0.makmal Diagnostik Reference 1

10/22/2015 CLINICALBIOCHEMISTRY(alsoknownasclinicalchemistryorchemicalpathology)isthelaboratoryserviceabsolutelyessentialformedicalpr
INTRODUCTION,ROLESOFBIOCHEMICALLABORATORY.
MECHANIZATIONANDAUTOMATIONINCLINICALBIOCHEMISTRY
CLINICALBIOCHEMICALANALYSESOFPROTEINS,PLASMAPROTEIN
SPECTRUM.ANALYTICALMETHODS.
ANALYSESUSEDINDIAGNOSTICSOFDIABETESMELLITUS.
CLINICALBIOCHEMISTRY(alsoknownasclinicalchemistryorchemicalpathology)isthelaboratoryservice
absolutelyessentialformedicalpracticeorbranchoflaboratorymedicineinwhichchemicalandbiochemicalmethods
areappliedtothestudyofdisease.
Theresultsofthebiochemicalinvestigationscarriedoutinaclinicalchemistrylaboratorywillhelpthecliniciansto
determinethediseases(diagnosis)andforfollowupofthetreatment/recoveryfromtheillness(prognosis).
Theuseofbiochemicaltests:
Biochemicalinvestigationsareinvolvedineverybranchofclinicalmedicine.
Theresultsofbiochemicaltestsmaybeofusein:
1.diagnosisandinthemonitoringoftreatment.
2.screeningfordiseaseorinassesingtheprognosis.
3.reseachintothebiochemicalbasisofdisease
4.clinicaltrialsofnewdrugs
Biochemicalinvestigationsholdthekeyforthediagnosisandprognosisofdiabetesmellitus,jaundice,myocardial
infarction,gout,pancreatitis,rickets,cancers,acidbaseimbalanceetc.Successfulmedicalpracticeisunimaginable
withouttheserviceofclinicalbiochemistrylaboratory.
Ingeneral,biochemicaltestscanbebroadlydividedintotwogroups:
Indiscretionaryorselectiverequesting,thetestsarecarriedoutonthebasisofanindividualpatient'sclinicalsituation.
Thecasefordiscretionaryrequestinghasbeenputadmirably(Asher,1954):
1.WhydoIrequestthistest?
2.WhatwillIlookforintheresult?
3.IfIfindwhatIamlookingfor,willitaffectmydiagnosis?
4.Howwillthisinvestigationaffectmymanage
mentofthepatient?
5.Willthisinvestigationultimatelybenefitthepatient?
Incontrast,screeningtestsareusedtosearchfordiseasewithouttherebeinganynecessaryclinicalindicationthat
diseaseispresent.
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ThesituationsinwhichdiscretionarytestrequestsareundertakenarelistedinTable1.2
Table1.2Testselectionforthepurposesofdiscretionarytesting
Category
Example
Toconfirmadiagnosis
Plasma(freeT4)and(thyroidstimulating
hormone,TSH)insuspected
hyperthyroidism
Toaiddifferentialdiagnosis
Todistinguishbetweendifferentformsof
jaundice
Torefineadiagnosis
UseofACTHtolocalizeCushing's
syndrome
Toassestheseverityofdisease
Plasma(creatinine)or(urea)inrenal
disease
Tomonitorprogress
Plasma(glucose)tofollowofpatientswith
diabetesmellitus
Todetectcomplicationsorsideeffects
ALTmeasurementsinpatientstreatedwith
hepatotoxicdrug
Tomonitortherapy
Plasmadrugconcentrationinpatients
treatedwithantiepilepticdrugs
Screeningmaytaketwoforms:1.Wellpopulationscreeninginwhichtypicallyaspectrumoftestsiscarriedouton
individualsfromanapparentlyhealthypopulationinanattempttodetectpresymptomaticorearlydisease.Thevalueof
wellpopulationscreeninghasbeencalledintoquestionandcertainlyshouldonlybeinitiatedundercertainspecific
circumstanceswhicharelistedinTable1.3.
Table1.3Requirementsforwellpopulationscreening
Thediseaseiscommonorlifethreatening
Thetestsaresensitiveandspecific
Thetestsarereadilyappliedandacceptabletothepopulationtobescreened
Clinical,laboratoryandotherfacilitiesareavailableforfollowup
Economicsofscreeninghavebeenclarifiedandtheimplicationsaccepted
2.Casefindingscreeningprogrammesperformappropriatetestsonapopulationsampleknowntobeathighriskof
aparticulardisease.
Theseareinherentlymoreselectiveandyieldahigherproportionofusefulresults(Table1.4).
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Table1.4Examplesoftestsusedincasefindingprogrammes.
Programmestodetectdiseasesin
Chemicalinvestigations
Neonates:
PKA(phenylketonuria)
Serum[phenylalanine]
Hypothyroidism
Serum[TSH]and/or[thyroxine]
Adolescentsandyoungadults:
Substance
Drugscreen
abuse
Pregnancy:
Diabetesmellitusinthemother
Plasmaandurine[glucose]
Openneuraltubedefect(NTD)inthefoetus
Maternalserum[afetoprotein]
Industry:
Industrialexposuretolead
Blood[lead]
Industrialexposuretopesticides
Plasmacholinesteraseactivity
Malnutrition
Plasma[albumin]and/or[pre
albumin]
Thyroiddysfunction
Plasma[TSH]and/or[thyroxine]
ADVANTAGESOFSCREENING
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First,anuncommonorunexpecteddiseasemaybefoundandcreated(Table1.5).Second,theearlyrequestingofa
batteryoftestsmightbeexpectedtoexpeditemanagementofthepatient.Moststudieshavenotshownthistobeso.
Table1.5Advantagesofscreeninginidentifyingunexpectedtestresults
Disease
Unexpectedabnormaltestresults
Hyperparathyroidism
Raisedplasmacalcium
Hypothyroidism
RaisedplasmaTSHand/oralowT4
Diabetesmellitus
Highrandomplasmaglucose
Renaltractdisease
Raisedplasmacreatinineorurea
Liverdisease
IncreasedplasmaALT,AST
DISADVANTAGESOFSCREENING
Itiseasytomisssignificantabnormalitiesinthe'flood'ofdatacomingfromthelaboratory,evenwhenthe
abnormalitiesare'flagged'insomeway.Mostoftheabnormalitiesdetectedwillbeoflittleornosignificance,yetmay
needadditionaltimeconsumingandoftenexpensiveteststoclarifytheirimportance(orlackofit).
Inotherinstances,tosimplifyrequesting,awiderangeoftestsareroutinelyrequestedonallpatientsinaparticular
category,forexample,admissionscreeningonallthoseadmittedthroughtheAccidentandEmergency(A&E)
Department.Mentionshouldalsobemadeofbatteriesoftestswhicharegenerallyrequestedonadiscre
tionarybasis
butwherethetestgroupcollectivelyprovidesinformationaboutanorgansystem(e.g.testsforliverdisease)ora
physiologicalstate(e.g.waterandelectrolytestatus).Manylaboratoriesanalyseandreportthesefunctionalororgan
relatedgroups.Forexample,a'liverfunctiontest'groupmightconsistofplasmabilirubin,alanineaminotransferase
(ALT),alkalinephosphatase(ALP),fglutamyltransferase(GGT)andalbuminmeasurements.
Clinicalbiochemicaltestscompriseoverofallhospitallaboratoryinvestigations.
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Corebiochemistry:Mostbiochemistrylaboratoriesprovidethe"coreanalyses",commonlyrequestedtestswhichare
ofvalueinmanypatients,onafrequentbasis.
Corebiochemicaltests:
Sodium,potassium,chlorideandbicarbonate
Ureaandcreatinine
Calciumandphosphate
Totalproteinandalbumin
Bilirubinandalkalinephosphatase
Alanineaminotransferase(ALT)andAspartateaminotransferase(AST)
Glucose
Amylase.
Specializedtests:
Noteverylaboratoryisequipedtocarryoutallpossiblebiochemistryrequests.
Largedepartmentsmayactasreferencecentreswherelesscommonlyaskedfortestsareperformed.
Specializedtests:
Hormones
Specificproteins
Traceelements
Vitamins
Drugs
Lipidsandlipoproteins
DNAanalyses
Theemergencylab:
Allclinicalbiochemistrylaboratoriesprovidefacilitiesforurgenttests.Anurgenttestisdesignatedasoneonwhichthe
clinicianislikelytotakeimmediateaction.Themainreasonforaskingforananalysistobeperformedonanurgent
basisisthatimmediatetreatmentdependsontheresult.
Emergencytests:
Ureaandelectrolytes
Bloodgases
Amylase
Glucose
Salicylate
Paracetamol
Calcium
TYPESOFLABORATORYTESTS:
Thebiochemicalinvestigations(onblood/plasma/serum)carriedoutintheclinicalbiochemistrylaboratorymaybe
groupedintodifferenttypes.
1.Discretionaryoronofftests:Mostcommonclinicalbiochemistryteststhataredesignedtoanswerspecific
questions,e.g.,doesthepatienthaveincreasedbloodurea/glucoseconcentration?Normally,thesetestsareusefulto
supportthediagnosis.
2.Biochemicalprofiles:Thesetestsarebasedonthefactthatmoreusefulinformationonthepatientsdiseasestatus
canbeobtainedbyanalysingzormoreconstituentsratherthanonee.g.,plasmaelectrolytes(Na+,K+,Cl,bicarbonate,
urea)liverfunctiontests(serumbilirubin,ALT,AST).
3.Dynamicfunctiontests:Thesetestsaredesignedtomeasurethebody'sresponsetoexternalstimuluse.g.,oral
glucosetolerancetest(toassessglucosehomeostasis):bromosulphthfeintest(toassessliverfunction).
4.Screeningtests:Thesetestsarecommonlyemployedtoidentifytheinbornerrorsofmetabolism,andtocheckthe
enteryoftoxicagents(pesticides,lead,mercury)intothebody.
5.Metabolicworkuptests:Theprogrammedintensiveinvestigationscarriedouttoidentifytheendocrinological
disorderscomeunderthiscategory.
Thetermemergencytestsisfrequentlyusedintheclinicallaboratory.Itreferstotheteststobeperformedimmediately
tohelptheclinicianforpropertreatmentofthepatiente.g.,bloodglucose,urea,serumelectrolytes.
Thereareover400differenttestswhichmaybecarriedoutinclinicalbiochemistrylaboratories.Theyvaryfromthe
verysimple,suchasthemeasurementofsodium,tothehighlycomplex,suchasDNAanalysis,screeningfordrugs,or
differentiationoflipoproteinvariants.Manyhighvolumetestsaredoneonlargeautomatedmachines.Lessfrequently
performedtestsmaybeconvenientlycarriedoutbyusingcommerciallypreparedreagentspackagedin"kit"form.
Someanalysesarecarriedoutmanually.
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Specimencollection:
Thebiologicalfluidsemployedintheclinicalbiochemistrylaboratoryincludeblood,urine,saliva,sputum,faeces,
tissueandcells,cerebrospinalfluid,peritonealfluid,synovialfluid,pleuralfluid,stones.
Amongthese,blood(directlyorintheformofplasmaorserum)isfrequentlyusedfortheinvestigationsintheclinical
biochemistrylaboratory.
Identificationofpatientsandspecimens
Thecorrectpatientmustbeappropriatelyiden
tifiedonthespecimenandrequestform,asfollows:
1.Patientidentificationdata(PID).Thisusuallycomprisesnameplusuniquenumber.
2.Testrequestinformation.Thisincludesrelevantclinicaldetails(includinganyriskofinfectionhazard),theteststo
beperformedandwherethereportistobesent.
3.Collectionofspecimens.Inthecorrecttubeandtheappropriatepreservative.
4.Matchingofspecimenstorequests.Eachspecimenmustbeeasilyandunequivocallymatchedtothecorresponding
requestforinvestigations.
Table1.1Somecommonercausesoferrorsarisingfromuseofthelaboratory.
