Noninvasive prenatal diagnosis of chromosomal aneuploidies by isolation and analysis of fetal cells from maternal blood.

The isolation and analysis of nucleated fetal cells (NFCs) from maternal blood may represent a new approach to noninvasive prenatal diagnosis. Although promising, these techniques require highly accurate separation of NFCs from nucleated cells of maternal origin; the two major problems limiting these techniques are the relative rarity of fetal cells in maternal blood and the need to establish their fetal origin. We now report a novel procedure that has allowed accurate separation of NFCs from maternal cells. The technique reported involves direct micromanipulator isolation of histochemically identified hemoglobin F-positive nucleated cells to obtain fetal nucleated red blood cells (FNRBCs) of high yield and purity. Using this technique, followed by cell-by-cell multicolor fluorescence in situ hybridization (FISH) analysis of purified FNRBCs, we were able to detect some of the most common human aneuploidies (including Down syndrome, Klinefelter syndrome, and trisomy 13) in 33 pregnant women referred for amniocentesis. The procedure used, which can be completed in <72 hrs, produced complete concordance with the results of amniocentesis. We also confirm findings of prior studies suggesting that the number of FNRBCs in maternal circulation is remarkably higher in abnormal pregnancies than in normal pregnancies, especially in women carrying a fetus with trisomy 21.     

Quantification of all fetal nucleated cells in maternal blood in different cases of aneuploidies

We quantified all fetal nucleated cells (FNCs) per unit volume of maternal blood in different aneuploid pregnancies using molecular cytogenetic techniques. Seven cases of male trisomy 18, two triploidies (69,XXX), two 47,XXX, one 47,XXY, one 47,XYY, one male trisomy 13, and one case of 47,XY,r(22),+r(22) were analyzed. Whole blood samples were obtained from 15 women between 17 and 29 gestational weeks and harvested without using fetal cell enrichment procedures. Fluorescence in situ hybridization and primed in situ labeling were performed to identify the FNCs. All slides were manually scanned to quantify those cells. We have identified 4-20 FNCs/ml of maternal blood in the cases of trisomy 18; 10 and 25 FNCs/ml in the two cases of triploidy; 16 and 14 FNCs/ml, respectively, in the two X trisomies; 19 FNCs/ml in the 47,XXY; 26 FNCs/ml in the 47,XYY; nine FNCs/ml in the trisomy 13; and 10 FNCs/ml in the case of r(22). To detect all FNCs in all aneuploid pregnancies, we have used a very simple method that minimizes the manipulation steps to avoid losing fetal cells. The number of FNCs identified in aneuploid pregnancies was 2-5 times higher than in normal pregnancies. This higher number of FNCs will favor the design of a non-invasive pre-natal test.     

异常妊娠孕妇外周血中胎儿细胞的数量研究

目的研究异常妊娠孕妇外周血中胎儿有核红细胞的数量变化。方法富集孕妇外周血中胎儿有核红细胞后计数,再用PEP—PCR技术鉴别其来源。比较中孕期妊高征组、胎儿畸形组、死胎组与正常妊娠组孕妇外周血中胎儿有核红细胞的数量有无差异。结果3组异常妊娠样本中的胎儿有核红细胞的数量均明显高于正常妊娠组（P〈0．05）。3组异常妊娠组的胎儿有核红细胞数量之间无明显差异（P〉0．05）。结论发生妊高征、胎儿畸形与死胎的孕妇外周血中胎儿有核红细胞的数量异常升高,起一定的标志作用。     

孕妇外周血中胎儿有核红细胞在性连锁遗传病产前诊断中的初步应用

目的：探讨利用母血胎儿有核红细胞进行性连锁遗传病产前诊断的可行性;方法：通过密度梯度离心富集母血胎儿有核红细胞,并经流式细胞仪鉴定,对富集后胎儿有核红细胞进行了ＳＲＹ 基因的套式ＰＣＲ检测;结果：５ 例有性连锁遗传病家族史的孕妇外周血中经扩增后３ 例阳性、２ 例阴性,所得结果均得到相应羊水、胎盘ＤＮＡＳＲＹ／ＰＣＲ 检测或出生胎儿生殖器检查结果的证实;结论：母血中胎儿有核红细胞可用于性连锁遗传病的早期产前诊断     

