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1、监测ABO不相容性肾移植的ABO血型抗体滴度约翰约翰 霍伯金斯医学院霍伯金斯医学院蔡伟蔡伟Background Informationn nJohns Hopkins Hospitaln nDepartment of Pathology Transfusion Medicine Division Transfusion Medicine Divisionn nSpecialist in Blood Bank Technology AABB (American Association of Blood Banks) AABB (American Association of Blood Bank
2、s)n nABO Incompatible Kidney Transplant Program 背背 景景 知知 识识约翰 霍伯金斯大学医院病理系 输血医学部美国血库协会 血库技术专家ABO 血型不相容性肾移植计划Crossing the ABO Barrier for Kidney TransplantsAs of May 25, 2007: 75,711 patients awaiting kidney As of May 25, 2007: 75,711 patients awaiting kidney transplantation transplantation In 2006, 1
3、7,092 kidney transplants were performed In 2006, 17,092 kidney transplants were performed (6,433 or 37.6% living donors):(6,433 or 37.6% living donors): Group O Group O 7,662 7,662 A A 6,354 6,354 B B 2,255 2,255 AB AB 821 821至至至至2007200720072007年年年年5 5 5 5月月月月25252525日止，日止，日止，日止， 有有有有75,71175,71175
4、,71175,711名病人在等待进行肾移植名病人在等待进行肾移植名病人在等待进行肾移植名病人在等待进行肾移植2006200620062006年年年年 进行了进行了进行了进行了17,90217,90217,90217,902个肾移植，其中个肾移植，其中个肾移植，其中个肾移植，其中6,4336,4336,4336,433约约约约37.6%37.6%37.6%37.6%是活是活是活是活体肾移植体肾移植体肾移植体肾移植 血型血型 肾移植肾移植肾移植肾移植 O 7,662O 7,662 A 6,354 A 6,354 B 2,255 B 2,255 AB 821 AB 821 克服ABO血型障碍进行的肾
5、移植Deaths on Waiting List In 2006, 4,056 patients In 2006, 4,056 patients died waiting for a kidney died waiting for a kidney transplant, or 11.1 transplant, or 11.1 patients died per daypatients died per day n n2,049 Group O2,049 Group On n1,250 Group A1,250 Group An n629 Group B629 Group Bn n128 Gr
6、oup AB128 Group AB在等待中死去的病人在等待中死去的病人 2006年，共有4,056名等待肾移植的病人死亡，即每天有11.1名病人在等待中死去。Cadaveric Kidney Transplant WaitAs of 5/25/07As of 5/25/07RegisteredRegistered5 or More 5 or More YearsYearsAll ABOAll ABO75,71175,7118,0798,079O O39,71339,7134,4594,459A A21,45721,4571,8441,844B B12,37912,3791,5921,592A
7、BAB2,1622,162184184等待尸肾移植的病人数及等待时间等待尸肾移植的病人数及等待时间至至0707年年5 5月月2525日日 登记登记 等待等待 5 5年或年或5 5年以上年以上 所有血型所有血型 75,711 8,079 O 39,713 4,459 A 21,457 1,844 B 12,379 1,592 AB 2,162 184Cadaveric Kidney Transplant Waitn nIf registered in 2001-2002, median wait times as of May 25, 2007 Blood GroupBlood GroupDay
8、s Days O O18361836A A11351135B B20322032ABAB7327322001-2002年间登记的病人，到2007年5月25日平均等待时间是血型血型天数天数O O18361836A A11351135B B20322032ABAB732732Objectivesn nExpand donor pool and availability of organs for transplantation Expand donor pool and availability of organs for transplantation n nDecrease time on o
9、rgan waiting list Decrease time on organ waiting list n nCircumvent hyperacute rejection and/or AMR (due to ABO and/or Circumvent hyperacute rejection and/or AMR (due to ABO and/or HLA antibodies) by developing immunomodulatory protocols HLA antibodies) by developing immunomodulatory protocols n nAB
10、O incompatibleABO incompatiblen nDonor-recipient with positive crossmatches (HLA) Donor-recipient with positive crossmatches (HLA) n nPosttransplant patients who develop AMR (HLA)Posttransplant patients who develop AMR (HLA)目目 的的n n扩大器官捐献群体及移植器官的来源扩大器官捐献群体及移植器官的来源n n减少器官移植的等待时间减少器官移植的等待时间n n通过免疫调节方案
