OBJECTIVES: To develop a non-invasive method for determining fetal RhD status in 
order to provide improved care for women most at risk.
DESIGN: A prospective study.
METHODS: Fetal erythroblasts were enriched from the peripheral circulation of 96 
RhD negative women with pregnancies at various stages in gestation using 
discontinuous density gradients. Amplification of RhD-specific mRNAs was carried 
out by reverse transcription-polymerase chain reaction assay. RNA, rather than 
DNA, was selected for amplification because it rarely contaminates samples, thus 
resulting in fewer false positives; moreover, its presence in multiple copies 
per cell should enhance the sensitivity of the assay, resulting in fewer false 
negatives. The study was prospective, relying on postnatal serological 
confirmation of RhD phenotype.
RESULTS: The assay was 75% accurate at predicting fetal RhD status, comparing 
favourably with standard genomic DNA-based assays. However, we found that 
accuracy dropped from 85% (29/34) in the third trimester of pregnancy, to 82% 
(32/39) in the second and 48% (11/23) in the first trimester. Discordant data 
were due to false negatives in the majority (78%) of cases.
CONCLUSIONS: We suggest that reverse transcription may be a useful and perhaps 
more sensitive alternative to standard genomic polymerase chain reaction in the 
majority of cases. However, under certain circumstances the absence or reduction 
of fetal erythroblasts or possibly RhD mRNA in some preparations may compromise 
the accuracy of the assay.