References:

y 1 UKGuidelinesfortheUseofThyroidFunction

Tests2008
y 2Tietz
2 i
textbookofClinicalChemistryand
b k fCli i lCh i
d
MolecularDiagnostics2006/Pathophysiology
andThyroidFunctionTesting
y 3 Qualitycontrolofthyroidfunctiontestsinvitro
y A.M.BOLDANDD.M.BROWNING
y FromtheClinicalChemistryDepartment,Queen
ElizabethHospital,Birmingham2003
y

Specimen Collection and Storage testosterone

SpecimenCollectionandStoragetestosterone
y Eitherserumorheparinized plasmaisusedtomeasuretotalorfree

testosterone.Testosteroneissubjecttoadiurnalvariation,reachinga
peakconcentrationbetween04hoursand08hours.Therefore,morning
k
i b
h
d 8h
Th f

i
specimensarepreferred.
y Specimensarestableforaweek(men)or3days(women)refrigeratedandfor
upto1yearfrozenat200C.
t
f
t C
y Nosteroids,thyroid,ACTH,estradiol,orgonadotropin medications
shouldbegivenfor48hoursbeforesampleCollection.
y Mostassaysarestandardizedforserumorheparinized plasma.Other
anticoagulantssuchasethylenediaminetetraacetic acid(EDTA)maygive
differentvalues.IncertainRIAassays,presenceofEDTAappearstocausea
10%decreaseintotaltestosteroneconcentrations.
%d
i t t lt t t

t ti

Specimen Collection and Storage DHEAS

SpecimenCollectionandStorageDHEAS
y Serumorplasma(preservedwithEDTA)issuitablefor

DHEAorDHEASimmunoassay
DHEAorDHEA Simmunoassay
y Nosteroids,ACTH,estradiol,orgonadotropin
medicationsshouldbegivenfor48hoursbeforesample
collection.

Earlymorningcollection,before10.30hours,is
preferredforDHEA.
f
df DHEA
Refrigeratedsamples(4Cto8C)arestableforupto14days,
thosefrozenat20Carestablefor>1years.
thosefrozenat20Carestablefor>1years

SpecimenCollectionandStorageEstriol
p
g
y Estriol serumorplasmaspecimensarestableatroomtemperaturefor24

hours,andcanberefrigeratedfor2daysandfrozenat20Cforupto1year.

Patientsshouldnotbefastingbeforespecimen
P ti t h ld tb f ti b f

collection.
y Comments:

Estrone determinationsarelimitedtodiagnosisof
postmenopausalbleedingandthemenstrualdysfunction
causedbyextraglandular
db
l d l estrone production.
d
i
y Normally,bloodestroneconcentrationsparallel
estradiol concentrationsthroughoutthemenstrualcycle,
butatslightlylowerconcentrations.Foraspecificanalysis
ofestrone,theinterestedreaderisdirectedtoother
references.
y

Specimen Collection and Storage progesterone

SpecimenCollectionandStorageprogesterone
y Serumorplasma(withheparinorEDTAasanticoagulant)

,
p
4
hasbeenused,butshouldbeseparatedwithin24hourS.

y Thepatientneednotbefasting,andnospecial

handlingproceduresarenecessary.
y Samplescanbestoredrefrigeratedforupto3daysat4Cto

8Corforupto1yearat200C.

y patientsshouldnotbeonanycorticosteroid,
i
h ld
b

i
id

ACTH,estrogen,orgonadotropin medication
foratleast48hoursbeforespecimencollection.
foratleast48hoursbeforespecimencollection

CollectionandStorageofSamples
catecholamine
catecholamine
y Theconditionsunderwhichplasmaorurinesamplesarecollectedcan

becrucialtothereliabilityandinterpretationoftestresults.
y Manycliniciansprefer24hourcollectionsofurineoverblood
samplingsincetheformeravoidsmanyoftherigidsampling
conditionsassociatedwithbloodcollectionsandismoreconvenient
f li i l t fft i l
forclinicalstafftoimplement.
t
y However,24hourcollectionsofurinearenotalwayseasily,
conveniently,orreliablycol1ectedbypatients,particularlypediatric
patients.Also,influencesofdietandsympatho
ti t Al i fl
fdi t d
th adrenalactivation
d
l ti ti
associatedwithphysicalactivityorchangesinposturearenotas
easilycontrolledforastheyareforbloodcollections.

y Becauseofthepossibleerrorsresultingfrom
B
f h
ibl

l i f

incomplete24hoururinecollectionsoruncontrolled
influencesofphysicalactivity,someinvestigators
p y
y
g
advocatespotorovernighturinecollections.

y C0rrectionfordifferencesindurationofcollectionis

achievedbynormalizingcatecholamineorcatecholamine
metaboliteexcretionagainsturinarycreatinine excretion.
excretion
y Additionalconsiderationsforurinecollectedunderthese
conditionsincludedietaryprotein,musclemass,levelof
physicalactivity,andtimeofday,allofwhichimpact
creatinine excretionandmayfurtherconfound
interpretationofresults.

y Studiesonthestabilityofcatecholamines inurineandplasmahaveyielded

d
l
h
bl
d
d
mixedresultswithvariablerecommendationsonappropriatepreservativesand
methodsofcollection.
y Elaboratetechniquesforsamplepreservationasrecommendedinearlieryears
nowappeartobelargelyunnecessary.
b l
l

y Variablefindingsmaybeexplainedbyautooxidation,

particularlyatalkalinepH,ordeconjugation,particularlyat
particularlyatalkalinepH
ordeconjugation particularlyat
lowpHtwoprocesseswithopposingeffectsonlevelsoffree
amines.
y Thegeneralrecommendationisthatcatecholamines inurinesamples

arebestpreservedwithhydrochloricacid(HCI)tomaintainurineacid.
Aliquotsarebeststoredfrozenoverprotractedperiodsoftimeat80Cto
f th i i i t
furtherminimizeautooxidation
id ti anddeconjugation.
dd
j
ti

InterferencesfromandInfluencesofDietandDrugs
and normetanephrine
norepinephrine andnormetanephrine

y Tricyclic antidepressantsinparticularareamajor
sourceof
ffalsepositiveresults
false positiveresultsformeasurements
f
t
ofnorepinephrine andnormetanephrine inplasmaor
urine.
y Presumablythisisduetotheprimaryinhibitoryactions
oftheseagentsonmonoaminereuptake.Theresultisan
increasedescapeofnorepinephrine fromsympathetic
nerveterminalsintothebloodstream.
y Ldopa,Sinemet,alphamethyldopa
Ld
Si
t l h
th ld
(Ald
(Aldomet),and
t) d
MAOinhibitors.Thisdrugscanthereforeinterfere
directlywithcatecholamineassaysandis
d
ect y w t catec o a
e assays a d s aalsoconvertedby
so co ve ted by
catecholaminesynthesizingandmetabolizingenzymestocatecholamine
productsandmetaboliteswithadditionalinterferingactions

