8.4 Complement

Overview: A Cascade That Converges on Lysis and Opsonization

The complement system is a cascade of roughly 30 plasma proteins that amplify innate and adaptive immune responses through sequential proteolytic activation. Three activation pathways -- classical, alternative, and lectin -- converge on the cleavage of C3 and proceed through a shared terminal pathway that assembles the membrane attack complex (MAC). The SBB exam focuses on the classical and alternative pathways and their downstream biologic effects, especially as they explain the mechanism of intravascular hemolysis.

The Classical Pathway

The classical pathway is triggered by antigen-antibody complexes -- specifically, antibody bound to antigen in a configuration that exposes a complement-binding site on the Fc region. IgM is the most efficient classical-pathway activator: because a single pentameric IgM molecule already presents multiple Fc regions in close proximity, one bound IgM molecule can activate complement. IgG activates complement far less efficiently and typically requires two IgG molecules bound in close proximity on the same cell surface -- and even then, only certain subclasses do so well: IgG1 and IgG3 fix complement efficiently, IgG2 weakly, and IgG4 essentially not at all.

Activation proceeds as follows: the C1 complex (C1q, which physically binds the antibody Fc region, plus the serine proteases C1r and C1s) is activated; activated C1s cleaves C4 and C2, and the fragments combine to form C4b2a, the classical-pathway C3 convertase. C4b2a cleaves C3 into C3a and C3b; C3b joins the complex to form C4b2a3b, the classical-pathway C5 convertase, which begins the terminal pathway shared by all three initiation routes.

The Alternative Pathway

The alternative pathway is antibody-independent and provides constant innate surveillance. It begins with low-level, spontaneous hydrolysis of C3 in plasma (often called "tickover"), generating a small amount of C3b that can bind covalently to nearby surfaces, including microbial cell walls. Factor B binds this surface-bound C3b and is cleaved by Factor D into Ba and Bb, forming C3bBb, the alternative-pathway C3 convertase. This convertase is inherently unstable but is stabilized by properdin, extending its half-life and allowing an amplification loop that generates large amounts of additional C3b. Addition of further C3b forms C3bBb3b, the alternative-pathway C5 convertase.

The Terminal Pathway and Intravascular Hemolysis

Both C5 convertases cleave C5 into C5a and C5b. C5b initiates assembly of the terminal complex: sequential binding of C6, C7, C8, and multiple copies of C9 builds the membrane attack complex (C5b-9, MAC), a ring-shaped structure that inserts into the lipid bilayer and creates a transmembrane pore. On a red cell, this pore allows uncontrolled influx of water and ions, causing osmotic lysis directly within the circulation -- the mechanistic definition of intravascular hemolysis. Because IgM is such an efficient complement activator, ABO-incompatible transfusion reactions (mediated by naturally occurring anti-A and anti-B) are the classic example of rapid, severe intravascular hemolysis; IgG1 and IgG3 antibodies that fix complement efficiently can also cause intravascular hemolysis, though typically less explosively than IgM-mediated activation. Laboratory findings of intravascular hemolysis include hemoglobinemia, hemoglobinuria, decreased haptoglobin, and elevated LDH, developing rapidly over minutes to hours.

This contrasts with extravascular hemolysis, in which red cells opsonized by IgG and/or C3b (without complete MAC assembly) are removed by macrophages of the mononuclear phagocyte system in the spleen and liver, a slower process (hours to days) that presents with rising indirect bilirubin and mild anemia rather than hemoglobinuria.

Biologic Properties: Anaphylatoxins, Opsonins, and Regulation

The cleavage fragments generated along the cascade have potent biologic activity independent of lysis. C3a and C5a act as anaphylatoxins, triggering mast cell and basophil degranulation, smooth muscle contraction, and increased vascular permeability; C5a is additionally a powerful chemotactic factor that recruits neutrophils to the site of activation. C3b functions as a major opsonin, coating targets so they are recognized and phagocytosed by macrophages bearing complement receptor 1 (CR1) -- the molecular basis of extravascular hemolysis and of immune-complex clearance by CR1-bearing red cells delivering complexes to the liver.

Complement activation is tightly regulated to protect host cells from bystander damage. Factor H and Factor I inactivate C3b in fluid phase and on host surfaces; decay-accelerating factor (DAF/CD55) dissociates C3/C5 convertases on the cell membrane; and membrane inhibitor of reactive lysis (CD59) blocks MAC pore assembly directly on the cell surface. Red cells that lose these GPI-anchored regulatory proteins through an acquired somatic mutation develop paroxysmal nocturnal hemoglobinuria (PNH), a disease defined by chronic complement-mediated intravascular hemolysis -- a direct, testable link between complement regulation and a named clinical disease.

Complement in the Antiglobulin Test and Autoimmune Hemolysis

Complement components deposited on red cells are a routine bench finding, not only a textbook abstraction. Polyspecific antihuman globulin (AHG) reagent contains anti-IgG and anti-C3d, so a positive polyspecific DAT must be followed by monospecific reagents to determine whether the cell is coated by IgG, C3d, or both -- a distinction with direct diagnostic weight. Warm autoimmune hemolytic anemia typically shows a DAT positive for IgG alone or IgG plus C3d, reflecting an IgG autoantibody that both opsonizes the cell for extravascular clearance and, in many cases, fixes complement. Cold agglutinin disease, by contrast, typically shows a DAT positive for C3d only: the causative IgM autoantibody itself elutes off the cell at body temperature during transport to the warm reticuloendothelial system, but the C3b/C3d it fixed remains covalently bound, so only complement -- not the triggering IgM -- is detected on the finished DAT. This pattern is a frequently tested example of how complement deposition can outlast the antibody that generated it, and why an SBB-level workup interprets DAT specificity patterns mechanistically rather than as a simple positive/negative call.

Complement measurement also has a quality dimension: hereditary deficiencies of early classical-pathway components (C1q, C4, C2) are associated with impaired immune-complex clearance and autoimmune disease, and C4 null alleles are common enough in the general population that an unexpectedly weak complement-mediated reaction should prompt consideration of a host complement deficiency rather than an assay failure alone.