Microsatellite analysis provides efficient confirmation of fetal trophoblast isolation from maternal circulation

Fetal trophoblasts can be found in maternal circulation from an early stage of pregnancy and thus provide a potential source of DNA for non-invasive prenatal diagnosis. We have developed a two-step method for trophoblast isolation between the 8th and 12th week of pregnancy. Blood was sampled from 14 women undergoing termination of pregnancy or spontaneous abortion. Immunomagnetic beads precoated with HLA class I and II, and with anti-cytokeratin-18 monoclonal antibodies, were used to remove CD8+ and other maternal cells, and to select for fetal trophoblasts, respectively. Microsatellite analysis was performed on DNA extracted from the isolated, maternal, paternal and placental cells after PCR amplification. Recovery of the trophoblasts was confirmed in 13/14 cases (93%) by the identification of an identical microsatellite pattern for fetal and placental cells. Further evidence was the presence of heterozygous alleles of both maternal and paternal origin. The correct prediction of gender in all five male fetuses was an additional confirmation of trophoblast recovery. We conclude that trophoblasts can be effectively isolated from maternal blood in the first trimester, and by using polymorphic microsatellite markers to confirm sample purity, this method has potential future application in prenatal diagnosis. Copyright © 2001 John Wiley & Sons, Ltd.     

Erythroid-specific antibodies enhance detection of fetal nucleated erythrocytes in maternal blood

Fetal nucleated erythrocytes (NRBC) in maternal blood are a non-invasive source of fetal DNA for prenatal genetic screening. We compared the effectiveness of three monoclonal antibodies for the separation of fetal cells from maternal blood by flow sorting. Mononuclear blood cells from 49 healthy pregnant women were incubated with antibody to CD 71, CD 36, and/or glycophorin A (GPA), employed singly or in combination with each other. These monoclonal antibodies recognize surface antigens on haematopoietic precursor cells. Successful isolation of fetal cells was defined as detection of Y chromosomal sequences in maternal blood from women carrying male fetuses, with absence of Y sequences when female fetuses were carried. Thus, gender prediction accuracy was used as a measure of fetal cell separation. Using anti-CD 71 to isolate fetal cells, gender prediction was 57 per cent correct; with anti-CD 36, it was 88 per cent correct. Anti-GPA, an erythrocyte-specific antigen, used alone or in combination with anti-CD 71 or 36, improved gender prediction to 100 per cent. We conclude that antibody to GPA improves the retrieval of fetal NRBC from maternal blood, permitting genetic analysis by the polymerase chain reaction.     

Enrichment of fetal cells from maternal blood by high gradient magnetic cell sorting (double MACS) for PCR-based genetic analysis

For simple and effective isolation of fetal cells from peripheral maternal blood, we combined depletion of maternal cells and enrichment of fetal cells by high-gradient magnetic cell separation (MACS). First CD45⁺ and CD14⁺ cells were depleted from maternal peripheral blood mononuclear cells by MACS. From the depleted fraction, CD71⁺ erythroid cells were enriched up to 80 per cent by MACS. This ‘double-MACS’ procedure yielded an average depletion rate of 780-fold and an average enrichment rate of 500-fold, with approximate recovery rates of 40–55 per cent. For paternity testing, cells from unseparated blood and the various fractions were analysed for polymorphism of the HLA-DQ-A1 locus and D1S80 locus by the polymerase chain reaction (PCR). In CD45⁻/CD71⁺ sorted cells from maternal blood, but not in unfractionated cells from maternal blood or CD45⁻/CD14⁻ cells, paternal alleles could be detected. In the CD45⁻/CD71⁺ fraction, the relative frequency of paternal alleles compared with maternal alleles ranged from 1 in 20 to 1 in 200 (determined by titration and depending on the quality of separation and biological variation). In 7 out of 11 cases, between weeks 12 and 25 of gestation, we could identify paternal alleles by PCR, either HLA-DQ-A1 or D1S80. This double-MACS procedure is simple, fast, efficient, and reliable for non-invasive prenatal diagnosis.     

