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.

Neutrophils activated by membrane attack complexes increase the permeability of melanoma blood vessels.

The microenvironment of malignant melanomas defines the properties of tumor blood vessels and regulates infiltration and vascular dissemination of immune and cancer cells, respectively. Previous research in other cancer entities suggested the complement system as an essential part of the tumor microenvironment. Here, we confirm activation of the complement system in samples of melanoma patients and murine melanomas. We identified the tumor endothelium as the starting point of the complement cascade. Generation of complement-derived C5a promoted the recruitment of neutrophils. Upon contact with the vascular endothelium, neutrophils were further activated by complement membrane attack complexes (MACs). MAC-activated neutrophils release neutrophil extracellular traps (NETs). Close to the blood vessel wall, NETs opened the endothelial barrier as indicated by an enhanced vascular leakage. This facilitated the entrance of melanoma cells into the circulation and their systemic spread. Depletion of neutrophils or lack of MAC formation in complement component 6 (C6)-deficient animals protected the vascular endothelium and prevented vascular intravasation of melanoma cells. Our data suggest that inhibition of MAC-mediated neutrophil activation is a potent strategy to abolish hematogenous dissemination in melanoma.

Expanding Horizons in Complement Analysis and Quality Control.

Complement not only plays a key role in host microbial defense but also modulates the adaptive immune response through modification of T- and B-cell reactivity. Moreover, a normally functioning complement system participates in hematopoiesis, reproduction, lipid metabolism, and tissue regeneration. Because of its powerful inflammatory potential, multiple regulatory proteins are needed to prevent potential tissue damage. In clinical practice, dysregulation and overactivation of the complement system are major causes of a variety of inflammatory and autoimmune diseases ranging from nephropathies, age-related macular degeneration (AMD), and systemic lupus erythematosus (SLE) to graft rejection, sepsis, and multi-organ failure. The clinical importance is reflected by the recent development of multiple drugs targeting complement with a broad spectrum of indications. The recognition of the role of complement in diverse diseases and the advent of complement therapeutics has increased the number of laboratories and suppliers entering the field. This has highlighted the need for reliable complement testing. The relatively rapid expansion in complement testing has presented challenges for a previously niche field. This is exemplified by the issue of cross-reactivity of complement-directed antibodies and by the challenges of the poor stability of many of the complement analytes. The complex nature of complement testing and increasing clinical demand has been met in the last decade by efforts to improve the standardization among laboratories. Initiated by the IUIS/ICS Committee for the Standardization and Quality Assessment in Complement Measurements 14 rounds of external quality assessment since 2010 resulted in improvements in the consistency of testing across participating institutions, while extending the global reach of the efforts to more than 200 laboratories in 30 countries. Worldwide trends of assay availability, usage, and analytical performance are summarized based on the past years' experiences. Progress in complement analysis has been facilitated by the quality assessment and standardization efforts that now allow complement testing to provide a comprehensive insight into deficiencies and the activation state of the system. This in turn enables clinicians to better define disease severity, evolution, and response to therapy.

Long-term data on two sisters with C3GN due to an identical, homozygous CFH mutation and autoantibodies.

C3 glomerulonephritis (C3GN) is a rare but severe form of kidney disease caused by fluid-phase dysregulation of the alternative complement pathway. Causative mutations in complement regulating genes as well as auto-immune forms of C3GN have been described. However, therapy and prognosis in individual patients remain a matter of debate and long-term data are scarce. This also applies for the management of transplant patients as disease recurrence post-transplant is frequent. Here, we depict the clinical courses of two sisters with the unique combination of an identical, homozygous mutation in the complement factor H (CFH) gene as well as autoantibodies with a clinical follow-up of more than 20 years. Interestingly, the sisters presented with discordant clinical courses of C3GN with normal kidney function in one (patient A) and end-stage kidney disease in the other sister (patient B). In patient B, eculizumab was administered immediately prior to and in the course after kidney transplantation, with the result of a stable graft function without any signs of disease recurrence. Comprehensive genetic work-up revealed no further disease-causing mutation in both sisters. Intriguingly, the auto-antibody profile substantially differed in both sisters: autoantibodies in patient A reduced the C3b deposition, while the antibodies identified in patient B increased complement activation and deposition of split products. This study underlines the concept of a personalized-medicine approach in complement-associated diseases after thorough evaluation of the individual risk profile in each patient.

