Prenatal diagnosis of trisomy 13 on fetal cells obtained from maternal blood after minor enrichment

In a pilot study to establish fetal nucleated red blood cell (NRBC) detection in maternal blood, trisomy 13 was diagnosed by FISH analysis at 11 weeks' gestation. The NRBCs were detected after a single-step ficoll density gradient enrichment. In blood samples taken both before and after CVS, 52 and 80 NRBCs, respectively, were found to be positive for fetal haemoglobin. In 47 per cent of these cells, FISH analysis for X and Y chromosomes confirmed the fetal sex. Moreover, 48 per cent of these NRBCs showed three fluorescent signals for a chromosome 13 probe, which confirmed the diagnosis of trisomy 13, previously detected at CVS karyotyping. This is the first report of non-invasive prenatal diagnosis of trisomy 13, i.e., pre-CVS, in the first trimester. The high number of fetal NRBCs detected indicates a connection with aneuploidy, probably due to early impairment of the feto-maternal barrier.     

Serum homocysteine level and protein intake are related to risk of microalbuminuria: the Hoorn Study

OBJECTIVE: Currently, prenatal diagnosis of chromosome abnormalities requires invasive techniques such as amniocentesis and chorionic villus sampling that carry small but finite risks of fetal loss. A noninvasive approach is to isolate fetal cells from maternal blood by flow sorting followed by genetic interphase analysis with fluorescence in situ hybridization. Because the ratio of fetal to maternal cells is relatively low after flow sorting and to detect 90% to 95% of fetal aneuploidies associated with serious birth defects, a 5-color fluorescent in situ hybridization strategy is necessary for simultaneous detection of chromosomes X, Y, 13, 18, and 21 in all flow-sorted nuclei recovered from a specimen. STUDY DESIGN: Fetal nucleated red blood cells were isolated from maternal blood in 40 cases (10.4 to 27.0 weeks' gestation) by flow cytometry on the basis of positive selection of CD71+ (transferrin receptor), CD45-, and LDS751 staining. Each case was evaluated for 5-color fluorescent in situ hybridization efficiency by determining the percentage of flow-sorted nuclei containing 8 hybridization signals for chromosomes X, Y, 13, 18, and 21. RESULTS: A total of 42,312 flow-sorted nuclei from maternal blood samples were analyzed. In 5 of 16 (31%) cases with a male fetus, 0.16% of nuclei scored were identified as fetal by the presence of 1 signal each for chromosomes X and Y. Fetal trisomy 21 nuclei were accurately detected in 2 cases with a female fetus, each of which was subsequently confirmed. CONCLUSIONS: Five-color interphase fluorescent in situ hybridization analysis can be used to effectively analyze rare fetal aneuploid nuclei in enriched flow-sorted cells isolated from maternal blood.     

True trisomy 15 mosaicism, detected by amniocentesis at 12 weeks of gestation and fetal echocardiography

A case of mosaicism of trisomy 15, with two-thirds of the cells trisomic, was detected at 12 weeks of gestation in amniotic fluid cell cultures obtained with the filtration technique. Ultrasound examination at 13 weeks showed a nodule protruding into the amniotic cavity which was speculated to be remnants of a co-twin, causing the trisomic cell line. At 20 weeks of gestation, a malformation scan (level III) was normal, but supplementary fetal echocardiography revealed a severe cardiac defect (mitral atresia and a ventricular septal defect). Fetal lymphocytes obtained by cordocentesis showed trisomy 15 mosaicism, but only in 5 per cent of the mitoses. After termination, the same percentage of trisomy 15 mosaicism was found in cells from skin and tendon as in the original early amniocentesis. No sign of earlier twinning was found in the placenta or membranes. We conclude that mosaicism in early amniotic fluid obtained by the filter technique in this case reflected the true karyotype accurately and that supplementary echocardiography added significantly to the interpretation of the clinical implications.     

Isolated choroid plexus cysts--the need for routine offer of karyotyping

The management of isolated fetal choroid plexus cysts remains controversial. We have prospectively studied 15,565 pregnancies at two large obstetric units for the presence of choroid plexus cysts. In all cases where cysts were present at 19 weeks' gestation or greater, and were multiple, bilateral or solitary and greater than 5 mm maximum diameter, women were offered amniocentesis or placental biopsy, irrespective of the presence or absence of other abnormalities. Choroid plexus cysts were present in 152 (0.98 per cent) of cases. Four cases (2.6 per cent) of autosomal trisomy (three of trisomy 18, one of trisomy 21) were detected on prenatal karyotyping. In all cases, choroid plexus cysts were the only detectable prenatal anomaly. This study and a review of other large studies do not support the view that isolated choroid plexus cysts are a benign variant, the risk of trisomy being 1 in 82. Until further evidence is available, we recommend that cases of isolated fetal choroid plexus cysts at 19 weeks' gestation or greater should be offered prenatal karyotyping.     

