Prenatal diagnosis in two cases of de novo complex balanced chromosomal rearrangements. Three-year follow-up in one case

We report two cases of apparently balanced complex de novo chromosomal rearrangements (BCCR) detected prenatally at 17 weeks and 10 weeks of gestation, respectively. Chromosomes were studied using GTG-banding and fluorescent in situ hybridization (FISH). In one case four chromosomes and in the other case three chromosomes were involved in the rearrangements. One of the pregnancies was terminated and no external or internal abnormalities were detected at autopsy. The other pregnancy continued to term. Level III ultrasound examination showed no abnormalities. The child is now 3 years old and has neither congenital anomalies nor evidence of delayed psychomotor development.     

Prenatal diagnosis of GM2-gangliosidosis B1 variant

Prenatal diagnosis in a pregnancy at risk for a juvenile B1 variant of GM₂-gangliosidosis was carried out. The biochemical study of the cultured amniocytes and the affected fetal brain is reported. The results obtained show that the sulphated artificial substrate can be used in the diagnosis of B1 variant, but not the neutral one. The accumulation of GM₂-ganglioside in the fetal brain of the B1 juvenile form and an infantile form of GM₂-gangliosidosis (0 variant) was compared.     

Flow sorting of fetal erythroblasts using intracytoplasmic anti-fetal haemoglobin: Preliminary observations on maternal samples

Monoclonal antibody to fetal haemoglobin (a₂γy₂) has been proposed as a fetal-specific reagent. We developed an intracellular staining protocol that combines fluorescein isothiocyanate or phycoerythrin conjugated anti-γ with the DNA binding dye Hoechst 33342 to identify and flow sort fetal erythroblasts from maternal blood. Our preliminary observations on anti-γ-positive cells sorted from four different pregnant women are described here, using fluorescence in situ hybridization (FISH) with chromosome-specific probes to identify fetal cells. Our data demonstrate that far fewer candidate fetal cells are sorted with this protocol than by current cell surface staining methods that employ the monoclonal antibody CD71. This results in increased fetal cell sorting purities. With this protocol, standard FISH techniques require modification due to the rigorous fixation with 4 per cent paraformaldehyde. Our initial data indicate the promise of this approach.     

An investigation of methods for enriching trophoblast from maternal blood

Trophoblast deportation is known to occur in normal human pregnancy, but it is not yet clear whether these cells routinely enter the maternal peripheral circulation and are available as a source of fetal DNA for non-invasive prenatal diagnosis of genetic disorders. To resolve this issue requires an efficient method of enriching trophoblast from maternal blood combined with a means to confirm its identity. Five different techniques were tested on ten retroplacental blood samples to determine the most sensitive and operator-efficient method. Lysis of red cells alone gave the best recovery of trophoblast but had to be discounted, together with Ficoll density gradient centrifugation, due to the very low purity and the excessive time required. Fluorescence-activated cell sorting (FACS) of pre-enriched trophoblast resulted in the lowest recovery rate (8 per cent) despite a 3250-fold enrichment and a very high purity. Immunomagnetic beads (Dynabeads) coated with anti-CD 16 antibody proved to be the best method for the subsequent immunocytochemical characterization of deported trophoblast. However, IO beads coated with anti-CD45 antibody may be more useful for isolating trophoblast for prenatal diagnosis due to the high purity, enrichment (32-fold), and recovery rate (78 per cent) obtained with this method.