External quality assurance program for diagnostic complement laboratories: evaluation of the results of the past seven years.

Introduction: The complement external quality assurance (EQA) program was first organized in 2010 by a group of researchers working in diagnostic complement laboratories. Starting in 2016, INSTAND e.V., a German, non-profit interdisciplinary scientific medical society dedicated to providing expert EQA programs for medical laboratories, started organizing the EQAs for complement diagnostic laboratories together with the same group of experienced scientists and doctors who also work as EQA experts. The aim of the current work is to provide descriptive analysis of the past seven years' complement EQA results and evaluate timeline changes in proficiency testing. Methods: Each year, in March and October, blinded samples (normal, pathological) were sent to the participating diagnostic laboratories, where complement parameters were evaluated exactly as in daily routine samples. Since no reference method/target values exist for these parameters, and participants used different units for measurement, the reported results were compared to the stable mean (Algorithm A) of the participants using the same method/measurement units. A reported result was qualified as "passed" if it fell into the 30-50% evaluation/target range around the mean of reported results (depending on the given parameter). Results: While the number of participating laboratories has increased in the past years (from around 120 to 347), the number of complement laboratories providing multiple determinations remained mostly unchanged (around 30 worldwide). C3, C4, C1-inhibitor antigen and activity determinations provided the best proficiency results, with >90% passing quotas in the past years, independent of the applied method. Determination of the functional activity of the three activation pathways was good in general, but results showed large variance, especially with the pathological samples. Complement factor C1q and regulators FH and FI are determined by only a few laboratories, with variable outcomes (in general in the 85-90% pass range). Activation products sC5b-9 and Bb were determined in 30 and 10 laboratories, respectively, with typical passing quotas in the 70-90% range, without a clear tendency over the past years. Conclusion: With these accumulated data from the past seven years, it is now possible to assess sample-, method-, and evaluation related aspects to further improve proficiency testing and protocolize diagnostic complement determinations.

Daratumumab therapy in a pediatric case of C3 nephritic factor-positive proliferative glomerulonephritis with monoclonal IgG deposits.

Proliferative glomerulonephritis with monoclonal immunoglobulin deposits (PGNMID) is an exceedingly rare cause of glomerulonephritis among children for which prognosis is generally poor, with low incidence of remission and high rates of recurrence after transplant. While there are more cases reported in the adult literature, substantial differences in pediatric vs. adult PGNMID render it essential that we further characterize pediatric cases to optimize management. We report the case of a 12-year-old male presenting initially with edema and hypertension who was subsequently diagnosed with IgG3/Kappa-dominant PGNMID. In the absence of any proven therapy and though without a detectable clone, he was empirically treated with daratumumab with positive effect to date. This is the first reported case of daratumumab monotherapy in pediatric PGNMID, as well as the first PGNMID case to detect presence of C3 nephritic factor.

Clinical Characteristics of Patients With Acquired Partial Lipodystrophy: A Multicenter Retrospective Study.

BACKGROUND: Barraquer-Simons syndrome (BSS) is a rare, acquired form of lipodystrophy characterized by progressive loss of upper body subcutaneous fat, which affects face, upper limbs, and trunk. The pathogenesis of the disease is not entirely known and may involve autoimmune mechanisms. AIM: This study aimed to provide a comprehensive picture of the clinical, immunological, and metabolic features of a large cohort of patients with BSS. Our primary objectives included the validation of existing diagnostic tools, the evaluation of novel diagnostic approaches, and the exploration of potential disease triggers or genetic predispositions. SUBJECTS AND METHODS: Twenty-six patients were diagnosed with BSS based on accepted criteria defined by international guidelines. Anthropometric parameters, biochemical tests, organ- and non-organ-specific autoantibodies, HLA status, and screening of the LMNB2 gene were performed. RESULTS: Patients were predominantly females (73%); fat loss occurred mostly during childhood (77%) at a median age of 8 years. Among various anthropometric measures, the ratio between the proportion of fat mass in upper limbs and lower limbs showed the best predictive value for diagnosis. A total of 11.5% of patients had diabetes, 34.6% dyslipidemia, and 26.9% hepatic steatosis. Seventy-five percent of children and 50% of adults had C3 hypocomplementemia; 76% of patients were positive for 1 or more autoantibodies. HLA-DRB1 11:03 had higher allelic frequencies compared with the general population. A single variant in the LMNB2 gene was found in 1 patient. CONCLUSION: BSS has a childhood onset and is often associated with autoimmune diseases. Skinfold thickness measurements and fat assessment by dual energy X-ray absorptiometry are useful tools to identify the disease. C3 hypocomplementemia and the presence of autoantibodies may be used as additional diagnostic supportive criteria but the prevalence of C3 hypocomplementemia may be lower than previously reported.

