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The presence of small numbers of fetal nucleated red cells in the maternal circulation has been a stimulus for the development of technologies for non-invasive prenatal genetic analysis. Our laboratory has been assessing the feasibility of density gradient centrifugation followed by magnetic activated cell sorting (MACS) of cells expressing CD32 and CD45, to deplete maternal nucleated blood cells. We have examined the efficiency of each of the steps of this procedure using cord blood from term pregnancies as a source of nucleated red blood cells. Cord blood was shown to contain highly variable numbers of nucleated red cells. Three different density gradients were examined. There was no major difference in the performances of the double and triple gradients. Density gradient centrifugation resulted in enrichments of nucleated red blood cells of about 1000-fold relative to the total cell count. However, it was apparent that the selection of the cell layers which were most enriched for these cells would result in significant losses of nucleated red cells in other layers. MACS sorting of cells using CD45 resulted in white cell depletions ranging from 7 to 34-fold. These data provide a foundation for comparison with other methods and for optimization of the MACS technique.

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.

A retrospective study to investigate the psychosocial sequelae of a second-trimester termination of pregnancy (TOP) for fetal abnormality (FA) is described. After appropriate consent was obtained, 84 women and 68 spouses were visited 2 years after the event and asked to complete an extensive questionnaire. Most couples reported a state of emotional turmoil after the TOP. There were differences in the way couples coped with this confusion of feelings. After 2 years about 20 per cent of the women still complained of regular bouts of crying, sadness, and irritability. Husbands reported increased listlessness, loss of concentration, and irritability for up to 12 months after the TOP. In the same period, there was increased marital disharmony in which 12 per cent of the couples separated for a while and one couple obtained a divorce. These problems could be attributed to a lack of synchrony in the grieving process. Confusing and conflicting feelings led to social isolation and lack of communication. Difficulties in coming to terms with the fetal loss were not found to be linked to the type of fetal abnormality or religious beliefs but were related to parental immaturity, inability to communicate needs, a deep-rooted lack of self-esteem before the pregnancy, lack of supporting relationships, and secondary infertility. Suggestions for improved management are given.84, or 87% of all women from West Scotland who had second trimester termination of pregnancy for fetal abnormalities in 1986, and 68 spouses, participated in a study of psychosocial sequelae. A structured questionnaire was administered during a home visit 2 years after the termination. Gestations ranged from 12 to 27 weeks (median 19). The women's ages ranged from 17 to 46 years (median 27), that of their partners was 17-48 (median 29). Indications for the abortions were tabulated. Only 5% of the women were without partners. 15% were unemployed. 33% of the pregnancies were the couples' 1st. 76% of the abnormal pregnancies were directed by the West of Scotland Alpha-fetoprotein screening program. 72% of the pregnancies had been planned, 26% had been unplanned but were welcome, and only 2% of women were still ambivalent. 2 couples regretted their decision. At 2 years 20% of women were having crying spells, sadness, and irritability. Husbands reported listlessness, poor concentration, and irritability up to 1 year afterward. 12% of couples marital problems resulting in separation of 2% and 1 divorce. Physical symptoms such as palpitations and panic attacks were only reported by those under 25. 1 woman and 1 man required in-patient treatment; 11 women received counseling; 5 took anti depressants. 3 men had counseling and 3 took antidepressants. A majority of men reported confusing and conflicting reactions to their wives' feelings which may have led to social isolation and lack of communication. 55% of the women and 58% of the men said they had not discussed their feelings with anyone. There were no specific psychological sequelae linked to specific fetal abnormalities or religious beliefs. Immaturity, inability to communicate needs, low self-esteem, lack of social support, and secondary infertility all were associated with dysfunctional grieving. It was concluded that such grief and emotional turmoil could be shortened by skilled preparatory counseling.

