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 (< 0.02%) inter-laboratory differences in the GPI(−) cell percentage. An analysis of 1210 patients with BMF revealed a GPI(−) cell population in 56.3% of patients with aplastic anemia and 18.5% of patients with myelodysplastic syndrome. The GPI(−) granulocyte percentages was 0.003–0.01% in 3.7% of patients. This FCM assay effectively identified an increase in the percentage of GPI(−) rare cells that are specific to BMF patients and allowed different laboratories to accurately detect 0.003–0.01% of pathological GPI(−) cells.