MXPA99008251A - Prenatal diagnostic methods - Google Patents

Abstract

A method of identifying embryonic or fetal red blood cells in a sample containing maternal blood cells and embryonic or fetal red blood cells or both, the method comprising determining which cell or cells contain or express and adult liver component. A method of isolating embryonic or fetal red blood cells from a sample containing maternal blood cells and embryonic or fetal red blood cells or both, the method comprising isolating the cells which contain or express an adult liver component. A method of determining a fetal abnormality the method comprising identifying or isolating embryonic or fetal cells according to the above methods and analysing said embryonic or early fetal cells for said abnormality. Use of a means for determining whether a cell contains or expresses an adult liver component for identifying or isolating an embryonic or fetal red blood cell.

Description

METHODS OF PRENATAL DIAGNOSIS DESCRIPTIVE MEMORY The present invention relates to diagnostic methods, in particular to methods of prenatal diagnosis and to reagents for use in such methods. The prenatal diagnosis is carried out widely in hospitals throughout the world. Existing procedures such as fetal, hepatic or chorionic biopsy to diagnose chromosomal disorders including Down syndrome, as well as individual gene defects including cystic fibrosis are very invasive and involve a considerable risk to the fetus and a small risk to the mother . For example, amniocentesis, involves inserting a needle into the matrix to collect cells from the embryonic tissue or fluid. The test, which can detect Down syndrome, carries a risk of abortion estimated at 1%. Fetal therapy is in its early stages and the possibility of early-stage testing for a wide variety of disorders could undoubtedly greatly increase the pace of research in this area. Current fetal surgical techniques have been improved, making fetal surgery possible for some genetic problems such as spina bifida and cleft palate. In addition, currently relatively simple effective fetal treatment is available for other disorders, for example deficiencies of 21-hydroxylase (treatment with dexamethasone) and holocarboxylase synthetase (treatment with biotin), while detection can take place at a sufficiently long stage. early In this way, relatively non-invasive prenatal diagnostic methods are an attractive alternative to the very invasive existing procedures. A method based on the mother's venipuncture would make the early stage diagnosis more widely available in the first trimester, increasing the options for parents and obstetricians (since genetic disorders could be detected at an earlier stage and more safe), and allowing the eventual development of a specific therapy for the fetus. The possibility of recovering fetal cells from the maternal circulation has excited a general interest as a possible, non-invasive means to the fetus, to diagnose abnormalities in the fetus (Simpson &Elias (1993) J. Am. Med. Assoc. 270, 2357 -2361). The initial interest was directed towards the trophoblastic detection systems but the isolation of these cells by flow cytometry has not been reliable since the maternal lymphocytes seem to absorb the proteins released by the trophoblastic cells (Mueller et al. (1990) Lancet 336, 197 -200; Covone and others (1984) Lancet 13 October edition, 841-843). More recently, attention has focused on the development of methods to separate blood cells from the fetus for cytogenetic analysis, particularly fetal nucleated erythrocytes, since their number exceeds that of fetal lymphocytes in the maternal circulation. It has been described the identification of erythrocytes from the fetus in the maternal blood, that is, in a male fetus, with probes for the centromere Y to identify the fetal cells or the amplification of specific DNA sequences for Y (Price and others ( 1991) \ / 77. J. Obstet, Gynecol 165, 1731-1735, Zheng et al. (1993) J. Med. Genet 30, 1051-1056, Hamada et al. (1993) Hum. Genet 91, 427- 432; Cheung et al. (1996) Nature Genetics 14, 264-268; and Williamson (1996) Nature Genetics 14, 239-249) or identification of the karyotype under trisomy conditions (for example, see Bianchi et al. (1992) Hum Genet, 90, 368-370). Hume et al. (1995) Early Human Development 42, 85-95 demonstrate that the microsomal glucose-6-phosphatase enzyme is present in fetal and human embryonic erythrocytes. Hume et al. (1995) Early Human Development 42, 85-95 attempts to identify fetal nucleated erythrocytes using immunocytochemistry using a human fetal hemoglobin antibody and a method of in situ hybridization using probes for the X and Y chromosomes. Hume et al. 1996) Blood 87, 762-770 describes a study of the expression of endoplasmic reticulum proteins in human embryonic and fetal erythrocytes. Wachtel et al. (1991) Human Reproduction 6, 1466-1469 describes the use of PCR to identify Y-specific DNA sequences in maternal cells isolated by cell sorting with transferrin receptor antibody and glycophorin A. Yeoh antibody and others ( 1991) Prenatal Diagnosis 11, 1 17-123 describes the detection of fetal cells in the maternal circulation by enzymatic amplification of a single copy gene that was specific to the fetus. Holzgreve et al. (1992) J. Reprod. Med. 37, 410-418 shows that the transferrin receptor antigen by itself is not sufficient for the enrichment of nucleated erythrocytes of the fetus and points out that the reproducibility and reliability of the techniques are still limited, mainly due to the lack of markers. of very specific cells. Zheng et al. (1993) J. Med. Genet. 30, 1051-1056 describes the use of a magnetically activated cell sorter (MACS) to enrich the nucleated erythrocytes of the fetus using mouse monoclonal antibodies specific for CD45 and CD32 to deplete leukocytes from maternal blood. The document indicates that there was significant maternal contamination even after enrichment with MACS preventing accurate analysis of fetal cells by in situ hybridization by fluorescence interface (FISH). Tomoda (1964) Nature 202, 910-91 1 describes the demonstration of fetal erythrocytes by immunofluorescent staining. There is a need for improved methods to identify fetus cells in maternal blood to properly perform prenatal diagnosis.

