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.     

Possible effect of gestational age on the detection of fetal nucleated erythrocytes in maternal blood

Maternal venous blood samples, obtained from six pregnant women, were used as a source of fetal nucleated erythrocytes (NRBC). Fetal cell enrichment was potentiated by flow sorting with the monoclonal antibodies Tf R, Leu-4, and Leu-M3. Single copy Y chromosomal DNA sequences were detected in samples obtained from two women at 11 and 12 weeks' gestation. Y DNA sequences were absent in a subsequent sample from one of these women at 19 weeks and in two other women at 16 and 20 weeks. All four women delivered males. Y DNA sequences were not detected in two women who delivered females. By combining these results with prior data on the detection of Y chromosomal DNA sequences in maternal blood from male-bearing pregnancies, a relationship between gestational age and feta-maternal transfer of NRBC is suggested.     

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.     

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.     

Enrichment of fetal nucleated cells from maternal blood: Model test system using cord blood

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.     

An investigation of methods for enriching trophoblast from maternal blood

Trophoblast deportation is known to occur in normal human pregnancy, but it is not yet clear whether these cells routinely enter the maternal peripheral circulation and are available as a source of fetal DNA for non-invasive prenatal diagnosis of genetic disorders. To resolve this issue requires an efficient method of enriching trophoblast from maternal blood combined with a means to confirm its identity. Five different techniques were tested on ten retroplacental blood samples to determine the most sensitive and operator-efficient method. Lysis of red cells alone gave the best recovery of trophoblast but had to be discounted, together with Ficoll density gradient centrifugation, due to the very low purity and the excessive time required. Fluorescence-activated cell sorting (FACS) of pre-enriched trophoblast resulted in the lowest recovery rate (8 per cent) despite a 3250-fold enrichment and a very high purity. Immunomagnetic beads (Dynabeads) coated with anti-CD 16 antibody proved to be the best method for the subsequent immunocytochemical characterization of deported trophoblast. However, IO beads coated with anti-CD45 antibody may be more useful for isolating trophoblast for prenatal diagnosis due to the high purity, enrichment (32-fold), and recovery rate (78 per cent) obtained with this method.     

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.     

Demonstration of spontaneously dividing male fetal cells in maternal blood by negative magnetic cell sorting and fish

We investigated a case of massive feto-maternal bleeding by using negative magnetic cell sorting (MACS) and fluorescent in situ hybridization (FISH). A 37-year-old pregnant woman had an uncomplicated amniocentesis for advanced maternal age at 16 weeks' gestation. The fetal karyotype was 46, XY. At 19 weeks' gestation, she had a minor car accident and slight vaginal bleeding. A subsequent Kleihauer-Betke test showed a 140 ml feto-maternal haemorrhage. Serial sonographic examinations indicated a normal fetus and placenta. We performed FISH analysis on maternal peripheral blood at 25 weeks. Anti-CD45 and MACS were used to deplete maternal leucocytes, enriching the proportion of fetal nucleated erythrocytes present. The isolated cells were analysed by using dual-colour FISH with X and Y specific probes. Approximately 65 800 nucleated cells were obtained after MACS depletion. A total of 234 cells were analysed by FISH. The results revealed that 70 of the nucleated cells (30 per cent) were male with one X and one Y signal. Among these cells, six male metaphases were observed in spontaneously dividing cells.     

The time of appearance and disappearance of fetal DNA from the maternal circulation

A single copy Y-chromosome DNA sequence was amplified using the polymerase chain reaction (PCR) from the peripheral blood of 30 women who had achieved a pregnancy through an in vitro fertilization (IVF) programme. The time of conception was known precisely and was confirmed by serial ultrasound scans. Conceptions were dated as the number of weeks after fertilization plus 2, to give a time equivalent to the obstetric menstrual dating of the pregnancy (LMP). Y-chromosome-specific DNA was detected in all pregnancies with a male fetus (18/30). The earliest detection was at 4 weeks and 5 days, and the latest at 7 weeks and 1 day. Y-chromosome-specific sequences were no longer detected in any of the male pregnancies 8 weeks after delivery. No Y-chromosome sequences were detected in any of the pregnancies where only female babies were delivered. This demonstrates that fetal DNA appears in the maternal circulation early in the first trimester, that it can be identified in all pregnancies tested by 7 weeks, that it continues to be present throughout pregnancy, and that it has been cleared from the maternal circulation 2 months after parturition. Early non-invasive prenatal diagnosis for aneuploidies and inherited disorders will be possible in all pregnancies if fetal cells can be isolated free from maternal contamination (or identified accurately in the presence of maternal cells) without problems of contamination from previous pregnancies.     

