The use of free DNA for fetal RHD genotyping in the Rh negative pregnant patient-the time has come.

Cell-free DNA to determine the fetal RHD genotype from the maternal circulation was first described in 1993. High throughput assays using polymerase chain reaction technology were introduced in Europe and gained widespread acceptance in the management of the Rhesus alloimmunized pregnancy. The specificity and sensitivity of these assays approached 99%. As confidence was gained with these results, Scandinavian countries began to employ cell-free DNA for fetal RHD typing as an integral component of their introduction of antenatal Rhesus immune globulin in non-alloimmunized pregnancies. Since 40% of RhD-negative pregnant women will carry an RhD-negative fetus, doses of Rhesus immune globulin were conserved. Recently 2 U.S. companies have introduced cell-free DNA assays for RHD as part of their noninvasive prenatal testing assays. Both utilize next generation sequencing and have developed methodologies to detect the aberrant RHD pseudogene and the hybrid RHD-CE-Ds genotype. In addition, excellent correlation studies with either neonatal genotyping or serology have been reported. The manufacturer of RhoGAM has recently announced a national shortage. Given the current availability of reliable cell-free DNA assays for determining the RHD status of the fetus, the time has come to implement this strategy to triage the antenatal use of Rhesus immune globulin in the U.S.

Molecular analysis of the RHD pseudogene by duplex real-time polymerase chain reaction.

OBJECTIVES: In this study, we aimed to present a strategy for the detection of the RHD pseudogene (RHDψ) based on a real-time polymerase chain reaction (PCR) assay. BACKGROUND: The D-negative phenotype is associated with many genetic alterations. In populations with African ancestry, this phenotype commonly results from the silent variant RHDψ. The evaluation of RHDψ is essential for correct inference of the RhD phenotype in order to avoid false-positive results. METHODS: We utilised a new method for the simultaneous detection of RHDψ and a fragment from exon 5 of the wild-type RHD gene based on duplex real-time PCR assay. RESULTS: The PCR assay allowed specific detection of RHDψ. There was complete agreement between the results generated by the new test and the results generated by molecular analysis performed using end-point PCR methods previously described. CONCLUSIONS: The assay developed is easy to execute and presents the potential for routine use at blood banks and other associated facilities where it is desired to determine the presence of RHDψ.