Monitoring anti-CD19 chimeric antigen receptor T cell population by flow cytometry and its consistency with digital droplet polymerase chain reaction

被引:11
作者
Cheng, Jiali [1 ]
Mao, Xia [1 ]
Chen, Caixia [1 ]
Long, Xiaolu [1 ]
Chen, Liting [1 ]
Zhou, Jianfeng [1 ]
Zhu, Li [1 ]
机构
[1] Huazhong Univ Sci & Technol, Tongji Hosp, Dept Hematol, Tongji Med Coll, Wuhan, Peoples R China
基金
中国国家自然科学基金;
关键词
CAR T cell therapy; CAR19 T cell; droplet digital PCR; flow cytometry; pharmacokinetics; rabbit anti-mouse FMC63 antibody; ACUTE LYMPHOBLASTIC-LEUKEMIA; QUANTIFICATION; EXPRESSION; CD19; PCR;
D O I
10.1002/cyto.a.24676
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
Anti-CD19 chimeric antigen receptor (CAR19) T cell therapy has produced impressive clinical efficacy in patients with relapsed or refractory B-cell malignancies. As a living drug, monitoring the pharmacokinetics of CAR T cells in vivo is an important part of clinical work, which provides valuable information for assessing therapeutic response and related side effects. However, no guidelines are available regarding the detection and quantification of CAR T cells. Flow cytometry is a convenient and commonly used method in monitoring CAR T cell kinetics, but its performance remains to be validated. By using a commercial anti-idiotype antibody that detects unique epitopes on the most popular CAR19 construct, we evaluated important performance parameters, including specificity, lower limit of detection, lower limit of quantification, and precision of flow cytometry in the detection and quantification of CAR19 T cells. Consistency between the results generated by flow cytometry and droplet digital PCR was then investigated in 188 pairs of clinical data and in cell line experiments. Rabbit anti-mouse FMC63 monoclonal antibody possesses high specificity in the detection of CAR19 positive cells by FCM with a cut-off value of 0.05%. The results produced by flow cytometry and ddPCR were well correlated in the clinical samples and in cell lines, but the correlation deteriorated as the abundance of CAR19 positive cells decreased. This was especially evident with less than 0.5% of lymphocytes in clinical data, possibly due to reduced precision (indicated by intra- and inter-assay coefficients of variability) of both droplet digital PCR and flow cytometry. We demonstrated that flow cytometry using anti-idiotype antibody is a reliable and robust approach in the detection and quantification of CAR19 T cells in vivo and has good consistency with droplet digital PCR in monitoring CAR19 T cell kinetics.
引用
收藏
页码:16 / 26
页数:11
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