Detection of fetal HLA-DQα sequences in maternal blood: A gender-independent technique of fetal cell identification

The objective of this study was to detect fetal HLA-DQα gene sequences in maternal blood. HLA-DQα genotypes of 70 pregnant women and their partners were determined for type A1. We specifically sought couples where the father, but not the mother, had genotype A1. In 12 women, maternal blood samples were flow-sorted. Candidate fetal cells were isolated and amplified by using PCR primers specific for a paternal HLA-DQα A1 allele. Fetal HLA-DQα A1 genotype was predicted from sorted cells; amniocytes or cheek swabs were used for confirmation. Six of twelve sorted samples had amplification products indicating the presence of the HLA-DQα A1 allele; 6/12 did not. Prediction of the fetal genotype was 100 per cent correct, as determined by subsequent amplification of amniocytes or cheek swabs. We conclude that paternally inherited uniquely fetal HLA-DQα gene sequences can be identified in maternal blood. This system permits the identification of fetal cells independent of fetal gender, and has the potential for non-invasive prenatal diagnosis of paternally inherited conditions.     

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.     

Extraction of DNA from amniotic fluid cells for the early prenatal diagnosis of genetic disease

Ten-ml samples of amniotic fluid were taken from pregnancies being terminated at 8–14 weeks' gestation. DNA was extracted from the amniotic cells by sequential centrifugation and analysed using the polymerase chain reaction (PCR). Fifteen samples were analysed for evidence of maternal contamination using Mfd5 oligo-nucleotide primers for repeat polymorphisms. Ten amniotic fluid samples were tested for the Delta-F₅₀₈ deletion characteristic of cystic fibrosis to demonstrate a diagnostic application for the technique. In each case, DNA extracted from fetal tissue from the same pregnancy was included in the controls. In 14 of the 15 cases tested with the Mfd5 primers, both the amniotic fluid DNA and the fetal DNA showed no evidence of contaminating DNA. In one case, neither the amniotic fluid cells nor the fetal cells yielded results. In nine of the ten cases tested with the Delta-F₅₀₈ primers, the amniotic fluid cell DNA provided accurate information about the genetic status of the fetus; in the tenth, the fetal DNA failed to amplify. The results indicate that adequate DNA can be extracted from amniotic fluid from 8 weeks' gestation onward and these samples are suitable for prenatal diagnosis using PCR.     

Isolating fetal nucleated red blood cells from maternal blood: The baylor experience—1995

In our previous work we have isolated fetal cells from maternal blood and used fluorescent in situ hybridization (FISH) for chromosome-specific probes to detect aneuploidy. Current efforts in the Baylor College of Medicine programme are focusing on obtaining consistency in flow-sorting methodology and on determining sensitivity and specificity. To this end, systematic evaluation of five glycophorin A (gly A) antibodies all produced agglutination, leading us to abandon the use of gly A antibodies for positive selection of fetal cells. Conversely, we have found LDS-751 to be useful for nuclear selection. CD45 negative selection can best be accomplished by the use of flasks coated with goat antibodies against mouse antibodies. Positive selection by flow sorting for either CD71⁺ cells or gamma-globin-positive cells seems to be successful. Using these two approaches, we have recently detected male (fetal) cells in pregnancies in which the fetus was 46, XY in 10 of 18 and in 12 of 14 cases, respectively.     