Low-level mosaicism for both trisomy 15 and monosomy-X in amniotic fluid cells confirmed in fetal tissues

We report here a case of true fetal mosaicism for both trisomy 15 and monosomy-X; the aberrant cell lines were initially detected at amniocentesis as low-level mosaicism (trisomy 15) and multiple-cell pseudo-mosaicism (monosomy-X). In the fetal lymphocytes, only metaphases with a normal chromosome complement were observed. After termination of the pregnancy, various fetal biopsies revealed both trisomy 15 and monosomy-X mosaicism, whereas, at autopsy, no external or internal abnormalities could be detected in the fetus. The karyotype can be described as 45,X[15]/47,XY,+15[3]/46,XY[27]. Our results implicate that an additional amniocentesis could be more helpful than fetal blood sampling in predicting the fetal karyotype after diagnosis of chromosome mosaicism at amniocentesis.     

NG-nitro-L-arginine methyl ester inhibits bone metastasis after modified intracardiac injection of human breast cancer cells in a nude mouse model

A retrospective evaluation of alpha-fetoprotein (AFP), human chorionic gonadotropin (hCG), and unconjugated oestriol (uE3) levels in maternal blood in the second trimester was conducted for cases of aneuploid pregnancies identified from a series of women who underwent amniocentesis. Blood samples were collected from 1078 women just before genetic amniocentesis was performed, mainly for individuals of advanced maternal age (greater than 35 years). Twenty-five maternal serum samples from pregnant women with an aneuploid fetus, including 14 with Down's syndrome, were available for analysis of all three parameters. An algorithm to detect Down's syndrome was used for this analysis with a risk of > or = 1:299 classified as screen-positive, this being found for 20.4 per cent of the cases (220/1078). The actual Down's syndrome detection rate was 85.7 per cent (12/14), whereas the detection rate for all aneuploidies was 72.0 per cent (18/25). Those that were not detected were two cases of trisomy 21, one trisomy 18, two trisomy 13, three sex chromosome abnormalities, and one case of an additional marker chromosome. The data indicate that this tri-analyte test should be provided after thorough genetic counselling and informed decision-making regarding maternal serum screening for women who wish for a prenatal diagnosis.     

Is serum thyroglobulin a useful marker for thyroid cancer in patients who have not had ablation of residual thyroid tissue?

Among 58,000 amniocenteses completed, our laboratories found one case of true cytogenetic trisomy 2 mosaicism in a fetus with multiple abnormalities. In contrast, 11 fetuses phenotypically normal at birth were found to have true trisomy 2 mosaicism in their chorionic villus cells among the 10,500 fetuses tested by chorionic villus sampling (CVS). In our single abnormal case, amniocentesis performed at 19 weeks after finding an elevated maternal serum AFP found two independent cultures with trisomy 2 karyotypes in 8 of 25 and 7 of 31 amniocytes, respectively. Although oligohydramnios was noted by ultrasound, the mother elected to continue the pregnancy. At 26 weeks the fetus had intrauterine growth retardation (IUGR), hydronephrosis, and cardiac abnormalities. When delivered by Cesarean section at 30 weeks, the infant had multiple anomalies and developed necrotizing enterocolitis and severe cholestasis. At 5 months coronal magnetic resonance imaging (MRI) displayed delayed myelination and abnormal brain morphology. The patient also exhibited significant growth failure and developmental delay. Although chromosomes were normal in blood, skin fibroblasts, and ascites fluid cells, 4 of 100 hepatic biopsy fibroblasts were 47,XY,+2. Molecular analysis excluded uniparental disomy (UPD) of chromosome 2 in the 46,XY cell line. This and other reports of rare phenotypically abnormal trisomy 2 mosaic fetuses identified by karyotyping amniocytes emphasizes the substantially higher fetal risk of abnormal development than when trisomy 2 is found only in chorionic villus cells.     

