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Akihiko Sekizawa - One of the best experts on this subject based on the ideXlab platform.
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Prenatal Screening of Single-Gene Disorders from Maternal Blood
American Journal of Pharmacogenomics, 2001Co-Authors: Akihiko Sekizawa, Hiroshi SaitoAbstract:Fetal cells and cell-free fetal DNA can be found circulating in Maternal Blood. Fetal cells recovered from Maternal Blood provide the only source of pure fetal DNA for noninvasive prenatal DNA diagnosis. Fetal nucleated erythrocytes (NRBCs) are considered the most suitable Maternally-circulating fetal cells for this purpose, because they are not commonly found in the peripheral Blood of healthy adults and are most abundant in the fetus during early gestation. Because fetal cells in Maternal Blood are extremely rare, a definitive separation method has not yet been established. Fetal NRBCs can be enriched from Maternal Blood via fluorescence- or magnetic-activated cell sorting, density gradients, immuno-magnetic beads or micromanipulation. Fetal cells are identified by Giemsa staining, hybridization with Y-chromosome specific probes, PCR-detection of a specific paternal allele, or immunostaining for fetal cell antigens. Amplification of fetal DNA sequences by primer extension preamplification and PCR has allowed prenatal screening for Duchenne muscular dystrophy and the fetal RhD Blood type. Sequence-specific hybridization has been used to detect sickle cell anemia and β-thalassemia prenatally in heterozygous carriers of these disorders. The use of cell-free fetal DNA in Maternal plasma for the diagnosis of single-gene disorders is limited to disorders caused by a paternally inherited gene or a mutation that can be distinguished from the Maternally inherited counterpart. At present, fetal gender can be determined from Maternal plasma. When a pregnant woman is a heterzygous carrier of an X-linked disorder, the determination of fetal gender is clinically very informative for first-step screening to avoid invasive amniocentesis. The non-invasive prenatal diagnosis of genetic disorders should be applied to pregnant women with a definite risk for a specific single-gene disorder.
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Prenatal screening of single-gene disorders from Maternal Blood.
American journal of pharmacogenomics : genomics-related research in drug development and clinical practice, 2001Co-Authors: Akihiko Sekizawa, Hiroshi SaitoAbstract:Fetal cells and cell-free fetal DNA can be found circulating in Maternal Blood. Fetal cells recovered from Maternal Blood provide the only source of pure fetal DNA for noninvasive prenatal DNA diagnosis. Fetal nucleated erythrocytes (NRBCs) are considered the most suitable Maternally-circulating fetal cells for this purpose, because they are not commonly found in the peripheral Blood of healthy adults and are most abundant in the fetus during early gestation. Because fetal cells in Maternal Blood are extremely rare, a definitive separation method has not yet been established. Fetal NRBCs can be enriched from Maternal Blood via fluorescence- or magnetic-activated cell sorting, density gradients, immuno-magnetic beads or micromanipulation. Fetal cells are identified by Giemsa staining, hybridization with Y-chromosome specific probes, PCR-detection of a specific paternal allele, or immunostaining for fetal cell antigens. Amplification of fetal DNA sequences by primer extension preamplification and PCR has allowed prenatal screening for Duchenne muscular dystrophy and the fetal RhD Blood type. Sequence-specific hybridization has been used to detect sickle cell anemia and β-thalassemia prenatally in heterozygous carriers of these disorders.
