胎儿游离DNA定量
Microfluidics Digital PCR Reveals a
Higher than Expected Fraction of Fetal
DNA in Maternal Plasma
Fiona M.F.Lun,1,2Rossa W.K.Chiu,1,2K.C.Allen Chan,1,2Tak Yeung Leung,3Tze Kin Lau,3
and Y.M.Dennis Lo1,2*
BACKGROUND:The precise measurement of cell-free fetal DNA in maternal plasma facilitates noninvasive prenatal diagnosis of fetal chromosomal aneu-ploidies and other applications.We tested the hy-pothesis that microfluidics digital PCR,in which in-dividual fetal-DNA molecules are counted,could enhance the precision of measuring circulating fetal DNA.
METHODS:We first determined whether microfluidics digital PCR,real-time PCR,and mass spectrometry produced different estimates of male-DNA concentra-tions in artificial mixtures of male and female DNA. We then focused on comparing the imprecision of mi-crofluidics digital PCR with that of a well-established nondigital PCR assay for measuring male fetal DNA in maternal plasma.
RESULTS:Of the tested platforms,microfluidics digi-tal PCR demonstrated the least quantitative bias for measuring the fractional concentration of male DNA.This assay had a lower imprecision and higher clinical sensitivity compared with nondigital real-time PCR.With the ZFY/ZFX assay on the microflu-idics digital PCR platform,the median fractional concentration of fetal DNA in maternal plasma was ?2times higher for all3trimesters of pregnancy than previously reported.
CONCLUSIONS:Microfluidics digital PCR represents an improvement over previous methods for quanti-fying fetal DNA in maternal plasma,enabling diag-nostic and research applications requiring precise quantification.This approach may also impact other diagnostic applications of plasma nucleic acids,e.g., in oncology and transplantation.
?2008American Association for Clinical Chemistry Conventional prenatal diagnostic methods for harvest-ing fetal materials for molecular analysis,such as am-niocentesis,are invasive and constitute a finite risk to the fetus.Much research has therefore been devoted to the development of new noninvasive methods for safe prenatal diagnosis.The discovery of circulating cell-free fetal DNA in maternal plasma in1997offered such new possibilities(1).A number of clinical diagnostic tests with impacts on clinical practice that have since been developed(2)include the determination of fetal RhD blood group status(3,4)and fetal sex determina-tion for sex-linked disorders(5).Furthermore,in-creased circulating fetal-DNA concentrations have been observed in certain pregnancy-associated disor-ders,including preeclampsia(6,7)and preterm labor (8).
Real-time PCR has become the most commonly used technology for the detection of fetal DNA in ma-ternal plasma(9),including both qualitative[e.g.,for fetal RhD genotyping(3,4)]and quantitative[e.g.,in preeclampsia(6)]applications.Because most of the DNA molecules in maternal plasma are derived from the pregnant woman,with only a minor proportion coming from the fetus,real-time PCR–based assays are generally directed toward fetal targets that either have no maternal counterparts[e.g.,Y chromosome se-quences(5)or the RHD4gene(Rh blood group,D antigen)in a RhD-negative woman(3,4)]or differ from the maternal counterparts at multiple DNA base pairs[e.g.,the4-nucleotide codon41/42deletion in
1Centre for Research into Circulating Fetal Nucleic Acids,Li Ka Shing Institute of Health Sciences,2Department of Chemical Pathology,and3Department of Obstetrics and Gynaecology,The Chinese University of Hong Kong,Shatin,New Territories,Hong Kong.
*Address correspondence to this author at:Department of Chemical Pathology, The Chinese University of Hong Kong,Prince of Wales Hospital,30–32Ngan Shing St.,New Territories,Hong Kong SAR,China.Fax?852********;e-mail
loym@https://www.wendangku.net/doc/4a16665513.html,.hk.
Received May27,2008;accepted July25,2008.
Previously published online at DOI:10.1373/clinchem.2008.111385
4Human genes:RHD,Rh blood group,D antigen;SRY,sex determining region Y; ZFX,zinc finger protein,X-linked;ZFY,zinc finger protein,Y-linked;HBB, hemoglobin,beta.
