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Table of Contents
Vol. 20, No. 4, 2005
Issue release date: July – August
Section title: Paper
Fetal Diagn Ther 2005;20:275–280
(DOI:10.1159/000085085)

Non-Invasive Fetal RHD Exon 7 and Exon 10 Genotyping Using Real-Time PCR Testing of Fetal DNA in Maternal Plasma

Hromadnikova I.a · Vechetova L.a · Vesela K.a · Benesova B.c · Doucha J.b · Kulovany E.b · Vlk R.b
a2nd Clinic of Paediatrics, bClinic of Obstetrics and Gynecology, and cBlood Bank, 2nd Medical Faculty, Charles University, University Hospital Motol, Prague, Czech Republic

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Article / Publication Details

First-Page Preview
Abstract of Paper

Received: January 15, 2004
Accepted: April 07, 2004
Published online: June 16, 2005
Issue release date: July – August

Number of Print Pages: 6
Number of Figures: 2
Number of Tables: 2

ISSN: 1015-3837 (Print)
eISSN: 1421-9964 (Online)

For additional information: http://www.karger.com/FDT

Abstract

Objective: In this prospective study, we assessed the feasibility of foetal RHD genotyping by analysis of DNA extracted from plasma samples of Rhesus (Rh) D-negative pregnant women using real-time PCR and primers and probes targeted toward exon 7 and 10 of RHD gene. Methods: We analysed 24 RhD-negative pregnant woman and 4 patients with weak D phenotypes at a gestational age ranging from 11th to 38th week of gestation and correlated the results with serological analysis of cord blood after the delivery. Results: Non-invasive prenatal foetal RHD exon 7 genotyping analyses of maternal plasma samples was in complete concordance with the serological analysis of cord blood in all 24 RhD-negative pregnant women delivering 12 RhD-positive and 12 RhD-negative newborns. RHD exon-10-specific PCR amplicons were not detected in 2 out of 12 studied plasma samples from women bearing RhD-positive foetus, despite the positive amplification in RHD exon 7 region observed in all cases. In 1 case red cell serology of cord blood revealed that the mother had D–C–E–c+e+ Cw– and the infant D+C–E–c+e+ Cw+ phenotypes. RhD exon 10 real-time PCR analysis of cord blood was also negative. These findings may reflect that DCw– paternally inherited haplotype probably possesses no RHD exon 10. In another case no cord blood sample has been available for additional studies. The specificity of both RHD exon 7 and 10 systems approached 100% since no RhD-positive signals were detected in women currently pregnant with RhD-negative foetus (n = 8). Using real-time PCR and DNA isolated from maternal plasma, we easily differentiated pregnant woman whose RBCs had a weak D phenotype (n = 4) from truly RhD-negative patients since the threshold cycle (CT) for RHD exon 10 or 7 amplicons reached nearly the same value like CT for control β-globin gene amplicons detecting the total DNA present in maternal plasma. However in these cases foetal RhD status cannot be determined.Conclusion:Prediction offoetal RhD status from maternal plasma is highly accurate and enables implementation into clinical routine. We suggest that safe non-invasive prenatal foetal RHD genotyping using maternal plasma should involve the amplification of at least two RHD-specific products.

© 2005 S. Karger AG, Basel


Article / Publication Details

First-Page Preview
Abstract of Paper

Received: January 15, 2004
Accepted: April 07, 2004
Published online: June 16, 2005
Issue release date: July – August

Number of Print Pages: 6
Number of Figures: 2
Number of Tables: 2

ISSN: 1015-3837 (Print)
eISSN: 1421-9964 (Online)

For additional information: http://www.karger.com/FDT


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Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher or, in the case of photocopying, direct payment of a specified fee to the Copyright Clearance Center.
Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.
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