11、，防止超急性排斥反应及通过免疫调节方案，防止超急性排斥反应及/ /或或因因ABOABO血型或血型或HLAHLA抗体介导的排斥反应抗体介导的排斥反应 ABOABO不相容性不相容性 供供- -受者交配反应阳性（受者交配反应阳性（HLAHLA） 病人移植后出现抗体介导的排斥反应病人移植后出现抗体介导的排斥反应(HLA) (HLA) ABO Incompatible Transplantationn nBased on Japanese experienceBased on Japanese experiencen nBased on our experience/protocol for cross
12、match positive Based on our experience/protocol for crossmatch positive donor/recipient pairs and antibody-mediated rejection donor/recipient pairs and antibody-mediated rejection n nFacilitated by laparoscopic donor nephrectomyFacilitated by laparoscopic donor nephrectomyn nPreparative regimen with
13、 intensive follow upPreparative regimen with intensive follow up pre/postoperative plasmapheresis pre/postoperative plasmapheresis IVIgG and immunosuppression IVIgG and immunosuppressionABOABO不相容的移植不相容的移植 基于日本的经验 基于我们对交配阳性的供-受者和抗体介导的 排斥反应的经验/方案 腹腔镜捐献者取肾促进了该类移植 强化随访的予处理方案 术前/后血浆置换 静脉注射IgG和免疫抑制Referen
14、ceReferenceABO不相容性肾移植的长期结果不相容性肾移植的长期结果ABO不相容性病人在亲属活体肾移植后抗不相容性病人在亲属活体肾移植后抗-A、B的滴度变化的滴度变化Our Program- InkTpn nSpecialized Incompatible Kidney Transplant service and clinicn nWeekly interdisciplinary rounds, composed of blood bank, apheresis service, HLA lab, nephrology, transplant surgery, pharmacist,
15、 psychologist, and social worker 我们的我们的不相容肾移植不相容肾移植项目项目 专业化的服务和临床 每周跨科的综合查房，包括血库、单采、HLA实验室、肾科、移植外科、药剂师、心理学家和社会工作者Our Protocoln nModified from the Japanese protocoln nInitial evaluationn ndonor and recipient medical, psychological donor and recipient medical, psychological and social work evaluations
16、and social work evaluationsn nHLA and ABO typing and titers HLA and ABO typing and titers n nReview at weekly meeting Review at weekly meeting n ndetermination of optimal donor (HLA vs ABO)determination of optimal donor (HLA vs ABO)n ntreatment plan establishedtreatment plan established我们的方案我们的方案n n
17、根据日本方案改进根据日本方案改进n n初步评价初步评价 捐献者和受者进行医学、心理和社会服务的 评估 HLAHLA和和ABOABO分型及抗体滴度分析分型及抗体滴度分析 每周会议的评述 根据根据HLAHLA与与 ABOABO相配情况决定最佳捐献者相配情况决定最佳捐献者 建立治疗方案建立治疗方案Treatment Plann nImmunosuppression, may include splenectomy n nAll patients vaccinated (pneumococcus, meningococcus, haemophilus influenzae), regardless o
18、f splenectomy status n nPheresis plan established (number of procedures pre and postop)治治 疗疗 方方 案案n 免疫抑制，可能包括脾切除n 不管是否脾切除，所有病人接种疫苗（肺炎球菌、脑膜炎球菌、流感嗜血杆菌 ）n n 建立置换计划（术前建立置换计划（术前/ /术后的置换程序和术后的置换程序和次数）次数）Plasmapheresisn nRemoves natural anti-A and/or anti-B antibodisn nBoth preop and postop proceduresn nRe
19、move 1 plasma volumen nReplace at 100% of volume removedn nReplace with 5% albuminn nexcept procedures immediately pre- and except procedures immediately pre- and post- surgery, replace with plasmapost- surgery, replace with plasma血血 浆浆 置置 换换n n去除天然的抗-A/抗-B抗体n n术前/后都进行置换n n去除一个血浆容量n n等量替换n n用5% 的白蛋白
20、置换 除术前/后立即的置换外，只用血浆置换ABO Antibody Titer Goalsn nPretransplant goal: titer 16 or lesstiter 16 or lessn nPosttransplantn nContinue to monitor ABO antibody titersContinue to monitor ABO antibody titersn nSignificance in prediction of antibody mediated Significance in prediction of antibody mediated rej
