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Bilirubin(serum,plasma)

1.1

1.2

1.3

1.4

1.5

2.1

2.2

Analyte
Nameofanalyte
Bilirubin
Alternativenames
None
NLMCcode
Descriptionofanalyte
Bilirubinisalineartetrapyrrole(MW585Da),thefinalproductofthe
breakdownofthecyclictetrapyrroleringofhaemoglobin,myoglobinand
thecytochromes.Bilirubinisinsolubleinwater.Inplasmaitispresentin
threeforms:
albuminbound(reversibly:unconjugatedbilirubin),normallythe
majorcomponent
conjugated
deltabilirubin(conjugatedbilirubincovalentlyboundtoalbumin).
Bilirubinistransportedinthebloodstreamtotheliverboundtoalbumin;
itistakenupbytheliverandrenderedwatersolublebyconjugationwith
glucuronicacid,principallytothediglucuronide,whichisexcretedinthe
bile.Smallamountsareabsorbedfromthegutandreexcreted.For
practicalpurposes,thepresenceofconjugatedbilirubinintheplasma
alwaysindicatesapathologicalprocess.Deltabilirubinbecomespresent
insignificantquantitiesonlyinprolongedcholestasis.
Onlyconjugatedbilirubincanbeexcretedinurine.Thusthedetectionof
bilirubinuriaalsoindicatesapathologicalprocess.
Notethatthewhiletheclearanceofconjugatedbilirubinfromtheplasma
israpid,thatofdeltabilirubindependsontheclearanceofalbumin,and
soislonger.
Functionofanalyte
Bilirubinisawasteproduct,destinedforexcretion.Ithasantioxidant
propertiesbutitisnotknownifthisisofphysiologicalsignificance.
Specimensandhandling
Bilirubinismeasuredinserumorplasma,althoughitsdetectioninurine
(usingdipsticks)isalsoinformative(see1.3).Noninvasive
transcutaneousphotometricmeasurementatpointofcareusinga
dedicatedinstrumentbilirubinometerispractisedininfantstoguidethe
needforbloodtobedrawntoguidetreatmentforneonataljaundice.
Bilirubinisphotosensitive,andspecimensshouldbeprotectedfromlight
whenhighlyaccuratemeasurementsarerequired.Thisisespecially
importantwithspecimenscollectedfromneonates.
Summaryofclinicalusesandlimitationsofmeasurements

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3.1

3.2

4.1

Uses
1.Intheinvestigationofjaundice(yelloworangediscolourationofthe
skinandscleraeduetothepresenceofbilirubin)
2.Asanindexofhepatobiliarydysfunction.Bilirubinistypically
measuredaspartofapanelofliverfunctiontests.Theexcessbilirubinin
hepatobiliarydisordersistypicallytotallyormainlyconjugated.

Limitations
1.Serum[bilirubin]canbeelevatedtoatleast50mol/Lwithout
jaundicebeingclinicallyapparent.
2.Neitherhepaticnorbiliarydiseaseinvariablycausesanincreasein
serum[bilirubin].
3.Anincreaseinserum[bilirubin]isnotspecifictohepatobiliarydisease;
itcanoccurinhaemolyticconditionsorconditionsofineffective
erythropoiesis.Theexcessbilirubinintheseinstancesisunconjugated.

Analyticalconsiderations

Analyticalmethods
1.Diazomethods
Thesearebasedonreactionwithdiazotisedsulfanilicacid(thediazo
reagent)toproducetwocolouredazodipyrrolesthatcanbemeasured
spectrophotometrically,eitherat530nmor,afteradditionofalkaline
tartrate,at530nmThereactionisacceleratedbyalcoholandavarietyof
other accelerators(e.g.sodiumbenzoate)thatcausethedissociationof
unconjugatedbilirubinfromalbumin.Inthepresenceofanaccelerant,
bothconjugated(includingdelta)andunconjugatedbilirubin(together
comprisingtotalbilirubin)aremeasured;thisisalsotermedindirect
bilirubin.Intheabsenceofanaccelerant,onlytheconjugated(direct)
bilirubinismeasured.Thedifferenceisconsideredtobeameasureof
unconjugatedbilirubin.Itisimportantthatnounconjugatedbilirubin
reactsindirectmethods:thiscanbeavoidedbymaintainingareaction
pHof~1.0.
2.Highperformanceliquidchromatography(HPLC)
HPLCmethodsarecapableofmeasuringthevariousbilirubinfractions
presentinplasmaseparately.ThiscanbeachievedusingaMicronex
RP30columnbutforpracticalpurposestheuseofdirectandindirect
diazotechniquesprovidessufficientclinicalinformationfordiagnostic
purposes.
3.Enzymatic
Thesemethodsemploytheenzymebilirubinoxidase(EC1.3.3.5)to
convertbilirubintobiliverdin.Thereactionisfollowedbymeasuringthe
fallinabsorbanceat425or460nm.Separatequantitationofthedifferent
speciesofbilirubinisachievedbyusingdifferentreactionpHconditions.
4.Spectophotometric
Thismethodinvolvesthemeasurementofabsorbanceat437nm,the
maximumabsorbanceofbilirubin.Thisisthebasisofthemethodusedin
bilirubinometers.

