Capillary electrophoresis mis-anchoring in a case of Hb Hope with HbE

Abstract

Background

Capillary electrophoresis is a widely used method for hemoglobin (Hb) fraction separation and relative quantitation, where pre-defined Hb peaks (typically HbA and HbA2) act as reference points to “anchor” the electropherogram and define migration zones. In cases lacking HbA or involving variants with migration patterns similar to HbA or HbA2, mis-anchoring can occur–leading to incorrect zoning of Hb variants. This presents a diagnostic challenge, where follow-up investigations, often including molecular testing, are required to establish an accurate diagnosis.

Case Report

We report a case of a 43-year-old Thai female who underwent hemoglobinopathy investigation for microcytic anemia. Capillary electrophoresis showed peaks in the HbA (70.8%), HbA2 (24.4%), and HbC (2.9%) zones, as well as two small peaks in the Z11 (0.9%) and HbD (1.0%) zones. Gel electrophoresis at acid pH showed a band in the HbA position and one slightly anodal to the HbF position and at alkaline pH showed a band in the HbC/E position and another slightly anodal to the HbA position. HBB sequencing identified heterozygosity for the pathogenic HbE and clinically benign Hb Hope variants. HBA PCR detected a single alpha globin gene deletion (αα/α-3.7), consistent with alpha thalassemia silent carrier. Reinterpretation of the electropherogram showed that Hb Hope and HbE mis-anchored as HbA and HbA2, respectively, due to their similar migration deltas.

Conclusion

This is the first documented case of compound heterozygosity for Hb Hope and HbE characterized by capillary electrophoresis. It highlights how beta chain variants with similar migration spacing to HbA and HbA2 can mis-anchor, emphasizing the need for molecular testing when results are unclear. Definitive testing helps avoid diagnostic misclassification and ensure accurate interpretation in complex hemoglobinopathy cases.