A Monoclonal Antibody That Provides a Model for C3 Nephritic Factors.

Complement is a major innate defense system that protects the intravascular space from microbial invasion. Complement activation results in the assembly of C3 convertases, serine proteases that cleave complement protein C3, generating bioactive fragments C3a and C3b. The complement response is rapid and robust, largely due to a positive feedback regulatory loop mediated by alternative pathway (AP) C3 convertase. C3 nephritic factors (C3NEFs) are autoantibodies that stabilize AP convertase, resulting in uncontrolled C3 cleavage, which, in principle, can promote critical tissue injury similar to that seen in certain renal conditions. Investigations of C3NEFs are hampered by a challenging issue: each C3NEF is derived from a different donor source, and there is no method to compare one C3NEF to another. We have identified a widely available mouse anti-C3 mAb that, similar to many C3NEFs, can stabilize functional AP convertase in a form resistant to decay acceleration by multiple complement regulators. The antibody requires the presence of properdin to confer convertase stability, and hampers the activity of Salp20, a tic salivary protein that accelerates convertase dissociation by displacing properdin from the convertase complex. This mAb can serve as an urgently needed standard for the investigation of C3NEFs. This study also provides novel insights into the dynamics of AP convertase.

Physiology and pathology of the C3 amplification cycle: A retrospective.

The C3 "Tickover" hypothesis, a mechanism whereby the host maintains constant surveillance of potential invading pathogens, targeting them for elimination through amplified C3b generation and C3-dependent effector mechanisms, was proposed by the late Professor Peter Lachmann in 1973. This unique insight came from a combined understanding of the complement system as it was then defined and the nature of the disease process in rare complement deficiencies and complement-driven diseases. In this review, I give a personal perspective of how understanding of "Tickover" has developed in the subsequent 50 years, culminating in the introduction into the clinic of therapeutic agents designed to combat amplification-driven disease.

Challenges in diagnostic testing of nephritic factors.

Nephritic factors (NeFs) are autoantibodies promoting the activity of the central enzymes of the complement cascade, an important first line of defense of our innate immune system. NeFs stabilize the complement convertase complexes and prevent their natural and regulator-mediated decay. They are mostly associated with rare complement-mediated kidney disorders, in particular with C3 glomerulopathy and related diseases. Although these autoantibodies were already described more than 50 years ago, measuring NeFs for diagnostic purposes remains difficult, and this also complicates our understanding of their clinical associations. In this review, we address the multifactorial challenges of NeF diagnostics. We describe the diseases NeFs are associated with, the heterogenic mechanisms of action of different NeF types, the different methods available in laboratories used for their detection, and efforts for standardization. Finally, we discuss the importance of proper NeF diagnostics for understanding the clinical impact of these autoantibodies in disease pathophysiology and for considering future complement-directed therapy.

Modeling C3 glomerulopathies: C3 convertase regulation on an extracellular matrix surface.

