Mass spectrometry in clinical chemistry: the case of newborn screening

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ContentslistsavailableatScienceDirect

Journal

of

Pharmaceutical

and

Biomedical

Analysis

jou rn al h om e p a g e :w w w . e l s e v i e r . c o m / l o c a t e / j p b a

Review

Mass

spectrometry

in

clinical

chemistry:

the

case

of

newborn

screening

Giancarlo

la

Marca

∗

DepartmentofNeurosciences,Psychology,PharmacologyandChildHealth,UniversityofFlorence,NewbornScreening,BiochemistryandPharmacology laboratory,MeyerChildren’sUniversityHospital,Florence,Italy

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received27March2014 Accepted28March2014 Availableonlinexxx Keywords: Newbornscreening LC–MS/MS

Tandemmassspectrometry Inbornerrorsofmetabolism Clinicalchemistry

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Newbornscreening(NBS)programisacomplexandorganizedsystemconsistingoffamilyandpersonnel education,biochemicaltests,confirmatorybiochemicalandgenetictests,diagnosis,therapy,andpatient followup.Theprogramidentifiestreatablemetabolicdisorderspossiblywhenasymptomaticbyusing driedbloodspot(DBS).Duringthelast20yearstandemmassspectrometry(TMS)hasbecometheleading technologyinNBSprogramsdemonstratingtobeversatile,sensitiveandspecific.Thereisconsistent evi-denceofbenefitsfromNBSformanydisordersdetectedbyTMSaswellasforcongenitalhypothyroidism, cysticfibrosis,congenitaladrenalhyperplasiabyimmune-enzymaticmethods.

RealtimePCRtestshavemorerecentlybeenproposedforthedetectionofsomeseverecombined immunodeficiences(SCID)alongwiththeuseofTMSforADAandPNPSCID;afirstevaluationoftheir cost-benefitratioisstillongoing.Avoidingfalsenegativeresultsbyusingspecificbiomarkersandreducing thefalsepositiveratebyusingsecondtiertests,isfundamentalforasuccessfulNBSprogram.Thefully integrationofNBSanddiagnosticlaboratorieswithclinicalserviceiscrucialtohavethebesteffectiveness inacomprehensiveNBSsystem.

Contents

1. Introduction... 00

2. Materialsandmethods... 00

2.1. Samplecollection... 00

2.2. Bloodcollectioninprematureinfantsandinnewbornsonparenteralnutritionortransfused... 00

2.3. Transportofsamplesandworkﬂowonlab... 00

3. NBSbyMS/MS... 00

3.1. Standardsandkits... 00

3.2. AnalysisbyMS... 00

4. ExpandingtheNBSpanelbyMS... 00

5. Diseasesincludedintheneonatalscreeningprogram... 00

5.1. Secondtiertests ... 00

5.2. Maternaldiseases... 00

5.3. Outcomemanagementprocedures... 00

6. Conclusions... 00

Acknowledgments ... 00

References... 00

∗ Correspondenceto:DepartmentofNeurosciences,Psychology,PharmacologyandChildHealth,UniversityofFlorence,VialePieraccini6,50139Florence,Italy. Tel.:+390555662988;fax:+390555662489.

E-mailaddresses:g.lamarca@meyer.it,giancarlo.lamarca@uniﬁ.it http://dx.doi.org/10.1016/j.jpba.2014.03.047

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1. Introduction

Newbornscreening(NBS) is knowntobea biochemical test enablingtheidentificationofmanyinbornerrorsofmetabolism (IEM)few daysafter birth. Iftheyare notdiagnosed and early treated,mostofthemcancausementaland/orgrowthretardation, severepermanentsequelaeandinsomecasedeath.Considering thatNBSrequiresexpertlabtechnicians,chemists,biologists, nutri-tionists,medicalspecialistsformetabolicdisorders,itcannotbe consideredonlyanusefulbiochemicaltest,butitshouldbe con-sideredasacomplexandintegratedprogram.Theobjectiveofa newbornscreeningprogramistodetectsomeIEMbeforethe clin-icalmanifestationoftheassociatedsymptoms.Asaconsequence, medicaldoctorscanstartthebestavailabletreatmentandhave thebest prognosisby modifyingthe natural courseof the dis-ease.ThehistoryofNBSaspopulation-basedtestdatesbackfrom thebeginning of1960swhenthemicrobiologistRobertGuthrie developedasimpleandinexpensivebacterialinhibitionassay(DBS based)abletoidentifythemostfrequentaminoacidopathia:the phenylketonuria[1].Inthefollowingdecade,someotherclinical labsbothintheUnitedStatesandinEurope,addedthe congen-italhypothyroidism(CH)totheirpanel,againbyusing asingle dropofwholebloodonpaper.Thefollowingdevelopmentof elec-trospraytandemmassspectrometryinmorerecentyears(1990s) haspermittedtheintroductionofthisnewtechnologyinclinical chemistrylaboratories,inparticularfornewbornscreening pur-poses[2].MS/MSisaversatile,specificandsensitivetechnology givingtechniciansthepossibilitytomeasuremanybiomarkersina singleandfastanalyticalrun.Peopleworkinginnewbornscreening fieldunderstoodthepossibilitytopassfromoneDBSforonetest andonedisordertooneDBSforonemultiplextestformany dis-orders.In fact,today MS/MScaneasilyidentifyand quantifyin a 2or less minutes’run – severalmetabolitessuchas acylcar-nitines,aminoacids,succinylacetone[3,4]andmorerecentlysome purines[5–8].Nowadays,includingpilotprojectsregionallyruled andstructurednationalNBSprograms,manylabsallaroundthe worldscreenfor morethan 30or moreIEM witha singletest. Thenumberofpotentialidentifiabledisordersisnota technolog-icalhamperbutitdependsonregionalornationalpublichealth strategies.

