C1q Positivity Research Articles

Association of Kidney Graft Loss With De Novo Produced Donor-Specific and Non-Donor-Specific HLA Antibodies Detected by Single Antigen Testing.

The association of donor-specific HLA antibodies (DSA) with kidney graft failure has been addressed previously; however, the majority of studies were based on small numbers of patients with graft failure. We investigated 83 patients with failed kidney transplants for a possible association of de novo development and persistence or loss of pre-existing DSA with graft failure. Single Antigen Bead assay-detected DSA and non-DSA antibodies were compared between patients with graft loss and matched controls with functioning grafts. The incidence of weak de novo DSA or non-DSA at a mean fluorescence intensity of 500 or higher was higher in the graft loss than in the nonrejector group (76% vs 40%, P < 0.001). Because of the low number of patients developing de novo DSA, the DSA results did not reach statistical significance (only 22% of patients with graft loss developed de novo DSA). However, at all cutoffs, there was a significantly higher rate of graft loss in patients with de novo non-DSA. The incidence of strong pretransplant DSA that persist after transplantation was higher in the graft loss group (10% vs 1%, P = 0.034). When C1q-binding ability in sera of rejectors and nonrejectors with posttransplant de novo or persistent DSA was compared, none of the nonrejectors demonstrated C1q positivity, whereas 43% of patients with graft loss showed C1q-positive antibodies, although not necessarily donor-specific (P < 0.001). Our data show that the posttransplant presence of persisting or de novo HLA antibodies, especially if C1q binding, is associated with graft loss, even if the antibodies are not specific for mismatched donor HLA.

Characteristics of donor-specific anti-HLA antibodies in renal transplant recipients

Introduction Antibody-mediated rejection (AMR) is the major complication of renal transplantation that significantly affects the renal allograft survival. Complement fixation mediated through donor specific anti-Human Leukocyte Antigen (HLA) antibodies (DSA) is the hallmark of AMR. Different methods have been developed to detect harmful DSAs. However, not all DSAs have the same detrimental effect, as different antibody characteristics might trigger distinct immune responses. Aim: In the present study, we assessed various biological (timing of appearance, strength and specificity) and functional (IgG subtype and c1q binding ability) characteristics of DSA in renal transplant patients. Methods A total of 315 kidney transplant recipients were accrued for this study and 39 patients with de novo DSA were retrospectively analysed for different DSA characteristics. The analyses were carried out using commercially available SAB assay for IgG determination, C1q assay for complement binding capability and modified SAB assay for IgG subclass determination (One Lambda) Results Important findings of the study were: (1) majority of the DSAs were denovo antibodies ( p = 0.0001); primarily against HLA class II antigen ( p = 0.0005); and typically developed after one year posttransplant ( p = 0.0001). (2) DSA with highest strength were routinely observed against DQB antigens in comparison to other HLA class I and II antigens ( p = 0.006). (3) IgG1 was the predominant IgG subclass (49.4%), followed by IgG3 (24.7%), IgG2 (16%), and IgG4 (9.9%). (4) DSA MFI and the IgG1 subclass were strongly correlated with C1q positivity ( p = 0.01 and p = 0.009, respectively). Conclusion Among the functional characteristics, higher MFI of DSA and IgG1 subclass were strongly correlated with C1q positivity. Among the biological characteristics, the majority of DSAs were against HLA class II antigens and developed frequently after one-year post transplantation. Further studies are needed to investigate the clinical relevance of these biological and functional DSA characteristics in predicting renal allograft outcomes.