Fetal cells in maternal blood of pregnancies with severe fetal growth restriction

The aim of this study was to examine whether, in pregnancies with severe early onset fetal growth restriction, the number of fetal erythroblasts in maternal blood is increased. The percentage of fetal erythroblasts in maternal blood, enriched by triple density gradient centrifugation and anti-CD71 magnetic cell sorting, was determined in 10 singleton pregnancies with severe intrauterine growth restriction in which there was Doppler ultrasound evidence of impaired placental perfusion. The values were compared to those of 10 normal pregnancies at the same gestational range of 22-26 weeks. In the growth restricted pregnancies the median number of fetal erythroblasts per 100 nucleated cells in maternal blood enriched for fetal cells was significantly higher than the median value in the control pregnancies (8.5% compared with 1%; P < 0.001). These data suggest that impaired uteroplacental perfusion and severe fetal growth restriction may be associated with placental damage leading to increased feto-maternal cell traffic. Alternatively the rate of transfer of fetal cells into the maternal circulation is not altered but in growth restriction the proportion of fetal erythroblasts in fetal blood is increased.     

用荧光原位杂交从母血中检测胎儿细胞

目的 从母血中分离胎儿细胞并确定其来自胎儿。方法 从孕早、中期各２０名、分娩后１５名母血中富集并分离有核细胞。用Ｙ特异性探针（ＰＹ３．４）行荧光原位杂交,从中识别胎儿细胞。结果 孕早、中期孕妇各怀１５名男胎。阳性细胞比例是１：６５２８．０及１：２７３．８。与同期１０名女胎阳性细胞相比,差别有高度显著性。分娩１周内的３名,阳必民孕中期的差别没有显著性。分娩３个月后的阳性率与比,差别有高度显著性。分娩     

Nucleated erythrocytes in maternal blood: quantity and quality of fetal cells in enriched populations

The discovery of nucleated erythrocytes in maternal circulationprovides a potential source for non-invasive prenatal diagnosis.We have evaluated the use of a three-stage procedure to determinethe number of cells that are of fetal rather than maternal origin.First, monoclonal antibodies specific for CD45 and CD14 wereused in conjunction with a magnetic (MACS) column to depleteunwanted leukocytes from maternal blood. This was followed bya positive MACS enrichment for nucleated erythrocytes, usingan anti-CD71 (transferrin receptor) monoclonal antibody. Todiscriminate between fetal nucleated erythrocytes and thoseof maternal origin, enriched fractions were simultaneously stainedwith an anti-fetal haemoglobin (HbF) antibody and hybridizedwith probes specific for X and Y chromosomes. Samples were thensubjected to blind analysis along with negative control samplesfrom non-pregnant volunteers. Using this dual analysis, we wereable to determine that less than one nucleated erythrocyte perml of maternal blood was of fetal origin. Small numbers of thesefetal cells were found in 87.5% of pregnancies, ranging from6 to 35 weeks gestational age. Comparison of HbF and X/Y probedata also suggests that the fetal cells are less suitable forfluorescence in-situ hybridization (FISH) analysis than similarpreparations from other sources. cell separation methods/fluorescence in-situ hybridization/hereditary diseases/polymerase chain reaction/pregnancy     

Detection of chromosomal aneuploidies in fetal cells isolated from maternal blood using single-chromosome dual-probe FISH analysis

