Large populations of non-clonogenic early apoptotic CD34-positive cells are present in frozen-thawed peripheral blood stem cell transplants

Apoptosis is the common cell death pathway which is initiated by a variety of different stimuli. The recognition of early apoptotic events would marke dly improve reliability and convenience of apoptosis assays. In the present study the vital stain Syto(R)16 in combination with the permeability marke r 7-amino actinomycin D, (7-AAD) has been used to identify an early stage o f apoptosis, not detected with trypan blue or 7-AAD alone or with conventio nal apoptosis tests and not consistently and only partly detected by the ea rly apoptosis marker annexin V. The method was established using solid tumo ur cell lines treated with TNF, Subsequently we applied it to determine apo ptotic populations in CD34(+) peripheral blood progenitor cells obtained fr om growth factor and/or chemotherapy mobilised patients and frozen/thawed a ccording to standard stem cell transplantation protocols. In a cell line mo del as well as CD34(+) progenitor cells, different subpopulations with decr eased SytoR16 fluorescence (Syto(R)16(int) or Syto(R)16(low), compared with the normal Syto(R)16(high)) appeared which are not, or only partly, apopto tic using conventional techniques including morphology or 7-AAD staining: e g percentages of Syto(R)16(int)/7-AAD(-) and Syto(R)16(low)/7-AAD(-) may am ount to the majority of cells present in a particular CD34(+) sample. Secon d, upon further incubation these subpopulations become late apoptotic/secon dary necrotic much faster than the unmodified Syto(R)16(high) population, a s determined with 7-AAD staining and morphology. Third, these cells have st rongly or completely reduced clonogenic capacity for committed (CFU-GM) and early (LTC-IC, determined only for CD34(+) cells) progenitors. This techni que needs the inclusion of a blocker of P-glycoprotein, which is highly act ive in CD34(+) progenitor cells. This prevents the interference of the dete ction of Syto(R)16(low) apoptotic cells by SytoR16(low) cells resulting fro m P-glycoprotein activity. By comparison with other apoptosis markers we fo und that early apoptotic subpopulations were detected in the order SytoR16> annexin V>7-AAD. In conclusion, the combination of Syto(R)16 and 7-AAD dete cts apoptotic events earlier than conventional apoptosis techniques or anne xin V, Compared to the presently available viability tests, it allows a muc h better estimation of the number of viable clonogenic CD34+ cells after fr eeze/thawing.