OR35 IgG subclass analysis in C4d+ DSA positive patients

Aim 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. The Luminex® Single Antigen (SA) assay is the current standard for identifying HLA IgG antibody specificities and determining if they are donor specific. As a means to improve the ability to discriminate between harmful and benign antibodies, the SA C1q assay was developed to detect antibodies that are capable of fixing complement. We also have adapted the standard SA IgG assay by utilizing IgG-subclass specific monoclonal antibodies. We sought to explore the detection capacity of the three SA assays and their correlation with renal transplant rejection. Methods 23 patients were selected who had received biopsies due to graft dysfunction and all had C4d deposition on the peritubular capillaries. Out of the 23 patients who presented with graft dysfunction, 5 proceeded to graft loss. Serum samples collected within a week of the biopsy were used for Luminex SA IgG, C1q and IgG subclass analysis. Results All 23 C4d+ patients had SA IgG DSAs with an average of 12,500 MFI (cumulative DSA MFI). 25 control patients with C4d− biopsies had average DSAs less than 500 MFI. The C1q assay showed 16 positive results out of the 23 DSA and C4d+ patients. The C1q assay appeared to be less sensitive particularly when multiple low level IgG DSAs occurred. There was no significant correlation to graft loss and C1q positivity. The IgG subclass assay showed complement fixing IgG1 in 20 of 23 patients. IgG2 DSAs were found in six patients and IgG3 DSAs were found in two patients, all in conjunction with IgG1 DSAs. Interestingly, IgG4 was seen in 10 of the 23 recipients’ sera, but always along with complement fixing IgG1. The detection of IgG4 subclass did not correspond to any enhanced graft survival outcome. Conclusions Cumulative DSA’s above 10,000 MFI were associated with C4d deposition and complement fixation. In this study of patients with C4d+ biopsies, 100% had IgG DSAs, 70% had C1q+ DSAs, and 83% had complement fixing IgG subclass antibodies. We could not demonstrate any enhanced benefit of IgG4. However, in this cohort of patients, IgG4 appeared coincidently to IgG1, and we have previously seen excellent function in patients when IgG4 DSA exists alone.

OR5 Igg subclass and concentration are determinants of HLA class I antibody capacity to fix complement in in vitro clinical and functional assays

It has been presupposed that “complement (C’) fixing” HLA antibodies (Ab) detected by C1qScreen or CDC are IgG1 or IgG3. We tested monoclonal HLA Ab of each human IgG subclass to evaluate the effect of subclass in in vitro complement assays. Chimeric HLA I Ab carrying the same variable region (pan HLA I) and human IgG1–4 constant regions were diluted (0.01–20 μg/mL) in human serum containing no HLA Ab, and tested in One Lambda LabScreen, C1qScreen, and Immucor LifeCodes Single Antigen and C3d Assays. C4d deposition on B cells and human endothelium was measured by flow cytometry, and cytotoxicity was measure using standard rabbit (rb-CDC) and human C’ (hum-CDC) assays. IgG-MFI, C1q-MFI, and C3d-MFI were dependent upon Ab concentration; however, the dynamic linear ranges of these assays was quite different. IgG-MFI reached saturation at lower Ab concentrations (500 ng/mL for IgG1) than C1q-MFI, while C1q-MFI became negative at Ab concentrations (0.125 ng/mL) still detectable by IgG-MFI. Lower amounts of Ab, including IgG2, were detectable by C3d assay than by C1qScreen. Ab concentration correlated well with C1q-MFI and C3d positivity. There was no linear relationship between IgG-MFI and C1q-MFI for any subclass, but C3d deposition did correlate with IgG-MFI. A threshold for C1q positivity was observed which differed for each subclass; for example, 15000 IgG1-MFI translated to C1q-MFI > 1000. Beads with lowest antigen density often had lowest IgG-MFI, C1q-MFI and C3d-MFI signals, with some false negative C’ results. Subclass-specific differences in C’ activation were observed. IgG2 triggered unanticipated strong C1q deposition in C1qScreen, and was nearly as potent as IgG1, but had lower potency in the C3d assay. Positive rb-CDC reactions were observed with IgG1, IgG3, and, unexpectedly, IgG2. In the hum-CDC, no chimeric subclass caused a positive reaction. Deposition of human C4d on the surface of cells could be detected, in a dose- and subclass-dependent manner. Our results highlight the dependence of C’ fixation and activation on Ab subclass and concentration, and illuminate important caveats to interpreting these assays. IgG-MFI did not directly correlate with C1q-MFI, there was a linear relationship between Ab concentration and C’ deposition in both C1qScreen and the C3d assay.

IgG Donor-Specific Anti-Human HLA Antibody Subclasses and Kidney Allograft Antibody-Mediated Injury.

