Background: The accuracy and reliability of detection of free fetal DNA in plasma of pregnant women can be significantly improved by increasing the overall DNA concentration following the isolation from maternal plasma. The aim of our study was to compare DNA concentration methods on samples with free fetal DNA. Materials and Methods: DNA isolated from plasma samples of pregnant women carrying a male fetus were concentrated by 3 different methods: vacuum concentration, centrifugal filters and spin columns. Their performance was evaluated using PCR-based Y-chromosomal short tandem repeat (Y-STR) genotyping of the fetus. Results: A statistically significant difference was found between the 3 tested methods (F = 15.57, p < 0.0001). Using vacuum concentration 85.3% of paternally inherited Y-STR alleles were correctly identified. A significantly smaller proportion of alleles was correctly identified in samples concentrated by centrifugal filters and spin columns - 75.9 and 66.5%, respectively. Discussion: The highest proportion of paternally inherited Y-STR alleles was found in samples concentrated with the use of vacuum concentration. This concentration procedure does not require further handling of the sample either, which is an advantage because it avoids potential sample contamination. On the other hand, when automation is considered, vacuum concentration is less suitable because of an uneven and unpredictable sample evaporation rate.

Keywords:
Cell-free fetal DNA, DNA concentration method, Free fetal DNA genotyping
1.
Mujezinovic F, Alfirevic Z: Procedure-related complications of amniocentesis and chorionic villous sampling: a systematic review. Obstet Gynecol 2007;110:687-694.
2.
Lo YM, Corbetta N, Chamberlain PF, Rai V, Sargent IL, Redman CW, Wainscoat JS: Presence of fetal DNA in maternal plasma and serum. Lancet 1997;350:485-487.
3.
Wright CF, Wei Y, Higgins JP, Sagoo GS: Non-invasive prenatal diagnostic test accuracy for fetal sex using cell-free DNA: a review and meta-analysis. BMC Res Notes 2012;5:476.
4.
Scheffer PG, van der Schoot CE, Page-Christiaens GC, de Haas M: Noninvasive fetal blood group genotyping of rhesus D, c, E and of K in alloimmunised pregnant women: evaluation of a 7-year clinical experience. BJOG 2011;118:1340-1348.
5.
Lam KW, Jiang P, Liao GJ, Chan KC, Leung TY, Chiu RW, Lo YM: Noninvasive prenatal diagnosis of monogenic diseases by targeted massively parallel sequencing of maternal plasma: application to beta-thalassemia. Clin Chem 2012;58:1467-1475.
6.
Chiu RW, Chan KC, Gao Y, Lau VY, Zheng W, Leung TY, Foo CH, Xie B, Tsui NB, Lun FM, Zee BC, Lau TK, Cantor CR, Lo YM: Noninvasive prenatal diagnosis of fetal chromosomal aneuploidy by massively parallel genomic sequencing of DNA in maternal plasma. Proc Natl Acad Sci USA 2008;105:20458-20463.
7.
Fan HC, Blumenfeld YJ, Chitkara U, Hudgins L, Quake SR: Noninvasive diagnosis of fetal aneuploidy by shotgun sequencing DNA from maternal blood. Proc Natl Acad Sci USA 2008;105:16266-16271.
8.
Zhong XY, Holzgreve W, Hahn S: The levels of circulatory cell free fetal DNA in maternal plasma are elevated prior to the onset of preeclampsia. Hypertens Pregnancy 2002;21:77-83.
9.
Lim JH, Kim MH, Han YJ, Lee DE, Park SY, Han JY, Kim MY, Ryu HM: Cell-free fetal DNA and cell-free total DNA levels in spontaneous abortion with fetal chromosomal aneuploidy. PLoS One 2013;8:e56787.
10.