Some expanded newborn screening programs are now screening notonly for PKU, CH,more recentlyfor cystic ﬁbro-sis(CF),congenitaladrenalhyperplasia(CAH)andgalactosemia, butalsofor othersaminoacidopathias,betaoxidation fattyacid defects,organicacidurias,ureacycledefectsand since2011for someseverecombinedimmunodeﬁciences (SCID).Onethe fun-damentalworldwide-approvedcriterion isthat oneIEM canbe screenedonlyifarelatedtreatmentisavailable.Foralldisorders includedinthenewbornscreeningprogramsatherapyshouldbe possibleevenif,insomecases,notcompletelycurative.Theterm “notcompletely”isonerelevantreasonwhynewbornscreening panelsarenotallthesameworldwide.

Theopinionofthiswriteristhatevenifatherapyisnot com-pletelycurative,theearlydetectionofthedisorderandafollowing immediatecorrecttreatmentshouldgivebabiesthepossibilityto havebetter qualityof life, toextendlifeexpectancy and when required, to allow a suitable geneticcounseling (especially for futureprenataldiagnoses).Moreover,anearlydiagnosisrelieves familieswithasevereillchildfromdifﬁcultdiagnosticiter.These simplecriteriarepresentthebasalevaluationreportedfromWilson andJungnerin1963toWorldHealthOrganization[9].

Therefore,theNBScannotbeconsideredonlyanefﬁcientand isolatedlabtestofpreventivemedicinebutitisamoreintegrated publichealthsysteminvolvingmanydifferentcategoriessuchas labtechnicians,chemists,biologists,biochemicalgeneticists(for bothprimaryand conﬁrmatorytests),administrativepersonnel,

nurses,dieticiansand/orpharmacologists,medicaldoctorsexpert inmetabolicdisordersandpediatricians.Asconsequence,onlyif laboratoryNBSprocedures,laboratoryconﬁrmatoryprocedures, clinicalserviceandcontinueeducationarefullyintegrated,areal effectivenessofaNBSprogramwillbereached.

2. Materialsandmethods

2.1. Samplecollection

Bloodsampleswerenormallyobtainedbyheelstickor hand-prick,spottedonﬁlterpaper(903,Whatmann,Milan,Italyor226 gradepaper,Ahlstrom,Helsinki,Finland)oftenreferredas“Guthrie cards”,driedatroomtemperatureandsentdailybycourierormail tothenewbornscreeninglaboratory. In mostpartof European countriesaswellasintheUSAandAustraliaaformalguideline forthesamplingprocedureisavailable.Inabouthalfthecountries theseguidelineshavebeendevelopedbyhealth authoritiesand intheotherhalfbyprofessionalgroups,eitherlocallyor nation-ally.

Thenumberofdropsisvariable(minimum3–4,maximum12) anditisgenerallydependingonthenumberoftests(bothﬁrstand secondtiertests).

Theexpansionofbloodonthepaperdependsonhaematocrit levelanddryingconditions.

Blood collectionfor newbornscreening in Italy (as in most partofcountries)isrecommendedbetween48and72hoflife.In Europe,thecollectionrangesfromthe36hinAustriaandCroatia to168hofGreece,NetherlandsandLuxembourg[10].Timingof thecollectionisacriticalissuebecausemarkerlevelscouldvary physiologicallyintheneonatalperiod[11].

