Susceptibility of lung transplants to preformed donor-specific HLA antibodies as detected by flow cytometry

BACKGROUND: Preformed anti-HLA antibodies are known to have the potential to induce early graft damage in organ transplant recipients. However, in lung transplant recipients, little information exists about the significance of preformed antibodies directed to either class I or class II HLA antigens. METHODS: A two-color flow cytometry cross-match was performed in 92 consecutive lung transplant recipients using serum obtained immediately before transplantation. The presence of preformed antibodies was correlated with the incidence of severe graft dysfunction manifested as pulmonary infiltrates and severe hypoxemia with onset in the first few hours after transplantation. RESULTS: Six patients (6.5%) had low-level anti-donor IgG antibodies detected by flow cytometry, four against T and two against B lymphocytes. Three patients (50%) developed severe graft dysfunction with pulmonary infiltrates and hypoxemia. Two patients responded to treatment, but the third, who had an antibody highly specific for HLA-DR11, died at 48 hr after transplant. Results of histopathologic studies in this patient are consistent with hyperacute rejection and support a pathogenic role of these antibodies. In contrast, of 86 (93.5%) cases with a negative flow cytometry cross-match, only 4 (5%) had severe but reversible early graft dysfunction with pulmonary infiltrates and hypoxemia, attributed to ischemia-reperfusion injury (P<0.005). CONCLUSIONS: Class II, and perhaps class I HLA antibodies at relatively low concentrations represent a risk factor for severe early pulmonary graft dysfunction, with the potential to progress to hyperacute rejection and death.     

Soluble HLA antigens, anti-HLA antibodies, and antiidiotypic antibodies in the circulation of renal transplant recipients

Chronic rejection represents the major threat to long-term survival of organ allografts. It is presumed that this form of rejection is mediated by antibodies against mismatched HLA antigens of the graft. The presence and specificity of anti-HLA-antibodies in posttransplantation sera are, however, difficult to document. We have explored the possibility that anti-HLA antibodies form immune complexes with soluble HLA antigens released from the injured graft and/or that they are blocked by antiidiotypic, anti-anti-HLA-antibodies. Our data demonstrate that the long-term survival of renal allografts is significantly lower in patients who develop anti-HLA-antibodies following transplantation than in patients who do not form antibodies. Following depletion of soluble HLA antigens by magnetic immunoaffinity, we could identify anti-HLA-antibodies in 57% of the sera obtained from patients undergoing chronic rejection of kidney allografts, compared with 41% prior to antigen depletion. In patients tolerating the graft for 4 years or more, the corresponding frequencies of antibody-positive sera was 2% and 5% prior and following depletion of HLA antigens. The presence of HLA antigen/anti-HLA-antibody immune complexes in patients' sera was positively associated with chronic humoral rejection (P less than 0.0001). Patients who tolerated the graft in spite of having developed antibodies against one of its mismatched HLA antigens show specific antiidiotypic (anti-anti-HLA-antibodies). Such antiidiotypic antibodies were not found in sera from patients with chronic rejection (P = 0.005). This indicates that antiidiotypic antibodies may delay the progression of chronic humoral rejection.     

The repercussions of implementing flow cytometry as a single HLA antibody screening technique in prospective renal transplant recipients

In an effort to replace the complement-dependent cytotoxicity test (CDC) with a more sensitive single technique we looked at flow cytometry as a possible replacement. The Flow PRA Bead technique (One Lambda) performed well in our laboratory. Although as expected this technique was more sensitive and specific than CDC, there remained 11 samples from eight patients which were flow negative, CDC positive. The results of various antibody identification tests on these samples prompted us to alter the positive selection criteria which we had been using on our routine screening with the Flow PRA Beads and persuaded us that the initial CDC result was correctly positive in nine of the 11 samples.     

Postoperative outcomes of lung transplant recipients with preformed donor-specific antibodies

