The complement system: analysis of components and their significance

The complement system is part of the innate humoral immune system that helps recognize and eliminate microorganisms, enhances the inflammatory response, and participates in opsonization and the formation of membrane attack complexes. Clinically, it is important not only for protection against infections but also for the control of immune complexes, autoimmune reactions, and a number of thrombotic and nephrological processes. [1]

Traditionally, the cascade is described as three activation pathways: classical, lectin, and alternative. All three pathways converge on the activation of C3, after which a common terminal stage is initiated involving C5, C6, C7, C8, and C9. It is this terminal stage that leads to the formation of the membrane-attack complex, which is capable of damaging the membranes of target cells. [2]

The classical pathway is most closely associated with immune complexes and components C1, C2, and C4. The lectin pathway is initiated by the recognition of microbial carbohydrate structures by mannose-binding lectin and associated proteases. The alternative pathway relies heavily on C3, factor B, factor D, and properdin and is particularly important in a number of complement-mediated kidney diseases. [3]

The complement system isn't designed to function without limitations, so it has regulators. These include, for example, factor H, factor I, C1 inhibitor, and cellular regulatory proteins. When a defect occurs not in the "combat" proteins of the cascade, but in the regulators, the result may not be immunodeficiency in the traditional sense, but rather excessive complement activation with inflammation, thrombosis, hemolysis, and kidney damage. [4]

This is why an article on the complement system must consider not only deficiencies but also consumption and dysregulation. In practice, a physician is more often confronted not with a "theoretical absence of a single protein," but with a specific laboratory pattern that must be linked to infections, angioedema, systemic lupus erythematosus, glomerulonephritis, atypical hemolytic uremic syndrome, or treatment with C5 inhibitors. [5]

Table 1. The main pathways of the complement system and their clinical significance

Path	Essential proteins	What is important for the clinic
Classical	C1, C2, C4	More commonly associated with immune complexes, systemic lupus erythematosus and early component deficiencies
Lectin	mannose-binding lectin, MASP-2, C2, C4	May be important in recurrent infections in children and in some secondary immunodeficiencies
Alternative	C3, factor B, factor D, properdin	Particularly important in alternative dysregulation, C3 glomerulopathy and atypical hemolytic uremic syndrome
Terminal	C5, C6, C7, C8, C9	Terminal component deficiencies dramatically increase the risk of Neisseria infection

Sources for the table. [6]

What complement system tests are actually used today?

In everyday practice, C3 and C4 are most commonly measured. These are the most common individual complement proteins used to assess immune activity, monitor autoimmune diseases, and as part of the search for complement disorders. However, even at this stage, it is important to understand that C3 and C4 alone are not sufficient if a congenital deficiency or atypical pathway dysregulation is suspected. [7]

The next key level is functional testing. Total hemolytic activity of complement CH50 evaluates the classical pathway and the final common stage. Alternative hemolytic activity AH50 evaluates the alternative pathway and the same terminal stage. These tests are considered first-line tests when complement deficiency is suspected. [8]

If CH50 and AH50 are abnormal, further testing is initiated: individual components, their concentrations or function are measured, and sometimes mannose-binding lectin for the lectin pathway, C1q, C1 inhibitor, factor B, factor D, properdin, factors H and I are added. This stepwise approach helps to understand where exactly the defect is located: in the early classical pathway, the alternative pathway, the terminal complex, or in regulation. [9]

In specific clinical situations, additional biomarkers are used. Soluble C5b-9, also known as soluble membrane attack complex, serves as a marker of terminal complement activation. It may be useful in certain autoimmune conditions, infections, trauma, and in monitoring the response to anti-C5 therapy, but remains a more specialized test. [10]

Monitoring targeted therapy deserves special attention. In patients receiving C5 inhibitors such as eculizumab and ravulizumab, CH50, AH50, and C5 functional activity are used to assess complement blockade. Here, the analysis focuses on a different approach: not looking for a deficiency, but rather understanding how completely the terminal pathway is blocked and whether treatment is effective. [11]

