Purpose: To examine the potential high throughput capability and efficiency of an automated DNA extraction system in combination with mass spectrometry for the non-invasive determination of the foetal Rhesus D status. Methods: A total of 178 maternal plasma samples from RHD-negative pregnant women were examined, from which DNA was extracted using the automated Roche MagNA Pure™ system. Presence of the foetal RHD gene was detected by PCR for RHD exon 7 and subsequent analysis using the Sequenom MassArray™ mass spectrometric system. Results: We determined that as little as 15pg of RHD-positive genomic DNA could be detected in a background of 585pg of RHD-negative genomic DNA. The analysis of the clinical samples yielded a sensitivity and specificity of 96.1 and 96.1%, respectively. Conclusion: Our study indicated that automated DNA extraction in combination with mass spectrometry permits the determination of foetal Rhesus D genotype with an accuracy comparable to the current approaches using real-time PC

Banzola, Irina

Grill, Simon

Hahn, Sinuhe

Holzgreve, Wolfgang

Legler, Tobias

Li, Ying

Müller, Sina

Rekhviashvili, Tea

Zhong, Xiao

RERO DOC Digital Library

Arch Gynecol Obstet (2009) 279:533–537DOI 10.1007/s00404-008-0774-5ORIGINAL ARTICLEHigh throughput non-invasive determination of foetal Rhesus D status using automated extraction of cell-free foetal DNA in maternal plasma and mass spectrometrySimon Grill · Irina Banzola · Ying Li · Tea Rekhviashvili · Tobias J. Legler · Sina P. Müller · Xiao Yan Zhong · Sinuhe Hahn · Wolfgang Holzgreve Received: 14 July 2008 / Accepted: 14 August 2008 / Published online: 28 August 2008©  Springer-Verlag 2008AbstractPurpose To examine the potential high throughputcapability and eYciency of an automated DNA extractionsystem in combination with mass spectrometry for the non-invasive determination of the foetal Rhesus D status.Methods A total of 178 maternal plasma samples fromRHD-negative pregnant women were examined, fromwhich DNA was extracted using the automated RocheMagNA Pure™ system. Presence of the foetal RHD genewas detected by PCR for RHD exon 7 and subsequent anal-ysis using the Sequenom MassArray™ mass spectrometricsystem.Results We determined that as little as 15 pg of RHD-pos-itive genomic DNA could be detected in a background of585 pg of RHD-negative genomic DNA. The analysis ofthe clinical samples yielded a sensitivity and speciWcityof 96.1 and 96.1%, respectively.Conclusion Our study indicated that automated DNAextraction in combination with mass spectrometry permitsthe determination of foetal Rhesus D genotype with anaccuracy comparable to the current approaches using real-time PCR.Keywords Rhesus D · RHD · MALDI-TOF · Non-invasive · Saber assayIntroductionThe non-invasive determination of the foetal RHD statusvia the analysis of cell-free foetal DNA in maternalplasma or serum by real-time PCR is well established andalready oVered as a clinical service in a number of coun-tries [2, 6]. This development can greatly assist with thescreening and monitoring of pregnancies at risk for hae-molytic disease of the foetus and newborn (HDN), andalso reduce the health care costs by avoiding unnecessaryprophylactic treatment in those cases where the foetus isRHD negative [7].It has recently been shown that MALDI-TOF (matrixassisted laser desorption ionization-time of Xight) massspectrometric devices, such as the Sequenom MassArray™system, can be used for the detection of foetal genetic lociby the analysis of cell-free foetal DNA in maternal plasmaor serum [11, 12]. The main advantage of this approachover conventional real-time PCR is that it permits the readydetection of foetal genetic loci which only diVer slightlyfrom the maternal counterpart, such as point mutations.Furthermore, it is possible to examine a large number ofmultiplex PCR products by mass spectrometry, implyingthat