Intracellular Cytokine Staining and Flow Cytometry: Considerations for Application in Clinical Trials of Novel Tuberculosis Vaccines.


Smith, SG; Smits, K; Joosten, SA; van Meijgaarden, KE; Satti, I; Fletcher, HA; Caccamo, N; Dieli, F; Mascart, F; McShane, H; Dockrell, HM; Ottenhoff, TH; TBVI TB Biomarker Working Group; (2015) Intracellular Cytokine Staining and Flow Cytometry: Considerations for Application in Clinical Trials of Novel Tuberculosis Vaccines. PLoS One, 10 (9). e0138042. ISSN 1932-6203 DOI: 10.1371/journal.pone.0138042

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Abstract

Intracellular cytokine staining combined with flow cytometry is one of a number of assays designed to assess T-cell immune responses. It has the specific advantage of enabling the simultaneous assessment of multiple phenotypic, differentiation and functional parameters pertaining to responding T-cells, most notably, the expression of multiple effector cytokines. These attributes make the technique particularly suitable for the assessment of T-cell immune responses induced by novel tuberculosis vaccines in clinical trials. However, depending upon the particular nature of a given vaccine and trial setting, there are approaches that may be taken at different stages of the assay that are more suitable than other alternatives. In this paper, the Tuberculosis Vaccine Initiative (TBVI) TB Biomarker Working group reports on efforts to assess the conditions that will determine when particular assay approaches should be employed. We have found that choices relating to the use of fresh whole blood or peripheral blood mononuclear cells (PBMC) and frozen PBMC; use of serum-containing or serum-free medium; length of stimulation period and use of co-stimulatory antibodies can all affect the sensitivity of intracellular cytokine assays. In the case of sample material, frozen PBMC, despite some loss of sensitivity, may be more advantageous for batch analysis. We also recommend that for multi-site studies, common antibody panels, gating strategies and analysis approaches should be employed for better comparability.

Item Type: Article
Faculty and Department: Academic Services & Administration > Academic Administration
Faculty of Infectious and Tropical Diseases > Dept of Immunology and Infection
Research Centre: TB Centre
Vaccine Centre
PubMed ID: 26367374
Web of Science ID: 361601100208
URI: http://researchonline.lshtm.ac.uk/id/eprint/2305243

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