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Alloimmune responses in kidney transplant (KT) patients previously hospitalized with COVID-19 are understudied. We analyzed a cohort of 112 kidney transplant recipients who were hospitalized following a positive SARS-CoV-2 test result during the first 20 months of the COVID-19 pandemic. We found a cumulative incidence of 17% for the development of new donor-specific antibodies (DSA) or increased levels of pre-existing DSA in hospitalized SARS-CoV-2-infected KT patients. This risk extended 8 months post-infection. These changes in DSA status were associated with late allograft dysfunction. Risk factors for new or increased DSA responses in this KT patient cohort included the presence of circulating DSA pre-COVID-19 diagnosis and time post-transplantation. COVID-19 vaccination prior to infection and remdesivir administration during infection were each associated with decreased likelihood of developing a new or increased DSA response. These data show that new or enhanced DSA responses frequently occur among KT patients requiring admission with COVID-19 and suggest that surveillance, vaccination, and antiviral therapies may be important tools to prevent alloimmunity in these individuals.

The new kidney allocation system (KAS) provides additional allocation points for candidates with broad HLA sensitization in an effort to increase transplant rates for this underserved population. Following the implementation of KAS, our center lowered the HLA antibody threshold for listing unacceptable antigens from a cytotoxicity crossmatch level to a flow cytometric crossmatch level increasing Calculated Panel Reactive Antibody (CPRA) values and allocation points, yet restricting acceptable donor HLA phenotypes. As a result, many sensitized candidates were transitioned from 50% to 98% CPRA categories into the 99% CPRA regional share and 100% CPRA national share categories. Exposure to these larger donor pools significantly increased transplantation with compatible donors for 100% CPRA candidates, but regional sharing was not sufficient to increase transplantation rates for our 99% CPRA candidates. Competition within the 100% CPRA cohort identified inequities for 99.99-100.0% CPRA candidates and highlighted the continued need for desensitization therapies to reduce immunological barriers and provide transplant opportunities for the most highly sensitized candidates.

Despite increased transplant rates for highly sensitized candidates, inequities for those with CPRA > 99.7% remain. This case highlights the efficacy of an endopeptidase (IdeS) to cleave IgG HLA-specific antibodies and complications for donor-specific HLA antibody (DSA) monitoring in the presence of therapeutic agents. DSA was evaluated using flow cytometric crossmatch tests (Flow-XM) and Luminex immunoassays (Lifecodes classes I and II single antigen; Immucor-Lifecodes, Stamford, CT and Single Antigen Beads; One Lambda, Canoga Park, CA). Sera were pretreated using Melon Columns™. A 43 year old male with a CPRA of 99.99% was entered into a clinical trial investigating the efficacy of IdeS to cleave IgG molecules and render them ineffective in eliciting complement activation or antibody-dependent cellular cytotoxicity. A deceased donor was identified to whom the patient had HLA-DP DSA yielding a positive prospective Flow-XM. Within 2 h of IdeS administration, immunoassays and a Flow-XM revealed no detectable DSA. Due to concern for DSA rebound, alternate day DSA monitoring and intense post-transplant immunosuppression were employed. Two single antigen panels confirmed DSA reappearance on POD7 prior to high dose immunoglobulin (IVIG) dosing. Administration of IVIG produced an increase in the negative and positive control beads and reactivity with self HLA-antigens (3400 mean fluorescent intensity, MFI). Utilization of multiple bead assays, Flow-XM tests, and HLA pattern analysis rather than MFI was needed to monitor further increases in DSA that would prompt therapeutic plasmapheresis (TPE). Cumulative DSA test analysis revealed a stabilization of DSA accompanied by declining serum creatinine values with no initiation of TPE. New therapeutics hold promise for removing DSA barriers; however, post transplant monitoring can be challenging due to test interference that accompany these biologics. This case illustrates the use of multiple assays and HLA pattern analysis to guide post transplant care. Download : Download high-res image (270KB) Download : Download full-size image A.M. Jackson: 3. Speaker’s Bureau; Company/Organization; Thermo Fisher Scientific.