Diagnosis of fetal cells in the maternal circulation will obviate the need for invasive sampling procedures. It is essential however, before this can be put into wide clinical practice, that a reliable isolation procedure and a simple, robust means of genotyping rare fetal cells be developed. A relatively inexpensive, sensitive diagnostic procedure which can detect single gene defects and/or aneuploidy is fluorescent PCR. This has been used extensively in molecular diagnostics including preimplantation diagnosis but, to our knowledge, has not been applied widely for the diagnosis of isolated fetal erythroblasts. Here we used cord blood as a model system for fetal cells in the maternal circulation: we isolated erythroblasts on a glass slide, employed a simple micromanipulation technique to place them into a PCR tube and then applied fluorescent PCR to diagnose sex and cystic fibrosis status in 10 patients. Nine samples had a normal cystic fibrosis status, one patient, however, had a mutant allele suggesting that this fetus was a carrier for cystic fibrosis. Fluorescent PCR has virtually limitless potential in the genotyping of rare fetal cells isolated from the maternal circulation.

Data on maternal serum alpha-fetoprotein (AFP) levels at 13-24 weeks' gestation in 46 twin pregnancies with open neural tube defects (22 with anencephaly, 24 with open spina bifida) and 169 unaffected twins were used to estimate the detection and false-positive rates associated with different cut-off levels. Using the conventional cut-off level of 2.5 multiples of the median (MoM) for unaffected singleton pregnancies of the same gestation and laboratory, the detection rate in twins was 99 per cent for anencephaly and 89 per cent for open spina bifida, with a false-positive rate of 30 per cent. Using a 5.0 MoM cut-off level to maintain a similar false-positive rate to that found among singleton pregnancies at 16-18 weeks' gestation (about 3 per cent), the detection rate was 83 per cent for anencephaly and 39 per cent for open spina bifida. Estimates are provided of the odds of having an affected twin pregnancy given a positive AFP result as well as the odds for individual women with a raised AFP level.

Fetal erythroblasts circulating in maternal blood are important candidate cells for non-invasive prenatal diagnosis. We have cultured erythroblasts from 16 maternal blood samples, both with and without prior enrichment by magnetic activated cell sorting (MACS), in a semi-solid medium containing growth factors. Individual colonies were examined by PCR with sex chromosome-specific primers and microsatellite marker primers. No conclusive Y-chromosome specific amplification could be demonstrated in any of the 16 cases, even when the mother was confirmed to be carrying a male fetus. All colonies tested by microsatellite marker PCR were of maternal origin. Our results suggest that the probability of obtaining fetal colonies from fetal erythroblasts circulating in maternal blood is very low and that approaches for culturing fetal erythroblasts in vitro cannot yet be used reliably for prenatal diagnosis using current methods for fetal cell enrichment. © 1998 John Wiley & Sons, Ltd.

Over a 2-year period from January 1991 to December 1992, second-trimester maternal serum screening for Down's syndrome using alpha-fetoprotein (alpha FP), human chorionic gonadotrophin (hCG), and unconjugated oestriol (uE3) was made available to five health districts in East Anglia, with a total population of 1.2 million. Amniocentesis was offered when the risk of Down's syndrome at term was 1:200 or greater. 25,359 singleton pregnancies were screened, representing an uptake of 77 per cent. The recall rate for the 24 per cent of women who had not had a dating scan prior to the test was 9.4 per cent compared with 3.9 per cent for those who had been scanned (P < 0.0005). Seventy-five per cent (36/48) of Down's syndrome pregnancies were detected for a false-positive rate of 4.0 per cent. Twenty-five out of 36 of detected Down's syndrome pregnancies were dated by scan prior to sampling, and in the 11 remaining cases, the dates were confirmed by scan after a high-risk result was obtained. The exclusion of uE3 from the screening protocol would have reduced the detection rate to 52 per cent (25/48) for the same false-positive rate. Eighty-five per cent of women identified at high risk accepted the offer of an amniocentesis. Other fetal abnormalities detected were trisomy 18 (3), trisomy 13 (2), 45,X (6), 69,XXX (5), other chromosome abnormalities (9), open neural tube defects (26), hydrocephalus (7), abdominal wall defects (4), and steroid sulphatase deficiency (6).

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.

