HLA在肾脏移植配型中的应用研究
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摘要
人类白细胞抗原(HLA)是人体主要组织相容性基因(MHC),介导实体器官移植中的超急性、加速性、急性和慢性排斥反应,是器官移植免疫反应中的首要角色。HLA配型的应用使得肾移植后超急性和加速性严重排斥反应罕见发生,急性排斥已经成为影响肾移植后人／肾近期和远期生存率的最重要因素。2001年美国联邦立法将HLA配型列入器官移植技术标准,2006年中国卫生部将HLA配型列入器官移植管理法规。
肾移植进行供受者HLA配型的必要性无容置疑,其目的就是测定供体与受体HLA相容性程度,力求使排斥反应减小到最低程度。同时,移植受者由于接触过同种HLA抗原而产生的抗-HLA抗体,必须通过PRA检测来反映受者体内抗-HLA抗体体液免疫状态,从而准确预测肾移植后超急性和加速等严重排斥反应的发生和排斥反应的强度,有效改善肾移植近期效果和远期效果。而目前国内外公认肾脏移植配型许可的标准为：供受者淋巴毒交叉配型阴性、通过PRA检测确认受者不存在不可接受的供者HLA基因、进行供肾与受者HLA相合(MM)程度的评估。其中,供受者淋巴细胞毒交叉配型为阳性或受者存在针对供者HLA的特异性抗体时,被视为肾脏移植的禁忌指标,但是供受者HLA相合程度的未见具体的标准。
尽管近年来对MICA、KIR、调节性T细胞(Treg)等与肾移植的相关研究十分热烈,但都远远不足于动摇HLA在获得最佳肾移植效果中无可替代的地位。主要体现在如下几方面：
1.HLA作为人体免疫系统的核心,是人类的免疫应答基因,同时也是遗传标记物,研究证实某些HLA基因与终末期肾脏病(ESRD)的存在易感关联,这种易感关联具有种族和地域差异。同时,ESRD患者的家族聚集性,也提示了存在相关的易感基因的可能。研究两者间的易感关联,有助于阐明ESRD的发病机制,而且HLA作为一种疾病发生的遗传标志可用于ESRD的易感预测、鉴别诊断、病因分类、治疗及预后分析。
2.HLA生物学功能体现在抗原识别、处理和递呈、约束免疫细胞之间的相互作用、参与T细胞活化通路的多个环节、特异性免疫调节、免疫应答(激活、抑制),将外来抗原(供者)提呈给相应T细胞受体,从而激发免疫细胞克隆、增殖和分化,导致移植排斥反应的发生最终导致移植物失功。研究发现,供受者HLA相合程度越高,则肾移植受者术后出现PRA阳性机会越低、移植物排斥反应几率越少、移植物有功能存活时间越长、移植后所需排斥药物的血药浓度水平越低。因而HLA相合率已经成为临床医师决定移植供受者的重要因素。
3.移植受体内存在HLA抗体(PRA)对临床肾移植有重要的影响。PRA作为一种IgG类型的体液免疫性抗体,能特异性地针对供者的HLA抗原,这种HLA抗体能够直接杀伤移植肾细胞,是引起超急性排斥、加速性排斥反应、急性排斥反应和慢性排斥反应和最终导致移植物失功的元凶。
但是,HLA与ESRD的发生、发展的相关性以及在肾移植中应用中仍存在许多问题有待解决。例如：(1)HLA与ESRD的易感关联,确切机制至今未明；HLA本身在肾移植中的作用,包括HLA与移植物失功的易感关联有待进一步研究。(2)已知HLA相合率对移植物长期生存有决定性的作用,而HLA相合率对移植近期效果的影响的研究较少。另外,供肾的匮乏与追求供肾的HLA高度相合之间存在矛盾,如何在两者间取得平衡,使得在尽量缩短受者移植轮候时间的同时,获得理想的HLA相合分数的移植有待研究。(3)PRA阳性受者存在不可接受的HLA基因必然导致其获得移植机会减少,等待时间相应增加,为使临床医师预先了解致敏受者与任何供者交叉配型阳性的可能性、为选择移植肾来源提供数据支持,建立一种能反映PRA阳性患者与供者不相容的的真实几率的校正PRA(CPRA)方法的需求,显得非常迫切。
第一章中国南方ESRD患者HLA易感关联研究
目的
(1)对中国南方ESRD患者中HLA的表达进行分析,探讨HLA与ESRD的易感风险。
(2)对肾移植术后5年内移植肾失功与HLA多态性进行相关性研究,探讨HLA在移植肾失功中的临床应用价值。
材料与方法
(1)收集2000年1月-2013年6月在南方医科大学南方医院、中国人民解放军第458医院等16家移植中心登记等候肾脏移植的ESRD患者,年龄、性别不限。通过病历复习选择HLA基因分型资料完整的ESRD患者4670例。健康对照组为2686例南方汉族对照来自广州市中心血站和深圳市中心血站的健康献血者。
(2)采集2000年1月～2009年2月肾移植手术病例且持续随访5年以上的受者896例(年龄、性别不限、HLA基因分型资料完整),其中术后移植肾5年内失功即血肌酐升至707μmol/L以上行透析治疗的的受者50例,列为移植肾失功组；术后5年肾功能仍然正常,血肌酐<150μmol/L的受者701例,列为对照组。
留取EDTA抗凝血3ml,采用PCR-SSP分型方法进行HLA-A、B、DR基因分型。HLA基因的表现型频率PF=n/N,基因频率GF=1-(1-n/N)1/2,采用卡方检验对病例组与对照组的HLA-A、B、DRB1基因频率逐一进行关联分析(当某一位点表现为两个相同的基因,即纯合子时,计作一个)。必要时采用Yates检验方法与Fisher确切概率法计算确切P值。多重比较采用Bonferroni方法校正检验水准α值,Pc值≤0.05具有统计学意义,同时按Woolf公式计算相对危险度(RR)的比值比(OR)及95%的可信区间(CI),均数比较用两样本t检验,率的比较用卡方检验,均为双侧检验,检验水准a=0.05。
结果
4670例ESRD患者中,男性3068人,女性1602人,男：女=2.04：1,平均年龄(43.74±13.50)岁。原发病按比例由多到少依次为：慢性肾小球肾炎尿毒症76.08%>糖尿肾病5.96%>其他原因5.91%>病因不详5.01%>多囊肾并尿毒症3.13%>高血压肾病2.16%>双肾多发性结石1.24%>红斑狼疮性肾病0.54%。比较HLA基因在ESRD组和健康对照组间的分布,经Pc校正之后两组间HLA-A1、A3、A11、A24、A30、A31、A32、B7、B8、B38、B44、B46、B52、 B56、B57、B60、B61、B62、B67、B71、B75、DR1、DR4、DR7、DR8、DR12、 DR13、DR14和DR16频率的差异有统计学意义(Pc<0.05)。01的有A11、A24、B38、B46、B56、B60、B75、DR4、DR12和DR14。
比较移植肾失功组与对照组HLA多态性分布的差异,失功组HLA-DR17的频率显著高于对照组(PF=0.372VS0.120, Pc=0.00, OR=4.50)。
结论
研究显示,HLA-A11、A24、B38、B46、B56、B60、B75、DR4、DR12和DR14基因可能为中国南方汉族ESRD的易感基因；HLA-A1、A3、A30、A31、 A32、HLA-B7、B8、B44、B52、B57、B61、B62、B67、B71、DR1、DR7和DR13基因可能为中国南方汉族ESRD的抵抗基因；HLA-DR17本身可能就是导致移植肾5年内失功的高危因素。HLA多态性可能是导致移植效果差异的原因之一,但是HLA的表达及其多态性在因各类肾脏疾病导致最终并发ESRD进程中的免疫遗传易感性及相关危险性作用机制,仍未明确,有待进一步研究。
第二章肾移植供受者HLA相合分析及其对肾移植效果的影响
目的
对肾移植供受者HLA相合分数进行分析,探讨供受者HLA-A、B、DR&DQ相合率对肾移植后3年效果的影响。
材料与方法
收集2006年1月～2011年12月期间在南方医科大学南方医院和中国人民解放军第四五八医院完成初次尸体肾移植的受者资料428例,受者需符合供受者HLA基因分型资料完整、受者术前PRA阴性、术后持续随访>37个月。采集术后发生急性排斥(AR)以及3年内受者有功能生存的情况。参照美国器官共享联合网络(UNOS)的策略,计算供受者间HLA-A、B、DR&DQ四个座位的相合率,同一座位的基因有1个相同记为1分,2个基因相同记为2分,无相同基因记为0分,四个座位分数相加即为HLA相合率。按照供受者HLA相合率分数,即0、1、2、3、4、5、6、7、8分,共分为9组,并以供受者HLA相合率0～8分为节点,分为0-8分组合组。对供受者之间HLA、B、DR&DQ的相合分数进行,并分析HLA相合分数与移植后3年内移植肾AR、受者有功能生存的相关性。采用SPSS13.0统计软件进行统计分析,组间均数比较用两样本t检验,率的比较用卡方检验,必要时采用Yates检验方法与Fisher确切概率法计算确切P值,均为双侧检验,检验水准α=0.05。
结果
