Validation of a flow cytometry-based method for quantitative viable lymphocyte- immunophenotyping in fresh and cryopreserved hematopoietic cellular products

Background \u0026 Aim Adoptive cellular therapy with immune effector cells (IEC) has shown remarkable toxicity against tumor cells and potential in immune regulation. Both variability in cell content of starting material and compromised cell viability during manufacturing necessitate a validated method for quantifying viable lymphocyte subtypes individually. Currently-commercialized immunophenotyping methods report cell viability in thawed products with significant error since they do not include any viability staining. We hereby report on the validation of a flow cytometry-based method for quantifying viable lymphocyte- immunophenotypes in fresh and cryopreserved hematopoietic cellular products. Methods, Results \u0026 Conclusion Using fresh or frozen cellular products and stabilized blood, we report on the validation parameters: accuracy, uncertainty, precision, sensitivity, robustness and carry-over for quantification of viable CD3+, CD4+ T cells, CD8+ T cells, CD3-CD56+CD16+/- NK cells, CD19+ B cells and CD14+ monocytes of relevance to hematopoietic products and immune effector cell (IEC) products on the Cytomics FC500 Beckman Coulter cytometer. The acceptance criteria set in the validation plan were all met. The method is able to accommodate the variability in absolute numbers of cells in starting materials collected or cryopreserved from patients or healthy donors (uncertainty of 10% at 3 different concentrations), stability over time (compliance over 3 years during regular inter-laboratory comparisons), and confidence in meaningful changes during cell processing and manufacturing (intra-assay and intermediate precision of 10% CV). Furthermore, the method can accurately report on the efficacy of cell depletion, since the lower limit of quantification was established (CD3+, CD4+ and CD8+ cells at 9, 8 and 8 cells/µl, respectively). The method complies with FACT Standards for IEC, FACT-JACIE Hematopoietic Cell Therapy Standards, ICH Q2(R1) and ISO15189 standards. Furthermore, it complies with LBAFG-AAPS, ICSH/ICCS and European Bioanalysis Forum recommendations for validating such methods. The implications of this effort include: standardization of viable cell immunophenotyping of starting material for cell manufacturing, cell selection, in-process or dosing of immune effector cells. This method also complies with all relevant standards, particularly FACT-JACIE Standards in terms of enumerating and reporting on the viability of the “clinically relevant cell populations”.