Clinical Complement Analysis—An Overview

YTMRV-50587; No of Pages 10 Transfusion Medicine Reviews xxx (xxxx) xxx

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Clinical Complement Analysis—An Overview Lillemor Skattum ⁎ Department of Laboratory Medicine, Section of Microbiology, Immunology and Glycobiology, Lund University, and Clinical Immunology and Transfusion Medicine, Region Skåne, Lund, Sweden

a r t i c l e

i n f o

Available online xxxx Keywords: Clinical complement analysis Complement activation/consumption Complement function Complement deficiency Complement diagnostics

a b s t r a c t The complement system plays an important role in varying types of disease, ranging from inflammatory and autoimmune disorders to immune deficiency states. In addition, new settings have emerged where complement analysis is of interest to monitor complement-directed therapy and aid identification of transplant complications. Therefore, it is critical that clinical laboratories offer optimized and timely complement analysis. This review presents a comprehensive overview of the most important complement analysis methods that are currently used. It also points to some areas within complement diagnostics where development is needed, for example, regarding certain analytes for which practical methods suitable for the routine laboratory are lacking. Furthermore, it contains a more detailed discussion on complement autoantibody assessment. The list of analyses providing clinically valuable information includes analysis of complement function, quantification of individual complement components and complement activation fragments, identification of autoantibodies to complement, as well as genetic complement analyses. There is still a shortage of commercially available methods suitable for highthroughput screening of complement deficiency and for assessment of complement activation, but development is under way. There is also ongoing work within the complement community to improve standardization of measurements, and recently, an extensive quality assurance program has been initiated. © 2019 Elsevier Inc. All rights reserved.

Contents Use of Complement Analysis in Clinical Practice . . . . Functional Analysis of Complement Activation Pathways Analysis of Individual Complement Components . . . . Complement Activation Assessment . . . . . . . . . Analysis of Autoantibodies to Complement Components Antibodies to C1q. . . . . . . . . . . . . . . . Antibodies to C1IINH . . . . . . . . . . . . . . Antibodies to C3 Convertases—Nephritic Factors . . Antibodies to Factor H. . . . . . . . . . . . . . Other Complement Autoantibodies . . . . . . . . Monitoring of Complement-Directed Treatments . . . . Analysis of Complement Genes . . . . . . . . . . . . Sample Material and Sample Conditions . . . . . . . . Standardization and quality assessment . . . . . . . . Declaration of Competing Interest . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . .

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Use of Complement Analysis in Clinical Practice

⁎ Corresponding author at: Lillemor Skattum, Clinical Immunology and Transfusion Medicine, Akutgatan 8, 221 85 Lund, Sweden. E-mail address: [email protected].

There are an increasing number of disease states in which complement analysis is important, both for diagnostic reasons and for assessment of disease activity. For decades, it has been known that the classical pathway of complement activation is activated in many

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conditions associated with immune complexes, including situations with formation of auto- or alloantibodies, as well as antibodies to microbial antigens. Important examples of diseases in which complement activation via the classical pathway are an important part of the pathogenesis are systemic lupus erythematosus, autoimmune hemolytic anemia (especially cold agglutinin disease), and cryoglobulinemic vasculitis [1-3]. It is also well known that the alternative pathway may be overactivated, like for example in invasive bacterial infections, poststreptococcal glomerulonephritis, and several other proinflammatory conditions. In more recent years, there has been an increasing understanding that the alternative pathway is dysregulated in different rare kidney diseases like complement-mediated (atypical) hemolytic uremic syndrome (aHUS) and C3 glomerulopathy [4,5]. Even more recently, the alternative pathway of complement has been shown to be involved in the pathogenesis of ANCA-associated vasculitis (reviewed in [6]). Heme is released into plasma during different situations characterized by intravascular hemolysis including hemolytic transfusion reactions and activates the alternative pathway, a mechanism that contributes to the toxic effects of free heme [7,8]. Meanwhile, the possible role for the lectin pathway in harmful complement activation—and links to other fluid phase protein cascades like the coagulation and kinin systems—has only just started to be elucidated [9,10]. Apart from excessive and thus detrimental complement activation, the complement system may also cause symptoms of disease by deficient functioning, leading to lack of activation when needed. Inherited complement deficiencies are rare and usually cause distinct phenotypes depending on which complement pathway or in some cases even depending on which complement protein is missing or deficient in functional activity [11,12]. Complement deficiency may also more commonly arise as a secondary state, usually due to increased consumption during different disease processes [13]. Analysis of complement in the clinical setting is aimed at assessing the state of the complement system by analyzing function and degree of complement activation. In specific settings, analysis of autoantibodies directed to complement proteins and complement genetics may also be of value. The main indications for complement analysis in current clinical practice are summarized in Table 1. Functional Analysis of Complement Activation Pathways The complement functional activity is usually estimated by analysis of the last step of the cascade, namely, the formation of the membrane attack complex (MAC). If MAC is detected upon activation, this ensures that all complement factors in the preceding cascade system are present and functional. Traditionally, hemolytic assays are used, which may be quantitative like the CH50 test (Fig 1a, [14]) or qualitative like the hemolysis-in-gel test (Fig 1b, [15]). Alternatively, quantitative hemolysis may be performed as a 1-tube test to save time and effort, but with equivalent results to the CH50 method [16]. Properdin deficiency is not readily detected by hemolytic complement function assays and thus requires additional measurement of properdin or modification of the hemolytic assay [17]. More recently, a semiquantitative enzymelinked immunosorbent assay (ELISA) method for all the 3 main activation pathways (the classical, alternative, and mannose-binding– initiated lectin pathways) has been commercialized and come into wide use (Fig 1c, [18]). In addition, a functional ELISA assay of the ficolin 3–activated lectin pathway has been developed [19] but is currently not commercially available. There are also liposome-based assays available that enable automatic high-throughput screening of complement function (Fig 1d, [20]). Complement functional assays are mainly used to rule out complement deficiency but are sometimes also used as an indirect measure in the assessment of degree of in vivo consumption of the respective activation pathways. Recently, complement function is in some cases also assessed as part of evaluation of new pharmaceutical compounds. In addition, it has been proposed that complement function should be used