4.2Referencemethod

Thereferencemethodisbasedonthediazomethod.

4.3Referencematerials
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4.4

4.5

5.1.1
5.1.2

Thereferencematerialishumanserumwithaddedpurifiedbilirubin
(SRM916a(NationalInstituteofStandardsandTechnology,
Gaithersburg,MD,USA)).
Interferingsubstances
Theonlymajorinterferingsubstanceishaemoglobin,butindividual
manufacturersassaysmaysufferinterferencewithcertaindrugs.
Sourcesoferror
Exposureofspecimenstolightmaycausephotolysisofbilirubinand
reduceitsconcentration.

Referenceintervalsandvariance

Referenceinterval(adults):25mol/L;conjugated2mol/L
Referenceintervals(others):25mol/L(though200mol/Lmay
occurinthefirst14daysoflife(peakat34days))asaresultof
physiologicaljaundice(see6.1(4)).
5.1.3 Extentofvariation
5.1.3.1 InterindividualCV:35%
5.1.3.2 IntraindividualCV:25%
5.1.3.3 Indexofindividuality:0.71
5.1.3.4 CVofmethod:13%
5.1.3.5 Criticaldifference:100%
5.1.4 Sourcesofvariation

Inhealthyindividuals,thedistributionofbilirubinconcentrationsis

skewedtotheright,reducingtheprecisionoftheupperreferencelimit.

ThehighprevalenceofGilbertssyndrome(see7.1(3))isanadditional

problem.

6
Clinicalusesofmeasurementandinterpretationofresults

6.1 Usesandinterpretation
1.Jaundice(adults)
Thedemonstrationthatanexcessofbilirubinintheserumisconjugated
(asmaybeinferredfromthedetectionofbilirubinuria)isindicativeof
hepatobiliarydiseasebutdoesnothelpdistinguishbetweenthepossible
causes.Thetransportofconjugatedbilirubinfromhepatocytesintothe
biliarysystemisanactiveprocess.Thusconjugatedhyperbilirubinaemia
canoccurin:
hepatocellulardamage(hepatitis)
intrahepaticcholestasis(e.g.cirrhosis)
extrahepaticcholestasis(e.g.duetoobstructionofthebileductsby
pancreaticcarcinoma).
Purelyunconjugatedhyperbilirubinaemiaisusuallyduetoa
haematologicaldisordercausingincreasedredcellbreakdown,butcan
occurasaresultofimpaireduptakebytheliverofdecreasedconjugation.
2.Hepatobiliaryfunction
Measurementsofbilirubincanbeusedtomonitorthecourseof
hepatobiliarydisease.Forexample,afallinconcentrationfollowinga
procedureaimedatrelievingobstructionsuggeststhatthishasbeen
successful.
3.Haematologicaldisorders
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6.2

Anelevated[unconjugatedbilirubin]ischaracteristicofhaemolytic
conditions(e.g.congenitalspherocytosis)andconditionsinwhichthereis
ineffectiveerythropoiesis(e.g.vitaminB12deficiency).Inadults,[total
bilirubin]veryrarelyexceeds100mol/Linsuchconditions.
4.Neonataljaundice
Considerationsregardingjaundiceinadultsarealsoapplicableto
neonates,butadditionalfactorsapply:
theneonatalliverisimmatureandhaemturnoverisincreased.This
cancausea(transient)increaseinplasma[unconjugatedbilirubin]
andphysiologicaljaundice
inconditionscausingunconjugatedhyperbilirubinaemia,plasma
bilirubincanexceed100mol/L.Concentrationsinexcessof300
mol/Lcarryariskofcausingbraindamage(kernicterus)
somecausesofjaundice(e.g.themoresevereinheriteddisordersof
bilirubinmetabolism)presentuniquelyorprincipallyonlyinthe
neonatalperiod
thepreceptthatbilirubinuriaindicatesanexcessofconjugated
bilirubininplasmaappliesinneonatesaswellasinadultsandit
shouldalwaysbefurtherinvestigated.