Trophoblast-like cells sorted from peripheral maternal blood using flow cytometry: A multiparametric study involving transmission electron microscopy and fetal dna amplification

Three monoclonal antibodies (MAbs) against trophoblast (GB17, GB21, and GB25) and flow cytometry were used to sort trophoblast-like cells (TLCs) from peripheral blood of pregnant women. Sorted TLCs were processed for electron microscopy and fetal DNA amplification of the Y-specific sequences from mothers carrying male fetuses. At the ultra-structural level, most of the nucleated cells had the morphology of leucocytes, suggesting maternal contaminants, and we did not find the characteristic features of the free inter-villous trophoblast cells. Nevertheless, polymerase chain reaction (PCR) analysis showed an amplification of Y-specific sequences in two out of three samples of sorted TLCs. These results suggest that besides the maternal leucocytes, sufficient trophoblast nucleated fetal cells can be obtained using cell enrichment by sorting. This sensitive method holds promise for non-invasive prenatal diagnosis of fetal sex and if sufficient Y(positive) nuclei are found, for the diagnosis of selected numerical chromosome abnormalities.     

Screening of monoclonal antibodies recognizing oncofetal antigens for isolation of trophoblasts from maternal blood for prenatal diagnosis

Many monoclonal antibodies have been produced against tumour-associated cell surface antigens for cancer therapy. They have therefore been selected for minimal reactivity with normal tissues and in particular for lack of binding to blood cells or serum components. Many of the antigens recognized are of fetal origin. These monoclonal antibodies may therefore be ideal candidates to recognize and sort fetal trophoblasts from maternal blood for prenatal diagnosis of genetic abnormalities. A panel of 19 anti-tumour antibodies were therefore screened for reactivity with early trimester placenta and two, 340 and 154, were shown to stain trophoblasts. If MAb 340 is linked to magnetic beads, it can efficiently sort trophoblast cell lines from whole blood.     

Prenatal diagnosis in advanced maternal age. Amniocentesis or CVS,a patient's choice or lack of information?

Ninety-six women of advanced maternal age were interviewed about the way they obtained information on prenatal diagnosis and about how the decision was made as to what procedure was to be performed (transabdominal chorionic villus sampling (TA-CVS) or amnio-centesis). In the CVS group, women visited their physician or midwife earlier in pregnancy (mean 7.1 weeks) than those in the amniocentesis group (mean 10.7 weeks). The availability of prenatal diagnosis was not mentioned during the first antenatal visit in 55 per cent of women from the amniocentesis group as opposed to 25 per cent from the TA-CVS group. Approximately 40 per cent of women eligible for prenatal diagnosis did not receive any information from the referring body prior to counselling at our centre. Only 29 per cent of women who underwent amniocentesis had actually chosen this procedure; 71 per cent were too late to undergo TA-CVS at 12 weeks. It is concluded that information to the patient must be improved in order to ensure early referral for prenatal diagnosis.     

Fetal erythroblasts from maternal blood identified with 2,3-bisphosphoglycerate (BPG) and in situ hybridization (ISH) using Y-specific probes

Different types of fetal nucleated cells can be found in maternal blood, providing the possibility of non-invasive prenatal diagnosis. For this purpose, we have studied fetal erythroblasts. We discovered that haemoglobin-containing cells treated with 2,3-bisphosphoglycerate (BPG) can be visualized by a peroxidase reaction, which at the same time visualizes an in situ hybridization (ISH) signal, specific for the X, Y or 21 chromosome. In order to prove that the BPG-positive cells were erythroid, an anti-glycophorin A (GPA) antiserum combined with a staphylococcal rosette technique was used. To enrich for erythroblasts, leukocytes were depleted from maternal blood by treatment with anti-CD45 monoclonal antibody and passage over an anti-mouse IgG-coated glass bead column. To evaluate the potential of the method for clinical use, we studied maternal blood samples from 18 women referred to us for prenatal diagnosis between 6 and 19 weeks of gestation. Erythroblasts were found in 13 out of 14 normal pregnancies. Erythroblasts with a Y-signal were found as early as 9 weeks of gestation, but at 6 weeks the Y-signal was seen in BPG-negative cells only. These cells showed an epithelioid morphology indicating that they were cytotrophoblasts. The BPG-ISH method provides a simple technique for identifying erythroblasts and simultaneously visualizing a desired probe.     

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.     

