Quantitation of RHD by real-time polymerase chain reaction for determination of RHD zygosity and RHD mosaicism/chimerism: an evaluation of four quantitative methods

BACKGROUND: Determination of RHD zygosity of the spouse is crucial in preconception counseling of families with history of hemolytic disease of the fetus and newborn caused by anti-D. RHD zygosity can be determined by quantitative real-time polymerase chain reaction (PCR) basically by determining RHD dosage, and this feature is relevant in investigating RHD mosaicism and chimerism. STUDY DESIGN AND METHODS: Monoplex and duplex real-time PCRs, uncalibrated and calibrated, were tested. RHD zygosity was determined for 72 samples and compared with serology-based prediction of RHD zygosity. Additionally, a range of constructed RHD dosages were compared with determined RHD dosages. Finally three samples, Days 1, 90, and 120, from a patient with loss of D were included. RESULTS: All setups enabled differentiation between hemi- and homozygous samples. Results from calibrated methods had the smallest variation and enabled differentiation at the mean ± 3SD. Incongruity between determined and predicted RHD zygosity was found in three samples. PCR–sequence-specific priming specific for the hybrid downstream Rhesus box was performed on those samples confirming the real-time PCR results. Determined RHD dosage equaled constructed RHD dosage in both calibrated real-time PCRs. Patient samples showed RHD with a mean of 1, 5, and 8 percent relative to GAPDH in all samples. CONCLUSION: Duplex calibrated real-time PCR was a robust and exact method for determination of RHD zygosity and applicable in evaluation of RHD mosaicism and chimerism. This was illustrated with patient samples, where this assay revealed for the first time signs of mosaicism in both expression of D antigens and in RHD dosage.