Confoundingfactors
Althoughhyperbilirubinaemiaischaracteristicofmanyconditions,itis
diagnosticofnone.Exceptwhenusedtomonitortheprogressofa
conditionalreadydiagnosed,thesignificanceofhyperbilirubinaemiacan
onlybeassessedinrelationtotheresultsofotherinvestigations(see
7.1.1)

Causesandinvestigationofabnormalresults

7.1
Highconcentrations
7.1.1Causes

Hyperbilirubinaemiahasbeenclassifiedinvariousways,e.g.prehepatic,
hepaticandposthepatic.Noneisentirelysatisfactory,asmorethanone
mechanismmaybeoperatinginindividualcases.

1.Increasedproductionofbilirubinmayexceedtheabilityoftheliverto
conjugateandexcretethepigment,causinganincreaseintheplasma
[unconjugatedbilirubin].However,thecapacityofthelivertoprocess
bilirubinexceedsthenormalrateofproduction,sothatincreased
productiondoesnotnecessarilycausehyperbilirubinaemia.Specific
causesinclude:
haemolysis(autoimmune,congenitalandotherhaemolyticanaemias)
ineffectiveerythropoesis
rhabdomyolysis(becauseofincreasedbreakdownofmyoglobin).
2.Decreasedhepaticuptakecanbecausedbyseveraldrugs,e.g.
rifampicin.
Althoughbilirubinuptakemaybedecreasedinhepatocellulardisease,
theexcessbilirubinintheplasmaisprimarilyconjugated,reflecting
decreasedsecretionintothebiliarysystem.
3.Theconjugationofbilirubinmayalsobeimpairedinhepatocellular
disease,butanyexcessbilirubinintheplasmaisprimarilyconjugated.
Twoinheritedconditionsofimpairedofbilirubinconjugationcancause
unconjugatedhyperbilirubinaemia.Theseare:
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Gilbertssyndrome(commonandbenign)typicallycausingonlymild
hyperbilirubinaemia(100mol/L)andsporadicjaundice,typically
inyoungadults
CriglerNajjarsyndromestypesIandII,causingsevere
hyperbilirubinaemiaandtypicallypresentingininfancy.
4.Impairedsecretionofbilirubinintothebiliarysystemcanoccurin
hepatocellulardiseases.Itcanalsobecausedbydrugs,including
chlorpromazine,carbamazepineanderythromycin.Impairedsecretion
bilirubinintothebiliarysystemisafeatureoftworareinherited
disorders,RotorsyndromeandDubinJohnsonsyndrome.
5.Biliaryobstructioncancausediffusionofconjugatedbilirubinfromthe
biliarysystemintothebloodstream.Suchobstructioncanbeintrahepatic
(e.g.cirrhosis)orextrahepatic(e.g.sclerosingcholangitis,carcinomaof
theheadofthepancrease,causingobstructionthroughexternalpressure
onthecommonbileduct.
7.1.2 Investigation
Bilirubinisusuallymeasuredaspartofapanelofliverfunctiontests,
includingalbumin,totalprotein,atransaminase(aminotransferase)and
alkalinephosphatase.Appropriateinvestigationwilldependonthe
resultsofthesetests.
1.Resultsofotherliverfunctiontestsarenormal
Valuesupto20%higherthantheupperlimitofnormal(ULN)maybe

statisticaloutliers.
Withvalues1.5xULN,theurineshouldbetestedforbilirubin.Ifthe
resultisnegative,itcanbeinferredthattheexcessbilirubinis
unconjugatedand themeasurementshouldberepeatedwithinthree
monthsunlessthereisclinicalsuspicionofdisease.
Ifavalueof1.5xULNisconfirmedoraninitialmeasurementis>1.5

