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W2028115197 abstract "Over the past 25 years the utilization of prenatal diagnosis by expectant couples and their physicians has expanded due primarily to two trends: smaller family size, with an increased emphasis on assurance of the ‘normalcy’ of each child, and advancing parental age. In the United States it is currently considered ‘standard of care’ to offer prenatal cytogenetic diagnosis to pregnant women who will be 35 years or older at the time of delivery (3). Such cytogenetic diagnoses are facilitated by obtaining fetal nucleated cells via an invasive technique such as chorionic villus sampling (CVS) or amniocentesis. 35 years of age was established as the threshold for these procedures because the liveborn incidence of an infant with the most common autosomal aneuploidy, trisomy 21, is roughly equal to the chance of a miscarriage secondary to these procedures (approximately 1 in 250). Despite the safety and accuracy of these techniques, to date there has been little impact upon the incidence of trisomy 21, which is still close to 1 per 1000 live births. A major reason for this is that prenatal diagnostic techniques are directed towards a minority of pregnant women. Although, individually, older pregnant women are at increased risk for having a baby with trisomy 21, as a group they are having a relatively small fraction of the total births. 80% of the newborn infants with Down syndrome are born to women under age 35, who are not offered the invasive prenatal diagnostic techniques because the risk of a procedural complication is greater than the incidence of Down syndrome in a given fetus. Therefore, over the past decade, increased attention has been paid to noninvasive techniques of screening for fetal trisomy 21 that can be offered to all pregnant women. Trisomy 21 is used as a benchmark because it is the most common liveborn aneuploidy associated with mental retardation and serious congenital anomalies. At present, screening for trisomy 21 consists initially of assessing the maternal age, because the risk of fetal chromosomal abnormalities due to nondisjunction increases as maternal age advances. In addition, second-trimester maternal serum screening for proteins such as human chorionic gonadotropin (hCG), oestriol, and alphafetoprotein (AFP) has been incorporated into routine obstetric care. Results are measured in absolute values, expressed in terms of multiples of the median, and interpreted as a mathematical risk for fetal trisomy 21. Women who have a test result that indicates a fetal Down syndrome risk of greater than 1 in 270 are offered amniocentesis. Because of the large-scale success of the serum-screening programmes which have been employed in the United States, Europe and Asia, research has been directed towards improving both their sensitivity and specificity. Current tests detect 60–70% of the cases of trisomy 21 with a calculated false positive rate of 5% (117). Addition of another marker such as dimeric inhibin A raises the sensitivity to 75% (118). More recently, a study measuring serum markers expressed during the first trimester, β-hCG and pregnancy-associated plasma protein A (PAPP-A), concluded that the sensitivity of screening at 10–13 weeks of gestation is nearly as good as the second-trimester test (38). An independent and newer method to screen for fetal trisomy 21 is the sonographic assessment of a fluid-filled space at the back of the fetal neck known as the nuchal translucency (NT) measurement. An increased NT measurement over that expected for gestational age is associated with an increased risk of fetal chromosome and cardiac abnormalities (73; 50). In a study of 96 127 singleton pregnancies, the combination of maternal age and NT measurement enabled detection of 77% of the fetuses with trisomy 21 in the 5% of patients with the largest NTs (99). However, by recommending amniocentesis or CVS following a positive screening test, 30 invasive tests are required to find one affected fetus. According to an editorial that accompanied the above study, antenatal screening research for Down syndrome is likely to place more emphasis on lowering the false-positive rate to 1% or less by discovering new markers and reconfiguring existing screening techniques (37). It is into the existing context of noninvasive screening for Down syndrome that fetal cells in maternal blood must be placed. Successful isolation of fetal cells from maternal blood represents a source of fetal chromosomes or DNA obtained non-invasively by maternal venepuncture. This test could be used as a primary screen, a secondary screen designed to be used in concert with the aforementioned screening tests (to reduce the 5% false-positive rate) (30), or, ultimately, as a diagnostic test. This review will summarize the key areas related to this field: the clinical implications of this test for the clinical practice of obstetrics and gynaecology, the surprising insights into the biology of pregnancy that have come from