Attempts were made to separate fetal and embryonic nucleated erythrocytes from maternal blood with the established immunomagnetic classification using sequentially anti-CD45 and anti-CD18 antibodies to remove the leukocytes and then anti-CD71 antibodies (transferrin receptor) to enrich the fetal nucleated erythrocytes. It was unpleasant to discover that the immunomagnetic classification, with the anti-CD71 antibody, did not purify the embryonic erythrocytes of the megaloblastic series. The advantage of purifying megaloblastic cells is that they are the predominant type of erythrocyte in the embryo and in the early stage fetus and that these are nucleated while the vast majority of adult erythrocytes are normocytic and non-nucleated. In subsequent conventional immunohistochemistry, it was found that the CD71 interactions with the megaloblasts were very weak, which presumably explains the poor purification of the embryonic cells with this antibody. This is a major problem in the current use of maternal blood for early diagnosis. It has been shown that nucleated megaloblastic series predominate in early stage development compared to nucleated normoblasts. This means that using conventional antibodies ie anti-CD71, it is very difficult to obtain pure nucleated hematopoietic cells arising from the early stage concept. Anti-CD71 does not react immunologically with most of these early stage cells making this technique possible in specialized research laboratories with groups of specialists and equipment, but it does not make it practical in routine service laboratories. The use of anti-CD71 is described in Cheung et al. (1996) Nature Genetics 14, 264-268 and is reviewed by Williamson (1996) Nature Genetics 14, 239-240. With this method it is a problem to look at the slide, among thousands of cells, for a few fetal cells, as well as the fact that anti-CD71 antibodies are not selective enough for fetal cells and they do not react with embryonic cells . An object of the present invention is to provide improved methods for identifying fetal cells in, and isolating, maternal blood. In particular, an object of the invention is to provide methods of identification and isolation of embryonic or early-stage fetus erythrocytes, and to analyze the cells for fetal abnormalities. A first aspect of the invention provides a method for identifying fetal or embryonic erythrocytes in a sample containing maternal blood and fetal or embryonic erythrocytes or both, comprising the method of determining which cell or cells contain or express an adult liver component. A second aspect of the invention provides a method for isolating fetal or embryonic erythrocytes from a sample containing maternal blood cells and embryonic or fetal erythrocytes or both, comprising the method of separating cells which contain or express an adult liver component . It has not previously been proposed that nucleated erythrocytes of early-stage fetuses or embryos function as adult liver cells while circulating in the bloodstream. Appropriately, the sample containing blood cells from the mother and the fetal or embryonic erythrocytes or both is a mixture of blood from a pregnant female. Preferably, the fetal erythrocyte is an early stage fetus erythrocyte. The pregnant female can be any mammal and in particular a mammal of commercial or agricultural importance or a domesticated mammal. Suitably, the mammal is a horse, cow, sheep, pig, goat, dog, cat or the like. The basic pattern of hematological development in the embryo and in the fetus is the same for all mammals. Preferably the pregnant female is human. A particular advantage of the present invention is that the methods identify, and can be used to isolate, early embryonic and fetus erythrocytes at an early stage of gestation. Therefore, it is preferred that the maternal blood sample be taken from the pregnant female at an early stage of pregnancy. In the case of a pregnant human female it is preferred that the sample be taken in the first trimester.

In general, whether for the potential therapy to the fetus or the option of cessation of pregnancy, the earlier the stage in pregnancy is better (ideally less than 10 weeks of gestation). An additional practical reason is that the means of termination of pregnancy is technically easier in early pregnancies and with fewer physical and psychological side effects. There is no upper limit for the intrauterine diagnosis of fetal abnormalities and the treatment of pregnancy in advanced stages may even be beneficial in utero or in the immediate newborn period. The ontogeny of embryonic and fetal nucleated cells clearly indicates that the percentage of these cells in the embryo / fetus is higher in the first trimester than in the later stages of pregnancy. However, the total fetal blood volume increases with gestation and the volumes of transfusion from fetus to mother may be proportionally greater. The detailed structure of the concept of human in development, sufficient to date accurately, has been described only in detail until 56 days after ovulation and the descriptive term, embryo, will be used as the convention for this procedure of determination of developmental stage (O'Rahilly &Muller (1987) Developmental Stages in Human Embryos, Publication 637, Washington: Camegie Institute of Washington). The descriptive term, fetus, will be used by the rest of the intraurethrin development of the human until its termination (> 37 completed weeks of gestation), being the age of development (until the nearest week) an estimate based on the size, including measurements crown-heel, crown-buttocks and heel-toes and menstrual history and determination of the date of pregnancy by ultrasound (Growth of the External! Dimensions of the Human Body in the Fetal Period, Minneapolis: Printing from the University of Minnesota,). The methods of the invention are particularly suitable for identifying, and can be used to isolate, embryonic and fetal erythrocytes from the nucleated megaloblastic series which predominate in the early stages of development compared to nucleated normoblasts. However, nucleated normoblasts can also be isolated or identified by the method with equal advantage. The proportion of megaloblasts is higher in the early stages of pregnancy. The morphology of the embryonic megaloblasto / fetus in the early stage differs fundamentally from the embryonic / fetal normoblasts and the small number of maternal normoblasts that may be present in the circulation. Megaloblasts and maternal megalocytes are extremely rare, but they can present deficiencies of vitamin B12 and a folate. For these reasons embryonic / early stage fetus megaloblasts are preferred but embryonic / early stage fetus normoblasts could also be used. The sample containing maternal blood cells and embryonic or fetal erythrocytes or both may be a sample from which certain maternal blood cells have been removed. For example, adult leukocytes can be removed from maternal blood sequentially using anti-CD45 and anti-CD18 antibodies although with relatively low efficiency. Enrichment of the samples can be achieved by centrifugation in density gradients (eg Ficoll gradients) but without separating the adult and fetal nucleated cells. The sample can be a sample that has been enriched in terms of fetal cells. For example, it could be a sample enriched by the use of an anti-transferrin receptor antibody as described in Cheung et al. (1996) Nature Genetics 14, 264-268. The sample (particularly when it is one in which embryonic or fetal cells are going to be identified in a more than isolated form) can be a sample that has been treated to be submitted to hematological analysis., biochemical, histochemical or molecular biological or similar. For example, the sample can be a blood sample or a sample of a blood fraction (such as that which has been enriched for fetal cells / or without maternal cells) which has been prepared for immunocytochemical analysis or for analysis of fluorescence in situ hybridization (FISH) or has simply been disseminated on a microscope slide. The sample containing the maternal blood cells and the embryonic or fetal erythrocytes or both may be any appropriate sample (including a fluid) containing said cells. For example, the sample may be urine from a patient with hematuria, amniotic fluid or fetal blood. By the term "adult liver component" is meant a component of an adult liver cell that is predominantly associated with the adult liver and, if found in other tissues of the adult, is found at low levels in the adult liver. that other tissue compared to the liver or the mass of the other tissue in which said component is found, compared to the liver mass, is smaller so that the total amount of the adult liver component is greater in the whole liver compared to the total amount of the other complete tissue. When the adult liver component is found at low levels in that other tissue, it is at least 10 times higher in the liver, preferably at least 100 times higher in the liver, compared to that other tissue. When the adult liver component is at levels similar to those of the liver in other tissues, that other tissue has 1/10 of the liver mass, preferably 1/25 of the liver mass. While the kidney has many functions unrelated to the liver, it can also perform, to a lesser degree, some of the functions of the liver such as gluconeogenesis. It is particularly preferred if, in the above definition of adult liver component, the "other tissue" is not the kidney. For example, glucose-6-phosphatase is a component of adult liver. The levels of glucose-6-phosphatase in the liver are much higher than the levels of gIucosa-6-phosphatase in the entire pancreas. However, the levels of gIucosa-6-phosphatase in islet cells (which are only a small proportion of cells in the pancreas) can be as high as in the liver. Similarly, GLUT2 is an adult liver component which is expressed in islet cells. Both are considered proteins that predominate in the liver in the sense that the total mass of the islets in the human body is small compared to the total mass of the liver. The adult liver component level is measured per unit cell fraction or per unit cell or per unit tissue. . The adult liver component is, therefore, typically, selective of the adult or specific liver of the adult liver. The adult liver component may be any of such appropriate components and may include proteins, RNA, carbohydrate entities and metabolites provided that these are predominantly associated with the adult liver and, if found in some other tissues of the adult. , they are either at low levels in that other tissue compared to the liver or the mass of the other tissue in which the component is found compared to the liver mass is low so that the total amount of the adult liver component it is greater in the whole liver compared to the total amount in the other complete tissue.