INTERFERENCES IN IMMUNOASSAYS
INTERFERENCESINIMMUNOASSAYS
y Immunologicalassaysarepronetointerferences,in

spiteoftheuseofhighlyspecificantibodiesfor
molecularrecognitionoftheanalyte
molecularrecognitionoftheanalyte.
y Falselylowresultscanoccurbecauseofthehook
effectathighantigenconcentrations.
effectathighantigenconcentrations

INTERFERENCESINIMMUNOASSAYS
y Falsenegativeorfalsepositiveresultsareencounteredifthe

samplecontainsantianimalantibodies.
y Forexample,inatwo
Forexample,inatwositesandwichassayforhCG
sitesandwichassayforhCG based

onmouseantibodies,anyhumanantimouse antibodies
(HAMA)presentinthespecimenwillrecognizethe
i
immobilizedmousecaptureandmouseconjugate
bili d

antibodiesandformacomplexthatisindistinguishable
fromanimmobilizedcaptureantibody:hCG :conjugate
complex.Thisleadstoafalsepositiveresult.
y AfalsenegativeresultwillbeobtainediftheHAMAreact
g
witheitherthecaptureantibodyorconjugatetosuchan
extentthatspecificantibodybindingtohCG isprevented.

INTERFERENCES IN IMMUNOASSAYS
INTERFERENCESINIMMUNOASSAYS
y Manydifferenttypesofcirculatingantianimal antibodies

havebeendetected(e.g.,humanantigoat,humanantibovine
h b
d
d(
h
i
h
ib i
antibodies)andshowntointerfereinimmunoassays.
y Inpractice,thistypeofinterferenceisminimizedby
Inpractice thistypeofinterferenceisminimizedby
includingadditivesintheimmunoassayreagents.
Nonimmune serumorIgG
g fromthespeciesusedtoraisethe
p
antibodieshasbeenapopularchoiceforthispurpose

Circadian Variation
CircadianVariation
y Manyconstituentsofbodyfluidsexhibitcyclicalvariations

throughouttheday.Factorscontributingtosuchvariations
includeposture,activity,foodingestion,stress,daylightor
darkness,andsleeporwakefulness.
y Thesecyclicalvariationsmaybequitelarge,andthereforethe
h
l l
b
l
d h f
h
drawingofthespecimenmustbestrictlycontrolled.For
example,theconcentrationofserumironmaychangebyas
p ,
y
g y
muchas50%from08to14,andthatofcortisol byasimilar
amountbetween08and16.
y Serumpotassiumhasbeenreportedtodeclinefrom5.4mmol/L
S
t i h b

t dt d li f

l/L
to4.3mmol/Lat0800 1400.

Circadian Variation
CircadianVariation
y Hormonesaresecretedinbursts,andthis,togetherwith

thecyclicalvariationtowhichmosthormonesare
subject,maymakeitverydifficulttointerprettheir
serumconcentrationproperly.
y Corticotropin secretionisinfluencedbycortisollike
steroids,butitisalsoaffectedbypostureandbylight,
,
yp
y g ,
darkncss,andstress.Itssecretionisincreasedthreefold
tofivefoldfromitsminimumbetweenafternoonand
midnighttoitsmaximumaroundwaking.
y Cortisol concentrationsaregreatestaround06to
08hoursandmaybetwiceashighasthoseobservedat
8h
d
b i hi h h b
d
midnight.

Circadian Variation
CircadianVariation

y Maximumrenin activitynormallyoccursearlyinthe

g
g
p;itsminimumoccurslateintheafternoon.
morningduringsleep
y Theplasmaaldosterone concentrationshowsasimilar
pattern.
y Glomerular
Gl
l filtrationrate(GFR)variesinverselywiththesecrction
fil
i
(GFR) i i
l i h h
i ofrenin.
f i

GFRisleastatthetimeatthetimeof
y maximumrenin secretionand20%greaterintheafternoonwhenrenin activity
i i i
isataminimum.

y Theexcretionof17ketosteroidsand17hydroxycorticosteroidsislowatnight
andreachesamaximumaboutmidafternoon.
y Thereisnocircadianvariationintheplasmaconcentrationsoffollicle
stimulatinghormone(FSH)andluteinizinghormone(LH)inmen,buta
20%to40%increaseofplasmatestosteroneoccursduringthenight.
y

Prolactin issecreted,likeotherhormones,inmultiple
bursts;prolactin concentrationisgreatestduringsleep

Circadian Variation
CircadianVariation
y Theserumthyroidstimulatinghormone(TSH)isata

maximumbetween02and04andataminimumbetween18
and22.Thevariationisoftheorderof50%.
y Therearealsovariationsintheserumthyroxine
concentration,buttheseappeartoberelatedtothechanges
b
h
b
l d
h h
inconccntration ofbindingproteinbroughtaboutby
changesinposture.Thesevariationsaremaximalbetween
g
p
10and14.

y Totalproteinconcentrationmayvarybyasmuchas

10%over24hours,butthevariationofindividual
proteinsmaybeevengreater.

Circadian Variation
CircadianVariation
y Growthhormonesecretionisgreatestshortlyaftersleep

commences.
y Conversely,basalplasmainsulinishigherinthemorningthan
laterintheday,anditsresponsetoglucoseisalsogreatestinthe
morningandleastaboutmidnight.
dl
b
d h
y Whenaglucosetolerancetestisgivenintheafternoon,higher
glucosevaluesoccurthanwhenthetestisgivenearlyintheday
glucosevaluesoccurthanwhenthetestisgivenearlyintheday.
Thehigherplasmaglucoseoccursinspiteofagreaterinsulin
response,whichisneverthelessdelayedandlesseffective.


circadian cycle /

) 6 ( 8



)9 3
( ).
150 /(. 200
/ 4
30 60 8 .
ACTH
6 10 ) 180 % 200 (
5 7 10 12 ).
1

2 ( % 100/
:
2 3 /
3 4 /
: .

3
13 15


2
13 15


circadian cycle /

4 8 % 50/

%60/ /
9 10 .
.

