Format

Send to

Choose Destination
See comment in PubMed Commons below
Cancer Immunol Immunother. 2015 May;64(5):585-98. doi: 10.1007/s00262-014-1649-1. Epub 2015 Feb 18.

Data analysis as a source of variability of the HLA-peptide multimer assay: from manual gating to automated recognition of cell clusters.

Author information

  • 1Department of Immunology, Institute for Cell Biology, Eberhard Karls University, Auf der Morgenstelle 15, 72076, Tübingen, Germany, cecile.gouttefangeas@uni-tuebingen.de.

Abstract

Multiparameter flow cytometry is an indispensable method for assessing antigen-specific T cells in basic research and cancer immunotherapy. Proficiency panels have shown that cell sample processing, test protocols and data analysis may all contribute to the variability of the results obtained by laboratories performing ex vivo T cell immune monitoring. In particular, analysis currently relies on a manual, step-by-step strategy employing serial gating decisions based on visual inspection of one- or two-dimensional plots. It is therefore operator dependent and subjective. In the context of continuing efforts to support inter-laboratory T cell assay harmonization, the CIMT Immunoguiding Program organized its third proficiency panel dedicated to the detection of antigen-specific CD8(+) T cells by HLA-peptide multimer staining. We first assessed the contribution of manual data analysis to the variability of reported T cell frequencies within a group of laboratories staining and analyzing the same cell samples with their own reagents and protocols. The results show that data analysis is a source of variation in the multimer assay outcome. To evaluate whether an automated analysis approach can reduce variability of proficiency panel data, we used a hierarchical statistical mixture model to identify cell clusters. Challenges for automated analysis were the need to process non-standardized data sets from multiple centers, and the fact that the antigen-specific cell frequencies were very low in most samples. We show that this automated method can circumvent difficulties inherent to manual gating strategies and is broadly applicable for experiments performed with heterogeneous protocols and reagents.

PMID:
25854580
PMCID:
PMC4528367
DOI:
10.1007/s00262-014-1649-1
[PubMed - indexed for MEDLINE]
Free PMC Article
PubMed Commons home

PubMed Commons

0 comments
How to join PubMed Commons

    Supplemental Content

    Full text links

    Icon for Springer Icon for PubMed Central
    Loading ...
    Support Center