Alternative pathway diagnostics

Abstract

Uncontrolled alternative pathway activation is the primary driver of several diseases, and it contributes to the pathogenesis of many others. Consequently, diagnostic tests to monitor this arm of the complement system are increasingly important. Defects in alternative pathway regulation are strong risk factors for disease, and drugs that specifically block the alternative pathway are entering clinical use. A range of diagnostic tests have been developed to evaluate and monitor the alternative pathway, including assays to measure its function, expression of alternative pathway constituents, and activation fragments. Genetic studies have also revealed many disease-associated variants in alternative pathway genes that predict the risk of disease and prognosis. Newer imaging modalities offer the promise of non-invasively detecting and localizing pathologic complement activation. Together, these various tests help in the diagnosis of disease, provide important prognostic information, and can help guide therapy with complement inhibitory drugs.

Keywords: alternative pathway; clinical; diagnostic; laboratory.

Figure 1.. Protein biomarkers of alternative pathway activation.

C3b can initially be generated by through the classical pathway (CP), alternative pathway (AP), or lectin pathway (LP). C3b then combines with factor B (FB) to form the alternative pathway C3 convertase (C3bBb). Cleavage of C3 and factor B generates C3a and Ba, respectively. Consequently, alternative pathway activation is associated with a reduction of C3 and factor B levels, and a simultaneous increase in C3a and Ba levels. The alternative pathway convertase is stabilized by properdin (P), and it is negatively regulated by factor H (FH). The factor H related proteins (FHRs) can block factor H. Expression levels of these positive and negative regulators (P, FH, and FHRs) can affect the risk of some diseases and the prognosis.

Figure 2.. Sources of complement activation fragments in urine.

Complement activation fragments in urine can come from several sources. A) Fragments that are generated systemically can filter into the urine, particularly small fragments such as C3a, C3d, C4a, Ba, and C5a. The concentration of systemically generated proteins may also increase if reabsorption of the fragments decreases due to tubular damage. B) Complement fragments generated by activation within the glomerular capillaries can freely enter the urine. C) Intact C3 and factor B that enter the urinary space can activate within the tubular lumen. D) Fragments generated within the tubulointerstitium can also leak into the urine.

Figure 3.. Cell-based complement assays.

A) In hemolytic assays, patient serum is added to erythrocytes, and complement activity is assessed by the degree of lysis. In classical pathway assays, antibodies reactive to the cells are used to activate complement. In alternative pathway assays, erythrocytes are used to which factor H does not bind, so the cells lyse even without the addition of antibodies. B) A modified hemolytic assay can also be used to test the function of factor H. Erythrocytes to which factor H ordinarily binds are used. If patient serum lyses the cells, it may indicate defective binding of factor H to the cell surface. C) Endothelial cell assays test the ability of patient serum to deposit C5b-9 on the surface of activated cells. Formation of C5b-9 on the cell surface may indicate alternative pathway dysregulation. D) Flow cytometry assays are used to measure deposition of complement proteins on erythrocytes, platelets, or extracellular vesicles. Deposition of complement fragments on these cells and cell fragments may indicate endovascular complement activation.

Figure 4.. Contribution of alternative pathway (AP) gene variants to disease.

Common variants in complement genes contribute to several common, polygenic diseases. Lower frequency variants contribute to several rare diseases. In general, the rarer the variant, the greater penetrance of disease in affected individuals. aHUS, atypical hemolytic uremic syndrome; C3G, C3 glomerulopathy; AMD, age related macular degeneration; AD, Alzheimer’s disease; IgAN, IgA nephropathy.