45

/
: /

/




/

/
/

TG

14 16 %10/
) %20(T3

 / /

%20/
4
)(%200

Lipoprotein

ACE
Prolactin
HDL

Renin/ MCV

/ALT/AST

Wbc/Hb/Hct

CEA/cu/pd/cadm
ium

selenium
b.carotenoid

SerumGHassay
y SerumGHisundetectableformostofthedayin

healthy,nonstressed individuals.Thisfactalong
withtheepisodicnatureofGHsecretionmakesa
singlesamplingdifficulttointerpret.Asaresult,
thediagnosisofGHdeficiencyismadeusingGH
h di
i fGHd fi i
i d i GH
measurementsfollowingpharmacologicstimulation,

y GHexcessisconfirmedbyfailureofGH

suppressionfollowinganoralglucoseload.

y GHiscommonlymeasuredby
GHi
l
db

chemiluminescent immunoassay

GrowthHormoneDeficiency
G o t o o e e c e cy
Idiopathicgrowthhormonedeficiencyisthemost
y
commoncauseofGHdeficiency(GHD)inchildren,
whereaspituitaryadenomaisthemostcommoncausein
adultonsetGHD.Thereisnosimple,reproduciblemethod
fordeterminingabnormalGHsecretory patterns.
patterns Inhealthy

individuals,7080%ofGHresultsarebelow1ng/mL
(<1g/L),butsecretory peakstypicallyreach2040
ng/mL
/ L (2040g/L).
(
/L)
Thus,inachildwithdecreasedgrowthvelocity,alowor
nondetectable GHdoesnotnecessarilyindicateGHD.Similar
toGH,IGF1 declineswithaging.

IGF1ismorediagnosticallyusefulinpatientsyounger
g
y
p
y
g
thanage40;however,itisstillnotsensitiveenoughtobe
usedasastandalonetesttomakethediagnosisofGHD

GrowthHormoneDeficiency
y Manipulationoftheendocrinesystemthroughstimulation
p
y
g

andsuppressionofthevariousaxesisoftenrequiredfor
diagnosingconditionsofhormonaldeficiencyandsurfeit.In
thisvein,
hi i GHDisdiagnosedbyshowingafailureof
GHDi di
db h i f il
f

GHtoincreaseadequatelyinresponseto
pharmacologicstimulation Theinsulintolerance
pharmacologicstimulation.Theinsulintolerance
test(ITT)haslongbeenconsideredthegold
standard'fordiagnosingGHD
g
g
;;howeveritismost
unpleasantforthepatient,requirestheattendanceofa
physicianthroughoutthetestingperiodandis
contraindicatedinthosewithahistoryofseizuresor,cardiac
t i di t di th ith hi t f i
di
orcerebrovasculardisease

GH/GrowthHormone

) (
) 0.5(

:

-1 ) /


(

-2

(

GH / Growth Hormone
GH/GrowthHormone



) (

1
%80


)

GH/GrowthHormone
:

) (

: /

:
-1 ) (

:




-2
/ /

/

/

GH/GrowthHormone
:

(
1

) (

10 100 1000

2-5

IGF-1 / Insulin Growth Factor / Somatomedine C

/

5-10

GrowthHormoneExcess
y Growthhormoneoverproductioncanresultinthecondition

called acromegaly.Ifdevelopedpriortoclosureofthe
epiphyses,theseindividualsmaybeexceedinglytall,'gigantism.
i h
th i di id l
b
di l t ll ' i
ti

y Thescreeningtestforclinicallysuspected acromegalyisa
randomlycollectedIGFII.
randomlycollectedIGF
y

IfthelevelofIGFIiselevatedinrespecttothe
appropriateage
pp p
g andgenderrelatedreference
g
range,thenitisnecessarytoconfirmthediagnosis
usinganoralglucosetolerancetest(OGTT).
g

Growth Hormone Excess

GrowthHormoneExcess
y The

OGTT isperformedbyorallyadministering 75gofglucose

andobtainingbloodsamplesatbaselineandevery30
d bt i i bl d
l tb
li d

minutesoverthenext2hoursforglucoseandGH.
y Anormal response is asuppressionofGH to<1ng/mL(1g/L)at

anytimeduringthetest. IfGHfailstodroptobelow1

ng/mL(1g/L),thesubjectisdiagnosedas
h i
havingacromegaly
l . Thedifficultycomesindiagnosingmild
h d ff l
d
ld
disease.
y Fredaetal.hadstudied60postoperativepatientswithacromegaly,22patientswith
activedisease(elevatedIGFI),38patientsinremission(normalIGFI)and25
healthycontrols.Thehighest nadirGHwas0.13g/Linthecontrolsand0.3g/Lin
thosewithactivedisease.FiftypercentofthosewithactivediseasehadGHvalues<
1g/L leadingtoamisclassificationoftheseindividualsasbeingnormalusingthe
1g/L,leadingtoamisclassificationoftheseindividualsasbeingnormalusingthe
abovestatedcriteria.Inamorerecentstudyof16patientswithmilddisease,the
majorityhavingmicroadenomas,. alsofoundthat50%ofthepatientswith
acromegalywereabletosuppresstheirGHlevelsto<1g/LfollowinganOGTT


Prolactin
y Prolactinisapolypeptideproducedbythelactotrophs

ofthepituitary;
y itisresponsiblefortheinitiationandmaintenance
oflactation.
y Itssecretionisnormallykeptatlowlevelsbythe
It
ti i
ll k t tl l l b th
inhibitoryactionsofdopamineproducedbythe
hypothalamus.
yp
y Aswithseveralpituitaryhormones,prolactinis
secretedinacircadianfashion,withthehighest
levelsbeingattainedduringsleepandanadir
occurringbetween10a.m. andnoon


Prolactin
y Prolactinissecretedinapulsatilefashion,theamplitude

andfrequencyofwhichnotonlyvariesthroughouttheday,
butisalsoimpacteduponbyavarietyofphysiologic
b ti l i
t d
b i t f h i l i
stimuli(i.e.,stress,postprandially,exercise).
y Becauseofthesefactorsandaserumhalflifeof2647
minutes,itisrecommendedthatwhenscreeninga

patientforhyperprolactinemiathree
specimensbeobtainedat20 to30minute
intervals.
y Thesesamplescaneitherbeanalyzedseparatelyand

theirresultsaveraged,
g , oralternatively,
y, anequal
q

aliquotfromeachsamplecanbepooledintoone
finalsamplethatisthenanalyzed.