The internal bony architecture of the sacrum

While the clinical features associated with full trisomy 13 have been well characterized, the clinical outcome associated with mosaic trisomy 13 is much less clear. The medical literature reports a broad range of possible clinical outcomes from severe mental retardation and birth defects to normal intelligence. There is no consensus about the typical phenotype in these cases. This makes genetic counselling after prenatal diagnosis of mosaic trisomy 13 particularly difficult. Some of the medical literature attempts to correlate the percentage of trisomic cells in peripheral blood leukocytes or skin fibroblasts with clinical outcome. There have not been case reports correlating the percentage of trisomic amniocytes and clinical outcome. We report the prenatal diagnosis of mosaic trisomy 13 by amniocentesis in which no prenatal ultrasound abnormalities were noted, and autopsy was normal with the exception of the presence of a small ventricular septal defect.     

A comparative evaluation of two day hospitals. Goal attainment scaling of behavior therapy vs. milieu therapy

A retrospective anatomical, family, and epidemiological study was made of 143 patients (81 female and 62 male) with diaphragmatic hernia who were born in the south-west of England between 1943 and 1974. Thirty-nine cases were stillborn. Seventy-five per cent of patients had a left-sided diaphragmatic defect, 22% had a right-sided defect, and 3% had a bilateral defect. Fifty per cent of the patients had other congenital malformations, most frequently of the nervous system. No maternal age or birth order effect was noted. Cases of diaphragmatic hernia without other malformations had in general a normal fetal growth rate. Eight per cent of the cases were illegitimate. There were two pairs of twins discordant for diaphragmatic hernia, one pair being dizygotic and the other monozygotic. In no case of diaphragmatic hernia was there a relative affected with a diaphragmatic hernia. The most common type of diaphragmatic defect was a posterolateral hernia (92%), followed in frequency by an eventration of the diaphragm (5%), the least common defect being a retrocostosternal hernia (2%). Diaphragmatic hernia appears to be aetiologically as well as anatomically heterogeneous. In this series there were two cases of trisomy 18, one case of trisomy 21, one case trisomic for a small part of chromosome 20, and two cases with the Pierre Robin syndrome. It seems likely that diaphragmatic hernia is a non-specific consequence of several teratological processes.     

A patient with myelodysplastic syndrome studied by GTG-banding and fluorescent in situ hybridization (FISH)

Molecular cytogenetics using fluorescent in situ hybridization (FISH) is an extremely useful adjunct technique to conventional cytogenetics via GTG-banding. The present paper illustrates the utility of FISH by describing a patient with myelodysplastic syndrome (MDS) who was initially studied using GTG-banding and whose bone marrow was found to be populated with hyperdiploid cells. FISH was used to delineate the numerical and structural chromosomal abnormalities. It revealed the presence of trisomy 8 and determined that the previously unidentifiable marker chromosome was of chromosome 10 origin. Although trisomy 8 is a frequent finding in MDS, the structural chromosomal abnormality of chromosome 10 as reported here is not a common finding.     

A simple electrode for metallic replication

The effect of ritodrine upon uterine artery blood flow (UtBF) and umbilical vein blood flow (UmBF) was investigated in near-term chronic sheep preparations. During intravenous ritodrine infusions to the ewe in sequential dose rates from 100 to 800 mug per minute, UtBF fell progressively to 43 per cent below control levels and mean maternal arterial pressure (MMAP) declined 20 per cent. During constant infusions of 100, 400, or 800 mug per minute of ritodrine to the ewe for 120 minutes,, UtBF decreased 10, 37, and 31 per cent, respectively, and MMAP decreased 6, 20 and 25 per cent respectively. Dose-related maternal tachycardia and hyperglycemia occurred. There were no significant changes in UmBF, mean fetal arterial pressure, or fetal heart rate. During all infusions, maternal and fetal arterial pH, PCO2, and PO2 remained within normal physiologic limits. Simultaneous infusion of ritodrine (400 mug per minute) and propranolol (100 mug per minute) blocked the maternal tachycardia, but decreases in UtBF, MMAP, and UmBF were observed. Ritodrine infusions to the fetus (25 mug per minute) resulted in fetal tachycardia and a variable increase in UmBF.     