Error
Consequence
Crossover of addressograph Thiscanleadtotwopatientseachwiththeother'ssetof
labelsbetweenpatients
results. Labels between patients Where the
patientisassignedacompletelywrongsetofresults,itis
important to investigate the problem in case there is a
secondpatientwithacorrespondingwrongsetofresults
Timingerror
Therearemanyexampleswheretimingisimportantbut
notconsidered.Sendinginabloodsampletooearlyafter
the administration of a drug can lead to misleadingly
high values in therapeutic monitoring. Interpretation of
some tests (e.g. cortisol) is critically dependent on the
timeofdaywhenthebloodwassampled
Sample
collection
tube Forsometeststhenatureofthecollectiontubeiscritical
error
whichiswhytheBiochemistryLaboratoryspecifiesthis
detail. For example, using a plasma tube with lithium
heparinastheanticoagulantinvalidatesthissampletube
for measurement of a therapeutic lithium level! Serum
electrophoresis requires a serum sample otherwise, the
fibrinogen interferes with the detection of any
monoclonalbands.Toppingupabiochemistrytubewith
a haematology (potassiumethylenediamine tetraacetic
acid (EDTA) sample) will lead to high potassium and
lowcalciumvaluesinthebiochemistrysample
Sample taken from close to Thebloodsamplewillbedilutedsothatallthetestswill
the site of an intravenous be correspondingly site of an intravenous (IV)
infusion
infusionlowwiththeexceptionofthosetestswhich
might be affected by the composition of the infusion
fluid itself. For example, using normal saline as the
infusingfluidwouldleadtoaloweringofalltestresults
butwithsodiumandchlorideresultswhicharelikelyto
beraised
Analyticalerror Althoughcomparativelyrare,thesedoinevitablyhappen
from time to time and any result which is unexpected
shouldleadtherequestingcliniciantodiscussthematter
further with the Laboratory. Transcription errors within
theLaboratoryareincreasinglylesscommonbecauseof
the electronic download of results to the Laboratory
computer as a source of the printout or results on the
VDU.MosterrorsgeneratedwithintheLaboratoryoccur
at the Reception as a result of mislabelling of samples
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withintheLaboratory
COLLECTIONOFBLOOD:
Venousbloodismostcommonlyusedforamajorityofbiochemicalinvestigations.Itcanbedrawnfromany
prominentvein(usuallyfromaveinonthefrontoftheelbow).
Capillaryblood(<0.2ml)obtainedfromafingerorthumb,islessfrequentlyemployed.
Arterialblood(usuallydrawnunderlocalanesthesia)isusedforbloodgasdeterminations.
Precautionsforbloodcollection:Useofsterile(preferablydisposable)needlesandsyringes,cleaningofpatients
skin,bloodcollectionincleananddryvials/tubesaresomeoftheimportantprecautions.
Biochemicalinvestigationscanbeperformedon4typesofbloodspecimenswholeblood,plasma,serumandred
bloodcells.Theselectionofthespecimendependsontheparametertobeestimated.
1.Wholeblood(usuallymixedwithananticoagulant)isusedfortheestimationofhemoglobin,carboxyhemoglobin,
pH,glucose,urea,nonproteinnitrogen,pyruvate,lactate,ammoniaetc.(Note:forglucosedetermination,plasmais
preferedinrecentyears).
2.Plasma,obtainedbycentrifugingthewholebloodcollectedwithananticoagulant,isemployedfortheparameters
fibrinogen,glucose,bicarbonate,chloride,ascorbicacidetc.
3.Serumisthesupernatantfluidthatcanbecollectedaftercentrifugingtheclottedblood.Itisthemostfrequentlyused
specimenintheclinicalbiochemistrylaboratory.Theparametersestimatedinserumincludeproteins
(albumin/globulins),creatinine,bilirubin,cholesterol,uricacid,electroylets(Na+,K+,Cl),enzymes(ALT,AST,
LDH,CK,ALP,ACP,amylase,lipase)andvitamins.
4.Redbloodcellsareemployedforthedeterminationofabnormalhemoglobins,glucose6phosphatedehydrogenase,
pyruvatekinaseetc.
Collectionandpreservationofbloodspecimens
Lackofthoughtbeforecollectingspecimensorcarelessnessincollectionmayadverselyaffecttheinterpretationor
impairthevalidityofthetestscarriedoutonthespecimens.Somefactorstoconsiderincludethefollowing:
1.DietDietaryconstituentsmayaltertheconcen
trationsofanalytesinbloodsignificantly(e.g.plasma[glucose]and
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[triglyceride]areaffectedbycarbohydrateandfatcontainingmeals,respectively).
2.DrugsManydrugsinfluencethechemicalcompo
sitionofblood.Sucheffectsofdrugtreatment,forexample,
antiepilepticdrugs,havetobetakenintoaccountwheninterpretingtestresults.Detailsofrel
evantdrugtreatmentmust
begivenwhenrequest
ingchemicalanalyses,especiallywhentoxicologicalinvestigationsaretobeperformed.
3.Diurnalvariation.Theconcentrationsofmanysubstancesinbloodvaryconsiderablyatdifferenttimesofday(e.g.
cortisol).Specimensfortheseanalysesmustbecollectedatthetimesspecifiedbythelaboratory,astheremaybeno
referencerangesrelatingtotheirconcentrationsinbloodatothertimes
Carewhencollectionbloodspecimens
Thepostureofthepatient,thechoiceofskincleansingagentandtheselectionofasuitablevien(orothersource)are
theprincipalfactorstocon
siderbeforeproceedingtocollecteachspecimen:
1.Theskinmustbecleanoverthesiteforcollect
ingthebloodspecimen.However,itmustberememberedthat
alconolandmethylatedspir
itscancausehaemolysis,andthattheiruseisclearlytobeavoidedifblood[ethanol]istobe
determined.
2.Limbsintowhichintravenousinfusionsarebeinggivenmustnotbeselectedasthesiteofvenepunctureunless
particularcareistaken.Theneedleorcannulamustfirstbethoroughlyflushedoutwithbloodtoavoiddilutionofthe
specimenwithinfusionfluid.
3.Venepuncturetechniqueshouldbestandardisedasfaraspossibletoenableclosercomparisonofsuccessiveresults
onpatients.
4.VenousbloodspecimensshouldbeobtainedwithminimalstasisProlongedstasiscanmarkedlyraisethe
concentrationsofplasmaproteinsandothernondiffusiblesubstances(e.g.proteinboundsubstances).Itisadvisableto
releasethetourniquetbeforewithdrawingthesampleofblood.
5.PostureshouldbestandardisedifpossibleWhenapatient'sposturechangesfromlyingtostanding,theremaybean
increaseofasmuchas13%intheconcentrationofplasmaproteinsorproteinboundconstituents,duetoredistribution
offluidintheextracellularspace.
6.Haemolysisshouldbeavoided,sinceitrendersspecimensunsuitableforplasmaK+,magne
siumandmanyprotein
andenzymeactivitymea
surements.
7.InfectionhazardHighriskspecimensrequirespecialcareincollection,andthisdangermustbeclearlyindicatedon
therequestform.
Careofbloodspecimensaftercollection
Bloodspecimensshouldbetransportedtothelab
oratoryassoonaspossibleaftercollection.Specialarrangementsare
neededforsomespecimens(e.g.foracidbasemeasurements,orunstablehormones)becauseoftheirlackofstability.
Mostotheranalytesarestableforatleast3hinwholeblood,orlongerifplasmaorserumisfirstsepa
ratedfromthe
cells.Asarule,wholebloodspecimensforchemicalanalysismustnotbestoredinarefrigera
tor,sinceionicpumps
thatmaintainelectrolytegradientsacrossthecellmembraneareinactiveatlowtemperatures.Conversely,separated
serumorplasmaisbestrefrigerated,tominimizechemicalchangesorbacterialgrowth.
Severalchangesoccurinwholebloodspecimensfollowingcollection.Thecommonerandmoreimportantchanges
thatoccurpriortotheseparationofplasmaorserumfromthecellsare:
1.Glucoseisconvertedtolactate:thisprocessisinhibitedbyfluoride
2.Severalsubstancespassthroughtheerythrocytemembrane,ormaybeaddedinsignificantamountstoplasmaasa
resultofredcelldestructioninsufficienttocausedetectablehaemolysis.ExamplesincludeK+andlactate
dehydrogenase
3.LossofCO2occurs,sincethePco2,ofbloodismuchhigherthaninair
4.Plasma[phosphate]increasesduetohydrolysisoforganicesterphosphatesintheredcells
5.Labileplasmaenzymeslosetheiractivity.
ANTICOAGULANTS
Certainbiochemicaltestsrequireunclottedblood.Serumfromcoagulatedbloodisthespecimenofchoiceformany
assaysystems.
Heparin(inhibitstheconvensionprothrmobintothrombin)isthemostwidelyusedanticoagulantforclinicalchemical
analysis.Heparinisanidealanticoagulant,sinceitdoesnotcauseanychangeinbloodcomposition.However,other
anticoagulantsarepreferedtoheparin,duetothecostfactor.
Ethylenediaminetetraaceticacid(EDTA)isachelatingagent,andisparticularlyusefulforhematological
examinationbecauseitpreservescellularcomponentsoftheBLOOD.Itchelateswithcalciumandblockscoagulation.
EDTAisemployedtocollectbloodforhematologicalexaminationsItmayaffectsomeoftheclinicalchemistrytests.
Sodiumfluorideisusuallyusedasapreservativeforbloodglucosebyinhibitingtheenzymesystemsinvolvedinthe
glycolysis.Withoutanantiglycolyticagent,thebloodglucoseconcentrationdecreasesabout10mg/dlperhourand
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falseresultsmaybeobtained.Fluorideisalsoanticoagulant.Itshouldnotbeusedforenzymeassays,aswellaswhen
thetestinvolvesenzymaticanalysis.
Citrateiswidelyusedforcoagulationstudies.
Oxalateinhibitsbloodcoagulationbyforminginsolublecomplexeswithcalciumions.Potassiumoxalatemaybeused
ataconcentrationof12mg/mlblood.Atconcentrationof3mg/ml,oxalatemaycausehemolysis.
Potassiumorsodiumoxalate:Thesecompoundsprecipitatecalciumandinhibitbloodcoagulation.Beingmore
soluble,potassiumoxalate(510mgper5mlblood)isprefered.
Potasiumoxalateandsodiumfluoride:Theseanticoagulantsareemployedforcollectingbloodtoestimateglucose.
Furthersodiumfluorideinhibitsglycolysisandpreservesbfoodglucoseconcentration.
Ammoniumoxalateandpotassiumoxalate:Amixtureofthesetwocompoundsintheratio3:2isusedforblood
collectiontocarryoutcertainhematologicaltests.
HEMOLOYSIS
TheruptureorlysisofRBC,releasingthecellularconstituentsinterfereswiththelaboratoryinvestigations.Therefore,
utmostcareshouldbetakentoavoidhemolysiswhenplasmaorserumareusedforbiochemicaltests.Useofdry
syringes,needlesandcontainers,allowingslowflowofbloodintosyringeareamongtheimportantprecautionstoavoid
hemolysis.
PRESERVATIONOFBLOODSPECIMENS
Plasmaorserumshouldbeseparatedwithin2hoursafterbloodcollection.Itisidealandadvisabletoanalyseblood,
plasmaorserum,immediatelyafterthespecimencollection.Thishowever,maynotbealwayspossible.Insuchacase,
thesamples(usuallyplasma/serum)canbestoredat4Cuntilanalysed.Forenzymeanalysis,thesamplearepreserved
at20C.
SamplingErrors:
1.Bloodsamplingtechnique.Difficultyinobtainingabloodspecimenmayleadtohaemolysiswithconsequentrelease
ofpotassiumandotherredcellconstituents.Resultsforthesewillbefalselyelevated.
2.Prolongedstasisduringvenepuncture.Plasmawaterdiffusesintotheinterstitialspaceandtheserumorplasma
sampleobtainedwillbeconcentrated.Proteinsandproteinboundcomponentsofplasmasuchcalciumorthyroxinewill
befalselyelevated.
3.Insufficientspecimen.Eachbiochemicalanalysisrequiresacertainvolumeofspecimentoenablethetesttobe
carriedout.
4.Errorsintiming.Thebiggestsourceoferrorinthemeasurementofanyanalyteina24hoururinespecimenisinthe
collectionofanaccuratelytimedvolumeofurine.
5.Incorrectspecimencontainer.Formanyanalysesthebloodmustbecollectedintoacontainerwithanticoagulantand
preservative.Forexample,samplesforglucoseshouldbecollectedintoaspecialcontainercontainingfluoridewhich
inhibitsglycolysisotherwisethetimetakentodeliverthesampletothelaboratorycanaffecttheresult.
6.Inappropriatesamplingsite.Bloodsamplesshouldnotbetakingdownstreamfromanintravenousdrip.Itisnot
unheardofforthelaboratorytoreceiveabloodglucoserequestonaspecimentakenfromthesamearmintowhich5%
glucoseisbeinginfused.