428例接受初次肾移植受者的平均年龄为(41.81±11.32)岁,HLA-A、B、DR、DQ的4个位点8个基因相合分数的平均值为(3.01±1.48)分,其中HLA-A、B、 DR, DQ位点相合分数的平均值分别(0.87±0.63)分、(0.59±0.58)分、(0.69±0.62)分和(0.85±-0.06)分,相合分数由高到低依次为A位点>DQ位点>DR位点>B位点。HLA-A、B、DR、DQ单个位点的相合分数为0分的比例,由高到低的顺序依次为B位点>DR位点>A位点>DQ位点,1分相合的百分比为DQ位点>A位点>DR位点>B位点,2分相合的百分比为A位点>DQ位>DR位点>B位点。HLA相合率0-8分组中比例最多的是3分组,占总对数的28.7%,其次是2分组和4分组,占23.1%和18.2%,相合数为8分仅为2对,占0.5%,HLA相合率的分布呈2-4分组较多,0分及7-8分少的趋势。428例肾移植受者术后AR率为17.76%,HLA相合分数0～8分组中AR率最高为0分组(33.33%),AR率从高到低排列依次为0分>2分>1分>7分>3分>6分>5分>4分>8分,分别0-9分组间的AR率的差异进行比较,其中HLA相合2分组与4分组之间AR率的差异存在统计学意义(P=0.03),其余组间比较差异均无统计学意义(P>0.05)。以供受者HLA相合分数0-8分为节点划分为8对0-8分的组合组,其中相合分数<3分的组合组的AR率均>30%,其中0-1分组合组AR率最高,为34.04%；相合分数1>3分的组合组,AR率均<10%。分别对8对组合组之间的AR率进行比较,0-1分组合组与2-8分组合组、0-2分组合组与3-8分组合组、0-3分组合组与4-8分组合组、0～4分组合组与5～8分组合组、0-5分组合组与6-8分组合组、0-6分组合组与7-8分组合组以及0-7分组合组与8分组之间AR率的差异均具有统计学意义(P<0.05),并以HLA相合率0～2分与3-8分组合组之间差异性最大(x2=26.40)；0分组与1-8分组合组之间AR率的差异无统计学意义(P>0.05)。428例肾移植受者移植1年人/肾存活率为95.33%,2年人/肾存活率为92.76%,3年人/肾存活率为91.12%。0-8分组按1年人/肾存活率从高到低排列,依次为HLA相合率8分=7分=6分>4分>1分>3分>5分>2分>0分组,3年人/肾存活率从高到低排列依次为8分=7分>4分>6分>5分>3分>1分>2分>0分组。
结论
由于HLA存在的高度多态性,导致供受者之间肾移植受者获得HLA-A、B、DR&DQ全相合供者的几率极低。本研究真实地反应了由移植医师选择肾移植供受者的HLA相合分数为(3.01±1.48)分。研究证实,HLA相合率对初次移植肾的近期效果具有显著影响,供受者间HLA相合率越高,术后发生排异反应等几率越低,尤其当HLA相合分数≥3分时,移植后AR率降低明显。研究提示,为了获得良好的肾移植近期效果,同时提高移植肾的长期存活率,初次肾移植受者应尽量选择供受者HLA相合率≥3分的供者,尽量避免选择供受者相合分数0-1分的供者。
第三章采用校正PRA方法分析HLA抗体阳性受者获得肾脏移植的几率
目的
建立一种分析PRA阳性患者与供者不相容几率的校正PRA(CPRA)方法,采用校正PRA(CPRA)方法分析临床肾移植中受者获得移植物的几率。
方法
收集2000年1月～2012年12月间在南方医科大学南方医院及中国人民解放军第四五八医院进行移植、且HLA基因分型资料完整供者共2004例；收集2008年1月～2012年12月间在南方医科大学南方医院及中国人民解放军第四五八医院等候肾移植的PRA阳性(PRA强度为峰值)的受者202例。采用PCR-SSP方法对2004例供者进行HLA-A、B、DR分型并建立供者HLA数据库,应用Arlequin软件3.11计算单个HLA-A、B、DR基因以及A-B、A-DR、B-DR和A-B-DR单倍型出现的频率,以此为基础建立了CPRA-Java计算软件,将PRA特异性结果作为不同接受的HLA基因,并在CPRA计算软件中输入各种基因组合时,即可获得一个CPRA的百分数,以代表不可接受HLA基因在该供者群体中所占的期望值百分比。采用ELISA方法对202例PRA阳性受者的血清进行HLA抗体确定实验,分析PRA强度(%)和HLA抗体特异性,将202例PRA特异性抗体作为不可接受的HLA基因,计算相应的CPRA值。按照PRA致敏程度,分别比较低度致敏(PRA>0-10%)、中度致敏(PRA>10～30%)和高度致敏(PRA>30%)与该致敏程度CPRA的平均强度的差异；比较PRA低、中、高致敏组中CPRA值(>0-10%、>10-30%和>30%)的分布情况,对PRA强度和CPRA值的均值进行一致性比较。CPRA值与PRA强度均值的比较采用独立样本t检验。CPRA值与PRA强度一致率的计算使用kappa参数。数据处理采用SPSS13.0软件,均为双侧检验,检验水准α=0.05。
结果
分析2004例供者的HLA-A、B、DR表达情况,将202例PRA阳性受者的HLA抗体特异性结果,输入CPRA-Java软件,获得含全部不可接受基因的供者期望值频数,即获得相应的CPRA值。202例PRA阳性受者平均强度为(23.12±17.96)%, CPRA均值为(46.07±23.30)%。低度致敏者(PRA>0-10%)占受者的30.69%(62/202),PRA平均强度为(6.87%±2.41)%,组中CPRA均值为(21.63±11.75)%,PRA平均强度与CPRA均值之间存在显著的统计学差异(P<0.05)；中度致敏者(10-30%)占受者的45.049%(91/202),平均强度为(20.15±5.17)%,组中CPRA均值为(50.56±16.86)%,PRA平均强度与CPRA均值之间存在显著的统计学差异(P<0.05)；高度致敏者(>30%)占受者的24.26%,平均强度为(49.20±15.17)%,组中CPRA均值为(68.64±15.35)%,PRA平均强度与CPRA均值之间无显著的统计学差异(P>0.05)。分别比较三种致敏程度中CPRA均值的差异,其中低度致敏组与中、高度致敏组的差异均存在统计学差异(P<0.05),中度致敏组和高度致敏组之间CPRA均值的差异则不存在统计学差异(P>0.05)。分析PRA低、中、高致敏组中CPRA值(>0-10%、>10～30%和>30%)分布的差异：低、中、高度致敏组PRA强度与CPRA值的一致率分别为19.36%、10.99%和100%,PRA强度与CPRA值的一致程度存在显著的统计学差异(Kappa=0.09, P<0.05)。
结论
本研究建立的CPRA方法,是通过分析供者HLA频率建立数据库计算PRA阳性受者不可接受的HLA基因组合在该供者群体中所占的百分比,即CPRA值。研究证明,应用PRA检测HLA抗体呈低度致敏时受者实际获得移植的几率可能比PRA强度反映的低；而PRA>30%时CPRA均>30%,证实PRA>30%为肾移植的高危因素。因此,高PRA受者选择供肾更需慎重,必须进行准确可靠的PRA特异性检测,避开既往曾出现过的全部不可接受HLA基因。传统的PRA强度分析反映了肾移植受者自身的致敏程度,代表了交叉配型时与供者不相容的HLA基因,准确可靠的PRA特异性检测其不可接受的HLA基因极为重要；在此基础上,应用CPRA方法则可以如实反映受者获得移植的几率,为临床医师预测受者等待时间、选择移植方式提供依据。
Human leukocyte antigen (HLA) is the major histocompatibility complex genes (MHC) of human, which mediated hyperacute rejection, accelerated rejection, acute rejection and chronic rejection and in played the primary role in immune response of solid organ transplantation. In2001, the United States federal legislation put HLA matching into organ transplant technical standards. In2006, National Healthy Ministry of China put HLA matching into organ transplant regulations. The application of HLA typing reduce the occurrence of hyperacute rejection and acceleration rejection after kidney transplantation, then acute rejection has become the most important factor affecting short and long term survival of transplant.
