Rapid prenatal diagnosis of trisomy 18 and triploidy in interphase nuclei of uncultured amniocytes by non-radioactive in situ hybridization

Two biotinylated chromosome-specific DNA probes were used to quantify the number of chromosomes 18 and 1 in uncultured amniocytes. Thirty-three samples of uncultured amniocytes were hybridized with a chromosome 18-specific DNA probe. Uncultured cells from two of the 33 samples were also hybridized with a chromosome 1-specific probe. Thirty of the samples were disomic with respect to chromosome 18; two samples were trisomic with respect to chromosome 18, and one sample was trisomic with respect to chromosomes 1 and 18. The two cases of trisomy 18 and the single case of triploidy were identified on uncultured celis within 48-72 h after amniocentesis. They were found among five samples from pregnant women who had amniocentesis because of an ultrasonographically identified fetal malformation. A trisomic karyotype could be diagnosed with certainty in uncultured amniocytes because the majority of the responding nuclei exhibited three hybridization signals. In normal cells, the majority of nuclei exhibited two signals. In no cases was there discordance between the genotype as predicted by in situ hybridization and that determined by cytogenetic analysis.     

Analysis of chromosome 21 copy number in uncultured amniocytes by fluorescence in situ hybridization using a cosmid contig

A comparison of the use of chromosome 21-specific libraries, DOP-PCR 21 paints, yeast artificial chromosome (YAC) clones, single cosmids, and a 21q cosmid contig as probes for the detection of the copy number of chromosome 21 in interphase cells by fluorescence in situ hybridization shows that the cosmid contig is a satisfactory probe for interphase analysis of chromosome 21. The contig cCMP21.a, which is 55 kb in length, is highly chromosome 21-specific and produces intense, compact signals in a high proportion of interphase cells. A retrospective blind analysis of coded uncultured amniotic fluid samples correctly detected four trisomy 21 cases out of 49 samples.     

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.     

Rapid prenatal diagnosis of aneuploidy from uncultured amniotic fluid cells using five-colour fluorescence in situ hybridization

In this paper we describe the use of five-colour fluorescence in situ hybridization for prenatal diagnosis of aneuploidy using uncultured amniotic fluid cells. The analysis is based on ratio mixing of dual-labelled probes and digital imaging for the detection and visualization of five different probes specific for the five target chromosomes, 13, 18, 21, X, and Y. A retrospective blind analysis of 30 coded uncultured amniotic fluid samples correctly detected fetal sex and five trisomy 21 cases. Multicolour fluorescence in situ hybridization used in this way allows rapid and simultaneous detection of the most frequent aneuploidies.     

Evaluation of X,Y, 18, and 13/21 alpha satellite DNA probes for interphase cytogenetic analysis of uncultured amniocytes by fluorescence in situ hybridization

The major aneuploidies diagnosed prenatally involve the autosomes 13, 18, and 21, and sex chromosomes. Fluorescence in situ hybridization (FISH) allows rapid analysis of chromosome copy number in interphase cells. This prospective study evaluated the use of four commercially available centromeric DNA probes (DXZ1, DYZ1, D18Z1, and D13Z1/D21Z1) for direct analysis of uncultured amniocytes. One hundred and sixteen amniotic fluid samples were analysed by FISH and standard cytogenetics. This evaluation demonstrated that FISH with, X, Y, and 18 alpha satellite DNA probes could accurately and rapidly detect aneuploidies involving these chromosomes and could be used in any prenatal clinical laboratory. In contrast, the 13/21 alpha satellite DNA probe hybridizing both chromosomes 13 and 21 was unreliable for prenatal diagnosis in uncultured amniocytes.     

