Diagnostic Utility of Isoelectric Focusing and High Performance Liquid Chromatography in Fetal Cord Blood Screening for Thalassemia and Non-Sickling Hemoglobinopathies,

Thalassemia and non-sickling hemoglobinopathies are highly prevalent in Southeast Asia. High performance liquid chromatography (HPLC), β-thalassemia short program, is the most common technique routinely used in this region, while IEF is rarely employed. However, HPLC, β-thalassemia short program, cannnot quantify HbBart's, as well as HbH. In addition, the acetylated HbF, which is frequently found in cord blood, interferes with the HbBart's and HbH peaks on HPLC. We primarily aim to compare diagnostic utilities of IEF and HPLC in fetal cord blood screening for prenatal diagnosis of thalassemia and non-sickling hemoglobinopathies in Thailand.

One-hundred and two cord bloods from the National Cord Blood Bank and fetuses of high risk couples were analyzed using IEF and HPLC, β-thalassemia short program, and correlated with red cell indices and DNA analyses. HbBart's was quantified only on the IEF method. The sensitivity and specificity to screen for α-globin and β-globin gene mutations were determined.

Of 88 cases showing HbBart's on hemoglobin analysis, IEF yielded 98.9% sensitivity to identify at least one α-globin gene defect (−α^−3.7, -α^−4.2, -α^CS, -α^PS, –^SEA, –^Thai, -α^−3.7/-α^−3.7 and -α^−3.7/-α^CS), while HPLC yielded only 83.7% sensitivity to detect at least one α-globin gene defect. All missing cases on HPLC were single α-globin gene deletion that had HbBart's less than 1.5% of total hemoglobin measured on IEF. Hb Constant Spring (CS) and Hb Pakse (PS) heterozygotes (N = 11) had small amount of HbBart's less than 3%, but neither HbCS nor HbPS was detectable on both tests. HbBart's ≥ 2% yielded 100% sensitivity to identify two α-globin gene defects, while HbBart's ≥ 3% yielded 100% specificity to diagnose two α-globin gene defects. MCV and MCH are the most helpful red cell parameter in screening for α-thalassemia. MCV ≤ 95 fL yielded 100% sensitivity to identify two α-globin gene deletions, while MCV ≤ 90 fL yielded 100% specificity to identify two α-globin gene deletions. In addition, MCH ≤ 30 pg yielded 100% sensitivity and 100% specificity to identify two α-globin gene deletions.

Thirty two cord bloods showed HbA₂/E, ranging from 1.9–15.6%. DNA analysis using a reverse dot blot technique for detecting common β-thalassemia mutations in Thailand demonstrated HbE mutation in all cases, while no β-thalassemia mutations were identified. Therefore, detectable HbA₂ in cord blood usually represents HbE, not HbA₂. Three HbE heterozygotes with two α-globin gene deletions demonstrated high HbBart's and low MCV, as well as low MCH, similar to those with two α-globin gene deletions alone. On the other hand, HbE heterozygotes with single α-globin gene defects could not be differentiated from pure HbE heterozygotes.

IEF is superior to HPLC β-short program in screening for α-globin gene defects in prenatal cord blood analysis. The amount of HbBart's, MCV and MCH are helpful for identifying α-thalassemia. The presence of HbA₂ in cord blood indicates HbE mutation, but not β-thalassemia.

Sutcharitchan:Novartis: Research Funding.