Enhancement of amniocyte growth on a precoated surface

This study investigates whether cell-free amniotic fluid facilitates cell attachment to the surface of culture plates and thereby promotes rapid amniocyte growth. Isolated or pooled cell-free amniotic fluid samples at different volumes were added to culture plates. Trypsinized subcultures, grown in Eagle's minimum essential alpha medium supplemented with fetal bovine serum (4–20 per cent), were monitored by cell counts. The results demonstrated growth stimulation on culture plates precoated with amniotic fluid. The minimal time for coating the culture plates was 6h. Maximal coating was observed after an overnight incubation with 2–3 ml of the fluid per culture vessel. No synergistic effect from addition of fetal bovine serum to amniotic fluid was observed. A freshly coated surface provided the best amniocyte growth. When primary cultures are grown on a precoated surface, there is an increase in colony counts in 80 per cent of the samples tested. This method may be used to improve amniocyte growth, especially in samples with relatively small numbers of cells.     

HUMAN CHORIONIC GONADOTROPHIN AND ALPHA-FETOPROTEIN LEVELS IN MATCHED SAMPLES OF AMNIOTIC FLUID,EXTRAEMBRYONIC COELOMIC FLUID,AND MATERNAL SERUM IN THE FIRST TRIMESTER OF PREGNANCY

Separately identified samples of amniotic fluid and extraembryonic coelomic fluid obtained by high resolution transvaginal ultrasound-guided amniocentesis from 32 women between 7 and 12 weeks of pregnancy were analysed for human chorionic gonadotrophin (hCG) and alpha-fetoprotein (AFP). There was a highly significant difference between the hCG levels in amniotic fluid (median level 6.3 U/ml; range 1.6–310.0 U/ml) and those in extraembryonic coelomic fluid (median level 400.0U/ml; range 135.0–2250.0U/ml) (p<0.001; Mann-Whitney U/–test). The levels of AFP were very similar in amniotic fluid (median 26.0 kU/ml; range 10.0–116.5 kU/ml) and extraembryonic coelomic fluid (median level 24.1 kU/ml; range 12.4–94.4 kU/ml).     

Isoelectric focusing pattern of human amniotic fluid α-fetoprotein

Isoelectric focusing (IEF) of amniotic fluid α-fetoprotein (AFP) in thin-layer polyacrylamide gels containing 8 M urea followed by immunoblotting reveals at least nine bands, band I lying next to the cathode. Compared with 298 amniotic fluid samples from normal pregnancies, we found that the density of band V was increased in seven cases of fetal death. In 16 amniotic fluid samples from pregnancies with open neural tube defects (ONTO), band V disappeared or was markedly decreased. In seven cases with elevated AFP and positive acetylcholines-terase (AChE) due to contamination with fetal blood, no difference in pattern was observed compared with samples from normal pregnancies. It is suggested that IEF of AFP and subsequent immunoblotting are an apparently diagnostic test for ONTD and intrauterine fetal death (IUFD).     

Amniotic fluid microvillar enzyme activities in the early detection of fetal abnormalities

Measurement of the microvillar enzymes, γ-glutamyltranspeptidase (GGTP), aminopeptidase M (APM) and alkaline phosphatase (ALP), in amniotic fluid supernatant has been proposed as a method for the early prenatal diagnosis of cystic fibrosis. The activities of these enzymes in a series of other fetal abnormalities have now been examined. GGTP activities were below the 5th percentile in 28 out of 54 cases of trisomy 21 and 9 of 14 cases of trisomy 18, while APM values were below this cut-off in 26 of 54 cases of trisomy 21 and 8 of 14 cases of trisomy 18. Abnormal ALP isoenzyme ratios were found in 6 of 54 cases of trisomy 21 and 4 of 14 cases of trisomy 18. If prenatal cytogenetic studies are routinely carried out on amniotic fluid cells, the occasional confounding effect of abnormal microvillar enzymes associated with fetal trisomies rather than with cystic fibrosis should be avoided.     

Prenatal diagnosis of inherited metabolic disorders by quantitation of characteristic metabolites in amniotic fluid: Facts and future

An attempt is made to summarize as completely as possible what is known about the prenatal diagnosis of amino– and organic acidurias by direct measurement of characteristic metabolites in amniotic fluid, and to indicate which disorders can potentially be diagnosed prenatally by direct quantitation of metabolites. Furthermore, the disorders are mentioned in which the prenatal diagnosis was proven to be unsuccessful by this approach. The prenatal diagnoses of a case of propionic acidemia and a case of tyrosinemia type I in the 11th and 12th week of gestational age, respectively, are reported and the prospects of performing amniocentesis in the first trimester for prenatal diagnosis are discussed.     