Trisomy 18 in chorionic villus sampling: Problems and consequences

Among 1547 patients undergoing first-trimester prenatal diagnosis, 100 fetal chromosome aberrations were detected. Thirteen of these involved chromosome 18. In two structural abnormalities of chromosome 18, the aberration could be excluded in amniotic fluid cells and two healthy infants were born. Trisomy 18 was not confirmed in amniotic fluid cells in three trisomy 18 mosaics. In eight non-mosaic trisomy 18 first-trimester diagnoses, the diagnosis was excluded by amniotic fluid cells or fetal cultures in four, and confirmed in the remaining four. Diagnosis of chromosome 18 aberrations in the direct preparation should be confirmed in the long-term culture of the chorionic villus sample or by amniotic fluid cultures.     

Prenatal detection of trisomy 21 in uncultured amniocytes by fluorescence in situ hybridization: A prospective study

A prospective study was undertaken to evaluate the use of fluorescence in situ hybridization (FISH) for the detection of trisomy 21 in interphase nuclei of uncultured amniotic fluid cells. Five hundred cases were analysed in situ and classified as normal or abnormal; the results were subsequently checked against the cytogenetic findings. Four hundred and ninety-three were correctly identified as normal with an 86·6 per cent average frequency of scored nuclei exhibiting two signals; six cases were correctly identified as trisomic for chromosome 21 with 81·7 per cent of scored nuclei exhibiting three signals; and one abnormal case involving an unbalanced chromosome 21·21 translocation was falsely scored as normal due to poor hybridization/detection efficiency. The method has been substantially improved and simplified so that it is suitable for the rapid detection of trisomy 21. As aneuploidy detection in interphase does not identify structural chromosome aberrations, it is not a substitute for fetal chromosome analysis.     

Amniotic fluid alpha-fetoprotein levels and prenatal diagnosis of autosomal trisomies

Pregnancies with fetal trisomy 21 have been associated with low amniotic fluid alpha-fetoprotein levels (AFAFP). This observation led to the suggestion that low AFAFP levels be used as a criterion for completion of a chromosomal analysis in patients who are not otherwise at increased risk for a fetal chromosome abnormality and in whom karyotyping might not have been completed for economic reasons. In order to assess the usefulness of such criteria, we reviewed the AFAFP levels of 90 cases of fetal trisomy 21, 23 cases of trisomy 18, and 10 cases of trisomy 13. These were compared with 2400 control samples with normal chromosome constitution. AFAFP levels were generally lower in pregnancies with trisomy 21, showing a median value of 0·72 MoM. However, 40 per cent of the trisomy 21 samples had AFAFP values greater than 0·8 MoM and 20 per cent were over 1·0 MoM. These data imply that over 50 per cent of Down syndrome cases might have been missed using a cut-off level of 0·70 MoM for completion of chromosome analysis. Using a higher cut-off level will leave only a small percentage of samples unkaryotyped. The distribution of AFP levels in trisomy 13 and 18 is no different from controls; we therefore believe that fetal karyotyping should be completed in every amniotic fluid sample obtained.     

Rapid detection of numerical aberrations of chromosomes 13, 18 and 21 in chorionic mesenchymal cells

We have devised and evaluated a rapid screening method for the detection of numerical aberrations of chromosomes13, 18 and 21 in chorionic villus cells. We used non-radioactive in situ hybridization (ISH) with three chromosome-specific probes on overnight-attached mesenchymal cells from chorionic villi. A blind study was performed of 47 karyotypically normal samples, one triploid sample, two samples trisomic for chromosome 21, and two samples from a fetus with putative mosaicism (46/47, +21). All samples were hybridized with the chromosome 18- and 21-specific probes. Thirty samples were additionally hybridized with the chromosome 13-specific probe. The test could be completed within 3-4 days of sampling. In samples disomic with respect to the probed chromosomes, an average of 2 per cent (range 0-9 per cent) had three hybridization signals. By contrast, in the samples trisomic for the probed chromosome(s), 57 per cent (chromosome 13), 51 per cent (chromosome 18), and an average of 74 per cent (55-86 per cent) (chromosome 21) of the nuclei exhibited three signals. In the putative mosaic samples, the number of nuclei with three chromosome 21-specific signals ranged from 41 to 69 per cent. We conclude that this technique rapidly and clearly distinguishes between normal and trisomic/triploid samples, and consequently may be of use in future prenatal diagnosis.     

