Monitoring hematopoietic stem and progenitor cells with Sysmex automated hematology analyzers

In this study, the behavior of CD34⁺ cells obtained from human umbilical cord blood (UCB) was investigated using a flow cytometry analyzer (FACScan) and the immature information (IMI) channels of the Sysmex SE-9000 and XE-2100 hematology analyzers. The methods that we used for CD34 cell isolation included positive selection with magnetic beads labeled by anti-human CD34 antibody, biotinylated CD34 antibody, and negative selection with multiple antibodies to remove lineage-positive cells. When the CD34⁺ cells were tested on the hematology analyzer, they were consistently found in a narrow area within the direct current/radio frequency (DC/RF) plot of the IMI channel. This detection area on an X-Y plot was used for cell enumeration and comparison with FACScan CD34⁺ cell counts. The samples were collected at different isolation stages containing various cells, including mononuclear cells obtained with a gradient-density method and enriched CD34⁺ cells, and were analyzed on both the FACScan and the hematology analyzer. The CD34⁺ cells suspended in phosphate-buffered saline (PBS) or mixed in peripheral blood at different concentrations were depicted with good linearity on both the FACScan and IMI channels of the hematology analyzer. Furthermore, when CD34⁺ cells were analyzed in the IMI channel, we found good sensitivity and specificity of results compared to those of flow cytometry using fluorescent-labeled CD34 monoclonal antibodies. Based on these findings, a hematopoietic progenitor cell (HPC) program was developed in the IMI channel for screening and monitoring hematopoietic stem and progenitor cells. The investigation revealed good correlation of HPC results from SE-9500 and XE-2100 hematology analyzers, suggesting that the stem cell-screening function of the IMI channel is identical in the 2 hematology analyzers. This article presents our research, describing how the purified CD34 cells were used as a reference for the establishment of the cell-counting program in the IMI channel of the hematology analyzers.