Abstract
BACKGROUND: Panel reactive antibodies (PRA) easily appear in the peripheral blood of organ transplant recipients sensitized by allogeneic human leukocyte antigen (HLA). How to enhance the success rate of renal transplantation and long-term survival rate of renal allografts in sensitized recipients should be further studied.
OBJECTIVE: This study was to detecthuman leukocyte antigen immunoglobulin G(HLA-IgG) antibody level and its specificity in renal transplant recipients, evaluate humoral immunity sensitization, and investigate the relationship of the acceptable mismatching of HLA cross-reactive group and survival rate of renal allograft.
DESIGN: A clinical observation.
SETTING: Zhujiang Hospital Affiliated to Southern Medical University.
PARTICIPANTS: A total of 1297 patients, 824 males and 473 females, averaging (42±16) years of age, received renal transplantation in the Department of Organ Transplantation, Zhujiang Hospital, Southern Medical University between January 1998 and December 2005, were recruited for this study. Among these patients, 165 were HLA-IgG antibody-positive recipients, 1132 were HLA-IgG antibody-negative ones, 1217 received renal transplantation for the first time, 77 received renal transplantation twice, 2 three times, and 1 four times. Written informed consent was obtained from each subject for related laboratory measurements and treatment. The protocol was approved by the Hospital's Ethics Committee. Reagents: Lambda antigen tray (LAT), Lambda antigen tray mixed (LATM), Special Monocloneal Tray-Asian HLA Class I, and Micro SSP? Generic HLA Class II were purchased from One Lambda Company, USA. Taq polymerase was purchased from PE Company, USA. DNA extract reagent was from Qiagen Company, Germany. Anti-human complement 4d (C4d) polyclonal antibody and chromogenic substrate DAB were purchased from Biomedica Company, Austria.
METHODS: Prior to operation, serum HLA-IgG antibody in the recipients was determined by an enzyme linked immunosorbent assay (ELISA). HLA-IgG antibody-positive serum was further detected by antigen tray (LAT1240 and LAT1HDS) for antibody-positive rate and specificity. HLA genotyping was performed by a sequence specific primer polymerase chain reaction (PCR-SSP). For 40 recipients who had elevated serum creatinine (Scr), anti-HLA antibody detection and renal transplant needle biopsy were conducted. At the same time, C4d deposition on the capillary wall around the renal tubule was observed by immunohistochemical staining. Survival rate of renal allografts in recipients 1, 3, and 5 years after transplantation, and relationships of gender and renal transplantation and antibody-positive rate were investigated. Survival rate of renal allograft in recipients that received different mismatch of HLA cross-reactive group was analyzed.
MAIN OUTCOME MEASURES: Prior to and after renal transplantation, HLA-IgG antibody-positive rate and HLA genotyping in renal transplant recipients. Characterization of C4d deposition on the capillary wall around the renal tubule in the renal transplant biopsy tissue. Difference of survival rate of renal allograft.
RESULTS: All 1297 recipients were included in the final analysis. Among them, 1132 were HLA-IgG antibody-negative recipients, 165 were HLA-IgG antibody-positive ones, 126 were anti-HLA class I IgG antibody-positive ones, 90 were anti-HLA class II IgG antibody-positive ones, 51 were anti-HLA class I and II IgG antibody-positive ones, and 94 were highly sensitized ones (antibody-positive rate >50%). Among 40 recipients with needle biopsy, C4d deposition was found in the 13 recipients, but not found in the 27 recipients. Ten out of thirteen C4d-positive recipients presented with anti-HLA antibody-positive in the peripheral circulation. The incidence for delayed graft function (DGF) was significantly higher in recipients with HLA-IgG antibody-positive than in recipients with HLA-IgG antibody-negative (P < 0.01). There was no significant difference in the survival rates of renal allografts between recipients with HLA-IgG antibody-positive and with HLA-IgG antibody-negative 1, 3, and 5 years after renal transplantation (P > 0.05). Antibody-positive rate was significantly higher in female recipients than in male recipients (P < 0.01). Antibody-positive rate was significantly higher in recipients that received renal transplantation for the second time than in recipients that received renal transplantation for the first time (P < 0.01). With HLA cross-reactive group mismatching increasing, survival rate of renal allograft presented a tendency of decline. One, three and five years after renal transplantation, the survival rate of renal allograft was respectively 97%, 94%, and 92% for recipients with no mismatching, and 91%, 82%, and 77% for recipients with two mismatches, which was respectively decreased by 6%, 12%, and 15% compared to recipients that received no mismatching. For recipients with three mismatches, the survival rate of renal allograft was respectively decreased by 9%, 15%, and 24% compared to recipients with no mismatching.
CONCLUSION: C4d deposition on the capillary wall around the renal tubule can be detected as an indicator of antibody-mediated humoral rejection. A good HLA matching can noticeably decrease the incidence of rejection and improve the survival of renal allograft.