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prenatal diagnosis of ornithine transcarbamylase deficiency by using a single nucleated erythrocyte from Maternal Blood
Human Genetics, 1998Co-Authors: Asuka Watanabe, Hiroshi Saito, Akihiko Sekizawa, Takumi Yanaihara, Atsushi Taguchi, Mitsunobu Shimazu, Ichiro MatsudaAbstract:We have developed a method that allows the prenatal DNA diagnosis of ornithine transcarbamylase (OTC) deficiency by using a single fetal nucleated erythrocyte (NRBC) isolated from Maternal Blood. OTC gene analysis of a male patient (TF) with early onset OTC deficiency was performed by single-strand conformation polymorphism (PCR-SSCP) and DNA sequencing. To investigate the possible prenatal diagnosis of OTC deficiency, Maternal Blood was obtained at 13 weeks of gestation of a subsequent pregnancy, from the mother of patient TF. NRBCs in the Maternal Blood were separated by using the density gradient method and then collected with a micromanipulator. The entire genome of a single NRBC was amplified by primer extension preamplification (PEP). The human leukocyte antigen (HLA)-DQ alpha genotype and sex were determined from small aliquots of the PEP product. The HLA-DQ alpha genotype of each of the parents of the male patient was also determined. Once a single NRBC had been identified as being of fetal origin, the OTC gene was analyzed by using the restriction fragment length polymorphism (RFLP) method. DNA analysis revealed a point mutation in exon 9 of the OTC gene in the OTC-deficient patient (TF). All NRBCs retrieved from Maternal Blood were successfully identified as being of fetal origin by HLA-DQ alpha genotyping and sex determination. RFLP analysis demonstrated that the fetal OTC gene was normal. This is the first study to successfully diagnose OTC deficiency prenatally, by using a single fetal NRBC from the Maternal circulation. Such prenatal DNA diagnosis is non-invasive and can be applied to other genetic diseases, including autosomal and X-linked diseases.
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Prenatal diagnosis of the fetal RHD Blood type using a single fetal nucleated erythrocyte from Maternal Blood
Obstetrics and gynecology, 1996Co-Authors: Akihiko Sekizawa, Hiroshi Saito, T Kimura, Akira Watanabe, Takumi Yanaihara, Sato TakeshiAbstract:Objective To develop a method that allows prenatal diagnosis of the fetal RhD Blood type from Maternal Blood. Methods Maternal Blood was obtained at 8–31 weeks' gestation, and nucleated erythrocytes were separated with Percoll using a discontinuous density gradient method, then collected individually by micromanipulation under microscopic observation. After whole genome amplification with primer extension pre-amplification, exon 7 of the RhD and RhCE as well as the ZFX/ZFY loci were further amplified by a nested polymerase chain reaction (PCR). Results Nucleated erythrocytes were detected in nine of ten Maternal Blood samples, and sex was determined in 13 of 21 nucleated erythrocytes. RhD genotype could be diagnosed in 12 of the 13 nucleated erythrocytes in which sex could be determined. The results of RhD Blood type and sex in nucleated erythrocytes obtained from Maternal Blood were identical with those of newborns. Fetal RhD Blood type could be determined in six of ten Maternal Blood samples. Conclusion A new method for noninvasive prenatal diagnosis of the fetal RhD Blood type using a single nucleated erythrocyte isolated from Maternal Blood was demonstrated. This diagnostic method offers extremely useful information for the management of Rh-negative pregnant women. Furthermore, this method of prenatal diagnosis can be applied to other genetic disorders and is expected to become the preferred method of noninvasive prenatal diagnosis of DNA.
Hiroshi Saito - One of the best experts on this subject based on the ideXlab platform.
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Prenatal Screening of Single-Gene Disorders from Maternal Blood
American Journal of Pharmacogenomics, 2001Co-Authors: Akihiko Sekizawa, Hiroshi SaitoAbstract:Fetal cells and cell-free fetal DNA can be found circulating in Maternal Blood. Fetal cells recovered from Maternal Blood provide the only source of pure fetal DNA for noninvasive prenatal DNA diagnosis. Fetal nucleated erythrocytes (NRBCs) are considered the most suitable Maternally-circulating fetal cells for this purpose, because they are not commonly found in the peripheral Blood of healthy adults and are most abundant in the fetus during early gestation. Because fetal cells in Maternal Blood are extremely rare, a definitive separation method has not yet been established. Fetal NRBCs can be enriched from Maternal Blood via fluorescence- or magnetic-activated cell sorting, density gradients, immuno-magnetic beads or micromanipulation. Fetal cells are identified by Giemsa staining, hybridization with Y-chromosome specific probes, PCR-detection of a specific paternal allele, or immunostaining for fetal cell antigens. Amplification of fetal DNA sequences by primer extension preamplification and PCR has allowed prenatal screening for Duchenne muscular dystrophy and the fetal RhD Blood type. Sequence-specific hybridization has been used to detect sickle cell anemia and β-thalassemia prenatally in heterozygous carriers of these disorders. The use of cell-free fetal DNA in Maternal plasma for the diagnosis of single-gene disorders is limited to disorders caused by a paternally inherited gene or a mutation that can be distinguished from the Maternally inherited counterpart. At present, fetal gender can be determined from Maternal plasma. When a pregnant woman is a heterzygous carrier of an X-linked disorder, the determination of fetal gender is clinically very informative for first-step screening to avoid invasive amniocentesis. The non-invasive prenatal diagnosis of genetic disorders should be applied to pregnant women with a definite risk for a specific single-gene disorder.
