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

Transfusion. 2013 Feb;53(2):353-62. doi: 10.1111/j.1537-2995.2012.03738.x. Epub 2012 Jun 13.

Abstract

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.

Study design and 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.

Conclusion: 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.

Publication types

  • Evaluation Study

MeSH terms

  • Amniotic Fluid / chemistry
  • Amniotic Fluid / metabolism
  • Base Sequence
  • Blood Group Antigens / analysis
  • Blood Group Antigens / genetics*
  • DNA / analysis
  • Female
  • Fetal Blood*
  • Gene Frequency
  • Genotype
  • Humans
  • Polymorphism, Single Nucleotide* / physiology
  • Pregnancy
  • Prenatal Diagnosis / methods
  • Prenatal Diagnosis / standards*
  • Real-Time Polymerase Chain Reaction
  • Reference Values
  • Rh-Hr Blood-Group System / blood
  • Rh-Hr Blood-Group System / genetics*
  • Sensitivity and Specificity

Substances

  • Blood Group Antigens
  • Rh-Hr Blood-Group System
  • DNA