Performance Evaluation of a Premier Resolution HPLC System for Detecting Hemoglobin Constant Spring, Hemoglobin...

Area of Science:

• Hematology
• Clinical Chemistry
• Genetics

Background:

• Hemoglobin Constant Spring (HbCS) and Hb Paksé are prevalent α-globin variants in Southeast Asia.
• These variants, when combined with α⁰-thalassemia, cause severe HbH disease.
• Their instability and low levels challenge conventional screening, leading to false negatives.

Purpose of the Study:

• To evaluate Premier Resolution High-Performance Liquid Chromatography (HPLC) for detecting HbCS and Hb Paksé.
• To compare its performance against capillary electrophoresis (CE).
• To characterize chromatographic patterns across different α- and β-globin genotypes.

Main Methods:

• A two-phase validation study involving 616 samples (Phase 1) and 2,032 individuals (Phase 2).
• Comparison of Premier Resolution HPLC with CE using DNA-confirmed cases.
• Molecular analysis served as the reference standard.

Main Results:

• Premier Resolution HPLC showed distinctive multi-peak patterns for HbCS/Hb Paksé.
• It demonstrated higher sensitivity (96.4%) and specificity (97.1%) than CE (92.8% sensitivity, 93.5% specificity).
• False negatives were reduced, particularly in heterozygotes, though heterozygous Hb Paksé remained undetectable by both methods.

Conclusions:

• Premier Resolution HPLC is a reliable and cost-effective method for HbCS detection in populations at risk for α-thalassemia.
• Its enhanced resolution aids routine screening in high-prevalence areas.
• Molecular confirmation is still essential for heterozygous cases and genetic counseling.