Aim The new Kidney Allocation System (KAS) resulted in a significant bolus in the transplantation of 99–100 CPRA candidates due to increased priority. We examined the impact of an individualized approach to maintain a high transplant rate for these candidates three years after KAS implementation. Methods The early post-KAS period (EPKAS) was defined as deceased donor transplants occurring in 2015 and 2 year post-KAS period (2YPKAS) as 2016 and 2017. Donor specific antibody positivity (DSA+) was defined by single antigen bead assays performed pre-transplant (±7 days) and 90 days post-transplant (±14 days). Flow cytometric crossmatches (FCXM) were performed using the pre-transplant serum, donor lymphocytes, and acquisition on a BD Canto instrument. Data from SRTR was used to assess outcome at 1 year for our center and nationally. Results Nationally, transplantation of 99–100 CPRA candidates rose sharply (17.7%) in the EPKAS period and has since tapered (10%) in the 2YPKAS period. At our center, we observed a similar increase in the EPKAS period (16%, n = 30) but this trend increased further during the 2YPKAS period (28%, n = 75). In the EPKAS period, 60% (n = 18) of 99–100 CPRA recipients were DSA+ and 39% (n = 7) of DSA+ recipients were also FCXM positive. 61% (n = 11) of DSA+ recipients during this period had persistent DSA at 90 + days post-transplant. In the 2YPKAS period, a similar percentage (57%, n = 43) of 99–100 CPRA recipients were DSA+ at time of transplant; however, fewer (8%, n = 6) DSA+ recipients were also FCXM+. Correspondingly, fewer (34%, n = 26) DSA+ recipients had persistent DSA at 90 + days post-transplant. A total of five (5%) candidates in the post-KAS era experienced a rise in DSA post-transplant that required intervention. The hazard ratio for 1 year graft survival was 1.05 for our center and 1.00 nationally. Conclusions Our center continues to successfully transplant 99–100 CPRA candidates at a rate higher than the national average. This has been achieved through a personalized approach for defining unacceptable antigens and assessing donor offers, transplanting across DSA barriers, and a stringent post-transplant monitoring protocol. Refinement in our approach in the 2 year post-KAS has resulted in more compatible transplants, less DSA persistence, and the maintenance of good clinical outcomes. A.M. Jackson: 3. Speaker’s Bureau; Company/Organization; Thermo-Fisher.

Identification of factors responsible for an increase in the breadth or strength of HLA-specific antibody (HSA) is critical to the continued successful management and transplantation of sensitized patients. A retrospective review of our HLA registry identified 107 patients with known HSA and sufficient information in their electronic patient record to determine the presence or absence of a proinflammatory event. The patients were stratified according to transplant status [sensitized and on the transplant waitlist (n = 65); immunosuppressed recipients of a positive crossmatch (+XM) transplant (n = 42)]. Eighty-three percent of waitlist candidates and 55% of sensitized kidney transplant recipients with a documented proinflammatory event had an associated increase in HSA. Interestingly, among patients with a culture-proven infection, 97% of the waitlist patients and 54.8% of +XM recipients had an associated rise in HSA. Overall, proinflammatory events were associated with a greater increase among waitlist patients than +XM recipients, 5.3-fold [IRR 5.25, (95% CI 4.03-6.85), p < 0.001] versus 2.5-fold [IRR 2.54, (95% CI 1.64-3.95), p < 0.001] increase in HSA. Therefore, sensitized patients known to have an infection or undergoing surgery should be monitored for expansion of HSA.

Virtual crossmatches have been performed for more than 40 years under the guise of unacceptable antigens. Today, solid-phase assays provide the opportunity for more accurate identification and more precise measurement of the strength of donor-specific antibodies. The process of performing a virtual crossmatch begins with establishing a correlation between the antibody testing assay and the results of actual crossmatches. We provide here data indicating that the identity and strength of DSA defined with solid-phase phenotype panels correlates significantly with the outcome of both cytotoxic (CDC; r = 0.83) and flow cytometric (r = 0.85) crossmatches. Based on the threshold established from these correlations, we were able to correctly predict the results of CDC and flow cytometric crossmatches in 92.8 and 92.4% of cases, respectively. The correlations with single antigen panels were substantially lower (82.6-47.9%) and may be caused by a variety of factors, including variability in the amount and condition of different antigens and extremely high sensitivity, which may make the test less robust. We demonstrate that adding additional information to the solid-phase results can increase the frequency of correct crossmatch prediction. We also present data demonstrating an additional use of the virtual crossmatch in posttransplant monitoring.

