Introduction
The complement is a vital part of the body's immune system that provides the destruction of invading microorganisms. Complement is activated by various pathways that lead to the membrane attack complex (MAC) formation for killing. Following complement activation, the involved proteins elicit many proinflammatory effects. Further, the inflammatory response is amplified by reactive oxygen species (ROS, highly reactive free radical molecules) and cytokines (inflammatory mediators). The complement system is also involved in numerous diseases and conditions, such as autoimmune (immunity against self) diseases, infections, cancer, organ transplantation, and inflammation.
What Is the Complement Test?
A complement blood test measures the complement protein amount and activity. The complement system comprises proteins (there are nine proteins, C1 to C9) that work with the immune system to identify and destroy pathogens such as viruses and bacteria. Complement proteins can be measured individually or in combination. C3 and C4 proteins are the most frequently tested complement proteins.
What Are the Clinical Indications for C3 and C4 Testing?
Clinical evidence indicates the prominent role of complement in immunodeficiency disorders, bacterial, viral, and fungal infections, systemic lupus erythematosus (SLE), and arthritis. C3 protein is the key molecule of the complement system and is found in the highest concentration. On the other hand, C4 plays a role in immunity, tolerance, and autoimmunity. The defects in the C3/ C4 proteins require C3/C4 testing in the following conditions.
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Bacterial Infections: C3 defects may be the cause of bacterial infections. Recurrent infection with Neisseria meningitidis (a bacterium that causes meningitis, that is, brain membrane inflammation) is associated with C3 deficiency.
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Membranoproliferative Glomerulonephritis: Membranoproliferative glomerulonephritis (MPGN) is a kidney disorder that involves inflammation and kidney cell alterations. Out of the two forms, MPGN I and MPGN II, many people suffer from type I. Also, MPGN II is less frequent and has a poorer prognosis than MPGN I.
In MPGN, there is C3 activation and consumption due to defective pathway regulation. Patients suffering from MPGN type II show low levels of C3. It results from a continuous C3 activation due to an autoantibody, termed C3 nephritic factor (C3NeF). The result is excessive C3 consumption leading to its functional deficiency. The C3 deficiency may also increase the risk of bacterial infections.
3. Autoimmune Disorders: Defects in the C4 protein are frequently associated with SLE. The strength of the association of C4 deficiency with SLE is 75 percent. In active SLE, low C4 concentration is verified by the detection of complement activation products. In patients with lupus nephritis (LN, which is kidney inflammation secondary to SLE), autoantibodies (antibodies against self) to C4 are found and can determine the prognosis (course of the disease).
Other indications for C4 testing include autoimmune hepatitis (liver inflammation), collagen vascular disease (an autoimmune disorder involving the body’s connective tissue), and scleroderma (an autoimmune disorder that leads to skin hardening).
What Are the Procedures and Interpretations for C3 and C4 Testing?
A clinician takes a blood sample using a small needle. After the needle insertion, a small amount of blood is collected into a test tube. The procedure takes less time. Various tests for C3 and C4 analysis are:
1. Quantitative Tests: Quantification of C3 and C4 can be performed by immunochemical assays. The measurements utilize the antigen-antibody immune complex formation (immune complex is formed by foreign antigens and antibodies in the body). Immune complexes affect various tissues and can cause autoimmune disorders. Following are the immunochemical assays for C3 and C4 testing.
- Nephelometry: Nephelometry is a chemistry technique based on light scatter from immune complexes. The intensity of light scatter is proportional to the number of complexes in the sample.
- Turbidimetry: Turbidimetry allows the quantification of antigens based on alteration in the transmitted light. An immune complex alters the transmission of light through the specimen that depends on the concentration of the C3 or C4.
- Radial Immunodiffusion: Radial Immunodiffusion (RID) is a versatile method allowing for the concentration and function of C3 and C4. RID uses an agarose gel (a medium) containing antibodies to C3 or C4. A precipitation ring is formed in case of a positive test. The diameter around the ring correlates to C3/C4 activity.
- Enzyme-Linked Immunosorbent Assay: Enzyme-Linked Immunosorbent Assay (ELISA) is a widely used method in antigen and antibody detection. In this technique, specific antibodies bind the target antigen (a harmful foreign particle) and detect the quantity of the binding antigens. Antigens are bound to a medium, and a sample containing antibodies for the disease binds to the antigens. Next, a second antibody with a marker is added, and a positive reaction is detected by a color change. ELISA can detect inherited C3 and C4 deficiency and the assessment of complement function. One must note that ELISA is the only available method for C3 and C4 activity measurement. However, the results of the ELISA vary between 58 and 79 percent.
2. Functional Tests: If a functional defect is suspected, the activity of C3 and C4 needs testing. A simple way to detect their activity is to test the sample's capability to reconstitute the complement function. It is done by adding fresh serum from the patient to check the activity. These functional techniques identify congenital deficiencies and monitor fluctuations in complement function (in SLE patients during exacerbations). These tests also provide information on a functional deficiency or lack of protein.
C3 and C4 measurements to monitor complement in an autoimmune disorder should be avoided. It is because the sensitivity and specificity of these measurements are low. If the results show increased activity of C3 and C4, a patient may have leukemia (blood cancer), non-Hodgkin lymphoma (NHL, lymphatic system cancer), and ulcerative colitis (inflammation of the large intestine). However, if a patient is undergoing treatment for SLE or another autoimmune disorder, increased activity of the complement proteins may mean the treatment is effective.
Conclusion
C3 and C4 analyses are useful in a limited number of conditions. Furthermore, the profile for each condition is different. Still, progress is ongoing in complement analysis to define disease pathology, severity, and response to therapy. Hence, with new tests and targeted complement therapy, several complement-mediated diseases will become more manageable.
