Evaluation of a flow cytometry-based method for determination of T-lymphocyte subtypes for quality assessment of cell therapy products.

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 (p = .004) and at room temperature (p = .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.