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Open Access Research

A critical assessment for the value of markers to gate-out undesired events in HLA-peptide multimer staining protocols

Sebastian Attig1, Leah Price2, Sylvia Janetzki3, Michael Kalos4, Michael Pride5, Lisa McNeil5, Tim Clay6, Jianda Yuan7, Kunle Odunsi8, Axel Hoos9, Pedro Romero10, Cedrik M Britten111* and the CRI-CIC Assay Working Group

Author Affiliations

1 Division of Translational and Experimental Oncology, Department of Internal Medicine III, University Medical Center of the Johannes Gutenberg-University, Mainz, Germany

2 Department of Biostatistics, New York University, New York, NY USA

3 ZellNet Consulting, Inc., Fort Lee, NJ USA

4 Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Abramson Family Cancer Research Institute, Philadelphia, PA USA

5 Vaccine Research East and Early Development, Pfizer Inc. Pearl River, NY USA

6 Surgery and Immunology, Duke University Medical Center, Durham, NC, USA

7 Ludwig Center for Cancer Immunotherapy, Memorial Sloan-Kettering Cancer Center, New York, NY USA

8 Departments of Gynecologic Oncology and Immunology, Roswell Park Cancer Institute, Buffalo, NY, USA

9 Bristol-Myers Squibb, Wallingford, CT USA

10 Translational Tumor Immunology Group, Ludwig Center for Cancer Research of the University of Lausanne, Switzerland

11 Research & Development, BioNTech AG, Mainz, Germany

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Journal of Translational Medicine 2011, 9:108  doi:10.1186/1479-5876-9-108

Published: 11 July 2011

Abstract

Background

The introduction of antibody markers to identify undesired cell populations in flow-cytometry based assays, so called DUMP channel markers, has become a practice in an increasing number of labs performing HLA-peptide multimer assays. However, the impact of the introduction of a DUMP channel in multimer assays has so far not been systematically investigated across a broad variety of protocols.

Methods

The Cancer Research Institute's Cancer Immunotherapy Consortium (CRI-CIC) conducted a multimer proficiency panel with a specific focus on the impact of DUMP channel use. The panel design allowed individual laboratories to use their own protocol for thawing, staining, gating, and data analysis. Each experiment was performed twice and in parallel, with and without the application of a dump channel strategy.

Results

The introduction of a DUMP channel is an effective measure to reduce the amount of non-specific MULTIMER binding to T cells. Beneficial effects for the use of a DUMP channel were observed across a wide range of individual laboratories and for all tested donor-antigen combinations. In 48% of experiments we observed a reduction of the background MULTIMER-binding. In this subgroup of experiments the median background reduction observed after introduction of a DUMP channel was 0.053%.

Conclusions

We conclude that appropriate use of a DUMP channel can significantly reduce background staining across a large fraction of protocols and improve the ability to accurately detect and quantify the frequency of antigen-specific T cells by multimer reagents. Thus, use of a DUMP channel may become crucial for detecting low frequency antigen-specific immune responses. Further recommendations on assay performance and data presentation guidelines for publication of MULTIMER experimental data are provided.