Kidney transplant candidates' perspectives on the implementation of a Canadian Willingness to Cross program: A strategy to increase access to kidney transplantation for highly sensitized patients.

BACKGROUND: Kidney transplantation is the optimal treatment for end-stage renal disease. However, highly sensitized patients (HSPs) have reduced access to transplantation, leading to increased morbidity and mortality on the waiting list. The Canadian Willingness to Cross (WTC) program proposes allowing transplantation across preformed donor specific antibodies (DSA) determined to be at a low risk of rejection under the adaptive design framework. This study collected patients' perspectives on the development of this program. METHODS: Forty-one individual interviews were conducted with kidney transplant candidates from three Canadian transplant centers in 2022. The interviews were digitally recorded and transcribed for subsequent analyses. RESULTS: Despite limited familiarity with the adaptive design, participants demonstrated trust in the researchers. They perceived the WTC program as a pathway for HSPs to access transplantation while mitigating transplant-related risks. HSPs saw the WTC program as a source of hope and an opportunity to leave dialysis, despite acknowledging inherent uncertainties. Some non-HSPs expressed concerns about fairness, anticipating increased waiting times and potential compromise in kidney graft longevity due to higher rejection risks. Participants recommended essential strategies for implementing the WTC program, including organizing informational meetings and highlighting the necessity for psychosocial support. CONCLUSION: The WTC program emerges as a promising strategy to enhance HSPs' access to kidney transplantation. While HSPs perceived this program as a source of hope, non-HSPs voiced concerns about distributive justice issues. These results will help develop a WTC program that is ethically sound for transplant candidates.

ABO-adjusted calculated panel reactive antibody (cPRA): A unified metric for immunologic compatibility in kidney transplantation.

Implementation of the kidney allocation system in 2014 greatly reduced access disparity due to human leukocyte antigen (HLA) sensitization. To address persistent disparity related to candidate ABO blood groups, herein we propose a novel metric termed "ABO-adjusted cPRA," which simultaneously considers the impact of candidate HLA and ABO sensitization on the same scale. An ethnic-weighted ABO-adjusted cPRA value was computed for 190 467 candidates on the kidney waitlist by combining candidate's conventional HLA cPRA with the remaining fraction of HLA-compatible donors that are ABO-incompatible. Consideration of ABO sensitization resulted in higher ABO-adjusted cPRA relative to conventional cPRA by HLA alone, except for AB candidates since they are not ABO-sensitized. Within cPRA Point Group = 99%, 43% of the candidates moved up to ABO-adjusted cPRA Point Group = 100%, though this proportion varied substantially by candidate blood group. Nearly all O and most B candidates would have elevated ABO-adjusted cPRA values above this policy threshold for allocation priority, but relatively few A candidates displayed this shift. Overall, ABO-adjusted cPRA more accurately measures the proportion of immune-compatible donors compared with conventional HLA cPRA, especially for highly sensitized candidates. Implementation of this novel metric could enable the development of allocation policies permitting more ABO-compatible transplants without compromising equity.

Transplantation of Patients With Long Dialysis Vintage in the Current Deceased Donor Kidney Allocation System (KAS).

RATIONALE & OBJECTIVE: In 2014 the wait-time calculation for deceased donor kidney transplantation in the United States was changed from the date of first waitlisting to the date of first maintenance dialysis treatment with the aim of minimizing disparities in access to transplantation. This study examined the impact of this policy on access to transplantation, patient survival, and transplant outcomes among patients treated with maintenance dialysis for a prolonged duration before waitlisting. STUDY DESIGN: Retrospective cohort study. SETTING & PARTICIPANTS: Patients identified in the US Renal Data System between 2008 and 2018 aged 18-70 years and in the 95th percentile of dialysis treatment duration (≥6.5 years) before waitlisting. EXPOSURE: Waitlisting for transplantation before versus after implementation of the policy. OUTCOME: Time from date of waitlisting to deceased donor transplantation and death, and from date of transplantation to all cause graft loss. ANALYTICAL APPROACH: Univariate and multivariable time to event analyses. RESULTS: Patients waitlisted after the policy change had a higher likelihood of deceased donor transplantation (HR, 3.12 [95% CI, 2.90-3.37]) and lower risk of death (HR, 0.74 [95% CI, 0.63-0.87]). The risk of graft loss was lower in the post-kidney allocation system (KAS) cohort (HR, 0.66 [95% CI, 0.55-0.80]). The proportion of adult patients treated with dialysis ≥6.5 years who were never waitlisted for transplantation remained high (73%) and did not decrease after the policy implementation. LIMITATIONS: Cannot determine causality in this observational study. CONCLUSIONS: The policy change was associated with an increase in deceased donor transplantation and marked improvement in patient survival for patients waitlisted after long periods of dialysis treatment without decreasing the utility of available deceased donor kidney supply. The policy was not associated with increased waitlisting of this disadvantaged population.