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Prenatal screening of single-gene disorders from Maternal Blood.
American journal of pharmacogenomics : genomics-related research in drug development and clinical practice, 2001Co-Authors: Akihiko Sekizawa, Hiroshi SaitoAbstract:Fetal cells and cell-free fetal DNA can be found circulating in Maternal Blood. Fetal cells recovered from Maternal Blood provide the only source of pure fetal DNA for noninvasive prenatal DNA diagnosis. Fetal nucleated erythrocytes (NRBCs) are considered the most suitable Maternally-circulating fetal cells for this purpose, because they are not commonly found in the peripheral Blood of healthy adults and are most abundant in the fetus during early gestation. Because fetal cells in Maternal Blood are extremely rare, a definitive separation method has not yet been established. Fetal NRBCs can be enriched from Maternal Blood via fluorescence- or magnetic-activated cell sorting, density gradients, immuno-magnetic beads or micromanipulation. Fetal cells are identified by Giemsa staining, hybridization with Y-chromosome specific probes, PCR-detection of a specific paternal allele, or immunostaining for fetal cell antigens. Amplification of fetal DNA sequences by primer extension preamplification and PCR has allowed prenatal screening for Duchenne muscular dystrophy and the fetal RhD Blood type. Sequence-specific hybridization has been used to detect sickle cell anemia and β-thalassemia prenatally in heterozygous carriers of these disorders.
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prenatal diagnosis of ornithine transcarbamylase deficiency by using a single nucleated erythrocyte from Maternal Blood
Human Genetics, 1998Co-Authors: Asuka Watanabe, Hiroshi Saito, Akihiko Sekizawa, Takumi Yanaihara, Atsushi Taguchi, Mitsunobu Shimazu, Ichiro MatsudaAbstract:We have developed a method that allows the prenatal DNA diagnosis of ornithine transcarbamylase (OTC) deficiency by using a single fetal nucleated erythrocyte (NRBC) isolated from Maternal Blood. OTC gene analysis of a male patient (TF) with early onset OTC deficiency was performed by single-strand conformation polymorphism (PCR-SSCP) and DNA sequencing. To investigate the possible prenatal diagnosis of OTC deficiency, Maternal Blood was obtained at 13 weeks of gestation of a subsequent pregnancy, from the mother of patient TF. NRBCs in the Maternal Blood were separated by using the density gradient method and then collected with a micromanipulator. The entire genome of a single NRBC was amplified by primer extension preamplification (PEP). The human leukocyte antigen (HLA)-DQ alpha genotype and sex were determined from small aliquots of the PEP product. The HLA-DQ alpha genotype of each of the parents of the male patient was also determined. Once a single NRBC had been identified as being of fetal origin, the OTC gene was analyzed by using the restriction fragment length polymorphism (RFLP) method. DNA analysis revealed a point mutation in exon 9 of the OTC gene in the OTC-deficient patient (TF). All NRBCs retrieved from Maternal Blood were successfully identified as being of fetal origin by HLA-DQ alpha genotyping and sex determination. RFLP analysis demonstrated that the fetal OTC gene was normal. This is the first study to successfully diagnose OTC deficiency prenatally, by using a single fetal NRBC from the Maternal circulation. Such prenatal DNA diagnosis is non-invasive and can be applied to other genetic diseases, including autosomal and X-linked diseases.