Flow sorting of fetal erythroblasts using intracytoplasmic anti-fetal haemoglobin: Preliminary observations on maternal samples

Monoclonal antibody to fetal haemoglobin (a₂γy₂) has been proposed as a fetal-specific reagent. We developed an intracellular staining protocol that combines fluorescein isothiocyanate or phycoerythrin conjugated anti-γ with the DNA binding dye Hoechst 33342 to identify and flow sort fetal erythroblasts from maternal blood. Our preliminary observations on anti-γ-positive cells sorted from four different pregnant women are described here, using fluorescence in situ hybridization (FISH) with chromosome-specific probes to identify fetal cells. Our data demonstrate that far fewer candidate fetal cells are sorted with this protocol than by current cell surface staining methods that employ the monoclonal antibody CD71. This results in increased fetal cell sorting purities. With this protocol, standard FISH techniques require modification due to the rigorous fixation with 4 per cent paraformaldehyde. Our initial data indicate the promise of this approach.     

First-trimester screening for fetal chromosomal abnormalities. Preliminary results

We have started a multicentre trial to study the possibilities of first-trimester maternal serum screening for fetal chromosomal abnormalities. Maternal blood samples were obtained before 13 weeks of gestation. We present the preliminary results of the first 950 patients on alpha-fetoprotein (AFP). Results on cancer antigen 125 (CA 125) in Down's syndrome and normal pregnancies are also presented. We conclude that the results on AFP are promising and that CA 125 might be predictive for fetal Down's syndrome.     

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.     

Incidence of abortion after genetic amniocentesis in twin pregnancies

Fetal outcome after genetic amniocentesis (AC) in viable twin pregnancies was analysed in a retrospective study at three centres in order to estimate the rate of fetal loss after AC. The maternal age ranged from 33 to 45 years (mean 36.7 years). The gestational age varied between 15 and 20 weeks of gestation (mean 17.1). In 98 viable twin pregnancies with complete follow-up, spontaneous abortion of both fetuses occurred within 28 completed weeks of gestation in eight pregnancies and six women aborted within 20 completed weeks of gestation after AC, corresponding to a rate of fetal loss of 8.1 and 6.1 per cent, respectively (excluding the loss of five twins with viable outcome of the co-twin in five pregnancies).     

Fetal blood group studies during mid-trimester pregnancy and the management of severe isoimmunization

Fetal blood samples collected at 14–22 weeks' gestation by fetoscopy have been analysed for 25 different red cell antigens and anti-A antibodies. The results obtained demonstrate that the technique can be used to determine fetal blood groups during mid-pregnancy and also to provide the opportunity to study the physiological and pathological developments of blood groups and their antibodies and to manage more rationally those pregnancies threatened by erythroblastosis fetalis.     

An audit of trisomy 16 in man

Sufficient information is now available from the literature to produce an audit of trisomy 16, in a theoretical cohort of 100 000 recognized pregnancies, from gametogenesis to term and onwards. Recent reports of premature separation of chromosome 16 bivalents during maternal meiosis I provide a novel mechanism for generation of this aneuploidy. Most, if not all, errors resulting in recognized mosaic and non-mosaic trisomy 16 pregnancies investigated using polymorphic DNA markers appear to originate at that stage. The incidence of this maternally derived trisomy 16 in the late first trimester is equivalent to 1500 cases in 100 000 recognized pregnancies, a figure which now corresponds very closely to the reinterpreted oogenesis data. Most trisomy 16 pregnancies are lost around 12 weeks' gestation, but of the order of 10 per cent (120–150 in this audit) undergo reduction to disomy, with 30 of these excluding aneuploidy from the fetal cell lineage (trisomic zygote rescue) and continuing into the second trimester. Maternal uniparental disomy (UPD) in one-third of this latter group is associated with loss later in pregnancy or severe intrauterine growth retardation, but can be compatible with a viable pregnancy. Adverse pregnancy outcomes are not restricted to those with UPD. Analysis of reports of confined placental mosaicism for chromosome 16 without associated UPD indicates that the presence of high levels of trisomic cells in the placenta alone consistently produces a more variable inhibition of fetal growth, which may also, in cases, be associated with late pregnancy loss.     

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.