Clinical utility of complement-dependent C3d assay in kidney recipients presenting with late allograft dysfunction.

The objective of this study was to evaluate the utility of a complement-dependent C3d assay to risk stratify donor-specific antibodies (DSA) in a multicenter cohort of kidney recipients presenting with new-onset clinical dysfunction. A total of 106 subjects with evidence of DSA at a mean period of 5.3 ± 5.0 years posttransplant underwent testing using C3d reagents. C3d positivity was strongly associated with both the peak and sum IgG DSA MFI, with 98.3% (n = 57/58) of strongly reactive sera (peak MFI > 10 000) eliciting a positive signal. Patients with C3d+ DSA had a higher creatinine (P = .03), more significant graft fibrosis (P = .035), and a faster rate of graft loss posttest compared to those with C3d- DSA (P = .05). Subanalysis of patients with low-moderate level DSA confirmed the inferior outcome associated with C3d positivity. Despite the prognostic value of C3d as a stand-alone test, the assay did not provide independent risk prediction after incorporation of graft fibrosis in a multivariate model (P = .94). Overall, C3d offered limited discriminatory value for strong DSA with peak IgG MFI > 10 000 and in patients where histologic data is available, but its utilization may be considered in those with low-moderate level DSA and where an allograft biopsy is not accessible.

Clinical applications of next-generation sequencing in histocompatibility and transplantation.

PURPOSE OF REVIEW: Next-generation sequencing (NGS) can overcome traditional methodological barriers to facilitate detailed studies of large genomes. Here, we summarize recent NGS-based developments in histocompatibility and transplantation, and highlight the dynamic range of clinical applications achievable on this platform. RECENT FINDINGS: Multiple NGS-based protocols have been established to achieve unambiguous human leukocyte antigen genotyping. These methods are presently engaged to serve the high-throughput demand of large bone marrow registries; however, the scalable nature of NGS makes it an equally attractive technology for select applications within solid organ transplantation. Recently, the exquisite sensitivity of NGS has been leveraged to perform noninvasive allograft monitoring by tracking the dynamics of donor-derived cell-free DNA. Further, NGS-based T-cell receptor and immunoglobulin heavy chain repertoire profiling appear to be useful in clarifying disease-specific diagnoses in certain complex allograft pathology; detecting/quantifying minimal residual disease following allogeneic stem cell transplantation; and tracking donor-reactive T cells to understand the mechanism of tolerance in kidney transplant recipients. SUMMARY: NGS is superior to classical Sanger sequencing in its throughput, sensitivity, and the ability to provide phase-defined sequence data. These unique properties allow its broad application to diverse areas in clinical transplantation.

Clinical relevance of HLA-DQ eplet mismatch and maintenance immunosuppression with risk of allosensitization after kidney transplant failure.

