Early Apoptosis Largely Accounts for Functional Impairment of CD34 + Cells in Frozen-Thawed Stem Cell Grafts

Abstract

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. Syto^high/7-AAD^- cells were defined as viable, Syto16^low/7-AAD^- cells as early apoptotic and Syto16^low/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 Syto16^high/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.