Complement System Assays

Definition

The complement system is a major component of innate and adaptive immunity; upon activation, the complement results in the formation of the membrane attack complex (MAC) that releases peptides called anaphylatoxins. About 90% of complement components are synthesized in the liver and are acute-phase proteins.
There are three pathways of activation: Classical (antibody-sensitized cells), alternative (early defense against microorganisms), and mannose-binding lectin (MBL) (recognizes microorganisms in the absence of antibodies that are replaced by a lectin, such as mannose-binding protein [MBP]). MBL is formed in the liver. It belongs to a family of molecules called collectins. Many components of the complement system interact through cascading mechanisms. The nine components of the classical pathway are designated by the upper case letter C, followed by a number indicating the order of appearance during the cascading sequence (the only exception is C4, which acts before C2). The components of the alternate pathway are called factors and are designated by upper-case letters (example: factor H, factor D). For the MBL pathway, the components are referred to by the abbreviations of the proteins names.
All three pathways of complement activation converge at the activation of C3 and assembly of the membrane attack complex, which is formed by components C5 " “C9.

Use

Complement should be investigated in patients with recurrent pyogenic infections (but with normal white cell counts and immunoglobulins), recurrent angioedema, cryoglobulinemic vasculitis, and with multiple family members with recurrent neisserial infections. Patients with deficiencies in the alternate pathway factors D, B, P, H, and I, or the late complement components C3, C5 " “C9, are particularly susceptible to infections with Neisseria meningitidis.
To determine the presence of deficiencies of individual components, and whether the deficiency is acquired or inherited.
The total hemolytic complement assay (CH50 or THC) is a global test; it is decreased if any of the components of the classical pathway are deficient. The AH50 assay screens for abnormalities in the alternate pathway. The use of CH50 and AH50 assays allows identification of abnormalities in both pathways. When the CH50 is very low, the next step is to measure specific complement components.

Normal Ranges

Complement levels are determined by immunologic and functional assays.
The most commonly used assays are C3, C4, CH50, and AH50 (see Complement Components in Plasma and Their Deficiencies table).

Interpretation

Decreased in acquired conditions (see Complement Components in Plasma and Their Deficiencies table). Decreases in complement in acquired conditions are usually only partial and affect several components of the system. They are most commonly due to complement consumption. ‚
Associated with arthritis ‚

Active systemic lupus erythematosus (SLE), especially if associated with renal disease. About 50% of patients with SLE have reductions in C3 and C4. Low complement levels correlate with more severe disease, especially with renal involvement. Normalization reflects good therapeutic results.
Hepatitis B or C
Essential mixed cryoglobulinemia
Serum sickness

Associated with vasculitis ‚

Rheumatoid vasculitis
Essential mixed cryoglobulinemia
Sj ƒ ¶gren syndrome
Hypocomplementemic vasculitis

Associated with nephritis ‚

Acute poststreptococcal GN (transient decline in C3)
IgA nephropathy
Membranous nephropathy
Types I and II membranoproliferative glomerulonephritis
SLE nephritis;
Tubulointerstitial nephritis
Dense deposit disease: C3 and factor B levels are low, and C4 is normal
Goodpasture syndrome

Associated with miscellaneous conditions ‚

The antiphospholipid antibody syndrome
Bacterial endocarditis
Cryoglobulinemia (decreased C2 and C4)
Acquired angioedema due to C1 inhibitor deficiency in B-cell lymphoproliferative diseases
Alcoholic liver disease: low C3 and C4 due to decreased synthesis
Acquired deficiency of decay accelerating factor (DAF) and CD59 " ”complement regulatory proteins that normally inhibit complement activation " ”develops in PNH, resulting in accelerated red cell hemolysis
Evans syndrome

Synovial Fluid

Depressed CH50 is found in the synovial fluid of joints in
- Seronegative rheumatoid arthritis
- SLE
- Pseudogout and gout
- Reiter syndrome
- Gonococcal arthritis

Decreased in Inherited Conditions (see Complement Components in Plasma and Their Deficiencies table)

Inherited deficiencies are characterized by the absence of single complement component. Most inherited disorders of the classical pathway are transmitted as autosomal recessive traits and are symptomatic in the heterozygous. Exception is partial C4 deficiency that predisposes to SLE. ‚

