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

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. © American Society for Clinical Pathology 2020. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.