Application, technical issues, and interpretation of C1q for graft outcome.

PURPOSE OF REVIEW: Significant interest and controversy surround the use of C1q for determining risk of antibody-mediated rejection (AMR) and graft loss. Alternate models for predicting outcomes have been proposed. This review focuses on the correlation of currently utilized assays for outcome, together with the technical and theoretical limitations, to distill current thinking. RECENT FINDINGS: Results demonstrate that C1q status is significantly correlated with AMR and graft loss. There is general consensus that C1q is more clinically relevant for graft outcome than neat IgG MFI. IgG titers, subclass, and other complement assays have now been studied to determine if they are more relevant. Only IgG3 and possibly C3d fixation have shown added value to C1q for outcome correlation. Direct parallel titer comparisons of C1q and IgG are lacking and the correlation is unknown. SUMMARY: Overall, results confirm the correlation with C1q+ donor-specific antibody (DSA) for AMR and graft loss. The association is stronger posttransplant. C1q+ de novo antibody appears to be especially detrimental portending graft loss in about 1-2.5 years post detection. Recommendations to biopsy and treat at time of de novo C1q+ antibody detection have been suggested by several groups.

HLA desensitization with bortezomib in a highly sensitized pediatric patient.

The proteasome inhibitor bortezomib has been used with variable success in the treatment of AMR following heart transplant. There is limited experience with this agent as a pretransplant desensitizing therapy. We report a case of successful HLA desensitization with a bortezomib-based protocol prior to successful heart transplantation. A nine-yr-old boy with dilated cardiomyopathy, not initially sensitized to HLA (cPRA of zero), required three days of ECMO, followed by implantation of a Heartmate II LVAD. Within six wk, the patient developed de novo class I IgG and C1q complement-fixing HLA antibodies with a cPRA of 100%. Two doses of IVIG (2 g/kg) failed to reduce antibody levels, although two courses of a novel desensitization protocol consisting of rituximab (375 mg/m(2) ), bortezomib (1.3 mg/m(2)  × 5 doses), and plasmapheresis reduced his cPRA to 0% and 87% by the C1q and IgG assays, respectively. He underwent heart transplantation nearly two months later. The patient is now >one yr post-transplant, is free of both AMR and ACR, and has no detectable donor-specific antibodies by IgG or C1q. Proteasome inhibition with bortezomib and plasmapheresis may be an effective therapy for HLA desensitization pretransplant.

Consensus guidelines on the testing and clinical management issues associated with HLA and non-HLA antibodies in transplantation.

BACKGROUND: The introduction of solid-phase immunoassay (SPI) technology for the detection and characterization of human leukocyte antigen (HLA) antibodies in transplantation while providing greater sensitivity than was obtainable by complement-dependent lymphocytotoxicity (CDC) assays has resulted in a new paradigm with respect to the interpretation of donor-specific antibodies (DSA). Although the SPI assay performed on the Luminex instrument (hereafter referred to as the Luminex assay), in particular, has permitted the detection of antibodies not detectable by CDC, the clinical significance of these antibodies is incompletely understood. Nevertheless, the detection of these antibodies has led to changes in the clinical management of sensitized patients. In addition, SPI testing raises technical issues that require resolution and careful consideration when interpreting antibody results. METHODS: With this background, The Transplantation Society convened a group of laboratory and clinical experts in the field of transplantation to prepare a consensus report and make recommendations on the use of this new technology based on both published evidence and expert opinion. Three working groups were formed to address (a) the technical issues with respect to the use of this technology, (b) the interpretation of pretransplantation antibody testing in the context of various clinical settings and organ transplant types (kidney, heart, lung, liver, pancreas, intestinal, and islet cells), and (c) the application of antibody testing in the posttransplantation setting. The three groups were established in November 2011 and convened for a "Consensus Conference on Antibodies in Transplantation" in Rome, Italy, in May 2012. The deliberations of the three groups meeting independently and then together are the bases for this report. RESULTS: A comprehensive list of recommendations was prepared by each group. A summary of the key recommendations follows. Technical Group: (a) SPI must be used for the detection of pretransplantation HLA antibodies in solid organ transplant recipients and, in particular, the use of the single-antigen bead assay to detect antibodies to HLA loci, such as Cw, DQA, DPA, and DPB, which are not readily detected by other methods. (b) The use of SPI for antibody detection should be supplemented with cell-based assays to examine the correlations between the two types of assays and to establish the likelihood of a positive crossmatch (XM). (c) There must be an awareness of the technical factors that can influence the results and their clinical interpretation when using the Luminex bead technology, such as variation in antigen density and the presence of denatured antigen on the beads. Pretransplantation Group: (a) Risk categories should be established based on the antibody and the XM results obtained. (b) DSA detected by CDC and a positive XM should be avoided due to their strong association with antibody-mediated rejection and graft loss. (c) A renal transplantation can be performed in the absence of a prospective XM if single-antigen bead screening for antibodies to all class I and II HLA loci is negative. This decision, however, needs to be taken in agreement with local clinical programs and the relevant regulatory bodies. (d) The presence of DSA HLA antibodies should be avoided in heart and lung transplantation and considered a risk factor for liver, intestinal, and islet cell transplantation. Posttransplantation Group: (a) High-risk patients (i.e., desensitized or DSA positive/XM negative) should be monitored by measurement of DSA and protocol biopsies in the first 3 months after transplantation. (b) Intermediate-risk patients (history of DSA but currently negative) should be monitored for DSA within the first month. If DSA is present, a biopsy should be performed. (c) Low-risk patients (nonsensitized first transplantation) should be screened for DSA at least once 3 to 12 months after transplantation. If DSA is detected, a biopsy should be performed. In all three categories, the recommendations for subsequent treatment are based on the biopsy results. CONCLUSIONS: A comprehensive list of recommendations is provided covering the technical and pretransplantation and posttransplantation monitoring of HLA antibodies in solid organ transplantation. The recommendations are intended to provide state-of-the-art guidance in the use and clinical application of recently developed methods for HLA antibody detection when used in conjunction with traditional methods.

