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1、Streamlining ABO Antibody Titers For Monitoring Streamlining ABO Antibody Titers For Monitoring ABO Incompatible Kidney TransplantsABO Incompatible Kidney TransplantsWei Cai, M.T. (ASCP)Wei Cai, M.T. (ASCP)Johns Hopkins Medical InstitutionJohns Hopkins Medical Institution监测ABO不相容性肾移植的ABO血型抗体滴度监测ABO不
2、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监测ABO不相容性肾移植的ABO血型抗体滴度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 Associati
3、on of Blood Banks)n nABO Incompatible Kidney Transplant Program 监测ABO不相容性肾移植的ABO血型抗体滴度背 景 知 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监测ABO不相容性肾移植的ABO血型抗体滴度Crossing the ABO Barrier for Kidney TransplantsAs of May 25, 2007: 75,711 patients awaiting kidney As of May 25, 2007: 75,711 patients aw
4、aiting kidney transplantation transplantation In 2006, 17,092 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监测ABO不相容性肾移植的ABO血型
5、抗体滴度至2007年5月25日止， 有75,711名病人在等待进行肾移植2006年 进行了17,902个肾移植，其中6,433约37.6%是活体肾移植 血型 肾移植 O 7,662 A 6,354 B 2,255 AB 821 克服ABO血型障碍进行的肾移植监测ABO不相容性肾移植的ABO血型抗体滴度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 transp
6、lant, 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 Group AB128 Group AB监测ABO不相容性肾移植的ABO血型抗体滴度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。监测ABO不相容性肾移植的ABO血型抗体滴度Cadaveric Kidney Transplant WaitAs of
7、 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 B12,37912,3791,5921,592ABAB2,1622,162184184监测ABO不相容性肾移植的ABO血型抗体滴度等待尸肾移植的病人数及等待时间至07年5月25日 登记 等待 5年或5年以上 所有血型 75,711 8,079 O 39,713 4,459 A 21
8、,457 1,844 B 12,379 1,592 AB 2,162 184监测ABO不相容性肾移植的ABO血型抗体滴度Cadaveric Kidney Transplant Waitn nIf registered in 2001-2002, median wait times as of May 25, 2007 Blood GroupBlood GroupDays Days O O18361836A A11351135B B20322032ABAB732732监测ABO不相容性肾移植的ABO血型抗体滴度2001-2002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
9、O O18361836A A11351135B B20322032ABAB732732监测ABO不相容性肾移植的ABO血型抗体滴度Objectivesn nExpand donor pool and availability of organs for transplantation Expand donor pool and availability of organs for transplantation n nDecrease time on organ waiting list Decrease time on organ waiting list n nCircumvent hyp
10、eracute 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 nABO incompatibleABO incompatiblen nDonor-recipient with positive crossmat
11、ches (HLA) Donor-recipient with positive crossmatches (HLA) n nPosttransplant patients who develop AMR (HLA)Posttransplant patients who develop AMR (HLA)监测ABO不相容性肾移植的ABO血型抗体滴度目目 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，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
12、HLAHLA抗体介导的排斥反应抗体介导的排斥反应n nABOABO不相容性不相容性n n供供- -受者交配反应阳性（受者交配反应阳性（HLAHLA）n n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) 监测ABO不相容性肾移植的ABO血型抗体滴度ABO Incompatible Transplantationn nBased on Japanese experienceBased on Japanese experiencen nBased on our experience/protocol for crossmatch positive Based
13、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 intensive follow upP
14、reparative regimen with intensive follow up pre/postoperative plasmapheresis pre/postoperative plasmapheresis IVIgG and immunosuppression IVIgG and immunosuppression监测ABO不相容性肾移植的ABO血型抗体滴度ABO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和免疫抑制监测ABO不相容性肾移植的ABO血型抗
15、体滴度Reference监测ABO不相容性肾移植的ABO血型抗体滴度ReferenceABO不相容性肾移植的长期结果不相容性肾移植的长期结果监测ABO不相容性肾移植的ABO血型抗体滴度监测ABO不相容性肾移植的ABO血型抗体滴度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监测ABO不相容性肾移植的ABO血型抗体滴度Our Program- InkTpn nSpecialized Incompatible Kidney Transplant service and clinicn nWeekly interdisciplinary round
16、s, composed of blood bank, apheresis service, HLA lab, nephrology, transplant surgery, pharmacist, psychologist, and social worker 监测ABO不相容性肾移植的ABO血型抗体滴度我们的不相容肾移植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监测ABO不相容性肾移植的ABO血型抗体滴度Our Protocoln nModified from the Japanese protocoln nIniti
17、al evaluationn ndonor and recipient medical, psychological and donor and recipient medical, psychological and social work evaluationssocial 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
