C1q Positivity Research Articles

#3041 DNAJB9 POSITIVE PRIMARY FIBRILLARY GLOMERULONEPHRITIS: TREATMENT WITH CORTICOSTEROID PULSES EXPERIENCE

Abstract Background and Aims Fibrillary glomerulonephritis (FGN) is a rare histological pattern. Electron microscopy is needed for diagnostic confirmation where predominantly mesangial fibrils are observed. To support the diagnosis; DNAJB9 is a biomarker that is 100% sensitive and 100% specific for FGN. To date, there is no defined therapeutic approach. Various immunosuppressants have been implemented to treat FGN showing poor renal response and approximately 50% progression to chronic kidney disease. Method Ambispective observational study. A protocol based on a bibliographic review was developed. The protocol included an induction therapy with methylprednisolone pulses (MTP), 500 mg a day for three days. Followed by oral prednisone at a dose of one mg/kg with an individual titration period considering the clinical evolution. Demographic, clinical, and pathological variables were extracted from the electronic health record. Results Case 1. Seventy-eight-year-old female. History of arterial hypertension (HT) treated with an angiotensin-converting enzyme inhibitor (ACEi). She debuted with a nephrotic syndrome characterized by proteinuria of 15 grams/day, hypoalbuminemia, and altered kidney function with creatinine (Cr) of 2.7 mg/dL. Secondary causes were rule out. The kidney biopsy showed mesangial expansion with mesangial deposition of IgG and C3 on the immunofluorescence. Congo red was negative. Positivity for DNAJB9 was observed. The electron microscopy (EM) showed 20-nanometer fibrils in the mesangium. The patient was initiated on the treatment protocol with a subsequent reduction of prednisone over a period of 24 weeks. Complete remission, defined as proteinuria <0.5g/day and reduction > 50% of Cr was achieved after 20 weeks of treatment. Case 2. Forty-seven-year-old female with a history of hypertension treated with ACEi. The patient presented with proteinuria 3.5g/d with a normal kidney function. Secondary causes were rule out. The kidney biopsy showed mesangial thickening with IgG, C3, C1q, Kappa, and lambda positivity on immunofluorescence. Congo red was negative. DNAJB9 was positive. An electron microscopy study showed randomly arranged mesangial fibrils. The treatment protocol was started. Prednisone was titrated over a period of 20 weeks. Partial remission was observed, defined by >50% reduction of proteinuria. Kidney function remained preserved. Conclusion Induction treatment based on steroid pulses followed by prednisone titration in patients with FGN with predominantly mesangial compromise was associated with a substantial reduction of proteinuria and stabilization of kidney function.

Efficacy of Plasmapheresis and Intravenous Immunoglobulin on the Reduction of Donor Specific Antibodies in Heart Transplant Recipients

<h3>Purpose</h3> Class II human leukocyte antigen (HLA) donor specific antibodies (DSA) have been associated with a high risk of graft loss and resistance to antibody removal therapy. We aimed to evaluate class II DSA reduction after plasmapheresis (PLEX) and intravenous immunoglobulin (IVIG) in heart transplant recipients (HTR). <h3>Methods</h3> All HTR who received IVIG and PLEX for class II DSAs from 1/1/2017-6/1/2021 were included. Multiorgan, retransplant, and transplant at other centers were excluded. Baseline demographics and mean immunodominant fluorescent intensity (MFI) of DSA were retrieved. The immunodominant DSA was defined as the DSA with highest MFI in any given serum sample. Median MFI reduction rates after PLEX and after IVIG +/- rituximab were calculated. <h3>Results</h3> Thirty encounters involving 21 HTR were identified. At baseline, the immunodominant class II DSA MFI was 9705 (1712,14337.3). Sixteen (76.2%) and five (23.8%) patients had DQ and DR immunodominant DSA, respectively. There was a median reduction of 5325.5 (1539.5,10003.3) in class II DSA MFI following treatment with PLEX from baseline and a subsequent increase of 1235.5 (0,5786) from post PLEX to post IVIG. Overall, there was a reduction from baseline to post PLEX and IVIG of 1864 (1034,3996.8). Four patients (19%) achieved DSA clearance after PLEX, with a median time to clearance of 34 days (26-60) from development. The patients who cleared were all non-African American, had DSAs to only one antigen, were not desensitized pre-transplant, did not have C1q positivity, and had immunodominant MFI <2000. Significantly more patients who failed to clear DSA were taking prednisone (0% vs 76.5%, p=0.005). <h3>Conclusion</h3> PLEX and IVIG can reduce the MFI of class II DSAs in some HTR. PLEX therapy is more efficacious to reduce class II DSA MFI than originally hypothesized. Results from this analysis suggest that DSA to DQ antigens may be less responsive to therapy with PLEX/IVIG at MFI >15,000 or when C1q positive.