INTRODUCTION

In the past few decades, studies of transplant rejection mainly focus on cellular rejection. Cyclosporin (CsA), tacrolimus (FK 506), mycophenolate mofefil (MMF) and other immunodepressants, and antilymphocytic globulin (ALG), antithymocyte globulin (ATG), OKT3 and other biological agents are developed and used for the prevention and treatment of cellular rejection [1-3]. With the development of transplant immunology and experimental technique, more and more studies have demonstrated that antibody-mediated humoral rejection is very important.for transplant injury [1,4]. Panel reactive antibody (PRA) easily appears in the peripheral blood of organ transplant recipients sensitized by allogeneic HLA. Anti-HLA IgG antibody is a key factor for hyperacute rejection, and it is also closely related to acute rejection, chronic rejection and transplant function delay [1]. The existence of anti-HLA antibody in the peripheral blood circulation and C4d deposition on the capillary wall around the renal tubule in the renal transplant biopsy tissue are the main evidence for the diagnosis of antibody-mediated humoral rejection. Clinically, C4d has been used as an important indicator for humoral rejection [5-7].
This study monitored HLA-IgG antibody levels and specificity in transplant recipients and evaluated humoral immunity sensitization to match sensitized patients according to the principle of match of HLA cross-reactive group and acceptable HLA mismatching formulated by United Network for Organ Sharing (UNOS) and in conjunction with the distribution characteristics of HLA for Chinese Han population. This study was in attempt to be away from the antigen corresponding to specific antibody in recipient to effectively avoid hyperacute rejection and protect transplant function.

SUBJECTS AND METHODS

Subjects
A total of 1297 patients, 824 males and 473 females, averaging (42±16) years of age, received renal transplantation in the Department of Organ Transplantation, Zhujiang Hospital, Southern Medical University between January 1998 and December 2005 and, were recruited for this study. Among these patients, 165 (48 males and 117 females) were HLA-IgG antibody-positive recipients, 1132 (776 males and 356 females) were HLA-IgG antibody-negative ones, 1217 received renal transplantation for the first time, 77 received renal transplantation twice, 2 three times, and 1 four times. Written informed consent was obtained from each subject for related laboratory measurements and treatment. The protocol was approved by the hospital's Ethics Committee.

Methods
Detection of HLA-IgG
Reagents: Lambda antigen tray (LAT) and Lambda antigen tray mixed (LATM) were purchased from One Lambda Company, USA. Prior to and after renal transplantation, the changes of HLA-IgG antibody level and specificity in the peripheral blood of renal transplant recipients were monitored by an enzyme linked immunosorbent assay (ELISA)[8].
HLA genotyping
Reagents: HLA-I monoclonal antibody tray (SMT72R)and HLA-Ⅱ DNA typing reagent were purchased from One Lambda Company, USA. Taq polymerase was purchased from PE Company, USA. DNA extract reagent was from Qiagen Company, Germany. According to previous report, HLA genotyping was performed [9].

Detection of C4d in the renal transplant needle biopsy tissue
Reagent: Anti-human C4d polyclonal antibody and chromogenic substrate DAB were purchased from Biomedica Company, Austria. The C4d deposition was detected by immunohistochemical staining. The paraffin sections of renal transplant needle biopsy tissue were dewaxed, then repaired with citric acid antigen retrieval buffers, digested with endogenous peroxydase and trypsin, and terminated by hydrogen dioxide. Next, they were incubated with anti-human C4d multiclonal antibody in the moist tray at 4 ℃, overnight, washed with phosphate buffer solution (PBS), developed with chromogenic substrate DAB, dehydrated and mounted. Finally, they were observed through a microscope.