Clinical Chemistry54:10
1664–1672(2008)
Molecular Diagnostics and Genetics 1664
?-thalassemia(10)].The detection of fetal-DNA tar-gets that differ from the maternal background by single bases has required more complex methods,such as size fractionation to enrich for the relatively shorter fetal-DNA targets(11)and mass spectrometry to detect the single-base variations(12).These methods have disad-vantages that include a susceptibility to contamination with current size-fractionation strategies(11)and the need for extensive optimization for mass spectrometry analysis of certain genomic targets(13).
For quantitative analyses,several investigators have reported higher imprecision with single-copy se-quences,such as the SRY gene(sex determining region Y)on the Y chromosome,for fetal-DNA quantification in maternal plasma(14).Furthermore,quantitative analysis with real-time PCR typically requires the use of calibrators that might vary between laboratories and between batches(14).In addition,new diagnostic ap-plications of plasma-DNA analysis,such as in detecting Down syndrome,require very precise quantification of circulating fetal DNA,which might be challenging with conventional technologies(15).
In view of these limitations,an investigation of new approaches to plasma-DNA analysis would be of clinical and scientific interest.We explored the use of microfluidics digital PCR for the detection and mea-surement of cell-free fetal DNA in maternal plasma. Digital PCR is an analytical strategy in which a nucleic acid sample is diluted and subjected to multiple PCR analyses so that most of the reactions contain either a single or no target molecule(16).The technology pro-vides a“digital”readout because any of these multiple PCR analyses will be either positive or negative,corre-sponding to the presence or absence of the target mol-ecule.Such presence/absence results are analogous to the“ones”and“zeros”in computer science.With ap-propriate statistical analyses,the proportion of positive and negative reactions would allow measurement of the number of target molecules in the input sample. Most published applications of this approach have been in the cancer-detection field(17).Recently,digi-tal PCR has been proposed as a possible strategy for the detection of fetal chromosomal aneuploidies in mater-nal plasma(15).
The main limitation of digital PCR is the labor-intensiveness of performing hundreds to thousands of reactions for each sample,but recent advances in mi-crofluidics technology have made possible the automa-tion of digital PCR(18,19).Microfluidics permits nanoliter aliquots of a nucleic acid sample to be chan-neled into nanoliter-scale amplification chambers where hundreds or thousands of real-time digital PCRs could be carried out(Fig.1A).In this study,we com-pared the performance of microfluidics digital PCR with existing methods for detecting fetal DNA in ma-ternal plasma.
Materials and Methods
STUDY PARTICIPANTS
Women with singleton pregnancies were recruited at the Prince of Wales Hospital,Hong Kong,with in-formed consent and Institutional Review Board ap-proval.Maternal peripheral blood samples were col-lected into EDTA-containing tubes during the first, second,and third trimesters before chorionic villus sampling,amniocentesis,and elective cesarean section, respectively.We recruited10pregnancies with male fetuses for each trimester and5first-trimester preg-nancies with female fetuses.As a positive control in the DNA-mixing experiments,we collected placental tis-sue from a healthy male baby at term immediately after elective cesarean section.Samples were processed as described in the Data Supplement that accompanies the online version of this article at http://www. https://www.wendangku.net/doc/4a16665513.html,/content/vol54/issue10.
COMPARISON OF DIFFERENT ANALYTICAL PLATFORMS
We compared3platforms in this study:real-time quantitative PCR(9),mass spectrometry(12,20),and microfluidics digital PCR(18,19).We prepared artifi-cial DNA mixtures consisting of0%,5%,10%,25%, 50%,75%,and100%male placental DNA in female blood cell DNA at a final concentration of1ng/?L(1 mg/L).These mixtures were prepared from100-ng/?L (100-mg/L)stock solutions of female blood cell DNA and male placental DNA.We then measured the quan-titative deviation of observed concentrations of male DNA from the expected concentrations by means of the3analytical platforms.To compare the analytical imprecisions of the real-time PCR and digital PCR platforms,we prepared an artificial mixture containing 7%male placental DNA in a background of female blood cell DNA and diluted the mixture to100pg/?L (100?g/L)to mimic extracted maternal plasma DNA from early pregnancy(9).
