多重抗体与高频率抗原对应抗体的鉴定技巧.ppt

按一下以編輯母片,第二層,第三層,按一下以編輯母片標題樣式,按一下以編輯母片標題樣式,按一下以編輯母片,第二層,第三層,第四層,第五層,*,*,*,血库工作人员在职训练,(,高级班,),台北荣总输血医学科 陈滢如医检师,2012,年,07,月,08,日,/2012,年,07,月,22,日,多重抗体与高频率抗原对应抗体的鉴定技巧,1,您已经熟悉,MP法、传统三相法 吸收冲出试验,不规则抗体鉴定,Cross-out,特异性分析,Selected Cells,通常从其他非常规使用的panel或Screening cells挑选，用以确认或排除某个抗体的特异性,2nd Panel,过期Panels,例：病人有,anti-c+?,c,Jka,Fyb,S,IS,37C,AHG,(1),0,+,0,0,0,0,0,(5),0,0,+,0,0,0,0,(9),0,0*,0*,+,0,0,1+,若各有两个以上的血球可供确认更佳,记得考虑剂量效应,&,老旧血球的影响,课程大纲,多重抗体的鉴定技巧,仔细判读反应价数，选择适当的鉴定方法,参考病人的,RBC,抗原分型,使用酵素,/DTT,处理之血球辅助鉴定,吸收及冲出试验,中和试验及其他方法,鉴定高频率抗原对应抗体（,Ab to high-prevalence Ag,）,啊全部都有反應.,抗体鉴定难题的初步分析,Antibody Screening(+),Autocontrol(-),與Panel cells反應 均(+)或大部份 cells反應為(+),反應期與反應強 度不致,與Panel cells 反應均(+),反應期與反應 強度致,疑为抗high-incidence antigen的异体抗体,疑为多重异体抗体,DAT(+),與Panel cells 反應均(+),反應期與反應 強度不致,Antibody Screening(+),Autocontrol(+),DAT(+),與Panel cells 反應均(+),反應期與反應 強度致,可能为自体抗体加上异体抗体,疑为自体抗体,(,但仍须仔细排除异体抗体的可能,),(,少数案例可见呈现特异性的自体抗体,),注意,：輸血後檢體可能為DAT(+,mf,),多重抗体鉴定,Skill 1,：,仔细判读反应价数,选择适当的鉴定方法（传统三相法通常优于,MP,法）,可针对每个,phase,，以排除法仔细分析特异性,可获知该抗体之血清学特性，辅助特异性鉴定,7,抗体鉴定方法的选择,传统三相法通常优于,MP,法,可获知抗体之血清学特性（温型,or,冷型），辅助特异性鉴定,可针对每个,phase,，以排除法仔细分析特异性,省略室温期的三相法或,CAT,可降低冷凝集素与冷型抗体的干扰,例1,MP,IS,37C,LISS,AHG,例2,MP,IS,37C,AHG,CAT,S I,2+,1+,1+,3+,S I,3+,4+,1+,2+,3,S II,2+,2+,0,2+,S II,3+,4+,1+,0,0,S III,2+,1+,0,2+,S III,3+,4+,1+,0,0,AC,0,0,0,0,AC,3+,4+,1+,2+,2,常见异体抗体之血清学特性,对应抗体,In-Vitro,Hemolysis,4C,22C,37C,AHG,Causes:,HDN,HTR,Anti-D,Some,Most,Most,Yes,Yes,Anti-C,Few,Most,Most,Yes,Yes,Anti-E,Some,Most,Most,Yes,Yes,Anti-c,Rare,Most,Most,Yes,Yes,Anti-e,Rare,Most,Most,Yes,Yes,Anti-K,Few,Some,Most,Yes,Yes,Anti-k,Few,Few,Most,Yes,Yes,Anti-Jka,Some,Few,Few,Most,Mild,Yes,Anti-Jkb,Some,Few,Few,Most,Mild,Yes,Anti-Fya,Rare,Rare,Most,Yes,Yes,Anti-Fyb,Rare,Rare,Most,Yes,Yes,Anti-Lea,Some,Most,Most,Some,Many,No,Few,Anti-Leb,Rare,Most,Most,Few,Some,No,No,Anti-M,Most,Some,Few,Few,Few,Few,Anti-N,Most,Few,Occ.,Occ.,Rare,No,Anti-S,Few,Some,Some,Most,Yes,Yes,Anti-s,No,Few,Few,Most,Yes,Yes,Anti-P1,Occ.,Most,Some,Occ,Rare,No,Rare,Anti-Mia,Some,Some,Most,Yes,Yes,Anti-Dia,Some,Some,Most,Yes,Yes,#摘自AABB Technical Manual 14th edition.Page288,317.,多重抗体鉴定,Skill 2,：,参考病人的,RBC,抗原分型,若病人之某抗原为阳性，则可排除其对应抗体的可能,病人的抗原,异体抗体？