First trimester screeningFor better maternal and fetal care

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Prenatal Screening for Down Syndrome

IntroductionRoughly estimated, every 800 th conception resulting in a viable baby is hit by an abnormal number of chromosomes, a so-called non-gonosomal aneuploidy. The most frequent is a trisomy of chromosome 21. The affected persons will remain handicapped and prone to diseases throughout their lives. Since there is knowledge about the etiology of the condition, there has been substantial effort to develop tools for its early preterm recognition allowing to advise parents timely. This brochure is about more recent findings in this field.

HistoryThe first report about trisomy 21 can be traced back to 1866. Theauthorofhisreport,JohnLangdonDown,wasthesuper-intendant of an asylum for retarded children. He observed two distinct populations amongst the children in his care: As is knowntoday,onegroupcomprisedchildrenwithacongenitalhypothyroidism,theotherchildrenwithatrisomy21,aconditionthatisreferredtotodayas“DownSyndrome”.

Roughly 40 years later a correlation of incidence and maternal age was reported 2(Fig.1).Inthe1930stherewasincreasingevidence,thatchromosomalabnormalitiesmaybethereasonfor the syndrome.3,4Finally,in1959Lajeune 5,6andJacobs 7 identifiedtrisomyofchromosome21tobeonecauseofit.Inthe1960smorechromosomalaberrations(translocation 8,9,mosaicism10) were reported also to result in the syndrome.

Today,theterm“DownSyndrome”isgenerallyacceptedtodescribe the clinical picture of people having an aneuploidy of chromosome 21.

Incidence and Etiology 11,12

The risk for the development of trisomy 21 correlates with maternal ageandrangesfromapprox.1:1600to1:5orevenhigher.Veryyoungmothers(15–18yrs)appeartohaveaslightlyincreasedriskaswell.Thepaternalageappearstohaveonlyasmall,ifanyinfluence.

Approximately97%ofallcasesaretheconsequenceofafaultycelldivision,90%ofwhichwouldaffectthematernaland5%thepaternalgenomeduringgametogenesis.Outofthese,2/3arethoughttobeduetoanimpairedfirstmeioticdisjunction,1/3toanincompletesecondmeioticdisjunction.Theremaining5%originateinapost-zygotic,mitoticcelldivisionerror(Fig.2).

In95%ofalltrisomy21cases,acompletethirdchromosome21 is present in all cells of the affected person. This condition is knownas“freetrisomy21”.

3%ofDownsyndromecasesareduetoanunbalancedtranslo-cation of parts of chromosome 21. There are 2 main types:

•14,21-translocations,50%ofwhichareinheritedfrombalancedcarriers,usuallyfromthemothers(familialtype).

•21,21-translocations,whichusuallyoccurdenovo.

AsmallpercentageofDownsyndromecasesresultfromso-calledmosaics,whichsometimesdonotpresentverypronouncedclinicalmanifestations and thus are often not discovered. Again there are 2 basic mechanisms that would cause a mosaic:

•Secondarysomaticlossofachromosome21fromaninitiallytrisomeric zygote.

•Mitoticdisjunctionerrorveryearlyduringembryodevelopment.

Fig. 1: Risk of Down syndrome depending on maternal age

15 20 25 30 35 40 45 50Age of Mother [Years]

Ris

k of

Dow

n S

yndr

ome

[1 t

o n]

1,600

1,200

800

400

1

Manifestation 12,17

TheclinicalpictureofDownSyndromeoriginatesfromanover-expressionofthegeneslocatedonchromosome21,bringingthe affected fetus out of genetic balance. The manifestation of signs and symptoms is not predictable and ranges from nearly not apparent to severely handicapping. Affected children often presentacharacteristicpatternofdysmorphisms,facultativemalformations,growthdeficiency,andmentalretardation.

Downbabiesareverysusceptibletoinfectiousdiseases.Beforetheavailabilityofantibiotics90%ofthemdiedearlyfrominfections.

BesidethedysmorphismsthereareseveralclinicalmanifestationsassociatedwiththeDownSyndrome.

On the clinical side there is an increased incidence of heart defects(50%),epilepsy,hypothyroidismandceliacdisease.

Bradycephaly Smallearswithfoldedhelix Macroglossy13,14 Upward slant to the eyes 15

Brushfieldirisspots Frequentlidinflammations Darkredtongue Epicanthal fold

Smallnosewithdepressedbridge15 Fissuredlips Irregularlypositionedteeth15 Predisposition for caries16

Shortandbroadneckwithexcessiveskin folds

Wide thorax Typical bowl configuration (X-ray) Shortbroadhandsandfingers

Transverse palmar crease Klinodactyly Brachymesophalangy(5 thfinger),only 1 inflexion crease

Excessive space between 1st and 2nd toe

Frequent dysmorphisms in Down syndrome

Atrioventricular septal defect TetralogyofFallot Duodenalstenosisoratresia

Omphalocele Preaxial polydactyly Hypothyroidism

Malformationofuppercervicalvertebrawithatlanto-occipital instability

Esophagal and anal atresia Aganglionic megacolon

Some facultative clinical manifestations in Down syndrome

Flattened nose and face, upward slanting eyes

Single palmarcrease, short fifth finger that curves inward

Widely separated first and second toes and increased skin creases

Fig. 2: Etiologies of Down Syndrome

n,21

Inherited

de novo

de novo

Cellularmosaicism

Tissue mosaicism

FreeDownsyndromePartial repair of

trisomeric zygote

Translocation

Downsyndrome

Non-disfunction

Paternal Maternal21,21

MeosisI MeosisII

Post-zygotic

Mosaicism

97 %

90 %5 %

3 %

50 %

50 %

2/3 1/3

5 %

14,21

Invasive methods in prenatal screening18

Amniocentesis Amniocentesis is used to collect amniotic fluid. A needle is insert-edthroughthemother’sabdominalwallintotheuterus,usingultrasound guidance. Approximately one ounce of fluid is taken for testing. Amniotic fluid contains fetal cells that can be exam-inedfortheirchromosomes.Ittakesabout2weekstodetermineifthefetushasDownsyndromeornot.