The optimal immunosuppression management in patients with a failed kidney transplant remains uncertain. This study analyzed the association of class II HLA eplet mismatches and maintenance immunosuppression with allosensitization after graft failure in a well characterized cohort of 21 patients who failed a first kidney transplant. A clinically meaningful increase in cPRA in this study was defined as the cPRA that resulted in 50% reduction in the compatible donor pool measured from the time of transplant failure until the time of repeat transplantation, death, or end of study. The median cPRA at the time of failure was 12.13% (interquartile ranges = 0.00%, 83.72%) which increased to 62.76% (IQR = 4.34%, 99.18%) during the median follow-up of 27 (IQR = 18, 39) months. High HLA-DQ eplet mismatches were significantly associated with an increased risk of developing a clinically meaningful increase in cPRA (p = 0.02) and de novo DQ donor-specific antibody against the failed allograft (p = 0.02). We did not observe these associations in patients with high HLA-DR eplet mismatches. Most of the patients (88%) with a clinically meaningful increase in cPRA had both a high DQ eplet mismatch and a reduction in their immunosuppression, suggesting the association is modified by immunosuppression. The findings suggest HLA-DQ eplet mismatch analysis may serve as a useful tool to guide future clinical studies and trials which assess the management of immunosuppression in transplant failure patients who are repeat transplant candidates.

The Benefits of Preemptive Transplantation Using High-Kidney Donor Profile Index Kidneys.

BACKGROUND: The Kidney Donor Profile Index (KDPI) is a percentile score summarizing the likelihood of allograft failure: A KDPI ≥85% is associated with shorter allograft survival, and 50% of these donated kidneys are not currently used for transplantation. Preemptive transplantation (transplantation without prior maintenance dialysis) is associated with longer allograft survival than transplantation after dialysis; however, it is unknown whether this benefit extends to high-KDPI transplants. The objective of this analysis was to determine whether the benefit of preemptive transplantation extends to recipients of transplants with a KDPI ≥85%. METHODS: This retrospective cohort study compared the post-transplant outcomes of preemptive and nonpreemptive deceased donor kidney transplants using data from the Scientific Registry of Transplant Recipients. 120,091 patients who received their first, kidney-only transplant between January 1, 2005, and December 31, 2017, were studied, including 23,211 with KDPI ≥85%. Of this cohort, 12,331 patients received a transplant preemptively. Time-to-event models for the outcomes of allograft loss from any cause, death-censored graft loss, and death with a functioning transplant were performed. RESULTS: Compared with recipients of nonpreemptive transplants with a KDPI of 0%-20% as the reference group, the risk of allograft loss from any cause in recipients of a preemptive transplant with KDPI ≥85% (hazard ratio [HR], 1.51; 95% confidence interval [CI], 1.39 to 1.64) was lower than that in recipients of nonpreemptive transplant with a KDPI ≥85% (HR, 2.39; 95% CI, 2.21 to 2.58) and similar to that of recipients of a nonpreemptive transplant with a KDPI of 51%-84% (HR, 1.61; 95% CI, 1.52 to 1.70). CONCLUSIONS: Preemptive transplantation is associated with a lower risk of allograft failure, irrespective of KDPI, and preemptive transplants with KDPI ≥85% have comparable outcomes with nonpreemptive transplants with KDPI 51%-84%.

Novel alleles in the era of next-generation sequencing-based HLA typing calls for standardization and policy.

Next-Generation Sequencing (NGS) has transformed clinical histocompatibility laboratories through its capacity to provide accurate, high-throughput, high-resolution typing of Human Leukocyte Antigen (HLA) genes, which is critical for transplant safety and success. As this technology becomes widely used for clinical genotyping, histocompatibility laboratories now have an increased capability to identify novel HLA alleles that previously would not be detected using traditional genotyping methods. Standard guidelines for the clinical verification and reporting of novelties in the era of NGS are greatly needed. Here, we describe the experience of a clinical histocompatibility laboratory's use of NGS for HLA genotyping and its management of novel alleles detected in an ethnically-diverse population of British Columbia, Canada. Over a period of 18 months, 3,450 clinical samples collected for the purpose of solid organ or hematopoietic stem cell transplantation were sequenced using NGS. Overall, 29 unique novel alleles were identified at a rate of ∼1.6 per month. The majority of novelties (52%) were detected in the alpha chains of class II (HLA-DQA1 and -DPA1). Novelties were found in all 11 HLA classical genes except for HLA-DRB3, -DRB4, and -DQB1. All novelties were single nucleotide polymorphisms, where more than half led to an amino acid change, and one resulted in a premature stop codon. Missense mutations were evaluated for changes in their amino acid properties to assess the potential effect on the novel HLA protein. All novelties identified were confirmed independently at another accredited HLA laboratory using a different NGS assay and platform to ensure validity in the reporting of novelties. The novel alleles were submitted to the Immuno Polymorphism Database-Immunogenetics/HLA (IPD-IMGT/HLA) for official allele name designation and inclusion in future database releases. A nationwide survey involving all Canadian HLA laboratories confirmed the common occurrence of novel allele detection but identified a wide variability in the assessment and reporting of novelties. In summary, a considerable proportion of novel alleles were identified in routine clinical testing. We propose a framework for the standardization of policies on the clinical management of novel alleles and inclusion in proficiency testing programs in the era of NGS-based HLA genotyping.

