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Complement

16 min

Skills you'll build4 skills
  • Distinguish classical, lectin, and alternative complement initiation
  • Match a complement fragment to its principal immune effect
  • Interpret paired CH50 and AH50 results to locate a complement defect
  • Check that a CH50 or AH50 specimen was separated and frozen promptly before accepting it

At the bench: Check specimen identity and suitability before analysis

Try first

Try first

Bacteria enter the blood of a person who has never met them, so no specific antibody exists yet. Which complement pathways can start on the bacterial surface?

Keep going to see the reasoning.

Right. Keep going to follow the reasoning.

Keep going to see the reasoning.

Keep going to see the reasoning.

Get the idea

Use the explanation to check your first decision.

Three pathways, one terminal pathway

Each pathway builds a C3 convertase, adds C3b to make a C5 convertase, and can continue through the shared terminal pathway.1

  • Classical. C1q binds antigen-bound IgM or clustered IgG. C1s cleaves C4 and C2 to form the C3 convertase C4b2a.
  • Lectin. Mannose-binding lectin or ficolins bind surface sugars. Their proteases cleave C4 and C2 to form the same C4b2a.
  • Alternative. Spontaneous C3 hydrolysis starts it. Factor B and factor D form the convertase C3bBb, which properdin stabilizes. This loop also amplifies C3b made by the other two pathways.

What the fragments do

C3b and C4b coat targets for phagocytes. C3a and C5a promote inflammation, and C5a strongly attracts leukocytes. C5b binds C6 through C9 to build C5b-9, the membrane attack complex, which forms a pore in susceptible membranes.1,2

CH50 and AH50

CH50 tests the classical pathway and the shared terminal pathway. AH50 tests the alternative pathway and the same shared terminal pathway.3,4,5

CH50AH50Likely defect
LowWithin intervalA classical-only component: C1, C4 or C2
Within intervalLowAn alternative-only component: factor B, factor D or properdin
LowLowShared C3 or C5 through C9, consumption, C5-directed therapy or loss during handling

When both screens are within their intervals, a lectin-pathway defect is still possible. Component assays identify the defect after the screens localize it.3

Specimen handling

Functional complement is labile. The performing laboratory's instructions govern collection. One reference laboratory asks for the following:4,5

  1. Place the whole-blood tube on wet ice at once and let it clot there.
  2. Centrifuge at 4 °C.
  3. Aliquot the serum.
  4. Freeze the serum within 30 minutes of centrifugation.

Warm, delayed handling consumes complement activity and generates activation fragments, so the functional result is falsely low. Before releasing a low CH50 or AH50, check how the specimen was handled and whether the patient receives complement-directed therapy.3

Follow the bench demonstration

Sources for this step
References
  1. Merle NS, Church SE, Fremeaux-Bacchi V, Roumenina LT. Complement system part I: molecular mechanisms of activation and regulation. Front Immunol. 2015;6:262. doi:10.3389/fimmu.2015.00262
  2. Merle NS, Noe R, Halbwachs-Mecarelli L, Fremeaux-Bacchi V, Roumenina LT. Complement system part II: role in immunity. Front Immunol. 2015;6:257. doi:10.3389/fimmu.2015.00257
  3. Willrich MAV, Braun KMP, Moyer AM, Jeffrey DH, Frazer-Abel A. Complement testing in the clinical laboratory. Crit Rev Clin Lab Sci. 2021;58(7):447-478. doi:10.1080/10408363.2021.1907297
  4. Mayo Clinic Laboratories. Complement, total, serum. Test ID COM. Accessed September 27, 2026. https://www.mayocliniclabs.com/test-catalog/Overview/8167
  5. Mayo Clinic Laboratories. Alternative complement pathway, functional, serum. Test ID AH50. Accessed September 27, 2026. https://www.mayocliniclabs.com/test-catalog/Overview/88676

Watch one

Read each decision and its explanation.

Serum for complement screens arrives frozen from an outpatient clinic. The collection record shows the tube clotted on wet ice, was centrifuged at 4 °C, and the serum was frozen 20 minutes after centrifugation. The requisition lists no complement-directed therapy.

Where in the pathways is the defect most likely?

TestResultReference intervalFlag
CH50Less than 10 U/mL Low38–90 U/mL38–90Low
AH5088 % of normal46 or more % of normal46 or more
C3124 mg/dL90–180 mg/dL90–180
C428 mg/dL10–40 mg/dL10–40

Specimen: H 3, L 5, I 1. Serum, handled on ice, frozen 20 minutes after centrifugation

  1. Check the handling: clotted on ice, spun cold, frozen 20 minutes after centrifugation.

    Warm, delayed handling lowers functional complement and would make any low result unreliable.

  2. Check the medications: no complement-directed therapy is listed.

    C5-directed therapy is expected to lower both screens.

  3. Read the pair: CH50 is low, and AH50 is within its interval.

    AH50 needs C3 and C5 through C9. A normal AH50 shows those shared components are functional.

  4. Read the concentrations: C3 and C4 are both within their intervals.

    Active consumption usually lowers C3 and C4. A normal C4 also argues against C4 deficiency.

