Aberrations in the complement system have been shown to be direct or indirect pathophysiological mechanisms in a number of diseases and pathological conditions such as autoimmune disease, infections, cancer, allogeneic and xenogeneic transplantation, and inflammation. Complement analyses have been performed on these conditions in both prospective and retrospective studies and significant differences have been found between groups of patients, but in many diseases, it has not been possible to make predictions for individual patients because of the lack of sensitivity and specificity of many of the assays used. The basic indications for serological diagnostic complement analysis today may be divided into three major categories: (a) acquired and inherited complement deficiencies; (b) disorders with complement activation; (c) inherited and acquired C1INH deficiencies. Here, we summarize indications, techniques, and interpretations for basic complement analyses and present an algorithm, which we follow in our routine laboratory.
The complement system is involved in numerous diseases and pathological conditions such as autoimmune disease, infections, cancer, allogeneic and xenogeneic transplantation, and inflammation [
The complement system has a primary function in host defense and clears the body of foreign cells, microorganisms, and cell debris, either by direct lysis or by recruitment of leukocytes that promote phagocytosis and cytotoxicity (recently reviewed in [
The nascent C3b molecule has the specific property of binding to proteins and carbohydrates via free hydroxyl or amino groups, resulting in covalent ester and amide bonds, respectively. The AP serves as a major amplification loop, so an initial weak stimulus mediated by any of the pathways may be markedly enhanced. The activation pathways converge in a common pathway to form the membrane attack complex (C5b-9), which elicits cell lysis by insertion itself into the lipid bilayer of cell membranes. The anaphylatoxins C3a and C5a activate and recruit leukocytes, while target-bound C3 fragments (C3b, iC3b, C3d,g) facilitate binding to and activation of the recruited cells (Figure
Overview of the complement system. Recognition by the lectin and classical pathways leads to the assembly of the C4b2a convertase, which cleaves C3. This reaction is greatly amplified by the alternative pathway, generating more C3b, and ultimately initiating the terminal sequence. The fluid phase anaphylatoxins, C3a and C5a, together with the cell-bound opsonins iC3b and C3d,g, facilitate phagocytosis. The main inhibitor of each step in the cascade is indicated in boxes: C1INH for initiation, C4BP for the classical pathway, factor H for the alternative pathway, and CD59 for the terminal sequence.
Excessive complement activation is part of the pathogenesis of a large number of inflammatory diseases. The pathologic effect may be due either to an increased and persistent activation, for example, caused by the presence of immune complexes (such as in systemic lupus erythematosus, SLE, and related disorders), or to a decreased expression or function of various complement inhibitors (see examples below), or to a combination of the two, as discussed in [
Ischemia, followed by reperfusion of an organ or blood vessel, occurs in a number of conditions, such as during heart infarction or stroke. It can also occur during medical treatment modalities such as cardiovascular surgery facilitated by cardiopulmonary bypass as well as after transplantation, in both allogeneic and xenogeneic settings, and can often be accompanied by ischemia/reperfusion (IR) injury. Complement activation and insufficient regulation play important roles in IR injury, and activation by all three pathways of complement has been implicated in the damage. The result is an multifunctional inflammatory process, involving generation of anaphylatoxins, upregulation of adhesion proteins and tissue factor on endothelial cells, and recruitment and extravasation of PMNs, as summarized in [
The net result of this dysregulation between initiators and inhibitors of complement activation in all these diverse conditions is a prolonged complement activation, which ultimately results in tissue damage.
