Platelets

Developmentalpathwayofplatelets
 Originandstructure
Megakaryocytes diameter35160m
Plateletdiameter23m
Containanirregularringoflobednuclei
Plateletsareformedwithinthecytoplasmof
megakaryocytes andreleasedintothecirculation
Plateletsurvivaltime812days
Destroyedmainlyinspleen
Bloodcount:1.54lakh/Lofblood
 Morphology
Wetpreparations:colourless,moderatelyrefractile,
discoidorelliptical
Darkfield illumination:translucent,sharpcontour,few
immobilegranules
Leishmans stainshowsafaintbluecytoplasmwith
distinctreddishpurplegranules
Electronmicroscopyrevealsacellmembrane6nm
thickwhichsurroundsacytoplasmic matrixcontaining
Golgiapparatus,endoplasmicreticulum,50100very
densegranules,mitochondria,microvesicles,
microtubules,filaments&granules
Structureofplatelet
 Structureofplatelet
Membranestructures:surfaceglycoproteins serveasreceptors,
facilitateplateletadhesion&contraction,anddetermineexpressionof
specificplateletantigensandantigenssharedwithotherformedelements
Canalicular system:numerousinvaginationsoftheplateletsurface
and,interspersedamongthesestructures,asetofnarrowerchannels
termedthedensetubularsystem
Densetubularsystem:isthemajorsiteforstorageofCa2+andthe
locationofcyclooxygenase
Cytoskeleton
Microtubules
Systemofcontractileproteins
Granules
 Plateletgranules
Densebody:ADP,ATP,GTP,GDP,serotonin,secretable
Ca++
Alphagranules:
Plateletspecificproteins:PF4,thromboglobulin,PDGF,
thrombospondin
Homologs ofplasmaproteins:fibrinogen,fibronectin,
albumin
Densetubularsystem:Prostaglandinconverting
enzymes,contractilecalcium
Peroxisomes:Catalase
Lysosomes:Acidhydrolase
 Function
Hemostasis
Bloodcoagulation
Phagocytosis
Storage&transportofsubstances
Eventsinhemostasis
Eventsinhemostasis.
 Hemostasis (Arrestofbleeding)
Formationofhemostatic plugdueto
Adhesviness todamagedliningofbloodvessels
PromotedbyCa++ions&ADP
Aggregationofplateletsleadstoformationof
whitebodiesormicrothrombiwhichmaygrow
untiltheyalmostfillthelumenofasmallvessel
Atfirsttheaggregationisreversible
Thenitbecomesirreversible,plateletgranulesare
discharged,leukocytesbegintoadhereto
platelets,fibrindepositionoccurs
 Suggestedsequenceofeventsin
hemostasis
Plateletadherencetocollagenindamaged
vesselswall
ATPisconvertedtoADPbyATPase
ADPisreleasedandpromotesaggregationof
passingplatelets:formationofplateletplug
Releaseoftissuefactorfromdamagedvessels
&phospholipidsfromplateletswillpromote
thrombinformation
Firmclotsealsthevesselpermanently
 Otherreactionsinhemostasis
Aftertheinitialdilatationthedamagedvesselsconstrictfor
about20minutes,5HTbeingimportantinthisprocess
Prostaglandinformingsystemisalsoinvolvedinplatelet
aggregation
Arachadonic acidisreleasedfromtheplateletcell
membranebyphospholipase andthenrapidlyoxidizedby
theenzymecyclo oxygenase toPGG2 &PGH2
Intheplatelets:PGG2 isconvertedtoTXA2 ahighlypotent
plateletaggregatorandconstrictorofarterialmuscle
Inthearterialendothelium:PGG2 &PGH2 areconvertedto
prostacyclin apotentinhibitorofplateletaggregationanda
vasodilator
 Bloodclotting
Inblooddeprivedofplatelets
Clottingtimeinglasstubeisprolonged
Activationofprothrombin isincomplete
Formedclotsdonotretract
Plateletsarenecessaryforintrinsicclotting
processbyreleasingPlateletfactor3which
causesconversionofprothrombin tothrombin
byfactorXandV
 Clotretraction
Clotretractionoccursbyshorteningoffibrin
fibersproducedbycontractionofattached
plateletpseudopodia,whichcontain
actomyosin likeprotein.
Agentswhichinhibitcellmetabolismor
enzymeactivityalsoinhibitclotretraction
Bloodallowedtoclotinaglasstubeat370 C
beginstoshowclotretractionafter30
minutes
 Phagocytosis
Carbonparticles,immunecomplexesand
virusesarephagocytosed byplatelets
Avestigialmechanismofclearingparticulate
materialfromtheblood
 Storage&transport
Storesof5HTandhistaminewhichare
releasedbyplateletdisintegration
Theyalsotakeup5HTbyactivetransport
Epinephrine&potassium
 Nonhematologicalfunctionsof
platelets
Inflammation
Plateletshave4featurescommontoall
inflammatorycells
Posssess widerangeofinfammatory mediators
Presenceofreceptorsforotherinflammatory
cells
Abilitytorespondtonoxiousstimuli
Cooperativewithotherinflammatorycells
Platelet&growthfactors
PDGF,EGF,FGFarereleasedfromalpha
granulesofplatelets
Platelets&neurotransmitters
Despitebeingofdifferentembryologicalorigin
ithasbeenshownthatplateletsbehavelike
serotonergic neuronsinCNS.Thusthey
provideaneuronalmodelforstudying
neurons
Platelets&metastasis
Promotetumour survivalinbloodstreamby
protectingtumour cellsfromattackbyNK
cells
Plateletsinasthma
ReleaseofPAF&PF4causebronchialhyper
reactivity
 Hemostasis
Physiologyofcoagulation
Testsforclotting
Clotretraction
Fibrinolysis
Anticoagulants
 Hemostasis (Preventionofbloodloss)
Mechanismsbywhichhemostasis isachieved
1. Vascularconstriction
Primaryhemostasis
2. Plateletplugformation
3. Bloodclotformation Secondaryhemostasis
4. Clotretractionandfibrinolysis Tertiaryhemostasis
 Primaryhemostasis
Vascularphase:Triphasic response
1. Immediatereflexvasoconstriction:Anintrinsic
responseofsmoothmusclesinsmallarterioles&pre
capillarysphincters
2. Transientvasodilation:DuetoPGE2,Histamine&
prostacyclin
3. Sustainedvascularcontraction:Due toreleaseoflocal
autocoid factorsfromthetraumatizedtissueand
platelets.Ex.TXA2,Fibrinopeptide B,epinephrine,
norepinephrine
 Primaryhemostasis
Plateletphaseofhemostasis:
Importantincontrollingbleedingfromcapillaries&smallvenules in
erosionofmucosalsurfaces
Adherencetoexposedsubendothelial structures&activation
Burstofmetabolicactivity
Shapechange
Plateletaggregation/plugs
ADPextrusion&otheractivatedsubtances
Activation&avalibility ofPF3&otherprocoagulants
Initiationofbloodclotting
Consolidationofplateletplugbyfibrin
Clotretraction
 Adhesion
Definedastheattachmentofplateletstonon
plateletsurfaces
Thefirstdetectableeventaftervascularinjury