H1N1 Infection Reduces Glucose Level in Human U937 Monocytes Culture.

Infection with influenza A (H1N1) virus contributes significantly to the global burden of acute respiratory diseases. Glucose uptake and metabolic changes are reported in different cell types after infections with different virus types, including influenza A virus. Alteration of glucose metabolism specifically in immune cells has major health consequences. The aim of this study was to monitor glucose concentration in unstimulated and stimulated U937 human monocytes with infectious or heat inactivated H1N1 or Staphylococcus aureus or in nonpathogenically stimulated monocytes with phorbol-12-myristate-13-acetate. Stimulated or unstimulated U937 human monocytes were subjected to H1N1 infection for different time points and the glucose profile in the growth medium was measured post infection. Results showed that regardless to whether the initial stimuli on U937 cells were of pathogen or nonpathogen origins, challenge infection by H1N1 causes a significant reduction of glucose levels 36 h post infection. In conclusion, H1N1 infection has a direct effect on the glucose uptake of U937 cells in vitro. This effect can be related to either H1N1 infection or cell differentiation status that might occur due to the exerted stimuli.

European Society for Immunodeficiencies (ESID) and European Reference Network on Rare Primary Immunodeficiency, Autoinflammatory and Autoimmune Diseases (ERN RITA) Complement Guideline: Deficiencies, Diagnosis, and Management.

This guideline aims to describe the complement system and the functions of the constituent pathways, with particular focus on primary immunodeficiencies (PIDs) and their diagnosis and management. The complement system is a crucial part of the innate immune system, with multiple membrane-bound and soluble components. There are three distinct enzymatic cascade pathways within the complement system, the classical, alternative and lectin pathways, which converge with the cleavage of central C3. Complement deficiencies account for ~5% of PIDs. The clinical consequences of inherited defects in the complement system are protean and include increased susceptibility to infection, autoimmune diseases (e.g., systemic lupus erythematosus), age-related macular degeneration, renal disorders (e.g., atypical hemolytic uremic syndrome) and angioedema. Modern complement analysis allows an in-depth insight into the functional and molecular basis of nearly all complement deficiencies. However, therapeutic options remain relatively limited for the majority of complement deficiencies with the exception of hereditary angioedema and inhibition of an overactivated complement system in regulation defects. Current management strategies for complement disorders associated with infection include education, family testing, vaccinations, antibiotics and emergency planning.

Increased Complement Factor B and Bb Levels Are Associated with Mortality in Patients with Severe Aortic Stenosis.

Inflammation is involved in initiation and progression of aortic stenosis (AS). However, the role of the complement system, a crucial component of innate immunity in AS, is unclear. We hypothesized that circulating levels of complement factor B (FB), an important component of the alternative pathway, are upregulated and could predict outcome in patients with severe symptomatic AS. Therefore, plasma levels of FB, Bb, and terminal complement complex were analyzed in three cohorts of patients with severe symptomatic AS and mild-to-moderate or severe asymptomatic AS (population 1, n = 123; population 2, n = 436; population 3, n = 61) and in healthy controls by enzyme immunoassays. Compared with controls, symptomatic AS patients had significantly elevated levels of FB (2.9- and 2.8-fold increase in population 1 and 2, respectively). FB levels in symptomatic and asymptomatic AS patients were comparable (population 2 and 3), and in asymptomatic patients FB correlated inversely with valve area. FB levels in population 1 and 2 correlated with terminal complement complex levels and measures of systemic inflammation (i.e., CRP), cardiac function (i.e., NT-proBNP), and cardiac necrosis (i.e., Troponin T). High FB levels were significantly associated with mortality also after adjusting for clinical and biochemical covariates (hazard ratio 1.37; p = 0.028, population 2). Plasma levels of the Bb fragment showed a similar pattern in relation to mortality. We concluded that elevated levels of FB and Bb are associated with adverse outcome in patients with symptomatic AS. Increased levels of FB in asymptomatic patients suggest the involvement of FB from the early phase of the disease.

Complement deficiencies and dysregulation: Pathophysiological consequences, modern analysis, and clinical management.

