Comparison of FACS and PCR for Detection of BCMA-CAR-T Cells
Chimeric-antigen-receptor (CAR)-T-cell therapy is already widely used to treat patients who are relapsed or refractory to chemotherapy, antibodies, or stem-cell transplantation. Multiple myeloma still constitutes an incurable disease. CAR-T-cell therapy that targets BCMA (B-cell maturation antigen)...
| Main Authors: | , , , , , , , , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2022-01-01
|
| Series: | International Journal of Molecular Sciences |
| Subjects: | |
| Online Access: | https://www.mdpi.com/1422-0067/23/2/903 |
| _version_ | 1797493224538898432 |
|---|---|
| author | Avinoam Reichman Alexander Kunz Jara J. Joedicke Uta E. Höpken Anna Keib Brigitte Neuber David Sedloev Lei Wang Genqiao Jiang Angela Hückelhoven-Krauss Franziska Eberhardt Carsten Müller-Tidow Martin Wermke Armin Rehm Michael Schmitt Anita Schmitt |
| author_facet | Avinoam Reichman Alexander Kunz Jara J. Joedicke Uta E. Höpken Anna Keib Brigitte Neuber David Sedloev Lei Wang Genqiao Jiang Angela Hückelhoven-Krauss Franziska Eberhardt Carsten Müller-Tidow Martin Wermke Armin Rehm Michael Schmitt Anita Schmitt |
| author_sort | Avinoam Reichman |
| collection | DOAJ |
| description | Chimeric-antigen-receptor (CAR)-T-cell therapy is already widely used to treat patients who are relapsed or refractory to chemotherapy, antibodies, or stem-cell transplantation. Multiple myeloma still constitutes an incurable disease. CAR-T-cell therapy that targets BCMA (B-cell maturation antigen) is currently revolutionizing the treatment of those patients. To monitor and improve treatment outcomes, methods to detect CAR-T cells in human peripheral blood are highly desirable. In this study, three different detection reagents for staining BCMA-CAR-T cells by flow cytometry were compared. Moreover, a quantitative polymerase chain reaction (qPCR) to detect BCMA-CAR-T cells was established. By applying a cell-titration experiment of BCMA-CAR-T cells, both methods were compared head-to-head. In flow-cytometric analysis, the detection reagents used in this study could all detect BCMA-CAR-T cells at a similar level. The results of false-positive background staining differed as follows (standard deviation): the BCMA-detection reagent used on the control revealed a background staining of 0.04% (±0.02%), for the PE-labeled human BCMA peptide it was 0.25% (±0.06%) and for the polyclonal anti-human IgG antibody it was 7.2% (±9.2%). The ability to detect BCMA-CAR-T cells down to a concentration of 0.4% was similar for qPCR and flow cytometry. The qPCR could detect even lower concentrations (0.02–0.01%). In summary, BCMA-CAR-T-cell monitoring can be reliably performed by both flow cytometry and qPCR. In flow cytometry, reagents with low background staining should be preferred. |
| first_indexed | 2024-03-10T01:16:57Z |
| format | Article |
| id | doaj.art-3db2c18fb74e4b93a9d4bd720be7894e |
| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-10T01:16:57Z |
| publishDate | 2022-01-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | International Journal of Molecular Sciences |
| spelling | doaj.art-3db2c18fb74e4b93a9d4bd720be7894e2023-11-23T14:06:15ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672022-01-0123290310.3390/ijms23020903Comparison of FACS and PCR for Detection of BCMA-CAR-T CellsAvinoam Reichman0Alexander Kunz1Jara J. Joedicke2Uta E. Höpken3Anna Keib4Brigitte Neuber5David Sedloev6Lei Wang7Genqiao Jiang8Angela Hückelhoven-Krauss9Franziska Eberhardt10Carsten Müller-Tidow11Martin Wermke12Armin Rehm13Michael Schmitt14Anita Schmitt15Department of Hematology, Oncology and Rheumatology, Heidelberg University Hospital, Im Neuenheimer Feld 410, 69120 Heidelberg, GermanyDepartment of Hematology, Oncology and Rheumatology, Heidelberg University Hospital, Im Neuenheimer Feld 410, 69120 Heidelberg, GermanyDepartment of Microenvironmental Regulation in Autoimmunity and Cancer, Max-Delbrück Center for Molecular Medicine (MDC), Robert-Rössle-Str. 10, 13125 Berlin-Buch, GermanyDepartment of Translational Tumor Immunology, Max-Delbrück Center for Molecular Medicine (MDC), Robert-Rössle-Str. 10, 13125 Berlin-Buch, GermanyDepartment of Hematology, Oncology and Rheumatology, Heidelberg University Hospital, Im Neuenheimer Feld 410, 69120 Heidelberg, GermanyDepartment of Hematology, Oncology and Rheumatology, Heidelberg University Hospital, Im Neuenheimer Feld 410, 69120 Heidelberg, GermanyDepartment of Hematology, Oncology and Rheumatology, Heidelberg University Hospital, Im Neuenheimer Feld 410, 69120 Heidelberg, GermanyDepartment of Hematology, Oncology and Rheumatology, Heidelberg University Hospital, Im Neuenheimer Feld 410, 69120 Heidelberg, GermanyDepartment of Hematology, Oncology and Rheumatology, Heidelberg University Hospital, Im Neuenheimer Feld 410, 69120 Heidelberg, GermanyDepartment of Hematology, Oncology and Rheumatology, Heidelberg University Hospital, Im Neuenheimer Feld 410, 69120 Heidelberg, GermanyDepartment of Hematology, Oncology and Rheumatology, Heidelberg University Hospital, Im Neuenheimer Feld 410, 69120 Heidelberg, GermanyDepartment of Hematology, Oncology and Rheumatology, Heidelberg University Hospital, Im Neuenheimer Feld 410, 69120 Heidelberg, GermanyNCT/UCC Early Clinical Trial Unit (ECTU), Medical Faculty C.