Isolating fetal cells in maternal circulation for prenatal diagnosis

Fetal cells unequivocally exist in and can be isolated from maternal blood. Erythroblasts, trophoblasts, granulocytes and lymphocytes have all been isolated by various density gradient and flow sorting techniques. Chromosomal abnormalities detected on isolated fetal cells include trisomy 21, trisomy 18, Klinefelter syndrome (47,XXY) and 47,XYY. Polymerase chain reaction (PCR) technology has enabled the detection of fetal sex, Mendelian disorders (e.g. β-globin mutations), HLA polymorphisms, and fetal Rhesus (D) blood type. The fetal cell type that has generated the most success is the nucleated erythrocyte; however, trophoblasts, lymphocytes and granulocytes are also considered to be present in maternal blood. Fetal cells circulate in maternal blood during the first and second trimesters, and their detection is probably not affected by Rh or ABO maternal-fetal incompatibilities. Emphasis is now directed toward determining the most practical and efficacious manner for this technique to be applied to prenatal genetic diagnosis. Only upon completion of clinical evaluations could it be considered appropriate to offer this technology as an alternative to conventional invasive and non-invasive methods of prenatal cytogenetic diagnosis.     

Detection and isolation of fetal cells from maternal blood using the flourescence-activated cell sorter (FACS)

The presence of fetal cells in the maternal circulation during pregnancy has been suggested by repeated observations of small numbers of cells containing Y chromatin or a Y chromosome in the blood of pregnant women. With the fluorescence-activitated cell sorter (FACS), we have used antibodies to a paternal cell surface (HLA) antigen, not present in the mother, to select fetal cells from the lymphocyte fractions of a series of maternal blood samples, collected as early as 15 weeks of gestation. These sorted cells have been examined for a second paternal genetic marker, Y chromatin. Y chromatin-containing cells were found among the sorted cells from prenatal maternal blood specimens in 8 pregnancies subsequently producing male infants whose lymphocytes reacted with the same antibodies to paternal antigen used for sorting with the FACS. In each of 17 pregnancies resulting in male infants who failed to inherit the antigen detected by the antibodies used for cell sorting, Y chromatin-containing cells were not found prenatally. The use of two paternal genetic markers, a cell surface antigen and nuclear Y chromatin, to identify fetal cells in maternal blood permits us to conclude that these cells are present in the mother's circulation, as early as 15 weeks gestation. Further development of the techniques reported here could lead to widespread screening of maternal blood samples during pregnancy for detection of fetal genetic abnormalities.     

Identification of triploid trophoblast cells in peripheral blood of a woman with a partial hydatidiform molar pregnancy

In a woman with a partial hydatidiform molar pregnancy with 69,XXY karyotype, the presence of male fetal cells of trophoblastic origin was demonstrated in maternal blood by X/Y-chromosome specific PCR and by immunostaining combined with FISH on two cell populations isolated from maternal blood. Blood was obtained three weeks prior to the detection of fetal demise, at 13 weeks' gestation. Results were confirmed on formalin-fixed paraffin-embedded molar tissue, removed at 16 weeks' gestational age for therapeutic reasons. The results indicate that both plasma and cells from maternal peripheral blood might be useful for non-invasive prenatal diagnosis of fetal aneuploidies, as described in the current case with a partial molar pregnancy. Copyright © 2001 John Wiley & Sons, Ltd.     

Prenatal testing for inherited immune deficiencies by fetal blood sampling

We attempted to develop a prenatal diagnosis in fetuses at risk for immunodeficiency by fetal blood sampling performed under fetoscopy at 18–22 weeks of gestation. In order to obtain normal values, we first investigated thirty-five control fetuses whose blood punctures were undertaken for the diagnosis of haemoglobinopathies. Surface markers and in vitro mitogen-induced proliferation of the fetal lymphocytes were studied using micromethods. We then examined two fetuses at risk for two different types of severe combined immunodeficiency and established their immunological integrity, hence avoiding an unjustified termination of pregnancy. This immunological integrity was confirmed after birth.     

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.     