Familial X centromere variant resulting in false-positive prenatal diagnosis of monosomy X by interphase FISH

Interphase fluorescent in situ hybridization (FISH) analysis performed on uncultured amniotic fluid cells from a female fetus revealed a single signal using an X chromosome alpha-satellite probe, and the absence of any signal using a Y chromosome alpha-satellite probe. This result was initially interpreted as monosomy for the X chromosome in the fetus. Subsequent chromosome analysis from the cultured amniotic fluid cells showed two apparently normal X chromosomes. FISH using the X alpha-satellite probe on metaphase spreads revealed hybridization to both X chromosomes, although one signal was markedly reduced compared to the other. The same hybridization pattern was observed in the mother of the fetus. This is the first report of a rare familial X centromere variant resulting in a false-positive diagnosis of monosomy X by interphase FISH analysis for prenatal diagnosis. Copyright © 2001 John Wiley & Sons, Ltd.     

False-positive diagnosis of trisomy 21 using fluorescence in situ hybridisation (FISH) on uncultured amniotic fluid cells

An amniocentesis was performed on a 22-week pregnancy following the detection of foetal abnormalities on ultrasound. A rapid aneuploid screen using fluorescence in situ hybridisation on uncultured amniotic fluid cells revealed 3 signals for chromosome 21, consistent with trisomy 21. In situ metaphase cultures revealed a 46,XY normal male karyotype. These discordant results may be explained by a sub-standard batch of the commercially available probe or alternatively, a very specific variation within the sample interacting with the probe. Alternative strategies are proposed in order to safeguard against inappropriate clinical action as a consequence of discordant results. Copyright © 2003 John Wiley & Sons, Ltd.     

A chromosome 21-specific cosmid cocktail for the detection of chromosome 21 aberrations in interphase nuclei

Fluorescent in situ hybridization (FISH) with a 21q11-specific probe (CB21c1) consisting of three non-overlapping cosmids has been applied to interphase amniocytes of pregnancies at increased risk for fetal aneuploidy (N = 78) and to interphase lymphocytes, cultured and uncultured, of patients referred for Down syndrome (N = 19 and 28, respectively). In the uncultured amniocytes, six chromosome aberrations were detected: three cases of trisomy 21, a triploidy, a de novo 46,XX,t(21q21q), and a mosaic 46,XY/47,XY,+dic(21)(q11)/48,XY,+dic(21)(q11), +del(21)(q11). In 15 cultured and 20 uncultured blood samples, FISH correctly diagnosed trisomy 21 (full or mosaic) at the interphase level, which was confirmed in all cases by subsequent karyotyping. Because of specific and strong signals in interphase nuclei, CB21c1 appears to be a useful tool for the rapid detection of chromosome 21 abnormalities.     

Rapid prenatal diagnosis of 14 cases of triploidy using fish with multiple probes

Fluorescence in situ hybridization (FISH) of chromosome-specific probes to interphase nuclei can rapidly identify aneuploidies in uncultured amniotic fluid cells. Using DNA probe sets specific for chromosomes 13, 18, 21, X, and Y, we have identified 14 fetuses where the hybridization pattern was consistent with a triploid chromosome constitution. In each case, the identification of fetal abnormalities by ultrasound examination initiated a request for rapid determination of ploidy status via prenatal FISH analysis of uncultured amniocytes. FISH produced a three-signal pattern for the three autosomes in combination with signals indicating an XXX or XXY sex chromosome complement. This hybridization pattern was interpreted to be consistent with triploidy. Results were reported to the physician within 2 days of amniocentesis and subsequently confirmed by cytogenetics. These cases demonstrate the utility of FISH for rapid prenatal identification of triploidy, particularly when fetal abnormalities are seen with ultrasonographic examination.     