Current models for allocation of kidneys from living non-directed donors Living non-directed (LND) donors, also known as altruistic, good Samaritan, anonymous, or benevolent community donors, are a new and rapidly growing source of solid organs for transplantation. The willingness of individuals to donate organs without a designated recipient has been unexpected, but has probably developed as a societal response to the growing crisis in organ availability. In the context of this shortage, health professionals have attempted to make the best use of kidneys from LND donors. We present a novel application of paired donation that has the potential to multiply the number of recipients who can benefi t from each LND donation. At present, there is no universally accepted system for allocation of organs from LND donors. Selection of recipients has been at the discretion of the transplant centres where LND donors have presented and has generally been guided by one of three models: donorcentric, recipient-centric, or sociocentric allocation. Each of these models is supported by valid ethical arguments. The main goal of donor-centric allocation is to ensure a successful outcome for the recipient. A good outcome provides justifi cation for medical professionals to assist a person who is not ill to put themselves in harm’s way to aid another. A positive result also gives an LND donor a sense that their eff ort was fruitful and worthwhile. However, this model dictates allocation to the healthiest patient on the transplant waiting list. These recipients are the most likely to have good outcomes on dialysis or with organs from deceased donors, and therefore are arguably the least in need. Recipient-centric allocation is based on the belief that society has a responsibility to protect its most vulnerable and disadvantaged members. Under this model, organs from LND donors are given to those patients in the greatest need, those for whom a kidney transplant might be truly life saving, or those disadvantaged under the existing system for allocation of kidneys from deceased donors. This model mainly benefi ts children, patients who have no vascular access, highly sensitised patients, and those with life-threatening medical illnesses related to dialysis. However, because the recipient-centric model accords priority to such patients, it tends to yield unacceptably poor transplant outcomes, and could lead to a negative public perception of LND donation. Under the third model, of sociocentric allocation, the LND donated organ is treated as a public resource that should be allocated in the fairest and most equitable way, irrespective of outcome or need. This rationale dictates that the recipient should be the patient at the top of the transplant waiting list administered by the United Network of Organ Sharing (UNOS). UNOS oversees the allocation of deceased donor organs in the USA, using a so-called match run algorithm that ranks potential recipients according to agreed criteria. The limitations of this model are that a patient at the top of the list will probably receive a kidney from a deceased donor in the near future, and that they will have already incurred the costs, and exposure to comorbidity, that result from a long period on dialysis. The waiting list for deceased donor kidneys can be circumvented by patients who fi nd a willing live donor. But direct donation might be complicated by diff erences in blood type and by HLA sensitivity. Some incompatible donor-recipient pairs enter into programmes that facilitate paired donation, also known as kidney paired donation. A donor and recipient who have incompatible blood groups or HLA sensitivity can be matched with another incompatible pair, to result in two compatible transplants (fi gure). Although there are many ways to match up a pool of incompatible pairs, the mathematical technique of optimisation helps to fi nd out which matches will yield the best results. Nevertheless, even in paired-donation programmes in which mathematical optimisation is applied, more than 50% of the incompatible pairs in the pool remain unmatched. In many cases, pools of incompatible donor-recipient pairs have a high proportion of patients with blood types that are hard to match and those with HLA sensitisation.

