Case Report False Negative Cell-Free DNA …downloads.hindawi.com/journals/crig/2016/7397405.pdfCase...

Case ReportFalse Negative Cell-Free DNA Screening Result ina Newborn with Trisomy 13

Yang Cao1 Nicole L Hoppman1 Sarah E Kerr1 Christopher A Sattler1

Kristi S Borowski2 Myra J Wick2 W Edward Highsmith1 and Umut Aypar1

1Department of Laboratory Medicine and Pathology Mayo Clinic Rochester MN 55905 USA2Department of Obstetrics and Gynecology Mayo Clinic Rochester MN 55905 USA

Correspondence should be addressed to Umut Aypar ayparumutmayoedu

Received 22 December 2015 Accepted 27 January 2016

Academic Editor Patrick Morrison

Copyright copy 2016 Yang Cao et alThis is an open access article distributed under theCreative CommonsAttribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Background Noninvasive prenatal screening (NIPS) is revolutionizing prenatal screening as a result of its increased sensitivityspecificity NIPS analyzes cell-free fetal DNA (cffDNA) circulating in maternal plasma to detect fetal chromosome abnormalitiesHowever cffDNA originates from apoptotic placental trophoblast therefore cffDNA is not always representative of the fetusAlthough the published data forNIPS testing states that the current technique ensures high sensitivity and specificity for aneuploidydetection false positives are possible due to isolated placental mosaicism vanishing twin or cotwin demise and maternalchromosome abnormalities or malignancy Results We report a case of false negative cell-free DNA (cfDNA) screening due tofetoplacental mosaicism An infant male with negative cfDNA screening result was born with multiple congenital abnormalitiesPostnatal chromosome and FISH studies on a blood specimen revealed trisomy 13 in 2020metaphases and 100 interphase nucleirespectively FISH analysis on tissues collected after delivery revealed extraembryonic mosaicism Conclusions Extraembryonictissue mosaicism is likely responsible for the false negative cfDNA screening result This case illustrates that a negative result doesnot rule out the possibility of a fetus affectedwith a trisomy as cffDNA is derived from the placenta and thereforemay not accuratelyrepresent the fetal genetic information

1 Background

Cell-free fetal DNA (cffDNA) circulating in maternal plasmawas initially identified approximately two decades ago how-ever the laboratory use of cffDNA to detect fetal chromo-some abnormalities was not available until 2011 [1] cfDNAscreening (also referred to as NIPS NIPT) analyzes cffDNAcirculating in maternal plasma Clinical utilization of cfDNAscreening has been rapidly incorporated into obstetric prac-tice as it offers improved sensitivity specificity and PPVwhen compared to first- and second-trimester screeningHowever cfDNA screening also has limitations The cffDNAoriginates from apoptotic placental trophoblast cells [2]therefore cffDNA may not always represent the chromoso-mal make-up of the fetus Although the genetic componentof placental and fetal tissue is identical in the vast majority ofpregnancies false positive or false negative resultsmay be due

to fetoplacental mosaicism In several reported cases follow-up amniocentesis based on positive cfDNA screening resultshas identified a normal karyotype suggesting a false positivecfDNA screening result [3ndash8] Even though the publisheddata indicates high sensitivity (ge99 for trisomy 21 ge92 fortrisomy 18 and ge87 for trisomy 13) and specificity (ge99for trisomy 21 18 and 13) for aneuploidy detection [9] falsepositive results have been reported for confined placentalmosaicism vanishing twin or cotwin demise fetal chromo-some rearrangement and maternal chromosome abnormal-ities or malignancy [10ndash14] Based on several reports falsenegative results for fetal aneuploidy are much less commonthan false positive results [4ndash6 15 16] It is generally acceptedthat false negative cfDNA screening results are primarily dueto a low level of cffDNA fraction in maternal plasma andtherefore could be overcome by technical improvement [17]However aside from the technical reasons a limited number

Hindawi Publishing CorporationCase Reports in GeneticsVolume 2016 Article ID 7397405 5 pageshttpdxdoiorg10115520167397405

2 Case Reports in Genetics

of false negative cfDNA screening cases due to fetoplacentalmosaicism andor structural chromosome rearrangementhave also been reported [13 18]

