Enumeration of CD34+ hematopoietic stem cells for reconstitution following myeloablative therapy.

The CD34+ cell fraction of bone marrow and blood contains the hematopoietic stem cells required for marrow reconstitution following myeloablative therapy. Because they are present in small numbers, accurate quantification is often difficult. We have developed a reproducible and sensitive flow cytometric method for CD34+ enumeration of both bone marrow harvests and peripheral blood stem cell collections. The total numbers of harvested cells are enumerated by particle counting. A measured aliquot is stained with two FITC-labeled anti-CD34 antibodies, one directed against 8G12 and the other against QBend epitope. To eliminate cells committed to mature lineages (lin+), the suspension is counterstained with a cocktail of PE-labeled antibodies including CD3 (T cells), CD19 (B cells), CD11b (neutrophils), and CD14 (monocytes). Particles < 6 microns in diameter are excluded by use of a standard bead gate. Regions are established using unstained U937 cells to set the vertical axis and PE stained U937 cells for the horizontal axis. Because of the low numbers of CD34+ cells, 20,000 events/sample are analyzed. Dilutions of KG-1A tumor cells (CD34+) in U937 cells showed a threshold of detection of 0.1% CD34+lin- cells. Duplicate samples varied by < 10%. Initial studies indicate that this procedure can be reliably used to measure CD34+lin- cells in blood, pheresis products, and bone marrow harvests. This CD34 enumeration procedure should result in increased consistency in enumerating this stem cell population.