10.4 Complement Pathways and Biologic Properties

10.4 Complement Pathways and Biologic Properties

Quick Answer: The classical pathway starts when C1q binds one IgM or two closely spaced IgG Fc regions (IgG1/IgG3). Alternative and lectin pathways do not need antibody. C3b is the opsonin; MAC (C5–C9) punches the hole of intravascular hemolysis. Polyspecific AHG = anti-IgG + anti-C3b/C3d; monospecific reagents split them. In-vivo DAT C3-only is the cold-AIHA (and some drug) pattern. Heat 56 °C for 30 minutes inactivates complement. EDTA chelates calcium, so plasma cannot fix complement in vitro — that is why serum versus plasma matters.

The June 9, 2026 BB outline lists complement at III.A.4. You are not drawing every factor for a complementologist. You are explaining intravascular ABO hemolysis, a C3-only DAT, and why yesterday’s EDTA sample will not show a Kidd hemolysin.

Three pathways, one C3, one MAC

All roads converge on C3, then on the membrane-attack complex.

Classical pathway (antibody-triggered):

1. C1q binds Fc. Geometry rule: one IgM is enough (pentameric Fc cluster). IgG needs two molecules close together — that is why IgG complement fixation prefers antigens with many sites, and why IgG1 and IgG3 (not IgG4) work.
2. C1r and C1s activate (C1qrs). Calcium is required to hold C1 together.
3. C4 and C2 are cleaved. C4b2a is the classical C3 convertase.
4. C3 → C3b (opsonin) + C3a (anaphylatoxin).
5. C5 convertase cleaves C5. C5b plus C6, C7, C8, and polymerized C9 form the MAC. Water and ions flood in. That is intravascular hemolysis.

Lectin pathway: mannose-binding lectin (MBL) plus MASP proteases tag microbial sugars, then join the classical path at C4. Antibody is not required. BB rarely asks MASP names; it asks that lectin is antibody-independent and still reaches C3.

Alternative pathway: spontaneous C3 “tickover,” Factor B, Factor D, properdin. Antibody is not required. Magnesium is required. The alternative path amplifies any C3b laid down by classical activation — once a Kidd IgG starts classical, alternative can pile on C3b.

Biologic properties the exam actually uses

• C3b (and iC3b): opsonins. Macrophage CR1/CR3 bind C3b-coated red cells → extravascular clearance, especially in the liver. A DAT that is C3-positive is a statement that this clearance path is available.
• C3a and C5a: anaphylatoxins. They explain bronchospasm, hypotension, and the shock picture of ABO incompatibility beyond simple anemia.
• MAC C5–C9: intravascular hemolysis — free hemoglobin, drop in haptoglobin, hemoglobinuria, possible DIC and renal injury. ABO is the everyday MAC disease. Some Kidd, Vel, P/PP1Pk, and H (Bombay) antibodies can do the same.
• C3d: the long-lived breakdown fragment that remains on the red cell after C3b is cleaved. Anti-C3 AHG reagents are often written anti-C3b/C3d because C3d is what you still find hours later.

IgG-only coating without much C3 is typically cleared in the spleen via Fc receptors (Rh antibodies classically). Heavy C3 coating adds hepatic clearance and, if MAC assembles, intravascular lysis.

AHG reagents: anti-IgG, anti-C3, polyspecific

A DAT workup that stops at “polyspecific positive” is incomplete. You must say whether the coating is IgG, C3, or both.

In-vitro complement binding (IAT in a system with intact complement) can make a complement-binding alloantibody show up with anti-C3 even when anti-IgG is weak. That is a test-system phenomenon. In-vivo, the pattern BB loves is DAT C3 only:

• Cold agglutinin disease — pathogenic IgM anti-I binds in cooler extremities, fixes C3, then dissociates at 37 °C. Washed DAT cells keep C3d, not the IgM. Anti-IgG is negative.
• Some drug reactions deposit C3 with little IgG.
• PCH (Donath-Landsteiner, biphasic IgG anti-P) can leave C3 on cells after the IgG has come off at warmer temperatures.

That is not the same as an in-vitro cold that never bound complement in the patient. Do not call every room-temperature anti-I a C3-only DAT.

Inactivation: heat and EDTA

Heat, 56 °C, 30 minutes destroys complement activity in serum. Use it when you need to remove complement (or a heat-labile interfering factor) but keep immunoglobulin. Do not invent other temperatures. Thirty-seven degrees for thirty minutes is incubation, not inactivation.

EDTA chelates calcium and magnesium. Without Ca2+, C1 cannot assemble. Without Mg2+, the alternative path stalls. Therefore:

• EDTA plasma is excellent for preventing in-vitro complement uptake and for DAT samples (stops complement from binding in the tube after the draw).
• EDTA plasma cannot demonstrate in-vitro complement-dependent hemolysis or C3 coating. A Kidd hemolysin or a complement-only IAT reaction may be invisible in plasma and visible in fresh serum.
• Serum from a clotted red-top contains complement if it is fresh (or properly frozen). Aged serum loses activity — complement is labile.

This is the whole “plasma versus serum” item. It is not a preference for prettier buttons. It is calcium-dependent C1.

Complement also explains why some laboratories still keep a serum tube when investigating unexplained hemolysis, and why a DAT specimen is drawn in EDTA: you want to stop complement at the moment of phlebotomy so the DAT reflects in-vivo coating only.

Exam traps

• Saying any single IgG molecule activates C1 as efficiently as IgM. Need two IgG; one IgM.
• Crediting IgG4 or IgA with strong classical activation.
• Calling MAC “C3b.” C3b is the opsonin; C5–C9 is the hole.
• Reporting a C3-only DAT in cold AIHA as “anti-IgG should have been positive because antibody is still on the cell.” The IgM has eluted.
• Using EDTA plasma to prove a complement-dependent hemolysin.
• Heating serum to 37 °C for 30 minutes and calling complement gone. The teaching number is 56 °C for 30 minutes.