-G. Carus, Technical University Dresden, Fetscherstraße 74, 01307 Dresden, GermanyDepartment of Microenvironmental Regulation in Autoimmunity and Cancer, Max-Delbrück Center for Molecular Medicine (MDC), Robert-Rössle-Str. 10, 13125 Berlin-Buch, GermanyDepartment of Hematology, Oncology and Rheumatology, Heidelberg University Hospital, Im Neuenheimer Feld 410, 69120 Heidelberg, GermanyDepartment of Hematology, Oncology and Rheumatology, Heidelberg University Hospital, Im Neuenheimer Feld 410, 69120 Heidelberg, GermanyChimeric-antigen-receptor (CAR)-T-cell therapy is already widely used to treat patients who are relapsed or refractory to chemotherapy, antibodies, or stem-cell transplantation. Multiple myeloma still constitutes an incurable disease. CAR-T-cell therapy that targets BCMA (B-cell maturation antigen) is currently revolutionizing the treatment of those patients. To monitor and improve treatment outcomes, methods to detect CAR-T cells in human peripheral blood are highly desirable. In this study, three different detection reagents for staining BCMA-CAR-T cells by flow cytometry were compared. Moreover, a quantitative polymerase chain reaction (qPCR) to detect BCMA-CAR-T cells was established. By applying a cell-titration experiment of BCMA-CAR-T cells, both methods were compared head-to-head. In flow-cytometric analysis, the detection reagents used in this study could all detect BCMA-CAR-T cells at a similar level. The results of false-positive background staining differed as follows (standard deviation): the BCMA-detection reagent used on the control revealed a background staining of 0.04% (±0.02%), for the PE-labeled human BCMA peptide it was 0.25% (±0.06%) and for the polyclonal anti-human IgG antibody it was 7.2% (±9.2%). The ability to detect BCMA-CAR-T cells down to a concentration of 0.4% was similar for qPCR and flow cytometry. The qPCR could detect even lower concentrations (0.02–0.01%). In summary, BCMA-CAR-T-cell monitoring can be reliably performed by both flow cytometry and qPCR. In flow cytometry, reagents with low background staining should be preferred.https://www.mdpi.com/1422-0067/23/2/903BCMA-CARpolymerase chain reactiondetection reagentflow cytometry |
| spellingShingle | Avinoam Reichman Alexander Kunz Jara J. Joedicke Uta E. Höpken Anna Keib Brigitte Neuber David Sedloev Lei Wang Genqiao Jiang Angela Hückelhoven-Krauss Franziska Eberhardt Carsten Müller-Tidow Martin Wermke Armin Rehm Michael Schmitt Anita Schmitt Comparison of FACS and PCR for Detection of BCMA-CAR-T Cells International Journal of Molecular Sciences BCMA-CAR polymerase chain reaction detection reagent flow cytometry |
| title | Comparison of FACS and PCR for Detection of BCMA-CAR-T Cells |
| title_full | Comparison of FACS and PCR for Detection of BCMA-CAR-T Cells |
| title_fullStr | Comparison of FACS and PCR for Detection of BCMA-CAR-T Cells |
| title_full_unstemmed | Comparison of FACS and PCR for Detection of BCMA-CAR-T Cells |
| title_short | Comparison of FACS and PCR for Detection of BCMA-CAR-T Cells |
| title_sort | comparison of facs and pcr for detection of bcma car t cells |
| topic | BCMA-CAR polymerase chain reaction detection reagent flow cytometry |
| url | https://www.mdpi.com/1422-0067/23/2/903 |
| work_keys_str_mv | AT avinoamreichman comparisonoffacsandpcrfordetectionofbcmacartcells AT alexanderkunz comparisonoffacsandpcrfordetectionofbcmacartcells AT jarajjoedicke comparisonoffacsandpcrfordetectionofbcmacartcells AT utaehopken comparisonoffacsandpcrfordetectionofbcmacartcells AT annakeib comparisonoffacsandpcrfordetectionofbcmacartcells AT brigitteneuber comparisonoffacsandpcrfordetectionofbcmacartcells AT davidsedloev comparisonoffacsandpcrfordetectionofbcmacartcells AT leiwang comparisonoffacsandpcrfordetectionofbcmacartcells AT genqiaojiang comparisonoffacsandpcrfordetectionofbcmacartcells AT angelahuckelhovenkrauss comparisonoffacsandpcrfordetectionofbcmacartcells AT franziskaeberhardt comparisonoffacsandpcrfordetectionofbcmacartcells AT carstenmullertidow comparisonoffacsandpcrfordetectionofbcmacartcells AT martinwermke comparisonoffacsandpcrfordetectionofbcmacartcells AT arminrehm comparisonoffacsandpcrfordetectionofbcmacartcells AT michaelschmitt comparisonoffacsandpcrfordetectionofbcmacartcells AT anitaschmitt comparisonoffacsandpcrfordetectionofbcmacartcells |