a large number of mutations or diVerent loci can beexamined in a single analysis [18]. A further attribute of theMassArray™ system is that it is highly amenable to highthroughput analysis of several thousand samples per day.Consequently, it has been proposed that this system may beS. Grill · I. Banzola · Y. Li · T. Rekhviashvili · X. Y. Zhong · S. Hahn · W. HolzgreveDepartment Biomedicine, University Women’s Hospital, Basel, SwitzerlandT. J. Legler · S. P. MüllerDepartment of Transfusion Medicine, University of Göttingen, Göttingen, GermanyW. Holzgreve (&)Laboratory for Prenatal Medicine and Gynaecological Oncology, Women’s Hospital, University of Basel, Hebelstrasse 20, 4031 Basel, Switzerlande-mail: hahns@uhbs.ch; wolfgang.holzgreve@unibas.ch123534 Arch Gynecol Obstet (2009) 279:533–537suitable for the analysis of cell-free foetal DNA in routineclinical diagnostic labs [5, 11, 12].As to date few large-scale studies have examined thesuitability of such a system for this purpose, we have nowperformed a study in which 178 clinical samples were ana-lysed in a blinded manner for the presence or absence of thefoetal RHD gene.Materials and methodsPlasma sample collection and processingThe study was approved by the review boards from both theinstitutions. After written informed consent, 15 mL EDTAblood samples were obtained from pregnant women at riskfor HDN caused by alloimmunization. The samples wereprocessed on site at the University of Göttingen, Germany,by twofold centrifugation (1: 10 min, 2,700£g; 2: 45 min12,500£g). The plasma was divided into 1 mL aliquots andstored at ¡80°C. One aliquot of each sample was frozenand sent to Basel for analysis.DNA isolation from maternal plasmaThe automatic DNA isolation system MagNA Pure™instrument (Roche Applied Science, Switzerland) was usedfor plasma DNA extraction. Following the “DNA LVBlood_1000.blk” protocol, DNA was extracted from 1 mLof RHD-negative maternal plasma with the Roche MagNAPure™ Pure LC DNA Isolation Kit-Large Volume. TheDNA was eluted into 200 L of elution buVer and stored at¡20°C for later use. A sample volume of 50 L was avail-able for analysis.Assay designThe assay design was kindly provided by Sequenom Inc.,USA. As only the foetal RHD gene is detected, and no cor-responding maternal allele, a SABER (single allele baseextension reaction) assay was used which exclusivelydetects the presence of the RHD 7 exon (see below). Alloligonucleotides were synthesised and HPLC puriWed byMicrosynth (Switzerland). AmpliWcation primers containeda 5–10 mer tag to keep them out of the analytical massrange.PCR ampliWcationPCR reactions were performed in a total volume of 12.5 Lcontaining 8 L of eluted cfDNA, 400 nM of each primer(PCR-RHD7-fw and PCR-RHD7-rev, Table 1), 3 mM Mg2+,0.5 U HotStart Taq DNA Polymerase (Qiagen, Switzer-land), 50 M dNTPs and 1£ PCR buVer. Thermal cyclingwas performed as follows: 15 min at 95°C followed by 50cycles of 95°C for 45 s, 57°C for 45 s and 72°C for 1 min.The reaction was completed with a Wnal extension at 72°Cfor 7 min.Shrimp alkaline phosphatase (SAP) treatmentFor the dephosphorylation of excess dNTPs, 4 L of a solu-tion containing 3.06 L ddH2O, 0.34 L hME buVer(Sequenom Inc.) and 0.6 L shrimp alkaline phosphatase(Sequenom Inc.) were added to 12.5 L of PCR product.After incubation at 37°C for 60 min, the enzyme was inacti-vated at 85°C for 5 min.Single allele base extension reactionFour microliters of a solution containing 3.12 L ddH2O,0.4 L Termination Mix C (containing 2,3-dideoxycytos-intriphosphate (ddCTP), Sequenom Inc.), 0.4 L 25 MRHD7 extension primer (Table 1) and 0.08 L (2.56 U)Thermo Sequenase (Sequenom Inc.) were added to 18.5 LSAP treated PCR product. Thermal cycling was performedas follows: 2 min at 94°C followed by 99 cycles of 94°C for5 s, 51°C for 5 s and 52°C for 5 s.Desalting and mass spectrometric analysisA