Amniocentesis is usually done between the 14th and 18th week of preg-nancy.Sideeffectstothemotherincludecramping,bleeding,infectionand leaking of amniotic fluid afterwards. There is a slight increase in the risk of miscarriage: the normal rate of miscarriage at this gestational ageis2to3%,andamniocentesisincreasesthatriskbyanadditional½to1%.Amniocentesisisnotrecommendedbeforethe14 th week of pregnancy due to a higher risk of complications and miscarriage.

Chorionic villus sampling (CVS)Chorionicvillussampling(CVS)isanotherinvasivemethodtocol-lectfetalcellsforchromosomeanalysis.Inthisprocedure,insteadofamnioticfluid,asmallamountoftissueistakenfromtheyoungplacenta (chorionic layer). The cells can be collected transabdominally like amniotic fluid. Another method is to insert a tube into the uterus transcervically. The method depends on the mother’s anatomy and the location of the placenta.

CVSisusuallydonebetweenthe10 th and 12thweekofpregnancy.Sideeffects to the mother are the same as with amniocentesis. The risk of miscarriageafterCVSisaround3–5%,slightlyhigherthanwithamnio-centesis.Inthepast,aftertheuseofCVSanumberofbabieswerebornwithmissingorshortenedfingersortoes.However,thatcouldbeattributedtotheuseofCVSbeforethe10 th week of pregnancy.

CordocentesisCordocentesisisalsoknownaspercutaneousumbilicalcordbloodsampling(PUBS).Itissimilartoamniocentesis,butinsteadofsamplingamnioticfluid,PUBScollectsfetalblood.Underultrasound guidance the needle is inserted through the mother’s abdomenanduterinewallintothefetalveinoftheumbilicalcord,whereafetalbloodsampleistaken.Becausethefetalveinisfragileearlyinpregnancy,PUBSisperformednoearlierthan17weeks into pregnancy.

PUBScannotonlydetectchromosomalabnormalities,butalsoblooddisorder,somemetabolicdisorders,infections,andotherproblems.

MiscarriageistheprimaryriskassociatedwithPUBS;theriskissimilartoCVS.Additionalpossiblecomplicationsincludebloodlossatthepuncturesite,infection,andprematureruptureofmembranes.

SincethediscoveryofthechromosomalbackgroundofDownSyndromeinthe1960stherehavebeencontinuouseffortsforanearlypre-termmethodtodetectDownpregnancies.Inthe1970samniocentesisandotherinvasivetechniqueswerethemethodsofchoice.However,havingacomplicationrateof1–2%,theseexaminationswererestrictedtotheriskgroup(womenbeyond35),sothatanoveralldetectionrateof30%onlycouldbeachieved.

Fig. 3: Amniocentesis

Fig. 4: Chorionic villus sampling (CVS)

Fig. 5: Cordocentesis

Whileamniocentesisstillisthe“ultimaratio”today,therecentyears saw the development of biochemical and imaging meth-ods to preselect women with a clear indication for invasive techniques in order to minimize the risk of complications in probably unaffected pregnancies.

Inthe1980stheso-calledtripletestwasstateoftheart.Here,the initial risk based on the maternal age was refined with find-ingsofAFP,hCGandunconjugatedE3.Thetripletestincreasesthedetectionrateto60%,however,isonlydiagnosticduringthe 2 ndtrimester,whichisrelativelylate.19,20,30,36,37,39,40,41,44

Embryos in the first trimester do not yet have fully function-ingexcretionorgans,sothattheyaccumulatesomeliquid.Thisliquid collects in a fold at the neck. Normal embryos have very littleliquid,thenuchalfoldisonly1–2mmthick.Embryoswithachromosomalaberration,however,mayhaveaverypro-nounced edema.24

The thickness of this neck vesicle is known as nuchal translu-cency (NT) and has meanwhile proven to be a very powerful markerforthepredictionofDownSyndrome.Butonlysincetheavailabilityofimprovedtechnologiesinthe1990sithasbeenpossibletomeasureNTbymeansofultrasound.Inthattime,NTwas used to refine the a priori age related risk to predict chro-mosomalaberrationsandadetectionrateofupto77%couldbe achieved.

Meanwhile,certainbiochemicalmarkerswereidentified,thathavepredictivepotentialforDownSyndromealreadyveryearlyinpreg-nancy. Routinely used today are pregnancy associated plasma pro-tein A (PAPP-A) and free b-hCG.UsedtogetherwithNTaprioririskofDownSyndromecanberefinedsignificantly,sothatadetectionaround90%isreached.Thisscreeningalgorithmisknownas“FirstTrimesterScreening”.Comparedwiththe“old”tripletestithasdistinctadvantages:

1.Supportsearlydecisionprocess: Applicableatagestationalageof10–14weeks(1 sttrimester),

leaving ample time for further clinical investigations.2. Higher detection rate:

Adetectionrateof90%atanacceptablespecificity(5%)ascomparedto60%forthetripletest.

The 1st trimester algorithm is currently state of the art for routine Down Syndrome screening.28-30,38

Non-invasive methods in prenatal screening

Normal

Abnormal

Fig. 6: Nuchal translucency (NT)

Risk Calculation 42-44

ThetargetofDownSyndromescreeningistoidentifypregnancieswhich are candidates for further investigations in order to avoid unnecessaryinvasiveexaminations,whichcarryalow,thoughconcrete risk of complications.