Embryonic or fetal cells can be identified or isolated according to the methods of the invention by detecting, or binding to, one or more adult liver components as defined. It is particularly preferred that the adult liver component be substantially absent in maternal blood cells of the maternal blood.

Preferably, compared to embryonic or fetal erythrocytes, the maternal cells of maternal blood contain less than 1% of the adult liver component on a single cell basis; preferably they contain less than 0.1% on a single cell basis. It is possible, when the liver has been severely damaged by trauma, that cells or groups of cells are spread in the bloodstream. Therefore, it is preferred that the sample is not a blood sample that comes from a female whose liver has been damaged in such a way that liver cells are released into the blood. It is also preferred that the sample is not any other sample containing adult liver cells. Liver damage is assessed by plasma estimation of routine liver function tests, for example alanine aminotransferase activity. Morphologically, the appearance of a circulating adult liver cell and an embryonic nucleated erythrocyte is sufficiently different to allow discrimination. Preferably, the adult liver component is a protein. Preferably, the adult liver protein is any of a microsomal enzyme glucose-6-phosphatase, which is another protein component of the gIucose-6-phosphatase system that includes phosphate or glucose or glucose-6-phosphate transporters, a uridine-diphosphate- glucuronosyltransferase (UDPGT), an isozyme of cytochrome P450 (P450), nicotinamide adenine dinucleotide phosphate (NADP), cytochrome P450-reductase (P450 reductase), glucose transporter 2 (GLUT2), a P glycoprotein, an MDRP (multiple drug resistance protein), an MRP (protein similar to the multiple drug resistance protein) Y-glutamyl transpeptidase, a lipoprotein receptor, an alkaline phosphatase, a bile salt transporter, a bile acid transporter, a hormone receptor, a multiple organic ion transporter (MOAT, equivalent to MRP), a bilirubin transporter (bilitranslocase) or conjugated bilirubin transporter (eg bilirubin glucuronide) (equivalent to MRP). The plasma membrane of the liver contains transporters for a wide variety of drugs, xenobiotic compounds and endogenous compounds because these are taken by the liver which is the main site of metabolism. Similarly, the conjugated metabolites are then returned through the plasma membrane of the liver. Conveyors, many of which are components of adult liver as defined, are suitable objectives for practicing the methods of the invention. It will be appreciated "that additional carriers of this type will be purified and their genes and cDNA molecules cloned in. The invention contemplates that additional carriers not yet known will be appropriate targets.

In the case where there are isozymes of a particular protein or class of protein that are not selective for the adult liver or specific for the adult liver, it is the specific isozymes of adult or selective liver of adult liver that are used. For example, certain cytochromes P450 are not selective of the adult liver or specific of the adult liver and therefore, in the practice of the invention, it is those cytochromes P450 that are selective of adult liver or specific of adult liver that are relevant.

Typically, the P450s that metabolize the xenobiotic compounds are liver-selective or liver-specific. Appropriately, particularly when said embryonic or fetal erythrocytes are to be isolated from the sample, the adult liver component is a component of the surface of the cell. However, it will be appreciated that such cell surface components are useful for the purposes of both isolation and identification, and cell surface components are preferred for the purposes of both isolation and identification. Preferably, the surface component of the cell is a plasma membrane protein that is predominantly associated with the adult liver plasma membrane and, if found in other tissues of the adult, is at low levels. Conveniently, the adult liver plasma membrane protein is any of GLUT2, a P glycoprotein, an MDRP, an MRP, β-glutamyl transpeptidase, a lipoprotein receptor, an alkaline phosphatase, a bile salt transporter, a transporter bile acid, a hormone receptor, a MOAT, a bilirubin transporter or a bilirubin conjugate transporter, all as defined above. If the transferrin receptor is an adult liver component as defined, preferably the adult liver component is not a transferrin receptor. To properly identify or isolate embryonic or fetal erythrocytes it is preferred that said sample is contacted with a binding portion whose binding portion binds to said adult liver component and said embryonic or fetal cell is identified in, or isolated from, , the sample thanks to joining the link portion. It will be appreciated that embryonic or fetal erythrocytes can be identified in other ways. For example, many of the adult liver components are enzymes and therefore the cells can be identified by the presence of the enzyme. Appropriately, histochemical stains for glucose-6-phosphatase can be used. In addition, the tests for UDP glucuronosyltransferase are appropriately useful. The invention also contemplates identifying the cells by detecting adult liver components which are molecules of MRNA using, for example, the reverse transcriptase polymerase chain reaction.

In a further preferred embodiment the adult liver component is detected intracellularly. For example, when the adult liver component is an enzyme, it is convenient to use a substrate (which enters the cell to which the cell may or may not be permeable depending on the substrate) and which, when metabolized by said enzyme, supplies a product with color or a fluorescent product or a product that can be easily identified. It will be appreciated that in this modality the embryonic or early stage fetus erythrocytes will be fluorescent or with color (or marked in some other way) thanks to the presence of said product produced by said enzyme. Fluorescent cells can be separated from non-fluorescent cells using a FACS classifier. Substrates for glucose-6-phosphatase that give rise to a product with color are known in the art. In addition to the individually established adult liver protein components, as potential sources of antibodies for the isolation and / or identification of embryonic and fetal cells, the following method is also useful. The plasma membranes of human liver and / or some other mammal are isolated by differential centrifugation from homogenized liver (or by other known techniques) and used either (a) directly to create antibodies or (b) subfractionation of the membranes Plasma liver is carried out before creating the antibodies against particular components. Such antibodies will be polyspecific polyclonal antibodies that bind to the adult liver plasma membrane and, in accordance with the invention, to the plasma membrane of embryonic and fetal erythrocytes. It will be appreciated that mixtures of defined antibodies directed to the adult liver plasma membrane components are also useful in the methods of the invention. Preferably, the antibodies bind to portions of the adult liver plasma membrane component that are exposed on the surface of the cell. Conveniently, said binding portion is an antibody or fragment or derivative thereof. The monoclonal antibodies that will bind to many of these antigens (whether they are protein antigens or non-protein antigens) are known beforehand, but in any case, with today's techniques in relation to monoclonal antibody technology, they can be prepare antibodies for most antigens. The portion that binds to the antigen may be a part of an antibody (e.g., a Fab fragment) or a synthetic antibody fragment (e.g., a single chain Fv fragment [ScFv]). Suitable monoclonal antibodies for selected antigens can be prepared by known techniques, for example those described in "Monoclonal Antibodies: A manual of techniques", H Zola (CRC Press, 1988) and in "Monoclonal Hybridoma Antibodies: Techniques and Applications", JGR Hurrell (CRC Press, 1982).