Significance of Glomerular C1q deposits in IgA Nephropathy

Introduction: Immunoglobulin A (IgA) nephropathy is the most common form of glomerulonephritis in young men, often presenting as gross or microscopic haematuria and accounts for approximately 10% of the patients with End-Stage Renal Disease (ESRD). The contribution of the complement system to amplify tissue injury in IgA nephropathy has been suggested but the precise pathways of complement activation especially the involvement of classical pathway remain largely unknown. Aim: To determine the prevalence of glomerular C1q deposition in IgA nephropathy to delineate the relationship of glomerular C1q positivity and different histological variables indicating disease activity and disease progression and also to determine the relationship of glomerular C1q positivity and the Oxford scoring system in IgA nephropathy. Materials and Methods: This was a prospective study conducted over a period of three years {January 2014- December 2016} in the Department of Pathology with the cooperation of the Department of Nephrology at Government Medical College, Kozhikode , Kerala, India. A total of 44 cases which were both biopsy and immunofluorescence proven as IgA nephropathy were included in the study. For light microscopy, the tissue received in buffered formalin was processed into paraffin blocks, stained with Haematoxylin and Eosin (H&amp;E) and histopathological changes analysed. For immunofluorescence, tissue received in normal saline was frozen in cryostat and 3 μm sections were stained using the Dako polyclonal Fluorescein Isothiocyanate (FITC) conjugated antibodies IgG, IgM, IgA, C3, C1q and studied for pattern of glomerular staining. Chi-square test was used for statistical analysis. Results: The prevalence of C1q deposits was 27.3%. Among histopathological variables, only fibrous crescent was found to have significant relationship with C1q positivity (p= 0.0472). On follow-up, 50% C1q positive patients and 11.76% C1q negative patients who were having normal renal functions at the start of the study went into renal insufficiency. Conclusion: The study revealed that there was a fairly high prevalence of C1q deposits in IgA nephropathy patients. Also, significant association was found between C1q deposits and fibrous crescent. Most significantly the study concluded that there is an increased tendency for the C1q positive patients to go into renal failure.

Noninvasive Assessment of the Alloimmune Response in Kidney Transplantation.

Transplantation remains the optimal mode of kidney replacement therapy, but unfortunately long-term graft survival after 1year remains suboptimal. The main mechanism of chronic allograft injury is alloimmune, and current clinical monitoring of kidney transplants includes measuring serum creatinine, proteinuria, and immunosuppressive drug levels. The most important biomarker routinely monitored is human leukocyte antigen (HLA) donor-specific antibodies (DSAs) with the frequency based on underlying immunologic risk. HLA-DSA should be measured if there is graft dysfunction, immunosuppression minimization, or nonadherence. Antibody strength is semiquantitatively estimated as mean fluorescence intensity, with titration studies for equivocal cases and for following response to treatment. Determination of invitro C1q or C3d positivity or HLA-DSA IgG subclass analysis remains of uncertain significance, but we do not recommend these for routine use. Current evidence does not support routine monitoring of non-HLA antibodies except anti-angiotensin II type 1 receptor antibodies when the phenotype is appropriate. The monitoring of both donor-derived cell-free DNA in blood or gene expression profiling of serum and/or urine may detect subclinical rejection, although mainly as a supplement and not as a replacement for biopsy. The optimal frequency and cost-effectiveness of using these noninvasive assays remain to be determined. We review the available literature and make recommendations.

Treatment of allosensitized patients receiving allogeneic transplantation

Donor-specific anti-HLA antibodies (DSAs) are a major cause of engraftment failure in patients receiving haploidentical stem cell transplantation (HaploSCT). Effective treatments are needed for these patients, who often have no other donor options and/or are in need to proceed urgently to transplantation. We studied a multimodality treatment with alternate-day plasma exchange (PE), rituximab, intravenous γ globulin (IVIg) and an irradiated donor buffy coat for patients with DSAs at 2 institutions. Thirty-seven patients with a median age of 51 years were treated with this desensitization protocol. Treatment outcomes were compared with a control group of HaploSCT patients without DSAs (n = 345). The majority of patients in the DSA group were female (83.8% vs 37.1% in controls, P < .001) and received stem cells from a child as the donor (67.6% vs 44.1%, P = .002). Mean DSA level before and after desensitization was 10 198 and 5937 mean fluorescence intensity (MFI), respectively, with mean differences of 4030 MFI. Fourteen of 30 tested patients (46.7%) had C1q positivity, while 8 of 29 tested patients (27.6%) remained positive after desensitization. In multivariable analysis, patients with initial DSA > 20 000 MFI and persistent positive C1q after desensitization had a significantly lower engraftment rate, which resulted in significantly higher non-relapse mortality and worse overall survival (OS) than controls, whereas graft outcome and survival of patients with initial DSA < 20 000 MFI and those with negative C1q after treatment were comparable with controls. In conclusion, treatment with PE, rituximab, IVIg, and donor buffy coat is effective in promoting engraftment in patients with DSAs ≤20 000 MFI.

