Quantitation of MHC tetramer-positive cells from whole blood: evaluation of a single-platform, six-parameter flow cytometric method.
BACKGROUND
Quantitation of antigen-specific T cells provides an insight into the development and dynamics of T-cell responses in tumor immunology and infectious diseases. Soluble major histocompatibility class I tetramers are widely used to monitor immune responses; however, variations due to handling and analysis are likely to confound comparisons between different experiments and laboratories.


METHODS
Whole blood from healthy donors was stained with HLA-A*0201/tetramers specific for an epitope of phosphoprotein 65, the immunodominant antigen in cytomegalovirus infection. With the help of Trucount tubes, 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.


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


CONCLUSIONS
The method is highly reliable and can be standardized for multiple experiments. It is therefore suitable for the direct ex vivo analysis of antigen-specific T cells in a variety of clinical settings such as infectious, autoimmune, or neoplastic diseases and can be implemented as a tool for multicenter studies.
Wölfl
Matthias
M
Children's Hospital, Department of Pediatric Hematology and Oncology, University of Cologne, Cologne, Germany. matthias.woelfl@medizin.uni-koeln.de
Schalk
Stefan
S
Hellmich
Martin
M
Huster
Katharina M
KM
Busch
Dirk H
DH
Berthold
Frank
F
D001773:Blood Cells / Q000166:cytology; D005434:Flow Cytometry / Q000295:instrumentation / Q000379:methods*; D015234:HLA-A Antigens / Q000032:analysis*; D006801:Humans; D010750:Phosphoproteins / Q000276:immunology; D013601:T-Lymphocytes / Q000737:chemistry / Q000166:cytology*; D014763:Viral Matrix Proteins / Q000276:immunology
D015234:HLA-A Antigens; D010750:Phosphoproteins; D014763:Viral Matrix Proteins; C065418:cytomegalovirus matrix protein 65kDa