Quantification of hemoglobin variants by capillary isoelectric focusing

Abstract

Capillary isoelectric focusing (cIEF) was used to identify and quantify major and minor hemoglobin (Hb) variants. Whole blood (~10 μL required) hemolysate was analyzed with a commercial instrument equipped with a 50 μm (i.d.) x 27 cm coated capillary filled with 20 g/L ampholytes (pH 6-8) in 4 g/L methylcellulose (MC). Cathode and anode solutions were 20 mol/L NaOH and 100 mol/L H3PO4 in MC, respectively. Samples (~40 nL) were applied via autosampler by low-pressure injection, focused for 3 min at 30 kV, and mobilized by simultaneous voltage and low pressure past the detector, where absorbance at 415 nm was analyzed by an automated data acquisition system. Blood from subjects with sickle cell trait, Hb S/C disease, and various β- thalassemias were analyzed by cIEF in <15 min. cIEF was used to separate Hb S from Hb D-Los Angeles. Assay precision determined with commercial controls gave CV <2% for Hb A and S, and 1-11% for minor Hb variants A2, F, and A(1c). Results obtained by cIEF for patients' samples agreed well with values determined by conventional assays (r2 >0.95). The results demonstrate that cIEF is a rapid, sensitive, high-resolution automated method for routine quantitative clinical analysis of Hb variants.