  5. Name the candidates for the component assays: C1 subcomponents and C2.

    The classical-only components left are C1 and C2.

The pattern suggests a deficiency of a classical-only component, C1 or C2. Component assays follow, including functional assays, because a component can be present as protein and still lack function.

Practice this skill

Your turn

Problem 1 of 3 · Hints available

CH50 and AH50 are both low in a correctly handled specimen from a patient receiving no complement-directed therapy. Which explanation fits this pattern?

Incorrect. C2 acts in the classical and lectin pathways, so its loss lowers CH50 and leaves AH50 within the reference interval.

Incorrect. Factor B acts in the alternative pathway, so its loss lowers AH50 and leaves CH50 within the reference interval.

Correct. Both screens need C3 and C5 through C9, so a defect there, or active consumption of shared components, lowers both results. Individual component assays then identify the deficient component.

Hint
  1. List the components CH50 needs and the components AH50 needs.
  2. Ask what a missing C2 would do to the AH50.

Review Laboratory assessment

Problem 2 of 3 · Hints available

Which complement product forms the pore that lyses a susceptible gram-negative bacterium?

C3b coats the bacterium so phagocytes can take it up. It also helps build the C5 convertase, and the pore forms later in the cascade.

Assigned membrane-pore formation to C3b

C3b coats targets for phagocytic clearance, C5a recruits inflammatory cells, and C5b-9, the membrane attack complex, forms the pore. Crediting C3b with lysis confuses opsonization with the terminal pathway.

C5a is released into the fluid and strongly attracts leukocytes. It does not insert into the membrane.

C4b coats targets and forms part of the classical and lectin C3 convertase.

C5b binds C6, C7, C8 and C9 to build the membrane attack complex, which inserts into the membrane as a pore.

Hint
  1. The pore forms at the end of the cascade.
  2. Follow what happens after the C5 convertase splits C5.

Review Complement effects and regulation

Problem 3 of 3 · On your own

A tube for CH50 and AH50 was drawn in a clinic at 08:00 and sent by courier at room temperature. It reaches the laboratory at 12:30 as unseparated clotted blood. The patient receives no complement-directed therapy. What do you do?

Four and a half hours of warm clotting consumes complement activity. A low result would report a handling artifact as deficiency or consumption, and a comment cannot correct the number.

Accepted a low activity result after warm, delayed handling

Functional complement is labile. A specimen left warm too long before separation and freezing loses activity and generates activation fragments. Accepting the low CH50 or AH50 reports a handling artifact as deficiency or consumption.

Freezing now prevents further loss. It cannot restore the activity lost during the warm delay.

The specimen did not meet the collection requirements for a labile analyte. A new specimen handled on ice gives a result that reflects the patient's complement activity.

Review Laboratory assessment

Use it

Read the case evidence, then make each decision. The step is complete without it.

  • Serum for CH50 and AH50 comes in for Priya Reade, 42, MRN 7730418.
  • The requisition lists a C5-inhibiting monoclonal antibody, last dose 6 days before the draw.
TestResultReference intervalFlag
CH50Less than 10 U/mL Low38–90 U/mL38–90Low
AH50Less than 10 % of normal Low46 or more % of normal46 or moreLow
C3118 mg/dL90–180 mg/dL90–180
C425 mg/dL10–40 mg/dL10–40

Specimen: H 2, L 4, I 1. Serum clotted on wet ice, centrifuged at 4 °C and frozen 15 minutes later

Decision 1 of 3

Is the specimen acceptable for testing?

The collection record documents ice, a cold spin and prompt freezing. The low results need another explanation.

The record meets each handling requirement, so the functional results reflect the patient's serum.

Complement screens are run on patients receiving these drugs. The laboratory needs the drug and dose timing to interpret the result.

Review Laboratory assessment

Decision 2 of 3

What best explains both screens reading low?

The C3 concentration is within its interval, and the requisition names a drug that lowers both screens. The paired screens and the history fit the therapy.

Read one low screen as an inherited deficiency

A low CH50 or AH50 can reflect a missing component, consumption, complement-directed therapy, or loss of activity from warm, delayed handling. Reading one low screen as an inherited deficiency skips the paired CH50 and AH50 and the check of specimen temperature and delay that separate these causes.

A classical-only defect lowers CH50 and leaves AH50 within its interval. Here both are low.

The handling record meets every requirement.

Both screens depend on terminal-pathway completion through C5. A C5 inhibitor lowers both, and C3 and C4 stay within their intervals.

Review Laboratory assessment

Decision 3 of 3

How are the results released?

The drug explains the pattern. Naming a deficiency would report the therapy's effect as a disease.

The ordering provider needs the agent, its target and the dose timing to interpret a low CH50 and AH50 on therapy.

The requisition already explains the pattern. Component assays are added when the history leaves the low screens unexplained.

Review Laboratory assessment

The drug on the requisition explains both low screens:

  • CH50 and AH50 both low
  • C3 and C4 within their intervals
  • A C5 inhibitor dosed 6 days before the draw

Both screens need C5, so a C5 inhibitor lowers both. The handling record rules out a warm-tube artifact, and the comment gives the ordering provider the drug and its timing.

For reference

Sources checked