the study of fetal cells in maternal blood, and the technical challenges associated with rare event cell separation. Several recent papers and chapters have discussed historical aspects of the study of fetal cells in the maternal circulation, beginning with Schmorl's 1893 description of the deportation of trophoblast sprouts into the pulmonary circulation of a woman who died of eclampsia (89; 39; 6). For many years the existence of fetal cells in maternal blood was considered controversial. Definitive proof was not possible until the development of sensitive molecular techniques of genetic analysis, such as the polymerase chain reaction (PCR) and fluorescence in situ hybridization (FISH). Today, a consensus exists that fetal cells are present in maternal blood but either their relative rarity or fragility make it difficult (but not impossible) to isolate and physically identify them. Many investigators have created novel approaches to distinguish between immature maternal and fetal cells when mixed in a maternal blood sample. The fetal leucocyte ushered in the modern era of fetal cells in maternal blood in a landmark study described by 119). These investigators demonstrated the presence of a Y chromosome in mitogen-stimulated lymphocytes obtained from pregnant women who were carrying male fetuses. In the early 1970s several other reports appeared, using primarily interphase analysis by quinacrine-staining of the Y body. Two papers demonstrated the presence of these apparent male lymphocytes in the blood of pregnant women 1 and 5 years after the birth of a male infant, respectively (88; 24). These studies implied that the fetal leucocyte could persist post-partum. Fetal leucocytes were used as a target for flow-sorting experiments described by 47). This group used monoclonal antibody to HLA-A2, and flow-sorted fetal HLA-A2+ cells in pregnant women who did not have this antigen. Fetal gender and HLA type were successfully predicted with a highly significant probability (51). Despite the apparently good results, clinical applications were considered to be somewhat impractical due to the necessity of performing HLA typing of both parents prior to flow sorting. Further studies of fetal leucocytes in the maternal circulation have been somewhat limited by the lack of monoclonal antibodies specific for uniquely fetal leucocyte antigens and the concern regarding whether a particular leucocyte originates from a previous pregnancy. Presently most investigators are studying cell types that are terminally differentiated, such as trophoblast sprouts and nucleated erythrocytes. Trophoblast sprouts are particularly attractive because of their unique morphology, which permits definitive microscopic identification. Trophoblasts are commonly shed into the maternal circulation extensively during the first trimester, but in a normal pregnancy they are cleared rapidly by the pulmonary circulation. Investigators who are focusing on the isolation of trophoblasts from the maternal circulation have confronted several difficulties, including the apparent paucity of monoclonal antibodies that are specific for trophoblast markers (25, 26; 5). Previous problems resulting from the adsorption of trophoblast antigens onto the cell surface of maternal leucocytes may be overcome with some of the newer markers, such as HASH-2, human placental lactogen, or HLA-G (68; 112; 54). A fundamental difficulty appears to be the fact that trophoblast sprouts are not generally detectable in maternal blood when the pregnancy is normal (85). Trophoblasts are detectable, however, when the mother has pre-eclampsia, although it is not known whether this is a cause or an effect of the maternal hypertension. A further concern with the use of trophoblasts is the fact that they are part of the placenta, which is known from chorionic villus sampling studies to have a 1% incidence of chromosomal mosaicism. Therefore genetic analysis of a placental trophoblast sprout might not be fully representative of the fetal karyotype. Despite these technical and biological concerns, some groups have successfully isolated trophoblasts from the maternal circulation and performed morphologic and genetic diagnoses (69; 44, 45). The need for a fetal cell target with a limited life-span and morphologically distinguishing characteristics from the counterpart maternal cell led to the choice of the nucleated erythrocyte (NRBC). Other reasons included the following: NRBCs have a full complement of nuclear genes, NRBCs are abundant in first-trimester fetal blood, during the yolk sac and liver phases of haemopoiesis, and the erythrocyte line develops earlier in gestation than the white cell line. If a given fetomaternal transfusion is reflective of fetal blood, a 1000 to 1 red cell to white cell ratio is present, theoretically making more fetal red cells available for isolation. In 1990 we flow-sorted fetal erythrocytes from the peripheral blood of pregnant women on the basis of CD71 (transferrin receptor) expression (7). We proved that