Detection of chromosomal aneuploidies using fetal cells isolated from maternal blood, for prenatal non-invasive genetic investigation, has been a long-sought goal of clinical genetics to replace amniocentesis and chorionic villous sampling to avoid any risk to the fetus. The purpose of this study was to develop a sensitive and specific new assay for diagnosing aneuploidy with circulating fetal cells isolated from maternal blood as previously reported using two novel approaches: (i) simultaneous immunocytochemistry (ICC) evaluation using a monoclonal antibody for i-antigen, followed by fluorescence in situ hybridization (FISH); (ii) dual-probe FISH analysis of interphase nuclei using two differently labeled probes, specific for different loci of chromosomes 21 and 18; in addition, short tandem repeats (STR) analysis on single cells isolated by micromanipulation was applied to confirm the presence of fetal cells in the cell sample enriched from maternal blood. Blood samples were obtained from women carrying trisomic fetuses, and from non-pregnant women and men as controls. Using ICC-FISH approach, a large heterogeneity in immunostaining pattern was observed, which is a source of very subjective signal interpretation. Differently, dual-probe FISH analysis provided for a correct diagnosis of all pregnancies: the mean percentage of trisomic cells was 0.5% (range, 0.36-0.76%), while the mean percentage of trisomic cells in the control group (normal pregnancies or non-pregnant women) was ≤0.20%. The application of the dual-probe FISH protocol on fetal cells isolated from maternal blood enables accurate molecular detection of fetal aneuploidy, thus providing a foundation for development of non-invasive prenatal diagnostic testing.     

Isolation of fetal nucleated red blood cell from maternal blood using immunomagnetic beads for prenatal diagnosis

The immunomagnetic beads method for isolation of fetal nucleated red blood cells (FNRBCs) from peripheral blood of 78 pregnant women for prenatal diagnosis was developed. The study subjects were classified into 8-10 and 11-14 weeks of gestation (n = 39 each). Peripheral blood cells were divided into two for the FNRBCs isolation using two protocols, one with anti-CD45 depletion followed by anti-CD71 and anti-GPA monoclonal antibodies and another without CD45 depletion. The use of CD45 depletion gave a slightly higher number of sorted cells but not significantly different (p > 0.05). The percentage of CD71+ and GPA+ cells obtained from 8-10 weeks and 11-14 weeks of gestation was not different (p > 0.05). The sensitivity in determining the sorted FNRBCs for male fetal sex by PCR using 8-10 and 11-14 weeks of gestation was generally 50 and 69%, respectively. The method so developed is simple and cost effective and may thus be applied for prenatal diagnosis.     

Investigation of maternal blood enriched for fetal cells: Role in screening and diagnosis of fetal trisomies

Prenatal diagnosis of chromosomal abnormalities relies on assessment of risk followed by invasive testing in the group with highest risk. Assessment of risk by a combination of maternal age and fetal nuchal translucency and invasive testing in the 5% of the population with the highest risk would identify about 80% of trisomy 21 pregnancies. Preliminary reports suggest that chromosomal abnormalities can also be diagnosed by fluorescent in situ hybridization (FISH) in maternal blood enriched for fetal cells. This study examines the potential role of this method on the prenatal diagnosis of fetal trisomies. Maternal blood was obtained before invasive testing in 230 pregnancies at 10–14 weeks of gestation. After enrichment for fetal cells, by triple density centrifugation and anti-CD71 magnetic cell sorting, FISH was performed and the proportion of cells with positive signals in the chromosomally normal and abnormal groups was determined. Fetal karyotype was normal in 150 cases and abnormal in 80 cases, including 36 with trisomy 21. Using a 21 chromosome-specific probe, three-signal nuclei were present in at least 5% of the enriched cells from 61% of the trisomy 21 pregnancies and in none of the normal pregnancies. For a cut-off of 3% of three-signal nuclei the sensitivity for trisomy 21 was 97% for a false positive rate of 13%. Similar values were obtained in trisomies 18 and 13 using the appropriate chromosome-specific probe. Examination of fetal cells from maternal blood may provide a noninvasive prenatal diagnostic test for trisomy 21 with the potential of identifying about 60% of affected pregnancies. Alternatively, this technique can be combined with maternal age and fetal nuchal translucency as a method of selecting the high-risk group for invasive testing. Potentially, 80% of trisomy 21 pregnancies could be identified after invasive testing in less than 1% of the pregnant population. Am. J. Med. Genet. 85:66–75, 1999. © 1999 Wiley-Liss, Inc.     

