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: 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.