Screening of umbilical cord blood hemoglobins by isoelectric focusing in capillaries

Abstract

Separation and quantitation of the three main hemoglobin components of umbilical cord blood (fetal, acetylated fetal and adult hemoglobins; Hb F, Fac, A) by capillary isoelectric focusing (IEF) in a pH 6-8 gradient is reported. Even in coated capillaries (with covalently bound chains of linear acrylamido derivatives, notably N-acryloylaminoethoxyethanol), no base line separation is obtained between Hb F and A, although this is routinely achieved in gel slab IEF. However, when the carrier ampholyte buffers were added to 3% short-chain liquid linear polyacrylamide, base line resolution and stabilization of peak transit times were obtained. This suggests that even in the best coating procedures, patches of the inner capillary surface could still be naked, so that the static coating is complemented by a dynamic coating on the unoccupied sites. An additional improvement in separation occurs if the above mixture, comprising 5% carrier ampholytes in the pH 6-8 range and 3% soluble polyacrylamide, is made to contain 50 mM beta-alanine, a "separator" known to flatten the pH gradient around pH 7. In the normal newborns analyzed (n = 30), the following average values were obtained: Hb F, 70.1% (range 65-75%); Hb A, 20.2% (range 15-25%); and HbFac, 9.5% (range 7-11%).

Dynamic analyte introduction and focusing in plastic microfluidic devices for proteomic analysis

“…Among various electrokinetic-based separation techniques, isoelectric focusing (IEF) not only contributes to a high-resolution protein separation with a pI difference as small as 0.005 pH unit [16] but also potentially allows the analysis of low-abundance proteins with a typical concentration factor of 50-100 times. To perform IEF, the entire channel is initially filled with a solution mixture containing proteins/peptides and carrier ampholytes for the creation of a pH gradient.…”

Quantitation of glycated hemoglobins in human adult blood by capillary isoelectric focusing

“…Only recently, Conti etal. [14] have been able to offer a validated capillary IEF method for screening of umbilical cord blood hemoglobins: reproducibility of peak elution times was obtained by a combination of covalent coating with added viscous polymers to the background electrolyte; simultaneously, ample peak separation was enforced by flattening the region of interest (around pH 7) with the aid of 50 mM P-alanine.…”

CE‐based analysis of hemoglobin and its applications in clinical analysis

“…Mario et al [31] admitted their method, by using the neutral (polyacrylamide)-coated capillary, but did not obtain the resolution as with dimethylpolysiloxane silica capillary. But it was claimed to be much better than that with a linear acrylamino-coated capillary, as reported by Conti et al [32], which did not allow baseline resolution between Hb F and Hb A. The N-acryloylaminoethoxyethanol and polyAAEEcoated capillaries were also developed and evaluated by Jenkins et al [33], which have given satisfactory results for the Hb's.…”