the cells were fetal in origin by demonstrating that they contained fetal haemoglobin and by PCR analysis using primers that amplified a section of the Y chromosome. Since then, many other groups have isolated fetal nucleated erythrocytes from maternal blood (32; 124; 95; 101). Initially, we erroneously believed that all NRBCs isolated from a maternal sample were fetal in origin. This was because reports such as that of 79) stated that NRBCs were rarely seen in peripheral blood samples from pregnant women. The newer and more sensitive techniques of enrichment used to detect fetal cells have uncovered a previously under-appreciated population of maternal NRBCs that circulate during pregnancy (98, 97). In contrast to the leucocyte, many cell surface and cytoplasmic markers exist that can differentiate between immature and mature erythrocytes (126). Markers such as embryonic haemoglobin, although completely specific for fetal cells, are only expressed during a narrow window of time (67). The widespread availability of monoclonal antibodies to erythrocyte antigens, the relative ease of NRBC enrichment protocols, and the demonstration of ‘proof of principle’, i.e. successful cytogenetic diagnosis in fetal NRBCs isolated from maternal blood (83; 9; 31; 94) have led to the NRBC as the target cell type of choice for most workers in this field. To date, almost all studies have focused on complete and intact fetal cells in the maternal circulation, so that their nuclei are available for either cell culture or FISH analysis. Recently, however, 59, 65) demonstrated surprisingly high mean concentrations (3.4–6.2%) of fetal DNA in maternal plasma and serum DNA at term, with mean fetal DNA concentrations increasing 12-fold over the course of the pregnancy. Results, expressed as copies of SRY, a single-copy Y chromosome-specific sequence, indicated that significantly more fetal DNA was present in the serum and plasma than prior studies using intact fetal cells would indicate. Mechanisms that could explain these findings include: continuous leakage of fetal cells across the placenta that are rapidly destroyed by the maternal immune system, leaving DNA remaining in the plasma or active remodelling of the placenta at the feto-maternal interface with continuous cell lysis and direct release of DNA into the maternal circulation. Additionally, developmentally-associated apoptosis of fetal cells may occur. It is somewhat surprising that fetal DNA is not immediately metabolized. However, tumour DNA can persist in the serum of patients affected with cancer, so perhaps a precedent exists for this finding. According to the report of 65), male DNA was not detected in the maternal plasma when the prior child was male but the current fetus was female; they concluded that the fetal DNA is eventually cleared from the circulation. The potential for persistence of fetal cells post partum is of concern because of the chance that diagnostic error might occur by performing genetic analysis on circulating cells that originated in a prior pregnancy. As stated above, the existence of male lymphocytes in maternal blood has been demonstrated 1–5 years after the birth of a male infant (88; 24). Using PCR-amplification of Y-chromosome-specific sequences in DNA extracted from the blood of women who had previously given birth to males, some investigators have found evidence of fetal DNA for several months post delivery; others have not (49; 42). Most of these groups did not use any techniques of fetal cell purification or enrichment prior to PCR. In contrast, by flow-sorting specific subpopulations of cells before amplification, the discovery was made that fetal cells can persist in the mother for as long as 27 years postpartum (14). The persistent cells were either lymphoid or myeloid progenitors that expressed CD34 or both CD34 and CD38. In one woman, male CD4+ cells were detected. The women studied were all healthy and had no history of blood transfusions. This study led to the speculation that normal pregnancy can lead to a physiological state of low-grade microchimaerism in a woman. 70) hypothesized that fetal cell microchimaerism played a role in the higher incidence of autoimmune disease that occurs in women after their childbearing years. This hypothesis was later tested in a blinded study that demonstrated statistically significantly increased amounts of male (presumed fetal) DNA detectable in the peripheral blood of women who suffered from the disease scleroderma, as compared to their healthy sisters or normal controls (72). These data suggested that fetal cell microchimaerism resulting from pregnancy, labour or delivery may play a role in the pathogenesis of scleroderma. In other work, 4) have published similar findings, and expanded their observations to include demonstration of male lymphocytes in the skin biopsies of women with scleroderma. Analysis of class II histocompatibility antigens in scleroderma patients revealed an increased incidence of compatibility between the affected person and