Specific chromosome changes associated with rabbit cell lines cultured in vitro

Thirty-three rabbit cell lines were established from various fetal tissues of the inbred strain III of the New Zealand rabbit (Oryctolagus cuniculus). None of these lines exhibited senescence during a growth period of more than 2 years. Karyologic studies of most cell lines at 10 to 20 cell-passage intervals revealed that the karyotype stability of the rabbit cells in vitro was correlated with the organs from which the cell lines were derived. Thus, lines derived from cornea, spleen, and kidney tissues usually contained high frequencies of polyploidy in their early passages, whereas most of those derived from lung and skin were found to retain the normal diploid karyotype for much longer periods of time. One line derived from fetal lung tissue, designated Lung 16, remained diploid up to 100 passages. In late passages of the majority of all the lines studied, the cells became pseudodiploid, hyperdiploid, or polyploid. Among the pseudodiploid and the hyperdiploid cell lines, the chromosomal changes followed three basic patterns: (1) a gain of one or more telocentric chromosomes; (2) a loss of one telocentric chromosome plus a metacentric marker chromosome (M); or (3) a gain of a long telocentric marker chromosome with or without changes in the number of telocentric D chromosomes. By the G-banding technique, the telocentric chromosome involved in these three patterns was identified as the D-group chromosome 18 and the M marker chromosome as an isochromosome of 18. These results suggest that chromosomal rearrangement in rabbit cells involving trisomy of 18 may be responsible for the longevity of these cell lines cultured in vitro.     

Prenatal ultrasonic diagnosis of obstructive bowel disease: a retrospective analysis

Fetal obstructive bowel disease was diagnosed in 29 patients at 22-37 weeks (median 32 weeks) of gestation, seven (24 per cent) of whom also displayed other anomalies. Polyhydramnios was present in 20/29 cases (69 per cent). An abnormal karyotype existed in 7/29 cases (24 per cent), of which six were diagnosed prenatally (trisomy 21, n = 5; 69,XXX, n = 1) and one postnatally (trisomy 21). There was always an association with the ultrasonic 'double bubble' sign. Obstructive bowel disease was confirmed postnatally in 20/29 (69 per cent) cases, i.e., oesophageal atresia (n = 1), duodenal obstruction (n = 12), and small bowel obstruction (n = 7). Other anomalies existed in 6/29 (21 per cent) cases, i.e., multicystic kidney (n = 1) and multiple congenital anomalies (n = 5). The perinatal mortality rate was 35 per cent (7/20).     

HELLP syndrome in the 21st week of pregnancy in mosaic trisomy 9

We report for the first time the case of postabortional HELLP-syndrome in the 21st week of gestation. In this case mosaic trisomy 9 was confirmed by amniocentesis prior to induction. Pertinent history, clinical course and pathoanatomical morphology are described. We emphasize the early onset of the HELLP-syndrome in association with trisomy 9 after abortion. The possibility of interconnections between trisomy 9 and the occurrence of HELLP-syndrome (sparse blood, vessels in the villi, circulatory deficit on the fetal side of the placenta, increased production of e.g. vasopressive substances) is discussed.     

Poly-FISH: a technique of repeated hybridizations that improves cytogenetic analysis of fetal cells in maternal blood

Prenatal diagnosis of fetal chromosomal abnormalities using interphase fetal nucleated erythrocytes (FNRBCs) separated from maternal peripheral blood can be technically challenging due to the limited number of FNRBCs available for analysis, the limited number of probes that can be used simultaneously, and low FISH efficiency on the formaldehyde-fixed and immunohistochemically stained interphase FNRBCs. We developed a technique of sequential FISH analysis that involves removal of the previous hybridized probe under denaturing conditions, and rehybridization with different probes to improve FISH efficiency. This technique facilitates the analysis of multiple chromosome-specific probes on the same nuclei. Results from our experiments show that FISH can be performed at least nine times on the same interphase nucleus and at least three different probes can be used simultaneously. Thus, theoretically, at least 24 different chromosomes can be analysed on a single interphase fetal cell isolated from maternal blood. We have termed this technique 'Poly-FISH', and have successfully diagnosed trisomy 21, triploidy, and other chromosome abnormalities in FNRBCs using this technique.     

Frequency of fetal cells in sorted subpopulations of nucleated erythroid and CD34+ hematopoietic progenitor cells from maternal peripheral blood

Fetal cells that circulate in maternal peripheral blood (PB) during pregnancy offer a potential source of nucleated fetal material for noninvasive prenatal diagnosis. Fluorescence-activated cell sorting was used to target two populations of fetal cells: nucleated erythroid cells (NECs; CD71/glycophorin-A+ CD45(lo-int) CD34-) and hematopoietic progenitor cells (CD34+ cells; CD34++ CD71/glycophorin-A- CD45(int)). Fetal cells were detected by fluorescence in situ hybridization (FISH) using directly conjugated chromosome X and Y probes in 65% (13 of 20) of the maternal PBs (fetal karyotype 46,XY). The frequency of fetal cells isolated from the NEC and CD34+ fractions was, respectively, 0 to 14 and 0 to 7 cells per 2 x 10(7) previously frozen maternal cells (approximately 20 mL of blood). In nonfrozen samples, the yield and recovery of fetal cells was moderately improved. Culturing the CD34+ sorted fractions in serum-free media with cytokines improved the quality of the FISH preparations and resulted in a slight expansion in detectable fetal cells. The frequency of fetal cells isolated from cultured CD34+ fractions was 0 to 35 and 0 to 93 cells per 2 x 10(7) previously frozen and nonfrozen maternal PB cells, respectively. These results document the isolation, characterization, and enumeration of fetal cells from the maternal periphery that appear to be present in most, but not all, samples analyzed.     

