Evaluation of single-nucleotide polymorphisms as internal controls in prenatal diagnosis of fetal blood groups.
BACKGROUND
Determination of fetal blood groups in maternal plasma samples critically depends on adequate amplification of fetal DNA. We evaluated the routine inclusion of 52 single-nucleotide polymorphisms (SNPs) as internal reference in our polymerase chain reaction (PCR) settings to obtain a positive internal control for fetal DNA.


METHODS
DNA from 223 plasma samples of pregnant women was screened for RHD Exons 3, 4, 5, and 7 in a multiplex PCR including 52 SNPs divided into four primer pools. Amplicons were analyzed by single-base extension and the GeneScan method in a genetic analyzer. Results of D screening were compared to standard RHD genotyping of amniotic fluid or real-time PCR of fetal DNA from maternal plasma.


RESULTS
The vast majority of all samples (97.8%) demonstrated differences in maternal and fetal SNP patterns when tested with four primer pools. These differences were not observed in less than 2.2% of the samples most probably due to an extraction failure for adequate amounts of fetal DNA. Comparison of the fetal genotypes with independent results did not reveal a single false-negative case among samples (n = 42) with positive internal control and negative fetal RHD typing.


CONCLUSIONS
Coamplification of 52 SNPs with RHD-specific sequences for fetal blood group determination introduces a valid positive control for the amplification of fetal DNA to avoid false-negative results. This new approach does not require a paternal blood sample. It may also be applicable to other assays for fetal genotyping in maternal blood samples.
DRK-Blutspendedienst NSTOB, Institut Bremen-Oldenburg, Oldenburg, Germany. doescher@bsd-nstob.de
Andrea
A
Doescher
Eduard K
EK
Petershofen
Franz F
FF
Wagner
Markus
M
Schunter
Thomas H
TH
Müller
D000653:Amniotic Fluid / Q000737:chemistry / Q000378:metabolism; D001483:Base Sequence; D001789:Blood Group Antigens / Q000032:analysis / Q000235:genetics*; D004247:DNA / Q000032:analysis; D005260:Female; D005312:Fetal Blood; D005787:Gene Frequency; D005838:Genotype; D006801:Humans; D020641:Polymorphism, Single Nucleotide / Q000502:physiology; D011247:Pregnancy; D011296:Prenatal Diagnosis / Q000379:methods / Q000592:standards*; D060888:Real-Time Polymerase Chain Reaction; D012016:Reference Values; D012204:Rh-Hr Blood-Group System / Q000097:blood / Q000235:genetics*; D012680:Sensitivity and Specificity
D001789:Blood Group Antigens; D012204:Rh-Hr Blood-Group System; D004247:DNA