Chimeric antibodies are discussed by Neuberger and others (1988, 8th International Biotechnology Symposium Part 2, 792-799). Polyclonal antibodies are useful in the methods of the invention. Monospecific polyclonal antibodies are preferred. Appropriate polyclonal antibodies can be prepared using methods well known in the art. Antibody fragments can also be used, such as Fab and Fab2 as well as genetically engineered antibodies and antibody fragments can be used. The heavy variable (V) and variable light (VL) domains of the antibody are involved in the recognition of the antigen, a fact recognized first by the initial experiments of protease digestion.

Subsequent confirmation was discovered by "humanization" of rodent antibodies. The variable domains of rodents can be fused to human constant domains so that the resulting antibody retains the antigenic specificity of the antibody obtained from rodents (Morrison et al. (1984) Proc. Nati. Acad. Sci. USA 81, 6851-6855). It is known that this antigenic specificity is conferred by the variable domains and is independent of the constant domains from experiments involving the expression in bacteria of antibody fragments, all containing one or more of the variable domains. These molecules include Fab-like molecules (Better et al. (1988) Science 240, 1041); Fv molecules (Skerra et al. (1988) Science 240, 1038); single chain Fv molecules (ScFv) wherein the associated VH and VL domains are linked by a flexible oligopeptide (Bird et al. (1988) Science 242, 423; Huston et al. (1988) Proc. Nati Acad. Sci. USA 85, 5879) and single domain antibodies (dAbs) comprising isolated V domains (Ward et al. (1989) Nature 341, 544). A general review of the techniques involved in the synthesis of antibody fragments that retain their specific binding sites is found in Winter & Milstein (1991) Nature 349, 293-299. By the term "ScFv molecules" is meant molecules wherein the associated VH and V domains are linked by a flexible oligopeptide. The Fab, Fv, ScFv and dAb antibody fragments can all be expressed in and secreted from E. coli, thus allowing the easy production of large amounts of said fragments. The whole antibodies, and the F (ab ') 2 fragments are "bivalent". By "bivalent" it is meant that said antibodies and F (ab ') 2 fragments have two antigen-combining sites. In contrast, the Fab, Fv, ScFv and dAb fragments are monovalent, having only one antigen-combining site. It will be appreciated that when the adult liver component is a receptor the recipient, and therefore the embryonic or fetal erythrocyte, can be identified using a ligand for the receptor. For example, the cognate hormone is a ligand for a hormone receptor.

Typically, in a method of identifying the embryonic or fetal erythrocyte, the binding portion is detectably labeled or at least detectable. For example, the binding portion is labeled with a radioactive atom or a molecule with color or a fluorescent molecule or a molecule that can be easily detected in any other form. The link portion can be directly marked with a detectable mark or can be marked indirectly. For example, the binding portion can be an unlabeled antibody which can be detected by another antibody which is itself labeled. Alternatively, the second antibody can be bound to biotin and the binding of labeled streptavidin to biotin is used to indirectly label the first antibody. Typically, in a method of isolating the embryonic or fetal erythrocytes, the binding portion is immobilized on a solid support so that embryonic or early-stage fetus erythrocytes can be separated by affinity binding. Conveniently, the solid support consists of any suitable matrix such as agarose, acrylamide, Sepharose (a trade name) and Sephadex (a trade name). The solid support can also be a solid substrate such as a microtiter plate or the like. Advantageously, the binding portion is magnetically marked (either directly or indirectly) so that, when attached, the embryonic or fetal cell can be separated from the rest of the sample by providing an appropriate magnetic field. The microbeads used for the magnetic classification of cells are often called super paramagnetic colloidal MACS microbeads. Fetal or embryonic cells marked in this way can be classified by a magnetically activated cell sorter (MACS). Appropriately, the binding portion is labeled with a fluorescent molecule (either directly or indirectly) and the embryonic or fetal erythrocytes are separated using a fluorescence activated cell sorter (FACS). Therefore, tools and methods have been developed that clearly identify, and show that the correct type of cell is being observed (ie, embryonic or fetal erythrocytes). A third aspect of the invention provides a method of determining a fetal abnormality comprising the method identifying or isolating the embryo or fetal cells according to the method of the first or second aspect of the invention and analyzing said embryonic or fetal cell for said fetal abnormality. In one embodiment, the cells are identified using a binding portion, or they are identified thanks to the presence of an enzyme as described above and the analysis of said fetal abnormality is performed directly on the identified cells. For example, the cells can be identified immunohistochemically using an appropriate binding portion, or using an appropriate enzyme detection system, and the analysis of fetal abnormality is performed in situ in the cell identified in this manner using techniques such as hybridization in fluorescence site (FISH) to detect chromosomal abnormalities. The polymer chain reaction can be used in situ. While fluorescent detection systems work well, it is also possible to use labeled probes and enzyme-linked detection systems. In a particularly preferred embodiment, the embryonic or fetal cells are isolated according to the second aspect of the invention. The cells isolated by this method are substantially all embryonic or fetal cells and, in particular, substantially no maternal cells are present. The analysis of the fetal abnormality involves the analysis of said embryonic or fetal cell in which the potential abnormality is investigated. Although the method can be used according to the invention, it is preferred that a defect in the glucose-6-phosphatase family is not detected, or that disorders of liver protein expression are not diagnosed when the cells are identified using a binding portion that binds to an intracellular component of adult liver such as glucose-6-phosphatase. Although the method can be used in accordance with the invention, it is also preferred that a genetic deficiency of an endoplasmic reticulum protein is not detected when the cells are identified using a binding portion that binds to an intracellular component of the adult liver. It is preferred that the fetal cell abnormality be determined by analyzing the genetic material. In particular, the constitution of the genomic DNA of the fetal cells isolated by the method of the second aspect of the invention will be the same as the constitution of the genetic DNA of the somatic cells of the fetus. In a preferred embodiment, chromosomal abnormalities are detected. By "chromosomal abnormality" is included any serious abnormality in a chromosome or in the number of chromosomes. For example this includes detecting trisomy on chromosome 21 which indicates Down Syndrome, trisomy 18, trisomy 13, chromosomal sex abnormalities such as Klinefelter syndrome (47, XXY), XYY or Turner syndrome, translocations and chromosomal deletions, a small proportion of patients with Down syndrome have translocation and chromosome deletion syndromes that include Pradar-Willi syndrome and Angelman syndrome, both of which involve deletions of parts of chromosome 15 and detection of mutations (such as deletions, insertions, transitions, transversions and other mutations) in individual genes. There are also other types of chromosomal problems such as Fragile X syndrome, which can be detected by DNA analysis. The following table indicates certain genes, whose mutations lead to particular genetic diseases.

