complement fixation

**Principle (two-stage assay):** 1. Test stage: Patient serum (heat-inactivated to destroy native complement) is mixed with a known antigen plus a measured amount of exogenous complement (usually guinea pig). If the serum contains specific antibody, antigen-antibody complexes form and activate the classical pathway (C1q binds IgG or IgM Fc), consuming (fixing) the complement. 2. Indicator stage: Sheep RBCs coated with anti-sheep RBC antibody (hemolysin/amboceptor) are added. - Complement consumed (antibody present): no lysis, RBCs settle as a button. This is a POSITIVE test. - Complement still free (no antibody): indicator RBCs lyse, and the solution is red/clear. This is a NEGATIVE test. - Memory aid: "No hemolysis = positive."

**Complement background (high-yield):** - Classical pathway: triggered by IgG (IgG1, IgG3) or IgM complexed with antigen. C1q, then C1r/C1s, C4, C2, C3 convertase (C4b2a), then C3b, then C5 convertase (C4b2a3b). - Alternative pathway: microbial surfaces (spontaneous C3 hydrolysis). Lectin pathway: mannose-binding lectin binds microbial mannose. - All pathways converge on C3, then C5b-C9 membrane attack complex (MAC), which lyses cells. - Key functions: C3b = opsonization; C3a, C4a, C5a = anaphylatoxins (C5a also a neutrophil chemotaxin); C5b-9 = MAC. - IgM is the most efficient complement fixer; IgG is next. IgA, IgD, and IgE do not fix complement by the classical pathway (IgA can activate the alternative/lectin pathways).

**Clinical uses and associations:** - Serologic diagnosis of Coccidioides immitis (CF titers correlate with disseminated disease; a rising titer suggests dissemination), Histoplasma, Blastomyces, Rickettsia/Coxiella (Q fever), Chlamydia (e.g., LGV, psittacosis), Mycoplasma, and some viruses (e.g., RSV, influenza, arboviruses). - Wassermann test for syphilis (historical; now replaced by nontreponemal tests like VDRL/RPR and treponemal tests like FTA-ABS). - Largely replaced by ELISA, PCR, and immunofluorescence because CFT is labor-intensive and less sensitive.

**Related clinical concepts:** - Immune complex diseases (type III hypersensitivity, e.g., SLE, post-streptococcal glomerulonephritis, serum sickness) fix complement in vivo and lower serum C3/C4 and CH50 (total hemolytic complement). - Classical pathway consumption: low C4, low C3 (e.g., SLE, immune-complex GN). Alternative pathway activation: low C3 with normal C4 (e.g., C3 glomerulopathy, post-infectious GN). - Deficiencies: C1 esterase inhibitor (hereditary angioedema; low C4; no urticaria; ACE inhibitors contraindicated); C3 (recurrent pyogenic sinopulmonary infections); C5-C9 (recurrent Neisseria infections); early classical components C1, C2, C4 (SLE-like disease and encapsulated organism infections); DAF/CD55 and CD59 (PNH, complement-mediated hemolysis). - Anti-CD20/rituximab and some therapeutic antibodies kill via complement-dependent cytotoxicity. Eculizumab (anti-C5) is used in PNH and aHUS, with a risk of Neisseria infections (vaccinate beforehand).

**Boards pearl:** If a question describes a serology test where sheep RBCs are used as an indicator and hemolysis does NOT occur, think complement fixation = patient has antibodies to the antigen tested.