Department of Transfusion Medicine and Transplantation Biology, University Hospital Centre Zagreb, Zagreb, Croatia.
University of Zagreb School of Medicine, Zagreb, Croatia.
Scand J Clin Lab Invest. 2024 Jul;84(4):273-277. doi: 10.1080/00365513.2024.2377961. Epub 2024 Jul 14.
Chimeric antigen receptor-T (CAR-T) cell therapy is currently the best-known type of immune effector cells therapy. For CAR T-cell therapy, the determination of CD3+ T cells is necessary for the quality control of fresh leukapheresis product as starting material. The aim was to validate analytical method for quantification of percentage and absolute count of T lymphocyte subtypes (CD3+, CD4+ and CD8+ cells) in fresh apheresis products using single-platform method on flow cytometer BD FACS Canto II. Validation study included determination of precision, trueness (bias), assessment of linearity, carryover, comparison of results obtained with two different protocols on flow cytometer for CD3+ cells determination and stability study. For between-run precision coefficients of variation (CVs) were <20%, as well as bias for all T-lymphocyte subtypes. For within-run precision, CVs were <10%, except for low CD8+ cell (percentage 10.51% and viable absolute count 12.37%). Comparison of results obtained with two different protocols for CD3+ cells determination shows no statistically significant difference. Statistically significant differences between results of the analysis of CD4+ cells in fresh samples and results obtained after storage at 4 °C ( = .004) and at room temperature ( = .018) were found. In conclusion, method for enumeration of T-lymphocyte subtypes can be used in routine work on BD FACS Canto II instrument for quality assessment of fresh cell products collected by leukapheresis procedure.
嵌合抗原受体 T (CAR-T) 细胞疗法是目前最知名的免疫效应细胞疗法之一。对于 CAR T 细胞疗法，作为起始材料的新鲜白细胞分离产品的质量控制需要确定 CD3+T 细胞。本研究旨在使用流式细胞仪 BD FACS Canto II 上的单平台方法验证分析方法，以定量新鲜白细胞分离产品中 T 淋巴细胞亚群（CD3+、CD4+和 CD8+细胞）的百分比和绝对计数。验证研究包括精密度、准确度（偏差）、线性评估、携带污染、两种不同方案在流式细胞仪上用于 CD3+细胞测定的结果比较以及稳定性研究。运行间精密度的变异系数（CVs）<20%，所有 T 淋巴细胞亚群的偏差均<20%。对于运行内精密度，除低 CD8+细胞（百分比 10.51%和活细胞绝对计数 12.37%）外，CVs<10%。两种不同方案用于 CD3+细胞测定的结果比较显示无统计学差异。在新鲜样本中分析 CD4+细胞的结果与在 4°C（=0.004）和室温（=0.018）下储存后获得的结果之间存在统计学显著差异。总之，该方法可用于 BD FACS Canto II 仪器上的常规工作，以评估通过白细胞分离程序收集的新鲜细胞产品的质量。
