Automated Enumeration of CD34 + Cells in Peripheral Blood and Bone Marrow

Abstract

We have developed a rapid and accurate method to enumerate the number of CD34⁺ cells in peripheral blood, bone marrow, and leukopheresis samples. The method consists of a two-tube assay and a dedicated software program for data acquisition and analysis. The first reagent combination consists of (a) a nucleic acid dye to identify nucleated cells, (b) a CD45 monoclonal antibody labeled with PE/CY5 to discriminate progenitor cells from mature lymphoid, neutrophil, erythroid, and monocytic cells, (c) an IgG₁ control antibody labeled with PE to establish the boundary between specific and nonspecific staining, and (d) a known number of fluorescent beads to determine an absolute count of cells. In the second reagent combination the IgG₁ control antibody is replaced by a CD34 antibody labeled with PE that is used to identify the CD34⁺ cells in the location established by the control reagent combination. The software program uses the fluorescent beads to adjust the forward light scatter, orthogonal light scatter, and three fluorescence detectors of the flow cytometer. The expected location of the CD34⁺ cells is then established with the control reagent combination followed by the enumeration of the CD34⁺ cells per microliter of sample with the reagent combination containing the CD34 antibody. This method is sensitive enough to detect CD34⁺ cells in peripheral blood of normal donors and can reliably determine an increase in CD34⁺ cells in the peripheral blood of patients treated with chemotherapy and/or growth factors. The method alleviates some of the difficulties encountered when small numbers of CD34⁺ cells are enumerated. The system allows for more precise evaluations of the grafts used for bone marrow transplantation.