OBJECTIVESFew studies have evaluated the outcomes of lung transplantation (LTx) in recipients with preformed donor-specific antibodies (DSAs). This study investigated the postoperative changes in preformed DSAs based on prospectively collected data of DSAs, and the influences of preformed DSAs on postoperative outcomes among LTx recipients. Open in a separate windowMETHODSBetween July 2010 and December 2019, 216 recipients underwent LTx (81 living-donor lobar lung transplants and 135 deceased-donor lung transplants). We reviewed 8 cases with preformed DSAs to determine postoperative changes in DSAs and compared postoperative outcomes between recipients with and without DSAs.RESULTSThe preoperative mean fluorescence intensity of preformed DSAs ranged from 1141 to 14 695. Two recipients experienced antibody-mediated rejection within 2 weeks after LTx. DSAs disappeared in 7 recipients; however, 1 recipient experienced the relapse of DSAs and died from chronic lung allograft syndrome (CLAD), whereas 1 recipient had persisting DSAs within the study period and died from CLAD. Neither overall survival (OS) nor CLAD-free survival was significantly different between recipients with and without DSAs (P = 0.26 and P = 0.17, respectively). However, both OS and CLAD-free survival were significantly lower in recipients with DSAs against HLA class II than in those without these antibodies {5-year OS: 25.0% [95% confidence interval (CI): 0.9–66.5%] vs 72.1% (95% CI: 63.8–78.9%), P = 0.030 and 5-year CLAD-free survival: 26.7% (95% CI: 1.0–68.6%) vs 73.7% (95% CI: 66.5–79.5%), P = 0.002}.CONCLUSIONSPrognosis in recipients experiencing the relapse of preformed DSAs and those with persisting DSAs may be poor. The recipients with anti-HLA class II preformed DSAs had a significantly worse prognosis.     

Detection of donor-specific anti-HLA antibodies with flow cytometry in eluates and sera from renal transplant recipients with chronic allograft nephropathy

BACKGROUND: Chronic allograft nephropathy (CAN), which remains the main cause of graft loss after kidney transplantation, is still poorly understood. Because anti-HLA antibodies may be involved in the pathogenesis of CAN, this study was performed to look for donor-specific antibodies (DSA) fixed onto renal transplants with CAN. METHODS: DSA were identified after elution with flow cytometric assay and/or flow cytometric crossmatches in 20 transplants removed after irreversible graft failure caused by CAN and in control samples from 2 transplants with relapsing glomerulopathy, 2 transplants lost after vascular thrombosis, and 4 normal kidneys. The results were compared with those obtained in the serum samples 1 year after grafting, at the time of transplantectomy, and within 2 months after transplantectomy. RESULTS: IgG anti-class I, anti-class II, or both DSA were identified in 70.6% of eluates versus 73.6% of posttransplantectomy serum samples (NS), 42.1% of 1-year postgrafting serum samples (P<0.05), and 31.6% of serum samples at the time of transplantectomy (P<0.05). Our data show a good correlation between the target of anti-HLA antibodies found in both eluates and posttransplantectomy serum samples, but the precise specificity of anti-HLA antibodies is more often assigned in posttransplantectomy serum samples than in eluates. This problem needs further evaluation. CONCLUSION: This study shows that testing for anti-HLA DSA in eluates from removed kidney transplants using flow cytometry can be achieved and is highly efficient. It already suggests that both anti-class I and anti-class II HLA antibodies can be involved in CAN. Further studies are now needed to evaluate the possibility of identifying such antibodies in the eluates of transplant biopsy specimens from recipients experiencing CAN.     

Assignment of C1q-binding HLA antibodies as unacceptable HLA antigens avoids positive CDC-crossmatches prior to transplantation of deceased donor organs

Soon, a virtual crossmatch shall replace the complement-dependent cytotoxicity (CDC) allocation crossmatch in the Eurotransplant region. To prevent positive CDC-crossmatches in the recipient centre, careful definition of unacceptable antigens is necessary. For highly sensitized patients, this is difficult by CDC alone. Assignment of all antibodies detected by sensitive assays, however, could prevent organ allocation. To assess the usefulness of the Luminex C1q-assay to prevent positive CDC-crossmatches, all CDC-crossmatches performed prior to deceased kidney transplantation in a 16-month-period were reviewed. Sera causing positive crossmatches were investigated by the C1q-assay. 31 out of 1432 crossmatches (2.2%) were positive. Sera involved in 26 positive crossmatches were available. C1q-binding donor-specific antibodies were detected in 19 sera (73.1%). The other sera were from recipients without any HLA antibodies detectable by CDC or common solid phase assays. Three patients had known Non-HLA antibodies causing positive CDC-results. Four crossmatches were only weak positive. Therefore, avoidance of donors with HLA antigens against whom C1q-binding antibodies were detected would have prevented all positive crossmatches due to HLA antibodies. Provided that all HLA specificities against which antibodies are detected by the Luminex C1q-assay are considered as unacceptable antigens, CDC-crossmatches prior to transplantation might safely be omitted in many patients. They should be maintained in highly immunized patients, however, for whom assignment of all C1q-positive antibodies as unacceptable antigens could lead to a significant delay or even prevention of transplantation.     

