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Waitlisted sensitised transplant recipients with HLA allele level antibodies to their own HLA antigen family are disadvantaged by current deficiencies in HLA typing for deceased donors. This is primarily because at time of organ allocation, HLA typing is provided at antigen level whereas solid phase assays provide allele level antibody definition. The gold standard for HLA allele typing is next generation sequencing (NGS), however time limitations with established NGS systems prevent NGS use for deceased donors. Instead, many labs use a real-time PCR (qPCR) antigen level result for deceased donors, which can disadvantage sensitised patients. Here, we compared assigning qPCR 2-field alleles to qPCR antigen level to determine the impact on virtual crossmatch (VXM) and discuss impact on donor-specific antibody (DSA) assignments. 244 consecutive deceased donors were HLA typed to allelic level by qPCR (LinkSeq SABR) and subsequently by NGS (One Lambda Alltype). The impact of qPCR allele assignments on potential DSA identification was investigated, by retrospectively investigating all 3904 VXMs, where recipient DSA assessments were assessed against donor HLA, was performed within the cohort. There was 96.3% concordance between qPCR and NGS for all allele level loci, with HLA-A; DQB1; and DPB1 having best agreement (99.4%, 98.4% and 99.4% respectively). Of the 3904 VXMs with qPCR allele assignment, there were 13 (

The field of tissue typing and crossmatching has undergone extensive growth over the last 20 years with increasing refinement in the definition of Human Leukocyte Antigens (HLA) and an increased understanding of the immunologically important parts of HLA molecules. At the same time, advances in techniques for the detection of anti-HLA antibodies in recipients have become increasingly sensitive and sophisticated. Currently, the interpretation of crossmatch data is frequently beyond the grasp of the general nephrologist and can require considerable input from a specialised tissue typing scientist (Mulley and Kanellis, Nephrology (Carlton) 16(2):125–133, 2011). Amongst this, deceased donor kidneys remain scarce, relative to demand, with novel strategies for determining the best allocation method emerging.

Tissue typing is the process by which an individual's human leukocyte antigens (HLA) are determined. In transplantation, this vital process allows the immunologic or rejection risk of a donor-recipient pairing to be assessed through reviewing their HLA matching and whether any anti-HLA antibodies present in recipient serum are donor specific. Tissue typing has increased in sophistication over time which has allowed a deeper appreciation of the antigenically important parts of HLA and increased the complexity of determining immunologic risk.

Antibody-mediated rejection, whereby transplant recipient B cells and/or plasma cells produce alloreactive anti-human leukocyte antigen (HLA) antibodies, negatively influences transplant outcomes and is a major contributor to graft loss. An early humoral immune response is suggested by the production of anti-HLA donor-specific antibodies (DSA) that can be measured using solid phase assays. We report the early posttransplant coexistence of a shared anti-HLA antibody profile in 5 solid organ transplant recipients who received organs from the same donor. Retrospective analysis of the donor's serum confirmed the presence of the same anti-HLA profile, suggesting the transfer of donor-derived anti-HLA antibodies, or the cells that produce them, to multiple solid organ transplant recipients. The time frame and extent of transfer suggest a novel variant of the passenger lymphocyte syndrome. These findings have important implications for the consideration of all posttransplant antibody measurements, particularly the interpretation of non-DSAs in the sera of transplant recipients.

Background: There are few data comparing patient-reported outcomes (PROs) in randomized trials of initial antiretroviral therapy. We present results from a substudy of the NEAT001/ ANRS143 trial. Methods: The randomized trial compared first-line DRV/r 800/100 mg once daily plus RAL 400 mg twice daily and DRV/r plus TDF/ FTC 245/200 mg once daily. Changes in PROs were assessed with 3 questionnaires: EuroQoL 5 domains (EQ-5D), Center for Epidemiologic Studies Depression (CES-D) scale, and HIV Treatment Satisfaction Questionnaire. Major depressive disorder (MDD) was defined as CES-D $ 16. General estimating equations were used to model change over 96 weeks in PROs from baseline. Results: Of the 805 participants, 797 (99%) contributed to the substudy. Baseline PRO data were similar for the 2 randomized groups. Health status improved over time with a mean increase in EQ-5D visual analogue scale (VAS) of 8.0 by W96 [95% confidence interval (CI): 6.5 to 9.4; P, 0.001], and no statistically significant differences between groups (difference of 0.3 on VAS score (95% CI: 21.7 to 2.3); P = 0.7, global P value $ 0.05 for all domains over follow-up). There was no significant difference between groups on CES-D [difference of 20.1 (95% CI: 21.3 to 1.1); P = 0.9], or MDD during follow-up, adjusted for baseline MDD (odds ratio = 0.98, 95% CI: 0.82 to 1.18; P = 0.9). RAL + DRV/r group had lower level of convenience (P = 0.03) and fitted less well into patients’ lifestyle (P = 0.007) than the TDF/FTC + DRV/r regimen, and was associated with lower treatment satisfaction [median score: 53 RAL + DRV/r vs 55 TDF/FTC + DRV/r (P = 0.001)]. Conclusion: PROs improved after starting antiretroviral therapy, with no statistically significant difference between groups. The lower satisfaction with RAL + DRV/r may be explained by twicedaily administration.

