Antenatal diagnosis of β‐thalassemia by isoelectric focusing in immobilized pH gradients

Abstract

A new method for antenatal diagnosis of thalassemias is reported based on the analysis of the major Hb components of fetal cord blood, sampled at week 18 of pregnancy under ultrasonic guidance, by isoelectric focusing in immobilized pH gradients (IPG). In an IPG gel encompassing a pH 6.7-7.6 span, HbA and HbFac are separated by a distance nine times greater than in a conventional carrier ampholyte pH 6-8 gel and three times greater than in an ampholine gel with separators (an equimolar mixture of beta-alanine and 6-amino caproic acid). Band evenness (in terms of uniform protein concentration within a zone) and straightness (in terms of parallel alignment of the bands to the electrodes), because of insensitivity of IPG gels to salt distortions, allows for accurate and reproducible quantitation of HbF, -A, and -Fac levels. The possibility of greatly overloading IPG matrices in total Hbs increases the sensitivity of the technique to the detection of only 0.5% HbA in the total Hb mixture, the lower limit of conventional IEF being only 2.5% HbA. Of 15 fetuses from couples at risk analyzed in the region of Ozieri, three were found to be homozygous beta-thalassemic, eight heterozygous, and four normal with no false-positives or -negatives.

Increased prevalence of false positive hemoglobinopathy newborn screening in premature infants

“…One of the possibilities that could explain the difficulty detecting hemoglobin A is that acetylated Hb F (Hb F ac ) and Hb A co‐migrate in electrophoresis. A good separation of HbA from HbF ac is achieved with several IEF variants 19, 20, 22, but not in conventional IEF. The IEF methodology used by the Florida Newborn Screening Laboratory is not able to differentiate Hb A from HbF ac ; therefore possibly missing minute amounts of Hb A; thus it is important to confirm the findings of IEF with HPLC before releasing screening results.…”

“…IEF is very sensitive to detect Hb S and C at the 0.6% concentration 21. The sensitivity of IEF in detecting low levels of Hb A is somewhat broader (0.5–2.5% level), depending on the IEF variant utilized 22–24. One of the possibilities that could explain the difficulty detecting hemoglobin A is that acetylated Hb F (Hb F ac ) and Hb A co‐migrate in electrophoresis.…”

DNA polymorphism and globin chain analysis in the prenatal diagnosis of β‐thalassaemia major in Taiwan

“…For direct fetal blood analysis, many methods have been reported, such as carboxymethyl chromatography (Weatherall, 1984), high performance liquid chromatography (Shelton et al, 1984), globin chain electrophoresis (Alter et al, 1981), and haemoglobin isoelectric focusing (Manca et al, 1986). Most of these procedures involve incubation of fetal reticulocytes with [3H]leucine culture medium and then determination of the synthetic ratio of the P / y chain.…”

Molecular diagnosis of inherited disorders: lessons from hemoglobinopathies

“…Finally, isoelectric focusing (IEF) on agarose gels can be used to separate different Hb fractions and variants [Ferrari et al, 1984, Manca et al, 1986 and globin chains [Righetti et al, 1979]. IEF is frequently the first analytical test used for the diagnosis of Hb fractions (Fig.…”