Successful Eradication of De Novo C1q-Binding Donor-Specific Antibodies with Rituximab Monotherapy

<h3>Introduction</h3> Antibody-mediated rejection (AMR) is an increasingly recognized cause of lung transplant allograft failure. Treatment of AMR involves combination therapy that is often poorly tolerated or unsuccessful with regimens involving the combination of intravenous immunoglobulins, plasmapheresis, and proteasome inhibitors, complement, or CD20 therapy. The development of DSAs alone, specifically complement activating C1q-binding DSAs, has been identified as an independent risk factor for solid organ transplant allograft dysfunction. Previous studies have examined the efficacy of combination therapy involving the anti-CD20 antibody rituximab to preemptively eliminate DSAs. Here we report on a case of successful eradication of de novo C1q-binding DSAs in a lung transplant recipient using rituximab. <h3>Case Report</h3> Our patient underwent double-lung transplantation for idiopathic pulmonary fibrosis in August 2019. His post-transplant course was notable for multiple episodes of acute cellular rejection (ACR) requiring burst-dose corticosteroids. DSA titers were monitored every three months post-transplant. At six months, de novo DSAs were detected (DQ7 with DQA1*05) at a mean fluorescence intensity (MFI) of 3623 (low-moderate) (Figure 1). These subsequently rose to an MFI of 11281 (strong) with development of C1q positivity that peaked at an MFI of 15038. Given his frequent episodes of ACR, the strong C1q positivity, and concerns regarding his ability to tolerate therapy for potential future AMR, he was preemptively treated with a single infusion of rituximab (375 mg per meter-squared body surface area). Follow-up DSA titers demonstrated near-total elimination of the C1q component (MFI 403). The patient tolerated the treatment without issue and continues to demonstrate improvement in his allograft function. <h3>Summary</h3> Rituximab monotherapy can be considered preemptively in patients with C1q-binding DSAs that may not tolerate traditional treatment of antibody-mediated rejection.

P0440EPIDEMIOLOGICAL AND CLINICAL CHARACTERSTICS OF IGA NEPHROPATHY PATIENTS IN TURKEY: TSN-GOLD WORKING GROUP

Abstract Background and Aims According to the data of the Turkish Society of Nephrology-Glomerular Diseases Working Group (TSN-GOLD Working Group), IgA nephropathy is the most common primary glomerular disease in Turkey. The purpose of this study was to investigate the epidemiological and clinical data of IgA nephropathy patients in Turkey. Method 4399 patients with primary glomerular diseases from 47 centers who were followed up between May 2009 and May 2019 were included in the study conducted by TSN-GOLD Working Group. 524 patients were excluded due to lack of pathological data. Among the remaining patients, demographic, clinical and laboratory data of 994 patients with IgA nephropathy were analyzed. Results The median age of the patients was 37 (28-47) years, and 37.3% of them were female. The laboratory and clinical data at the time of diagnosis is presented in Figure-1, and biopsy indications are described in Figure-2. The median number of glomeruli was 16 (IQR: 3.5-4.3), sclerotic glomeruli was 2 (IQR: 1-5), and segmental sclerotic glomeruli was 1 (IQR: 1-2). Exudative changes, subendothelial and subepithelial deposition were present in 566 patients (56.9%), 46 patients (4.6%) and in 38 patients (3.8%), respectively. 662 (66.1%) and 611 of the patients (61.4%) had tubular atrophy and interstitial fibrosis in varying degrees, respectively. 672 (%67.6) and 416 patients (%41.9) had interstitial inflammation and vascular changes, respectively. In immunofluorescence staining, 18%, 30.1%, 4.4%, 68% of the patients had IgG, IgM, C1q and C3 positivity, respectively. Crescentic glomeruli were detected in 227 patients (3.3 ± 3.1 glomeruli). Patients with crescentic glomeruli had significantly higher proteinuria and lower eGFR than the patients without [2203 mg/day (15-26078) vs 1807 mg/day (15-29112); p=0.001; 55.3 ml/min/1.73 m2 (3.72-141.9) vs 72 ml/min/1.73 m2 (3.84-150.81); p&amp;lt;0.001, respectively]. Oxford classification was applied to 544 patients. Endocapillary hypercellularity (E1), mesengial hypercellularity (M1), tubular atrophy and interstitial fibrosis (T1 and T2), segmental sclerosis (S1) were present in 126 (13%), 425 (42.8%), 306 (30.8%) and 325 patients (%32.7), respectively. Proteinuria levels were higher in patients with endocapillary hypertrophy, mesengial hypercellularity, tubular atrophy-interstitial fibrosis and segmental sclerosis. eGFR levels were lower in patients with endocapillary hypertrophy, tubular atrophy-interstitial fibrosis and segmental sclerosis (Figure-3). Conclusion In this study we found that, the most common presentation of IgA nephropathy patients in our country was asymptomatic urinary abnormalities followed by nephritic and nephrotic syndrome. Higher proteinuria and lower eGFR values in patients with crescentic glomeruli, support the adoption of crescentic lesions in the new Oxford classification (MEST-C) to predict more precise outcome of IgA nephropathy patients. The high number of patients to whom the Oxford classification was applied provided us with the opportunity to examine the clinical reflections of pathological features. Evaluation of the follow-up data of the patients will give us the possibility to reveal the effect of initial clinical and pathological features on clinical findings and renal outcome.

