Linda Cantwell

Review article: Luminex technology for HLA antibody detection in organ transplantation.

Since its inception in the early 1960s, the serologically based complement‐dependent cytotoxicity (CDC) assay has been the cornerstone technique for the detection of human leucocyte antigen (HLA) antibodies, not only in pre‐transplant renal patients, but also in other forms of organ transplantation. Recently, solid phase assays have been developed and introduced for this purpose, and in particular the Flow‐based bead assays such as the Luminex system. This latter assay has proved to be far more sensitive than the CDC assay and has revealed pre‐sensitization in potential transplant recipients not detected by other methods of HLA antibody detection. However, the clinical implications of this increased sensitivity have not been convincingly demonstrated until recently. This technology for HLA antibody detection permits the evaluation of the clinical importance of antibodies directed at, for example, HLA‐DPB1 and HLA‐DQA1, which has not been possible to date. There are Luminex issues, however, requiring resolution such as the ability to distinguish between complement fixing and non‐complement fixing antibodies and determination of their relative clinical significance. Luminex technology will permit a re‐evaluation of the role of HLA antibodies in both early and late antibody‐mediated rejection.

Solid phase HLA antibody detection technology – challenges in interpretation

The introduction into routine diagnostic laboratories of solid phase assays for human leukocyte antigen (HLA) antibody detection has resulted in the application of new laboratory matching algorithms in clinical organ transplantation which have improved pre-transplant detection of immunization, in turn resulting in avoidance of rejection in many cases which until their introduction would not have been possible using the historical complement dependent serological techniques. There have been two generations of solid phase assays introduced into routine practice, namely, the enzyme-linked immunosorbent assay (ELISA) technique and the use of fluorescent beads with HLA molecules bound to their surface which can either be used in conventional flow cytometry or in conjunction with Luminex instrumentation, the latter having become the most popular approach. The use of the fluorescent bead techniques has raised interesting questions both with respect to technical performance and the interpretation of the results obtained. The advantages of bead technology for HLA antibody determination and the technical issues requiring resolution are the subject of this review.

High transplant rates of highly sensitized recipients with virtual crossmatching in kidney paired donation

Background In kidney paired donation (KPD), flexibility in the allocation of incompatible pairs is required if a critical mass of pairs to efficiently find matches cannot be reached. Methods In the Australian KPD program, virtual crossmatch is used for the allocation of suitable donors to registered recipients. Matching is based on acceptable mismatches, and donors are excluded from matching to recipients with donor-specific antibodies (DSAs) greater than 2000 mean fluorescence intensity (MFI). Match and transplant rates in the first year of the program were reviewed with respect to recipient and donor characteristics, including blood group distribution, level of recipient’s sensitization, and postallocation crossmatches. Results Four quarterly match runs were performed, which included 53 pairs and 2 altruistic donors. Human leukocyte antigen incompatibility accounted for 90% of the listed pairs. In the second run, the DSA threshold was increased to greater than 8000 MFI, because no matches were found with standard allocation. Optional ABO-incompatible matching was introduced from run 3. Matches were identified in 37 (70%) patients, of whom 92% had a negative crossmatch with their matched donor. Crossmatch positive results were found only in recipients with DSAs greater than 2000 MFI in the second run. In 4 cases immunological reasons and in 4 cases other reasons resulted in breakdown of chains and 17 patients not progressing to transplantation. Eventually, 20 (38%) patients received a KPD transplant, and 35% of these had a calculated panel-reactive antibody greater than 90%. Conclusions KPD using virtual crossmatch is a valid and effective solution for patients with immunologically incompatible donors even in the context of highly sensitized recipients.

Evolving experience of treating antibody-mediated rejection following lung transplantation.

BACKGROUND The importance of antibody-mediated rejection (AMR) following lung transplantation remains contentious. In particular, the diagnostic criteria suggested to define AMR, namely the presence of donor-specific antibodies (DSA), C4d immunoreactivity, histological features and allograft dysfunction are not always readily applicable or confirmatory in lung transplantation. METHODS In a retrospective single-center study of 255 lung transplant recipients (LTR), we identified 9 patients in whom a clinical diagnosis of AMR was made within 12months of transplant, and define the immunological, histological, clinical features, as well as the therapeutic response of this cohort. RESULTS Nine LTR with AMR underwent combination therapy with high-dose intravenous corticosteroid, intravenous immunoglobulin, plasmapheresis and rituximab. Following therapy, while the total number of the original DSA dropped by 17%, and the median value of the mean fluorescence intensity (mfi) of the originally observed DSA decreased from 5292 (IQR 1319-12,754) to 2409 (IQR 920-6825) (p<0.001), clinical outcomes were variable with a number of patients progressing to either chronic lung allograft dysfunction or death within 12month. CONCLUSION AMR in lung transplantation remains both a diagnostic and therapeutic challenge, but when clinically suspected is associated with a variable response to therapy and poor long-term outcomes.