MICROFLUIDICS DIGITAL PCR ANALYSIS
Two87-bp amplicons of the ZFX(zinc finger protein, X-linked)and ZFY(zinc finger protein,Y-linked)loci were coamplified with the same primer set and distin-guished with chromosome-specific TaqMan probes (Applied Biosystems;Table1).We carried out all digi-tal experiments on the BioMark System(Fluidigm)us-ing the12.765Digital Arrays(Fluidigm).Each Digital Array consists of12panels,each of which is further partitioned into765reaction chambers(Fig.1B).The reaction for one panel was set up with the2?TaqMan Universal PCR Master Mix Kit(Applied Biosystems)in
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1 panel
Fig.1.Application of microfluidics chip for digital analysis.
(A),Frontal view of a microfluidics digital array.The chip in the center is divided into12panels,and each is connected to a sample inlet(on the top frame).(B),Digital readout accuracy experiment.Each panel is compartmentalized into reaction wells.Red-and blue-colored dots represent wells that are positive for ZFY and ZFX signals,respectively. Black-colored dots represent wells with no reaction.percentage next to each panel denotes the fractional male-DNA concentration of the input mixture of artificial DNA in particular panel.
Table1.Oligonucleotide sequences for the ZFX and ZFY assays.a
Digital PCR and nondigital real-time PCR
Forward primer5?-CAAGTGCTGGACTCAGATGTAACTG-3?
Reverse primer5?-TGAAGTAATGTCAGAAGCTAAAACATCA-3?
ZFX TaqMan probe5?-(VIC)TCTTTAGCACATTGCA(MGBNFQ)-3?
ZFY TaqMan probe
Mass spectrometry
Forward primer
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a reaction volume of10?L,inclusive of a dead volume of5.4?L.A3.5-?L volume of input DNA was loaded onto each panel.We used2reaction panels to measure the male-DNA concentration in each artificial DNA mixture in the experiment to measure the quantitative biases across the different analytical platforms.We used12reaction panels on all of the plasma samples and the artificial DNA mixture containing7%male DNA to assess the imprecision of the digital PCR assay. For digital analysis of plasma DNA to measure frac-tional fetal-DNA concentrations,12reaction panels would allow a total of19.32?L of plasma DNA to be analyzed,after the dead volume of the chip had been taken into account.This volume was comparable to the total volume of plasma DNA analyzed for the conven-tional real-time SRY/HBB assay(see below)in which two5-?L plasma-DNA aliquots were used for dupli-cate analyses for each of SRY and HBB(hemoglobin, beta),for a total of20?L of plasma DNA per reportable fractional fetal-DNA concentration(9).For digital analysis of plasma DNA to qualitatively determine the fetal sex,we scored only the first6reaction panels, amounting to9.66?L of plasma DNA.This volume of maternal plasma DNA is comparable to the10-?L total volume of plasma DNA used for the duplicate SRY real-time PCR(9).Details of the digital assay are de-scribed in the online Data Supplement.
We counted the number of wells that were positive for ZFY or ZFX amplification for each sample.Accord-ing to the Poisson distribution,the original number of molecules derived from chromosomes X and Y can be calculated with the following equations:
ZFY??ln??N?Y?/N??N;
ZFX??ln??N?X?/N??N,
where ZFY is the number of ZFY molecules,N is the total number of wells counted,Y is the number of Y-positive wells,ZFX is the number of ZFX molecules, and X is the number of X-positive wells.The fraction of ZFY molecules of the total zinc finger protein DNA sequences(i.e.,ZFY plus ZFX)can be calculated as: ZFY/(ZFY?ZFX).
Because each male fetal cell contains a copy each of ZFX and ZFY and each background maternal cell con-tains2copies of ZFX and no ZFY,the proportion of fetal DNA in a maternal plasma sample[i.e.,the per-centage of the total genome equivalents(GEs)of DNA in the maternal plasma that was fetus derived]is calcu-lated as:(2?ZFY)/(ZFY?ZFX)?100.
REAL-TIME QUANTITATIVE PCR
Apart from the real-time SRY and HBB assays,which have been widely used in previous studies(9),we also designed real-time PCR assays targeting ZFX and ZFY for comparison.The SRY/HBB and ZFY/ZFX assays were performed on an ABI7300Real-Time PCR Sys-tem(Applied Biosystems)with5?L of DNA sample per reaction.The ZFX and ZFY primer and probe se-quences were the same as on the digital platform,but the probes were used separately.Reaction conditions are summarized in the online Data Supplement.