Usefulness of chromosome examination in the diagnosis of malignant pleural effusions

To determine whether chromosome analysis could facilitate the diagnosis of malignant pleural effusions, we examined chromosomes in effusions from 104 unselected patients. An effusion was regarded as malignant if at least three of 30 metaphase cells were hyperdiploid or contained a marker chromosome. Results were compared with standard cytologic diagnoses. All 22 benign effusions were diagnosed correctly by cytologic examination, but one nosed correctly by cytologic examination, but one (acute rheumatoid lung disease) was misclassified as positive by chromosome criteria. Of the 82 malignant effusions, 53 (65 per cent) were diagnosed correctly by cytologic tests, as compared with 58 (71 per cent) by chromosome analysis (P greater than 0.2). Among patients with malignant neoplasms, 13 had leukemia or lymphoma; only four of these (31 per cent) were diagnosed by cytologic tests as compared with 11 (85 per cent) by chromosome analysis (P less than 0.01). The combination of standard cytologic and chromosome analyses correctly identified 83 per cent of the neoplasms, a result significantly better than that with either technic alone (P less than 0.01).     

Charge flow separation: quantification of nucleated red blood cells in maternal blood during pregnancy

We set out to ascertain the numbers of fetal cells that enter the maternal blood stream during pregnancy. Samples of 15-16 ml of whole blood were collected from 225 women--mostly 10-18 weeks pregnant--and then processed by charge flow separation, a novel method based on free flow electrophoresis in a buffer counterflow gradient. After their recovery in four different separation instruments, nucleated red blood cells (NRBC) were enumerated histologically. In some cases fetal NRBC were identified and enumerated by fluorescence in situ hybridization with probes for the X and Y chromosomes and fetal haemoglobin mRNA. Recoveries were consistent among the four separation instruments: the median numbers of NRBC obtained were 4190, 1590, 2805 and 3860. Our data show that approximately 30 per cent of those cells were fetal. Thus, recent reports on the separation of fetal NRBC by other methods, give underestimates of their frequency in maternal blood.     

Hepatoblastoma

Hepatoblastoma is the most frequently occurring liver tumor in children, accounting for over 25% pediatric hepatic tumors and nearly 50% of those that are malignant. Histologically, the tumor can be divided into the following six patterns: (1) fetal epithelial; (2) embryonal and fetal epithelial; (3) macrotrabecular; (4) small cell undifferentiated; and (5) mixed epithelial and mesenchymal type with teratoid features or (6) without teratoid features. Immunohistochemical studies display a wide variety of immunostaining with monoclonal antibodies particularly those specific for epithelial-derived components. Tumor cytogenetics show a high incidence of trisomy 20 and trisomy of all or part of chromosome 2. The developing liver displays many features similar to those seen in hepatoblastoma, including uniform hepatocytes and cords two cells thick separated by sinusoids displaying hematopoiesis. Hepatoblastomas display only minimal ductular differentiation, similar to the fetal development of the liver that does not display significant ductular development until well into the second trimester.     

Morphologic and functional characterization of human aortic endothelium. III. Growth in culture and colony formation at low seeding density

HLA-G is a nonclassical, class I HLA gene that is primarily expressed by fetal cells at the maternal-fetal interface and is thought to play a key role in the induction of tolerance in pregnancy. This paper reports the identification of a single base pair deletion at position 1597 (1597delC) in exon 3 (encoding the alpha2-domain) of HLA-G on 20 of 272 (7.4 per cent) African American chromosomes, three of 102 (2.9 per cent) Hispanic chromosomes, and none of 134 Caucasian chromosomes. This relatively common frameshift mutation results in amino acid substitutions in all of the residues in the second half of exon 3 including the conserved cysteine at codon 164. An adult individual was identified who was homozygous for this 'null' allele, and a first trimester placenta that was homozygous for 1597delC had no detectable HLA-G1 protein. These data indicate that expression of HLA-G1 protein is not essential for fetal survival.