Analysis of single erythrocytes by injection-based capillary isoelectric focusing with laser-induced native fluorescence detection

A modified version of capillary isoelectric focusing (cIEF) was developed to separate hemoglobin variants contained within single human erythrocytes. Laser-induced native fluorescence with 275 nm excitation was used to detect the separated hemoglobins. In this method, baseline fluctuations were minimized and detection sensitivity was improved by using dilute solutions of anolyte, catholyte, and carrier ampholytes (with methylcellulose). Since electroosmotic flow was used for mobilization of the focused bands, separation and detection were integrated into a single step. The capillary was first filled with only ampholyte solution, and the cell (or standard) was injected as in capillary zone electrophoresis. The similar to 90 fl injection volume for individual cells is 7 x 10(4) times lower than those previously reported. Adult (normal and elevated A(I)), sickle (heterozygous), and fetal erythrocytes were analyzed, with the amounts of hemoglobins A(0), A(Ic), S and F determined. The pH gradient for cIEF was linear (r(2) = 0.9984), which allowed tentative identification of Hb F-ac. Variants differing by as little as 0.025 pI units were resolved.