Rituximab may improve graft survival in renal acute antibody-mediated rejection (AMR), but data confirming efficacy and optimal dosing is lacking. High-dose regimens may be associated with significant rates of infective complications. We therefore conducted a pilot study of a single low-fixed dose (500 mg) of rituximab in seven consecutive patients with AMR resistant to standard therapy. After a mean follow-up of 21 months (range, 9.5-33 months), graft and patient survival were 100% with serum creatinine levels significantly lower than peak rejection levels (171+/-73 micromol/L vs. 559+/-358 micromol/L, P=0.028). B cells were undetectable in all patients for more than or equal to 6 months and in six of seven patients for more than or equal to 12 months after rituximab. Three patients encountered a significant infective complication including cytomegalovirus reactivation, viral pneumonia, and polyoma viral nephropathy. All have since resolved. A single low-fixed dose of rituximab may help improve graft survival in AMR and offers the potential advantage of reduced infective complications.

The causes of cryopreserved allograft heart valve degeneration are poorly understood. We investigated HLA mismatch and other factors implicated in allograft valve degeneration. For this study we recruited 110 adult recipients of allograft heart valves who underwent surgery between June 1998 and March 2003 in the state of Victoria, Australia. Recipients and donors were HLA typed using serological and molecular methods. Valve function at most recent echocardiographic follow-up was examined for an association with the following variables using univariate and multivariate methods: HLA-A,-B, and -DR donor-recipient mismatch; HLA class I mismatch; total HLA mismatch; valve ischemic time; recipient age; donor age; ABO blood group donor-recipient match; and allograft size. Mean recipient age was 45 years (18-75 years), 75% were men. Seventy-four pulmonary (62 Ross procedure) and 36 aortic allografts were examined. Median valve ischemic time was 31 hours, range 20-48 hours. Echocardiographic follow-up was complete at a mean of 41 (+/-18) months, range 6-85 months. At univariate analysis longer ischemic time and younger recipient age were associated with valve dysfunction. HLA-A, -B, or DR mismatch, HLA class I mismatch, total HLA mismatch, donor age, ABO mismatch, and allograft size were not associated with valve dysfunction. Only younger recipient age remained significant at multivariate analysis. In conclusion, longer ischemic times and younger patient age predicted valve dysfunction at a mean of 3 years follow-up. Recipient age remained the strongest predictor of valve dysfunction. These results indicate that allograft ischemic times should be minimized.

Aim To evaluate the effect of the change in cPRA calculation in the NOMS from March 1st 2016 on kidney transplant allocation, in particular with respect to highly sensitised patients. Methods Analysis of data from the NOMS describing kidney transplants performed over 2 equal time periods: 1 March 2015–29 February 2016 (prior to the change in cPRA calculation) and 1 March 2016–28 February 2017 (after the change in cPRA calculation). Recipients of multiple organ transplants (apart from multiple kidneys) were excluded as the allocation process differs in these patients. P values were calculated from chi-square analysis, with the null hypothesis of no change in proportions between the time periods. Results The change in systems resulted in a mean increase of 12% in the “PRA” considered in the allocation algorithm, although the median change was 0 (as the majority of patients had no antibodies). Of patients on the waiting list at the time of the change, 41 (3.3%) had a documented PRA over 80%, which is the threshold for increased points according to the algorithm. With the changed method in the same group, 199 (16%) had a PRA over 80%, a 4-5-fold increase. The change resulted in a number of “hidden” sensitised patients becoming “visible” to the algorithm- eg 53 patients (4.3%) increased their PRA by 80% or more with the change, meaning that they would qualify for bonus allocation points even if their PRA was 0 by the previous system. Data on actual transplants revealed that the number of patients transplanted who were “highly sensitised” by the cPRA calculation rose from 24/201 (11.9%) to 56/240 (23.3%) (p Conclusions The change in cPRA calculation method has resulted in a significant increase in transplantation for highly sensitised patients. Significant variation was found between ABO blood groups in recipients, which warrants further investigation.