18、donor (HLA vs ABO)determination of optimal donor (HLA vs ABO)n ntreatment plan establishedtreatment plan established监测ABO不相容性肾移植的ABO血型抗体滴度我们的方案根据日本方案改进初步评价 捐献者和受者进行医学、心理和社会服务的 评估 HLA和ABO分型及抗体滴度分析 每周会议的评述 根据HLA与 ABO相配情况决定最佳捐献者 建立治疗方案监测ABO不相容性肾移植的ABO血型抗体滴度Treatment Plann nImmunosuppression, may includ
19、e splenectomy n nAll patients vaccinated (pneumococcus, meningococcus, haemophilus influenzae), regardless of splenectomy status n nPheresis plan established (number of procedures pre and postop)监测ABO不相容性肾移植的ABO血型抗体滴度治 疗 方 案免疫抑制，可能包括脾切除 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 ） 建立置换计划（术前/术后的置换程序和次数）监测ABO
20、不相容性肾移植的ABO血型抗体滴度Plasmapheresisn nRemoves natural anti-A and/or anti-B antibodisn nBoth preop and postop proceduresn nRemove 1 plasma volumen nReplace at 100% of volume removedn nReplace with 5% albuminn nexcept procedures immediately pre- and post- except procedures immediately pre- and post- surge
21、ry, replace with plasmasurgery, replace with plasma监测ABO不相容性肾移植的ABO血型抗体滴度血血 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% 的白蛋白置换n n除术前/后立即的置换外，只用血浆置换监测ABO不相容性肾移植的ABO血型抗体滴度ABO Antibody Titer Goalsn nPretransplant goal: titer 16 or lesstiter 16 or lessn nPosttransplantn nContinue to mo
22、nitor ABO antibody titersContinue to monitor ABO antibody titersn nSignificance in prediction of antibody mediated Significance in prediction of antibody mediated rejection is uncertainrejection is uncertain监测ABO不相容性肾移植的ABO血型抗体滴度ABOABO抗体滴度目标抗体滴度目标n n移植前目标：移植前目标：n n滴度滴度1616n n移植后移植后n n继续监测继续监测ABOABO抗
23、体滴度抗体滴度n n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监测ABO不相容性肾移植的ABO血型抗体滴度Plasmapheresis ProceduresABO Antibody ABO Antibody Titer (AHG)Titer (AHG)Pretransplant Pretransplant Treatments (#)Treatments (#)Posttransplant Posttransplant Treatments (#)Treatments (#) 1610241024 15 155 5监测ABO不相容性肾移植的ABO血型抗体滴
24、度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 5监测ABO不相容性肾移植的ABO血型抗体滴度ABO Antibody TitersConventional test tube methodConventional test tube methodn nAABB technical manualAABB technical manual 15th ed. Bethe
25、sda: 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 (Immucor, Norcross, GA) of the appropriat
26、e ABO type were Pooled indicator cells (Immucor, Norcross, GA) of the appropriate ABO type were addedaddedn n30 minutes 22C incubation test phase30 minutes 22C incubation test phasen n30 minutes 37C incubation test phase30 minutes 37C incubation test phasen nAHG test phase using monospecific anti-Ig
27、G (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 reciprocal of the highest dilution demonstrating
28、 AHG titer endpoint was the reciprocal of the highest dilution demonstrating macropscopic (score 3) agglutinationmacropscopic (score 3) agglutination 监测ABO不相容性肾移植的ABO血型抗体滴度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（血型指示细胞（ Immucor I
29、mmucor ，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释倍数监测ABO不相容性肾移植的AB
30、O血型抗体滴度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 监测ABO不相容性肾移植的ABO血型抗体滴度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
31、时间n n我们的经验表明只有我们的经验表明只有AHGAHG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监测ABO不相容性肾移植的ABO血型抗体滴度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 wher
32、e the room temperature temperature incubation phase was eliminatedincubation phase was eliminated 2) An anti-IgG gel microcolumn method 2) An anti-IgG gel microcolumn method监测ABO不相容性肾移植的ABO血型抗体滴度目目 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
33、) 2) 应用抗应用抗-IgG-IgG凝胶微柱法凝胶微柱法监测ABO不相容性肾移植的ABO血型抗体滴度Material and methods Revised test tube titration 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, N
34、orcross, GA) of the Pooled indicator cells (Immucor, Norcross, GA) of the appropriate ABO type 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 incub
35、ated for 30 minutes 37C and then converted to the AHG test phase using monospecific anti-IgG (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 sco
36、ring systemsystemn nAHG titer endpoint was the reciprocal of the highest dilution AHG titer endpoint was the reciprocal of the highest dilution demonstrating macropscopic (score 3) agglutinationdemonstrating macropscopic (score 3) agglutination监测ABO不相容性肾移植的ABO血型抗体滴度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