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Prenatal diagnosis of the fetal RHD Blood type using a single fetal nucleated erythrocyte from Maternal Blood
Obstetrics and gynecology, 1996Co-Authors: Akihiko Sekizawa, Hiroshi Saito, T Kimura, Akira Watanabe, Takumi Yanaihara, Sato TakeshiAbstract:Objective To develop a method that allows prenatal diagnosis of the fetal RhD Blood type from Maternal Blood. Methods Maternal Blood was obtained at 8–31 weeks' gestation, and nucleated erythrocytes were separated with Percoll using a discontinuous density gradient method, then collected individually by micromanipulation under microscopic observation. After whole genome amplification with primer extension pre-amplification, exon 7 of the RhD and RhCE as well as the ZFX/ZFY loci were further amplified by a nested polymerase chain reaction (PCR). Results Nucleated erythrocytes were detected in nine of ten Maternal Blood samples, and sex was determined in 13 of 21 nucleated erythrocytes. RhD genotype could be diagnosed in 12 of the 13 nucleated erythrocytes in which sex could be determined. The results of RhD Blood type and sex in nucleated erythrocytes obtained from Maternal Blood were identical with those of newborns. Fetal RhD Blood type could be determined in six of ten Maternal Blood samples. Conclusion A new method for noninvasive prenatal diagnosis of the fetal RhD Blood type using a single nucleated erythrocyte isolated from Maternal Blood was demonstrated. This diagnostic method offers extremely useful information for the management of Rh-negative pregnant women. Furthermore, this method of prenatal diagnosis can be applied to other genetic disorders and is expected to become the preferred method of noninvasive prenatal diagnosis of DNA.
Diana W. Bianchi - One of the best experts on this subject based on the ideXlab platform.
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pcr quantitation of fetal cells in Maternal Blood in normal and aneuploid pregnancies
American Journal of Human Genetics, 1997Co-Authors: Diana W. Bianchi, Lisa M. Sullivan, John M. Williams, Frederick W Hanson, Katherine W Klinger, Anthony P ShuberAbstract:Summary Fetal cells in Maternal Blood are a noninvasive source of fetal genetic material for prenatal diagnosis. We determined the number of fetal-cell DNA equivalents present in Maternal whole-Blood samples to deduce whether this number is affected by fetal karyotype. Peripheral Blood samples were obtained from 199 women carrying chro-mosomally normal fetuses and from 31 women with male aneuploid fetuses. Male fetal-cell DNA-equivalent quantitation was determined by PCR amplification of a Y chromosome-specific sequence and was compared with PCR product amplified from known concentrations of male DNA run simultaneously. The mean number of male fetal-cell DNA equivalents detected in 16-ml Blood samples from 90 women bearing a 46, XY fetus was 19 (range 0–91). The mean number of male fetal-cell DNA equivalents detected in 109 women bearing a 46, XX fetus was 2 (range 0-24). The mean number of male fetal-cell DNA equivalents detected when the fetus was male compared with when the fetus was female was highly significant ( P = .0001). More fetal cells were detected in Maternal Blood when the fetus was aneuploid. The mean number of male fetal-cell DNA equivalents detected when the fetal karyotype was 47, XY, +21 was 110 (range 0.1–650), which was significantly higher than the number of male fetal-cell DNA equivalents detected in 46, XY fetuses ( P = .0001). Feto-Maternal transfusion of nucleated cells appears to be influenced by fetal karyotype. The sixfold elevation of fetal cells observed in Maternal Blood when the fetus had trisomy 21 indicates that noninvasive cytogenetic diagnosis of trisomy 21 should be feasible.
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Prenatal diagnosis by analysis of fetal cells in Maternal Blood.
The Journal of pediatrics, 1995Co-Authors: Diana W. BianchiAbstract:The data accumulated thus far indicate that fetal NRBCs are the target cell type of choice in Maternal Blood for most investigators, although some groups continue to work with the trophoblast. Reports of persistent circulation of hematopoietic stem cells, lymphoid/myeloid progenitors, and lymphocytes mandate that removal of these cell types must occur before clinical diagnosis of the current pregnancy can be made. In selected cases, accurate detection of fetal aneuploidy has been made from fetal cells in Maternal Blood; the clinical evaluation sponsored by the National Institute of Child Health and Human Development will determine the sensitivity and specificity of cytogenetic diagnosis in a larger group of pregnant women, but this information will not be available for several years. At present, detection of uniquely fetal, paternally inherited gene polymorphisms or mutations such as the Rh(D) antigen is possible only because the mother lacks these genes; hence, Maternal cell contamination does not hinder diagnosis. Currently the presence of large numbers of Maternal cells in enriched samples precludes single-gene diagnosis for conditions in which the mother carries a mutant gene, because her cells are preferentially amplified and difficult to distinguish from those of the fetus. It is likely, however, that as techniques of individual fetal cell isolation are perfected, Maternal cell contamination will no longer be an issue, and the entire fetal genome will become available for diagnosis and therapy. Pediatricians need to be aware of the progress of research in this field, because fetal cell isolation from Maternal Blood not only could change prenatal diagnosis but would change the amount of genetic information that arrives with a newborn infant at birth. The ultimate goal of this work is to diagnose noninvasively, in the first trimester, the common fetal aneuploidies and single-gene disorders, to permit in utero treatment, or to allow low-risk pregnant women carrying an abnormal fetus an opportunity for reproductive choice.