21、ection is uncertainrejection is uncertainABOABO抗体滴度目标抗体滴度目标n n移植前目标：移植前目标： 滴度滴度1616n n移植后移植后 继续监测继续监测ABOABO抗体滴度抗体滴度 对于预测抗体介导的排斥反应的意义仍不确定对于预测抗体介导的排斥反应的意义仍不确定Plasmapheresis ProceduresABO Antibody ABO Antibody Titer (AHG)Titer (AHG)Pretransplant Pretransplant Treatments (#)Treatments (#)Posttransplant
22、Posttransplant Treatments (#)Treatments (#) 1610241024 15 155 5血浆置换过程血浆置换过程ABOABOABOABO抗体滴度抗体滴度抗体滴度抗体滴度移植前治疗次数移植前治疗次数移植前治疗次数移植前治疗次数移植后治疗次数移植后治疗次数移植后治疗次数移植后治疗次数 16 16 161024102410241024 15 15 15 155 5 5 5ABO Antibody TitersConventional test tube methodConventional test tube methodn nAABB technical ma
23、nualAABB technical manual 15th ed. Bethesda: American Association of Blood Banks, 2005. 15th ed. Bethesda: American Association of Blood Banks, 2005. n nSerial dilutions of each sample were prepared in 0.9% salineSerial dilutions of each sample were prepared in 0.9% salinen nPooled indicator cells (
24、Immucor, Norcross, GA) of the appropriate ABO type Pooled indicator cells (Immucor, Norcross, GA) of the appropriate ABO type were addedwere addedn n30 minutes 22C incubation test phase30 minutes 22C incubation test phasen n30 minutes 37C incubation test phase30 minutes 37C incubation test phasen nA
25、HG test phase using monospecific anti-IgG (Immucor, Norcross, GA) AHG test phase using monospecific anti-IgG (Immucor, Norcross, GA) n nAgglutination was scored using the Marsh 0 to 12 scoring systemAgglutination was scored using the Marsh 0 to 12 scoring systemn nAHG titer endpoint was the reciproc
26、al of the highest dilution demonstrating AHG titer endpoint was the reciprocal of the highest dilution demonstrating macropscopic (score 3) agglutinationmacropscopic (score 3) agglutination ABOABO抗体滴定抗体滴定常规的试管法常规的试管法n nAABBAABB技术手册技术手册n n样品用样品用0.9%0.9%的盐水进行连续稀释的盐水进行连续稀释 n n加入适量的混合加入适量的混合ABOABO血型指示细胞
27、（血型指示细胞（ Immucor Immucor ，Norcross, GA Norcross, GA ）n n2222度孵育度孵育3030分钟分钟n n3737度孵育度孵育3030分钟分钟n n单特异性抗单特异性抗-IgG (Immucor, Norcross, GA) -IgG (Immucor, Norcross, GA) 的抗人球蛋的抗人球蛋白（白（AHGAHG）实验阶段）实验阶段n n用用Marsh 0Marsh 0到到1212打分系统给凝集打分打分系统给凝集打分n nAHGAHG最终滴度是样品出现肉眼可见的（最终滴度是样品出现肉眼可见的（3 3分）凝集的最高稀分）凝集的最高稀释倍数
28、释倍数ABO Antibody Titersn nIt is time consumingn nTurn around time is criticaln nOur experience indicates that only the AHG titer values are criticaln nTiter endpoint is demonstrated with a macroscopic agglutination ABOABO抗体滴度抗体滴度n n这是一个花费时间的实验这是一个花费时间的实验n n关键是出现结果的时间关键是出现结果的时间n n我们的经验表明只有我们的经验表明只有AHG
29、AHG滴度是关键值滴度是关键值n n最终滴度是由肉眼可见的凝集决定最终滴度是由肉眼可见的凝集决定Objectivesn nThe goal of the present study was to streamline ABO antibody titer determinations by assessing AHG titers using 1) A revised test tube method where the room 1) A revised test tube method where the room temperature temperature incubation ph
30、ase was eliminatedincubation phase was eliminated 2) An anti-IgG gel microcolumn method 2) An anti-IgG gel microcolumn method目目 的的n n本研究的目的是通过评估本研究的目的是通过评估AHG AHG 滴度，系列滴度，系列测定测定ABOABO抗体抗体 1) 1) 应用不需要室温孵育过程的改良试管法应用不需要室温孵育过程的改良试管法 2) 2) 应用抗应用抗-IgG-IgG凝胶微柱法凝胶微柱法Material and methods Revised test tube ti
