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

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.

Fig. 1

The figure shows eight selected examples (a–h) of dot plots where gating led to reporting of increased number of events in the upper right quadrant. All dot plots show the CD8-staining on the x-axis and the staining with the HLA-peptide multimer on the y-axis. Dot plots were chosen from laboratories ID01, ID10, ID28 and ID30. Under each dot plot the expected versus the reported (underlined) frequency of multimer-positive CD8 cells is indicated

Fig. 2

Figures a–e show representative dot plots from centers ID05, 17, 20, 21 and 22 reported for staining samples from donor1 with the Influenza-M1 multimer. *Center ID21 set the analytical gate in such a way that multimer-negative cells are shown in the upper right quadrant. **Center ID22 used an atypical gating strategy in which CD8-negative cells were removed at an earlier step of the analysis. The inserted table (f) shows reported values for non-specific binding of the Melan-A/Mart-1-specific multimer in samples from triplicate analysis (T1, T2 and T3) of donor 1 performed by centers ID05, 17 and 20