Non-Invasive Detection of Fetal Rhesus D Status: A Comparison between Polymerase Chain Reaction and Flow Cytometry

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Abstract
Objective: A non-invasive prenatal determination of the fetal RhD status might be useful for the management of pregnancies in RhD-negative women whose partners are RhD positive. Methods: Maternal peripheral blood of 32 RhD-negative women (17–24 weeks of gestation) was collected, and circulating fetal cells were enriched by CD71 mini-magnetic activated cell sorting. The RhD status of the fetuses was assessed using multiparametric flow cytometry, and results were compared to those of reverse transcriptase (RT)-polymerase chain reaction (PCR), or PCR, which acted as control. Flow-cytometric study of fetal cells employed monoclonal antibodies directed against CD71, glycophorin A (GPA) and RhD antigens. Results: The median percentage of CD71- and RhD-positive cells was 0.83% (range 0.14–6.44%), and that of CD71 and GPA-positive cells was 10.07% (range 0.52–45.84%). Flow-cytometric analysis correlated with RT-PCR results of RNA obtained from whole maternal blood. In 1 case, an incorrect result was due to the failure of the amplification of the specific RhD band on RNA extracted from the CD71-positive fraction. In two instances, we observed false-positive results for RhD in PCR of DNA obtained from maternal plasma. Conclusion: Based on our results, flow-cytometric analysis might be proposed as a clinical tool for the non-invasive prenatal determination of the fetal RhD status independently of fetal gender.