Parental mosaic trisomy 21 detected following maternal cell contamination of an amniotic fluid specimen from a normal male pregnancy

We report a case of maternal mosaic trisomy 21 ascertained at prenatal diagnosis as a result of maternal cell contamination of an amniotic fluid sample. A 34 year old female was referred for karyotyping because of a previous trisomy 21 pregnancy. Chromosome analysis of primary in situ cultures showed a karyotype of 47,XX, + 21[6]/46,XY[32]/46,XX[2]. Molecular testing demonstrated maternal cell contamination of the amniotic fluid sample and G-banded karyotyping of maternal blood showed that 3/200 cells had trisomy 21, consistent with the mother being a Down syndrome mosaic. A normal male baby with a 46,XY chromosome complement was delivered at 30 weeks. This case emphasises the need for close collaboration between cytogenetic and molecular genetics laboratories in resolving unusual cases of mosaicism. Copyright © 2007 John Wiley & Sons, Ltd.     

Characterization of i(18p) in prenatal diagnosis by fluorescence in situ hybridization

A case is presented in which chorionic villus direct preparation and cultured chorionic villus cells revealed a 47,XX, + mar karyotype. The marker was a small metacentric chromosome and appeared to be i(18p)—isochromosome 18p. Follow-up studies in both amniotic fluid and fetal fibroblasts confirmed the karyotype. In order to characterize the marker, a panel of biotinylated DNA probes was used, including a whole chromosome 18 probe, chromosome 18-specific alpha satellite DNA, Yac clones, and a pan-telomeric probe. These studies show that the marker is a monocentric i(18p) in which about 80 per cent of chromosome 18 alpha satellite DNA has been lost.     

Prenatal detection of structural abnormalities of chromosome 18: associations with interphase fluorescence in situ hybridization (FISH) and maternal serum screening

We describe two cases of prenatally ascertained isochromosome 18. Case 1 included both an isochromosome 18p and an isochromosome 18q, while Case 2 involved only an isochromosome 18q. Both of these cases were associated with a positive maternal serum triple screen trisomy 18 risk (greater than 1 in 100 risk). In addition, fluorescence in situ hybridization (FISH) was performed on uncultured amniotic fluid interphase cells in both cases looking for aneuploidy for chromosomes 13, 18, 21, X and Y. The results of the interphase analyses support the common knowledge that careful interpretation of interphase FISH analysis is necessary and that results should be followed by full cytogenetic analysis. To our knowledge these are the first reported cases of structurally abnormal chromosomes 18 being associated with a positive maternal serum triple screen for trisomy 18. Copyright © 2002 John Wiley & Sons, Ltd.     

Retrospective study of trisomy 18 in chorionic villi with fluorescent in situ hybridization on archival direct preparations

Trisomy 18 in direct chorionic villus preparations needs further investigation since the chromosome abnormality may be confined to the placenta and may not represent the actual fetal karyotype. We performed, retrospectively, fluorescent in situ hybridization (FISH) with the chromosome 18 centromere probe (L1.84) on interphase nuclei of destained slides of all cases of full trisomy 18 (n=22) and mosaic trisomy 18 (n=8) detected among 7600 first-trimester chorionic villus samples during an 8-year period (1985–1992). More nuclei displaying three signals were encountered in cases of full and mosaic trisomy 18 confirmed in fetal tissue than in non-confirmed cases. FISH can be useful for the verification of trisomy 18 in direct chorionic villus preparations.     

Alkaline phosphatase activity in amniotic fluid in pregnancies with fetal disorders

Alkaline phosphatase (ALP) activity was determined in 255 amniotic fluid samples collected by amniocentesis between 15 and 39 weeks of gestation. The samples were originally used for chromosomal analysis and/or alpha-fetoprotein measurements. The mean ALP activity in early amniotic fluid from pregnancies with fetal trisomy 18 and 21 syndromes was half of that found in the controls. Highly elevated ALP activity (over 10 times the median level) was found in 14 samples. Two of these pregnancies had normal outcome. Three samples were from pregnancies with intrauterine fetal death. Fetal disorders, including abdominal wall defect (four cases), Meckel's syndrome (two), hydrops fetalis syndrome (two) and genital anomaly (one), were observed in nine cases. Moderately elevated ALP activity (over three times the median) was found in 10 cases, including five pregnancies with a preterm labour shortly after the sample collection. The results indicate that elevated ALP activity in the third trimester amniotic fluid is often associated with fetal disorders.     