Table 1 Indications for clinical complement analysis Diagnosis of inherited and acquired complement deficiency Symptom(s)/disease

Complement aberration(s)

Recurrent bacterial infections (especially invasive infections with Streptococcus pneumoniae, Haemophilus influenzae, and meningococci) Meningococcal infection

Deficiency of factor I, CP, AP, or TP components

Angioedema SLE or SLE-like disease

aHUS

C3G, PLD, MPGN 1, and 3

PNH

AGN Eculizumab treatment AMD

Deficiency of properdin, factor B, factor D or TP components Deficiency of C1INH; HAE and AAE, autoantibodies to C1INH Deficiency of CP components/complement consumption through CP, autoantibodies to C1q Mutations (heterozygous or compound heterozygous), hybrid genes, etc, in genes of AP components and complement regulators; autoantibodies to factor H Autoantibodies to C3 convertase and/or C5 convertase, in some cases mutations or rare variants in genes of AP components or complement regulators Clonal somatic mutation in the PIGA gene leading to loss of membrane regulators CD55 and CD59 Temporary consumption through the AP No formation of TCC or C5a Polymorphisms and rare variants in the genes of factor H, C2, factor B, factor I and C9

AAE, acquired angioedema; AGN, acute poststreptococcal glomerulonephritis; aHUS, atypical hemolytic uremic syndrome; AMD, age-related macular degeneration; AP, alternative pathway; C1INH, C1 inhibitor; CP, classical pathway; C3G, C3 glomerulopathy; HAE, hereditary angioedema; HUVS, hypocomplementemic urticarial vasculitis syndrome; MPGN, membranoproliferative glomerulonephritis; PNH, paroxysmal nocturnal hemoglobinuria; PLD, acquired partial lipodystrophy; SLE, systemic lupus erythematosus; TCC, terminal complement complex (sC5b-C9); TP, terminal pathway.

to evaluate the clinical significance of alloreactive antibodies to blood group antigens. Cunnion et al have described hemolytic assays using human erythrocytes to assess the risk of causing hemolytic transfusion reactions in different blood group incompatible transfusion settings [21]. Analysis of Individual Complement Components Several different immunochemical methods are used for quantification of complement components in clinical samples. Common examples are gel precipitation assay, and nephelometry/turbidimetry, all of which use polyclonal antisera to form quantifiable immune complexes. Different forms of enzyme immunoassays (EIAs) may also be used; however, these commonly use monoclonal antibodies for detection, which may cause difficulties to identify proteins that have been immunogenically altered by disease processes or by genetic variations. Important, but not widely recognized, is the fact that analysis of C1q is not well suited to be performed with nephelometry/turbidimetry probably due to interference from the affinity of the C1q globular head region for immunoglobulin (Ig) Fc regions [22]. C1q should preferably be quantified using a solid phase technique like gel precipitation or EIA. Multiplex complement component analysis has hardly been used in clinical diagnostic laboratories, but development of new assays with high-throughput potential is under way, which might enable inclusion of complement deficiency tests into screening programs [22,23]. Flow cytometry can be used to analyze expression of membranebound complement inhibitors and complement receptors on different blood cells. In paroxysmal nocturnal hemoglobinuria (PNH), the clonal expansion of hematopoietic cells lacking GPI anchor was previously assessed by analyzing complement-regulating cellular membrane proteins CD55 (also called decay-accelerating factor) and CD59 on

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a)

1.

