Validation of Gating and Leukocyte Classification on Sysmex XE Series Automated Cell Counters

− 1 − Leukocyte classification on Sysmex XE series hematology analyzers involves staining leukocytes with proprietory Polymethine Dye reagents that have affinity for nucleic acids. The outputs are two-dimensional scatter diagrams of side scatter and fluorescence intensity (DIFF scattergrams) subsequent to flow cytometric analysis of the stained cells. Leukocyte classification is based on the location of treated cells in the DIFF scattergram. Comparative methods for leukocyte typing can be done using monoclonal antibodies to CD antigens expressed on the cell surfaces. The present study investigates the relationship between cell-classification by means of CD antibodies versus cell classification on the Sysmex hematology analyzer. This was achieved by double staining cells with both CD antibodies and Sysmex cell counting reagents. Granulocytes, T and B lymphocytes, and monocytes were prepared using density gradient centrifugation and negative selection using magnetic cell sorting methods. Samples were peripheral blood samples from healthy human subjects. Isolated cells were stained with FITC-conjugated CD antibodies. Subsequently the same cells were incubated in Sysmex cell counting reagents in a way that mimics conditions used in the hematology cell counters. Post treatment, the cells were analyzed by flow cytometry (FCM), confocal laser scanning microscopy (CLSM), and electron microscopy (EM). FCM analysis revealed that most CD3and CD19-; CD14-; and CD16b-positive cells appeared in the lymphocyte; monocyte; and neutrophil gates on DIFF scattergrams. CLSM and EM observation shows characteristic morphological changes for each type of leukocyte, related to the degree of membrane disruption and nucleic acid distribution within the various cell types. This experimental approach appears to validate the method for classification of leukocytes on DIFF scattergrams versus classification on the basis of surface antigen labeling.