2 Case Presentation

A 19-year-old gravida 2 para 1 female underwent obstetricultrasound at 19 57 weeks of gestation which identifiedmul-tiple fetal anomalies including hypoplastic left heart bilateralcleft lip bilateral echogenic kidneys with hydronephrosisechogenic bowel and bowed right femur Genetic consul-tation was provided and risks benefits and alternativesof further genetic evaluation including amniocentesis andcfDNA screening were discussed The patient expressedconcerns regarding the risks of invasive testing and opted toproceed with cfDNA screening Limitations of cfDNA in thissetting were reviewed cfDNA screening was performed at 20weeks of gestational age A negative cfDNA screening resultwas issued for chromosomes 13 18 21 X and Y Althoughthe fetal fraction (percentage of fetal DNA among all DNA inmaternal plasma) was not included in the final report laterinquiries to the testing laboratory revealed a fetal fractionof 85 Genetic counseling was provided to the patientat 24-57 weeks of gestation during which amniocentesiswith cytogenetic analyses was further discussed The patientagain declined invasive testing After induction of labor dueto multiple fetal anomalies a male infant was deliveredvaginally at 38-47 weeks of gestational age Apgar scoreswere 8 7 and 9 at one five and ten minutes respectivelyPhysical examination revealed multiple anomalies includingcutis aplasia on the scalp cleft lip and palate polydactylyand cryptorchidism postnatal echocardiogram confirmedhypoplastic left heart The newborn also had respiratoryinsufficiency and was intubated due to signs of airwayobstruction A peripheral blood specimen was collected atbirth and sent to the Cytogenetics Laboratory for postnatalevaluation

Chromosome analysis was performed on 20 metaphaseswhich identified additional chromosome 13 in each meta-phase (47 XY +13) suggesting nonmosaic trisomy 13 (Fig-ure 1(a)) FISH analysis was also performed and 100of nuclei indicated three signals of probes for chro-mosome 13 also consistent with nonmosaic trisomy 13(Figure 1(b)) Unfortunately the infant passed away four daysafter birth

We proposed that the discordant cfDNA and postnatalcytogenetic results were due to fetoplacental mosaicismTo test this hypothesis we further evaluated extraembry-onic tissue samples The placenta was of normal weight(588 grams) and gross appearances of the placenta andumbilical cord were normal for 38 weeks of gestation Thechorionic villi showed mild villous enlargement and edemawith increase in Hofbauer cells but no other morphologicabnormalities Representative areas of the umbilical cordamnion villous trophoblast villous stroma and intermediatetrophoblast were identified from placental sections for ane-uploidy analysis by FISH As shown in Figure 1(c) all fivetypes of extraembryonic tissues demonstrated different levelsof extraembryonic mosaicism of trisomy 13 (58 57 32

1 2 3 4 5

6 7 8 9 10 11 12

13 14 15 16 17 18

19 20 21 22 X Y47 XY +13

(a)

Arrows indicate 3 LSI 13 signals

(b)

White arrows indicate cells with 3 LSI 13 signals Yellow arrows indicate cells with 2 LSI 13 signals

(c)

Figure 1 Postnatal aneuploidy detection by chromosome and FISHanalysis (a) G-banded karyotype on a blood specimen of thenewborn shows trisomy 13 (b) FISH analysis on a blood specimenof the newborn shows three signals of LSI 13 (RB1) probe targetedon 13q14 (green) and two signals of LSI 21 (D21S341) probe targetedon 21q2213-q222 (orange) Cells are stained with DAPI (blue)to visualize nuclei (c) FISH analysis for aneuploidy detection indifferent tissue types of placental specimen shows mosaic trisomy13 Signals of LSI 13 (RB1) probe targeted on 13q14 are shown ingreen Signals of LSI 21 (D21S341) probe targeted on 21q2213-q222are shown in orange Cells are stained with DAPI (blue) to visualizenuclei

46 and 64 of nuclei with trisomy 13 in umbilical cordamnion villous trophoblast villous stroma and intermedi-ate trophoblast resp data listed in Table 1) These resultssuggest that mosaicism of the extraembryonic tissues wasresponsible for the false negative cfDNA screening result inthis case

3 Materials and Methods

31 G-Banding Peripheral blood lymphocytes were culturedfor 72 h in PB-Max plus excess thymidine and were har-vested according to standard cytogenetic protocols Meta-phases were dropped in aThermotron chamber and baked for