The necessity of HLA matching between kidney transplant donor and recipient is undoubtedly, the purpose is the detect the HLA compatibility between donor and recipient, and strive to make the rejection reduced to a minimum. Meanwhile, transplant recipients might produce anti-HLA antibodies by touched allo-HLA antigen. Therefore, recipients had to have PRA test to detect anti-HLA antibody in vivo to prevent hyperacute rejection and other serious acceleration rejection accurately post-renal transplant, and then to improve long-term and short-term effect of kidney transplantation. In now days, kidney transplant matching license criteria werer:donor and recipient Complement dependent cytotoxicity(CDC) test was negative, recipients did not have any unacceptable donor specificity HLA antibody by PRA test, and assess HLA-matched (MM) rate between donor and recipient.Among them, if CDC test between donor and recipient was positive nor recipient had presence of specific antibodies against the donor's HLA antigen, each of that was considered as taboo of kidney transplant, but HLA match degree between donor and recipient do not have any concrete standard yet.
Although the rearch of MICA, KIR, regulatory T cells (Treg), and other related research and transplantation is very popular in recent years, but the irreplaceable position of HLA never in the shake to get the best kidney transplant. Mainly reflected in the following aspects:
1. HLA is the core and the response genes of human immune system, but also the genetic marker. Some studies confirmed the presence of susceptibility associated between certain HLA genes and uremia with racial and geographical differences. Meanwhile, the familial aggregation of ESRD patients, also suggests the existence of susceptibility genes. To study the association between them maight be good for elucidate the pathogenesis of ESRD, but also as a genetic marker occurrence of some disease that can be used to predict susceptibility, differential diagnosis, etiology, treatment and prognosis of ESRD.
2. The biological functions of HLA is reflected in recognition, processing and presenting antigen, constrained interaction between immune cells, involved in multiple aspects of T cell activation pathway, specific immune regulation, immune response(activation/inhibition). HLA presents foreign(donor) antigen to appropriate T cell receptors, so as to stimulate the clones, proliferation or differentiation of immune cells. That may lead to transplant rejection and eventually turn into graft dysfunction. Studies found that when donor/recipient's HLA match rate was higher, the lower chance of kidney transplant recipients has PRA positive after transplant, the less opportunities of graft rejection, the longer functional graft survival, the lower concentration levels of immunosuppression drugs were needed. The rate of HLA match has become an important factor for clinicians to determine a transplant donor.
3. The HLA antibodies (PRA) in recipients before thansplant has a significant impact on clinical renal transplantation. PRA as a type of humoral immune IgG antibodies that specifically for the donor's HLA antigens. The anti-HLA antibodies can be directly against donor's kidney cells. That maight cause hyperacute rejection, accelerated rejection, acute rejection and chronic rejection and ultimately lead to graft dysfunction culprit.
However, the correlation between HLA and the occurrence and development of ESRD, and the role of HLA in renal transplantation there are still exists lots of mysteries. For example:(1) The exact mechanisms of susceptibility association between ESRD and HLA is still unknown. How dose HLA use to prognosis, pathogenesis, diagnosis, etiology, treatment or prognosis in ESRD that needs to be further study.(2) It's known that HLA match rate is playing a decisive role in long-term survival, and studies of the impact of HLA match rate in transplant recent effect is not much. In addition, the contradiction between lack of donor and height requirements of HLA match is exist. How to strike a balance between them, how to choose the best HLA-matched kidney transplant scores has not be illustrated.(3) PRA positive recipients exist unacceptable HLA genes inevitably lead to the less opportunities to access transplantation and waiting time increase. PRA reflects renal transplant recipients himself of sensitized level, so that the reliable detection of HLA specificity antibody is extremely important. After that, CPRA provide evidence to respond the truthfully probability of access to transplant, also assist clinicians to predict waiting time and to select transplant method for PRA positive recipients.
Chapter One Investigation of HLA polymorphism
susceptible in Southern Chinese patients with ESRD
Objective
(1) To analyze the expression of HLA in South Chinese ESRD patients, and discussed risk of HLA susceptible with ESRD.
(2) To study the association between HLA polymorphism and graft failure after kidney transplantation in5years, and to investigate the clinical value of HLA in renal graft failure.
Materials and Methods
(1) We collected ESRD patients with any age or gender, who registered for waiting kidney transplant in16transplant centers from January2000to June2013., incluing Nanfang Hospital,458Hospital of PLA,and so on. By medical record review, we select4670cases of ESRD patients that has HLA genotyping data Guangzhou Blood Center and Shenzhen Blood Center as healthy control group.
(2) Seven hundreds and fifty one cases of recipients that follow-up>5years from Jan.2000to Feb.2009after transplant were collected, including50cases that had graft failure after kidney transplantation in5years as graft failure group and other701with good function graft as control group.
EDTA tube was used to collect3ml anticoagulant blood from each samples, and PCR-SSP technique was used to analysis HLA-A, B, DRB1genotyping. By direct counting method to calculate the frequency of each gene in HLA-A, B, DRB1locus (homozygous considered as one). HLA antigen phenotype frequencies(PF)=n/N, gene frequency(GF)=1-(1-n/N)1/2. Chi-square test was used to calculate the frequency of each gene in HLA-A, B, DRB1locus (homozygous considered as one). Yates test methods and Fisher exact test was used to calculate the exact P value when necessary. Multiple comparisons used Bonferroni method to correct the a value, Pc value≤0.05was statistically significant. Meanwhile, ratio (OR) of Woolf relative risk (RR) and95%confidence intervals (CI) was calculated. Mean value used two-sample t test, comparison of rate used chi-square test, both of them were two-sided test, test level α=0.05.
Result
In4670cases of ESRD patients, male were3068,female were1602, male: female=2.04:1, the average age was (43.74±13.50). Primary disease from more to less proportionally as follows:chronic glomerulonephritis uremia(76.08%)> diabetic nephropathy(5.96%)> for other reasons(5.91%)> unknown etiology(5.01%)> polycystic kidney disease and uremia(3.13%)> hypertensive nephropathy(2.16)%> multiple renal calculi (1.24%)> lupus nephropathy(0.54%). Comparison of HLA frequency between ESRD group and healthy control group after Pc correction, the frequency of HLA-A1, A3, All, A24, A30, A31, A32, B7, B8, B38, B44, B46, B52, B56, B57, B60, B61, B62, B67, B71, B75, DR1, DR4, DR7, DR8, DR12, DR13, DR14and DR16was statistically significant different between two groups (Pc<0.05). HLA-A1, A3, A30, A31, A32, B7, B8, B44, B52, B57, B61, B62, B67, B71, DR1, DR7and DR13was0OR<1, and All, A24, B38, B46, B56, B60, B75, DR4, DR12and DR14was OR>1.
Compared the HLA frequencies between graft failure group and control group, the frequency of HLA-DR17was significantly higher in graft failure group (PF=0.213VS0.062, Pc=0.00, OR=4.50).
Conclusion
Research shows that HLA-A11, A24, B38, B46, B56, B60, B75, DR4, DR12and DR14might be the susceptibility genes to ESRD in Southern Chinese Han; HLA-A1, A3, A30, A31, A32, HLA-B7, B8, B44, B52, B57, B61, B62, B67, B71, DR1, DR7and DR13may be resistance gene in the southern Chinese Han ESRD. HLA-DR17itself may be the susceptibility genes that lead to graft failure in5years. HLA polymorphism may be one of the causes of differen effect in transplant.
d whether to carry HLA susceptibility genes can be used as one of the factors to consider early entry into the transplant waiting list in order to reduce the waiting time for its transplantation. However, further studies due to various types of renal disease process leading to the final concurrency ESRD immune genetic mechanisms that susceptibility and risk associated to highly polymorphism expression of HLA were still unknow, worthy of further study.
