Clinical Application of Capillary Isoelectric Focusing on Fused Silica Capillary for Determination of Hemoglobin Variants

Hemoglobin is composed of two α and two β polypeptide chains. Any modifications in the amino acid sequence, which may either be congenital or acquired, affects the oxygen-carrying ability of hemoglobin, resulting in a series of hemoglobinopathies. To date, >600 structurally different human hemoglobins are known (1)(2). Identification of abnormal hemoglobin is very important in the differential diagnosis of hemoglobinopathies. Currently, most clinical laboratories have a battery of hemoglobin assays that includes gel electrophoresis, ion-exchange or affinity chromatography, and isoelectric focusing (1)(3). Of all the available methods, computer-operated cation-exchange HPLC is the most widely used for identifying and quantifying major and minor hemoglobins (4)(5)(6)(7). The main disadvantage of a HPLC system is expensive instrumentation and the high cost of columns and reagents. Although an excellent resolution is achieved with gel isoelectric focusing (8), it is labor intensive, time consuming, and not readily amenable for routine screening. The use of capillary electrophoresis (CE) for identification of hemoglobin variants has been reported by several authors (9)(10)(11)(12). In 1994, Hempe and Craver demonstrated the applicability of CE for quantification and identification of hemoglobin variants in clinical samples (13). Capillary isoelectric focusing (cIEF) was performed on a dimethylpolysiloxane (DMS)-coated fused silica capillary having an i.d. of 50 μm. The method is rapid, requires low sample volume, and gives excellent resolution for all major and most of the minor hemoglobin variants. The main drawback of this method is the use of DMS-coated capillary. In our laboratory we found that coated capillaries are not very stable and show considerable lot-to-lot variation, thereby resulting in irreproducible migration times. cIEF can also be done on a fused silica capillary provided electroosmotic flow (EOF) is eliminated or reduced. This can be achieved by addition of hydrophilic polymers such as methyl cellulose (MC) (11). MC (2 g/L) reduces EOF significantly but not to the extent seen in a neutral DMS-coated capillary, thus resulting in poor precision for migration times.