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Donor-specific alloantibodies (DSA) to HLA-DP may cause antibody-mediated rejection (AMR), especially in re-transplants. We describe the immunization history of a patient who received 3 kidney transplants; the 3rd kidney was completely matched except at DPA1 and DPB1. Prior to the 3rd transplant, single antigen bead analysis (SAB) showed DSA reactivity against DPA1 shared by the 1st and 3rd donors, but B and T flow crossmatch (FXM) results were negative. Within 11 days the 3rd transplant underwent acute C4d+ AMR which coincided with the presence of complement (C1q)-binding IgG1 DSA against donor DPA1 and DPB1. Using HLAMatchmaker and SAB, we provide evidence that eplet (epitope) spreading on DPA1 and eplet sharing on differing DPB1 alleles of the 1st and 3rd transplants was associated with AMR. Since weak DSA to DPA1/DPB1 may induce acute AMR with negative FXM, donor DPA1/DPB1 high resolution typing should be considered in sensitized patients with DP-directed DSA.

Aim Computer-identified possible Kidney Paired Donation (KPD) exchanges are presented as offers to participating transplant programs and eventually converted into transplants. We evaluated reasons why these offers failed to progress into successful transplants. Methods/Results The Alliance for Paired Donation (APD) has an optimization algorithm to generate possible KPD exchanges, which are presented to transplant centers for their review as offers. Each offer may be composed of one to multiple 1-way exchanges. We tracked the progress in moving from offers to transplants and recorded the reason for failure. Between January 2008 and August 2014, the APD made 981 offers. Of these offers 181 resulted in transplants while 800 failed, namely 268 failed for specific reasons while 529 failed as they were dependent on the same 268 1-ways. Overall, specific reasons accounted for 57% of all failures with: 25% positive flow crossmatch (FXM); 13% competing offer outside the APD; and, 19% declined of donors by transplant centers (Fig. 1). Our detail analysis showed that the 25% positive FXM with negative virtual crossmatch (VXM) correlated with an increased number of highly sensitized patients: the pool of sensitized patients with a PRA >80% has increased from 31% in 2008 to 47% in 2014. Since many patients choose to enroll in multiple KPD registries some offers resulted in futile exchanges with patients already committed to alternative exchanges. Finally, donors were declined by transplant centers. To avoid these specific reasons for failure, the APD has begun pre-review consulting about donors and recipients prior to making a formal offer. Conclusions To prevent specific reasons for failure the APD has implemented the following changes: (1) testing of all donors for HLA-DQA, DPA and DPB; (2) expansion of the donor pool to increase options for highly sensitized patients; and (3) improved coordination with other KPD programs to avoid competing offers. Download : Download full-size image

Aim To perform a retrospective analysis of the 3rd kidney transplant rejection with exclusive disparity at HLA-DP. Methods The 1st transplant, nearly completely mismatched, functioned well for 7 years but was rejected due to chronic allograft nephropathy. The 2nd transplant, completely matched except at DRB3 and DPB1, failed after 1 year with acute and chronic rejection. A 3rd kidney was completely matched except at DPA1*02:01 and DPB1*01:01. Although single antigen bead (SAB) analysis showed donor-specific alloantibodies (DSA) directed to DPA1*02:01 (3496-4024 MFI), both B and T flow crossmatches (FXM) were negative. Within 10 days the re-transplant underwent aggressive C4d+ acute antibody-mediated rejection (AMR; Banff 2a). Despite thymoglobulin therapy and 4 cycles of plasmapheresis, kidney failed within 6 months. At 30 days post-transplant, SAB assay revealed that DSA (directed to donor DPA1*02:01 and DPB1*01:01 bead) was IgG (9511 MFI) of IgG1 subclass (8664 MFI) with C1q binding function (22800 MFI). Results Epitope analysis showed that the 1st transplant shared DPA1*02:01 with the 3rd transplant while the 1st transplant DPB1*11:01 shared two epitopes (35YA, 84DEAV) with the 3rd donor DPB1*01:01. DSA response directed to just one epitope (111R) on DPA1*02:01 spread to other epitopes (83A, 51RA, 127P)(Fig. 1). Conclusions (1) DPA1 and DPB1 alleles may induce an aggressive acute AMR resulting in graft failure; (2) DSA directed to donor DPA1 allele may be undetected by FXM; (3) DPA1 and DPB1 alleles should be identified in donors of re-transplanted patients with DPA1/DPB1-directed Abs; and 4) epitope spreading and epitope sharing enhance FXM-undetectable DSA during acute AMR [Fig. 1].

