5.

The Complement
System
MEDT 21 – Immunology and Serology

References
• Stevens, C. D., & Miller, L. E. (2016). Clinical Immunology and
 Serology: A Laboratory Perspective. FA Davis.
• Turgeon, M. L. (2013). Immunology & Serology in Laboratory
Medicine. Elsevier Health Sciences.
• Abbas, A. K., Lichtman, A. H., & Pillai, S. (2014). Cellular and
molecular immunology. Elsevier Health Sciences.
• McPherson, R. A., & Pincus, M. R. (2017). Henry's Clinical
Diagnosis and Management by Laboratory Methods. Elsevier
Health Sciences.
• Sales, M.E.H. (n.d.). A Lecture Module in Clinical Immunology and

Serology. Unpublished.
MEDT 21 – Immunology and Serology

Learning Outcomes
After the completion of the chapter, students will be able to:
[Link] the roles of the complement system.
[Link] between the classical, alternative, and lectin
pathways and indicate proteins and activators involved in each.
[Link] regulators of the complement system and their role in
the complement system.
[Link] the complement-related diseases and applicable
testing MEDT 21 – Immunology and Serology

The Complement System

I. INTRODUCTION AND NOMENCLATURE
II. CLASSICAL PATHWAY
III. ALTERNATIVE PATHWAY
IV. LECTIN PATHWAY

• YouTube Videos
• Assignment
 MEDT 21 – Immunology and Serology

Biological Roles of Complement

Host Defense against Infections
[Link] cell lysis
[Link] of Phagocytosis
• Pro-inflammatory functions
• Anaphylatoxins – C5a, C3a, C4a
• Chemotaxins – C5a
 • Opsonization – C4b, C3b, iC3b, C3dg
Link between Innate and Adaptive Immunity
1. Augmentation of Antibodies
• In association with opsonization
2. Enhanced Immune Response
• Receptors in both APCs and B cells
3. Enhancement of Immunologic Memory
MEDT 21 – Immunology and Serology

Biological Roles of Complement

Waste Disposal
[Link] of Immune Complexes from Tissues
• Complement receptor 1 (CR1) mediates the transport of C3b
coated immune complexes to the liver and spleen
2. Clearance of Apoptotic Cells

MEDT 21 – Immunology and Serology

Complement System Controls

• Activation of complement could cause tissue damage and have
devastating systemic effects if it could proceed uncontrolled •
Ensures that infectious agents and not self-antigens are
destroyed, and that the reaction remains localized
• Majority are aimed to halting C3b

MEDT 21 – Immunology and Serology

Complement System Controls

C1 Inhibitor
• Inactivates C1 by binding to the active sites of C1r and C1s,
dissociating them from C1q
• Also inactivates MASP-2
• Deficiency: associated with hereditary angioedema

MEDT 21 – Immunology and Serology

Complement System Controls

Regulators of C3 and C5 Convertase
[Link] I
• Serine protease that inactivates C3b and C4b upon binding
• Acts in harmony with the regulators #2-6
• Deficiency: recurrent pyogenic infections
2.C4-binding protein (C4BP)
• Cofactor of Factor I
• Can bind both fluid-phase or membrane-bound C4b
[Link] Receptor 1 (CR1)
• Cofactor of Factor I
• Binds C3b and C4b
• Also mediates the transport of C3b-coated immune complexes to the liver
and spleen
[Link] Cofactor Protein (MCP) / CD46
• Most efficient cofactor for Factor I-mediated cleavage of C3b inhibiting
formation of C5 convertase for all pathways
• Can serve as cofactor for binding C4b

MEDT 21 – Immunology and Serology

Complement System Controls
Regulators of C3 and C5 Convertase
[Link] Accelerating Factor (DAF) / CD 55
• Can dissociate the C3 convertase of both classical/lectin and
alternative pathways
• Can bind to both C3b and C4b in the same manner as CR1
• Protects cells from bystander lysis.
• Deficiency: associated with paroxysmal nocturnal
hemoglobinuria
[Link] H
• Principal soluble regulator of the alternative pathway
• Binds C3b to prevent binding of Factor B
o C3b has 100x more affinity for Facor H than Factor B • Also
accelerate the dissociation of the membrane-bound C3bBb
complex.
• Also acts as cofactor of I to degrade C3b
• Deficiency: recurrent pyogenic infections
MEDT 21 – Immunology and Serology