Donor-recipient HLA matching
According to HLA class I cross-reactive group matching criteria and HLA class II acceptable mismatch principle, donor-recipient HLA matching was performed using a rapid HLA matching software developed automatically.

Statistical analysis
Frequency distribution analysis and survival analysis were conducted by the second author using SPSS (version 13.0). Chi-square test was used. A value of P < 0.05 was considered significant.

RESULTS

Quantitative analysis of participants
All 1297 patients were included in the final analysis.

Detection results of HLA-IgG antibody in PRA positive recipients
Among 165 PRA positive recipients, 126 had anti-HLA class I IgG antibody, with an average positive rate of (50.3±27.2)% (range 11%-100%) and 90 had anti-HLA class II IgG antibody, with an average positive rate of (65.1±30.1)% (range 17%-100%) (Table 1).

Serum epitope analysis was performed in 126 anti-HLA class I IgG antibody-positive recipients. Results showed that anti-public epitope antibodies were not found in 14 recipients whose PRA was less than 20%, but found in 9(17.3%) out of 52 recipients whose PRA was between 20%-50% and in 49 (81.7%) out of 60 recipients whose PRA was greater than 50% (Table 2).

Effects of renal transplantation and gender on HLA-IgG antibody
The positive rate of HLA-IgG antibody in the peripheral blood was 11.3% for the recipients that received renal transplantation for the first time, and 33.8% for the recipients that received renal transplantation for the second time. There was a significant difference between the two groups (P < 0.01). The positive rate of HLA-IgG antibody in the peripheral blood was higher in female recipients than in male recipients (24.7% vs. 5.8%, P < 0.01) (Table 3).

Effects of HLA-IgG antibody on acute rejection, DGF and survival rate of renal allograft
For HLA-IgG antibody-positive recipients, the incidence of acute rejection was 15.2%, and survival rate of renal allograft was 95%, 88%, and 80% 1, 3 and 5 years after renal transplantation, respectively. For HLA-IgG antibody-negative recipients, the incidence of acute rejection was 12.9%, and survival rate of renal allograft was 97%, 89%, and 85% 1, 3 and 5 years after renal transplantation, respectively. There was no statistical difference between the two groups (P > 0.05). DGF incidence was higher in HLA-IgG antibody-positive recipients than in HLA-IgG antibody-negative ones (33.9% vs. 11.9%, P < 0.01).

Correlation of anti-HLA antibody to C4d deposition on the capillary wall around the renal tubule
C4d deposition was found in 10 (52.6%) out of 19 PRA positive recipients and in 3 (14.3%) out of 21 PRA negative recipients. There was a significant difference between the two groups (P < 0.01). It indicates that C4d deposition is closely related to PRA in the peripheral blood circulation during renal transplantation.

Effect of HLA matching on survival rate of renal allograft
With mismatch of HLA cross-reactive group increasing, survival rate of renal allograft presented a tendency of decline. One, three and five years after renal transplantation, the survival rate of renal allograft was respectively 97%, 94%, and 92% for recipients with no mismatch, and 91%, 82%, and 77% for recipients with two mismatches, which was respectively decreased by 6%, 12%, and 15% compared to recipients with no mismatching. For recipients with three mismatches, the survival rate of renal allograft was respectively decreased by 9%, 15%, and 24% compared to recipients with no mismatching (Figure 1).