Prolactin
y Themajorcirculatingformofprolactinisthe

nonglycosylatedmonomer.Anumberofotherformscan
occurincludingbig' prolactinandmacroprolactin
l i
(big, big'prolactin),consideredtobeprolactin
coupledwithimmunoglobulin.
l d ithi
l b li
y Becausetheseformsallreactwithimmunoassaysthey
canresultinfalselyhighPRLresultsinpatientsin
whomapathologicalelevationofPRLisnotsupported
byCTorMRI.Variousanalyticalmethodshavebeen
y
y
developedtoeliminatethisconfusion,includingthe
performanceofimmunoassayfollowingpolyethyleneglycol
extractionandcentrifugalultrafiltration.
i d
if
l l fil
i


Prolactin
y Insomeinstancesofprolactinoma,thevaluesof
prolactinmaybeextremelyelevated.
y Becauseusuallyonlyasingledilutionisperformed
whenassayingforprolactin,extremelyhigh
concentrationsmaysaturatethebindingsites
t ti

t
t th bi di it
resultinginafalselylowresult(Barkan,1998).
Thishook'effect
hook effect mayresultinmisdiagnosingthe

lti i di
i th
patientashavinganonfunctioningchromophobe
adenoma.

y Ifthepretestprobabilityofthepatienthavinga

macroprolactinomaishigh,itisrecommendedthatthe
serumsamplebesubjectedtoatleasta1:100dilution.


Prolactin
Prolactinactsonbreasttissue,whereinthesettingofestrogen
priming,itstimulateslactation.Prolactinalsoactsatthe
hypothalamustoinhibitthesecretionofgonadotropin
h
th l
t i hibitth
ti f
d t i
releasinghormone(GnRH).InhibitionofGnRHresultsina
decreaseinthereleaseofLHandFSHfromtheanterior
pituitary.Infemales,thisleadstoadecreaseinestrogenand
progesteronesynthesisandsecretionbytheovariesanda
failureofovarianfollicularmaturation(ovulation).Inmales,a
(
)
deficiencyofFSHandLHcausesadecreaseintesticular
productionandsynthesisoftestosteroneandahaltin
spermatogenesis.Inadditionthereissomesuggestionthat
hyperprolactinemiamayalsostimulateadrenalandrogen
productionaswellashaveaneffectonimmune
responsiveness


Prolactin
y Thereferencevalueforserumprolactin is125ng/mL (125

g/L)forwomenand120ng/mL (120g/L)formen.
y Thehigherprolactin levelsseeninfemalesbeginspost
pubertyandarepresumablyduetothestimulatory
effectofestrogen .Duringpregnancythereisaprogressive
Duringpregnancythereisaprogressive
riseinserumprolactin withlevelsreportedlyreachingas
highas500ng/mL bythethirdtrimester.Thisislargely
duetoanincreaseinnumberofprolactinsecretingcellsand
canbeassociatedwithadoublingorevengreaterincreasein
pituitaryglandsize Prolactin levelsfallbacktobaseline
pituitaryglandsize.Prolactin
about3weekspostpartum inwomenwhoarenot
breastfeeding.Innursingmothersbasalprolactin
g
g
p
levels
remainmoderatelyelevatedandwithepisodicburstsin
secretioninresponsetosuckling.


Prolactin
y Prolactinlevelsareincreasedbymanyphysiologicand

pathologicfactorsaswellasbyawidevarietyof
medication.
di ti
y Elevationsinprolactinresultingfromphysiologicand
pharmacolo icstimulirarelyexceed200ng/mL.
pharmacologicstimuli
rarelyexceed200ng/mL


Prolactin
y Prolactindeficiencycanbeseenwithpituitarynecrosisor

infarctionandinsomecasesofpseudohypoparathyroidism.
Inwomenwithcompleteprolactindeficiency,menstrual
disordersandinfertilityhavebeenfound.
y Itisprolactinexcessthatisassociatedwithclinical
Iti l ti
th ti
i t d ith li i l
pathology.HyperprolactinemialeadstoinhibitionofGnRH
secretion,whichtypicallymanifestsassexualdysfunction
,
yp
y
y
andinfertilityinbothmenandwomen.Womenmay
presentwithlutealphaseabnormalities,
oligomenorrheaorfrankamenorrhea,withorwithout
li
h f
k
h ith ith t
galactorrhea.Menwillpresentwith
hypoandrogenemia,decreasedlibidoandimpotence.
yp
g
,
p


Prolactin
y Hyperprolactinemia existsin2040%ofpatientswith

acromegaly;thisisdueeithertothepresenceofamixed
tumor(containingbothlactotrophs andsomatotrophs)orto
interferencewiththenormallyactive,prolactininhibitory
mechanisms.
mechanisms
y Anotherimportantcauseofhyperprolactinemia is
hypothyroidism.Thyrotropinreleasinghormone(TRH)not
yp
y
y
p
g
(
)
onlystimulatesTSHsecretionbutitalsostimulatesprolactin
secretionaswell,thusexplainingthemildhyperprolactinemia
seeninbothprimary(thyroid)andsecondary(pituitary)
i b th i
(th id) d
d ( it it )
hypothyroidism.Treatmentwiththyroidhormone
replacementwillusuallyreturntheprolactin
p
y
p
backtonormal.
Rarely,hyperprolactinemia maybecausedbyectopic
hormoneproduction.


Prolactin
Iti i
t tt
l t allpatients
ll ti t discoveredto
di
dt
y Itisimportanttoevaluate
haveanabnormally elevatedprolactin.Thyroid
(
y
[ 4]]andTSH)arealways
)
y
functiontests(freethyroxine[FT
indicatedtoruleouthypothyroidism.Since
hyperprolactinemiacanbefoundinupwardsof40%ofcases
ofacromegaly,itisappropriatetomeasureinsulinlike
f
l iti
i t t
i
li lik
growthfactor(IGF1)discussedbelow.Otherhormonesthat
maybeassayedincludeFSH,LH,testosterone,estradioland
ay be assayed c ude S ,
, testoste o e, est ad o a d
ifclinicallyindicated,testsofadrenalaxisfunction.All
patientsshouldundergoeitherCTorMRIofthesella,with
andwithoutcontrast.MRIprovidesbettercontrastand
d i h
MRI
id b

d
anatomicdetailandisbetterforvisualizingmicroadenomas.
Formalvisualfieldexaminationisalsoakeymonitoringtool
inmanagingpatientswithpituitarytumorsandshouldbe
doneatleastyearlyinpatientswithstabledisease.