Introduction: C3 glomerulopathies (C3G) are ultra-rare complement-mediated diseases that lead to end-stage renal disease (ESRD) within 10 years of diagnosis in ~50% of patients. Overactivation of the alternative pathway (AP) of complement in the fluid phase and on the surface of the glomerular endothelial glycomatrix is the underlying cause of C3G. Although there are animal models for C3G that focus on genetic drivers of disease, in vivo studies of the impact of acquired drivers are not yet possible. Methods: Here we present an in vitro model of AP activation and regulation on a glycomatrix surface. We use an extracellular matrix substitute (MaxGel) as a base upon which we reconstitute AP C3 convertase. We validated this method using properdin and Factor H (FH) and then assessed the effects of genetic and acquired drivers of C3G on C3 convertase. Results: We show that C3 convertase readily forms on MaxGel and that this formation was positively regulated by properdin and negatively regulated by FH. Additionally, Factor B (FB) and FH mutants impaired complement regulation when compared to wild type counterparts. We also show the effects of C3 nephritic factors (C3Nefs) on convertase stability over time and provide evidence for a novel mechanism of C3Nef-mediated C3G pathogenesis. Discussion: We conclude that this ECM-based model of C3G offers a replicable method by which to evaluate the variable activity of the complement system in C3G, thereby offering an improved understanding of the different factors driving this disease process.

Long-term follow-up including extensive complement analysis of a pediatric C3 glomerulopathy cohort.

BACKGROUND: C3 glomerulopathy (C3G) is a rare kidney disorder characterized by predominant glomerular depositions of complement C3. C3G can be subdivided into dense deposit disease (DDD) and C3 glomerulonephritis (C3GN). This study describes the long-term follow-up with extensive complement analysis of 29 Dutch children with C3G. METHODS: Twenty-nine C3G patients (19 DDD, 10 C3GN) diagnosed between 1992 and 2014 were included. Clinical and laboratory findings were collected at presentation and during follow-up. Specialized assays were used to detect rare variants in complement genes and measure complement-directed autoantibodies and biomarkers in blood. RESULTS: DDD patients presented with lower estimated glomerular filtration rate (eGFR). C3 nephritic factors (C3NeFs) were detected in 20 patients and remained detectable over time despite immunosuppressive treatment. At presentation, low serum C3 levels were detected in 84% of all patients. During follow-up, in about 50% of patients, all of them C3NeF-positive, C3 levels remained low. Linear mixed model analysis showed that C3GN patients had higher soluble C5b-9 (sC5b-9) and lower properdin levels compared to DDD patients. With a median follow-up of 52 months, an overall benign outcome was observed with only six patients with eGFR below 90 ml/min/1.73 m2 at last follow-up. CONCLUSIONS: We extensively described clinical and laboratory findings including complement features of an exclusively pediatric C3G cohort. Outcome was relatively benign, persistent low C3 correlated with C3NeF presence, and C3GN was associated with higher sC5b-9 and lower properdin levels. Prospective studies are needed to further elucidate the pathogenic mechanisms underlying C3G and guide personalized medicine with complement therapeutics.

Complete Renal Recovery in Pediatric Patient with C3 Glomerulonephritis: A Case Report.

C3 glomerulonephritis (C3GN) is a rare kidney disease resulting from dysregulation of the alternative complement cascade. Without treatment, approximately 70% of affected children and 30-50% of affected adults will develop worsening of proteinuria and progress to end-stage renal disease within 10 years of diagnosis. Here, we describe a 9-year-old Sudanese girl with no significant past medical history who presented to the Emergency Department with a 2-month history of fatigue, poor oral intake, and worsening facial and lower extremity edema, and subsequently found to have anemia, hypoalbuminemia, microscopic hematuria, and proteinuria. Additional laboratory testing revealed that the patient had low C3, high C3 nephritic factor (C3NeF), and high factor H. Renal function was normal. The diagnosis of C3GN was confirmed by renal biopsy. The patient was treated with ACE inhibitor, mycophenolate mofetil (600 mg per m2 per dose, every 12 h), in combination with "pulse" methylprednisolone at 30 mg/kg/day IV bolus (maximum 1 g) for 3 consecutive days, followed by 2 months of daily oral prednisolone (2 mg/kg/day) and alternate-day prednisolone weaning from 1 mg/kg to 0.1 mg/kg for additional 12 months. Mycophenolate was continued throughout her treatment course and for maintenance therapy. In response to treatment, anemia, microscopic hematuria, hypoalbuminemia, and proteinuria resolved. Complete complement profile before and at 6 months therapy showed normalization of C3NeF, complement regulatory factor H and C3. This present case provides evidence of the full responsiveness of a rare form of complement dysregulation C3GN to a combination of mycophenolate and corticosteroids. The disease has NOT recurred in >2 years after initial presentation.

