Chapter 20 An Additional Pre-amplification Step for the Early Determination of Fetal RHD from Maternal Plasma Tadeja Dovˇ c-Drnovšek, Nataša Toplak, Irena Bricl, Tanja Blejec, Minka Kovaˇ c, and Primož Rožman Abstract The determination of the fetal RhD blood group is essential for the pre- vention of haemolytic disease of the newborn; prenatally it can be determined by genotyping fetal DNA. The advantage of fetal RHD genotype determination from maternal plasma compared to classical fetal sampling is enormous, since there is no risk for either the mother or the fetus. In the last few years, sensitive methods such us qPCR, have been developed. In our study, the newly developed qPCR assay was used and a new pre-analytical step was included to improve the robustness of the test and the reliability of the results. This pertains especially to the determination of the fetal RHD status in early pregnancy when the amount of cell-free fetal DNA in the mother’s blood is extremely low. The new pre-analytical step increased 512-fold the amount of detected cell free DNA and cell free fetal DNA from maternal plasma. Keywords Cell-free DNA · Maternal plasma · Pre-amplification · Prenatal diagnosis Abbreviations cfDNA Cell-free DNA cffDNA Cell-free fetal DNA qPCR Real-time PCR RhD Rhesus D Introduction Rhesus D (RhD) blood group incompatibility between an RhD-negative mother and an RhD-positive fetus followed by allosensitation can result in haemolytic disease of the newborn (HDN) and even fetal death due to anti-D antibodies (Mollison et al. T. Dovˇ c-Drnovšek (B ) Blood Transfusion Centre of Slovenia, Ljubljana, Slovenia e-mail: tadeja.dovc-drnovsek@ztm.si 147 P.B. Gahan (ed.), Circulating Nucleic Acids in Plasma and Serum, DOI 10.1007/978-90-481-9382-0_20, C  Springer Science+Business Media B.V. 2011