Original Article

Cancer Immunology, Immunotherapy

, Volume 58, Issue 10, pp 1701-1713

First online:

Open Access This content is freely available online to anyone, anywhere at any time.

Harmonization guidelines for HLA-peptide multimer assays derived from results of a large scale international proficiency panel of the Cancer Vaccine Consortium

  • Cedrik Michael BrittenAffiliated withTumor Immunology Group, Department for Immunohematology and Blood Transfusion, Leiden University Medical Centre Email author 
  • , Sylvia JanetzkiAffiliated withZellNet Consulting, Inc.
  • , Leah Ben-PoratAffiliated withDepartment of Biostatistics, New York University
  • , Timothy M. ClayAffiliated withSurgery and Immunology, Duke University Medical Center
  • , Michael KalosAffiliated withClinical Immunobiology Correlative Studies Laboratory, City of Hope
  • , Holden MaeckerAffiliated withBD Biosciences
  • , Kunle OdunsiAffiliated withDepartments of Gynecologic Oncology and Immunology, Roswell Park Cancer Institute
  • , Michael PrideAffiliated withVaccines Early Phase Programs, Wyeth Research
  • , Lloyd OldAffiliated withLudwig Institute for Cancer Research, New York Branch, Memorial Sloan-Kettering Cancer Center
    • , Axel HoosAffiliated withBristol-Myers Squibb
    • , Pedro RomeroAffiliated withDivision of Clinical Onco-Immunology, Lausanne Branch, Ludwig Institute for Cancer Research, University Hospital (CHUV) Email author 
    • , for the HLA-peptide Multimer Proficiency Panel of the CVC-CRI Immune Assay Working Group



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.

Experimental design

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.


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.


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.


HLA-peptide multimer Tumor immunity Flow cytometry CTL