Cell-free fetal DNA
Traditionally, invasive diagnostic procedures were needed to obtain material for analyzing the fetal genome. The discovery of cell-free fetal (cff) DNA in the blood of pregnant women in 1997 presented a noninvasive, and thus safe, method to determine the fetal genotype. CffDNA is released from trophoblastic cells undergoing apoptosis. Within the maternal circulation, cffDNA is present among an overwhelming background of maternal cell-free DNA, predominately of hematopoietic origin . It can be detected as early as 5 weeks of gestation, and gradually increases during pregnancy, from 10 genome equivalents (GE) per ml to around 300 GE late in pregnancy. After birth, cffDNA is cleared from the maternal circulation within several hours, with an observed half-life of 16 minutes. Within the so-called EU-granted SAFE -Network, 50 laboratories have collaborated between 2004 and 2009 on the development and standardization of DNA isolation methods and non-invasive diagnostic assays. Although the final aim of this network was to develop assays for many diseases, including chromosomal aneuploidies, fetal RHD typing served as a model system for non-invasive prenatal diagnostics.
Nationwide screening of all D-negative pregnant women
RhD incompatibility between a pregnant women and her fetus can result in alloimmunisation and consequently hemolytic disease of the fetus and newborn (HDFN). Postnatal immune-prophylaxis and more recently antenatal immunoprophylaxis, has successfully decreased the incidence of HDFN. Postnatal immunoprophylaxis is given based on cord blood serology, since only 60% of the newborns of a D-negative mother are D-positive. But as long as the fetal blood group is not known, antenatal prophylaxis is administered to all D-negative women. To restrict antenatal prophylaxis to women carrying D-negative fetuses, we have developed a fully-automated fetal RHD genotyping assay using cff DNA isolated from maternal blood and showed that implementation of this assay was cost-effective. Based on these results, the Dutch government decided to implement fetal RHD typing in the 27th week of pregnancy. The false negative rate of this program is now below 0.05%. Also postnatal immunoprophylaxis is given based on the PCR result and standard cord blood serology is no longer performed.
Application of cell free fetal DNA for chromosomal abnormalities
The fetal RHD genotyping was the first diagnostic application of cell free fetal DNA. However, numerous studies have shown that assays based on next generation sequencing, can reliably detect chromosomal abnormalities like trisomy 21 in the fetus. In many countries this diagnostic service is already offered and hopefully the Netherlands will follow soon.
Ellen van der Schoot, Sanquin Research