Enumeration of antigen-specific CD8+ T lymphocytes by single-platform, HLA tetramer-based flow cytometry: A European multicenter evaluation

Background: HLA class I peptide tetramers represent powerful diagnostic tools for detection and monitoring of antigen-specific CD8+ T cells. The impetus for the current multicenter study is the critical need to standardize tetramer flow cytometry if it is to be implemented as a routine diagnostic assay. Hence, the European Working Group on Clinical Cell Analysis set out to develop and evaluate a single-platform tetramer-based method that used cytomegalovirus (CMV) as the antigenic model. Methods: Absolute numbers of CMV-specific CD8+ T cells were obtained by combining the percentage of tetramer-binding cells with the absolute CD8+ T-cell count. Six send-outs of stabilized blood from healthy individuals or CMV-carrying donors with CMV-specific CD8+ T-cell counts of 3 to 10 cells/μl were distributed to 7 to 16 clinical sites. These sites were requested to enumerate CD8+ T cells and, in the case of CMV-positive donors, CMV-specific subsets on three separate occasions using the standard method. Results: Between-site coefficients of variation of less than 10% (absolute CD8 + T-cell counts) and ∼ 30% (percentage and absolute numbers of CMV-specific CD8+ T cells) were achieved. Within-site coefficients of variation were ∼ 5% (absolute CD8+ T-cell counts), ∼ 9% (percentage CMV-specific CD8+ T cells), and ∼ 17% (absolute CMV-specific CD8+ T-cell counts). The degree of variation tended to correlate inversely with the proportion of CMV-specific CD8+ T-cell subsets. Conclusions: The single-platform MHC tetramer-based method for antigen-specific CD8+ T-cell counting has been evaluated by a European group of laboratories and can be considered a reproducible assay for routine enumeration of antigen-specific CD8+ T cells.

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