But the expression of HLA polymorphism in various types of kidney disease that eventual due to ESRD, how does HLA process the genetic susceptibility and immune-related risk mechanism of action were still unknow, worthy of further study.
Chapter Two Analysis of HLA match rate between kidney transplant donor/recipient and its effects on renal transplant
Objective
To study HLA-A, B, DR&DQ match rate between kidney transplant recipient/donor and its affect on three years after kidney transplantation effect.
Method
Four hundred and twenty-eight cases of transplant recipients who accepted the initial renal transplant in Nanfang Hospital and458Hospital of People's Liberation Army from January2006to December2011, PRA tested to be negative and had integrated donor and recipient HLA genotyping data were choosed, and postoperative continuous follow-up of>37months. Time of first occurrence of acute rejection(AR) after transplantation, method of processing, restoration and renal function of AR were collected. Referring to the United States for Organ Sharing United Network (UNOS) strategy, the calculation HLA-A, B, DR&DQ four seats match rate of donor/recipient, one gene in the same of a locus as1point, two genes in the same of a locus as2point, no same genes as0point, sum four locus score is HLA match rate. According to HLA match rate,428recipients divided into nine groups that from0to8points'group in total of9groups. And based on0to8points as nodes to divided in0-8point combination group. Then analyzed donor/recipient's HLA-A,B,DR&DQ match rate and its association with acute rejection after transplantation and the survival rate of recent renal transplantation. SPSS13.0statistical software was uesd for statistical analysis. Compared mean value between two groups using two-sample t-test, and compared rate in two group with the chi-square test, Yates calculated using Fisher's exact test methods and exact P value method, when necessary, both of them were two-sided test, test level α=0.05.
Result
The average age of428recipients who accepted renal transplant for the first time has mean age of (41.81±11.32) year, HLA-A, B, DR, DQ eight genes average score matched was (3.01±1.48) points, single loci of HLA-A, B, DR, DQ matched mean score was (0.87±0.63) points,(0.59±0.58) points,(0.69±0.619) points, and (0.85±0.06) points, and the match rate from high to low was A locus> DQ locus> DR locus> B locus. In0-8point group, maximum proportion of them was3point group(28.74%), followed by2groupand4point group(18.22%), there were only2pairs in8point group(0.27%), distribution as of HLA match in2to4point the most and the less in0point and7to8group. In428cases of renal transplant recipients, rate of AR was17.76%. In0-8groups, highest rate of AR was0point group (33.33%), AR rate from highest to lowest order of0point>2point>1point>7point>3point>6point>5point>4point>8point. AR rates were compared between0to9point groups, there was statistically significant difference between2point and4point group(P=0.03), there were no difference statistically significant between the rest of the group(P>0.05). To the donor and recipient HLA match0-8points as nodes to divided into0to8points combined group. AR rate was>30%in<3point combined group, and AR was the highest(34.04%) in0-1combined group, and AR rate was <10%in>3point combined group. Comparing the AR rate in8pairs of combined group, there was statistically significant difference between0-1point and2-8combined group,0-2point and3-8combined group,0-3point and4-8combined group,0-4point and5-8combined group,0-5point and6-8combined group,0-6point and7-8combined group and0-7point and8combined group(P<0.05). And the value of difference between0-2point and3-8combined group was maximum(χ2=26.40). In428cases of renal transplant recipients,1-year patient/graft survival rate was95.33%,2-year patient/graft survival rate was92.76%,3-year patient/graft survival rate was91.12%.1year graft survival rate from highest to lowest in0-8point groups was8point=7point=6point>4point>1point>3point>5point>2point>0point, and3year graft survival rate from highest to lowest was8point=7point>4point>6point>5point>3point>1point>2point>0point.
Conclusion
Due to the presence of highly polymorphic HLA, the chance of renal transplant recipients accept a HLA-A, B, DR&DQ full match kidney is very low. In this study, truly reflects choice by transplant physicians that kidney transplant donor/recipients of HLA match rate is (3.01±1.48) points.
It confirmed that HLA match rate had a significantly effect on short-term of primary renal transplantation, the higher rate of HLA match between donor and recipient, the lower the probability of postoperative rejection, while rate of HLA match was over3point, AR rate would significantly decreased. In order to get a good short-term effect of renal transplantation, and improve the long-term graft survival, we believe that primary ranal transplant recipients should choose a kidney that HLA-matches>3point and avoid to choose a kidney that HLA-matches0to1point.
Chapter3Application of calculated PRA (CPRA) methods to analyze the probability of access to transplant in kidney transplantation recipients
Objective
To establish an calculated PRA (CPRA) method to analysis the incompatible rate to donors with PRA-positive patients, and to analyze the probability of access to transplant in kidney transplantation recipients.
Methods
Two thousand and four cases of donor that transplant in Nanfang Hospital and NO.458Hospital of People's Liberation Army were collected from January2000to December2012, which already had complete HLA genotyping data of both donor and recipient.202cases of PRA positive patients (PRA peak strength) who were waiting kidney transplant in Nanfang Hospital and NO.458Hospital of People's Liberation Army from January2008to December2012were collected. PCR-SSP method was used to perform for HLA-A, B, DR typing in2004cases of donors then establish an donor HLA database by excel. Arlequin software(3.11) was used to calculate the frequency of single HLA-A,B,DR gene and HLA-A-B, A-DR, B-DR and A-B-DR haplotype. Based on2,004cases of transplant donors' HLA data to establish donor HLA-A, B, DR database, and set up, to calculator CPRA expectation value percentage to represent an unacceptable risk of donor HLA once the HLA-specific antibodies of PRA-positive recipients was typed in this software. By using ELISAto test202cases of PRA positive recipients for HLA antibodies in serum, and analyzed PRA strength(%) and HLA antibody specificity. The PRA-specific antibodies of202cases as unacceptable HLA genes used CPRA-Java calculator software to calculate its corresponding CPRA value. According to the degree of PRA sensitization, compared the difference between low sensitized (PRA>0～10%), moderate sensitization(PRA>10-30%) or highly sensitized (PRA>30%) with their corresponding CPRA value, the the distribution of CPRA value(>0-10%,>10～30%and>30%) in low, medium and high sensitized groups. Compared the intensity value between CPRA and PRA. Independent samples t test was used to compare mean value of CPRA and PRA. Kappa parameter was used to compare the concordance of CPRA and PRA strength. SPSS13.0statistical software was uesd for statistical analysis. Both of them were two-sided test, test level a=0.05.
Result
Analyze HLA-A, B, DR expression in2004donors. Entered HLA-specific antibodies202cases of PRA positive recipients into CPRA-Java calculator, then the result as a percentage of CPRA that represents the expectation value of unacceptable HLA in donor group. Mean value of PRA strength in202positive recipients was (23.12±17.83)%, and mean value of CPRA was (46.07±23.30)%.Low sensitized (PRA>0-10%)30.69%of the recipients(62/202), which mean PRA value was (6.87%±2.41)%and mean CPRA value was (21.63±11.75)%, there were statistically significant differences between them(P<0.05). Moderate sensitized (10-30%) representing45.05%of recipients (91/202), which mean PRA value was (20.27±5.71)%and mean CPRA value was (50.56±16.86)%, there were statistically significant differences between them(P<0.05). Highly sensitized(>30%) representing24.26%of the recipients, which mean PRA value was (48.90±15.18)%and mean CPRA value was (68.64%±15.35)%, there were no statistically significant differences between them (P>0.05). Compared the difference mean CPRA value in three sensitization degree, there were significant difference between low sensitized group and moderate or highly sensitized group (P<0.05), but no significant difference between moderate sensitized group and highly sensitized group(P>0.05). Analyzed the difference of CPRA (>0～10%,>10～30%and>30%) distribution in PRA low, moderate and highly group, concordance rate of PRA value and CPRA value in Low sensitized group was19.35%(P<0.05),in moderately sensitized group was10.99%(P <0.05),highly sensitized group wsas100%(P>0.05). There were significant difference of concordance rate between PRA value and CPRA value(Kappa=0.09, P<0.05).
Conclusions
CPRA method was established in this study, by analyzing the frequency of HLA donor, to calculate the percentage of PRA positive recipient's unacceptable HLA gene combinations in the donor database, namely CPRA value. Studies have shown that the chance that sensitized transplant recipients actually get may be lower than PRA strength might shown. Studies have shown that the probability of access to transplant in sensitized recipients might lower than PRA%shows. CPRA was>30%while PRA>30%, either, that proved PRA>30%was a risk factor for kidney transplantation. Studies have shown that, PRA reflects renal transplant recipients himself of sensitized level, so that the reliable detection of HLA specificity antibody is extremely important. After that, CPRA provide evidence to respond the truthfully the probability of access to transplant in kidney transplantation recipients to assist clinicians to predict waiting time and to select transplant method for recipient.