her offspring compared with controls (71; 4). The current working hypothesis is that a major fetomaternal transfusion occurring at the time of delivery includes some fetal cells with proliferative potential. When the fetus and the mother are antigenically similar, fetal cells can migrate to lymphopoietic organs and proliferate. Subsequently, a graft-versus-host response can occur, which may result in the development of autoimmune disease. Data obtained from studies of umbilical cord blood samples using DNA polymorphisms indicate that in 4–40% of cases small numbers of maternal cells can be detected in the fetal circulation (41; 60; 100). Maternal cell microchimaerism could potentially explain the rarer occurrence of scleroderma in men. Studies such as these have expanded interest in the phenomenon of bidirectional fetal and maternal cell trafficking (81, 80; 60; 108). In the past, controversy over whether fetal cells were present in maternal blood chiefly centred on the inconsistent ability to detect them. At the time it was unclear whether this was due to biology (that is, the cells were physiologically rare in the blood samples) or technology (sufficiently sensitive methods were not available to prove their presence). Our initial results obtained from many flow-sorting experiments suggested the former, i.e. that a low number of fetal cells was present (10). However, loss could also occur as a result of enrichment techniques needed to detect the fetal cells. We therefore developed a whole blood quantitative PCR (qPCR) method to assay the number of fetal cells present in different types of samples to determine if the low numbers were due to intrinsic biology of the pregnancy, or secondary to cell loss following cell sorting. The Y chromosome was used as the fetal cell marker because it is the easiest way to distinguish between maternal and fetal cells. Thus, quantitative information could only be obtained from women carrying male fetuses. The study encompassed peripheral blood samples from 230 pregnant women, of whom 199 carried fetuses with a normal karyotype and 31 fetuses had aneuploidy (12). qPCR was performed using primers that amplified a sequence from the long arm of the Y chromosome. Reaction products were subjected to phosphorimage analysis. Numerical values were calculated by comparing the counts per minute in reaction products of standard male and female DNA samples versus the maternal samples. In 90 maternal samples obtained from women carrying 46,XY fetuses, the mean number of nucleated male cells detected in a 16 ml blood sample was 19. The range was 0.1–91 cells. Approximately 25% of male fetuses had 0–5 cells detected, 25% had >5–10 cells detected, 25% had >10–30 cells detected, and 25% had >30–91 cells detected. The reason for this distribution is currently unknown, but may be influenced by factors related to the pregnancy. In 109 samples obtained from women carrying 46,XX fetuses, the mean number of male cells detected was 2 (range 0–24 cells). The male cells detected in the cases in which the current fetus was female probably represent cells circulating from a prior pregnancy. The number of male cells detected in 46,XY versus 46,XX fetuses was significant (P=0.0001). Importantly, in the 18 samples studied from women carrying male fetuses with trisomy 21 the mean number of fetal cells detected was elevated 6-fold compared with the normal fetuses. This was also statistically significant (P=0.001). Blood samples obtained from women carrying fetuses with other chromosome abnormalities also suggested that aneuploidy resulted in increased feto-maternal transfusion. The results of this study demonstrated that a relatively low number of fetal cells is present when the fetus has a normal karyotype: approximately one fetal cell per ml of maternal whole blood. This assay detects all nucleated cells and not only NRBCs. The relative rarity of fetal cells has been confirmed by other investigators using a variety of different methods (84; 43; 22; summarized in 74). Whereas the baseline number of fetal cells is low in normal pregnancies, evidence is accumulating to suggest that they are higher in abnormal pregnancies. This is probably due to abnormalities in the placental barrier, whether secondary to vascular changes or the development of villus oedema. A higher number of fetal cells is present in the maternal circulation when pre-eclampsia is present (23; 33; 48) and when the fetus is aneuploid (29; 94; 31; 12). Other factors likely to influence the number of fetal cells present in the mother include gestational age (56; 106), blood group incompatibility and prior sensitization, and whether or not an invasive procedure has been previously performed (52). Because we now know that the fetal cells are so rare in maternal blood samples a variety of strategies have evolved to find them. One strategy is to perform positive selection based on uniquely fetal characteristics. Another is to negatively select or deplete cells that have maternal characteristics. Considerations