Optimization of whole blood antigen-specific cytokine assays for CD4(+) T cells.

Important note: Information in this article was accurate in 2000. The state of the art may have changed since the publication date.

Optimization of whole blood antigen-specific cytokine assays for CD4(+) T cells.

Cytometry. 2000 May 1;40(1):60-8. Unique Identifier : AIDSLINE MED/20218907
Nomura LE; Walker JM; Maecker HT; Biological Research and Development, BD Biosciences,; Immunocytometry Systems, San Jose, CA 95131, USA.

Abstract: BACKGROUND: The analysis of cytokine production is a valuable component of studies of immune response to stimulation such as pathogens, vaccines, and other immunological challenges. One highly sensitive method of cytokine evaluation involves three-color flow cytometric analysis of cytokine production in individual CD4(+) T cells. METHODS: We present four methods to enhance the acquisition and analysis of cells secreting the cytokines interferon gamma (IFNgamma), tumor necrosis factor alpha (TNFalpha), interleukin-2 (IL-2), and interleukin-4 (IL-4). Using cytomegalovirus (CMV) as the antigenic model, titration and kinetic experiments were carried out in whole blood from CMV-seropositive donors. RESULTS: CMV is most effective as a stimulating antigen when used at a dose of 5 microg/ml and for a period of at least 6 h, the first 2 h in the absence of 10 microg/ml Brefeldin A. This period of incubation can be made more convenient by the use of a "timed cooling" device, whereby the samples are automatically cooled and held at 4 degrees C at the end of incubation. Such timed cooling does not affect backgrounds or the proportion of responding cells. For certain samples, a high background can be reduced by adding fourth-color reagents. They identify and allow for elimination of monocytes and activated platelets, which contribute to false positive staining. CONCLUSIONS: These optimizations make the assay both convenient for use in whole blood samples and highly reproducible (intra-assay variability is less than 10%; interassay variability is less than 25%). Copyright 2000 Wiley-Liss, Inc.

Keywords: JOURNAL ARTICLE Antigens, Viral/IMMUNOLOGY Blood Donors Cold Cytomegalovirus/IMMUNOLOGY CD4 Lymphocyte Count/*METHODS CD4-Positive T-Lymphocytes/CHEMISTRY/*CYTOLOGY/VIROLOGY False Positive Reactions Flow Cytometry/*METHODS/STANDARDS Fluorescent Antibody Technique Human Interferon Type II/ANALYSIS Interleukin-2/ANALYSIS Interleukin-4/ANALYSIS Kinetics Reproducibility of Results Titrimetry Tumor Necrosis Factor/ANALYSIS

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