Insomecountries,suchasFinlandandMalta,cordblood samp-ling is used for newborn screening testing according to early maternalandnewborndischarge[12,13],butithasbeen evalu-atedthat betterresultsare obtainedfromsamplescollectedby heel/handprickatalaterstage[14,15].

Useofurine collectedontoanabsorbentpaperplacedinthe baby’snappycouldbeaveryusefultoolasadditionalbutnot alter-nativeNBStest.Evenifnotcommon,someNBSprogramscontinued thepracticeofurinescreening[16]andinsomecasestheurineDBS hassigniﬁcantlycontributedtotheIEMdetectionupto44%oftotal cases[17].

Animportantparameterforthequalityoftheprogramisthe completenessofsampling,preferably100%butreachingthisvalue ispracticallyimpossiblebecauseinmostcountriesitshouldbe pos-sibleforparentstorefuse participationof theirchildrentoNBS programs.Ifinformedconsentistakenseriously,theoptiontostay outwillsometimestakeplace.

2.2. Bloodcollectioninprematureinfantsandinnewbornson parenteralnutritionortransfused

Prematurity,birthweight,parenteralnutrition,transfusionsand typeoffeedscanallpotentiallyinﬂuenceNBSresults.

In orderto decreaseparental stressrelated toretesting and toensurethatimportantinformation for interpretingscreening resultsisgiven,establishedprotocolsareuseful.Theseprotocols shouldproviderepeatedtestsduringtheearlypostnatalperiodfor infantrequiringblood/plasmatransfusion,for prematurebabies or term/preterm babies onparenteral nutrition at the time of screeningsamplecollection.

Insomecountries[18,19]forprematureinfants(birthweight <1800g),theﬁrstDBSsampleiscollectedonthe3rd–5thdayof life,thentwoadditionalsamplesat15and30days.Forbabieson parenteralnutrition,includingprematurebabies,asecondsample at48haftertheendingofparenteralnutritioniscollected.Inall

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transfusednewborns,anewsampleistakenbetween7and15days aftertheendoftransfusion.

2.3. Transportofsamplesandworkﬂowonlab

Somemarkersarerelativelyunstableandheat,humidityand delaysintransportcancausedegradationandpotentialfalse neg-ativesresults. Therefore, whenmail service is notefﬁcient,the samplesshouldbesenttothelabbycourier.

For the same reason the best practical workﬂow is seven days/weekand24h/daybutitisquiterare.Atypicallaboratory activityis5/6daysperweekand12hbasedshiftworks.

Test turnaround time within the laboratory should be kept short;amaximumof48hisrecommended.

3. NBSbyMS/MS

3.1. Standardsandkits

Labeledinternalstandards ofacylcarnitinesand aminoacids areavailablefromdifferentmanufactureslikeCambridgeIsotope Laboratories,Andover,MA,USA;a typicalstocksolutionis pre-pared in methanol. The standard concentrations may vary but normallyitisintherange500–2500␮mol/Lforaminoacids,and 7.6–152␮mol/Lforacylcarnitines.Allchemicalsandsolventsmust beofthehighestpurityavailablefromcommercialsourcesandthey arenormallyusedwithoutanyfurtherpurification.Some laborato-riesusecommerciallyIVDkitssuchasNeoGram®_/NeoBase®_from PerkinElmer(Waltham,MA,USA)andMassChrom®_from Chrom-systems(Munich,Germany).Itisimportanttohighlightthat“useof certifiedkitmaterial”doesnotmeanthatamassspectrometercan beusedwithoutanyknowledgeorspecificeducation,especiallyfor newbornscreeningordiagnostictests.Amassspectrometerisnota commonbenchtopmachinenormallyusedinagenerallaboratory ora“blackbox”.Trainingandeducationinmassspectrometryfor newbornscreeninglabpeopleisafundamentalissue,itcannotbe delayedanditcannotbeoverruledbyusingcertifiedkitmaterial. 3.2. AnalysisbyMS

Screeninganalysesisworldwide performedaccordingtothe conventionalmethodaspreviouslyreported[2,20]withsome mod-iﬁcations.

Originally,itwasfoundthatcarnitineandacylcarnitineswere easilymeasuredfromplasmasamplesbyfastatombombardment orthermospray[21,22]forthedetectionoffattyaciddisorders.

Lateron,advancementsinMS/MSandresearchdemonstrated thattheanalysisofacylcarnitinescouldbeusedtoidentifyfatty acidoxidationdefectsinaneonatalpopulation.Carnitineandits esterswereperfectcandidatesforaMS/MSscreeningtechnique sincetheyallpresentedacommonproduction.Regardlessofthe attachedfattyacidester,acommonfragment(85Th)that con-tainsthequaternaryammoniumfunctioncouldbedetectedwith highsensitivityandspeciﬁcity(Fig.1).Thesestructural character-isticsenabledthedetectionofapanelofsaturated,unsaturatedand hydroxylateacylcarnitinesinaneasyandcheapway.

