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

Abstract

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.