Flow cytometric analysis of granulocytes in PNH using a combination of anti-CD15 FITC, anti-CD24 PE, and anti-CD16 PE:Cy5. Flow cytometry plots (A-B) show the gating strategy for identifying a pure population of granulocytes based on light scatter characteristics and a transmembrane lineage marker (CD15). An initial region (R1) is set on CD15a⁺SSC^High events (A). A second region (R2) is then drawn around the FSC/SSC characteristics of these cells to further refine the gating procedure (B). Only events that meet both these criteria are analyzed. (C-E) Using this combination, normal eosinophils (blue) can be clearly separated from PNH cells (red) and normal granulocytes (green). Plots are from 3 representative patients with PNH. (C) A small PNH granulocyte clone of 0.18% of total granulocytes in a patient with aplastic anemia (PNH-sc/aplastic anemia). (D) A larger PNH granulocyte clone of 8.7% in a patient with aplastic anemia (PNH/aplastic anemia). (E) Plot is from a patient with classic PNH and shows a large PNH granulocyte pool (98.8%). In this instance, the majority of hematopoiesis is derived from PNH stem cells. There is some residual normal granulocyte production (the 1% of cells that are CD16⁺CD24⁺) and this population serves as an internal positive control for antibody staining. Illustration enhanced by A. Y. Chen.