Serially diluted serum

3

b)

Mix with Sensized sheep erythrocytes

2. Incubate at 37°C for 30 min

3. Measure hemolysis

4. Idenfy amount of serum that lyses 50% of defined amount of erythrocytes

c)

Classical

Lecn

Alternave

d)

Substrate Complement

1. Addion of diluted serum

* 2. Incubaon at 37°C

3. Detecon of C9 deposion

Enzyme in liposome

Enzyme released aer complement-mediated lysis

*Lecn pathway; C1q blocked by specific anbody

Fig 1. a-d, Overview of complement functional assays. a, Simplified scheme of the CH50 assay. b, Picture of representative results of hemolysis-in-gel assay, by permission from Prof Lennart Truedsson. NHS, normal human serum; C2D, C2 deficiency; C4D, C4 deficiency; C3D, C3 deficiency, etc; HAE, hereditary angioedema. c, Overview of EIA for function of the classical, lectin, and alternative pathways, respectively, as indicated. d, Principle of the liposome-based assay for classical pathway function assessment.

monocytes and neutrophils from peripheral blood. Instead, the current recommendation is to analyze expression of other, non–complementrelated molecules in the diagnostic workup of PNH, reviewed in [24]. In aHUS, low expression of CD46 on, for example, neutrophils and monocytes may indicate disease-associated mutations in the corresponding gene [25]. Normal expression of complement components in the circulation or on cells does not exclude the presence of a dysfunctional protein due to, for example, gene mutations or blocking autoantibodies. It may therefore be necessary to investigate the function of individual components. Most of the proteins of the activation pathways can be assessed by hemolytic assays by utilizing serum that is naturally lacking the component or that has been artificially depleted of it. Also, the different complement inhibitor proteins may be analyzed by specific methods. One important example is the analysis of C1 inhibitor (C1INH) function in the workup of patients with hereditary or acquired C1INH deficiency. The preferable commercially available assays assess the capacity of C1INH to inactivate its substrate, C1s [26]. Different functional tests for the investigation of patients with aHUS have also been described, including hemolytic and other assays for factor H function [27,28], and a modified form of the Ham test [29]. However, the clinical value of these tests is currently uncertain, and they have not come into general clinical use. Methods for assessment of factor I function (the function being degradation of C3b and C4b in the presence of its different cofactors) have also been published [30-32]. Complement Activation Assessment When complement is activated in vivo, the native forms of the complement components are decreased in concentration, and instead, complement activation fragments and complexes are formed.

Demonstration of lowered concentrations of native components is commonly used for detection of increased complement activation. However, ongoing complement activation may be missed in a situation where increased protein synthesis (the acute phase reaction) is compensating for increased consumption. Therefore, quantification of complement activation products is a more reliable way of assessing the degree of complement activation—provided that the correct sample type and sample conditions are used (see “Sample Types and Sample Conditions”). To specifically detect complement activation fragments, in separation from their corresponding native components, special measures have to be taken, as the 2 types of proteins usually share many antigenic epitopes. For example, monoclonal antibodies that detect neoepitopes found only in the activation fragments may be used [33,34]. Alternatively, the cross-reacting fragments that are not to be quantified may have to be separated from the activation product prior to analysis [17,35]. Depending on which activation pathway is predominantly affected, different fragments form in the circulation. In some situations, it is sufficient to determine the degree of global complement activation (the sum of all activation pathways), whereas in other situations, it is more valuable to have information on involvement of 1 or more specific pathways. The complement activation product that is most commonly analyzed in clinical practice is C3dg, which is the fragment remaining when activated C3 has been completely degraded by factor I by help of its cofactors [17]. The concentration of C3dg gives information of the sum of activation of all pathways up to the level of C3 convertases. Measurement of the sample content of soluble terminal complement complexes (soluble MAC, sC5b-C9) is another fairly common means to assess global complement activation. In clinical practice, it is still not common to analyze activation pathway-specific complement fragments, but the field is developing toward more diversified monitoring,