Theadditionofaminoacidstonewbornscreeningpaneldates backtothebeginningofnineties’withthefirstendeavorto mea-surephenylalanineusingfastatombombardment(FAB)[20].The procedurerequiredesterification(asbutylesters)toenhance sur-faceionizationinaglycerolmatrix.Thebutylation“quenched”the negativechargeontheacidmoietyof phenylalanineincreasing ionizationefficiencyandsensitivity.

Inaﬁrststage,methylesteriﬁcation wasusedpreviouslyfor analysisofacylcarnitinesinplasma[23]butlateronthemostpart oflaboratoriesswitchedofftobutylation.

Duringtheﬁrsttestsbyusingphenylalaninebutylesterforthe detectionofPKUotheraminoacidswereidentiﬁed,deducingthat acommonneutralproduct,butylformatepresentingwithamass of102Da.Sincethen,thecommonneutral lossof102Dais the basisfortheMS/MSassayofseveralkeyaminoacidsinnewborn screening(Fig.1)[24].

Whilebutylesteriﬁcationassaysbecomeroutine,inthelastten yearsnewmethodsweredevelopedabletodetectAAsandACsas theirnativefreeacids(underivatized)tosimplifyanalytical opera-tionsandtominimizetheuseofcorrosivecompounds.

Thecollisioninduceddissociationofaminoacidsand acylcar-nitinesasbutylestersandfreeacidsisreportedinFig.1.

Nowadaysclassical routine MS/MSanalysisis performed on one/two(dependingonthenumberofthesamples)instruments normallytriplequadrupolesandoneadditionalforback-up. Quan-titationisperformedbyusingthe“isotopicdilution”technique.The analyte concentrationsarecalculated automaticallyusing prop-ertysoftware.TuscanyregionsasanexampleuseABISciex(API 4000,3200(ABSciex,FosterCity,CA,USA))orShimadzu(8040, Kyoto,Japan)LC-MS/MSsystemsandChemoview(ABSciex, Fos-terCity,CA,USA)orNeonatalSolution(Shimadzu,Kyoto,Japan). Other similar and equally efﬁcient systems are commercially available.

4. ExpandingtheNBSpanelbyMS

Newmethodsarenowavailableandtheysupplementthe cur-rentscreeningpanel.Theclassicalunderivatizedandderivatized samplepreparationprocedureswerefurthermodiﬁedtoquantify succinylacetone(speciﬁcmarkerfortyrosinaemiatypeI)byadding anewcomponent:hydrazineoritsderivatives.Thesample prepa-rationcanbeperformedincludingtheextractionofsuccinylacetone ashydrazonederivative,alongwithothermetabolitesinasingle stepsince2008[3].Lateronalternativemethodsrequiringtwo dif-ferentextractionprocedures[4]andcleaningsteps[25]havebeen developed.

In 2011and 2014two newcheapmethods abletoquantify somepurinesalongwithothermetaboliteshavebeendeveloped [5,26].Themeasurementsofthesenewmetabolitesvia underiva-tizedmethod,allowthedetectionofadenosinedeaminase(ADA) andpurinenucleosidephosphorylase(PNP)deﬁcienciesatbirth. Somewidelyusedsamplepreparationproceduresaredescribedin Fig.2.

ADAandPNParefatalautosomalrecessiveformsofSCIDcaused by an inherited disorder of purine metabolism. Both ADA and PNP-SCIDcomplywithallthecriteriaforinclusioninanewborn screeningprogram.TheSecretary’sAdvisoryCommitteeon Heri-tableDisordersinNewbornsandChildrenin2010andtheEuropean EUtenderin2011recommendtheinclusion ofallSCIDsin the newbornscreeningpanels.Since2008apolymerasechain reac-tion(PCR)basedtestisavailableforthedetectionofallT-SCID. Thismethodpresentstheindubitableadvantagetoscreenforall theT-SCIDpanelbutitisquiteexpensive(RT-PCRmachines,kits andmolecularbiologistsareneeded)withrespecttoMS; more-overithasbeenrecentlydemonstratedthatitisnotsensitivefor late/delayedADA-SCIDforms andthatitcandetectsome disor-dersnotrespondingtothefuturestobeincludedinaNSpanel [6,27,28].