Case Reports in Genetics 3

Table 1 Summary of tissue specific trisomy 13 mosaicism

Tissue type of nuclei with disomy 13 of nuclei with trisomy 13 of total nuclei of nuclei with trisomy 13Umbilical cord 21 29 50 58Amnion 18 24 42 57Intermediate trophoblast 26 46 72 64Villous trophoblast 28 13 41 32Villous stroma 23 20 43 46

1 hour and 30 minutes at 100∘C G-banding was performedaccording to standard cytogenetic methods using trypsinand Leishman stain Twenty GTL-banded metaphases wereevaluated

32 Fluorescence In Situ Hybridization (FISH) Initial FISHfor newborn aneuploidy detection was performed on aperipheral blood sample Additional postpartum FISH anal-ysis for aneuploidy detection was performed on formalinfixed paraffin-embedded tissue We used FISH probes thathybridize to theX centromere (DXZ1) Y centromere (DYZ3)13q14 (Rb1) 13q34 (LAMP1) 18 centromere (D18Z1) and21q22 (D21S341) Bacteria artificial chromosomes (BACs)located within the critical regions listed above were used forFISH probe development Briefly each BAC was extractedfrom E coli using the Qiagen Plasmid Maxi kit accordingto the manufacturerrsquos instructions and was then labeledwith SpectrumOrange SpectrumGreen or SpectrumAqua(Abbott Molecular) using the Nick Translation Kit (AbbottMolecular) according to the manufacturerrsquos instructions AFISH probe working solution was made by adding 3120583Lof labeled BAC to 7 120583L of LSIWCPreg hybridization buffer(Abbott Molecular) Slides were pretreated according tostandard cytogenetic protocols followed by application of3 120583L of probe working solution to the hybridization siteSlides were denatured at 75∘C for 5min and hybridized at37∘C for 70 h followed by washing for 2min at 72∘C with04x saline-sodium citrate (SSC) and rinsed in 01 NP-402xSSC for 1min at room temperature 10 120583L counterstain[10 410158406-diamidino-2-phenylindole (DAPI)] was added tothe hybridization area One hundred nuclei were scored foreach specimen for aneuploidy detection

4 Discussion

cfDNA screening is revolutionizing prenatal screening as aresult of its robust test performance with increased sensitivityand specificity for the detection of common autosomal aneu-ploidies (trisomies 13 18 and 21) compared to other prenatalaneuploidy screening methodologies Since the introductionin the clinical setting there have been several reports ofcfDNA performance in high-risk population (indicated byadvanced maternal age screen positive on first- or second-trimester serum biochemical screening the presence of afetal abnormality on ultrasound or a personal or familyhistory of a chromosomal abnormality) as well as in low-risk population (general population) [4 5 9] In high-riskpregnancies cfDNA screening PPV for common autosomal

aneuploidies varies from 909 to 100 based on differentstudies while NPV remains as 999ndash100 [4 5 9] In low-risk pregnancies PPV for common autosomal aneuploidieshas been reported as 853 with 999 NPV based on146958 pregnancies [4]Thus although clinical performanceof cfDNA screening is widely accepted and desirable forscreening utility it is not appropriate for use as a diagnostictest

Some of the reasons for false positive and negativecfDNA screening results include fetoplacental mosaicism avanishing twincotwin demisematernal chromosome abnor-mality and maternal metastatic disease Several publishedreports of false positive and false negative cfDNA screeningcases have implicated fetoplacental mosaicism [3 8 1318] The most common type of fetal-placental mosaicismconfined placental mosaicism (CPM) is a conception withnormal fetus and placenta mosaic for a chromosome abnor-mality [19] However false negative cfDNA screening resultscannot be explained by CPM Instead it may be due toanother type of fetoplacental mosaicism in which there is anaffected (possibly nonmosaic) fetus and mosaic or normalplacenta Although false negative cfDNA screening result fortrisomy 13 has been reported at least once the causativemechanism of false negative result was not identified atthat time [20] This is the first report of a case with falsenegative cfDNA screening result for trisomy 13 that is causedby fetoplacental mosaicism For trisomies 18 and 21 thismechanism has been recently described A false negativetrisomy 18 cfDNA screening result due to 48 XXX +18placentalmosaicism and two caseswith false negative trisomy21 due to placental mosaicism have been reported [3 13]Potential contribution of fetoplacental mosaicism to discor-dant cfDNA screening results has been reported this studysuggested that the sensitivity or specificity of cfDNA screen-ing will never reach 100 due to the nature of fetoplacentalmosaicism

