Complement Binding Capacity Research Articles

#1390 Differences in the detection of anti-HLA antibodies between two single antigen bead methods in kidney transplantation patients

Abstract Background and Aims Two semiquantitative single-antigen bead (SAB) methods are currently available for the detection of anti-HLA antibodies and the estimation of their activity, depending on complement binding. However, data on accordance of the two methods on specificities detection and their reactivity evaluation are scarce. The purpose of this study is to compare the two methods in terms of anti-HLA antibodies specificities detection and of their complement binding capacity Method Sera from 97 patients with a positive Panel Reactive Antibody Test (>5%) was tested by two SAB methods, Immucor (IC) and One-lambda (OL), for class I and II anti-HLA antibody specificities and their ability to bind complement. The patients were either on a waiting list for kidney transplantation or transplanted patients tested routinely or after a clinical event. Statistical analysis of the results and comparison of the two methods followed. Results OL detected more class I positive specificities per patient [25 (9-34) vs. 18 (7-28) with IC, p < 0.001], while the two methods showed no difference in class II anti-HLA antibodies. IC detected 1608 out of 8148 and 1136 out of 7275 totally examined, positive anti-HLA class I and II antibody specificities respectively, with a median mean fluorescence intensity (MFI) of 4195 (1995-11272) and 6706 (2647-13184) respectively. Similarly, OL detected 1942/8148 and 1247/7275 positive specificities of anti-HLA class I and II antibodies respectively, with MFI 6185 (2855-12099) and 9498 (3630-17702)] respectively. In the IC method, 428/1608 [MFI 13900 (9540-17999)] and 409/1136 [MFI 11832 (7128-16531)] positive class I and II specificities, respectively, bound the C3d fraction of complement. Similarly, OL detected 485/1942 [MFI 15452 (9369-23095)] and 298/1247 [MFI 18852 (14415-24707)] class I and II specificities respectively that bound the C1q fraction of complement. In the analysis of the specificities that were positive by both methods, MFI showed a strong positive linear correlation between the two methods (r = 0.61, p < 0.001 and r = 0.54, p = 0.001 for classes I and II respectively). Accordingly, data were analyzed with Principal Components Analysis, in which negative results were also included. In the projection of the plane defined by the first two principal components the vector corresponding to the IC method for class I antibodies was congruent with the vectors of the complement binding detection methods, C3d and C1q. In contrast, the OL vector was nearly perpendicular to the other vectors, indicating the existence of non-complement-binding specificities that are not detected by IC. A similar picture was found for antibodies against HLA class II (Figure). Conclusion OL was more sensitive for detecting class I and II anti-HLA antibodies despite the fact that there was no difference in the number of class II specificities per patient. However, the additional specificities detected by OL, potentially do not bind complement. The MFI of the complement binding specificities were higher in both methods. The two methods were equivalent in detecting complement-binding class I specificities, however the C3d IC method was more sensitive in detecting complement-binding class II anti-HLA antibodies.

Desensitization of Highly Immunized Kidney Transplant Recipients Awaiting Transplantation—Polish Single-Center Experience

IntroductionThe increasing number of highly immunized patients waiting for kidney transplantation is a significant problem in Europe as the proportion of such patients has doubled in the last decade. Transplantation in this group is enabled by desensitization methods, i.e., intravenous pharmacotherapy with human immunoglobulin (IVIG), anti-CD20 monoclonal antibody (rituximab), and plasma exchange. The objective was to evaluate the efficacy and safety of this protocol. Material and MethodsThe inclusion criteria: presence of established anti-HLA antibodies with complement-binding capacity, i.e., anti-HLAC1q+ (>MFI 15,000 for the most common antigens), no renal transplantation within 1 year after activation on the waiting list. Thirteen patients were selected for the procedure. IVIG was administered twice (2 g/kg-maximum 140 g/dose). Between IVIG doses, patients received rituximab (375 mg/m2). Anti-HLA was tested after 1 and 2 months after completion of the procedure. ResultsAll patients have completed the protocol. No significant changes after desensitization in the amount/profile of alloantibodies were observed. However, with negative vCM for HLA-A/B/DR (no DSA against the reported donor) and negative CM-CDC, according to the allocation system, patients were given priority on the recipient list. Seven out of 13 patients received a transplant within 12 months after treatment (mean 11.5 weeks). Renal graft function was good (mean creatinine level after 1 month: 1.5 mg/dL). No incidents of acute rejection were reported. The most common complications were infections (especially pneumonia). ConclusionThe desensitization protocol (IVIG + rituximab) allows highly immunized patients to undergo organ transplantation. In short-term analysis, no acute rejection was observed, graft function was satisfactory. Desensitization was associated with an increased risk of infection.