Aim The new Kidney Allocation System (KAS) provides additional allocation points for sensitized candidates in an effort to increase transplant rates for this underserved population Following the implementation of KAS, our center lowered the HLA antibody threshold for listing unacceptable antigens from a cytotoxicity level to a flow cytometric crossmatch level. The aim of this study was to assess the effect of KAS following CPRA optimization for our renal waitlist candidates. Methods We examined renal transplants that occurred 11 months prior and post CPRA optimization. We investigated transplant numbers per CPRA category; the average number of Donor Specific Antibodies (DSAs) at transplant; the number of post-transplant specimens submitted, and Virtual Crossmatch (VXM) requests for high CPRA (>97) patients. Results During an 11 month period prior to CPRA optimization our center performed 105 deceased donor transplants. Of these, 84.76% were patients with a CPRA value of 0, while 1.04% were patients with high CPRA values (>97%). Of the 175 deceased donor transplants performed after our laboratory’s CPRA optimization, 26.38% were patients with a CPRA >97%. Transplanted individuals with a 0% CPRA decreased to 57.67%. Patients transplanted pre-optimization versus post-optimization submitted an average of 12 and 48.5 samples per month, respectively. Patients transplanted pre-optimization and post-optimization had an average of 0.46 and 0.47 donor specific antibodies, respectively. Our instances of VXM requests for high CPRA patients more than tripled following the implantation of KAS. VXM numbers: October 2014 (0), April 2015 (10), October 2015 (29), April 2016 (36). Conclusions More high CPRA patients are being transplanted following implementation of KAS. Our laboratory has experienced an increase in VXM assessments and post-transplant DSA testing received while experiencing similar numbers of DSAs prior to transplant. Download : Download high-res image (72KB) Download : Download full-size image

Providing transplantation opportunities for patients with incompatible live donors through kidney paired donation (KPD) is seen as one of the important strategies for easing the crisis in organ availability. It has been estimated that an additional 1000-2000 transplants per year could be accomplished if a national KPD program were implemented in the United States. While most of these transplants could be arranged within the participants' local or regional area, patients with hard-to-match blood types or broad HLA sensitization would benefit from matching across larger geographic areas. In this case, either patients or organs would need to travel in order to obtain maximum benefit from a national program. In this study, we describe how a triple KPD enabled a highly sensitized patient (PRA 96%) to receive a well-matched kidney from a live donor on the opposite coast. The kidney was removed in San Francisco and transported to Baltimore where it was reperfused 8 h later. The patient had prompt function and 1 year later has a serum creatinine of 1.1 mg/dl. This case provides a blueprint for solving some of the complexities that are inherent in the implementation of a national KPD program in a large country like the United States.

The algorithm for determining CPRA values that incorporate two or more of the HLA-A, -B, -C, -DRB1, -DRB3-5, -DQA, and -DQB loci requires information about the frequencies of alleles at the various loci as well as of the haplotypes comprised of the loci to which a patient has antibodies. Increasing awareness of the relevance of antibodies to HLA-DP has made it desirable to include DP in the CPRA calculation. DPB alleles sort independently from HLA alleles telomeric to DPA and therefore, do not require the frequencies of DP haplotypes for incorporation into CPRA. Calculations of CPRA that include DP incompatibilities (CPRA-DP) were performed according to the following algorithm: CPRA-DP = 1 − p2 (1-CPRA) where p is the sum of the frequencies of compatible DP alleles and CPRA is the currently used CPRA calculation. DP alleles frequencies were determined from 1082 deceased donors typed in our laboratory. CPRA-DP was determined for patients grouped by traditional CPRA values for varying degrees of DP incompatibility. As expected, the impact of DP incompatibility was greatest for patients with the lowest CPRA values and decreased as CPRA values increased. Depending on the specificity of the DP antibodies, including DP in the CPRA was shown to make a substantial difference in all but the highest CPRA categories. Given the addition of a sliding point scale for CPRA and prioritization for the highest CPRA candidates in the new kidney allocation system (KAS), the addition of DP to the CPRA calculation would benefit all candidates with DP antibodies. Download full-size image