Some methods were developed for thedetection of lysoso-mal storage disorders (LSDs) and even if for some of them a reliableandrelativelysimpletestisavailable,areasonable asso-ciatedtherapy couldbestillnot available[29–38].It isnot the taskofthiswritertoproducea statementthattheLSDsshould be included in newborn screening panels, but some of these disordersseemtomeetthecriteriaforinclusion,includingbasal availabilityofasimpletest,acombinationtherapythatmodiﬁes

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Fig.1. SchematicrepresentationoffragmentationinMSMSofbutylatedandunbutylatedacylcarnitine(panelA)andaminoacidresidues(panelB).

thenaturalcourseofthedisease,earlydiagnosisthatallowsgenetic counselingandprenataldiagnosisinfamilieswithanaffectedbaby.

5. Diseasesincludedintheneonatalscreeningprogram

Thepanelusedforneonatalscreeningisdifferentfromregion toregionandfromcountrytocountry.

TheAmericanCollegeforMedicalGeneticsinthe2012decided foranationalandcommonNBSpanelfortheUSAnewborns.It con-tains55disorders(includingtestsbyimmune-enzymaticsystems): 30IEMforthenamedcorepaneland25assecondarytarget.

AnationalscreeningprogramdoesnotexistinCanada.Health careandscreeningprogramsfallunderprovincialandterritorial

jurisdiction.Thereisawidespectruminthenumberofdisorders for which screening is offered,rangingfrom 5 disorders to38, dependingonthejurisdiction.TheAustralianNewbornScreening PolicyandGuidelinesDocumentin2011reportedon25disorders detectedbymeansofNS(evenif“otherdisorderscouldbedetected duringthetest,includingmaternaldisorders”).InJapan,wherea nationalpilotprojectisﬁnishedin2012,thebeginningofanational NSprogrambyMSwillstartthenextApril2014.TheEuropean situationisthemorecomplicatedandevenifcommon recommen-dationsareavailable,manyrelevantdifferencesarestillpresent. Themostrelevantdifferencesarethetimeofthedrawingofthe bloodperiod(between36 and168hafterbirth),thenumberof newbornscreeningcenterspercountryrangingfrom1tomorethan

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Fig.2.Schematicrepresentationsofsamplepreparationprocedures.PanelAshowsderivatizedprocedurefortheassayofacylcarnitines,aminoacidsandsuccinylacetone andunderivatizedprocedurefortheassayofacylcarnitines,aminoacids,succinylacetoneandpurines.PanelBshowsthe2stepproceduresfortheassayofacylcarnitines, aminoacidsasbutylestersandsuccinylacetoneandpurinesinnaïveforms

20.MoreoverandprobablymostlyimportantacommonNSpanel doesnotexistduetopublichealthsystemsfallingunderregional jurisdictions.

InItalytheexpandedNBSbyMSisregionallyruledevenifa nationallawhasbeenpromulgatedattheendofDecember2013. Anationalexpertcommissionisnowevaluatingthediseasepanel andtheminimalnumberofnewbornscreeningcenters.

Table1liststhesuspecteddiseaseincludedinTuscanyregion NBS panel in the presence of one or more changes, the cut-offvaluesandtheratiosforeachanalyte.Twenty-fourdisorders consistofprimarypanel,22secondarypanel.Somediseases iden-tiﬁableby expanded newbornscreening havebeen includedin the panel as conditions that are part of the differential diag-nosis of a primary panel condition, such as IBD defect versus SCADdeﬁciency.Formiminoglutamicaciduria,whichmayappear asafalse butyrrylcarnitine(C4)alteration[39]byusing deriva-tizedsamplepreparationprocedures,isnolongersubjecttorecall becauseitisconsidereda benigncondition.Someauthorshave

consideredretaining3-methyl-crotonylglycinuriainthescreening panel,despiteitbeingconsideredabiochemicalconditionrather thanadisease[40,41].Recentlysomeauthorsreporteddata con-ﬁrmingthatMCCdeﬁciency,despitelowpenetrance,mayleadto asevereclinicalphenotyperesemblingclassicalorganicacidurias [42].

Someauthorsbelieveit mayberelevanttomaintainglycine inthescreeningpanelbecauseitisasecondarymarkerinsome organic acidaemias[43]. Moreover,even ifonly in somecases [44],glycinecouldpermitthediagnosisofnonketotic hyperglyci-naemia[45]which,althoughthediseaseiscurrentlynottreatable, allows appropriate genetic counseling and possible prenatal diagnosis.

Tyrosineisananalytefrequentlyresponsibleforlargenumberof recalls.TyrosinecanbeelevatedintyrosinaemiatypesI,IIandIII, butitalsocanbetransientlyincreasedinmanynewborns, espe-cially thoseborn prematurely.In thecase of tyrosinaemiatype I, false-negatives mayoccur if thecut-off is toohigh or if the

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Table1

ConditionstestedintheTuscany/Umbrianewbornscreeningprogram,primaryandsecondaryrelatedbiomarkers,andusedratios.

