Complement Deficiencies

Test for Complement Deficiencies
For the evaluation of particular pathways of the complement system, functional tests have been developed. The ‘total haemolytic complement’ or CH50 is based on a haemolytic assay in which series of patient serum dilutions is incubated with sheep erythrocytes covered with specific IgM, activating the classical complement pathway (Wen L et al). The reciprocal of the serum dilution which causes the lysis of 50% of the erythrocytes is calculated to quantify the amount of active complement. For example, if 50% of the sheep erythrocyte are lysed at a serum dilution of 1:200, this equal toa CH50 value of 200 units/ml. Since the formation of membrane attack complexes (MACs) is necessary for the lysis of the erythrocytes and requires all components of the classical and common terminal complement pathway (C1 through C9), a deficiency or defect in any of these components would result in a significantly lowered CH50. Several variants of this test are in use example one based on liposomes containing an enzymes as read-out to simplify the quantification of lysis using a standard analyser (Wen et al). Thus, reference values of CH50 may vary depending on the system that is used.
A similar test, the AH50 is used to assess the alternative complement pathway (properdin, factor B, D and C3) in conjunction with the common terminal pathway (C5 through C9) (Wen et al). Serum dilutions are incubated with rabbit erythrocytes in a buffer which inhibits the classical or lectin pathway. If the alternative pathway is intact, a properdin-stabilised C3 convertase (C3bBb) can form on the surface of the erythrocytes and allow the assembly of C5 convertase and MACs, resulting in lysis which can be quantified.
Using both CH50 and AH50 can help to map the complement deficiency to a specific pathway. If the classical pathway is affected, the CH50 value will be low and the AH50 value will be normal. The opposite is true for a defect in the alternative pathway and if both CH50 and AH50 are low, the abnormal component is likely to be part of the common terminal pathway. However, CH50 and AH50 can also be low due to errors in sample handling because several complement components are unstable (Lock RJ et al). In case of such a result, the quality of the specimens should be controlled and the test might need to be repeated if in doubt.
To test the lectin pathway, a mannan-based ELISA can be used. MBL from the patient’s serum binds the mannan-coated wells, leading to MASP-activation and cleavage of C4. The fragments C4b and C4d can then be quantified using enzymes-linked antibodies (Wen et al).
In order to eventually identify the deficiency, serum levels of individual complement factors can be measured by immunochemical assays based on immunoprecipitation and nephelometry (measurement of turbidity caused by antigen-antibody complexes). Such test are readily available for C3 and C4. However, quantification of other complement components might require the help pf specialised laboratories. One major drawback of this method is the dependence on the quality of antibodies and standards used. Moreover, functional defects i.e non- functional complement factors with normal serum levels are not detected. Genetic testing by sequencing exons coding for complement components avoids these problems.

Complement Deficiencies Complement Deficiencies 02/19/2016
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