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Validation of immunomonitoring methods for application in clinical studies: the HLA-peptide multimer staining assay

Validation of immunomonitoring methods for application in clinical studies: the HLA-peptide multimer staining assay
Validation of immunomonitoring methods for application in clinical studies: the HLA-peptide multimer staining assay

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.

assay validation, biomarker, HLA-multimer, immunotherapy, mmunomonitoring
1552-4949
342-353
Chandran, P. Anoop
743e0536-da55-46c5-adb9-0b7dc4cbe50e
Laske, Karoline
8cdca8cb-bf0c-415f-8eb5-51acd6c65f47
Cazaly, Angelica
944b91cc-ab19-463c-ad08-a6b04c6e628d
Rusch, Elisa
1c4a2527-294d-43cf-8a36-c543420d6d13
Schmid-Horch, Barbara
2077758e-f741-4ea5-960a-f343007dc474
Rammensee, Hans Georg
d88e7677-da87-4fa3-b899-5384d5ff24bd
Ottensmeier, Christian H.
42b8a398-baac-4843-a3d6-056225675797
Gouttefangeas, Cécile
e06644af-a88f-4d60-b11c-debfaffefd0d
Chandran, P. Anoop
743e0536-da55-46c5-adb9-0b7dc4cbe50e
Laske, Karoline
8cdca8cb-bf0c-415f-8eb5-51acd6c65f47
Cazaly, Angelica
944b91cc-ab19-463c-ad08-a6b04c6e628d
Rusch, Elisa
1c4a2527-294d-43cf-8a36-c543420d6d13
Schmid-Horch, Barbara
2077758e-f741-4ea5-960a-f343007dc474
Rammensee, Hans Georg
d88e7677-da87-4fa3-b899-5384d5ff24bd
Ottensmeier, Christian H.
42b8a398-baac-4843-a3d6-056225675797
Gouttefangeas, Cécile
e06644af-a88f-4d60-b11c-debfaffefd0d

Chandran, P. Anoop, Laske, Karoline, Cazaly, Angelica, Rusch, Elisa, Schmid-Horch, Barbara, Rammensee, Hans Georg, Ottensmeier, Christian H. and Gouttefangeas, Cécile (2018) Validation of immunomonitoring methods for application in clinical studies: the HLA-peptide multimer staining assay. Cytometry Part B - Clinical Cytometry, 94 (2), 342-353. (doi:10.1002/cyto.b.21397).

Record type: Article

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.

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More information

Accepted/In Press date: 28 June 2016
e-pub ahead of print date: 1 July 2016
Published date: 1 March 2018
Keywords: assay validation, biomarker, HLA-multimer, immunotherapy, mmunomonitoring

Identifiers

Local EPrints ID: 419340
URI: https://eprints.soton.ac.uk/id/eprint/419340
ISSN: 1552-4949
PURE UUID: 24b7d84b-c1de-46ec-9be4-786070d5bc86

Catalogue record

Date deposited: 11 Apr 2018 16:30
Last modified: 08 Aug 2018 16:31

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