Please cite this article as: L. Skattum, Clinical Complement Analysis—An Overview, Transfusion Medicine Reviews, https://doi.org/10.1016/j. tmrv.2019.09.001

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to the Fc region of antibodies of the IgG and IgM classes. Thus, to avoid interference from immune complexes in anti-C1q analysis, it has been recommended to use the isolated collagenous region of C1q as antigen or to use buffer with high ionic strength to abolish binding between the C1q globular head region and the Fc part of Igs [42]. For unknown reasons, anti-C1q with affinity for separated C1q protein chains B and C are common in HUVS, whereas anti-C1q from SLE patients mostly recognize whole C1q or the intact collagenous region [43,44]. These differential binding patterns can be demonstrated by Western blot analysis.

alongside the development of treatments that affect the complement system. Figure 2 shows complement activation products in the setting of an overview of the complement system. Analysis of Autoantibodies to Complement Components There are several examples of different clinically important autoantibodies with specificity for complement proteins, which can be analyzed to aid diagnostics and/or disease activity monitoring. Autoantibodies to complement may be present at low concentrations in healthy individuals, similar to other autoantibodies. Thus, it is important to establish relevant cutoff levels for each assay. This is commonly achieved by analysis of a sufficient number of samples from healthy persons, with a small proportion allowed to be autoantibody positive for statistical reasons (usually less than, or approximately equivalent to, 5% of blood donors).

Antibodies to C1IINH In the diagnostic workup of patients with C1INH deficiency, analysis of autoantibodies to C1INH (anti-C1INH) may help to differentiate between hereditary (HAE) and acquired (acquired angioedema, AAE) forms of deficiency, as anti-C1INH are prevalent in AAE but not in HAE. AAE is often associated with lymphoproliferative diseases, with or without presence of monoclonal gammopathy (M component) [45]. In AAE patients with an M component, anti-C1INH are fairly often of the same Ig class as the M component itself. Thus, as opposed to other autoantibodies, anti-C1INH of other Ig classes than IgG (ie, IgA and IgM) are clinically relevant and should be analyzed. Some anti-C1INH may cause disease by binding to the reactive center loop of C1INH, thereby blocking its function. Another possible effect of anti-C1INH is more rapid clearance of C1INH from the circulation as it becomes part of an immune complex. This effect might not be compensated for by synthesis of new molecules. There are to the best of the author's knowledge no commercial assays for anti-C1INH available, and consequently, these are typically analyzed by different in-house ELISA methods. An ELISA method for detection of the C1INH-blocking capacity of anti-C1INH has been described [46].

Antibodies to C1q Antibodies to the recognition molecule of the classical pathway, C1q (anti-C1q), are found at increased concentrations in many conditions. Although not specific for any diagnosis, anti-C1q are commonly detected in systemic lupus erythematosus (SLE), in particular, patients with renal involvement, where their concentration also is known to correlate with disease activity [36]. Anti-C1q are also reported to be found in N95% of patients with the SLE-like hypocomplementemic urticarial vasculitis syndrome (HUVS [37]). Other disease associations are different forms of primary and secondary glomerulonephritis including membranoproliferative glomerulonephritis (MPGN) and poststreptococcal glomerulonephritis, as well as RA vasculitis and other chronic immune complex-associated conditions [38-40]. Commercial assays are available for analysis of anti-C1q, which are used alongside with different in-house methods, mainly ELISAs. Originally, anti-C1q detected in SLE patients were reported to be specific for the collagenous portion of the C1q molecule and not to have affinity for the globular head region [41]. During initiation of the classical pathway, the globular head region is the part of the C1q molecule that binds

Antibodies to C3 Convertases—Nephritic Factors The C3 and C5 convertases of the complement system may become autoantigens. Autoantibodies to the convertases often stabilize them against intrinsic and extrinsic decay, a phenomenon that leads to

C4a C4d

Classical pathway C1q C1r C1s

Lecn pathway C4 C2

MBL/ficolin MASP

Alternave pathway C3b

C3bBbP

B D P

C1INH-C1s-C1r-C1INH

C3 C4b2b C3bBb

Ba Feedback loop

C3b

Bb

C3a

C5 sC5b-C9

C4b2b3b C3b2Bb

C3dg

C5a

Fig 2. Overview of the complement system with complement activation complement products. Graph of the 3 main complement activation pathways. Complement activation products in yellow boxes and circles.