肾移植进行供受者HLA配型的必要性无容置疑,其目的就是测定供体与受体HLA相容性程度,力求使排斥反应减小到最低程度。同时,移植受者由于接触过同种HLA抗原而产生的抗-HLA抗体,必须通过PRA检测来反映受者体内抗-HLA抗体体液免疫状态,从而准确预测肾移植后超急性和加速等严重排斥反应的发生和排斥反应的强度,有效改善肾移植近期效果和远期效果。而目前国内外公认肾脏移植配型许可的标准为：供受者淋巴毒交叉配型阴性、通过PRA检测确认受者不存在不可接受的供者HLA基因、进行供肾与受者HLA相合(MM)程度的评估。其中,供受者淋巴细胞毒交叉配型为阳性或受者存在针对供者HLA的特异性抗体时,被视为肾脏移植的禁忌指标,但是供受者HLA相合程度的未见具体的标准。
尽管近年来对MICA、KIR、调节性T细胞(Treg)等与肾移植的相关研究十分热烈,但都远远不足于动摇HLA在获得最佳肾移植效果中无可替代的地位。主要体现在如下几方面：
1.HLA作为人体免疫系统的核心,是人类的免疫应答基因,同时也是遗传标记物,研究证实某些HLA基因与终末期肾脏病(ESRD)的存在易感关联,这种易感关联具有种族和地域差异。同时,ESRD患者的家族聚集性,也提示了存在相关的易感基因的可能。研究两者间的易感关联,有助于阐明ESRD的发病机制,而且HLA作为一种疾病发生的遗传标志可用于ESRD的易感预测、鉴别诊断、病因分类、治疗及预后分析。
2.HLA生物学功能体现在抗原识别、处理和递呈、约束免疫细胞之间的相互作用、参与T细胞活化通路的多个环节、特异性免疫调节、免疫应答(激活、抑制),将外来抗原(供者)提呈给相应T细胞受体,从而激发免疫细胞克隆、增殖和分化,导致移植排斥反应的发生最终导致移植物失功。研究发现,供受者HLA相合程度越高,则肾移植受者术后出现PRA阳性机会越低、移植物排斥反应几率越少、移植物有功能存活时间越长、移植后所需排斥药物的血药浓度水平越低。因而HLA相合率已经成为临床医师决定移植供受者的重要因素。
3.移植受体内存在HLA抗体(PRA)对临床肾移植有重要的影响。PRA作为一种IgG类型的体液免疫性抗体,能特异性地针对供者的HLA抗原,这种HLA抗体能够直接杀伤移植肾细胞,是引起超急性排斥、加速性排斥反应、急性排斥反应和慢性排斥反应和最终导致移植物失功的元凶。
但是,HLA与ESRD的发生、发展的相关性以及在肾移植中应用中仍存在许多问题有待解决。例如：(1)HLA与ESRD的易感关联,确切机制至今未明；HLA本身在肾移植中的作用,包括HLA与移植物失功的易感关联有待进一步研究。(2)已知HLA相合率对移植物长期生存有决定性的作用,而HLA相合率对移植近期效果的影响的研究较少。另外,供肾的匮乏与追求供肾的HLA高度相合之间存在矛盾,如何在两者间取得平衡,使得在尽量缩短受者移植轮候时间的同时,获得理想的HLA相合分数的移植有待研究。(3)PRA阳性受者存在不可接受的HLA基因必然导致其获得移植机会减少,等待时间相应增加,为使临床医师预先了解致敏受者与任何供者交叉配型阳性的可能性、为选择移植肾来源提供数据支持,建立一种能反映PRA阳性患者与供者不相容的的真实几率的校正PRA(CPRA)方法的需求,显得非常迫切。
第一章中国南方ESRD患者HLA易感关联研究
目的
(1)对中国南方ESRD患者中HLA的表达进行分析,探讨HLA与ESRD的易感风险。
(2)对肾移植术后5年内移植肾失功与HLA多态性进行相关性研究,探讨HLA在移植肾失功中的临床应用价值。
材料与方法
(1)收集2000年1月-2013年6月在南方医科大学南方医院、中国人民解放军第458医院等16家移植中心登记等候肾脏移植的ESRD患者,年龄、性别不限。通过病历复习选择HLA基因分型资料完整的ESRD患者4670例。健康对照组为2686例南方汉族对照来自广州市中心血站和深圳市中心血站的健康献血者。
(2)采集2000年1月～2009年2月肾移植手术病例且持续随访5年以上的受者896例(年龄、性别不限、HLA基因分型资料完整),其中术后移植肾5年内失功即血肌酐升至707μmol/L以上行透析治疗的的受者50例,列为移植肾失功组；术后5年肾功能仍然正常,血肌酐<150μmol/L的受者701例,列为对照组。
留取EDTA抗凝血3ml,采用PCR-SSP分型方法进行HLA-A、B、DR基因分型。HLA基因的表现型频率PF=n/N,基因频率GF=1-(1-n/N)1/2,采用卡方检验对病例组与对照组的HLA-A、B、DRB1基因频率逐一进行关联分析(当某一位点表现为两个相同的基因,即纯合子时,计作一个)。必要时采用Yates检验方法与Fisher确切概率法计算确切P值。多重比较采用Bonferroni方法校正检验水准α值,Pc值≤0.05具有统计学意义,同时按Woolf公式计算相对危险度(RR)的比值比(OR)及95%的可信区间(CI),均数比较用两样本t检验,率的比较用卡方检验,均为双侧检验,检验水准a=0.05。
结果
4670例ESRD患者中,男性3068人,女性1602人,男：女=2.04：1,平均年龄(43.74±13.50)岁。原发病按比例由多到少依次为：慢性肾小球肾炎尿毒症76.08%>糖尿肾病5.96%>其他原因5.91%>病因不详5.01%>多囊肾并尿毒症3.13%>高血压肾病2.16%>双肾多发性结石1.24%>红斑狼疮性肾病0.54%。比较HLA基因在ESRD组和健康对照组间的分布,经Pc校正之后两组间HLA-A1、A3、A11、A24、A30、A31、A32、B7、B8、B38、B44、B46、B52、 B56、B57、B60、B61、B62、B67、B71、B75、DR1、DR4、DR7、DR8、DR12、 DR13、DR14和DR16频率的差异有统计学意义(Pc<0.05)。0
比较移植肾失功组与对照组HLA多态性分布的差异,失功组HLA-DR17的频率显著高于对照组(PF=0.372VS0.120, Pc=0.00, OR=4.50)。
结论
研究显示,HLA-A11、A24、B38、B46、B56、B60、B75、DR4、DR12和DR14基因可能为中国南方汉族ESRD的易感基因；HLA-A1、A3、A30、A31、 A32、HLA-B7、B8、B44、B52、B57、B61、B62、B67、B71、DR1、DR7和DR13基因可能为中国南方汉族ESRD的抵抗基因；HLA-DR17本身可能就是导致移植肾5年内失功的高危因素。HLA多态性可能是导致移植效果差异的原因之一,但是HLA的表达及其多态性在因各类肾脏疾病导致最终并发ESRD进程中的免疫遗传易感性及相关危险性作用机制,仍未明确,有待进一步研究。
第二章肾移植供受者HLA相合分析及其对肾移植效果的影响
目的
对肾移植供受者HLA相合分数进行分析,探讨供受者HLA-A、B、DR&DQ相合率对肾移植后3年效果的影响。
材料与方法
收集2006年1月～2011年12月期间在南方医科大学南方医院和中国人民解放军第四五八医院完成初次尸体肾移植的受者资料428例,受者需符合供受者HLA基因分型资料完整、受者术前PRA阴性、术后持续随访>37个月。采集术后发生急性排斥(AR)以及3年内受者有功能生存的情况。参照美国器官共享联合网络(UNOS)的策略,计算供受者间HLA-A、B、DR&DQ四个座位的相合率,同一座位的基因有1个相同记为1分,2个基因相同记为2分,无相同基因记为0分,四个座位分数相加即为HLA相合率。按照供受者HLA相合率分数,即0、1、2、3、4、5、6、7、8分,共分为9组,并以供受者HLA相合率0～8分为节点,分为0-8分组合组。对供受者之间HLA、B、DR&DQ的相合分数进行,并分析HLA相合分数与移植后3年内移植肾AR、受者有功能生存的相关性。采用SPSS13.0统计软件进行统计分析,组间均数比较用两样本t检验,率的比较用卡方检验,必要时采用Yates检验方法与Fisher确切概率法计算确切P值,均为双侧检验,检验水准α=0.05。
结果