Aim Donor HLA-specific antibody represents a risk for rejection or engraftment failure following HLA mismatched solid organ or hematopoietic stem cell transplantation. We have examined the immunogenicity of Bw4 subtype mismatches in 211 renal recipients post-transplant. Methods HLA typing was performed using Luminex based rSSOP (LAB Type, One Lambda). HLA single antigen panels (LABScreen, One Lambda) were used to identify HLA-Bw4 sensitization. Results Bw4 epitopes present on HLA-B and HLA-A antigens can be divided into 8 subtypes based on amino acid differences at positions 77–83 (Voorter et al. Tissue Antigens, 2000). Furthermore, residues 80–83 have been shown to impact the electrostatic charge and explain observed serological Bw4 patterns (Kosmoliaptsis et al. Human Immunol. 2011). Bw4 motifs TALR and TLLR (expressed on HLA-B∗44:02, B∗13:01 and B∗37:01, B∗27:05, B47:01, respectively) have similar electrostatic potentials, but are uniquely different from the IALR Bw4 motif (expressed on HLA-B∗57:01, 58:01, 49:01, 51:01, 53:01, 15:16, 15:13, 38:01 59:01 and HLA-A23:01, 24:02, 24:03, 25:01,32:01). Previously, using serum/cell adsorption experiments, we have shown that sensitization to the IALR can occur in recipients possessing TALR or TLLR. The incidence of sensitization to these different Bw4 motifs is shown in Table 1.The IALR motif evoked the highest incidence of sensitization in recipients possessing Bw6, Bw4-TALR, or Bw4-TLLR in their own phenotype. In contrast, mismatches involving TALR or TLLR provoked little sensitization in recipients possessing IALR. Conclusions Structural and electrostatic potential differences between Bw4 subtypes appear sufficient to elicit alloimmune responses. Exposure to IALR, the most common Bw4 motif, can provoke a substantial alloantibody barrier to prospective donors (CPRA of 54%). Moreover, Bw4 sensitization may be relevant when selecting HLA allele mismatched donors for antigens that contain multiple Bw4 subtypes (i.e. HLA-B27, B38, B44, and B53). Download : Download full-size image

The number of renal transplants can be increased by implementing an exchange program involving donor-recipient pairs for whom the donors are each incompatible with their original patient but compatible with each other's patient. The number can be further increased if the exchanges are not limited to ABO incompatible pairs or combinations of two donor-recipient pairs. However, as the number of donor-recipient pairs willing to participate in such a program increases, there is a substantial increase in both the time taken to identify such matches and the potential for error. We have developed a computer program that accounts for ABO and HLA compatibility and is not limited to two-way exchanges. With our database of 60 patients and 83 donors, we have been able to identify 122 two-way and 1230 three-way exchanges with an average run time of 30 s.

Aim Use of the virtual crossmatch (XM) has increased the efficiency of organ allocation and reduced the number of organs declined based on unexpected positive XMs (Cecka JM., et al. Am J Transplant. 2011). Despite these benefits, a cell based XM still provides a final safeguard before transplantation. False negative XM predictions may arise when reactivity is due to both antigen specific antibodies and antibodies to crossreactive epitopes. Methods HLA-specific antibodies were assessed using class I or class II phenotype panels and IgG or C1Q single antigen bead assays. (Lifecodes Immucor; Labscreen, One Lambda,Inc). One wash CDC B cell XMs were performed. Results Luminex IgG tests yielded reactivity with DR53 phenotypes ranging from 3,000-10,000 MFI with DR53 heterozygotes and >10,000 MFI with homozygotes. Reactivity with DR10 phenotypes were 2,000-3,000 MFI. Single antigen beads yielded reactions with DR53 of 20,000 MFI with undiluted serum and 17,000 MFI with serum diluted 1:8. Reactions with DR10 were 15,000 and 2,000 MFI with undiluted and 1:8 diluted serum, respectively. MFI values given above would predict negative XMs with DR10+/DR53- cells and positive XMs with DR10-/DR53+ cells. The C1q assay yielded MFI values of 28,000 with DR53 and 300 with DR10. These C1Q results substantiate the prediction of a negative CDC XM result with a DR10 cell. CDC XM tests yielded the following results: + with a titer of 2 with a DR10+/DR53- cell, + with a titer of 32 with a DR10-/DR53 homozygote cell, and negative with DR53 heterozygotes and with cells bearing other antigens crossreactive with DR10 (DR1 and DR15/51). Conclusions We propose that the CDC+ XM with the DR10 cell was due to contributions from the DR10 antibodies and the much stronger antibodies to DR53 that shares one or more epitopes with DR10. These data show that accurate prediction of crossmatches must take into account the possible contributions of antibodies positive for antigens crossreactive with the target antigen.

