Rapid and Accurate Quantitation of Hb Bart's and Hb H Using Weak Cation Exchange High Performance Liquid Chromatography: Correlation with the α-Thalassemia Genotype

Abstract

The Bio-Rad Variant™ Hemoglobin testing system is an automated high performance liquid chromatography analyzer marketed with a β-thalassemia short program to quantify Hbs F and A₂, and assist in detecting Hbs A, S, D, C., and E. Although the two hemoglobins present in Hb H disease, Hb Bart's and Hb H, are separated by the system, they are not quantitated. In this study we modified the β-thalassemia short program in order to facilitate quantitation of Hb Bart's and Hb H. Blood samples from 60 patients with Hb H disease, with various underlying genotypes, were studied. Analyses were performed on the day of blood collection or on hemolysates stored at -80°C in cyanide or carbomonoxy forms. The mean sum of Hb Bart's and Hb H levels in all patients was found to be 12% (range 1.8-35%). Patients with nondeletional mutations (or association of α⁰ deletion and nondeletional mutations) had notably higher Hb Bart's and Hb H levels when compared to patients with deletional genotypes.