There are several software solutions available that support the consolidation of the single risk factors into one total risk. The risksareusuallyexpressedasodds,andatotalvaluehigherthan1:250(1:300)definesahigh-riskpregnancy.

The algorithm uses the risk related to the maternal age as a prioriodds,whicharemultipliedwithallpositivelikelihoodratiosderivedfromtheGaussianmarkers(NT,PAPP-A,freeb-hCG)andthestaticonesforotherriskfactorstoyieldtherefined a posteriori odds.

While the likelihood ratios for the static risk factors are provided asalistfromthesoftware,thelikelihoodratiosfortheGaussianmarkers are calculated from mathematical approximations of the distribution curves for affected and unaffected pregnancies using theindividualmeasuredMoM-values,whicharecalculatedas(see the graphs below):

LR+ = Detection Rate (DR) / False Positive Rate (FPR)

The current recommendation is that women with a risk of having a childwithDownsyndromeof1in250orgreatershouldbeofferedamniocentesis. There is controversy over whether to use the risk at the timeofscreening(“riskatscreeningdate”)orthepredictedriskatthetimeofbirth(“riskatterm”).TheriskatscreeningdateishigherthantheriskattermsincemanyfetuseswithDownsyndromeabortspon-taneouslyaroundthetimeofscreeningorafterwards(20–40%).18

However,non-invasivescreeningmethodssupporttheeffortstomini-mize unnecessary invasive investigations with higher risk for mother and fetus.

Q oddsvalue,e.g.1:500

LR + positive likelihood ratio

n number of likelihood ratios to be considered

priori before testing

posteriori after testing

n

Q posteriori = Q priori • P LRi

+

i=1

Nuchal translucency 25,28-30,36,37

NTbelongstotheso-calledGaussianmarkersinDownSyndromescreening.Thismeans,thatthevaluesfoundforunaffectedandaffectedfetusesfollowGaussiandistributioncurves.Obviously,themeans (maxima) of these curves depend on the gestational age (GA).ItiscommonpracticetodivideameasuredNTvaluebytheNTmeanofthegivengestationalweektoget“MultiplesoftheMedian”(MoM),whichmakesthedistributionindependentfromGAandfacilitatesfurtherdataprocessing.

ThepracticalexecutionofNTmeasurementisdifficult,becausethedistancestobedeterminedareusuallysmall(1–2mm)and1mmmoreorlessdoesmakeadifferenceinrisk.Ifnotaper-fectsagittalviewisachievedwhilesonographingtheembryo,theresultmaybeuselessifnotmisleading.Itisforthisreasonthatsomeofficialbodiesdemandthesonographers,whoperformsuchexaminations,toprovetheirskillsandbecertified.

Moreover,NThastobeinterpretedincontextwiththesizeoftheembryoexpressedas“CrownRumpLength”(CRL).Forfurthercalculations,bothvalueshavetobecollected.

PAPP-A 26-37,39

PAPP-Aisahighmolecularweightglycoprotein(187kDa)andbelongstothemetzincinsuperfamilyofzincmetalloproteinases.Itsgeneisinchromosomeband9q33.1.Amongstothersites,PAPP-Ais produced by trophoblasts in the placenta and released into the maternalcirculation.Itisnotpresentinamnioticfluid.

PAPP-Acleavesinsulin-likegrowthfactorbindingprotein-4(IGFBP-4)andreducesdramaticallytheaffinityofIGFBP-4forIGF-IandIGF-II.AssuchitisaregulatorofIGFbioactivityinseveralsystems,includingthehumanovaryandthecardiovascularsystem.BiologicalactivePAPP-Aexistsasa2:2heterodimerwithmajorbasicprotein(MBP).

PAPP-Aincreasescontinuouslyasofweek5duringnormalpreg-nancieswithaboostinthelasttrimester.Concentrationsarelowerinaneuploidpregnancies(0.5MoM),however,itsdiagnosticefficiencydeclinesafterweek14.Decreasedvaluesarealsoseenincaseofintrauterinegrowthrestriction,prematuredelivery,pre-eclampsia,stillbirth.

LikeNT,PAPP-AbelongstotheGaussianmarkersandisnormalizedtoMoMsbeforefurtherprocessing.

0.5

DR

1 2 3 4 15105Nuchal Translucency [MoM]

Unaffected Down Syndrome

FPR

0.1

DR

0.2 0.5 1 105432PAPP-A [MoM]

UnaffectedDown Syndrome

FPR

Fig. 8: The Gaussian distribution of pregnancy associated plasma protein A (PAPP-A) measurement in affected and unaffected pregnancies (DR: detection rate; FPR: false-positive rate)

Fig. 7: The Gaussian distribution of nuchal translucency (NT) measurement in affected and unaffected pregnancies (DR: detection rate; FPR: false-positive rate)

Free b-hCG 26-30,35-37,39,45-47

Thewell-knownproteohormonehCGconsistsoftwoproteinsub-units,a-hCGandb-hCG,whichmaketheholo-hormone.Theholo-hormone can be cleaved into the free chains by elastase activity,however,theamountoffreeb-hCGinbloodisusuallylow(1%oftotal),butcanincreaseundercertainpathologicalcondi-tions.

Incaseofaneuploidpregnanciesfreeb-hCGisincreased(2.0MoMin the 1sttrimester).ThedifferencegrowswithGA.

Freeb-hCGisalsoaGaussianmarkerandisnormalizedtoMoMsbefore further processing.