Evaluation of Carfilzomib for Antibody Mediated Rejection in Lung Transplant Recipients

Though definition and recognition of antibody mediated rejection (AMR) in lung transplant (LTx) has improved over the past few years, treatment of AMR remains poorly defined. Data supporting use of carfilzomib (CFZ) for treatment of AMR in combination with plasmapheresis and intravenous immunoglobulin (IVIG) suggest positive outcomes through donor specific antibody (DSA) depletion or conversion to noncomplement-activating antibodies. The aim of this retrospective analysis was to describe our center's experience treating AMR with CFZ. All adult LTx recipients who received CFZ for AMR from 2014-2018 were included. Patients were excluded if they received CFZ prior to transplant for desensitization. Per institution protocol, all patients received induction with basiliximab followed by maintenance immunosuppression with tacrolimus, mycophenolate, and prednisone. Acute rejection was diagnosed on trans-bronchial lung biopsy at week 2 and months 1, 2, 3, 6, 8, and 12 post-transplant, or when bronchoscopy was performed for clinical indication. Frequency of DSA checks and decision to use CFZ was per clinician discretion. A positive response to CFZ was defined as: 1) loss of DSA C1q-fixing ability after last CFZ dose, 2) clearance of de novo DSA (dnDSA) or 3) decrease in dnDSA MFI of >3000. Data on pulmonary function, infections, and incidence of acute kidney injury were also collected. During the study period, 21 patients with 24 AMR episodes were treated with CFZ in combination with plasmapheresis and IVIG. A positive response to CFZ was observed in 70.8% of AMR episodes and 76.1% of patients. This response was driven by loss of C1q-fixation (69.2%), elimination of class I DSAs (60.0%), and reduction in class II DSA MFI (median 3621, 26% reduction from baseline). Analysis of responders to CFZ revealed that delaying therapy with CFZ for ≥30 days after dnDSA development was associated with lack of response to therapy (p=0.002). Our cohort is the largest to date describing use of CFZ for treatment of AMR in LTx. The positive response the majority of our patients experienced was determined by decrease in both class I and II DSA MFI, elimination of dnDSAs, and reversal of C1q positivity. Larger prospective studies investigating the ideal time from dnDSA development to initiating AMR treatment, and defining the most appropriate time to utilize CFZ are needed.

Desensitization Therapy for Patients with DSA Receiving Haploidentical (Haplo) Stem Cell Transplantation

