Prenatal genotyping ofRHDandSRYusing maternal blood

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Abstract
Background and Objectives The aim of the study was to perform fetalRHDgenotyping in maternal plasma using a fluorescent polymerase chain reaction (PCR) technique. Duplex PCR, amplifyingRHDandSRYin the same tube, was undertaken. The effect of varying storage temperatures on the concentration of fetal DNA was investigated in a separate study involving 10 RhD‐negative pregnant women.Materials and Methods Primers and probes for theRHDgene's exon 7 and the sex‐determining region, Y, were designed, and monoplex and duplex PCR were performed. Blood samples from 10 RhD‐negative women were split into four and treated in four different ways before measuring the concentration of fetal DNA by quantitative PCR.Results DNA extracted from the plasma of 114 RhD‐negative pregnant women was tested for the presence of fetalRHD. The discrepancy between genotyping and serological RhD typing of the babies postpartum was 8% when counting one positive replicate as a positive result. Duplex PCR, amplifyingRHDandSRYin the same tube, showed a reduced sensitivity for amplification of theSRYgene segment. There was a statistically significant reduction of fetal DNA in blood samples stored at room temperature for 48 h compared with the same sample stored at a temperature of < 10 °C for the same length of time.Conclusions This method is not suitable for routine analysis because of the lack of a positive control forRHD‐negative female fetuses and a decrease in PCR sensitivity when performing duplex PCR. Fetal DNA in maternal plasma is better preserved when the blood sample is kept cool.