Quantification of all fetal nucleated cells in maternal blood between the 18th and 22nd weeks of pregnancy using molecular cytogenetic techniques

Different types of nucleated fetal cells (trophoblasts, erythroblasts, lymphocytes, and granulocytes) have been recovered in maternal peripheral blood. In spite of many attempts to estimate the number of fetal cells in maternal circulation, there is still much controversy concerning this aspect. The numbers obtained vary widely, ranging from 1 nucleated cell per 104 to 1 per 109 nucleated maternal cells. The purpose of our project was to determine the absolute number of all different types of male fetal nucleated cells per unit volume of peripheral maternal blood. Peripheral blood samples were obtained from 12 normal pregnant women known to carry a male fetus between 18 and 22 weeks of pregnancy. Three milliliters (3 ml) of maternal blood has been processed without any enrichment procedures. Fluorescence in situ hybridization (FISH) and primed in situ labeling (PRINS) were performed, and fetal XY cells were identified (among maternal XX cells) and scored by fluorescent microscopy screening. The total number of male fetal nucleated cells per milliliter of maternal blood was consistent in each woman studied and varied from 2 to 6 cells per milliliter within the group of normal pregnancies. The number of fetal cells in maternal blood, at a given period, is reproducible and can therefore be assessed by cytogenetic methods. This confirms the possibility of developing a non-invasive prenatal diagnosis test for aneuploidies. Furthermore, we demonstrate that it is possible to repeatedly identify an extremely small number of fetal cells among millions of maternal cells.     

Isolating fetal cells in the maternal circulation

Fetal cells exist in maternal blood and can be utilized forprenatal genetic diagnosis. The use of polymerase chain reaction(PCR) technology on maternal blood has enabled the detectionof fetal sex, Mendelian disorders (e.g. (rß-globinmutations), HLA polymorphisms and fetal Rhesus (D) blood type.Enrichment for erythro-blasts and trophoblasts by various densitygradient and flow sorting techniques followed by fluorescencein-situ hybridization (FISH) with chromosome-specific DNA probeshas allowed detection of trisomy 21, trisomy 18, Klinefeltersyndrome (47,XXY) and 47,XYY. The fetal cell type that has generatedthe most success is the nucleated erythrocyte; however, trophoblasts,lymphocytes and granulocytes are also considered to be presentin maternal blood. Fetal cells circulate in maternal blood duringthe first and second trimesters, with frequency increasing asgestation advances. Emphasis is thus now directed toward determiningthe most practical and efficacious manner for this techniqueto be applied to prenatal genetic diagnosis. A large scale collaborationof clinical evaluations is underway in the USA, upon completionof which assessment can be made of whether this technology canserve as an alternative to conventional invasive and non-invasivemethods of prenatal cytogenetic diagnosis.     

Prenatal diagnosis from maternal blood: simultaneous immunophenotyping and FISH of fetal nucleated erythrocytes isolated by negative magnetic cell sorting.

Fetal nucleated cells in the maternal circulation constitute a potential source of cells for the non-invasive prenatal diagnosis of fetal genetic abnormalities. We have investigated the use of the Magnetic Activated Cell Sorter (MACS) for enriching fetal nucleated erythrocytes. Mouse monoclonal antibodies specific for CD45 and CD32 were used to deplete leucocytes from maternal blood using MACS sorting, thus enriching for fetal nucleated erythrocytes which do not express either of these antigens. However, significant maternal contamination was present even after MACS enrichment preventing the accurate analysis of fetal cells by interphase fluorescence in situ hybridisation (FISH). To overcome this problem, we used simultaneous immunophenotyping of cells with the mouse antifetal haemoglobin antibody, UCH gamma, combined with FISH analysis using chromosome X and Y specific DNA probes. This approach enables selective FISH analysis of fetal cells within an excess of maternal cells. Furthermore, we have confirmed the potential of the method for clinical practice by a pilot prospective study of fetal sex in women referred for amniocentesis between 13 and 17 weeks of gestation.     

