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 markedly improve reliability and convenience of apoptosis assays. In the present study the vital stain SytoR 16 in combination with the permeability marker 7-amino actinomycin D, (7-AAD) has been used to identify an early stage of apoptosis, not detected with trypan blue or 7-AAD alone or with conventional apoptosis tests and not consistently and only partly detected by the early apoptosis marker annexin V. The method was established using solid tumour cell lines treated with TNF. Subsequently we applied it to determine apoptotic populations in CD34(+) peripheral blood progenitor cells obtained from growth factor and/or chemotherapy mobilised patients and frozen/thawed according to standard stem cell transplantation protocols. In a cell line model as well as CD34(+) progenitor cells, different subpopulations with decreased SytoR 16 fluorescence (SytoR 16int or SytoR 16low, compared with the normal SytoR 16high) appeared which are not, or only partly, apoptotic using conventional techniques including morphology or 7-AAD staining: eg percentages of SytoR 16(int)/7-AAD(-) and SytoR 16(low)/7-AAD(-) may amount to the majority of cells present in a particular CD34(+) sample. Second, upon further incubation these subpopulations become late apoptotic/secondary necrotic much faster than the unmodified SytoR 16high population, as determined with 7-AAD staining and morphology. Third, these cells have strongly or completely reduced clonogenic capacity for committed (CFU-GM) and early (LTC-IC, determined only for CD34(+) cells) progenitors. This technique needs the inclusion of a blocker of P-glycoprotein, which is highly active in CD34(+) progenitor cells. This prevents the interference of the detection of SytoR16(low) apoptotic cells by SytoR 16low cells resulting from P-glycoprotein activity. By comparison with other apoptosis markers we found that early apoptotic subpopulations were detected in the order SytoR 16 > annexin V > 7-AAD. In conclusion, the combination of SytoR 16 and 7-AAD detects apoptotic events earlier than conventional apoptosis techniques or annexin V. Compared to the presently available viability tests, it allows a much better estimation of the number of viable clonogenic CD34(+) cells after freeze/thawing.