title: Harmonization guidelines for HLA-peptide multimer assays derived from results of a large scale international proficiency panel of the Cancer Vaccine Consortium.
abstract: OBJECTIVE
  The Cancer Vaccine Consortium of the Cancer Research Institute (CVC-CRI) conducted a multicenter HLA-peptide multimer proficiency panel (MPP) with a group of 27 laboratories to assess the performance of the assay.
  
  
  METHODS
  Participants used commercially available HLA-peptide multimers and a well characterized common source of peripheral blood mononuclear cells (PBMC). The frequency of CD8+ T cells specific for two HLA-A2-restricted model antigens was measured by flow cytometry. The panel design allowed for participants to use their preferred staining reagents and locally established protocols for both cell labeling, data acquisition and analysis.
  
  
  RESULTS
  We observed significant differences in both the performance characteristics of the assay and the reported frequencies of specific T cells across laboratories. These results emphasize the need to identify the critical variables important for the observed variability to allow for harmonization of the technique across institutions.
  
  
  CONCLUSIONS
  Three key recommendations emerged that would likely reduce assay variability and thus move toward harmonizing of this assay. (1) Use of more than two colors for the staining (2) collect at least 100,000 CD8 T cells, and (3) use of a background control sample to appropriately set the analytical gates. We also provide more insight into the limitations of the assay and identified additional protocol steps that potentially impact the quality of data generated and therefore should serve as primary targets for systematic analysis in future panels. Finally, we propose initial guidelines for harmonizing assay performance which include the introduction of standard operating protocols to allow for adequate training of technical staff and auditing of test analysis procedures.
authors.affiliation: Tumor Immunology Group, Department for Immunohematology and Blood Transfusion, Leiden University Medical Centre, Leiden, The Netherlands. cebritten@web.de
authors.forename: Cedrik Michael
authors.initials: CM
authors.lastname: Britten
authors.forename: Sylvia
authors.initials: S
authors.lastname: Janetzki
authors.forename: Leah
authors.initials: L
authors.lastname: Ben-Porat
authors.forename: Timothy M
authors.initials: TM
authors.lastname: Clay
authors.forename: Michael
authors.initials: M
authors.lastname: Kalos
authors.forename: Holden
authors.initials: H
authors.lastname: Maecker
authors.forename: Kunle
authors.initials: K
authors.lastname: Odunsi
authors.forename: Michael
authors.initials: M
authors.lastname: Pride
authors.forename: Lloyd
authors.initials: L
authors.lastname: Old
authors.forename: Axel
authors.initials: A
authors.lastname: Hoos
authors.forename: Pedro
authors.initials: P
authors.lastname: Romero
mesh_terms: D001681:Biological Assay; D019496:Cancer Vaccines / Q000276:immunology*; D019411:Clinical Laboratory Techniques / Q000592:standards*; D017408:Guidelines as Topic; D015789:HLA-A2 Antigen / Q000276:immunology / Q000378:metabolism; D006801:Humans; D007391:International Cooperation; D010446:Peptide Fragments / Q000276:immunology / Q000378:metabolism*; D055503:Protein Multimerization
chemical_list: D019496:Cancer Vaccines; D015789:HLA-A2 Antigen; D010446:Peptide Fragments