Complement defects are associated with an enhanced risk of a broad spectrum of infectious as well as systemic or local inflammatory and thrombotic disorders. Inherited complement deficiencies have been described for virtually all complement components but can be mimicked by autoantibodies, interfering with the activity of specific complement components, convertases or regulators. While being rare, diseases related to complement deficiencies are often severe with a frequent but not exclusive manifestation during childhood. Whereas defects of early components of the classical pathway significantly increase the risk of autoimmune disorders, lack of components of the terminal pathway as well as of properdin are associated with an enhanced susceptibility to meningococcal infections. The impaired synthesis or function of C1 inhibitor results in the development of hereditary angioedema (HAE). Furthermore, complement dysregulation causes renal disorders such as atypical hemolytic uremic syndrome (aHUS) or C3 glomerulopathy (C3G) but also age-related macular degeneration (AMD). While paroxysmal nocturnal hemoglobinuria (PNH) results from the combined deficiency of the regulatory complement proteins CD55 and CD59, which is caused by somatic mutation of a common membrane anchor, isolated CD55 or CD59 deficiency is associated with the CHAPLE syndrome and polyneuropathy, respectively. Here, we provide an overview on clinical disorders related to complement deficiencies or dysregulation and describe diagnostic strategies required for their comprehensive molecular characterization - a prerequisite for informed decisions on the therapeutic management of these disorders.

C3-Glomerulopathy Autoantibodies Mediate Distinct Effects on Complement C3- and C5-Convertases.

C3 glomerulopathy (C3G) is a severe kidney disease, which is caused by defective regulation of the alternative complement pathway. Disease pathogenesis is heterogeneous and is caused by both autoimmune and genetic factors. Here we characterized IgG autoantibodies derived from 33 patients with autoimmune C3 glomerulopathy. Serum antibodies from all 33 patients as well as purified IgGs bound to the in vitro assembled C3-convertase. Noteworthy, two groups of antibodies were identified: group 1 with strong (12 patients) and group 2 with weak binding C3-convertase autoantibodies (22 patients). C3Nef, as evaluated in a standard C3Nef assay, was identified in serum from 19 patients, which included patients from group 1 as well as group 2. The C3-convertase binding profile was independent of C3Nef. Group 1 antibodies, but not the group 2 antibodies stabilized the C3-convertase, and protected the enzyme from dissociation by Factor H. Also, only group 1 antibodies induced C3a release. However, both group 1 and group 2 autoantibodies bound to the C5-convertase and induced C5a generation, which was inhibited by monoclonal anti-C5 antibody Eculizumab in vitro. In summary, group 1 antibodies are composed of C3Nef and C5Nef antibodies and likely over-activate the complement system, as seen in hemolytic assays. Group 2 antibodies show predominantly C5Nef like activities and stabilize the C5 but not the C3-convertase. Altogether, these different profiles not only reveal a heterogeneity of the autoimmune forms of C3G (MPGN), they also show that in diagnosis of C3G not all autoimmune forms are identified and thus more vigorous autoantibody testing should be performed.

Increased compensatory kidney workload results in cellular damage in a short time porcine model of mixed acidemia - Is acidemia a 'first hit' in acute kidney injury?

Acute kidney injury (AKI) corrupts the outcome of about 50% of all critically ill patients. We investigated the possible contribution of the pathology acidemia on the development of AKI. Pigs were exposed to acidemia, acidemia plus hypoxemia or a normal acid-base balance in an experimental setup, which included mechanical ventilation and renal replacement therapy to facilitate biotrauma caused by extracorporeal therapies. Interestingly, extensive histomorphological changes like a tubular loss of cell barriers occurred in the kidneys after just 5 hours exposure to acidemia. The additional exposure to hypoxemia aggravated these findings. These 'early' microscopic pathologies opposed intra vitam data of kidney function. They did not mirror cellular or systemic patterns of proinflammatory molecules (like TNF-α or IL 18) nor were they detectable by new, sensitive markers of AKI like Neutrophil gelatinase-associated lipocalin. Instead, the data suggest that the increased renal proton excretion during acidemia could be an 'early' first hit in the multifactorial pathogenesis of AKI.

Author Correction: Immune evasion of Borrelia miyamotoi: CbiA, a novel outer surface protein exhibiting complement binding and inactivating properties.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Immunological Alterations due to Hemodialysis Might Interfere with Early Complications in Renal Transplantation.

