Non-invasive Testing For The Determination Of The Fetal RhCcE Factor In Maternal Plasma

Background:Rh blood group incompatibility between mother and child is one of the most common causes of hemolytic disease of the fetus and newborn (HDFN). Prenatal diagnosis of fetal blood group is the primary means in prevention and treatment of HDFN. At present, the routine methods of prenatal diagnosis are amniocentesis and Chorionic villus, however, the procedures for obtaining these materials are invasive and not benign, and are associated with a higher risk of spontaneous abortion. Therefore, it will be beneficial if a non-invasive method is developed for determining fetal RH type in all pregnant women.Objective:To establish a Fluorescent Quantitative PCR (FQ-PCR) method to detect fetal RhCcE factor in maternal plasma, optimized and validated the effectiveness of fluorescence quantitative PCR to detect fetal RhCcE factor from maternal plasma at various gestational ages.Methods:Free DNA were extracted from pregnant women at8-38weeks of gestation. The fetal RhCcE factor were genotyped by FQ-PCR and validated with serological testing from umbilical cord blood of infant.Results:1. A FQ-PCR method was developed to detect fetal RhCcE factor from maternal plasma;2. The accuracy rate of this method detected fetal RhC、c、E factor in maternal plasma is94%、100%、100%, respectively;3. Comparison results between FQ-PCR from maternal plasma and serological testing from umbilical cord blood of infant in different gestational weeks,the accuracy rate of C genotyping is94.1%、91.6%、100%, respectively, the omission ratio is5.9%、8.3%、0%(the ratio were1/17、2/24、0/18); the accuracy rate of c genotyping is97.6%、100%、100%, respectively, the omission ratio is2.4%、0%、0%(the ratio were1/42、0/55、0/30); the accuracy rate of E genotyping is97.8%、98.4%、100%, respectively, the omission ratio is2.1%、1.6%、0%(the ratio were1/47、1/61、0/35);4. This experiment suggested higher yield of free fetal DNA of QIAamp DNA Blood Midi Kit in comparison with the QIAamp DNA Blood Mini Kit.Conclusions:FQ-PCR can be used to detect fetal RhCcE factor in early gestation, can be used for prenatal diagnosis of fetal and newborn hemolytic disease, the accuracy rate of FQ-PCR to detect fetal RhCcE factor increased with the gestation. The method of free DNA extraction from maternal plasma is an important factor to sensitivity of diagnosis.