,阳性,不可能！,阴性,不能排除,（无法下定论）,12,RBC,抗原分型的难题,病人近日输过血,找出输血前的检体测试,分离病人自身血球测试,基因分型,IgG,或,IgM,抗体附着的血球,IgM,附着：须移除自发性凝集,IgG,附着：多不影响分型，除非须用抗球蛋白试剂,若输注单位不多，仔细观察,mix-field,反应的有无，仍可得到可信的分型结果,简易离心法(Simple Centrifugation)分离自体红血球,血球以,saline,洗三次，最后一次离心,1000g 5-15,分钟，于不破坏,buffy,coat,层下尽量移除上清液。,充分混合均匀后，填满十根毛细管,(,不含抗凝剂,),，一端封口。,注意！充填每支毛细管前均须再次充份混合！,离心,15,分钟。,割下十支毛细管顶端,5mm,，集中于试管，加入,saline,。,以滴管将红血球全冲出后，移至另一乾净试管。,洗三次后泡成,2-5%,红血球悬浮液,（参考资料：AABB Technical Manual 15th edition,Method 2.15）,新生自体红血球比重较低，可利用离心法将之与输入的血球分离,注1:至少须输血后3天的检体才有较佳的分离效果,注2:此法不适用于reticulocytes生成数较低的病人，亦不适用于有Hemoglobin S或spherocytic disorders 病人,注3:有些抗原在reticulocytes的表现比在老旧的血球为弱，测E,e,c,Fya,Jka与Ge抗原要特别留意,Anemia:Hypoproliferative(Low Reticulocyte Count),Nutrient Deficiency,Iron deficiency,Vitamin B12 deficiency,Folate deficiency,Other,Anemia of Chronic Disease,Chronic infection,Chronic inflammatory diseases,Malignancy,Chronic renal disease,Chronic liver disease,Endocrine dysfunction,Bone Marrow Disorders,Hereditary sideroblastic anemias,Pure red cell aplasia,Leukemia,acute and chronic,Hodgkins disease,Non-Hodgkins lymphoma(NHL),Myeloproliferative disorders,Myelodysplastic syndromes(MDS),.,处理,/,测试,IgM,附着的血球,因有cold autoagglutinin引起，有的检体甚至在室温有自发性凝集反应。,将病人红血球悬浮液置37加热15分钟，再以温的saline洗数次，通常可消除此cold autoagglutinin。,若仍无法消除，可以DTT(dithiothreitol)或2-ME(2-mercaptoethanol)处理，破坏IgM。,但此法处理过的血球主要用于测ABO、Rh与DAT。某些,抗原会被0.01M DTT破坏。,待处理红血球以,PBS,泡成,50%.,加入等量,0.01 M DTT,，于,37C,反应,15 minutes.,以,NS,洗涤三次后泡成,2-5%,悬浮液。,处理,/,测试,IgG,附着的血球,可能为自体免疫溶血型贫血病人，或有,HDFN,的新生儿等。亦可能见于近期内曾输入不合血品的病人。,大部分的单株抗体试剂（非用抗球蛋白试验法），即使测的是,DAT,阳性血球，也能得到可信的结果。,但某些高蛋白厚度抗血清有可能造成非特异性凝集。,若必须使用抗球蛋白试验，可以热或酸或酵素处理血球，使其,DAT,变为阴性或减弱后测试。,处理后的血球较脆弱易溶血，甚至抗原无预期地被破坏，故操作时要求以已知分型血球平行测试。,注意某些抗原会被酸或酵素破坏。,Gentle Heat Elution:45,加热,1015,分钟,Heat Elution:56,加热,35,分钟,Gamma EGA Kit,DAT(+),红血球以,saline,洗三次，泡成,3-5%,悬浮液。,取一试管，滴入,30,滴,3-5%,红血球，离心,1,分钟，尽量移除上清液。,另取一试管，加入,4,滴,EGA Solution 1,与,16,滴,EGA Solution 2,，配制成,EDTA,glycine,-acid solution,。,立即将此,EDTA,glycine,-acid solution,滴入前述红血球，混合均匀，设置计时器，于室温反应不超过,2,分钟。,若处理过的血球明显变色或形成团块，需缩短处理时间。,立即加,4,滴,EGA Solution 3,，混合均匀，离心,30,秒。,去除上清液，以,saline,洗三次后泡成,3-5%,红血球悬浮液，立即检测,DAT,。