total of 29.5 L H2O and 6 mg of SpectroCLEAN resin(Sequenom Inc.) were added to 20.5 L extension product.The plate was rotated for 10 min at room temperature fol-lowed by centrifugation at 3,000£g for 3 min to pellet theresin.A total of 15 nL of each desalted sample were dispensedonto a 384-format SpectroCHIP using a MassARRAYTable 1 Oligonucleotides for PCR and extension reaction and corresponding masses used for the RHD exon 7 mass spectrometry assayPCR-RHD7-fw 5-ACGTTGGATGGGGTGTTGTAACCGAGTGCTG-3PCR-RHD7-rev 5-ACGTTGGATGCCGGCTCCGACGGTATC-3RHD7 extension primer 5-ATTCCCCACAGCTCCAT-3SABER-assay terminator 2,3-dideoxycytosintriphosphate (ddCTP)Mass of extension primer 5050.3 DaMass of extension product (Call: G)5323.5 Da123Arch Gynecol Obstet (2009) 279:533–537 535nanodispenser (Sequenom Inc.) and subsequently analysedby MassARRAY Analyzer Compact. The data wererecorded and interpreted by MassARRAY TYPER (Seque-nom Inc.) software.ResultsValidation of the mass spectrometry Assay for RHD exon 7To evaluate the sensitivity of the SABER assay, we seriallydiluted RHD-positive genomic DNA into RHD-negativegenomic DNA, such that the overall quantity of DNA was600 pg per reaction. This is the equivalent amount to thatnormally present in cell-free DNA preparations. Underthese conditions, we were able to detect 15 pg of RHD-positivegenomic DNA in a background of 585 pg of RHD-negativegenomic DNA (Fig. 1). This is almost equal to the threecopies of the RHD gene in a single reaction, indicating thatthe RHD assay exhibits a degree of sensitivity and speciWc-ity suitable for the analysis of cell-free foetal DNA.Detection of the foetal RHD exon 7 in maternal plasmaA total of 178 samples were analysed in a blinded manner.The results were conWrmed by those obtained from serolog-ical analysis using cord blood in Göttingen. The presenceor absence of the foetal RHD exon 7 was determined fol-lowing the analysis of the samples in duplicates. If theresult was unambiguously positive or negative, the sampleswere scored accordingly. If the result was equivocal, thesample was re-analysed in duplicate. The sample was onlyFig. 1 Sensitivity and speciWcity of the RHD assay using the MALDI-TOF MS based SABER approach. RHD-positive genomic DNA wasdiluted into RHD-negative genomic DNA. The total DNA amount perreaction was 600 pg. a 600 pg RHD-positive genomic DNA. b 30 pgRHD-positive DNA in 570 pg RHD-negative background. c 15 pgRHD-positive DNA in 585 pg RHD-negative background. d 600 pgRHD negative genomic DNA. UEP unextended primer, G RHD allele,PP pausing peak123536 Arch Gynecol Obstet (2009) 279:533–537scored as RHD positive if at least two positive signals weredetected amongst the two sets of duplicates, else it wasscored as RHD negative.Following a comparison of our results with thoseobtained by classical serology of the cord blood, we ascer-tained that out of 178 samples we had examined, Wve sam-ples had been incorrectly scored as RHD negative and twosamples incorrectly determined as being RHD positive(Table 2). This equates to a sensitivity of 96.1% and a spec-iWcity of 96.1%. Our data show 96.1% concordance tothose obtained by quantitative real-time PCR.DiscussionNon-invasive prenatal RHD analysis is currently most fre-quently performed using real-time PCR, either in conjunc-tion with manual or automated procedures for theextraction of cell-free DNA from the maternal plasma/serum samples [4, 9, 13, 15, 16, 19]. In this study, weanalysed the foetal RHD status by mass spectrometry, incombination with automated system for the extraction ofcell-free DNA from maternal plasma.The validation of the assay showed that we were able todetect as little as 2.5% of RHD-positive genomic DNA in abackground of RHD-negative genomic DNA, indicatingthat the assay is very sensitive. A similar sensitivity (20 pgmutant DNA in 580 pg wildtype DNA) has recently