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 of carbamoyl-phosphate synthetase deficiency by fetal liver biopsy

In a pregnancy at risk for carbamoyl-phosphate synthetase (CPS) deficiency, prenatal diagnosis was attempted by fetal liver biopsy, performed at 18 weeks of gestation. CPS activity was absent and the diagnosis was confirmed after termination of the pregnancy. The technique employed for fetal liver biopsy is described together with an evaluation of its possible role in prenatal diagnosis.     

Fetal cells in the uterine cervix: a source for early non-invasive prenatal diagnosis

Various non-invasive techniques for prenatal diagnosis have been under investigation. We evaluated the success of fetal sexing using a non-invasive technique for obtaining fetal cells, uterine cervix brushing, in combination with FISH. Thirty pregnant women who completed between 6 and 10 weeks of gestation and who were scheduled to undergo pregnancy termination were included in the study. A Pap smear cytobrush was inserted through the external os to a maximum depth of 2 cm and removed while rotating it a full turn. The material that was caught on the brush was spread on four microscope slides. Two-color FISH was used for fetal sexing. Following pregnancy termination, a placental sample was used for full karyotyping. In 29/30 cases FISH diagnosis was confirmed by chromosomal analysis. The only male case in which a Y chromosome was not seen was from a pregnancy of 6 weeks 6 days gestational age. One case was mosaic of 46,XY/47,XXY (25%). In most cases (7/13) the Y chromosome was already identified in the first analyzed slide. With the use of a cytobrush fetal cells can be easily obtained for the purpose of prenatal diagnosis of chromosomal disorders. Copyright © 2001 John Wiley & Sons, Ltd.     

Huntington disease–unaffected fetus diagnosed from maternal plasma using QF-PCR

The discovery of fetal DNA in maternal plasma from early pregnancies has led to new opportunities for clinical application. In the last few years there have been numerous reported applications, mainly fetal gender and RhD genotyping. The prenatal diagnosis of some inherited genetic diseases such as Huntington disease is also very frequently required in the prenatal diagnosis routine. We have successfully diagnosed, with a non-invasive procedure, an unaffected HD fetus at the 13th week of gestation using fetal DNA from maternal plasma and the quantitative fluorescent PCR method, which is one of the most sensitive ways to detect fetal DNA in maternal plasma at such an early time of gestation. Copyright © 2003 John Wiley & Sons, Ltd.     

Prenatal diagnosis of congenital Chagas' disease (American trypanosomiasis)

The prenatal diagnosis of congenital transmission of Chagas' disease in a pregnant woman with the indeterminate form of the disease is reported. Sonography revealed fetal hydrops at 31 weeks' gestation. Anti-Trypanosoma cruzi IgM and IgG antibodies were negative in the fetal blood sampled by cordocentesis, but T. cruzi trypomastigotes were found in its buffy coat. Owing to anemia, in utero exchange transfusion was undertaken, but fetal demise ensued. Labor was induced and a stillborn infant weighing 2030 g was delivered. The pathological examination revealed placentitis and meningoencephalitis, myocarditis and splenitis in the stillborn fetus. Amastigotes were found in the myocardium, brain and placenta. Copyright © 2004 John Wiley & Sons, Ltd.     

Prenatal diagnosis of haemoglobin disorders by cordocentesis at 12 weeks' gestation

Prenatal diagnosis of haemoglobin disorders is accepted to be a useful procedure to avoid births of infants with homozygous diseases. Advances in sampling and molecular techniques, such as polymerase chain reaction (PCR) and chorionic villus sampling (CVS), have made earlier and safer first-trimester prenatal diagnosis possible. However, these procedures need previous studies of at-risk couples, which can be very time-consuming when a number of different β-thalassaemia mutations occur in the region. We describe the possibility of making a first-trimester prenatal diagnosis by cordocentesis and fetal blood analysis at the 12th week of gestation. We found no statistically significant difference (p>0.05) between β/γ values in fetuses at the 12th and 18th weeks of gestation. In seven affected fetuses aborted at the 12th week of gestation, the diagnosis was confirmed in all cases by PCR analysis. These findings suggest that early cordocentesis could be an alternative procedure to CVS and PCR analysis.     