Table 2. Basic laboratory tests of the complement system

Test	What does it show?	When it is especially useful
C3	Concentration of component C3	In autoimmune and nephrological diseases, if alternative dysregulation is suspected
C4	Concentration of component C4	In systemic lupus erythematosus, C1 inhibitor deficiency, classical pathway
CH50	Function of the classical pathway and the common terminal stage	First step when complement deficiency is suspected
AH50	Function of the alternative pathway and the common terminal stage	First step when suspecting an alternative defect
Mannose-binding lectin	Lectin pathway marker	With normal CH50 and AH50, if suspicion persists
C1q	Component C1	Particularly useful in differentiating between hereditary and acquired angioedema.
C1 inhibitor, quantity and function	Regulator of the classical and lectin pathways	For recurrent angioedema without urticaria
Soluble C5b-9	Terminal activation marker	In complement-mediated conditions and monitoring of anti-C5 therapy

Sources for the table. [12]

Table 3. Which tests are usually sufficient at the first stage?

Clinical situation	What do people usually order first?
Suspected systemic lupus erythematosus or immune complex disease	C3 and C4
Suspected congenital complement deficiency	CH50 and AH50
Recurrent angioedema without urticaria	C4, C1 inhibitor, C1 inhibitor function, C1q
Recurrent Neisseria infections	CH50 and AH50
Suspected alternative complement nephropathy	C3, C4, CH50, AH50, factor B and regulators as indicated
Monitoring of C5 inhibitors	CH50, AH50, functional C5

Sources for the table. [13]

How to correctly interpret the combinations C3, C4, CH50 and AH50

Isolated low C3 and isolated low C4 are not the same thing. Low C3 with normal C4 is now considered an important clue toward alternative dysregulation. This pattern is particularly concerning in C3 glomerulopathy and atypical hemolytic uremic syndrome, although a definitive diagnosis requires additional laboratory and often genetic data. [14]

When several components, especially C3 and C4, are simultaneously reduced, the physician more often considers complement consumption due to an active immune complex process rather than isolated hereditary deficiency. ARUP explicitly emphasizes that consumption is distinguished by the simultaneous reduction of several components, and C3 and C4 are widely used in the diagnosis and monitoring of autoimmune diseases, including systemic lupus erythematosus. [15]

A low C4 with a normal C3 is particularly important in the context of C1 inhibitor deficiency or dysfunction. In hereditary angioedema types 1 and 2, C4 is typically low even between attacks, and further confirmation comes from the level and function of C1 inhibitor. If C1q is also low, this suggests an acquired rather than a hereditary variant. [16]

Functional tests yield even more useful patterns. A normal AH50 with a low CH50 suggests an early classical pathway, such as C1, C2, or C4. A normal CH50 with a low AH50 is more consistent with an alternative pathway defect. A simultaneous decrease in CH50 and AH50 indicates either a terminal defect or severe complement consumption, and the combination of low CH50 and AH50 with normal C3 and C4 is particularly suggestive of a C5-C9 deficiency. [17]

If CH50 and AH50 are normal and the clinical suspicion of complement deficiency is still high, the next step is usually not endlessly repeating the same tests, but moving on to assessing the lectin pathway or another immune system. A modern review of complement disorders explicitly states that with normal CH50 and AH50, further testing is often not required at all unless there is a very specific suspicion. [18]

Table 4. How to interpret combinations of CH50 and AH50

CH50	AH50	What is most often assumed
Short	Normal	Early classical pathway defect, such as C1, C2, C4
Normal	Short	Alternative pathway defect, e.g. factor B, factor D, properdin
Short	Short	Terminal defect, C3 deficiency or severe complement consumption
Short	Low with normal C3 and C4	Particularly suspicious for deficiency of C5, C6, C7, C8 or C9
Normal	Normal	A significant defect in the classical and alternative pathways is less likely; if suspicion persists, the lectin pathway is considered.

