Background and Objectives: In peripheral blood stem cell transplantation, the number of CD34+ cells infused is considered a predictor of haematopoietic engraftment. However, the currently accepted minimal threshold of CD34+ cells/kg was determined by counting CD34+ cells before freezing, and the loss of viable CD34+ cells during freezing, cryopreservation or thawing prior to reinfusion has not been assessed. Materials and Methods: Total and viable CD34+ cells were quantified using single platform flow cytometry and viability dye, 7-amino actinomycin D (7-ADD), at the time of collection and prior to reinfusion in 46 peripheral haematopoietic stem cell grafts from 36 patients. The time to engraftment of neutrophil and platelet was assessed by routine peripheral blood cell counts performed daily. Results: The median number of viable CD34+ cells harvested was 3.6 × 106/kg (range 0.05-21.2), and the median viability was 98% (range 70-100%) before freezing. After thawing, the median number of viable CD34+ cells was reduced to 2.2 × 10 6/kg (range 0.04-14.8) and the median viability was reduced to 71% (range 31-89%). The number of viable CD34+ cells/kg before freezing and after thawing significantly correlated with engraftment of neutrophils (P < 0.0001 both) and platelets (P = 0.007 and 0.006, respectively). Although the minimum dose for engraftment (2.0 × 106 CD34+ cells/kg) was harvested in 37 of 46 cases (85%), only 25 cases (54%) met this threshold at the time of reinfusion. For platelet engraftment, determination of viable CD34+ cells prior to reinfusion was more important than enumeration at the time of collection. Conclusion: Quantification of post-thaw viable CD34+ cells better represents the actual composition of the graft and may be a more accurate predictor of haematopoietic engraftment than post-thaw total CD34+ cell counts, or prefreeze determinations, especially for platelet engraftment. It is necessary to develop good quality controls for freezing and thawing procedures to minimize variance in cell viability.
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