V V V
GP + W GP W GP W
F F F

VWFbinding Adhesion
HighmolecularweightsubunitofvWF actsasaninitialligand
betweenplatelets&subendothelial structuresi.e.collagen
 VonWillebrand factor
Aproteinsynthesised byvascularendothelialcells&megakaryocytes
Belongstoaclassofadhesiveproteins.
AdhesiveproteinscontainasequenceofaminoacidsArgGlyAsp(RGD)
sequence
Thissequenceallowsadhesiveproteinstobindtointegrins (cellsurface
proteins)
GPIIb &GPIIIa aretwointegrins presentonplateletsurface
Secretedintoplasmaandalsoabluminally intothesuperficiallayersofsub
endothelium
SubendothelialvWF contributestoplateletadhesionbutisinsufficient
AfterendothelialcelldisruptiontheplasmavWF bindstothe
subendothelium afterwhichitcanbindtosurfaceofunstimulated
plateletsandlinkthemtothesubendothelium
 Adhesion
Fibronectin
Synthesised byendothelialcells
Stored&secretedbyplatelets
Canbindcollagen
 Plateletshapechange
Occurswithinsecondsofexposureto
activatingagents
Fromflatteneddiscstosphereswithmultiple
projectingpseudopods
Polymerizationofplateletactin
Microtubulesthatarenormallyatthe
peripherygotothecentersurroundingthe
granulesthathavegonetothecenterpriorto
release
 Plateletaggregation
Definedastheattachmentofactivatedplateletstoeachother
Primaryaggregation:ADP&thrombincausedirect
aggregation
GPIIbIIIa isanintegrin thatrecoginzes twoRGDsequences
presentonfibrinogen
Fibrinogenthusformsabridgebetweentwoopposing
activatedplatelets
Secondaryaggregation:substancescausingaggregationby
releaseofADPorPG
Thrombospondin fromalphagranulesleadstoireversible
aggregationbybindingtofibrinogen&GPIVonplatelet
surface
 Plateletactivation
Seriesofprogressiveoverlappingevents
Shapechange
Aggregation
Liberation&oxidationofarachadonic acid
Secretionofalpha&densegranules
Reorganizationofsurfacemembrane
phospholipids
Orientedcentripital contractionofactomyosin
 Plateletactivation
Primaryagoniststriggeringplateletactivation
Thrombinformedatinjurysite
Sequenceofcollagenonsubendothelium
Maintenance&amplificationofplatelet
activation
ADP
Arachadonic acidoxidationproducts
 Raisedcytosolic Ca++ triggers
Phosphorylation ofmyosinlightchainkinase.
Requiredforreorientationofcytoskeletal
proteinsneededforplateletshapechange,
secretionandcontraction
Activationofacalciumdependentprotease
calledcalpain whichthroughproteolysis
activatesotherplateletenzymes
Activationofphospholipase A2
Prostaglandinthromboxane system
PLA2
MembranePL AA Lipoxygenase HPETE GPX HETE
Cycloxygenase
Aspirin Prostacyclin Inhibitionofrelease
Aggregation
PGG2+PGH2
PGF2,PGD2,PGE2
TXsynthase
Initiationofrelease
Aggregation
Malonaldihyde TXA2
 Roleofaspirin
Aspirinirreversiblyinactivatesplatelet
cycloxygenase
ThussynthesisofPGH2 &TXA2 isprevented
Prolongationofbleedingtimemayoccur
Therapeuticroleinpatientsofcoronaryartery
disease
 PGH2:Actsasacofactorenhancingcollagensabilityto
functionasaplateletagonist
TXA2:Bindstoaspecificplateletmembranereceptor
withresultantactivationofphospholipase C&
amplificationofplateletactivationthroughfurther
generationofDAG&IP3
Prostacyclin:Secretedbyendotheliumhelpstokeep
plateletsinanunstimulated statethroughactivationof
plateletadenyl cyclase &aresultantriseinplatelet
cAMP levels
PGD2:Canalsoactivateplateletadenyl cyclase
 Phosphoinositol metabolism
Plateletactivation

