Caner Süsal

Consensus guidelines on the testing and clinical management issues associated with HLA and non-HLA antibodies in transplantation.

Background The introduction of solid-phase immunoassay (SPI) technology for the detection and characterization of human leukocyte antigen (HLA) antibodies in transplantation while providing greater sensitivity than was obtainable by complement-dependent lymphocytotoxicity (CDC) assays has resulted in a new paradigm with respect to the interpretation of donor-specific antibodies (DSA). Although the SPI assay performed on the Luminex instrument (hereafter referred to as the Luminex assay), in particular, has permitted the detection of antibodies not detectable by CDC, the clinical significance of these antibodies is incompletely understood. Nevertheless, the detection of these antibodies has led to changes in the clinical management of sensitized patients. In addition, SPI testing raises technical issues that require resolution and careful consideration when interpreting antibody results. Methods With this background, The Transplantation Society convened a group of laboratory and clinical experts in the field of transplantation to prepare a consensus report and make recommendations on the use of this new technology based on both published evidence and expert opinion. Three working groups were formed to address (a) the technical issues with respect to the use of this technology, (b) the interpretation of pretransplantation antibody testing in the context of various clinical settings and organ transplant types (kidney, heart, lung, liver, pancreas, intestinal, and islet cells), and (c) the application of antibody testing in the posttransplantation setting. The three groups were established in November 2011 and convened for a “Consensus Conference on Antibodies in Transplantation” in Rome, Italy, in May 2012. The deliberations of the three groups meeting independently and then together are the bases for this report. Results A comprehensive list of recommendations was prepared by each group. A summary of the key recommendations follows. Technical Group: (a) SPI must be used for the detection of pretransplantation HLA antibodies in solid organ transplant recipients and, in particular, the use of the single-antigen bead assay to detect antibodies to HLA loci, such as Cw, DQA, DPA, and DPB, which are not readily detected by other methods. (b) The use of SPI for antibody detection should be supplemented with cell-based assays to examine the correlations between the two types of assays and to establish the likelihood of a positive crossmatch (XM). (c) There must be an awareness of the technical factors that can influence the results and their clinical interpretation when using the Luminex bead technology, such as variation in antigen density and the presence of denatured antigen on the beads. Pretransplantation Group: (a) Risk categories should be established based on the antibody and the XM results obtained. (b) DSA detected by CDC and a positive XM should be avoided due to their strong association with antibody-mediated rejection and graft loss. (c) A renal transplantation can be performed in the absence of a prospective XM if single-antigen bead screening for antibodies to all class I and II HLA loci is negative. This decision, however, needs to be taken in agreement with local clinical programs and the relevant regulatory bodies. (d) The presence of DSA HLA antibodies should be avoided in heart and lung transplantation and considered a risk factor for liver, intestinal, and islet cell transplantation. Posttransplantation Group: (a) High-risk patients (i.e., desensitized or DSA positive/XM negative) should be monitored by measurement of DSA and protocol biopsies in the first 3 months after transplantation. (b) Intermediate-risk patients (history of DSA but currently negative) should be monitored for DSA within the first month. If DSA is present, a biopsy should be performed. (c) Low-risk patients (nonsensitized first transplantation) should be screened for DSA at least once 3 to 12 months after transplantation. If DSA is detected, a biopsy should be performed. In all three categories, the recommendations for subsequent treatment are based on the biopsy results. Conclusions A comprehensive list of recommendations is provided covering the technical and pretransplantation and posttransplantation monitoring of HLA antibodies in solid organ transplantation. The recommendations are intended to provide state-of-the-art guidance in the use and clinical application of recently developed methods for HLA antibody detection when used in conjunction with traditional methods.