Other risk factorsBesidestheGaussianmarkers,manymoreprenatalriskfactorshavebeenidentifiedandtheirlikelihoodratiosarepublished.Ifunequivocallyidentifiedornotidentified,theirlikelihoodratios(LR+) can be used to further refine the probability for the develop-ment of an aneuploid pregnancy. 21-25

Risk factor LR+

Pyelectasis 6.77

Shortfemur 7.94

Absent nasal bone 26

Echogenic cardiac foci 6

Echogenic bowel 21.17

Majordefect 32.96

Shorthumerus 22.76

SummaryDown Syndrome screening is another example of how the application of state of the art diagnostic tools and algorithms improves the reliability of screening methods. Starting with a detection rate of 30 % thirty years ago, first trimester screening today achieves 90 % and more at a false positive rate of 5 %. Today, not only the sensitivity is improved, but the results are also available significantly earlier. This allows for more time for confirmation and subsequent professional counseling.

0.1 0.5 1 15105432free �-hCG [MoM]

Unaffected Down Syndrome

DR

FPR

Alpha

hCG subunits

Beta

Fig. 9: The Gaussian distribution of free b-human chorionic gonadotropin

(free b-hCG) measurement in affected and unaffected pregnancies

a posteriori (probability) The refined probability of an event to happen after further knowledge about the nature of the event was collected.

a priori (probability) The estimated probability of an event to happen before further knowledge about the nature of the event has been collected.

AFP Alpha-1 fetoprotein.

Aganglionic Absence of normal enteric nerves.

Amniocentesis Puncture of the amniotic sac through abdominal wall and uterus for the collection of amniotic fluid.

Amniotic fluid The nourishing and protecting liquid contained by the amniotic sac of a pregnant woman. The amnioticsacgrowsandbeginstofill,mainlywithwater,aroundtwoweeksafterfertilization.Afterafurther10weekstheliquidcontainsproteins,carbohydrates,lipidsandphospholipids,ureaandelectrolytes,allofwhichaidinthegrowthofthefetus.Inthelatestagesofgestationmuchoftheamniotic fluid consists of fetal urine.

Amniotic sac A two-layered membrane that surrounds the embryo or fetus in the uterus. The amniotic sac is filled with fluid in which the embryo or fetus is suspended.

Aneuploidy Conditionwithanabnormalnumberofchromosomes.

Atlanto Concerningtheatlas.

Atlas Thefirstcervicalvertebra(C1).

Atresia Conditioninwhichabodyopeningorpassageinthebodyisabnormallyclosedorabsent.

Atrioventricular septal defect Aholeinthewall,thatseparatestherightfromtheleftsideoftheheart(AVSD).

Balanced (translocation) Translocation,thatdoesnotincreasetheoverallnumberofgenesinacell.

Brachymesophalangy Congenitalshorteningofthemiddleboneinafinger.

Bradycephaly Smallerthanusualheadcircumference.

Brushfield iris spots Pigmentationparticularityoftheiris,notinfluencingvision.

Chromosome OrganizedstructureofDNAandproteinsfoundincellscontaininggenes,regulatoryelementsandother nucleotide sequences.

Celiac disease Autoimmune disorder of the small intestine.

Cervical vertebrae The7vertebraethatarelocateddirectlyundertheskull(C1–C7).

Glossary

Chorionic layer The fetal part of the placenta.

Chorionic villus sampling Collectionoftissuefromthefetalpartoftheplacenta.Thematerialcontainsfetalcells,whicharesubjecttoe.g.chromosalanalysis.

Congenital Influenceonordamagetoadevelopingembryoorfetus(preterm).

Cordocentesis Collectionoffetalbloodfromtheumbilicalvein.Thesampleissubjecttosubsequentexamination,e.g.chromosomalanalysisofthefetalcellscontainedherein,orthedetectionofinfectiveagents.

Crown Rump Length (CRL) Thesizeofanembryofromthetopoftheheadtothebottom,usuallyobtainedsonographically.

Detection rate The relative number of true positive results obtained from a collective of positive samples (a.k.a. sensitivity).

Disjunction Duringtheprocessofcelldivisionthechromosomeshavetoduplicateatacertainstage.Theinitial product of this step is a pair of identical chromatids which are held together at a special positionwithintheDNAchain(centromer)yieldingthewellknownX-shape(metaphase).Subse-quently,thismetaphasicchromosomehastobecleavedinordertoliberatethechromatids,whichare then moved by means of the spindle apparatus to opposite poles of the dividing cell.

Down Syndrome The clinical picture that results from a trisomy of chromosome 21.

Duodenal Concerningtheduodenum,whichisthefirstpartofthesmallintestineinmosthighervertebrates.

Dysmorphism An anatomical malformation.

E3 Estriol.

Embryo Theearlieststageofdevelopmentofamulticellulardiploideukaryote.Inhumansthetimefromfertilization to about 8 weeks of gestation.

Epicanthal fold Askinfoldoftheuppereyelid,fromthenasalbonetotheinferiorsideoftheeyebrow,coveringthe inner corner of the eye.

Esophagus “Entranceforeating”.Themainpartoftheconnectionbetweenmouthandstomach.

Etiology The background or cause of a clinical condition.

Expression (genes) Normally,genesarenotactive.Onlyunderdistinctconditions–whenthecorrespondingproteinisneeded–ageneisactivated.ThisactivationprocessincludesaphysicalexpressionofthegenefromtheDNA-helix in order to make it accessible to the intra-cellular mechanisms of transcription and translation.

Facultative Notcompulsory,theoppositeofobligatory.

Fetus Gestationalstagefromabout9weeksofgestatationuntildelivery.

Gametogenesis Process of gamete (ovums and sperms) production.

Gene A piece of the genome that contains information e.g. for the production of a protein.

Genome The complete genetic material of an organism.

Gestational age (GA) Durationofapregnancysinceconception.

Gonosomal Referring to the gonosomes.

Gonosomes Inhumansthe23rdpairofchromosomes(genderchromosomes):XXorXY.

hCG Human chorionic gonadotropin.