7.Incorectspecimenstorage.Abloodsamplestoredovernightbeforebeingsenttothelaboratorywillshowfalsely
highpotassium,phosphateandredcellsenzymessuchaslactatedehydrogenase,becauseofleakageintothe
extracellularfluidfromthecells.
Manyhormonesshowcircardianrhythm.Forexample,ACTHhasmaximumpeakatearlymorning,and
minimumlevelatafternoon.Maximumlevelofgrowthhormoneisduringnightandminimumisinthedaytime.Many
referencevaluesareagerelatede.g.,levelsofureaandcholesterolaremoreingeriatricpatients.Exercisewillincrease
theleveloftransaminasesandcreatinine.Triglyceridelevelistobedoneinfastingcondition.Caffeine(coffeeandtea)
willincreasethelevelsoffreefattyacid,glycerol,totallipidsandglucose.SmokingwillincreasethelevelsofGH,
cortisolandtriglycerides.
COLLECTIONOFURINE:
An early morning fasting specimen is generally the most concentrated specimen. Therefore, this is preferred for
microscopicexaminationandforthedetectionofproteins,betachorionicgonadotropinandothermetabolites.
Urine, containing the metabolic waste products of the body in water is the most important excretory fluid. For
biochemical investigations, urine can be collected as a single specimen or for 24 hours. Single specimens of urine,
normally collected in the morning, are useful for qualitative tests e.g., sugar, proteins. Twenty four hour urine
collections(donebetween8AMto8AM)areemployedforquantitativeestimationofcertainurinaryconstituentse.g.,
proteins,hormones,metabolites.
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Dependingonthetest,eitherarandomoracom
pletetimedcollectionofurineisneeded.Thetimedcollectionis
obtainedasfollows:
1.Justbeforethecollectionperiodisduetostart,thepatientemptieshis/herbladder.Thisurinemustbediscarded.
2.Thereafter,fromthestart(e.g.at8AM)totheendofthecollectionperiod,allurinepassedbythepatientmustbe
addedtothecontainer.Ifthiscon
tainspreservative,thespecimenmustbemixedgentlyeachtimemoreurineispassed
andaddedtothecollection.
3.Attheendoftheperiod(e.g.8AMthenextday,inthecaseofa24hcollection),thepatientemp
tieshis/herbladder.
Thisurinemustbeincludedinthecollection.
4.Theperiodoverwhichthecollectionwasmademustberecordedandwrittenonthespecimencontainerandthe
requestform.
Forlargevolumes,analiquot(e.g.25mL)maybesenttothelaboratory,butthecompletespeci
menmustfirstbemixed
anditsvolumerecordedonthecontainerandtherequestform.
Urinespecimenstendtodeteriorateunlessthecorrectpreservativeisaddedfromthestart,orthespecimenis
refrigeratedthroughoutthecollectionperiod.Thechangesinclude:
1.destructionofglucosebybacteria
2.conversionofureatoammonia,bybacte
ria,withfallin[H+]andprecipitationofphosphates
3.oxidationofurobilinogentourobilinandporphobilinogentoporphyrins.
Preservativesforurine:Thepreservativesareused(1)toreducebacterialaction(2)tominimisechemical
decomposition,and(3)todecreaseatmosphericoxidationofunstablecompounds.Themostsatisfactoryformof
preservationofurinespecimenistorefrigerateitduringthecollection.Formalin,thymol,chloroform,toluene,
concentratedHCIandglacialaceticacidarethecommonlyusedurinepreservatives.
Forthecollectionof24hrurinesamples,preservativeshavetobeusedorelseurineundergoeschangesduetobacterial
action.Hydrochloricacid,toluene,lightpetroleum,thymol,formalinetc.,areamongthecommonpreservativesused.
TimedUrineSpecimen
Usually,urinesampleiscollectedforthe24hourperiod.Thiswillminimisetheinfluenceofshorttermbiological
variationsanddiurnalrhythms.Generally,collectionofurinesamplesaredonefrom6AMtonext6AM.Thebladder
shouldbeemptiedwhenthecollectionisstarted(6AM),andthisurineisdiscarded.Thereafteralltheurineshouldbe
collected.Thenextdayurineisvoidedat6AMandthissampleisalsocollected.
CEREBROSPINALFLUID:
CSFisafluidofthenervoussystem.Itisformedbyaprocessofselectivedialysisofplasmabythechoroidplexuses
oftheventriclesofthebrain.ThetotalvolumeCSFis100200ml.
CollectionofCSF:CSFiscollectedbypuncturingtheinterspacebetweenthe3rdandthe5thnumbervertabrae,under
asepeticconditionsandlocalanesthesia.
BiochemicalinvestigationsonCSF:Protein,glucoseandchlorideestimationsareusuallyperformedintheclinical
biochemistrylaboratory.
Theinterpretationofresults:
Mostbiochemicalanalysesarequantitative.Manytestsmeasuretheamountoftheanalyteinasmallvolumeofthe
sample(blood,plasma,serum,urineorsomeotherfluidortissue).Thetestsresultsarecommonlyexpressedinmolar
units.Amoleofanycompoundalwayscontains6*1023molecules.Describinghowmuchofananalyteispresentin
molesindicateshowmanymoleculesofthesubstancearepresent.Molarunitscanbeconvertedtomassunits:onemole
isthemolecularweightofthesubstanceingrams.Resultsarereportedasconcentrations,usuallyintermsofthe
numberofmolesinonelitre(mol/l).
Molarunits:
Mole
Abbreviation
Definition
Milimole
mmol
*103ofamole
Micromole
mol
*106ofamole
Nanomole
nmol
*109ofamole
Picomole
pmol
*1012ofamole
Femtomole
fmol
*1015ofamole
Enzymesarenotusuallyexpressedinmolesbutasenzymeactivityin"units".Largemoleculessuchasproteinsare
reportedasgramsormilligrams.Bloodgasresults(PCO2orPO2)areexpressedinkilopascals(kPa),theunitsinwhich
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partialpressuresaremeasured.
Biologicalfactorsaffectingtheinterpretationofresults:
1.Sexofthepatient.Referencerangesforsomeanalytessuchasserumcreatininearedifferentformenandwomen.
2.Ageofthepatient.Theremaybedifferentreferencesrangesforneonates,children,adultsandtheeldery.
3.Effectofdiet.Thesamplemaybeinappropriateiftakenwhenthepatientisfastingorafterameal.
4.Timewhensamplewastaken.Theremaybevariationsduringthedayandnight.
5.Stressandanxiety.
6.Postureofthepatient.Redistributionoffluidmayeffecttheresult.
7.Effectsofexercise.Strenuousexercisecanreleaseenzymesfromtissues.
8.Medicalhistory.Infectionortissueinjurycanaffectbiochemicalvaluesindependentlyofthediseaseprocess
beinginvestigated.
9.Pregnancy.Thisalterssomereferencesranges.
10.Menstrualcycle.Hormonemeasurementwillvarythroughthemenstrualcycle.
11.Drughistory.
Theclinicianmaywellaskthefollowingquestionsonreceivingabiochemistryreport:
1.DoestheresultfitinwithwhatIexpectedonthebasisoftheclinicalexaminationandhistoryofthepatient?
2.IftheresultisnotwhatIexpected,canIexplainthediscrepancy?
3.HowcantheresultchangemydiagnosisorthewayIammanagingthepatient?
4.WhatshouldIdonext?
QUALITYCONTROL:
Qualitycontrolinclinicalbiochemistrylaboratoryreferstothereliabilityofinvestigativeservice.Anyerrorinthe
laboratorywilljeopardizethelivesofpatients.Itisthereforeutmostimportantthatthelaboratoryerrorsareidentified
andrectified.
Qualitycontrolcomprisesoffourinterrelatedfactorsnamelyprecision,accuracy,specificityand
sensitivity.
Precisionreferstothereproducibilityoftheresultwhenthesamesampleisanalysedondifferentoccasions(replicate
measurements)bythesameperson.Forinstance,theprecisionisgood,ifthebloodglucoselevelis78,80and82mg/dl
onreplicates.
Accuracymeanstheclosenessoftheestimatedresulttothetruevaluee.g.,iftruebloodurealevelis50mg/dl,the
laboratoryreporting45mg/dlismoreaccuratethantheonereporting35mg/dl.
Specificityreferstotheabilityoftheanalyticalmethodtospecificallydetermineaparticularparametere.g.,glucose
canbespecificallyestimatedbyenzymaticglucoseoxidasemethod.
Sensitivitydealswiththeabilityofaparticularmethodtodetectsmallamountsofthemeasuredconstituent.
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METHODSOFQUALITYCONTROL
1.Internalqualitycontrolreferstotheanalysisofthesamestoredsampleondifferentdaysinalaboratory,theresults
shouldvarywithinanarrowrange.
2.Externalqualitycontroldealswiththeanalysisofasamplereceivedfromoutside,usuallyfromanationalor
regionalqualitycontrolcentre.Theresultsobainedarethencompared.
Laboratoryanalyticalperformance:
Anumberoftermsdescribebiochemicalresults.Theseinclude:
1.Precisionandaccuracy
Precisionisthereproducibilityofananalyticalmethod.Accuracydefineshowclosethemeasuredvalueistotheactual
value.
2.Sensitivityandspesificity
Sensitivityofanassayisameasureofhowlittleoftheanalytethemethodcandetect.Spesificityofanassayrelatesto
howgoodtheassayisatdiscriminatingbetweentherequestedanalyteandpotentiallyinterferingsubstances.
3.Qualityassurance
Everylaboratorytakesgreatpainstoensurethatthemethodsinusecontinuetoproducereliableresults.Laboratory
staffmonitorperformanceofassaysusingqualitycontrolsamplestogivereassurancethatthemethodisperforming
satisfactolilywiththepatients'specimens.
Theseareinternalqualitycontrolswhichareanalysedeverydayoreverytimeanassayisrun.Theexpectedvalues
areknownandtheactualresultsobtainedarecomparedwithpreviousvaluestomonitorperformance.Inexternal
qualityassuranceschemes,identicalsamplesaredistributedtolaboratoriesresultsarethencompared.
Qualitycontrolshouldbecomeanintegralpartoftheoperationofaclinicalchemistrylaboratory.Thepurposeof
qualitycontrolistoensurethereliabilityofeachmeasurementperformedonasample.
Acentralreferencelaboratorysendsaserumsamplecontainingknownquantityofasubstancethisisanalysedina
peripheral(small)laboratory.Iftheresultobtainedintheperipherallabisthesameasthatofthereferencelaboratory,
thenthearrangementsavailableintheperipherallabissaidtobereliable.Thisiscalledexternalqualitycontrol.This
typeofcheckingisusuallydoneonceortwiceinamonth.Moreover,theperipherallaboratoryitselfmakesareference
standardserumsample,andcheckstheresultsonthedailybasisthisiscalledinternalqualitycontrol.
Accuracy
Itistheclosenessofaresulttothetruevalue.Forexample,ifonetechnicianperformsatestonaserumwhichis
knowntocontain5,0mmol/Lglucoseandobtainsaresultof4.9mmol/L.
Asecondtechniciandoesthesametestonthesamesample,andgetstheresultof4.5mmol/L.Thenthevalueofthe
firsttechnicianisconsideredasaccurate.Valuesfartherawayfromthetruevaluearelessaccuratethanthosecloser.
Precision
Thisreferstothereproducibilityoftheresult.Ifonetechnicianperformsglucoseanalysisonthesamesampleonthree
differentoccasionsandobtains4mmol/L,3.9mmol/L,and4.1mmol/L,thentheresultshavebeenreproducedvery
well,andtheprecisionisverygood.Precisiondependsonthetechnique,thereagents,aswellasonthetechnician.
Specificity
Specificityofareactiondenotesthatonlyonesubstancewillanswerthatparticulartest.Forexample,inthecaseof
glucoseoxidasemethod,onlyglucosemoleculesareassayed.Soitisaveryspecificmethod.Butifthereducing
propertyoftheglucoseisutilisedfortheassaypurpose(e.g.,NelsonSomogyimethod),thenotherreducingagentsin
thebloodwillinterfereinthereaction,andhencespecificityislowered.Specificityisdeterminedbythemethodofthe
analysis.
Sensitivity|
Itindicatesthatwhetherthemethodcouldbeutilisedtotestaverydilutesolution.Forexample,biuretmethodisused
forsolutionshavingafewgofprotein/dl.Spectrophotometricmethodisusefultodetectafewmgofprotein/dl,while
ELISAmethodisemployedifthesolutionhasonlymicrogramofprotein/dl.ThusELISAmethodismostsensitive.
