SCREENING OF UMBILICAL-CORD BLOOD HEMOGLOBINS BY ISOELECTRIC-FOCUSING IN CAPILLARIES

Separation and quantitation of the three main hemoglobin components of umbilical cord blood (fetal, acetylated fetal and adult hemoglobins; Hb F, F-ac,F- 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 HbF(ac,) 9.5% (range 7-11%).