Antibodies may have different pathogenicities according to IgG subclass. We investigated the association between IgG subclasses of circulating anti-human HLA antibodies and antibody-mediated kidney allograft injury. Among 635 consecutive kidney transplantations performed between 2008 and 2010, we enrolled 125 patients with donor-specific anti-human HLA antibodies (DSA) detected in the first year post-transplant. We assessed DSA characteristics, including specificity, HLA class specificity, mean fluorescence intensity (MFI), C1q-binding, and IgG subclass, and graft injury phenotype at the time of sera evaluation. Overall, 51 (40.8%) patients had acute antibody-mediated rejection (aABMR), 36 (28.8%) patients had subclinical ABMR (sABMR), and 38 (30.4%) patients were ABMR-free. The MFI of the immunodominant DSA (iDSA, the DSA with the highest MFI level) was 6724±464, and 41.6% of patients had iDSA showing C1q positivity. The distribution of iDSA IgG1-4 subclasses among the population was 75.2%, 44.0%, 28.0%, and 26.4%, respectively. An unsupervised principal component analysis integrating iDSA IgG subclasses revealed aABMR was mainly driven by IgG3 iDSA, whereas sABMR was driven by IgG4 iDSA. IgG3 iDSA was associated with a shorter time to rejection (P<0.001), increased microcirculation injury (P=0.002), and C4d capillary deposition (P<0.001). IgG4 iDSA was associated with later allograft injury with increased allograft glomerulopathy and interstitial fibrosis/tubular atrophy lesions (P<0.001 for all comparisons). Integrating iDSA HLA class specificity, MFI level, C1q-binding status, and IgG subclasses in a Cox survival model revealed IgG3 iDSA and C1q-binding iDSA were strongly and independently associated with allograft failure. These results suggest IgG iDSA subclasses identify distinct phenotypes of kidney allograft antibody-mediated injury.

LBP35: STRENGTH OF DE NOVO HLA DONOR SPECIFIC ANTIBODY IS A STRONG PREDICTOR OR ITS C1Q BINDING CAPABILITY

Introduction Antibody mediated rejection (AMR) is the major complication of renal transplantation that significantly affects the renal allograft survival. Complement fixation mediated through Donor specific anti-Human Leukocyte Antigen (HLA) antibodies (DSA) is the hallmark of AMR. Different methods have been developed to detect DSA including Luminex single antigen beads (SAB) assay. However, it is still unclear how to define harmful DSA that causes poor graft outcomes. Aim We analyzed C1q assay positivity at different posttransplant time points and evaluated its relationship with different DSA characteristics including time of occurrence (early or late posttransplant), HLA class (class I, II or both) and Ab titre (mean fluorescence intensity, MFI). Method A total of 315 kidney transplant recipients between January 2007 and December 2012 were retrospectively analyzed for the presence of DSAs and their MFI using IgG SAB assay (One Lambda Inc., USA). Out of those, 39 patients with de novo DSA were further characterized using C1q assay. All 39 patients were followed for 2 years and their sera collected consequently with the protocol and/or diagnostic biopsies were analyzed by C1q assay (One Lambda Inc.). Results Most patients had at least 3 posttransplant sera during the 2-year follow up. The high MFI of HLA-IgG in all biopsy samples correlated significantly with C1q positivity. The mean MFI values of HLA-IgG for the negative and positive C1q were 2547 and 6424 ( p = 0.003), 2481 and 6130 ( p = 0.001) and 2200 and 8400 ( p = 0.009) for the sera collected at 6, 12 and 18-month posttransplant, respectively (Fig. 1). No other DSA characteristic correlated with C1q positivity. Conclusion The C1q assay is a potentially useful tool in allograft monitoring after transplantation. Our data indicates that Ab strength strongly correlates with the capacity to bind C1q. Correlation with clinical outcomes will further elaborate the relevance of C1q positivity on the renal allograft outcome.