Fetal nucleated red blood cells from CVS washings: an aid to development of first trimester non-invasive prenatal diagnosis

Fetal nucleated red blood cells (n-rbc) occur in the maternal circulation from 7 weeks of pregnancy. The enrichment of these cells from maternal blood will depend upon their stage of differentiation, which changes during development. We characterised the fetal n-rbc from chorionic villus sample (CVS) washings and used them to model first trimester non-invasive prenatal diagnosis. The ratio of ε- to γ-globin-producing cells declined rapidly from 10 to 13 weeks, as did the ratio of nucleated to non-nucleated rbc. By 13 weeks the great majority of cells containing γ- or ε-globin are anucleate. The fetal n-rbc were highly variable in size and density and sedimented over a wide density range with a high proportion (>80%) at a density overlapping with that of maternal rbc. We have devised an enrichment procedure using Orskoff lysis to differentially lyse the maternal cells followed by density centrifugation and separation using magnetic beads. This simple protocol allowed recovery of 70% (69±22%) of fetal cells when added at approximately 10 fetal cells/ml maternal blood. When 1 fetal cell/ml millilitre maternal blood was added (total volume 10 ml) the recovery was more variable but remained at approximately 70% (72±47%), with at least one fetal cell recovered in all cases. Copyright © 2001 John Wiley & Sons, Ltd.     

Prenatal diagnosis by chorionic villus sampling: Lessons of the first 600 cases

Chorionic villus sampling (CVS) has emerged as a first trimester alternative to amniocentesis for the prenatal detection of genetic disorders. We report our experience in 600 consecutive CVS procedures to better delineate the safety, efficacy and reliability of this new method of prenatal diagnosis. Adequate samples were obtained at the initial visit in 97 per cent of the cases, and successful cultures were established in 98.7 per cent of these patients. Chromosome abnormalities were detected in 5.9 per cent of those pregnancies tested because of advanced maternal age (≥ 35 years). A discrepancy between the villus karyotype and that of the fetus was found in 2.0 per cent of cases, and most commonly consisted of mosaicism in the villus sample for a chromosomal abnormality that was not found in fetal samples. The risk of spontaneous abortion following the procedure was 6.3 per cent. We conclude that chorionic villus sampling is an acceptably safe and reliable procedure, but further investigation is needed before it can become an established technique in prenatal diagnosis.     

Reproductive behaviour and prenatal diagnosis following genetic termination of pregnancy in women of advanced maternal age

One hundred and fifty-one women of advanced maternal age who underwent genetic termination of pregnancy (TOP) were studied for their reproductive behaviour and the type of procedure for prenatal diagnosis in a subsequent pregnancy. A total of 59 women (39 per cent) had a further pregnancy. In all continuing pregnancies prenatal diagnosis was performed, of which 75 per cent consisted of chorionic villus sampling (CVS). Reproductive behaviour following a genetic termination was negatively correlated with maternal age and parity. Both reproductive behaviour and the choice to undergo a diagnostic procedure in the next pregnancy were independent of the type of diagnostic procedure in the previous affected pregnancy.     

New technique using galactose-specific lectin for isolation of fetal cells from maternal blood

To isolate fetal cells from maternal blood, we developed a new method based on galactose-bearing conjugation. Nucleated red blood cells (NRBCs), which highly express galactose on their surface, were selectively attached to a substrate coated with a galactose-containing polymer via soybean agglutinin (SBA), a galactose-specific lectin. Cord blood samples were used to evaluate enrichment efficacy of NRBCs by this method. Blood samples were obtained from 131 pregnant women between 6 and 27 gestational weeks. After preliminary condensation of fetal cells by Ficoll gradient centrifugation, NRBCs were enriched using galactose-positive selection by adjusting SBA concentration. We isolated one to severalhundred NRBCs (mean±SD, 7.8±8.5) in 2.3 ml of peripheral blood samples from 96% of pregnantwomen. The isolated NRBCs were analyzed by a Y-chromosome FISH probe in eight cases carrying male fetuses. Y-signals were detected in all eight cases and more than half of the NRBCs wereoffetal origin. The study demonstrates that our new method using galactose-specific lectin provides effective enrichment of fetal NRBCs allowing non-invasive prenatal diagnosis. Copyright © 2002 John Wiley & Sons, Ltd.     

Ethical problems in prenatal diagnosis: A cross-cultural survey of medical geneticists in 18 nations

In order to provide a basis for international discussion of ethics among geneticists, we surveyed the responses of medical geneticists to a questionnaire describing 14 hypothetical cases that posed ethical dilemmas. The cases were selected through discussions with leading geneticists in 12 nations, as representative of the most difficult problems of moral choice experienced in practice. Six cases involved indications for prenatal diagnosis or disclosure of information. All medical geneticists in each of 18 nations were included. Of 1053 asked to participate, 677 (64 per cent) responded. A large majority (83 per cent) would perform prenatal diagnosis for parents who oppose abortion but request the service. A smaller majority (63 per cent) would do so for maternal anxiety, and a minority (25 per cent) would perform for sex selection in the absence of X-linked disease. Most would disclose conflicting controversial, or ambiguous test results, and two-thirds would disclose colleagues' differences of opinion.     