HLA-B57 and HLA-B58 are major histocompatibility class (MHC)-I allotypes that are potentially predictive of important clinical immune phenotypes. HLA-B*5701 is strongly associated with hypersensitivity to the HIV drug abacavir, liver toxicity from the antibiotic flucloxacillin and is a marker for slow progression of HIV AIDS. HLA-B*5801 is associated with hypersensitivity to allopurinol used to treat hyperuricaemia and recurrent gout. Here we describe a monoclonal antibody (mAb) specific for HLA-B57 and HLA-B58 that provides an inexpensive and sensitive screen for these MHC-I allotypes. The usefulness of HLA-B57 screening for prediction of abacavir hypersensitivity was shown in three independent laboratories, including confirmation of the mAb sensitivity and specificity in a cohort of patients enrolled in the PREDICT-1 trial. Our data show that patients who test negative by mAb screening comprise 90%-95% of all individuals in most human populations and require no further human leukocyte antigen (HLA) typing. Patients who test positive by mAb screening should proceed to high-resolution typing to ascertain the presence of HLA-B*5701 or HLA-B*5801. Hence, mAb screening provides a low-cost alternative to high-resolution typing of all patients and lends itself to point-of-care diagnostics and rapid ascertainment of low-risk patients who can begin immediate therapy with abacavir, flucloxacillin or allopurinol.

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.

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.

As the cause of allograft heart valve degeneration is poorly understood, the study aim was to investigate the host antibody response to allograft valve implantation.Sera were obtained from 92 recipients of allograft heart valves (61 pulmonary, 31 aortic). Sera were tested for anti-HLA class I antibodies by ELISA and complement-dependent cytotoxicity (CDC) methods, and anti-HLA class II antibodies by ELISA. Specificities of recipient anti-HLA class I antibodies were defined by standard CDC testing against a panel of T lymphocytes from 80 blood donors. Donor valve HLA typing was performed on stored donor DNA samples using molecular methods. The presence of donor-specific anti-HLA class I antibodies was hence defined in recipient sera. The presence of anti-HLA antibodies and donor-specific anti-HLA class I antibodies were correlated with function of allograft valves at the most recent echocardiographic follow up.At a mean of 3.0 years (range: 0.3-5.4 years) after allograft implantation, 96% (87/92) and 82% (75/92) of patients were positive for anti-HLA class I and II antibodies, respectively, by ELISA testing. Some 68% (61/90) of patients were positive for anti-HLA class I antibody (PRA5%) by CDC testing. PRA levels decreased with greater postoperative interval (r = -0.31, p = 0.003). In 68 recipients where donor HLA type was defined, 54% (37/68) of patients had antibodies specific to at least one donor HLA class I antigen. In 87 patients with a recent echocardiographic examination available for analysis (at a mean of 3.5 +/- 1.6 years postoperatively), there was no association between valve dysfunction and antibody status.Anti-HLA class I and II antibodies were detected by ELISA methods in most patients after allograft implantation extending to 5.4 years. The clinical significance of these findings is unclear, as no correlation was found between the prevalence of anti-HLA antibody and echocardiographic parameters of valve dysfunction at a mean of 3.5 years follow up.

Purpose Solid phase assays detect anti-HLA antibodies with high accuracy and sensitivity, however debate continues on how these results should be interpreted in the setting of lung transplantation. Methods and Materials We performed a retrospective analysis of pre-transplant anti-HLA antibody levels detected using a solid-phase HLA microbead assay (Luminex) in 80 patients undergoing lung transplantation at a single centre between 1/1/09 and 1/5/10. All patients underwent prospective T- and B-cell crossmatches (complement-dependent cytotoxicity assays) prior to transplantation. Outcome variables analyzed included acute cellular and antibody-mediated rejection, BOS and survival. Results Pre-transplant class I and class II anti-HLA antibodies were present in 31/80 (38%) and 13/80 (10%) patients, respectively, but did not predict a positive T- or B-cell crossmatch which was weekly positive in 1/80 and 14/80 patients, respectively. HLA class I and II donor-specific antibodies (DSA) were present pre-transplant in 13 and 7 patients, respectively. The presence pre-transplant of class I DSA, irrespective of a negative prospective T- and B-cell crossmatch predicted for a poor outcome (Odds ratio for BOS/death 4.6, p = 0.03). Pre-transplant DSA was not associated with the subsequent development of either acute cellular or antibody-mediated rejection. Only two patients developed clinically significant antibody-mediated rejection. Both developed DSA following transplant having not been present pre-transplant. Conclusions Identifying DSA pre-transplant predicts poor outcomes post-transplant and provides prognostic information beyond that which has been historically provided by a negative prospective crossmatch. Future studies need to focus on defining the level of pre-transplant DSA as measured using solid phase assays that predict for adverse outcomes following lung transplantation.