BSHI/BTS guidance on crossmatching before deceased donor kidney transplantation.

All UK H&I laboratories and transplant units operate under a single national kidney offering policy, but there have been variations in approach regarding when to undertake the pre-transplant crossmatch test. In order to minimize cold ischaemia times for deceased donor kidney transplantation we sought to find ways to be able to report a crossmatch result as early as possible in the donation process. A panel of experts in transplant surgery, nephrology, specialist nursing in organ donation and H&I (all relevant UK laboratories represented) assessed evidence and opinion concerning five factors that relate to the effectiveness of the crossmatch process, as follows: when the result should be ready for reporting; what level of donor HLA typing is needed; crossmatch sample type and availability; fairness and equity; risks and patient safety. Guidelines aimed at improving practice based on these issues are presented, and we expect that following these will allow H&I laboratories to contribute to reducing CIT in deceased donor kidney transplantation.

Using an Integrated -Omics Approach to Identify Key Cellular Processes That Are Disturbed in the Kidney After Brain Death.

In an era where we are becoming more reliant on vulnerable kidneys for transplantation from older donors, there is an urgent need to understand how brain death leads to kidney dysfunction and, hence, how this can be prevented. Using a rodent model of hemorrhagic stroke and next-generation proteomic and metabolomic technologies, we aimed to delineate which key cellular processes are perturbed in the kidney after brain death. Pathway analysis of the proteomic signature of kidneys from brain-dead donors revealed large-scale changes in mitochondrial proteins that were associated with altered mitochondrial activity and morphological evidence of mitochondrial injury. We identified an increase in a number of glycolytic proteins and lactate production, suggesting a shift toward anaerobic metabolism. Higher amounts of succinate were found in the brain death group, in conjunction with increased markers of oxidative stress. We characterized the responsiveness of hypoxia inducible factors and found this correlated with post-brain death mean arterial pressures. Brain death leads to metabolic disturbances in the kidney and alterations in mitochondrial function and reactive oxygen species generation. This metabolic disturbance and alteration in mitochondrial function may lead to further cellular injury. Conditioning the brain-dead organ donor by altering metabolism could be a novel approach to ameliorate this brain death-induced kidney injury.

The UK Pancreas Allocation Scheme for Whole Organ and Islet Transplantation.

In order to develop a national allocation scheme for donor pancreases, factors affecting waiting time and transplant outcomes in the United States (US) and United Kingdom (UK) were analyzed and compared. Blood group, sensitization, dialysis requirement, and whether the patient was waiting for a kidney and pancreas or pancreas alone affected waiting time in both countries; ethnicity and body mass index (BMI) also affected waiting time in the US. Ninety-day pancreas survival was similar in the UK and US, and was poorer for patients receiving a pancreas alone, with older donors, higher BMI and longer duration of ischemia in both countries. Factors affecting outcome, together with published data on factors affecting islet transplantation, informed the development of a points based allocation scheme for deceased donor pancreases in the UK providing equitable access for both whole organ and islet recipients through a single waiting list. Analysis of the allocation scheme 3 years after its introduction in December 2010 showed that the results were broadly as simulated, with a significant reduction in the number of long waiting patients and an increase in the number of islet transplants. There remains a surplus of highly sensitized patients in the waiting list, which the scheme should address in time.

Genetic variation in caveolin-1 correlates with long-term pancreas transplant function.

Pancreas transplantation is a successful treatment for a selected group of people with type 1 diabetes. Continued insulin production can decrease over time and identifying predictors of long-term graft function is key to improving survival. The aim of this study was to screen subjects for variation in the Caveolin-1 gene (Cav1), previously shown to correlate with long-term kidney transplant function. We genotyped 435 pancreas transplant donors and 431 recipients who had undergone pancreas transplantation at the Oxford Transplant Centre, UK, for all known common variation in Cav1. Death-censored cumulative events were analyzed using Kaplan-Meier and Cox regression. Unlike kidney transplantation, the rs4730751 variant in our pancreas donors or transplant recipients did not correlate with long-term graft function (p = 0.331-0.905). Presence of rs3801995 TT genotype (p = 0.009) and rs9920 CC/CT genotype (p = 0.010) in our donors did however correlate with reduced long-term graft survival. Multivariate Cox regression (adjusted for donor and recipient transplant factors) confirmed the association of rs3801995 (p = 0.009, HR = 1.83;[95% CI = 1.16-2.89]) and rs9920 (p = 0.037, HR = 1.63; [95% CI = 1.03-2.73]) with long-term graft function. This is the first study to provide evidence that donor Cav1 genotype correlates with long-term pancreas graft function. Screening Cav1 in other datasets is required to confirm these pilot results.

