qf-pcr in substitution of direct method for the analysis of the … · 2015-04-22 · qf-pcr in...

QFQF--PCR in substitution of direct method for PCR in substitution of direct method for

the analysis of the chorionic the analysis of the chorionic villivilli in prenatal in prenatal

diagnosis: limits, advantages and valuations diagnosis: limits, advantages and valuations

from an experience of 44727 prenatal from an experience of 44727 prenatal

diagnoses on CVSdiagnoses on CVS

Francesca R. Grati, Francesca R. Grati, Ph.DPh.D..

ResearchResearch & & DevelopmentDevelopment, cytogenetics, , cytogenetics, molecularmolecular cytogenetics and cytogenetics and molecularmolecular biologybiology

TOMA, Advanced Biomedical Assays, S.p.A. TOMA, Advanced Biomedical Assays, S.p.A.

[email protected]@tomalab.com

�� Gold standard of Gold standard of fetalfetal karyotyping on karyotyping on chorionicchorionic villi (CVS)villi (CVS)

ItIt requiresrequires: :

�� a a complexcomplex cytogeneticcytogenetic lab lab organizationorganization

�� standardizationstandardization of of cytogeneticcytogenetic protocolsprotocols

�� expert expert operatorsoperators in in cellcell colturescoltures & & karyotypekaryotype analysisanalysis

SimoniSimoni, , TerzoliTerzoli e Rossella, 1990; e Rossella, 1990; SimoniSimoni e Rossella, 1986; e Rossella, 1986; VejerslevVejerslev and and MikkelsenMikkelsen, , 19891989

HahnemannHahnemann and and VejerslevVejerslev, 1997, 1997

PREMESSAPREMESSABACKGROUNDBACKGROUND

�� ThisThis combinedcombined approachapproach isis the the mostmost reliablereliable methodmethod and and withwith the best the best

diagnosticdiagnostic yieldyield forfor CVS CVS analysisanalysis consideringconsidering allall indicationsindications forfor invasive invasive prenatalprenatal

diagnosisdiagnosis

Direct or Short Direct or Short TermTerm Culture Culture

(STC)(STC)

Long Long TermTerm Culture, LTCCulture, LTC

IntroductionIntroduction of Quantitative of Quantitative fluorescentfluorescent

polymerasepolymerase chainchain reactionreaction (QF(QF--PCR)PCR)

�� can can bebe appliedapplied toto the detection of the detection of chromosomechromosome copy copy numbernumber byby

amplificationamplification of of repeatrepeat sequencessequences at at polymorphicpolymorphic lociloci

MICROSATELLITEMICROSATELLITE

�� tandem tandem repeatsrepeats of 2of 2--33--4 nt 4 nt ieie: (CA)n : (CA)n

�� manymany allelesalleles, , veryvery informativeinformative

�� distributeddistributed uniformlyuniformly alongalong the the genomegenome

everyevery 100 100 KbKb

TCAT repeat unitTCAT repeat unit

5 repeats5 repeats

8 repeats8 repeats

TCATTCAT

5 repeats

8 repeats

��TheseThese repeatrepeat sequencessequences are are amplifiedamplified byby PCR PCR usingusing primersprimers flankingflanking

the the repeatedrepeated regionregion

D21S11D13S317 D18S51

AMEL

D21S1435

D21S1270

D13S634 D18S535

D18S978

D21S1411D18S499

D13S628

D18S391

D18S386

D13S742

D13S305

MAOA

P39

SRY

DXS981

DXS996

DXS1283

DXS6854

DXS8051

X22

HPRT

QFQF--PCR DESCRIPTIONPCR DESCRIPTION

��PrimerPrimer pairspairs forfor the the polymorphicpolymorphic loci (loci (markersmarkers) are ) are multiplexedmultiplexed

togethertogether toto givegive a a rapidrapid, , efficientefficient and and inexpensiveinexpensive diagnosticdiagnostic test test forfor

trisomytrisomy 13, 18, 21, sex 13, 18, 21, sex chromosomechromosome aneuploidiesaneuploidies and and triploidytriploidy

