Flow cytometric measurement of apoptosis and necrosis in cryopreserved PBPC concentrates from patients with malignant diseases

The number of viable precursor cells actually reinfused into patients after high-dose chemotherapy is one of the most clinically important variables determining graft success or failure. A modified, previously described flow cytometric method based on annexin V staining was therefore applied to assess the degree of apoptosis and necrosis in cryopreserved PBPC concentrates from patients with malignant diseases. Twenty-two samples of unmanipulated cryopreserved PBPC concentrates were analyzed by flow cytometry. The samples were triple-stained with anti-CD34 PE, annexin V-FITC and actinomycin D, which enabled the separation of viable, early apoptotic and late apoptotic/necrotic CD34(+) precursor cells. Apotosis and necrosis were also measured in the total cell population of the concentrates. Eighty-one percent (range 49-97) of the CD34(+) cells were viable, while 7% (range 1-15) were early apoptotic and 12% (range 2-36) were late apoptotic/necrotic after freeze/thaw. There was no difference in apoptosis and necrosis in CD34(+) cells harvested from mildly pretreated patients with multiple myeloma and heavily pretreated patients with non-Hodgkin's lymphoma. Apoptosis and necrosis were higher in the total mature cell population of the concentrates. Thirty-two percent (range 7-69) of the cells were apoptotic and 33% (range 1260) were necrotic. We conclude that flow cytometric analysis of annexinV/actinomycin D binding in PBPC concentrates is a simple technique that can give additional information of the viability status of the cells post thaw. The present study confirms the relative robustness of human CD34(+) precursor cells concerning the freeze/thaw procedure, which are carried out in daily clinical practice.