Klin Padiatr 2004; 216 - 46
DOI: 10.1055/s-2004-828591

Evaluation of a single-platform, MHC tetramer based, six-parameter flow cytometric method for immunmonitoring

S Schalk 1, M Wölfl 1, M Hellmich 2
  • 1Children's Hospital, Department of Pediatric Hematology and Oncology, University of Cologne, Cologne, Germany
  • 2Institute of Medical Statistics, Informatics and Epidemiology, University of Cologne, Cologne, Germany

A major setback in tumor vaccine therapy is the lack of an adaequate and easy to use immune monitoring system. The quantitation of antigen-specific T-cells would provide an insight into the development and dynamics of the induced T-cell responses.

In order to establish the monitoring system, we used pp65, immundominant epitope of CMV as a model antigen. Whole blood from healthy donors was stained with HLA-A*0201/tetramers that stain the pp65 specific T-cell receptor. A single-platform, four-color flow cytometric assay was established to obtain absolute counts of tetramer-positive cells. Various staining and gating strategies were evaluated.

The no-wash method was a quick procedure for the quantitation of tetramer-positive events from whole blood. The level for background staining was low. The information about the intra-assay-related variation and the physiologic variation allows for the validation and interpretation of data in future studies.

The method is highly reliable and can be standardized for multiple experiments. It is suitable for the direct ex vivo analysis of antigen-specific T cells in a variety of clinical settings.

However the expected numbers of tumor specific T-Cells after tumor vaccination are 10folds lower than the estimated overall variation of this assay. Therefore it is less suitable for immunmonitoring in tumor immunology.