�� dosagedosage limitedlimited toto chrchr 13, 18, 21, X/Y13, 18, 21, X/Y

��ThisThis diagnosticdiagnostic approachapproach waswas first first suggestedsuggested in 1993 (in 1993 (MansfieldMansfield, ,

1993) and 1993) and prospectiveprospective studiesstudies werewere carriedcarried out in the midout in the mid--1990s (1990s (PertlPertl

etet al, 1994) al, 1994) followedfollowed byby the the developmentdevelopment ofof a a QFQF--PCRPCR--basedbased test test forfor sex sex

chromosomechromosome imbalanceimbalance ((PertlPertl etet al, 1997; al, 1997; CiriglianoCirigliano etet al, 1999).al, 1999).

��ItIt waswas notnot untiluntil 2001 2001 thatthat the first report of the first report of clinicalclinical applicationapplication of of thisthis

test test appearedappeared ((CiriglianoCirigliano etet al, 2001)al, 2001)

��Best Best practicepractice guidelinesguidelines havehave beenbeen developeddeveloped byby a a collaborationcollaboration

betweenbetween the the associationassociation of of ClinicalClinical CytogeneticsCytogenetics (ACC) (ACC)

(http://www.cytogenetics.org.uk/) and the (http://www.cytogenetics.org.uk/) and the ClinicalClinical MolecularMolecular GeneticsGenetics

Society (CMGS) (Society (CMGS) (http://www.cmgs.orghttp://www.cmgs.org), and a 2012 ), and a 2012 versionversion hashas beenbeen

ratifiedratified byby bothboth councilscouncils

�� CECE--IVD commercial IVD commercial kitskits are are nownow availableavailable fromfrom a a numbernumber of of differentdifferent companiescompanies

�� In In manymany labslabs, , assaysassays are are designeddesigned toto test test forfor bothboth sex sex chromosomechromosome

aneuploidyaneuploidy and the and the viableviable trisomiestrisomies, and , and thusthus, , autosomeautosome and sex and sex

chromosomechromosome markersmarkers are are multiplexedmultiplexed togethertogether..

�� In the majority of UK NHS In the majority of UK NHS laboratorieslaboratories, a , a

separate sex separate sex chromosomechromosome assayassay isis appliedapplied onlyonly in in

a a minorityminority of of casescases referredreferred withwith anan indicationindication of of

monosomymonosomy X; X;

�� thisthis isis becausebecause testingtesting, , whichwhich revealsreveals incidentalincidental

findingsfindings of of debatabledebatable clinicalclinical significancesignificance suchsuch asas

triple X and XYY in triple X and XYY in prenatalprenatal samplessamples, , isis generallygenerally

notnot consideredconsidered toto bebe justifiedjustified..


�� In some In some CountriesCountries QFQF--PCR PCR forfor common common

aneuploidiesaneuploidies isis appliedapplied asas ““stand alonestand alone”” test test

((withoutwithout karyotypekaryotype and sex and sex chrchr investigationinvestigation) in ) in

pregnanciespregnancies thatthat are are referredreferred forfor raisedraised riskrisk of of

trisomytrisomy ((increasedincreased MSS MSS forfor DS, AMA and DS, AMA and AnxietyAnxiety

<35y) and <35y) and withoutwithout fetalfetal ultrasound ultrasound

abnormalitiesabnormalities (Hills (Hills etet al, 2010)al, 2010)

��SeveralSeveral largelarge data data setssets havehave beenbeen publishedpublished

60 670 AF and 33 266 CV 60 670 AF and 33 266 CV samplessamples

��DifferencesDifferences betweenbetween QFQF--PCR PCR resultsresults and and karyotypekaryotype resultsresults forfor

the the chromosomeschromosomes testedtested byby QFQF--PCR PCR havehave beenbeen reportedreported, ,

usuallyusually wherewhere one test one test detectsdetects full full trisomytrisomy, , whereaswhereas the the otherother

detectsdetects mosaicismmosaicism; ; suchsuch differencesdifferences are are likelylikely toto bebe due due toto

differencesdifferences in material in material testedtested

��RegardingRegarding AFsAFs, , therethere havehave beenbeen no no reportedreported complete complete

discrepanciesdiscrepancies betweenbetween a a diagnosticdiagnostic QFQF--PCR and PCR and karyotypekaryotype

resultresult forfor autosomalautosomal trisomiestrisomies


��The The incidenceincidence of of suchsuch discrepantdiscrepant resultsresults isis likelylikely toto bebe

determineddetermined byby severalseveral factorsfactors: :

11-- The The qualityquality and and sizesize of the of the originaloriginal CV CV biopsybiopsy. .