Complement and Non-Complement Binding Anti-HLA Antibodies Are Differentially Detected with Different Antigen Bead Assays in Renal Transplant Recipients.

Two semi-quantitative, Luminex-based, single-antigen bead (SAB) assays are available to detect anti-HLA antibodies and evaluate their reactivity with complement binding. Sera from 97 patients with positive panel reactive antibody tests (>5%) were analyzed with two SAB tests, Immucor (IC) and One-Lambda (OL), for anti-HLA antibody detection and the evaluation of their complement-binding capacity. IC detected 1608/8148 (mean fluorescent intensity (MFI) 4195 (1995-11,272)) and 1136/7275 (MFI 6706 (2647-13,184)) positive anti-HLA class I and II specificities, respectively. Accordingly, OL detected 1942/8148 (MFI 6185 (2855-12,099)) and 1247/7275 (MFI 9498 (3630-17,702)) positive anti-HLA class I and II specificities, respectively. For the IC assay, 428/1608 (MFI 13,900 (9540-17,999)) and 409/1136 (MFI 11,832 (7128-16,531)) positive class I and II specificities bound C3d, respectively. Similarly, OL detected 485/1942 (MFI 15,452 (9369-23,095)) and 298/1247 (MFI18,852 (14,415-24,707)) C1q-binding class I and II specificities. OL was more sensitive in detecting class I and II anti-HLA antibodies than IC was, although there was no significant difference in the number of class II specificities per case. MFI was higher for complement vs. non-complement-binding anti-HLA antibodies in both assays. Both methods were equal in detecting complement-binding anti-HLA class I antibodies, whereas the C3d assay was more sensitive in detecting complement-binding anti-HLA class II antibodies.

A Case of Chronic Active Antibody-Mediated Rejection Caused by a Pre-Existing Anti-DQ Donor-Specific Antibody in a Systemic Lupus Erythematosus Recipient Without History of Sensitization: A Case Report

Systemic lupus erythematosus (SLE) is reported to produce anti-HLA antibodies. We report a case of chronic active antibody-mediated rejection caused by pre-existing donor-specific antibody (DSA) in a patient with SLE without a history of sensitization. The case was a 29-year-old man with end-stage renal disease due to lupus nephritis. Cross-match with the mother was negative, but low titer anti-DQ DSA was detected, although he had no prior history of sensitization. After desensitization with rituximab and mycophenolate mofetil, a living donor kidney transplant was undergone, and his early postoperative period was uneventful. However, his renal function started to decline at 2 years post-transplant. Although there was no rejection on the biopsy at 2.5 years post-transplant, his renal function continued to decline after that. At 7 years, he had failed his graft due to chronic active antibody-mediated rejection. Retrospective analysis of human leukocyte antigen antibody tests revealed that anti-DQ DSA had disappeared at 1 year post-transplant, but high titer DSA was detected again with complement-binding capacity at 2 years and after that. Careful monitoring might be warranted in an SLE patient with pre-existing DSA, even though the titer was low and without any prior histories of sensitization events.

Complement Binding Anti-HLA Antibodies and the Survival of Kidney Transplantation.

Antibody-mediated rejection (AMR) is one of the most important challenges in the context of renal transplantation, because the binding of de novo donor-specific antibodies (dnDSA) to the kidney graft triggers the activation of the complement, which in turn leads to loss of transplant. In this context, the objective of this study was to evaluate the association between complement-fixing dnDSA antibodies and graft loss as well as the possible association between non-complement-fixing antibodies and transplanted organ survival in kidney transplant recipients. Our study included a cohort of 245 transplant patients over a 5-year period at Virgen de las Nieves University Hospital (HUVN) in Granada, Spain. dnDSA was observed in 26 patients. Of these patients, 17 had non-complement-fixing dnDSA and 9 had complement-fixing dnDSA. Our study demonstrated a significant association between the frequency of rejection and renal graft loss and the presence of C1q-binding dnDSA. Our results show the importance of the individualization of dnDSA, classifying them according to their ability to activate the complement, and suggest that the detection of complement-binding capacity by dnDSA could be used as a prognostic marker to predict AMR outcome and graft survival in kidney transplant patients who develop dnDSA.