Cutting through the weeds: Evaluation of a novel adsorption with crossmatch cells and elution protocol to sharpen HLA antibody identification by the single antigen bead assay.

The single antigen bead (SAB) assay is the most used test for the identification of HLA specific antibodies pre- and post-transplant. Nevertheless, detection of spurious reactivities remains a recognized assay limitation. In addition, the presence of weak reactivity patterns can complicate unacceptable antigen assignment. This work presents the evaluation of the adsorption with crossmatch cells and elution (AXE) technique, which was designed to help differentiate weak HLA specific antibodies targeting native antigens from spurious and background SAB assay reactivity. The AXE protocol uses selected donor cells to adsorb HLA specific antibodies from sera of interest. Bound antibodies are then eluted off washed cells and identified using the SAB assay. Only antibodies targeting native HLA are adsorbed. Assay evaluation was performed using five cell donors and pooled positive control serum. AXE efficiency was determined by comparing SAB reactivity of adsorbed/eluted antibody to that of the antibodies in unadsorbed sera. A robust efficiency was seen across a wide range of original MFI for donor specific antibodies (DSA). A higher absorption/elution recovery was observed for HLA class I antigens vs. class II. Locus-specific variation was also observed, with high-expression HLA loci (HLA-A/B/DR) providing the best recovery. Importantly, negligible reactivity was detected in the last wash control, confirming that AXE eluates were not contaminated with HLA antibody carry-over. Donor cells incubated with autologous and DSA-containing allogeneic sera showed that AXE selectively adsorbed HLA antibodies in a donor antigen-specific manner. Importantly, antibodies targeting denatured epitopes or other non-HLA antigens were not detected by AXE. AXE was particularly effective at distinguishing weak HLA antibodies from background reactivity. When combined with epitope analysis, AXE enhanced precise identification of antibody-targeted eplets and even facilitated the characterization of a potential novel eplet. Comparison of AXE to flow cytometric crossmatching further revealed that AXE was a more sensitive technique in the detection of weak DSA. Spurious reactivities on the current SAB assay have a deleterious impact on the assignment of clinically relevant HLA specificities. The AXE protocol is a novel test that enables users to interrogate reactive patterns of interest and discriminate HLA specific antibodies from spurious reactivity.

Use and Outcomes of Induction Therapy in Well-Matched Kidney Transplant Recipients.

BACKGROUND AND OBJECTIVES: The optimal induction treatment in low-immune risk kidney transplant recipients is uncertain. We therefore investigated the use and outcomes of induction immunosuppression in a low-risk cohort of patients who were well matched with their donor at HLA-A, -B, -DR, -DQB1 on the basis of serologic typing. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: Our study was an observational study of first adult kidney-only transplant recipients in the United States recorded by the Organ Procurement and Transplant Network. RESULTS: Among 2976 recipients, 57% were treated with T cell-depleting antibodies, 28% were treated with an IL-2 receptor antagonist, and 15% were treated without induction. There was no difference in allograft survival, death-censored graft survival, or death with function between patients treated with an IL-2 receptor antagonist and no induction therapy. In multivariable models, patients treated with T cell-depleting therapy had a similar risk of graft loss from any cause, including death (hazard ratio, 1.19; 95% confidence interval, 0.98 to 1.45), compared with patients treated with an IL-2 receptor antagonist or no induction. The findings were consistent in subgroup analyses of Black recipients, patients grouped by calculated panel reactive antibody, and donor source. The incidence of acute rejection at 1 year was low (≤5%) and did not vary between treatment groups. CONCLUSIONS: Use of induction therapy with T cell-depleting therapy or IL-2 receptor antagonists in first kidney transplant recipients who are well matched with their donor at the HLA-A, -B, -DR, -DQB1 gene loci is not associated with improved post-transplant outcomes.

