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

D. R. Sutherland, A. Keating, R. Nayar, S. Anania, Alexander Keith Stewart

Research output: Contribution to journalArticle

180 Scopus citations

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 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.

Original languageEnglish (US)
Pages (from-to)1003-1010
Number of pages8
JournalExperimental Hematology
Volume22
Issue number10
StatePublished - 1994
Externally publishedYes

Keywords

  • CD34 cells
  • Clinical samples
  • Cord blood
  • Enumeration
  • Peripheral blood

ASJC Scopus subject areas

  • Cancer Research
  • Cell Biology
  • Genetics
  • Hematology
  • Oncology
  • Transplantation

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