Anti-pan-neurofascin antibodies induce subclass-related complement activation and nodo-paranodal damage.

Autoimmune neuropathy associated with antibodies against pan-neurofascin is a new subtype of nodo-paranodopathy. It is relevant because it is associated with high morbidity and mortality. Affected patients often require intensive care unit treatment for several months, and data on the reversibility and long-term prognosis are limited. The pathogenicity including IgG subclass-associated mechanisms has not been unravelled, nor directly compared to anti-neurofascin-155 IgG4-related pathology. Understanding the underlying pathology might have a direct impact on treatment of these severely affected patients. By a multicentre combined prospective and retrospective approach, we provide clinical data of a large cohort of patients with anti-neurofascin-associated neuropathy (n = 18) including longitudinal titre and neurofilament light chain assessment via Ella® and relate clinical data to in vitro pathogenicity studies of anti-neurofascin antibodies. We assessed antibody binding characteristics and the pathogenic effects of anti-pan-neurofascin versus neurofascin-155 antibodies on living myelinating dorsal root ganglia co-cultures. Additionally, we analysed the IgG subclass profile and the complement binding capacity and effector functions considering the effects of intravenous immunoglobulin preparations via enzyme-linked immunosorbent and cell-based assays. In contrast to chronic neurofascin-155 IgG4-associated neuropathy, anti-pan-neurofascin-associated disease presented with a high morbidity and mortality, but as a monophasic and potentially reversible disorder. During follow-up, antibodies were no longer detectable in 8 of 11 patients. Anti-pan-neurofascin had direct access to the nodes of Ranvier in myelinating cultures titre-dependently, most probably inducing this severe phenotype. Antibody preincubation led to impaired paranode formation, destruction of paranodal architecture and alterations on paranodal myelin and sensory neurons in the cultures, with more severe effects than neurofascin-155 antibodies. Besides IgG4, subclass IgG3 was detected and associated with complement binding and cytotoxic effects in vitro. As a possible correlate of axonal damage in vivo, we detected highly increased serum neurofilament light chain levels (sNF-L), correlating to serum C3a. Still, sNF-L was not identified as a marker for poor prognosis, but rather as an intra- and interindividual marker for acuteness, severity and course, with a strong decrease during recovery. Our data provide evidence that anti-pan-neurofascin antibodies directly attack the node and induce severe and acute, but potentially reversible, nodo-paranodal pathology, possibly involving complement-mediated mechanisms. Screening for autoantibodies thus is crucial to identify this subset of patients who benefit from early antibody-depleting therapy. Titre and sNF-L might serve as valuable follow-up parameters. The prospect of a favourable outcome has high relevance for physicians, patients and relatives during months of critical care.

DSA in solid organ transplantation: is it a matter of specificity, amount, or functional characteristics?

The present review describes the clinical relevance of human leukocyte antigen (HLA) donor-specific antibodies (HLA-DSAs) as biomarkers of alloimmunity and summarizes recent improvements in their characterization that provide insights into immune risk assessment, precision diagnosis, and prognostication in transplantation. Recent studies have addressed the clinical utility of HLA-DSAs as biomarkers for immune risk assessment in pretransplant and peritransplant, diagnosis and treatment evaluation of antibody-mediated rejection, immune monitoring posttransplant, and risk stratification. HLA-DSAs have proved to be the most advanced immune biomarkers in solid organ transplantation in terms of analytical validity, clinical validity and clinical utility. Recent studies are integrating multiple HLA-DSA characteristics including antibody specificity, HLA class, quantity, immunoglobulin G subclass, and complement-binding capacity to improve risk assessment peritransplant, diagnosis and treatment evaluation of antibody-mediated rejection, immune monitoring posttransplant, and transplant prognosis evaluation. In addition, integration of HLA-DSAs to clinical, functional and histological transplant parameters has further consolidated the utility of HLA-DSAs as robust biomarkers and allows to build new tools for monitoring, precision diagnosis, and risk stratification for individual patients. However, prospective and randomized-controlled studies addressing the clinical benefit and cost-effectiveness of HLA-DSA-based monitoring and patient management strategies are required to demonstrate that the use of HLA-DSAs as biomarkers can improve current clinical practice and transplant outcomes.