We ran duplicate DNA samples and reported the mean in the results.For absolute quantification,we ran a calibration curve consisting of serially diluted male blood cell DNA(1–1000GE per reaction)in parallel and in duplicate with each analysis.We used a conver-sion factor of6.6pg DNA/cell.Amplification data were analyzed with Sequence Detection Software(version 1.2.3;Applied Biosystems).The same calibration curve was used for the real-time SRY/HBB assay and the non-digital ZFY/ZFX assay.
For the real-time SRY/HBB assay,the percentage of male DNA per reaction was given by:(SRY GE)/ (HBB GE)?100.
To calculate the percentage of male DNA in the real-time ZFY/ZFX assay,we used the same equation as for the digital version of the assay.
MASS SPECTROMETRY
We performed MALDI-TOF mass spectrometry anal-ysis with a standard homogenous MassEXTEND assay (Sequenom).An82-bp region in ZFX and ZFY was coamplified with one primer set(Table1).The respec-tive amplicons were identified by a primer-extension reaction that targets the base differences between ZFX and ZFY(Table1).Further details of the assay are pro-vided in the online Data Supplement.The extension products were dispensed onto a SpectroCHIP(Seque-nom)by a MassARRAY Nanodispenser S(Sequenom). Data acquisition from the SpectroCHIP was done in the MassARRAY Analyzer Compact Mass Spectrome-ter(Sequenom).
An inherent property of mass spectrometry is that products with higher molecular masses are usually at-tenuated on the mass spectrum.To control for the peak skewing,we included a calibration curve consisting of mixtures of male and female blood cell DNA(0%,10%, 20%,30%,40%,and50%of male DNA)for peak fre-quency correction.We carried out2homogenous MassEXTEND reactions for each test sample and dis-pensed each homogenous MassEXTEND product twice onto the SpectroCHIP.We therefore obtained4 mass spectra for each case.The percentage of fetal DNA in maternal plasma was quantified by the relative peak frequencies of ZFY to ZFX in the mass spectrum.The percentage of male DNA for each case was the mean of 4data points:2?(observed ZFY frequency)?100. This value was further corrected for peak skewing by dividing by the slope-correction value(1.024in this
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Clinical Chemistry54:10(2008)1667
experiment):Percent reported male DNA?(Percent observed male DNA)/1.024.
Results
We determined whether biases existed with measure-ments made on the3tested analytical platforms by comparison with the expected concentrations of male DNA.The observed biases were expressed graphically in a difference plot vs the expected male-DNA concen-tration(Fig.2)(21).Of the3assays,the microfluidics digital PCR assay most correctly reflected the actual sample compositions and produced the smallest per-centage bias.The nondigital real-time SRY/HBB assays (9),which have been widely used in the field,showed a negative bias,underestimating the fractional male-DNA concentration by approximately20%.The non-digital real-time ZFY/ZFX assay exhibited a positive bias that worsened as the fractional concentration of male DNA decreased.The mass spectrometry–based assay exhibited a positive bias at higher fractional male-DNA concentrations but had a negative bias as the frac-tional male-DNA concentration decreased.
We focused subsequent experiments on compar-ing the microfluidics digital PCR ZFY/ZFX system and the nondigital real-time PCR SRY/HBB assay(9),be-cause the latter has been one of the standard assays used in the field.We used an artificial sample mixture of7% male DNA to compare the analytical imprecision of these2systems for measuring the fractional male-DNA concentration.We carried out20analyses of this sam-ple with20different microfluidics chips(1reportable result per chip)on the digital PCR platform.For the conventional nondigital PCR system,we measured this sample20times with20plates(1reportable result per plate),with the SRY and HBB assays carried out in the same plate for each measurement.The CVs of the dig-ital and nondigital assays were16%and49%,respec-tively(Fig.3),indicating that the digital assay was3.1 times more precise than the nondigital assay.
