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

Authors

  • Andrea Doescher,

    Corresponding author
    1. From the DRK-Blutspendedienst NSTOB, Institut Bremen-Oldenburg, Oldenburg, Germany; the DRK-Blutspendedienst NSTOB, Institut Springe, Springe, Germany; and St Josefs-Hospital, Wiesbaden, Germany.
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  • Eduard K. Petershofen,

    1. From the DRK-Blutspendedienst NSTOB, Institut Bremen-Oldenburg, Oldenburg, Germany; the DRK-Blutspendedienst NSTOB, Institut Springe, Springe, Germany; and St Josefs-Hospital, Wiesbaden, Germany.
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  • Franz F. Wagner,

    1. From the DRK-Blutspendedienst NSTOB, Institut Bremen-Oldenburg, Oldenburg, Germany; the DRK-Blutspendedienst NSTOB, Institut Springe, Springe, Germany; and St Josefs-Hospital, Wiesbaden, Germany.
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  • Markus Schunter,

    1. From the DRK-Blutspendedienst NSTOB, Institut Bremen-Oldenburg, Oldenburg, Germany; the DRK-Blutspendedienst NSTOB, Institut Springe, Springe, Germany; and St Josefs-Hospital, Wiesbaden, Germany.
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  • Thomas H. Müller

    1. From the DRK-Blutspendedienst NSTOB, Institut Bremen-Oldenburg, Oldenburg, Germany; the DRK-Blutspendedienst NSTOB, Institut Springe, Springe, Germany; and St Josefs-Hospital, Wiesbaden, Germany.
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Andrea Doescher, DRK-Blutspendedienst NSTOB, Institut Bremen-Oldenburg, Brandenburger Strasse 21, 26133 Oldenburg, Germany; e-mail: doescher@bsd-nstob.de.

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.

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