PNH clone distribution in aplastic anaemia: heterozygous germline carriers exhibit immune-mediated clone patterns...

Distinguishing immune-mediated aplastic anaemia (AA) from inherited bone marrow failure syndromes (IBMFS) is critical but challenging. The diagnostic value of paroxysmal nocturnal haemoglobinuria (PNH) clones in this context remains uncertain. We prospectively studied 222 AA patients (median age 21.5 years, range 0.8-67) who underwent high-sensitivity PNH flow cytometry (≥0.01%) and next-generation sequencing. Patients were classified as category 1 (pathogenic/likely pathogenic variants fully explaining phenotype; confirmed IBMFS), category 2 (single heterozygous variants), and categories 3/4 (variants of uncertain significance or no variants). Of 222 cases, PNH clones ≥0.01% were detected in 124 (55.9%), ≥1% in 73 (32.9%), and ≥10% in 27 (12.2%). Category 1 IBMFS was identified in 17 of 222 cases (7.7%). Among category 1 patients, only one of 17 (5.9%) had clones ≥1%, and, critically, none had clones ≥10%. At the ≥1% threshold, PNH detection showed 94.1% specificity and 98.3% positive predictive value (PPV) against IBMFS (odds ratio 9.2, p=0.013). Category 2 patients (15/222, 6.8%) demonstrated PNH frequencies (40.0% at ≥1%) indistinguishable from those of acquired AA (36.4%; p=0.785), supporting an immune-mediated disease mechanism on a genetically permissive background. In this comprehensive single-centre cohort, PNH clones ≥1% demonstrated high specificity (94.1%) and PPV (98.3%) for immune-mediated disease. Notably, heterozygous germline carriers exhibited PNH clone distributions indistinguishable from those in acquired AA. These findings warrant validation in larger multicentre cohorts to establish generalisable diagnostic thresholds.