Other genetic disorders are known that can be detected by DNA analysis such as 21-dehydroxylase deficiency or holocarboxylase synthetase deficiency, aspartylglucosaminuria, metachromic leukodystrophy, Wilson's disease, steroid sulfatase deficiency, X-related adrenoleukodystrophy, phosphorylase kinase deficiency (type VI glycogen storage disease) and debranching enzyme deficiency (glycogen storage disease type III).

These and other genetic disorders are mentioned in The Metabolic and Molecular Basis of Inherited Disease, 7a. Ed., Vol. 1,11 and III, Scriver, C.R., Beaudet, A.L., Sly, W.S. and Valle, D. (eds), McGraw Hill, 1995. Clearly, any genetic disease wherein the gene has been cloned can be analyzed and mutations detected, by this embodiment of the method of the invention. Genetic testing methods include standard techniques of restriction fragment length polymorphism tests and PCR-based tests, as well as other methods described below. The test may involve any appropriate method for identifying mutations or polymorphisms, such as: determination of the DNA sequence in one or more of the relevant positions; differential hybridization of an oligonucleotide probe designed to hybridize at the relevant positions to any of the wild type or mutant sequences; denaturing gel electrophoresis followed by digestion with a suitable restriction enzyme preferably, following the amplification of the relevant DNA regions; analysis of the nuclease sequence S1; non-denaturing gel electrophoresis, preferably following the amplification of the relevant DNA regions; conventional tests RFLP (restriction fragment length polymorphism), selective amplifications of DNA using oligonucleotides which are complemented for the wild-type sequence and not complemented for the mutant sequence or vice versa, or the selective introduction of a restriction site using an initiator PCR (or similar) supplemented for the wild-type or mutant genotype, followed by a restriction digestion.The test may be indirect, ie it may allow detecting a mutation in another position or gene known to be linked to one or more of the mutant positions Probes and primers can be DNA fragments obtained from natural sources or can be synthetic A non-denaturing gel can be used to detect lengths that differ from the fragments resulting from digestion with an appropriate restriction enzyme DNA is usually amplified before digestion, for example by using l Polymerase chain reaction (PCR) method and modifications thereof. DNA amplification can be achieved by the PCR method established as described by Saiki et al (1988) Science 239, 487-491 or by developments thereto or alternatives such as the ligase chain reaction, QB replicase and amplification based on the nucleic acid sequence.

An "appropriate restriction enzyme" is one that will recognize and cut the wild type sequence and not the mutated sequence or vice versa. The sequence that is recognized and cut by the restriction enzyme (or not as might be the case) may be present as a consequence of the mutation or it may be introduced into the mutant or normal allele using poorly complemented oligonucleotides in the PCR reaction. It is convenient if the enzyme cuts DNA only infrequently, in other words if it recognizes a sequence that occurs only on rare occasions. In another method, a pair of PCR primers is used which complement (i.e., hybridize to) either wild type genotype or mutant genotype but not both. Whether or not amplified DNA is produced will then indicate the wild type genotype or the mutant genotype (and hence the phenotype). Nevertheless, this method partially relies on a negative result (ie the absence of amplified DNA) that could be due to a technical failure. It is therefore less reliable and / or requires additional control experiments. A preferable method uses similar PCR primers but, as well as hybridizing to only one of the wild-type or mutant sequences, these are introduced into a restriction site that is not otherwise present in any of the wild-type or mutant To properly facilitate the subsequent cloning of amplified sequences, the primers could have restriction enzyme sites attached to their 5 'ends. In this manner, all nucleotides of the primers are obtained from the sequence of the gene of interest or the sequences adjacent to that gene except for the few nucleotides necessary to form a restriction enzyme site. Such enzymes and sites are well known in the art. The primers themselves can be synthesized using techniques that are well known in the art. In general, primers can be made using synthesizing machines that are commercially available. A fourth aspect of the invention provides a kit of parts for determining a fetal abnormality comprising (a) means for determining whether a cell contains or expresses an adult liver component and (b) means for analyzing a cell as an abnormality . The means for determining whether a cell contains or expresses an adult liver component comprises the aforementioned binding portions and reagents (e.g. substrate) for detecting said enzymes when the liver component is an enzyme and reagents such as PCR primers. , deoxynucleotides and a DNA polymerase to detect said mRNA when the adult liver component is an mRNA. Antibodies, or fragments or derivatives thereof, are preferred for adult liver components. It is particularly preferred that the adult liver component be a cell surface component as described above. The means for analyzing a cell for an abnormality include any such means. It is particularly preferred that the means be means for detecting a genetic abnormality. Therefore such appropriate methods include nucleic acid molecules, such as PCR primers and probes, which selectively hybridize to a gene of interest, i.e., one in which a genetic abnormality is sought. A fourth aspect of the invention provides the use of a binding portion which binds to an adult liver component in a method for determining a fetal abnormality comprising the method of identifying or isolating embryonic or fetal cells in accordance with the method of the first or second aspect of the invention and analyzing said embryonic or fetal cell with respect to said fetal abnormality. Preferably, the binding portion is used in a method wherein the abnormality of the fetal cells is determined by analyzing the genetic material, for example as to abnormalities or chromosomal mutations in the DNA. A further aspect of the invention provides the use of means for determining whether the cell contains or expresses an adult liver component to identify or isolate an embryonic or fetal erythrocyte. The means for determining whether the cell contains or expresses an adult liver component are those described in the fourth aspect of the invention. The invention will now be described in greater detail with reference to the following examples and figure wherein: Figure 1 (95chorion aGLUT2 1; 100X40RH) shows a human chorionic hair on day 56 post-ovulatory showing intense GLUT 2 alpha immunoreactivity in a megaloblasto (arrow) and shows no reactivity in a normocyte (arrow head) within a chorionic blood vessel of the fetus. The syncytiotrophoblastic (s) and cytotrophoblastic (c) layers are poorly immunoreactive.

