Evaluation of single nucleotide polymorphisms as internal controls in prenatal diagnosis of fetal blood groups

Ziel: Determination of fetal blood groups in plasma of pregnant women is increasingly utilized as an additional diagnostic method. However, test interpretation can fail, if insufficient amplification of fetal DNA occurs (a negative test result is expected, if the fetus is negative but may also occur if the amplification of the fetal DNA fails). To discriminate fetal and maternal DNA we evaluated the use of 52 SNPs as individual internal positive controls (IPC) in our PCR settings for prenatal blood group typing.

Methodik: DNA from 224 plasma samples (fetal & maternal DNA) was screened for 52 SNPs divided into four primer pools in a multiplex PCR setting. Detection of amplicons were done using SBE (single base extension) and the GeneScan Method in an ABI310. SNP frequencies were calculated and compared to ALFRED (allele frequency database).

Ergebnis: 97.8% of all samples showed differences in maternal and fetal SNP patterns when tested with four primer pools, 2.2% lacked these differences because of identical genotype or due to failure of fetal DNA extraction. Comparison with the independent results did not reveal a single false-negative case among samples with positive internal control and negative fetal RHD typing. Co-amplification with sequences for RHD,RHc,RHE,KEL and HPA 1 was successful, other systems are still in progress.

Schlussfolgerung: Negative results in fetal blood group determination can be confirmed with SNPs as a positive control for the presence of fetal DNA, even if a paternal blood sample is not available. Detection of amplicons with single base extension gives also the opportunity to include several blood group specific sequences.