Stimulation of human amniotic fluid cell proliferation and colony formation by cell plating on a naturally produced extracellular matrix

Human amniotic fluid cells exhibit a higher cloning efficiency and rate of cell proliferation when maintained on dishes coated with a naturally produced extracellular matrix (ECM) in comparison with the regular tissue culture plastic. In 22 out of 31 amniotic fluid samples there was by plating the cells on ECM a 2–6 fold increase in number and size of colonies and in the cell density per colony as detected by actual staining and viewing of each colony. These effects yielded, in 21 of 41 additional samples, a reduction ranging from 2–8 days, in the culture time elapsing between amniocentesis and the first harvesting of cells for chromosomal analysis. An even greater effect was obtained with primary cells that failed to attach to plastic surfaces and stayed floating in the medium but did attach and proliferate when seeded on ECM. Cells that were left firmly attached to ECM after the first trypsinization and harvesting of cells for chromosomal analysis yielded colonies ready for second karyotyping in less than half the time required for cells maintained on plastic. Studies with secondary cultures of human amniotic fluid cells have demonstrated a 5–10 fold decrease in serum requirement of cells cultured on ECM as compared with plastic. Addition of fibroblast growth factor (FGF) to the cultures further potentiated the effects of ECM. The ECM induced stimulation of cell attachment and proliferation was not associated with any chromosomal anomalies, nor did it interfere with the handling procedure. ECM coated dishes may be useful to reduce the time interval between amniocentesis and diagnosis, in particular when the amniotic cells exhibit an exceedingly slow rate of proliferation on plastic or when large quantities of cells are required for enzymatic studies.     

Prenatal detection of cystic fibrosis; comparative study of maltase and alkaline phosphatase activities in amniotic fluid

The potential value of microvillar enzymes in the prenatal diagnosis of cystic fibrosis (CF) has previously been demonstrated and is corroborated in the present comparative study. Maltase and alkaline phosphatase (ALP) activities were studied in the amniotic fluids of 57 pregnancies with a 1 in 4 risk for CF or with a known CF outcome and in 489 controls. A simple assay for maltase activity (MU-maltase) with the fluorogenic substate 4-methylumbelliferyl α-glucoside, offers great technical advantages and an at least equal detection rate of CF, when compared to the previously used test with maltose as substrate. Intestinal ALP was estimated either as phenylalanine inhibitable activity (PI-ALP) or as the proportions of residual activity in the presence of the inhibitors phenylalanine or homoarginine. MU-maltase and PI-ALP appeared the most successful methods: both tests were able to detect 14 of the 16 (88 per cent) pregnancies with fetal CF. Each of the two tests alone also allowed a correct prediction in 24 of the 25 pregnancies at risk but with normal outcome; however all 25 cases could be correctly predicted by a combined evaluation. It is suggested that more than one intestinal enzyme activity should be evaluated to allow optimal results in the prenatal monitoring of pregnancies at high risk for CF.     

Chemical analysis of succinylacetone and 4-hydroxyphenyllactate in amniotic fluid using selective ion monitoring

A method for the measurement of the concentration of succinylacetone and 4-hydroxyphenyllactic acid in amniotic fluid was developed for the prenatal diagnosis of hereditary tyrosinemia. Succinylacetone was converted to 5-methyl-3-isoxazolepropionic acid and isolated with 4-hydroxyphenyllactic acid by liquid partition chromatography and the trimethylsilyl derivatives quantified by ammonia chemical ionization selected ion monitoring gas chromatography-mass spectrometry with 2-hydroxy-n-caproic acid as the internal standard. The concentration of 4-hydroxyphenyllactic acid in normal amniotic fluid was 1.97±0.75 (S.D.) μmol/l while succinylacetone was undetectable. A pregnancy at risk for tyrosinemia type II was monitored. The concentration of 4- hydroxyphenyllactic acid was within the normal range and a healthy child was born.     

The association of congenital skin disorders with acetylcholinesterase in amniotic fluid

We describe a fetus with epidermolysis bullosa dystrophica and a fetus with aplasia cutis congenita who were normal by careful ultrasound examination but whose midtrimester amniotic fluids exhibited elevated concentrations of alpha-fetoprotein and presence of acetyl-cholinesterase. These cases show that serious fetal skin pathology can be a source of amniotic fluid acetylcholinesterase and elevated alpha-fetoprotein concentration and should be considered as part of the differential diagnosis of these amniotic fluid findings.     

Discordance between prenatal cytogenetic diagnosis and outcome of pregnancy

From 1.3.73 to 30.9.80 5580 women had an amniocentesis performed here or elsewhere; fetal chromosome analyses were carried out in this laboratory. We found 112 abnormal karyotypes (2 per cent) out of 5591 chromosome analyses. In 40 women (0.7 per cent) no cytogenetic diagnosis was obtained. Follow-up was successful in 99.5 per cent. Nine cases are reported in detail: Three cases had discrepancy between the karyotype in amniotic fluid and peripheral blood after delivery, two of these cases turned out to be 46,XX (male) while the third was prenatally determined as trisomy 21, but had a 46,XX karyotype at birth. Six cases had discrepancy between the karyotype in amniotic fluid and the phenotypic outcome at birth/abortion. One case was a prenatally undetected 45,X/46,XY mosaicism; one case was an unexplained 45,X male fetus; two cases were prenatally determined as trisomy 21, but at abortion a normal karyotype was determined and in two cases maternal cells were probably examined. The incidence of cytogeneric errors in this study was very low.