若,DAT,阴性则可继续以,IAT,法检测抗原。,若,DAT,仍为阳性，可试着再处理一次,利用EDTA glycine-acid 使红血球上的IgG脱离，以检测抗原分型,注,1:,处理过的血球其,Kell,血型、,Era,与,Bg,抗原被破坏。,注,2:,此法亦可应用于处理,DAT(-),血球，破坏,Kell,血型抗原，辅助抗体鉴定。,5 min,多重抗体鉴定,Skill 3,：,使用酵素处理之血球辅助鉴定,比较病人血清与酵素处理,/,未处理血球的反应消长,同理，亦可使用,DTT,处理之血球辅助鉴定,21,例：病人血清只与筛检细胞,SI,与,panel,的,3,号细胞有反应，怀疑有,Anti-E,。,0,0,1+w,0,0,0,0,0,0,0,0,0,0,0,2+,0,0,2+,0,0,0,0,2+,Papain,MP,AC,酵素处理细胞组应用,(1),辅助检测弱反应抗体,1+,3+,2+,3+,2+,1+,3+,1+,3+,2+,3+,AGT,AC,其他抗原分型：,R2R2,Jk(a+b+),酵素处理细胞组应用,(2),区分多重抗体,例：病人已知有Anti-Fyb，但与所有Fyb(-)血球都有反应，是否有其他抗体?特异性?,2+,2+,0,2+,2+,2+,2+,2+,2+,2+,2+,Papain,再找Fyb(-)e(-)血球来作用，以排除其他抗体,Effects of Enzymes and Chemicals on Antigens,Ficin/,Papain,0.2M DTT,Possible Specificity,Neg,Pos,M,N,S,s*;Xga;Fya,Fyb;Ch/Rg,Neg,Neg,Indian;JMH,Pos,Weak,Cromer;Knops;Lutheran;Dombrock;AnWj;MER2,Variable,Neg,Yta,Pos,Neg,Kell;LW;Scianna,Pos,Enhance,Kx,Pos,Pos,A,B;H;P1;Rh;Lewis;Kidd;Fy3;Diego;Co;Ge3;Oka;I,i;P,LKE;Ata;Csa;Era;Jra;Lan;Vel;Sda,*s variable with ficin/papain,Blood Group Antigens&Antibodies (2007)Page 194,DTT,在血库实验的应用,处理,IgM,附着的血球，去除自发性凝集,去除红血球上的某些抗原，辅助抗体特异性鉴别,破坏血清中,IgM,抗体，分清某种特异性抗体属,IgM,或,IgG,待測血球與對照血球以,PBS,洗滌。,各加四份,0.2 M DTT,，於,37C,反應,3045 min,以,PBS,洗滌四次後泡成,2-5%,懸浮液。,0.01 M,0.2 M,0.01 M,取2支試管，各加入1mL待測血清或血漿,“Control”管加入1mL pH7.3 PBS；“Test”管加入1mL,0.01M,DTT,，,於,37C反應3060 min,s,。,使用DTT试剂区别血清中的IgM与IgG抗体Using Sulfhydryl Reagents to Distinguish IgM From IgG Antibodies,取,2,支试管，各加入,1mL,待测血清或血浆,取其一标示“,Control,管，加入,1mL pH7.3 PBS,另一管标示“,Test,，加入,1mL 0.01M DTT,混合均匀，于,37C,反应,3060 minutes.,以两管血清平行测试，记录反应价数,（参考资料：AABB Technical Manual 16th edition,Method 3-8）,DTT,会破坏,Kell,抗原，故若作,Kell,抗体的调查，需改用其他方法。,DTT,处理过久或厚度错误会造成检体呈胶状。胶状血清的蛋白均已被破坏，不能再使用。,補充,多重抗体鉴定,Skill 4,：,吸收与冲出试验,将,12,种抗体以吸收法去除，鉴定余下的抗体,可再合并冲出法收集被吸收掉的抗体作多重（异体）抗体鉴定。,吸收技术经常用于移除病人血清中的自体抗体,30,Combined Adsorption-Elution,RBC,Fy,a,(-),c(-),選擇細胞,異體吸收,沖出,Ab Identification:,anti-S,-Jka,Ab Identification:,anti-c,-Fya,anti-c,-Fya,?,应用：分离单一检体中的多重抗体,证实血球上弱表现抗原的存在,辅助鉴定弱反应抗体,Adsorption Procedure 吸收试验,（参考资料：AABB Technical Manual 15th edition,Method 3.12）,取适量血球，以,saline,洗涤三次；最后次离心至少,5,分钟，尽可能移除上清液。（保留少许血球作备用）,以剪成细条的滤纸，可将上清液移除较完全,以适量,packed,RBCs,与血清（一般用,1:1,）作用，于适当温度反应,3060,分钟。期间不时将之混合均匀。,离心至少,5,分钟，将上清液（即吸收后血清）移至乾净试管。