Aim Interpretation of HLA antibody reactivity should consider different HLA-Bw4 epitopes associated with HLA-B and HLA-A alleles. Methods HLA typing was performed using Luminex based rSSOP (LAB Type, One Lambda) or sequence based typing (AlleleSEQR, Celera). HLA antibody testing used HLA phenotype (LIFECODES, Gen-Probe) and single antigen (LAT, One Lambda) bead panels on a Luminex platform. Results Bw4 is an epitope present on several HLA-A and HLA-B antigens that can be divided into 8 subtypes based on amino acid differences (Tissue Ag 2000, 56:363). Recently, residues 80-83 have been shown to influence electrostatic potential of the Bw4 epitope, explaining differences in serological patterns (Human Immunol 2011, 72:1049). We present two patients with Bw4 present in their own phenotype yet they generated antibody to a different Bw4 subtype. Patient 1 typed as a B ∗ 44:03, containing Bw4 residues 80 T -81 A -82 L -83 R . She was sensitized through pregnancy to her daughter who typed as a HLA-B ∗ 27:02 containing Bw4 residues 80 I -81 A -82 L -83 R . This daughter was a potential HLA mismatched stem cell donor. Patient 2 was a renal patient who typed as a HLA-B ∗ 37:01 containing Bw4 residues 80 T -81 L -82 L -83 R . This patient was sensitized to HLA-B51 and -B53, both contain Bw4 residues 80 I -81 A -82 L -83 R . Patient 2 is being evaluated for retransplantation. For both patients, the strongest antibody reactivity was toward beads containing the Bw4 motif 80 I -81 A -82 L -83 R , to which they were both sensitized. Moreover, there were differences in antibody strength toward HLA antigens that differed only by the 80 I -81 A -82 L -83 R epitope (B49 vs B50 and B38 vs B39). Cell adsorptions are underway to confirm the presence of Bw4 subtype-specific antibodies. Conclusions Sensitization to Bw4 subtypes may be relevant when selecting HLA allele mismatched donors for antigens that contain multiple Bw4 subtypes. Accurate characterization of antibody specificity permits identification of incompatible alleles not represented on HLA bead panels.

ContextFirst proposed 2 decades ago, live kidney paired donation (KPD) was considered a promising new approach to addressing the shortage of organs for transplantation. Ethical, administrative, and logistical barriers initially proved formidable and prevented the implementation of KPD programs in the United States.ObjectiveTo determine the feasibility and effectiveness of KPD for the management of patients with incompatible donors.Design, Setting, and PatientsProspective series of paired donations matched and transplanted from a pool of blood type or crossmatch incompatible donors and recipients with end-stage renal disease (6 conventional and 4 unconventional KPD transplants) at a US tertiary referral center (between June 2001 and November 2004) with expertise in performing transplants in patients with high immunologic risk.InterventionKidney paired donation and live donor renal transplantation.Main Outcome MeasuresPatient survival, graft survival, serum creatinine levels, rejection episodes.ResultsA total of 22 patients received transplants through 10 paired donations including 2 triple exchanges at Johns Hopkins Hospital. At a median follow-up of 13 months (range, 1-42 months), the patient survival rate was 100% and the graft survival rate was 95.5%. Twenty-one of the 22 patients have functioning grafts with a median 6-month serum creatinine level of 1.2 mg/dL (range, 0.8-1.8 mg/dL) (106.1 μmol/L [range, 70.7-159.1 μmol/L]). There were no instances of antibody-mediated rejection despite the inclusion of 5 patients who were highly sensitized to HLA antigens due to previous exposure to foreign tissue. Four patients developed acute cellular rejection (18%).ConclusionsThis series of patients who received transplants from a single-center KPD pool provides evidence that recipients with incompatible live donors, even those with rare blood type combinations or high degrees of HLA antigen sensitization, can receive transplants through KPD with graft survival rates that appear to be equivalent to directed, compatible live donor transplants. If these results can be generalized, broader availability of KPD to the estimated 6000 patients with incompatible donors could result in a large expansion of the donor pool.