Donor-specific antibody characteristics, including persistence and complement-binding capacity, increase risk for chronic lung allograft dysfunction

Chronic lung allograft dysfunction (CLAD) is the major complication limiting long-term survival in lung transplant recipients (LTRs), with those developing donor-specific anti-human leukocyte antigen (HLA) antibodies (DSAs) previously found to have increased risk for CLAD. However, as DSA responses vary in timing of development, specificity, breadth, persistence, and complement-binding capacity, we hypothesized that these characteristics would impact CLAD and survival outcomes. We retrospectively analyzed DSA characteristics and outcomes in a single-center cohort of 582 LTRs who had serum samples collected prospectively from 2010 to 2016. Luminex-based single antigen bead assays were performed to assess DSA. DSAs were detected in 247 LTRs (42%), of which 124 (21.3%) were de novo DSAs and 53 (9.1%) were complement-binding (C1q+). CLAD developed in 208 LTRs (35.7%) during the follow-up period, with 67.8% determined as bronchiolitis obliterans syndrome phenotype and 32.2% as restrictive allograft syndrome phenotype. We found a shorter time to CLAD in LTRs with persistent DSAs (p = 0.04) and HLA-DQ-specific DSAs (p = 0.03). LTRs who developed C1q+ DSAs had significantly shorter time to CLAD (p < 0.001), with 100% of C1q+ DSAs being persistent and no differences between CLAD phenotypes. CLAD-free survival was significantly reduced in LTRs who developed C1q+ DSAs (p = 0.001), HLA-DQ-specific DSAs (p = 0.03), and multiple DSAs (p = 0.02). Together, our findings demonstrate that DSA characteristics of persistence, HLA-DQ specificity, and C1q+ DSAs are associated with shorter time to CLAD. Additionally, C1q+, HLA-DQ-specific, and multiple DSAs are associated with decreased CLAD-free survival. These characteristics may improve DSA risk stratification for deleterious outcomes in LTRs.

Characteristics and predictive value for graft fibrosis of the complement-binding capacity of donor-specific human leukocyte antigen antibodies after pediatric liver transplantation.

Donor-specific HLA antibodies (DSAs) have detrimental effects on short- and long-term outcomes after organ transplantation. Despite evidence that the complement-binding capacity of DSAs has predictive power in kidney transplantation, its clinical impact during long-term follow-up after LT remains unclear. In this study, we assessed the complement-binding capacities of DSAs and their association with histological findings. In total, 72 patients who underwent pediatric LT at our institution between July 1991 and October 2013 were retrospectively reviewed. A subgroup analysis of histological findings was performed for 37 subjects who underwent liver graft biopsy. Patients were divided into two groups based on the degree of graft fibrosis, and clinical characteristics were assessed. All anti-class I DSAs were C1q-negative. Anti-DR and anti-DQ DSAs were identified in 34% and 41% of patients, respectively; however, only three of 25 patients with anti-DR DSAs exhibited a positive C1q-binding assay, whereas, 25 of 29 anti-DQ DSAs showed C1q-binding capacity. MFI values for DSA were significantly higher for patients with C1q-binding capacity than for those without (P<.0001). Complement-binding anti-DR DSA was relatively rare in both groups. Regarding anti-DQ DSA, there were no differences between fibrosis and non-fibrosis groups, irrespective of complement-binding capacity. The association between anti-DR DSA and liver fibrosis, which was supported in this cohort, was not strengthened but rather impaired when accounting for complement-binding capacity due to low positive detection. Further studies of the association between complement-binding anti-DQ DSA and histological findings in LT are needed.

Role of C1q-binding anti-HLA antibodies as a predictor of lung allograft outcome.