Identification of Highly Responsive Kidney Transplant Recipients Using Pretransplant Soluble CD30

The identification of high immunologic responders is desirable for the selection of appropriate immunosuppressive regimens. With the collaboration of 29 transplant centers in 15 countries, we investigated whether the pretransplant serum content of soluble CD30 (sCD30), a marker for the activation state of Th2-type cytokine producing T cells, is a useful predictor of kidney graft outcome. Pretransplant sera of 3899 cadaver kidney recipients were tested for serum sCD30 concentration using a commercially available enzyme-linked immunosorbent assay kit. Subsequent kidney graft survival was analyzed. The 5-yr graft survival rate in 901 recipients with a high pretransplant serum sCD30 (> or =100 U/ml) was 64 +/- 2%, significantly lower than the 75 +/- 1% rate in 2998 recipients with low sCD30 (<100 U/ml) (P < 0.0001). High sCD30 was associated primarily with graft loss and not with patient death. The sCD30 effect on graft survival was evident in first transplants as well as in retransplants, in presensitized patients with lymphocytotoxic antibodies as well as in nonsensitized patients, and in patients who received HLA well-matched kidneys as well as in patients who received poorly matched grafts. Recipients with a high pretransplant sCD30 needed significantly more rejection treatment after the first posttransplant year and continued to lose grafts at a higher rate during the 5-yr follow-up period, indicating that pretransplant sCD30 predicts not only the risk of acute rejection but also of chronic allograft nephropathy.

Kidney graft failure and presensitization against HLA class I and class II antigens

Background. It is well known that kidney transplant recipients with preformed lymphocytotoxic antibodies against HLA antigens have an increased graft rejection rate. However, the individual contribution of anti-HLA class I and class II antibodies to this phenomenon is poorly understood. We investigated the clinical relevance of preformed anti-HLA class I and class II antibodies on graft outcome in more than 4000 kidney recipients. Methods. Pretransplant sera of 4136 cadaver kidney recipients from 28 transplant centers were tested in ELISA for IgG-anti-HLA class I and IgG-anti-HLA class II antibodies. The influence of antibody reactivity on graft survival was analyzed. Results. Four hundred eighty of the anti-HLA class I antibody-positive recipients had a graft survival rate at 2 years of 77±2%, compared with an 84±1% rate in 3656 anti-HLA class I antibody-negative recipients (P <0.0001), and 770 anti-HLA class II-positive recipients had a graft survival rate of 79±2%, compared with an 84±1% rate in 3366 anti-HLA class II-negative patients (P <0.0001). Importantly, good 2-year graft survival rates of 85±3% and 84±2%, respectively, were observed in 206 anti-HLA class I-positive/class II-negative and 496 anti-HLA, class I-negative/class II-positive recipients. In contrast, the 274 recipients positive for both types of antibodies showed a poor graft survival rate of 71±3% (P <0.0001). Among 853 patients who received a well-matched kidney (0 or 1 HLA-A+B+DR mismatch), sensitization against either class I or class II, or both, had no deleterious effect. However, in 113 class I and class II antibody-positive patients who received a kidney with ≥3 HLA-A+B+DR mismatches, the 2-year graft survival rate was only 60±5%. Conclusion. Presensitization of first kidney transplant recipients against either HLA class I or class II is of no clinical consequence, whereas sensitization against both HLA class I and class II results in increased rejection of HLA mismatched grafts.

Serial peripheral blood perforin and granzyme B gene expression measurements for prediction of acute rejection in kidney graft recipients.

In the present study we investigated whether peripheral blood gene expression measurements may serve as an early and non‐invasive tool to predict renal allograft rejection. Peripheral blood was collected twice weekly after transplantation and gene expression was measured using real‐time polymerase chain reaction (PCR). Recipients with acute rejection (n = 17) had higher levels of perforin and granzyme B transcript on days 5–7, 8–10, 11–13, 17–19, 20–22, and 26–29, as compared to patients without rejection (n = 50, p < 0.05 in all cases). Rejection diagnosis using gene expression criteria, determined with receiver operating characteristic (ROC) curves, was possible 2–30 days before traditional diagnosis (median 11 days). The best diagnostic result was obtained from samples taken on days 8–10, with a specificity of 90% and a sensitivity of 82% for perforin, and a specificity of 87% and sensitivity of 72% for granzyme B. Decreases in perforin (p < 0.01) and granzyme B expression (p < 0.05) were observed after initiation of anti‐rejection therapy. Our data indicate that gene expression measurement is a useful tool for the recognition of graft rejection in its earliest stages. Serial measurements could be implemented as a monitoring system to highlight patients at higher risk of rejection, making them candidates for biopsy or pre‐emptive anti‐rejection therapy.