Functional Hemolytic Test for Complement Alternative Pathway Convertase Activity.

The complement system is a key part of innate immunity. However, if the system becomes dysregulated, damage to healthy host cells can occur, especially to the glomerular cells of the kidney. The convertases of the alternative pathway of the complement system play a crucial role in complement activation. In healthy conditions, their activity is strictly regulated. In patients with diseases caused by complement alternative pathway dysregulation, such as C3 glomerulopathy and atypical hemolytic uremic syndrome, factors can be present in the blood that disturb this delicate balance, leading to convertase overactivity. Such factors include C3 nephritic factors, which are autoantibodies against the C3 convertase that prolong its activity, or genetic variants resulting in a stabilized convertase complex. This chapter describes a method in which the activity and stability of the alternative pathway convertases can be measured to detect aberrant serum factors causing convertase overactivity.

Detection of Complement Factor B Autoantibodies by ELISA.

Antibodies to autoantigens are implicated in a large number of diseases. Such autoantibodies may cause pathological activation of complement, an ancient humoral recognition and effector system of innate immunity; in addition, complement components or regulators may be target of autoantibodies and cause abnormal complement activation or function. Autoantibodies to complement proteins are in particular involved in kidney diseases. Those binding to complement convertase enzymes can cause enhanced stability of convertases and their increased resistance to regulation, thus promoting complement turnover. Here, we describe an ELISA method to detect factor B autoantibodies that bind to and stabilize the alternative complement pathway C3 convertase enzyme, C3bBb.

Detection of C3 Nephritic Factor by Hemolytic Assay.

C3 nephritic Factor (C3NeF) is autoantibody that binds neoepitopes of the C3 convertase C3bBb, resulting in a stabilization of the enzyme. First functional characterizations of C3NeF were performed by hemolytic assays using preactivated sheep erythrocytes (bearing C3b). Sheep erythrocytes are beforehand sensitized with an anti-sheep red blood cell stroma antibody produced in rabbit (hemolysin). Sensitized sheep erythrocytes will initiate cascade complement activation via the classic pathway, followed by alternative pathway amplification loop, resulting in C3b covalent binding to cell surface. Sheep erythrocytes bearing C3b permit the alternative pathway exploration, in particular decay of alternative pathway C3 convertase.

Evidence of ongoing complement activation on adipose tissue from an 11-year-old girl with Barraquer-Simons syndrome.

Barraquer-Simons syndrome (BSS), a form of acquired partial lipodystrophy, is a rare condition characterized by gradual loss of adipose tissue from the upper body, keeping intact the white adipose tissue of the lower extremities. The etiology of BSS is not well understood, and clinical follow-up studies have not been assessed in these patients. Moreover, no histological studies have been conducted during the active phase of the disease, and complement system activation products have not been sought in the affected areas. The objective of this work was to analyze the clinical, immunological and histological events in an 11-year-old girl with BSS over a 5-year follow-up period. Clinical data were collected during six regular visits for a time period of 5 years. The circulating levels of C3, C3adesArg (a product released upon C3 activation), C4 and immunoglobulins (Ig) were quantified in serum while fat tissue from lipoatrophic areas was examined by immunohistochemical and immunofluorescence approaches. In her regular visits, no clinical or laboratory abnormalities had been observed in the patient, except for the progression of lipoatrophy linked to the C3 hypocomplementemia and the occurrence of C3 nephritic factor. Adipose tissue from the patient showed atrophied and dead adipocytes, an abnormal production of extracellular matrix, and a remarkable accumulation of infiltrating CD68 macrophages and adipocyte precursors (marked by c-Kit positiveness). Simultaneous detection of IgG, C3, C5a and C5b-9 proved the ongoing complement activity and complement-directed injury within the adipose tissue. Our results showed the first evidence that the complement system hyperactivation occurs within the adipose tissue and is linked with fat loss in patients with BSS.

