Abstract
Background: Routine diagnosis of paroxysmal nocturnal hemoglobinuria (PNH) is based on flow cytometric measurement of PNH clone in RBC and granulocyte with sensitivity of 1%, and recent introduction of high-sensitive flow cytometry (FCM) makes it possible to detect 0.01% clone. However, the clinical significance of small PNH clone has not been elucidated, so even the necessity to treat or not is controversial. We investigated whether quantitative results of PNH clone size measured by FCM correlate with mutant burden of PIG gene.
Methods: A total of 44 specimens from 20 patients whose PNH clone size was >1% in either RBC or granulocyte by routine FCM were enrolled (classical PNH n=10, AA/PNH n=8, MDS/PNH n=2). To detect small quantity of cells with PIG gene mutation, we performed ultra-deep sequencing (average depth 3000X) for PIGA, PIGM, PIGT, and PIGX genes on these 44 consecutive specimens.
Results: Sixteen patients (80.0%) were found to harbor PIG gene mutations: 15 patients had PIGA mutation and 1 patient had PIGM mutation. Granulocyte PNH clone size and variant allele frequency (VAF) of PIG gene mutation showed higher correlation (Spearman's r=0.73, p=0.0002) than that of RBC (Spearman's r=0.61, p=0.0073). All patients harboring PIG gene mutation showed more than 10% PNH clone by FCM. In contrast, 4 patients who did not have PIG gene mutation showed less than 10% PNH clone by FCM.
Conclusion: Considering the mechanism of PNH development, the presence of PIG gene mutation is a definitive evidence supporting PNH diagnosis. PNH granulocyte clone size more than 10% by FCM seems to be clinically significant in relevance to PIG gene mutation. We suggest that neither high sensitivity FCM nor the detection of PIG gene mutation is a requisite for routine clinical diagnosis of PNH.
No relevant conflicts of interest to declare.