,若能于反应温度离心尤佳，可避免抗体解离,血球可保留进行冲出试验。,取吸收后血清与用于吸收的血球反应，若无反应则表示吸收完全，若仍有反应则再取,packed,RBCs,，重复吸收步骤。,血球血清的接触面积大吸收效能较佳，至少用,13mm,以上的试管。,通常吸收二次以上较完全，但太多次吸收可能稀释抗体。,使用酵素处理的血球可增加某些抗体的吸收效能，但处理过的血球较不宜用于冲出试验。,针对,DAT,阳性的检体做进步调查,WAIHA,案例,溶血性输血反应,新生儿溶血症,药物引起溶血性反应,厚缩与纯化特定抗体,证实弱表现抗原的存在,辅助多重抗体的特异性鉴定,制备,antibody-free,的红血球,抗原分型,自体吸收,冲出技术的应用,通常需并用吸收技术,注意,(1),某些冲出方法会破坏特定抗原,(2),若作抗原分型，宜选择适当的血球，同步处理后当对照组。,DAT(+),DAT(+),Ab Identification,Elution,冲出试验方法选择,IgM or IgG？,红血球被破坏 or 保持完整？,试剂可取得？,操作容易？,No single elution method is ideal in all situations!,ELU KITTM PLUS 酸冲出试剂组For Acid Elution of Antibodies from Intact Red Cells,Wash Solution Concentrate:以ddH2O稀释10倍爲Working wash solution，洗去未结合的抗体。,Solution I,Acid Elution Solution:为low pH glycine buffer，含pH指示剂。,Solution II,Base Buffering Solution:含TRIS与albumin，可中和冲出液。,Store at 15-30C.,Do not freeze.,DAT(+),检体：,保存于,4C,，,72,小时内测试。,冲出试验的结果分析,与所有试剂血球反应：,极有可能为自体抗体,(尤其当病人近期内未输过血）,与部分试剂血球反应：,在,HDFN,或溶血性输血反应案例，冲出液中往往可测得血清中测不到的不规则抗体,与所有试剂血球均无反应：,Anti-A,或,anti-B,低频率抗原的对应抗体,药物相关的抗体,非特异性吸附的球蛋白（例：,-globulin level,）,新产生的抗体可能需,1421,天才可在血清中测得,最后次洗涤液：,须为阴性！,（参考资料：AABB Technical Manual 15th edition,p456-8）,冲出试验结果分析的其他考量,若希望增加冲出液的反应强度，可选择使用：,与enzyme-treated cells反应,使用polyethylene glycol(PEG)加强反应,改变fluid-to-cell ratio,以厂制设备将eluate再厚缩,用low-ionic-strength saline(LISS)或cold wash solution洗涤冲出前的血球,由于某些冲出法对特定抗体效果较差，当病人明显有RBCs被破坏的临床徵状，但冲出液却无反应时，改用不同冲出法调查也许有帮助。,若serum与eluate与所有细胞均无反应，且病人有接受血管内注射高剂量的penicillin或其他药物治疗，应作drug-related antibodies的考量与实验调查。,（参考资料：AABB Technical Manual 15th edition,p456-8）,多重抗体鉴定,Skill 5,：,中和试验,利用可溶性抗原物质，去除特定抗体的反应,间接证实某种抗体存在,于多重抗体案例，将某种抗,体去除，鉴定余下的抗体,38,Antigens in Soluble Form,Human source(from antigen-positive people),A,B and H(in secretors),Lea and Leb,Saliva,serum/plasma,I,Human milk,serum/plasma(low levels),Sda,Urine(Tamm-Horsfall glycoprotein),Ch and Rg,Serum/plasma,Cromer antigens,Urine,serum/plasma(low levels),Inb,Knops antigens,Serum/plasma(low levels),Nonhuman source,P1,Pigeon egg white and hydatid cyst fluid,Sda,Guinea pig urine,（参考资料：Blood Group Antigens&Antibodies (2007)Page 195）,HTLA,1+,3+,2+,0,2+,0,3+,0,0,2+,3+,0,IS,AC,中和试验于多重抗体鉴定的应用,例：怀疑病人有Anti-P1，还有其他抗体!?特异性?,参考：抗原分型、历史鉴定纪录、,Selected cells,反应结果,必要时才利用中和法，移除,anti-P1,，测试剩余的抗体特异性,0,2+,1+,0,1+,1+,2+,1+,0,1+,2+,0,37C,1+,2+,2+,1+,3+,1+,3+,1+,1+,2+,3+,AGT,PART 2.,鉴定高频率抗原对应抗体 Investigating Antibodies to High-Prevalence Antigens,41,抗体鉴定难题的初步分析,Antibody Screening(+),Autocontrol(-),與Panel cells反應 均(+)或大部份 cells反應為(+),反應期與反應強 度不致,與Panel cells 反應均(+),反應期與反應 強度致,疑为抗high-incidence antigen的异体抗体,疑为多重异体抗体,DAT(+),與Panel cells 反應均(+),反應期與反應 強度不致,Antibody Screening(+),Autocontrol(+),DAT(+),與Panel cells 反應均(+),反應期與反應 強度致,可能为自体抗体加上异体抗体,疑为自体抗体,(,但仍须仔细排除异体抗体的可能,),(,少数案例可见呈现特异性的自体抗体,),注意,：輸血後檢體可能為DAT(+,mf,),本课程中提及之高频率抗原,System,Antigens,System,Antigens,001,ABO,016,LW,LWa,LWab,002,MNS,U,Ena,ENKT,N,ENEP,ENEH,ENAV,017,CH/RG,Ch1Ch6,Rg1,Rg2,003,P,018,H,H,004,RH,Hr0,Hr,Rh29,HrB,Nou,Sec,Dav,MAR,019,XK,Kx,005,LU,Lub,Lu3Lu8,Lu11,Lu12,Lu13,Lu16,Lu17,Lu20,020,GE,Ge2,Ge3,Ge4,006,KEL,k,Kpb,Ku,Jsb,K11K14,k-like,K18,K19,Km,K22,TOU,RAZ,021,CROM,Cra,Tca,Dra,Esa,IFC,WESb,UMC,007,LE,022,KN,Kna,McCa,Sla,Yka,008,FY,Fy3,Fy5,Fy6,023,IN,Inb,INFI,INJA,009,JK,Jk3,024,OK,Oka,010,DI,Dib,Wrb,025,RAPH,MER2,011,YT,Yta,026,JMH,JMH,012,XG,027,I,I,013,SC,Sc1,Sc3,STAR,SCER,SCAN,028,GLOB,P,014,DO,Gya,Hy,Joa,029,GIL,GIL,015,CO,Coa,Co3,030,RHAG,Duclos,DSLK,Updated August 2008,High Incidence Antigens,(901 series),Lan,Ata,Jra,Emm,AnWj,Sda,PEL,MAM,调查对抗高频率抗原的异体抗体（,1,）,分清病人抗体为异体抗体,自体对照与DAT为阴性,若病人近期输过血，自体对照与DAT可呈现mixed-field反应,测试病人的ABO血型,Anti-H或A-HI常见于A1型或A1B型者,孟买型者(例：Oh)产生Anti-H异体抗体,测试抗体的反应期（Phase of reactivity）,以传统三项法取代MP法测试，可区分抗体为温型或冷型,若于室温即有反应，可能为anti-Ena,-Vel,-Sda,-P,-PP1Pk,与抗体筛检及鉴定细胞均有反应、自体对照无反应、与,ABO,血型相同之血球无反应,(,非异体抗体！,),若用新鲜serum检体，有试管内溶血现象,可能为anti-Vel,-P,-PP1Pk,-Jk3,调查对抗高频率抗原的异体抗体（,2,）,询问病人之籍贯（人种）,某些高频率抗原阴性者，在特定人种中相对较可能出现；可优先列入测试考量,Caucasian：Kpb,Coa,Yta,and Lub,Black:Hr0,Hr,U,Fy5,Jsb,Hy,Ata,hrB,and hrS,Japanese:Dib,Jra,Mongolian descent/Mexicans:Dib,Polynesian/Filipino:Jk3,Swedish:PP1Pk,Melanesians:Ge,Jewish/Arab/Druse:Yta,Selected Rare Phenotypes in Certain Populations(1/2),Ag negative,Population,Oh(Bombay),Indians Japanese any,U S-s-,Blacks,Ena,Finns Canadians English Japanese,k,Whites Blacks any,Kpb,Whites Japanese,Jsb,Blacks,K0(Knull),All,Fy(a-b-),Blacks Arabs/Jews Mediterranean