CD19-靶向嵌合抗原受体T细胞（CAR-T）疗法已显示出在B细胞复发/难治性急性淋巴细胞白血病（R/R-ALL）和B细胞非霍奇金淋巴瘤患者中取得了前所未有的治疗效果，这些患者没有其他可治愈的选择。CAR-T细胞动力学的体内监测对于了解CAR-T细胞扩增和持久性与治疗反应和毒性发展之间的相关性至关重要。本研究的目的是使用流式细胞术定义一种稳健、敏感和通用的CAR-T细胞检测方法。我们建立并比较了基于商业化试剂的三种CD19 CAR-T细胞检测方法。所有方法均使用CAR构建物的单链变量片段识别的重组人CD19蛋白片段。间接染色试剂( CD19his + APC-偶联的抗组氨酸抗体和 CD19bio + APC-偶联的抗生物素抗体)与CD19-FITC的直接染色相比，敏感性和特异性更好，而且CD19his具有更好的成本效益概况。我们通过与平行定量实时PCR数据的验证，证明了CD19his检测的CAR与之间的强烈正相关性。我们还展示了CD19his染色可以轻松地包含在多色流式细胞术面板中，以获取有关CAR-T细胞阳性亚群的细胞表型的附加信息。最后，该方法可用于不同的抗CD19 CAR-T细胞产品和不同的样本来源。这些数据表明，通过CD19his流式细胞术染色检测CAR-T细胞是一种可靠、稳健且广泛适用的用于CAR-T细胞体内监测的工具。本文受版权保护。版权所有。

CD19-targeted Chimeric Antigen Receptor T (CAR-T) cell therapy has shown unprecedented results in patients with B cell relapsed/refractory acute lymphoblastic leukemia (R/R-ALL) and B cell non-Hodgkin lymphomas where no other curative options are available. In vivo monitoring of CAR-T cell kinetics is fundamental to understand the correlation between CAR-T cells expansion and persistence with treatment response and toxicity development. The aim of this study was to define a robust, sensitive, and universal method for CAR-T cell detection using flow cytometry. We set-up and compared with each other three assays for CD19 CAR-T cell detection, all based on commercially available reagents. All methods used a recombinant human CD19 protein fragment recognized by the single chain variable fragment of the CAR construct. The two indirect staining assays (CD19his + APC-conjugated anti-histidine antibody and CD19bio + APC-conjugated anti-biotin antibody) showed better sensitivity and specificity compared to the direct staining with CD19-FITC, and CD19his had a better cost-effective profile. We validated CAR detection with CD19his with parallel quantitative real time PCR data and we could demonstrate a strong positive correlation. We also showed that CD19his staining can be easily included in a multicolor flow cytometry panel to achieve additional information about the cell phenotype of CAR-T cell positive subpopulations. Finally, this method can be used for different anti-CD19 CAR-T cell products and for different sample sources. These data demonstrate that detection of CAR-T cells by CD19his flow cytometry staining is a reliable, robust, and broadly applicable tool for in vivo monitoring of CAR-T cells. This article is protected by copyright. All rights reserved.This article is protected by copyright. All rights reserved.