Immunological features of patients affected by Barraquer-Simons syndrome.

BACKGROUND: C3 hypocomplementemia and the presence of C3 nephritic factor (C3NeF), an autoantibody causing complement system over-activation, are common features among most patients affected by Barraquer-Simons syndrome (BSS), an acquired form of partial lipodystrophy. Moreover, BSS is frequently associated with autoimmune diseases. However, the relationship between complement system dysregulation and BSS remains to be fully elucidated. The aim of this study was to provide a comprehensive immunological analysis of the complement system status, autoantibody signatures and HLA profile in BSS. Thirteen subjects with BSS were recruited for the study. The circulating levels of complement components, C3, C4, Factor B (FB) and Properdin (P), as well as an extended autoantibody profile including autoantibodies targeting complement components and regulators were assessed in serum. Additionally, HLA genotyping was carried out using DNA extracted from peripheral blood mononuclear cells. RESULTS: C3, C4 and FB levels were significantly reduced in patients with BSS as compared with healthy subjects. C3NeF was the most frequently found autoantibody (69.2% of cases), followed by anti-C3 (38.5%), and anti-P and anti-FB (30.8% each). Clinical data showed high prevalence of autoimmune diseases (38.5%), the majority of patients (61.5%) being positive for at least one of the autoantibodies tested. The HLA allele DRB1*11 was present in 54% of BSS patients, and the majority of them (31%) were positive for *11:03 (vs 1.3% in the general population). CONCLUSIONS: Our results confirmed the association between BSS, autoimmunity and C3 hypocomplementemia. Moreover, the finding of autoantibodies targeting complement system proteins points to complement dysregulation as a central pathological event in the development of BSS.

The Immunopathology of Complement Proteins and Innate Immunity in Autoimmune Disease.

The complement is a powerful cascade of the innate immunity and also acts as a bridge between innate and acquired immune defence. Complement activation can occur via three distinct pathways, the classical, alternative and lectin pathways, each resulting in the common terminal pathway. Complement activation results in the release of a range of biologically active molecules that significantly contribute to immune surveillance and tissue homeostasis. Several soluble and membrane-bound regulatory proteins restrict complement activation in order to prevent complement-mediated autologous damage, consumption and exacerbated inflammation. The crucial role of complement in the host homeostasis is illustrated by association of both complement deficiency and overactivation with severe and life-threatening diseases. Autoantibodies targeting complement components have been described to alter expression and/or function of target protein resulting in a dysregulation of the delicate equilibrium between activation and inhibition of complement. The spectrum of diseases associated with complement autoantibodies depends on which complement protein and activation pathway are targeted, ranging from autoimmune disorders to kidney and vascular diseases. Nevertheless, these autoantibodies have been identified as differential biomarkers for diagnosis or follow-up of disease only in a small number of clinical conditions. For some autoantibodies, a clear relationship with clinical manifestations has been identified, such as anti-C1q, anti-Factor H, anti-C1 Inhibitor antibodies and C3 nephritic factor. For other autoantibodies, the origin and the functional consequences still remain to be elucidated, questioning about the pathophysiological significance of these autoantibodies, such as anti-mannose binding lectin, anti-Factor I, anti-Factor B and anti-C3b antibodies. The detection of autoantibodies targeting complement components is performed in specialized laboratories; however, there is no consensus on detection methods and standardization of the assays is a real challenge. This review summarizes the current panorama of autoantibodies targeting complement recognition proteins of the classical and lectin pathways, associated proteases, convertases, regulators and terminal components, with an emphasis on autoantibodies clearly involved in clinical conditions.

