Overview of non-invasive fetal blood group genotyping

Non-invasive prenatal testing (NIPT) is commonly used to identify feto-maternal blood group allele discrepancy. Fetal cell-free DNA (cfDNA) in maternal blood sample has two important uses: firstly, in genotyping for the diagnosis of hemolytic disease of the fetus and newborn (HDFN) and secondly, in targeting routine antenatal anti-D prophylaxis (RAADP) at women carrying an RHD -positive fetus. The aim of the current paper is to review technical aspects of cell-free fetal blood group genotyping including methods, controls, and accuracy of different assays. As a highly accurate technique, real -time PCR is overwhelmingly the most commonly used method for RHD screening tests. The antenatal RHD screening assays are exceedingly sensitive allowing reliable to target anti-D prophylaxis solely at women bearing an RHD -positive fetus, also after childbirth. Droplet digital PCR (ddPCR), capillary electrophoresis and massively parallel sequencing are powerful methods for fetal genotyping, and assays for blood groups within the blood group systems ABO, Rh, Kell, Fy, Jk and MNS have been implemented with reliable results. The massive abundance of maternal DNA in a sample, the small size of circulating cfDNA fragments and blood group genetics pose challenges for designing robust assays. Marker allele panels, epigenetic markers, total DNA and process controls are used to confirm the presence of fetal DNA in these assays.