Abstract
Glycosylphosphatidylinositol-anchored protein (GPI-AP)-deficient “PNH-type” cells are well known to be present in some patients with bone marrow (BM) failure who show no signs of hemolysis, such as those with aplastic anemia(AA) and myelodysplastic syndrome (MDS). However, whether or not a PNH-type cell population of ≤0.1% has any prognostic value remains controversial, mainly due to the lack of a reliable flow cytometry (FCM) assay capable of discriminating significant PNH-type cell populations from non-significant ones in healthy individuals. We herein established a new FCM assay using fluorescein-labeled aerolysin (FLAER). The borderline that separates the GPIAP- from GPI-AP+ cells on the scattergram was positioned by testing at least 50 healthy individuals. In a nationwide observational study to determine the prevalence of PNH-type cells, a pair of positive samples containing 0.02% PNH-type cells and a negative sample were sent to six different laboratories in a blind manner every six months for a quality control evaluation. This method allowed us to define ≥0.003% of CD11b+FLAER- granulocytes and ≥0.005% of glycophorin A+CD55-CD59- erythrocytes as significant PNH-type cell populations. Longitudinal cross-validation studies showed minimal (<0.02%) inter-laboratory differences in the percentage of PNH-type cells among 6 different laboratories. Our FCM method revealed six different dot patterns for the PNH-type cells in individual patients. Our assay was useful for identifying significant PNH-type cell populations in BM failure patients and allowed different labs to accurately detect 0.003% to 0.1% of PNH-type cells.