Complement deficiencies are associated with an increased risk of infections and, in some cases, autoimmunity [
Eculizumab is the first approved complement inhibitor in clinical use. It is a humanized monoclonal antibody that binds to complement component C5, hindering its proteolytic activation, and thereby inhibiting the generation of the anaphylatoxin C5a as well as the initiation of the lytic C5b-9 complex. The main indication for this complement inhibitor is for the treatment of paroxysmal nocturnal hemoglobinuria, PNH [
Paroxysmal nocturnal hemoglobinuria (PNH) is a rare hematologic disorder in which the afflicted patients suffer from hemolysis with acute exacerbations that lead to anemia, as well as from an increased risk of venous thrombosis. The disease is caused by acquired somatic mutations of the X-chromosomal gene
SLE and urticarial vasculitides belong to the group of autoimmune immune complex diseases [
Membranoproliferative glomerulonephritis (MPGN) types II are associated with C3 nephritic factors (C3Nef) [
In individuals suffering from poststreptococcal glomerulonephritis (PSGN), C3 and C5 may be consumed and sC5b-9 generated, in the rehabilitation period, up to 6 months after the infection [
Atypical hemolytic uremic syndrome (aHUS) is a disease that appears in the childhood and is characterized by microangiopathic hemolytic anemia, thrombocytopenia, and acute renal failure resulting from membranoproliferative glomerulonephritis. The cause of this disease is dysregulated complement activation following a mutation in factor H, factor I, MCP, or factor H-related proteins (FHR) 1, 3, or 5, that impairs the functioning of these inhibitors. In addition, mutations in C3 and factor B that lead to dysregulated activation have also been described [
The cells that are affected are erythrocytes, platelets, and endothelial cells, including those of the mesangium of the kidney. Mutations within the factor H gene are the most common cause of aHUS. The majority of these mutations are localized in the C-terminally located short consensus repeats (SCRs) 19-20, which are involved in the binding of factor H to the cell surface. Factor H binds to carbohydrates, and heparan sulfate and sialic acid are common ligands for the protein. As in the case of factor H deficiency, engagement of the AP leading to consumption of C3 and generation of C3d,g (or other C3 fragments) and C5b-9 may be seen. In rare cases, the mobility in SDS/PAGE followed by western blotting may differ from that of normal factor H. Patients with suspected aHUS should be handled by laboratories specialized in determining mutations in all activators and soluble and cell bound regulators of the AP.
Hereditary angioedema (HAE) and acquired angioedema (AEE) are rare disorders that are caused by a C1INH deficiency and, in rare cases, by mutations of the contact system proteins [
Acquired deficiencies of C1INH may occur in lymphoproliferative and autoimmune diseases, due to formation of paraproteins, for example, M-components and autoantibodies against C1INH, respectively, which result in consumption of the protein [
Available complement assays have recently been comprehensively reviewed [
The concentration of individual proteins is determined by various types of immunoassays. The most common approach in clinical practice is to use immunoprecipitation assays, today mainly nephelometry and turbidimetry. In the latter techniques, polyclonal antibodies against the protein of choice (e.g., C1q, C1INH, C4, C3, or factor B) are added to the sample, forming complexes that will distort a detecting light beam that is passed through the sample. These techniques, which use polyclonal antibodies to detect the total amount of the antigen in question, are relatively robust with regard to the effects of suboptimal sample handling, such as proteolytic cleavage or denaturation of the target proteins. For example, the polyclonal antibodies raised against C3c used in such assays will recognize C3c-fragment containing intact, nonactivated C3 as well as its inactive proteolytic fragments C3b, iC3b, and C3c, on an equimolar basis. Similarly, anti-C4c antibodies will detect the corresponding forms of C4. If the sample is poorly treated, resulting in fragmentation of the intact protein, determination of the c-fragment (C3c or C4c) ensures that the determined concentration is similar to that
Activation and consumption of complement
A number of complement proteins are activated and inactivated by sequential proteolytic cleavages that are accompanied by conformational alterations. These reactions have been studied most extensively for C3. Therefore, the strategy used to demonstrate that complement activation has occurred
Complement activation gives rise to products with different properties than those of the zymogen molecules. Therefore, assays for the determination of complement activation products generally work according to one of two principles: either (1) the zymogen molecules and products are fractionated according to size before being detected by polyclonal antibodies, as described above for C3d,g (below), or (2) monoclonal antibodies specific for amino acid sequences that are hidden in the native zymogen molecule but exposed upon activation (so-called neoepitopes) are used. Since only the activation product and not the zymogen molecule will be detected in this assay, it is not necessary to include a precipitation step. Most available assays for C3a, C3b/iC3b/C3c, and C5b-9 (below) are based on neoepitope monoclonal antibodies [
C3d,g is detected by nephelometry/turbidimetry or enzyme immunoassays (EIA) using polyclonal antibodies (see Section
The final step in the complement cascade is the formation of the C5b-9 complex, which is inserted into cell membranes, thereby causing cell damage and/or lysis. sC5b-9, the soluble form of this high molecular weight complex, can be quantified in the fluid phase as a marker of full complement activation, by using an EIA with a monoclonal antibody specific for a neoepitope in C9, which is exposed in complex-bound but not intact C9. Detection of the formed complexes is performed by using polyclonal antibodies against C5 or C6 (i.e., another protein present in the same macromolecular complex) [
Since all these activation markers can be rapidly produced by complement activation
The function of each of the complement activation pathways is dependent on the integrity of each of the participating components, and therefore a deficiency in a single component will affect the activity of the whole cascade. One major advantage of functional tests that monitor a whole activation pathway from initiation to the effector phase (lysis) is that they will detect both deficiencies in complement components and consumption-related decrease of complement activity, thereby combining information obtained using the types of assays described above.