Generallyspeaking,asthesensitivityisincreased,specificityisdecreased.
LimitofErrorsAllowableinLaboratory:
Inalaboratory,errormaynotbetotallyavoidedbutshouldbekeptataminimum.Thelimitsaredenotedbytheterm,
percentageerror.Thepercentageofallowableerrorinanassayisgivenbytheformula:
Differencebetweenmaximumandminimumofnormalrange
x100
Meanofthenormalrangex4
Thepercentageerror,therefore,willvaryfromtesttotest.Totakeanexample,inthecaseofbloodglucose
analysis,thenormalrangeis70to110mg/dl.Ifthesevaluesaresubstitutedintheformula:
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(110minus70)
x100=10%
90x4
Now,inthecaseofbloodurea(normalrangeof2040mg/dl),thepercentageerrorwillbe
(40minus20)
x100=16%
30x4
Biochemicaltestingoutsidethelaboratory:
Themethodsformeasuringsomebiologicalcompoundsinbloodandurinehavebecomesorobustandsimpletouse
thtmeasurementscanbemadeawayfromthelaboratorybythepatient'sbedside,intheward,inthehomeoreven
intheshopingcentre.
Testsperformedawayfromthelaboratory:
Themostcommonbloodtestoutsidethelaboratoryisthedeterminationofglucoseconcentration,inafingerstab
sample,athomeorintheclinic.Diabeticpatientswhoneedtomonitortheirbloodglucoseonaregularbasiscando
soathomeoratworkusingoneofmanycommerciallyavailablepocketsizedinstruments.
Aportablebenchanalyser.Thisinstrumentmaybeusedtomonitorpatients'glucoseandcholesterol,andits
frequentlyusedinmanyoutpatientclinicsandinscreeningcentres.
Commontestsonbloodperformedawayfromthelaboratory
Analyte
Usedwheninvestigating
A
Bloodgases
Acidbasestatus
Glucose
Diabetesmellitus
Urea
Renaldisease
Creatinine
Renaldisease
Bilirubin
Neonataljaundise
Therapeuticdrugs
Complianceoftoxicity
Salicylate
Detectionofpoisoning
Paracetamol
Detectionofpoisoning
Glucose
Diabeticmonitoring
B
Cholesterol
Coronaryheartdiseaserisk
Alcohol
Fitnesstodrive/confusion,
C
coma
Commontestsonurineperformedawayfromthelaboratory
Analyte
Usedwheninvestigating
A
Ketones
Diabeticketoacidosis
Protein
Renaldisease
Redcells/haemoglobin
Renaldisease
Bilirubin
Liverdiseaseandjaundise
Urobilinogen
Jaundise/haemolysis
pH
Renaltubularacidosis
Glucose
Diabetesmellitus
B
hCG
Pregnancytest
Thetestscommonlyperformedawayfromthelaboratorycanbecategorizedasfollows:
A.Testsperformedinmedicalornursingsettings.
Theyclearlygivevaluableinformationandallowthepractitionertoreassurethepatientorfamily,orinitiatefuther
investigationsortreatment.
B.Testsperformedinthehome,shoppingcentreorclinicalsetting.
Theycangivevaluableinformationwhenproperlyandappropriatelyused.
C.Alcoholtests.
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Methodologyofsideroomtests
Itisafeatureofmanysideroomteststhattheirsimplicitydisguisestheuseofsophisticatedmethodology.Ahome
pregnancytestmethodinvolvesanelegantapplicationofmonoclonalantibodytechnologytodetectthehuman
chorionicgonadotropin(hCG)whichisproducedbythedevelopingembryo.Thetestissimpletocarryout:afew
dropsofurineareplacedinthesamplewindow,andtheresultisshownwithin5minutes.Theadditionoftheurine
solubilizesamonoclonalantibodyforhCGwhichiscovalentlyboundtotinybluebeds.Asecondmonoclonal,
specificforanotherregionofthehCGmolecule,isfirmlyattachedinalineattheresultwindow.IfhCGispresent
inthesampleitisboundbythefirstantibody,formingabluebeadantibodyhCgcomplex.Astheurinediffuses
throughthestrip,anyhCGpresentbecomesboundatthesecondantibodysiteandthisconcentratesthebluebead
complexinalineapositiveresult.Athirdantibodyrecognizestheconstantregionofthefirstantibodyandbinds
theexcess,thusprovidingacontroltoshowthatsufficienturinehadbeenaddedtotheteststrip,themostlikely
formoferror.
Generalproblems
Theobviousadvantagesintermsoftimesavingandconveniencetobothpatientandclinicianmustbebalancedbya
numberofpossibleproblemsintheuseofthesetests.Theyinclude:
1.Cost.Manyofthesetestsareexpensivealternativestothetraditionalmethodsusedinthelaboratory.
2.Responsibility.Thepersonperformingtheassayoutwiththelaboratory(theoperator)mustassumeanumberof
responsibilitieswhichwouldnormallybethoseofthelaboratorystaff.Thereistheresponsibilitytoperformthe
assayappropriatelyandtoprovideananswerthatisaccurate,preciseandmeaningful.
Analyticalproblems
Themostcommonlyencounteredanalyticalerrorsarisebecauseoffailureto:
1.Calibrateaninstrument
2.Cleantheinstrument
3.Usequalitycontrolmaterials
4.Storereagentsorstripsinappropriateconditions
Alloftheseproblemscanbereadilyovercomebyfollowinginstructionscarefully.
Pointofcaretesting(NearPatientTesting)
Incontrasttotheanalysisofbiochemicaltestsinalargehospitalclinicalbiochemistrylaboratory,itisalsopossibleto
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carryoutanincreasingrangeofbiochemicaltestsonsmall,benchtopanalysersorusingdipsticksmethodologycloseto
thepatient,eitheratthebedside,intheclinicorsurgeryoreveninthehome.
Table1.6listssomeofthetestswhichcanbeanalysedonthisbasis.
Table1.6ExamplesofPOCT
POCT
Example
application
Stillwidelyusedforscreeningpurposesin
Sideroomtestsonurineandfaeces
primary and secondary care. This includes
the 'multistix' range of tests which include
glucose, protein, ketones, bilirubin and
urobilinogen, etc. The technology of
dipstick testing can now also be extended
toDrugsofAbuseandothertestsandmay
haveaplaceinanappropriateclinicsetting
Theseareincreasinglywidelyusedinacute
Bloodgasanalysers
settingsinhospitalsuchasA&E,Acute
AdmissionsUnitandIntensiveCareUnit.
Usingappropriatelytrainedstaffand
payingattentiontoqualityissues,results
aregenerallyexcellentandallowmultiple
samplingonfreshbloodsamplesinacutely
unwellpatientsorwherefrequent
monitoringisnecessaryformanagement
purposes(e.g.cardiothoracicoperations)
Smallchemistryanalysers(oftenproviding Smallinstrumentswhichprovideawider
repertoireoftestsareincreasingly
bloodgasresultsandcommonanalytes
availableandmostbloodgasanalysersdo
suchaselectrolytes,glucose,lactate
nowofferthiswiderrepertoireoftests.The
bilirubinorvariouscombinationsoftests
additionaltestsprovidedcan,tosome
results
extent,betailoredtothelocationofthe
instrument.Foroftest
results)example,a
NeonatalUnitwouldrequireaninstrument
whichdealswithsmallvolumesofsamples
andpreferablyprovideselectrolyteresults,
includingcalcium,aswellaslactateand
bilirubin.
Bloodglucoseanalysers
Thesearewidelyusedbothinprimaryand
secondarycareandbypatientsthemselves.
Again,withappropriatetraining,resultsare
accurateandcomparewellwith
correspondingmainLaboratoryresults.
Fromtheindividualpatientperspective,an
extendedprofileofglucoseresultsallows
rationaladjustmentoftreatmenttooptimise
bloodglucosecontrol,particularlyinthe
insulindependentdiabetic
Aparticularlyimportantbenefitofpointofcaretesting(POCT)relatestotherapidavailabilityofresultstooptimally
managetheacutelyillpatient.ExampleswouldincludethemeasurementofbloodgasesintheIntensiveCareUnitto
assistventilationstatusortheregularmonitoringofdiabeticpatientsusingbloodglucosetestingontheward.Tests
performedintheOutpatientorGPsurgerycanprovideanimmediacyofresponseallowingforreassuranceorthe
initiationoffurtherconfirma
torytests.Usedwithinthehome,themonitoringofbloodglucosecanallowthediabetic
patienttomakelifestyleadjustmentsappropriatetooptimaldiabeticcare,aswellasprovidingarecordforinformed
decisionsontreatment.
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Pointofcaretestingisnotwithoutitsdisadvan
tages.Testcostsareinvariablyconsiderablyhigherthanthose
undertakenbyacentrallaboratory.Whenundertakenoutsideofthelaboratorycon
text,thereisariskofoperatorerror,
especiallyiftrainingisinadequate.Issuesalsosurroundtherequirementstomaintainandcalibrateinstru
mentsto
ensuremeaningfulresults,aswellastheneedtoaddressqualitycontrolandmattersofhealthandsafety.Whereitis
appropriatetointro
ducepointofcaretesting,itiscriticalthatmattersoftraining,analyticalperformance,qualitycon
trol,andhealthandsafetyareproperlyaddressedandthecentralclinicalbiochemistrylaboratoryoftenhasanimportant
parttoplayinthisaspectofpointofcaretesting.
ThefutureThereisnodoubtthatinfuture,biochemicaltestingofpatientsoutsidethelaboratorywillbecomepractical
formanyoftheanalytescurrentlymeasuredinthelaboratory.
BIOCHEMICALINVESTIGATIONOFBLOODPLASMAPROTEINS
Totalbloodvolumeisabout4.5to5litres.
Ifbloodismixedwithananticoagulantandcentrifuged,thecellcomponents(RBCandWBC)areprecipitated.The
supernatantiscalledplasma.About5560%ofbloodismadeupofplasma.
Ifbloodiswithdrawnwithoutanticoagulantandallowedtoclot,afterabouttwohoursliquidportionisseparatedfrom
the clot. This defibrinated plasma is called serum, which lacks coagulation factors including prothrombin and
fibrinogen.
Plasmacontainsabouthundredofdifferentproteins.
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Totalproteincontentofnormalplasmais6to8g/100ml(6585g/l).Asafirststepofstudy,theplasmaproteinsmay
beseparatedintoAlbumin(3550g/l),Globulins(2535g/l)andfibrinogen(24g/l).
Thealbumin:globulinratioisusuallybetween1.2:1to1.5:1.
Almostallplasmaproteins,exceptimmunoglobulinsaresynthesizedinliver.
Inclinicallaboratory,totalproteinsinserumorplasmaofpatientsareestimatedbyBiuretmethod.
Inclinicallaboratory,electrophoresisisemployedregularlyforseparationofserumproteins.
Thetermelectrophoresisreferstothemovementofchargedparticlesthroughanelectrolytewhensubjectedtoan
electricfield.
The positively charged particles (cations) move to cathode and negatively charged ones (anions) to anode. Since
proteins exist as charged particles, this method is widely used for the separation of proteins in biological fluids. The
techniquewasfirstusedbyTiseliusin1937nameditasmovingboundaryorfrontalelectrophoresis(NobelPrizein
1948).
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FactorsAffectingElectrophoresis
Therateofmigration(separationofparticles)duringelectrophoresiswilldependonthefollowingfactors:
1.Netchargeontheparticles
2.Massandshapeoftheparticles.
3.ThepHofthemedium.
4.Strengthofelectricalfield.
5.Propertiesofthesupportingmedium.
6.Temperature.
TypesofElectrophoresis
Therearemainlytwotypesofelectrophoresishorizontalandvertical.
Different types of support media are used in horizontal electrophoresis, e.g. filter paper, cellulose acetate, agar gel,
agarosegel,starchgel,etc.Theverticalelectrophoresismainlyusespolyacrylamidegel.Thenatureofthesupporting
mediumwillalsoinfluencethemobility.
ElectrophoresisApparatus
The electrophoresis system basically consists of the electrophoresis tank to hold the buffer and fitted with the
electrodes, as well as a power pack to supply electricity at constant current and voltage. When the electrophoresis is
carriedout,thebufferischoseninsuchawaysoastoensureeffectiveseparatumofthemixtureofproteins.ThepH
andionicstrengthandnatureofthebuffermaybevariedaccordingtotheproteinstobeseparated,e.g.serumproteins
areseparatedatapHof8.6usingbarbitonebuffer.AtthispHallserumproteinswillhaveanetnegativechargeand
willmigratetowardstheanode.
