Noninvasive prenatal diagnosis of fetal RhD status using cell-free fetal DNA in maternal plasma with TaqMan® real-time PCR assay

摘要

Prenatal diagnosis is now part of established obstetric practice in many countries.udHowever, conventional methods of prenatal diagnosis of obtaining fetal tissues forudgenetic analysis, including amniocentesis and chorionic villus sampling, are invasive andudconstitute a finite risk to the unborn fetus1. At present, it is widely accepted that bothudintact fetal cells as well as cell-free fetal DN A are present in the maternal circulation andudcan be recovered for non-invasive prenatal genetic diagnosis2,96,97. However, the rarity ofudcirculating fetal cells has limited the development and practical use of this approach.udFetal DNA is present in maternal plasma in much higher fractional concentration thanudfetal DNA in the cellular fraction of maternal blood. This important feature has made theudrobust detection of fetal DNA possible, even without special enrichment procedures.udAs a result of these deve lopments, fetal DNA in maternal plasma has been used for theudnoninvasive prenatal diagnosis of sex-linked disorders and single gene disorders such asudbeta-thalassemia, congenital adrenal hyperplasia and achondroplasia3-5. In addition,udquantitative aberrations of cell-free fetal DNA have also been found in variousudpregnancy-associated disorders, including preeclampsia, preterm labor and fetal trisomyud216,7.udMaternal plasma DNA analysis is also useful for the noninvasive prenatal determinationudof fetal RhD blood group status in RhD - negative pregnant women8. For the managementudof Hemolytic disease of the fetus and newborn (HDFN) a reliable non-invasive approachudto determine fetal RhD genotype would be of great clinical value. RhD genotyping wouldududalso eliminate the routine administration of Rh-immunoglobulin in RhD-negativeudpatients.udThis approach has been shown by many groups to be highly accurate (See Table 1), andudhas been introduced as a routine service in several centers in Europa49,53.udIn this thesis, the detail protocol of noninvasive detection of fetal RhD status usingudTaqManâ real-time polymerase chain reaction (PCR) technique is going to be described.