Detection of both the normal and mutant alleles in single cells of individuals heterozygous for the sickle cell mutation—prelude to preimplantation diagnosis

As a preliminary step to preimplantation diagnosis of sickle cell disease in unfertilized eggs or 8-cell embryos of heterozygous parents, we established quality control for detection of the mutant and normal alleles of the beta-haemoglobin gene using single buccal cells. Efficient polymerase chain reaction (PCR) amplification of a 680 base pair sequence of the beta-globin gene spanning the site of the sickle cell mutation was obtained for 79 per cent of single heterozygous cells. In 71 per cent of cases, both alleles were detected. With this current efficiency, we predict that a clinical preimplantation diagnosis at the 8-cell embryo stage could be carried out safely and reliably for a couple at risk of transmitting sickle cell disease to their children.     

Derivated fetal haemoglobin as a marker for red cell age in the human fetus reflecting stimulated or impaired red blood cell production

We have determined whether derivated fetal haemoglobin (dHbF, consisting of glycated and acetylated HbF) can be used as a cell age marker for fetal red blood cells (RBCs). Cord blood was obtained between 19 and 39 weeks of gestation from 28 alloimmunised anaemic fetuses (23 RhD+ and 5 Kell) and from 20 non-anaemic fetuses and newborns (controls). Density gradient centrifugation was applied to 36 samples (20 RhD+, 15 controls and 1 Kell) to obtain fractions of increasing cell age. Blood samples were used for measurements of mean cellular volume (MCV), mean cell haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), pyruvate kinase activity (PK) and derivated fetal haemoglobin (dHbF) by cation-exchange HPLC. Reticulocytes were counted only in the whole blood samples. In all density gradient separated RBC fractions, the values for MCV, MCH and PK activity decreased and those of MCHC and dHbF increased with increasing density (equivalent to increasing cell age). The mean density was lower for RBCs of the anaemic RHD group (1.072±0.007 g/ml) than for the non-anaemic controls (1.077±0.005 g/ml) (p<0.05) The RBC density of the Kell sensitised fetus did not differ from those of the controls. In the control group, the values of the cell age markers in whole blood changed significantly with the gestational age, showing an increase of mean age of the erythrocyte population. The best linear relationship was found for dHbF (y=6.28+0.17*weeks; r=0.84; p<0.001). In the anaemic RhD+ fetuses, the RBC age markers did not change with gestational age; the dHbF percentages were lower, and the MCV, MCH, PK values and the reticulocyte counts were higher than in the controls (0.05<p<0.001). The dHbF values of the Kell sensitised fetuses were above (p<0.01) and the reticulocyte counts were below normal (p<0.05) for gestational age. For the anaemic fetuses, a significant number of the dHbF values (86%) and of the reticulocyte counts (78%) differed from the values of the controls (p<0.01). The dHbF percentages in RhD+ fetuses showed the best correlation with the Hb deficit, which is a measure for anaemia (r=−0.81, p<0.0001). We conclude that the percentage derivated HbF may indicate whether the RBC production is normal for gestational age. It may in that sense reflect stimulated or impaired erythropoiesis in alloimmunised haemolytic anaemia. Copyright © 2001 John Wiley & Sons, Ltd.     

Mitomycin C induced chromosome damage in fetal blood cultures and prenatal diagnosis of fanconi's anaemia

We report the use of fetal blood for the prenatal diagnosis of Fanconi anaemia (FA). The clastogenic action of Mitomycin C (MMC) is compared in blood cultures from different fetuses, normal controls and FA heterozygotes. The fetus at risk is shown to suffer from FA on the grounds of excessive chromosome breakage, both spontaneous and MMC induced.     

Altered fetal cardiac flow patterns in pure red cell anaemia (the Blackfan-Diamond syndrome)

In a case of fetal anaemia due to pure red cell anaemia (Blackfan-Diamond syndrome), two-dimensional fetal Doppler echocardiography revealed an altered blood flow velocity pattern with entire incorporation of the atrial contraction component in the early passive filling phase of the right ventricle. Intracardiac blood velocities were increased, whereas cardiac output was only moderately increased. The fetal heart rate was normal. It is concluded that in fetal anaemia the compensatory mechanisms are limited and restricted to an increase in stroke volume. The hypothesis that chronic fetal anaemia is associated with ‘high output cardiac failure’ corresponds well with the present findings. The technique described may prove to be useful in the early diagnosis of fetal anaemia.     

First trimester studies of A fetus at risk for triose phosphate isomerase deficiency

A first trimester prenatal diagnosis was offered to a mother whose child had died of haemolytic anaemia and multisystem disease caused by TPI deficiency. The deficiency state was characterized by greatly reduced TPI activity in both erythrocytes and peripheral lymphocytes. Specific activity of TPI in trophoblast homogenates from the index fetus was about 30 per cent less than in the controls, but the heat stability test showed overlap. These data were confirmed in uncultured and cultured amniotic cells, where glycolytic intermediate concentrations DHAP, GAP and FDP fell in the range of controls. These results suggested that the fetus was a TPI heterozygote. This prenatal prediction was confirmed by RBC and haematological studies at birth.     

First trimester monitoring of a pregnancy at risk for glucose phosphate isomerase deficiency

First trimester prenatal diagnosis was offered to the mother of a child affected by severe haemolytic anaemia due to glucose phosphate isomerase (GPI) deficiency. The mutant enzyme was characterized by an increased thermal lability. Both parents had 50 per cent normal red cell GPI activity. We have shown that the homozygous and heterozygous genotypes can be clearly distinguished from each other and controls by combinations of the measurement of enzyme activity and enzyme thermal lability. Examination of trophoblast cells obtained at 9 weeks of gestation led to the diagnosis of a GPI heterozygous fetus. The result was confirmed by analysis on uncultured and cultured amniotic fluid cells sampled at 16 weeks and by red blood cell studies of the healthy newborn. Prenatal diagnosis of GPI deficiency is indicated in families with previous cases resulting in severe haemolysis and mainly with the conservative view of arranging appropriate therapeutic measures for affected fetuses.