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SupportMediumforElectrophoresis
FilterPaper
CelluloseAcetateMembrane
AgarorAgarose
StarchGel
Polyacrylamidegelelectrophoresis(PAGE)
VisualisationofProteinBands
Aftertheelectrophoreticruniscompleted,theproteinsarefixedtothesolidsupportusingafixativesuchasacetoneor
methanol.Thenitisstainedbyusingdyes
(AmidoSchwartz,naphthaleneblack,PonceauSorCoomassieBlue)andthendestainedbyusingdiluteaceticacid.The
electrophoretogramcanbescannedusingadensitometerandeachbandquantitated.Inthedensitometer,lightispassed
through the agar gel plate the absorption of light will be proportional to the quantity of protein present on a band.
Anothermethodisthatthestainmaybeelutedfromthesupportandeachfractionquantitatedcolourimetrically.
NormalValuesandInterpretations
Inagargelelectrophoresis,normalserumwillbeseparated
intofivebands.Theirrelativeconcentrationsaregiven
below:
Albumin:5565%
Alpha1globulin:24%
Alpha2globulin:612%
Betaglobulin:812%
Gammaglobulin:1222%
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ALBUMIN
Thenameisderivedfromthewhiteprecipitateformedwheneggisboiled(Latin,albus=white).Albuminconstitutes
themajorpartofplasmaproteins.Ithasonepolypeptidechainwith585aminoacidsand17disulfidebonds.Ithasa
molecular weight of 69,000. It is synthesisedby hepatocytes therefore, albumin level in blood is decreased in liver
cirrhosis.Estimationofalbuminisaliverfunctiontest.Halflifeofalbuminisabout20days.Liverproducesabout12
gofalbuminperday,representingabout25%oftotalhepaticproteinsynthesis.Albumincancomeoutofvascular
compartment.SoalbuminispresentinCSFandinterstitialfluid.Over80allelicformsofalbumin,allwithnormal
function, but with altered electrophoretic mobilities are described. Albumin is coded by a gene on the long arm of
chromo
somenumber4.
FunctionsofAlbumin
1.Itcontributestothecolloidosmoticpressureofplasma.
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Thetotalosmolalityofserumis278305mosmol/kg(about5000mmofHg).Butthisisproducedmainlybysalts,
whichcanpasseasilyfromintravasculartoextravascularspace.Therefore,theosmoticpressureexertedbyelectrolytes
inside and outside the v a s c u l a r compartments will cancel each other. But proteins cannot easily escape out of
bloodvessels,andtherefore,proteinsexertthe'effectiveosmoticpressure'.Itisabout25 mm Hg, and 80% of it is
contributedbyalbumin.Themaintenanceofbloodvolumeisdependentonthiseffectiveosmoticpressure.According
to Starling's hypothesis, at the capillary end the blood pressure (BP) or hydrostatic pressure expels water out, and
effectiveosmoticpressureEOP)takeswaterintothevascularcompartment.Atarterialendofthecapillary,BPis35
mmHgandEOPis25mmthuswaterisexpelledbyapressureof10mmHg.Atthevenousendofthecapillary,
EOPis25mmandBPis15mm,andthereforewaterisimbibedwithapressureof10mm.Thus,thenumberofwater
moleculesescapingoutatarterialsidewillbeexactlyequaltothosereturnedatthevenoussideandthereforeblood
volumeremainsthesame.Ifproteinconcentrationinserumisreduced,theEOPiscorrespondinglydecreased.Then
return of water into blood vessels is diminished, leading to accumulation of water in tissues. This is called edema.
Edemaisseeninconditionswherealbuminlevelinbloodislessthan2g/dl.
2.Anothermajorfunctionofalbuministotransportvarioushydrophobicsubstances.
3.Beingawaterymedium,bloodcannotsolubiliselipidcomponents.Bilirubinandnonesterifiedfattyacids
are specifically transported by albumin. Drugs (sulpha, aspirin, salicylates, dicoumarol, phenytoin),
steroidhormones,thyroxine,calcium,copper
and heavy metals are nonspecifically carried by albumin. Only the unbound fraction of drugs is
biologicallyactive.Thehistidineresidueatposition3ofalbuminbindscopper.
4. All proteins have buffering capacity. Because of its high concentration in blood, albumin has maximum
bufferingcapacity.Albuminhasatotalof16histidineresidueswhichcontributethisbufferingaction.
5.Alltissuecellscantakeupalbuminbypinocytosis.Itisthenbrokendowntoaminoacidlevel.Soalbumin
maybeconsideredasthetransportformofessentialaminoacidsfromlivertoextrahepaticcells.
ClinicalApplications
1.Albuminfattyacidcomplexcannotcrossbloodbrainbarrierandhencefattyacidscannotbetakenupbybrain.
Thebilirubinfromalbuminmaybecompetitivelyreplacedbyaspirinandsuchotherdrugs.Innewborns,bilirubinis
already high, and if such drugs are given, there is a probability that free bilirubin is deposited in brain leading to
kernicterusandmentalretardation.
2.Whentwodrugshavinghighaffinitytoalbuminareadministeredtogether,theremaybecompetitionforthe
available sites, with consequent displacement of one drug. Such an effect may lead to clinically significant drug
interactions,e.g.phenytoindicoumarolinteraction.
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3.Proteinboundcalciumisloweredinhypo
albuminemia.Thus,eventhoughtotalcalciumlevelinbloodis
lowered,ionisedcalciumlevelmaybenormal,andsotetanymaynotoccur.Calciumisloweredby0.8mg/dlfora
fallof1g/dlofAlbumin.
4.Humanalbuministherapeuticallyusefultotreatburns,hemorrhageandshock..
Normal Value and Interpretation: Normal level of Albumin is 3.55 g/dl. Lowered level of albumin
(hypoalbuminemia)hasimportantclinicalsignificance.
Hypoalbuminemia
1.Incirrhosisofliverandinchronicliverfailure,albumin
synthesisisdecreasedandsobloodlevelislowered.
2. In malnutrition and in malabsorption syndrome the availability of amino acids is reduced and so albumin
synthesisisaffected.
3.Innephroticsyndrome,kidneyglomerularfiltrationisdefectivesothatalbuminisexcretedinlargequantities.
Increasedloss,toacertainextent,iscompensatedbyincreasedsynthesisbutbloodlevelofalbuminisdecreased.
4.Presenceofalbumininurine(albuminuria)isalwayspathological.Largequantities(severalgramsperday)of
albumin is lost in urine in nephrotic syndrome. Small quantities are lost in urine in acute nephritis, and other
inflammatory conditions of urinary tract. Detection of albumin in urine is done by heat and acetic acid test. In
microalbuminuriaorminimalalbuminuriaorpaucialbuminuria,smallquantityofalbumin(50300mg/d)isseenin
urine (Paucity = small in quantity). However, microalbuminuria is also clinically important, as it is a predictor of
futurerenaldiseases.
5.Inburns,albuminislostthroughtheunprotectedskinsurface.
6.Inproteinlosingenteropathylargequantitiesofalbuminislostthroughintestines.
7. Hypoalbuminemia will result in tissue edema. It may be seen in malnutrition, where albumin synthesis is
depressed {generalized edema), in nephrotic syndrome, where albumin is lost through urine {facial edema) or in
cirrhosis of liver (mainly ascites). In chronic congestive cardiac failure, venous congestion will cause increased
hydrostaticpressureanddecreasedreturnofwaterintocapillariesandsopittingedemaoffeetmayresult.
8.Albuminisanegativeacutephaseproteinlevelofalbuminfallsmildlyinpresenceofinflammatorycytokines
suchasinterleukin6.
9.Analbuminemia{absenceofalbumin)isaveryraregeneticallydeterminedcondition.
AlbuminGlobulinRatio:Inalltheabovementionedconditionsofhypoalbuminemia,therewillbeacompen
satory increase in globulins which are synthesised by the reticuloendothelial system. Albuminglobulin ratio (A/G
ratio)isthusalteredorevenreversed.Thisagainleadstoedema.
Hypoproteinemia: Since albumin is the major protein present in the blood, any condition causing lowering of
albumin will lead to reduced total proteins in blood (hypoproteinemia). So it is observed in cirrhosis, nephrotic
syndrome,malnutritionandmalabsorptionsyndromes.
Hypergammaglobulinemias
1.Whenalbuminlevelisdecreased,bodytriestocompensateitbyincreasingtheproductionofglobulinsfrom
reticuloendothelialsystem.Thus,allcausesfor
hypoalbuminemiawillresultinalbumin:globulinratioreversal,andcorrespondingincreaseinpercentageinglobulins.
2.Inchronicinfections,thegammaglobulinsareincreased,buttheincreaseissmoothandwidebased.
3.Drasticincreaseinglobulinsareseeninpara
proteinemias,whenasharpspikeisnotedinelectro
phoresis.This
istermedasMband.Thisisduetomonoclonaloriginofimmunoglobulinsinmultiplemyeloma.
Hyperbetaglobulinemia:It is associated with hyper
lipoproteinemia, atherosclerosis and other hyperlipidemic
conditions.
Hyperalphaglobulinemia: In nephrotic syndrome, small molecular weight proteins (including albumin) leak
outthroughurine.Butproteinswithlargermolecularweightremainsinbloodsothereisanincreaseinalphaglobulin
fraction,whichcontainsalpha2macroglobulin.
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TRANSPORTPROTEINS
Blood is a watery medium so lipids and lipid soluble substances will not easily mix in the blood. Hence such
moleculesarecarriedbyspecificcarrierproteins.Albuminisanimportanttransportprotein,whichcarriesbilirubin,
freefattyacids,calciumanddrugs.
1.Prealbuminissonamedbecauseofitsfastermobilityinelectrophoresisthanalbumin.Itismoreappropriately
namedasTransthyretinorThyroxinbindingprealbumin(TBPA),becauseitcarriesthyroidhormones,thyroxin(T4)
andtriiodothyronineT3).Itcanbindlooselywithallsubstanceswhicharecarriedbyalbumin.Itsmolecularweightis
lesserthanthatofalbumin.Itisrichintryptophan.Itshalflifeinplasmaisonlyoneday.
2.Retinolbindingprotein(RBP)carriesvitaminA.Itisalowmolecularweightprotein,andsoisliabletobe
lost in urine. To prevent this loss, RBP is attached with prealbumin the complex is big and will not pass through
kidney glomeruli. It is a negative acute phase protein. Zinc is required for RBP synthesis, and so RBP level and
vitaminAlevelmaybeloweredinzincdeficiency.
3.Thyroxinebindingglobulin(TBG)isthespecificcarriermoleculeforthyroxineandtriiodothyronine.TBG
levelisincreasedinpregnancybutdecreasedinnephroticsyndrome.
4. Transcortin, otherwise known as Cortisol binding globulin (CBG) is the transport protein for Cortisol and
corticosterone..
5.Haptoglobin(forhaemoglobin),Hemopexin(forheme)andTransferrin(foriron)areimportanttopreventloss
ofironfrombody.
6.Cholesterolinbloodiscarriedbylipoproteins,HDLandLDLvarieties.
POLYMORPHISM
Thetermpolymorphismisappliedwhentheproteinexistsindifferentphenotypesinthepopulationbutonlyone
form is seen in a particular person. Haptoglobin, transferrin, ceruloplasmin, alpha1antitrypsin and
immunoglobulins exhibit polymorphism. For example, Haptoglobin (Hp) exists in three forms, Hp11, Hp21, and
Hp22.Twogenes,designatedHp1andHp2areresponsibleforthesepolymorphicforms.Theirfunctionalcapabilities
are the same. These polymorphic forms are recognised by electrophoresis or by immunological analysis. Study of
polymorphismisusefulforgeneticandanthropologicalstudies.
ACUTEPHASEPROTEINS
The level of certain proteins in blood may increase 50 to 1000folds in various inflammatory and neoplastic
conditions. Such proteins are acute phase proteins. Interleukins (ID, especially IL1 and IL6, released by
macrophages and lymphocytes, are the primary agents which cause induction and release of these acute phase
proteins.ImportantacutephaseproteinsareCreactiveprotein,ceruloplasmin,haptoglobin,a1acidglycoprotein,a1
antitrypsinandfibrinogen.
