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.