HLA Matching at the Eplet Level Protects Against Chronic Lung Allograft Dysfunction

Donor selection in lung transplantation (LTx) is historically based upon clinical urgency, ABO compatibility, and donor size. HLA matching is not routinely considered; however, the presence or later development of anti‐HLA antibodies is associated with poorer outcomes, particularly chronic lung allograft dysfunction (CLAD). Using eplet mismatches, we aimed to determine whether donor/recipient HLA incompatibility was a significant predictor of CLAD. One hundred seventy‐five LTx undertaken at the Alfred Hospital between 2008 and 2012 met criteria. Post‐LTx monitoring was continued for at least 12 months, or until patient death. HLA typing was performed by sequence‐based typing and Luminex sequence‐specific oligonucleotide. Using HLAMatchmaker, eplet mismatches between each donor/recipient pairing were analyzed and correlated against incidences of CLAD. HLA‐DRB1/3/4/5+DQA/B eplet mismatch was a significant predictor of CLAD (hazard ratio [HR] 3.77, 95% confidence interval [CI]: 1.71–8.29 p < 0.001). When bronchiolitis obliterans syndrome (BOS) and restrictive allograft syndrome (RAS) were analyzed independently, HLA‐DRB1/3/4/5 + DQA/B eplet mismatch was shown to significantly predict RAS (HR 8.3, 95% CI: 2.46–27.97 p < 0.001) but not BOS (HR 1.92, 95% CI: 0.64–5.72, p = 0.237). HLA‐A/B eplet mismatch was shown not to be a significant predictor when analyzed independently but did provide additional stratification of results. This study illustrates the importance of epitope immunogenicity in defining donor–recipient immune compatibility in LTx.

Providing better-matched donors for HLA mismatched compatible pairs through kidney paired donation

Background Participation of compatible pairs (CP) in kidney paired donation (KPD) could be attractive to CPs who have a high degree of HLA mismatch, if the CP recipient will gain a better HLA match. Because KPD programs were not designed to help CP, it is important to define allocation metrics that enable CP to receive a better-matched kidney, without disadvantage to incompatible pairs (ICP). Methods Simulations using 46 ICPs and 11 fully HLA-mismatched CPs were undertaken using the Australian KPD matching algorithm. Allocations were preformed adding 1 CP at a time or all 11 CPs at once, and with and without exclusion of unacceptable antigens selected to give a virtual calculated panel-reactive antibody ranging 70% to 80% to improve HLA matching in CP recipients. Results On average, most CP recipients could be matched and had a lower eplet mismatch (EpMM) with the matched donor (57 ± 15) than with their own donor (78 ± 19, P < 0.02). However, only recipients who had an EpMM to own donor greater than 65 achieved a significant reduction in the EpMM with the matched donor. The gain in EpMM was larger when CPs were listed with unacceptable antigens. Furthermore, inclusion of 1 CP at a time increased matching in ICP by up to 33%, and inclusion of all 11 CPs at once increased ICP matching by 50%. Conclusions Compatible pair participation in KPD can increase match rates in ICP and can provide a better immunological profile in CP recipients who have a high EpMM to their own donor when using allocation based on virtual crossmatch.

Application of an epitope-based allocation system in pediatric kidney transplantation