CausesofHyperprolactinemia
y Physiologic:

Sleep stress postprandially pain

Sleep,stress,postprandially,pain
Coitus,pregnancy,nipplestimulationornursing

y Systemic.disorders:
y
y yp
y
Primaryorsecondaryhypothyroidism
Adrenalinsufficiency
Chronicrenalfailure
Cirrhosis/Chestwallorthoracicspinalcordlesions

y Medications:
Psychiatricmedications:phenothiazines,haloperidol,thioxanthines,buspirone,
olanzapine,risperidone,domperidone,monoamineoxidase inhibitors,fluoxetine,
amitriptylene
Metoclopramide
Antihypertensives:labetolol,methyldopa,reserpine,verapamil
Cimetidine,ranitidine
Estrogens,oralcontraceptives,oralcontraceptivewithdrawal
Opiates:heroin methadone morphine apomorphine
Opiates:heroin,methadone,morphine,apomorphine

Prolactinsecretingpituitarytumor:
y

prolactinoma,acromegaly
Macroadenoma (compressingthepituitarystalk)
Macroprolactinemia
Pressureonortransection ofthepituitarystalk interruptingthetransmissionofdopaminetotheD2receptors
onthelactotrophs

Ectopicsecretionofprolactin bynonpituitary tumors

Idiopathic/Polycysticovariandisease /Epilepticseizures

Thyroid
Disease
y

The Hidden Health Problem

Thyroid
Disease- The Hidden Health Problem
y

Hyperthyroidism

Hypothyroidism

Too muchh th
T
thyroid
id
hormone
Metabolism speeds
up

Nott enoughh thyroid

N
th id
hormone
Metabolism slows
down

Hyperthyroidism
yp y
Overproduction of thyroid hormone, causing
metabolism acceleration
Affects roughly
g y 1% of the U.S. ppopulation
p
or two
million Americans
Graves Disease
Most common form of hyperthyroidism
Autoimmune disorder

Hypothyroidism
yp y

Underproduction of thyroid hormone, causing metabolism to

slow
Affects up to 25 million Americans, approximately twelve million
remain
i undiagnosed
di
d
Hashimotos Thyroiditis
Most common thyroid disease in the U.S.
US
Autoimmune disease

Two Common Types of Thyroid Disease

TwoCommonTypesofThyroidDisease

y Hyperthyroidism

y Hypothyroidism

What are thyroid function tests?

Whatarethyroidfunctiontests?
y Thyroidfunctiontests(TFTs)areagroupofteststhat

arerequestedtogethertohelpevaluatethyroidgland
arerequestedtogethertohelpe aluateth roidgland
functionandtohelpdiagnosethyroidglanddisorders.
y TFTsincludeameasureoftheamountofthyroidhormones,
TFTsincludeameasureoftheamountofthyroidhormones
Thyroxine (T4)orTriiodothyronine (T3)inyourblood.

Currently,themajorityofUKlaboratoriesmeasure
thefreeformofthehormones FreeT4 (FT4)or
FreeT3 (FT3).
y thyroidFunctionTestsusuallyincludesomecombinationof:
th
idF
ti T t
ll i l d

bi ti f
y TSH(thyroidstimulatinghormoneorthyrotropin) totestfor

hypothyroidism,hyperthyroidism andtomonitorthyroidreplacement
therapy
th
y T4orFT4 totestforhypothyroidismandhyperthyroidism
y T3orFT3 totestforhyperthyroidism

Howarethyroidfunctiontestsused?
y TFTsareisusedtohelpdiagnosehypo
p
g
yp andhyperthyroidism
yp
y

y
y

whichcanbeduetovariousthyroiddiseases,oroccasionally,
disordersofthepituitarygland.
Insomelaboratories,theinitialbloodtestforthyroid
I
l b
i h i i i lbl d
f h
id
disordersisaTSHtest.IfyourTSHconcentrationis
abnormal,itwillusuallybefollowedupwithaFT4
y
p
4
measurement(oroccasionallytotalT4).InsomecasesFT3
(ortotalT3) willalsobeperformed.
Often thelaboratorywilldothisfollow uptesting
Often,thelaboratorywilldothisfollowuptesting
automatically,andthisisknownasreflextesting.
Thissavesyourdoctortimefromhavingtowaitfortheresultsof
y
g
theinitialtestandthenrequestingtheadditionaltestingto
confirmorclarifyadiagnosis.
F ll
Followuptestsareoftenperformedontheoriginalsamplethat
t t ft f
d th i i l
l th t
wassubmittedwhentheinitialtestwasrequested.

the following table summarises test results and their potential meaning.

TSH

T4

T3

InterpRetation

High
Hi h

Normal
N
l

Normal
N
l

(
)
Mild(subclinical)
hypothyroidism

High

Low

Lowornormal

Hypothyroidism

Low

Normal

Normal

Mild(subclinical)
hyperthyroidism

Low

Highornormal

Highornormal

Hyperthyroidism

Lowornormal

Nonthyroidal illness;
Rarely
hypothyroidism
duetopituitary
disease
di

Low

Lowornormal

CharacterizationofThyroidDisordersAccordingtoResultsofThyroidFunctionTests
Disorder

TSH

T4

T3

FT4

Tg

TBG

rT3

ATPO

ATG

TBII

TSI

TBA

Primary
hypothyroidism

N or

N or

N or

N or

N or

n or

Transient
neonatal
hypothyroidism
Hashimoto
thyroiditis
hypothyroidism
Graves' disease

N or

N or

N or N or N or

n or

n or

n or

Neonatal Graves'
disease

n or

n or

n or

TSH deficiency

N or

Thyroid

dishormonogene
sis
Thyroid hormone N or
resistance

N, or

N
V

V
N or

N
V

N
N

V+
N

V
N or

n
n

n
n

n
n

n
n

n
n

or

or

or

TSH-dependent
hyperthyroidism
T4 protein-binding
abnormalities[*]
Nonthyroidal
illness
Subacute
thyroiditis[]

or or or

ThyroidStimulatingHormone(TSH)
y TheradioimmunoassayformeasuringTSHwasfirst

developedbyOdellandcolleaguesin1965.
sensitive immunometric TSHmethodusingeither
y sensitive'immunometric
monoclonalorpolyclonalantibodieswasdevelopedwhich
hadanimprovedsensitivityto0.10.2mU/L.
y Athirdgenerationnonisotopic immunometric TSHassay
usingachemiluminescent labelwasdevelopedinthe
1990s;thisistheassaymethodwhichiscurrentlyin
commonuse.
y Itssensitivitywasreducedtoabout0.005mU/L,

whichis100timesmoresensitivethanthemost
sensitive'TSHassayand1000timesmoresensitive
thanradioimmunoassaymethods.

