Detection of Hemoglobin Constant Spring: A Comparison of Capillary Electrophoresis Versus High-Performance Liquid Chromatography

Introduction: In Malaysia, Hemoglobin Constant Spring (Hb CS) is the most common non-deletional alpha thalassemia, caused by a mutation at the termination codon of the alpha 2-globin gene (TAA>CAA). Detection typically involves identifying an abnormal peak at zone 2 on capillary electrophoresis (CE) or a small peak at the C-window on high-performance liquid chromatography (HPLC), indicative of Hb CS. Objective: This study aimed to investigate the correlation between HPLC and CE in detecting Hb CS, evaluating their respective diagnostic accuracies and limitations. Methods: A cross-sectional study was conducted at Hospital Sultanah Nur Zahirah involving secondary school students (Form 4) from Terengganu who participated in a thalassemia screening program conducted by the Ministry of Health (MOH) from January 2019 to December 2022. Blood samples from subjects showing a positive peak in zone 2 of CE and a small peak at the C-window of HPLC were selected. Molecular studies of these samples were performed to confirm the presence of Hb CS. For the statistical analysis, the Pearson correlation coefficient was employed to assess the relationship between CE and HPLC results. Results: Hb CS was confirmed in all samples by molecular studies, revealing 92.3% heterozygous, 7.2% compound heterozygous, and 0.5% homozygous cases. CE detected 92.3% of heterozygous Hb CS cases, while HPLC detected only 48.2%. For compound heterozygous Hb CS, CE detected 100%, whereas HPLC detected 89.3%. Both homozygous cases were detected by CE and HPLC. The Pearson correlation coefficient test showed a significant linear relationship (p<0.001) between CE's zone 2 peak values and HPLC's Cwindow peaks (n=389). Conclusion: These findings highlight the efficacy of CE as a reliable method for Hb CS detection, suggesting its potential superiority over traditional HPLC in clinical settings.