Whites,Lu(a-b-),All,Jk(a-b-),Polynesian Finns Japanese any,Dib,S.Americans Native Americans Japanese,LWa,Baltic Sea region,LW(a-b-),(Transient)Canadians,（参考资料：Blood Group Antigens&Antibodies (2007)/AABB Technical Manual 16th edition）,Selected Rare Phenotypes in Certain Populations(2/2),Ag negative,Population,Yta,Arabs Jews any,Jra,Japanese Mexicansany,Kna,Whites Blacks any,McCa,Blacks Whites any,Sla,Blacks Whites any,Yka,Whites Blacks any,Inb,Indians,Oka,Japanese,Jra,Japanese Mexicansany,P,Japanese Scandinavians Israelis any,PP1Pk,Japanese Swedes Israelis any,AnWj,Israeli Arabs (Transient in all),（参考资料：Blood Group Antigens&Antibodies (2007)/AABB Technical Manual 16th edition）,测试病人的RBC次要抗原分型,若病人输过血，需找出输血前的检体，或分离病人自身血球测试（例：简易离心法）；若有基因分型技术亦可利用,基本的次要抗原分型,D,C,E,c,e:if all(-)Rhnull Anti-Hr0(-Rh17),-Hr(-Rh18)?.,Jka,Jkb:if Jk(a-b-)Anti-Jk3?,Fya,Fyb:if Fy(a-b-)Anti-Fy3?,S,s:if S+s-Anti-s?,if S-s-Anti-U?,Dia:if(+)Dib:if(-)Anti-Dib?,(K),k:if K+k-Anti-k?,if K-k-K0 phenotype Anti-Ku(-KEL5)?,或 McLeod phenotype Anti-Kx+-Km(-KEL20)?,P1:if(-)不能排除为 p phenotype anti-PP1Pk(-Tja)?,调查对抗高频率抗原的异体抗体（,3,）,Kx Antigen(XK1),McLeod Syndrome,and McLeod Phenotype,McLeod syndrome,X-linked condition,Acanthocytosis(棘型红血球),Variety of late-onset muscular,neurologic,and psychiatric symptoms.,McLeod phenotype,Kell antigens are expressed weakly and Km(KEL20)as well as Kx are absent.,When transfused,CGD(chronic granulomatous disease,慢性肉芽组织病)patients with McLeod syndrome usually produce anti-Kx plus anti-Km making it almost impossible to find compatible donors.It is recommended,therefore,that transfusion of males with CGD and McLeod syndrome be avoided.,鉴别anti-Kx+anti-Km(anti-KL)的方法:(其实不太容易),Anti-Kx:与K0血球反应较强，正常血球反应较弱,Anti-Km:与正常血球反应，但不与K0及McLeod血球反应,用K0血球吸收掉anti-Kx后，鉴定出Anti-Km。另从冲出液鉴定anti-Kx。,与proteases(例：ficin,papain)处理的血球反应,if all(-)anti-Ch?Rg?Inb?-JMH?.,与硫氢还原剂(例：DTT,AET)处理的血球反应,if all(-)anti-Kell?anti-LW?-Yta,-JMH?