We next investigated the diagnostic sensitivity of the digital and nondigital assays for detecting male fetal DNA in the plasma of10first-trimester(12–14weeks gestation)pregnant women carrying male fetuses.We scored the first6of the12panels of each chip to deter-mine fetal sex by digital PCR and scored all12panels of each chip to quantitatively measure the fractional fetal-DNA concentration.We used this procedure to ensure that we fairly compared the digital and nondigital PCR systems with similar input volumes of plasma DNA (see Materials and Methods).For the nondigital PCR system,we carried out duplicate amplifications for each sample,as originally described(9),with at least one SRY-positive signal for the2amplifications being
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scored as positive for a male fetus.The diagnostic sen-sitivities of the digital and nondigital assays for the de-tection of male fetuses were100%and90%,respec-tively.The diagnostic specificities of the digital(12 panels)and nondigital assays were confirmed with plasma samples from5first-trimester(12–14weeks gestation)pregnant women carrying female fetuses. The digital and nondigital assays detected no ZFY and SRY signals,respectively.
We used both the digital and nondigital assays to measure the fractional fetal-DNA concentrations in10plasma samples each from pregnant women who were in their first trimester(as described above),second trimester(17–22weeks gestation), and third trimester(38–39weeks gestation)and car-rying male fetuses.The median fractional fetal-DNA concentrations measured with the digital PCR assay were9.7%,9.0%,and20.4%for the first,second, and third trimesters,respectively(Fig.4).The cor-responding values for the nondigital PCR assays were4.8%,4.1%,and7.6%,respectively.Thus,the median fractional fetal-DNA concentrations mea-sured with the digital PCR assay were2.0,2.2,and 2.7times higher than those obtained with the non-digital PCR system for the respective gestational ages.
Discussion
We explored microfluidics digital PCR as a tool for the detection and measurement of fetal DNA in maternal plasma.Digital PCR is approximately3.1times more precise than conventional nondigital real-time PCR (Fig.3).We also demonstrated that microfluidics dig-ital PCR revealed the least bias in measuring the frac-tional concentration of male DNA,compared with as-says based on conventional nondigital real-time PCR and mass spectrometry(Fig.2).Digital PCR is expected a priori to be more correct and precise than nondigital PCR formats of PCR because by analyzing a sample in a multitude of aliquot volumes containing less than a single copy,digital PCR transforms the analog output of conventional PCR to an“all-or-nothing”(i.e.,digi-tal)readout of individual amplifications.This ap-proach allows the counting of the number of individual positive amplifications and calculation of the concen-
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tration on the basis of mathematical principles,i.e.,the Poisson distribution.The physical nature of the quan-titative data obviates the use of calibrator solutions. This fact is evident by the smaller bias value for the digital ZFY/ZFX assay than for the nondigital version of the assay(Fig.2).
In contrast,the nondigital real-time PCR assays are dependent on the use of a series of calibrators to correlate the fluorescence signal with the nucleic acid concentration.One source of inaccuracy may occur when a new set of calibrators is instituted or when dif-ferent laboratories prepare their own calibrator solu-tions.Furthermore,different assays may generate dif-ferent dose–response curves for a series of calibrators, thus producing different degrees of bias.This point is illustrated by the difference plots for the SRY/HBB and ZFY/ZFX real-time PCRs in which the same calibrators were used for both systems(Fig.2).The former assay showed a negative bias,whereas the latter showed a positive bias.
More specifically,the SRY/HBB assay,which was the first real-time PCR system to be developed for mea-suring fractional fetal-DNA concentrations in mater-nal plasma(9),underestimated the proportion of male DNA by approximately20%in experiments involving artificial mixtures of male and female DNA(Fig.2).In experiments with plasma samples from pregnant women carrying male fetuses,the digital PCR assay re-vealed median fractional fetal-DNA concentrations that were approximately2-fold higher than previously reported(9).Apart from the expected better quantita-tive performance of the digital PCR assay,another fac-tor that might have contributed to the higher fractional fetal-DNA concentration with the digital assay is the fact that the87-bp ZFY amplicon is shorter than the 137-bp SRY amplicon(9).Previous work has indicated that fetal DNA in maternal plasma is shorter than the background maternally derived DNA(22).