Complement activation by the CP is studied in hemolytic assays utilizing sheep erythrocytes coated with rabbit antibodies (IgM with or without IgG). When serum is added, the C1 complex will bind, leading to formation of the CP convertase, which activates C3. Activation of C3 then initiates the assembly of the C5b-9 complex, which ultimately results in erythrocyte lysis [
Complement activation by the AP is studied by using rabbit or guinea pig erythrocytes, which are spontaneous activators of the human AP. When the cells are incubated in serum with the addition of EGTA to chelate
Hemolytic assays can be performed in different ways; the original assays, the so-called CH50 and AH50, are based on titration of the amount of serum necessary to lyse 50% of specified amount of cells [
More recently, a method comprising three separate EIAs which for the first time enables the simultaneous determination of all three activation pathways (including the LP) has been reported. The assay can best be described as a solid-phase functional test, since it comprises recognition molecules specific for each pathway (IgM for the CP, mannan or acetylated bovine serum albumin, BSA, for the LP, and LPS for the AP). These molecules are coated onto ELISA plate wells, and then serum is incubated under conditions in which only one pathway is operative at a given time and the other two pathways are blocked. The final step in each EIA is the detection of the resulting C5b-9 complex by a monoclonal antibody against a neoepitope in complex-bound C9 [
These functional techniques are particularly useful for (1) identifying congenital deficiencies and (2) monitoring fluctuations in complement function, for example, in SLE patients during exacerbations. A tentatively identified deficiency can be confirmed by concentration determination using a protein-specific assay and by experiments in which the patient sample is reconstituted with the relevant protein. (Most plasma complement components are commercially available). These analyses will provide information whether it is a functional deficiency or a total lack of the protein (Figure
Algorithm for complement analyses. The aims are to diagnose complement deficiency in patients with recurrent bacterial infections (a), diagnose the cause of their persistent complement activation (b), and to dissect the cause of C1INH deficiency (c). See text (Section
C3Nef are autoantibodies that bind to components of the AP convertase, thereby prolonging its functional
Recent efforts to improve detection of C3Nef include construction of ELISA-based functional assays using nickel-stabilized C3bBb, and real-time monitoring of the formation and decay of C3-convertase formation using surface plasmon resonance, SPR [
Anti-C1q autoantibodies are found in several autoimmune conditions and also in healthy controls. The assay is performed as follows: coating ELISA plates with purified C1q, incubation of patient serum and binding of true anti-C1q autoantibodies to the collagen part of C1q, and detection of bound IgG antibodies using antihuman IgG antibodies. In order to avoid that IgG-containing immune complexes in the samples bind to the coated C1q it is necessary to perform the assay in the presence of high concentrations of NaCl, typically 0.5–1.0 M, which dissociates the binding of C1q to IgG-containing immune complexes. The role of anti-C1q autoantibodies and methodological considerations are discussed in detail in [
Immunoconglutinins (IKs) are autoantibodies against fragments of C3 or C4 that affect the functioning of these components and are found in inflammatory states and autoimmune diseases, including SLE. IKs can be detected by EIA, using C3-coated wells for capture and polyclonal antihuman IgG, IgA, or IgM antibodies for detection [