Sources for the table. [19]

Table 5. How to interpret combinations C3 and C4

C3	C4	What is most often assumed
Short	Normal	Alternative dysregulation, such as C3 glomerulopathy or atypical hemolytic uremic syndrome
Short	Short	Complement consumption in systemic immune complex processes
Normal	Short	Deficiency or dysfunction of C1 inhibitor, part of the classic complement disorders
Normal	Normal	Does not rule out path deficiency if functional tests are abnormal
Normal or high	Normal or high	It can also occur during an active process if the analysis is taken outside the consumption phase or against the background of acute phase proteins.

Sources for the table. [20]

In what diseases is the complement system most often examined?

One of the most important clinical scenarios is recurrent infections, especially meningococcal infections. Deficiencies of the terminal components C5-C9 dramatically increase the risk of Neisseria meningitidis infection and disseminated Neisseria gonorrhoeae. Properdin, which belongs to the alternative pathway, is also associated with severe meningococcal infections. Therefore, an adult or child with repeated unexplained Neisseria infections is a classic candidate for complement system evaluation. [21]

Early components of the classical pathway have a different clinical profile. Deficiencies of C1, C2, and C4 are more often associated not only with infections but also with autoimmune diseases, particularly systemic lupus erythematosus and other immune complex syndromes. Therefore, low C3 and C4 levels in a patient suspected of having lupus are important for both diagnosis and monitoring disease activity. [22]

A separate, important area is nephrology. In C3 glomerulopathy and atypical hemolytic uremic syndrome, dysregulation of the alternative pathway is particularly significant. These conditions typically feature decreased C3 with normal C4, and further testing may include factor B, factor H, factor I, anti-factor H antibodies, and genetic testing. [23]

Another distinct scenario is angioedema without urticaria. Here, the complement system is needed not to detect an infectious immunodeficiency, but to identify a deficiency or dysfunction of the C1 inhibitor. Hereditary angioedema is typically characterized by low C4 and reduced C1 inhibitor function, while low C1q helps distinguish the acquired variant from the hereditary one. Importantly, antihistamines and glucocorticosteroids are usually ineffective in this form, because the mechanism is related to bradykinin, not histamine. [24]

Finally, the complement system is also being studied in modern targeted therapy. In patients receiving C5 inhibitors, complement testing helps monitor the completeness of the cascade blockade. This is no longer a diagnostic for a classic deficiency, but an example of how the complement system has moved from fundamental immunology to routine monitoring of modern biological drugs. [25]

Table 6. Which clinical scenarios most strongly suggest complement system disorders?

Clinical situation	Most likely complement block
Recurrent meningococcal meningitis	Terminal components C5-C9 or properdin
Early recurrent capsulated bacterial infections in a child	C3 or early components of the classical pathway
Systemic lupus erythematosus and other immune complex diseases	C3 and C4 consumption or congenital early deficiencies
Angioedema without urticaria	C1, C4, C1q inhibitor
Unexplained glomerulonephritis with low C3 and normal C4	Alternative complement dysregulation
Monitoring anti-C5 therapy	CH50, AH50, functional C5

Sources for the table. [26]

How to take tests correctly and what to do after abnormal results

Pre-analysis is especially important for the complement system. Functional tests are sensitive to temperature, processing delays, and improper storage. Mayo and ARUP emphasize that degradation and spontaneous consumption of components can lead to falsely low function, and for AH50, they generally recommend freezing samples immediately after collection. [27]

Therefore, the first step after an unexpectedly low result is not to rush to a diagnosis. It is necessary to clarify how the sample was processed, whether there was a delay, whether it was stored correctly, whether plasmapheresis was performed, and whether there is a significant inflammatory process. If the clinical picture is questionable or does not match the clinical picture, functional tests should be repeated on a new sample. [28]

If a persistent abnormality is detected, the next step depends on the pattern. If CH50 is low and AH50 is normal, the classical pathway and components C1, C2, and C4 are examined. If CH50 is normal and AH50 is low, factors of the alternative pathway are examined. If CH50 and AH50 are low, the terminal components and C3 are assessed together, and complement consumption is also considered. This algorithm allows for a logical approach rather than "checking everything." [29]