Diagnosing and preventing inherited disease: Nucleated erythrocytes in maternal blood: quantity and quality of fetal cells in enriched populations

The discovery of nucleated erythrocytes in maternal circulationprovides a potential source for non-invasive prenatal diagnosis.We have evaluated the use of a three-stage procedure to determinethe number of cells that are of fetal rather than maternal origin.First, monoclonal antibodies specific for CD45 and CD14 wereused in conjunction with a magnetic (MACS) column to depleteunwanted leukocytes from maternal blood. This was followed bya positive MACS enrichment for nucleated erythrocytes, usingan anti-CD71 (transferrin receptor) monoclonal antibody. Todiscriminate between fetal nucleated erythrocytes and thoseof maternal origin, enriched fractions were simultaneously stainedwith an anti-fetal haemoglobin (HbF) antibody and hybridizedwith probes specific for X and Y chromosomes. Samples were thensubjected to blind analysis along with negative control samplesfrom non-pregnant volunteers. Using this dual analysis, we wereable to determine that less than one nucleated erythrocyte perml of maternal blood was of fetal origin. Small numbers of thesefetal cells were found in 87.5% of pregnancies, ranging from6 to 35 weeks gestational age. Comparison of HbF and X/Y probedata also suggests that the fetal cells are less suitable forfluorescence in-situ hybridization (FISH) analysis than similarpreparations from other sources.     

孕妇外周血胎儿有核红细胞的分离与检测

目的从孕妇血循环中分选出有核红细胞（ＮＲＢＣｓ），并证实其是胎儿来源的。方法选用红细胞特异单克隆抗体ＧｌｙｃｏｐｈｏｒｉｎＡ（ＧＰＡ）结合流式细胞术成功地从３例妊娠１６～２４周的孕妇血中分离出ＮＲＢＣｓ，并用Ｙ－染色体特异重复序列ＤＮＡ探针（ＰＹ３．４）荧光原位杂交及聚合酶链反应技术检测Ｙ－染色体特异序列（ＳＲＹ）结果ＦＩＳＨ结果，３例分选出的ＧＰＡ阳性细胞中ＰＹ３．４杂交阳性率分别是３１．４％、０、３８％；其ＰＣＲ结果，分别为阳性、阴性、阳性，并在引产后得到性别证实。结论孕妇外周血中确实存在胎儿有核红细胞，采用非创伤性方法可以分离并检测它们     

Fetal cells in maternal blood

Fetal lymphocytes, trophoblasts, and nucleated red blood cells have each been separated from maternal blood by methods such as flow cytometry, magnetic cell sorting, and charge flow separation. The frequency of fetal cells among circulating maternal mononuclear cells remains to be ascertained. Current estimates range from about 10-5 to 10-7, but the numbers may be increased in women carrying aneuploid fetuses. Fetal cells separated from maternal blood have been studied by methods such as polymerase chain reaction and fluorescence in situ hybridization. Among fetal conditions so far identified are sex; human leukocyte antigen and Rh blood types; trisomy 13, 18 and 21; triploidy; and sickle cell anemia and thalassemia. Thus, fetal cell separation might one day be used for screening of the common aneuploidies and, ultimately, for prenatal diagnosis. Individual fetal erythroid precursors have been cultured after separation in some laboratories. Culturing and karyotyping of separated fetal cells might enable diagnosis of a spectrum of chromosomal and genetic disorders. Further development will be required, however, before regular clinical application of these methodologies.     

母血中胎儿有核红细胞计数与宫内感染的关系

目的探讨母血中胎儿有核红细胞数量与巨细胞病毒宫内感染之间的关系。方法收集54例巨细胞病毒宫内感染孕妇和68例非感染孕妇的外周血样本,富集胎儿有核红细胞后计数。每例样本取2个胎儿有核红细胞并用PEP-PCR技术证实其胎源性。结果宫内感染组的胎儿有核红细胞数量与100个有核细胞数的比值显著高于非感染组(P<0.05)。结论巨细胞病毒宫内感染孕妇外周血中的胎儿有核红细胞数量比正常妊娠者增高,对进一步阐明该病的病生机制具有一定的意义。     

Fetomaternal cell trafficking: A new cause of disease?