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Prenatal genetic diagnosis by isolation and analysis of fetal cells circulating in Maternal Blood.
Seminars in perinatology, 1994Co-Authors: Geifman-holtzman O, Robert N. Blatman, Diana W. BianchiAbstract:In recent years, considerable progress has been achieved in the genetic analysis of fetal cells isolated from Maternal Blood. The goal of clinical diagnosis of either fetal chromosome abnormality or Mendelian DNA mutation has already been realized in an ever-increasing number of cases. The remaining challenges involve increasing the yield and visual identification of fetal cells while decreasing the remaining numbers of Maternal cells. It is presently unknown whether the frequency of fetal cells in Maternal Blood differs in cytogenetically abnormal pregnancies as compared with normal pregnancies. Although fetal cells in Maternal Blood have a practical role in prenatal diagnosis, their very existence and persistence in the mother raise many new questions regarding Maternal-fetal interaction.
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Erythroid‐specific antibodies enhance detection of fetal nucleated erythrocytes in Maternal Blood
Prenatal diagnosis, 1993Co-Authors: Diana W. Bianchi, Gretchen K. Zickwolf, Melissa C. Yih, Alan F. Flint, Ossie H. Geifman, Marlena S. Erikson, John M. WilliamsAbstract:Fetal nucleated erythrocytes (NRBC) in Maternal Blood are a non-invasive source of fetal DNA for prenatal genetic screening. We compared the effectiveness of three monoclonal antibodies for the separation of fetal cells from Maternal Blood by flow sorting. Mononuclear Blood cells from 49 healthy pregnant women were incubated with antibody to CD 71, CD 36, and/or glycophorin A (GPA), employed singly or in combination with each other. These monoclonal antibodies recognize surface antigens on haematopoietic precursor cells. Successful isolation of fetal cells was defined as detection of Y chromosomal sequences in Maternal Blood from women carrying male fetuses, with absence of Y sequences when female fetuses were carried. Thus, gender prediction accuracy was used as a measure of fetal cell separation. Using anti-CD 71 to isolate fetal cells, gender prediction was 57 per cent correct; with anti-CD 36, it was 88 per cent correct. Anti-GPA, an erythrocyte-specific antigen, used alone or in combination with anti-CD 71 or 36, improved gender prediction to 100 per cent. We conclude that antibody to GPA improves the retrieval of fetal NRBC from Maternal Blood, permitting genetic analysis by the polymerase chain reaction.
Sato Takeshi - One of the best experts on this subject based on the ideXlab platform.
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Prenatal diagnosis of the fetal RHD Blood type using a single fetal nucleated erythrocyte from Maternal Blood
Obstetrics and gynecology, 1996Co-Authors: Akihiko Sekizawa, Hiroshi Saito, T Kimura, Akira Watanabe, Takumi Yanaihara, Sato TakeshiAbstract:Objective To develop a method that allows prenatal diagnosis of the fetal RhD Blood type from Maternal Blood. Methods Maternal Blood was obtained at 8–31 weeks' gestation, and nucleated erythrocytes were separated with Percoll using a discontinuous density gradient method, then collected individually by micromanipulation under microscopic observation. After whole genome amplification with primer extension pre-amplification, exon 7 of the RhD and RhCE as well as the ZFX/ZFY loci were further amplified by a nested polymerase chain reaction (PCR). Results Nucleated erythrocytes were detected in nine of ten Maternal Blood samples, and sex was determined in 13 of 21 nucleated erythrocytes. RhD genotype could be diagnosed in 12 of the 13 nucleated erythrocytes in which sex could be determined. The results of RhD Blood type and sex in nucleated erythrocytes obtained from Maternal Blood were identical with those of newborns. Fetal RhD Blood type could be determined in six of ten Maternal Blood samples. Conclusion A new method for noninvasive prenatal diagnosis of the fetal RhD Blood type using a single nucleated erythrocyte isolated from Maternal Blood was demonstrated. This diagnostic method offers extremely useful information for the management of Rh-negative pregnant women. Furthermore, this method of prenatal diagnosis can be applied to other genetic disorders and is expected to become the preferred method of noninvasive prenatal diagnosis of DNA.