ThyroidStimulatingHormone(TSH)
y AlthoughafourthgenerationTSHassayhas

recentlybeendevelopedwithasensitivityof
0.0004mU/L,inadditiontoitnotbeingwidely
0 0004mU/L inadditiontoitnotbeingwidely
availablethethirdgenerationassaysprovide
sufficientsensitivityforthevastmajorityof
clinicalapplications.
y TheAmericanThyroidAssociation(ATA)
recommendationsstatethatthirdgeneration
assaysshouldbeabletoquantitate TSHinthe
0.010.02mU/Lrangeonaninterassay basis
withacoefficientofvariationof20%orless..
ith
ffi i t f i ti f % l

ThyroidStimulatingHormone(TSH)

Reference range TSH

Prematures, 28-36 wk (1st week of life)
Cord blood (>37 wk)
Birth to 4 days
2-20 wk
21 wk-20 yr
Adults 21-54 yr
Adults 55-87 yr
Pregnancy
First trimester

mlU/L
0.7-27.0
2.3-13.2
1.0-39.0
1.7-9.1
0.7-64.0
0.4-4.2
0.5-8.9
0.3-4.5

Pregnancy
Second trimester

0.5-4.6

Pregnancy
Third trimester

0 8-5 2
0.8-5.2

T4:Thyroxine

TBG:Thyroxine BindingGlobin

.

) (






.

T4:Thyroxine

T4
) (
T3

T4

Reference range T4
Cord
Children
C
d e 1-3
3 days
d ys
Children 1-2 wk
Children 1-4 mo
Children 4-12
4 12 mo
Children 1-5 yr
Children 5-10 yr
Children 10-15 yr
Adults (15-60 yr) Males

Micro g/dL
7.4-13.0
11.8-22.6
.8 .6
9.8-16.6
7.2-14.4
7.8-16.5
7 8 16 5
7.3-15.0
6.4-13.3
5.6-11.7
4.6-10.5

nmol/L
95-168
152-292
5 9
126-214
93-186
101-213
101 213
94-194
83-172
72-151
59-135

Adults (15-60 yr) Females

5.5-11.0

65-138

Adults >60
60 yr

5.0 10.7
5.0-10.7
>7.5

65 138
65-138
>97

>6.5

>84

Newborn screen
(whole blood)
1-5 days
Newborn
N
b
screen
(whole blood)

6 days

FT4:FreeThyroxine

.
y MostT4inbloodisattachedtoaprotein;lessthan1%is

unattached.Thebloodtestcanmeasureeitherthetotal
(bothboundandunattached,TT4)orfree(unattached,
FT4)T4hormoneinyourblood.
y Scientistsbelievethatfreehormoneisresponsibleforall
p
theeffectsofthyroidhormone.

Binding of Thyroid Hormones

BindingofThyroidHormones
99
g y
y Morethan99%ofcirculatingthyroidhormonesare
boundtoserumproteins
y Thyroxinebindingglobulin(TBG)
y Thyroxinebindingprealbumin (TBPA)
y Albumin(TBA)

y T4ismoreextensivelyboundthanT3
l b
d h
y 0.04%oftotalT4iffree
y 0.4%oftotalT3isfree
% ft t lT i f


/



/
/

)(%15-30

) ((%15-30

Reverse T3

)
(

) Thyroglobin ( TG

30

:
-1
-2 )

-3

%20

:
.

Thyroid antibody testing

Thyroidantibodytesting
y Thyroidantibodytestingisprimarilyrequestedtohelp

diagnoseanautoimmunethyroiddiseaseandtoseparateit
fromotherformsofthyroiditis andthyroiddisease.
y Itmaybeusedtohelpinvestigatethecauseofanenlargedthyroid
gland(goitre)and/orperformedasafollowupwhenother
l d(
) d
f
d
f ll
h
h
thyroidtestresults(suchasT3,T4,and/orTSH)showsignsof
thyroiddysfunction.
y
y
y Thyroidantibodytestsmayalsoberequestedifapersonwitha
knownnonthyroidrelatedautoimmunecondition,suchas
systemiclupuserythematosus,rheumatoidarthritis,orpernicious
t i l
th
t
h
t id th iti
i i
anaemia,developssymptomsthatsuggestthyroidinvolvement.
Thisinvolvementmayoccuratanytimeduringthecourseofthe
othercondition(s).

Thyroidantibodytesting

When is it requested?

ThyroidAntibody

Acronym

Presentin

Whenordered
Whenpatienthassymptomsortest

Thyroidperoxidase antibody

Autoimmunethyroid
resultssuggestinghypothyroidism;when
doctorisconsideringstartingapatienton
d t i
id i t ti ti t
disease:
di
adrugtherapy,suchaslithium,
Hashimotosthyroiditis (95%);
amiodarone,interferonalspha,or
TPOAb
primarymyxoedema (90%); interleukin2,thathasassociatedrisksof
developinghypothyroidismwhenTPOAb
p g yp y
Gravesdisease(18%)
(
)
arepresent

Thyroglobulin antibody

TgAb
Tg
Ab

Testedaspartofmonitoringof
treatmentforthyroidcancer.
Al
Alsopresentinautoimmune
i
i

thyroiddisease.

Thyroidstimulatinghormone
receptorantibody
TRAb Gravesdisease

NotasreliableasTPOAb intestingfor
autoimmunethyroid
disease. Thyroglobulin antibodytogether
withthyroglobulin levelsareusedat
regularintervalsafterthyroidcancer
Th
f h
l b li
treatment. Thepresenceofthyroglobulin
antibodiesmayinterferewiththetestfor
thyroglobulin whichisalsotestedasa
tumour marker.

Whenpatienthassymptoms
of hyperthyroidism;tomonitor
effectivenessofantithyroidtherapy


Anti TSH Receptor
:
-1 -2

:

-1
) (
-2







)
(




:
2

4 6

) ( .

24
) ( 3 6

) (

TSHscreening
y TSHscreeningisroutinelyperformedintheUnited

Statesonnewborns aspartofeachstate'snewborn
screeningprogram.
i

y TheAmericanThyroidAssociationrecommendsthat
adultsolderthanage35 bescreenedforthyroid
diseasewithaTSHtesteveryfiveyears,although
otherorganizations,suchastheU.S.Preventive
ServicesTaskForce,challengethisrecommendation.
y Severalorganizationsrecommendinsteadscreening
womenover50

orthoseathighriskforthyroid
th thi h i kf th id
disorders,suchaspregnantandpostpartumwomen

y Doctorsdonotgenerallytest

y p
women,butthosewith
asymptomatic
symptomsand/oraknownthyroid
disorder maybetestedatintervals
y
recommendedbythedoctortodetect
yp
y
or
andevaluatehyperthyroidism
hypothyroidism bothduringpregnancy
andafter

Interferance thyroidpannel tests

y Manymedications includingaspirinandthyroid

hormonereplacementtherapy mayaffect
thyroidglandfunctiontestresultsandtheiruseshould
bediscussedwiththedoctorpriortotesting.
y Whenadoctoradjustsaperson'sthyroidhormone
Whenadoctoradjustsaperson sthyroidhormone
replacementdosage,itisimportanttowaitat
leastonetotwomonthsbeforecheckingtheTSH
againsothatthenewdosecanhaveitsfulleffect.

y Extremestressandacute illnessmayalso

affectTSHtestresults,andresultsmaybelow
ff tTSHt t
lt d
lt
b l
duringthefirsttrimesterofpregnancy.