This report describes a case of false negative cfDNAresults for trisomy 13 due to fetoplacental mosaicism Cytoge-netics analysis demonstrated that the infant was nonmosaicfor trisomy 13 with placental tissue mosaic for trisomy 13This mosaicism was demonstrated in at least five differentextraembryonic tissues including umbilical cord amnionvillous trophoblast villous stroma and intermediate tro-phoblast In this case the level of trisomy 13 mosaicism inthe various tissues investigated ranged from 32 to 64The level of fetal fraction required for robust detection oftrisomies varies slightly among commercial providers but isgenerally in the range of 4 In the case presented here


the proportion of the 85 fetal fraction that derived fromtrisomy 13 positive extraembryonic tissues was likely lessthan half thus decreasing the functional fetal fraction to lessthan the level required Similar patterns of mosaicism havebeen reported in cases with false negative cfDNA screeningresults [3 13 18] This type of fetoplacental mosaicism isdifferent from CMP which is more commonly reportedetiology for discordant cfDNA and fetalinfant cytogeneticresults Cytogenetic analyses of infant cord blood as wellas physical examination suggested nonmosaic trisomy 13for the infant However because our studies were limitedto a single specimen type from the infant we cannot ruleout the possibility that the infant may have had mosaicismin other tissues This case along with other reports ofdiscordant cfDNA screening findings demonstrates thatproviders should understand and patients should be coun-seled regarding the limitations of cfDNA screening In thesetting of a positive cfDNA screening result confirmatorydiagnostic testing is highly recommended This case reportalso underscores the importance of diagnostic testing inthe setting of multiple fetal anomalies with negative cfDNAresults Pre- and posttest genetic counseling are importantin such cases to educate about the benefits and limitationsof cfDNA screening to ensure that patients are able to makeinformed decisions

5 Conclusions

Here we report a case with negative cfDNA screeningresults that had a postnatal diagnostic test result of nonmo-saic trisomy 13 FISH analysis on placental tissues revealedextraembryonic mosaicism of trisomy 13 which is likelyresponsible for the false negative cfDNA screening resultThis case illustrates the limitation of cfDNA screening as cell-free fetal DNA is derived from the placenta and thereforemay not accurately represent the fetal genetic informationTherefore in the setting of multiple fetal anomalies withnegative cfDNA screening results diagnostic testing is rec-ommended

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

References

[1] R W K Chiu R Akolekar Y W L Zheng et al ldquoNon-invasiveprenatal assessment of trisomy 21 by multiplexed maternalplasma DNA sequencing large scale validity studyrdquo BritishMedical Journal vol 342 Article ID c7401 2011

[2] E Flori B Doray E Gautier et al ldquoCirculating cell-free fetalDNA in maternal serum appears to originate from cyto- andsyncytio-trophoblastic cells Case reportrdquo Human Reproduc-tion vol 19 no 3 pp 723ndash724 2004

[3] Y Wang J Zhu Y Chen et al ldquoTwo cases of placental T21mosaicism challenging the detection limits of non-invasiveprenatal testingrdquo Prenatal Diagnosis vol 33 no 12 pp 1207ndash1210 2013

[4] H Zhang Y Gao F Jiang et al ldquoNoninvasive prenatal testingfor trisomies 21 18 and 13mdashclinical experience from 146958pregnanciesrdquoUltrasound inObstetrics ampGynecology vol 45 no5 pp 530ndash538 2015

[5] P JWillems H Dierickx E Vandenakker et al ldquoThe first 3000Non-Invasive Prenatal Tests (NIPT) with the harmony test inBelgium and the Netherlandsrdquo Facts Views amp Vision in ObGynvol 6 no 1 pp 7ndash12 2014

[6] T K Lau S W Cheung P S Lo et al ldquoNon-invasive prenataltesting for fetal chromosomal abnormalities by low-coveragewhole-genome sequencing of maternal plasma DNA reviewof 1982 consecutive cases in a single centerrdquo Ultrasound inObstetrics amp Gynecology vol 43 no 3 pp 254ndash264 1982