Helix (ear) The prominent rim of the outer ear.

Heterodimer A chemical or biological entity consisting of two structurally different subunits.

Holo Greekprefixmeaningwhole,all,total.

Hypothyroidism Conditionwithalackofthyroidhormones.

Incidence Rateofoccurrenceofnewcasesperpopulationduringagiventimeperiod.Conveysinformationabout the risk of developing a certain clinical condition.

Klinodactyly Congenitalsidewaysdeviationofafinger.

Likelihood ratio Statisticalpropertydefininghowmuchmoreprobableatruepositiveresultiscomparedtoafalsepositive(sens/(1-spec)).

Macroglossy Pathologic enlargement of the tongue.

Megacolon Pathologic enlargement of the colon (last part of the digestive system).

Meiosis Celldivisionmechanismthatproduces4haploidcellsoutofoneparentcell,occurringforgametogen-esisinovariesandtesticles.Meiosiscomprisestwodisjunctionsteps(1st and 2ndmeioticdisjunction).

Mitosis The“normal”typeofcelldivision.Mitosisproducestwoidenticaleuploidcellsoutofoneparentcell.Mitosiscomprisesonedisjunctionstep.

Mosaicism The coexistence of two or more genetically distinct cell populations derived originally from a single zygote.Mosaicsmayariseatanystageofdevelopment,fromthetwo-cellstageonward,orinanytissue which actively proliferates thereafter. The phenomenon may be caused by somatic mutation orchromosomalnondisjunction.

Nuchal Referring to the back of the neck.

Nuchal translucency (NT) Thicknessoftheneckfoldinafetus,obtainedsonographically.NTisapowerfulpredictorofchromosomal aberrations.

Occipital bone Boneatthebottomoftheskullthatconnectswiththeatlas.

Omphalocele Atypeofabdominalwalldefectinwhichtheintestines,liver,andoccasionallyotherorgansremainoutside of the abdomen in a sac because of a defect in the development of the muscles of the abdominal wall.

Over-expression (genes) Too many copies of a gene are made available for transcription with the effect that the corre-spondingproteinisoverproducedwhichmaynotbeinlinewiththeorganismsneeds.Mayleadtopathological conditions.

Ovum The female gamete (egg).

Palmar Concerningtheinnersideofthehand.

Physiognomy Characteristicfacialexpression.

Polydactyly Abnormalnumber(>5)offingersortoes.

Preaxial polydactyly Polydactyly affecting the thumbs.

Sagital (plane) Animaginaryplanethattravelsverticallyfromthetoptothebottomofthebody,dividingitintoleftand right portions.

Somatic (cells) All cells that are not gametes.

Specificity The relative number of true negative results obtained from a collective of negative samples.

Stenosis Abnormal narrowing in a tubular structure.

Tetralogy of Fallot Congenitalheartdefect,themostcommoncauseforthebluebabysyndrome.

Transcervical Through the neck of the uterus.

Translocation The rearrangement of genetic material within the same chromosome or the transfer of a segment of one chromosome to another nonhomologous one.

Trisomy Aneuploidyinwhichtherearethreecopies,insteadofthenormaltwo,ofaparticularchromosome.

Trophoblasts The first cells that differentiate from the fertilized egg. They provide nutrients to the embryo and develop into a large part of the placenta.

Umbilical cord Connectionbetweenfetusorembryowiththeplacenta.

Unbalanced (translocation) Duplicationofasegmentofgeneticmaterialandadditionofthecopytoahomologousornonho-mologous chromosome.

Vertebrae The bones that the backbone is composed of.

Zygote Thecellformedbytheunionoftwogametes,especiallyafertilizedovumbeforecleavage.

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22SSDWLabSoftware.www.sbpsoftware.com

23Smith-Bindman,etal.(2001).SecondtrimesterultrasoundtodetectfetuseswithDownsyndrome.

JAMA 285(5):1044-55.

24Rotmensch,S.etal(1997).Prenatalsonographfindingsin187fetuseswithDownsyndrome.

Prenat Diagn 17(11):1001-9.

25Stewart,T.I.,Malone,F.D.(1999).Firsttrimesterscreeningforaneuploidy:nuchaltranslucency

sonography. Semin Perinatol 23(5):26-381.

26Haddow,J.E.etal.(1998).ScreeningofmaternalserumforfetalDown’ssyndromeinthefirst

trimester. NEJM 338(14): 955-61.

27Powell,K.J.,Grudzinskas,J.G.(1995).ScreeningforDownsyndromeinthefirsttrimester.Reprod

FertilDev7:1413-17.

28Nikolaides,K.H.(2004).The11th–13+6scan.Fetal Medicine Foundation, London.

www.fetalmedicine.com[Accessed23-July-2010].

29Spencer,K.etal.(1999).AscreeningprogramforTrisomy21at10–14weeksusingfetalnuchal

translucency,maternalserumfreebeta-humanchorionicgonadotropinandpregnancyassociated

plasma protein-A. Ultrasound Obstet Gynecol 13: 231-7.

30Wald,N.etal.(2003)FirstandsecondtrimesterantenatalscreeningforDown’sSyndrome:The

resultsoftheSerumUrine,andUltrasoundScreeningStudy(SURUSS). J Med Screen 10:56-104.

31Lin,T.M.,Halbert,S.P.,Kiefer,D.,Spellacy,W.N.,Gall,S.(1974).Characterizationoffourhuman

pregnancy-associated plasma proteins. Am J Obstet Gynecol 118(2): 223-36.

32Bischof,P.(1992).Pregnancy-AssociatedPlasmaProtein-A.Seminars in Reproductive Endocrinology

10(2):127-35.

33Rosen,S.W.(1986).Newplacentalproteins:Chemistry,physiologyandclinicaluse.Placenta 7: 575-

94.