DISCUSSION

Since professor Terasaki invented complement-dependent cytotoxicity (CDC) test, which is used for detecting anti-HLA antibody, in 1960s, people have performed considerable experimental and clinical studies on antibody-mediated humoral rejection, and obtained plentiful outcomes. According to the theory of transplant rejection humoral immunity, antibody is an important cause for allogeneic transplant rejection, mainly presented in the following aspects: ① HLA-IgG antibody causes the hyperacute rejection of renal allograft; ② HLA-IgG antibody causes C4d deposition on the capillary wall around the renal tubule and renal corpuscle; ③ HLA-IgG antibody is a good indicator for acute rejection of renal allograft; ④ Antibody exists in most of recipients with allograft rejection; ⑤ HLA-IgG antibody is closely related to the chronic rejection of kidney, heart, lung and liver allograft. During the peripheral circulation, HLA-IgG antibody often appears prior to renal allograft rejection and bronchiole inflammatory occlusion [1,10-12].
It is known to all that hyperacute rejection easily occurs during renal transplantation in HLA humoral immunity sensitized recipient and leads to the failure of transplantation, even threatens recipient's life.
Martin et al [13-14] reported that anti-exogenous HLA homologous antibody existing in PRA positive sensitized recipients was a high risk factor for renal transplantation. Thus, it was difficult to search for cross-match test negative donor, making recipients wait for longer time prior to transplantation. Moreover, the transplantation effects in PRA positive sensitized recipients were inferior to those in PRA negative sensitized recipients. Therefore, measurement of PRA has been included in the routine laboratory items for tissue matching of clinical organ transplantation.
Many clinicians have paid more attention to PRA monitoring than to HLA matching. Using CDC test, ELISA and flow cytometry, scholars monitored the PRA and donor specific antibody (DSA) pre-preserved in the peripheral blood circulation of recipients and scientifically evaluated humoral immunity sensitization of recipient prior to transplantation. Thus, anti-HLA antibodies specificity pre-preserved in the sensitized recipients were avoided, and hyperacute and other severe rejections could be also effectively avoided [4, 15-17].
In the present study, we identified and analyzed HLA-IgG antibody positive rate, specificity and epitope in the peripheral blood of 165 PRA positive recipients. Results demonstrated that anti-public epitope antibodies existed in the serum of 49 (81.7%) out of 60 highly sensitized recipients with positive rate of anti-HLA class I IgG antibody > 50%, and at the same time, specific antibodies corresponding to many epitopes existed in the serum of some individuals; however, anti-public epitope antibodies were not found in the 14 recipients with PRA < 20%; and among 52 recipients with PRA between 20%-50%, only 9 (17.3%) had anti-public epitope antibodies. It indicates that with the increase of humoral immunity sensitization, antibodies aiming at HLA antigen epitope increase uninterruptly. Because recipients are widely sensitized, so it is difficult to find cross- cytotoxicity test-negative donors for highly sensitized recipients.
In the present study, according to antigenic specificity in negative reactive well and recipient HLA phenotype in the PRA monitoring, we gave out " acceptable mismatch antigen window" of highly sensitized recipients or searched for acceptable mismatched antigen in the cross-reactive group, in which HLA antigen exists, in the highly sensitized recipients, and finally we had found HLA coincided and cross-matching negative donors for 165 PRA positive humoral immunity sensitized recipients (60 highly sensitized recipients with PRA > 50% included) and all successfully performed renal transplantation without hyperacute rejection.
In recent years, with rapidly increasing number of renal transplant recipients, more and more recipients need retransplantation due to allograft dysfunction. Most of these recipients were PRA positive, and retransplantation is more difficult. Therefore, how to boost the success rate of renal transplantation and long-term survival rate of renal allograft in highly sensitized recipients is a challenge for transplant surgeons.
Clinical experimental data have demonstrated that pregnancy, blood transfusion, blood platelet perfusion and organ transplantation all can lead to the production of HLA-IgG antibodies in the recipients [1,18-19].
Our results of HLA-IgG antibody level and specificity in renal transplant recipients showed that among 1297 renal transplant recipients, 165 (48 males and 117 females) were HLA-IgG antibody-positive, 1132 (776 males and 356 females) were HLA-IgG antibody-negative, and HLA-IgG antibody positive rate was higher in female recipients than in male ones (24.7% vs. 5.8%, P < 0.001).