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patient IgG in combination with Ig-coated sheep erythrocytes on which convertases are built using purified human complement components [59]. Different ELISA methods have also been described [55,60,61]. Advantages of these are that the inherent variability of erythrocytes is avoided and that they admit direct confirmation that the serum components that bind convertases are indeed antibodies. Most ELISA methods use purified complement components to build C3 or C5 convertases on the plastic surface in a convertase-stabilizing buffer. Patient serum is then incubated, allowing C3/C5NeF to bind if present, with subsequent detection of bound antibodies. C4NeF are measured at very few clinical laboratories, mostly with hemolytic methods [62]. Figure 3 shows the stabilization of C3 convertases by C3NeF and C4NeF, respectively, on sheep test erythrocytes.

increased consumption of C3 and or C5 and, in some cases, consumption also of other complement components further downstream in the cascade [47,48]. Autoantibodies to the alternative pathway convertase C3bBb are called C3 nephritic factors (C3NeF) and are the most extensively studied and probably most common of these autoantibodies. Other convertase autoantibodies are C5 nephritic factors (C5NeF) that recognize the alternative pathway C5 convertase and C4 nephritic factors (C4NeF) that bind to the classical pathway C3 convertase C4b2b [49,50]. All types of nephritic factors are found mainly in association with the chronic glomerulonephritides that are collectively termed C3 glomerulopathy (C3G [51]). C3NeF are also very common in the rare disease acquired partial lipodystrophy [52], and both C3NeF and C4NeF may lead to increased susceptibility for invasive bacterial infections due to the low levels of circulating native C3 molecules resulting from persistent C3 consumption [53]. Whether or not C3NeF and other convertase antibodies are pathogenic in relation to C3G is unclear [54]. Several different methods for detection of nephritic factors have been described. Analysis is complicated because the autoantigens consist of complexes of 2 or more activated complement components and need to be presented in the right conformation(s) to be recognized by the autoantibodies, which are known to be extensively heterogeneous. In consequence, it has been recommended to use more than 1 method to assess presence of C3NeF [55]. The simplest form of C3NeF analysis detects the extent of C3 consumption after incubation of patient serum mixed with serum from a healthy donor in a buffer blocking the classical pathway [56,57]. C3 breakdown may be detected by, for example, crossed immunoelectrophoresis or Western blot. This test shows the C3-consuming effect of C3NeF but does not exclude other causes like, for example, mutated complement proteins. Different hemolytic methods are also commonly used for C3NeF analysis, using either whole serum and unsensitized sheep erythrocytes [58], or purified

Antibodies to Factor H In 2005, autoantibodies to the alternative pathway complement inhibitor factor H (anti-FH) were demonstrated in the serum of patients with aHUS [63]. Such autoantibodies were later found to be strongly associated with a deletion in the gene encoding factor H-related proteins 1 and 3 (CFHR1/3). This deletion, although common in healthy persons at up to 20% in different populations, is found in more than 75% of patients with anti-FH–associated aHUS. Anti-FH in aHUS are directed to the C-terminal part of factor H and may therefore block binding of factor H to host cells, thus rendering them unprotected from complement attack via the alternative pathway. Anti-FH–associated aHUS comprises 5%-13% of European aHUS patients, and analysis of anti-FH is considered mandatory in suspected aHUS [64]. Patients with anti-FH–associated aHUS may benefit from treatment with plasma exchange combined with immunosuppressive drugs, as opposed to other forms of aHUS where C5-inhibiting therapy is needed [4]. Anti-FH may also be present in some patients with C3G, mainly antibodies with specificity

Factor H

Factor D

C3NeF C3b

B

C3b

B

C3b

Bb

C3b

Bb C3b

ShE

ShE

ShE

ShE

ShE

Hemolysis

ShE

C4BP C1q

C4NeF C4b C2b

ShE

C4b C2b

ShE

C4b C2b

ShE

C4b C2b C3b

ShE

C4b C2b C3b

ShE

Fig 3. Hemolytic assay for C3 nephritic (C3NeF) and C4 nephritic factors (C4NeF): stabilization of C3 convertases. Upper panel shows stabilization of the alternative pathway C3 convertase by C3NeF. Lysis of unsensitized sheep erythrocytes [49]. Lower panel shows stabilization of the classical pathway C3 convertase by C4NeF. Lysis of sensitized sheep erythrocytes (for description of analysis method, see [53]).

