Quality assessment of stem cell grafts is usually performed by flow cytometric CD34+ enumeration or assessment of clonogenic output of fresh material. Previously, we identified the occurrence of early apoptosis, not detectable with the permeability marker 7-amino actinomycin D (7-AAD), in purified frozen-thawed CD34+ cells, using the vital stain Syto16. Sytohigh/7-AAD- cells were defined as viable, Syto16low/7-AAD- cells as early apoptotic and Syto16low/7-AAD+ as dead. This was confirmed in a subsequent study using frozen-thawed transplants of lymphoma patients. In the present study on grafts from multiple myeloma and lymphoma patients, we investigated the functional consequences of the early apoptotic process. The mean Syto16-defined viability was 41 and 42%, respectively, for both graft groups, compared to 78% and 72%, respectively, using 7-AAD only. The established early apoptosis marker annexin V missed roughly 50% of the early apoptosis detected with Syto16. In contrast, viability of CD34+ cells in nonmanipulated whole blood transplants from a matched group of lymphoma patients, after 72 h of storage at 4°C, was more than 90%, even with the Syto16 assay. CFU recovery (median 26-33%) after cryopreservation matched CD34+ recovery after Syto16, but not 7-AAD correction. In contrast, colony-forming unit (CFU) recovery in the whole blood transplant was close to 100%. Furthermore, early apoptotic CD34+ cells had lost migratory ability toward stromal cell derived factor-1α (SDF-1α). The establishment of a Syto16high/7-AAD- proportion of CD34+ cells offers a new approach for a more correct determination of the number of viable nonapoptotic CD34+ cells in stem cell grafts. Further development of this assay should allow its incorporation into the routine CD34+ assessment of post-thawed samples in clinical flow cytometry laboratories.
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