Analytical performance of a standardized single-platform MHC tetramer assay for the identification and enumeration of CMV-specific CD8+ T lymphocytes

Abstract

Major histocompatibility complex (MHC) multimers that identify antigen-specific T cells, coupled with flow cytometry, have made a major impact on immunological research. HLA Class I multimers detect T cells directed against viral, tumor, and transplantation antigens with exquisite sensitivity. This technique has become an important standard for the quantification of a T cell immune response. The utility of this method in multicenter studies, however, is dependant on reproducibility between laboratories. As part of a clinical study using a standardized two-tube three-color single-platform method, we monitored and characterized performance across multiple sites using tetramers against the T cell receptors (TCR) specific for MHC Class I, A*0101--VTEHDTLLY, A*0201--NLVPMVATV and B*0702--TPRVTGGGAM CMV peptides. We studied the analytical performance of this method, focusing on reducing background, maximizing signal intensity, and ensuring that sufficient cells are enumerated to provide meaningful statistics. Inter and intra-assay performance were assessed, which included inherent variability introduced by shipping, type of flow cytometer used, protocol adherence, and analytical interpretation across a range of multiple sample levels and specificities under routine laboratory testing conditions. Using the described protocol, it is possible to obtain intra- and interlab CV's of <20%, with a functional sensitivity for absolute tetramer counts of 1 cell/microL and 0.2% tetramer+ percent for A*0101, A*0201, and B*0702 alleles. The standardized single-platform MHC tetramer assay is simple, rapid, reproducible, and useful for assessing CMV-specific T cells, and will allow for reasonable comparisons of clinical evaluations across multiple centers at clinically relevant thresholds (2.0-10.0 cells/microL).