肾移植长期存活受者致敏状态的检测及其临床意义

目的 检测肾移植长期存活受者的致敏状态,并分析其与移植肾功能的关系。方法 在定期随访的肾移植受者中,选取存活３年以上的受者６６例,其中肾功能正常者４６例,慢性肾功能减退者２０例,用Ｌａｍｂｄａ Ａｎｔｉｇｅｎ Ｔｒａｙ（ＬＡＴ）酶联免疫试剂盒检测血清中的可溶性抗ＨＬＡ抗体,分析抗体的类型和特异性、致敏与肾功能及移植后时间的关系。结果 肾功能正常组轻度、中度、高度致敏分别为９例、０例、２例,肾功能减     

肾移植受者血清可溶性HLA-I类抗原的动态监测

目的　了解血清可溶性ＨＬＡＩ类抗原（ｓＨＬＡＩ） 与肾移植受者发生急性排斥反应及感染的关系。　方法　应用酶联免疫法动态监测３６ 例肾移植受者ｓＨＬＡＩ水平。　结果　尿毒症组ｓＨＬＡＩ水平为（２－９４±０－３４）μｇ／Ｌ,显著高于正常对照组（０－７６±０－３３）μｇ／Ｌ（Ｐ＜ ０－０５） 。移植后功能稳定组ｓＨＬＡ降至（０－６３±０－３１ ）μｇ／Ｌ,显著低于尿毒症组（ Ｐ＜０－０５）。ｓＨＬＡＩ在发生排斥反应前３ 天及感染后５～７ 天显著升高。　结论　ｓＨＬＡＩ可作为监测肾移植受者急性排斥反应和感染的参数。     

肾移植受者血清可溶性HLA—Ⅰ类抗原的动态监测

目的 了解血清可溶性ＨＬＡ－Ⅰ类抗原（ｓＨＬＡ－Ⅰ）与肾移植受发生急性排斥反应及感染的关系。方法 应用酶联免疫法动态监测３６例肾移植受ｓＨＬＡ－Ⅰ水平。结果尿毒症组ｓＨＬＡ－Ⅰ水平为（２．９４±０．３４）μｇ／Ｌ,显高于正常对照组（０．７６±０．３３）μｇ／Ｌ（Ｐ〈０．０５）。移植后功能稳定组ｓＨＬＡ降至（０．６３±０．３１）μｇ／Ｌ,显低于尿毒症组（Ｐ〈０．０５）。ｓＨＬＡ－Ⅰ在发生排斥     

Development of an antibody specific to major histocompatibility antigens detectable by flow cytometry after lung transplant is associated with bronchiolitis obliterans syndrome

BACKGROUND: Chronic allograft rejection manifested as bronchiolitis obliterans syndrome (BOS) is the leading cause of late death after lung transplantation. Although increasing evidence suggests an association between anti-human leukocyte antigens (HLA) antibodies and chronic rejection of kidney or heart allografts, the clinical significance of anti-HLA antibodies in lung recipients is less clear, especially in previously unsensitized recipients. The use of flow cytometry based panel reactive antibody (flow-PRA) provides a highly sensitive means to identify the development of de novo anti-HLA antibodies in lung recipients. METHODS: Flow-PRA testing was used to analyze the pre- and posttransplant sera in stable BOS free lung recipients who survived at least 6 months. Patients without prior sensitization as defined by a negative pretransplant flow-PRA were analyzed posttransplant for the presence of anti-HLA antibodies by flow-PRA. A proportional hazards model was used to determine the impact of anti-HLA antibody on BOS risk. RESULTS: Sera from 90 recipients at Duke University with negative pretransplant flow-PRA were tested by flow-PRA at various time points after transplant. Sera from 11% (10/90) of recipients were found to contain anti-HLA antibodies detectable by flow-PRA. Nine patients (90%) developed anti-HLA antibodies specific for donor antigens, and one patient developed anti-HLA class II antibodies, not specific to donor antigens. Among the nine patients with donor antigen specific antibodies, flow-PRA specificity analysis demonstrated eight were specific for class II antigens and one for class I antigens. In a multivariate model that controls for other BOS risk factors, a positive posttransplant flow-PRA was significantly associated with BOS grades 1,2, or 3 (hazard ratios [HR] 3.19; 95% confidence interval [CI]: 1.41-7.12, P=0.005) and BOS grade 2 or 3 (HR 4.08; 95% CI: 1.66-10.04, P=0.002). Four patients with de novo anti-HLA antibodies died during follow-up; all four had BOS. Among BOS patients, the presence of anti-HLA antibodies was associated with a significantly worse survival (P =0.05, log-rank test). CONCLUSIONS: Although uncommon, previously unsensitized lung transplant recipients can develop anti-HLA antibodies to donor class II antigens. The development of de novo anti-HLA antibodies significantly increases the risk for BOS, independent of other posttransplant events. Furthermore, de novo anti-HLA antibodies identify BOS patients with significantly worse survival. Additional studies are needed to determine if class II-directed anti-HLA antibodies contribute mechanistically to the chronic rejection process in lung recipients.     