The enhanced analytical performance of microflu-idics digital PCR could have an important impact on the use of fetal DNA in maternal plasma for noninva-sive prenatal diagnosis.For example,many investiga-tors have attempted to develop methods for increasing the fractional concentrations of fetal DNA extracted from maternal plasma,either through size fraction-ation(11)or suppression of the background maternal DNA through formaldehyde treatment(23).The con-troversy surrounding the latter approach in particular has illustrated the importance of precise and correct measurements of circulating fetal-DNA concentra-
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tions,because one area of dispute has concerned the imprecision of the serial-dilution approach used in the original version of the formaldehyde-treatment method(23–25).
Furthermore,the number of counted molecules required to detect Down syndrome has been demon-strated to be inversely related to the fractional fetal-DNA concentration(15).Our demonstration that the median fractional concentration of circulating fetal DNA obtained with digital PCR might be2-fold higher than previously recognized(Fig.4)suggests that the technical challenge for detecting Down syndrome via plasma-DNA analysis is less than has previously been assumed.Indeed,for every2-fold increase in the frac-tional fetal-DNA concentration,the number of ana-lyzed molecules required to diagnose Down syndrome decreases by a factor of approximately4(15).
Microfluidics digital PCR also has benefits for ap-plications requiring only the qualitative detection of circulating fetal DNA.For example,we have demon-strated that microfluidics digital PCR improves the de-tection of male fetal DNA in maternal plasma,com-pared with conventional real-time PCR.In addition, microfluidics digital PCR would have an advantage for detecting fetal-DNA targets that differ from maternal-DNA sequences by one or a small number of nucleo-tides,because digital PCR operates at target concentra-tions at which most positive reactions would contain only a single target molecule(15,16).Thus,fetal-and maternal-DNA targets that would be cross-amplified with the same primer set would now be separately am-plified in different digital PCRs,with the correspond-ing fetal and maternal amplicons being identified by probes labeled with different reporters.Although we based this study on the quantification of male fetal DNA in maternal plasma,the advantage mentioned above also indicates that fetal-DNA concentrations for female fetuses could be measured via digital PCR quan-tification of fetus-specific,paternally inherited alleles of a panel of single-nucleotide polymorphisms.
One current drawback of microfluidics digital PCR is the cost of the chips.In this study,one chip was consumed for each maternal plasma sample.The en-hanced analytical performance of digital PCR could be realized in nonmicrofluidics-based digital real-time PCR or in digital primer-extension reactions with mass spectrometry(15).For investigators who prefer to use nondigital PCR approaches for cost reasons,there may be room for improvement in the quantitative perfor-mance of the nondigital platforms,for example via the addition of internal calibrators to each PCR reaction (26).
In conclusion,this study has demonstrated that microfluidics digital PCR is a useful new tool that al-lows improved measurement of circulating cell-free fe-tal DNA and potentially other nucleic acid species in plasma,such as tumor-derived DNA(27)and donor-derived DNA(28)in the plasma of cancer patients and transplant recipients,respectively.
Author Contributions:All authors confirmed they have contributed to the intellectual content of this paper and have met the following3requirements:(a)significant contributions to the conception and design,acquisition of data,or analysis and interpretation of data;(b)drafting or revising the article for intellectual content;and(c)final approval of the published article.
Authors’Disclosures of Potential Conflicts of Inter-est:Upon submission,all authors completed the Disclo-sures of Potential Conflict of Interest form.Potential con-flicts of interest:
Employment or Leadership:None declared. Consultant or Advisory Role:Y.M.Dennis Lo,Seque-nom
Stock Ownership:Y.M.Dennis Lo,Sequenom Honoraria:None declared.
Research Funding:Rossa W.K.Chiu,University Grants Committee of the Government of the Hong Kong Special Administration Region,China,under the Areas of Excellence Scheme(AoE/M-04/06);Y.M. Dennis Lo,Sequenom.
Expert Testimony:None declared.
Other:Rossa W.K.Chiu,Patent applications have been filed on the detection of fetal nucleic acids in maternal plasma for noninvasive prenatal diagnosis.Part of this patent portfolio has been licensed to Sequenom. Role of Sponsor:The funding organizations played no role in the design of study,choice of enrolled patients, review and interpretation of data,or preparation or approval of manuscript.
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