Soluble CD30 as a predictor of kidney graft outcome.

Background. In the present study, we investigated whether the soluble form of CD30 (sCD30), a marker for T helper 2‐type cytokine‐producing T cells, is increased in sera of potential kidney graft recipients. We also investigated whether the pretransplantation serum sCD30 content is related to kidney graft survival. Methods. Pretransplantation sera of 844 cadaver kidney recipients from three transplant centers in Germany were tested for serum sCD30 content using a commercially available ELISA kit. Results. Kidney graft recipients showed a significantly higher serum sCD30 content than healthy controls (P<0.0001). High sCD30 serum content was associated with graft rejection. The 2‐year graft survival rate in recipients with a high pretransplantation serum sCD30 was 68±6%, significantly lower than the 86±1% rate in recipients with a low sCD30 (P<0.0001). Importantly, high sCD30 was indicative of an increased risk of graft loss even in recipients without lymphocytotoxic alloantibodies. Conclusion. These data show that an elevated pretransplantation serum sCD30 reflects an immune state that is detrimental for kidney graft survival.

No Association of Kidney Graft Loss With Human Leukocyte Antigen Antibodies Detected Exclusively by Sensitive Luminex Single-Antigen Testing: A Collaborative Transplant Study Report

Background. It is unclear whether kidney transplant recipients with preformed donor-specific human leukocyte antigen (HLA) antibodies (DSA) detectable only in the highly sensitive Luminex single-antigen (LSA) assay are at an increased risk of graft failure. Methods. We studied 3148 patients who received a deceased donor kidney graft between 1996 and 2008 and were enrolled in the prospective serum project of the Collaborative Transplant Study. There were 118 patients with graft loss during the first 3 years after transplantation on whom recipient and donor DNA was available for complete HLA typing. We compared the incidence of LSA-detected DSA in these patients with graft failure and matched controls with functioning grafts. All patients were found negative in the less-sensitive complement-dependent lymphocytotoxicity and enzyme-linked immunosorbent assays. Results. When mean fluorescence intensity (MFI) of greater than or equal to 1000 was used as a cutoff for Luminex positivity, 118 patients with graft loss did not show a higher incidence of DSA against HLA-A, -B, -C, -DRB1/3/4/5, -DQA1, -DQB1, -DPA1, or -DPB1 antigens than 118 matched controls without graft loss (for all loci P not significant). The incidence of strong DSA (MFI ≥2000 or MFI ≥3000) detected only by LSA was low (for all loci between 0% and 5%) and did not identify unacceptable antigens that were relevant for graft loss within the first 3 years after transplantation. Conclusion. We conclude that, given currently practiced crossmatch procedures and immunosuppressive regimens, exclusion of donor organs carrying “unacceptable” HLA based exclusively on sensitive LSA antibody testing is not justified.

Evaluation of posttransplantation soluble CD30 for diagnosis of acute renal allograft rejection1

Background. Posttransplantation measurement of soluble CD30 (sCD30) may be useful for identifying kidney graft recipients at risk of impending graft rejection in the early posttransplantation period. Methods. We measured plasma sCD30 levels and evaluated the levels in relation to the diagnosis of rejection. Results. Receiver operating characteristic curves demonstrated that on posttransplantation days 3 to 5, sCD30 allowed a differentiation of recipients who subsequently developed acute allograft rejection (n=25) from recipients with an uncomplicated course (n=20, P <0.0001) (area under the receiver operating characteristic curve 0.96, specificity 100%, sensitivity 88%) and recipients with acute tubular necrosis in the absence of rejection (n=11, P =0.001) (area under the receiver operating characteristic curve 0.85, specificity 91%, sensitivity 72%). Conclusions. sCD30 measured on posttransplantation days 3 to 5 offers a noninvasive means for differentiating patients with impending acute allograft rejection from patients with an uncomplicated course or with acute tubular necrosis.

