Background:Routine diagnosis of paroxysmal nocturnal hemoglobinuria (PNH) is based on flow cytometric measurement of PNH clone in RBC and granulocyte. However there is no well‐established consensus on the clinically significant size of PNH clone.Aims:So we investigated whether quantitative results of PNH clone size measured by flow cytometry (FCM) correlate with mutant burden of PIG gene.Methods:A total of 89 specimens from 63 patients whose PNH clone size was ≥0.1% by FCM was enrolled. We performed ultra‐deep sequencing for PIGA, PIGM and PIGX genes on these 89 consecutive specimens. Also, we followed up 6 patients who were treated with Eculizumab and analyzed their laboratory changes including hemoglobin, lactate dehydrogenase level, PNH flow cytometry and PIG gene mutant burden. Treatment response was evaluated every 6 months.Results:Sixteen out of 63 patients had >10% granulocyte PNH clone and they all had PIG gene mutation. Fifteen (93.8%) had PIGA mutation and one (6.3%) PIGM mutation. Patients with >15% RBC PNH clone (n = 12) all had PNH‐related symptoms (p <0.001). Granulocyte clone size showed better correlation with VAF than RBC clone size (Granulocyte r = 0.61, p = 0.01, RBC r = 0.39, p = 0.14). On the other hand, RBC clone size was better reflective of PNH‐related symptoms with a cut‐off of 15% (p <0.001). We found no significant laboratory parameter for prediction of Eculizumab treatment response.Summary/Conclusion:Patients with >10% granulocyte PNH clone by FCM were always accompanied by PIG gene mutation. Those with >15% RBC PNH clone by FCM all had PNH‐related symptoms. Weak correlation between granulocyte clone size and VAF of PIG gene mutation implies that granulocyte clone reflects the real PNH clonal burden.image

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