Disorders Primarymarkers

(cutoff␮mol/L)

Secondarymarkers (cutoff␮mol/L)

Ratios Diagnoses

Corepanel

ClassicPKU Phe↑(n.v.<100) Tyrn/↓(n.v.10–250) Phe/Tyr 18

HPAtypeIIandIII Phe↑(n.v.<100) Tyrn/↓(n.v.10–250) Phe/Tyr 47

TyrosinemiatypeI SA↑(n.v.<2) Tyrn/↑(n.v.10–250) 3

CitrullinemiatypeI Cit↑(n.v.3–30) 7

Argininosuccinicaciduria Asa↑(n.v.<1) Cit↑(n.v.3–30) 2

Homocystinuria(cystathionine␤synthasedeﬁciency) Met↑(n.v.6–36) Maplesyrupurinedisease Val↑(n.v.<170);

Ile/Leu↑(n.v.<237)

Propionicacidaemia C3↑(n.v.<3.3) Gly↑(n.v.<260) C3/C0;C3/C4;C3/C16 3 Metylmalonicaciduriamut C3↑(n.v.<3.3) Gly↑(n.v.<260) C3/C0;C3/C4;C3/C16 8 MetylmalonicaciduriaCblAandB C3↑(n.v.<3.3) Gly↑(n.v.<260) C3/C0;C3/C4;C3/C16

Isovalericacidemia C5↑(n.v.<0.56) C5/C3;C5/C4;C5/C8 3

Multiplecarboxylasedeﬁciency C5OH↑(n.v.<0.7) C3n/↑(n.v.<3.3)

3-Methylcrotonylglycinuria C5OH↑(n.v.<0.57) 6

4maternal GlutaricacidemiatypeI C5DC↑(n.v.<0.18) C5DC/C4;C5DC/C8;

C5DC/C12;C5DC/C3DC 2 3-OH-3methylglutaryl-CoAlyasedeﬁciency C5OH↑(n.v.<0.57) C6DCn/↑(n.v.<0.7)

␤Ketothiolasedeﬁciency C5:1↑(n.v.<0.06) C5OHn/↑(n.v.<0.57) 1

Primarycarnitinedeﬁciency C0↓(n.v.5.5–45) Ctot↓(v.n.21–73) 2

2maternal Mediumchainacyl-CoAdehydrogenasedeﬁciency C6↑(n.v.<0.25);C8↑

(n.v.<0.40);C10:1↑ (n.v.<0.5)

C10↑(n.v.<0.36) C6/C8↑;C8/C10↑ 16

Verylong-chainacyl-CoAdehydrogenasedeﬁciency C14:1↑(n.v.<0.44) C14n/↑(n.v.<0.57; C16n/↑(n.v.0.48–5.6); C18:1n/↑(0.39–2.43)

C14:1/C4;C14:1/C5; C14:1/C8

6

Long-chainhydroxyacyl-CoAdehydrogenasedeﬁciency C16-OH↑(n.v.<0.1); C18:1OH↑(n.v.<0.05)

1 Trifunctionalproteindeﬁciency C16-OH↑(n.v.<0.1);

C18:1OH↑(n.v.<0.05)

1 Adenosinedeaminasedeﬁciency Ado↑(n.v.<2)

Deoxyado↑(n.v.<0.1)

1

Secondarytargets

TyrosinemiatypeIIandIII Tyr↑(n.v.<250)

CitrullinemiatypeII Cit↑(n.v.3–30) 2

Argininemia Arg↑(n.v.<34)

BH4defects Phe↑(n.v.<100) Tyrn/↓(n.v.10–250) Phe/Tyr 1

MTHFRdeﬁciency Met↓(n.v.6–36)

Methionineadenosyltransferasedeﬁciency Met↑(n.v.6–36) 2

MetylmalonicaciduriaCblCandD C3↑(n.v.<3.3) Met↑(n.v.<6) C16:1OH↑(n.v.<0.09)

C3/C0;C3/C4;C3/C16 14 19maternal MalonicAciduria C3DC↑(n.v.<0.7)

2-Methyl3-OH-butyryl-CoAdehydrogenasedeﬁciency C5OH↑(n.v.<0.57)

Shortchainacyl-CoAdehydrogenasedeﬁciency C4↑(n.v.<0.92) 10 GlutaricacidemiatypeII(MAD) C4–C18↑

Shorthydroxylacyl-CoAdehydrogenasedeﬁciency C4OH↑(n.v.<0.50)

