监测ABO不相容性肾移植的ABO血型抗体滴度.ppt

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 nJohns Hopkins Hospital nDepartment of Pathology Transfusion Medicine Division nSpecialist in Blood Bank Technology AABB (American Association of Blood Banks) nABO 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 performed (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 克服克服ABOABO血型障碍进行的肾移植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 died per day n2,049 Group O n1,250 Group A n629 Group B n128 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 As of 5/25/07 Registered5 or More Years All ABO75,7118,079 O39,7134,459 A21,4571,844 B12,3791,592 AB2,162184 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 nIf registered in 2001- 2002, median wait times as of May 25, 2007 Blood GroupDays O1836 A1135 B2032 AB732 2001-20022001-2002年间登记的病人，年间登记的病人， 到到20072007年年5 5月月2525日平均等待时间是日平均等待时间是 血型天数 O1836 A1135 B2032 AB732 Objectives nExpand donor pool and availability of organs for transplantation nDecrease time on organ waiting list nCircumvent hyperacute rejection and/or AMR (due to ABO and/or HLA antibodies) by developing immunomodulatory protocols nABO incompatible nDonor-recipient with positive crossmatches (HLA) nPosttransplant patients who develop AMR (HLA) 目 的 n扩大器官捐献群体及移植器官的来源 n减少器官移植的等待时间 n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 nBased on Japanese experience nBased on our experience/protocol for crossmatch positive donor/recipient pairs and antibody-mediated rejection nFacilitated by laparoscopic donor nephrectomy nPreparative 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 nSpecialized Incompatible Kidney Transplant service and clinic nWeekly interdisciplinary rounds, composed of blood bank, apheresis service, HLA lab, nephrology, 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 nModified from the Japanese protocol nInitial evaluation ndonor and recipient medical, psychological and social work evaluations nHLA and ABO typing and titers nReview at weekly meeting ndetermination of optimal donor (HLA vs ABO) ntreatment plan established 我们的方案 n根据日本方案改进 n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 nImmunosuppression, may include splenectomy nAll patients vaccinated (pneumococcus, meningococcus, haemophilus influenzae), regardless of splenectomy status nPheresis plan established (number of procedures pre and postop) 治 疗 方 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 建立置换计划（术前/术后的置换程序和 次数） Plasmapheresis nRemoves natural anti-A and/or anti-B antibodis nBoth preop and postop procedures nRemove 1 plasma volume nReplace at 100% of volume removed nReplace with 5% albumin nexcept procedures immediately pre- and post- surgery, replace with plasma 血 浆 置 换 n去除天然的抗-A/抗-B抗体 n术前/后都进行置换 n去除一个血浆容量 n等量替换 n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 nPretransplant goal: titer 16 or less nPosttransplant nContinue to monitor ABO antibody titers nSignificance in prediction of antibody mediated rejection is uncertain ABO抗体滴度目标 n移植前目标： 滴度16 n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 ABO Antibody Titer (AHG) Pretransplant Treatments (#) Posttransplant Treatments (#) 1024 155 血浆置换过程 ABO抗体滴度移植前治疗次数移植后治疗次数 1024 155 ABO Antibody Titers Conventional test tube method nAABB technical manual 15th ed. Bethesda: American Association of Blood Banks, 2005. nSerial dilutions of each sample were prepared in 0.9% saline nPooled indicator cells (Immucor, Norcross, GA) of the appropriate ABO type were added n30 minutes 22C incubation test phase n30 minutes 37C incubation test phase nAHG test phase using monospecific anti-IgG (Immucor, Norcross, GA) nAgglutination was scored using the Marsh 0 to 12 scoring system nAHG 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 nAABB技术手册 n样品用0.9%的盐水进行连续稀释 n加入适量的混合ABO血型指示细胞（ Immucor ， Norcross, GA ） n22度孵育30分钟 n37度孵育30分钟 n单特异性抗-IgG (Immucor, Norcross, GA) 的抗人球蛋 白（AHG）实验阶段 n用Marsh 0到12打分系统给凝集打分 nAHG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 nIt is time consuming nTurn around time is critical nOur experience indicates that only the AHG titer values are critical nTiter endpoint is demonstrated with a macroscopic agglutination ABO抗体滴度 n这是一个花费时间的实验 n关键是出现结果的时间 n我们的经验表明只有AHG滴度是关键值 n最终滴度是由肉眼可见的凝集决定 Objectives 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 temperature incubation phase was eliminated 2) An anti-IgG gel microcolumn method 目 的 n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 method nSerial dilutions of each sample were prepared in 0.9% saline nPooled indicator cells (Immucor, Norcross, GA) of the appropriate ABO type were added n30 minutes room temperature was omitted nTiters were incubated for 30 minutes 37C and then converted to the AHG test phase using monospecific anti-IgG (Immucor, Norcross, GA) nAgglutination was scored using the Marsh 0 to 12 scoring system nAHG 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 n每个样品用0.9%的盐水进行系列稀释 n加入适当的ABO血型的混合指示细胞（ Immucor ， Norcross, GA ） n省略室温30分钟孵育 n37度孵育30分钟后用单特异性抗-IgG 进行AHG实验 n用Marsh打分系统对凝集进行打分 nAHG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 method nSerial dilutions of each sample were prepared nTwenty-five microliters of each plasma dilution and 50 microliters of 0.8% indicator red cells prepared in MTS Diluent 2 were added to the gel card microcolumns nAfter incubation at 37C for 15 minutes, the gel cards were centrifuged for 10 minutes nThe 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 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, 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 nSerial EDTA plasma samples from patients in our ABO INKT program nFifty samples with anti-A and/or Anti-B antibodies nAHG titers ranging from 2 to 512 n18 of the 50 (36%) samples were selected with titers of 16 病人样品 n我们 ABO不相容的肾移植项目的病人 EDTA血浆标本 n50个有抗-A/抗-B抗体的标本 nAHG 滴度在2-512之间 n50个中有18个样本滴度为16 Patient samples Number of Samples TitersAnti-AAnti-BAnti-A/Anti-BTotal 2-862210 16125118 32-649312 128-256729 51211 Total3413350 病 人 样 本 样 本 数 滴度抗-A抗-B抗-A/抗-B总计 2-862210 16125118 32-649312 128-256729 51211 总计3413350 Results 结 果 Results (table 1) nComparison of antiglobulin (AHG) titer results by test tube methods with and without a room temperature (RT) incubation phase Conventional test tube method Revised Test tube method (RTT) N=Identical to CTT AHG titer CTT AHG titer传统 AHG滴度 CTT AHG titer传统AHG滴度 RTT AHG titer改良试管法滴度 改良试管法滴度 211 4321 866 1618162 32761 6455 128642 256321 51211 总计5043 (86%)5 (10%)2 (4%) Results nAll the AHG titer results (100%) varied no more than one standard dilution wh