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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References
Wagner JE, Barker JN, DeFor TE, Baker KS, Blazar BR, Eide C et al. Transplantation of unrelated donor umbilical cord blood in 102 patients with malignant and nonmalignant diseases: influence of CD34 cell dose and HLA disparity on treatment-related mortality and survival. Blood 2002; 100: 1611–1618.
Gluckman E, Rocha V, Arcese W, Michel G, Sanz G, Chan K-W et al. Factors associated with outcomes of unrelated cord blood transplant: guidelines for donor choice. Exp Hematol 2004; 32: 397–407.
Schuurhuis GJ, Muijen MM, Oberink JW, de Boer F, Ossenkoppele GJ, Broxterman HJ . Large populations of non-clonogenic early apoptotic CD34-positive cells are present in frozen-thawed peripheral blood stem cell transplants. Bone Marrow Transplant 2001; 27: 487–498.
Sparrow RL, Tippett E . Discrimination of live and early apoptotic mononuclear cells by the fluorescent SYTO 16 vital dye. J Immunol Methods 2005; 305: 173–187.
De Boer F, Dräger AM, Pinedo HM, Kessler FL, van der Wall E, Jonkhoff AR et al. Extensive early apoptosis in frozen-thawed CD34-positive stem cells decreases threshold doses for haematological recovery after autologous peripheral blood progenitor cell transplantation. Bone Marrow Transplant 2002; 29: 249–255.
De Boer F, Dräger AM, Pinedo HM, Kessler FL, Muijen MM, Weijers G et al. Early apoptosis largely accounts for functional impairment of CD34+ cells in frozen-thawed stem cell grafts. J Hematother Stem Cell Res 2002; 11: 951–963.
Koenigsmann MP, Koenigsmann M, Notter M, Neuloh M, Mücke C, Thiel E et al. Adhesion molecules on peripheral blood-derived CD34+ cells: effects of cryopreservation and short-term ex vivo incubation with serum and cytokines. Bone Marrow Transplant 1998; 22: 1077–1085.
De Boer F, Dräger AM, Van der Wall E, Pinedo HM, Schuurhuis GJ . Changes in L-selectin expression on CD34-positive cells upon cryopreservation of peripheral blood stem cell transplants. Bone Marrow Transplant 1998; 22: 1103–1110.
Hattori Y, Kato H, Nitta M, Takamoto S . Decrease of L-selectin expression on human CD34+ cells on freeze-thawing and rapid recovery with short-term incubation. Exp Hematol 2001; 29: 114–122.
Zheng Y, Watanabe N, Nagamura-Inoue T, Igura K, Nagayama H, Tojo A et al. Ex vivo manipulation of umbilical cord blood-derived hematopoietic stem/progenitor cells with recombinant human stem cell factor can up-regulate levels of homing-essential molecules to increase their transmigratory potential. Exp Hematol 2003; 31: 1237–1246.
Beck C, Nguyen XD, Klüter H, Eichler H . Effect of recombinant human deoxyribonuclease on the expression of cell adhesion molecules on thawed and processed cord blood hematopoietic progenitors. Eur J Haematol 2003; 70: 136–142.
Koenig JM, Baron S, Luo D, Benson NA, Deisseroth AB . L-selectin expression enhances clonogenesis of CD34+ cord blood progenitors. Pediatr Res 1999; 45: 867–870.
Dercksen MW, Gerritsen WR, Rodenhuis S, Dirkson MKA, Slaper-Cortenbach ICM, Schassberg WP et al. Expression of adhesion molecules on CD34+ cells: CD34+L-selectin+ cells predict a rapid platelet recovery after peripheral blood stem cell transplantation. Blood 1995; 85: 3313–3319.
Watanabe T, Dave B, Heimann DG, Jackson JD, Kessinger A, Talmadge JE . Cell adhesion molecule expression on CD34+ cells in grafts and time to myeloid and platelet recovery after autologous stem cell transplantation. Exp Hematol 1998; 26: 10–18.
Stanworth S, Warwick D, Fehily D, Persaud C, Armitage S, Navarrete C et al. An international survey of unrelated umbilical cord blood banking. Vox Sang 2001; 80: 236–243.
Donaldson C, Armitage WJ, Denning-Kendall PA, Nicol AJ, Bradley BA, Hows JM . Optimal cryopreservation of human umbilical cord blood. Bone Marrow Transplant 1996; 18: 725–731.
Sutherland DR, Anderson L, Keeney M, Nayar R, Chin-Yee I . The ISHAGE guidelines for CD34+ cell determination by flow cytometry. J Hematother 1996; 5: 213–226.
Yang H, Acker JP, Hannon J, Miszta-Lane H, Akabutu JJ, McGann LE . Damage and protection of UC blood cells during cryopreservation. Cytotherapy 2001; 3: 377–386.
Xiao M, Dooley DC . Assessment of cell viability and apoptosis in human umbilical cord blood following storage. J Hematother Stem Cell Res 2003; 12: 115–122.
Kim H, Whartenby KA, Georgantas RW, Wingard J, Civin CI . Human CD34+ hematopoietic stem/progenitor cells express high levels of FLIP and are resistant to Fas-mediated apoptosis. Stem Cells 2002; 20: 174–182.
Tao W, Hangoc G, Hawes JW, Si Y, Cooper S, Broxmeyer HE . Profiling of differentially expressed apoptosis-related genes by cDNA arrays in human cord blood CD34+ cells treated with etoposide. Exp Hematol 2003; 31: 251–260.
Barker JN, Weisdorf DJ, DeFor TE, Blazar BR, McGlave PB, Miller JS et al. Transplantation of two partially HLA-matched umbilical cord blood units to enhance engraftment in adults with hematologic malignancy. Blood 2005; 105: 1343–1347.
Kern PM, Keilholz L, Forster C, Stach C, Beyer TD, Gaipl US et al. UVB-irradiated T-cells undergoing apoptosis lose L-selectin by metalloprotease-mediated shedding. Int J Radiat Biol 2000; 76: 1265–1271.
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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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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DOI: https://doi.org/10.1038/sj.bmt.1705405
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