Complement Testing - Complement Deficiency and Anticomplement Therapeutic Response Monitoring

Medical Experts

Contributor

Nandakumar

Vijayalakshmi (Viji) Nandakumar, PhD, MS

Former Medical Director, Immunology, ARUP Laboratories

Contributor

Saadalla

Abdulrahman Saadalla, MB, BCh

Assistant Professor (Clinical), University of Utah School of Medicine

Medical Director: Immunology Core Lab and Coagulation

The complement system is composed of over 50 soluble and cell-bound proteins that play a major role in host immune defense and inflammation. Complement proteins can be broadly classified as effector and regulatory proteins. The effector proteins act as a cascade activated via three distinct pathways initiated by different triggers: the classical pathway (CP), which is activated by antigen-antibody complexes; the alternative pathway (AP), which is activated autonomously and on contact with negatively charged surfaces; and the lectin pathway (LP), which is triggered by pathogen recognition molecules. Activation of any of the three pathways results in the formation of C3 convertases that proteolytically cleave the complement component 3 (C3) protein. The C3 convertases are then modified to C5 convertases, which proteolytically cleave C5. The cleaved C5b fragment binds to the terminal complement pathway proteins to form the membrane attack complex (MAC; C5b-9), which punctures cellular and pathogen membranes to exert the immune cytotoxic functions of complement.

Deficiency in any of the effector or regulatory complement proteins may lead to recurrent infections (eg, Neisseria) or inappropriate immune responses. Laboratory testing for complement deficiencies includes primary testing to determine which pathway is affected, followed by secondary testing to identify the deficient complement protein and/or its regulators to reach a definitive diagnosis. Laboratory testing may also be used to monitor complement blockage in patients treated with anticomplement therapeutics.

Quick Answers for Clinicians

Which laboratory tests can be used to monitor complement blockage in individuals treated with complement component 5 (C5) inhibitors?

The inhibiting effects of anti-C5 therapeutics (eg, eculizumab, ravulizumab) can by monitored by measuring total complement activity (CH50), alternative pathway complement activity (AH50), and complement component 5 (C5) functional activity.^, Comparisons to pretreatment activation levels may be helpful to determine therapeutic efficacy and monitor response to anti-C5 inhibitor treatment. Soluble C5b-9 is a marker of terminal complement pathway activation and may be also be used to monitor responses to anti-C5 monoclonal therapeutics. Refer to the Testing to Monitor Anti-C5 Therapeutic Response section for more information.

When is testing for complement function, rather than complement concentration, preferred?

Patients with congenital deficiencies in any components of the terminal pathway (C5-C9) may be susceptible to recurrent infections (eg, Neisseria) and inappropriate immune responses. A small number of individuals with these findings may have normal protein concentrations but defective protein functions. In these cases, functional assays may be useful to detect deficiency. Complement functional test results can be impacted when the patient is being treated with complement inhibitory drugs (eg, eculizumab or ravulizumab).

Which complement tests are useful in the evaluation of systemic lupus erythematosus?

Complement consumption often occurs in active systemic lupus erythematosus (SLE), which leads to low concentrations of classical pathway complement proteins, particularly complement component 3 (C3) and C4. Complement testing, including total complement testing and testing for C3 and C4, may therefore be useful to support a diagnosis of SLE. Complement tests are also recommended every 3-6 months to monitor disease activity in patients with SLE. Decreased C3 and C4 concentrations and the presence of anti-C1q autoantibodies have been associated with lupus nephritis in patients with SLE. For more information, refer to the ARUP Consult Systemic Lupus Erythematosus topic.

What is the role of complement testing in hereditary angioedema?

The complement component 1 (C1) esterase inhibitor protein is a regulatory protein that is deficient in hereditary angioedema and may lead to complement defects. Laboratory testing for hereditary angioedema includes testing for C1 esterase antigen, C1 esterase function, and C4 concentrations.

What is the role of complement testing in diagnosing hemolytic uremic syndrome?

Thrombotic microangiopathies (TMAs) have been associated with numerous underlying etiologies, including autoimmunity and complement dysregulation. Atypical hemolytic uremic syndrome (aHUS) is a rare form of TMA that occurs primarily due to complement dysregulation and/or overactivation.^, Acquired autoantibodies to the complement regulatory protein factor H, loss of function variants in genes encoding complement regulators (eg, factor H, factor I, CD46), or gain of function variants in complement component 3 (C3) or factor B can all predispose susceptible individuals to aHUS.^, Although nonspecific, testing to measure total complement activity (CH50) and alternative pathway complement activity (AH50), and markers of terminal complement pathway activation (soluble complement component 5b-9 complex [sC5b-9]), can aid in the diagnosis of aHUS. Decreased levels of C3, C4, and C5 can also indicate complement consumption as a result of overactivation. Testing to assess complement activation is also suggested before initiating complement inhibitor therapy. However, treatment should not be delayed due to pending test results.