Phosphatidyl inositol bis phosphate(PIP2)

Diacyl glycerol(DG)+Inositol
Cofactorfor
triphosphate(IP3)
Proteinkinase C
Mobilises Ca++from
Phosphatidic acid(PA) intracellularstores
Mobilises Ca++from
intracellularstores
 Plateletreleasereaction
granules
Plateletproteins
Thrombospondin:Paltelet aggregation
PF4:Neutrlises anticoagulantactivityofheparin,competeswith
antithrombin IIIforbondingsitesonheparan sulphate presenton
endothelialcells,chemoattractantforWBC,smoothmusclecells&
fibroblasts
PDGF:chemo attractantforWBC,smoothmusclecells&fibroblasts
TGF:chemoattractantforWBC,smoothmusclecells&fibroblasts
Analogus ofplasmaproteins:
Albumin,IgG noknownhemostatic function
Fibrinogen,vWF,FactorV knownhemostatic function
Densegranules:ADP,ATP,Calcium,serotonin
Lysosomal granules:
Compaction&stabilizationofplatelet
plug
TheGPIIbIIIa hetrodimer traversesthe
plateletmembrane
Whenfibrinogenbindstotheexternaldomain
ofGPIIbIIIa thecytoplasmic domainisaltered
andbindstoactin filaments
Thisorientscentripetalcontraction
 Bloodcoagulation
Secondaryhemostasis
 Bloodclottingfactors
FactorI(Fibrinogen):asolubleplasmaprotein.In
afibrinoginemia clottingdoesnotoccur
FactorII(Prothrombin):Inactiveprecursorofthrombin
FactorIII(Tissuefactor/Tissuethromboplastin):converts
prothrombin tothrombininpresenceoffactorsV,VII,
X,Ca++ andphospholopids
FactorIV(Calcium):Essentialforclotting.
FactorV(Labilefactor/proaccelerin):
FactorVII(Stablefactor/proconvertin):Deficiencyinduced
byoralanticoagulants
 Bloodclottingfactors
FactorVIII(Antihemophilic globulin/factorA):
Congenitaldefficiency causesClassicalHemophilia
FactorIX (Christmasfactor/antihemophilic factorB):
Congenitaldeficiencyleadstoahemorrhagicstate
resemblingHemophila(Christmasdisease)
FactorX(StuartPrower factor)
FactorXI(Plasmathromboplastin antecedent/
Antihemophilic factorC)
FactorXII(Hagemanfactor,contactfactor)
FactorXIII(fibrinstabilizingfactor)
 Bloodclottingfactors
HMWK:Highmolecularweightkininogens
PreKa:prekallikrein/Fletcherfactor
Ka:Kallikrein
PL:Plateletphospholipid
 FactorsII,VII,IX,X,proteinC&proteinS
areformedintheliver
VitaminKisnecessaryforsomepost
translationalmodificationsinthesefactors
InvitaminKdefficiency or
inhibitionbyoralanticoagulant,Warfarin
theplasmalevelsofthesefactorsarelow
Basicreactionsinvolvedincoagulation