beenreported by our group for the mass spectrometry basedgenotyping of the foetal KEL1 blood group from cell-freeDNA in maternal plasma [11].Out of 178 samples, Wve cases were incorrectly scored asRHD negative and two were incorrectly scored as RHDpositive. Of those Wve false negative samples, three wereobtained during the third trimester, while the remaining twowere from the second trimester of pregnancy. The two sam-ples that were scored false positive were from the secondand third trimester or pregnancy. These remained to beassessed incorrectly, even after several rounds of re-analy-sis, indicating that the sample may have been contaminated.As the samples stem from a Caucasian population, a falsepositive result caused by the inactive allele RHD [10, 16]is unlikely, but can only be excluded convincingly byexamining for the presence of this allele.In those cases where the foetal RHD gene cannot bedetected, it is unclear, whether the foetus is Rhesus-nega-tive or if the amount of foetal DNA in the sample is belowthe detection limit of the system. For the exclusion of thelatter case, the detection of paternally inherited singlenucleotide polymorphisms [12], the detection of a Y-chro-mosome speciWc sequence [8], or the detection of an epige-netic marker such as the foetal hypermethylated RASSF1Asequence [3] can be performed. In our current study, wewere unable to perform such additional analyses due tovery limited amount of sample available.A recent review of large-scale studies reporting on thenon-invasive determination of foetal RHD status by thereal-time PCR analysis of cell-free DNA indicated thataccuracies between 95 and 99% could be attained [14]. Assuch, our data yielding an overall accuracy of 96.1% concurfavourably with those obtained by real-time PCR.In future studies we aim to make better use of the Seque-nom MassArray™ mass spectrometric system, by perform-ing multiplex analyses. In this manner it will be possible todetect variant RHD alleles such as the RHD gene [1, 17],as well as polymorphic markers indicating whether cell-free foetal DNA was present in the sample or not. Thisadded information will increase the value of the analysis,and by being able to perform these numerous examinationsin a single analytic run, help to reduce costs.In this manner, the combination of an automated DNAextraction system and mass spectrometry may emerge as acompetitive alternative to the current commonly used real-time PCR approaches used to determine non-invasively thefoetal RHD status.Acknowledgments Funding from the European Commission for theSpecial Non-invasive Advances in Fetal and Neonatal Evaluation(SAFE) Network of Excellence (LSHB-CT-2004-503243) and theSwiss National Science Foundation (SNSF 3200B0-107697/1), fromwhich this study was partially funded, is gratefully acknowledged.References1. Avent ND (2008) RHD genotyping from maternal plasma: guide-lines and technical challenges. Methods Mol Biol 444:185–201.doi:10.1007/978-1-59745-066-9_142. Bianchi DW, Avent ND, Costa JM, van der Schoot CE (2005)Noninvasive prenatal diagnosis of fetal Rhesus D: ready forprime(r) time. Obstet Gynecol 106:841–8443. Chan KC, Ding C, Gerovassili A, Yeung SW, Chiu RW, LeungTN et al (2006) Hypermethylated RASSF1A in maternal plasma:a universal fetal DNA marker that improves the reliability of non-invasive prenatal diagnosis. Clin Chem 52:2211–2218.doi:10.1373/clinchem.2006.0749974. Costa JM, Giovangrandi Y, Ernault P, Lohmann L, Nataf V, El HNet al (2002) Fetal RHD genotyping in maternal serum during theTable 2 The results of foetal RHD genotyping in maternal plasmausing the MALDI-TOF based SABER assayGestational age No. of casesRHD positiveRHD negativeSensitivity/speciWcity (%)First trimester 5 4/4 1/1 100/100Second trimester 127 91/93 33/34 97.8/97.1Third trimester 32 18/21 10/11 85.7/90.1Unknown 15 10/10 5/5 100/100Total 178 122/127 