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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.

SUMMARYThe results of 200 antenatal diagnoses in pregnancies at risk for homozygous r-thalassaemia, carried out on fetal blood samples obtained by placental aspiration in the second trimester, are described. Globin chain synthesis in the fetuses was measured by means of 3H-leucine incorporation and separation of the chains on carboxy-methyl-cellulose columns. Fetal red cell enrichment was performed by NH4Cl-N`H4HCO3 differential lysis of maternal cells or anti-i differential agglutination. Sufficient fetal blood for analysis was obtained in 97.5 % of the cases. The overall fetal loss rate was 6.5 %, but it declined from 10% in the first consecutive 100 cases to 3 % in the last 100 cases. Fetal loss was the result of early or late intrauterine death or spontaneous abortion. Forty-two homozygous fetuses had no 3-chain synthesis and one had a very low 3/ry ratio (0.005). Of the pregnancies, 37 were terminated at parental request and four aborted spontaneously. Absence of 5-chain radioactivity was confirmed in 12 abortuses with suitable cord blood samples for analysis. Two pregnancies with homozygous fetuses were not terminated, as one member of each couple was a devout Catholic. As expected, both infants developed Cooley's anaemia. Follow-up of the 146 infants, diagnosed in utero as non-homozygotes, showed cerebral palsy in one and a small cutaneous needle injury in three. None of these developed homozygous P-thalassaemia. Even P-thalassaemia trait with a F3/y ratio of 0.046 i 0-012 can be distinguished from normal, showing a r3/y ratio of 0-086 ± 0.019 with a high degree of certainty.

Prenatal diagnosis was attempted in 133 pregnancies at risk for beta thalassemia (132 cases) or sickle-cell beta 0 thalassemia (1 case). Of these, 76 couples requested diagnosis because they already had children affected with homozygote beta thalassemia (72 cases) or beta+ thalassemia (4 cases). The others were probably at risk for beta 0 thalassemia since this is by far the predominant thalassemia type in Sardinia. Sufficient fetal blood for analysis was obtained by placental aspiration at 18--24 weeks gestation in 130 cases. Ten fetal losses occurred. The pregnancies were followed and no relevant complications were seen. Of the newborns delivered, 45 were followed from birth with particular attention to congenital malformation, neurological, growth, and maturity assessement. No major adverse effect of placentocentesis on child growth and development was observed. Placental samples were analyzed by globin chain synthesis analysis on carboxylmethylcellulose columns. When the placental samples contained more than 20% maternal red cells, fetal red cell enrichment was carried out by anti-i (53 cases) or anti-AB (2 cases) differential agglutination or NH4Cl-NH4HCO3 differential lysis of maternal cells (17 cases). Of the 130 cases, 32 fetuses had no beta-chain radioactivity and one had a beta/gamma ratio of 0.005. These were presumed to be homozygous and all but one were electively aborted. Absence of beta-chain radioactivity was confirmed in 10 abortuses with suitable cord blood samples. A total of 91 infants have been born and are nonhomozygous. Genotype assessment at 6 months after birth in 33 infants showed that there was only a slight overlap between the ranges of normal (0.095 +/- 0.016) and heterozygous (0.05 +/- 0.01) fetal beta/gamma globin chain synthesis ratios.