Prothrombin(FactorII)
Ca++, factorsderivedfromdamagedtissues,
disintegratingplatelets,plasma
Thrombin
Fibrinogen Fibrin
(FactorI)
 Physiologyofclottingprocess
Thrombinfibrinogenreaction
Formationoffibrinclotistheonlyvisible&measurable
partofclottingprocess
Typeofreaction
Proteolysis
Thrombin
Fibrinogen Fibrinmonomers+Peptides
Polymerization
Fibrinmonomers Fibrinpolymers
(Solublefibrinclot)
Clotting
Fibrinpolymers FactorXII
Ca++
Insolublefibrinclot
 Conversionofprothrombin tothrombin
requires
Prothrombin activatororthromboplastin
Prothrombin activatoris formedin2mainways
Asresultoftissuedamage(Extrinsicsystem)
Activationofbloodconstituents(Intrinsic
system)
Keyreaction:FactorX FactorXa
Extrinsicpathwayforinitiatingbloodclotting .
 Extrinsicsystem
Keyeventtriggeringbloodcoagulationduring
hemostasis isexposureofbloodtotissue
factor
Italsoactsasacofactorforactivationoffactor
VII
Intrinsicpathwayforinitiatingbloodclotting .
 Intrinsicsystem
Morecomplicated
Moreprolonged
Fibrinolytic andkinin formingsystemsarealso
activated
Enzymecascadehypothesis(Macfarlane):
Surfacecontactinducesasequenceofchangesin
whichaninactiveprecursorisconvertedintoan
activeenzymewhichthenactsonthenext
precursortoformthenextactiveenzyme
Anamplifyingsystem
 Intrinsicsystem
FactorXII,prekallikrein,Factor XI&High
molecularweightkininogen areknownasthe
contactactivationfactors
FactorXII,prekalikrein &HMWKareessentialfor
triggeringbloodcoagulationinaglasstesttube
Howevertheyplaynoroleinnormalhemostasis
sincepatientswithisolateddeficienciesofeach
ofthesedonotbleedabnormally
 OnceXa isformedclottingoccurswithin
seconds
Contactwithaforeignsubstancei.e.glassor
urate crystalproducesclottingonlyafter48
minutes
Overviewofclotting
 Effectsofthrombin
Positivefeedbackeffectonfactorsconcerned
withformationofprothrombin activator
ActivatesfactorVIII
IncreasesactivityoffactorV&XIII
Promotesaggregationofplatelets
Increasestheamountofavailablephosphlipids
Afteractivatingthesemechanismssoon
inactivatesthem
 Regulationofbloodcoagulation
Adsorptionofthrombinontofibrin
Neutralizationofthrombinbyplasmaproteinase
inhibitors,Antithrombin III,2macroglobulin&
heparincofactorII
Antithrombin IIIalsoinhibitstheactivityofkey
intermediateenzymes,FactorIXa &Xa
ActivatedproteinCinhibitsthecofactorsfor
FactorVIIIa &Va
Extrinsicpathwayinhibitor(EPI)&factorXa
neutralizethecatalyticactivityofFactor
VIIa/tissuefactorcomplex
 Clotretraction

Plateletcontractileproteinscontract.

Squeezesfluid(serum)outoftheclot.

Drawstheedgesofthetornbloodvesseltogether.

Setsthestageforrepair.

PDGF

VEGF
 Clotretraction
Freshlyformedfibrinthreadsareextremelysticky
andadheretoeachother,bloodcells,tissues&
foreignsubstances
Freshlyshedbloodsetsinasoftjellylikemass
Graduallyitcontractsdown(retracts)to40%ofits
originalvolume,squeezingoutserum
Finalclotismoretougherandsolid
Clotretractionisimpairedifplateletsare
removedfromblood
 Fibrinolysis (Dissolutionofclot)
Exrtinsic /Intrinsic
Plasminogen Plasminogen Plasmin
activators

Fibrin Plasmin
Smallpeptides
(Fibrindegradationproducts)
Intrinsicplasminogen activatorsgetactivatedby:
Bodyormentalstress,operation,violentexercise,adrenalin
injection
Extrinsicactivators:Widelydistributedthroughoutthecell
&bodyfluids
Tissueactivatorsoccurinmicrosomes,urokinase inurine
 Fibrinolytic system&itsregulationby
proteinC
Endothelialcells
(Thrombomodulin thrombincomplex)

ProteinC ActivatedproteinC
+ProteinS
VIII inactiveVIIIa V inactiveVa

InactivatesinhibitorsoftPA

Plasminogen Plasmin
Thrombin
tPA,uPA
Lysesfibrin
Plasminogen (fibrinolytic)system