include yield, or absolute number of fetal cells recovered, and purity, the relative numbers of fetal and maternal cells remaining after enrichment (10). Yield is paramount; one cannot afford to lose any of the fetal cells. The absolute number of fetal cells can be increased by haemopoietic cell culture. A relative increase in yield can be achieved by enrichment. Purity will influence the efficiency of the test, which can be improved through better and faster recognition of fetal cells through automated scanning (86; 105; 76). Many approaches have been designed to recover fetal cells from maternal blood. These include density gradient centrifugation (76; 96; 90), carbonic anhydrase inhibition (86), magnetic activated cell sorting (MACS) (19; 32, 31), immunomagnetic bead separation (8; 121), ferrofluid suspension with magnet (102; 66), avidin-conjugated columns with biotinylated antibodies (40), micromanipulation of individual cells (104, 91, 93; 120), and fluorescence-activated cell sorting (FACS) (47; 51; 7; 83; 114; 107; 55; 101). The advantage of FACS, as in other areas of haematological research, relies on its ability to achieve high purity by simultaneous analysis of multiple crtieria in an individual cell. Many groups, however, prefer the MACS because it is a bench-top technique, costs relatively little, and it is easy to use. The major disadvantage of the MACS is that selection is based upon only one criterion, which results in significant contamination by maternal cells. A new method, charged flow separation, has been described, which permits differentiation of cell types according to their characteristic surface charge densities using a horizontal cross-flow fluid gradient. Investigators who have applied this technique to fetal cells in maternal blood have reported a significantly higher recovery of nucleated erythrocytes than observed by other workers using older methods (115, 116). It is presently unclear, however, as to whether all of the NRBCs isolated by this technique are of fetal origin. The aim of fetal cell isolation is to identify unique characteristics of the rare fetal cell that permit distinction from the more abundant maternal cells. Ironically, this challenge has resulted in a contribution of knowledge regarding haematological changes that occur in the mother during pregnancy, an area that is relatively underappreciated (13). Several enzymes have been used to highlight differences between fetal and immature maternal cells; these include 2,3-biphosphoglycerate (BPG), carbonic anhydrase and thymidine kinase (TK). In an initial study, 113) used BPG as a marker of the presence of fetal haemoglobin. BPG lowers the oxygen affinity to haemoglobin and binds to deoxyhaemoglobin. The BPG–haemoglobin complex, when exposed to peroxidase, creates a coloured precipitate. The presence of different developmentally-specific isoenzymes of carbonic anhydrase can also be exploited to cause selective lysis of maternal RBCs (86). In addition, it has been recently noted that fetal, but not maternal, cells express high levels of TK (46). By using fluorescent thymidine analogues and FACS, fetal cells can be distinguished from maternal cells. Extensive study of surface differences between fetal and maternal cells has identified several promising reagents which, even if not completely specific for fetal cells, result in significant enrichment (126). A majority of researchers worldwide have had experience using monoclonal antibody (mAb) to the transferrin receptor, anti-CD71 (7; 32; 55; 101). CD71 is expressed on nearly all first-trimester fetal nucleated blood cells (126). Its expression declines with gestational age but is increased in fetuses with an abnormal karyotype. The disadvantage of CD71 is that it is expressed on a subpopulation of maternal cells, which results in low purity (13; 126). The second most commonly used mAbs for fetal cell identification are those that recognize the various fetal and embryonic globins (124, 125; 78; 84; 28; 55; 22; 76, 77; 67). The embryonic globins, although unique to fetal cells, are expressed only during a narrow window of time during gestation. Gamma globin is expressed in most fetal cells over a wide range of gestational ages. Although some maternal cells may make gamma globin (79), in practice we have found that most gamma-globin-positive cells in maternal blood prove to be fetal by cytogenetic studies (125). The only exception to this appears to be when a pregnant woman is a carrier of a β-thalassaemia mutation we have found an increased production of maternal gamma-positive cells (90). Other mAbs used for identification of fetal NRBCs include the thrombospondin receptor, CD36 (15), glycophorin A (83; 114; 55), I/i blood group antigens, HAE9 (87), erythropoietin receptor (109), FB3-2, 2-6B/6 and H3-3 (126). Mabs that have been described for the depletion of maternal cells include CD45 (11), CD3, CD4, CD19, CD13 and CD32, and CD14 (55; 102). Many groups use a combination of the above techniques, for