Influence of panel-reactive antibodies on posttransplant outcomes in lung transplant recipients

BACKGROUND: Panel-reactive antibody (PRA) is used to estimate the degree of humoral sensitization in the recipient before transplantation. Although pretransplant sensitization is associated with increased complications in other solid organ transplant recipients, less is known about the outcome of sensitized lung transplant recipients. Therefore, we sought to determine the impact of elevated pretransplant PRA on clinical outcomes after lung transplantation. METHODS: The records of the first 200 lung transplant operations performed at Duke University Medical Center were reviewed. The outcomes of sensitized patients, PRA greater than 10% before transplantation (n = 18), were compared with the outcomes of nonsensitized patients. RESULTS: Sensitized patients experienced a significantly greater number of median ventilator days posttransplant (9 +/- 8) as compared with nonsensitized recipients (1 +/- 11; p = 0.0008). There were no significant differences between the number of episodes of acute rejection; however, there was a significantly increased incidence of bronchiolitis obliterans syndrome occurring in untreated sensitized recipients (56%) versus nonsensitized (23%; p = 0.044). In addition, there was a trend towards decreased survival in the sensitized recipients, with a 2-year survival of 58% in sensitized recipients as compared with 73% in the nonsensitized patients (p = 0.31). CONCLUSIONS: Sensitized lung transplant recipients experience more acute and chronic complications after transplantation. These patients probably warrant alternative management strategies.     

A retrospective flow cytometric crossmatch study in transplant recipients with autoreactive lymphocytotoxic antibodies

Abstract Our previous data shows renal transplant recipients with autoreactive lymphocytotoxic antibodies to have a reduced transplant survival when compared to patients without autoantibodies. This could have been due to the presence of weak IgG antibodies inhibited by the dithiothreitol used to remove IgM antibodies in the pretransplant cytotoxicity cross-match. That possibility was investigated in a retrospective study of 52 recipients of 57 renal transplants who were recrossmatched using a more sensitive flow cytometry crossmatch (FCXM) to detect recipient IgG antibodies to donor T and/or B cell splenic lymphocytes. Fourteen of the 57 (24%) transplants failed. Six losses were within the 1st month posttransplant and four of these were immunological failures. None of the transplant failures had a positive pretransplant FCXM. These results showed that the recipients with autoantibodies did not have pretransplant IgG anti-donor antibodies. The transplant failures did not, therefore, relate to the presence of antibodies undetected by the dithiothreitol-treated cytotoxicity crossmatch.     

单中心肾移植受者HLA抗原分布频率分析

目的分析肾移植受者HLA-A、B、DR和DQ抗原分布频率。 方法收集2017年1月至2018年6月在解放军总医院第八医学中心接受肾移植的791例受者HLA抗原检测数据,分析HLA-A、B、DR和DQ抗原种类和分布频率。 结果HLA-A抗原共出现18种,其中A2、A11和A24(9)抗原分布频率较高,分别为30.59%、17.83%和15.68%;A2403、A34(10)和A66(10)抗原分布频率最低,均为0.06%。HLA-B抗原共出现42种,其中分布频率最高的是B13(12.64%),分布频率最低的是B44、B41和40∶40(均为0.06%)。HLA-DR抗原共出现16种,其中DR15(2)、DR4和DR12(5)分布频率较高,分别为14.60%、13.65%和13.40%;DR6分布频率最低(0.19%);DR15(2)与DQ6(1)具有强连锁反应(90.48%)。HLA-DQ抗原共出现7种,DQ7(3)抗原分布频率最高(24.91%),DQ8(3)分布频率最低(5.50%)。 结论肾移植受者HLA-A、B、DR和DQ抗原中,A2、B13、DR15(2)和DQ7(3)分别为分布频率最高的抗原。分析HLA抗原分布频率有利于寻找HLA匹配的供者。