Kringles :Lysinebindingsites

tPA:ProducedbyrecombinantDNAtechnology
Usedinmyocardialinfarctionandstroke
 Testsfordefectsinbloodclotting
Screeningforadequacyofhemostatic plug
formation
Plateletcount
Bleedingtime:forconditionsotherthan
thrombocytopeniathatcanimpairthe
formationofhemostatic plug
Testsfordefectsinbloodclotting
Screeningfortheadequacyofbloodcoagulation
Prothrombin time:Measurestheadequacyof
reactionsthatclotplasmawhenaveryhigh
concentrationoftissuefactorispresent.
Teststheadequacyofextrinsicsystem
Activatedpartialthromboplastin time:Measures
theadequacyofclottingreactionsthatclot
plasmawhenareagentoptimizingthecontact
activationreactions&providingprocoagulant
phospholipid ispresent
Testsadequacyofintrinsicsystem
Activatedpartialthromboplastin time
 Disordersaffectingformationof
plateletplug
Thrombocytopenia
Physiological:Newborn,Menstruation
Pathological:
Decreasedmarrowproduction:Tumours,fibrosis,
aplasia,drugs(thiazide diuretics)
Spleenic sequestration:Spleenomegaly
Increaseddestruction:Autoantibodyformation,
aspirin,insecticides,cardiacvalves,Sepsis,
vasculitis
 Disordersaffectingformationof
plateletplug
Functionalplateletdefects
Disorderedadhesion
VonWillebrands disease:vWF,FactorVIIIactivity,BT
Mostcommonhereditaryhemostatic disorder
Disorderedaggregation
Thrombasthenia(Glanzmanns syndrome)
Afibrinoginemia
Disorderedgranulerelease
Chediak Higashisyndrome
Cardiopulmonarybypass
Myeloproliferative disorders
Aspirin&otherNSAIDS
 Plateletcount
>1lakh:Asymptomatic,Normalbleedingtime
500001lakh:Bleedingoccursaftersevere
trauma,Bleedingtimeslightlyprolonged
<50000:Easybruisingmanifestedbyskin
purpura afterminortrauma
<20000:Spontaneousbleeding,usuallyhave
petechiae,intracranial/spontaneousbleeding
 Disordersaffectingbloodcoagulation
Hereditarydisorders
FactorVIIIdeficiency(HemophiliaA) Xlinked
FactorIXdefficiency(HemophiliaB) recessivedisorders
FactorXIdeficiency:Autosomal recessive
Acquireddisorders
VitaminKdeficiency
Liverdisorders:FallinallfactorsexceptfactorVIII
Disseminatedintravascularcoagulation
Acquiredantibodiesagainstclottingfactors
TransmissionofHemophilia
Normal Carrier
male female
TransmissionofHemophilia
Diseased Carrier
male female
 Anticoagulantsinvivo
Antithrombin III:Serineproteaseinhibitor
Heparin
Thrombomodulin:Thrombinbindingprotein
Thrombomodulinthrombincomplex:Activates
proteinC
ActivatedproteinCandS
Plasmin
Dicumarol &Warfarin:InhibitactionofvitaminK
 Anticoagulantsinvitro

