(F)Utility of the physical crossmatch for living donor evaluations in the age of the virtual crossmatch

Flow cytometric crossmatches (FCXM) are routinely performed to support living-donor renal transplantation. While long a laboratory mainstay, a physical crossmatch is costly, time consuming, and frequently poses interpretative conundrums with both false-positive and false- negative results. Given the increased utilization of the virtual crossmatch (vXM) in the deceased donor setting, our aim was to assess its utility in living donor evaluations. We reviewed 100 living donor FCXMs and retrospectively performed a vXM for each pair. Seventy-five (75) cases were concordant, (i.e., FCXM?/vXM? or FCXM+/vXM+) while 25 cases were discordant; Five were vXM+/FCXM? and 20 were FCXM+/vXM?. Since donor-specific antibodies (DSA) were not detected in the 20 FCXM+/vXM? cases, these were interpreted as false-positive, i.e., due to non-HLA antibodies. Importantly, none of these patients, when transplanted across a positive FCXM, experienced early antibody mediated rejection or subsequently developed HLA DSA. These data reveal that, for the vast majority of living donor evaluations, a vXM is an acceptable vetting procedure.     

Understanding solid-phase HLA antibody assays and the value of MFI

As the practice of medicine becomes more reliant on imaging and laboratory tests, medical decisions will be increasingly based on numbers. Accordingly, following the introduction of solid-phase testing to the HLA testing repertoire, laboratory directors and physicians have employed preset mean fluorescence intensity (MFI) thresholds as the basis for decisions in the management of transplant patients. However, what do MFI values mean? The literature is rife with reports detailing numerous factors that influence antibody assessment including (but not limited to) sensitization history of the patient, level of mismatch between donor and recipient, presence of interfering substances in the serum, whether the antigen on multiplex beads is native or denatured, day-to-day and technologist variability, and the historical performance of an assay in a given institution. How are these variables incorporated into the interpretation of MFI values? Herein, the pitfalls and complexities of single antigen bead (SAB) testing and interpretation are discussed with specific attention to what can and cannot be inferred by MFI.     

Early acute antibody-mediated rejection of a negative flow crossmatch 3rd kidney transplant with exclusive disparity at HLA-DP

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.     

HLA compatibility assessment and management of highly sensitized patients under the new kidney allocation system (KAS): A 2016 status report from twelve HLA laboratories across the U.S.

Twelve HLA laboratories were surveyed to assess the methods and operational issues involved to define highly sensitized patients and to assess HLA compatibility under the new kidney allocation system (KAS) in the U.S. All laboratories used single antigen bead assays both pre- and post-KAS to define both broad and allele-specific HLA antibodies. The methods and threshold used to list HLA unacceptable antigens in UNet for virtual crossmatch (vXM) and the criteria used for determining HLA compatibility varied among laboratories. Laboratories reported several limitations of the current assays including the accuracy of quantifiable antibody fluorescence values, inadequate coverage of common alleles on the bead panels, and challenges in calibrating the vXM. The new KAS has resulted in a significant surge of deceased donor organ offers requiring vXM evaluation under tight time constraints. In the post-KAS period, eight of twelve laboratories (67%) indicated that their center did not proceed to transplant based on vXM without a prospective lymphocyte crossmatch. In conclusion, HLA laboratories play a critical role in deceased donor allocation for highly sensitized patients under the new KAS. Significant opportunities exist to improve the methods used in the assessment of HLA compatibility to safely transplant highly sensitized patients.     

The ABCs (DRDQDPs) of the prozone effect in single antigen bead HLA antibody testing: Lessons from our highly sensitized patients

Accurate identification of HLA antibodies using the single antigen bead (SAB) assay is critical for assessment of pre/post-transplant immunological risk and successful virtual crossmatching. Unfortunately, high titer HLA antibodies can be missed or underestimated in the SAB assay as a result of interference with the detection of IgG. This so called prozone effect has been attributed to both complement- and IgM-dependent mechanisms and can be minimized with serum dilution or treatment with heat, EDTA, or DTT. In this study we describe the frequency, nature, and degree of prozone in a cohort of highly sensitized patients (cPRA ≥ 95%), in whom accurate detection of HLA antibodies and virtual crossmatching is of paramount importance. Sera were tested by the SAB assay ± EDTA treatment, ±1:10 dilution to identify the prozone effect. The relative contribution of complement vs IgM to prozone was assessed using anti-C3d and anti-IgM reporter antibodies, respectively. We found that prozone was very frequent in highly sensitized patients (80%), especially those with a history of previous transplantation (87%). Class I HLA specificities were more commonly affected than class II and the susceptibility to prozone was locus dependent with HLA-A(31%), -B(29%) and -DQ(26%) being affected more frequently than HLA-DP(17%), -C(16%) and -DR(5%) antigens. Interestingly, the presence of prozone could be predicted by C3d positivity (MFI ≥ 4000; sensitivity = 95.2%, specificity = 97.2%) and the degree of prozone correlated directly with the extent of C3d deposition. The role of IgM was less clear. However, serum dilution studies suggested that IgM may contribute to interference in a small subset of prozone positive specificities. Our study underscores the importance of serum treatment to inhibit complement activation and minimize prozone in the SAB assay, especially in highly sensitized patients.     

Accelerated humoral renal allograft rejection due to HLA-C14 mediated allosensitization to HLA-Bw6

Objectives

To investigate immunological mechanisms underlying accelerated antibody-mediated rejection (AMR) of a living-related renal allograft in a patient with no detectable antibodies to donor human leukocyte antigens (HLA) in pre-transplant sera.

