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.     

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.     

Aseptically collected calf serum as an effective alternative to fetal calf serum in the culture of amniotic fluid cells

The growth-promoting activities of three different bovine sera have been compared in primary and secondary amniotic fluid cell cultures. In secondary amniotic fluid cell micro- cultures, aseptically collected calf serum (CS) was slightly, though not significantly more effective than fetal calf serum (FCS) in promoting DNA synthesis, while newborn calf serum (NCS) was significantly less effective than either CS or FCS. All three sera were optimally effective at concentrations of 10 per cent (v/v). Significant variation in quality occurred within four batches of each of CS and FCS, but not within four batches of NCS. A selected batch of CS was significantly more effective in promoting the growth of primary amniotic fluid cell cultures than were a number of batches of FCS then in routine laboratory use. It is suggested that CS may serve as an effective and economical alternative to FCS in the culture of amniotic fluid cells, thereby expanding the scope of serum batch testing. A possible explanation for the varying growth-promoting activities of different sera is discussed.     

In vitro characteristics of human fetal cells obtained from chorionic villus sampling and amniocentesis

In vitro characteristics of human fetal cells have been investigated after chorionic villus sampling at the first trimester and amniocentesis at the second trimester of pregnancy. Light microscopy revealed heterogeneous morphology of cell types in both the chorionic villus culture and the amniotic fluid cultures. Based on the experiments performed, chorionic villus cells are more sensitive to pronase, trypsin, and versene during subculture and have a higher DNA content per single cell and release more [¹²⁵I]-Beta-human chorionic gonadotropin into culture medium than those found in amniotic fluid cells. The practical applications of this study are discussed.     

Cultivated cells from mid-trimester amniotic fluids. IV. Cell type identification via one and two-dimensional electrophoresis of clonal whole cell homogenates

Clones of cultivated amniotic fluid cells that have distinct morphologic and growth characteristics (F, AF and E-type) were examined by one-dimensional SDS polyacrylamide gel electrophoresis (SDS-PAGE) and by two-dimensional electrophoresis employing isoelectric focusing and SDS-PAGE (IEF-PAGE). No qualitative differences in band pattern were observed in SDS-PAGE between the various amniotic fluid cell types, but consistent quantitative differences in the ratios of four bands of presumed filamentous proteins provided good distinction between amniotic fluid cells and postnatal skin fibroblast-like cells. By adding separation on the basis of electrical charge to that of molecular size (IEF-PAGE), we observed reproducible qualitative differences in the protein spot patterns between F and both AF and E-type amniotic fluid cells. At least eight discrete proteins appear not to be synthesized by prenatal F-type cells in comparison with their isogenic AF and E counterparts under identical culture conditions. The two-dimensional electrophoretic patterns thus confirm that F and AF amniotic cells, in spite of their morphologic and growth kinetic similarities, are developmentally distinct cell types that retain their differentiated states in culture.     

Cultured amniotic fluid cells for prenatal diagnosis of lysosomal storage disorders: A methodological study

The influence of culture conditions on the ultrastructure and enzyme activities of amniotic fluid cells are reported. Morphological changes were determined as a function of the number of lysosomal-like inclusion bodies per cell, and these results correlated to the activity of Thiexosaminidase, a-mannosidase, β-glucuronidase, arylsulphatase C and 5′ nucleotidase. The parameters examined were pH of the culture media, type of media, increasing cell passage and day of harvest. Our results indicate that enzyme activities are less sensitive to changes in culture conditions as compared to ultrastructural changes. We therefore recommend that in order to obtain reliable ultrastructural results for the diagnosis of storage disorders, cultures should be grown in MEM as the culture medium, the pH of the medium carefully monitored to remain below pH 7·4, examining the cultures no later than the eighth cell passage and no later than the 10th day after subculture.     