<h3>Introduction</h3> The effect of desensitization therapy on patients with Donor-specific Anti-HLA Antibodies (DSA) for patients receiving Haplo transplantation are unknown. <h3>Methods</h3> Data of all 51 patients with DSA receiving Haplo transplantation between 11/2010-1/2019 at MD Anderson Cancer Center (N=41) and City of Hope (N=10) were included. Patients received desensitization with plasma exchange (PE), rituximab (R), and IVIg +/- buffy coat (BC) infused on day-1, as previously described by us (Ciurea SO. BBMT.2018:53:521). We analyzed transplant outcomes for DSA treated patients vs. those without DSA receiving Haplo transplant in the same period of time (control; N=345). <h3>Results</h3> The median age of patients with DSA was 51 years (range 19-69) and 47 years (range 18-72) in control group (p=0.15). There were no significant differences in diagnosis, remission status at transplant, DRI and ABO matching between 2 groups. RIC conditioning was used in 28% and 43% in DSA and control group, respectively (p=0.03) whereas 67% and 82% respectively (p=0.02) received marrow stem cell. Median levels of DSA on single antigen assay before desensitization was 5556 MFI (range 2360-28688). Of 51 patients with DSA, 37 (72.5%) DSA patients received desensitization with PE, R and IVIg, and 27/37 (73%) patients also received BC infusion before transplant. Twenty of 37 (54%) desensitized patients had DSA >5000 MFI and 14 out of them had complement binding DSA suggested by a positive C1q assay. Cumulative incidence (CI) of neutrophil engraftment at 28 days for patients who had DSA>5000 MFI and received desensitization was 75% compared with 91% in those without DSA (p=0.12) (Figure 1A) while 1-year NRM was 32% vs. 29% (p=0.57) (Figure 1B) and 1-year OS was 54% vs. 58%, respectively (p=0.18) (Figure 1C). The engraftment rate is significantly improved with desensitization treatment as our previous study demonstrated that only 46% of patients with DSA>5000 MFI achieved engraftment (Ciurea SO. BBMT.2015;21:1392). Remarkably, patients with DSA 2000-10000 who received treatment had 83% engraftment vs. 91% (p=0.86). Among 14 patients who had C1q+ DSA, 8 patients had persistently positive C1q after desensitization and only 4 engrafted, 2 had primary graft failure and 2 died before engraftment with CI of engraftment at 28 days of 43% (p=0.09 compared with control group). Persistent C1q positivity was associated with significantly higher NRM (37% vs. 29% at 1 year, p=0.01) and lower OS (33% vs. 58% at 1 year, p=0.002) compared with control group (Figure 1D). These significances persisted after adjusting for other factors in multivariable analysis. <h3>Conclusions</h3> Treatment with PE, R, IVIg and BC infusion is an effective strategy to desensitize patients with DSA before haploidentical transplantation. Patients who remain C1q+ at transplantation have high risk for engraftment failure and should not proceed to transplantation.

EDTA Treatment for Overcoming the Prozone Effect and for Predicting C1q Binding in HLA Antibody Testing.

The Luminex-based single antigen bead (SAB) assay is widely used to detect HLA antibody in transplant recipients. However, one limitation of the SAB assay is the prozone effect, which occurs mostly as a result of complement interference. We investigated the efficacy of EDTA treatment for overcoming the prozone effect and predicting C1q binding of HLA antibody. We subjected 27 non-treated (naïve) and EDTA-treated serum samples from highly sensitized patients to IgG-SAB assays, and we confirmed the prozone effect in 53% and 31% of class I and class II antibody tests, respectively, after EDTA treatment. When we conducted additional assays after dithiothreitol treatment and serum dilution, EDTA was the most efficacious in eliminating the prozone effect. Reducing the prozone effect by EDTA treatment strengthened the correlation between IgG mean fluorescence intensity (MFI) and C1q MFI values (ρ=0.825) as compared with the naïve sera (ρ=0.068). Although C1q positivity was dependent on the concentration of HLA antibody in EDTA-treated sera, the correlations varied individually. Overall, our results confirmed the efficacy of EDTA treatment for overcoming the prozone effect. EDTA treatment showed a positive effect on the correlation between IgG MFI and C1q MFI values.

Long-term outcomes of eculizumab-treated positive crossmatch recipients: Allograft survival, histologic findings, and natural history of the donor-specific antibodies.

We aimed to determine the long-term outcomes of eculizumab-treated, positive crossmatch (+XM) kidney transplant recipients compared with +XM and age-matched negative crossmatch (-XM) controls. We performed an observational retrospective study and examined allograft survival, histologic findings, long-term B-cell flow cytometric XM (BFXM), and allograft-loss-associated factors. The mean (SD) posttransplant follow-up was 6.3 (2.5) years in the eculizumab group; 7.6 (3.5), +XM control group; 7.9 (2.5), -XM control group. The overall and death-censored allograft survival rates were similar in +XM groups (P=.73, P=.48) but reduced compared with -XM control patients (P<.001, P<.001). In the eculizumab-treated group, 57.9% (11/19) of the allografts had chronic antibody-mediated rejection, but death-censored allograft survival was 76.6%, 5years; 75.4%, 7years. Baseline IgG3 positivity and BFXM ≥300 were associated with allograft loss. C1q positivity was also associated with allograft loss but did not reach statistical significance. Donor-specific antibodies appeared to decrease in eculizumab-treated patients. After excluding patients with posttransplant plasmapheresis, 42.3% (9/21) had negative BFXMs; 31.8% (7/22), completely negative single-antigen beads 1year posttransplant. Eculizumab-treated +XM patients had reduced allograft survival compared with -XM controls but similar survival to +XM controls. BFXM and complement-activating donor-specific antibodies (by IgG3 and C1q testing) may be used for risk stratification in +XM transplantation.