CreactiveProtein(CRP):
It is thus named because it reacts with Cpolysaccharide of capsule of pneumococci. CRP consists of five
polypeptidesubunitstoformadiscshapedcyclicpolymer.Ithasamolecularweightof115140kD.Itissynthesised
in liver. It can stimulate complement activity and macrophage phagocytosis. When the inflammation has subsided,
CRPquicklyfalls.CRPlevelhasapositivecorrelationinpredictingtheriskofcardiovasculardisease.
Ceruloplasmin
Ceruloplasminisblueincolour(Latin,caeruleus=blue).Itisanalpha2globulinwithmolecularweightof160,000.It
is synthesised in liver. It contains 6 to 8 copper atoms per molecule. Ceruloplasmin is also called Ferroxidase, an
enzymewhichhelpsintheincorporationofironintotransferrin.Ninetypercentofcoppercontentofplasmaisbound
with ceruloplasmin, and 10% with albumin. Copper is bound with albumin loosely, and so easily exchanged with
tissues.HencetransportproteinforcopperisAlbumin.Ceruloplasminisanenzyme.Itisanimportantantioxidantin
plasma.
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ClinicalApplication:
Normal blood level of ceruloplasmin is 2550 mg/dl. It is estimated either by its oxidative property on phenylene
diamine,orbyradialimmunodiffusion.ThislevelisreducedinWilson'shepato
lenticulardegeneration.Ceruloplasmin
level less than 20 mg/dl is pathognomonic of Wilson's disease. It is an inherited autosomal recessive condition.
Incidence of the disease is one in 50,000. The defect is associated with chromosome No.13. The basic defect is a
mutationinageneencodingacopperbindingATPaseincells,whichisrequiredforexcretionofcopperfromcells.So,
copperisnotexcretedthroughbile,andhencecoppertoxicityisseen.Increasedcoppercontentinhepatocyteinhibits
theincorporationofcoppertoapoceruloplasmin.Soceruloplasminlevelinbloodisdecreased.Accumulationinliver
leadstohepatocellulardegenerationandcirrhosis.Depositsinbrainbasalganglialeadstolenticulardegenerationand
neurologicalsymptoms.Anothercommonfindingiscopperdepositsasgreenorgoldenpigmentedringaroundcornea
thisiscalledKayserFleischerring.Copperdepositsinkidneymaycauserenalfailure,andinbonemarrowleadsto
hemolyticanemia.TreatmentconsistsofadietcontaininglowcopperandinjectionofDpenicillaminewhichexcretes
copperthroughurine.Sincezincdecreasescopperabsorption,zincisusefulintherapy.
Loweredlevelsofceruloplasminisalsoseeninmalnutrition,nephrosis,andcirrhosis.Ceruloplasminisanacutephase
protein.Soitslevelinbloodmaybeincreasedinallinflammatoryconditions,collagendisordersandinmalignancies.
Alpha1Antitrypsin(AAT)
AAT is otherwise called aantiproteinase or protease inhibitor (Pi). It inhibits all serine proteases (proteolytic
enzymes having a serine in their active centre), such as plasmin, thrombin, trypsin, chymotrypsin, elastase, and
cathepsin.SerineproteaseinhibitorsareabbreviatedasSerpins.Bindingofthisinhibitortoproteaseisverytightonce
bounditisnotreleased.Normally,about95%oftheantiproteaseactivityinplasmaisduetoAAT.Itissynthesisedin
liver.Ithasamolecularweightof50,000andhas3polypeptidechains.Itformsthebulkofmoleculesjnserumhaving
a1 mobility. It is estimated by radial immunodiffusion method. Normal serum level is 75200 mg/dl.
Electrophoretically, multiple allelic forms can be separated, the most common variety is PiMM determined by the
genotypeMM.Morethan75variantsareknown,outofwhichabout30geneticvariantsshowdecreasedorverylow
serumconcentrations.Geneislocatedonthesmallarmofchromosomenumber14.
AATdeficiencycausesthefollowingconditions:
1.Emphysema:Thedeficiencyisinheritedasacodominanttrait.TheincidenceofAATdeficiencyis1in1000,and
isoneofthemostcommoninbornerrors.About5%oftotalpopulationcarrytheabnormalgeneinheterozygousstate.
The total activity of a1AT is reduced in these individuals. Bacterial infections in lung attract macrophages which
release elastase. In the a1AT deficiency, unopposed action of elastase will cause damage to lung tissue, leading to
emphysema. About 5% of emphysema cases are due to a1AT deficiency. The alpha1 band on electrophoresis is
reduced or absent. The methionine residue at 358 position of a1AT is important in the enzyme binding. This
methionine may be oxidised to methionine sulfoxide by smoking. So emphysema is very common in smokers with
normala1ATlevelandsmokingwillworsenthesituationina1ATdeficientpersons.
2.Cirrhosis:AATdeficiencyisalsoseeninpersonswithPiZZgenes.Thisgeneticmakeupisassociatedwith
cirrhosis of liver. The ZZ protein has a substitution of glutamic acid by lysine at position 342. The protein is
unsialylatedandisnotreleasedfromhepatocytes,causingdeathofcellswithconsequentfibrosisandcirrhosis.
3.InNephroticsyndrome,AATmoleculesarelostinurine,andsoAATdeficiencyisproduced.
Alpha2Macroglobulin (AMG): AMG is a tetrameric protein with molecular weight of 725,000. It is the major
componentof2proteins.Geneislocatedinthelongarmofchromosomenumber12.Itissynthesisedbyhepa
tocytes
andmacrophages.AMGinactivatesallproteases,andthusitisanimportantinvivoanticoagulant.Proteasescleavethe
"bait" region of AMG, releasing a small unit, to provoke conformational changes in AMG, which then "traps" the
enzyme. So proteolytic enzymes cannot function. AMGprotease complexes are internalised by a receptor mediated
endocytosisbymacrophages,andthendegraded.AMGisthecarrierofmanygrowthfactorssuchasplateletderived
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growthfactor(PDGF).Normalserumlevelis130^300mg/dl.AMGcontributesabout1%ofalltotalplasmaproteins.
Itsconcentrationismarkedlyincreased(upto23g/dI)inNephroticsyndrome,becauseotherproteinsarelostthrough
urineinthiscondition.
Alpha1AcidGlycoprotein:ItisotherwiseknownasOrosomucoid.Ithasamolecularweightof44,000andhas
ahighcontentofabout45%ofcarbohydrates.ItsisoelectricpHis0.73.5.Itissynthesisedbyhepatocytes.Itbinds
lipophilicsubstancesandvariousdrugs.Itbindswithprogesteronetightly.Normalserumlevelis55140mg/dl,andits
halflifeisfivedays.Itisincreasedinpregnancy.Itisalsoanacutephaseprotein.Itisareliableindicatorofclinical
activityofulcerativecolitis.
NEGATIVEACUTEPHASEPROTEINS
During an inflammatory response, some proteins are seen to be decreased in blood these are called negative acute
phaseproteins.Examplesarealbumin,transthyretin(pre
albumin),retinolbindingproteinandtransferrin.
Transferrin:Itisaspecificironbindingprotein.Itisanegativeacutephaseproteinsothebloodlevelisdecreased
inacutediseases.Ithasahalflifeof710daysandisusedasabetterindexofproteinturnoverthanalbumin.
IMMUNOGLOBULINS
The terms gamma globulin and immunoglobulin are not synonymous. Antibodies are chemically called immuno
globulinsNintypercentofthesemoleculesmoveinthegammaregiononelectrophoresis,butsomemoleculeshave
beta and some even have alpha mobility. Immunoglo
bulin is a functional term, while gammaglobulin is a physical
term.ImmunoglobulinsaregenerallyabbreviatedasIg.Theyareclassifiedintofivemajorclasses.Theircharacteristics
aredescribedindetailin.
Plasmacontainsmanyenzymesandproteinhormone.Acomprehensivelistofnormalvaluesforthesubstances
presentinbloodisgivenintheAppendixII.
CLOTTINGFACTORS
ThewordcoagulationisderivedfromtheGreekterm,coagulare=tocurdle.Thebiochemicalmechanismofclottingis
atypicalexampleofcascadeactivation.Thecoagulationfactorsarepresentincirculationasinactivezymogenforms.
Theyareconvertedtotheiractiveformsonlywhentheclottingprocessisinitiated.Thiswouldpreventunnecessary
intravascularcoagulation.Activationprocessleadstoacascadechemicalamplificationeffect,inwhichonemoleculeof
precedingfactoractivates1000moleculesofthenextfactor,andsoon.Thuswithinseconds,millionsortrillions of
moleculesoffinalfactorsareactivated.
Several of these factors require calcium for their activation. The calcium ions are chelated by the gamma carboxyl
group of glutamic acid residues of the factors, prothrombin, VII, IX, X, XI and XII The gamma carboxylation of
glutamic acid residues is dependent on vitamin K, and occurs after synthesis of the protein (posttranslational
modification).
Prothrombin
It is a single chain zymogen with a molecular weight of 69,000 and plasma concentration around 1015 mg/dl. The
prothrombinisconvertedtothrombinbyFactorXa,bytheremovalofNterminalfragment.Thethrombinisaserine
proteasewithmolecularweightof34,000.TheCa++bindingofprothrombinisessentialforanchoringtheprothrombin
ontheanionphospholipidonthesurfaceofplatelets.Whentheterminalfragmentiscleavedoff,thecalciumbinding
sitesareremovedandso,thrombinisreleasedfromtheplateletsurface
Fibrinogen
The conversion of fibrinogen to fibrin occurs by cleaving of ArgGly peptide bonds of fibrinogen. Fibrinogen has a
molecularweightof340,000andissynthesisedbytheliver.Normalfibrinogenlevelinbloodis200400mg/dl.The
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fibrin monomers formed are insoluble. They align themselves lengthwise, aggregate and precipitate to form the soft
clot.Fibrinogenisanacutephaseprotein.Thefinalstepinclottingistheconversionofsoftclottohardclotbyfactor
XIIIorFibrinstabilisingfactor.ThisoccursbyformationofcrosslinksbetweenLysineandGlutamineresidues.The
amide group of glutamine is replaced by epsilon amino group of Lysine by factor XIII which acts as a
transglutaminase.
Platelets
Plateletsformaplugininjuredvessels.PlateletsalsoprovidefactorXIIIandphospholipidsandplateletfactorIV.The
serine proteases, and thereby the clotting mechanism is normally inhibited by heparin antithrombin complex. The
plateletfactornowreleasedwillremovethisinhibition,andcausesinitiationofthrombosis.Theplateletsthatadhere
totheendotheliallayerareactivatedandtheysecreteADPandThromboxaneA2whichfavourplateletaggregation
andformationoftheplug.
Anticoagulants
In vitro anticoagulants removes Ca++ which is essential for several steps on clotting, e.g. oxalates, citrate, EDTA.
Heparin and antithrombin III are the major in vivo anticoagulants. They exert an inhibitory effect on the serine
proteasewhichactivatestheclottingfactorsbypartialproteolysis.Heparinisalsousedindialysisandinthetreatment
of intravascular thrombosis. Since vitamin K is essential for coagulation, antagonists to vitamin K are used as
anticoagulantsespeciallyfortherapeuticpurposes,e.g.Dicoumarol,Warfarinsodium.Alpha2macroglobulinalsohas
anticoagulantactivity.
Fibrinolysis
Unwanted fibrin clots are continuously dissolved in vivo by Plasmin, a serine protease. Its inactive precursor is
plasminogen(90kD).Itiscleavedintotwopartstoproducetheactiveplasmin.Plasmininturn,isinactivatedbyalpha
2antiplasmin. Tissue plasminogen activator (TPA) is a serine protease present in vascular endothelium. TPA is
released during injury and then binds to fibrin clots. Then TPA cleaves plasminogen to generate plasmin, which
dissolvestheclots.Urokinaseisanotheractivatorofplasminogen.Urokinaseissonamedbecauseitwasfirstisolated
from urine. Urokinase is produced by macrophages, monocytes and fibroblasts. Streptokinase, isolated from
streptococciisanotherfibrinolyticagent.
Clinicalsignificance:Thrombosisincoronaryarteryisthemajorcauseofmyocardialinfarction(heartattack).If
TPA,urokinaseorstreptokinaseisinjectedintravenouslyintheearlyphaseofthrombosis,theclotmaybedissolved
andrecoveryofpatientispossible.