EDTA is the only anticoagulant that completely inhibits any complement activation
In this section we summarize the indications for complement analyses and present an algorithm, which we follow in our laboratory when we perform complement diagnostics. The algorithm is shown in Figure
Complement deficiencies may be due to treatment with complement-inhibiting drugs such as the newly introduced eculizumab. Quantitative functional assays can be used to test the effect of the drug
The initial step in our algorithm to determine and assess the cause of complement activation (Figure
The cause of a C1INH deficiency (Figure
Activation of the complement system both
Use of the commonly employed combination of C3 and C4 concentrations to monitor complement in immune complex disease should be avoided, since both the sensitivity and specificity of these measurements are low. For example, SLE patients may have inherently low concentrations of C4 as a result of a low gene copy number [
Complement activation and hemolytic function during an SLE exacerbation. C3, C4, factor B, and other components are consumed, leading to a depression in hemolytic function (red line). The resulting activation products, C3d,g, C3a, and sC5b-9 (green line), peak concomitantly with the SLE disease index (SLEDAI).
As an illustration of what was discussed in the previous section we have constructed Table
Complement function of the CP and AP, plasma concentrations of C3, C4, factor B, and C3d,g, and the C3d,g*1000/C3 ratio in one patient with SLE and one patient with a C2 deficiency.
CP (%) | AP (%) | C3 (g/L) | C4 (g/L) | Factor B |
C3d,g |
C3d,g*1000/C3 | |
---|---|---|---|---|---|---|---|
SLE | 15 | 45 | 0.53 | 0.07 | 0.18 | 7.0 | 13.2 |
C2 def | 5 | 97 | 0.80 | 0.14 | 0.30 | 3.5 | 4.4 |
Reference interval | 80–120 | 50–150 | 0.67–1.29 | 0.13–0.32 | 0.16–0.44 | <5.3 | <5.3 |
In summary, complement analyses for individual patients are useful in a relatively limited number of conditions, which are summarized in Table
Complement pathology, differential diagnostics.
Analyses | HAE/AAE | SLE, urticarial vasculitis | PSGN | MPGN |
Complement |
---|---|---|---|---|---|
CP | N | L | L | L | * |
AP | N | N (L) | L | L | * |
C1INH (conc) | L | N | N | N | * |
C1INH (funct) | L | N | N | N | * |
C1q | (L) | L | N | N | * |
C4 | L | L | N | N | * |
C3 | N | L | L | L | * |
C5 | N | — | L | (L) | * |
Factor B | N | N | L | N | * |
Properdin | N | — | L | N | * |
C3d | N | H | H | H | N |
C3d,g/C3 | N | H | H | H | N |
*Variable (dependent on which component is defective); N: normal; L: low; H: high.
Acquired angioedema
Atypical hemolytic uremic syndrome
Alternative pathway of complement
Bovine serum albumin
C1 inhibitor
C3 nephritic factor
C4b-binding protein
Classical pathway of complement
Complement receptor 1 (CD35)
Decay acceleration factor (CD55)
Enzyme immunoassay
Factor H-related protein
Glycosylphosphatidylinositol
Hereditary angioedema
Hypocomplementemic urticarial vasculitis syndrome
Immunoconglutinins
Ischemia/reperfusion
Lectin pathway of complement
Lipopolysaccharide
Mannan-binding lectin
Membrane cofactor protein (CD46)
Membranoproliferative glomerulonephritis
Polyethylene glycol
paroxysmal nocturnal hemoglobinuria
poststreptococcal glomerulonephritis
regulator of complement activation
systemic lupus erythematosus
SLE disease index
half-life.
The authors thank Professor Ulf R. Nilsson for valuable contributions and discussions over the years concerning the presented concept and Dr. Deborah McClellan for excellent editorial assistance. This work was supported by grants from the Swedish Research Council (VR) 2009-4675, 2009-4462, and by faculty grants from the Linnæus University.