EXAMPLE 1 Antibodies directed against adult liver components Antibodies directed against testosterone / 4-nitrophenol UDPGT from rat liver are created in Suffolk Cross Blackface sheep by a combination of intradermal and subcutaneous injection. IgG is prepared from the antiserum by a combination of precipitation with ammonium sulfate and chromatography in diethylaminoethyl cellulose (Burchell et al. (1984) Biochem. Soc. Trans. 12, 50). Typically the antibody preparation of testosterone / 4-nitrophenol UDPGT from sheep antirata liver (RAL 1) inhibits the activity of UDPGT towards bilirubin, testosterone, 1-naphthol, androsterone, estrone and morphine and the immunoblotting confirms a broad spectrum of cross-reactivity to multiple isoforms in liver microsomes of fetus and adult of human and rat. The monospecific polyclonal antiserum to the catalytic subunit of the microsomal system of glucose 6-phosphate, T2 and T3 are each created in Cheviot sheep by 3 subcutaneous injections of 80 μg of purified protein and the complete Freund's adjuvant as described (Burchell &Waddell: Genetic deficiencies of the hepatic microsomal glucose-6-phosphatase system, in Randle et al. (Eds): Genetics and Human Nutrition, London, UK, Libbey, 1990, p93; Waddell et al. (1991) Biochem. J. 275, 363; Burchell & Cain (1985) Diabetologia 28, 852). The pre-immune serum is obtained from each lamb before the injection with antigen. The enzyme glucose-6-phosphatase, T2 and T3 used as antigens are also all isolated from fasting Wistar rat liver microsomes. The antisera are further purified by fractionation with (NH4) 2S04 and affinity purification using protein G columns. It has been shown many times that antibody preparations, although created against rat liver proteins, each cross-react well with the respective human proteins as judged by immunoblot analysis after gel electrophoresis. of sodium dodecylsulfate-polyacrylamide. Antisera coupled to bovine serum albumin to methyl-moximada PGEM (PGEM-MOX) are created in rabbits (Kelly et al. (1986) Prostaglandins Leukot, Med. 24, 1). In the radioimmune test, the PGEM-MOX antiserum has only 0.05% cross-reactivity with PGE2. Cross-reactions of PGEM-MOX antiserum to other methyl-moximate PGs are typically less than 0.02%, except for 5-keto-PGE2 (12%) and 15-keto-PGE2a (0.9%). The percentage of cross reactivities of PGEM-MOX antiserum to other PGs are typically less than 0.02%, except for 6,15-di-13,14-dihydro-PGE2a (0.35%); 13,14-dihydro-PGE2a (2%); Y -keto-PGF2a (4%). Cross-reactivity of Gemeprost to the antiserum PGEM-MOX is typically less than 0.03% and toward the PGFM-MOX antiserum 0.03%. The antibody specificity of the PGEM-MOX antiserum for immunohistochemistry is shown by selective uptake of immunostaining by PGEM-MOX (Hume et al. (1993) Exp. Lung Res. 19, 361).

Anti-PGHS-1, a goat polyclonal IgG fraction cross-reactive towards PGHS-1 from a number of mammalian species but with negligible reactivity for PGHS-2, and an antibody to monoclonal 15-hydroxyprottaglandin dehydrogenase are purchased from Oxford Biomedical Research, Ine (Oxford, Ml). PGE2 is conjugated to limpet hemocyanin and injected intradermally in sheep. The resulting antiserum for PGE2, by applying a radioimmunological test, is highly selective, with a minimum cross-reactivity between the F and E series of the PG. The cross-reactivity of Gemeprost for the PGE2 antiserum is typically less than 1%. The antiserum specificity for immunohistochemistry is tested by the selectivity of absorption of PGE2 immunostaining in human fetus lung (Hume et al. (1992) Exp. Lung Res. 18, 259). The antibodies for the purified cytochrome P450s are isolated as described in Wolf et al. (1984) Carcinogenesis 5, 993. The specific character towards isozyme of the antiserum thus made has been demonstrated by immunoblot analysis with recombinant human cytochrome P450 proteins. expressed (Forrester et al. (1992) Biochem. J. 281, 359). The NADPH-cytochrome P450 oxidoreductase is purified (Wolf et al. (1984) Carcinogenesis 5, 993) and it is shown that the antibodies created in rabbits cross-react with the human enzyme in sodium dodecylsulfate-polyacrylamide gel electrophoresis (Smith et al. (1994) Proc. Nati Acad. Sci. USA 91, 8710). In addition to the antibodies made using isolated proteins as an antigen, the antipeptide antibodies are also useful. Anti-peptide antibodies are synthesized on the basis of known sequence information and / or large portions of proteins that have been generated using portions of the cDNA or gene linked to appropriate sequences that facilitate isolation after over-expression in an appropriate system, for example bacteria. A series of anti-peptide antibodies have been prepared against portions of the human enzyme glucose-6-phosphatase (CSHIHSIYNASLKKY (SEQ ID NO: 1); CMNVLHDFGIQSTHY (SEQ ID NO: 2); CLAQVLGQPHKKSL (SEQ ID NO: 3); CLSRIYLAAHFPHQ (SEQ ID No. 4)) as follows. These peptides were conjugated to limpet hemocyanin and injected into sheep by intradermal and subcutaneous route. It has been shown that the resulting antipeptide antiserum cross-reacts and can be used for the immunohistochemical detection of cells containing glucose-6-phosphatase including fetal and embryonic cells.

EXAMPLE 2 Combined analyzes of immunocytochemistry and in situ hybridization by fluorescence of a maternal blood sample to detect trisomy 21 Blood sample and cell preparation. Peripheral venous blood samples (EDTA) were obtained from a pregnant woman in the first trimester. Aliquots of 5 ml of blood are carefully deposited in layers on 3.5 ml aliquots of Polymorphoprep (Nycomed, Norway) in 15 ml tubes which are then centrifuged at 500 g for 30 minutes at room temperature. The mononuclear cells at the plasma / Polymorphoprep interface (upper portion of the two obtained bands) are harvested using a Pasteur pipette and dispensed in a clean tube. The cells are washed three times using 5 ml of cold phosphate buffered saline (PBS) containing 0.5% bovine serum albumin (BSA) and 5 mM ethylenediaminetetraacetic acid (EDTA) followed by a 10 minute centrifugation at 400 g each time. The cell pellets are finally suspended again in PBS / BSA / EDTA at a concentration of 106 cells / ml.

Preparation of the slides. The aliquots (100 μl) of blood mononuclear cells are cytocentrifuged on glass slides (Cytospin, Shandon) and air-dried overnight.

