Complement System Assays

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Subject: Complement System Assays

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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

  • Sjögren 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*

Suggested Readings

Botto  M, Kirshfink  M, Macor  P Complement in human diseases: lessons from complement deficiencies. Mol Immunol.  2009;46:2774–2783.
Nester  CM, Thomas  CP. Atypical hemolytic uremic syndrome: what is it, how is it diagnosed, and how is it treated? Hematology Am Soc Hematol Educ Program.  2012;2012:617–625.
 
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