Performance Evaluation of a Premier Resolution HPLC System for Detecting Hemoglobin Constant Spring, Hemoglobin Paksé, and Coexisting α‐ and β‐Thalassemia Mutations

ABSTRACT Background Hemoglobin Constant Spring (HbCS) and Hb Paksé are clinically significant α‐globin variants prevalent in Southeast Asia that cause severe non‐deletional HbH disease when combined with α 0 ‐thalassemia deletions. Their inherent instability and low circulating levels (0.1%–1.0%) present analytical challenges, often yielding false negatives with conventional screening methods. This study aimed to evaluate Premier Resolution HPLC performance in detecting HbCS and Hb Paksé vs. capillary electrophoresis (CE), and to characterize chromatographic patterns across α‐ and β‐globin genotype combinations. Methods A two‐phase validation study was conducted. Phase 1 analyzed 616 samples (307 DNA‐confirmed HbCS/Hb Paksé cases) to characterize chromatographic patterns and compare Premier Resolution with CE. Phase 2 prospectively validated the system in 2032 consecutive individuals undergoing routine health screening. Molecular analysis served as the reference standard. Results Premier Resolution HPLC demonstrated distinctive multi‐peak patterns spanning the S and C chromatographic windows for HbCS/Hb Paksé, with quantification limited to homozygous and α 0 ‐thalassemia co‐inherited cases. It outperformed CE with 96.4% sensitivity, 97.1% specificity, and 96.8% accuracy vs. CE (92.8% sensitivity, 93.5% specificity, 93.2% accuracy). False negatives occurred mainly in heterozygotes (Premier Resolution: 5/73 6.8% vs. CE: 14/73 19.2%); heterozygous Hb Paksé remained undetectable by both techniques (3/3, 100%). Clinical validation confirmed excellent performance with 94.9% sensitivity, 99.6% specificity, and 99.36% accuracy. Conclusions Premier Resolution HPLC provides a reliable and cost‐effective method for HbCS detection in α‐thalassemia–risk populations. Its enhanced resolution and characteristic chromatographic patterns support routine thalassemia screening in high‐prevalence regions, though molecular confirmation remains necessary in heterozygous cases for genetic counseling.