Harmonization guidelines for HLA-peptide multimer assays derived from results of a large scale international proficiency panel of the Cancer Vaccine Consortium |
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Authors: | Cedrik Michael Britten Sylvia Janetzki Leah Ben-Porat Timothy M Clay Michael Kalos Holden Maecker Kunle Odunsi Michael Pride Lloyd Old Axel Hoos Pedro Romero |
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Institution: | 1. Tumor Immunology Group, Department for Immunohematology and Blood Transfusion, Leiden University Medical Centre, Leiden, The Netherlands 2. ZellNet Consulting, Inc., Fort Lee, NJ, USA 3. Department of Biostatistics, New York University, New York, NY, USA 4. Surgery and Immunology, Duke University Medical Center, Durham, NC, USA 5. Clinical Immunobiology Correlative Studies Laboratory, City of Hope, Duarte, CA, USA 6. BD Biosciences, San Jose, CA, USA 7. Departments of Gynecologic Oncology and Immunology, Roswell Park Cancer Institute, Buffalo, NY, USA 8. Vaccines Early Phase Programs, Wyeth Research, Pearl River, NY, USA 9. Ludwig Institute for Cancer Research, New York Branch, Memorial Sloan-Kettering Cancer Center, New York, NY, USA 10. Bristol-Myers Squibb, Wallingford, CT, USA 11. Division of Clinical Onco-Immunology, Lausanne Branch, Ludwig Institute for Cancer Research, University Hospital (CHUV), Lausanne, Switzerland
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Abstract: | Purpose 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.
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.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. |
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Keywords: | HLA-peptide multimer Tumor immunity Flow cytometry CTL |
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