22-- StrategyStrategy forfor processing processing biopsiedbiopsied material. material.

33-- AnalysisAnalysis skillskill ((OgilvieOgilvie & Mann, Prenatal & Mann, Prenatal DiagnosisDiagnosis 2012)2012)

��DiagnosesDiagnoses forfor CVS are more CVS are more problematicproblematic, , withwith reportsreports of of

completelycompletely discrepantdiscrepant QFQF--PCR and PCR and karyotypekaryotype resultsresults due due toto

placentalplacental mosaicismmosaicism and feto and feto placentalplacental discrepanciesdiscrepancies fromfrom a a

numbernumber of of centrescentres (Allen (Allen etet al, 2006; al, 2006; WatersWaters etet al, 2006 and al, 2006 and

2007; 2007; HolgadoHolgado etet al, 2011; Mann al, 2011; Mann etet al, 2001)al, 2001)

��IncidenceIncidence of of discrepantdiscrepant resultsresults: : highlyhighly variablevariable 1/815 1/815

((HolgadoHolgado etet al, 2011) al, 2011) --> <1/10000 (Mann & > <1/10000 (Mann & OgilvieOgilvie, in press), in press)

QFQF--PCR on CVSPCR on CVS

�� ThisThis approachapproach shouldshould decreasesdecreases the TAT and the TAT and simplifysimplify the the

laboratorylaboratory organizationorganization, , neverthelessnevertheless itit isis necessarynecessary toto

evaluateevaluate and and quantifyquantify on on cytogeneticcytogenetic retrospectiveretrospective auditsaudits

limits and limits and advantagesadvantages of of thisthis new new methodmethod comparedcompared toto the the

traditionaltraditional methodmethod beforebefore eestablishingstablishing a diagnostic servicea diagnostic service

�� RecentlyRecently some some laboratorieslaboratories proposedproposed the replacement of the replacement of

chromosomal analysis of chorionic chromosomal analysis of chorionic villivilli (CV) direct preparation (CV) direct preparation

samples (DIR) by quantitative fluorescence PCR (QFsamples (DIR) by quantitative fluorescence PCR (QF--PCR) (QFPCR) (QF--

PCR+LTC instead of STC+LTC)PCR+LTC instead of STC+LTC)

ChristopoulouChristopoulou etet al., 2009al., 2009

�� BasingBasing on on thesethese false false negtivenegtive and positive and positive resultsresults some some

laboratorieslaboratories put put intointo effecteffect strategiesstrategies toto minimizeminimize the the riskrisk of of

false false resultsresults

WatersWaters etet al., 2007; al., 2007; CiriglianoCirigliano etet al., 2009al., 2009

BACKGROUNDBACKGROUND

ExploreExplore limits and limits and advantagesadvantages of the of the proposedproposed

QFQF--PCR+LTC PCR+LTC approachapproach comparedcompared toto the gold the gold

standard standard methodmethod ((STC+LTCSTC+LTC) and ) and quantifyquantify the the

associatedassociated riskrisk of false of false resultsresults

AIMAIM

MATERIALS AND METHODSMATERIALS AND METHODS

�� RetrospectiveRetrospective auditaudit of 44.727 consecutive of 44.727 consecutive prenatalprenatal

diagnosesdiagnoses on CVS on CVS donedone byby TOMA lab TOMA lab combiningcombining STC+LTCSTC+LTC

�� homogeneoushomogeneous diagnosticdiagnostic proceduresprocedures and and diagnostcdiagnostc

criteriacriteria

�� in agreement in agreement withwith the the ItalianItalian and and EuropeanEuropean guidelinesguidelines

