Clinical implementation of a one-step no-wash flow cytometry method allows for real-time monitoring of patients treated with autologous CAR-T cells.

[BACKGROUND] The best way to monitor Chimeric Antigen Receptor (CAR)-T cells persistence and expansion after infusion, and the significance of observed fluctuations over time remains controversial. As living drugs, these therapies do not follow classical pharmacokinetic patterns. Therefore, reliable quantification of circulating CAR-T cells is essential to explore the relations between expansion and persistence on one hand, tumor response and occurrence of side-effects on the other hand; this work will prepare integration of this information in harmonized post-CAR-T Cells intervention algorithms. Conventional flow cytometry protocols rely on multi-step wash procedures that increase processing time and may reduce sensitivity. We here developed and validated a one-step no-wash flow cytometry assay for routine CAR-T monitoring in patients treated with approved autologous CAR-T Cells.

[METHODS] CAR-T cell monitoring was implemented between 2021 and 2024 in patients treated with autologous CD19-directed CAR-T therapies at our institution with a classical two steps and wash flow cytometry method. Analytical validation included determination of detection and quantification limits, linearity, precision, and inter-laboratory reproducibility. In 2024, the classical method was considerably optimized to a one-step no-wash format to reduce manual handling and improve sensitivity. Clinical relevance was assessed in a cohort of 29 patients treated with axicabtagene ciloleucel, correlating CAR-T expansion metrics with clinical endpoints.

[RESULTS] The optimized one-step no-wash assay markedly improved analytical sensitivity, achieving a limit of detection of 0.3 cells/µL and a lower limit of quantification of 1.0 cells/µL, versus 2.0 and 5.0 cells/µL, respectively, when using the previous two steps and wash protocol, while demonstrating a strong concordance (r² = 0.984). Inter-assay coefficients of variation remained below 9%, confirming maintained precision despite workflow simplification. In 29 patients treated with axicabtagene ciloleucel (axicel), peak CAR-T expansion significantly correlated with objective responses (p< 0.01) and occurrences of immune effector cell-associated neurotoxicity syndrome (ICANS) (p = 0.02), supporting the clinical relevance of flow cytometry-based CAR-T monitoring in routine practice.

[CONCLUSION] This one-step no-wash flow cytometry assay combines enhanced sensitivity with improved operational efficiency and provides clinically informative CAR-T cell monitoring in standard-of-care settings.