Judith Worthington

Posttransplantation production of donor HLA-specific antibodies as a predictor of renal transplant outcome

Background. This study aimed to determine whether the production, in renal transplant recipients, of antibodies directed against donor HLA mismatches is predictive of transplant failure. Methods. The failure study group comprised 112 adult recipients of primary renal transplants who had re-entered the transplant waiting list after failure of the first graft. A control group of 123 recipients with functioning transplants was selected from transplantations performed during the same time period, in which patients had equivalent HLA matching and immunosuppression and a minimum of 5 years of follow-up. Sera taken before transplantation and at 1, 3, and 6 months and annually after transplantation were tested by enzyme-linked immunoabsorbent assay (ELISA) for the presence of HLA class I- and class II-specific antibodies. Antibody specificity was defined by a combination of cytotoxicity, ELISA, and flow cytometry techniques to determine whether the antibodies were directed against donor mismatches. Results. All recipients were negative for donor HLA-specific antibodies before transplantation. After transplantation, 57 (50.9%) of the 112 patients in the failure group produced donor HLA-specific antibodies compared with 2 (1.6%) of the 123 controls (P <0.0001; odds ratio [OR]=64.98; confidence interval [CI], 14.78–399.51). For 60% of the donor-specific antibody-positive patients, antibodies were detected before transplant failure. In 17 cases, these were class I specific; in 14 cases, class II specific; and in 3 cases, specific for both class I and II. Conclusions. This study has demonstrated that the production of posttransplantation antibodies directed against donor HLA-A, -B, -Cw, -DR, and -DQ mismatches are all strongly predictive of transplant failure.

Association between C4d staining in renal transplant biopsies, production of donor-specific HLA antibodies, and graft outcome.

Background. We carried out a retrospective study of C4d staining in paraffin sections from renal transplant biopsies to determine the association between C4d staining, donor-specific antibodies (DSA), histological features, and graft outcome. Methods. We studied 92 patients who had been biopsied for graft dysfunction. Biopsies were classified using Banff 97 criteria and features suggestive of antibody-mediated rejection were noted. Paraffin sections were stained with a polyclonal antibody using an immunoperoxidase technique. The presence of DSA in concurrent sera was determined by enzyme-linked immunosorbent assay and clinical data were reviewed. Results. Of the 92 cases, 15% showed diffuse and 24% showed focal C4d positivity. The grafts failed in 36% of the diffuse (P<0.025), 23% of the focal, and 7% of the negative group at between one month and 15 years posttransplantation. Only patients in the group with diffuse C4d positivity had concurrent DSA (five cases, P<0.001). Of the five DSA-positive patients, three had type II acute rejection and two of these transplants subsequently failed. The remaining two had chronic allograft nephropathy with features of alloimmune injury. Only two of the nine DSA-negative/C4d-positive transplants had failed at the time of writing, in one case due to recurrent disease. Conclusion. We demonstrated a significant association between diffuse C4d staining, production of DSA, and graft failure. Although the concurrent detection of DSA and C4d positivity is uncommon in our patients, these results indicate that outcome in this group is poor and they may benefit from therapies directed at the humoral response.

A comparison of enzyme-linked immunoabsorbent assays and flow cytometry techniques for the detection of HLA specific antibodies.

LATM, Quikscreen (QS), and B-Screen (QSB) are ELISA-based tests for the detection of HLA specific antibodies. FlowPRA beads are microparticles coated with HLA antigens for the detection of HLA specific antibodies by flow cytometry. The aim of this study was to evaluate the sensitivity and specificity of the LATM, QS, QSB, and FlowPRA screening tests. One hundred sixty-three sera from renal transplant patients were tested using LATM, FlowPRA, QS, and QSB. Discrepant results were further investigated using complement dependent cytotoxicity, QuikID, and PRA-STAT. When QS was compared with LATMI and FlowPRAI for the detection of HLA class I specific antibodies the overall concordance was 82.8% with no particular specificity missed by any one test. Comparing QSB with LATMII and FlowPRAII, for the detection of HLA class II specific antibodies, there was 90.7% concordance. Although the overall concordance was better for class II specific antibodies, QSB failed to detect antibodies to HLA-DQ in a number of samples from different patients. Of the methods tested, flow cytometry using FlowPRA beads appeared to be the most sensitive and specific, missing the least number of specificities. However, the ELISA methods offer the advantage of being more suitable for testing large numbers of samples in a more time- and cost-effective manner.

DETECTION OF HLA-SPECIFIC ANTIBODIES BY PRA-STAT AND THEIR ASSOCIATION WITH TRANSPLANT OUTCOME

OBJECTIVE The aim was to investigate the correlation between renal transplant outcome and the presence of HLA-specific antibodies detected using the ELISA kit PRA-STAT as compared with complement-dependent cytotoxicity (CDC). METHOD 295 sera from 95 renal transplant recipients (99 transplants) were investigated for the presence of HLA-specific antibodies using both PRA-STAT and CDC. The patients were divided into group I (49 transplants failed within 1 month) and group II (50 successful transplants). RESULTS The concordance between PRA-STAT and CDC for the detection of HLA class I-specific antibodies was 87.8% (259 of 295). For 19 sera, antibodies were detected only by PRA-STAT; for 17 sera, antibodies were detected only by CDC. No donor-specific antibodies were detected by either technique for patients in group II. For four group I patients (six sera), donor-specific IgG antibodies were detected only by PRA-STAT (one before, three after transplant) and all four transplants failed. For five other group I patients (six sera), donor HLA-specific antibodies were detected only by CDC (one before, four after transplant) and all five transplants failed. The antibodies detected before transplant by CDC were shown to be IgM alloantibodies. CONCLUSION This study showed that PRA-STAT could detect HLA-specific IgG antibodies relevant to transplant outcome that were not detected by CDC. However, it could not detect IgM alloantibodies that were also shown to be important. PRA-STAT is therefore a useful addition to a histocompatibility laboratorys screening repertoire only when used in conjunction with other techniques.

