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.     

Body stalk anomaly associated with maternal cocaine abuse

A case of body stalk anomaly diagnosed prenatally by ultrasound during the 24th week of pregnancy in a cocaine abusing mother is presented. Accurate visualization of the fetal organs was difficult due to the severe oligohydramnios caused by premature rupture of membranes, probably related to the cocaine use. The sonographic findings were an omphalocoele, fetal attachment to the placenta, kyphoscoliosis, and absence of a floating umbilical cord. The prenatal diagnosis of the syndrome and the possible relationship with cocaine abuse are discussed.     

Detection of fetal HLA-DQα sequences in maternal blood: A gender-independent technique of fetal cell identification

The objective of this study was to detect fetal HLA-DQα gene sequences in maternal blood. HLA-DQα genotypes of 70 pregnant women and their partners were determined for type A1. We specifically sought couples where the father, but not the mother, had genotype A1. In 12 women, maternal blood samples were flow-sorted. Candidate fetal cells were isolated and amplified by using PCR primers specific for a paternal HLA-DQα A1 allele. Fetal HLA-DQα A1 genotype was predicted from sorted cells; amniocytes or cheek swabs were used for confirmation. Six of twelve sorted samples had amplification products indicating the presence of the HLA-DQα A1 allele; 6/12 did not. Prediction of the fetal genotype was 100 per cent correct, as determined by subsequent amplification of amniocytes or cheek swabs. We conclude that paternally inherited uniquely fetal HLA-DQα gene sequences can be identified in maternal blood. This system permits the identification of fetal cells independent of fetal gender, and has the potential for non-invasive prenatal diagnosis of paternally inherited conditions.     

DNA-based carrier detection and prenatal diagnosis of tyrosinase-negative oculocutaneous albinism (OCA1A)

We describe molecular prenatal diagnosis and carrier detection of tyrosinase-negative oculocutaneous albinism (OCA1A) in two families. In one family, we carried out DNA-based prenatal diagnosis of OCA1A. In the other family, mutation analysis and carrier detection obviated the need for prenatal diagnosis. Molecular analysis is safer and probably more accurate than fetoscopy and fetal scalp biopsy, and should become the method of first choice for prenatal diagnosis of OCA1.     

Flow cytometric method to measure urea-resistant alkaline phosphatase from blood neutrophils for use in the prenatal screening of Down's syndrome

The histochemical measurement of urea-resistant alkaline phosphatase from maternal blood neutrophils is known to have a high detection rate for the prenatal detection of Down's syndrome pregnancies. However, because the histochemical method is laborious and subjective to use, it has not gained widespread acceptance in prenatal screening programmes. We present a simple and objective method for the measurement of urea-resistant alkaline phosphatase by flow cytometry. The method should allow the design of larger studies aimed at evaluating the role of neutrophil urea-resistant alkaline phosphatase in the prenatal screening for Down's syndrome.     

Hepatic calcifications in a fetus with trisomy 9 that underwent cordocentesis

Foci of calcification were observed at autopsy in the liver of a fetus with complete trisomy 9 on which two cordocenteses had been performed. It is suggested that liver calcifications are a possible complication of the procedure. As several other cases of calcifications in the liver and other organs of fetuses with autosomal trisomies have been described without a history of cordocentesis, further studies should be carried out to determine whether fetuses with chromosomal anomalies are more prone to thrombus formation and embolization.     

Prenatal diagnosis of neurofibromatosis type 1: From flanking rflps to intragenic microsatellite markers

Even though the neurofibromatosis type 1 (NF1) gene was cloned more than 3 years ago, the process of identifying mutations has not been fruitful, and genetic counselling is mainly based on the use of linked markers. Since 1990, we have analysed 130 NF1 families and have performed six prenatal diagnoses. In each case, genetic counselling has relied on linked markers and informativity was achieved in all of them. The use of intragenic microsatellite polymorphisms (IVS27AAAT2.1, IVS27AC28.4, IVS27AC33.1, and IVS38GT53.0) has increased the informativeness in our series of NF1 families to an average of 90 per cent, providing accurate diagnosis and confirmation of the disease status.     

On the response of 500 gal propane tanks to a 25% engulfing fire

This paper presents detailed data on the thermal response of two 500 gal ASME code propane tanks that were 25% engulfed in a hydrocarbon fire. These tests were done as part of an overall test programme to study thermal protection systems for propane-filled railway tank-cars.

The fire was generated using an array of 25 liquid propane-fuelled burners. This provided a luminous fire that engulfed 25% of the tank surface on one side. The intent of these tests was to model a severe partially engulfing fire situation.

The paper presents data on the tank wall and lading temperatures and tank internal pressure. In the first test the wind reduced the fire heating and resulted in a late failure of the tank at 46 min. This tank failed catastrophically with a powerful boiling liquid expanding vapour explosion (BLEVE). In the other test, the fire heating was very severe and steady and this tank failed very quickly in 8 min as a finite rupture with massive two-phase jet release. The reasons for these different outcomes are discussed. The different failures provide a range of realistic outcomes for the subject tank and fire condition.     

On the thermal rupture of 1.9 m propane pressure vessels with defects in their thermal protection system

This paper describes the results from a series of fire tests that were carried out to measure the effect of defects in thermal protection systems on fire engulfed propane pressure vessels.

In North America thermal protection is used to protect dangerous goods rail tank-cars from accidental fire impingement. They are designed so that a tank-car will not rupture for 100 min in a defined engulfing fire, or 30 min in a defined torching fire. One common system includes a 13 mm blanket of high-temperature ceramic fibre thermal insulation covered with a 3 mm steel jacket. Recent inspections have shown that some tanks have significant defects in these thermal protection systems. This work was done to establish what levels of defect are acceptable from a safety standpoint.

The tests were conducted using 1890 l (500 US gallon) ASME code propane pressure vessels (commonly called tanks in the propane industry). The defects tested covered 8% and 15% of the tank surface. The tanks were 25% engulfed in a fire that simulated a hydrocarbon pool fire with an effective blackbody temperature of 870 °C.

The fire testing showed that even relatively small defects can result in tank rupture if the defect area is engulfed in a severe fire, and the defect area is not wetted by liquid from the inside. A wall failure prediction technique based on uniaxial high-temperature stress rupture test data has been developed and agrees well with the observed failure times.