Among 165 HLA-IgG antibody-positive recipients, 138 received renal transplantation for the first time, 27 (16.4%) for the second time; while among 1132 HLA-IgG antibody-negative recipients, 1079 received renal transplantation for the first time, and 53 (4.7%) for the second time. The positive rate of HLA-IgG antibody was significantly higher in recipients with renal transplantation for the second time than those receiving renal transplantation for the first time (33.8% vs. 11.3%, P < 0.001). Recently, with further understanding and study of HLA humoral immunity sensitization in renal transplant recipients, less recipients received blood transfusion prior to transplantation and female recipients are often sensitized by pregnancy and transplantation. Therefore, pregnancy and organ transplantation are two important causes for HLA humoral immunity sensitization in recipients.
Long-term follow-up findings of renal transplant recipients showed that DGF incidence was significantly higher in anti-HLA-IgG antibody-positive recipients than in anti-HLA-IgG antibody-negative ones. Effects of HLA-IgG antibody on transplant rejection and survival rate of renal allograft are closely related to donor-recipient HLA matching, i.e. a good HLA matching can completely avoid hyperacute rejection, noticeably decrease acute rejection incidence and enhance long-term survival rate of renal allograft. In the present study, acute rejection incidence and long-term survival rate of renal allograft in 165 HLA-IgG antibody-positive recipients were closer to those in HLA-IgG antibody-negative recipients, and no statistical difference existed between the two groups. This may be because of more strict tissue matching in PRA positive recipients, thus, donor-recipient HLA matching degree is much higher.
Cardarelli et al [7, 11-12] reported that donor specific anti-HLA antibody appearing in the peripheral blood circulation and C4d deposition in the renal transplant needle biopsy tissue are the important causes for acute humoral rejection, chronic rejection and allograft dysfunction. DSA directly aiming at donor mismatched HLA antigen leads to irreversible damage to transplant functions and decreases long-term survival rate of renal allograft.
Our findings also demonstrated that among 38 recipients, in which anti-HLA antibody appeared within 6-36 months after transplantation, 22 (57.9%) presented with functional damage of renal allograft and obviously increased serum creatinine level, 5 presented with complete functional loss of renal allograft and needed hemodialysis again. In 16 recipients, anti-HLA antibodies existed prior to transplantation and completely disappeared after transplantation, serum creatinine level always kept normal, and renal function damage was not found. In addition, by detecting HLA antibody and C4d deposition in 40 recipients who had abnormal functions of renal allograft, it was found that C4d deposition existed in 10 (52.6%) out of 19 recipients who had anti-HLA antibodies in the peripheral blood circulation, while it existed in only 3 (14.3%) out of 21 recipients who had no anti-HLA antibodies in the peripheral blood circulation. There was a significant difference between the two groups (P < 0.01). It indicates that C4d deposition in the renal allograft is closely related to PRA in the peripheral blood circulation. It can be used as a marker of antibody-mediated humoral rejection, and is an important factor for damaging allograft function and decreasing the survival rate of renal allograft[20].
With rapid development of transplantation immunology and tissue matching technique, more and more scholars have realized the significance of HLA matching in the organ transplantation [21]. Panigrahi et al [22-23] reported that there was a lower rejection incidence and a better survival rate of renal allograft in good HLA matching recipients than in poor matching ones. Our data demonstrated that donor-recipient HLA matching degree was closely related to renal allograft rejection and survival rate of renal allograft. With the number of mismatch of HLA-cross reactive group increasing, incidence of allograft rejection noticeably increased, while survival rate of renal allograft remarkably decreased. Compared to recipients with no mismatch, the survival rate of renal allograft was respectively decreased by 6%, 12%, and 15% in recipients with two mismatches and by 9%, 15%, and 24% in recipients with three mismatches one, three and five years after renal transplantation. Therefore, a good donor-recipient HLA matching is very significant for reducing rejections, and enhancing success rate of renal transplant and long-term survival rate of renal allograft.

