USE OF FLOW CYTOMETRIC CD34 ANALYSIS TO QUANTIFY HEMATOPOIETIC PROGENITOR CELLS

This review summarizes our experiments on flow cytometric analysis of CD34 positive mono-nuclear cells (MNC) and on colony formation of myel oid hematopoietic progenitor cells in the clonogenic assay. We examine d MNC isolated by density centrifugation of bone marrow, cord blood an d peripheral blood. The latter samples originated either from patients recovering from myelosuppressive treatment who received no growth fac tors or from patients treated with G-CSF or GM-CSF. We attempted to co rrelate the results obtained by CD34 analysis with the cloning efficie ncy determined after a 14 day culture period in the methylcellulose-ba sed clonogenic assay. The highest cloning efficacy (60%-100%) was obse rved in cord blood, however, a good correlation was found in both untr eated and GM-CSF treated peripheral blood samples in which a mean of 5 0% and 20% of the number of CD34 positive MNC gave rise to myeloid col onies. In bone marrow, the cloning efficacy was generally lower and ra nged between 5% and 15%. The lowest values were observed in G-CSF trea ted peripheral blood in which colonies were grown from only 1%-9% of t he CD34+ MNC. Due to the variable numbers of CD34+ lymphoid and/or mor e committed myeloid precursors which form either no colonies or only c lusters, there was a greater variation and a lower cloning efficiency in the latter two cell sources. In conclusion, one colour CD34 analysi s of cord blood MNC and untreated or GM-CSF treated peripheral blood M NC provides reliable results with respect to the content of myeloid pr ogenitors. Analysis of bone marrow MNC and G-CSF treated peripheral bl ood MNC requires two colour staining using CD34 and CD45RA. Exclusion of the CD34+/CD45RA++ cell fraction will improve the correlation and g ive results similar to those obtained with untreated blood MNC.