Prenatal aneuploidy detection in interphase cells by fluorescence in situ hybridization (Fish)

FISH is a quick, inexpensive, accurate, sensitive and relatively specific method for aneuploidy detection in samples of uncultured chorionic villus cells and amniotic fluid cells. FISH allows detection of the autosomal trisomies 13, 18 and 21 and X and Y abnormalities and any other chromosome abnormality for which a specific probe is available. The detection rate of these abnormalities is high in informative samples which have a concordance of > 99.5% with cytogenetic results. A relatively high number of abnormal cases are found in uninformative samples. However, such samples should be regarded as samples to be investigated further. Clinical experience with the use of FISH for prenatal diagnosis is now beyond 10,000 cases; a number of clinical protocols and smaller trials have also been carried out, resulting in 90% of attempted analyses giving informative results with a high detection rate and extraordinarily low false-positive and false-negative rates Unsolved problems remain, such as occasional technical failures, admixtures of maternal blood and up to 20% uninformative scoring results, especially for abnormal specimens. FISH is at present used as an adjunct to classical cytogenetic analysis. However, this should not be interpreted as meaning that FISH could not be used as a methodology in its own right. If FISH were to be considered a Diagnostic test then this might be the case, due to the risk of false-negative and false-positive results and the fact that FISH does not allow a diagnosis of certain structural abnormalities. If, on the other hand, FISH is considered a screening test, which means that in all abnormal (or indeterminate) cases, classical cytogenetic analysis would follow the abnormal screening test, the accuracy which is potentially higher than for other screening methods, for example in cases of trisomy 21, justifies FISH as a prenatal screening test in its own right.     

Fluorescence in situ hybridization (FISH) for rapid detection of aneuploidy: experience in 911 prenatal cases†

Fluorescence in situ hybridization (FISH) was performed with probes specific for chromosomes 13, 18, 21, X and Y on 911 of 11123 (8.2%) amniotic fluid samples submitted to the present authors' laboratory for cytogenetic analysis over an 8-year period. Altogether 3516 hybridizations were performed with an interpretable FISH result on all chromosomes requested in 884/911 (97%) of cases. An uninformative FISH result occurred in 44 hybridizations among 27 cases (3%). Of a total of 89 karyotypically proven cases with aneuploidy that might have been detected by FISH, the overall detection rate was 84%. An inconclusive or incomplete FISH result occurred in 9/89 (10%) of these proven aneuploid cases. In the remaining 80 informative proven aneuploid cases, correct detection of aneuploidy was accomplished in 75/80 (94%) of samples. A false-negative result occurred in the remaining 5/80 (6%) of such informative cases. Eighteen cases had karyotypically proven abnormalities that could not have been detected by the targeted FISH. Aside from these 18 cases, FISH allowed correct detection of normal disomy in 785/804 (98%) of such cases. An incomplete FISH result occurred in 18 normal disomic cases. There was a single possible ‘false-positive’ FISH result for chromosome 21. Interphase FISH analysis of uncultured amniotic fluid cells has been shown to be a useful laboratory tool for rapid fetal aneuploidy screening during pregnancy. As with all clinical laboratory diagnostic tests, incomplete or inconclusive results (or even interpretive errors) occur in a small percentage of cases. Nevertheless, FISH results accompanied by other data and by appropriate counseling provide clinicians and patients with valuable information for clinical decision-making surrounding family planning and pregnancy management. Copyright © 2001 John Wiley & Sons, Ltd.