Whatisa3rdgenerationTSH
g
andanultrasensitiveTSH?
y Bothofthesetermsrelatetotheevolutionof

gy
theTSHtest.Overtime,increasingly
sensitive andspecific TSHtestshavebeen
p
p
developedandadopted.Mostlaboratories
nowusethe3rdgeneration/ultrasensitive
TSHtestastheir"TSHtest."Thisnewest
versionisabout100timesmoresensitive
thanthefirstgenerationTSHtest.

ThyroidStimulating
Hormone (TSH)
Thyroid
StimulatingHormone(TSH)
y TSHisaglycoproteinconsistingoftwomono

covalentlylinkedalphaandbetasubunits.
covalentlylinkedalphaandbetasubunits
y Thealphasubunithasthesameaminoacidsequences
asluteinizinghormone(LH),folliclestimulating
g
( ),
g
hormones(FSH)andhumanchorionicgonadotropin
(HCG).
y Itisthebetasubunitthatcarriesthespecific
informationtothebindingreceptorsfor
expressionofhormonalactivities.
expressionofhormonalactivities

 Hypothyroidism

::
-1
-2 )

(

3 ) (
-4

-5

:



.
:
/



/
/

 Hyperthyroidism


:

(
)

:


.


6-8

.
/

/

/

/

)





(
) (

)
(..

:



) (
)
(..



:
-1

)
-2
(
-3

4 )

-5

-6
-7

-8
/
/
/
...

-9

..


(1
) Biorad ) (SERO

(
(2 ) (

ReconfirmofabnormalresultofElISA* methodwithELFA
(Vidas)orECL

(3
(
Recheckedwithnew

sample

(4

) ( :


(1

Recheckedwithsamesample


(5 Patientmeancontrol


T3/TSH/Ferritin

: Delta.Check
Delta Check

(6
)
(
(7 : Correlation.Check

ANA RF...




(8

(Daily&weekly&monthlyPM)preventivemaintenance



:

(9
Verification& validationofnewmethod&equipment




-1

-2 ) (

) (

-3

) (...
) (...
) / (...
( ) (
(

-4
( 3 ) (
4

(
) (

) PCR Western blot
PCR, (
) (Validate
) -( ) (Validate
) (Mantle Lab
) (Validate

-
) (Validate

)(
-
-

) ((.
) (

-
) (

) (Batch

- - ) (

-70c , -20c

) (Radial immunodiffusion - -
-
-

A
1
/
2

3

4

5


6
) (
7

30
10
10
10
10
10

15
15

15

10

B
11

20

12

13

20

14

20

15

20

16

20

17

20

18

20

19

10

20

300

%60-70 ) 180-210( ) (
) %70 - %80 210-240( ) (
) %80 240(

:



.

Chemiluminesence

:
)
/ / (...

Chemiluminesence

-1
:

) (10 -21 /

(
-2

-3
-4

-5

-6
-7

-8


CL & ECL


ECL

430

Classification of Chemiluminesence

Flash method
0.5 3 2 4

) (

GLOWmethod
2 7 20 30

Chemiluminesence


-1

) (
)
-2
/ (

-3

-4
-5

-6 ) (10 -15

LuminescentSystems
y Chemiluminescence,electroluminescence, andbioluminescence

areallbiochemicalsystemsthatproducelight.
y Inachemiluminescent system, achemicalreaction(usually
oxidation)generatesanorganicmoleculeinanelectronically
excitedstate,whichemitsphotonsuponreturntothe
groundstate.
y Thechemicalreactionisinitiatedbytheadditionofanoxidizing
agent.Lightemissionbyelectroluminescenceisaccomplishedby
generatinganelectronicallyexcitedstateofanappropriate
moleculethroughtheapplicationofanelectricpotential.
y .Eachofthesesystemsoffersthefollowingadvantages:

LuminescentSystems
Eachofthesesystemsoffersthefollowingadvantages:
y .Eachofthesesystemsoffersthefollowing
.1 Becausesampleradiationisnotrequired,thehigh
g
g
g
p
backgroundsduetolightscatteringandnonspecific
excitationareeliminated,allowinggreatersensitivity and
dynamicrange.
2 Improvedsignaldetectioncoupledwiththepowerof
2
immunoassaytechnologyprovidesbetterspecificity.
3
3 Luminescentassaysoftenoccuronsolidphaseswhich
y
p
capturetheanalyte andwashawayinterferingsubstances,
enhancingsensitivityandspecificity.
4 Luminescentreagentsandconjugatesaregenerallystableand
4
Luminescentreagentsandconjugatesaregenerall stableand
nontoxic.

AssayDescription
y
p
y Assayformatsaretypicallymonoclonal/polyclonalsandwich

assaysonsolidphases.Bothdirectandindirectcompetitive
immunoassayprotocolspredominateinchemiluminescent
i
t
l d i t i h il i
t
systems.
y Quantitativelightemissionmayresultfromdirectchemical
cleavageofanacridinium esterforexample;orfromtheaction
ofanenzymeorenzymaticsystemonaluminescentsubstrate
(e g luciferin/luciferase oraluminol/enzymepair); orfrom
(e.g.,luciferin/luciferase
anelectronicallyexcitedstateofanappropriatemolecule,
resultingfromtheapplicationofanelectricalpotential
differencetothereaction.

Limitations and Precautions

LimitationsandPrecautions

y Becausephotondetectionisaverysensitivemethod,

contaminatingsignalsmayresultfromavarietyofsources.
Biologicalorganismsproduceluminescentmaterials;
cleaningsolutionsandlaboratorydustmaycontain
substa ces t at p oduce g t; etc.
substancesthatproducelight;etc.
y Thevaliditycriteriaforbackgroundmeasurementsandassay
measurementsaresafeguardsagainstmanyinterfering
substances.
b
y Themaintenanceproceduresprovidedbythemanufacturer
addresssystemdecontaminationandcleanliness,butthe
importanceofGoodLaboratoryPracticescannotbe
overstatedwhenworkingwiththistechnology

ELISA TROUBLESHOOTING
ELISATROUBLESHOOTING
y Dr. mehrdad vanaki
y 88/10/17


ELISA
y
EnzymeLinkedImmuno
SorbentAssayy
y

:


-1 :

)

(
-2 :


)
(
.


