Stem cell doses necessary for engraftment after myeloablative therapy as defined for fresh transplants vary largely. Loss of CD34+ cell quality after cryopreservation\ud might contribute to this variation. With a new early apoptosis assay including the vital stain Syto16, together with the permeability marker 7-AAD, CD34+ cell viability in leucapheresis samples of 49 lymphoma patients receiving a BEAM regimen was analysed. After freeze–thawing large numbers of non-viable, early apoptotic cells appeared, leading to only 42% viability\ud compared to 72% using 7-AAD only. Based on this Syto16 staining in the frozen–thawed grafts, threshold numbers for adequate haematological recovery of 2.8–\ud 3.0 x 106 CD34+ cells/kg body weight determined for fresh grafts, now decreased to 1.2–1.3 x 106 CD34+ cells/kg. In whole blood transplantation of lymphoma patients (n = 45) receiving a BEAM-like regimen, low\ud doses of CD34+ cells were sufficient for recovery (0.3– 0.4 x 106 CD34+ cells/kg). In contrast to freeze–thawing of leucapheresis material, a high viability of CD34+ cells\ud was preserved during storage for 3 days at 4ºC, leaving threshold doses for recovery unchanged. In conclusion, the Syto16 assay reveals the presence of many more non-functional stem cells in frozen–thawed transplants than presumed thus far. This led to a factor 2.3-fold adjustment downward of viable CD34+ threshold doses for haematological recovery
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