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. Sytohigh/7-AAD2 cells were defined as viable, Syto16low/7-AAD2 cells as early apoptotic and Syto16low/7-AAD1 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-1a (SDF-1a). The establishment of a Syto16high/7-AAD2 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.