Complement System Controls

Regulators of the Membrane Attack Complex
[Link] S / Vitronectin
• Binds with C5b67 / C5b-7 to inhibit binding to cell membranes
[Link] Inhibitor of Reactive Lysis (MIRL) / CD59 •
Also blocks the formation of MAC
• Protects cells from bystander lysis
• Deficiency: associated with paroxysmal nocturnal

hemoglobinuria MEDT 21 – Immunology and Serology

In Vitro Complement
Inactivation
[Link]
• 50oC for 20 minutes – inactivates Factor B
• 56oC for 30 minutes – destroys C1, C2 and some C4
• 56oC for 10 minutes – re-inactivation
[Link]
• EDTA – chelation of calcium and/or magnesium
• Heparin – inhibits the cleavage of C4 by C1s
[Link] Storage
• Alters C4

MEDT 21 – Immunology and Serology

Complement Receptors (CR)
1.CR1 / CD35
• Previously discussed
2.CR2 / CD21
• With CD19, binds complement-coated antigens which cross-links it to
membrane antibody to activate B cells
• Receptor for Epstein-Barr virus entry
3.CR3 / CD11b/CD18
• Binds particles opsonized with iC3b
• Mediates phagocytosis
4.CR4 / CD11c/CD18
• Like CR3, also binds particles opsonized with iC3b and mediates
phagocytosis
[Link] receptors
• Bind to the collagen portion of C1q
• Enhances the binding of C1q to Fc receptors
• Mediates phagocytosis

MEDT 21 – Immunology and Serology

Complement Receptors (CR)

6. DAF / CD55 – previously discussed
7. MIRL / CD59 – previously discussed
8. MCP / CD46 – previously discussed
 MEDT 21 – Immunology and Serology

Complement and Diseases

• Complement can contribute to tissue damage and even death
• Conditions where complement causes harm:
o Large scale systemic activation (e.g., Gram-negative
 septicemia) o Activation by tissue necrosis (e.g., myocardial
infarction) o Causing lysis of erythrocytes (e.g., cold autoimmune
hemolytic anemia)
• Complement deficiencies
o C2 deficiency – most common
o C3 deficiency – most severe
• Atypical Hemolytic uremic syndrome (aHUS)
o Associated with autoantibodies to Factor H and Factor I o
Associated with genetic mutations of Factor B, H and I, MCP, and
thrombomodulin
MEDT 21 – Immunology and Serology

Laboratory Assays for Complement

Abnormalities
[Link] assays – cannot distinguish if the individual
 components are functionally active
[Link] immunodiffusion (RID)
[Link]

MEDT 21 – Immunology and Serology

Laboratory Assays for Complement

Abnormalities
2. Qualitative assays – measures lysis, which is the functional endpoint
of complement activation
a. Hemolytic Titration / CH50 Assay
• Most commonly used assay for classical pathway
• Measures the amount of patient serum required to lyse 50% of a
standardized concentration of antibody-sensitized sheep
erythrocytes
• For classical pathway
b. Radial Hemolysis
• For classical pathway
• Uses agarose gel
c. Enzyme-linked immunosorbent assay (ELISA)
• For both classical and alternative pathway
MEDT 21 – Immunology and Serology
Laboratory Assays for Complement
Abnormalities
2. Qualitative assays (cont.)
d. Hemolytic titration / AH50 assay
• For alternative pathway
• Performed in the same manner as the CH50, except
magnesium chloride and ethylene glycol tetraacetic acid (EGTA)
are added to the buffer and calcium is left out
• Rabbit RBCs are used as the indicator because they provide an
ideal surface for alternative pathway activation
e. One test system using strips
• Can determine activity of all three pathways using three different
strips
MEDT 21 – Immunology and Serology

End of Lecture
Thank you for listening!

MEDT 21 – Immunology and Serology