Quality Assessment of Cryopreserved Peripheral Blood Stem Cell Products: Evaluation of Two Methods for Flow Cytometric Viability Testing

Introduction: The standard flow cytometry method for viability testing using 7- aminoactinomycin D (7- AAD) determines cells in necrosis and late apoptosis. The colony- forming unit (CFU) assay, which evaluates the proliferation ability of HSCs, is also used in graft quality assessment despite known deficiencies that make this assay impractical in routine clinical settings. The aim was to compare the effectiveness of the flow cytometry 7- AAD/annexin V method with the 7- AAD method in assessing the quality of HSCs in autologous and allogeneic peripheral blood stem cell (PBSC) products. Methods: Thirty autologous and 30 allogeneic fresh and thawed cryopreserved PBSC products were included in this study. The viability of HSCs was determined using the 7- AAD method and 7- AAD/annexin V method on a flow cytometer, while their clonogenic capacity was assessed by CFU assay. Results: There was an excellent correlation for CD34+ cell viability between the 7- AAD and the 7- AAD/annexin V method for fresh samples (Rs = 0.930, p < 0.001) and a good correlation for thawed PBSC samples (Rs = 0.739, p < 0.001). Excellent correlation was observed for post- thaw CD34+ cell recovery between the two methods for viability (Rs = 0.980, p < 0.001). Statistical analysis showed a weak correlation between CFU- GM recovery and CD34+ cell recovery, regardless of which viability testing method was used (7- AAD method p = 0.021, Rs = 0.298; 7- AAD/annexin V method p = 0.029, Rs = 0.282). Conclusions: Results of this study showed that in the quality assessment of cryopreserved PBSC product viability, the 7- AAD/ annexin V method had no added value compared to the 7- AAD method, which was suitable enough for routine quality control of cryopreserved autologous and allogeneic PBSC samples.