CD34 progenitor cell subset analyses in normal human bone marrow and marrow harvested after intermediate-dose chemotherapy.

Abstract

Previous attempts to characterize harvested marrow and peripheral blood stem cell (PBSC) in order to predict time to and quality of engraftment post autologous bone marrow transplant (autoBMT) have included use of in vitro colony forming unit (CFU) assays. These assays are hampered by interlaboratory variability and are not uniformly predictive. CD34 quantification by flow cytometric technique has also been used to assess the quality of harvested marrow and PBSC. However, a lack of standardization has hampered direct comparison of published reports. We sought to characterize these early lineage-committed CD34+ progenitor cells from non-ficolled harvested marrow with six progenitor cell (PC) panels containing CD34 antibody plus two additional early lineage markers, using multiparameter flow cytometry. The specific gating technique including simultaneous CD34-PE vs. side scatter and forward vs. side scatter, was verified using morphologic analyses of sorted CD34+ cells. An ungated file was initially acquired to assess total CD34+ content. A second file using a CD34 threshold was then acquired to resolve lineage-committed subsets. The % CD34+ cells as well as cells/microliter of bone marrow was calculated using cell counts at the time of marrow harvest. Bone marrow (mean total cell dose = 3.8 x 10(5)/kg), obtained from 42 normal donors for allogeneic transplantation was first analyzed. CD34+ cells comprised a mean 1.3% of non-ficolled marrow, with 328 CD34+ cells/microliter, and mean CD34+ cells collected was 4.8 x 10(6)/kg. While no significant differences in total cells harvested nor proportion of CD34+ cells was found, a significant decrease in CD34 cells/microliter (= 233, P = .0012) was found in cancer patients. The percentage of CD19+ and CD38+ progenitor cells was significantly increased, while CD5+ and CD71+ cells were decreased. The proportions of all other early lineage-committed CD34 subsets were not different. Measurement of lineage-committed CD34 progenitor cells is a useful technique to characterize harvested marrow and PBSC, and may be applied to predict time and quality of engraftment post ablative conditioning regimens.