Immunocytochemistry The following polyclonal and monoclonal antibodies are used in the immunocytochemistry procedure: (a) a mouse monoclonal antibody directed to the enzyme glucose-6-phosphatase (b) rabbit anti-mouse IgG (DAKO) at 1:25, and (c) ) PAP (DAKO) at 1: 100. All antibodies are diluted in TBS containing 20% of normal rabbit serum (Serotec) and the antibody incubations are carried out in a humid chamber at room temperature. The dry cell centrifuges are fixed in acetone for 3 minutes and washed for 5 minutes in two changes of saline solution regulated with Tris (TBS) standard PAP technique (Sternberger et al., 1970, J. Histochem, Cytochem, 18, 315- 333) is modified. Briefly, the endogenous peroxidase activity is blocked using 0.3% hydrogen peroxide in TBS for 30 minutes after which the slides are rinsed in two TBS changes for 5 minutes. Cell centrifuges are incubated with normal 20% rabbit serum in TBS for 5 minutes to block non-specific binding sites. After removal of most of the normal rabbit serum, the cell centrifuges are incubated for 30 minutes with anti-G6Pase nonooclonal antibody. After washing the slides in TBS as above, followed by incubation with normal 20% rabbit serum in TBS for 5 minutes, cell centrifuges are incubated with rabbit anti-mouse IgG for 30 minutes. The slides are washed as indicated above and incubated with normal rabbit serum at % TBS for 5 minutes followed by 30 minutes with mouse PAP. The slides are then washed as indicated above and two to three drops of 3,3-diaminobenzidine (DAB) substrate solution are added and incubated at room temperature for 7 minutes. The DAB solution is prepared by dissolving 2.5 mg of DAB (Sigma) in 5 ml of TBS followed by the addition of 0.1 ml of 1% hydrogen peroxide prepared just before use. The slides are washed to the tap of the key for 2 minutes followed by a 5 minute incubation in copper sulfate solution (0.4 g of copper sulfate, 0.72 g of sodium chloride, 100 ml of distilled water) and rinsed again to the jet of the key. To check again the morphology of the cells after the immunocytochemistry, the slides can be counterstained with Mayer's hematoxylin (Sigma) for 20 minutes, dehydrated with alcohol gradients and checked using a Zeiss Axioskop 20 microscope. Immunohistochemistry in sections of tissues is performed using anti-G6Pase monoclonal antibody and a standard PAP technique (Stemberger et al., 1970, J. Histochem, Cytochem, 18, 315-333). The sections are lightly counterstained with hemotoxylin, dehydrated with alcohol gradients and rinsed in xylene before placing the coverslip in synthetic resin.

In situ fluorescence hybridization (FISH). After immunocytochemistry, the cell centrifuges are fixed in glacial acetic acid methane solution 3: 1 (v / v) for 30 minutes at room temperature. The fixation is repeated with a fresh preparation of the same fixative for another 30 minutes and with glacial acetic acid solution: water 70:30 (v / v) for 90 seconds. The slides are washed for 5 minutes in two PBS changes and then dehydrated through 70%, 85% and 100% alcohol and air-dried at room temperature. FISH analysis is performed using human-specific probes for human chromosome 21 directly labeled with fluorescein isothiocyanate (FITC). Briefly, the hybridization fluid is pipetted into each of the cell centrifuges and a coverslip coated with the fluorescent probes is placed on top and sealed with rubber solution. In the presence of the hybridization fluid, the probes are eluted from the coverslip and after denaturation of both the probe and the target DNA which is presented under the coverslip by heating the slides at 70 ° C, the probes are allowed to hybridize to the objectives for 25 minutes at 37 ° C. After several subsequent hybridization washes, the slides are mounted in an anti-fading solution containing diamidino-2-phenyl-indole dihydrochloride (DAPI).

Microscopy Slides are analyzed using a Zeiss microscope Axioskop 20 equipped with a microscope illuminator with HBO 50W short-arc mercury vapor lamp and the appropriate Zeiss filter combinations (02 for DAPI, 09 for FITC). The immunopositive cells are located using the microscope in the visible light mode, after which the visible light source is blocked and the microscope is switched to the fluorescence mode and the slides are analyzed using each set of filters.

The cells are photographed with Fujichrome Provia 400 color film (Fuji Photo Film Co., Tokyo, Japan) in both the visible light mode and the fluorescence mode. Then black and white and negative prints are made from the color film.

Results Three positive fluorescent spots from chromosome 21 are present in embryonic or fetal cells obtained from an affected fetus. In contrast, normal maternal cells and normal fetus cells give two positive fluorescent spots for chromosome 21. The cells of the fetus and the mother are distinguished by immunoreactivity towards gIucosa-6-phosphatase, ie the fetus erythrocytes are immunopositive , maternal blood cells are immunonegative.

EXAMPLE 3 Isolation of fetus cells and PCR analysis for sickle cell anemia and thalassemia Figure 1 (95chorion aGLUT2 1; 100X40RH) shows human chorionic hair at 56 days after ovulation showing intense immunoreactivity for alpha GLUT 2 in a megaloblasto (arrow) and no reactivity in a normocyte (arrowhead) within a chorionic blood vessel of a fetus. The syncytiotrophoblastic (s) and cytotrophoblastic (c) layers are poorly immunoreactive. Fetal cells from maternal blood taken in the first trimester are isolated as follows, and are subjected to PCR analysis for sickle cell anemia and thalassemia.

Blood sample and cell preparation Peripheral blood (16-18 ml) is collected from a pregnant woman in the first trimester in EDTA Vacutainer tubes (Becton Dickinson, Rutherford, NJ). The blood is then diluted 1: 2 with phosphate buffered saline (PBS), with each 15 ml deposited in layers on 10 ml of Ficoll-paque plus (density 1.077 g / ml, Pharmacia Biotech, Piscatawa, NJ) and centrifuged at 400 g for 30 minutes at room temperature. The cells of the interface are then carefully removed, washed twice with PBS supplemented with 5 mM EDTA and 0.5% bovine serum albumin (BSA), and resuspended in 80 μl of PBS / EDTA / BSA for every 107 cells.

