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1、Streamlining ABO Antibody Titers For Monitoring ABO Incompatible Kidney Transplants,Wei Cai,M.T.(ASCP)Johns Hopkins Medical Institution,监测ABO不相容性肾移植的ABO血型抗体滴度约翰 霍伯金斯医学院蔡伟,Background Information,Johns Hopkins HospitalDepartment of Pathology Transfusion Medicine DivisionSpecialist in Blood Bank Techno
2、logy AABB(American Association of Blood Banks)ABO Incompatible Kidney Transplant Program,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,Crossing the ABO Barrier for Kidney Transplants,As of May 25,2007:75,711 patients awaiting kidney transplantation In 2006,17,092 kidney transplants were p
3、erformed(6,433 or 37.6%living donors):Group O 7,662 A 6,354 B 2,255 AB 821,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,Deaths on Waiting List,In 2006,4,056 patients died waiting for a kidney transplant,or 11.1 patients
4、died per day,2,049 Group O1,250 Group A629 Group B128 Group AB,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 Wait,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 21,457 1,844 B 12,379 1,592 AB 2,162 184,Cadaveric Kidney Transplant Wait,I
5、f registered in 2001-2002,median wait times as of May 25,2007,2001-2002年间登记的病人，到2007年5月25日平均等待时间是,Objectives,Expand donor pool and availability of organs for transplantation Decrease time on organ waiting list Circumvent hyperacute rejection and/or AMR(due to ABO and/or HLA antibodies)by developing
6、immunomodulatory protocols ABO incompatibleDonor-recipient with positive crossmatches(HLA)Posttransplant patients who develop AMR(HLA),目 的,扩大器官捐献群体及移植器官的来源减少器官移植的等待时间通过免疫调节方案，防止超急性排斥反应及/或因ABO血型或HLA抗体介导的排斥反应ABO不相容性供-受者交配反应阳性（HLA）病人移植后出现抗体介导的排斥反应(HLA),ABO Incompatible Transplantation,Based on Japanese
7、 experienceBased on our experience/protocol for crossmatch positive donor/recipient pairs and antibody-mediated rejection Facilitated by laparoscopic donor nephrectomyPreparative regimen with intensive follow up pre/postoperative plasmapheresis IVIgG and immunosuppression,ABO不相容的移植,基于日本的经验基于我们对交配阳性的
8、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,Reference,Reference,ABO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-A、B的滴度变化,Our Program-InkTp,Specialized Incompatible Kidney Transplant service and clinicWeekly interdisciplinary rounds,composed of blood bank,apheresis service,HLA lab,nephro
9、logy,transplant surgery,pharmacist,psychologist,and social worker,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,Our Protocol,Modified from the Japanese protocolInitial evaluationdonor and recipient medical,psychological and social work evaluationsHLA and ABO typing and titers
10、Review at weekly meeting determination of optimal donor(HLA vs ABO)treatment plan established,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,Treatment Plan,Immunosuppression,may include splenectomy All patients vaccinated(pneumococcus,meningococcus,ha
11、emophilus influenzae),regardless of splenectomy status Pheresis plan established(number of procedures pre and postop),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）,Plasmapheresis,Removes natural anti-A and/or anti-B antibodisBoth preop and postop proceduresRemove 1 pl
12、asma volumeReplace at 100%of volume removedReplace with 5%albuminexcept procedures immediately pre-and post-surgery,replace with plasma,血 浆 置 换,去除天然的抗-A/抗-B抗体术前/后都进行置换去除一个血浆容量等量替换用5%的白蛋白置换除术前/后立即的置换外，只用血浆置换,ABO Antibody Titer Goals,Pretransplant goal:titer 16 or lessPosttransplantContinue to monitor
13、 ABO antibody titersSignificance in prediction of antibody mediated rejection is uncertain,ABO抗体滴度目标,移植前目标：滴度16移植后继续监测ABO抗体滴度对于预测抗体介导的排斥反应的意义仍不确定,Plasmapheresis Procedures,血浆置换过程,ABO Antibody Titers,Conventional test tube methodAABB technical manual 15th ed.Bethesda:American Association of Blood Ban
14、ks,2005.Serial dilutions of each sample were prepared in 0.9%salinePooled indicator cells(Immucor,Norcross,GA)of the appropriate ABO type were added30 minutes 22C incubation test phase30 minutes 37C incubation test phaseAHG test phase using monospecific anti-IgG(Immucor,Norcross,GA)Agglutination was