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for the N-terminal part of factor H, instead of the C-terminal part which is the common pattern in aHUS [65]. In 2014, an initiative to standardize measurement of anti-FH that involved several complement laboratories in Europe and the United States led to the establishment of control samples with a defined concentration of anti-FH, expressed in arbitrary units, and agreement on a recommended ELISA protocol [66]. Commercial assays are also available. Other Complement Autoantibodies Several other complement autoantibodies have been described in association with different diseases. For example, factor B antibodies may be found in serum of patients with different types of C3G and Igrelated GN [67,68]. The lectin pathway initiator ficolin 3 (also called Hakata antigen or H-ficolin) was originally detected as an autoantigen in SLE patients [69], and antibodies to MBL have been reported in RA patients. However, the clinical value of these and more complement autoantibodies as diagnostic or disease activity markers is uncertain. Table 2 shows examples of typical combinations of results of complement analyses in association with different diseases. The main clinical complement analysis methods are listed in Table 3, with comments regarding their properties. Monitoring of Complement-Directed Treatments Currently, there are only 2 types of complement-inhibiting pharmaceuticals available: monoclonal antibodies to C5 (eculizumab and its successor ravulizumab) that inhibit activation of C5 and thereby the formation both of the MAC complex and of the anaphylatoxin C5a [70], and several compounds of C1INH (plasma-purified and recombinant). Eculizumab is approved for treatment of aHUS, PNH, and treatmentrefractory myasthenia gravis, whereas C1INH is used for treatment of HAE and AAE [71]. These and a number of other complement-directed drugs are under trial for treatment of many other immune-mediated and inflammatory conditions like, for example, ANCA-associated vasculitis, antibody-mediated transplant rejection, neurodegenerative diseases, and thrombotic microangiopathies [72,73]. Thus, there is an increasing demand for monitoring of these complement-targeting therapies, most importantly follow-up of eculizumab treatment in aHUS. Eculizumab treatment in aHUS is aimed at achieving sufficient inhibition of MAC formation at the endothelium to prevent cell damage. Activity of the complement activation pathways is used as a surrogate measure, and it is assumed that absence of MAC formation in vitro guarantees sufficient

complement inhibition in situ at the target organ. This is most commonly tested by performing hemolytic assays or EIA for the classical and alternative pathways, where the goal is usually activity b10% of normal, although probably higher activity may also be innocuous [74]. It has been reported that hemolytic assay of the classical pathway and EIA may fail to detect residual MAC formation [75,76]. Analysis of eculizumab trough levels and free C5 has also been used for monitoring. An optimization of treatment monitoring might provide the means to reduce treatment costs while avoiding potentially harmful breakthrough terminal pathway activation [77,78]. Analysis of Complement Genes Currently, genetic complement analysis is mainly indicated for the investigation of patients with aHUS and for patients with inherited complement deficiency. In aHUS, many different mutations have been detected, predominantly affecting genes of complement regulating proteins. Typical findings are combinations of heterozygous and compound heterozygous mutations, and even combinations of common polymorphisms in different complement genes, so-called complotypes. The clinical penetrance of detected mutations is often variable within a family. If aHUS is suspected, it is recommended to sequence the following complement-related genes: CFH, CFI, CD46, C3, CFB, CLU, and THBD. In addition, the gene for diacylglycerol kinase ε should be investigated, as well as copy number variations and hybrid genes involving CFH and the genes of the complement factor H-related proteins [4,79]. The interpretation of the clinical association of gene variants found in aHUS patients is complicated, and it is thus recommendable to perform these at specialized centers. Patients with inherited complement deficiencies are very rare, and the deficiency is usually caused by a homozygous mutation. Each case or family typically presents with a unique genetic aberration, which implies that sequencing of the full gene is required to establish the cause of the defect. An exception is C2 deficiency in the white population, which in around 90% of cases is caused by a 28–base pair deletion in the C2 gene, a deletion that is associated with a particular HLA haplotype [80,81]. This deletion can be detected by polymerase chain reaction in combination with gel electrophoresis. Hereditary angioedema types I and II (C1INH) differ from other monogenic complement deficiency states because they are an autosomal dominant trait [82]. C4 isotype deficiency, caused by lack of gene copies of C4A (C4A0) or C4B (C4B0), is fairly common [83]. C4A deficiency is associated with an increased risk of developing SLE [84], and like the C2 and factor B genes, the C4

Table 2 Typical complement analysis results in different diagnoses Condition

CP

AP

LP

C3

C4

C1q

C1INH

P

C3d

TCC

aC1q

C3NeF/C4NeF

aFH

aC1INH

SLE, active disease HUVS aHUS C3G/PLD HAE AAE AGN AMR, AIHA CP deficiency AP deficiency LP deficiency TP deficiency