3-Methylglutaconyl-CoAhydratasedeﬁciency C5OH↑(n.v.<0.57) 1

Isobutyryl-CoAdehydrogenasedeﬁciency C4↑(n.v.<0.92) 14

Carnitineacylcarnitynetranslocasedeﬁciency C16↑(n.v.0.48–5.6) CarnitinepalmitoyltransferaseIIdeﬁciency C16↑(n.v.0.48–5.6)

C18:1↑(n.v.0.39–2.43)

C2↓(n.v.6.3–48) (C16+C18:1)/C2 within10daysoflife CarnitinepalmitoyltransferaseIdeﬁciency C16↓(n.v.0.48–5.6)

C18↓(n.v.0.23–1.77)

C0n/↑(8–45) C0/(C16+C18)within 10daysoflife

HHHsyndrome Orn↑(n.v.25–150)

Gyrateatrophyofthechoroidsandretina Orn↑(n.v.25–150) Purinenucleosidephosphorylasedeﬁciency Deoxyguanosine↑

(n.v.<0.09) Deoxyinosine↑ (n.v.<2.85)

Guanosine↑(n.v.<1.7) Inosine↑(n.v.<33)

screeningisperformedintheﬁrst2–5daysoflife.However,the

useofalowcut-offlevelincreasesthefalse-positiverate.Inrecent

years,theuseofsuccinylacetoneasnewbiomarkerhasallowed

peopleworkinginNSﬁeldtoavoidfalse-negativeresultsfor

tyrosi-naemiatypeI.

Attentionshould be paid to the higher cut-off of citrulline,

capableofdiagnosingcitrullinaemiatypesIandII,and,assecond

marker,argininosuccinicaciduria.Weshouldalsotoevaluatethe

citrullinelowercut-offasapossiblesignofdefectsintheﬁrstand

secondstepsoftheureacycleaswellasmitochondrialdiseases

suchasNARP.

5.1. Secondtiertests

Someimportantmetabolitesinexpandednewbornscreening

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Fig.3.RecallrateofexpandednewbornscreeninginTuscany,2002–2013.

speciﬁcity,causingacriticalimpactonthepopulationduetothe highnumberofrecalls.Thebestwaytodecreasetherecallrate, duetofalsepositiveresultsintheprimaryscreeningtest,is rep-resentedbytheintroduction ofthesecondtier test,performed onthesameNBSspecimenwithnonecessitytoadditionally con-tactpatient’family.Secondtiertestresultoverrulestheprimary result.

Sinceforsomemetabolitesthereisanoverlapbetween con-trolsandpatientsranges,secondtiertestproveditssuperiority indiscriminatingthetwopopulationsthankstothehigh speci-ﬁcity.

ThechoiceofreferencevaluesforNBStestshouldbeagood compromisebetweensensitivityandspeciﬁcity;agoodsensitivity couldresultinahighfalsepositiveratewhilehavinghighspeciﬁcity couldcausefalsenegativeresults.Adjustingcutoffvaluesforeach metaboliteshouldconsidertheavailabilityofasecondtiertest;if itisavailable,primarytestcut-offshouldbereducedtocapture presumptivepositivecases[46].

SecondtiertestsareusuallyperformedbyHPLC–MS/MSbut in recent years a second tier genetic testing couldbe used to detectcysticfibrosis[47].ForCAHscreening,theprimarytestis performedbyimmunoassayandpositiveresultsareretestedby HPLC–MS/MStoobtainamorespecificanddetailedsteroidprofile [48].

Someauthorsreportedabouttheuseofsuccinylacetoneonly assecondtiertesttoimprovetherelativelypoorspeciﬁcityofthe primaryscreeningusingtyrosine[49,50].

In2011inTuscanyregion,ADASCIDbiomarkerswereincluded inthepanelofexpandedNBS;alsointhiscaseasecondtiertestcan beperformedwhenelevatedconcentrationofpurinesmetabolites aredetected[51].

Howeversecondtiertestusuallyidentifyadditionalanalytes, notdetectableattheprimaryNBStest,forbetterinterpretationof abnormalﬁndings.

Thiskindofapproachiswellrepresentedbypropionylcarnitine (C3),primarymarkerformethylmalonicacidurias(MMA)and pro-pionicacidurias(PA).Inaretrospectivestudyourgroupshowed thaton564totalrecalls,124(22%)wereforC3ofwhomonly5 (4%)weretrulyaffected:3methylmalonicaciduriasand2PAs.The developmentofasecondtiertesttodetectfreediagnosticacids allowedtodistinguishingtruepositivesfromfalsepositivesrising thepositivepredictivevalueforC3to100%[51].