ABNORMALITIESINCOAGULATION
HemophiliaA(ClassicalHemophilia):ThisisaninheritedXlinkedrecessivediseaseaffectingmalesandtransmitted
byfemales.Malechildrenofhemophiliapatientsarenotaffectedbutfemalechildrenwillbecarriers,whotransmit
thediseasetotheirmaleoffspring.ThisisduetothedeficiencyoffactorVIM(AHG).Itisthemostcommonofthe
inheritedcoagulationdefects.Therewillbeprolongationofclottingtime.Hence,eventrivialwoundssuchastooth
extraction will cause excessive loss of blood. Patients are prone to internal bleeding into joints and gastrointestinal
tract.TreatmentconsistsofadministrationofAHG,preparedfrompooledseraeverythreemonths.Sincethisisnot
generallyavailable,theusualtreatmentistotransfusebloodperiodically,whichmayleadtoeventualironoverload,
hemochromatosis. Several hemophilia patients, receiving repeated transfusions became innocent victims of AIDS.
PureAHGisnowbeingproducedbyrecombinanttechnologyandisthetreatmentofchoice.
Hochstetter (1635) and Banyer (1743) had described hemophilialike diseases but the first clear account was
givenbyJohnOttoin1803.ThediseasehasgainedimportancebecauseitwaspresentintheroyalfamiliesofEurope.
ThegeneforAHGhasbeencloneditisoneofthelargestgeneswith186kbinsize.Thegenelocusisinthelongarm
ofXchromosomeatXq24qsite.PrenataldiagnosisbyDNAanalysisofcellsfromamniocentesisisnowpossible.
HemophiliaBorChristmasDisease:ItisduetofactorIXdeficiency.TheChristmasdiseaseisnamedafterthe
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firstpatientreportedwiththisdisease.SimilardeficienciesoffactorsXandXIarealsoreported.Inthesediseases,the
manifestationswillbevariationsofclassicalhemophilia.
VonWillebrandt'sDisease:Thisisanautosomaldominantdefectleadingtofailureofplateletstoadhere.Thedefect
is in the glycoprotein platelet adherence factor (PAF) present in the plasma and platelets. The bleeding time is
prolonged.
Otherdisorders:Acquiredhypofibrinogenemiaorafibrinogenemiamayoccurasacomplicationofprolongedlabour
(abruptioplacenta).Proteolyticthromboplasticsubstancesmayenterfromplacentatomaternalcirculationwhichsetoff
the clotting cascade (disseminated intravascular coagulation or DIC). But the clots are usually degraded
immediatelybyplasminolysis.Continuationofthisprocessleadstoremovalofallavailableprothrombinandfibrinogen
moleculesleadingtoprofusepostpartumhemorrhage.Insomecasesofcarcinomaofpancreas,trypsinisreleasedinto
circulation leading to intravascular coagulation. This may be manifested as fleeting thrombo
phlebitis. Trousseau
diagnosedhisownfataldiseaseascancerofpancreaswhenhedevelopedthrombophlebitis.
The combination of carcinoma of pancreas, migratory thrombophlebitis and consumption coagulopathy is
termedasTrousseau'striad.
Proteinuria:
Richard Bright established proteinuria as an indicator of renal disease in 1827. Proteinuria is an important index of
renaldiseases.Innormalurine,proteinconcentrationisverylow(lessthan100mgperday),whichcannotbedetected
bytheusualtests.Theseproteinsaresecretedbythetubularepithelialcells.
Proteinuriamaybeclassifiedas:
a)Glomerularproteinuria:Theglomeruliofkidneyarenotpermeabletosubstanceswithmolecularweightmore
than69,000andsoplasmaproteinsareabsentinnormalurine.Whenglomeruliaredamagedordiseased,theybecome
morepermeableandplasmaproteinsmayappearinurine.Thesmallermoleculesofalbuminpassthroughdamaged
glomeruli more readily than the heavier globulins. So, when proteins appear in the urine, the albumin fraction
predominates.Albuminuriaisalwayspathological.Largequantities(afewgramsperday)ofalbuminarelostinurine
innephrosis.Smallquantitiesareseeninurineinacutenephritis,strenuousexerciseandpregnancy.
Microalbuminuria or minimal albuminuria or paucialbuminuria) is identified, when small quantities of albumin
(50300mg/day)isseeninurine.Itisseenasacomplicationofdiabetesmellitusandhypertension.Itisanindicatorof
futurerenalfailure.
b)Overflowproteinuria:Whensmallmolecularweightproteinsareincreasedinblood,theyoverflowintourine.
Forexample,hemoglobinhavingamolecularweightof67,000canpassthroughnormalglomeruli,andtherefore,itit
exists in free form (as in haemolytic conditions ), hemoglobin can appear in urine (hemoglobinurial). Similarly,
myoglobinuriaisseenfollowingmusclecrushinjury.YetanotherexampleistheBenceJonesproteinuria.Inabout20%
casesofmultiplemyeloma,thelightchainsofimmunoglobulinsareproducedabnormally.Beingofsmallermolecular
weight, they are excreted in urine. These are called BenceJones Proteins (monoclonal light chains produced by
plasmacytomas).Whentheurineisheated,at45Ctheystartprecipitating,at60Cthereismaximumprecipitation,at
80Ctheseproteinsstartredissolving,andwillformaclearsolutionat100C.Theprecipitatereformsoncooling.
c)Nephronlossproteinuria:Thisoccurswhenfunctionalnephronsarereduced,GFRisdecreasedandremaining
nephronsareoverworking.
d) Tubular proteinuria: It is seen when tubular reabsorption mechanism is impaired, and low molecular weight
proteinsareexcreted.
e)Urogenicproteinuria:Thisisduetoinflammationoflowerurinarytract,whenproteinsaresecretedintothe
tract.
Measurementofurinaryproteinsmaybecarriedout:
a)toestablishthepresenceorabsenceofrenaldisease
b)todefinethenatureofrenaldiseasec)todefinethedegreeofrenaldysfunctionandd)tomonitortheresponseto
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treatment.
Theproteinuriaiscommonlyassessedbytheheatandaceticacidtest(FrederickDekkers,1694).Filltwothirdsofa
testtubewiththeurinesampleandheatthetopofthecolumntoboiling.Add1%aceticacid(3drops).Comparethe
topportionwiththelower.Acloudinessonthetopshowsthepresenceofproteins.Albuminiscoagulatedwhenheated,
whichisprecipitatedattheisoelectricpoint,whenaceticacidisadded.Calciumandmagnesiumphosphatesarealso
precipitatedonboiling,buttheyaresolubleinacidicmedium.
Esbach'sAlbuminometerisusedforroughquantitativeestimationofproteinsintheurine.Addurineuptothemark'U'
andtheEsbach'sreagentuptothemark'R'oftheapparatus.Mixwell.Keepfor24hours.Readtheamountofprotein
precipitatedonthetubeingramsofproteinperliterofurine.Esbach'sreagentispicricacidandcitricacid.
Microalbuminuriaisdetectedbyradialimmunodiffusionorbyenzymelinkedimmunosorbentmethods.
Laboratorydiagnosticsofdiabetesmellitus
Diabetesisthemostcommonmetabolicdisorder,anditsincidenceisincreasing.Biochemicalmeasurementsare
particularlyimportantindetectingit,monitoringitscontrolandtreatingitsmetaboliccomplications.Hypoglycaemia
occursininsulintreateddiabeticpatients,butisother
wiserare.However,itisanimportantdiagnosistomakebecause
ofitspossibleconsequences.Otherdisordersofcarbohydratemetabolismareuncommon.
Dietarycarbohydrateisdigestedinthegastrointestinaltracttosimplemonosaccharideswhicharethenabsorbed.Starch
providesglucosedirectly,whilefructose(fromdietarysucrose)andgalactose(fromdietarylactose)areabsorbedand
alsoconvertedintoglucoseintheliver.Glucoseisthecommoncarbohydratecurrencyinthebody.
Insulinistheprincipalhormoneaffectingbloodglucoselevels.Itisasmallproteinsynthesizedinthebetacellsofthe
isletsofLangerhansofthepancreas.Itactsthroughmembranereceptorsanditsmaintargettissuesareliver,muscleand
adiposetissue.theoveralleffectsofinsulinistopromotecellularuptakeandstorageofmetabolicfuels.Insulinis
synthesisedinthecellsoftheisletsofLangerhansinthepancreas.Itisformedaspreproinsulin,whichisrapidly
cleavedtoproinsulin.TheproinsulinispackagedintosecretorygranulesintheGolgiapparatusandcleavedtoinsulin
andCpeptide.InsulinandCpeptidearelaterreleasedintothecirculationinequimolaramounts.Ariseinblood
[glucose]isthemainstimulusforinsulinsecretion.Someaminoacids(e.g.leucine),fattyacidsandketonebodiesalso
promoteinsulinsecretion.ThereleaseofinsulininresponsetohyperglycaemiaisenhancedbythepresenceofGIPor
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glucagon.GIPisprobablythemostimpor
tantfactorinthelargerreleaseofinsulinthatoccursinresponsetoanoral
glucoseload,com
paredwiththesamedoseofglucosegivenintra
venously.Vagalstimulationalsopromotesinsulin
release.Theinsulinreceptorislocatedonthecellsurfaceandisinternalisedafterinsulinbinding.Withindif
ferent
organs,targetenzymeshavebeenidentifiedthatservetoexplaintheknowneffectsofinsulinonintermediary
metabolism.Forinstance,activationofglucosetransport,inductionofhexokinase(orglucokinase)andactivationof
phosphofructokinase,pyruvatekinaseandpyruvatedehydrogenaseintheliverareallconsistentwiththeactionsinsulin
inpromotingincreasedglucoseuptakeandglycolyticbreakdown.Stimulationofglycogensynthaseaccordswiththe
effectsofinsulinonglycogenformationintheliver.
Theeffectsofinsulinareopposedbyotherhormones,glucagon,adrenaline,glucocorticoidsandgrowthhormone.The
bloodglucoseconcentrationistheresultofabalancebetweenthesedifferentendocrineforces.
Diabetesmellitusmaybedefinedasasyndromecharacterizedbyhyperglycemiaduetoanabsoluteorrelativelackof
insulinand/orinsulinresistance.
Primarydiabetesmellitusissubclassifiedintoinsulindependentdiabetesmellitus(IDDMortypeI)andnoninsulin
dependentdiabetesmellitus(NIDDMortypeII).
ThecontrastingfeaturesofIDDMandNIDDMareshowninTable1:
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Secondarydiabetesmellitusmayresultfrompancreaticdisease,endocrinediseasesuchasCushing'ssyndrome,drug
therapy,andinsulinreceptorabnormalities.
Insulindependentdiabetesmellitus(IDDMortypeI)accountsforapproximately15%ofalldiabetics.Itcanoccurat
anyage,butismostcommonintheyoung,withapeakincidencebetween9and14yearsofage.Theabsolutelackof
insulinisaconsequenceoftheautoimmunedestructionofinsulinproducingbetacells.
Noninsulindependentdiabetesmellitus(NIDDMortypeII)accountsforapproximately85%ofalldiabeticsand
canoccuratanyage.Itismostcommonbetween40and80years.Inthisconditionthereisresistanceofperiferaltissues
totheactionsofinsulin,sothattheinsulinlevelmaybenormalorevenhigh.Obesityisthemostcommonlyassociated
clinicalfeature.
Latecomplicationsofdiabetesmellitus
Diabetesmellitusisnotonlycharacterizedbythepresenceofhyperglycemiabutalsobytheoccurrenceoflate
complications:
1.Microangiopathyisdefinedasabnormalitiesinthewallsofsmallbloodvessels,themostprominentfeatureof
whichisthickeningofthebasementmembrane.
2.retinopathymayleadtoblindnessbecauseofvitreoushaemorrhagefromproliferatingretinalvessels,and,
maculopathyasaresultofexudatesfromvesselsoroedemaaffectingthemacula.
3.Nephropathyleadsultimatelytorenalfailure.Intheearlystagethereiskidneyhyperfunction,associatedwithan
increasedGFR,increasedglomerularsizeandmicroalbuminuria.Inthelatestage,thereisincreasingproteinuria
andamarkeddeclineinrenalfunction,resultinginuraemia.
4.Neuropathymaybecomeevidentasdiarrhoea,posturalhypotension,impotence,neurogenicbladderand
neuropathicfootulcersduetomicroangiopathyofnervebloodvesselsandabnormalglucosemetabolisminnerve
cells.
5.Macroangiopathyleadstoprematurecoronaryheartdisease.Theexactmechanismsforincreasedsusceptibilityto
atherosclerosis.
Clinicalsymptomsofhyperglycemiainclude:
polyuria
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polydipsia
lassitude
weightloss
pruritusvulvae
balanitis
These symptoms are common to both NIDDM and IDDM but are more pronounced in IDDM. It is important to
rememberthatpatientswithNIDDMmaybecompletelyasymptomatic.