BACKGROUND: Chronic or intercurrent alterations of the immune system in patients with end-stage renal disease (CKD) and intermittent hemodialysis (CKD5D, HD) have been attributed to an acute rejection of renal allograft. METHODS: Leukocyte subsets in flow cytometry, complement activation, and concentrations of TGFß, sCD30 (ELISA), and interleukins (CBA) of fifteen patients eligible for renal transplantation were analyzed before, during, and after a regular HD. RESULTS: Before HD, the median proportion of CD8+ effector cells, CD8+ CCR5+ effector cells, and HLA-DR+ regulatory T cells as well as the median concentration of soluble CD30 increased and naive CD8+ T cells decreased. During HD, there was a significant decrease in CD4- CD8- T cells (p < 0.001) and an increase in CD25+ T cells (p = 0.026), sCD30 (p < 0.001), HLA-DR+ regulatory T cells (p = 0.005), and regulatory T cells (p = 0.003). TGFß and sCD30 increased significantly over time. The activity of the classical complement pathway started to slightly increase after the first hour of HD and lasted until fifteen minutes after finishing dialysis. The decrease in the functional activity of the alternative pathway was only transient and was followed by a significant increase within 15 minutes after finishing the treatment. CONCLUSION: HD might interact with the allograft outcome by influencing T cell subsets and activation of the complement system in a biphasic course.

Complement C5b-9 and Cancer: Mechanisms of Cell Damage, Cancer Counteractions, and Approaches for Intervention.

The interactions of cancer cells with components of the complement system are highly complex, leading to an outcome that is either favorable or detrimental to cancer cells. Currently, we perceive only the "tip of the iceberg" of these interactions. In this review, we focus on the complement terminal C5b-9 complex, known also as the complement membrane attack complex (MAC) and discuss the complexity of its interaction with cancer cells, starting with a discussion of its proposed mode of action in mediating cell death, and continuing with a portrayal of the strategies of evasion exhibited by cancer cells, and closing with a proposal of treatment approaches targeted at evasion strategies. Upon intense complement activation and membrane insertion of sufficient C5b-9 complexes, the afflicted cells undergo regulated necrotic cell death with characteristic damage to intracellular organelles, including mitochondria, and perforation of the plasma membrane. Several pro-lytic factors have been proposed, including elevated intracellular calcium ion concentrations and activated JNK, Bid, RIPK1, RIPK3, and MLKL; however, further research is required to fully characterize the effective cell death signals activated by the C5b-9 complexes. Cancer cells over-express a multitude of protective measures which either block complement activation, thus reducing the number of membrane-inserted C5b-9 complexes, or facilitate the elimination of C5b-9 from the cell surface. Concomitantly, cancer cells activate several protective pathways that counteract the death signals. Blockage of complement activation is mediated by the complement membrane regulatory proteins CD46, CD55, and CD59 and by soluble complement regulators, by proteases that cleave complement proteins and by protein kinases, like CK2, which phosphorylate complement proteins. C5b-9 elimination and inhibition of cell death signals are mediated by caveolin and dynamin, by Hsp70 and Hsp90, by the mitochondrial stress protein mortalin, and by the protein kinases PKC and ERK. It is conceivable that various cancers and cancers at different stages of development will utilize distinct patterns of these and other MAC resistance strategies. In order to enhance the impact of antibody-based therapy on cancer, novel precise reagents that block the most effective protective strategies will have to be designed and applied as adjuvants to the therapeutic antibodies.

IgM cleavage by Streptococcus suis reduces IgM bound to the bacterial surface and is a novel complement evasion mechanism.

