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We have developed a two-colour immunocytochemical staining method for the detection of fetal and embryonic haemoglobin in erythroid cells. The method was applied to study these haemoglobin types in fetal red cells. Specimens from fetal blood (10 weeks), cord blood and fetal liver (14 weeks) as well as chorionic villus samples (10-13 weeks) were stained for gamma and epsilon chains using CY3 and FITC labelled antibodies. Morphometric analysis was applied to determine cell size. Samples from organs involved in early embryonic development contained relatively large erythroblasts expressing the epsilon globin chain (megaloblasts); later in gestation the gamma chain was co-expressed by the same cells which ultimately became smaller and contained HbF (alpha 2 gamma 2) only. This phenomenon was confirmed in CVS samples in which all cell types were abundantly present. Since fetal erythroblasts are considered candidate cells for non-invasive prenatal diagnosis using FISH, we studied the phenotype of erythroblasts circulating in the maternal blood. The majority of erythroblasts in maternal blood appeared to be of the relatively small gamma globin-containing cell type. However, careful screening of the same maternal blood samples also revealed erythroblasts expressing epsilon or epsilon and gamma globins simultaneously, although at low frequency. Control specimens from non-pregnant women did not show nucleated red cells expressing either of the haemoglobin types. These observations may contribute to the better recognition of fetal cells in the maternal blood for prenatal diagnosis.

Sonographically directed placental aspiration has been investigated as a method for obtaining fetal erythrocytes. The technique is generally successful and the fetal red cells are generally usuable for the prenatal diagnosis of the hemoglobinopathies.

Two pregnancies at risk for X-linked recessive Duchenne's muscular dystrophy were studied at 18 and 20 weeks. Fetal blood was obtained by placental aspiration for measurement of plasma creatine phosphokinase activity. Activity in the first fetus was 96 IU per liter, as compared to a control range of 0 to 150 IU per liter in 16 pregnancies not at risk for the disorder. The pregnancy continued, and the infant was normal after birth. In the second fetus creatine phosphokinase activity was significantly elevated to 540 IU per liter (P less than 0.001). Fetal blood also showed considerable hemolysis, an unusual observation in placental blood sampling. After abortion, examination of fetal muscle by light, phase and electron microscopy showed characteristic features of Duchenne's muscular dystrophy, including wide variation in muscle-fiber diameter and reduction in the number of fibers per fasciculus. These cases illustrate the potential usefulness of fetal plasma for prenatal diagnosis and, specifically, of creatine phosphokinase activity for diagnosis of muscular dystrophy.

The development of fetoscopy and the ability to obtain fetal blood samples have made possible the prenatal diagnosis of diseases not diagnosable by amniocentesis. Foremost among these disorders are the hemoglobinopathies. We report our experience in establishing the first program for fetoscopy and fetal blood sampling in the Middle East. The results demonstrate that the experience and training required for a fetoscopy and fetal blood sampling program can be obtained in about one year. During that year, 23 couples who fetuses were at risk for a hemoglobinopathy learned the genetic status of their fetuses.

To examine the potential for prenatal diagnosis of genetic lipoprotein metabolic defects (e.g. abetalipoproteinemia. Tangier disease) we determined the normal concentrations of apolipoproteins (apo) A-I, A-II, B, and E in mid-trimester amniotic fluid and fetal plasma. The concentrations of apo A-I and apo A-II in amniotic fluid were 1--2 per cent of the respective levels in the mother's plasma, whereas apo B and apo E were undetectable in amniotic fluid. In contrast to amniotic fluid, all four apolipoproteins were detectable in fetal plasma, and the levels of apo A-I, apo B and apo E were in the range observed in the mothers: 160.2 +/- 103.1, 59.8 +/- 35.7 and 5.7 +/- 3.5 mg/dl respectively (mean +/- SD, n = 13). The fetal plasma level of apo A-II (28.3 +/- 12.4 mg/dl) was two-thirds that observed in the mother's plasma. The normal levels of these apolipoproteins in fetal plasma are well above the sensitivity of the methods, and their quantification requires only 10--20 microliters of fetal plasma. Determination of apolipoproteins in fetal blood obtained by fetoscopy thus may provide a method for the prenatal diagnosis of congenital apolipoprotein deficiencies.

The amount of fetal-maternal transfusion during invasive intrauterine diagnostic instrumentation was determined by measuring the increase in maternal serum alpha-fetoprotein (delta AFP) caused by the procedure. Fetal liver biopsy or fetoscopy for purposes other than blood sampling caused a mean delta AFP of 11.4 ng/ml and 34.2 ng/ml, respectively. Fetoscopy with fetal blood sampling produced a mean delta AFP of 211.8 ng/ml, while fetoscopy followed by placentesis caused a mean delta AFP of 462.8 ng/ml (representing a 1.07 ml fetal-maternal transfusion). Although this magnitude of fetal-maternal transfusion is an acceptable risk for the fetus, it is a sufficient transfusion to cause blood cell antigen sensitization.

To evaluate paternity in a case where both rape and conjugal coitus had occurred close to the time of conception, fetoscopy with fetal blood sampling was performed at 20 weeks gestation. Detailed blood group typing of the wife, husband, and fetus, and the presence of a very long Y chromosome in the last two, indicated a 99.9% chance that the fetus was fathered by the husband and only a 0.1% chance that it was fathered by "some other male Caucasian." The couple elected to continue the pregnancy. Neonatal testing verified the prenatal findings.