Cell loss and recovery in umbilical cord blood processing: a comparison of postthaw and postwash samples

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Abstract
Engraftment after umbilical cord blood (UCB) transplantation is highly dependent on nucleated cell (NC) and CD34+ cell content. Current standard postthaw (PT) processing includes a wash step to remove dimethyl sulfoxide (DMSO), lysed red cells, and stroma. The contribution of the wash step to cell loss and ultimately the dose of cells available for transplant have yet to be systematically reported. This study examines the effect of the wash step as well as that of PT storage on various quality control variables of UCB units. Ten units were thawed and washed based on the New York Blood Center method. Samples were removed from each unit at six time points: prefreeze (PF), immediately PT, immediately postwash (PW), and 1, 2, and 5 hours PW. On each sample, total nucleated cell (TNC) count, CD34+ cell enumeration, colony-forming unit (CFU)-granulocyte-macrophage, and viability assays (fluorescence microscopy [acridine orange/propidium iodide, or AO/PI] and flow cytometry [7-aminoactinomycin]) were obtained. TNC counts decreased PT and at subsequent time points; the PT TNC recovery was 89 percent compared to 82 percent PW (p < 0.01). TNC recovery decreased to 90 percent of PW (82% of PT) values (p < 0.01) and 83 percent of PW (76% of PT) values (p < 0.001), at 2 and 5 hours PW, respectively. CD34+ cell loss PT was not significant. Viability by AO/PI decreased PT and plateaued over time. In contrast, viability by flow cytometry remained higher and increased slightly over time. CFUs were significantly lower PT, recovering PW. Our data indicate that the thawing and washing results in a substantial loss of cells, with TNC loss approaching 20 percent when compared with PF counts; the wash step was responsible for nearly half of the cell loss. The reduced PT viability was expected. Elapse of time PW resulted in further loss of NCs but no detectable significant changes in CD34+ cell content and viability and/or CFU.