Comparison of PCR methods for detecting fetal RhDin maternal plasma.

Background: Aim of this study was to establish the method yielding the highest sensitivity routinely used to determine fetal RhD type and gender from maternal cell-free plasma DNA in different periods of gestation.

Methods: Plasma DNA concentrations were measured from 46 pregnant women in different gestational periods and tested for RhD using three different PCR methods on exon 7: Thermal Cycler, Taqman method on LightCycler, and melting curve analysis on LightCycler. In addition, fetal gender was determined by PCR. Cell-free plasma DNA was measured in 100 healthy volunteers as a reference group.

Results: The mean value of cell-free plasma DNA in the reference group was 10.9 pg/microL mean, (standard deviation (SD): 3.66) in 50 healthy women and 12.7 pg/microL (SD: 8.2) in 50 healthy men. In the first trimester of pregnancy cell-free plasma DNA was 14.9 pg/microL mean, (SD: 4.2), in the second trimester 15.4 pg/microL mean, (SD: 4.96), and the maximum was achieved in the third trimester of pregnancy 15.6 pg/microl mean, (SD: 6.49). TaqMan probes had the same accuracy, when compared with Thermal Cycler technology (46 samples, 6 failures). Using real-time PCR with melting curve analysis 12 of 17 samples were correctly tested. Gender determination was correctly in 41 of 46 samples.

Conclusion: RhD determinations with TaqMan and Thermal Cycler technology are useful methods for fetal RhD prediction. To increase the accuracy of RhD determination it is necessary to test on other exons in addition.