Abstract 16877: Correlation of Antibody Binding Strength to Complement Fixation

Background: In patients (pts) who are highly sensitized, desensitization therapy is commonly applied to reduce antibody (Abs) load and to reduce potential morbidity and mortality following heart transplant (HTx). It is not known as to what level of antibody binding is needed to initiate desensitization therapy. It is believed that high binding Abs may have the ability to fix complement and lead to cytotoxicity. Methods: Between 2011 and 2012 we assessed 13 highly sensitized pts who were noted to have standardized fluorescence intensity (SFI) &gt;100,000 pretransplant. A complement binding assay (C1q binding assay) was then applied to these Abs to assess for a correlation of binding to cytotoxicity. Correlation statistics were used to derive significance between higher antibody binding and capacity to bind complement. Results: There seems to be no correlation between the different binding strength and C1q positivity. However, when evaluating Luminex 1:8 test, there seems to be correlation between class I binding strength with C1q positivity. There was C1q positive rate of 10.8% in SFI 200,000 range. Conclusions: Undiluted antibodies do not correlate to C1q binding ability. However the 1:8 dilution test seems to provide an better prediction of complement binding. 1:8 dilutions should be performed to accurately assess the ability for antibodies to bind complement.

Determinants of C1q binding in the single antigen bead assay.

A modified single antigen bead (SAB) assay measuring C1q binding to human leukocyte antigen antibodies has recently been introduced. The aim of this study was to investigate the determinants of C1q binding on SAB. Sera from 73 sensitized patients were analyzed by the generic IgGpan as well as IgG subclass specific SAB assays and correlated with the standard and an anti-human globulin (AHG) enhanced C1q test. Among 2,665 SABs with positive IgGpan results (mean fluorescence intensity [MFI]>500), strong complement-binding IgG1 and IgG3 subclasses accounted for a median of 99% (interquartile range, 76%-100%) of the total IgG amount. IgGpan MFI alone showed a very strong association with standard C1q positivity (r=0.72), which was superior to a model including all IgG subclass MFI (r=0.62). Combining all IgG subclass MFI and IgGpan MFI only marginally improved the prediction of standard C1q positivity compared with IgGpan MFI alone (Δr=0.02). IgGpan MFI greater than 14,154 predicted standard C1q positivity, with 92% sensitivity and 96% specificity. Notably, 1,840 (93%) of the 1,974 C1q-negative SABs contained human leukocyte antigen antibodies with strong complement-binding IgG1 and IgG3 subclasses. Anti-human globulin significantly enhanced the signal in the C1q assay, but the association of AHG C1q positivity with IgGpan MFI was less strong (r=0.51). C1q binding on SAB is strongly associated with IgGpan MFI. IgG subclass information only marginally improves prediction of C1q binding likely because complement-binding IgG1 and IgG3 subclasses dominate regarding frequency and relative amounts. A negative C1q assay result does not indicate the absence of strong complement-binding IgG subclasses.

Pan HLA I Chimeric Human IgG Reveal Subclass Differences in Complement Fixing Assays.

Purpose: The purpose of this study was to compare hIgG1 and hIgG2 HLA I antibodies in their capacity to activate complement in in vitro assays, and to determine whether any correlation existed between antibody strength (MFI in single antigen assay) and ability to fix C1q. Methods: We generated chimeric pan HLA I antibodies by fusing W6/32 variable regions to human IgG1 and human IgG2 constant regions. Binding to HLA I and activation of complement was assessed by solid phase (SAB Labscreen and C1qScreen (OneLambda); C3d bead assay (Immucor/Lifecodes) and cell-based assays (flow cytometry, cell-based ELISA). Results: HLA I hIgG1 and hIgG2 bound highly to all alleles and loci of HLA I on single antigen (SAB) beads. While the distribution differed among alleles, depending on antigen density (with Cw15 having the highest, and Cw4 the lowest, MFI), there was no significant difference between subclasses (R2=0.967). Both chimers also bound to native antigen on endothelial cells (EC). HLA I hIgG1 caused C1q deposition on all LSA1 beads in a concentration dependent manner. There was an association between SAB MFI and C1q binding that varied depending on HLA locus; on B locus beads (R2>0.9) but not C locus beads. C1q positivity (MFI>1000) correlated with MFI in SAB tests of >12000 MFI. C1q was also deposited on all HLA I beads in response to high dose HLA I hIgG2, although hIgG2 was significantly less potent than hIgG1 at fixing C1q. The degree of C1q deposition by HLA I hIgG2 varied significantly (MFI >25000 to <1000) from bead to bead at the same concentration of antibody, particularly on C locus beads. hIgG2 showed poor correlation between SAB MFI and C1q-fixing capacity. Both HLA I hIgG1 and hIgG2 also resulted in functional complement activation, as measured by C3d deposition on single antigen beads, C3b deposition on EC, and CDC-XM positivity in a concentration-dependent manner. hIgG1 was significantly more effective at complement activation than hIgG2. Conclusions: hIgG2, while less potent than hIgG1, fixed complement in C1q, C3b and CDC assays at high concentrations, challenging the notion that hIgG2 DSA is less pathogenic. Moreover, C1q deposition in solid phase assays was quite variable from bead to bead, at the same concentration of pan HLA I antibody, suggesting that antigen density or denatured state of antigen significantly influences sensitivity in complement assays. In general, HLA-C locus beads were the most variable in assays measuring hIgG and C1q binding. DISCLOSURE:Reed, E.: Grant/Research Support, Pfizer, Novartis, True North Therapeutics, Immucor.