�� mosaicmosaic in CV in CV --> > confirmatoryconfirmatory amniocentesisamniocentesis (AF)(AF)

�� CPM CPM involvinginvolving imprintedimprinted chromosomeschromosomes: UPD : UPD esclusionesclusion on on

amniocytesamniocytes byby allelicallelic segragationsegragation fromfrom parentsparents toto fetusfetus of of

microsatellitemicrosatellite markersmarkers mappedmapped toto the the imprintedimprinted chrchr

involvedinvolved in the in the trisomictrisomic cellcell lineline

(Grati (Grati etet al, 2006)al, 2006)

RESULTSRESULTS

False negative False negative evaluationevaluation riskrisk associate associate toto QFQF--

PCR+LTC PCR+LTC approachapproach

�� Compare Compare toto the the traditionaltraditional gold standard gold standard methodmethod STC+LTCSTC+LTC, the QF, the QF--

PCR+LTC PCR+LTC approachapproach associatesassociates toto twotwo mainmain typestypes of false negative of false negative

riskrisk::

11-- relatedrelated toto placentalplacental discrepanciesdiscrepancies::

AA-- TrueTrue fetalfetal mosaicismmosaicism typetype IV (TFM IV (TFM typetype IV) IV) withwith anan

abnormalabnormal cytotrophoblastcytotrophoblast ((mosaicmosaic and non and non mosaicmosaic) )

and a and a normalnormal mesenchymemesenchyme

BB-- UniparentalUniparental disomiesdisomies (UPD) (UPD) forfor imprintedimprinted

chromosomeschromosomes

22-- relatedrelated toto the the presencepresence of a of a maternalmaternal cellcell contaminationcontamination (MCC) in (MCC) in

the LTCthe LTC

[1a] False negative [1a] False negative casescases relatedrelated toto TFM TFM typetype IV IV

CVS cases from May, 2000 to July 31st, 2011 44.727

Mosaicism on CVS 979 (2.2%)

Follow up at amniocentesis 768

True fetal mosaicism confirmed on AF 95 (12.3%)

TROPHOBLAST MESENCHYME

(direct) (culture)

I CPM Abnormal Normal Normal 34,63% (266/768)

II CPM Normal Abnormal Normal 43,09% (331/768)

III CPM Abnormal Abnormal Normal 9,89% (76/768)

IV TFM Abnormal Normal Abnormal 1.82% (14/768)

V TFM Normal Abnormal Abnormal 5,33% (41/768)

VI TFM Abnormal Abnormal Abnormal 5,20% (40/768)

TYPE NATURE AMNIOCYTES RELATIVE FREQUENCIES

�� DistributionDistribution and and typetype of the of the abnormalabnormal cellcell line line presentpresent in the in the

cytotrophoblastcytotrophoblast of the 14 TFM IV of the 14 TFM IV casescases

Table 1: Description of 14 TFM type IV cases

ID case IndicationAbnormal

cell line

#abnormal/#tot

metaphases on

direct prep.

Level of

mosacism in

cytotrophoblast

LTC AF

TFM IV-1 AMA 47,XX,+13 3/26 12% Normal 47,XX,+13

TFM IV-2 AMA 45,X 6/52 12% Normal mos 45,X[50]/47,XXX[3]

TFM IV-3 Increased MSS 45,X 5/25 20% Normal 45,X

TFM IV-4 AMA 45,X 8/40 20% Normal mos 45,X[21]/46,XY[55]

TFM IV-5 AMA 47,XX,+mar 5/10 50% Normal mos 47,XX,+mar[7]/46,XX[33]*

TFM IV-6 AMA 47,XX,+mar 17/23 75% Normal mos 47,XX,+mar[6]/46,XX[99]**

TFM IV-7 AMA 47,+mar 5(+G)+3(+F)/20 40% Normal

mos

47,XX,+i(12)(p10)[19]/46,XX[28]

.ish(12)(p10)(pter++)

TFM IV-8 AMA 47,XX,+21 20/20 100% Normal 47,XX,+21

TFM IV-9 AMA 45,X 20/20 100% Normal mos 45,X[13]/46,XX[37]

TFM IV-10 AMA 45,X 17/17 100% Normal 46,X,inv(Y)(p11q12)

