title: Quantitation of MHC tetramer-positive cells from whole blood: evaluation of a single-platform, six-parameter flow cytometric method.
abstract: 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.
authors.affiliation: Children's Hospital, Department of Pediatric Hematology and Oncology, University of Cologne, Cologne, Germany. matthias.woelfl@medizin.uni-koeln.de
authors.forename: Matthias
authors.initials: M
authors.lastname: Wölfl
authors.forename: Stefan
authors.initials: S
authors.lastname: Schalk
authors.forename: Martin
authors.initials: M
authors.lastname: Hellmich
authors.forename: Katharina M
authors.initials: KM
authors.lastname: Huster
authors.forename: Dirk H
authors.initials: DH
authors.lastname: Busch
authors.forename: Frank
authors.initials: F
authors.lastname: Berthold
mesh_terms: 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
chemical_list: D015234:HLA-A Antigens; D010750:Phosphoproteins; D014763:Viral Matrix Proteins; C065418:cytomegalovirus matrix protein 65kDa