34Overgaard,M.T.etal.(2003).ComplexofPregnancy-associatedPlasmaProtein-Aandtheproform

ofeosinophilmajorbasicprotein.J Biol Chem 278(4): 2106-117.

35Canick,J.A.etal(2006).Comparisonofserummarkersinfirst-trimesterDownSyndromescreen-

ing. Obstetrics & Gynecology 108(5):1192-9.

36Wald,N.J.,Rodeck,C.,Hackshaw,A.K.,Walters,J.,Chitty,L.,Mackinson,A.M.(2003).Firstandsecond

trimesterantenatalscreeningforDown’sSyndrome:theresultsoftheSerum,UrineandUltrasound

ScreeningStudy(SURUSS).Health Technol Assess 7(11).

37Malone,F.D.etal.(2005).First-trimesterorsecond-trimesterscreening,orboth,forDown’sSyn-

drome. NEJM 353(19): 2001-11.

38Nicolaides,K.H.etal.(2005).Multicenterstudyoffirst-trimesterscreeningfortrisomy21in75821

pregnancies: results and estimation of the potential impact of individual risk-orientated two-stage

first-trimester screening. Ultrasound Obstet Gynecol 25: 221-6.

39Spencer,K.etal.(2002).Temporalchangesinmaternalserumbiochemicalmarkersoftrisomy21

across the first and second trimester of pregnancy. Ann Clin Biochem 39: 567-76.

40Cuckle,H.S.,Wald,N.J.,Thompson,S.G.(1987).Estimatingawoman’sriskofhavingapregnancy

associatedwithDown’ssyndromeusingherageandserumalpha-fetoproteinlevel.Brit J Obstet

Gynecol 94: 387-402.

41Palomaki,G.E.,Haddow,J.E.(1987).Maternalserumalpha-fetoprotein,age,andDownSyndrome

risk. Am J Obstet Gynecol 156: 460-3.

42Reynolds,T.M.,Penney,M.D.(1989).Themathematicalbasisofmultivariateriskscreening:with

specialreferencetoscreeningforDown’ssyndromeassociatedpregnancy.AnnClinBiochem

27:452-8.

43Bray,I.,Wright,D.E.,Davies,C.,Hook,E.B.(1998).JointestimationofDownsyndromeriskand

ascertainmentrates:Ameta-analysisofninepublisheddatasets.PrenatDiagn18:9-20.

44Benn,P.A.(2002).AdvancesinprenatalscreeningforDownsyndrome:I.Generalprinciplesand

second trimester testing. Clin Chim Acta 323:1-6.

45Berger,P.etal.(2002).TheISOBMTD-7WorkshoponhCGandRelatedMolecules.Tumor Biol 23:

1-38.

46Cole,L.A.(1997).Immunoassayofhumanchorionicgonadotropin,itsfreesubunits,andmetabo-

lites. Clin Chem 43(12):2233-43.

47 Alfthan,H.,Stenman,U.H.(1996).Pathophysiologicalimportanceofvariousmolecularformsof

human choriogonadotropin. Mol Cell Endocrinol 125: 107-20.

COBAS,COBASC,COBASEandLIFENEEDSANSWERS are trademarks of Roche.

©2010 Roche

RocheDiagnosticsLtd.CH-6343RotkreuzSwitzerlandwww.roche.com

06

425

9170

01 a

091

0-

First trimester screeningFor better maternal and fetal care

The aim of pregnancy examinations is the early detection of specific risks. “Risk screening” using multiple markers has achieved wide acceptance in routine antenatal care. Non-invasive screening methods in the first and second trimester for detection of fetal chromosome abnormalities have been developed over the last 15 years. Due to benefits of the earlier decision process there is a clear trend towards first trimester screening. Additionally, the highest detection rates for trisomy 21 can be achieved in the first trimester with maternal serum markers PAPP-A and free b-hCG in combination with nuchal translucency (NT) results.1,2,3

Proven Elecsys performance with highly accurate and reliable assay results4,5 •Highassayqualityandexcellentprecisionduetostate-of-the-

art electro-chemiluminescence (ECL) technology •Assayexcellencewithover20yearsofdevelopmentexpertise

leads to overachievement of FMF accreditation targets•Premiumqualityofcobasanalyticalplatforms:highsafetyand

reliability standards …for improved service to laboratory customers

Flexible solutions for consolidated testing•Integrationintoroutinetestingwithefficiency,costandwork-

flow improvements without compromising performance•Flexibleriskcalculationsoftwareoffering•Broadsystemplatformportfolioforeverylabsizewithstandard-

izedreagentsacrosstheplatforms

•Comprehensiveassaymenuincludinginnovativemarkersformaternal,fetalandneonatalhealthmonitoring:anemia,diabetes, endocrinology, fertility, infectious diseases, inflam-mation,preeclampsia,thyroidfunction,TORCHandothers

…for streamlined lab organization with efficiency and cost gains

FMF accreditation for certified quality supporting early decision process•Highqualityresultsleadtoeffectiveinformationsupporting

early decision making •Excellentdetectionratesfortrisomy21•Minimizeinvasiveinvestigationswithhigherriskformother

and fetus …for better maternal and fetal care

COBASandLIFENEEDSANSWERSaretrademarksofRoche.All other trademarks are property of their respective owners.

©2010Roche

Roche Diagnostics Ltd.CH-6343RotkreuzSwitzerlandwww.roche.com

References

1 Nikolaides,K.H.(2004).The11th-13+6Scan,FetalMedicineFoundation,London.

2 Spencer,K.etal(1999).AscreeningprogramforTrisomy21at10-14weeksusingfetalnuchal

translucency, maternal serum free-beta-human chorionic gonadotropin and pregnant-associated

plasma protein-A. Ultrasound Obstet. Gynecol. 13:231-7.

