Capillary Zone Electrophoresis for the Diagnosis of Congenital Hemoglobinopathies

Abstract

The analysis of human hemoglobins (Hbs) is of medical importance in a number of congenital defects. The hemoglobinopathies are grouped into defective variants of Hb, such as Hb S and >600 other variants, and thalassemias characterized by abnormal expression of the genes for normal globin chains (1)(2)(3). Alkaline electrophoresis, performed historically on cellulose acetate and currently on agarose, combined with citrate agar electrophoresis at acidic pH is still widely used (4)(5)(6)(7), but high-performance cation-exchange chromatography (HPCEC) offers superior resolution, speed, and automation (8)(9)(10). Capillary electrophoresis uses numerous separation principles and shares with HPLC the advantages of high resolution and automation, with on-line detection and direct quantification (11)(12)(13)(14)(15). Capillary isoelectric focusing on coated capillaries can be used to study Hb (16)(17)(18)(19)(20)(21), but is slower than HPCEC (21). The first reported assays based on capillary zone electrophoresis (CZE) gave poor resolution of Hbs or were not quantitative (22)(23)(24). The aim of the present work was to evaluate a rapid CZE assay with dynamic coating of the fused-silica at alkaline pH to detect abnormal Hbs and to quantify Hbs for the diagnosis of thalassemias, and at acidic pH to confirm the identity of Hb variants. Reagents were obtained from Analis. The alkaline kit A2 contained a hemolyzer, an initiator consisting of a polycation, and an arginine buffer (pH 8.8) containing a polyanion. The acidic kit A1 has been described elsewhere for Hb A1c measurement (25). Adult and newborn samples were collected in EDTA-containing tubes at the maternity unit of the Hopital Saint-Antoine (AP-HP, Paris, France) and received in the laboratory for Hb analysis. Whole blood samples …