example some sort of primary bulk separation followed by fetal cell identification using gamma globin and automated scanning. Genetic analysis of the identified fetal cells has relied primarily on two techniques, fluorescence in situ hybridization (FISH) using chromosome-specific probes and polymerase chain reaction (PCR) to amplify uniquely fetal gene sequences. FISH has had an enormous impact on this field because it does not require the presence of a dividing cell. Therefore the major fetal cell conditions associated with an abnormality in chromosome number can be readily detected. To date, almost all of the significant fetal aneuploidies have been detected in fetal cells isolated from maternal blood (83; 29; 9; 20; 31; 94; 125; 82; 75). These cases include all of the autosomal trisomies, some of the sex chromosome abnormalities, and triploidy. Recent technical advances in FISH analysis of the highly condensed erythrocyte nucleus include the capability to perform simultaneous multicolour FISH (16) and sequential assays that permit analysis of all chromosome pairs (Poly-FISH) (123). Interestingly, fetal NRBCs isolated from maternal blood may be more representative of the fetal karyotype than chorionic villi or amniocytes obtained through traditional invasive techniques. In a case report, 17) identified seven XXY and two XY fetal nuclei in a flow-sorted sample. In cultured chorionic villi obtained from the same woman, only four XXY nuclei were seen out of 250 total nuclei analysed in one culture; none were seen in the other culture. The development of PCR also significantly affected this field, because the rare number of fetal cells present in a maternal sample no longer was a limiting factor. Initially Y chromosome-specific PCR was used to prove the existence of fetal cells in maternal blood (64, 63; 7; 53; 103; 1). Subsequently the PCR was used to prove the presence of paternally-inherited fetal genes that were absent in the mother, including β globin mutations, HLA DR and DQ alpha genes, Rhesus D and Rhesus C (21; 44; 35, 34, 36; 122; 58, 62). Micromanipulation of individual or pooled NRBCs, followed by PCR, has been advocated as a means of fetal genotyping for conditions in which the mother carries a mutant allele. This technique was first described by 104) but was applied to the diagnosis of Duchenne muscular dystrophy, Rhesus D (RhD), HLA-DQ alpha genotype, and ornithine transcarbamylase deficiency by 91, 93) and 120). 22) identified fetal cells by a combination of MACS and anti-gamma or anti-zeta staining, and pooled several NRBCs to prevent the complication of allele dropout. In this manner they were able to exclude fetal inheritance of sickle cell anaemia, and β thalassaemia. The practical applications of these techniques include potential non-invasive testing for fetal RhD genotype. In one report, 34) sorted fetal NRBCs from the blood of 18 RhD-negative pregnant women, and amplified a 261 base pair sequence within the RhD gene. One woman had twins. Of the 19 fetuses, the RhD genotype was correctly predicted in 16. Three cases were incorrect due to a false negative result, as a result of a lack of amplification product. This may have been due to a lack of fetal cells in the reaction. In the clinical setting of an RhD-negative pregnant woman with an RhD heterozygous positive partner, if an amplification product is detected in a sample obtained from maternal blood, the fetus may be presumed to be RhD positive, thus obviating invasive procedures such as amniocentesis. The protocol published by 34), although far from optimized, already had a 100% positive predictive value and a 67% negative predictive value. If no RhD gene amplification product is observed, an amniocentesis can be subsequently performed. If the fetus is truly RhD negative, then no additional risk for sensitization occurs as a result of the procedure. More recently, a reverse transcriptase PCR for the presence of fetal messenger ribonucleic acid in maternal blood was shown to be more sensitive than PCR using genomic DNA for the non-invasive prenatal diagnosis of fetal RhD blood type (2). The relative rarity of fetal cells in maternal blood could be overcome by culture, which could improve the absolute number of fetal cells present. Unfortunately, maternal stem and progenitor cells are also present in any given sample; techniques designed to stimulate fetal cells have a similar effect on maternal cells, which predominate in culture. Despite these limitations, several groups have reported the successful identification of fetal cells in cultured maternal blood samples (61; 57; 110, 109, 111). More recently, 18) described a novel method for distinguishing between fetal and maternal cells in culture based upon their respective kinetics of fetal haemoglobin production. Successful selective culture of fetal cells from a maternal sample will ultimately depend upon the identification of fundamental but perhaps subtle differences in the biology of these two cell types. The initial success in the