37、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 ）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
38、的倒数监测ABO不相容性肾移植的ABO血型抗体滴度Material and methods Anti-IgG microcolumn gel methodn nSerial dilutions of each sample were preparedSerial dilutions of each sample were preparedn nTwenty-five microliters of each plasma dilution and Twenty-five microliters of each plasma dilution and 50 microliters of 0.8%
39、indicator red cells prepared in 50 microliters of 0.8% indicator red cells prepared in MTSMTS Diluent 2 were added to the gel card microcolumns Diluent 2 were added to the gel card microcolumnsn nAfter incubation at 37C for 15 minutes, the gel cards After incubation at 37C for 15 minutes, the gel ca
40、rds were centrifuged for 10 minuteswere centrifuged for 10 minutesn nThe titer endpoint was the reciprocal of the highest The titer endpoint was the reciprocal of the highest dilution demonstrating 1+dilution demonstrating 1+监测ABO不相容性肾移植的ABO血型抗体滴度实验材料和方法 抗抗-IgG-IgG微柱凝胶法微柱凝胶法n n每个样品系列稀释每个样品系列稀释n n在微柱
41、凝胶卡中加入在微柱凝胶卡中加入2525微升每一稀释度的血浆和微升每一稀释度的血浆和5050微升应用微升应用MTSMTS稀释液稀释液2 2制备的制备的0.8%0.8%的指示红细胞。的指示红细胞。n n3737孵育孵育1515分钟后将凝胶卡离心分钟后将凝胶卡离心1010分钟分钟n n最终滴度是显示最终滴度是显示1+1+凝集的最高稀释度的倒数凝集的最高稀释度的倒数监测ABO不相容性肾移植的ABO血型抗体滴度Anti-IgG Microcolumn Gel Cards Micro Typing Systems, MTS, Micro Typing Systems, MTS, Ortho Clinica
42、l Diagnostics, Raritan, NJ, USAOrtho Clinical Diagnostics, Raritan, NJ, USA监测ABO不相容性肾移植的ABO血型抗体滴度抗抗-IgG-IgG微柱凝胶卡片微柱凝胶卡片Micro Typing Systems, MTS, Micro Typing Systems, MTS, Ortho Clinical Diagnostics, Raritan, NJ, USAOrtho Clinical Diagnostics, Raritan, NJ, USA 微型血型仪微型血型仪监测ABO不相容性肾移植的ABO血型抗体滴度Patien
43、t samplesn nSerial EDTA plasma samples from 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监测ABO不相容性肾移植的ABO血型抗体滴度病人样品n n我们我们 ABO ABO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
44、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2-5122-512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1616监测ABO不相容性肾移植的ABO血型抗体滴度Patient 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
45、 31212128-256128-2567 72 29 95125121 11 1TotalTotal343413133 35050监测ABO不相容性肾移植的ABO血型抗体滴度病病 人人 样样 本本样样 本本 数数滴度滴度抗抗-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 35050监测ABO不相容性肾移植的ABO血型抗体滴度Results监测ABO不相容性肾移植的ABO血型抗
46、体滴度结结 果果监测ABO不相容性肾移植的ABO血型抗体滴度Results (table 1)n nComparison of antiglobulin (AHG) titer results by test tube methods with Comparison of antiglobulin (AHG) titer results by test tube methods with and without a room temperature (RT) incubation phase and without a room temperature (RT) incubation phas
47、e 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 titer CTT AHG titer CTT AHG titer CTT AHG titer传统AHG滴度 CTT AHG titer CTT AHG titer CTT AHG titer 传统传统AHGAHG滴度滴度 RTT AHG titer RTT
48、 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总计总计505043 (86%)43 (86%)5 (10%)5 (10%)2 (4%)2 (4%)监测ABO不相容性肾移植的ABO血型抗体滴度Resultsn nAll the AHG titer results (100%) varied no more than
49、one standard dilution which is within the acceptable limits for titration methods Technical Manual. 15th ed. Bethesda: American Association of Blood Banks, 2005. Technical Manual. 15th ed. Bethesda: American Association of Blood Banks, 2005. Yes!监测ABO不相容性肾移植的ABO血型抗体滴度结 果n n 所有AHG滴度测定的结（100%）的变化上下不超过
50、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） 监测ABO不相容性肾移植的ABO血型抗体滴度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 methods for monitoring ABO antibody titers. acceptable alternative methods
51、 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 are stable, batch titers can be better accommodated Because the rea
52、ctions 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 implementing ABO antibody titers by the IgG GEL method which will enable us t
53、o 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. 监测ABO不相容性肾移植的ABO血型抗体滴度讨 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
54、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， IgG IgG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。 监测ABO不相容性肾移植的ABO血型抗体滴度监测ABO不相容性肾移植的ABO血型抗体滴度监测ABO不相容性肾移植的ABO血型抗体滴度