Post‐Transplant sCD30 and Neopterin as Predictors of Chronic Allograft Nephropathy: Impact of Different Immunosuppressive Regimens

Immunological monitoring for chronic allograft nephropathy (CAN) is of great potential interest. We assessed serum soluble CD30 (sCD30) together with in vitro Th2‐type responses (IL‐4, IL‐10, CD4 helper activity) and neopterin in a prospective study of 84 renal transplant recipients with 2‐year follow‐up. Patients were randomized to CsA/Aza, CsA/MMF and Tacr/Aza, respectively, to analyze the effect of immunosuppression on posttransplant sCD30 and neopterin. ATG induction and acute rejections did not alter sCD30 levels whereas CMV disease was associated with transient upregulation of sCD30 (p = 0.003 at 4 months) and sustained upregulation of neopterin (corrected for graft function (Neo/CR) p = 0.005 at 2 years). Tacr versus CsA treatment proved to be an independent variable associated with downregulation of 1‐year sCD30, which was positively related to Neo/CR (p = 0.007 and 0.01, respectively; logistic regression). Importantly, increased 1‐year sCD30 and Neo/CR were associated with decreased glomerular filtration rate at 2 years (p = 0.02 and p < 0.0005, respectively) and evidence of CAN (p < 0.0005). High 1‐year sCD30 could not be attributed to enhanced Th2‐type responses and was not associated with HLA antibody formation. Our data suggest that elevated sCD30 and neopterin predict graft deterioration by CAN. Tacr effectively downregulates these responses and might be of advantage in patients with elevated sCD30 or neopterin.

Current role of human leukocyte antigen matching in kidney transplantation.

Purpose of review With graft survival rates steadily improving during the recent years, there is debate whether donor kidneys should still be allocated according to compatibility for human leukocyte antigens (HLA). Recent findings Recent studies argue for continued kidney exchange efforts for achieving better HLA compatibility. In this modern era of immunosuppression, better HLA matching is associated not only with better graft survival, but also with the administration of lower dosages of immunosuppressive agents, a lower incidence of side-effects of immunosuppression such as non-Hodgkin lymphoma, hip fractures, and death from infection, and a lower grade of sensitization if a patient has lost a kidney graft and is relisted for a retransplant. Summary Despite the overall improved graft survival rates in the recent years, the data continue to support organ sharing based on HLA matching in kidney transplantation.

Presensitized kidney graft recipients with HLA class I and II antibodies are at increased risk for graft failure: A Collaborative Transplant Study report

We have previously reported that kidney transplant recipients with enzyme-linked immunoabsorbent assay-reactive human leukocyte antigen (HLA) class I and II antibodies are at an increased risk for graft failure. To determine whether positivity for both classes of HLA antibodies before transplantation can serve as an indicator for identification of high-risk patients, the impact of preformed HLA antibodies on graft survival was analyzed in a new series of 5315 kidney transplantations performed between 2000 and 2008. In line with our previous findings, 121 first transplant recipients positive for both HLA class I and II antibodies by enzyme-linked immunoabsorbent assay had a poor 2-year graft survival rate of 76.5% +/- 4.0%, compared with a rate of 87.5% +/- 0.5% in 4175 recipients who were negative for both antibody classes (log-rank p < 0.001). Good survival rates of 82.7% +/- 3.2% and 86.1% +/- 2.7%, respectively, were observed in 149 HLA class I-positive/class II-negative recipients and in 183 HLA class I-negative/class II-positive recipients. Importantly, graft survival was good in HLA class I-positive and class II-positive patients when they received a kidney with a 0-1 HLA-A+B+DR mismatch. These data confirm our previous observation that kidney transplant recipients who simultaneously possess HLA class I and II antibodies in their pretransplantation serum are at increased risk for graft failure and require special attention.