Harmonization guidelines for HLA-peptide multimer assays derived from results of a large scale international proficiency panel of the Cancer Vaccine Consortium.
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.
Britten
Cedrik Michael
CM
Tumor Immunology Group, Department for Immunohematology and Blood Transfusion, Leiden University Medical Centre, Leiden, The Netherlands. cebritten@web.de
Janetzki
Sylvia
S
Ben-Porat
Leah
L
Clay
Timothy M
TM
Kalos
Michael
M
Maecker
Holden
H
Odunsi
Kunle
K
Pride
Michael
M
Old
Lloyd
L
Hoos
Axel
A
Romero
Pedro
P
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
D019496:Cancer Vaccines; D015789:HLA-A2 Antigen; D010446:Peptide Fragments