fetal rhd genotyping using real-time polymerase chain reaction analysis of cell-free fetal dna in pregnancy of rhd negative women in south of iran

objective: maternal-fetal rhd antigen incompatibility causes approximately 50% of clinically significant alloimmunization cases. the routine use of prophylactic anti-d immunoglobulin has dramatically reduced hemolytic disease of the fetus and newborn. recently, fetal rhd genotyping in rhd negative pregnant women has been suggested for appropriate use of anti-d immunoglobulin antenatal prophylaxis and decrease unnecessary prenatal interventions. materials and methods: in this prospective cohort study, in order to develop a reliable and non-invasive method for fetal rhd genotyping, cell free fetal dna (cffdna) was extracted from maternal plasma. real-time quantitative polymerase chain reaction (qpcr) for detection of rhd exons 7, 5, 10 and intron 4 was performed and the results were compared to the serological results of cord blood cells as the gold standard method. sry gene and hypermethylated ras-association domain family member 1 (rassf1a) gene were used to confirm the presence of fetal dna in male and female fetuses, respectively. results: out of 48 fetuses between 8 and 32 weeks (wks) of gestational age (ga), we correctly diagnosed 45 cases (93.75%) of rhd positive fetuses and 2 cases (4.16%) of the rhd negative one. exon 7 was amplified in one sample, while three other rhd gene sequences were not detected; the sample was classified as inconclusive, and the rhd serology result after birth showed that the fetus was rhd-negative. conclusion: our results showed high accuracy of the qpcr method using cffdna for fetal rhd genotyping and implicate on the efficiency of this technique to predict the competence of anti-d immunoglobulin administration.