If a hereditary deficiency is suspected, consultation with a clinical immunologist and genetic testing are helpful. Recent reviews emphasize that after laboratory confirmation of the defect, consideration should be given not only to the patient but also to relatives, as family identification allows for early discussion of vaccination, prophylaxis, and management of severe infections. [30]

Finally, abnormal complement system results cannot be treated "by analysis." Treatment is not based on the low C3 level itself, but on the underlying problem: lupus, angioedema, infectious predisposition, nephrological complement dysregulation, or the consequences of anti-C5 therapy. This is what distinguishes the modern interpretation of the complement system from the outdated model, which attempted to draw conclusions based on C3 or C4 alone. [31]

Table 7. What most often interferes with the correct interpretation of complement tests

Problem	Why is this important?
Incorrect storage of the sample	May give falsely low functional results
Delay in blood processing	Promotes degradation and consumption of components
Blood collection after plasmapheresis	May not reflect the actual complement status.
Rating only C3 and C4 without CH50 and AH50	It's easy to miss a pathway-specific defect
Functional test assessment without clinical consideration	False diagnostic conclusions are possible
Mistaking complement consumption for a congenital deficiency	Requires distinction across multiple components and clinical context

Sources for the table. [32]

FAQ

What is the complement system in simple terms?
It is a cascade of innate immune proteins that helps tag microbes, enhance inflammation, and destroy target cells. Clinically, the complement system is important not only for protection against infections, but also for autoimmune, nephrological, and angioedematous conditions. [33]

What tests are most often prescribed first?
Most often, they start with C3 and C4, and if a congenital deficiency is suspected, with CH50 and AH50. In case of angioedema without urticaria, C4, C1 inhibitor, its function, and C1q are added. [34]

How does CH50 differ from AH50?
CH50 reflects the function of the classical pathway and the common endpoint. AH50 reflects the function of the alternative pathway and the same endpoint. Their combination allows us to understand the likely location of the defect. [35]

Does low C3 always indicate kidney disease
? No. Low C3 can occur with alternative complement dysregulation, but it also occurs with complement consumption in autoimmune processes and other conditions. The combination with C4, CH50, AH50, and the clinical picture is significant. [36]

Does low C4 always indicate hereditary angioedema
? No. Low C4 is indeed important in C1 inhibitor deficiency, but it alone is not enough. C1 inhibitor levels and function should be measured, and if an acquired variant is suspected, C1q should also be measured. However, in some patients, C4 may be normal between attacks. [37]

When terminal component deficiencies are especially important:
When a patient has had recurrent meningococcal infections or disseminated Neisseria gonorrhoeae infections, especially without another obvious cause of immunodeficiency. Such cases are characterized by defects in C5-C9, as well as properdin. [38]

Can a complement disorder be ruled out if C3 and C4 are normal
? No. Normal C3 and C4 do not rule out a pathway-specific defect. For example, with a deficiency of the early classical or terminal pathway, an abnormal result may appear specifically in CH50 or AH50. If both CH50 and AH50 are normal, further testing is usually not required, except in special cases where the lectin pathway is suspected. [39]

Why is C1q measured in angioedema?
C1q helps differentiate inherited from acquired C1 inhibitor deficiency. In the inherited variant, C1q is usually normal, while in the acquired variant, it is often decreased. [40]

Why do complement tests sometimes have to be repeated?
Because functional tests are very sensitive to sample collection, transportation, and freezing. If the sample is processed incorrectly, a false decrease can be obtained and a severe complement defect can be mistakenly suspected. [41]

Where the complement system is particularly important in modern medicine
Today, it is particularly important in four major areas: immunodeficiencies with recurrent infections, systemic autoimmune diseases, complement-mediated nephropathy, and monitoring of targeted anti-C5 therapy. It is in these scenarios that complement assays truly change clinical decisions. [42]