Outcome of C3 glomerulopathy patients: largest single-centre experience from South Asia.

BACKGROUND: C3 glomerulopathy (C3G) is related to dysfunction of alternative complement pathway (ACP) because of its hyperactivation. Triggering factors and genetic profile are likely to be different in developing countries as compared to the Western world. Data regarding C3G from South Asian is scanty. STUDY DESIGN: In the present study, 115 patients of C3G from 2012 to 2017 were analyzed. Clinical details were reviewed; serological levels of C3, C4, complement factor H or B and autoantibody testing was done by nephelometry/ELISA. Limited genetics workup for CFH and CFHR5 genes was done. RESULTS: The prevalence of C3G was 1.52%. There was no difference in demographic and histopathologic profiles of C3G patients. Majority of patients had low functional assay and C3 levels. C3 nephritic factor was present in 47.5% of DDD and 38.6% of C3GN. Autoantibodies to CFH were present more often in the patients of C3GN (29.5%) than DDD (12.5%). Autoantibodies to CFB were equally common in both groups. Past history of infections was present in one-third patients and monoclonal paraproteins were present only in two patients. No pathogenic variants were noted in CFH/CFHR5 gene. On follow-up (3.2 + 1.6 years), complete and partial remission was achieved in one-fourth patients and 26% had resistance disease. About 40% progressed to ESRD and 18 underwent renal transplantation of which nine had a post-transplant recurrence. CONCLUSIONS: Indian cohort had some differences in the immunological and genetic profile when compared to the Western literature; most significant was the absence of monoclonal immunoglobulins as a trigger for C3G.

Clinicopathological features of C3 glomerulopathy in children: a single-center experience.

BACKGROUND: C3 glomerulopathy (C3G) is defined by dominant glomerular deposition of C3 and minimal or no immunoglobulin, with two subtypes-dense deposit disease (DDD) and C3 glomerulonephritis (C3GN)-distinguished by features on electron microscopy (EM). Given that this rare disease has generally unfavorable yet highly variable outcomes, we sought out to review the histopathology, complement/genetic studies, and renal outcomes of pediatric patients with C3G at our institution. METHODS: All native kidney biopsies performed in a single pediatric hospital over a 10-year period were reviewed for features of C3G. Of 589 biopsy reports, we identified 9 patients fulfilling the diagnostic criteria for C3G and retrospectively reviewed their clinical chart and renal biopsy findings. RESULTS: We identified 4 patients with DDD, 4 with C3GN, and 1 indeterminate case, with features of both C3GN and DDD. Five patients were positive for one or more nephritic factors (C3NeF, C4NeF, C5NeF) with 1 patient additionally positive for complement factor H (CFH) autoantibody. Genetic testing done in 5 of the 9 patients failed to identify any causative mutations. Three patients showed progressive renal dysfunction over a mean follow-up period of 33 months. CONCLUSIONS: Complement and genetic studies are now routinely recommended for patients with a histopathological diagnosis of C3G. Careful interpretation of these studies and their prognostic and therapeutic implications in conjunction with biopsy findings is needed to further understand the pathophysiology of this rare disease in children.

C4 nephritic factor in patients with immune-complex-mediated membranoproliferative glomerulonephritis and C3-glomerulopathy.