Detection of fetal cells in maternal blood

We report the detection of fetal cells in the maternal circulation by enzymatic amplification of a single copy gene sequence that was fetal-specific. Fetal HLA-A2-positive cells were sorted from maternal HLA-A2-negative cells by flow cytometry and confirmed by demonstration of a fetal-specific HLA-DR4 sequence. However, this sequence could not be detected in unenriched maternal DNA prepared at 28 and 32 weeks' gestation. The sensitivity of detection was 1 HLA-DR4-positive cell in 10⁵ HLA-DR4-negative cells. We conclude that prenatal diagnosis of paternally inherited autosomal-dominant genetic defects may be possible by selective gene amplification of maternal peripheral blood. However, preliminary enrichment for fetal cells may be necessary.     

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 findings on ultrasound and X-ray in a case of overgrowth syndrome associated with increased nuchal translucency

A case of prenatal diagnosis of an overgrowth syndrome at 30 weeks of gestation is reported. The diagnosis was suggested on the basis of increased fetal growth from 16 weeks onwards, advanced bone age, and characteristic facial features such as hypertelorism, broad forehead and small chin. The fetus presented at 12 weeks with a markedly increased nuchal translucency thickness and generalized skin edema, but normal karyotype. Serial ultrasound scans revealed brain abnormalities including mild unilateral ventriculomegaly and a cyst in the cavum septi pellucidi. The pregnancy was terminated at the parents' request at 32 weeks of gestation and postmortem examination confirmed the prenatal findings. This case demonstrates the possibility of prenatal diagnosis of early overgrowth syndromes and highlights the dilemma arising from the prenatal diagnosis of a non-lethal condition associated with an uncertain prognosis and poorly documented in utero. Copyright © 2001 John Wiley & Sons, Ltd.     

Transabdominal chorionic villus sampling: Fetal loss rate in relation to maternal and gestational age

In this paper we report the fetal loss rate in relation to both maternal and gestational age in 1764 pregnant women who underwent transabdominal chorionic villus sampling (TA-CVS) between January 1986 and August 1990. The fetal loss rate, considered as a proportion of continuing pregnancies, decreased with advancing gestational age at sampling from 4.3 per cent before 9 weeks to 0.4 per cent at or after 13 weeks, the difference being statistically significant (p <0.025). The fetal loss rate increased from 1.6 per cent in women under 30 to 2.4 per cent in women of 40 years or over, but the difference was not statistically significant. Considering that the total fetal loss rate before 28 weeks' gestation was on average 1.91 percent (1.3 per cent under 35 years and 2.8 per cent in women of 35 or over), we believe that TA-CVS is a safe and effective technique for prenatal diagnosis of genetic diseases.     

Tetrasomy 12p (Pallister-Killian syndrome): Ultrasound indicators and confirmation by interphase fish

Tetrasomy 12p (Pallister-Killian syndrome) is a mosaic aneuploidy syndrome in which the isochromosome is present in amniocytes with a much greater percentage than fetal lymphocytes. Two new cases identified by prenatal diagnosis are reported. Indications for prenatal diagnosis were advanced maternal age and fetal anomalies. The most consistent reported prenatal ultrasound findings for tetrasomy 12p include polyhydramnios with short femurs and a diaphragmatic hernia. Recognition of congenital malformation patterns prenatally may allow appropriate selection of tissue for chromosome analysis. Molecular cytogenetic analysis using fluorescence in situ hybridization was used retrospectively to confirm the presence of the isochromosome 12p in various formalin-fixed fetal tissues. The levels of mosaicism detected in fetal and placental tissues were lower than those detected prenatally.     