REFERENCES

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肾移植受者人类白细胞抗原体液免疫致敏状态的监测及其临床意义

李留洋，刘占国，陈剑荣，钱 俊，孙尔维，赵明
南方医科大学附属珠江医院器官移植科，广东省广州市 510282
李留洋，男，1965年生，江西省宜春市人，汉族，1988年上海医科大学毕业，副主任技师，主要从事器官移植组织配型和移植免疫学研究。
摘要
背景：器官移植受者在受到同种抗原人类白细胞抗原（HLA）免疫致敏后，外周血中容易产生群体反应性抗体，如何提高致敏受者肾移植的成功率和移植物长期存活率值得深入研究。
目的：通过检测肾移植受者的HLA-IgG抗体水平及其特异性，评估受者体液免疫致敏状态，并观察HLA交叉反应组配型可接受性错配情况与移植肾存活率的关系。
设计：临床观察。
单位：南方医科大学附属珠江医院。
对象：选择1998-01/2005-12在南方医科大学附属珠江医院器官移植科接受肾移植1297例患者，男824例，女473例，平均（42±16）岁，其中HLA-IgG抗体阳性受者165例，阴性受者1 132例。初次肾移植受者1217例，2次移植77例，3次移植2例，4次移植1例。所有患者在接受本实验相关检测及治疗前均签署知情同意书，本实验经过医院伦理委员会批准许可。试剂：莱姆德抗原板和混合抗原板购自美国One Lambda公司。HLA-Ⅰ类单克隆抗体湿板和HLA-Ⅱ类DNA分型试剂购自美国One Lambda公司；Taq多聚酶购自美国PE公司；DNA抽提试剂购自德国QIAGEN公司。抗人C4d多克隆抗体和显色底物DAB购自奥地利Biomedica公司。
方法：①通过酶联免疫吸附试验筛查受者术前血清中的HLA-IgG抗体，对阳性血清进一步用抗原板（LAT1240和LAT1HDS）检测抗体阳性率及其特异性，采用序列特异性引物聚合酶链反应技术进行HLA基因分型。②对40例Scr升高的受者进行抗HLA抗体检测和移植肾穿刺活检，并通过免疫组织化学染色观察肾小管周围毛细血管壁上C4d的沉积。③移植受者术后1，3，5年移植肾存活率及性别、移植与抗体阳性率的关系，并分析不同HLA交叉反应组错配数受者移植肾存活率的差别。
主要观察指标：①肾移植患者手术前后HLA-IgG抗体阳性率及HLA分型。②移植肾活检组织中肾小管周围毛细血管壁上C4d的沉积特征。③移植肾存活率差别。
结果：患者1297例均进入结果分析。①HLA-IgG抗体阴性受者1132例，阳性受者165例，I类HLA-IgG抗体阳性受者126例，II类HLA-IgG抗体阳性受者90例，51例受者同时存在I类和II类HLA-IgG抗体，抗体阳性率 > 50％的高致敏受者94例。②40例移植肾穿刺活检标本中，13例患者观察到肾小管周围毛细血管壁上存在C4d的沉积，27例未见C4d沉积；13例C4d阳性受者中10例外周循环中抗HLA抗体阳性。③抗体阳性受者移植物功能延迟恢复的发生率显著高于阴性受者（P < 0.01）。抗体阳性受者的1，3及5年移植肾存活率与抗体阴性受者无明显学显著性差异（P > 0.05）。女性受者中抗体阳性率明显高于男性受者（P < 0.01），再次移植受者中抗体阳性率显著高于初次移植受者（P < 0.01）；随着HLA交叉反应组错配数的增加，移植肾存活率呈下降趋势，0错配受者的1，3，5年移植肾存活率分别为97%，94%和92%，2错配受者1，3，5年移植肾存活率分别为91%，82%和77%，3错配受者较0错配受者下降更为明显，分别为9%，15%和24%。
结论：肾小管周围毛细血管壁上C4d的沉积可作为抗体介导体液性排斥反应的客观指标，供受者间良好的HLA配型能显著降低排斥反应发生率和改善移植物存活。
关键词：组织配型；体液免疫；群体反应性抗体；肾移植
中图分类号: R617 文献标识码: A 文章编号: 1673-8225(2008)18-03561-06
李留洋，刘占国，陈剑荣，钱俊，孙尔维，赵明.肾移植受者人类白细胞抗原体液免疫致敏状态的监测及其临床意义[J].中国组织工程研究与临床康复，2008，12(18):3561-3566
[www.zglckf.com/zglckf/ejournal/upfiles/08-18/18k-3561(ps).pdf]
(Edited by Stefania Lorenzini/Song LP/Wang L)