TFM IV-11 AMA 45,X 17/17 100% Normal mos 45,X[28]/46,XX[22]

TFM IV-12 AMA 45,X 15/15 100% Normal mos 45,X[11]/46,XY[62]

TFM IV-13 AMA 47,XXX 15/15 100% Normal 47,XXX

TFM IV-14 AMA 47,XY,+mar 18/18 100% Normalmos 47,XY,+mar[33]/46,XY[20]***

*Small, metacentric, not satellited

**Minute, not satellited, QFQ+

***Small, satellited

MOSAIC ABNORMAL CELL LINE IN CYTOTROPHOBLAST

HOMOGENEOUS ABNORMAL CELL LINE IN CYTOTROPHOBLAST


1.1. ConstitutionConstitution of the of the villousvillous treetree: : mesenchymemesenchyme componentcomponent

representsrepresents 4040--50% of the pool of DNA 50% of the pool of DNA derivedderived fromfrom direct CVS direct CVS

(Mann (Mann etet al, 2007) al, 2007)

2.2. QFQF--PCR PCR hashas a a mosaicismsmosaicisms detection detection thresholdthreshold of of

~20% (~20% (DonaghueDonaghue etet al, 2005)al, 2005)

3.3. In UK approach sex chromosomes are In UK approach sex chromosomes are

investigated only in cases referred with an investigated only in cases referred with an

indication of indication of monosomymonosomy X (X (Hills Hills etet al, 2010): al, 2010):

routinelyroutinely a QFa QF--PCR PCR restrictedrestricted toto autosomesautosomes isis

appliedapplied

DNA DNA forfor QFQF--PCRPCR40-50%

60-50%


BasingBasing on the on the descriptiondescription of the 14 TFM of the 14 TFM typetype IV IV casescases withwith QFQF--PCR+LTC PCR+LTC

approachapproach can can bebe associatedassociated withwith a false negative in a false negative in casescases withwith::

�� anan abnormalabnormal mosaicmosaic cellcell line in the line in the cytotrophoblastcytotrophoblast withwith anan aneuploidyaneuploidy

of of chrchr 13, 18, 21, X/Y 13, 18, 21, X/Y isis presentpresent in in ≤≤50% of the 50% of the cellscells (n=4; TFM IV(n=4; TFM IV--11--

>4);>4);

�� anan abnormalabnormal homogeneoushomogeneous karyotypekaryotype 45,X o 47,XXX in 45,X o 47,XXX in cytotrophoblastcytotrophoblast

whenwhen sex sex chromosomeschromosomes are are notnot investigatedinvestigated (n=5; TFM IV(n=5; TFM IV--99-->13); >13);

�� a a mosaicmosaic (n=3; TFM IV(n=3; TFM IV--5, 6, 7) or 5, 6, 7) or homogeneoushomogeneous (n=1; TFM IV(n=1; TFM IV--14) 14)

sSMCsSMC notnot containingcontaining chromosomechromosome regionsregions of of chrchr 13, 18, 21, X/Y 13, 18, 21, X/Y

investigatedinvestigated byby QFQF--PCRPCR

P (false negative) P (false negative) relatedrelated toto TFM tipo IV:TFM tipo IV:

~1/3000~1/3000 (QF(QF--PCR PCR onlyonly forfor autosomesautosomes + LTC; 13/39533*) + LTC; 13/39533*)

& &

~1/5000~1/5000 (QF(QF--PCR + LTC; 8/39533*)PCR + LTC; 8/39533*)

*total*total normalnormal casescases: 44727 : 44727 –– incomplete incomplete casescases -- casescases withoutwithout risultatrisultat

(MCC) (MCC) -- pathologicpathologic casescases


[1b] False negative [1b] False negative casescases due due toto UPD for imprinted UPD for imprinted

chromosomeschromosomes

�� 44.727 44.727 prenatalprenatal diagnosesdiagnoses on CVS: 5 UPD/230 on CVS: 5 UPD/230 casescases withwith a a mosaicmosaic cellcell line in line in