Presence of anti-human leukocyte antigen donor-specific antibodies (DSAs) is associated with poor outcome after lung transplantation. Currently, DSAs are detected using the Luminex technique, which may be overly sensitive. The new C1q assay allows for the exclusive detection of complement (C1q)-binding antibodies, involved in antibody-mediated rejection. We investigated whether early detection of complement-binding DSAs is associated with chronic lung allograft dysfunction (CLAD) and survival.From 2009 to 2012, lung transplant recipients from three transplantation centres were screened for the presence of DSA and their complement-binding capacity during the 6-12 months post-transplantation in a stable condition.The analysis included 168 patients. The 3-year rates of freedom from CLAD and graft survival were lower for patients with complement-binding DSAs (33.6% and 53.7%, respectively), as compared with patients with non-complement-binding DSAs (61.9% and 77.4%, respectively) and patients without DSA (70% and 84.9%, respectively) (p<0.001 and p=0.001, respectively). Detection of complement-binding DSA was associated with a risk of graft loss that was nearly tripled after adjustment for clinical, functional, histological and immunological factors (hazard ratio 2.98, 95% CI 1.33-6.66; p=0.008).Assessment of the C1q-binding capacity of DSA appears to be useful in identifying stable lung transplant recipients at high risk of lung allograft loss.

The Clinical Significance of Pretransplant C1q Assay Status in Positive Crossmatch Kidney Transplantation

Background Patients with a pretransplant donor-specific antibodies (DSA) are at higher risk for antibody-mediated rejection (AMR). However, not all DSA have the same pathogenetic potential. The novel assay for complement binding capacity may help to stratify the risk for AMR. The aim of this study was to investigate the association between complement binding ability of DSA and clinical outcomes in patients with a positive crossmatch (XM). Methods We analyzed 59 patients with a positive XM who underwent living donor kidney transplantation between 2011 and 2016 with desensitization therapy (12 complement-dependent cytotoxic [CDC] XM and 47 flow cytometric XM). We analyzed clinical outcomes according to XM and C1q status.Results Biopsy-proven AMR developed in 18 patients (30.5%). CDC XM patients suffered more AMR than flow cytometric XM patients (P=0.004). Although the incidence of AMR was higher in C1q-positive DSA than in C1q-negative DSA, the difference was not statistically significant (P=0.067). The XM techniques and C1q positivity did no correlated with graft loss. C1q positivity was associated with higher IgG titer (11181 ± 3997 vs. 4823 ± 6132, P=0.002).Conclusion C1q-positive DSA in patients with positive crossmatch was unable to predict AMR, but CDC XM compared to flow cytometric XM did. The C1q binding activity in patients with positive XM largely reflects differences in antibody strength.

Combining Sensitive Crossmatch Assays With Donor/Recipient Human Leukocyte Antigen Eplet Matching Predicts Living-Donor Kidney Transplant Outcome

IntroductionDespite the different assays available for immune-risk stratification before living-donor kidney transplantation (LDKT), the precise type and number of tests to perform remain uncertain.MethodsIn a cohort of 330 consecutive LDKT patients, all of which were complement-dependent cytotoxicity (CDC)−crossmatch negative, we retrospectively analyzed the impact on main clinical outcomes of most sensitive immunoassays (complement-dependent cytotoxicity−panel-reactive antibody [CDC-PRA], flow cytometry crossmatch [FC-XM], donor-specific antibodies [DSAs], and their complement-binding capacity DSA-C3d]), together with donor/recipient HLA eplet matching. Mean follow-up was 67 months (range 24−190 months).ResultsOf 330 patients, 35 (11%) showed a CDC-PRA >20%; 17 (5%) FC-XM+; 30 (9%) DSA+, 18(5%) DSA-C3d+, with low overlapping results (10 patients positive in all donor-specific tests). Unlike HLA allele compatibility, the mean number of HLA class II eplet mismatches was higher in LDKT patients with positive baseline test results. DSA-C3d+ showed higher mean fluorescence intensity (MFI) DSA, with a cut-off MFI of 6192 accurately predicting complement fixation (area under the curve = 0.85, P = 0.008). Although all assays were associated with acute rejection (AR), only DSA-C3d+ (odds ratio [OR] = 6.64, P = 0.038) or high MFI-DSA (OR = 7.54, P = 0.038) independently predicted AR. Likewise, poorly HLA class II eplet−matched patients were at higher risk for AR, particularly patients with negative baseline test results (OR = 1.14, P = 0.019). Finally, previous AR and FC-XM+/DSA+, regardless of C3d positivity, independently predicted graft loss.ConclusionCombining FC-XM and solid-phase assays with the evaluation of donor/recipient HLA eplet mismatches, are most accurate tools for immune-risk stratification prior LDKT.