428例接受初次肾移植受者的平均年龄为(41.81±11.32)岁,HLA-A、B、DR、DQ的4个位点8个基因相合分数的平均值为(3.01±1.48)分,其中HLA-A、B、 DR, DQ位点相合分数的平均值分别(0.87±0.63)分、(0.59±0.58)分、(0.69±0.62)分和(0.85±-0.06)分,相合分数由高到低依次为A位点>DQ位点>DR位点>B位点。HLA-A、B、DR、DQ单个位点的相合分数为0分的比例,由高到低的顺序依次为B位点>DR位点>A位点>DQ位点,1分相合的百分比为DQ位点>A位点>DR位点>B位点,2分相合的百分比为A位点>DQ位>DR位点>B位点。HLA相合率0-8分组中比例最多的是3分组,占总对数的28.7%,其次是2分组和4分组,占23.1%和18.2%,相合数为8分仅为2对,占0.5%,HLA相合率的分布呈2-4分组较多,0分及7-8分少的趋势。428例肾移植受者术后AR率为17.76%,HLA相合分数0～8分组中AR率最高为0分组(33.33%),AR率从高到低排列依次为0分>2分>1分>7分>3分>6分>5分>4分>8分,分别0-9分组间的AR率的差异进行比较,其中HLA相合2分组与4分组之间AR率的差异存在统计学意义(P=0.03),其余组间比较差异均无统计学意义(P>0.05)。以供受者HLA相合分数0-8分为节点划分为8对0-8分的组合组,其中相合分数<3分的组合组的AR率均>30%,其中0-1分组合组AR率最高,为34.04%；相合分数1>3分的组合组,AR率均<10%。分别对8对组合组之间的AR率进行比较,0-1分组合组与2-8分组合组、0-2分组合组与3-8分组合组、0-3分组合组与4-8分组合组、0～4分组合组与5～8分组合组、0-5分组合组与6-8分组合组、0-6分组合组与7-8分组合组以及0-7分组合组与8分组之间AR率的差异均具有统计学意义(P<0.05),并以HLA相合率0～2分与3-8分组合组之间差异性最大(x2=26.40)；0分组与1-8分组合组之间AR率的差异无统计学意义(P>0.05)。428例肾移植受者移植1年人/肾存活率为95.33%,2年人/肾存活率为92.76%,3年人/肾存活率为91.12%。0-8分组按1年人/肾存活率从高到低排列,依次为HLA相合率8分=7分=6分>4分>1分>3分>5分>2分>0分组,3年人/肾存活率从高到低排列依次为8分=7分>4分>6分>5分>3分>1分>2分>0分组。
结论
由于HLA存在的高度多态性,导致供受者之间肾移植受者获得HLA-A、B、DR&DQ全相合供者的几率极低。本研究真实地反应了由移植医师选择肾移植供受者的HLA相合分数为(3.01±1.48)分。研究证实,HLA相合率对初次移植肾的近期效果具有显著影响,供受者间HLA相合率越高,术后发生排异反应等几率越低,尤其当HLA相合分数≥3分时,移植后AR率降低明显。研究提示,为了获得良好的肾移植近期效果,同时提高移植肾的长期存活率,初次肾移植受者应尽量选择供受者HLA相合率≥3分的供者,尽量避免选择供受者相合分数0-1分的供者。
第三章采用校正PRA方法分析HLA抗体阳性受者获得肾脏移植的几率
目的
建立一种分析PRA阳性患者与供者不相容几率的校正PRA(CPRA)方法,采用校正PRA(CPRA)方法分析临床肾移植中受者获得移植物的几率。
方法
收集2000年1月～2012年12月间在南方医科大学南方医院及中国人民解放军第四五八医院进行移植、且HLA基因分型资料完整供者共2004例；收集2008年1月～2012年12月间在南方医科大学南方医院及中国人民解放军第四五八医院等候肾移植的PRA阳性(PRA强度为峰值)的受者202例。采用PCR-SSP方法对2004例供者进行HLA-A、B、DR分型并建立供者HLA数据库,应用Arlequin软件3.11计算单个HLA-A、B、DR基因以及A-B、A-DR、B-DR和A-B-DR单倍型出现的频率,以此为基础建立了CPRA-Java计算软件,将PRA特异性结果作为不同接受的HLA基因,并在CPRA计算软件中输入各种基因组合时,即可获得一个CPRA的百分数,以代表不可接受HLA基因在该供者群体中所占的期望值百分比。采用ELISA方法对202例PRA阳性受者的血清进行HLA抗体确定实验,分析PRA强度(%)和HLA抗体特异性,将202例PRA特异性抗体作为不可接受的HLA基因,计算相应的CPRA值。按照PRA致敏程度,分别比较低度致敏(PRA>0-10%)、中度致敏(PRA>10～30%)和高度致敏(PRA>30%)与该致敏程度CPRA的平均强度的差异；比较PRA低、中、高致敏组中CPRA值(>0-10%、>10-30%和>30%)的分布情况,对PRA强度和CPRA值的均值进行一致性比较。CPRA值与PRA强度均值的比较采用独立样本t检验。CPRA值与PRA强度一致率的计算使用kappa参数。数据处理采用SPSS13.0软件,均为双侧检验,检验水准α=0.05。
结果
分析2004例供者的HLA-A、B、DR表达情况,将202例PRA阳性受者的HLA抗体特异性结果,输入CPRA-Java软件,获得含全部不可接受基因的供者期望值频数,即获得相应的CPRA值。202例PRA阳性受者平均强度为(23.12±17.96)%, CPRA均值为(46.07±23.30)%。低度致敏者(PRA>0-10%)占受者的30.69%(62/202),PRA平均强度为(6.87%±2.41)%,组中CPRA均值为(21.63±11.75)%,PRA平均强度与CPRA均值之间存在显著的统计学差异(P<0.05)；中度致敏者(10-30%)占受者的45.049%(91/202),平均强度为(20.15±5.17)%,组中CPRA均值为(50.56±16.86)%,PRA平均强度与CPRA均值之间存在显著的统计学差异(P<0.05)；高度致敏者(>30%)占受者的24.26%,平均强度为(49.20±15.17)%,组中CPRA均值为(68.64±15.35)%,PRA平均强度与CPRA均值之间无显著的统计学差异(P>0.05)。分别比较三种致敏程度中CPRA均值的差异,其中低度致敏组与中、高度致敏组的差异均存在统计学差异(P<0.05),中度致敏组和高度致敏组之间CPRA均值的差异则不存在统计学差异(P>0.05)。分析PRA低、中、高致敏组中CPRA值(>0-10%、>10～30%和>30%)分布的差异：低、中、高度致敏组PRA强度与CPRA值的一致率分别为19.36%、10.99%和100%,PRA强度与CPRA值的一致程度存在显著的统计学差异(Kappa=0.09, P<0.05)。
结论
本研究建立的CPRA方法,是通过分析供者HLA频率建立数据库计算PRA阳性受者不可接受的HLA基因组合在该供者群体中所占的百分比,即CPRA值。研究证明,应用PRA检测HLA抗体呈低度致敏时受者实际获得移植的几率可能比PRA强度反映的低；而PRA>30%时CPRA均>30%,证实PRA>30%为肾移植的高危因素。因此,高PRA受者选择供肾更需慎重,必须进行准确可靠的PRA特异性检测,避开既往曾出现过的全部不可接受HLA基因。传统的PRA强度分析反映了肾移植受者自身的致敏程度,代表了交叉配型时与供者不相容的HLA基因,准确可靠的PRA特异性检测其不可接受的HLA基因极为重要；在此基础上,应用CPRA方法则可以如实反映受者获得移植的几率,为临床医师预测受者等待时间、选择移植方式提供依据。
Human leukocyte antigen (HLA) is the major histocompatibility complex genes (MHC) of human, which mediated hyperacute rejection, accelerated rejection, acute rejection and chronic rejection and in played the primary role in immune response of solid organ transplantation. In2001, the United States federal legislation put HLA matching into organ transplant technical standards. In2006, National Healthy Ministry of China put HLA matching into organ transplant regulations. The application of HLA typing reduce the occurrence of hyperacute rejection and acceleration rejection after kidney transplantation, then acute rejection has become the most important factor affecting short and long term survival of transplant.
