title: Sensitive detection and enumeration of CD34+ cells in peripheral and cord blood by flow cytometry.
abstract: 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 CD45 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 analyze 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.
authors.affiliation: Oncology Research Laboratories, Toronto Hospital, Ontario, Canada.
authors.forename: D R
authors.initials: DR
authors.lastname: Sutherland
authors.forename: A
authors.initials: A
authors.lastname: Keating
authors.forename: R
authors.initials: R
authors.lastname: Nayar
authors.forename: S
authors.initials: S
authors.lastname: Anania
authors.forename: A K
authors.initials: AK
authors.lastname: Stewart
mesh_terms: D015703:Antigens, CD / Q000097:blood*; D018952:Antigens, CD34; D001781:Blood Component Removal; D001853:Bone Marrow / Q000276:immunology; D001854:Bone Marrow Cells; D005312:Fetal Blood / Q000166:cytology / Q000276:immunology*; D005434:Flow Cytometry / Q000379:methods*; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology; D006801:Humans
chemical_list: D015703:Antigens, CD; D018952:Antigens, CD34; D016178:Granulocyte-Macrophage Colony-Stimulating Factor