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

Abstract

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 <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. © 1994 Wiley‐Liss, Inc. Copyright © 1994 Wiley‐Liss, Inc.