Sensitivity and Specificity of CD19.CAR-T Cell Detection by Flow Cytometry and PCR

Chimeric-antigen-receptor-T (CAR-T) cells are currently revolutionizing the field of cancer immunotherapy. Therefore, there is an urgent need for CAR-T cell monitoring by clinicians to assess cell expansion and persistence in patients. CAR-T cell manufacturers and researchers need to evaluate transd...

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Autores principales:	Nicola Schanda, Tim Sauer, Alexander Kunz, Angela Hückelhoven-Krauss, Brigitte Neuber, Lei Wang, Mandy Hinkelbein, David Sedloev, Bailin He, Maria-Luisa Schubert, Carsten Müller-Tidow, Michael Schmitt, Anita Schmitt
Formato:	article
Lenguaje:	EN
Publicado:	MDPI AG 2021
Materias:	CD19.CAR-T cells flow cytometry (FACS) polymerase chain reaction (PCR) detection reagent Biology (General) QH301-705.5
Acceso en línea:	https://doaj.org/article/2c6aaa8ebc984dbc97c671d6ef05193c
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spelling	oai:doaj.org-article:2c6aaa8ebc984dbc97c671d6ef05193c2021-11-25T17:12:54ZSensitivity and Specificity of CD19.CAR-T Cell Detection by Flow Cytometry and PCR10.3390/cells101132082073-4409https://doaj.org/article/2c6aaa8ebc984dbc97c671d6ef05193c2021-11-01T00:00:00Zhttps://www.mdpi.com/2073-4409/10/11/3208https://doaj.org/toc/2073-4409Chimeric-antigen-receptor-T (CAR-T) cells are currently revolutionizing the field of cancer immunotherapy. Therefore, there is an urgent need for CAR-T cell monitoring by clinicians to assess cell expansion and persistence in patients. CAR-T cell manufacturers and researchers need to evaluate transduction efficiency and vector copy number for quality control. Here, CAR expression was analyzed in peripheral blood samples from patients and healthy donors by flow cytometry with four commercially available detection reagents and on the gene level by quantitative polymerase chain reaction (qPCR). Flow cytometric analysis of CAR expression showed higher mean CAR expression values for CD19 CAR detection reagent and the F(ab’)2 antibody than Protein L and CD19 Protein. In addition, the CD19 CAR detection reagent showed a significantly lower median background staining of 0.02% (range 0.007–0.06%) when compared to the F(ab’)2 antibody, CD19 protein and Protein L with 0.80% (range 0.47–1.58%), 0.65% (range 0.25–1.35%) and 0.73% (range 0.44–1.23%). Furthermore, flow cytometry-based CAR-T cell frequencies by CD19 CAR detection reagent showed a good correlation with qPCR results. In conclusion, quality control of CAR-T cell products can be performed by FACS and qPCR. For the monitoring of CAR-T cell frequencies by FACS in patients, CAR detection reagents with a low background staining are preferable.Nicola SchandaTim SauerAlexander KunzAngela Hückelhoven-KraussBrigitte NeuberLei WangMandy HinkelbeinDavid SedloevBailin HeMaria-Luisa SchubertCarsten Müller-TidowMichael SchmittAnita SchmittMDPI AGarticleCD19.CAR-T cellsflow cytometry (FACS)polymerase chain reaction (PCR)detection reagentBiology (General)QH301-705.5ENCells, Vol 10, Iss 3208, p 3208 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	CD19.CAR-T cells flow cytometry (FACS) polymerase chain reaction (PCR) detection reagent Biology (General) QH301-705.5
spellingShingle	CD19.CAR-T cells flow cytometry (FACS) polymerase chain reaction (PCR) detection reagent Biology (General) QH301-705.5 Nicola Schanda Tim Sauer Alexander Kunz Angela Hückelhoven-Krauss Brigitte Neuber Lei Wang Mandy Hinkelbein David Sedloev Bailin He Maria-Luisa Schubert Carsten Müller-Tidow Michael Schmitt Anita Schmitt Sensitivity and Specificity of CD19.CAR-T Cell Detection by Flow Cytometry and PCR
description	Chimeric-antigen-receptor-T (CAR-T) cells are currently revolutionizing the field of cancer immunotherapy. Therefore, there is an urgent need for CAR-T cell monitoring by clinicians to assess cell expansion and persistence in patients. CAR-T cell manufacturers and researchers need to evaluate transduction efficiency and vector copy number for quality control. Here, CAR expression was analyzed in peripheral blood samples from patients and healthy donors by flow cytometry with four commercially available detection reagents and on the gene level by quantitative polymerase chain reaction (qPCR). Flow cytometric analysis of CAR expression showed higher mean CAR expression values for CD19 CAR detection reagent and the F(ab’)2 antibody than Protein L and CD19 Protein. In addition, the CD19 CAR detection reagent showed a significantly lower median background staining of 0.02% (range 0.007–0.06%) when compared to the F(ab’)2 antibody, CD19 protein and Protein L with 0.80% (range 0.47–1.58%), 0.65% (range 0.25–1.35%) and 0.73% (range 0.44–1.23%). Furthermore, flow cytometry-based CAR-T cell frequencies by CD19 CAR detection reagent showed a good correlation with qPCR results. In conclusion, quality control of CAR-T cell products can be performed by FACS and qPCR. For the monitoring of CAR-T cell frequencies by FACS in patients, CAR detection reagents with a low background staining are preferable.
format	article
author	Nicola Schanda Tim Sauer Alexander Kunz Angela Hückelhoven-Krauss Brigitte Neuber Lei Wang Mandy Hinkelbein David Sedloev Bailin He Maria-Luisa Schubert Carsten Müller-Tidow Michael Schmitt Anita Schmitt
author_facet	Nicola Schanda Tim Sauer Alexander Kunz Angela Hückelhoven-Krauss Brigitte Neuber Lei Wang Mandy Hinkelbein David Sedloev Bailin He Maria-Luisa Schubert Carsten Müller-Tidow Michael Schmitt Anita Schmitt
author_sort	Nicola Schanda
title	Sensitivity and Specificity of CD19.CAR-T Cell Detection by Flow Cytometry and PCR
title_short	Sensitivity and Specificity of CD19.CAR-T Cell Detection by Flow Cytometry and PCR
title_full	Sensitivity and Specificity of CD19.CAR-T Cell Detection by Flow Cytometry and PCR
title_fullStr	Sensitivity and Specificity of CD19.CAR-T Cell Detection by Flow Cytometry and PCR
title_full_unstemmed	Sensitivity and Specificity of CD19.CAR-T Cell Detection by Flow Cytometry and PCR
title_sort	sensitivity and specificity of cd19.car-t cell detection by flow cytometry and pcr
publisher	MDPI AG
publishDate	2021
url	https://doaj.org/article/2c6aaa8ebc984dbc97c671d6ef05193c
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Sensitivity and Specificity of CD19.CAR-T Cell Detection by Flow Cytometry and PCR

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