DIRECT MEASUREMENT OF CD34(-CELL ABSOLUTE COUNTS BY FLOW-CYTOMETRY() BLOOD STEM)

For the collection of adequate numbers of peripheral blood stem cells (PESC) for PBSC transplantation, an accurate quantification of circula ting CD34(+) stem cells is required for deciding the optimal time of t he collection. To enumerate peripheral blood (PB) CD34(+) stem cells, the percentage of CD34(+) cells in the gated PB mononuclear cells shou ld be multiplied by the percentage of the gated mononuclear cells amon g white blood cells (WBC) and by the total WBC count. Accordingly, a m inor difference in the measured percentage of the CD34(+) cells can le ad to a major difference in the PB CD34(+) cell concentration. In the present study, me measured the concentration of PB CD34(+) stem cells with a flow cytometer designed to provide direct absolute counts of ce ll subsets from a single instrument. Whole blood mas stained with a ph ycoerythrin-conjugated anti-CD34 monoclonal antibody, and, after the l ysis of red blood cells, CD34(+) cells mere counted in a fraction of t he lymphocyte and monocyte gate. The accuracy of our method was demons trated in an experiment in which various dilutions of known numbers of CD34(+) leukemic cells mere mixed with normal blood; the predicted va lue of the CD34(+) cell count was observed. The concentration of CD34( +) cells in leukapheresis products mas measured both by our direct ass ay and an indirect assay that calculates the number from the percentag e of CD34(+) cells in mononuclear cells, and our assay was shown to pr oduce less variation. Further, our assay showed a significant correlat ion between the concentration of mobilized CD34+ cells in the PB and t he number of harvested CD34(+) cells in leukapheresis. These findings indicate that the monitoring of the concentration of PB CD34(+) cells by the present method can be used to predict the number of stem cells collected in leukapheresis. This procedure is easy to perform and can be applied to daily monitoring to decide the appropriate timing for ha rvest of mobilized stem cells.