↓ ↓/→ → → ↓b ↓b → ↓ ↓ → → ↓

↓/→ → ↓/→ ↓ → → ↓ → → ↓ → ↓

→ → → → → → → → ↓e/→ → ↓ ↓

↓/→ ↓/→ ↓/→ ↓ → → ↓ ↓/→

↓ ↓/→ → → ↓ ↓ → ↓

↓/→ ↓ → → → ↓ → ↓/→

→ → → → ↓ ↓ → →

→ → → →/(↓) → → ↓ →

↑ ↑/→ ↑/→ ↑ → ↑/→ ↑ ↑

↑/→ →/↑ ↑/→ ↑/→ → ↑/→ ↑/→ ↑/→

+/− + − +/− − − +/− −

-a − − + − − (+)d/− −

-a n.k. +c/− −/+ n.k. n.k. n.k. −

-a − − − −/(+) +/− − −

AAE, acquired angioedema; AGN, acute poststreptococcal glomerulonephritis; aHUS, atypical hemolytic uremic syndrome; AHIA, autoimmune hemolytic anemia; AMR, antibody-mediated transplant rejection; AP, alternative pathway; C1INH, C1 inhibitor; CP, classical pathway; C3G, C3 glomerulopathy; HAE, hereditary angioedema; HUVS, hypocomplementemic urticarial vasculitis syndrome; MPGN, membranoproliferative glomerulonephritis; n.k., not known; PLD, acquired partial lipodystrophy; SLE, systemic lupus erythematosus; TCC, terminal complement complex (sC5b-C9) ; TP, terminal pathway. a May be present. b Dependent of assay method. c Positive in a subgroup of aHUS. d May be positive initially in a minority. e LP low in C2 and C4 deficiency; normal in C1 deficiency.

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Table 3 Complement assays List of main complement assays, not including genetic analysis or quantification of native complement components Assay

Features

Functional assays CH50/AP50

Hemolytic, quantitative assays. Tube titration. − Cumbersome. Blood donor animal-dependent variation. Properdin deficiency not readily detected (AP50). + Easy to perform. Properdin deficiency will be detected. − Considerable CV. + Easy to perform, high precision, high throughput. − Only CP. + Easy to perform, inexpensive, high throughput − Only qualitative/semiquantitative. Properdin deficiency not readily detected. + Accurate results. − Automation requires specialized instruments. + Easy to perform. − Possibly not sufficient sensitivity for hereditary angioedema26. + Easy to perform, inexpensive. − Unknown sensitivity for detection of disease-causing genetic variants. Cell viability test; effect of serum on cells from cell line with mutated PIGA, to detect aHUS. Not known to the author whether the analysis is available at clinical laboratories. − Cumbersome.

EIA for CP/AP/MBL-LP Liposome-based CP assay Hemolysis-in-gel, CP and AP C1INH functional assay, chromogenic C1INH functional assay, complex EIA Factor H functional assay, hemolytic Modified Ham-test

Activation markers C3d, nephelometry/turbidimetry C3d, double-decker rocket immunoassay TCC/sC5b-C9 by EIA C5a, C3a by EIA Eculizumab treatment monitoring Complement pathway functional assays (see above) Eculizumab trough levels, EIA Complement autoantibody assays C3NeF, hemolytic assay

C3NeF, hemolytic assay according to Rother [58]

C3NeF, assessment of C3 breakdown

C3NeF, EIA

Anti-C1q, EIA Anti-C1q, Western blot Anti-C1INH, EIA Anti-C1INH, neutralizing antibodies EIA Anti-factor H, EIA

Commercially available/not

46

+ High throughput. − Requires pretreatment of samples (removal of C3b and native C3). + No need for pretreatment of samples. − Low throughput. + Easy to perform. − More sensitive to transport/storage than C3d. Due to short half-life mainly suitable for research purposes. Easy to perform.

Both EIA and CH50/AP50 are used. AP50 more sensitive to residual complement activity [75,76].

No Yes Yes No Yes Yes No No

Yes

Yes Yes

Varying

Both in-house and commercial methods exist.

Yes

C3NeF-dependent stabilization of C3 convertase built from purified complement components on ShE. Gold standard. + No influence from complement proteins of patient serum when purified IgG is tested. − Cumbersome. C3NeF-dependent stabilization of C3 convertase deposited by whole serum on ShE. Equal parts of patient and normal serum mixed. + Easy to perform, inexpensive. − Several possible sources of errors: eg, defective complement protein/s in patient serum. Detection of formation of C3 activation products after incubation of patient serum together with normal serum (by Western blot/crossed immunoelectrophoresis/other method). + Easy to perform, inexpensive. − Not specific for C3NeF. Several methods described; functional methods detecting, eg, Bb, or nonfunctional methods detecting bound IgG specific for C3 convertase. + Possibility to directly show patient IgG binding to C3 convertase. − Cumbersome. + Easy to perform + Can aid in differentiation between SLE and HUVS − Uncertain sensitivity in relation to different diagnoses. + Easy to perform. − No information on functional consequences of detected antibodies. + Informs about functional consequences regarding C1INH. + Easy to perform. − No information on functional consequences of detected antibodies.

