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.