Evaluation of Pretransplant Donor-Specific Alloantibodies With Different Crossmatch Techniques

Donor-recipient crossmatching for kidney transplantation is an obligatory step for the transplant work-up process. Attention is currently put on single bead assay (SBA) that enables virtual crossmatching. In contrast, methods developed for complement binding capacity are still not routinely established. We compared modified, cytolytic flow cytometry crossmatch (cFC-XM) with complement-dependent serological crossmatch (CDC-XM), SBA, and flow cytometry crossmatch (FC-XM) to verify whether newly developed techniques may be beneficial for transplant immunological matching. Also, the cutoff value for donor-specific alloantibodies levels currently used for virtual crossmatch was verified. Serum from 22 sensitized patients was crossmatched with surrogate donors by CDC-XM, FC-XM, and cFC-XM, while anti-HLA antibodies were measured by SBA. In all cases, virtual crossmatch was positive at 5000 mean fluorescence intensity cutoff. Among 22 tested sera with donor-specific alloantibodies above 5000 mean fluorescence intensity, the positive CDC-XM result was noted only in 41% of patients (n = 9), but positive FC-XM was noted in 86% (n = 19), and further lytic antibodies (cFC-XM) were confirmed in 27% of cases (n = 6). Our results suggest that donor-recipient immunological matching for kidney transplantation requires different methods to verify the importance of alloantibodies, and improvement in laboratory investigation is needed. This is especially important for immunized patients that have many types of alloantibodies and virtual crossmatching used as a tool for deceased donor allocation should balance between the likelihood of transplantation and risk of positive crossmatch result.

The Complement Binding and Inhibitory Protein CbiA of Borrelia miyamotoi Degrades Extracellular Matrix Components by Interacting with Plasmin(ogen).

The emerging relapsing fever spirochete Borrelia (B.) miyamotoi is transmitted by ixodid ticks and causes the so-called hard tick-borne relapsing fever or B. miyamotoi disease (BMD). More recently, we identified a surface-exposed molecule, CbiA exhibiting complement binding and inhibitory capacity and rendering spirochetes resistant to complement-mediated lysis. To gain deeper insight into the molecular principles of B. miyamotoi-host interaction, we examined CbiA as a plasmin(ogen) receptor that enables B. miyamotoi to interact with the serine protease plasmin(ogen). Recombinant CbiA was able to bind plasminogen in a dose-dependent fashion. Moreover, lysine residues appear to play a crucial role in the protein-protein interaction as binding of plasminogen was inhibited by the lysine analog tranexamic acid as well as increasing ionic strength. Of relevance, plasminogen bound to CbiA can be converted by urokinase-type plasminogen activator (uPa) to active plasmin which cleaved both, the chromogenic substrate S-2251 and its physiologic substrate fibrinogen. Concerning the involvement of specific amino acids in the interaction with plasminogen, lysine residues located at the C-terminus are frequently involved in the binding as reported for various other plasminogen-interacting proteins of Lyme disease spirochetes. Lysine residues located within the C-terminal domain were substituted with alanine to generate single, double, triple, and quadruple point mutants. However, binding of plasminogen to the mutated CbiA proteins was not affected, suggesting that lysine residues distant from the C-terminus might be involved in the interaction.

Clinical utility of C3d binding donor-specific anti-human leukocyte antigen antibody detection by single antigen beads after kidney transplantation-a retrospective study.

Development of donor-specific antibodies (DSA) after renal transplantation is known to be associated with worse graft survival, yet determining which specificities in which recipients are the most deleterious remains under investigation. This study evaluated the relationship of the complement binding capacity of post-transplant de novo anti-human leukocyte antigen (HLA) antibodies with subsequent clinical outcome. Stored sera from 265 recipients previously identified as having de novo DSA were retested for DSA and their C3d binding capacity using Luminex-based solid-phase assays. Most recipients had anti-HLA class II-reactive DSA (class I = 12.5%, class II = 68.7%, class I and class II = 18.9%). The recipients that had C3d binding DSA (67.5%) had a significantly higher incidence of antibody-mediated rejection and any rejection. They also had significantly lower kidney survival, with the lowest survival in those that had both anti-HLA class I and class II C3d binding DSA. Concurrent biopsy comparison revealed a 96.2% positive predictive value and 47.4% negative predictive value for C4d peritubular capillary (Ptc) deposition. Anti-HLA class I and class II C3d binding DSA carried a twofold and 1.5-fold increased risk of kidney loss, respectively, in multivariate analysis.

