Non-invasive fetal ABO genotyping in maternal plasma using real-time PCR

Background: Fetal-maternal ABO incompatibility is a frequent cause of hemolytic disease of the fetus and newborn (HDFN). The routine serological testing of maternal IgG antibody level to predict HDFN shows low reliability. Non-invasive fetal ABO genotyping could provide a new avenue for predicting ABO-HDFN in early pregnancy. The aim of our study is to investigate the feasibility of fetal ABO genotyping in maternal plasma with real-time PCR. Methods: Plasma samples were collected from a total of 73 blood group O pregnant women between 12 and 25 weeks of gestation, and then DNA was extracted from the maternal plasma containing cell-free fetal DNA (cffDNA). TaqMan-based real-time PCR was performed after methylation-sensitive restriction enzyme to detect hypermethylated RASSF1A sequences of fetal origin in maternal plasma. Fetal ABO genotypes were determined by SYBR-based real-time PCR with allele-specific primers. The performance of the fetal ABO genotyping was assessed by the blood group serology results of the newborns. Results: The fetal RASSF1A sequences were detectable in all the 73 plasma samples, which confirmed the successful extraction of cffDNA. The diagnostic accuracy of fetal ABO genotyping was 93.2%, in which the accuracy of fetal genotype OO, OA and OB was 100%, 83.3% and 96.8%, respectively. Conclusions: We have developed a rapid and reliable protocol for fetal ABO genotyping in maternal plasma using real-time PCR. This protocol is suitable for routine prenatal diagnose of HDFN and forensic analysis.