Prenatal genotyping of RHD and SRY using maternal blood
Article first published online: 24 NOV 2003
Volume 85, Issue 4, pages 300–306, November 2003
How to Cite
Randen, I., Hauge, R., Kjeldsen-Kragh, J. and Fagerhol, M. K. (2003), Prenatal genotyping of RHD and SRY using maternal blood. Vox Sanguinis, 85: 300–306. doi: 10.1111/j.0042-9007.2003.00371.x
- Issue published online: 24 NOV 2003
- Article first published online: 24 NOV 2003
- Received: 22 May 2003, revised 24 September 2003, accepted 26 September 2003
- DNA preservation;
- duplex PCR;
- fetal RHD genotyping
Background and Objectives The aim of the study was to perform fetal RHD genotyping in maternal plasma using a fluorescent polymerase chain reaction (PCR) technique. Duplex PCR, amplifying RHD and SRY in 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 the RHD gene'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 fetal RHD. 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, amplifying RHD and SRY in the same tube, showed a reduced sensitivity for amplification of the SRY gene 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 for RHD-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.