Mortality in diabetes: pancreas transplantation is associated with significant survival benefit

BACKGROUND Pancreas transplantation in complicated type 1 (insulin dependent) diabetes mellitus improves the quality of life, increases longevity and stabilizes diabetic complications. There may be clinician reticence due to perceived poor outcomes with published associated mortality rates of 5-8% due to significant co-morbidities, particularly cardiovascular impairment. METHODS Retrospective analysis was performed on patients undergoing pancreas transplantation in a single centre since the programmes initiation [simultaneous pancreas kidney (SPK) = 148, pancreas after kidney (PAK) = 33 and pancreas transplant alone (PTA) = 11] compared with a control group accepted contemporaneously onto the waiting list. The primary endpoint was patient mortality. The risk factors including medical and diabetic history, demographics, transplant type and waiting time were analysed. RESULTS The waiting list mortality was 30% (35 of 120) compared with a mortality of 9% (20 of 193) post-transplantation (P < 0.001). Deaths on the waiting list compared with transplantation up to 1 year had a relative risk of 2.67 (95% CI: 0.81-3.51; P = 0.19), whilst those surviving >1 year had a relative risk of 5.89 of dying on the waiting list (95% CI: 1.70-3.20; P < 0.0005). There were no differences in terms of cardiovascular or renal-associated risk factors, nor in other potential confounding factors other than duration of diabetes (P = 0.02). Median survival from listing was shorter in younger patients (<50; P < 0.0001). CONCLUSIONS Type 1 diabetics with renal failure listed for pancreas transplantation are at a significant risk of mortality even without surgery. Transplantation offers considerable survival benefits, despite associated surgical and immunosuppressive risks. In selected patients, pancreas transplantation remains the benchmark treatment for type 1 diabetes mellitus.

The detection and definition of IgM alloantibodies in the presence of IgM autoantibodies using flowPRA beads

We have developed a flow cytometry-based screening method using FlowPRA (One Lambda) human leukocyte antigen (HLA) class I panel beads and FlowPRA (One Lambda) HLA class I specificity beads for the detection and definition of immunoglobulin (Ig)M HLA-specific antibodies in the presence of IgM autoantibodies. Forty-six autoantibody-positive patients who were on the waiting list for a renal transplant (56 sera) were tested in parallel with FlowPRA (One Lambda) HLA class I beads and FlowPRA (One Lambda) control beads. Sera that were positive for IgM HLA class I antibodies were subsequently tested with FlowPRA HLA class I specificity beads to determine the HLA specificities. Thirteen of the 46 patients were positive for IgM HLA class I-specific antibodies. Eleven of the 13 had previous failed transplants and 2 were awaiting a primary transplant. For 9 of the 13 positive patients, IgM HLA class I specificities were defined. We have demonstrated the presence of IgM HLA-specific antibodies in patients with IgM autoantibodies. This study demonstrates the value of FlowPRA HLA class I panel and specificity beads for the detection and definition of IgM HLA class I-specific antibodies.

Pediatric kidney recipients may benefit from monitoring for donor-specific antibodies.

There are limited data regarding the presence of DSAs and their effect on graft function in pediatric renal transplantation. The role for serial DSA monitoring in routine clinical practice is unclear. All patients attending a regional transplant clinic were tested for DSAs, measured using Luminex single/mixed antigen beads. Any patient having a positive result subsequently underwent historic testing on samples previously obtained. DSA‐positive patients underwent prospective monitoring of DSAs, and correlation with clinical events was studied. Nine of a total of 50 patients (18%) were DSA‐positive, of whom six had graft dysfunction. The DSA‐positive cohort had significantly increased episodes of AR (p = 0.01). There were two graft losses in the DSA‐positive group and none in the DSA‐negative group. Eight of the DSA‐positive group had potentially reduced exposure to IS because of either adherence issues or clinical indications. DSAs were associated with increased risk of rejection. There appears to be a role for serial monitoring of DSAs in patients where there has been a reduced exposure to IS so that early intervention with optimized IS can be considered.

Antibody screening and crossmatching in retransplantation

Classical immunological dogma stipulates that reexposure to antigen results in an immediate and forceful response by the adaptive immune system. In the context of organ retransplantation this response is influenced by several factors, including antibody specificity of the primary response [1], affinity and isotype of preformed antibody, dose and immunogenicity of the antigen, immune responsiveness and health of the recipient. The pioneering work of Kissmeyer-Nielsen in Europe and Terasaki in the USA in the mid 1960s established that kidney transplants could be rejected hyperacutely by thrombosis of the transplant due to binding of preformed recipient antibody to donor antigen. It is now accepted that the most frequent target antigens in such instances are HLA antigens, coded for by the class I (HLA-A, -B, -Cw) or class II (HLA-DR,-DQ) genes of the major histocompatibility complex. Primary sensitization in transplant recipients usually occurs by one of three routes: blood transfusions, pregnancy and organ transplantation [2].