Flow cytometry-based monitoring of chimeric antigen receptor (CAR) T cells: Reagent selection, assay design, and clinical utility

Accurate quantification of chimeric antigen receptor (CAR) T cells is essential for monitoring post-infusion CART expansion and persistence and for real-time clinical decision-making. Multiparameter flow cytometry (MFC) enables rapid, live-cell detection with absolute quantification and concurrent immunophenotypic characterization. This review focuses on the practical and technical aspects of flow cytometry–based CAR T-cell monitoring, including selection of CAR detection reagents (target-specific, construct-specific, and target-agnostic strategies), assay optimization, purpose-driven panel design, and matrix-appropriate validation for peripheral blood and other clinically relevant specimens. We also address assay considerations unique to gene-edited allogeneic CAR T-cell products, including the use of surrogate immunophenotypic approaches when construct-specific reagents are unavailable. Finally, we discuss the role of MFC in identifying CAR T-cell clonal expansions and in evaluating suspected secondary hematolymphoid neoplasms in the post-CAR T setting.