Hemoglobin E Associated with Hemoglobin Constant Spring: Diagnostic Challenges in Heterozygous and Homozygous States.

Abstract

Hemoglobin E (HbE) is a common structural hemoglobin variant with a β+-thalassemic effect. Hemoglobin Constant Spring (HbCS) is the most prevalent non-deletional α-thalassemia variant and is characterized by low expression and marked instability, making detection difficult. The coexistence of both variants may produce atypical hematologic and electrophoretic profiles, complicating diagnosis. We describe two patients with combined HbE and HbCS: a 12-month-old girl heterozygous for HbE and a 58-year-old man homozygous for HbE. Both presented with microcytosis and characteristic patterns on capillary electrophoresis and high-performance liquid chromatography, which were performed simultaneously as part of routine diagnostic testing. Molecular analysis using StripAssay, MLPA (multiplex ligation-dependent probe amplification), and next-generation sequencing (NGS) confirmed heterozygous HbCS in both cases, with no additional alterations in the α- and β-globin gene clusters. The association of HbE and HbCS poses significant diagnostic challenges. These cases highlight the limitations of phenotypic techniques used in isolation and underscore the value of an integrated diagnostic approach that incorporates complementary molecular methods, such as NGS-based platforms.