Should HLA mismatch acceptability for sensitized transplant candidates be determined at the high-resolution rather than the antigen level?

Defining HLA mismatch acceptability of organ transplant donors for sensitized recipients has traditionally been based on serologically defined HLA antigens. Now, however, it is well accepted that HLA antibodies specifically recognize a wide range of epitopes present on HLA antigens and that molecularly defined high resolution alleles corresponding to the same low resolution antigen can possess different epitope repertoires. Hence, determination of HLA compatibility at the allele level represents a more accurate approach to identify suitable donors for sensitized patients. This approach would offer opportunities for increased transplant rates and improved long term graft survivals.

Killer immunoglobulin-like receptor gene repertoire influences viral load of primary human cytomegalovirus infection in renal transplant patients.

Killer cell immunoglobulin-like receptors (KIR) are highly polymorphic members of the immunoglobulin superfamily, which influence the response of natural killer cells and some T-lymphocyte subsets. Analysis of a cohort of previously human cytomegalovirus (HCMV)-negative patients, who developed primary HCMV infection following HCMV-positive renal transplant (n=76), revealed an increase in the frequency of KIR genes located on the telomeric region of B haplotypes (Tel B). The presence of Tel B in combination with the KIR ligand HLA-C2 was significantly more frequent in this subgroup. These genetic factors were associated with resistance to HCMV infection in a second cohort (n=65), where the Tel B genes KIR2DL5, -2DS1, 2DS5 and -3DS1 were all significantly associated with high viral loads. Furthermore, the KIR haplotype Tel A when in combination with the KIR ligand HLA-C1 was significantly protective against the development of severe infection. Our results suggest that KIR are a significant factor in the control of primary HCMV infection, and that determination of KIR gene repertoire may help in detection of renal transplant patients who were most at risk.

De novo donor-specific HLA antibodies: biomarkers of pancreas transplant failure.

This study assesses the role of posttransplant HLA antibody monitoring in the surveillance of pancreas transplant recipients. Four hundred thirty-three pancreas transplants were performed at the Oxford Transplant Centre 2006-2011 (317 simultaneous pancreas kidney [SPK] and 116 isolated pancreas [IP]). HLA antibody monitoring was performed at 0, 6 and 12 months and annually and during clinical events. There was no association between pancreas graft failure and recipient or donor characteristics. Posttransplant antibody status, available for 354 (81.8%) of recipients, demonstrated that 141 (39.8%) developed de novo HLA antibodies, of which 52 (36.9%) were de novo donor-specific HLA antibodies (DSA) (34 SPK, 18 IP). The development of antibodies to donor HLA, but not to nondonor HLA, was significantly associated with poorer graft outcomes, with 1- and 3-year graft survival inferior in SPK recipients (85.2% vs. 93.5%; 71.8% vs. 90.3%, respectively; log-rank p = 0.002), and particularly in IP recipients (50.0% vs. 82.9%; 16.7 vs. 79.4%, respectively; log-rank p = 0.001). In a multivariate analysis, development of de novo DSA emerged as a strong independent predictor of pancreas graft failure (hazard ratio 4.66, p < 0.001). This is the largest study to examine de novo HLA antibodies following pancreas transplantation and clearly defines a high-risk group in need of specific intervention.

