qf-pcr in substitution of direct method for the analysis of the … · 2015-04-22 · qf-pcr in...
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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..
QFQF--PCR DESCRIPTIONPCR DESCRIPTION
�� 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
QFQF--PCR DESCRIPTIONPCR DESCRIPTION
��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
[1a] False negative [1a] False negative casescases relatedrelated toto TFM TFM typetype IV IV
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%
[1a] False negative [1a] False negative casescases relatedrelated toto TFM TFM typetype IV IV
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
[1a] False negative [1a] False negative casescases relatedrelated toto TFM TFM typetype IV IV
[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
2* 15 20 Disomico,XY
3* 16 12 Disomico,XY
4* 22 50 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..
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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
polymerasepolymerase chainchain reactionreaction (QF(QF--PCR)PCR)
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
polymerasepolymerase chainchain reactionreaction (QF(QF--PCR)PCR)
TRISOMY 21 PROFILETRISOMY 21 PROFILE
D21S1435 D21S11 D21S1270 D13S634 D18S535
D18S978 D21S1411 D18S499 D13S628
D18S391D18S386
D13S742
D13S305
D13S252
D13S762
D18S1002D21S226
IFNAR (CHR21)