31、tration methodRevised test tube titration methodn nSerial dilutions of each sample were prepared in 0.9% salineSerial dilutions of each sample were prepared in 0.9% salinen nPooled indicator cells (Immucor, Norcross, GA) of the Pooled indicator cells (Immucor, Norcross, GA) of the appropriate ABO ty
32、pe were addedappropriate ABO type were addedn n30 minutes room temperature was omitted30 minutes room temperature was omittedn nTiters were incubated for 30 minutes 37C and then converted to Titers were incubated for 30 minutes 37C and then converted to the AHG test phase using monospecific anti-IgG
33、 (Immucor, the AHG test phase using monospecific anti-IgG (Immucor, Norcross, GA) Norcross, GA) n nAgglutination was scored using the Marsh 0 to 12 scoring Agglutination was scored using the Marsh 0 to 12 scoring systemsystemn nAHG titer endpoint was the reciprocal of the highest dilution AHG titer
34、endpoint was the reciprocal of the highest dilution demonstrating macropscopic (score 3) agglutinationdemonstrating macropscopic (score 3) agglutination实验材料和方法实验材料和方法 改良的试管法改良的试管法n n每个样品用每个样品用0.9%0.9%的盐水进行系列稀释的盐水进行系列稀释n n加入适当的加入适当的ABOABO血型的混合指示细胞（血型的混合指示细胞（ Immucor Immucor ，Norcross, GA Norcross, GA
35、 ）n n省略室温省略室温3030分钟孵育分钟孵育n n3737度孵育度孵育3030分钟后用单特异性抗分钟后用单特异性抗-IgG -IgG 进行进行AHGAHG实验实验n n用用MarshMarsh打分系统对凝集进行打分打分系统对凝集进行打分n nAHGAHG最终滴度是样品出现肉眼可见的（最终滴度是样品出现肉眼可见的（3 3分）凝集的最高分）凝集的最高稀释度的倒数稀释度的倒数Material and methods Anti-IgG microcolumn gel methodn nSerial dilutions of each sample were preparedSerial dilu
36、tions of each sample were preparedn nTwenty-five microliters of each plasma dilution Twenty-five microliters of each plasma dilution and 50 microliters of 0.8% indicator red cells and 50 microliters of 0.8% indicator red cells prepared in MTSprepared in MTS Diluent 2 were added to the gel card Dilue
37、nt 2 were added to the gel card microcolumnsmicrocolumnsn nAfter incubation at 37C for 15 minutes, the gel After incubation at 37C for 15 minutes, the gel cards were centrifuged for 10 minutescards were centrifuged for 10 minutesn nThe titer endpoint was the reciprocal of the The titer endpoint was
38、the reciprocal of the highest dilution demonstrating 1+highest dilution demonstrating 1+实验材料和方法 抗-IgG微柱凝胶法n每个样品系列稀释n在微柱凝胶卡中加入25微升每一稀释度的血浆和50微升应用MTS稀释液2制备的0.8%的指示红细胞。n37孵育15分钟后将凝胶卡离心10分钟n最终滴度是显示1+凝集的最高稀释度的倒数Anti-IgG Microcolumn Gel Cards Micro Typing Systems, MTSMicro Typing Systems, MTS, , Ortho Cli
39、nical Diagnostics, Raritan, NJ, USAOrtho Clinical Diagnostics, Raritan, NJ, USA抗抗-IgG-IgG微柱凝胶卡片微柱凝胶卡片Micro Typing Systems, MTSMicro Typing Systems, MTS, , Ortho Clinical Diagnostics, Raritan, NJ, USAOrtho Clinical Diagnostics, Raritan, NJ, USA 微型血型仪微型血型仪Patient samplesn nSerial EDTA plasma samples f
40、rom patients in our ABO INKT programn nFifty samples with anti-A and/or Anti-B antibodiesn nAHG titers ranging from 2 to 512n n18 of the 50 (36%) samples were selected with titers of 16病人样品n n我们我们 ABOABO不相容的肾移植项目的病人不相容的肾移植项目的病人EDTAEDTA血浆标本血浆标本n n5050个有抗个有抗-A/-A/抗抗-B-B抗体的标本抗体的标本n nAHG AHG 滴度在滴度在2-512