Getting on target: Development of the novel,prozone-resistant,dual antibody rapid test (DART) for the LABScreen single antigen bead (SAB) assay

A major limitation of the single antigen bead (SAB) assay is the so called prozone effect, whereby the detection of high titer complement fixing HLA antibodies is compromised due to complement split product (from C3 and C4 components) deposition and interference with the reporter anti-IgG-PE antibody binding. Strategies to minimize prozone include serum titration or treatment with heat, dithiotreitol (DTT), or ethylenediaminetetraacetic acid (EDTA). While effective, these treatments may compromise HLA antibody binding and detection. Here we describe the Dual Antibody Rapid Test (DART), a modified version of the rapid optimized SAB (ROB) protocol, in which we use an IgG-PE/C3d-PE antibody cocktail to simultaneously detect bead bound IgG and C3d, which allows for detection of HLA antibodies independent of the prozone effect. Twenty prozone positive sera (10 class I and 10 class II), identified by titration, were tested by the ROB protocol, with or without EDTA pre-treatment, using three reporter antibody cocktails: (1) IgG-PE, (2) C3d-PE, or (3) IgG-PE/C3d-PE (DART). Mean fluorescence intensity (MFI) values were then compared. IgG negative (n = 735) vs IgG positive (n = 1185) reactions were identified using a 1000 MFI IgG EDTA cutoff. IgG positive reactions were classified based on ΔMFI (IgG EDTA – IgG) as follows: (1) prozone negative (ΔMFI < 3000; n = 737), (2) slight prozone (ΔMFI 3001–5000; n = 49), (3) moderate prozone (ΔMFI 5001–10,000; n = 93), and (4) marked prozone (ΔMFI > 10,001; n = 306). No C3d deposition was present on IgG negative beads, and the majority of prozone positive specificities (438/448; 98%) fixed complement and were detected with the C3d-PE reporter. Interestingly, C3d-PE MFI was directly proportional to the degree of prozone (mean C3d-PE MFI = 4419.5 ± 1606.3 for slight, 5991.0 ± 2302.7 for moderate, and 12,417.4 ± 2969.9 for marked prozone specificities). Interestingly, EDTA treatment was found to have a negative impact on MFI of up to 15% of prozone negative specificities. Importantly, the DART protocol detected all prozone positive specificities while MFI for prozone negative specificities correlated well with those seen with the IgG-PE reporter alone (R² = 0.97). In conclusion, the DART protocol accurately detects HLA antibodies independent of the prozone effect. Implementation of DART is an easy way to overcome the prozone effect without compromising HLA antibody detection.     

Frequent development of subclinical chronic antibody-mediated rejection within 1 year after renal transplantation with pre-transplant positive donor-specific antibodies and negative CDC crossmatches

Although short-term graft survival has been improved by recent desensitization protocols including B cell depletion therapy, little is known about risk factors of chronic antibody-mediated rejection (CAMR) in HLA-incompatible (HLA-I) renal transplantation (RTx). Twenty-six HLA-I RTx with positive donor-specific antibodies (DSA) and negative T cell cytotoxic crossmatches were compared with 88 ABO-incompatible (ABO-I) and 207 ABO-identical/compatible (ABO-Id/C) RTx. The desensitization therapy consisted of mycophenolate mofetil, rituximab and double-filtration plasmapheresis. Protocol biopsies within 1 year revealed subclinical CAMR in 36% of HLA-I, 5% of ABO-I and 3% of ABO-Id/C, although clinical acute AMR was observed in 8%, 3% and 1%, respectively. The incidence of CAMR was not different between class I and class II DSA. Most of class I DSA (94%) changed to negative 1 year after RTx, whereas 77% of class II DSA remained positive. In addition, the remaining DRB ± DQB DSA caused CAMR in 80% of patients, while DQB DSA alone did not. The progress of subclinical CAMR within 1 year could not be circumvented by rituximab. Sustained class II (DRB ± DQB) DSA detection after RTx may pose a potential risk for developing CAMR, but negative change in class I DSA could also elicit CAMR.     

Correlations between donor-specific antibodies and non-adherence with chronic active antibody-mediated rejection phenotypes and their impact on kidney graft survival

Chronic-active antibody-mediated rejection (CAABMR) is associated with poor kidney graft survival and has no clear effective treatment. Forty-one cases of CAABMR were detected in indication graft biopsies and evaluated according to current Banff classification. We investigated the impact of concurrent donor-specific antibodies (DSA) and their characteristics, together with non-adherence regarding immunosuppression on CAABMR histopathological phenotypes and prognosis. Twenty-four (59%) patients had detectable DSA at biopsy, with 15 of them being considered non-adherent. Graft function at biopsy was similar in DSA (+) and (?) patients. DSA (+) patients had significantly higher tubulointerstitial inflammation (i and ti) and acute humoral (g+ptc+v+C4d) composite score than DSA (?). DSA (+)/non-adherent cases presented additionally with increased microvascular inflammation (ptc and v), besides having a distinctively lower ah score. C1q DSA strength was higher (P?=?.046) in non-adherent patients and correlated closely with C4d score (P?=?.002). Lower graft function and ah score, higher proteinuria and ci?+?ct score, and, separately per each model, DSA (+) (HR?=?2.446, P?=?.034), DSA (+)/non-adherent (HR?=?3.657, P?=?.005) and DSA (+)/C1q (+) (HR?=?4.831, P?=?.003) status were independent predictors of graft failure. CAABMR with concomitant DSA pose a higher risk of graft failure. Adherence should be evaluated, and histopathological phenotyping and DSA characterization may add critical information.