Extensive early apoptosis in frozen–thawed CD34-positive stem cells decreases threshold doses for haematological recovery after autologous peripheral blood progenitor cell transplantation

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Abstract
Stem cell doses necessary for engraftment after myeloablative therapy as defined for fresh transplants vary largely. Loss of CD34(+) cell quality after cryopreservation 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 compared to 72% using 7-AAD only. Based on this Sytol.6 staining in the frozen-thawed grafts, threshold numbers for adequate haematological recovery of 2.8-3.0 x 106 CD34(+) cells/kg body weight determined for fresh grafts, now decreased to 1.2-1.3 X 10(6) CD34(+) cells/kg. In whole blood transplantation of lymphoma patients (n = 45) receiving a BEAM-like regimen, low doses of CD34(+) cells were sufficient for recovery (0.3-0.4 X 10(6) CD34(+) cells/kg). In contrast to freeze-thawing of leucapheresis material, a high viability of CD34(+) cells was preserved during storage for 3 days at 4degreesC, 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 recover

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