Rapid Molecular Detection for Differentiation of Homozygous HbE and ß0-Thalassemia/HbE in Samples Related With HbE >80% and Variable HbF Levels

Abstract

Abstract Objective To validate a novel rapid molecular testing method for differentiation of homozygous hemoglobin (Hb)E and HbE/β 0-thalassemia genotypes using multiplex melt curve combined with high-resolution melt (HRM) analysis in a single test tube. Methods All 10 genotypes contained (β N/β N; n = 95), (β N/β 3.5-kb; n = 71), (β N/β 45-kb; n = 28), (β N/β E; n = 10), (β E/β 3.5-kb; n = 6), (β E/β 45-kb; n = 4), (β E/β 41/42; n = 28), (β E/β 17; n = 9), (β E/β IVSI#1; n = 6), and (β E/β E; n = 76) were recruited for validation. A proposed strategy for rapid differentiation of β 0-thalassemia/HbE disease and homozygous Hb E in specimens with HbE greater than 80% and variable HbF levels was demonstrated. Results In the validation method, all genotypes showed 100% concordance, compared with the conventional reverse dot blot (RDB) and gap–polymerase chain reaction (PCR) methods. Conclusions Our newly developed method could be useful in routine laboratory settings. The method is rapid, simple, and cost effective; does not require a post-PCR step; and can be applied in routine settings.