3 Wald,N.etal(2003).FirstandsecondtrimesterantenatalscreeningforDown’sSyndrome:The

resultsoftheSerumUrine,andUltrasoundScreeningStudy(SURUSS).J. Med. Screen. 10:56-104.

4 PackageinsertElecsysPAPP-A.RocheDiagnostics.

5 Package insert Elecsys free ß-hCG. Roche Diagnostics.

6 RochemulticenterEvaluationStudy.Dataonfile,2009.

First trimester screening with Elecsys PAPP-A and free b-hCG For better maternal and fetal care

Max. total CV in % [medium conc.]

0 1 432 5 6

FMF requirement

7 98 10

DELFIA® Xpress /AutoDELFIA®

IMMULITE®

KRYPTOR /K. compact

cobas eanalyzers

free �-hCG PAPP-A

Max. total CV in % [medium conc.]

0 1 432 5 6

FMF requirement

7 98 10

DELFIA® Xpress /AutoDELFIA®

IMMULITE®

KRYPTOR /K. compact

cobas eanalyzers

free �-hCG PAPP-A IncludingnuchaltranslucencydatafromFMFcertifiedcentresthedetectionratecanbeincreasedupto90%.1,2,3

Detection rate of trisomy 21 (without NT)

0 10 403020 50 60 70 80 90 100

cobas e analyzers

KRYPTOR 65.6 %

75.0 %

False positive rate of trisomy 21 (without NT)

0 1 432 5 6 7 8 9 10

cobas e analyzers

KRYPTOR 7.7 %

5.8 %

Figure 2: Detection rates (%) generated with PAPP-A and free b-hCG, n=1047

Figure 3: False positive rates (%) obtained with PAPP-A and free b-hCG; n=326Figure 1: Comparison of precision (CV= coefficient of variation). Ref.: Package inserts.

Elecsys® free b-hCGElectro-chemiluminescence immunoassay (ECLIA) for the in-vitro determination of human free b-hCG in serum

IndicationHuman chorionic gonadotropin (hCG) is a glycoprotein hormone (~37 kDa) composed of two non-covalently linked subunits – the a- and b-chain. During the early weeks of pregnancy, the protein is produced by trophoblast tissue and serves to maintain the corpus luteum. In addition, it also influences steroid production. Physiologically, hCG appears only in blood and urine of pregnant women. The serum of pregnant women mainly contains intact hCG. However, a minor fraction of the subunit circulates in an unbound form. The pro-portion of free b-hCG averages ~1% compared to intact hCG. It is now well established that the free b-hCG concentration in serum is a reliable marker for fetal aneuploidy. Free b-hCG, in combination with serum pregnancy-associated plasma protein A (PAPP-A) and the sonographic determination of nuchal translucency (NT), are markers of choice to identify women at an increased risk of carrying a fetus affected with Down syndrome during the first trimester (week 11-14) of pregnancy. Using this marker combination, detection rates of up to 70% (serum markers only) and 90% (combined with NT) have been described at a false positive rate of 5%. Median maternal serum free b-hCG levels in affected pregnancies are higher compared to the median of non affected pregnancies. Based on the maternal age, the risk for having a Down syndrome pregnancy can be calculated using an algorithm based on likelihood ratios.

Elecsys technology ECL (ElectroChemiLuminescence) is Roche’s technology for immunoassay detection. Based on this technology and combined with well-designed, specific and sensitive immunoassays, Elecsys delivers reliable results. The development of ECL immunoassays is based on the use of a ruthenium-complex and tripropylamine (TPA). The chemiluminescence reaction for the detection of the reaction complex is initiated by applying a voltage to the sample solution resulting in a precisely controlled reaction. ECL technology can accommodate many immunoassay principles while providing superior performance.

Test principle: one-step sandwich assay

Ru Ru Ru

Biotinylated monoclonalantibody against human �-hCG (epitope hidden in holo-hCG)

free �-hCG in the sample

holo-hCG in the sample

9 min 9 min

Streptavidin microparticle

Ruthenylated monoclonal antibody against human �-hCG

Ru

RuMeasurement

COBAS, COBAS E, LIFE NEEDS ANSWERS and ELECSYS are trademarks of Roche.

©2010 Roche

Roche Diagnostics Ltd.CH-6343 RotkreuzSwitzerlandwww.roche.com

* Each laboratory should investigate the transferability of the expected values to its own patient population and if necessary determine its own reference ranges. For prenatal testing it is recommended that the median values should be re-evaluated periodically.

** Multicenter evaluation data

Elecsys® free b-hCG test characteristics

Assay time 18 min

Test principle One-step sandwich assay

Calibration 2 point

Sample material Serum

Sample volume 10 μL

Detection limit 0.1 IU/L

Functional sensitivity < 0.5 IU/L

Measuring range 0.1 – 190 IU/L

Traceability NIBSC 75/551

Total imprecision (NCCLS) cobas e 411, E2010: 1.2 – 2.9 % (7.56 - 101 IU/L)cobas e 601/ e 602, E170: 1.6 – 2.7 % (7.1 – 98.2 IU/L)

Expected values*

Week 11: median 49.9 IU/L (n = 206)Week 12: median 40.6 IU/L (n = 623)Week 13: median 33.6 IU/L (n = 1384)Week 14: median 28.8 IU/L (n = 1057)

Certificates Fetal Medicine Foundation (FMF) accreditation

Free b-hCG levels in affected pregnancies:Median maternal serum free b-hCG levels in affected pregnancy are higher, compared to the median of non-affected pregnancies.