[7] H Choi T K Lau F M Jiang et al ldquoFetal aneuploidy screeningby maternal plasma DNA sequencing lsquofalse positiversquo due toconfined placental mosaicismrdquo Prenatal Diagnosis vol 33 no2 pp 198ndash200 2013

[8] A L Hall H M Drendel J L Verbrugge et al ldquoPositive cell-free fetal DNA testing for trisomy 13 reveals confined placentalmosaicismrdquo Genetics in Medicine vol 15 no 9 pp 729ndash7322013

[9] R P Porreco T J Garite KMaurel et al ldquoNoninvasive prenatalscreening for fetal trisomies 21 18 13 and the common sexchromosome aneuploidies from maternal blood using mas-sively parallel genomic sequencing of DNArdquo American Journalof Obstetrics and Gynecology vol 211 no 4 pp 365e1ndash365e122014

[10] T Futch J Spinosa S Bhatt E de Feo R P Rava and A JSehnert ldquoInitial clinical laboratory experience in noninvasiveprenatal testing for fetal aneuploidy frommaternal plasmaDNAsamplesrdquo Prenatal Diagnosis vol 33 no 6 pp 569ndash574 2013

[11] H Yao L Zhang H Zhang et al ldquoNoninvasive prenatalgenetic testing for fetal aneuploidy detects maternal trisomy XrdquoPrenatal Diagnosis vol 32 no 11 pp 1114ndash1116 2012

[12] C M Osborne E Hardisty P Devers et al ldquoDiscordantnoninvasive prenatal testing results in a patient subsequentlydiagnosed with metastatic diseaserdquo Prenatal Diagnosis vol 33no 6 pp 609ndash611 2013

[13] Y Gao D Stejskal F Jiang and W Wang ldquoFalse-negativetrisomy 18 non-invasive prenatal test result due to 48XXX+18placental mosaicismrdquo Ultrasound in Obstetrics amp Gynecologyvol 43 no 4 pp 477ndash478 2014

[14] M Pan F T Li Y Li et al ldquoDiscordant results between fetalkaryotyping and non-invasive prenatal testing by maternalplasma sequencing in a case of uniparental disomy 21 due totrisomic rescuerdquo Prenatal Diagnosis vol 33 no 6 pp 598ndash6012013

[15] M Smith K M Lewis A Holmes and J Visootsak ldquoA case offalse negative NIPT for down syndrome-lessons learnedrdquo CaseReports in Genetics vol 2014 Article ID 823504 3 pages 2014

[16] J C Wang T Sahoo S Schonberg et al ldquoDiscordant nonin-vasive prenatal testing and cytogenetic results a study of 109consecutive casesrdquo Genetics in Medicine vol 17 no 3 pp 234ndash236 2015

[17] J A Canick G E Palomaki E M Kloza G M Lambert-Messerlian and J E Haddow ldquoThe impact of maternal plasmaDNA fetal fraction on next generation sequencing tests forcommon fetal aneuploidiesrdquo Prenatal Diagnosis vol 33 no 7pp 667ndash674 2013

[18] Q Pan B Sun X Huang et al ldquoA prenatal case with discrepantfindings between non-invasive prenatal testing and fetal genetictestingsrdquoMolecular Cytogenetics vol 7 article 48 2014


[19] D K Kalousek and F J Dill ldquoChromosomal mosaicism con-fined to the placenta in human conceptionsrdquo Science vol 221no 4611 pp 665ndash667 1983

[20] R Hochstenbach G C Page-Christiaens A C van Oppen etal ldquoUnexplained false negative results in noninvasive prenataltesting two cases involving trisomies 13 and 18rdquo Case Reports inGenetics vol 2015 Article ID 926545 7 pages 2015

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Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


of false negative cfDNA screening cases due to fetoplacentalmosaicism andor structural chromosome rearrangementhave also been reported [13 18]