Pretransplant Calculated Panel Reactive Antibody in the Absence of Donor-Specific Antibody and Kidney Allograft Survival.

BACKGROUND AND OBJECTIVES: Panel reactive antibody informs the likelihood of finding an HLA-compatible donor for transplant candidates, but has historically been associated with acute rejection and allograft survival because testing methods could not exclude the presence of concomitant donor-specific antibodies. Despite new methods to exclude donor-specific antibodies, panel reactive antibody continues to be used to determine the choice of induction and maintenance immunosuppression. The study objective was to determine the clinical relevance of panel reactive antibody in the absence of donor-specific antibodies. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: Retrospective observational study of kidney allograft survival among 4058 zero HLA-A-, B-, DR-, and DQB1-mismatched transplant recipients without antibodies to donor kidney antigens encoded by these HLA gene loci. RESULTS: Among 4058 first and repeat transplant recipients, patients with calculated panel reactive antibody (cPRA) 1%-97% were not at higher risk of transplant failure, compared with patients with cPRA of 0% (death censored graft loss: hazard ratio, 1.07; 95% confidence interval, 0.82 to 1.41). Patients with cPRA ≥98% had a higher risk of graft loss from any cause including death (hazard ratio, 1.39; 95% confidence interval, 1.08 to 1.79) and death censored allograft failure (hazard ratio, 1.78; 95% confidence interval, 1.27 to 2.49). In stratified analyses, the higher risk of graft loss among patients with cPRA ≥98% was only observed among repeat, but not first, transplant recipients. In subgroup analysis, there was no association between cPRA and graft loss among living related transplant recipients. CONCLUSIONS: Calculated panel reactive antibody is poorly associated with post-transplant immune reactivity to the allograft in the absence of donor-specific antibody. PODCAST: This article contains a podcast at https://www.asn-online.org/media/podcast/CJASN/2021_01_25_CJN13640820_final.mp3.

Comprehensive Immune Profiling of a Kidney Transplant Recipient With Peri-Operative SARS-CoV-2 Infection: A Case Report.

To date there is limited data on the immune profile and outcomes of solid organ transplant recipients who encounter COVID-19 infection early post-transplant. Here we present a unique case where the kidney recipient's transplant surgery coincided with a positive SARS-CoV-2 test and the patient subsequently developed symptomatic COVID-19 perioperatively. We performed comprehensive immunological monitoring of cellular, proteomic, and serological changes during the first 4 critical months post-infection. We showed that continuation of basiliximab induction and maintenance of triple immunosuppression did not significantly impair the host's ability to mount a robust immune response against symptomatic COVID-19 infection diagnosed within the first week post-transplant.

Clinical Utility of Complement Dependent Assays in Kidney Transplantation.

Formation of antibodies against polymorphic HLA molecules on donor endothelium is central to the pathogenesis of antibody-mediated rejection, the dominant cause of long-term kidney allograft loss. Although introduction of the single-antigen bead assay has greatly facilitated the immune risk assessment of transplant recipients, it is recognized that not all IgG HLA antibodies detected using this method are equally relevant. In recent years, novel assays (C4d, C1q, C3d) have been developed to interrogate the complement-activating potential of anti-HLA antibodies in vitro, with the hypothesis that complement-fixing antibodies are more immediately injurious to the graft compared with noncomplement-binding antibodies. Although initial studies demonstrated the potential of these assays to risk-stratify antibodies beyond the conventional limited metric of mean fluorescence intensity values, new data from recent analyses challenge some of these early findings. In this review, we examine the technical aspects of these assays and key studies that evaluated the discriminant capacity of these tests to predict numerous outcomes in kidney transplantation. We discuss conflicting data and emerging controversies in the context of recent experimental evidence which offer new insights into the major factors that influence complement activation. Finally, we provide our perspective on the current role and utility of complement diagnostic assays as 1 variable in the multifactorial risk assessment and management of kidney transplant recipients.