EDTA
Oxalate
Heparin
SodiumCitrate
SodiumFluoride/PotassiumOxalate
 Characteristics
Itmustnotalterthesizeoftheredcells
Itmustnotcausehemolysis
Itmustminimizeplateletaggregation
Itmustminimizedisruptionofthestaining
andmorphologyofleukocytes
Itmustbereadilysolubleinblood
 EDTA
(Ethylenediaminetetraacetic acid)
Bestmeetstheaboverequirements,andisusedmostfrequently.
Thetripotassium salt(K3EDTA),andthedisodiumsalt(Na2EDTA).
CalciumEDTAisnotusedasananticoagulant,butinthe
treatmentofleadpoisoning
Modeofaction:Itformsinsolublecalciumsaltsbychelation
Uses:Makingabloodsmearforcellmorphologystudies,testfor
microfilaria,performcellcounts,BUN,plasmaprotein,fibrinogen,
glucose,determinationofCoomb's Test
EDTApreservesthestainingandmorphologicalcharacteristicsof
leukocytes
Disadvantages
ExcessiveconcentrationsofEDTAwillcauseshrinkageofRBC'sand
erroneousPCV,MCVandMCHCresults
 Oxalate
Amixtureofdryammoniumoxalateandpotassiumoxalate inthe
ratioof3:2isused
Modeofaction:ItcombineswithcalciumtoforminsolubleCa
oxalate.
Potassiumoxalatealonecausesredcellstoshrink;ammonium
oxalatealonecausesredcellstoswell.Usedtogether,littlecellular
distortionoccursinthefirsthouraftercollection
Uses:Theoxalatemixturemaybeusedforhematological
sedimentationstudies.Potassiumoxalatealoneisvalidfor
immediateglucosedeterminations.Ammoniumoxalateisusedasa
diluent insomemethodsformanuallycountingWBCsandplatelets
Disadvantages
1)Itdoesnotpreventplateletaggregationinvitro aseffectivelyas
EDTA.
2)Itispoisonousandshouldnotbeusedforbloodtransfusion.
 Heparin
Chemicalstructure:Apolysaccharidederived
fromglucosamineandglucuronic acid.
Containsmanysulphate groups.
Molecularweightaverages1500018000
Anticoagulantactionduetoitsstrong
electronegativechargedueitssulphuric acid
group
Antagonists:Toludene blue&protamine by
antagonizingthenegativechargeofheparin
 Heparin
FirstisolatedfromLiver
Subsequentlydemonstratedinextractifmany
otherorgans
Inhibitsbloodcoagulationbothinvitro&invivo
Mechanismofaction:
Preventsactivationofprothrombin tothrombin
Neutralizesactionofthrombinonfibrinogen
Facilitatestheactionofantithrombin III
 Heparin
Heparinistheanticoagulantofchoiceforblood
pHandbloodgasanalysisforacidbasebalance.
Disadvantages
1)Itcausesclumpingofleukocytes.
2)Itinterfereswiththestainingofleukocytes.
3)Itisthemostexpensiveoftheanticoagulants.
4)Bloodwillclotin812hoursbecauseclottingisonly
delayedandnotprevented.
5)Itisnotsuitableforagglutinationtests,coagulation
studies(prothrombin timetests)orplasmafibrinogen
determination
 Sodiumcitrate
Acidcitratedextrose (ACD)ispreparedfromdisodiumhydrogen
citrateandistheanticoagulantofchoiceforbloodtransfusions.
usedintheratioof1partACDto4partsofblood
Modeofaction:Itcombineswithcalciumtoformaninsolublesalt
ofcalciumcitrate.
Sodiumcitrateistheanticoagulantofchoiceforstudiesofplatelet
functionandmorphology
e.Disadvantages:
1)Itinterfereswithmanychemicaltests.
2)Usedaloneitpreservesbloodforonlyafewhours.
3)Ithasatendencytoshrinkcells.
4)Becauseofa10%dilutionofblood,Nacitrateisgenerallynotused
forCBC.
Bloodgroups
 Bloodgroups
Agglutinogen :TheantigenpresentonRBC
surface
Major0ABbloodtypes
BloodgroupA:AantigenApresentonsurface
BloodgroupB:Bantigenpresentonsurface
BloodgroupAB:A&Bantigenpresentonsurf.
BloodgroupO:Noantigenpresentonsurface
 Geneticdeterminationof
agglutinogens
GenesthatdeterminetheA&Bphenotypes
arefoundonchromosome9p
Theyareexpressedinamendelian co
dominantmanner
Thegeneproductsareglycosyl transferases,A
orB,whichconfertheenzymaticabilityof
attachingthespecificantigeniccarbohydrate
groups
 Geneticdeterminationof
agglutinogens
Glycosyl transferase Adirectstheformationof
antigenAonRBCsurface
Glycosyl transferase Bdirectstheformationof
antigenBonRBCsurface
WhenA&Btransferases areabsentthenno
antigenispresentonRBCsurfaceandblood
groupissaidtobeO
 Agglutinins
AntibodiesdirectedagainstRBCantigenmay
resultfromnaturalexposureparticularlyto
carbohydratethatmimicsomebloodgroup
antigens
 Bloodtypeswiththeirgenotypes

