Apoptotic lymphocytes and CD34+ cells in cryopreserved cord blood detected by the fluorescent vital dye SYTO 16 and correlation with loss of L-selectin (CD62L) expression

Abstract

Discrimination between live and apoptotic cells is important for accurate determination of viable CD34(+) cells in hematopoietic stem cell transplant products. SYTO16 is a sensitive fluorescent dye for discriminating live from apoptotic leukocytes. The incidence of apoptotic leukocytes in paired samples of fresh and cryopreserved-thawed cord blood (CB) was determined by the SYTO16/7-AAD flow cytometric assay. Cell migration and expression of the cell homing molecule L-selectin (CD62L) was determined in relation to SYTO16 staining. SYTO16 detected significant proportions of apoptotic lymphocytes and CD34(+) cells in fresh and thawed CB buffy-coat samples that were not detected by 7-AAD. Compared to fresh CB, the proportion of apoptotic lymphocytes and CD34(+) cells significantly increased following thawing. Significantly higher proportions of live SYTO16(bright) lymphocytes and CD34(+) cells were found in the migrated cell population compared to the non-migrated population. Significantly fewer lymphocytes and CD34(+) cells expressed CD62L following thawing. Absence of CD62L expression was strongly correlated with apoptotic/SYTO16(dim) lymphocytes and CD34(+) cells. Cryopreserved-thawed CB contains significant proportions of apoptotic lymphocytes and CD34(+) cells that are not detected by 7-AAD. SYTO16 offers a sensitive method for discrimination of live from apoptotic leukocytes and assists in accurate assessment of CB quality and suitability for use in clinical transplantation.