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

Prenatal diagnosis is now part of established obstetric practice in many countries.
However, conventional methods of prenatal diagnosis of obtaining fetal tissues for
genetic analysis, including amniocentesis and chorionic villus sampling, are invasive and
constitute a finite risk to the unborn fetus1. At present, it is widely accepted that both
intact fetal cells as well as cell-free fetal DN A are present in the maternal circulation and
can be recovered for non-invasive prenatal genetic diagnosis2,96,97. However, the rarity of
circulating fetal cells has limited the development and practical use of this approach.
Fetal DNA is present in maternal plasma in much higher fractional concentration than
fetal DNA in the cellular fraction of maternal blood. This important feature has made the
robust detection of fetal DNA possible, even without special enrichment procedures.
As a result of these deve lopments, fetal DNA in maternal plasma has been used for the
noninvasive prenatal diagnosis of sex-linked disorders and single gene disorders such as
beta-thalassemia, congenital adrenal hyperplasia and achondroplasia3-5. In addition,
quantitative aberrations of cell-free fetal DNA have also been found in various
pregnancy-associated disorders, including preeclampsia, preterm labor and fetal trisomy
216,7.
Maternal plasma DNA analysis is also useful for the noninvasive prenatal determination
of fetal RhD blood group status in RhD - negative pregnant women8. For the management
of Hemolytic disease of the fetus and newborn (HDFN) a reliable non-invasive approach
to determine fetal RhD genotype would be of great clinical value. RhD genotyping would
also eliminate the routine administration of Rh-immunoglobulin in RhD-negative
patients.
This approach has been shown by many groups to be highly accurate (See Table 1), and
has been introduced as a routine service in several centers in Europa49,53.
In this thesis, the detail protocol of noninvasive detection of fetal RhD status using
TaqManâ real-time polymerase chain reaction (PCR) technique is going to be described.