Alemtuzumab and sirolimus in renal transplantation: six-year results of a single-arm prospective pilot study.

mTOR inhibitors avoid calcineurin nephrotoxicity, but sirolimus de novo is associated with unacceptable side effects and higher rejection rates. We have investigated a modified strategy: alemtuzumab induction with tacrolimus and mycophenolate maintenance, switching from tacrolimus to sirolimus at 6 months and stopping mycophenolate at 12 months. Here, we report the 6-year follow-up of 30 patients prospectively recruited to this single-arm pilot study and compare outcomes to a matched contemporaneous control group of 30 patients who received standard induction and calcineurin-inhibitor-based immunosuppression.Six-year patient and graft survival were 83% and 80%(alemtuzumab) versus 77% and 70% (control). Rejection rates in the first 6 months were similar in alemtuzumab (6.6%) and control groups (10%). A higher than expected incidence of rejection in the alemtuzumab group following cessation of mycophenolate at 1 year (17%) was mitigated in later patients by retaining low dose mycophenolate. Mean eGFR was higher in the alemtuzumab group at all time points but not significantly (p»0.16). Tacrolimus levels in the first 6 months were significantly higher in the contemporaneous control group (p<0.001). Alemtuzumab induction with initial treatment with tacrolimus enables conversion to sirolimus without the side effects and incidence of acute rejection seen in earlier protocols.

HLA-A, -B, -DR allele group frequencies in 7007 kidney transplant list patients in 27 UK centres.

This observational study aims to determine the HLA specificity frequencies of patients on the UK renal transplant list, which can be used as a resource for those laboratories that support the UK renal transplant programme. Whilst the HLA specificity frequencies may differ from that of the general population, it is the individuals on the transplant list who are in need of a new kidney, which has to be provided from the general population. Any differences in protein allele frequencies between this patient population and the general population are likely to be minimal because of the very large number of patients included. The HLA-A, -B and -DR allele group frequencies from 7007 patients on the UK kidney transplant list (August, 2009) were analysed. HLA types had been submitted to NHSBT to register patients on the UK deceased donor kidney waiting list. The data were submitted from 27 different registering centres throughout the UK. Within this data set, 25 different HLA-A, 50 HLA-B and 18 HLA-DR allele groups were present. The most common allele groups at each locus were -A2 (phenotype frequency 42.6%), -B44 (phenotype frequency 23.3%) and -DR4 (phenotype frequency 29.8%). The least common allele groups at each locus were -A19, - A43, -B16, -B21, -B22, -B83 and -DR5. Reports of HLA frequency (protein allotype) data from populations as large as this are not readily available adding value to this observational study.

Pancreas transplantation from donors after circulatory death from the United Kingdom.

This study reports the comparative short-term results of pancreas transplantation from donors after circulatory death (DCD) (Maastricht III & IV), and pancreases from brainstem deceased donors (DBD). Between January 2006 and December 2010, 1009 pancreas transplants were performed in the United Kingdom, with 134 grafts from DCD and 875 from DBD. DCD grafts had no premortem pharmacological interventions performed. One-year pancreas and patient survival was similar between DCD and DBD, with pancreas graft survival significantly better in the DCD cohort if performed as an SPK. Early graft loss due to thrombosis (8% vs. 4%) was mainly responsible for early graft loss in the DCD cohort. These results from donors with broader acceptance criteria in age, body mass index, premortem interventions, etc. suggest that DCD pancreas grafts may have a larger application potential than previously recognized.

British Society for Histocompatibility & Immunogenetics and British Transplantation Society guidelines for the detection and characterisation of clinically relevant antibodies in allotransplantation.

Ongoing technological developments in antibody detection and characterisation allowing relative quantitation of HLA-specific antibody levels, combined with crossmatch results, now allow a graded assessment of patient potential donor immunological risk for allotransplantation, rather than a simple 'positive' or 'negative' categorization of crossmatch results. These developments have driven a thorough revision of the British Society for Histocompatibility & Immunogenetics and British Transplantation Society Guidelines for the Detection and Characterisation of Clinically Relevant Antibodies in Allotransplantation. These newly published revised Guidelines contain a number of recommendations as to best practice for antibody detection and crossmatching for the transplantation of a wide range of solid organs and tissues. These recommendations are briefly summarized in this article.

Association study of MICA and MICB in Alzheimer's disease.

Following the replication of the association of the human leucocyte antigen (HLA) allele, HLA-B*07, with Alzheimer's disease (AD) in the cohort of the Oxford Project to Investigate Memory and Ageing (OPTIMA) in a previous study, we examined whether that association could be due to linkage disequilibrium with MICA or MICB alleles. We found a possible association of MICA*00801 heterozygotes with AD in subjects positive for the epsilon 4 allele of apolipoprotein E. This finding was supported by Hardy-Weinberg analysis, by stratified association analysis and by interaction analysis, but did not survive correction for multiple testing. In any case, these results do not explain our previously reported association of HLA-B*07 with AD.

