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Harmonization of the intracellular cytokine staining assay

Harmonization of the intracellular cytokine staining assay
Harmonization of the intracellular cytokine staining assay
Active immunotherapy for cancer is an accepted treatment modality aiming to reinforce the T-cell response to cancer. T-cell reactivity is measured by various assays and used to guide the clinical development of immunotherapeutics. However, data obtained across different institutions may vary substantially making comparative conclusions difficult. The Cancer Immunotherapy Immunoguiding Program organizes proficiency panels to identify key parameters influencing the outcome of commonly used T-cell assays followed by harmonization. Our successes with IFN?-ELISPOT and peptide HLA multimer analysis have led to the current study on intracellular cytokine staining (ICS). We report the results of three successive panels evaluating this assay. At the beginning, 3 out of 9 participants (33 %) were able to detect >6 out of 8 known virus-specific T-cell responses in peripheral blood of healthy individuals. This increased to 50 % of the laboratories in the second phase. The reported percentages of cytokine-producing T cells by the different laboratories were highly variable with coefficients of variation well over 60 %. Variability could partially be explained by protocol-related differences in background cytokine production leading to sub-optimal signal-to-noise ratios. The large number of protocol variables prohibited identification of prime guidelines to harmonize the assays. In addition, the gating strategy used to identify reactive T cells had a major impact on assay outcome. Subsequent harmonization of the gating strategy considerably reduced the variability within the group of participants. In conclusion, we propose that first basic guidelines should be applied for gating in ICS experiments before harmonizing assay protocol variables.
0340-7004
967-978
Welters, Marij J.P.
ece995e7-8d4c-431d-83a9-fc0a24f6fdf4
Gouttefangeas, Cécile
e06644af-a88f-4d60-b11c-debfaffefd0d
Ramwadhdoebe, Tamara H.
da9361db-4d0c-440f-9c30-a72be7aa972a
Letsch, Anne
6ec5772a-5a2c-4978-8deb-f544a748b84c
Ottensmeier, Christian H.
42b8a398-baac-4843-a3d6-056225675797
Britten, Cedrik M.
a303deac-ffba-4a55-9a7e-fa5e158c0f8d
van der Burg, Sjoerd H.
1e880617-4966-4046-b70c-0ef451c2c2ab
Welters, Marij J.P.
ece995e7-8d4c-431d-83a9-fc0a24f6fdf4
Gouttefangeas, Cécile
e06644af-a88f-4d60-b11c-debfaffefd0d
Ramwadhdoebe, Tamara H.
da9361db-4d0c-440f-9c30-a72be7aa972a
Letsch, Anne
6ec5772a-5a2c-4978-8deb-f544a748b84c
Ottensmeier, Christian H.
42b8a398-baac-4843-a3d6-056225675797
Britten, Cedrik M.
a303deac-ffba-4a55-9a7e-fa5e158c0f8d
van der Burg, Sjoerd H.
1e880617-4966-4046-b70c-0ef451c2c2ab

Welters, Marij J.P., Gouttefangeas, Cécile, Ramwadhdoebe, Tamara H., Letsch, Anne, Ottensmeier, Christian H., Britten, Cedrik M. and van der Burg, Sjoerd H. (2012) Harmonization of the intracellular cytokine staining assay. Cancer Immunology Immunotherapy, 61 (7), 967-978. (doi:10.1007/s00262-012-1282-9). (PMID:22714399 )

Record type: Article

Abstract

Active immunotherapy for cancer is an accepted treatment modality aiming to reinforce the T-cell response to cancer. T-cell reactivity is measured by various assays and used to guide the clinical development of immunotherapeutics. However, data obtained across different institutions may vary substantially making comparative conclusions difficult. The Cancer Immunotherapy Immunoguiding Program organizes proficiency panels to identify key parameters influencing the outcome of commonly used T-cell assays followed by harmonization. Our successes with IFN?-ELISPOT and peptide HLA multimer analysis have led to the current study on intracellular cytokine staining (ICS). We report the results of three successive panels evaluating this assay. At the beginning, 3 out of 9 participants (33 %) were able to detect >6 out of 8 known virus-specific T-cell responses in peripheral blood of healthy individuals. This increased to 50 % of the laboratories in the second phase. The reported percentages of cytokine-producing T cells by the different laboratories were highly variable with coefficients of variation well over 60 %. Variability could partially be explained by protocol-related differences in background cytokine production leading to sub-optimal signal-to-noise ratios. The large number of protocol variables prohibited identification of prime guidelines to harmonize the assays. In addition, the gating strategy used to identify reactive T cells had a major impact on assay outcome. Subsequent harmonization of the gating strategy considerably reduced the variability within the group of participants. In conclusion, we propose that first basic guidelines should be applied for gating in ICS experiments before harmonizing assay protocol variables.

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Accepted/In Press date: 2 May 2012
Published date: July 2012
Organisations: Cancer Sciences

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Local EPrints ID: 396351
URI: https://eprints.soton.ac.uk/id/eprint/396351
ISSN: 0340-7004
PURE UUID: 5a493d10-03fe-4c11-9960-c2cf88e7c88f

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Date deposited: 08 Jun 2016 15:31
Last modified: 15 Jul 2019 20:23

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Contributors

Author: Marij J.P. Welters
Author: Cécile Gouttefangeas
Author: Tamara H. Ramwadhdoebe
Author: Anne Letsch
Author: Cedrik M. Britten
Author: Sjoerd H. van der Burg

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