Application of High-Throughput Next-Generation Sequencing for HLA Typing on Buccal Extracted DNA: Results from over 10,000 Donor Recruitment Samples.

BACKGROUND: Unambiguous HLA typing is important in hematopoietic stem cell transplantation (HSCT), HLA disease association studies, and solid organ transplantation. However, current molecular typing methods only interrogate the antigen recognition site (ARS) of HLA genes, resulting in many cis-trans ambiguities that require additional typing methods to resolve. Here we report high-resolution HLA typing of 10,063 National Marrow Donor Program (NMDP) registry donors using long-range PCR by next generation sequencing (NGS) approach on buccal swab DNA. METHODS: Multiplex long-range PCR primers amplified the full-length of HLA class I genes (A, B, C) from promotor to 3' UTR. Class II genes (DRB1, DQB1) were amplified from exon 2 through part of exon 4. PCR amplicons were pooled and sheared using Covaris fragmentation. Library preparation was performed using the Illumina TruSeq Nano kit on the Beckman FX automated platform. Each sample was tagged with a unique barcode, followed by 2×250 bp paired-end sequencing on the Illumina MiSeq. HLA typing was assigned using Omixon Twin software that combines two independent computational algorithms to ensure high confidence in allele calling. Consensus sequence and typing results were reported in Histoimmunogenetics Markup Language (HML) format. All homozygous alleles were confirmed by Luminex SSO typing and exon novelties were confirmed by Sanger sequencing. RESULTS: Using this automated workflow, over 10,063 NMDP registry donors were successfully typed under high-resolution by NGS. Despite known challenges of nucleic acid degradation and low DNA concentration commonly associated with buccal-based specimens, 97.8% of samples were successfully amplified using long-range PCR. Among these, 98.2% were successfully reported by NGS, with an accuracy rate of 99.84% in an independent blind Quality Control audit performed by the NDMP. In this study, NGS-HLA typing identified 23 null alleles (0.023%), 92 rare alleles (0.091%) and 42 exon novelties (0.042%). CONCLUSION: Long-range, unambiguous HLA genotyping is achievable on clinical buccal swab-extracted DNA. Importantly, full-length gene sequencing and the ability to curate full sequence data will permit future interrogation of the impact of introns, expanded exons, and other gene regulatory sequences on clinical outcomes in transplantation.

Impact of three Illumina library construction methods on GC bias and HLA genotype calling.

Next-generation sequencing (NGS) is increasingly recognized for its ability to overcome allele ambiguity and deliver high-resolution typing in the HLA system. Using this technology, non-uniform read distribution can impede the reliability of variant detection, which renders high-confidence genotype calling particularly difficult to achieve in the polymorphic HLA complex. Recently, library construction has been implicated as the dominant factor in instigating coverage bias. To study the impact of this phenomenon on HLA genotyping, we performed long-range PCR on 12 samples to amplify HLA-A, -B, -C, -DRB1, and -DQB1, and compared the relative contribution of three Illumina library construction methods (TruSeq Nano, Nextera, Nextera XT) in generating downstream bias. Here, we show high GC% to be a good predictor of low sequencing depth. Compared to standard TruSeq Nano, GC bias was more prominent in transposase-based protocols, particularly Nextera XT, likely through a combination of transposase insertion bias being coupled with a high number of PCR enrichment cycles. Importantly, our findings demonstrate non-uniform read depth can have a direct and negative impact on the robustness of HLA genotyping, which has clinical implications for users when choosing a library construction strategy that aims to balance cost and throughput with data quality.