Sensitive detection and enumeration of CD34⁺ cells in peripheral and cord blood by flow cytometry

Peripheral blood stem cell autografts are increasingly used to reconstitute hematopoiesis after intensive, potentially marrow-ablative therapy. Assessment of autograft adequacy by enumeration of hematopoietic progenitors in colony-forming assays is handicapped by lack of reproducibility and prolonged assay time. Alternative approaches of graft assessment by flow-cytometric enumeration of stem/progenitor cells bearing the CD34 antigen can be hampered by low specificity and sensitivity. Here, we report a rapid and reliable multiparameter flow-cytometric approach to accurately enumerate CD34⁺ cells in peripheral blood (PB) mononuclear cells (MNCs). Total nucleated white blood cells (WBCs) are quantified by staining with fluorescein isothiocyanate (FITC)-conjugated GD45 antibody. Simultaneous staining by phycoerythrin (PE)-conjugated CD34 antibody defines an approximate number for the CD34⁺ progenitor/stem cell subfraction. When starting CD34⁺ cell numbers are low (0.01-0.5%), other nonspecifically stained leukocytes make accurate enumeration impossible. However when the CD34⁺ fraction is analyzed for CD45 expression vs. side scatter (granularity)/true CD34⁺ blast cells form a discrete cluster exhibiting low-density CD45 expression and low side-scatter characteristics. Cells within this 'blast region' can be readily distinguished from lymphocytes, monocytes, granulocytes, and other events that can contaminate the CD34⁺ population. Here, we used this sensitive procedure to enumerate CD34⁺ cells in steady-state PB samples (0.03-0.09%), normal bone marrow (BM) aspirates, and umbilical cord blood collections (0.33-1.98%). This approach thus provides a means to ana lyze CD34⁺ cells in specimens from patients who have been extensively treated with chemotherapy and those undergoing PB stem cell mobilization with cytokines. Additionally, it is useful for assessment of CD34⁺ cells in a variety of clinical samples exhibiting perturbations of the hematopoietic progenitor/stem cell compartments.