Cytogenetic studies of amniotic fluid taken before the 15th week of pregnancy for earlier prenatal diagnosis: A report of 114 consecutive cases

One hundred and fourteen samples of amniotic fluid taken before 15 weeks of gestation were cultured for cytogenetic studies. The results of culturing these early amniotic fluid (EAF) samples were compared with the results of culturing 114 standard amniotic fluid (SAP) samples taken after 15 weeks of gestation matched for maternal age and received in the laboratory within the same week. Cell culture was successful in all 114 of the EAF samples and in 111 SAP samples. There was no significant difference in the days to harvesting and days to reporting in the two groups. Three samples of SAP failed to grow and two EAF samples produced tetraploid karyotypes, so that in these five cases amniocentesis had to be repeated. These problems were attributed to toxicity of a fungicide used in the culture medium. Pseudomosaicism was noted in two EAF samples and one SAP sample; and maternal cell contamination was noted in one EAF and one SAP sample. Thus, culturing and karyotyping cells harvested from EAF and SAP are similar, indicating that EAF samples from 12–14-week pregnancies could be used for prenatal diagnosis.     

The effect of oxygen tension on colony formation and cell proliferation of amniotic fluid cellsin vitro

The effects of reduced oxygen concentrations in the gas phase over the culture medium on colony formation and cell proliferation were investigated in high and low cell density primary and secondary cultures of amniotic fluid cells. Using two standard culture methods (25 cm² plastic flasks and Leighton type tubes) a significantly reduced culture time was observed at high cell density for mass cultures by incubation within a low oxygen tension gas phase (2.5 per cent to 7.5 per cent O₂) instead of conventional air (18 per cent O₂). At low cell density colony formation was significantly enhanced in cultures grown at reduced oxygen tension. Using gas permeable membranes as support, lowering the oxygen tension from 7.5 per cent to 2.5 per cent yielded an increase in plating efficiency of cells from approximately 5 per cent to 25 per cent, whereas plating efficiency was less than 2 per cent for cells grown at ambient 18 per cent O₂. It is suggested that low oxygen tension in the gas phase is an effective means of enhancing clonal growth in amniotic fluid cell cultures, thereby reducing both culture time and risk of culture failure.     

Evaluation of amniotic fluid cell filtration: An experimental approach to early amniocentesis

Prior to a prospective application of amniotic fluid (AF) cell filtration to early amniocentesis, we tested the technique on a surplus from mid-trimester samples. By using the same sample size of 5 ml in experiments with a filter and in routine diagnostic procedures (control), we evaluated an optimal filter system. The prolonged culture time of filtered cells and the reduced number of clones are most probably due to mechanical stress (filtration pressure), whereas loss of the cells by adhesion to the filter system, and an AF-free culture medium (growth factors) are suggested to be less important. The AF cells are very sensitive to mechanical stress. Slow filtration (⩽3 ml AF/min) through filters with a high porosity and the largest possible pore size should be preferred. A mixed cellulose ester filter membrane with a pore size of 5·0 μm proved to be the most efficient, allowing harvest of the filtered cells after only a slight prolongation of the culture time (+2·4 days) compared with unfiltered aliquots. A filter set with a bypass connected by three-way taps allows cell filtration during either aspiration or reinjection of the AF. Cell filtration after amniocentesis and consecutive reverse flushing of the membrane with the appropriate amount of culture medium proved to be the best with regard to easy handling and reducing the risk of bacterial contamination.     

Maternal contamination of amniotic fluid demonstrated by DNA analysis

DNA from 16 sets of samples comprising DNA from uncultured amniotic fluid cells, cultured amniotic fluid cells, fetal tissue, and maternal blood was analysed by the polymerase chain reaction (PCR) with AC-repeat primers. The analysis was performed to investigate the presence of contaminating maternal cells in amniotic fluid which would affect the reliability of DNA studies for prenatal diagnosis. In three sets, maternal contamination of uncultured amniotic fluid cells was detected. In one of the three sets, maternal contamination was present in both uncultured and cultured amniotic fluid cells. The use of amniotic fluid cells as a source of DNA for prenatal diagnosis should be limited to cases where the purity of the DNA can be demonstrated prior to the diagnostic test being performed. This limitation in the use of amniotic fluid DNA also extends to other forms of diagnosis relying on the purity of amniotic fluid samples, particularly the new in situ hybridization methods currently being developed.     