Genotypes Bloodtypes Agglutinogen Agglutinin

OO O Antia&Antib
OAor AA A A Antib
OBorBB B B Antia
AB AB A&B
Relativefrequenciesofdifferentblood
groups
Westerncountries India
O 47% 32%
A 41% 20%
B 9% 40%
AB 3% 8%
 Landsteinerslaw
Statesthatifanagglutinogen ispresentinthe
RBCsofanindividualthecorresponding
agglutininmustbeabsentintheplasma
Orconverselyifanagglutinogen isabsentin
theRBCsofanindividualthecorresponding
agglutininmustbepresentintheplasma
 Rh systemofbloodgrouping
RBCofrhesusmonkeywheninjectedintoarabbit,the
rabbitdevelopedantibodiestotherhesusRBC
Furtheritwasdiscoveredthatrabbitserumcontaining
antirhesusantibodiescouldagglutinatenotonly
rhesusRBCbutalsohumanRBCin85%cases
TheRBCsofthesehumanshaveontheirsurfacean
antigenidenticaltoorsimilartorhesusantigen
ThushumanshavingthisantigenweretermedasRh
positive
 Rh bloodgroup
ThreeRh genesarepresent
Rh genesarelocatedonchromosome1
TypesofRh antigens/factor:C,D,E
Dismostantigenic
ThetermRh positiveisgenerallyusedto
meanthattheindividualhasagglutinogen D
85%CaucasiansareRh positive
99%AsiansareRh positive
 DifferencebetweenABOandRh
groups
ABOantigensarepresentoncellsotherthan
RBCswhereastheRh antigenisnotpresenton
anyothercellexceptRBCs
IntheABOsystemtheplasmaantibodies
responsiblefortransfusionreactiondevelop
spontaneouslywhereasintheRh system
antibodiesonlydevelopinRh negative
personswhentheyhavebeenpreviously
sensitized
 Knowledgeofthemodeofinheritanceof
bloodgroupsisusefulincasesofdisputed
paternity
Thenegativeverdictisdefinitive:Itrulesout
thepossibilityofagivenmanbeingthefather
Childs Childs Mothers Possible Possible Father Father
blood genotype blood contribution contribution might couldnot
group group ofmother offather havebeen havebeen
A AA AorAB A A AorAB Oor B
AO B O A AorAB OorB
B BB BorAB B B BorAB OorA
BO O O B BorAB OorA
AB AB A A B B OorA
AB B B A AorAB OorB
O OO A O O AorBor AB
OO B O O O AB
 Rh incompatibility
IfaRh negativepersonistransfusedwithRh
positivebloodforthefirsttime:Noimmediate
adversereaction
Ifthesameindividualreceivesasecond
transfusionlaterantiRh antibodiesare
synthesizedpromptlyinlargeamounts
 Rh incompatibilityinpregnancy
Rh negativefemale
Rh positivemale
FetusisRh positive
Chancesofanapparentabnormalityresulting
fromRh incompatibilityare
Negligibleinfirstpregnancy
3%duringsecondpregnancy
10%duringthirdpregnancy
 Rh incompatibilityinpregnancy
Erythroblastosis foetalis/Hemolyticdiseaseofnewborn
FetalRBCsundergomassivehemolysis
Compensatoryincreaseinerythropoietic activityleading
toimmaturecellsincirculation
Enlargementofextramedullary sitesofhematopoiesis
leadstohepatomegaly &spleenomegaly
Childisanemic,Jaundice(Hemolytic)ispresent,edema
Hydrops fetalis:Deathinutero
Kernicterus: Depositionofunconjugated bilirubin inthe
basalganglia
Ifbilirubin >20mg%thenbraindamage
 Erythroblastosis fetalis
Preventivetreatment:GivingantiRh antibodiesto
Rh negativepregnantmother
MechanismbywhichantiRh antibodyprevent
thecomplicationofRh incompatibility
Theydestroyfetalredcellswhichcrossoverto
thematernalcirculation.Thusantigenicstimulus
isreduced
Highantibodylevelsinhibitproductionofthe
sameantibodymolecules
Definitivetreatment:ExchangetransfusionwithRh
negativeblood
 Otherbloodgroupsystems
Lewissystem
ISystem
Psystem
MNSSUsysetm
Kell system:Immunogenicityisthirdbehind
theABO&Rh systems
Duffysystem
Kiddsystem
 Bloodtransfusion:Compatibility
testing
ABORh typing:Antibodiesaredirectedagainst
thoseantigensthatlackintheindividualsown
blood
Crossmatching:Atrialtransfusionwithinatest
tubeinwhichdonorRBCaremixedwithrecipient
serumtodetectapotentialforserious
transfusion
Antibodyscreen:Atrialtransfusionbetweenthe
receipients serum&commerciallysuppliedRBCs
thatarespecificallyselectedtocontainoptimal
numberofRBCantigens
 Storageofblood
CPDA1
Citrate:Anticoagulant
Phosphate:Buffer
Dextrose:energysource
Adenine:Forresynthesis ofATPsoastoextendthe
storagetimefrom21to35days
AS1:(Adsol):Adenine,glucose,mannitol &NaCl
Increasesshelflifeto42days
AS3:(Nutricel):Containsadenine,glucose,citrate,
phosphate,NaCl
 Storageofblood
Durationofstorage
SetbytheUSfederalregualtion
Atleast70%ofthetransfusedRBCsremainin
circulationfor24hoursafterinfusion
SinceRBCsthatsurvive24hoursafter
transfusiondisappearfromcirculationatthe
normalrate
Bloodtransfusion
 Storageofblood
Threeproblems
Anticoagulation
Preservationofcellviability
Preservationofcellfunction

Criteriaforassessingtheadequacyofbanked
blood
70%survivalat24hours
 RBCstoragelesion
StoredRBCsundergoaseriesofbiochemical&structural
changes
RBCnucleotidedepletion
ATP

