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The complement cascade comprises over 20 serum proteins that form part of the innate immune system. The complement sequence consists of classical and alternate (Properdin) pathways which may be activated sequentially by a number of different causes. In general, the classic pathway is activated by antigen-antibody and cell‑antibody complexes and the alternate pathway by bacteria, fungi and some immune complexes. The sequence of activation in the classical pathway is C1, C4, C2, C3 and C5 to C9. In the alternate pathway C1, C4 and C2 are bypassed and C3 is activated by an initiating factor (IF), and two substances called Properdin Factors D and B. Complement acts in a number of ways to help clear invading organisms, with a major function being the lysis of bacteria through formation of the membrane attack complex (MAC).

The most frequently occurring alterations of complement are increased levels, since most complement proteins are acute phase reactants. The main clinical application of complement assays is the detection of decreased levels, which may indicate an on‑going immunological disorder. The complex interactions of the complement cascade mean that functionality of the MAC cannot necessarily be inferred by apparently normal levels of any single complement component. Total hemolytic complement (CH50) is the best functional assay of the complete complement sequence.

The traditional method for measuring total complement activity in serum is the CH50 test, which is a measure of total complement activity. The original CH50 method was based on complement mediated hemolysis of antibody sensitized erythrocytes. CH50 is the dilution of serum (quantity of complement) needed to lyse 50% of sheep RBCs sensitized with rabbit antibody.

Newer automated methods rely on liposomes, encapsulating glucose-6-phosphate dehydrogenase (G6PDH), to mimic an invading microorganism. On addition of sample, antibodies in the reagent combine with dinitrophenyl groups on the surface of the liposomes. The resultant complex activates complement in the sample, which lyses the liposomes, releasing G6PDH to react with glucose-6-phosphate and NAD in the reagent. The change in absorbance can be measured and is proportional to the complement activity in the sample. Comparison to a calibration curve gives a value for the unknown patient sample.

CH50 is often decreased in SLE, glomerulonephritis and other immune complex diseases. A normal CH50 level indicates that all the components, C1 through C9, are present. However, individual complement factors may be depleted 50 to 80% without affecting CH50 activity. Depletion of alternative factors is not detected. For this reason, it may be necessary to measure individual complement components.

Body fluid CH50 activity should normally be approximately one third to one half of the serum value. Decreased CH50 titers and complement protein levels may be seen in the joint fluid of patients with rheumatoid arthritis, gout, pseudogout, Reiter's syndrome and gonococcal arthritis. Serum levels in these patients may be normal or increased.

Sample integrity is the single most important consideration to insure accurate CH50 results. Preanalytical issues include:

  • In vitro Complement activation
  • Degradation of complement factors at room temperature.
  • Adherence of complement proteins to membranesInteraction with immunoglobulins
  • Treatment with eculizumab, a monoclonal antibody against C5

Adult reference range is 42 to 95 U/mL. A low CH50 suggests the possibility of a complement factor deficiency.

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