A Case of Classic Polyarteritis Nodosa Resembling Lupus Nephritis

Classic polyarteritis nodosa (cPAN) is a systemic necrotizing vasculitis of medium-sized muscular arteries. Glomerular involvement is not expected in the course of cPAN. Herein, we describe a case of cPAN with glomerular and multiple arterial involvement. The patient presented with severe abdominal pain and high fever. Urine analysis showed hematuria and 1g/day proteinuria. Kidney biopsy showed fibrinoid necrosis of arterioles and IgG, IgA and C1q positivity raising a suspicion of lupus nephritis. However digital subtraction angiography revealed typical multiple micro-aneurysms in the coronary, mesenteric, splenic and renal arteries establishing the diagnosis of cPAN. Kidney biopsy in cPAN may reveal non-specific immune-deposits and fibrinoid necrosis of arterioles mimicking lupus nephritis and microscopic polyangiitis. c-PAN should be carefully differentiated from these entities.

C1q nephropathy in children: clinical characteristics and outcome

C1q nephropathy has been suggested as a separate disease entity. C1q positivity has also been described in association with nephrotic syndrome (NS) as a potential marker for worse outcome. The aims of this study were to describe the clinical characteristics, laboratory parameters and outcomes of 35 children whose renal histology revealed predominant mesangial C1q deposition and to investigate if the experience at our institution supports the above hypothesis. Clinical and pathological characteristics of all children whose kidney biopsies showed positive C1q staining were retrospectively recorded. The outcome of children with minimal change nephrotic syndrome (MCNS) and predominant mesangial C1q deposition based on C1q staining was compared with that of a concurrent group of children with MCNS with no such immune staining. The median age of the patient cohort was 4.5 years (range 6 months to 16 years), 69 % were boys and 88 % presented with nephrotic syndrome (NS). Children with C1q staining and MCNS had more relapses (p = 0.001) and shorter relapse-free periods (p = 0.033) than those with negative immunostaining, but the long-term outcomes were similar in both groups. Our data do not support C1q nephropathy as a separate diagnostic category. Children with MCNS and mesangial C1q deposition (staining) showed more relapses but no difference in long term renal outcome.

62-OR

Aim The presence of C1q antibodies may be important for assessing the risk of antibody mediated rejection (AMR) in solid organ transplants. We studied the correlation between the C1q-single antigen bead assay (C1q-SAB) with the long-incubation complement-dependent lymphocytotoxicity assay (CDC) and the IgG-single antigen bead assay (IgG-SAB). Methods 60 reference sera (38 Class I and 22 Class II) from the UCLA serum exchange program were characterized using the CDC, Luminex PRA (Gen-probe Inc.) and IgG-SAB (One Lambda Inc.) assays. We also tested pre- and post-transplant sera from 16 renal allograft recipients. All patients had persistent posttransplant DSA as demonstrated by the IgG-SAB assay. Results The C1q-SAB assay showed 85% concordance with the CDC assay as 15% of the CDC positive antibodies were negative in the C1q test. Correlation between the SAB MFI and C1q positivity showed that 90% of sera with the MFI of >18,000 in the IgG-SAB assay were C1q binding. A retrospective study of 16 renal transplant recipients developing post-transplant donor specific antibodies (DSA) showed 5 of 9 patients diagnosed as C4d+ AMR displayed C1q positive DSA (P = 0.145). One out of 7 patients without AMR displayed C1q binding DSA. Conclusions Our data indicate that antibody strength correlates with the capacity to bind C1q (Fig. 1). However we identified a subset of antibodies that displayed high MFI values and CDC positivity, that failed to bind C1q. The development of C1q positive DSA was not significantly associated with positive C4d staining on the graft. Additional studies are required using a larger cohort of patients to verify these findings [Fig. 1]. Download : Download full-size image

C1q nephropathy- unity in diversity.