Hereditary angioedema an autosomal dominant disease; patients have very low C4, but normal C3
C1q deficiency: more than 90% develop SLE
Deficiency of C1r or C1s as well as partial deficiency of C4 predispose to the development of SLE
Deficiency of C4a or C4b is associated with the development of other diseases (scleroderma, IgA nephropathy, childhood diabetes, etc.)
Familial Mediterranean fever (C5a inhibitor)
Urticarial vasculitis and recurrent infections (C3)
Severe combined immunodeficiency (C1q)
X-linked hypogammaglobulinemia (C1q)
Recurrent neisserial infections (C5, C6, C7, C8, C9, and the alternate pathway)
Congenital deficiencies of C2 or C4 may present with lupus-like or other autoimmune disorder involving:
- Arthritis
- Nephritis
- Rashes
- Pneumococcal infections
Heterozygous mutations in plasma factors H and I result in consumption of C3, predisposing patients to infections and glomerulonephritis.
A genetic deficiency of complement regulatory proteins has been identified in 40 " “80% of cases of atypical hemolytic uremic syndrome (aHUS), a rare syndrome of microangiopathic hemolysis, thrombocytopenia, and renal failure. A deficiency of factor H (the most frequent abnormality in aHUS), C3 mutations, and deficiencies of membrane cofactor protein, factor I, factor B, and thrombomodulin.
Properdin (alternate pathway) deficiency is transmitted as an X-linked condition, and these males are affected by Neisseria meningitidis, often of unusual types.
Lectin pathway deficiencies are rare: deficiencies of MBL, MBL-associated protease 2, and ficolin-3 are associated with pyogenic infections, especially with encapsulated bacteria.

Increased

In inflammatory conditions that increase acute-phase reactants. ‚

Complement may be normal or increased in:
- Juvenile rheumatoid arthritis
- Palindromic arthritis
- Pseudogout and gout
- Reiter syndrome
- Gonococcal arthritis

Pitfalls

Complement activity is unstable and heat-labile, and it is reduced after a few hours in samples kept at room temperature. Serum samples should not be kept at room temperature for more than 6 hours or refrigerated for more than 7 days prior to analysis. ‚

Specimens left to clot at refrigerated temperature lose activity because of complement activation.
Complement components deteriorate in samples that are repeatedly thawed and frozen.
Severe hemolysis, lipemia, or bilirubinemia may interfere with accurate measurement.

‚
Complement Components in Plasma and Their Deficiencies*View LargeComplement Components in Plasma and Their Deficiencies* Protein Concentration in Plasma, mg/mL Clinical Correlations Global testsCH50: 60 " “144 CEA units AH50 (screens for abnormalities of the alternate pathway): 59% or greater CEA units Classical Pathway 150, 50, 50 GN, SLE C4 300 " “600 Scleroderma, SLE, IgA nephropathy, membranous GN C2 20 SLE, juvenile RA, GN Alternative Pathway Factor B 200 Neisseria meningitidis infections Factor D 2 Recurrent pyogenic infections Properdin 25 Recurrent pyogenic infections, fulminant meningococcemia MBL Pathway MBL Per specialized labs Recurrent infections MASP-1 1.5 " “13 MASP-2 Unknown Common to All Pathways C3 1,200 " “1,300 Recurrent pyogenic infections, GN Membrane Attack Complex C5 80 Recurrent disseminated neisserial infections, SLE C6 45 Recurrent disseminated neisserial infections C7 90 Recurrent disseminated neisserial infections, Raynaud disease C8 55 Recurrent disseminated neisserial infections C9 60 None Fluid-Phase Control Proteins C1-Inhibitor 240 Hereditary angioedema, autoimmune diseases C4b 250 Angioedema, Behchets-like syndrome Factor I 35 Recurrent pyogenic infections Factor H 300 " “450 Recurrent pyogenic infections, GN, age-related macular degeneration Cell-Bound Proteins CR1 Anemia and SLE CR3 Leukocyte adhesion deficiency-1, recurrent pyogenic infections, leukocytosis DAF/CD59/HRF (acquired) PNH *Modified from Massey ‚ HD, McPherson ‚ RA. In: Henry's Clinical Diagnosis and Management by Laboratory Methods, Table 47-3, p.920, and Table 47-4, p.921 22nd ed. Saunders, 2011.

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