Fetal cells in the maternal circulation: feasibility for prenatal diagnosis (1999)

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 explain the of scleroderma in such as these have expanded in the of fetal and maternal cell 60; In the over whether fetal cells were present in maternal blood on the to detect them. At the time it was whether this was due to biology the cells were rare in the blood or sensitive were not available to their obtained from many flow-sorting experiments suggested the i.e. that a of fetal cells was present However, could occur as a result of enrichment techniques to detect the fetal We a blood PCR method to the of fetal cells present in types of samples to the numbers were due to biology of the pregnancy, or secondary to cell following cell sorting. The Y chromosome was used as the fetal cell marker because it is the to distinguish between maternal and fetal could only be obtained from women carrying male fetuses. The study peripheral blood samples from pregnant of fetuses with a normal and fetuses had aneuploidy was performed using primers that amplified a from the long of the Y chromosome. were to analysis. were calculated by the per in reaction of male and DNA samples the maternal In maternal samples obtained from women carrying the mean of nucleated male cells detected in a blood sample was The was of male fetuses had cells had cells had cells and had cells detected. The reason for this is currently but may be by related to the pregnancy. In samples obtained from women carrying the mean of male cells detected was The male cells detected in the cases in which the current fetus was cells circulating from a prior pregnancy. The of male cells detected in fetuses was significant in the samples studied from women carrying male fetuses with trisomy 21 the mean of fetal cells detected was compared with the normal fetuses. This was statistically significant samples obtained from women carrying fetuses with other chromosome abnormalities suggested that aneuploidy in increased feto-maternal The of this study demonstrated that a relatively of fetal cells is present when the fetus has a normal one fetal cell per of maternal This all nucleated cells and not only The relative rarity of fetal cells has been by other investigators using a of in the of fetal cells is in normal pregnancies, evidence is to that they are higher in This is due to abnormalities in the placental whether secondary to or the development of villus A higher of fetal cells is present in the maternal circulation when is present and when the fetus is 31; Other likely to the of fetal cells present in the mother include gestational age blood group and prior and whether or not an invasive has been previously performed Because we that the fetal cells are so rare in maternal blood samples a of have to find them. is to positive on uniquely fetal is to or cells that have maternal include or absolute of fetal cells and the relative numbers of fetal and maternal cells remaining after enrichment is one to any of the fetal The absolute of fetal cells can be increased by cell A relative in can be by will the of the test, which can be and of fetal cells Many approaches have been designed to fetal cells from maternal These include cell with with antibodies of cells and cell 101). The of as in other areas of on to high by analysis of in an Many however, the because it is a relatively and it is to The major of the is that is upon only one which in significant by maternal A new flow has been which permits of cell types to their surface using a Investigators who have this technique to fetal cells in maternal blood have a significantly higher of nucleated erythrocytes than by other workers using older It is however, as to whether all of the NRBCs isolated by this technique are of fetal origin. The of fetal cell isolation is to identify unique characteristics of the rare fetal cell that from the more abundant maternal this has in a of regarding that occur in the mother during pregnancy, an that is relatively Several have been used to between fetal and immature maternal these include and In an study, used as a marker of the presence of fetal the to haemoglobin and to The when to a The presence of of can be to cause lysis of maternal In addition, it has been that but not cells high of using and fetal cells can be from maternal study of surface between fetal and maternal cells has several not completely specific for fetal cells, result in significant enrichment (126). A of have had using monoclonal antibody to the 101). CD71 is expressed on nearly all first-trimester fetal nucleated blood cells (126). expression with gestational age but is increased in fetuses with an karyotype. The of CD71 is that it is expressed on a of maternal cells, which in The most commonly used for fetal cell are that the fetal and embryonic The embryonic although unique to fetal cells, are expressed only during a narrow window of time during is expressed in most fetal cells over a of gestational some maternal cells may make in practice we have found that most cells in maternal blood to be fetal by cytogenetic studies The only to this appears to be when a pregnant woman is a of a we have found an increased of maternal cells Other used for of fetal NRBCs include the A (83; blood group antigens, and (126). that have been described for the of maternal cells include and and Many groups use a combination of the above techniques, for some of primary by fetal cell using and analysis of the