Clinical performance data of free b-hCG and PAPP-A assays

B. Detection rate in confirmed trisomy 21 pregnancies, without NT (n = 32)

In 32 affected samples the Roche methods showed a detec-tion rate of 75% (24/32) in comparison to 65.6% (21/32) obtained with the competitor methods. ** Including NT data, the detection rate can be increased up to 90 %.

Detection rate0 10 403020 50 60 70 80 90 100

cobas e analyzersCompetitvesystem 65.6 %

75.0 %

Order information

Elecsys® free b-hCG 100 tests 04854071 200

free b-hCG CalSet 4 x 1 mL 04854101

PC Maternal Care 1,2&3 2 x 2 mL each 04899881

A. Concordance analysis in unaffected pregnancies (n = 1047)

In 1047 unaffected samples the Roche methods correctly classified 986 samples (specificity: 94.2%) in comparison to 966 (specificity: 92.3%) correctly classified by the competitor methods. Roche methods showed a false positive rate of 5.8% in comparison to 7.7% obtained with the competitor methods. **

Specifity0 10 403020 50 60 70 80 90 100

cobas e analyzersCompetitvesystem

94.2 %

92.3 %

False positive rate0 1 432 5 6 7 8 9 10

cobas e analyzersCompetitvesystem 7.7 %

5.8 %

Elecsys® PAPP-AElectro-chemiluminescence immunoassay (ECLIA) for the in-vitro determination of human pregnancy associated plasma protein A (PAPP-A) in serum

IndicationHuman pregnancy-associated plasma protein A (PAPP-A) is a large glycoprotein with a molecular weight of 200 kDa. PAPP-A was first isolated from the serum of pregnant women, where its concentration increases steadily until term. It is now well established that the PAPP-A concentration in serum is a reliable marker for fetal aneuploidy. PAPP-A, in combination with free b-hCG and the sonographic determination of nuchal translucency (NT), are the markers of choice to identify women at increased risk of carrying a fetus affected with Down syndrome during the first trimester (week 11-14) of pregnancy. Using this marker combination, detection rates of up to 70% (serum markers only) and 90% (combined with NT) have been described at a false positive rate of 5%. Median maternal serum PAPP-A levels in affected pregnancy are lower, compared to the median of non-affected pregnancies. Based on the maternal age, the risk for having a Down syndrome pregnancy can be calculated using an algorithm based on likelihood ratios.

Elecsys technology ECL (ElectroChemiLuminescence) is Roche’s technology for immunoassay detection. Based on this technology and combined with well-designed, specific and sensitive immunoassays, Elecsys delivers reliable results. The development of ECL immunoassays is based on the use of a ruthenium-complex and tripropylamine (TPA). The chemiluminescence reaction for the detection of the reaction complex is initiated by applying a voltage to the sample solution resulting in a precisely controlled reaction. ECL technology can accommodate many immunoassay principles while providing superior performance.

Test principle: one-step sandwich assay

Biotinylated monoclonalantibody against human PAPP-A

Ruthenylated monoclonal antibody against human PAPP-A

PAPP-A inthe sample

9 min

Streptavidin microparticle

9 minRu

Ru

Ru Measurement

COBAS, COBAS E, LIFE NEEDS ANSWERS and ELECSYS are trademarks of Roche.

©2010 Roche

Roche Diagnostics Ltd.CH-6343 RotkreuzSwitzerlandwww.roche.com

** Multicenter evaluation data

Elecsys® PAPP-A test characteristics

Assay time 18 min

Test principle One-step sandwich assay

Calibration 2 point

Sample material Serum

Sample volume 15 μL

Detection limit 4 mIU/L

Functional sensitivity < 20 mIU/L

Measuring range 4 – 10000 mIU/L

Traceability Commercially available assay – IRP 78/610 (WHO)

Total imprecision (NCCLS) cobas e 411, E2010: 1.1 – 2.3 % (144 - 6630 mIU/L)cobas e 601/ e 602, E170: 1.4 – 2.8 % (141 – 6335 mIU/L)

Expected values*

Week 11: median 1337 mIU/L (n = 206)Week 12: median 1919 mIU/L (n = 623)Week 13: median 2926 mIU/L (n = 1384)Week 14: median 4358 mIU/L (n = 1057)

Certificates Fetal Medicine Foundation (FMF) accreditation

PAPP-A levels in affected pregnanciesMedian maternal serum PAPP-A levels in affected pregnancy are lower, compared to the median of non-affected pregnancies.

Order information

Elecsys® PAPP-A 100 tests 04854098 200

PAPP-A CalSet 4 x 1 mL 04854101

PC Maternal Care 1,2&3 2 x 2 mL each 04899881

B. Detection rate in confirmed trisomy 21 pregnancies, without NT (n = 32)

In 32 affected samples the Roche methods showed a detec-tion rate of 75% (24/32) in comparison to 65.6% (21/32) obtained with the competitor methods.** Including NT data, the detection rate can be increased up to 90 %.

Detection rate0 10 403020 50 60 70 80 90 100

cobas e analyzersCompetitvesystem 65.6 %

75.0 %

A. Concordance analysis in unaffected pregnancies (n = 1047)

In 1047 unaffected samples the Roche methods correctly classified 986 samples (specificity: 94.2%) in comparison to 966 (specificity: 92.3%) correctly classified by the competitor methods. Roche methods showed a false positive rate of 5.8% in comparison to 7.7% obtained with the competitor methods.**

Specifity0 10 403020 50 60 70 80 90 100

cobas e analyzersCompetitvesystem

94.2 %

92.3 %

False positive rate0 1 432 5 6 7 8 9 10

cobas e analyzersCompetitvesystem 7.7 %

5.8 %

* Each laboratory should investigate the transferability of the expected values to its own patient population and if necessary determine its own reference ranges. For prenatal testing it is recommended that the median values should be re-evaluated periodically.

Clinical performance data of PAPP-A and free b-hCG assays