Implication for health policy/practice/research/medical educationC1q nephropathy refers to a disorder in which C1q deposits are seen in mesangium on immunofluorescence microscopy and mesangial electron dense deposits on electron microscopy. The diagnosis of C1q nephropathy is based on demonstration of intense C1q (dominant or co-dominant) positivity, mainly in the mesangium on immunofluorescence microscopy. Electron dense deposits of C1q in C1q nephropathy are confirmatory of the diagnostic entity.

35-OR

Aim Sensitized heart transplant candidates are evaluated for donor specific HLA antibody (DSA) by Luminex single antigen bead (SAB) testing to assess donor suitability and predict a positive complement dependent cytotoxicity (CDC) crossmatch (XM) by virtual XM (VXM). However, SAB testing imperfectly correlates with VXM, and antibody thresholds for XM prediction are center-specific. A novel Luminex SAB-based assay detecting C1q-binding HLA antibodies adds functional data to SAB testing, but the relationship between SAB strength and C1q positivity is unclear. In this retrospective study, we examine the relationship between HLA antibody titer and complement-binding ability in cardiac allograft recipients and correlate C1q DSA to antibody-mediated rejection (AMR). Methods We identified 15 sensitized pediatric and adult heart recipients with a calculated panel reactive antibody >50%. Sera were tested by conventional SAB antibody testing using undiluted or 1:16 diluted sera and were compared to C1q results. Pre- and post-transplant DSA by both methods were correlated with XM and early AMR. Results In 9 samples, individual Class I antibodies measured by SAB testing in undiluted sera correlated poorly with C1q reactivity (R 2 = 0.07). In contrast, using sera diluted 1:16, conventional SAB results correlated better with C1q testing ( R 2 = 0.73). Receiver operator characteristic curve analysis also showed superior area under the curve (AUC) for predicting C1q positive results in sera diluted 1:16 than in undiluted sera (AUC = 0.99 vs 0.82). Low MFI by conventional SAB testing in alleles with high C1q MFI increased upon dilution or heat-treating sera and correlated with C1q results. Clinically, in 13 recipients C1q DSA predicted CDC-XM positivity and early AMR (positive predictive value (PV) 87.5%; negative PV 100%). Conclusions In conclusion, SAB titer correlated with and predicted C1q reactivity. The risk for positive crossmatch and early AMR in sensitized heart recipients strongly correlated with C1q binding DSA.

74-P

Aim Clinical management of renal transplant patients is often a clinical dilemma once they have developed donor specific antibodies (DSA). One of the mechanisms of graft injury by DSA is considered complement-mediated. We evaluated complement-fixing ability of DSA during post-transplant monitoring. Methods Frozen serum samples (N = 17) were selected from 6 patients in whom DSA was detected after kidney transplant, including collections from rising and stable creatinine periods. HLA antibody (IgG) was tested by LABScreen PRA and LABScreen Single Antigen beads (SAB, One Lambda). Complement fixing ability was tested by C1q assay (One Lambda). Donor/recipient HLA typing was tested by serology or SSO/SSP. DSA (IgG), DSA (C1q), creatinine, and biopsy results were correlated. Results From 6 patients (3 pediatrics and 3 adults), 17 serum samples are from 7 points during increasing creatinine and 10 points during stable creatinine. In these patients, the first detection of DSA was from 1.2 to 6 years ago. Specificity was class II only in 4 patients and both class I/II in 2 patients. All 17 data points detected maximum DSA (IgG) at >7000 MFI by SAB, and were also equally positive by C1q assay, regardless the creatinine trend or biopsy results. Corresponding biopsy results were available in 12 samples; C4d and T-cell rejection (N = 3), C4d only (N = 5), T-cell rejection only (N = 4), and neither (N = 0). Thus, 8 of 12 serum samples correlated C1q positivity and C4d positivity (66%). Among the 6 patients with C1q positive DSAs, 4 patients had stable creatinine, chronic antibody rejection was recognized 18 months after C1q detection, and one developed rising creatinine due to T-cell rejection. Conclusions All 17 DSA (IgG) positive samples were also positive by C1q assay, regardless of the creatinine or C4d biopsy status. In vivo responses and consequences of DSA are regulated by complex immune mechanisms, which may not correlate to an in vitro complement fixing assay.