Isolation of fetal cells from maternal blood is under active investigation as a noninvasive method of prenatal diagnosis. In the context of studying cell surface antigens expressed on fetal cells we discovered that fetal cells from a prior pregnancy also could be detected. This led to the appreciation of the persistence of fetal cells in maternal blood for as long as 27 years postpartum, and the realization that following pregnancy, a woman becomes a chimera. Quantitative polymerase chain reaction analyses have shown that a term pregnancy is not required for the subsequent development of fetal cell microchimerism. As many as 500,000 fetal nucleated cells are transfused following an elective first trimester termination of pregnancy. The relationship between fetal cell microchimerism and maternal disease is currently being explored. During pregnancy, fetal cells in the maternal skin are related to polymorphic eruptions of pregnancy and increased fetomaternal trafficking is detectable in cases of preeclampsia. After delivery, more male DNA of presumed fetal origin is present in the blood and skin of women with scleroderma as compared with healthy controls. Scleroderma is of particular interest because it shows a strong female predilection and it is an autoimmune disease with clinical similarities to graft‐versus‐host disease. Fetomaternal cell trafficking provides a potential explanation for the increased prevalence of autoimmune disorders in adult women following their childbearing years. Am. J. Med. Genet. 91:22–28, 2000. © 2000 Wiley‐Liss, Inc.     

Reproductive behaviour following spontaneous loss of a pregnancy after prenatal diagnosis

One hundred and fifty-eight women of advanced maternal age with complete follow up who experienced spontaneous fetal loss after prenatal diagnosis were studied for reproductive behaviour as well as prenatal diagnosis in a subsequent pregnancy. A higher rate of subsequent pregnancies amongst women who experienced an early spontaneous abortion after chorionic villus sampling (CVS) was expected compared with women who lost a pregnancy later during pregnancy after amniocentesis. Of the 92 women who underwent CVS in a previous pregnancy, 57 (62%) became pregnant again. Of the 66 women who underwent amniocentesis in the pregnancy that ended in fetal loss, 34 women (52%) had a subsequent pregnancy. The cumulative incidence of subsequent pregnancies was significantly influenced by maternal age but not by parity or the method of prenatal testing. Most women who decided on a new pregnancy opted for prenatal diagnosis. There was a preference for amniocentesis if the patient had previously undergone CVS. However, the reverse was not the case.     

Screening for fetal Down's syndrome in pregnancy by measuring maternal serum alpha-fetoprotein levels

Although the risk of Down's syndrome increases with maternal age, women under 35 bear about 80 percent of the infants born with this condition. We prospectively investigated the utility of measuring maternal serum alpha-fetoprotein during the second trimester in women under 35 in order to identify pregnancies in which the fetus was affected with Down's syndrome. Over a two-year period, 34,354 women in this age group were screened. Amniocentesis was offered when the risk of Down's syndrome, calculated as a function of maternal age and maternal serum alpha-fetoprotein concentration adjusted for maternal weight and race, was 1:270 or higher, the risk for a 35-year-old woman. This threshold was exceeded in 1451 women in whom gestational age was confirmed by ultrasound; 9 women in this group had a fetus with the syndrome. In three women whose fetuses had trisomy 18 and one whose fetus had trisomy 13, the calculated risk of Down's syndrome was 1:270 or higher. Thus, among women in whom the risk exceeded our cutoff point, 1 in 161 were found to have a pregnancy in which the fetus was affected with Down's syndrome; the figure was 1 in 112 for all autosomal trisomies. Eighteen pregnancies involving Down's syndrome, three involving trisomy 18, and two involving trisomy 13 were not associated with a calculated risk above the cutoff point. The available data indicate that in our population, using a cutoff for risk at which 5 percent of women under 35 are offered amniocentesis, we will detect one quarter to one third of pregnancies in which the fetus has Down's syndrome.