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Evaluation of Two Real-Time Multiplex PCR Screening Assays Detecting Fetal RHD in Plasma from RhD Negative Women to Ascertain the Requirement for Antenatal RhD Prophylaxis

Authors
  • Clausen, Frederik Banch
  • Krog, Grethe Risum
  • Rieneck, Klaus
  • Råsmark, Emma Elin Frida
  • Dziegiel, Morten Hanefeld
Type
Published Article
Journal
Fetal Diagnosis and Therapy
Publisher
S. Karger AG
Publication Date
Nov 11, 2010
Volume
29
Issue
2
Pages
155–163
Identifiers
DOI: 10.1159/000321347
PMID: 21071922
Source
Karger
Keywords
License
Green
External links

Abstract

Objective: To evaluate two different multiplex real-time PCR assays detecting fetal RHD for screening of RhD negative women in relation to antenatal RhD prophylaxis. Methods: We designed a duplex assay for the detection of RHD exon 7 and 10 and a triplex assay for the detection of RHD exon 7, 10 and 5. We used the same fluorescent dye for the exon 7 and 10 probes to increase sensitivity; exon 5 was VIC labeled. We evaluated possible inhibition of DNA amplification with dilution experiments. We then tested the two multiplex assays with DNA extracted from 97 plasma samples from 38 RhD negative women in gestational weeks 6–37. Results: Dilution experiments revealed no inhibition of amplification in the multiplex assays. For plasma samples, the duplex assay was significantly more sensitive than the triplex assay (p < 0.0001). For the duplex assay (exon 7/10), accuracy was 99.0%. For the triplex assay (exon 7/10), accuracy was 94.2%. Detection of exon 5 was less reliable. Conclusion: The duplex assay using exon 7/10 was the most reliable for prenatal prediction of fetal RhD type as a candidate assay for screening of RhD negative women in relation to antenatal RhD prophylaxis. The triplex assay needs further optimization.

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