用于 CAR-T 细胞定量的全血双平台流式细胞术方案的初步评估：迈向认证与临床常规应用

Chimeric Antigen Receptor T-cells, commonly known as CAR-T cells, have represented a major scientific breakthrough in cancer cell therapy revolutionizing the treatment of haematological disorders. Pharmacokinetic studies highlighted the importance of peripheral blood CAR-T cell monitoring to predict therapeutic outcomes and manage side effects.

This study aimed to establish and evaluate on three different flow cytometers (DxFLEX, Navios and Navios EX) the performances of a dual-platform protocol for CAR-T quantitation method using flow cytometry in a context of hospital routine use. Fresh whole blood was stained using a biotinylated CD19 or BCMA recombinant protein and a PE-coupled anti-biotin antibody. CAR-T cells were identified among CD45+ CD3+ cells after doublets elimination, using a control tube to define CAR-T positive expression threshold among T lymphocytes. Results were expressed as percentages of CAR T cells among CD3+ T lymphocytes.

Optilyse C and Versalyse red blood cell lysis reagents (Beckman Coulter) gave comparable results in terms of CAR T cell percentage among T lymphocytes as well as using 0. 5 L CAR detection reagent compared to the 2 L recommended by the manufacturer. Using this protocol, we did not observe any cross-reactivity between CD19 and BCMA CAR detection reagents or decreased signal even up to >3000 circulating CAR-T cells/ L.

The coefficient of variations for repeatability and intermediate precision were systematically below 10 % whatever the flow cytometer, the type of CAR reagent detection used and the proportion of circulating CAR-T cells. All three flow cytometers returned correlated and comparable results.

Finally, concerning whole blood sample and stained cell storage duration, we observed a significant decreased of CAR-T percentages when staining was delayed for more than one day after sampling, and after three days of stained cells storage at 4 C.

In conclusion, we propose a dual-platform protocol for CAR-T cell enumeration which demonstrated consistent and reliable performances for BCMA and CD19 CAR-T cell quantification.