Aim To identify HLA antibody (Ab) reactivity in a patient undergoing acute Ab-mediated rejection (AMR) following renal transplantation from a zero-mismatched HLA-A/B/C/DR/DQ donor. Methods We report here a patient who received 3 transplants based on the negative flow crossmatch (FXM). HLA sequencing methods were used to retrospectively identify recipient and donor HLA alleles. Luminex-based single antigen bead (SAB) and SAB-C1q assays were used to measure HLA- and HLA-C1q-binding Ab. HLAMatchmaker was used to evaluate epitope reactivity. Results A pediatric patient was immunized by a 1st transplant with multiple class I/II HLA mismatches and blood transfusions; this transplant lasted 10 years and was lost to chronic nephropathy. The HLA-matched (except for DRB3*02:02) 2nd transplant failed within 1 year due to acute and chronic rejection. The 3rd transplant was matched for HLA-A/B/C/DR/DQ but mismatched for HLA-DPA1*02:01 and -DPB1*01:01, *04:02. Although prior to the 3rd transplant the patient had DPA1*02:01-reactive Ab (DSA) in SAB (3518 MFI) and SAB-C1q (4900 MFI) assays, the FXM results were negative. Within 10 days post-transplant, the patient had elevated creatinine (3 mg/dL) and C4d+ biopsy-confirmed AMR (Banff 2a). This correlated with reactivity against DPA1*02:01 in SAB (18483 MFI; Figure) and SAB-C1q+ (25940 MFI). During the next 4 months the patient had additional AMR episodes confirmed by DSA and C4d+ biopsy, and was treated with plasmapheresis, IVIg, and rituximab. HLAMatchmaker analysis revealed that the DSA reactivity [directed to one epitope (111R) on DPA1*02:01 shared by the 1st and 3rd donors] was present prior to and peaked during rejection episodes post-transplant. Although a historical serum had weak DPB1 Ab reactivity, there was none immediately pre-transplant. Post-transplant sera showed SAB-positive but SAB-C1q negative DPB1 Ab reactivity. Conclusions These findings suggest that SAB-C1q reactive DPA1-specific DSA may mediate acute AMR.

Isolating sufficient B cells for DR typing is often a problem, particularly in patients with low lymphocyte and/or B cell counts. This paper describes a simple method for preparing highly purified B cells using monoclonal antibodies and complement to kill the majority of non-B cells. Mononuclear cells are isolated by standard Ficoll-Hypaque techniques and incubated at 0 degrees C for 10 min with the following monoclonal antibodies: OKT3 (pan T cell), OKT11 (pan T and null cell), and Leu11b (NK cell). Rabbit complement is added and the mixture is incubated at 37 degrees C for 20 min. Dead cells are then removed by Percoll gradient centrifugation. This procedure results in cell preparations containing 90.1 +/- 1.3% B cells as measured by two-colow immunofluorescence with an Epics C flow cytometer. A yield of 52% can routinely be obtained. In contrast, nylon wool adherence results in preparations with only 44 +/- 9.1% B cells with a yield of 31%. B cells prepared by the monoclonal antibody procedure always show stronger and clearer reactions on DR typing trays. These results are consistent with the flow cytometric analyses of the B cell preparations. In summary, this procedure has the following advantages: increased accuracy of DR typing, decreased sample size (only 1.0 X 10(7) mononuclear cells are required), reduced time of cell preparation, and reduction in the number of repeat typings necessitated by poor yields and/or results.