Prenatal diagnosis of congenital adrenal hyperplasia due to 21-hydroxylase deficiency by amniotic fluid steroid analysis

The concentration of 17OH-progesterone was measured in second trimester amniotic fluid samples from 12 mothers who previously had had an infant with congenital adrenal hyper-plasia due to 21-hydroxylase deficiency. In 4 affected pregnancies, the concentrations were more than 2 S.D. higher than those determined in 44 samples from normal pregnancies (mean ± S.D., 8·1 ± 2·4 nmol/1). The remaining 8 pregnancies were predicted to be unaffected based on the results of amniotic fluid concentrations within the normal range. In each instance, the infant was normal. The results indicate that measurement of amniotic fluid 17OH-progester-one concentrations during the second trimester is an accurate prenatal test for 21-hydroxylase deficiency. The results should be supplemented with determination of fetal sex by karyotype analysis on the amniotic fluid cells.     

Correct prenatal diagnosis of a hurler fetus where amniotic fluid cell cultures were of maternal origin

Contamination of amniotic fluid cell cultures by maternal cells can be expected to lead to misdiagnosis of fetal genotype in 0·1 to 0·5/100 cultures, when assays are carried out directly on cultured cells. Chemical analysis of the cell-free amniotic fluid supernatant may overcome this source of error and has the added advantages of speed and independence from amniotic cell culture failure. We describe a pregnancy at risk for Hurler's disease where amniotic cells cultured at amniocentesis had a female karyotype and an α-iduronidase activity towards both phenyl and 4-methylumbelliferyl substrates at the lower end of the normal range, suggesting a heterozygous fetus. An affected fetus was predicted, however, because of a high concentration of dermatan sulphate in the amniotic fluid. The discrepancy between these findings was shown to be due to maternal cell contamination of amniotic fluid cell cultures by the birth of a male infant with Hurler's disease.     

Mosaicism or pseudomosaicism: The problem of hypermodal cells in amniotic fluid cell culture

A series of 2029 consecutive amniotic fluid specimens studied for prenatal genetic diagnosis were reviewed and reassessed so as to evaluate the frequency and clinical significance of hypermodal cells in amniotic fluid cell cultures. Hypermodal cells were defined as those with more than 46 chromosomes, and were characterized by an additional structurally normal or structurally abnormal chromosome. Of 2029 specimens, 47 (2.31 per cent) contained a total of 167 hypermodal cells. True fetal mosaicism was detected in three cases (0.14 per cent). All had hypermodal cells in more than one culture flask or colony which contained the same aberrant chromosome complement. In all but one case the babies were normal when only one cell was hypermodal, or when several cells were hypermodal but present in only one colony or one culture vessel. One case had an extra No. 20 chromosome in one cell. Although the child had multiple anomalies, they were not characteristic of trisomy 20, and subsequent chromosomal study on the baby postnatally revealed a 46,XX karyotype. The in situ coverslip technique is recommended as the preferred method for prenatal diagnosis, and it is useful as an aid in differentiating true mosaicism from pseudomosaicism.     

Variables influencing growth and morphology of colonies of cells from human amniotic fluid

Growth of cells from amniotic fluid was studied with respect to cell concentration in the inoculum, blood contamination of the fluid, fluid colour, fluid clarity, gestational age of the pregnancy, and growth factors. Dependent variables measured were colony formation, colony size, and colony morphology after 7, 11, and 14 days of culture. The following conclusions were established from these studies: small sample volumes are the most efficient for producing colonies; cells from very bloody or dark brown fluids have a slower rate of growth; growth of cells from cloudy (noncontaminated) fluids is better than growth of cells from clear fluids; the proportion of colonies that are epithelioid varies with gestational age; the stimulating effect of 100 ng/ml fibroblast growth factor on cells from amniotic fluid was confirmed.     