Clinical impact of complement (C1q, C3d) binding De Novo donor-specific HLA antibody in kidney transplant recipients.

IntroductionComplement binding activity of donor-specific HLA antibodies (DSA) has been suggested as a new tool to stratify immunologic risk in kidney transplantation (KT). The objective of this study was to evaluate the clinical implication of C1q/C3d binding activity of de novo DSA (dnDSA) in KT recipients.Material and methodsA total of 161 pretransplant DSA-negative recipients were monitored for dnDSA at the time of biopsy. C1q/C3d binding activities of dnDSA were assessed using C1qScreen assay (One lambda, USA) and Lifecodes C3d detection assay (Immucor, USA), respectively. Clinical outcomes including biopsy-proven antibody mediated rejection (AMR), C4d detection and post-biopsy graft survival were investigated.ResultsDe-novo DSAs were detected in fifty-four (33.5%) patients (HLA class I only, n = 19; class II only, n = 29; both class I and II, n = 6). Of them, complement binding activities were detected in 26 (48.1%) patients, including 17 C1q+ and 24 C3d+ patients. Both C1q and C3d positivity were associated with increased mean fluorescence intensity values of dnDSA. Complement binding activity of dnDSA enhanced the incidence of AMR (25.0% in C1q-C3d-, 36.4% in C1q+/C3d- or C1q-/C3d+, and 60.0% in C1q+/C3d+ patients) (P <0.001). The incidence of AMR was not different between patients with C1q+ and those with C3d+ dnDSA (64.7%, 11/17 versus 45.8%, 11/24, P = 0.238). In comparison between C1q and C3d assay according to HLA specificity, C1q+ HLA class I ± II dnDSA was the best predictor for AMR (odds ratio: 27.2). C1q+/C3d+ dnDSA was associated with more C4d deposition in allograft tissue and inferior post-biopsy graft survival. Clinical outcomes were not significantly different between C1q+ and C3d+ dnDSA-positive patients.ConclusionDetection of complement binding activity using both C1q and C3d assays can be a further prognostic marker for predicting AMR and allograft outcome in dnDSA+ kidney transplant patients.

P116 Complement component 1q assay lacks sensitivity and trends with de novo donor-specific antibody titers

Aim Despite major advancements in immunosuppression, long-term allograft survival rates from deceased and living donors remain poor following post-kidney transplantation. The presence of de novo donor-specific antibodies (dnDSA) and complement component 1q (C1q) following transplantation contributes to repetitive microvascular injury that correlates with worsening graft function and graft failure. Immune monitoring post-transplantation of dnDSA and C1q is commonly employed to assess patient graft failure risk and can guide clinical decision making regarding the patient’s baseline immunosuppression. In this investigation, we sought to determine if the C1q assay provided added clinically relevant data as C1q positivity was often associated with dnDSA titer and the use of pooled patient serum for quality control varied greatly among different runs of the same lot number. Methods Kidney transplant patients were monitored for the presence of human leukocyte antigen (HLA) class I and class II antibodies using the Labscreen Single Antigen Antibody Detection Tests and the C1qScreen (One Lambda, Inc., Canoga Park, CA). C1q risk scores were delineated as N = C1q negative, 2 = C1q weak positive, 3 = C1q moderate positive, 4 = C1q strong positive, 5–6 = very strong positive based on C1q mean fluorescent intensity (MFI) cutoff ranges. Results DSA titration (1:16, MFI = 3136) trended as N = C1q negative. DSA titration (1:256, MFI = 13136) trended as 5–6 = C1q very strong positive. Poor delineation of C1q risk at intermediate titrations may be due to confounding quality control variability in the daily pooled plasma serum measurements, which can alter baseline risk scoring of C1q MFI values. Conclusions C1q and dnDSA are often tracked to make risk assessments regarding kidney allograft health post-transplantation; however, C1q MFI measurements may not be additionally informative for clinicians as it often tracked with dnDSA antibody titer and the C1q assay lacks sensitivity at lower concentrations and potentially makes C1q risk scores ambiguous or misleading.

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.