15、 scored using the Marsh 0 to 12 scoring systemAHG titer endpoint was the reciprocal of the highest dilution demonstrating macropscopic(score 3)agglutination,ABO抗体滴定,常规的试管法AABB技术手册样品用0.9%的盐水进行连续稀释 加入适量的混合ABO血型指示细胞（Immucor，Norcross,GA）22度孵育30分钟37度孵育30分钟单特异性抗-IgG(Immucor,Norcross,GA)的抗人球蛋白（AHG）实验阶段用Mar
16、sh 0到12打分系统给凝集打分AHG最终滴度是样品出现肉眼可见的（3分）凝集的最高稀释倍数,ABO Antibody Titers,It is time consumingTurn around time is criticalOur experience indicates that only the AHG titer values are criticalTiter endpoint is demonstrated with a macroscopic agglutination,ABO抗体滴度,这是一个花费时间的实验关键是出现结果的时间我们的经验表明只有AHG滴度是关键值最终滴度是由
17、肉眼可见的凝集决定,Objectives,The 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 temperature incubation phase was eliminated 2)An anti-IgG gel microcolumn method,目 的,本研究的目的是通过评估AHG 滴度，系列测定ABO抗体 1)应用不需要室温孵
18、育过程的改良试管法 2)应用抗-IgG凝胶微柱法,Material and methods,Revised test tube titration methodSerial dilutions of each sample were prepared in 0.9%salinePooled indicator cells(Immucor,Norcross,GA)of the appropriate ABO type were added30 minutes room temperature was omittedTiters were incubated for 30 minutes 37C
19、and then converted to the AHG test phase using monospecific anti-IgG(Immucor,Norcross,GA)Agglutination was scored using the Marsh 0 to 12 scoring systemAHG titer endpoint was the reciprocal of the highest dilution demonstrating macropscopic(score 3)agglutination,实验材料和方法,改良的试管法每个样品用0.9%的盐水进行系列稀释加入适当的
20、ABO血型的混合指示细胞（Immucor，Norcross,GA）省略室温30分钟孵育37度孵育30分钟后用单特异性抗-IgG 进行AHG实验用Marsh打分系统对凝集进行打分AHG最终滴度是样品出现肉眼可见的（3分）凝集的最高稀释度的倒数,Material and methods,Anti-IgG microcolumn gel methodSerial dilutions of each sample were preparedTwenty-five microliters of each plasma dilution and 50 microliters of 0.8%indicato
21、r red cells prepared in MTS Diluent 2 were added to the gel card microcolumnsAfter incubation at 37C for 15 minutes,the gel cards were centrifuged for 10 minutesThe titer endpoint was the reciprocal of the highest dilution demonstrating 1+,实验材料和方法,抗-IgG微柱凝胶法每个样品系列稀释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
22、2制备的0.8%的指示红细胞。37孵育15分钟后将凝胶卡离心10分钟最终滴度是显示1+凝集的最高稀释度的倒数,Anti-IgG Microcolumn Gel Cards,Micro Typing Systems,MTS,Ortho Clinical Diagnostics,Raritan,NJ,USA,抗-IgG微柱凝胶卡片,Micro Typing Systems,MTS,Ortho Clinical Diagnostics,Raritan,NJ,USA 微型血型仪,Patient samples,Serial EDTA plasma samples from patients in ou
23、r ABO INKT programFifty samples with anti-A and/or Anti-B antibodiesAHG titers ranging from 2 to 51218 of the 50(36%)samples were selected with titers of 16,病人样品,我们 ABO不相容的肾移植项目的病人EDTA血浆标本50个有抗-A/抗-B抗体的标本AHG 滴度在2-512之间50个中有18个样本滴度为16,Patient samples,病 人 样 本,Results,结 果,Results(table 1),Comparison of
24、 antiglobulin(AHG)titer results by test tube methods with and without a room temperature(RT)incubation phase,结果（表1）,有、无室温孵育的试管法抗人球（AHG）滴度实验结果的比较,Results(table 2),Comparison of conventional test tube AHG titers that included a room temperature test phase to the titers performed with anti-IgG gel card
25、s(IgG GEL),结果（表2）,有室温孵育的传统试管法与抗-IgG凝胶卡法 AHG滴度的比较,Results(table 3),Comparison of revised test tube AHG titer results with the IgG GEL titers,结果（表3）,改良试管法和IgG凝胶法AHG滴度的比较,Results,All the AHG titer results(100%)varied no more than one standard dilution which is within the acceptable limits for titration
26、 methods Technical Manual.15th ed.Bethesda:American Association of Blood Banks,2005.Yes!,结 果,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）,Discussion,This study shows that both the revised test tube and IgG GEL are acceptable alternative methods for monitoring ABO antibody titers.Th
27、e IgG GEL method offers the best turnaround time and requires less hands-on time.Because the reactions are stable,batch titers can be better accommodated by IgG GEL.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 to provide titer values in a more timely-manner for patients in the ABO INKT program.,讨 论,本研究表明改良的试管法和IgG凝胶法均可用于监测ABO抗体滴度IgG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由于反应稳定，IgG凝胶法的批间滴度更好。因此，我们实验室在用IgG凝胶法监测ABO抗体滴度，这使我们能为ABO 不相容的肾移植病人提供更及时的ABO抗体滴度。,