Microculture of fetal blood for prenatal cytogenetic studies from blood-stained amniotic fluid

An alternative method to the culture of amniotic fluid cells for prenatal diagnosis of chromosome disorders is proposed. Microculture of fetal blood can be used when fetal blood is drawn at amniocentesis through accidental puncture of the placenta. An easy discrimination of fetal red cells, a good response of fetal lymphocytes to PHA and the possibility of identification of the fetal karyotype from the maternal one are the technical bases of this method. This technique offers some undoubted advantages: a reduced need for repeating amniocentesis because of a lack of growth of AF cells due to massive contamination with red cells; a result may be obtained sooner. Thirty-seven cases out of 1092 amniocenteses were processed in this way (3·4 per cent). In two cases no mitoses were obtained but in the others the diagnosis was confirmed by the results of AF cell culture and/or by the outcome of pregnancy.     

46,XX/46,XY chromosome complement in amniotic fluid cell culture followed by the birth of a normal female child

Experience indicates that the most likely explanation for a mixture of 46,XX/46,XY cells in an amniotic fluid sample is that of maternal cell contamination and that a normal male child is to be expected at birth. We report the birth of a normal female child following prenatal diagnosis of such a mixture. Extensive postnatal studies failed to reveal an XY cell line. The possible sources of the XY cell line are discussed, as are the various techniques that were applied in an effort to discover it's origin. Cross-contamination of samples could be ruled out and there was no evidence of an unsuspected twin pregnancy. It is clear from this case that not all 46,XX/46,XY results obtained in amniotic fluid can be assumed to represent maternal cell contamination and some effort should be made to eliminate other potential sources for such a mixture.     

Preparation of high resolution chromosomes from amniotic fluid cells

A relatively simple method of obtaining high resolution chromosomes from amniotic fluid cells is described. The elongated chromosomes are achieved by adding ethidium bromide (5μg/ml) to the culture 4 1/2 hours before harvesting and the high resolution banding can be produced by usual banding procedures.     

Pseudomosaicism,true mosaicism,and maternal cell contamination in amniotic fluid processed with in situ culture and robotic harvesting

This study was designed to test the usefulness of the common definitions for maternal cell contamination, true mosaicism, and pseudomosaicism for amniotic fluid specimens processed by in situ culture and robotic harvesting. We prospectively studied 4309 consecutive amniotic fluid specimens processed with these methods and found that 0.84 per cent had maternal cell contamination, 0.28 per cent had true mosaicism, and 5.4 per cent had pseudomosaicism. Although the frequencies of maternal cell contamination and true mosaicism were comparable to those in similar published studies, the frequency of pseudomosaicism was more than twice as high as that in previous reports. This finding is most likely not due to the method, but rather to a more accurate estimate of the actual frequency of pseudomosaicism in amniotic fluid cultures than reported heretofore. Follow-up clinical information was available on 72 per cent of the cases. In three cases of true mosaicism involving structural anomalies, the results of cytogenetic follow-up studies on the neonates were normal. None of the pseudomosaic cases involving trisomy 8, 13, 18, or 21; triple X; or monosomy X were associated with newborns who had birth defects.     

First trimester amniocentesis between the seventh and 13th weeks: Evaluation of the earliest possible genetic diagnosis

Amniocentesis performed at the 12th week and later gives reliable results. The procedure can be performed using regimens developed for mid-trimester amniotic fluid (AF) cells. Extension to the 10th–11th week is, in principle, feasible. However, the high cytogenetic failure rate is a difficulty and despite a high clone count, the culture time is prolonged. The problem of the relatively high loss of AF could be overcome by cell filtration techniques and replacement of the fluid. Because of the short turnover rate of the AF, this may be unnecessary or replacement with an isotonic solution may be sufficient. (Pseudo)mosaicism appears to occur more frequently in early than in late amniocentesis. As yet, data are too sparse to allow a comparison with chorionic villus sampling. There are no reliable follow-up data from which to estimate the abortion rate and the number of embryonic malformations.     

Prenatal diagnosis of mucolipidosis type II on first-trimester amniotic fluid

We investigated the possibility of prenatal diagnosis of mucolipidosis type II (ML II) by lysosomal enzyme determination on amniotic fluid obtained at 11 weeks of gestation in three pregnancies at risk. Diagnosis of ML II was made in one case on the basis of increased levels of five lysosomal enzymes tested. The diagnosis was confirmed on cultured chorionic cells, their cultured medium, 17–week amniotic fluid, and fetal plasma obtained for confirmation prior to the termination of pregnancy.