Prenatal genotyping of RHD and SRY using maternal blood.
OBJECTIVE
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.


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 degrees 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.
Randen
I
I
Department of Immunology and Transfusion Medicine, Ullevål University Hospital, Oslo, Norway. ingrid.randen@ioks.uio.no
Hauge
R
R
Kjeldsen-Kragh
J
J
Fagerhol
M K
MK
D000328:Adult; D001788:Blood Grouping and Crossmatching / Q000379:methods*; D001793:Blood Preservation; D004247:DNA / Q000097:blood* / Q000235:genetics; D005260:Female; D005331:Fetomaternal Transfusion / Q000097:blood*; D024282:Genes, sry; D005838:Genotype; D006801:Humans; D007231:Infant, Newborn; D008297:Male; D016133:Polymerase Chain Reaction; D011247:Pregnancy; D011264:Pregnancy Trimesters / Q000097:blood*; D011296:Prenatal Diagnosis / Q000379:methods*; D015203:Reproducibility of Results; D012204:Rh-Hr Blood-Group System / Q000097:blood / Q000235:genetics*; D012680:Sensitivity and Specificity; D012732:Sex Determination Analysis; D013048:Specimen Handling / Q000379:methods; D013696:Temperature
D012204:Rh-Hr Blood-Group System; C026410:Rho(D) antigen; D004247:DNA