49/51 96.1/96.1123Arch Gynecol Obstet (2009) 279:533–537 537Wrst trimester of pregnancy. Br J Haematol 119:255–260.doi:10.1046/j.1365-2141.2002.03780.x5. Ding C (2008) Maldi-TOF mass spectrometry for analyzing cell-free fetal DNA in maternal plasma. Methods Mol Biol 444:253–267. doi:10.1007/978-1-59745-066-9_206. Finning K, Martin P, Daniels G (2004) A clinical service in the UKto predict fetal Rh (Rhesus) D blood group using free fetal DNAin maternal plasma. Ann N Y Acad Sci 1022:119–123.doi:10.1196/annals.1318.0197. Finning K, Martin P, Summers J, Massey E, Poole G, Daniels G(2008) EVect of high throughput RHD typing of fetal DNA inmaternal plasma on use of anti-RhD immunoglobulin in RhD neg-ative pregnant women: prospective feasibility study. BMJ336:816–818. doi:10.1136/bmj.39518.463206.258. Finning KM, Chitty LS (2008) Non-invasive fetal sex determina-tion: impact on clinical practice. Semin Fetal Neonatal Med13:69–75. doi:10.1016/j.siny.2007.12.0079. Harper TC, Finning KM, Martin P, Moise KJ Jr (2004) Use ofmaternal plasma for noninvasive determination of fetal RhD sta-tus. Am J Obstet Gynecol 191:1730–1732. doi:10.1016/j.ajog.2004.06.09810. Legler TJ, Lynen R, Maas JH, Pindur G, KulenkampV D, Suren Aet al (2002) Prediction of fetal Rh D and Rh CcEe phenotype frommaternal plasma with real-time polymerase chain reaction. Trans-fus Apher Sci 27:217–223. doi:10.1016/S1473-0502(02)00068-X11. Li Y, Finning K, Daniels G, Hahn S, Zhong X, Holzgreve W(2008) Noninvasive genotyping fetal Kell blood group (KEL1) us-ing cell-free fetal DNA in maternal plasma by MALDI-TOF massspectrometry. Prenat Diagn 28:203–208. doi:10.1002/pd.193612. Li Y, Wenzel F, Holzgreve W, Hahn S (2006) Genotyping fetalpaternally inherited SNPs by MALDI-TOF MS using cell-freefetal DNA in maternal plasma: inXuence of size fractionation.Electrophoresis 27:3889–3896. doi:10.1002/elps.20060008413. Lo YM, Hjelm NM, Fidler C, Sargent IL, Murphy MF, Chamber-lain PF et al (1998) Prenatal diagnosis of fetal RhD status bymolecular analysis of maternal plasma. N Engl J Med 339:1734–1738. doi:10.1056/NEJM19981210339240214. Maron JL, Bianchi DW (2007) Prenatal diagnosis using cell-freenucleic acids in maternal body Xuids: a decade of progress. Am JMed Genet C Semin Med Genet 145:5–17. doi:10.1002/ajmg.c.3011515. Rouillac-Le SC, Puillandre P, Gillot R, Baulard C, Metral S, LeVan KC et al (2004) Large-scale pre-diagnosis study of fetal RHDgenotyping by PCR on plasma DNA from RhD-negative pregnantwomen. Mol Diagn 8:23–31. doi:10.2165/00066982-200408010-0000416. van der Schoot CE, Hahn S, Chitty LS (2008) Non-invasive prena-tal diagnosis and determination of fetal Rh status. Semin FetalNeonatal Med 13:63–68. doi:10.1016/j.siny.2007.12.01217. van der Schoot CE, Soussan AA, Koelewijn J, Bonsel G,Paget-Christiaens LG, de HM (2006) Non-invasive antenatal RHDtyping. Transfus Clin Biol 13:53–57. doi:10.1016/j.tracli.2006.02.02118. Xiu-Cheng FA, Garritsen HS, Tarhouny SE, Morris M, Hahn S,Holzgreve W et al (2008) A rapid and accurate approach to iden-tify single nucleotide polymorphisms of mitochondrial DNA usingMALDI-TOF mass spectrometry. Clin Chem Lab Med 46:299–305. doi:10.1515/CCLM.2008.07119. Zhong XY, Holzgreve W, Hahn S (2001) Risk free simultaneousprenatal identiWcation of fetal Rhesus D status and sex by multi-plex real-time PCR using cell free fetal DNA in maternal plasma.Swiss Med Wkly 131:70–74123

High throughput non-invasive determination of foetal Rhesus D status using automated extraction of cell-free foetal DNA in maternal plasma and mass spectrometry

http://doc.rero.ch/record/320850/files/404_2008_Article_774.pdf

High throughput non-invasive determination of foetal Rhesus D status using automated extraction of cell-free foetal DNA in maternal plasma and mass spectrometry

Abstract

Similar works

Full text

Available Versions

RERO DOC Digital Library