Theintroductionofsecond-tiertestsprovedtobeanexcellent strategytocopewithfalsepositiveproblem.Forthisreason,todate, anincreasingnumberofpublicationsrelatetothedevelopmentof second-tiertestisreported[52–56].

Fig.3illustratestherecallratetrendfrom2002toFebruary2014 inTuscany.Atthebeginning,duringthepilotproject,cut-offsof mean_±2SDforallanalyteswereestablished.Recallrateincreased in2004withtheincreaseinthenumberofanalytesconsidered. Cut-offvaluesweremodifiedforspecificanalytes,upto4.5SDto minimizethefrequencyoffalse-positiveresults.Adjustmentof cut-offs,theintroductionofspecificratios,theuseofthe“postanalytical tools”ofR4Scollaborativeprojectandthedevelopmentofsome second-tiertestallowedareductionintherecallrateinrecentyears from1.47%to0.3%).Thepositivepredictivevalueincreasedfrom 3.5%in2004upto19.4%in2013.

5.2. Maternaldiseases

Insomenewborns,screeningresultscanbefalselypositivefor maternaldefects,resultingintransplacentalpassageof metabo-lites.Inourexperience,outof400000babies,weobservedlow valuesofC0,C2andtotalcarnitinesassociatedwithamaternal pri-marycarnitinedefect;twotwinswithhighvaluesofC5OHwithan asymptomaticmotheraffectedby3-methylcrotonylglycinuria;an infantwithhighC3andmethylmalonicaciddetectedby second-tiertestingwithamotherwithadefectofgastrin;andtwoinfants withelevatedlevelsofphenylalaninewithamotheraffectedby undiagnosedmildhyperphenylalaninaemia.

5.3. Outcomemanagementprocedures

Whenapositivescreening(ﬁrstscreeningandsecondtiertest, ifavailable)resultoccurs,twodifferentsubsequentproceduresare followed:

(a)In thecase of disorders that are at risk of acute metabolic decompensationduringtheneonatalperiod,ametabolic spe-cialist immediately recalls the baby; clinical examinations, conﬁrmatorytests,includingplasmaaminoacid,plasma acyl-carnitineandurineorganicacidproﬁlesareperformed; (b)Inthecaseofallotherdisorders,thenurseryiscontactedto

provideforaseconddriedbloodspot.Iftherepeatedtestshows apositiveresult,clinicalexaminationsandconﬁrmatorytests areperformed(seeFig.4).

Inbothcases,ifresultsfromtheconﬁrmatorytestsare posi-tive,themetaboliccliniciansprovidemanagement,counselingand follow-up.

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Fig.4. TuscanyNewbornScreeningalgorithmformetabolicdisorders.AfteranabnormalconcentrationsofC3isdetectedasecondtiertestforMMA/PAisperformed toidentifytruepositivenewbornsaffectedbymethylmalonicorpropionicacidurias.Anothersecond-tiertest,tomeasurehomocysteine(HCY)levelsisperformedifthe initialscreeningshowsMETlevelbeloworabovenormalcutofftoidentifyMTHFRdefectorhomocysteinuria,respectively.Abnormalscreeningresultsarere-analyzedfor conﬁrmationbeforerequiringnewspecimen.

6. Conclusions

MShasbeenusedinexpandedNBSprogramsforover20years andit hasbeenshownbeneﬁcial impactsonpublichealth care comparedtotraditionalprograms.CurrenttrendsinNBSaimed atexpandingtestingpanelscoveringawiderangeofconditions. ResearchtoincludemoretestsinNBShavemadehugesteps for-wardinimprovingsurvivalandinpreventinglong-termpermanent damageinaffectedpatients.However,theexpansionofNBSraises someethicalimplicationsrelating tovariabilityamongstates in conditionsincluded,informedconsentforstorageanduseof resid-ualDBS,whetherornot maybeappropriate toidentifycarrier statusoradultonsetconditions.

Datacollectedfrompilotstudiescanbeusedtocreate guide-linesthatcansigniﬁcantlyhelpdevelopNBSprogramsandcreate opportunities.

NBSprogramisacomplexprocessdependingona multidisci-plinaryteamincludingpediatricians,IEMspecialists,geneticists, dieticiansaswellasbiologistsandchemistswithexpertiseinmass spectrometry.Thecollaborationofalltheseprofessionalshas con-tributedtothissuccess.

Acknowledgments

IthankDrSabrinaMalvagia,DrSilviaFunghini,DrElisa Gio-caliere,DrDanielaOmbroneandDrGiuliaFornifromtheNewborn Screening,BiochemistryandPharmacologyLabofMeyerchildren’s UniversityHospitalofFlorenceforconstructivediscussion,for typ-ingthemanuscriptandfortheartwork.

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