Do C1q-Positive Donor Specific Antibodies have Prognostic Effect in Patients with Resistant Antibody-Mediated Rejection Treated with Bortezomib? Single-Centre Experience

Introduction and Aims Donor-specific antibodies (DSAs) examination is a crucial part of antibody-mediated rejection (AMR) diagnosis. C1q-positive (+) DSAs are associated with poor graft survival. We analyzed results of patients (pts) treated for acute resistant AMR by a bortezomib-based regimen with retrospectively performed C1q assay. The aim of this work was to analyze the treatment effect on DSA levels and potential effect of C1q+ DSAs on graft survival. Methods We retrospectively analyzed documentation of 772 pts who underwent renal transplantation (Tx) between 1/2012-6/2015. Novel therapeutic approach to resistant acute AMR in kidney transplant recipients was applied in 23 pts (3%) based on administration of bortezomib (B) [1 cycle of 4 doses of B (1.3 mg/m2)], small doses of i.v. steroids, plasmapheresis (PP) and a dose of rituximab (375mg/m2). This protocol was administered after conventional treatment had failed. Resistant AMR was defined as persisting deterioration or non-function of renal allograft in pts with histological verification of AMR, positive C4d staining and detection of DSA receiving standard antirejection treatment with PP + IVIG. C1q assay was performed 3 times – before Tx, at the time of diagnosis and after AMR treatment. Pts were followed for minimum of 24 months. Results Therapy of resistant acute AMR was administered to 23 pts after kidney Tx with median peak PRA 52%, actual PRA 36%, mean HLA mismatch in HLA-A 1.2 ± 0.4, HLA-B 1.7 ± 0.5, HLA-DR 1.3 ± 0, with median of 5.8 years on dialysis. 3 pts underwent 1st kidney Tx, while 20 pts reTx. All pts received induction therapy with antithymocyte globulin (n=22) or basiliximab (n=1), and maintenance immunosuppression with tacrolimus, mycophenolate mofetil/enteric-coated mycophenolate sodium and corticosteroids. Diagnosis of resistant acute AMR was made on 14th POD (7- 60 days). Using bortezomib regimen in treating resistant acute AMR led to decrease in DSA quantity in HLA especially in class I (p=0.005), class II (p = 0.015), but not in DQ (p= 0.2). No significant improvement of renal function was observed during the follow-up. The pts whose levels of serum creatinine increased more than 25% of baseline level in 6 months after administration of protocol with B, are progressors (n=11). The progressors graft survival was 57% in 2 years. The treatment protocol was not effective in decreasing C1q+ DSAs class I (p=0.25), class II (p=0.69), DQ (p=0.58). We detected C1q+ in 11 out of 11 progressors, compared with 3 out of 12 non-progressors (p<0.001). C1q+ DSAs correlated with C4d+ in graft biopsies at the time of AMR diagnosis (p=0.0012). Conclusions Bortezomib-based regimen was effective against class I and II DSAs, but it did not affect DQ DSAs. Pts with any C1q+ DSAs were more likely to have progressive graft dysfunction compared to those with C1q-negative DSAs. C1q positivity is a promising prognostic marker in pts with resistant AMR.

Relationship between Mean Fluorescence Intensity and C1q/C3d-fixing capacities of anti-HLA antibodies

BackgroundComplement-binding assays are proposed to better stratify the risk of antibody-mediated rejection associated-graft failure. Despite promising clinical results, some have suggested that the MFI of anti-HLA antibodies may influence these tests. MethodsWe investigated the impact of Abs MFI reduction, induced by plasmapheresis, on C1q- and C3d-binding assays. Sera provided from 7 sensitized kidney transplant patients were analyzed. ResultsFour hundreds and thirty-three SABs were analyzed. Before plasmapheresis, when compared to C1q− SABs, C1q+ SABs had a higher median MFI [17397 (IQR: 14851–18794) vs. 2745 (IQR: 1125–6476), p<0.01]. SABs that remained C1q+ after plasmapheresis had a higher median MFI. Regarding the C3d assay, results were strictly comparable. MFI value was a powerful predictor of both C1q and C3d positivity [AUC 0.97 (CI95% 0.95–0.99) and 0.96, (CI95% 0.93–0.98), respectively]. ConclusionOur data suggest that both C1q- and C3d-binding assays are intimately linked to the MFI of anti-HLA Abs.

Renal Transplant Patients Biopsied for Cause and Tested for C4d, DSA, and IgG Subclasses and C1q: Which Humoral Markers Improve Diagnosis and Outcomes?

The association between donor specific antibodies (DSA) and renal transplant rejection has been generally established, but there are cases when a DSA is present without rejection. We examined 73 renal transplant recipients biopsied for transplant dysfunction with DSA test results available: 23 patients diffusely positive for C4d (C4d+), 25 patients focally positive for C4d, and 25 patients negative for C4d (C4d−). We performed C1q and IgG subclass testing in our DSA+ and C4d+ patient group. Graft outcomes were determined for the C4d+ group. All 23 C4d+ patients had IgG DSA with an average of 12,500 MFI (cumulative DSA MFI). The C4d− patients had average DSA less than 500 MFI. Among the patients with C4d+ biopsies, 100% had IgG DSA, 70% had C1q+ DSA, and 83% had complement fixing IgG subclass antibodies. Interestingly, IgG4 was seen in 10 of the 23 recipients' sera, but always along with complement fixing IgG1, and we have previously seen excellent function in patients when IgG4 DSA exists alone. Cumulative DSA above 10,000 MFI were associated with C4d deposition and complement fixation. There was no significant correlation between graft loss and C1q positivity, and IgG subclass analysis seemed to be a better correlate for complement fixing antibodies in the C4d+ patient group.

