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Pre-Clinical Studies

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 SYTO16bright 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/SYTO16dim 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.

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Acknowledgements

We thank Dr Simon Bol, Dr Annette Trickett and staff of the BMDI Cord Blood Bank-Melbourne for providing cord blood units; Dr Gerritt Schuurhuis (Vrije Universiteit Medical Center, Amsterdam) for SYTO 16 technical advice; Geraldine Healey for technical assistance and Associate Professor Neil Boyce for critical review of the manuscript. This work was supported in part by a project grant from the Inner Wheel Australia Cord Blood Research Fund.

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Correspondence to R L Sparrow.

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Sparrow, R., Komodromou, H., Tippett, E. et al. 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. Bone Marrow Transplant 38, 61–67 (2006). https://doi.org/10.1038/sj.bmt.1705405

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