No

No

No

No

Yes No No No Yes

aHUS, atypical hemolytic uremic syndrome; AP, alternative pathway; C1INH, C1 inhibitor; C3NeF, C3 nephritic factor; CP, classical pathway; CV, coefficient of variation; EIA, enzyme immunoassay; HUVS, hypocomplementemic urticarial vasculitis; LP, lectin pathway; MBL, mannose-binding lectin; ShE, sheep erythrocytes; SLE, systemic lupus erythematosus; TCC, terminal complement complex.

genes are part of the HLA locus on chromosome 6. Analysis of C4 genes is not readily performed by, for example, large-scale next-generation sequencing platforms because the C4 region contains gene duplications with substantial copy number variation as well as common mutations, factors that also to some extent influence the individual C4 concentration in the circulation. Real-time quantitative polymerase chain reaction is more suitable for analysis of C4 genes than traditional sequencing or next-generation sequencing [85]. The genetics of mannose-binding lectin (MBL) are also somewhat complicated, as the great variation of

serum MBL concentrations in the normal population arises due to a combination of different polymorphisms in both the structural MBL2 gene and its promotor region [86]. Genetic analysis is often not indispensable in the clinical diagnostics of complement deficiency but may be useful for family investigation, especially of newborn infants where the complement system may not have been fully developed. In cases of dysfunctional protein variants, genetic analysis may also be necessary to determine the deficiency state.

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Sample Material and Sample Conditions For the assessment of complement activation pathway function, serum is the suitable sample type, whereas accurate estimation of in vivo complement activation requires addition of ethylenediaminetetraacetic acid that chelates Ca 2+ and Mg 2+, which minimizes further complement activation after the sample has been drawn. In situations where anticoagulation is required while preserving complement function, addition of, for example, specific thrombin inhibitors like lepirudin is useful [87]. Although addition of ethylenediaminetetraacetic acid lowers the risk of in vitro complement activation, there is still a substantial risk that has to be considered. The factors that influence degree of activation are temperature, time, and circumstances deriving from the sample itself. All samples for complement analysis should be frozen to −70°C or lower if prolonged storage is needed. Partly depending on which parameters are to be analyzed, requirements may differ, but in general, the results of all types of complement analyses will be affected if samples are not frozen within 24 hours from sampling. In fact, many complement activation products will start to increase after even shorter time. It is also not recommended to freeze to −20°C, as that will result in longer freezing time, allowing ongoing complement activation [17]. Repeat thaw-freeze circles must be avoided, and transportation of frozen samples should be carried out on dry ice. It is also important to keep in mind that several other factors apart from in vivo and in vitro complement consumption influence serum and plasma concentrations of complement components. Liver failure will lead to decreased production of most of the complement components except for C1q, properdin, and factor D, which are produced by other cells than hepatocytes. For largely unknown reasons, hypogammaglobulinemia is associated with isolated low concentration of C1q, whereas factor D concentration increases in patients with decreased glomerular filtration rate because of retention of this small molecule. Both C3 and C4 levels correlate to BMI, and increased levels are associated with the metabolic syndrome [88].

Standardization and quality assessment There are few available international calibrators for complement parameters. The certified reference material standard ERM-DA470k/IFCC from the European Commission Institute for Reference Materials and Measurements has assigned values for C3 and C4, and there is an international WHO standard for C1INH function that can be purchased from the National Institute for Biological Standards and Control, UK. Control samples with a defined content of antibodies to factor H can also be obtained [66]. The previous international calibrator for C3dg is no longer available. With the aim to promote standardization of analysis of other complement parameters including complement activation products, a collaborative project was formed to produce complement standard preparations [89]. Information about the International Union of Immunological Societies/International Complement Society Committee for the Standardization and Quality Assessment of Complement Measurements is available at the International Complement Society Web site, www.complement.org. For clinical laboratories, it is highly recommendable to participate in 1 or more quality control programs to ascertain accuracy of their analysis methods. Also, it is necessary to use internal quality controls so that reproducibility of measurements can be monitored effectively. The Web site of the European Complement Network lists the main complement diagnostic laboratories in Europe (www.ecomplement.org).

Declaration of Competing Interest None.

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