Donor–recipient HLA mismatch remains a leading cause for sensitization and graft loss in kidney transplantation. HLA compatibility at an epitope level is emerging as an improved method of matching compared with current HLA antigen allocation. A novel epitope‐based allocation approach to prospectively exclude donors with high‐level mismatches was implemented for pediatric KTRs on the DD waiting list. Nineteen consecutive transplants were followed for 12 months, including eight DD KTRs listed with eplet exclusions, as well as three DD KTRs and eight LD KTRs without exclusions. KTRs with eplet exclusions had estimated GFR of 78.5 mL/min/1.73 m2, no episodes of rejection, and time to transplant 6.55 months. HLA‐A, HLA‐B, HLA‐DR antigen mismatches were similar between all groups. KTRs with exclusions had significantly lower class II eplet mismatches (20.4) than the contemporary DD KTRs without exclusions (63.7) and DD KTRs transplanted in the preceding decade (46.9). dnDSAs were identified in two of eight DD KTRs with exclusions, two of three DD KTRs without exclusions and five of eight LD KTRs. Epitope‐based allocation achieved timely access to transplantation, low class II eplet mismatches, and low rates of dnDSAs in the first year. This strategy requires longer follow‐up and larger numbers, but has the potential to reduce anti‐HLA sensitization and improve both graft survival and opportunities for future retransplantation.

Four years of experience with the Australian kidney paired donation programme

New approaches to increase kidney transplantation rates through expansion of live donor kidney transplantation have become necessary due to ongoing shortage of deceased donor organs. These strategies include desensitization in antibody‐incompatible transplants to overcome the barrier of blood group incompatibility or human leucocyte antigen antibodies between recipient and donor and kidney paired donation (KPD) programmes. In KPD, a kidney transplant candidate with an incompatible live donor joins a registry of other incompatible pairs in order to find potentially compatible transplant solutions. To match the largest possible number of donor–recipient pairs while minimizing immunologic risk, KPD programmes use sophisticated algorithms to identify suitable matches with simultaneous two‐way or more complex multi‐way exchanges as well as including non‐directed anonymous donors to start a chain of compatible transplantations. Because of the significant immunologic barriers when fewer donor options are available, the optimal solution for difficult‐to‐match, highly sensitized patients is access to more potential donors using large multi‐centre or national KPD registries. This review focuses on the first 4 years of experience with the Australian multi‐centre KPD programme that was established in October 2010.

Optimising Outcomes in Pediatric Renal Transplantation through the Australian Paired Kidney Exchange Program

Kidney paired donation (KPD) programs offer the opportunity to enable living kidney donation when immunological and other barriers prevent safe directed donation. Children are likely to require multiple transplants during their lifetime; therefore, high‐level histocompatibility and organ quality matching are key priorities. Details are given for a cohort of seven pediatric renal transplantations performed through the Australian Kidney Exchange (AKX), including barriers to alternative transplantation and outcomes after KPD. Reasons for entering the KPD program were preformed donor‐specific antibodies to their registered donor in five cases, ABO mismatch, and avoidance of the risk of exposure to hepatitis B virus. Four recipients were highly sensitized. All patients received transplants with organs of lower immunological risk compared with their registered donors. HLA eplet mismatch scores were calculated for donor–recipient pairs; three patients had improved eplet mismatch load with AKX donor compared with their registered donor. All grafts are functioning, with a mean estimated glomerular filtration rate of 77 mL/min/1.73 m2 (range 46–94 mL) and a follow‐up range of 8–54 months, and no patient experienced clinical or histological rejection. KPD is a viable strategy to overcome many barriers to living donation for pediatric patients who have an otherwise suitable donor and provides an opportunity to minimize immunological risks.

Hidden perils in a highly sensitized kidney transplant recipient

Highly sensitised patients are at increased risk for antibody mediated rejection (AMR) and reduced graft survival. Highly sensitive assays for detecting recipient preformed anti‐HLA antibodies have been developed and identify high immunological risk donors. A 62yo male with end stage renal failure secondary to glomerulonephritis received a T‐cell crossmatch negative, deceased donor, renal transplant mismatched at 3 of 6 HLA loci. A donor specific antibody (DSAb) to DR17 (MFI 2073) was present. Given his advancing age, multiple medical comorbidities and broad HLA sensitisation the transplant was accepted, however, shortly before transplantation two atypical results were made available. Firstly a B‐cell crossmatch was performed and found to be negative in current serum but strongly positive in peak serum, secondly a further potential DSAb was predicted based on linkage disequilibrium with known donor HLA typing. The donor HLA typing would not be clarified until after the transplant. Despite the increased risk of AMR the transplant proceeded with pre‐emptive plasma exchange. The patient developed severe AMR requiring extensive therapy. Incomplete prospective donor HLA typing can generate uncertainty in the interpretation of the virtual crossmatch performed for deceased donor transplants. This may result in clinically relevant sequelae. Advances in antibody detection techniques need to be matched by timely donor HLA typing for its full benefit to be realised.