Use of Flow Cytometric CD34 Analysis to Quantify Hematopoietic Progenitor Cells

Abstract

This review summarizes our experiments on flow cytometric analysis of CD34 positive mono-nuclear cells (MNC) and on colony formation of myeloid hematopoietic progenitor cells in the clonogenic assay. We examined MNC isolated by density centrifugation of bone marrow, cord blood and peripheral blood. The latter samples originated either from patients recovering from myelosuppressive treatment who received no growth factors or from patients treated with G-CSF or GM-CSF. We attempted to correlate the results obtained by CD34 analysis with the cloning efficiency determined after a 14 day culture period in the methylcellulose-based clonogenic assay. The highest cloning efficacy (60%-100%) was observed in cord blood, however, a good correlation was found in both untreated and GM-CSF treated peripheral blood samples in which a mean of 50% and 20% of the number of CD34 positive MNC gave rise to myeloid colonies. In bone marrow, the cloning efficacy was generally lower and ranged between 5% and 15%. The lowest values were observed in G-CSF treated peripheral blood in which colonies were grown from only l%-9% of the CD34⁺ MNC. Due to the variable numbers of CD34⁺ lymphoid and/or more committed myeloid precursors which form either no colonies or only clusters, there was a greater variation and a lower cloning efficiency in the latter two cell sources. In conclusion, one colour CD34 analysis of cord blood MNC and untreated or GM-CSF treated peripheral blood MNC provides reliable results with respect to the content of myeloid progenitors. Analysis of bone marrow MNC and G-CSF treated peripheral blood MNC requires two colour staining using CD34 and CD45RA. Exclusion of the CD34⁺/CD45RA⁺⁺ cell fraction will improve the correlation and give results similar to those obtained with untreated blood MNC.

Key Words:

• CD34
• Flow cytometry
• Clonogenic assay
• Correlation bone marrow
• Cord blood
• Peripheral blood