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Cytometry B Clin Cytom. 2018 Mar;94(2):342-353. doi: 10.1002/cyto.b.21397. Epub 2016 Jul 22.

Validation of Immunomonitoring Methods for Application in Clinical Studies: The HLA-Peptide Multimer Staining Assay.

Author information

1
Department of Immunology, Institute for Cell Biology, Eberhard Karls University, and German Cancer Consortium (DKTK), German Cancer Research Center (DKFZ) Partner Site Tuebingen, Tuebingen, Germany.
2
Cancer Sciences Unit, Faculty of Medicine, University of Southampton, Southampton General Hospital, Southampton, SO16 6YD, United Kingdom.
3
Center for Clinical Transfusion Medicine GmbH, University Hospital, Tuebingen.

Abstract

BACKGROUND:

Validated assays are essential to generate data with defined specificity, consistency, and reliability. Although the process of validation is required for applying immunoassays in the context of clinical studies, reports on systematic validation of in vitro T cell assays are scarce so far. We recently validated our HLA-peptide multimer staining assay in a systematic manner so as to qualify the method for monitoring antigen-specific T cell responses after immunotherapy.

METHODS:

Parameters of the assay, specificity, precision, linearity, sensitivity, and robustness were assessed systematically. Experiments were designed to address specifically each parameter and are detailed.

RESULTS:

Nonspecific multimer staining was below the acceptance limit of 0.02% multimer(+) CD8(+) cells. The assay showed acceptable precision in all dimensions it was repeated (CV < 10%) and also demonstrated a linear detection (R2  > 0.99) of antigen specific cells.

CONCLUSIONS:

We succeeded in validating the HLA-multimer staining assay in a systematic manner. Additionally, we propose a technical framework and recommendations that can be applied for validating other T cell assessment methods. © 2016 International Clinical Cytometry Society.

KEYWORDS:

HLA-multimer; assay validation; biomarker; immunotherapy; mmunomonitoring

PMID:
27363684
DOI:
10.1002/cyto.b.21397
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