Risks appropriate for mid-trimester prenatal screening for autosomal trisomies have been estimated from a combination of maternal age and maternal serum (MS) alpha-fetoprotein (AFP) levels at 16–20 weeks gestation. Published data on the frequency of Down's syndrome births relative to maternal age were modified to include the additional age-related frequency of trisomy 18 and trisomy 13 cases to provide an overall risk for an autosomal trisomy at midtrimester. MSAFP results from a retrospective study of 142 affected (114 trisomy 21, 19 trisomy 18, and 9 trisomy 13)and 113 000 unaffected pregnancies were converted to multiples of the appropriate gestational median (MOM). The AFP levels in the autosomal trisomy pregnancies were found to be significantly reduced at 0.72 MOM of the unaffected pregnancies. Risks (likelihood ratios) were derived from the overlapping log Gaussian distributions for affected and unaffected pregnancies and combined with maternal age risks to give the overall odds of an affected pregnancy. A mid-trimester cut-off risk of 1:280 gave an estimated 37 per cent detection rate for autosomal trisomies in the west of Scotland population for a follow-up (false-positive) rate of 6.6 per cent. These figures compare with a 30 per cent detection and 6.7 per cent false-positive rate if age 35 years and over is used as the sole criterion for selection of at-risk pregnancies.

A retrospective study was designed to examine the perception of care in women who had experienced a second-trimester termination of pregnancy (TOP) for a neural tube defect. Women were identified over a 3-year period, 1983–1985. After appropriate consent, 166 women were visited at home between 4 weeks and 7 months post-TOP and interviewed by one experienced interviewer using a structured questionnaire with open and closed questions. The majority (137, 82 per cent) felt satisfied with the care received during screening, prenatal diagnosis, and during the TOP (126, 76 per cent). Patients were less satisfied (63, 38 per cent) with post-TOP care in hospital. On leaving hospital, the post-termination sequelae were mentioned to only 25 (15 per cent) patients, which left 135 (81 per cent) confused and bewildered by the post-partum reactions of their bodies, and by their strong emotions. After-care was perceived as unsatisfactory by 113 (68 per cent). One-quarter (42, 25 per cent) did not have, and were not invited for, a post-termination appointment and thus did not have an opportunity to ask questions or to discuss the fetus. Eighty-six (51-8 per cent) had no visit from any member of the primary health-care team, yet most would have appreciated such a visit. Suggestions for improved management are presented.

Alpha-fetoprotein (AFP) concentration and gamma-glutamyltranspeptidase (GGT) activity have been analysed in amniotic fluid from a series of 65 pregnancies with autosomal trisomies. AFP values were reduced on average to 60 per cent of normal in cases of trisomy 21, but were not significantly different from normal in cases of trisomies 18 and 13. GGT activities were uniformly lower (44 per cent of normal) for all types of autosomal trisomy. A review of the literature indicates that over 85 per cent of Down's pregnancies but only 39 per cent of trisomy 18 and 13 pregnancies have amniotic fluid AFP levels below the normal median value, while the corresponding figures for GGT are 91 per cent for Down's syndrome and 96 per cent for trisomies 18 and 13.

Prenatal diagnosis of trisomy 20 mosaicism was made in two pregnancies by chromosome analysis of cultured amniotic fluid cells. In both cases, the pregnancy continued to term and a healthy male infant was delivered. Regular assessments up to the age of 6.5 years revealed normal physical and intellectual development in both children.

Maternal age specific rates for all major chromosome aberrations have been determined in 52 965 pregnancies in mothers 35 years of age and over at the time of amniocentesis. Rates increase exponentially with advancing maternal age for trisomies 21, 18 and 13, and for the XXX and XXY syndromes, but in the autosomal trisomies this rise appears to be followed by a levelling off at the upper end of the age range. A significant inverse relationship with maternal age is found for 45,X cases. It is postulated that these various patterns are the result of the interaction of three principal factors: a maternal age effect acting particularly on first meiotic nondisjunction: a higher spontaneous abortion rate with advancing maternal age for aneuploid as compared to euploid conceptions; and an increased probability of spontaneous abortion before the time of amniocentesis for conceptions with more extensive chromosome imbalance. A stepwise logistic regression analysis of 13 299 pregnancies in which both parental ages are known shows that the father's age does not influence these maternal age specific rates, with the possible exception of the 47,XXY syndrome.