The identification of three novel MICA alleles by sequence-based typing.

During a study of MICA frequency in a healthy population and a cohort of patients suffering with inflammatory bowel disease, three DNA samples produced unusual reactivity patterns using polymerase chain reaction sequence-specific primers (PCR-SSP). These samples were subsequently characterized by sequence-based typing (SBT). Here, we report the sequence of these three novel MICA alleles.

A novel major histocompatibility complex class I-related chain allele.

We report the identification of a novel major histocompatibility complex class I-related chain (MICB) allele, provisionally designated as MICB-0114 pending the WHO Nomenclature Classification for the MICB locus. This new allele is identical to MICB-0103101v except for a single mutation of G to A in exon 4 that translates into an amino acid substitution from glutamic acid to lysine.

Identification of a novel MICA allele: MICA*051.

We have identified a novel MICA allele, MICA*051, detected by the polymerase chain reaction using sequence-specific primers and characterized by sequence-based typing. MICA*051 appears to be the result of a recombination between MICA*00801 and MICA*00701 at intron 2.

Human platelet alloantigens (HPAs): PCR-SSP genotyping of a UK population for 15 HPA alleles.

Alloimmunization to human platelet alloantigens (HPAs) is responsible for neonatal alloimmune thrombocytopenia (NAIT), post-transfusional purpura (PTP) and platelet transfusion refractoriness. HPAs may also have a role as histocompatibility antigens in transplantation as well as associations with cardiac disease. We have developed a polymerase chain reaction-sequence-specific primer (PCR-SSP) assay capable of detecting 15 HPA allelic variants. As part of the validation of the assay, 134 UK renal donors were genotyped to determine HPA allele frequencies in the UK population. The HPA allele frequencies obtained are consistent with those of the other European studies: GP1A*1 (HPA-5a) and GP1A*2 (HPA-5b), 0.914 and 0.086, respectively; GP1BA*1 (HPA-2a) and GP1BA*2 (HPA-2b), 0.925 and 0.075; GP2B*1 (HPA-3a) and GP2B*2 (HPA-3b), 0.627 and 0.373; GP3A*1 (HPA-1a) and GP3A*2 (HPA-1b), 0.840 and 0.161. The rare alleles GP2B*3 (HPA-9bw) and GP3A*3 to *8 (HPA-4b, -6b, -7bw, -8bw, -10bw and -11bw, respectively) were all absent. This comprehensive HPA genotyping assay allows rapid, accurate and reproducible results at low cost.

Characterization of a novel HLA-C allele, Cw*0315.

The presence of an unusual HLA class I reactivity pattern was detected in a Caucasoid-Asian individual by PCR-sequence specific primer (PCR-SSP) typing. Exons 2 and 3 were characterized using PCR-sequence-based typing (PCR-SBT) and were found to contain a novel Cw*03 sequence, Cw*0315. In the region studied, Cw*0315 was comprised mainly of the Cw*0302 sequence, but at four positions it contained nucleotides normally only found in other HLA Cw locus alleles. These positions each resulted in an amino acid substitution.

HLA-DR and DQ polymorphisms in bullous pemphigoid from northern China.

Bullous pemphigoid (BP) is an autoimmune disease mediated by autoantibodies against hemidesmosome components. This study used PCR-sequence-specific primers to genotype polymorphisms in HLA-DR and DQ in 25 BP patients and 57 normal controls from northern China. We found lower frequencies of DRB1*08 (DR8) and DRB1*08/DQB1*06 (DR8/DQ6) haplotypes in BP patients than in controls (4.08% vs. 15.19% and 1.54% vs. 13.82%, respectively; P < 0.05), suggesting a protective role for DR8 and DR8/DQ6 haplotypes in BP patients from northern China; there were no statistical differences among other alleles tested. This result is strikingly different from previous reports that DQB1*0301 is associated with BP in Caucasian patients and DRB1*1101, DQB1*0302, DRB1*04/DQA1*0301/DQB1*0302 and DRB1*1101/ DQA1*0505/DQB1*0302 with Japanese BP patients. Ethnic differences in the polymorphic composition of the HLA-DR and DQ genes may influence genetic susceptibility to BP.