CVS CVS involvinginvolving anan imprintedimprinted chrchr ((trisomytrisomy or or sSMCsSMC) () (incidenceincidence 2.2%) 2.2%)

�� In 2/5 a CPM In 2/5 a CPM typetype I I waswas presentpresent withwith a a trisomytrisomy 14 in 14 in cytotrophoblastcytotrophoblast. . ThisThis

chromosomchromosom isis notnot investigatedinvestigated byby QFQF--PCR so PCR so thesethese casescases wouldwould havehave beenbeen

undetectedundetected withwith the QFthe QF--PCR+LTC PCR+LTC approachapproach

P (false negative) P (false negative) relatedrelated toto UPD UPD causingcausing imprinting imprinting

syndromessyndromes: ~1/20.000 (2/39533): ~1/20.000 (2/39533)

Imprinted related chr

20

1616

15

1414

7

2

RelatedRelated

UPDUPD

55230230TotalTotal

-0/45Mar, rearr

-0/23trisomy 20

2 (CPM III)+1(TFM VI)2 (CPM III)+1(TFM VI)3/163/16trisomy 16trisomy 16

-0/19trisomy 15

2 (CPM I)2 (CPM I)2/82/8trisomy 14trisomy 14

-0/60trisomy 7

-0/59 trisomy 2

TypeType of CPM or TFMof CPM or TFMNo UPD/No of No UPD/No of

studiedstudied casescasesChromosomeChromosome

AbnormalitiesAbnormalities

TOTAL CASES INVOLVING

IMPRINTED CHR230

UPD CASES 5

FREQUENCY (%) 2.17

UPD CONFIRMATIONS

[2] False negative [2] False negative casescases due due toto MCCMCC

�� 15.025 15.025 monitoredmonitored diagnosesdiagnoses on CVS : 135 on CVS : 135 casescases withwith MCC in male sex LTC MCC in male sex LTC

(1.8%)(1.8%)

N Incidence (%)

Total Monitored cases

(Jan2009 - May2011)15.025 //

Total cases w MCC

(XX cell line in a male sample)135x2 1.80%

Cases with Dir XY and LTC w MCC

(2-90% XX cell line) 128x2 1.7% (~1:60)

Cases with Dir XY and LTC w MCC

(100% XX); 5*x2 0.06% (~1:1600)

Only LTC w MCC (100%); 2**x2 0.026% (~1:3800)

��In 7/135 the In 7/135 the karyotypekaryotype fromfrom LTC LTC waswas femalefemale

ID sample Dir (# XY metaphases)LTC (# XX

metaphases)

QF-PCR on

native DNA

1* 16 20 Disomico,XY




5* 15 50 (1met 46,XY) Disomico,XY

6** // 26 Disomico,XX

7** // 20 Disomico,XX

�� MCC1MCC1-->5: QF>5: QF--PCR PCR appliedapplied on DNA on DNA fromfrom direct CVS direct CVS showedshowed a a normalnormal disomicdisomic

male pattern male pattern unmaskingunmasking, , asas the STC the STC methodmethod doesdoes, the MCC, the MCC

�� MCC6MCC6--7: STC 7: STC withoutwithout spontaneousspontaneous metaphasesmetaphases ((probablyprobably due due toto the the presencepresence of of

the decidua the decidua onlyonly) and the ) and the followingfollowing US US investigationsinvestigations showedshowed a male a male fetusfetus

�� The The retrospectiveretrospective MCC MCC comparingcomparing the the fetalfetal and and maternalmaternal allelesalleles on DNA on DNA fromfrom

direct CVS direct CVS showedshowed a a normalnormal disomicdisomic fenalefenale pattern pattern corrispondingcorrisponding toto the the maternalmaternal

componentcomponent indicatingindicating a a substantialsubstantial MCCMCC

1) The 1) The failurefailure of the STC of the STC combinedcombined withwith a a femalefemale karyotypekaryotype in LTC in LTC

couldcould bebe the the ““alarmalarm bellbell”” of the of the presencepresence of a massive MCC of a massive MCC thatthat shouldshould

bebe investigatedinvestigated byby a MCC a MCC esclusionesclusion test test

2) 2) WithWith a routine a routine approachapproach QFQF--PCR+LTC the MCC PCR+LTC the MCC

esclusionesclusion test test withwith the the comparisoncomparison of the of the fetalfetal and and

maternalmaternal allelesalleles isis notnot performedperformed. In case of . In case of disclosuredisclosure

of a of a femalefemale fetusfetus byby sonographicsonographic investigationsinvestigations, the MCC , the MCC

wouldwould havehave beenbeen undetectedundetected

P (false negative) due P (false negative) due toto MCC: ~1/7500 MCC: ~1/7500

3) 3) PreferencePreference of LTC: of LTC:

P (false P (false resultresult) due ) due toto MCC=MCC=

1/1100 (7x2/15025);1/1100 (7x2/15025);

4) MCC 4) MCC esclusionesclusion isis advisableadvisable withwith the the comparisoncomparison of of fetalfetal and and

maternalmaternal allelesalleles on DNA on DNA fromfrom culture media culture media

PoorPoor amountamount CVSCVS

[2] False negative [2] False negative casescases due due toto MCCMCC

3) 3) ConfirmationsConfirmations on AFon AF

�� EachEach abnormalabnormal resultresult byby QFQF--PCR (PCR (mainlymainly in in casescases withoutwithout US US

abnormalitiesabnormalities) ) albeitalbeit in in combinationcombination withwith anan abnormalabnormal non non mosaicmosaic

mesenchymemesenchyme hashas toto bebe confirmedconfirmed byby amniocentesisamniocentesis

�� WithoutWithout a a cytogeneticcytogenetic resultresult on on cytotrophoblastcytotrophoblast, QF, QF--PCR PCR cannotcannot toto

discriminate a discriminate a homogeneoushomogeneous trisomytrisomy fromfrom high high levellevel trisomytrisomy (>50%) (>50%)

(CPM (CPM typetype II & III and TFM II & III and TFM typetype V & VI) V & VI)

ThisThis conditioncondition happenshappens forfor cr13, 18, 21 in ~1/2600 cr13, 18, 21 in ~1/2600

casescases (15/39533) (15/39533)

QF-PCR LTC Follow-up

Disomic AM/ANM

Trisomic AM/ANM

Trisomic Normal

Confirmatory AF is required

Possib

le r

esu

lts

4) 4) CostsCosts evaluationevaluation

�� staff staff costscosts ((techniciantechnician//ancillaryancillary staff staff –– biologistbiologist//supervisorsupervisor))

�� reagentsreagents and and disposablesdisposables

�� instrumentsinstruments ((rentalrental reagentsreagents, , purchasepurchase, , maintenancemaintenance))

�� lab lab rentrent and and additionaladditional lab lab costscosts

�� TypeType of lab of lab organizationorganization ((horizzontalhorizzontal or or verticalvertical))

�� TypeType of the test: CEof the test: CE--IVD o IVD o homehome--brewbrew

��LevelLevel of of skillsskills of the staffof the staff

FromFrom thisthis evaluationevaluation, , consideringconsidering a a horizontalhorizontal

organizationorganization of of bothboth protocolsprotocols, the , the costcost/sample of QF/sample of QF--

PCR CEPCR CE--IVD (IVD (notnot homehome--brewbrew) ) isis similarsimilar toto the one of the one of

STCSTC

5) 5) TurnTurn--aroundaround time (TAT)time (TAT)

�� STC: 36STC: 36--48h48h

�� QFQF--PCR: 24PCR: 24--36h36h

Due Due toto the the fetofeto--placentalplacental discrepanciesdiscrepancies bothboth resultsresults (of (of

QFQF--PCR and of STC) are PCR and of STC) are notnot definitive definitive withoutwithout a a

karyotypekaryotype fromfrom LTC LTC whichwhich requiresrequires 1010--12 12 daysdays

6) Staff training6) Staff training

CytogeneticsCytogenetics

�� TechnicalTechnical//ancillaryancillary staff (staff (cellcell culturescultures-->>imageimage capturecapture): 1 working ): 1 working monthmonth

�� BiologistBiologist//SupervisorSupervisor ((countcount-->>analysisanalysis-->>reportingreporting): 4 working ): 4 working monthsmonths