Complement-fixing donor-specific antibodies identified by a novel C1q assay are associated with allograft loss.

Long-term outcomes following renal transplantation remain disappointing. Recently, interest has focused on the antibody-mediated component of allograft injury and the deleterious effects of DSA. We applied a novel C1q solid-phase assay in parallel with the standard IgG SAB assay to identify DSA with the potential to activate complement by binding C1q. Among 193 consecutive renal transplants at our center, 19.2% developed de novo DSA following transplantation. Of the patients with DSA, 43% had antibodies that bound C1q in vitro [C1q+ DSA]. Patients with C1q+ DSA were more likely to develop allograft loss than patients with DSA that did not bind C1q (46.7% vs. 15%; p = 0.04); patients with C1q+ DSA were nearly six times more likely to lose their transplant than those with C1q- DSA. Additionally, patients with C1q+ DSA who underwent allograft biopsy were more likely to demonstrate C4d deposition (50% vs. 8%; p = 0.03) and meet criteria for acute rejection (60% vs. 17%; p = 0.02) when compared with patients with DSA that did not bind C1q. These data suggest that DSA with the ability to activate complement, as determined by this novel C1q assay, are associated with greater risk of acute rejection and allograft loss.

C1q-fixing human leukocyte antigen antibodies are specific for predicting transplant glomerulopathy and late graft failure after kidney transplantation.

BACKGROUND: Human leukocyte antigen (HLA) antibodies, especially those that fix complement, are associated with antibody-mediated rejection and graft failure. The C1q assay on single antigen beads detects a subset of HLA antibodies that can fix complement and precede C4d deposition. The aim of this study was to determine whether C1q-fixing antibodies distinguish de novo donor-specific antibodies (DSA) that are clinically relevant and harmful. METHODS: We retrospectively studied 31 of 274 kidney transplant recipients who had pretransplant and concurrent biopsy and serum specimens, 13 with C4d-positive and 18 with C4d-negative staining. We measured IgG and C1q DSA pretransplant and at the time of biopsy using single antigen bead assays. We identified 13 recipients who developed de novo DSA by IgG or C1q and examined associations with C4d deposition, transplant glomerulopathy, and graft failure. RESULTS: Testing for DSA by IgG is more sensitive for C4d deposition (IgG: 100%, 95% confidence interval [CI] 0.60-1; C1q: 75%, 95% CI 0.36-0.96). Testing for DSA by C1q is more specific for transplant glomerulopathy (C1q: 81%, 95% CI 0.57-0.94; IgG: 67%, 95% CI 0.43-0.85) and graft loss (C1q: 79%, 95% CI 0.54-0.93; IgG: 63%, 95% CI 0.39-0.83). Absence of de novo DSA by IgG and C1q has a high negative predictive value for the absence of C4d deposition (IgG: 100%, 95% CI 0.73-1; C1q: 88%, 95% CI 0.62-0.98), transplant glomerulopathy (IgG: 100%, 95% CI 0.73-1; C1q: 100%, 95% CI 0.77-1), and graft failure (IgG: 86%, 95% CI 0.56-0.97; C1q: 88%, 95% CI 0.62-0.98). CONCLUSION: Monitoring patients with the C1q assay, which detects antibodies that fix complement, offers a minimally invasive means of identifying patients at risk for transplant glomerulopathy and graft loss.

Consensus opinion from the antibody working group on the diagnosis, reporting, and risk assessment for antibody-mediated rejection and desensitization protocols.

During the past few decades, much of the experimental and clinical effort in solid-organ transplantation has been directed toward ameliorating or abrogating T-cell-mediated responses. As a result, universally understood and accepted nomenclature and diagnostic criteria have evolved. Humoral immunity in transplantation has yet to undergo a similar renaissance. Readers of transplant journals regularly find it difficult and often impossible to interpret data on the diagnosis and management of antibody-mediated rejection. The Antibody Working Group was assembled in an attempt to provide guidelines for the standardization of nomenclature, diagnostic criteria, reporting, antibody profiling, and risk assessment.