The necessity of HLA matching between kidney transplant donor and recipient is undoubtedly, the purpose is the detect the HLA compatibility between donor and recipient, and strive to make the rejection reduced to a minimum. Meanwhile, transplant recipients might produce anti-HLA antibodies by touched allo-HLA antigen. Therefore, recipients had to have PRA test to detect anti-HLA antibody in vivo to prevent hyperacute rejection and other serious acceleration rejection accurately post-renal transplant, and then to improve long-term and short-term effect of kidney transplantation. In now days, kidney transplant matching license criteria werer:donor and recipient Complement dependent cytotoxicity(CDC) test was negative, recipients did not have any unacceptable donor specificity HLA antibody by PRA test, and assess HLA-matched (MM) rate between donor and recipient.Among them, if CDC test between donor and recipient was positive nor recipient had presence of specific antibodies against the donor's HLA antigen, each of that was considered as taboo of kidney transplant, but HLA match degree between donor and recipient do not have any concrete standard yet.
Although the rearch of MICA, KIR, regulatory T cells (Treg), and other related research and transplantation is very popular in recent years, but the irreplaceable position of HLA never in the shake to get the best kidney transplant. Mainly reflected in the following aspects:
1. HLA is the core and the response genes of human immune system, but also the genetic marker. Some studies confirmed the presence of susceptibility associated between certain HLA genes and uremia with racial and geographical differences. Meanwhile, the familial aggregation of ESRD patients, also suggests the existence of susceptibility genes. To study the association between them maight be good for elucidate the pathogenesis of ESRD, but also as a genetic marker occurrence of some disease that can be used to predict susceptibility, differential diagnosis, etiology, treatment and prognosis of ESRD.
2. The biological functions of HLA is reflected in recognition, processing and presenting antigen, constrained interaction between immune cells, involved in multiple aspects of T cell activation pathway, specific immune regulation, immune response(activation/inhibition). HLA presents foreign(donor) antigen to appropriate T cell receptors, so as to stimulate the clones, proliferation or differentiation of immune cells. That may lead to transplant rejection and eventually turn into graft dysfunction. Studies found that when donor/recipient's HLA match rate was higher, the lower chance of kidney transplant recipients has PRA positive after transplant, the less opportunities of graft rejection, the longer functional graft survival, the lower concentration levels of immunosuppression drugs were needed. The rate of HLA match has become an important factor for clinicians to determine a transplant donor.
3. The HLA antibodies (PRA) in recipients before thansplant has a significant impact on clinical renal transplantation. PRA as a type of humoral immune IgG antibodies that specifically for the donor's HLA antigens. The anti-HLA antibodies can be directly against donor's kidney cells. That maight cause hyperacute rejection, accelerated rejection, acute rejection and chronic rejection and ultimately lead to graft dysfunction culprit.
However, the correlation between HLA and the occurrence and development of ESRD, and the role of HLA in renal transplantation there are still exists lots of mysteries. For example:(1) The exact mechanisms of susceptibility association between ESRD and HLA is still unknown. How dose HLA use to prognosis, pathogenesis, diagnosis, etiology, treatment or prognosis in ESRD that needs to be further study.(2) It's known that HLA match rate is playing a decisive role in long-term survival, and studies of the impact of HLA match rate in transplant recent effect is not much. In addition, the contradiction between lack of donor and height requirements of HLA match is exist. How to strike a balance between them, how to choose the best HLA-matched kidney transplant scores has not be illustrated.(3) PRA positive recipients exist unacceptable HLA genes inevitably lead to the less opportunities to access transplantation and waiting time increase. PRA reflects renal transplant recipients himself of sensitized level, so that the reliable detection of HLA specificity antibody is extremely important. After that, CPRA provide evidence to respond the truthfully probability of access to transplant, also assist clinicians to predict waiting time and to select transplant method for PRA positive recipients.
Chapter One Investigation of HLA polymorphism
susceptible in Southern Chinese patients with ESRD
Objective
(1) To analyze the expression of HLA in South Chinese ESRD patients, and discussed risk of HLA susceptible with ESRD.
(2) To study the association between HLA polymorphism and graft failure after kidney transplantation in5years, and to investigate the clinical value of HLA in renal graft failure.
Materials and Methods
(1) We collected ESRD patients with any age or gender, who registered for waiting kidney transplant in16transplant centers from January2000to June2013., incluing Nanfang Hospital,458Hospital of PLA,and so on. By medical record review, we select4670cases of ESRD patients that has HLA genotyping data Guangzhou Blood Center and Shenzhen Blood Center as healthy control group.
(2) Seven hundreds and fifty one cases of recipients that follow-up>5years from Jan.2000to Feb.2009after transplant were collected, including50cases that had graft failure after kidney transplantation in5years as graft failure group and other701with good function graft as control group.
EDTA tube was used to collect3ml anticoagulant blood from each samples, and PCR-SSP technique was used to analysis HLA-A, B, DRB1genotyping. By direct counting method to calculate the frequency of each gene in HLA-A, B, DRB1locus (homozygous considered as one). HLA antigen phenotype frequencies(PF)=n/N, gene frequency(GF)=1-(1-n/N)1/2. Chi-square test was used to calculate the frequency of each gene in HLA-A, B, DRB1locus (homozygous considered as one). Yates test methods and Fisher exact test was used to calculate the exact P value when necessary. Multiple comparisons used Bonferroni method to correct the a value, Pc value≤0.05was statistically significant. Meanwhile, ratio (OR) of Woolf relative risk (RR) and95%confidence intervals (CI) was calculated. Mean value used two-sample t test, comparison of rate used chi-square test, both of them were two-sided test, test level α=0.05.
Result
In4670cases of ESRD patients, male were3068,female were1602, male: female=2.04:1, the average age was (43.74±13.50). Primary disease from more to less proportionally as follows:chronic glomerulonephritis uremia(76.08%)> diabetic nephropathy(5.96%)> for other reasons(5.91%)> unknown etiology(5.01%)> polycystic kidney disease and uremia(3.13%)> hypertensive nephropathy(2.16)%> multiple renal calculi (1.24%)> lupus nephropathy(0.54%). Comparison of HLA frequency between ESRD group and healthy control group after Pc correction, the frequency of HLA-A1, A3, All, A24, A30, A31, A32, B7, B8, B38, B44, B46, B52, B56, B57, B60, B61, B62, B67, B71, B75, DR1, DR4, DR7, DR8, DR12, DR13, DR14and DR16was statistically significant different between two groups (Pc<0.05). HLA-A1, A3, A30, A31, A32, B7, B8, B44, B52, B57, B61, B62, B67, B71, DR1, DR7and DR13was0OR<1, and All, A24, B38, B46, B56, B60, B75, DR4, DR12and DR14was OR>1.
Compared the HLA frequencies between graft failure group and control group, the frequency of HLA-DR17was significantly higher in graft failure group (PF=0.213VS0.062, Pc=0.00, OR=4.50).
Conclusion
Research shows that HLA-A11, A24, B38, B46, B56, B60, B75, DR4, DR12and DR14might be the susceptibility genes to ESRD in Southern Chinese Han; HLA-A1, A3, A30, A31, A32, HLA-B7, B8, B44, B52, B57, B61, B62, B67, B71, DR1, DR7and DR13may be resistance gene in the southern Chinese Han ESRD. HLA-DR17itself may be the susceptibility genes that lead to graft failure in5years. HLA polymorphism may be one of the causes of differen effect in transplant.
d whether to carry HLA susceptibility genes can be used as one of the factors to consider early entry into the transplant waiting list in order to reduce the waiting time for its transplantation. However, further studies due to various types of renal disease process leading to the final concurrency ESRD immune genetic mechanisms that susceptibility and risk associated to highly polymorphism expression of HLA were still unknow, worthy of further study.
But the expression of HLA polymorphism in various types of kidney disease that eventual due to ESRD, how does HLA process the genetic susceptibility and immune-related risk mechanism of action were still unknow, worthy of further study.
Chapter Two Analysis of HLA match rate between kidney transplant donor/recipient and its effects on renal transplant
Objective
To study HLA-A, B, DR&DQ match rate between kidney transplant recipient/donor and its affect on three years after kidney transplantation effect.