-Inb?,Scianna;Cromer;Knops;Lutheran;,Dombrock;AnWj,与cord RBCs反应,if all(-)anti-Ch?Rg?AnWj,利用简单的方法排除部分抗体，缩小鉴定范围,节省珍贵的稀有血球及特殊抗血清资源,调查对抗高频率抗原的异体抗体（,4,）,Effects of Enzymes and Chemicals on Antigens,Ficin/Papain,0.2M DTT,Possible Specificity,Neg,Pos,M,N,S,s*;Xga;Fya,Fyb;Ch/Rg#,Neg,Neg,Indian;JMH#,Pos,Weak,Cromer;Knops#(Kn,McC,Sla,Yka),;Lutheran;Dombrock;AnWj;MER2,Variable,Neg,Yta,Pos,Neg,Kell;LW;Scianna,Pos,Variable,Vel,Pos,Enhance,Kx,Pos,Pos,A,B;H;P1;Rh;Lewis;Kidd;Fy3;Diego;Co;Ge3;Oka;I,i;P,LKE;Ata;Csa#;Era;Jra;Lan;Sda;PEL;MAM;ABTI,*s variable with ficin/papain,#High-Titer,Low-Avidity HTLA antibodies,Usefulness of Cord RBCs,Negative,Lea,Leb;Sda;Ch,Rg,AnWj,Weak,A,B,H,I,P1,Lua,Lub,Yta,Vel,Bg,McCa,Yka,Sla,Csa,Hy,Gy,Joa,Doa,Dob,Fy3,Strong,LW system antigens;i antigen;Leab(Lex),Antigens in Soluble Form,Human source(from antigen-positive people),A,B and H(in secretors),Lea and Leb,Saliva,serum/plasma,I,Human milk,serum/plasma(low levels),Sda,Urine(Tamm-Horsfall glycoprotein),Ch and Rg,Serum/plasma,Cromer antigens,Urine,serum/plasma(low levels),Inb,Knops antigens,Serum/plasma(low levels),Nonhuman source,P1,Pigeon egg white and hydatid cyst fluid,Sda,Guinea pig urine,（参考资料：Blood Group Antigens&Antibodies (2007)Page 195）,HTLA,HTLA Antigens,High Titer Low Avidity (high titer,low antigen density),High titer:1:64 or greater,Low Avidity:1+or less through all titer dilutions,Occur with high frequency,Antibodies are weak and are not clinically significant,The antigens detected by antibodies formerly called HTLA are now well characterized and have been assigned to their own Blood Group System.,ISBT No.,System,HTLA Antigen,017,Chido/Rodgers,Ch,Rg,022,Knops,Kn,McC,Sla,Yka,026,John Milton Hagen,JMH,Cost collection,Csa,補充,Investigation of HTLA cases,If you suspect an HTLA Ab you can do,Titer patient serum:HTLA antibodies will titer out a long way(1:64)while others will not then you can,Chemical treatment of test RBCs,Neutralize patient serum with normal fresh plasma,it will remove HTLA antibody(anti-Ch/anti-Rg).,Ficin/Papain,0.2M DTT,Specificity,Neg,Pos,Ch/Rg,Neg,Neg,JMH,Pos,Weak,Knops(Kn,McC,Sla,Yka),Pos,Pos,Csa,補充,使用血浆抑制法鉴别anti-Ch与-Rg Using Plasma Inhibition To Distinguish Anti-Ch and-Rg from other Antibodies with Similar Characteristics,将待测病人血清以,NS,作系列稀释（例如,1:21:512,）。,取试管依序加入,2d,稀释血清，需准备两组。,其中一组各管加入,2d pooled plasma,（测试组），另一组各管加入,6%albumin,（对照组）。,混合均匀，于室温反应至少,30 min,。