P001 Changes in anti-HLA antibody complement-binding capacity reflect the efficacy of antibody-mediated rejection therapy and predict kidney allograft survival

Aim We investigated wether the kinetics of donor-specific anti-HLA antibody (DSA) characteristics might predict kidney allograft survival in patients with antibody-mediated rejection (AMR) receiving standard-of-care therapy. Methods We prospectively enrolled 2666 kidney recipients transplanted between 2004 and 2012 and we included in this study all patients diagnosed with active AMR who received standardized therapy including plasma exchanges (×4–5) and intravenous immune globulins (2 g/kg × 3). Patients were systematically assessed at diagnosis and 3 months post-therapy for circulating anti-HLA DSA (specificity, mean fluorescence intensity, C1q-binding capacity) using Luminex SAB, clinical data (eGFR and proteinuria) and histology (allograft biopsy). Results 278 patients with biopsy-proven acute or chronic active AMR received standard-of-care therapy. At rejection, 76 (27%) patients had class I DSA, 127 (46%) class II DSA and 75 (27%) class I and II DSA. Dominant DSA was C1q binding in 85 (31%) patients. The MFImax decreased from 5223 ± 318 to 3611 ± 280 after AMR therapy ( P P = 0.001). Patients showing a reduction of DSA MFImax after therapy ( N = 207) had a better 5-year graft survival than those who had stable or increasing MFImax (N = 71): 80% vs. 62%; P N = 177) and those with pre-therapy positive/post-therapy negative C1q-DSA ( N = 51) showed a better 5-year graft survival (82% and 94%, respectively) than those with C1q-DSA before and after therapy ( N = 34) and those with pre-therapy negative/post-therapy positive C1q-DSA ( N = 16) (36% and 38%, respectively; P Conclusions The kinetics of circulating anti-HLA DSA C1q-binding capacity in kidney recipients with AMR more accurately identify responders to standard-of-care therapy than DSA MFI level, independently of clinical and histological parameters.

Donor-Specific Antibodies in Kidney Transplant Recipients.

Donor-specific antibodies have become an established biomarker predicting antibody-mediated rejection. Antibody-mediated rejection is the leading cause of graft loss after kidney transplant. There are several phenotypes of antibody-mediated rejection along post-transplant course that are determined by the timing and extent of humoral response and the various characteristics of donor-specific antibodies, such as antigen classes, specificity, antibody strength, IgG subclasses, and complement binding capacity. Preformed donor-specific antibodies in sensitized patients can trigger hyperacute rejection, accelerated acute rejection, and early acute antibody-mediated rejection. De novo donor-specific antibodies are associated with late acute antibody-mediated rejection, chronic antibody-mediated rejection, and transplant glomerulopathy. The pathogeneses of antibody-mediated rejection include not only complement-dependent cytotoxicity, but also complement-independent pathways of antibody-mediated cellular cytotoxicity and direct endothelial activation and proliferation. The novel assay for complement binding capacity has improved our ability to predict antibody-mediated rejection phenotypes. C1q binding donor-specific antibodies are closely associated with acute antibody-mediated rejection, more severe graft injuries, and early graft failure, whereas C1q nonbinding donor-specific antibodies correlate with subclinical or chronic antibody-mediated rejection and late graft loss. IgG subclasses have various abilities to activate complement and recruit effector cells through the Fc receptor. Complement binding IgG3 donor-specific antibodies are frequently associated with acute antibody-mediated rejection and severe graft injury, whereas noncomplement binding IgG4 donor-specific antibodies are more correlated with subclinical or chronic antibody-mediated rejection and transplant glomerulopathy. Our in-depth knowledge of complex characteristics of donor-specific antibodies can stratify the patient's immunologic risk, can predict distinct phenotypes of antibody-mediated rejection, and hopefully, will guide our clinical practice to improve the transplant outcomes.