Takumi Yanaihara - One of the best experts on this subject based on the ideXlab platform.
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prenatal diagnosis of ornithine transcarbamylase deficiency by using a single nucleated erythrocyte from Maternal Blood
Human Genetics, 1998Co-Authors: Asuka Watanabe, Hiroshi Saito, Akihiko Sekizawa, Takumi Yanaihara, Atsushi Taguchi, Mitsunobu Shimazu, Ichiro MatsudaAbstract:We have developed a method that allows the prenatal DNA diagnosis of ornithine transcarbamylase (OTC) deficiency by using a single fetal nucleated erythrocyte (NRBC) isolated from Maternal Blood. OTC gene analysis of a male patient (TF) with early onset OTC deficiency was performed by single-strand conformation polymorphism (PCR-SSCP) and DNA sequencing. To investigate the possible prenatal diagnosis of OTC deficiency, Maternal Blood was obtained at 13 weeks of gestation of a subsequent pregnancy, from the mother of patient TF. NRBCs in the Maternal Blood were separated by using the density gradient method and then collected with a micromanipulator. The entire genome of a single NRBC was amplified by primer extension preamplification (PEP). The human leukocyte antigen (HLA)-DQ alpha genotype and sex were determined from small aliquots of the PEP product. The HLA-DQ alpha genotype of each of the parents of the male patient was also determined. Once a single NRBC had been identified as being of fetal origin, the OTC gene was analyzed by using the restriction fragment length polymorphism (RFLP) method. DNA analysis revealed a point mutation in exon 9 of the OTC gene in the OTC-deficient patient (TF). All NRBCs retrieved from Maternal Blood were successfully identified as being of fetal origin by HLA-DQ alpha genotyping and sex determination. RFLP analysis demonstrated that the fetal OTC gene was normal. This is the first study to successfully diagnose OTC deficiency prenatally, by using a single fetal NRBC from the Maternal circulation. Such prenatal DNA diagnosis is non-invasive and can be applied to other genetic diseases, including autosomal and X-linked diseases.
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Prenatal diagnosis of the fetal RHD Blood type using a single fetal nucleated erythrocyte from Maternal Blood
Obstetrics and gynecology, 1996Co-Authors: Akihiko Sekizawa, Hiroshi Saito, T Kimura, Akira Watanabe, Takumi Yanaihara, Sato TakeshiAbstract:Objective To develop a method that allows prenatal diagnosis of the fetal RhD Blood type from Maternal Blood. Methods Maternal Blood was obtained at 8–31 weeks' gestation, and nucleated erythrocytes were separated with Percoll using a discontinuous density gradient method, then collected individually by micromanipulation under microscopic observation. After whole genome amplification with primer extension pre-amplification, exon 7 of the RhD and RhCE as well as the ZFX/ZFY loci were further amplified by a nested polymerase chain reaction (PCR). Results Nucleated erythrocytes were detected in nine of ten Maternal Blood samples, and sex was determined in 13 of 21 nucleated erythrocytes. RhD genotype could be diagnosed in 12 of the 13 nucleated erythrocytes in which sex could be determined. The results of RhD Blood type and sex in nucleated erythrocytes obtained from Maternal Blood were identical with those of newborns. Fetal RhD Blood type could be determined in six of ten Maternal Blood samples. Conclusion A new method for noninvasive prenatal diagnosis of the fetal RhD Blood type using a single nucleated erythrocyte isolated from Maternal Blood was demonstrated. This diagnostic method offers extremely useful information for the management of Rh-negative pregnant women. Furthermore, this method of prenatal diagnosis can be applied to other genetic disorders and is expected to become the preferred method of noninvasive prenatal diagnosis of DNA.