Flow cytometry using annexin V can detect early apoptosis in peripheral blood stem cell harvests from patients with leukaemia and lymphoma

Abstract

Quantifying progenitor cells in peripheral blood stem cell (PBSC) harvests by flow cytometric enumeration of CD34⁺ cells does not account for cell viability. Cell membrane asymmetry in early apoptosis exposes phosphatidylserine on the cell surface. This can be detected by staining with annexin V FITC. Apoptosis in 30 autologous PBSC harvests mobilised by cyclophosphamide + G-CSF or standard chemotherapy + G-CSF was analysed immediately after collection by dual-colour flow cytometry with CD34 PE and annexin V FITC. Harvests contained a median of 3.4 × 10⁶/kg (range 0.3–91.8) CD34⁺ cells. Of these 87.6% (range 30–96.5) were annexin V−. In 10% of harvests more than 50% of CD34⁺ cells were apoptotic. Differences in PBSC mobilisation or collection could not explain the variation in annexin V binding. Cyclophosphamide + G-CSF significantly increased the yield of CD34⁺ cells but did not increase apoptosis. Comparison of consecutive harvests showed no difference in the numbers of CD34⁺ cells collected but found a significant decrease in apoptotic CD34⁺ cells through multiple collections. Analysis of annexin V binding in PBSC harvests is a simple flow cytometry technique which gives additional information on the status of CD34⁺ progenitor cells.