ADP

AMP

IMP

Hypoxanthine
 RBCstoragelesion
RBCmembranechanges
Normaldiscshaped

Sphericalwithsurfaceprojections

Lossofmembraneproteins
Lossofdeformability
 RBCstoragelesion
2,3DPGdepletion
Earliestdetectablechange
Withacidcitratedextran(ACD)
DPGdropsto<50%within48hours
Withcitratephosphatedextran(CPD)
DPGfallssignificantlyafter2weeks
 RBCstoragelesion
DecreasedDPG
IncreasedaffinityofHb foroxygen
IncreasedHb butnoproportionateincreasein
oxygenavability
 RBCstoragelesion
Accumulationoflactate
AccumulationofH+ ions
DecreaseinpH
LossofK+andgainofNa+
Increasedosmoticfragility
Somecellsundergolysis
 Useofanticoagulant
Citrate
Mostimportantanticoagulant
Modeofaction
Aftertransfusionitisreadilymetabolizedbythe
recipient
PreservesRBCsinaviablestatefor1weekonly
 ACD(Acidcitratedextran)
Glucoseispresent
RBCstoragefor3weeks
CPD(Citratephosphatedextran):ModifiedACDwith
addedNaH2PO4
Viabilityafterfirst24hoursisbetter
At4oCbloodmaybestoredfor21days
CPDA1:CPDsupplementedwithadenineandwith
25%moreglucosethanCPD(Standardanticoagulant
preservative)
RBCstorageupto35days
 2,3DPGpreservation
InACD,CPD,CPDA1,2,3DPGdisappearsby2
weeks
Chemicalagentscapableofmaintaining
normallevelsduringstorage
dihydroxyacetone,pyruvate,ascorbicacid
 Indicationsofbloodtransfusion
Hypovolemia:Bloodloss<20%oftotalblood
volumedoesnotrequireRBCtransfusion
fluidreplacementissufficient
Surgery
Anemia:Hb levels<6gm%require
transfusion
 Pretransfusiontesting
Pretransfusiontestingofapotentialrecipientconsistsof
thefollowing
Forwardtype:DeterminestheABO&Rh phenotypeof
therecipient
Reversetype:Detectsisoagglutinins inthepatients
serumandshouldcorrelatewiththeforwardtype
testing.
Thealloantibodyscreenidentifiesantibodiesdirected
againstotherRBCantigens
Crossmatching:DonorRBCsaremixedwithrecipient
plasmaonaslideandcheckedforagglutination
 Adverseeffectsoftransfusion
Immunemediatedreactions
Acutehemolytictransfusionreactions
Occurduetopreformedantibodiesthatbind&lyse
donorRBC
ABOisoagglutinins areresponsibleforthemajority
ofthesereactions
Presentation:Hypotension,tachypnea,tachycardia,
fever,chills,hemoglobinemia,hemoglobinuria,
chest/flankpain,discomfortatcatheterinfusion
site
 Adverseeffectsoftransfusion
Immunemediatedreactions
Delayedhemolytic&serologicaltransfusion
reaction:
OccursinpatientspreviouslysensitizedtoRBC
alloantigenwhohaveanegativealloantibody
screenduetolowantibodylevels.
Whentransfusedwithantigenpositivebloodan
anamnestic responseresultsintheearly
productionofalloantibodythatbindstodonor
RBC
 Adverseeffectsoftransfusion
Immunemediatedreactions
Febrilenonhemolytictransfusionreaction
Mostfrequentreaction
S&S;Chills&rigor&10Cormorerisein
temperature
Adiagnosisofexclusion
AntibodiesagainstdonorWBCs&HLA
antigens
 Adverseeffectsoftransfusion
Immunemediatedreactions
Allergicreactions
Urticarial reactionscharacterizedbypruritic
rash,edema,headache&dizziness
Relatedtoplasmaproteinsfoundin
transfusedcomponents
 Adverseeffectsoftransfusion
Immunemediatedreactions
Anaphylacticreactions
S&S:difficultyinbreathing,coughing,nausea
&vomiting,hypotension,bronchospasm,
respiratoryarrest,shock&lossof
consciousness
Treatment;Epinephrine,glucocorticoids
 Adverseeffectsoftransfusion
Immunemediatedreactions
Graftversushostdisease
MediatedbydonorTlymphocytesthat
recognizehostHLAasforeignandmountan
immuneresponse
S&S:fever,cutaneous erruptions,diarrhoea &
liverabnormalities
Clinicalmanifestationsoccur810dayspost
transfusion
 Adverseeffectsoftransfusion
Immunemediatedreactions
Transfusionrelatedacutelunginjury
Uncommonreaction
Duetotransfusionofdonorplasmathatcontain
hightitre antiHLAantibodythatbind
correspondingantigenonrecipientleukocytes.
Theseleukocytesthenaggregateinpulmonary
vasculature&releasemediatorscausingan
increaseincapillarypermeability
 Adverseeffectsoftransfusion
Immunemediatedreactions
Posttransfusionpurpura
Presentsasthrombocytopenia710daysafter
platelettransfusion
Duetoproductionofantibodiesthatreactto
bothdonor&recipientplatelets
 Adverseeffectsoftransfusion
Nonimmunereactions
Fluidoverload
Hypothermia
Electrolytetoxicity
Ironoverload
 Adverseeffectsoftransfusion
Infectiouscomplications
Viral:HCV(Mostcommon)
HBV,HIV,CMV,HTLV(I),ParvovirusB19
Bacterial:Pseudomonas&Yersenia(Cangrowa
160C)
Parasitic:Malaria,Babesiosis,Chagas disease
 Adverseeffectsoftransfusion
LowfactorV&VIII