Molecular evidence of fetal-derived chromosome 21 markers (STRs) in transcervical samples

Transcervical cells (TCCs), collected by flushing or aspiration at 8–13 weeks of gestation, were analysed for the presence of fetal-derived DNA sequences. DNA extracted from maternal peripheral blood, TCC samples, and placental tissue was amplified by the polymerase chain reaction (PCR) to detect small tandem repeat (STR) markers specific to chromosome 21. STR products of fetal origin could be clearly observed in four TCC samples. TCC samples collected by flushing or aspiration were also analysed by fluorescent in situ hybridization (FISH) using X and Y probes simultaneously: 46,XY cells could be detected in all TCC samples obtained from mothers with male fetuses.     

Detection and enumeration of colonic mucosal cells in amniotic fluid using a colon epithelial-specific monoclonal antibody

Since its introduction, prenatal diagnosis of chromosomal and metabolic disorder by mid-trimester amniocentesis has relied upon the use of a mixture of fetal cells obtained from amniotic fluid. Little knowledge has been gained in the sorting of these cells for diagnosis of tissue-specific disorders. In an attempt to determine the contribution of fetal colonic mucosal cells to the overall amniocyte population, we used the colonic epithelial-specific monoclonal antibody (MC-Ab) 7E₁₂H₁₂, IgM isotype. Specimens of the small intestine, colon, buccal mucosa, kidney, urinary bladder, and umbilical cord were obtained from electively aborted normal fetuses of 12–28 weeks' gestation. All of these specimens were examined with 7E₁₂H₁₂ by the immunoperoxidase technique. The MC-Ab reacted with the colonic epithelial cells but not with any of the other tissues. In addition, 40 amniotic fluid samples obtained from women between 16 and 18 weeks of gestation, who underwent amniocentesis because of advanced maternal age, were tested using a fluorescent activated cell sorter. Among the amniotic fluid specimens examined, 18·4 ± 10·3 percent cells reacted with 7E₁₂H₁₂. Double immunofluorescence studies revealed that all Mc-Ab-stained cells contained secretory component, confirming that they were epithelial in origin. All fetuses whose amniotic fluid was analysed had normal karyotypes and amniotic fluid alpha-fetoprctein levels that were also normal. This study demonstrates that cell-specific Mc-Ab can be used to detect colon cells in the amniotic fluid and that colon cells contribute significant numbers in the mixture of amniotic fluid cells. This technique could be helpful in the prenatal diagnosis of disorders in which the flow of amniotic fluid through the fetal intestine is impaired, such as cystic fibrosis, imperforate anus, Hirschsprung aganglionic megacolon, and intestinal atresia.     

Prenatal diagnosis of lissencephaly

We report two cases of prenatal detection of lissencephaly by high-resolution ultrasound. The first case studied was referred for high-risk obstetrical management and serial antenatal ultrasounds because of a family history of lissencephaly in an unresolved chromosomal abnormality. Diagnosis of a smooth gyral pattern consistent with lissencephaly was made at 32 weeks' gestation. The second case was referred for prenatal ultrasound because of a size versus dates discrepancy. The ultrasound examination showed a smooth gyral pattern at 31.5 weeks. In light of this ultrasound finding, a fetal blood sample was obtained and a chromosomal abnormality reported, confirming the diagnosis. To our knowledge, these cases represent the first report of the sonographic prenatal diagnosis of cerebral agyria or lissencephaly.     

Embryonic and fetal globins are expressed in adult erythroid progenitor cells and in erythroid cell cultures

The understanding of human hemoglobin ontogeny during development is of biological and clinical importance. Molecular and immunocytological techniques were used to study the expression of embryonic zeta (ζ), epsilon (ε), and fetal gamma (γ) globin genes in newborn cord blood, peripheral blood from men, pregnant and non-pregnant women, and in vitro mononuclear cell cultures. We have shown that embryonic and fetal globin mRNA and peptides are expressed in cultured erythroid cells and in circulating blood cells from newborns, adult non-pregnant women and from men. The findings suggest that during erythroid cell differentiation in newborns and adults, there is a transient recapitulation of sequential globin chain expression as found during embryonic and fetal development. Furthermore, these findings underscore the need for caution in using embryonic and fetal globin chains as markers to identify erythroid cells of fetal origin in maternal circulation for prenatal diagnosis. Copyright © 2001 John Wiley & Sons, Ltd.