detection of abnormalities of chromosome number in fetal cells isolated from maternal blood led the United States' National Institutes of Health to sponsor a multicentre clinical study known as the NIFTY trial (National Institutes of Health FeTal Cell StudY) designed to evaluate the accuracy of this method of non-invasive prenatal diagnosis (27). This is a non-intervention trial that began in 1994; study patients are considered to be at high risk for fetal chromosomal abnormalities, and have already agreed to a procedure such as amniocentesis or chorionic villus sampling. Study subjects do not receive the results of their fetal cell isolation test and make no clinical decisions based on the experimental results. The study design involves isolation of fetal cells from maternal blood by a variety of methods. DNA probes are then used to search for aneuploidies involving chromosomes 13, 18, 21, X or Y. The results of interphase fetal cell chromosome analysis are then compared to the results of conventional karyotyping on the amniocentesis specimen or chorionic villus sample. An additional aspect of the study is an investigation of women's attitudes towards non-invasive prenatal diagnosis. Study subjects answer a questionnaire designed to ascertain whether pregnant women would feel coerced into having a blood test that would detect fetal chromosome abnormalities. Although the study is ongoing, preliminary data analysis suggests several trends. First of all, the sensitivity of detection of aneuploidy is on the order of 40–50%, which is not as high as with maternal serum screening, but certainly within the same arena. A major advantage of fetal cells is the lower false positive rate, at present approximately 1% or lower. A trend towards an increased number of fetal cells being present in the sample when the fetus is aneuploid has been observed, but this has not yet achieved statistical significance. An increased feto-maternal transfusion when the fetus has aneuploidy may be related to structural abnormalities in the placenta that are associated with these conditions. As stated in the beginning of this paper, fetal cells in maternal blood must be placed in the context of other available non-invasive screening tests. Each of the serum screening markers, as well as the NT measurement, appear to function independently of each other. It is entirely feasible that fetal cell isolation may be used as another independent marker to further refine the risk for Down syndrome, either as part of a primary package, or as a secondary non-invasive test when serum screening or NT measurement is abnormal. The New England Medical Center's perinatal diagnostic group has recently applied for grant funding of a very large clinical study comparing all available noninvasive testing for Down syndrome. This so-called FASTER trial (First And Second Trimester Evaluation of Risk for aneuploidy) would encompass 50 000 low-risk pregnant women. Furthermore, in geographic areas in which there exists a high prevalence of a certain monogenic disorder, fetal cells could also be used for noninvasive molecular genetic diagnosis. With the advent of sensitive molecular genetic analytic techniques, the existence of fetal nucleated cells in maternal blood samples has been conclusively demonstrated. Quantitative PCR studies performed on maternal whole blood and plasma/serum samples have shown that in most pregnancies, fetal cells are rare in number. A larger feto-maternal transfusion occurs when the fetus is aneuploid, or when pre-eclampsia develops. Detection of aneuploidy is likely to be facilitated by the increased number of fetal cells detectable in the mother, as well as the fact that blood cells from aneuploid fetuses are immature for gestational age and express many antigens at higher density than normal fetal or immature maternal cells. The major strategies being used to detect fetal cells include enrichment, cell culture, and improved recognition through automated microscope scanning. At present, almost all fetal aneuploidies have been diagnosed in maternal blood using FISH. Many single gene mutations have also been demonstrated. Conditions in which the mother is also a carrier of a mutant gene can be addressed by micromanipulation and single-cell PCR. Recent data regarding the postpartum development of fetal cell microchimaerism in the mother and its possible relationship to subsequent onset of ‘autoimmune’ disorders may enhance our understanding of the fetus as an allograft and engender a new appreciation of the biology of human pregnancy. This work was supported in part by a grant from Genzyme Genetics, NIH contract number N01-HD-4-3204, NIH grant number P01-HD-18658." @default.
W2028115197 created "2016-06-24" @default.
W2028115197 creator A5081715355 @default.
W2028115197 date "1999-06-01" @default.
W2028115197 modified "2023-10-10" @default.
W2028115197 title "Fetal cells in the maternal circulation: feasibility for prenatal diagnosis" @default.
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