Establishment of a flow cytometry assay for detecting paroxysmal nocturnal hemoglobinuria-type cells specific to patients with bone marrow failure

Minor populations of glycosylphosphatidylinositol-anchored protein-deficient (GPI[−]) cells in the peripheral blood may have a prognostic value in bone marrow failure (BMF). Our objective is to establish the optimal flow cytometry (FCM) assay that can discriminate GPI(−) populations specific to BMF from those of healthy individuals. To identify a cut-off that discriminates GPI(−) rare cells from GPI(+) cells, we determined a position of the borderline that separates the GPI(−) from GPI(+) cells on a scattergram by testing more than 30 healthy individuals, such that no GPI(−) dot fell into the upper left quadrant where fluorescein-labeled aerolysin (FLAER)−CD11b+ granulocytes and CD55−CD59− glycophorin A+ erythrocytes were positioned. This method allowed us to define ≥ 0.003% CD11b+FLAER− granulocytes and ≥ 0.005% glycophorin A+CD55−CD59− erythrocytes to be specific to BMF patients. Longitudinal cross-validation studies showed minimal (