Classification of magnetically activated cells Nucleated erythrocytes were enriched by MiniMACS (Miltenyi Biotech, Inc., Sunnyvale, CA) following the manufacturer's protocol. MACS microbeads conjugated with anti-GLUT2 antibody are added to the resuspended cells in a ratio of 20 μl per 10 7 cells. The mixture is then incubated for 15 minutes at 6-12 ° C in a refrigerator. The suspension of cells labeled with magnetic beads is pipetted onto a MiniMACS column and the unlabeled cells are then collected by pushing them out of the column using 1 ml of buffer and a plunger. The cells isolated in this manner are substantially all embryonic nucleated erotrocytes or early stage fetuses. PCR analysis is performed as described (Saiki et al. (1988) Science 239, 487-491) in 50 μl reaction volume using a Perkin-Elmer DNA thermal cycler. Each amplification cycle consists of 1 minute at 95 ° C, 1 minute at 55 ° C and 1 minute at 72 ° C, with a final extension of 10 minutes at 72 ° C in the last cycle. DNA from fetal or embryonic cells eluted from the MiniMACS column is amplified first by 40 cycles and one fifth of the products is examined on an 8% acrylamide gel. If specific or very weak PCR products are not seen, an aliquot of 10 μl of the first round of PCR products is amplified for another 20-30 cycles using the same conditions. The primers used to amplify the ß-globin sequences are 'pco3' with biotin (5'-Biotin-ACACAACTGTGTTCACTAGC-3 '(SEQ ID No: 5)),' ß110 'with biotin (5'-Biotin-AAAATAGACCAATAGGCAGA-3' (SEQ ID No: 6)) and 'China 2 with biotin '(5'-Biotin-TGCAGCTTGTCACAGTGCAGCTCACT-3' (SEQ ID No: 7)). The 248 bp DNA amplified by 'pco3' and 'β1 10' is used for the detection of the falciform gene. The 460 bp DNA amplified by 'pco3' and 'China 2' is used for the detection of β-thalassemia. • 10 inverted dot blot hybridization Membrane strips containing multiple spots of immobilized oligonucleotide probes and normal ß-globin sequences are prepared as described (Maggio et al. (1993) Blood 81, 239-242; Cai et al. (1994) Human Mutation 3, 59-63). The sequences of these oligonucleotide probes for detecting the mutations are as follows: to detect the falciform gene, the normal probe is 5'-TGACTCCTGAGGAGAAGT-3 '(SEQ ID No: 8) and the mutant probe is 5'- CAGACTTCTCCACAGGA-3 '(SEQ ID No: 9); to detect the ß39 mutation, the The normal probe is 5'-CTTGGACCCAGAGGTTCTT-3 '(SEQ ID No: 10) and the mutant probe is 5'-AGAACCTCTAGGTCCAAGG-3' (SEQ ID No: 1 1); and to detect the β110 mutation, the normal probe is 5'- GAAAATAGACCAATAGGCAGA-3 '(SEQ ID NO: 12), and the mutant probe is 5'-CTGCCTATTAGTCTATTTTC-3' (SEQ ID NO: 13). The PCR products of the fetal cells are added to 0.8 ml of solution containing 2 x SSC / 0.1% SDS. The strips containing the oligonucleotide probes are added and the DNA molecules are denatured by boiling them in a water bath for 5 minutes. Hybridization is carried out in a water bath at 42 ° C overnight. The strips are then washed in 0.5 x SSC / 0.1% SDS at 42 ° C for 10 minutes, conjugated with streptavidin-HRP at room temperature for 15 minutes and the color developed with tetramethyl benzidine solution containing the chromogenic substrate, citrate of sodium and H2O2 at room temperature until the colors were visible and can be detected by this method whether or not the fetal cells have sickle cell mutation or thalassemia.

Claims (22)

NOVELTY OF THE INVENTION CLAIMS

1. - A method for identifying embryonic or fetal erythrocytes in a sample containing blood cells from the mother and erythrocytes embryonic or fetal erythrocytes or both, the method comprising determining which cell or cells contain or express an adult liver component, characterized in that the component of Adult liver is not a transferrin receptor.

2. A method for isolating embryonic or fetal erythrocytes from a sample containing blood cells from the mother and embryonic or fetal erythrocytes or both, the method comprising isolating the cells that contain or express an adult liver component, also characterized in that the Adult liver component is not transferrin receptor.

3. A method according to claim 1 or claim 2, further characterized in that said sample is a blood sample from a pregnant female.

4. A method according to claim 3, further characterized in that the pregnant female is a human female and the sample is taken in the first trimester.

5. - A method according to claim 1 or 2, further characterized in that the embryonic or fetal erythrocytes are of the nucleated megaloblastic series.

6. A method according to any of claims 1 to 5, further characterized in that the component is a protein.

7. A method according to any of claims 1 to 6, further characterized in that the component is substantially absent from maternal cells of the blood of the mother.

8. A method according to any of claims 1 or 2, further characterized in that the protein is any of a microsomal enzyme glucose-6-phosphatase, another protein component of the glucose-6-fostatase system that includes phosphate transporters or glucose or glucose-6-phosphate, a uridine diphosphate-glucuronosyltransferase, a cytochrome P450 isozyme, nicotinamide adenine dinucleotide phosphate, cytochrome P450 reductase, glucose transporter 2, a P glycoprotein, an MDRP, an MRP,? -glutamyl transpeptidase , a lipoprotein receptor, an alkaline phosphatase, a bile salt transporter, a bile acid transporter, a hormone receptor, a multiple organ transporter, a bilirubin transporter or a bilirubin conjugate transporter.

9. A method according to any of the preceding claims, further characterized in that the adult liver component is a component exposed on the surface of the cell.

10. - A method according to claim 9, further characterized in that the component exposed on the surface of the cell is a protein of the plasma membrane of adult liver.

11. A method according to claim 10, further characterized in that the adult liver plasma membrane protein is either glucose transporter 2, a glycoprotein P, a MDRP, an MRP,? -glutamyl transpeptidase, a lipoprotein receptor, an alkaline phosphatase, a bile salt transporter, a bile acid transporter, a hormone receptor, a multiple organic ion transporter, a bilirubin transporter or a transporter of bilirubin conjugate.

12. A method according to any of the preceding claims, further characterized in that said sample is contacted with a binding portion whose portion is linked to said adult liver component and said embryonic or fetal cell is identified in or isolated of the sample thanks to joining the link portion.

13. A method according to claim 1 to 11, further characterized in that said sample is contacted with a substrate for an enzyme, said enzyme being an adult liver component, and said embryonic or fetal cell is identified in or isolates from the sample thanks to the product formed by the action of said enzyme on said substrate.

14. - A method according to claim 12, further characterized in that said binding portion is an antibody or fragment or derivative thereof.

15'- A method for isolating embryonic or fetal erythrocytes from a sample according to claim 12 or 14, further characterized in that said binding portion is immobilized on a solid support.

16. A method according to claim 12 or claim 14, further characterized in that said binding portion is detectably labeled or capable of detection.

17- A method for isolating embryonic or fetal erythrocytes from a sample according to claim 16, further characterized in that said mark facilitates the isolation of the cells.

18. A method according to claim 13, further characterized in that said product is fluorescent or has color.

19. A method for determining an abnormality in the fetus, the method comprising identifying or isolating embryonic or fetal cells according to the method of any of the preceding claims and analyzing said embryonic or fetal cell with respect to said fetal abnormality.

20. A method according to claim 19, further characterized in that the fetal cell abnormality is determined by analysis of the genetic material.

21. - A method according to claim 20, further characterized in that abnormalities are detected in the chromosomes.

22. A method according to claim 20, further characterized in that the mutations in the DNA are detected. 23.- A team of parts to determine a fetal abnormality , ie * comprising a) means for determining whether a cell contains or expresses an adult liver component and (b) means for analyzing a cell for an abnormality. 24.- The use of a link portion as defined in the 10 claim 12 in a method according to any of claims 19 to 22. 25.- The use of a means to determine whether a cell contains or expresses an adult liver component to identify or isolate an embryonic or fetal erythrocyte.