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P $P $ 0  0$$c.0P $$Ifantigenantibodyreactionoccurs,thecellsadheretothesidesofthewell,ifnoreaction 4 occursthecellssettletothebottomofthewell.d P $P $ D.0  PrincipleandVariablesInvolvedInAgglutinationReactions$$ 0  1.0$$Twostagesinvolvedinagglutinationreaction.l$$ 0  0$$a.0P $$The firststage ofagglutinationis sensitization ,attachmentofantibodyontothe t correspondingantigensiteontheRBCmembrane.NP $P $ 0   $$ 0  0$$b.0P $$The secondstage istheformationofbridgesbetweenthesensitizedcells.P $P $ 0  0$$0P $$1)0 P $P $IgGantibodieshavetwoantigenbindingsites,onesitewillbindtoantigenonan X RBCandtheothersitewillbindtoantigenonadifferentRBCresultinginthe 0 formationofalattice.`  $ $ 0  0$$0P $$2)0 P $P $ Latticeformation resultsin_hemagglutination_." $ $ 0  2.0$$Variablesaffectingfirststageofagglutination. j$$$ 0  0$$a.0P $$ Antigenantibodyratio (serumtocellratio)isverycritical.r" &P $P $ 0  0$$0P $$1)0 P $P $Optimalamountofserummustbedetermined.&$!( $ $ 0  0$$0P $$2)0 P $P $Increasingamountofserumincreasesnumberofantibodymoleculescoatingthecell.$") $ $ 0  0$$b.0P $$ pH Thereactivityofthemajorityofbloodgroupantibodiesisoptimalata pH of6.57.5, &V$+ forroutinebloodbankprocedures,apHof7.0shouldbeused.'2%,P $P $ 0  0$$c.0P $$ Ionicstrengthofsuspendingmedium :)&.P $P $ 0  0$$0P $$1)0 P $P $Salinepartiallyneutralizesoppositelychargedsitesonantigenandantibody *(0 moleculeswhichhinderstheassociationofantigensandantibodies.+n)1 $ $ 0  0$$0P $$2)0 P $P $Lowionicstrengthsalineisasalinesolutionwithalowersaltconcentrationwhich v-+3 enhancesuptakeofantibodyontothecells.N.+4 $ $ Ї0  0$$0P $$3)0 P $P $Albuminunlessusedunderlowionicconditionsdoeslittletoaffectantibodyuptake X ontothecell,ratheritinfluencesthesecondstageof_hemagglutination_.0 $ $ 0  0$$d.0P $$ Temperaturerequirements differdependinguponantibodyclassinvolved.P $P $ 0  0$$0P $$1)0 P $P $AntibodiesoftheIgGclassattachtoantigensoptimallyat37C.< $ $ 0  0$$0P $$2)0 P $P $IgMantibodiesattachtoantigenspreferentiallyatRTorbelow(422C).D $ $ 0  0$$e.0P $$ Incubationtime   P $P $ 0  0$$0P $$1)0 P $P $Antigenantibodyreactionshaveanoptimaltimeofincubationthatfavorsmaximal  P  bindingofantibodytoantigen. (  $ $ 0  0$$0P $$2)0 P $P $Enhancementagentsincreaseantibodyuptakeontothecelltherebydecreasingthe X  incubationtime.0  $ $ 0  4.0$$Variablesaffectingthesecondstage. $$ 0  0$$a.0P $$ Immunoglobulinclass 8P $P $ 0  0$$0P $$1)0 P $P $IgGisasmallmonomerwiththeabilitytosensitize_RBCs_,butnotagglutinate.D $ $   0  0P $$2)0 P $P $IgMisalarge_pentamer_Ԁwith10antigenbindingsites,veryeasilyagglutinates_RBCs_. $ $ 0  0$$b.0P $$ AntigenSites antigensitesmaybesparselydistributedorpresentinlownumberswhichmay t resultinnoagglutination.PP $P $ 0  0$$c.0P $$ ElectrostaticRepulsionForces XP $P $ 0  0$$0P $$1)0 P $P $Latticeformationdependsuponovercomingtheelectrostaticforcesof_RBCs_.  $ $ 0  0$$0P $$2)0 P $P $The zetapotential isthetermusedtodescribetheelectricalforceswhichkeep_RBCs_  awayfromeachotherserologicmethodsareaimedatreducingthezetapotential.f $ $ E.0  Principleofthe_Antiglobulin_ԀTestn $$ 0  1.0$$_Hemagglutination_Ԁisthemostcommonmethodusedinbloodbankingforthedetectionand " identificationofbloodgroupantigensandantibodiesbutinsomecasestheshortIgGantibody # moleculecansensitize_RBCs_Ԁbutcannotproduceagglutination. v$$$ 0  0$$a.0P $$ThesmallsizeoftheIgGantibodymoleculesmakesthemunabletoovercometheforcesthat ~"& & cause_RBCs_Ԁtorepeleachother.V# 'P $P $ 0  0$$b.0P $$Ifundetected,IgGantibodiescancausecoatinganddestructionoftransfused_RBCs_.%")P $P $ 0  2.0$$In1945_Coombs_,_Mourant_ԀandRacedescribedatestfordetecting_nonagglutinating_,coatingantibodies &^$+ nowcalledthe antihumanglobulin(_AHG_Ԁor_Coomb_'s) test.'6%,$$ 0  0$$a.0P $$AreagentantibodytoIgG(antiIgG)and/orcomplementisproduced(_AHG_Ԁserum)andis @)&. addedtotestcellafterincubationofthecellwithserumorantisera.*'/P $P $ 0  0$$b.0P $$Ifthecellshavebeensensitized(coated)withIgGantibodiesduringtheincubationperiodthe +p)1 AHGwillattachedthesecondantigenbindingsiteoftheIgGmoleculescoatingthecells, ,H*2 causingagglutination.x- +3P $P $  P.+4  H H H$H$ H H H H kY&7'#` p0` (-1E+> Pk H H H H @ H H H H  H H H H  H 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Hu Hu Hu Hu  H$H$Hu Hu 0  G.0$$TypesofAntiHumanGlobulin(AHG)Reagents$")$$     1.0 P PolyspecificAHG &N$+P $P $ 0  0$$0P $$a.0 P $P $PolyspecificAHGconsistofapoolofantihumanIgGandantiC3bandC3d.Z(&- $ $ 0  0$$0P $$b.0 P $P $Maybeproducedbyhybridomas,rabbits,oramixture.2)&. $ $ 0  0$$0P $$c.0 P $P $Usedforroutinecompatibilitytesting,antibodyidentificationandDAT. *'/ $ $ 0  0$$0P $$d.0 P $P $MostimportantfunctionistodetectIgGantibodiescoatingthecells.*(0 $ $ 0  0$$0P $$e.0 P $P $TheimportanceofthepresenceofanticomplementinAHGserumisvery +b)1 controversialforroutinecompatibilitytesting.,:*2 $ $ 0  0$$0P $$f.0 P $P $Antibodieswhicharedetectableonlybytheirabilitytobindcomplementarerare,so j-+3 it'snotasusefulforcompatibilitytestingasmanyfalsepositivereactionsoccur.B.+4 $ $    /,5 Ї0  0$$2.0P $$ AntiIgG XP $P $   0  0P $$a.0 P $P $Majorcomponentisantibodytohumangammachains(antiIgG)containsnoanti   complement. $ $ 0  0$$0P $$b.0 P $P $UsedasalternativetopolyspecificAHGinroutinebloodbankprocedures.d $ $ 0  0$$0P $$c.0 P $P $AdvantageofutilizingantiIgGisthatiteliminatesthefalsepositivesfrequently < obtainedwhenusingpolyspecificAHGduetocomplementandnonspecificcold l reactingantibodiesyetwilldetectclinicallysignificantalloantibodies.D $ $ 0  0$$3.0P $$ AntiC3b,AntiC3d   P $P $ 0  0$$0P $$a.0 P $P $Containsanticomplementantibodiestodetectcomplementcoatingthecells. P  $ $ 0  0$$0P $$b.0 P $P $UsedforclassifyingthecoatingproteinsonRBCstodetermineifIgG,complement,  (  orbotharepresent.X  $ $ 0  0$$0P $$c.0 P $P $Primaryimportanceisforclassificationofautoimmunehemolyticanemiasanddrug 0  inducedhemolysisofRBCs.  $ $ 0  0$$0P $$ P $P $ 0  H.0$$AntiglobulinTechniques`$$ 0  0$$1.0P $$ DirectAntiglobulinTest(DAT) isusedtodemonstrateordetect invivo coatingofRBCswith h globulin,particularlyIgGand/orC3.DP $P $ 0  0$$0P $$a.0 P $P $Onedropofapatient'scellsuspensioniswashed3timestoremoveallcontaminating  proteins.t $ $ 0  0$$0P $$b.0 P $P $AHGisadded directly tothewashedcells,centrifugedandobservedfor |$ agglutination,whichisindicativeofIgGantibodiescoatingthecells,theAHGserum X willbindtotheIgGonthecellsforminglattices=agglutination.0 $ $ 0  0$$2.0P $$ IndirectAntiglobulinTest(IAT) detects invitro sensitizationwithantibodycomplementor  both.bP $P $ 0  0$$0P $$a.0 P $P $Patientserum(orspecialtypesofantiserums)areaddedtocellsandincubatedat37 j  C(bodytemperature)foraspecifiedtime.B! $ $ 0  0$$0P $$b.0 P $P $ReadafterincubationbecausesomeIgGantibodiesmaycoattheRBCsheavily # enoughafterincubationtocauseagglutination. r$ $ $ 0  0$$0P $$c.0 P $P $TheRBCsarewashedfreeofcontaminatingproteinandAHGserumisadded,ifthe z"" & cellshavebeencoatedwithIgGduringtheincubationphasetheantiantibodyinthe R# ' AHGwillformlatticeswhichresultinginagglutination.*$!( $ $ 0  0$$3.0P $$Whenapatienthasanantibodyidentifiedintheirserumitiscriticaltofinddonorunitswhich %#* lacktheantigen,typingthedonormayrequiretheIAT.&Z$+P $P $ 0  0$$4.0P $$ThecrossmatchprocedurealsorequirestheIAT,patientserumisincubatedwithdonorcells, b( &- washedandtestedwithAHGserum.Agglutinationindicatesthattheunitcannotbegivento :)&. thepatient(itisincompatible).*'/P $P $   J.+4 0  I.0$$ FalsenegativeresultsintheDATand_IAT_. 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L P $P $ 0  0$$7.0P $$Prozonereactions| $ P $P $ 0  0$$8.0P $$NegativeDATmaynotnecessarilymeanabsenceofcoatingglobulins., P $P $ 0  0$$0P $$a.0 P $P $PolyandantiIgGwilldetectapproximately200moleculesofIgGpercell.  $ $ 0  0$$0P $$b.0 P $P $Presenceofweakantibodymaygoundetected.\ $ $ 0  0$$9.0P $$Ifanegativereactionisobtained,IgGcoatedreagent_RBCs_Ԁ(checkcells),areaddedtothetest d  system,agglutinationensuresthatthe_AHG_Ԁserumisactive.<P $P $ 0  0$$10.0P $$IntheDATcomplementcoatingmaynotbeapparentasagglutinationuponimmediate  reading.lP $P $ 0  0$$11.0P $$Falsenegativesinthe_IAT_Ԁ(_IAT_ԀandDATaredifferent).tP $P $ 0  0$$0P $$a.0 P $P $Redcellsandserumlosereactivityifimproperlystored.$ $ $   0  0P $$b.0 P $P $Occasionalexamplesofanti_Jka_andanti_Jkb_maybedetectedonlyinthepresenceof  activecomplement.| $ $ 0  0$$0P $$c.0 P $P $Temperatureandincubationtimeaffectattachmentofantibodyorcomplementtothe T redbloodcells., $ $   0  0P $$d.0 P $P $Optimalproportionofserumtocellsshouldbeachieved.\  $ $ Ѐ0  J.0$$ Falsepositiveresults intheDATand_IAT_. 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P $P $ 0  0$$3.0P $$Complementcoatedredcellsmayormaynotproceedtohemolysis.0P $P $      P     X      `      h  !      P     X      `