fetal cells has primarily on two techniques, fluorescence in situ hybridization using chromosome-specific and polymerase chain reaction (PCR) to uniquely fetal FISH has had an impact on this because it not the presence of a Therefore the major fetal cell associated with an in chromosome can be detected. To date, almost all of the significant fetal have been detected in fetal cells isolated from maternal blood (83; 9; 31; These cases include all of the autosomal some of the chromosome and technical in FISH analysis of the highly erythrocyte include the to FISH and that analysis of all chromosome fetal NRBCs isolated from maternal blood may be more representative of the fetal than chorionic or obtained invasive In a and two fetal nuclei in a flow-sorted sample. In chorionic obtained from the woman, only nuclei were seen of total nuclei in one were seen in the other The development of PCR significantly affected this because the rare of fetal cells present in a maternal sample no was a Y chromosome-specific PCR was used to the existence of fetal cells in maternal blood the PCR was used to the presence of fetal that were in the including HLA and genes, and 35, of or by has been as a of fetal for in which the mother a This technique was first described by but was to the diagnosis of and by and fetal cells by a combination of and or and several NRBCs to the complication of In this they were to fetal of cell and The applications of these techniques include potential for fetal In one fetal NRBCs from the blood of pregnant and amplified a the woman had the the was predicted in cases were due to a false as a result of a lack of This may have been due to a lack of fetal cells in the In the clinical of an pregnant woman with an positive an is detected in a sample obtained from maternal blood, the fetus may be to be invasive procedures such as amniocentesis. The published by although from had a positive and a If no is an amniocentesis can be If the fetus is no risk for occurs as a result of the More recently, a PCR for the presence of fetal in maternal blood was to be more sensitive than PCR using DNA for the prenatal diagnosis of fetal blood type The relative rarity of fetal cells in maternal blood could be overcome by which could the absolute of fetal cells maternal and cells are present in any given techniques designed to fetal cells have a similar effect on maternal cells, which in Despite these several groups have the successful of fetal cells in maternal blood samples More recently, described a novel method for distinguishing between fetal and maternal cells in culture upon their of fetal haemoglobin Successful culture of fetal cells from a maternal sample will upon the of fundamental but perhaps in the biology of these two cell The success in the detection of abnormalities of chromosome in fetal cells isolated from maternal blood led the United of to a clinical study known as the of designed to the accuracy of this method of prenatal diagnosis This is a that in study patients are considered to be at high risk for fetal chromosomal and have to a such as amniocentesis or chorionic villus not the of their fetal cell isolation test and make no clinical on the The study isolation of fetal cells from maternal blood by a of DNA are used to for chromosomes 21, or The of interphase fetal cell chromosome analysis are compared to the of on the amniocentesis or chorionic villus sample. An of the study is an of towards prenatal a designed to whether pregnant women would into having a blood test that would detect fetal chromosome the study is data analysis several of the sensitivity of detection of aneuploidy is on the of which is not as high as with maternal serum but the A major of fetal cells is the false positive at present 1% or A towards an increased of fetal cells present in the sample when the fetus is has been but this has not An increased feto-maternal transfusion when the fetus has aneuploidy may be related to abnormalities in the placenta that are associated with these As stated in the beginning of this fetal cells in maternal blood must be in the context of other available screening of the serum screening markers, as as the NT to of each It is that fetal cell isolation may be used as another independent marker to further the risk for Down either as part of a primary or as a secondary test when serum screening or NT measurement is The diagnostic group has for of a clinical study all available noninvasive for Down This of for would pregnant women. in areas in which there exists a high of a fetal cells could be used for noninvasive molecular genetic the of sensitive molecular genetic techniques, the existence of fetal nucleated cells in maternal blood samples has been PCR studies performed on maternal blood and samples have that in most pregnancies, fetal cells are rare in A feto-maternal transfusion occurs when the fetus is or when of aneuploidy is likely to be facilitated by the increased of fetal cells detectable in the as as the fact that blood cells from fetuses are immature for gestational age and many antigens at higher than normal fetal or immature maternal The major used to detect fetal cells include cell and At present, almost all fetal have been in maternal blood using Many have been in which the mother is a of a can be by and PCR. data regarding the postpartum development of fetal cell microchimaerism in the mother and possible to of may of the fetus as an and a new of the biology of human pregnancy. This was in part by a from