2 Case Presentation

A 19-year-old gravida 2 para 1 female underwent obstetricultrasound at 19 57 weeks of gestation which identifiedmul-tiple fetal anomalies including hypoplastic left heart bilateralcleft lip bilateral echogenic kidneys with hydronephrosisechogenic bowel and bowed right femur Genetic consul-tation was provided and risks benefits and alternativesof further genetic evaluation including amniocentesis andcfDNA screening were discussed The patient expressedconcerns regarding the risks of invasive testing and opted toproceed with cfDNA screening Limitations of cfDNA in thissetting were reviewed cfDNA screening was performed at 20weeks of gestational age A negative cfDNA screening resultwas issued for chromosomes 13 18 21 X and Y Althoughthe fetal fraction (percentage of fetal DNA among all DNA inmaternal plasma) was not included in the final report laterinquiries to the testing laboratory revealed a fetal fractionof 85 Genetic counseling was provided to the patientat 24-57 weeks of gestation during which amniocentesiswith cytogenetic analyses was further discussed The patientagain declined invasive testing After induction of labor dueto multiple fetal anomalies a male infant was deliveredvaginally at 38-47 weeks of gestational age Apgar scoreswere 8 7 and 9 at one five and ten minutes respectivelyPhysical examination revealed multiple anomalies includingcutis aplasia on the scalp cleft lip and palate polydactylyand cryptorchidism postnatal echocardiogram confirmedhypoplastic left heart The newborn also had respiratoryinsufficiency and was intubated due to signs of airwayobstruction A peripheral blood specimen was collected atbirth and sent to the Cytogenetics Laboratory for postnatalevaluation

Chromosome analysis was performed on 20 metaphaseswhich identified additional chromosome 13 in each meta-phase (47 XY +13) suggesting nonmosaic trisomy 13 (Fig-ure 1(a)) FISH analysis was also performed and 100of nuclei indicated three signals of probes for chro-mosome 13 also consistent with nonmosaic trisomy 13(Figure 1(b)) Unfortunately the infant passed away four daysafter birth

We proposed that the discordant cfDNA and postnatalcytogenetic results were due to fetoplacental mosaicismTo test this hypothesis we further evaluated extraembry-onic tissue samples The placenta was of normal weight(588 grams) and gross appearances of the placenta andumbilical cord were normal for 38 weeks of gestation Thechorionic villi showed mild villous enlargement and edemawith increase in Hofbauer cells but no other morphologicabnormalities Representative areas of the umbilical cordamnion villous trophoblast villous stroma and intermediatetrophoblast were identified from placental sections for ane-uploidy analysis by FISH As shown in Figure 1(c) all fivetypes of extraembryonic tissues demonstrated different levelsof extraembryonic mosaicism of trisomy 13 (58 57 32

1 2 3 4 5

6 7 8 9 10 11 12

13 14 15 16 17 18

19 20 21 22 X Y47 XY +13

(a)

Arrows indicate 3 LSI 13 signals

(b)

White arrows indicate cells with 3 LSI 13 signals Yellow arrows indicate cells with 2 LSI 13 signals

(c)

Figure 1 Postnatal aneuploidy detection by chromosome and FISHanalysis (a) G-banded karyotype on a blood specimen of thenewborn shows trisomy 13 (b) FISH analysis on a blood specimenof the newborn shows three signals of LSI 13 (RB1) probe targetedon 13q14 (green) and two signals of LSI 21 (D21S341) probe targetedon 21q2213-q222 (orange) Cells are stained with DAPI (blue)to visualize nuclei (c) FISH analysis for aneuploidy detection indifferent tissue types of placental specimen shows mosaic trisomy13 Signals of LSI 13 (RB1) probe targeted on 13q14 are shown ingreen Signals of LSI 21 (D21S341) probe targeted on 21q2213-q222are shown in orange Cells are stained with DAPI (blue) to visualizenuclei

46 and 64 of nuclei with trisomy 13 in umbilical cordamnion villous trophoblast villous stroma and intermedi-ate trophoblast resp data listed in Table 1) These resultssuggest that mosaicism of the extraembryonic tissues wasresponsible for the false negative cfDNA screening result inthis case

3 Materials and Methods

31 G-Banding Peripheral blood lymphocytes were culturedfor 72 h in PB-Max plus excess thymidine and were har-vested according to standard cytogenetic protocols Meta-phases were dropped in aThermotron chamber and baked for






4 Discussion







5 Conclusions




References



























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















4 Discussion







5 Conclusions




References



























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















5 Conclusions




References



























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Case Report False Negative Cell-Free DNA …downloads.hindawi.com/journals/crig/2016/7397405.pdfCase...

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