For additional information on the diagnostic workup for possible HUS, refer to the Thrombotic Microangiopathies - Thrombotic Thrombocytopenic Purpura and Hemolytic Uremic Syndrome topic.

What is the soluble complement component 5b-9 complex (sC5b-9), and when should sC5b-9 testing be performed?

The soluble complement component 5b-9 complex (sC5b-9) is a marker of terminal complement pathway activation. Also referred to as the soluble membrane attack complex (sMAC), the sC5b-9 complex is comprised of complement component 5b (C5b), C6, C7, C8, and C9. Testing for sC5b-9 is potentially useful in the evaluation of many conditions, including autoimmune conditions, complement deficiencies, infections, traumas, and others. Testing for sC5b-9 may also be useful in monitoring response to anti-C5 therapeutics. In transplant-associated thrombotic microangiopathy, sC5b-9 testing may be used in diagnosis and for risk stratification.

Laboratory Testing

Primary Testing for Complement Deficiency

Initial evaluation for suspected complement deficiency is used to identify the affected pathway and should include testing for CP and terminal pathway (TP) activity (using the total complement activity [CH50] assay for total hemolytic complement) and AP activity (using the alternative complement activity [AH50] assay for alternative pathway hemolytic activity).

The initial evaluation may also include testing for LP function (using a mannose-binding lectin [MBL] test), depending on the clinical circumstance.

Secondary Testing for Complement Deficiency

The results from the initial evaluation guide secondary testing decisions to identify the deficient component or factor. Complement deficiency may present similarly to complement consumption (eg, due to autoimmune disease); complement consumption can be distinguished by the simultaneous reduction in multiple complement component concentrations. Secondary testing may include direct measurement of protein concentrations or assessment of protein function. Genetic testing may be useful if a hereditary deficiency of a particular component or regulatory factor is suspected.

Primary and Secondary Testing for Complement Deficiency^a
Primary Testing			Defect Implicated	Secondary Testing
CH50	AH50	MBL	Defect Implicated	Secondary Testing
Low or absent	Normal	Normal	CP defect or complement consumption^b	Complement concentrations or functional testing: C1, C2, C4
Normal	Low or absent	Normal	AP or regulatory factor deficiency	Properdin concentration or functional testing Factor levels: B, D
Low or absent	Low or absent	Normal	TP defect, regulatory factor deficiency, or complement consumption^b	Complement component concentrations or functional testing: C3, C5, C6, C7, C8, C9 Factor levels: H, I
Normal	Normal	Low	LP defect	LP components
^aRefer to the Complement Deficiency Testing Algorithm for a visual representation of the suggested testing sequence. ^bThe simultaneous reduction in concentrations of multiple components (eg, C3 and C4) suggests a disorder of complement consumption. Source: Bonilla, 2015

Testing to Monitor Anti-C5 Therapeutic Response

The activation of C5 results in a proinflammatory response and cell lysis, which plays a role in the pathophysiology of several complement-associated diseases. Complement-targeted therapy, specifically selective inhibition of C5 activation and the subsequent activation of C5a and C5b, is an ongoing area of research and drug development. Few C5 inhibitors (eg, eculizumab and ravulizumab) have been approved by the FDA and are prescribed for various conditions (eg, atypical hemolytic uremic syndrome, myasthenia gravis, neuromyelitis optica spectrum disorder, and paroxysmal nocturnal hemoglobinuria); other C5 inhibitors are being evaluated in clinical trials.

Personalized monitoring is strongly recommended for individuals receiving anti-C5 therapeutics because the clearance of these drugs is variable based on disease activity and renal state. Treatment with C5 inhibitors is associated with side effects such as increased susceptibility to Neisseria infection and may be costly. Therefore, complement testing, in conjunction with clinical evaluation, may be useful to monitor and optimize treatment efficacy.^, In patients with partial clinical response, assessing complement blockage may be helpful to escalate therapy. Similarly, a select group of patients with less severe pathology may benefit from testing to assess the appropriateness of dose de-escalation or drug discontinuation.

C5 inhibitors will impact the complement functional assays that depend on the activation of C5 and the formation of the membrane attack complex. CH50, AH50, and C5 functional assays all rely on this activation and may be useful,^, although C5 functional assays provide the most specific measurement of C5 blockage by C5 inhibitors.