41、2-512之间之间n n5050个中有个中有1818个样本滴度为个样本滴度为1616Patient samples Number of SamplesNumber of SamplesTitersTitersAnti-AAnti-AAnti-BAnti-BAnti-A/Anti-BAnti-A/Anti-BTotalTotal2-82-86 62 22 21010161612125 51 1181832-6432-649 93 31212128-256128-2567 72 29 95125121 11 1TotalTotal343413133 35050病病 人人 样样 本本样样 本本 数数
42、滴度滴度抗抗-A-A抗抗-B-B抗抗-A/-A/抗抗-B-B总计总计2-82-86 62 22 21010161612125 51 1181832-6432-649 93 31212128-256128-2567 72 29 95125121 11 1总计总计343413133 35050Results结结 果果Results (table 1)n nComparison of antiglobulin (AHG) titer results by test tube methods with Comparison of antiglobulin (AHG) titer results by
43、test tube methods with and without a room temperature (RT) incubation phase and without a room temperature (RT) incubation phase Conventional test Conventional test tube methodtube methodRevised Test tube method (RTT)Revised Test tube method (RTT)N=N=Identical to CTT AHG titerIdentical to CTT AHG ti
44、ter CTT AHG titer CTT AHG titer CTT AHG titer 传统传统AHGAHG滴度滴度 CTT AHG titer CTT AHG titer CTT AHG titer 传统传统AHGAHG滴度滴度 RTT AHG titer RTT AHG titer RTT AHG titer 改良试管法滴度改良试管法滴度 改良试管法滴度改良试管法滴度 2 21 11 14 43 32 21 18 86 66 61616181816162 232327 76 61 164645 55 51281286 64 42 22562563 32 21 15125121 11 1
45、总计总计505043 (86%)43 (86%)5 (10%)5 (10%)2 (4%)2 (4%)Resultsn nAll the AHG titer results (100%) varied no more than one standard dilution which is within the acceptable limits for titration methods Technical Manual. 15th ed. Bethesda: American Association of Blood Banks, Technical Manual. 15th ed. Beth
46、esda: American Association of Blood Banks, 2005. 2005. Yes!结 果n n 所有AHG滴度测定的结（100%）的变化上下不超过一个稀释度，均在可接受范围内（2005年第15版美国AABB技术手册标准）Discussionn nThis study shows that both the revised test tube and IgG GEL are This study shows that both the revised test tube and IgG GEL are acceptable alternative method
47、s for monitoring ABO antibody titers. acceptable alternative methods for monitoring ABO antibody titers. n nThe IgG GEL method offers the best turnaround time and requires less The IgG GEL method offers the best turnaround time and requires less hands-on time. hands-on time. n nBecause the reactions
48、 are stable, batch titers can be better accommodated Because the reactions are stable, batch titers can be better accommodated by IgG GEL.by IgG GEL.n nAs a result of this study, our laboratory is in the process of implementing As a result of this study, our laboratory is in the process of implement
49、ing ABO antibody titers by the IgG GEL method which will enable us to ABO antibody titers by the IgG GEL method which will enable us to provide titer values in a more timely-manner for patients in the ABO INKT provide titer values in a more timely-manner for patients in the ABO INKT program.program.
50、 讨 论n n本研究表明改良的试管法和本研究表明改良的试管法和IgGIgG凝胶法均可用于监凝胶法均可用于监测测ABOABO抗体滴度抗体滴度n nIgGIgG凝胶法提供了出现最佳结果的时间，所需手工凝胶法提供了出现最佳结果的时间，所需手工操作的时间更少操作的时间更少n n由于反应稳定，由于反应稳定， IgGIgG凝胶法的批间滴度更好。凝胶法的批间滴度更好。n n因此，我们实验室在用因此，我们实验室在用IgGIgG凝胶法监测凝胶法监测ABOABO抗体滴抗体滴度，这使我们能为度，这使我们能为ABO ABO 不相容的肾移植病人提供不相容的肾移植病人提供更及时的更及时的ABOABO抗体滴度。抗体滴度。