QFQF--PCRPCR

�� TechnicalTechnical//ancillaryancillary staff (DNA staff (DNA estractionestraction-->>electrophoresiselectrophoresis) (CE) (CE--IVD): 2 IVD): 2

working working weeksweeks

�� BiologistBiologist//SupervisorSupervisor ((electrpherogramelectrpherogram interpretationinterpretation-->>reportingreporting): 3 working ): 3 working

weeksweeks

CytogeneticCytogenetic staff training staff training requiresrequires 55--fold time fold time comparedcompared

toto QFQF--PCR staff trainingPCR staff training

CONCLUSIONSCONCLUSIONS

4) 4) QFQF--PCR staff training requires much less time than PCR staff training requires much less time than

cytogenetic staff trainingcytogenetic staff training

3) 3) ConsideringConsidering a a horizontalhorizontal organizationorganization, , QFQF--PCR CEPCR CE--IVD (not IVD (not

homehome--brew) and STC has similar to the cost/sample and brew) and STC has similar to the cost/sample and

identical TATidentical TAT

1) QF1) QF--PCR+LTC PCR+LTC comparedcompared toto STC+LTCSTC+LTC in CVS in CVS analysisanalysis

associatedassociated toto a a cumulative cumulative riskrisk of false negative of false negative resultresult of of

~1/2000~1/2000 mainlymainly due due toto the TFM the TFM typetype IV, UPD IV, UPD conditionsconditions

associatedassociated toto imprinting imprinting syndromessyndromes in the in the fetusfetus and and toto

erroneouserroneous diagnosisdiagnosis consequentconsequent toto MCC. MCC.

2) 2) EachEach abnormalabnormal QFQF--PCR PCR resultresult, , albeitalbeit combinedcombined toto anan

abnormalabnormal non non mosaicmosaic LTC LTC karyotypekaryotype, , requiresrequires a a confirmationconfirmation

on on amniocytesamniocytes toto discriminate high discriminate high levellevel CPM (>50%) CPM (>50%) fromfrom

TFM: TFM: thisthis happenshappens inin 1/2600 1/2600 casescases..

THANK YOU THANK YOU

D3 FGAvWA 5-FAM (blue)

D13D5 D7 NED (yellow)

A D8 D21 D18 JOE (green)

ROX (red)

100 bp 400 bp300 bp200 bp

Size Separation

Col

or S

epar

atio

n

D3 FGAvWA 5-FAM (blue)D3 FGAvWA 5-FAM (blue)D3 FGAvWA 5-FAM (blue)

��The The differentlydifferently labelledlabelled productsproducts are are separatedseparated byby gel gel electrophoresiselectrophoresis whichwhich resolvesresolves

the allele the allele productsproducts on the on the basisbasis ofof the allele the allele sizesize//numbernumber ofof repeatsrepeats

IntroductionIntroduction ofof Quantitative Quantitative fluorescentfluorescent


An allele pattern An allele pattern ofof the the investigatedinvestigated

polymorphicpolymorphic locus locus ofof::

�� twotwo peakspeaks withwith equalequal areasareas isis diagnosticdiagnostic ofof

twotwo copiescopies ofof the target the target regionregion

�� threethree peakspeaks withwith equalequal areasareas ((ratioratio 1:1:1) or 1:1:1) or

twotwo peakspeaks withwith a a ratioratio ofof 2:1 or 1:2 are 2:1 or 1:2 are

indicative indicative ofof trisomytrisomy forfor the target the target regionregion

�� INTERPRETATION OF QFINTERPRETATION OF QF--PCR PROFILES:PCR PROFILES:

IntroductionIntroduction ofof Quantitative Quantitative fluorescentfluorescent


TRISOMY 21 PROFILETRISOMY 21 PROFILE

D21S1435 D21S11 D21S1270 D13S634 D18S535

D18S978 D21S1411 D18S499 D13S628

D18S391D18S386

D13S742

D13S305

D13S252

D13S762

D18S1002D21S226

IFNAR (CHR21)

qf-pcr in substitution of direct method for the analysis of the … · 2015-04-22 · qf-pcr in...

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