Streptococcus suis (S. suis) causes meningitis, arthritis and endocarditis in piglets. The aim of this study was to characterize the IgM degrading enzyme of S. suis (IdeSsuis) and to investigate the role of IgM cleavage in evasion of the classical complement pathway and pathogenesis. Targeted mutagenesis of a cysteine in the putative active center of IdeSsuis abrogated IgM cleavage completely. In contrast to wt rIdeSsuis, point mutated rIdeSsuis_C195S did not reduce complement-mediated hemolysis indicating that complement inhibition by rIdeSsuis depends on the IgM proteolytic activity. A S. suis mutant expressing IdeSsuis_C195S did not reduce IgM labeling, whereas the wt and complemented mutant showed less IgM F(ab')2 and IgM Fc antigen on the surface. IgM cleavage increased survival of S. suis in porcine blood ex vivo and mediated complement evasion as demonstrated by blood survival and C3 deposition assays including the comparative addition of rIdeSsuis and rIdeSsuis_C195S. However, experimental infection of piglets disclosed no significant differences in virulence between S. suis wt and isogenic mutants without IgM cleavage activity. This work revealed for the first time in vivo labeling of S. suis with IgM in the cerebrospinal fluid of piglets with meningitis. In conclusion, this study classifies IdeSsuis as a cysteine protease and emphasizes the role of IgM cleavage for bacterial survival in porcine blood and complement evasion though IgM cleavage is not crucial for the pathogenesis of serotype 2 meningitis.

Cisterno-lumbar gradient of complement fractions in geriatric patients with suspected normal pressure hydrocephalus.

BACKGROUND: The complement system is a functional link between the innate and adaptive immune system and present in all compartments of the body. The composition of the cerebrospinal fluid (CSF) differs between the ventricular, cisternal and lumbar space. Usually, concentrations of blood-derived CSF proteins increase from ventricular to lumbar fractions. METHODS: In 20 geriatric patients with suspected normal pressure hydrocephalus (NPH) [13 women, 7 men, age 80.5 (75/85) years; median (25th/75th percentile)] a lumbar spinal tap of 40 ml was performed, and 10 ml of serum was drawn. CSF, sequentially collected in 8 fractions of 5 ml (1st fraction: lumbar CSF; 8th fraction: cisterna magna-near CSF), was analyzed for complement protein C3, and the activation products C3a and sC5b-9 by enzyme immunoassay. RESULTS: The concentrations of the complement factors measured in fractions 1 and 8 of each individual patient were strongly correlated: C3 (Spearman's rank correlation coefficient rS = 0.75, p = 0.0002); C3a (rS = 0.93, p < 0.0001); sC5b-9 (rS = 0.64, p = 0.002). CSF complement concentrations were lower in the cistern-near fraction 8 than in the lumbar fraction 1 (C3: p = 0.005; C3a: p = 0.0009; sC5b-9: p = 0.0003, Wilcoxon signed rank test). The concentrations of complement factors in CSF were two orders of magnitude lower than those in serum. C3 levels in the lumbar CSF strongly correlated with the lumbar CSF/serum albumin concentration quotient (QAlb) as a measure of the functionability of the blood-CSF barrier and the velocity of CSF flow (rS = 0.84, p < 0.0001) suggesting diffusion of C3 from blood to CSF. The lumbar and cistern-near concentrations of C3a did not significantly correlate with QAlb (rS = 0.26) pointing to a local conversion of C3 to C3a. The lumbar concentrations of sC5b-9 moderately correlated with QAlb (rS = 0.62, p = 0.004). Plotting the CSF/serum quotient of C3 and sC5b-9 versus the QAlb revealed an approx. 50% local synthesis of C3, but a strong production of sC5b-9 in the CNS. CONCLUSIONS: The increase of the complement concentrations from cisternal to lumbar CSF and the strong correlation of C3 with QAlb suggest that (1) a substantial portion of complement C3 in CSF originates from blood and (2) the complement system is mildly activated in the CSF of NPH patients.

Complement analysis in the era of targeted therapeutics.

Complement immunobiology, and with it complement analysis, has undergone a renaissance in the past decade. Classically, complement analysis was limited number of testing C3, C4 in a routine laboratory with the possible addition of CH50 with all other analysis being performed at only few highly esoteric laboratories. This diagnostics expanding beyond specialized laboratories is the result of the growing recognition of the role played by complement dysfunction in many more diseases and disorders and the concomitant increase in interest in complement targeting therapeutics. In response, laboratories specializing in complement analysis have joined with the International Complement Society and the IUIS to coordinate efforts to standardize and improve complement testing, ongoing efforts that have already borne fruit. A recognition of the power of complement analysis has brought forward new testing but also realization of the importance of post-draw specimen handling to limit ex vivo activation, as well as the sometimes large difference between testing laboratory results. The increased usefulness of complement analysis and efforts to standardize and expand it means the future is strong for complement analysis.