1. Comparison of FACS and PCR for Detection of BCMA-CAR-T Cells.
- Author
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Reichman A, Kunz A, Joedicke JJ, Höpken UE, Keib A, Neuber B, Sedloev D, Wang L, Jiang G, Hückelhoven-Krauss A, Eberhardt F, Müller-Tidow C, Wermke M, Rehm A, Schmitt M, and Schmitt A
- Subjects
- B-Cell Maturation Antigen genetics, Biomarkers, Humans, Immunophenotyping, Immunotherapy, Adoptive methods, Immunotherapy, Adoptive standards, Real-Time Polymerase Chain Reaction, Receptors, Chimeric Antigen genetics, Reproducibility of Results, Sensitivity and Specificity, T-Lymphocytes immunology, B-Cell Maturation Antigen metabolism, Flow Cytometry methods, Flow Cytometry standards, Polymerase Chain Reaction methods, Polymerase Chain Reaction standards, Receptors, Chimeric Antigen metabolism, T-Lymphocytes metabolism
- Abstract
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.
- Published
- 2022
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