A multiplex PCR for non-invasive fetal RHD genotyping using cell-free fetal DNA.

AIM To design a protocol for non-invasive prenatal diagnosis of fetal Rhesus D (RhD) status. MATERIALS AND METHODS A total of 112 single lymphocytes were used to test the efficiency of the assay. The protocol was validated using blood samples from 84 RhD-negative pregnant women at 7-24 weeks of gestation. Cell-free DNA (cfDNA) was enzymatically digested using AciI and analyzed by a polymerase chain reaction (PCR) that allowed simultaneous amplification of RHD exons 7 and 10, SRY, RASFF1A and ACTB. RESULTS On the one genome-equivalent level, the efficiency of the protocol was ≥ 94.6% for each locus amplified. Conclusive results from the first set of PCRs were obtained for 79 cases with one false-positive. In five cases the analysis was repeated and, subsequently, all cases were accurately diagnosed. CONCLUSION The proposed protocol is rapid, applicable in most molecular diagnostic laboratories and provides the basis for non-invasive examination of fetal RhD with 96.7% specificity and 100% sensitivity.