title: A critical assessment for the value of markers to gate-out undesired events in HLA-peptide multimer staining protocols.
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.
authors: Division of Translational and Experimental Oncology, Department of Internal Medicine III, University Medical Center of the Johannes Gutenberg-University, Mainz, Germany.
authors: Sebastian
authors: S
authors: Attig
authors: Leah
authors: L
authors: Price
authors: Sylvia
authors: S
authors: Janetzki
authors: Michael
authors: M
authors: Kalos
authors: Michael
authors: M
authors: Pride
authors: Lisa
authors: L
authors: McNeil
authors: Tim
authors: T
authors: Clay
authors: Jianda
authors: J
authors: Yuan
authors: Kunle
authors: K
authors: Odunsi
authors: Axel
authors: A
authors: Hoos
authors: Pedro
authors: P
authors: Romero
authors: Cedrik M
authors: CM
authors: Britten
mesh_terms: D015415:Biomarkers / Q000378:metabolism*; D018414:CD8-Positive T-Lymphocytes / Q000166:cytology / Q000276:immunology; D002470:Cell Survival; D004396:Coloring Agents / Q000378:metabolism; D019295:Computational Biology; D006680:HLA Antigens / Q000378:metabolism*; D006801:Humans; D018655:Lymphocyte Count; D010455:Peptides / Q000378:metabolism*; D011485:Protein Binding; D055503:Protein Multimerization; D015203:Reproducibility of Results; D013194:Staining and Labeling / Q000379:methods*; D013347:Subcellular Fractions / Q000378:metabolism
chemical_list: D015415:Biomarkers; D004396:Coloring Agents; D006680:HLA Antigens; D010455:Peptides