Elisa Sandwich
AbsandwichorAgCapture


PSA/TSH/FSH/LH/HCG

AgSandwichorAbCapture

.


Elisa Competetive & Blocking
Elisa Competetive :

(
)

Elisa Blocking :


(

)

) (

ELISATROUBLESHOOTING
NoSignalorWeakSignal
HighBackground
PoorDuplicate
PoorStandardCurve
P
S
d dC
PoorLinearity
PoorAssaySensitivity
PoorCorrelation
UnexpectedClinicalClassification
ApparentShiftinReferenceInterval
A
tShifti R f
I t
l

ELISAStage

No Signal or Weak Signal

Expire
E i date,
d t
incorrect storage,
reagent not to RT

Omission of key
reagents

Collection system

Inactivation, Incomplete
or incorrect preparation
of chromogen

Scratch

Wrong filter

incubation
Incorrect time or temperature
Inactivation of
conjugate

Wash too
stringent

No Signal or Weak Signal

-1
) (
(

-2

-3
-4

No Signal or Weak Signal

-5





-6
-7

-8 ) (

)
(

HIGHBACKGROUND
Wrong filter in ELISA
reader

Contaminating
enzyme present
in sample

Improperly set up blank

Chromogen exposed to light

Cross
contamination from
other specimen or
positive control

Contaminated substrate
solution with metal ions
or oxidizing reagent

Incorrect assay temperature

Evaporation of well
during incubation

Concentration of
conjugate too high

Contamination of pipette , dispenser

or substrate with conjugate

Inadequate washing

HIGHBACKGROUND


-1

-2

-3




-4
-5 )
(




)
(

HIGHBACKGROUND
-6 )
(

-7

-8 /

-9
) (
) (

Poor Duplicate
PoorDuplicate
Dirty microwell
Particulate or
precipitate
in sample
Edge effect
Plate scratch
I
Incorrect
t dispensing
di
i off reagents
t
Pipetting error of standard
sample

Transfer liquid from well to well

Poor Duplicate

)
(

-1 )
(

-2
-3
-4
-5
-6

Poor Standard Curve

PoorStandardCurve

Reagent from
different kit or
different lot

Plate not clean

Pipetting error
poor dilution

Poor mixing of reagents

Improper , insufficient washing
Plate scratch

PoorStandardCurve
-1

-2
...

-3
-4

-5
-6


CalibrationcurveorDoseResponseCurve
)
(

)
( )
(
:

-1 : ) (
-2 : ) (

Poor Assay Sensitivity

PoorAssaySensitivity
Kit lot at fault

Incomplete mixing
of reagents

Poor kit design or

optimization

Kit reagents not

allowed to RT

Dirty tube or
wells
Variation of
wells
Incomplete mixing of
zero calibrator

Deterioration of kit
during shelf life or
exposure to extreme
temperature

Insufficient slop of
curve at very low
concentration

Poor Assay Sensitivity

-1

-2
-3

-4
..

-5
-6


-7

-8

Poor Correlation Between Two Kits

PoorCorrelationBetweenTwoKits
Incorrect
I
t

Kit lot at fault

Standardization of Kit
Instability of
reference
standard

Use of different
units or
international
standard

Used of old
samples

Equipment fault
Limited range of
concentration used

Limited number of patient

sample used for study

Curve fit bias

C
bi with
ith
either or both kits
Only one or two assay
performed

Poor Correlation Between Two Kits

-1
-2
-3
-4
-5
10





-6 /

-7
-8

U
UnexpectedorInconsistentClinicalClassification
t d I
i t t Cli i l Cl ifi ti
Unusual
off
U
l type
t

Change in

patient sample

protocol

High dose hook

effect

Kit lot at fault

Error or instability
in store calibration
curve

Interference by
cross reactant
(drug metabolites)
Incomplete mixing or
warming up of reagents
Incorrect storage of
sample or reagents

Equipment fault
Wrong sample tested or
wrong label

P
Poor
assay design
d i
Use incorrect unite

Unexpected or Inconsistent Clinical Classification

/
-1 ) /
(...
-2 / /
-3 /
-4 /
-5
-6
-7
-8


-9
-10 )
/
/ /

Apparent Shift in Reference Interval

ApparentShiftinReferenceInterval
Change
in
Ch
i
technician

Use of expired
kit

Change in sample

Error or instability
in sto
store
e ca
calibration
b at o
curve

collection method

Change in laboratory
temp/humidity

Equipment fault
Change in protocol

Poorly calibrated or
unstable calibrator

Change in curve fit

program


-1
-2
-3
-4
-5
-6
-7
-8

ALLWELLYELLOW
y Contaminatedsubstrate
y Contaminatedstopsolution
y Incorrectdilution
y Inefficientwashing
ffi i

hi

ALLWELLCLEAR
y ELISAnotperformedcorrectly
y Contaminatedconjugate
y Incorrectstorageofkit
y Overwashingofplate


-1 37 5
)


-2 /

-3 48
2 8 48
20
-4










-5

-6 %70
-7 ) (

20 25

-8
.


-9 37 -+2 .
Edgeeffect

37

(

)

.

-10


-11

. .
-12

-13 :(



(
(

-14


-15

-16
-17

.
.


-18
.
-19 )

( ) (
-20 ) 8(


soakingTime
-21 : 20
40
.
-22 ) 20(

-23

-24





.


Welltowellcontamination
-25 :

.











.

.
H k ff t
Hookeffect
-26 :
)
/
/




(

/TSH PSA/CA125




.
:
) (

-1
-2


highbackgroundnoise
-26 : ) (
0.1 ) 0.05( /
/
0.2 ) (%1




(

)




27
) (

-28

)
(


DRIFT -29 :
/
/

:
-1
.



-2
-3

-4

-5
-6


-1


-2
-3

-4

-5
)

(
-6
-7
-8 200 ) (
((%5

%3
)


-9 ) 2 5
(
-10


-1 y

.
: / /
/

-2 y

/
/

-3 y

-4 y

) y
-5
(
y


-1



-2 ) (
. 7.2
.

-3

-4
.
-5

 ) (

1200
16 32

)

TSH&Hbs
S & bs Ag
g


/
Diagnostic sensivity / Diagnostic Specifity
y Diagnostic sensivity
y

(

)
y Diagnostic Specifity
y

y
(


)
y :
-1 ) ( y
-2 y
-3 y


/
Analytical sensivity / Analytical Specifity
:Analytical sensivity

:Analytical Specifity

. :