《临床输血学实验》PPT课件.ppt

TestingSpecimenRequirement2mlEDTAwholebloodFillinthetubelabel(s)withname,临床输血学实验TransfusionMedicine(bloodtransfusionlaboratories)上海市第六人民医院ShanghaiSixthPeopleHospital输血科TransfusionDepartment,HistoryAncienttimes敬畏血液-生命的源泉-饮血疗法Theperiod1500-1800治疗精神病血液有毒放血疗法-人体血液循环，为输血奠定基础-血液的携氧功能，合理治疗手段Thediscoveryofbloodgroups血型之父-Landsteiner,人类血型的发现，为安全输血提供重要保证。而且，在遗传学、人类学、法医学、免疫学、部分疾病的发病机制探讨上也具有重要意义。,Whilebloodcellantigens:RedcellantigensHLAantigensGranulocytes-specificantigensPlatelets（22）,PartoneRedbloodcellgroup,目的要求：掌握ABO血型的鉴定熟悉Rh血型的鉴定,ABOSYSTEMPrincipleofprocedure,红细胞上具有A抗原者为A型-RedbloodcellspossessAbloodgroupantigens有B抗原者为B型-RedbloodcellspossessBbloodgroupantigensA和B抗原都没有者为O型-RedbloodcellslackAandBbloodgroupantigensA和B抗原都有者为AB型-RedbloodcellspossessAandBbloodgroupantigens,RhSYSTEM,ThemostcomplexintheredcellsantigenIndividualswhopossesstheDantigenareRhpositive,wholackitareRhnegative.TheDantigeniscapableofstimulatingproductionofAnti-DinpersonslackingtheDantigen.Anti-DisaclinicallysignificantantibodycapableofcausingRBCdestructionandmayresultinhemolyticdiseaseofthenewbornandtransfusionreaction.,已发现的抗原40多个。涉及临床主要是5个抗原：C、c、D、E、e及其相应的特异性抗体。Fourotherantigens(C、c、E、e)accountforalmostalloftheRh-relatedtransfusionproblems.TheyarelessantigenicthanD,and/ortheantibodiesarelessclinicallydangerous.Buttheycancausethetransfusionreaction.,MaterialsRequired,Centrifuge0.9%salinepipetterreagents:Themonoclonalantibody(IgM)includeAnti-A、Anti-B、Anti-D5%StandardRBC-Affirmagen,Methods,SolidphaseadherencetestTubetestGeltestManualSemi-automatedsystemAutomatedsystem,PROCEDURE,SpecimencollectionandpreparationRedbloodcellsuspensions(4%RBC)canbepreparedusingthefollowingcombinationsofsalineandpackedredbloodcells:SalineVolume-2mlPackedRBCVolume-100ul被检者RBC用生理盐水配成2-5%悬液待用(2ml0.9%NS+100ul压积红细胞）,TESTPROCEDURE-tubetest,正定型：取小试管三只，分别用记号笔标记后加抗A、抗B、抗D血清各一滴，然后加受检者RBC悬液一滴。bloodgroupingAddonedropofAnti-A、Anti-B、Anti-DtothreetubesseparatelyAddonedropofa2-5%RBCsuspensiontothreetubesseparately,TESTPROCEDURE,反定型：取小试管三只，分别用记号笔标记后加受检者血清一滴，然后加标准A、B及O型RBC各一滴。reversegroupingAddonedropofserum/plasmatothreetubesseparatelyAddonedropof5%standardRBCtothreetubesseparately,将上述混匀后静置数分钟或3000r/m离心15s肉眼判断凝集度。Shakegentlytomixcontentsofreactiontubes.Centrifugesixtubeswith3000r/s,15m.Shakegentlywith45-60angles.Evaluatesixtubesforagglutinationand/orhemolysisupontestcompletion.Recordthereactionstrengthonthepaper.,Evaluatetheagglutinationstrength,Interpretationofresults,Groupinganomalies,Coldauto-antibodiesAcquiredBUnexpectedmixedfieldreactionsPartialorweakDDnegativeIntrauterinetransfusions,RhDnegativeverification,Reagents:threedifferentAnti-Dhuman(IgG)monoclonalantibodyMethodIndirectantiglobulintest(IAT)Genetest,Indirectantiglobulintest,IncubatingthedonorsredcellswiththeRhtypingseraWashingthecellsthreetimesAddingAHGCentrifugingthemixtureObservingitforagglutination,TherelationshipbetweenABOgroupandpersonality,ABOgroupandamativeness,female,male,ParttwoRedcellcompatibilitytesting-Crossmatch,目的要求：掌握交叉配血试验的原理熟悉交叉配血试验的操作,CROSSMATCHING,Thecrossmatchisdefinedasaproceduretoexcludeincompatibilitybetweendonorandrecipient.Crossmatchingwillpickupincompatibilitiesbetweenthedonorandrecipientthatwillnotbeevidentonbloodtyping(asbloodtypingisnotavailableagainsteverybloodgroup,justthemajorones).Inaddition,thecrossmatchprocedurewillnotpickuplowtiterantibodiesandthuswillnotpreventdelayed-typehemolytictransfusionreactions,Immediatespincrossmatch,主侧配血-Majorcrossmatch(mostimportant)-comparingdonoreryghrocytestorecipientserumantibodiesinrecipientserumagainstdonorerythrocytes.次侧配血-MinorcrossmatchThiscomparesdonorserumtorecipienterythrocytesandchecksforpreformedantibodiesindonorserumthatcouldhemolyserecipientredcells,Choiceoftechniques,盐水介质-saline酶介质-enzyme+liss抗球蛋白介质-IAT,Crossmatchprocedure,Majorcrossmatch:Add1dof5%suspensionofdonorRBCsand1drecipientplasma/serumintothetube取受血者血清1-2滴+献血员2-5%红细胞盐水悬液1滴MinorcrossmatchAdd1dof5%suspensionofrecipientRBCsand1ddonorplasma/serumintothetube,取受血者2-5%红细胞盐水悬液1滴+献血员血清1-2滴,将上述混匀后静置数分钟或3000r/m离心15s肉眼判断凝集度。Centrifugesixtubeswith3000r/s,15m.Shakesoftlywith45-60angles.Evaluatetwotubesforagglutinationunderthemicroscope.,Result,Whenthereisanincompatiblereactiononthemajorcrossmatch,thedonorbloodshouldnotbetransfusionunderanycircumstances.Whenthereisanincompatiblereactionontheminorcrossmatch,thetransfusioncangoahead.However,ifthedonatedserumislikelytocontributesubstantiallytotheplasmavolumeoftherecipient,theserumshouldberemovedfromthedonorwholeblood.,Selectionofblood,Redcellcomponents-thesameABOandRhDgroupasthepatientIfABOidenticalbloodisnotavailable-groupORBC+groupABplasmaIfRhD-negativebloodarelimited-RhDpositivebloodmaybeselectedPatientwithclinicallysignificantredcellantibodyPatientwithautoimmunehaemolyticanaemiaMassivebloodtransfusion,PartthreeCoombstest,目的要求：掌握直接、间接抗人球蛋白试验的原理熟悉直接、间接抗人球蛋白试验的操了解直接、间接抗人球蛋白试验的临床应用,一、抗人球蛋白试验的原理,倘若用提纯的或在人的全血清内的人的球蛋白给动物（如兔和山羊）注射，动物对外来的蛋白质将会产生抗体，即抗人球蛋白。,抗人球蛋白血清，在适当处理之后将会与人球蛋白发生特异性反应。如果球蛋白分子，或者抗体或补体附着于红细胞膜上，抗球蛋白血清将会与球蛋白结合。因为球蛋白附着于红细胞上，抗球蛋白血清与球蛋白分子的结合就引起红细胞凝集反之，将不会被凝集。,直接抗球蛋白试验证实体内被致敏的红细胞。洗涤后的患者红细胞可直接进行抗人球蛋白血清检查。间接抗球蛋白试验证实体外被红细胞的致敏。,血清与红细胞孵育，然后用生理盐水洗涤红细胞以除去未结合到红细胞上多余的抗体。若加入抗人球蛋白血清导致的红细胞凝集，可证明是红细胞表面具有与血清中的抗体相同特异性的表面抗原。,意义：检查受检红细胞是否在体内已被抗体或补体致敏。用于：新生儿溶血病溶血性输血反应自身免疫溶血性贫血药物诱导溶血性贫血,二、操作,1、直接抗球蛋白试验操作取一试管放一滴受检者血液，用生理盐洗涤3-4次，末次洗涤后，配成2-5%RBC悬液待用。取一试管放1-2滴多价抗人球蛋白血清（IgG+C3），再加待用RBC一滴混匀，3000r/min离心1