Transformation of membranous nephropathy into antiglomerular-basement membrane glomerluonephritis.

Sir, Further to the recently published case of membranous nephropathy with superimposed anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis [1], we report a case of membranous nephropathy (MN) with crescentic transformation secondary to antiglomerular basement membrane (antiGBM) antibodies. AntiGBM disease is associated with ANCA-positive vasculitis in one-third of the cases but very rarely seen in association with MN. Individual cases or small series have been reported with the occurrence of antiGBM disease preceding, coexistent with or following MN [2–5]. A 63-year-old Caucasian male was referred to the renal services following detection of proteinuria on routine testing by the primary care physician. He was hypertensive for 11 years, well controlled with atenolol and bendroflumethazide. Other remarkable past medical history included open ureterolithotomy for renal stone disease. Initial laboratory tests showed serum urea 6.1 mmol/L, creatinine 105 μmol/L, albumin 31 g/L and proteinuria (4 g/24 h). Ultrasound showed a large left renal cyst and normal right kidney. ANA was weakly positive; serum immunoglobulins and complement levels were normal. HbsAg and antiHCV were negative. Renal biopsy was performed and revealed 21 glomeruli, all of which showed marked thickening of the capillary walls. There was minimal tubular atrophy with normal blood vessels. Silver staining revealed spikes on the glomerular basement membrane and immunohistochemistry showed granular IgG, IgM and C1q positivity. Subepithelial deposits were seen on electron microscopy (Figures 1 and ​and2)2) anti-neutrophil cytoplasmic antibody (ANCA) (Figures 3 and 4). A diagnosis of idiopathic membranous nephropathy was made and he was commenced on ramipril and simvastatin. Fig. 1. (A) Renal biopsy (silver stain ×10), (B) silver stain shows spikes (×40), (C) immunohistochemistry reveals granular IgG deposition in glomerular basement membrane (×40) and (D) immunohistochemistry reveals granular C1q deposition ... Fig. 2. (A) Haemotoxylin and eosin stain reveals crescents in the glomeruli (×1.6), (B) silver stain shows crescents in the glomeruli (×10), (C) silver stain shows crescent in the glomerulus (×40) and (D) immunohistochemistry shows IgG ... Three months later, he presented non-specifically unwell with anorexia, dark urine and loin pain. Laboratory investigations revealed severe renal failure with urea 32.9 mmol/L, serum creatinine 503 μmol/L with low albumin 22 g/L. Magnetic resonance angiography performed on suspicion of renovascular disease revealed patent renal vessels with no evidence of thrombosis or stenosis. Serologic tests came back strongly positive for antiGBM antibodies (>100 ELISA units, 0–2.5 normal) and negative for ANCA. Chest X-ray was normal with no evidence of pulmonary haemorrhage. Repeat renal biopsy revealed four glomeruli and all showed segmental necrosis with active crescents (Figure 2). Silver staining revealed typical spikes on the glomerular basement membrane, with granular IgG, IgM, C3 and C1q on immunohistochemistry and subepithelial deposits on electron microscopy. Immunofluoroscence was not performed. The biopsy picture was consistent with coexistent membranous nephropathy and antiGBM disease. He was treated with haemodialysis, intravenous pulse cyclophosphamide, steroids and plasma exchange. With advanced renal injury on biopsy and dialysis dependency at presentation, he did not recover renal function. Serial antiGBM titres became undetectable and immunosuppression was tapered to stop by 2 months of discharge. The patient remained on CAPD for 2 years and received a successful living related renal transplant from his daughter.