Anti-HLA-DQ antibodies are highly and independently related to the C1q-binding capacity of HLA antibodies

The complement-binding capacity of anti-HLA antibodies (HLAabs) is recognized as a key pathogenic factor. The aim of this study is to describe the patient characteristics associated to the presence of C1q+ as well as those of the Abs per se when associated to C1q binding. MethodologyThis is a cross-sectional, observational, descriptive study of patients with previous sensitizing factors and awaiting a kidney transplant (KT). We determined anti-HLA antibodies and their C1q binding capacity with the C1q assay. ResultsAmong the 55 included patients, 26 (47.2%) had at least one C1q+ anti-HLAab. A previous transplant history, a greater number of HLAabs, a greater % of class I or class II PRA, the average MFI of all HLAabs, the MFI of the dominant HLAab and the HLAab antigenic specificities against HLA-B, -C and -DQ, all proved to be risk factors associated to the presence of C1q binding HLAabs (C1q+). In the total population, were detected 1268 HLAabs, 230 (18.1%) of which were C1q+. On multivariate analysis, both HLAabs against the HLA-DQ antigenic specificity (OR 9.82 95% CI 5.4–17.6, p<0.001) and the MFI documented by LABScreen®SAB (OR 1.2% CI 1.22–1.3, p<0.001), proved to be risk factors. ConclusionAnti-HLA-DQ antibodies and the MFI (LABScreen®SAB) are highly and independently related to the C1q-binding capacity of HLA antibodies.

From Humoral Theory to Performant Risk Stratification in Kidney Transplantation.

The purpose of the present review is to describe how we improve the model for risk stratification of transplant outcomes in kidney transplantation by incorporating the novel insights of donor-specific anti-HLA antibody (DSA) characteristics. The detection of anti-HLA DSA is widely used for the assessment of pre- and posttransplant risks of rejection and allograft loss; however, not all anti-HLA DSA carry the same risk for transplant outcomes. These antibodies have been shown to cause a wide spectrum of effects on allografts, ranging from the absence of injury to indolent or full-blown acute antibody-mediated rejection. Consequently, the presence of circulating anti-HLA DSA does not provide a sufficient level of accuracy for the risk stratification of allograft outcomes. Enhancing the predictive performance of anti-HLA DSA is currently one of the most pressing unmet needs for facilitating individualized treatment choices that may improve outcomes. Recent advancements in the assessment of anti-HLA DSA properties, including their strength, complement-binding capacity, and IgG subclass composition, significantly improved the risk stratification model to predict allograft injury and failure. Although risk stratification based on anti-HLA DSA properties appears promising, further specific studies that address immunological risk stratification in large and unselected populations are required to define the benefits and cost-effectiveness of such comprehensive assessment prior to clinical implementation.

Abstract 12052: Non-complement-Binding Donor Specific Antibodies and Cardiac-Allograft Vasculopathy in Pediatric Heart Transplant Recipients

Aim: We hypothesized that the non-complement binding of de novo donor specific antibodies (DSA) detected after heart transplant (HT) confer a lower risk for acute rejections and coronary artery vasculopathy (CAV). The purpose of this study was to compare the clinical outcomes between pediatric HT recipients with C1q+ and C1q- fixing de novo DSA Methods: 127 patients from January 2005 to December 2014 were analyzed in this study to evaluate the outcomes including acute rejections and CAV of pediatric HT recipients who had C1q+ DSA compared to those with C1q- DSA. Serum samples from patients with circulating DSA were analyzed for C1q fixing according to the manufacturer’s protocol (C1qScreen, One Lambda). Results: Out of 127 patients transplanted between 2005 and 2014, 59 (46.4%) patients developed de novo DSA, of those 37 (62.7%) had C1q+ DSA. No difference in baseline characteristics: age at HT, gender, pre-HT diagnosis of CHD, ischemic time, number of HLA mismatch and pre-HT PRA &gt;10. We found that DSA strength is significantly associated with C1q+ (C-statistics 0.89 and p &lt;0.05) and at cut off 7000 MFI has 80% sensitivity and 80% specificity. C1q positive rate varied according to DSA class type: Class I (25% C1q+), class II (66% C1q +) and Class I and II (67% C1q+). C1q+ DSA patients had 2.4 times higher rate of acute rejection episodes compared to those with C1q- DSA. C1q+ DSA patients had significantly higher rates of CAV at 10-yr post-HT compared to C1q- DSA patients (72% vs 28%) (Figure) (P&lt;0.001). C1q+ DSA patients had a trend towards poor allograft survival at 10-yr post-HT compared to C1q-DSA patients (70% vs 52%) (p =0.15). Conclusions: De novo DSA which are C1q+ have significantly greater deleterious effect on long-term allograft outcome in pediatric heart transplant recipients. Thus, measuring DSA and determining their complement binding capacity is helpful in assessing the risk of cardiac allograft vasculopathy. Figure: