Fetal RhD Genotyping using Cell Free Fetal DNA from Maternal Plasma in RhD Negative Women in Pakistan

Abstract

Background: The Rhesus (Rh) blood group system is one of the most hemogenic and polymorphic system in the body. RhD negative pregnant women carrying RhD positive fetus are always at the risk of hemolytic disease of the fetus and newborn. Prenatal fetal RhD genotyping leads to appropriate management and targeted anti-D immunoprophylaxis in RhD-positive pregnancies only. Objective: To determine the accuracy and diagnostic feasibility of non-invasive fetal RhD genotyping by analysis of cell free fetal DNA based on real time polymerase chain reaction (RT-PCR). Materials and Methods: A total of 100 EDTA-blood samples from pregnant women between 10-32 weeks of gestation were collected. The cell free fetal DNA was extracted from maternal plasma followed by PCR analysis for SRY, RhD (exons 5,7,10) and RASSF1A genes using specific primers. Fetus was considered as RhD-positive when amplification of all targeted RhD exons were observed and in case, where no amplification for RhD exons was detected but Beta-globin gene and SRY gene was amplified, fetus were characterized as RhD-negative. When one or two RhD exons were amplified, the results were predicted to be inconclusive and RT-PCR assay with new extracted DNA samples was repeated. Results: Out of 100 pregnancies, none of the sample was found to be inconclusive for fetal RhD status. No false- positive and falsenegative results were registered. The sensitivity, specificity and diagnostic concordance was predicted to be 100%. Conclusion: The protocol we applied was highly accurate for non-invasive fetal RhD determination. We therefore recommend implementation of non-invasive RhD genotyping in prenatal screening. Keywords: Cff DNA; Fetal RhD genotyping; Maternal plasma