P044 C1q status and titer of de novo donor specific antibodies are not predictors of allograft survival

De novo donor-specific antibodies ( dn DSA) that develop after renal transplantation are independent predictors of allograft loss. However, it is unknown if dn DSA C1q status or titer at the time of first detection can independently predict allograft outcomes. In 70 renal transplant recipients who developed dn DSA we performed LABScreen single antigen bead assays using: neat, neat with ethylenediaminetetraacetic acid (EDTA), three dilutions after EDTA treatment (1:16, 1:64, 1:1024) and the C1q assay (neat sera). Histologic and clinical outcomes were correlated with the C1q assay or dn DSA titer. C1q positivity correlated with dn DSA titer (p dn DSA (p dn DSA development, but did not predict T-cell mediated rejection (TCMR) or antibody mediated rejection (ABMR). De novo DSA titer correlated with glomuerulitis, interstitial inflammation, tubulitis, peritubular capillaritis, and C4d scores, and predicted TCMR (p dnDSA development or other known predictors of post-dnDSA graft loss (i.e. clinical phenotype or non-adherence) neither C1q status nor dn DSA titer were independent predictors of a composite outcome of a 50% decline in eGFR or allograft loss (Fig. 1). Therefore, at present the additional cost and time associated with these approaches is not justified in routine practise. However, in cases where a renal biopsy is not possible dn DSA titer correlates with TCMR, and AMBR, and may assist in risk stratification. Download high-res image (239KB) Download full-size image

Association of C1q Binding Status With De Novo HLA Antibody Clinical Features and Allograft Function in Kidney Transplantation Patients During Eight Years of Dynamic Follow-up

C1q-binding donor-specific antibody (DSA) is detrimental to transplanted kidney function. However, the factors that affect C1q binding status are unclear. A total of 519 samples from 129 consecutive kidney transplantation patients during 8 years of dynamic follow-up were collected for HLA antibody (Ab) screening and C1q detection. Among the detected HLA Abs, the majority were class II, and the DQ subtypes composed the highest proportion. The C1q-binding Abs were all HLA-II, and the DQ subtypes had the highest rate of C1q positivity. With a cutoff mean fluorescence intensity (MFI) value of 7349, the sensitivity and specificity of detecting C1q-binding Abs from all HLA-II Abs were 84.48% and 83.56%, respectively. Additionally, C1q is more likely to be bound by DSA than non-donor-specific antibody (NDSA). Compared with free DSA/NDSA, the MFI values of C1q-binding DSA/NDSA are more closely correlated with serum creatinine levels and reflect the effect of anti-antibody-mediated rejection treatment more sensitively. HLA-II Abs (particularly DQ subtypes), high titers of Abs, and DSA are important relevant factors of C1q positivity. The MFI value of C1q-binding DSA may be a useful clinical indicator of HLA antibody-mediated graft injury before the appearance of histologically typical humoral rejection.

Prognostic significance of C1q deposition in serial biopsies for predicating the long-term outcome in patients with proliferative lupus nephritis.

Lupus nephritis (LN) is characterized by a highly variable clinical course. It has been reported that histopathologic lesions are risk factors for the progression of LN. The aim of this study is to investigate the relationship among the co-deposition of C1q, clinicopathological features, and renal outcomes in patients with LN. The clinical and histological parameters were studied in patients with International Society of Nephrology/Renal Pathology Society Class III or IV LN, who underwent two kidney biopsies. The patients were divided into two groups based on the glomerular C1q deposits: C1q-positive and C1q-negative. The impact of C1q status and longterm renal outcome on the doubling of serum creatinine and the rate of remission in the two groups were further investigated. Fifty-three patients had pure proliferative nephritis and 37.7% of these had a co-deposition of C1q. Doubling of serum creatinine was observed in 25% of patients with C1q-positive and 24.2% of patients with C1q-negative deposits. There was no difference between the two groups in terms of achieving complete or partial remission. The renal survival in the two groups was similar (P = 0.75). Upon repeat biopsy, the persistence of C1q positivity was associated with a poor outcome (P = 0.007). C1q deposition in the glomerulus in the baseline biopsy was not associated with a poor renal outcome or severe pathologic features in patients with proliferative LN. However, the persistence of C1q positivity in repeat kidney biopsy is associated with a poor renal outcome.