Faas BH, de Ligt J, Janssen I, Eggink AJ, Wijnberger LD, van Vugt JM, Vissers L, Geurts van Kessel A: Non-invasive prenatal diagnosis of fetal aneuploidies using massively parallel sequencing-by-ligation and evidence that cell-free fetal DNA in the maternal plasma originates from cytotrophoblastic cells. Expert Opin Biol Ther 2012;12(suppl 1):S19-S26.
11.
Illanes S, Denbow M, Kailasam C, Finning K, Soothill PW: Early detection of cell-free fetal DNA in maternal plasma. Early Hum Dev 2007;83:563-566.
12.
Lun FM, Chiu RW, Allen Chan KC, Yeung Leung T, Kin Lau T, Dennis Lo YM: Microfluidics digital PCR reveals a higher than expected fraction of fetal DNA in maternal plasma. Clin Chem 2008;54:1664-1672.
13.
Lo YM, Tein MS, Lau TK, Haines CJ, Leung TN, Poon PM, Wainscoat JS, Johnson PJ, Chang AM, Hjelm NM: Quantitative analysis of fetal DNA in maternal plasma and serum: implications for noninvasive prenatal diagnosis. Am J Hum Genet 1998;62:768-775.
14.
Legler TJ, Liu Z, Mavrou A, Finning K, Hromadnikova I, Galbiati S, Meaney C, Hulten MA, Crea F, Olsson ML, Maddocks DG, Huang D, Fisher SA, Sprenger-Haussels M, Soussan AA, van der Schoot CE: Workshop report on the extraction of foetal DNA from maternal plasma. Prenat Diagn 2007;27:824-829.
15.
Fleischhacker M, Schmidt B, Weickmann S, Fersching DM, Leszinski GS, Siegele B, Stotzer OJ, Nagel D, Holdenrieder S: Methods for isolation of cell-free plasma DNA strongly affect DNA yield. Clin Chim Acta 2011;412:2085-2088.
16.
Repiska G, Sedlackova T, Szemes T, Celec P, Minarik G: Selection of the optimal manual method of cell free fetal DNA isolation from maternal plasma. Clin Chem Lab Med 2013;51:1185-1189.
17.
Zimmermann B, El-Sheikhah A, Nicolaides K, Holzgreve W, Hahn S: Optimized real-time quantitative PCR measurement of male fetal DNA in maternal plasma. Clin Chem 2005;51:1598-1604.
18.
Johnson CL, Warren JH, Giles RC, Staub RW: Validation and uses of a Y-chromosome STR 10-plex for forensic and paternity laboratories. J Forensic Sci 2003;48:1260-1268.
19.
Kimura M, Hara M, Itakura A, Sato C, Ikebuchi K, Ishihara O: Fragment size analysis of free fetal DNA in maternal plasma using Y-STR loci and SRY gene amplification. Nagoya J Med Sci 2011;73:129-135.
20.
Rong Y, Gao J, Jiang X, Zheng F: Multiplex PCR for 17 Y-chromosome specific short tandem repeats (STR) to enhance the reliability of fetal sex determination in maternal plasma. Int J Mol Sci 2012;13:5972-5981.
21.
Li Y, Zimmermann B, Rusterholz C, Kang A, Holzgreve W, Hahn S: Size separation of circulatory DNA in maternal plasma permits ready detection of fetal DNA polymorphisms. Clin Chem 2004;50:1002-1011.
22.
Dhallan R, Au WC, Mattagajasingh S, Emche S, Bayliss P, Damewood M, Cronin M, Chou V, Mohr M: Methods to increase the percentage of free fetal DNA recovered from the maternal circulation. JAMA 2004;291:1114-1119.
23.
Devaney SA, Palomaki GE, Scott JA, Bianchi DW: Noninvasive fetal sex determination using cell-free fetal DNA: a systematic review and meta-analysis. JAMA 2011;306:627-636.
© 2014 S. Karger AG, Basel
2014
Copyright / Drug Dosage / Disclaimer
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.
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.
Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.
You do not currently have access to this content.