Diagnosisandmonitoringofdiabetesmellitus
Thediagnosisofdiabetesmellitushasseriousconsequences.Itconfersariskoflongtermdiabeticcomplications,
includingblindness,renalfailureandamputations,aswellasanincreasedriskofcardiovasculardisease.Italsomeansa
lifetimeofdietaryrestrictionandmedicationsandcanseri
ouslycurtaillifestyleandemploymentprospects.The
diagnosismaybesuggestedbythepatient'shistory,orbytheresultsofdipsticktestsforglu
coseonurinespecimens.
However,urineglucosemeasurementsbythemselvesareinadequatefordiagnosingdiabetes.Theypotentiallyyield
falsepositiveresultsinsubjectswithalowrenalthresh
oldforglucose,andinapatientwithdiabetes,theymayyield
falsenegativeresultsifthepatientisfasting.Aprovisionaldiagnosisofdiabetesmellitusmustalwaysbeconfirmedby
glucosemeasurementsonbloodspecimens.
Themostrecentcriteriaforthediagnosisofdia
betesmellitushavebeenlaiddownbytheWorldHealthOrganization
(WHO)in1998,andbytheAmericanDiabetesAssociationin1997.Thesearebroadlysimilarbutdifferinsome
details.Itislikelythatthepreciserequirementsforthediagno
sisofdiabetesandthestatesofimpairedglucose
regulationwillcontinuetoevolveasknowledgeoftherelationbetweenglucoseregulationandthefuture
developmentofcomplicationsaccumulates.ThefollowingdescriptionsadheretotheWHOrecommendations.
Separatecriteriaaredescribeddependingonwhethervenousorcapillarywholeblood,orvenousorcapillaryplasma
specimensareused.Inpractice,resultsforthisimportantdiagnosiswillusuallycomefromaclinicallabora
toryand
usevenousplasma.Accordingtothecriteria,arandomvenousplasma[glucose]of11.1mmol/Lormore,orafasting
plasma[glucose]of7.0mmol/Lormore,establishesthediagnosis.Asingleresultissufficientinthepresenceof
typicalhyperglycaemicsymptomsofthirstandpolyuria.Intheirabsence,avenousplasma[glucose]inthediabetic
rangeshouldbedetectedonatleasttwoseparateoccasionsondifferentdays.Wherethereisanydoubt,anOGTT
shouldbeperformed,andifthefastingorrandomvaluesarenotdiagnostic,the2hvalueshouldbeused.Inprac
tice,
thediagnosisisoftenobviousclinically,and[glucose]isonlyneededforconfirmationandisunequivocallyhigh.The
diagnosisshouldneverbemadeonthebasisofasingletestinapatientwithoutsymptoms.
AtpresentaraisedHbA1cshouldnotbeusedtomakeadiagnosisofdiabetes.StandardisationofHbA1canalysishas
beenaprobleminthepastalthoughthisisnowlessofanissue.Thishasmeantthatithasnotbeenpossibletoreliably
determineasinglecutofflevelthatwoulddiag
nosediabetesinalllaboratories.
Glucosuria allows for a good firstline screening test for diabetes mellitus normally glucose does not appear in the
urineuntiltheplasmaglucoserisesabove10mmol/l(renalthresholdforglucosethemaximumamountofglucosein
blood,whichiscompletelyreabsorbedinkidneytubuls).
Ketone bodies may accumulate in the plasma of a diabetic patient. Ketones may be present in a normal subject as a
resultofsimpleprolongedfasting.Dryreagentstripswhichdetectacetoacetatemightthereforeprovideanunderstimate
ofketonaemia/ketonuria.
Bloodglucose
Glucose is routinely measured in the laboratory on blood specimen which have been collected into tubes containing
fluoride,aninhibitorofglycolysis.Becouseoftheneedsometimestoobtainrapidbloodglucoseresultsandthewide
spreadselfmonitoringofdiabeticpatients,bloodglucoseisalsoassesedoutsidethelaboratoryusingteststrips.
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TheWorldHealthOrganizationhaspublishedguidelinesforthediagnosisofdiabetesmellitusonthebasisofblood
glucoseresultsandtheresponsetoanoralglucoseload.
Randombloodglucose(RBG) is the only test required in an emergency. An RBG of less than 8 mmol/l should be
expectedinnondiabetics.
RBGhigherthan11mmol/lusuallyindicatesdiabetesmellitus.
Fasting blood glucose (FBG) is measured after overnight fast (at least 10 hours). An FBG is better than RBG for
diagnostic purposes. In nondiabetics it is usually lower than 6 mmol/l. Fasting values of 68 mmol/l should be
interpretedasborderline.
Oralglucosetolerancetest(OGTT)
Abaselinebloodsampleisfirsttakenafteranovernightfast.Thepatientisthengiven75gofglucoseorally,inabout
300mlofwater,tobedrunkwithin5minutes.Plasmaglucoselevelsaremeasuredevery30minfor2hours.Urinemay
also be tested for glucose at time 0 and after 2 hours. The patient should be sitting comfortably throughout the test,
shouldnotsmokeorexerciseandshouldhavebeenonnormaldietforatleast3dayspriortothetest.
Indications:
borderlinefastingorpostprandialbloodglucose
persistentglycosuria
glycosuriainpregnantwomen
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pregnant women with a family history of diabetes mellitus and those who previously had large babies or
unexplainedfetalloss
InterpretationofanOGTT
If the patient has a normal fasting plasma glucose and only the 2 h value in the diabetic range, the test should be
repeatedafterapproximately6weeks.Impairedglucosetolerance(IGT)shouldnotberegardedasadisease.Itsignals
that the patient is at an intermediate stage between normality and diabetes mellitus and is at an increased risk of
developingdiabetes.Suchpatientsshouldbefollowedyearly,anddietarytreatmentmaybeused.
Longtermindicesofdiabetescontrol
AhighconcentrationofglucoseintheECFleadstoitsnonenzymaticattachmenttothelysineresiduesofavarietyof
proteins.Thisiscalledglycation.Theextentofthisprocessdependsontheambientglucoselevel.Theconcentrationof
glycatedproteinisthereforeareflectionofameanbloodglucoselevelprevailingintheextracellularfluidduringthe
lifeofthatprotein.
HaemoglobinA1corglycatedhaemoglobin
Glycatedhaemoglobinreflectsthemeanglycaemiaover2monthpriortoitsmeasurement,thehalflifeofhaemoglobin.
Thistestisacceptedasagoodindexofdiabeticcontrolandisusedroutinelyinmostdiabeticsclinicstocomplement
theinformationfromsinglebloodglucoselevels,orindeedapatient'slogofhisorherownbloodglucose
measurements.
Fructosamine
Manyotherproteinsinadditiontohaemoglobinareglycatedwhenexposedtoglucoseintheblood.Asindicationofthe
extentofthisglycationcanbeobtainedbymeasuringfructosamine,theketoamineproductofnonenzymaticglycation.
Asalbuministhemostabundantplasmaprotein,glycatedalbuministhemajorcontributortoserumfructosamine
measurements.Asthisproteinhasashorterhalflifethanhaemoglobin,fructosaminemeasurementsarecomplementary
toHbA1cprovidingtheindexofglucosecontroloverthe3weekspriortoitsmeasurement.
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Microalbuminuria
Itmaybedefinedasanalbuminexcretionrateintermediatebetweennormality(2.525mg/day)and
macroalbuminuria(>250mg/day).Theimportanceofmicroalbuminuriainthediabeticpatientsisthatitisasignalof
early,reversiblerenaldamage.
Diabeticketoacidosis
AllmetabolicdisturbancesseeninDKAaretheindirectordirectconsequencesofthelackofinsulin.Decreased
glucosetransportintotissuesleadstohyperglycemiawhichgivesrisetoglycosuria.Increasedlipolysiscausesover
productionoffattyacids,someofwhichareconvertedintoketones,givingketonaemia,metabolicacidosisand
ketonuria.Glucosuriacausesanosmoticdiuresis,whichleadstothelossofwaterandelectrolytessodium,potassium,
calcium,magnesium,phosphateandchloride.Dehydration,ifsevere,producesprerenaluraemia,andmayleadto
hypovolaemicshock.Theseveremetabolicacidosisispartiallycompensatedbyanincreasedventilationrate(Kussmaul
breathing).Frequentvomitingisalsousuallypresentandaccentuatesthelossofwaterandelectrolytes.
Laboratoryinvestigations
Urine(ifavailable)shouldbetestedforglucoseandketones,andbloodcheckedforglucoseusingateststrip.Venous
bloodshouldbesenttothelaboratoryforplasmaglucoseandserumsodium,potassium,chloride,bicarbonate,ureaand
creatinin.Anarterialbloodsampleshouldalsobesentformeasurementofbloodgases.Bloodglucoseshouldbe
monitoredhourlyatthebedsiteuntillessthan15mmol/l.Thereafterchecksmaycontinue2hourly.Theplasmaglucose
shouldbeconfirmedinthelaboratoryevery24hours.
Treatment
ThemanagementofDKArequirestheadministrationof3agents:
Fluids.PatientswithDKAareusuallyseverelyfluiddepletedanditisessentialtoexpandtheirECFwithsaline
torestoretheircirculation.
Insulin.Intravenousinsulinismostcommonlyused.
Potassium.
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Hypoglycemiaisalaboratorydiagnosiswhichisusuallytakentomeanabloodglucoselevelbelow2.5mmol/l.
Alowbloodglucoselevelnormallyleadstothestimulationofcatecholaminesecretionandstimulationofglucagon,
cortisolandgrowshormone.
Symptomsmosrcommonlyseeninhypoglycaemia:
sweating
shaking
tachycardia
nausea
weakness
Laboratoryinvestigation
Bloodglucose.Thedetectionofhypoglycaemiaisbybloodglucosetesting.Urinetestingcannotdetecthypoglycaemia.
Plasmainsulin.Insulinmeasurementscanleadtothediagnosisorexclusionofinsulinoma.
Insulin/glucoseratio.
PlasmaCpeptide.insulinsecretionininsulintreateddiabeticscannotbeassesedbythemeasurementofplasmainsulin
sincetheinsulingiventherapeuticallywillalsobemeasured.However,insulinanditsassociatedconnectingpeptide(or
Cpeptide)aresecretedbytheisletcellsinequimolaramountsandthusmeasurementofCpeptidelevelstogetherwith
insulincandifferentiatebetweenhypoglycaemiaduetoinsuloma(highCpeptide)andthatduetoexogenousinsulin
(lowCpeptide).
Specificcausesofhypoglycaemia
Over99%ofallepisodesofhypoglycaemiaoccurininsulindependentdiabeticpatients.
Reasonsforhypoglycaemiainthediabeticsinclude:
insufficientcarbohydrateintake
excessofinsulin
strenuousexercise
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excessivealcoholintake
Othercausesofhypoglycaemiamaybedividedintwogroups:thosewhichproducehypoglycaemiainthefasting
patient,andthoseinwhichthelowglucoseconcentrationisaresponsetoastimulus(reactivehypoglycaemia).
Fastinghypoglycaemia
Causesoffastinghypoglycaemiainclude:
Insulinoma.cellislettumorsofthepancreasmayproduceinsulinbothinappropriatelyandinexcess.
Cancer.hypoglycaemiaisassociatedwithadvancedmalignancy.
Hepaticdisease.
Addison'sdisease
Sepsis
Reactivehypoglycaemia
Inreactivehypoglycaemiapatientsmaybecomehypoglycemicinresponseto:
Drugs.
Food:postprandialhypoglycaemia.Acceleratedgastricemptyingaftergastricsurgery(dumpingsyndrome)may
giverisetothiscondition.
Alcohol.
Neonatalhypoglycaemia
Thediagnosisandtreatmentofhypoglycaemiaintheneonateisparticularlyimportantbecauseofthehighriskof
hypoglycaemicbraindamage.Thereareanumberofimportantcauses:
Babiesofdiabeticmothers.Afetusthatisexposedtomaternalhyperglycaemiawillhavepancreaticisletcell
hyperplasiaandelevatedinsulinlevels.Afterdeliverytheneonateisunabletosuppressitsinappropriatelyhighinsulin
levelsandwilldevelophypoglycaemia.
Intrauterineretardation.Smallfordatesbabiesmayhaveinadequateliverglycogenstores.
Inbornerrorsofmetabolism.Galactosaemiaandglycogenstoragediseaseareexamples.
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