Maternal cell contamination in cultured chorionic villi: Comparison of chromosome Q-polymorphisms derived from villi,fetal skin,and maternal lymphocytes

Maternal cell contamination of chorionic villi (CV) samples used for first trimester prenatal diagnosis can cause obvious and/or unrecognized diagnostic dilemmas. The purpose of this investigation is to assess the frequency of maternal cell contamination (MCC) in chorionic villus samples and to evaluate selected parameters which might predict where contamination is more likely to have occurred. Maternal lymphocytes, chorionic villi from ultrasonically directed transcervical catheter aspiration, and fetal tissue were obtained at 8–11 weeks gestation from 45 patients undergoing elective termination. Quinacrine (Q) banded metaphases were compared from duplicate direct preparations of chorionic villi; cultured chorionic villi, fetal fibroblast tissue cultures, and maternal lymphocyte cultures. Q-polymorphisms in metaphase chromosomes were 100 per cent concordant between fetal tissue and direct CV preparation. However, evidence for maternal cell contamination occurred in 13.1 per cent of cultured chorionic villi preparations where polymorphisms were found to be identical between maternal and cultured CV and both distinct from fetal tissue preparations. Where MCC was identified, it was noted that CV cell cultivation interval was prolonged (24.2±6.8 days) compared with non-contaminated cultures (14.1±4.4 days) (p <0.05). We conclude that maternal cell contamination is a significant problem with chorionic villus sampling. Where direct preparations are not employed or when cultures are ‘slow growing’, MCC may be a significant and unrecognized complication re: fetal diagnosis. Direct preparations, multiple cultures, quinacrine banding, and maternal Q-polymorphism comparisons can minimize diagnostic dilemmas secondary to maternal cell contamination. Q-polymorphism comparisons between maternal and fetal chromosomes should be included in all instances where cultured chorionic villi are utilized for fetal diagnosis and where direct preparations are not available.     

Prenatal diagnosis of thalassemia major by fetal blood analysis: Experience with 1000 cases

In this report we have summarized our experience with the prenatal diagnosis of β-thalassemia in 1000 pregnancies followed at least until 12 months after birth. In the majority of these cases, the thalassemia lesion was the nonsense mutation at the codon corresponding to amino acid 39, which produces the hematological phenotype of β-thalassemia. Fetal blood sampling was carried out by placental aspiration, by which a sufficient amount of fetal blood for analysis was obtained in the majority of cases (99 per cent). The fetal mortality associated with fetal blood sampling was 6·3 per cent. Those placental samples contaminated by maternal cells were successfully purified by Ørskov lysis. Fetal blood was analysed by globin chain synthesis on CM–52 columns, which gave reliable results. Two misdiagnoses (0·2 per cent) have been made of which one was due to a non-globin protein co-migrating with the β-chains while the other resulted from a misclassification of the type of thalassemia segregating in the family.     

Prenatal cytogenetic diagnosis after transabdominal chorionic villus sampling in the first trimester

First trimester prenatal cytogenetic diagnosis was attempted in 350 pregnancies after trans-abdominal chorionic villus sampling. The cytogenetic investigation was performed using both a short-term method (24 h incubation) and cell culture. Adequate samples were obtained in 99·1 per cent and in all these cases the fetal karyotype was established. A chromosome abnormality was found in 2·0 per cent of cases. A discrepancy between the karyotype obtained after 24 h incubation and the karyotype in cell culture was observed in 2·3 per cent. Maternal cell contamination in the cultures was confirmed in 13 of 181 cases where the 24 h incubation revealed a male karyotype. Studies of culture morphology showed that colonies of convoluted cells may serve as a marker for contamination with maternal cells in culture. For the present, we recommend using a short-term method as well as cell culture for cytogenetic investigation until the problems with karyotype discrepancy and maternal cell contamination have been further clarified.     

Prenatal diagnosis for the detection of down syndrome: Why are so few eligible women tested?