but3xULN,thepossibilityofahaematologicalcauseshouldbe

investigatedbyperformingafullbloodcount(includingreticulocytes),

examiningaperipheralbloodfilmandperformingaCoombstest.If

haemolysisisexcluded,themostlikelydiagnosisisGilbertssyndrome.If

required,thiscanbeconfirmedbyagenetictest.
Values>3xULNshouldbeinvestigatedfurtherregardlessofwhether

otherliverfunctiontestsareabnormal.Iftheconjugatedfractionis

>50%ofthetotal,ultrasoundimagingofthehepatobiliarysystemis

indicated;iftheunconjugatedfractionis>70%ofthetotal,investigations

forhaemolysisshouldbeperformed.
2.Resultsofotherliverfunctiontestsareabnormal
Theinvestigationofhyperbilirubinaemiawithabnormalliverfunction
testsislikelytobeinformedbyanyaccompanyingclinicalabnormalities.
However,predominantelevationinserumaminotransferase
(transaminase)activitiesincomparisonwithserumalkalinephosphatise
activitysuggestshepatocellulardysfunction.Thisshouldbeinvestigated
byviralandautoimmuneserology.Apredominantincreaseinalkaline
phosphatasesuggestsbiliaryobstruction(eitherintraorextrahepatic)
andshouldbeinvestigatedinitiallybyultrasoundexamination,further
investigationdependingonthefindings.

7.2 Lowconcentrations
7.2.1 Causes

Thelowerlimitofthereferencerangeforbilirubiniszero.Pathologically

lowvaluesdonotoccur.
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7.2.1 Investigation

Notapplicable

7.3 Note
Jaundiceiscommoninneonates.Ifitdevelopsmorethan24hafterbirth,
lastsnotmorethan14daysandisdueonlytounconjugatedbilirubin,it
maybebenignphysiologicaljaundice.Ahigh[bilirubin](>300mol/L)
carriesariskofbraindamageandrequiresintervention.Conjugated
hyperbilirubinaemiaisalwayspathological,asishyperbilirubinaemia
presentinginthefirst24horpersistingafter14days.

8
Performance

8.1
Sensitivity,specificityetc.forindividualconditions

Sensitivityofserumbilirubinlevelsinselectingpatientsformagnetic

resonancecholangiopancreatography.HarounAA,AlHadidiAM,

TarawanehISetal.Hepatogastroenterology2007;54:995998.Increased

[conjugatedbilirubin]reportedtohave77%sensitivityand80%specificity

forpredictingabnormalfindingsonMRCP.

9
Systematicreviewsandguidelines

9.1
Systematicreviews

Allsystematicreviewsidentifiedrelatetoneonataljaundiceandthe

efficacyofproceduresforpredictingpossiblebilirubinencephalopathy

(kernicterus);theconsensusappearstobethatacombinationofearly

measurementofserumtotalbilirubinwithassessmentofotherrisk

factorscannotreliablypredictthoseinfantsathighriskofthecondition.

E.g.TrikalinosTA,ChungM,LauJ,IpS.Systematicreviewofscreeningfor

bilirubinencephalopathyinneonates.Pediatrics.2009;124:11621171.

9.2
Guidelines

NICEClinicalGuideline98(2010)NeonatalJaundice.

http://publications.nice.org.uk/neonataljaundicecg98(accessed

25.iv.2012)

Containscomprehensiveguidanceontheinvestigationandmanagementof

jaundiceininfants.

Noguidelinesidentifiedinrelationtojaundiceinadults.

9.3
Recommendations
Recommendationsidentifiedcomplementtheadviceprovidedin
guidelineswithregardtojaundiceininfants.Nospecific
recommendationsidentifiedinrelationtojaundiceinadults.
Recommendationsgivenfortheinvestigationofisolatedincreasesin
serum[bilirubin]arebasedonexpertopinion,notclinicalevidence.

10. Links

10.1Relatedanalytes

None

10.2 Relatedtests
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Measurementofotherindicesofliverfunction,includingthestandard
liverfunctiontests(see7.1.1),isoftenvaluableintheinterpretationof
anabnormal[bilirubin].Ingeneral,however,laboratorytestsontheir
ownrarelyprovideafinaldiagnosis:theirresultstendtoindicatetypesof
pathologicalprocess.

Author:WilliamMarshall

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