BACKGROUND: Acquired or genetic abnormalities of the complement alternative pathway are the primary cause of C3glomerulopathy(C3G) but may occur in immune-complex-mediated membranoproliferative glomerulonephritis (IC-MPGN) as well. Less is known about the presence and role of C4nephritic factor(C4NeF) which may stabilize the classical pathway C3-convertase. Our aim was to examine the presence of C4NeF and its connection with clinical features and with other pathogenic factors. RESULTS: One hunfe IC-MPGN/C3G patients were enrolled in the study. C4NeF activity was determined by hemolytic assay utilizing sensitized sheep erythrocytes. Seventeen patients were positive for C4NeF with lower prevalence of renal impairment and lower C4d level, and higher C3 nephritic factor (C3NeF) prevalence at time of diagnosis compared to C4NeF negative patients. Patients positive for both C3NeF and C4NeF had the lowest C3 levels and highest terminal pathway activation. End-stage renal disease did not develop in any of the C4NeF positive patients during follow-up period. Positivity to other complement autoantibodies (anti-C1q, anti-C3) was also linked to the presence of nephritic factors. Unsupervised, data-driven cluster analysis identified a group of patients with high prevalence of multiple complement autoantibodies, including C4NeF. CONCLUSIONS: In conclusion, C4NeF may be a possible cause of complement dysregulation in approximately 10-15% of IC-MPGN/C3G patients.

Complement dysregulation in glomerulonephritis.

Glomerulonephritis (GN) refers to a group of renal diseases affecting the glomeruli due to the damage mediated by immunological mechanisms. A large proportion of the disease manifestations are caused by disturbances in the complement system. They can be due to genetic errors, autoimmunity, microbes or abnormal immunoglobulins, like modified IgA or paraproteins. The common denominator in most of the problems is an overactive or misdirected alternative pathway complement activation. An assessment of kidney function, amount of proteinuria and hematuria are crucial elements to evaluate, when glomerulonephritis is suspected. However, the cornerstones of the diagnoses are renal biopsy and careful examination of the complement abnormality. Differential diagnostics between the various forms of GN is not possible based on clinical features, as they may vary greatly. This review describes the known mechanisms of complement dysfunction leading to different forms of primary GN (like IgA glomerulonephritis, dense deposit disease, C3 glomerulonephritis, post-infectious GN, membranous GN) and differences to atypical hemolytic uremic syndrome. It also covers the basic elements of etiology-directed therapy and prognosis of the most common forms of GN. Common principles in the management of GN include treatment of hypertension and reduction of proteinuria, some require immunomodulating treatment. Complement inhibition is an emerging treatment option. A thorough understanding of the basic disease mechanism and a careful follow-up are needed for optimal therapy.

Novel Assays to Distinguish Between Properdin-Dependent and Properdin-Independent C3 Nephritic Factors Provide Insight Into Properdin-Inhibiting Therapy.

C3 glomerulopathy (C3G) is an umbrella classification for severe renal diseases characterized by predominant staining for complement component C3 in the glomeruli. The disease is caused by a dysregulation of the alternative pathway (AP) of the complement system. In more than half of C3G patients C3 nephritic factors (C3NeFs) are found. These autoantibodies bind to the AP C3 convertase, prolonging its activity. C3NeFs can be dependent or independent of the complement regulator properdin for their convertase-stabilizing function. However, studies to determine the properdin-dependency of C3NeFs are rare and not part of routine patient workup. Until recently, only supportive treatments for C3G were available. Complement-directed therapies are now being investigated. We hypothesized that patients with properdin-dependent C3NeFs may benefit from properdin-inhibiting therapy to normalize convertase activity. Therefore, in this study we validated two methods to distinguish between properdin-dependent and properdin-independent C3NeFs. These methods are hemolytic assays for measuring convertase activity and stability in absence of properdin. The first assay assesses convertase stabilization by patient immunoglobulins in properdin-depleted serum. The second assay measures convertase stabilization directly in patient serum supplemented with the properdin-blocking agent Salp20. Blood samples from 13 C3NeF-positive C3G patients were tested. Three patients were found to have properdin-dependent C3NeFs, whereas the C3NeF activity of the other ten patients was independent of properdin. The convertase-stabilizing activity in the samples of the patients with properdin-dependent C3NeFs disappeared in absence of properdin. These data indicate that inhibition of properdin in patients with properdin-dependent C3NeFs can normalize convertase activity and could represent a novel therapy for normalizing AP hyperactivity. Our assays provide a tool for identifying C3G patients who may benefit from properdin-inhibiting therapy and can be incorporated into standard C3G laboratory investigations.