Postpartum women ≧ 33 years were interviewed about their attitudes to and knowledge and use of prenatal diagnosis. Overall, 68 per cent had heard of prenatal diagnosis; nevertheless, only 30 per cent of those ≧ 35 had actually been tested. The only significant difference between eligible women who were tested and those who were not was maternal age. Of those tested, half requested it for themselves; conversely, only two-thirds of women requesting the procedure actually received it. Among women not tested, 82 per cent were never offered the procedure by the physician. Expressed attitudes to prenatal diagnosis were strongly positive among all women, with 75 per cent continuing to want testing after learning both their age-specific risk of having an affected child and the possible risks of amniocentesis. The data document a potential demand for amniocentesis far in excess of current use and present service facilities. They suggest, moreover, that underuse may reflect professional hesitation and underreferral more than consumer lack of demand or reluctance to be tested.     

Revised estimates and projections of down syndrome births in the United States,and the effects of prenatal diagnosis utilization, 1970–2002

The objectives of this study were to: (1) calculate revised estimates and projections of United States annual Down syndrome (DS) births for 1970–2002, and (2) estimate the effects of amniocentesis on these baseline DS birth projections. Three models of amniocentesis utilization among 30–34 and ≧ 35-year-old women were considered. The recently revised Census Bureau birth projections, and new single year maternal age DS risk rates estimated from a 1970–1983 Ohio data set, were used. Data from all three Census Bureau projection series were analysed; series II was considered in depth since it is consistent with recent fertility levels. Assuming no use of amniocentesis, total estimated DS births dropped from about 4770 in 1970 to 4120 in 1980 (a 14 per cent decline), but are projected to a plateau of about 5100 by the year 1990 (a 24 per cent increase). DS births to women ≧ 35 would increase dramatically from about 1050 in 1980 to 1900 in 2000 (an 81 per cent increase). Assuming 1983 Ohio prenatal diagnosis ratios for women aged 30–34 (1.7 per cent) and ≧ 35 (23.4 per cent) are used nationally, an annual reduction of about 7 per cent of DS births in 1986 and 9 per cent in 2002 would result. Fifty and 70 per cent utilization among women 30–34 and 235, respectively, would reduce DS births by about 33 per cent in 1986 and 38 per cent in 2002. Therefore, if the projected increase in DS births is to be averted, utilization of prenatal diagnosis by ≧ 30-year-old women must increase substantially.     

Chromosomal prenatal diagnosis: Study of 936 cases of intrauterine abnormalities after ultrasound assessment

Nine hundred and thirty-six prenatal chromosomal analyses were performed by four cytogenetic centres after ultrasound diagnosis of fetal abnormalities, amniotic fluid disorders, fetal growth retardation, and fetal or placental abnormalities. During the same period, 6515 fetal karyotypes were analysed because of maternal age. Frequencies of chromosomal aberrations in each case were respectively 4·4, 6·7 and 15·8 per cent, compared with 3·18 per cent when the fetal karyotype was performed because of maternal age. High rates of chromosomal aberrations are observed in cases of cervical hygroma, limb abnormalities, omphaloceles, duodenal stenosis, hydrocephalus, and facial abnormalities. In the case of polymalformations, this rate was 29·2 per cent. When malformations were seen together with an amniotic fluid disorder or growth retardation, 21·5 per cent chromosomal aberrations were observed. This frequency was 10·4 per cent when growth retardation was associated with an amniotic fluid disorder. Trisomy 13, 18, 21 and monosomy X accounted for 4/5 of all abnormalities in which we observed a high rate of triploidies (4·9 per cent) and balanced (3·3 per cent) or unbalanced (9·8 per cent) non-Robertsonian structural abnormalities. Sonographic ascertainment of these aberrations and prenatal characteristics of major anomalies are discussed.     

Phenotypic analysis of fetal blood leucocytes: Potential for prenatal diagnosis of immunodeficiency disorders

Recent technological advances allow the detection and quantitation of subsets of leucocytes using monoclonal antibodies. We have taken advantage of this to study the ontogeny of fetal blood leucocytes, using very small blood samples obtained at fetoscopy. By 14 weeks gestation T cells represent 35 per cent or more of fetal leucocytes and the distribution of the helper/inducer and suppressor/cytotoxic subsets is similar to that of adults. B lymphocytes before 161/2 weeks are low (4–20 per cent), but rise to a mean of 28 per cent in 17–26 week fetuses. Granulocytic cells, many of which are phenotypically immature, represent 18–34 per cent of total leucocytes. The methodology employed is very reliable and offers the opportunity for the prenatal diagnosis of some immunodeficiency disorders, since using the same reagents we have diagnosed children with severe combined immunodeficiency shortly after birth.