokine flow cytometry assays

BMC Immunol. 2005 Jun 24:6:13. doi: 10.1186/1471-2172-6-13.

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% IFNgamma + T cells, and highest (57-82%) for samples with a mean of < 0.1% IFNgamma + 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.

Publication types

  • Comparative Study
  • Evaluation Study
  • Multicenter Study
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Blood Preservation
  • Cryopreservation
  • Cytokines / blood*
  • Cytomegalovirus Infections / blood
  • Cytomegalovirus Infections / immunology
  • Enzyme-Linked Immunosorbent Assay
  • Flow Cytometry / methods
  • Flow Cytometry / standards*
  • Freeze Drying
  • Humans
  • Indicators and Reagents
  • Laboratories
  • Lymphocytes / chemistry
  • Phosphoproteins / blood
  • Reproducibility of Results
  • Specimen Handling
  • T-Lymphocytes / chemistry*
  • Viral Matrix Proteins / blood

Substances

  • Cytokines
  • Indicators and Reagents
  • Phosphoproteins
  • Viral Matrix Proteins
  • cytomegalovirus matrix protein 65kDa