Method
Four hundred and twenty-eight cases of transplant recipients who accepted the initial renal transplant in Nanfang Hospital and458Hospital of People's Liberation Army from January2006to December2011, PRA tested to be negative and had integrated donor and recipient HLA genotyping data were choosed, and postoperative continuous follow-up of>37months. Time of first occurrence of acute rejection(AR) after transplantation, method of processing, restoration and renal function of AR were collected. Referring to the United States for Organ Sharing United Network (UNOS) strategy, the calculation HLA-A, B, DR&DQ four seats match rate of donor/recipient, one gene in the same of a locus as1point, two genes in the same of a locus as2point, no same genes as0point, sum four locus score is HLA match rate. According to HLA match rate,428recipients divided into nine groups that from0to8points'group in total of9groups. And based on0to8points as nodes to divided in0-8point combination group. Then analyzed donor/recipient's HLA-A,B,DR&DQ match rate and its association with acute rejection after transplantation and the survival rate of recent renal transplantation. SPSS13.0statistical software was uesd for statistical analysis. Compared mean value between two groups using two-sample t-test, and compared rate in two group with the chi-square test, Yates calculated using Fisher's exact test methods and exact P value method, when necessary, both of them were two-sided test, test level α=0.05.
Result
The average age of428recipients who accepted renal transplant for the first time has mean age of (41.81±11.32) year, HLA-A, B, DR, DQ eight genes average score matched was (3.01±1.48) points, single loci of HLA-A, B, DR, DQ matched mean score was (0.87±0.63) points,(0.59±0.58) points,(0.69±0.619) points, and (0.85±0.06) points, and the match rate from high to low was A locus> DQ locus> DR locus> B locus. In0-8point group, maximum proportion of them was3point group(28.74%), followed by2groupand4point group(18.22%), there were only2pairs in8point group(0.27%), distribution as of HLA match in2to4point the most and the less in0point and7to8group. In428cases of renal transplant recipients, rate of AR was17.76%. In0-8groups, highest rate of AR was0point group (33.33%), AR rate from highest to lowest order of0point>2point>1point>7point>3point>6point>5point>4point>8point. AR rates were compared between0to9point groups, there was statistically significant difference between2point and4point group(P=0.03), there were no difference statistically significant between the rest of the group(P>0.05). To the donor and recipient HLA match0-8points as nodes to divided into0to8points combined group. AR rate was>30%in<3point combined group, and AR was the highest(34.04%) in0-1combined group, and AR rate was <10%in>3point combined group. Comparing the AR rate in8pairs of combined group, there was statistically significant difference between0-1point and2-8combined group,0-2point and3-8combined group,0-3point and4-8combined group,0-4point and5-8combined group,0-5point and6-8combined group,0-6point and7-8combined group and0-7point and8combined group(P<0.05). And the value of difference between0-2point and3-8combined group was maximum(χ2=26.40). In428cases of renal transplant recipients,1-year patient/graft survival rate was95.33%,2-year patient/graft survival rate was92.76%,3-year patient/graft survival rate was91.12%.1year graft survival rate from highest to lowest in0-8point groups was8point=7point=6point>4point>1point>3point>5point>2point>0point, and3year graft survival rate from highest to lowest was8point=7point>4point>6point>5point>3point>1point>2point>0point.
Conclusion
Due to the presence of highly polymorphic HLA, the chance of renal transplant recipients accept a HLA-A, B, DR&DQ full match kidney is very low. In this study, truly reflects choice by transplant physicians that kidney transplant donor/recipients of HLA match rate is (3.01±1.48) points.
It confirmed that HLA match rate had a significantly effect on short-term of primary renal transplantation, the higher rate of HLA match between donor and recipient, the lower the probability of postoperative rejection, while rate of HLA match was over3point, AR rate would significantly decreased. In order to get a good short-term effect of renal transplantation, and improve the long-term graft survival, we believe that primary ranal transplant recipients should choose a kidney that HLA-matches>3point and avoid to choose a kidney that HLA-matches0to1point.
Chapter3Application of calculated PRA (CPRA) methods to analyze the probability of access to transplant in kidney transplantation recipients
Objective
To establish an calculated PRA (CPRA) method to analysis the incompatible rate to donors with PRA-positive patients, and to analyze the probability of access to transplant in kidney transplantation recipients.
Methods
Two thousand and four cases of donor that transplant in Nanfang Hospital and NO.458Hospital of People's Liberation Army were collected from January2000to December2012, which already had complete HLA genotyping data of both donor and recipient.202cases of PRA positive patients (PRA peak strength) who were waiting kidney transplant in Nanfang Hospital and NO.458Hospital of People's Liberation Army from January2008to December2012were collected. PCR-SSP method was used to perform for HLA-A, B, DR typing in2004cases of donors then establish an donor HLA database by excel. Arlequin software(3.11) was used to calculate the frequency of single HLA-A,B,DR gene and HLA-A-B, A-DR, B-DR and A-B-DR haplotype. Based on2,004cases of transplant donors' HLA data to establish donor HLA-A, B, DR database, and set up, to calculator CPRA expectation value percentage to represent an unacceptable risk of donor HLA once the HLA-specific antibodies of PRA-positive recipients was typed in this software. By using ELISAto test202cases of PRA positive recipients for HLA antibodies in serum, and analyzed PRA strength(%) and HLA antibody specificity. The PRA-specific antibodies of202cases as unacceptable HLA genes used CPRA-Java calculator software to calculate its corresponding CPRA value. According to the degree of PRA sensitization, compared the difference between low sensitized (PRA>0～10%), moderate sensitization(PRA>10-30%) or highly sensitized (PRA>30%) with their corresponding CPRA value, the the distribution of CPRA value(>0-10%,>10～30%and>30%) in low, medium and high sensitized groups. Compared the intensity value between CPRA and PRA. Independent samples t test was used to compare mean value of CPRA and PRA. Kappa parameter was used to compare the concordance of CPRA and PRA strength. SPSS13.0statistical software was uesd for statistical analysis. Both of them were two-sided test, test level a=0.05.
Result
Analyze HLA-A, B, DR expression in2004donors. Entered HLA-specific antibodies202cases of PRA positive recipients into CPRA-Java calculator, then the result as a percentage of CPRA that represents the expectation value of unacceptable HLA in donor group. Mean value of PRA strength in202positive recipients was (23.12±17.83)%, and mean value of CPRA was (46.07±23.30)%.Low sensitized (PRA>0-10%)30.69%of the recipients(62/202), which mean PRA value was (6.87%±2.41)%and mean CPRA value was (21.63±11.75)%, there were statistically significant differences between them(P<0.05). Moderate sensitized (10-30%) representing45.05%of recipients (91/202), which mean PRA value was (20.27±5.71)%and mean CPRA value was (50.56±16.86)%, there were statistically significant differences between them(P<0.05). Highly sensitized(>30%) representing24.26%of the recipients, which mean PRA value was (48.90±15.18)%and mean CPRA value was (68.64%±15.35)%, there were no statistically significant differences between them (P>0.05). Compared the difference mean CPRA value in three sensitization degree, there were significant difference between low sensitized group and moderate or highly sensitized group (P<0.05), but no significant difference between moderate sensitized group and highly sensitized group(P>0.05). Analyzed the difference of CPRA (>0～10%,>10～30%and>30%) distribution in PRA low, moderate and highly group, concordance rate of PRA value and CPRA value in Low sensitized group was19.35%(P<0.05),in moderately sensitized group was10.99%(P <0.05),highly sensitized group wsas100%(P>0.05). There were significant difference of concordance rate between PRA value and CPRA value(Kappa=0.09, P<0.05).
Conclusions
CPRA method was established in this study, by analyzing the frequency of HLA donor, to calculate the percentage of PRA positive recipient's unacceptable HLA gene combinations in the donor database, namely CPRA value. Studies have shown that the chance that sensitized transplant recipients actually get may be lower than PRA strength might shown. Studies have shown that the probability of access to transplant in sensitized recipients might lower than PRA%shows. CPRA was>30%while PRA>30%, either, that proved PRA>30%was a risk factor for kidney transplantation. Studies have shown that, PRA reflects renal transplant recipients himself of sensitized level, so that the reliable detection of HLA specificity antibody is extremely important. After that, CPRA provide evidence to respond the truthfully the probability of access to transplant in kidney transplantation recipients to assist clinicians to predict waiting time and to select transplant method for recipient.
引文
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