IMMUNE HEMOLYSIS & COMPLEMENT
Complement helps fight off bacterial and viral
infections, eliminates protein complexes, and helps in the immune response
complex. The three major roles of complement are:
- promoting acute inflammation process that allow
white cells and macrophages to migrate to the source of the problem
- altering the cell surfaces to encourage
- modifying the cell surface that will eventually
lead to cell lysis
Characteristics of Complement
Complement is a series of proteins found in fresh, normal serum.
It is in the beta region on protein electrophoresis and is categorized as a
beta globulin. The complement component that is found in the
highest concentration is C3. Terminology commonly used for the various
- Complement components are identified by C and their
number: C1, C2,
- Complement products resulting from the splitting of
these proteins during the activation process are followed by a lower case
letter: C3a, C3b,
- If complement complexes develop that have enzymatic
activity are written with a bar above the top: C5b678
Complement controls various biological process:
- Cell destruction through lysis
- Cell destruction through opsonization (enhanced
phagocytosis) especially with C3b
- Chemotaxis via certain split products acting as
chemical signals to the phagocytic cells
- Anaphylaxis again through the split products (C5a
and C3a), which promote inflammation. C5a and C3a can bind with mast
cells and basophils leading to the release of histamine. This is
- Increases vascular permeability
- Smooth muscle contraction to preserve blood for
- Increases cellular membrane adhersion
Complement is normally inactive in serum or if
activate the activation pathways are inhibited by control mechanisms of
other complement proteins. It becomes activated by:
- Antigen-antibody reactions - most often IgM (classical pathway)
- Bacterial polysaccharides, virus particles, enzymes, endotoxins (alternate
and lectin pathway)
Complement Cascade - (Classical Pathway)
Two adjacent antigen sites bound by antibody
capable of binding complement:
- IgM molecule since it has 5 immunoglobulin subunits
- Certain IgG molecues that are capable of binding
with complement. (only IgG 1, 2, or 3) Complement needs 2 IgG molecules
bound to antigen sites that are within 30-40 nm of each other.
The steps of the classical pathway
- Antigen-Antibody reaction occurs
- Complement senses adjacent Fc receptors
- Complement is activated and forms C1qrs complex
- C1qrs activates C4
- Activated C4 breaks down to C4a and C4b
- C4b activates C2 to form C4b2a complex
- C4b2a complex also known as C3 convertase
- In presence of Ca+, C4b2a activates C3
- Activated C3 breaks down to C3a and C3b
- C3b attaches to rbc membrane - is sensed by RE system and removed
from circulation (extravascular hemolysis)
- Activated C3a activates many C5 molecules that
leads to the development of the Membrane Attack
- Each activated C5 activates many more C6 and C7 molecules
- C6 and C7 conversion proceed rapidly to C8 and C9 activation
- Activated C8 activates many more C9 molecules
- Activated C9 attacks red cell membrane, boring holes in it
Contents of red cell leak out into plasma leading to intravascular hemolysis.
As the complement cascade results in biologic side-effects such as
chemotaxis, opsonization, and anaphylaxis.
What Determines Whether or Not Complement Will Be Activated:
Nature of antibody coating the cell
IgM antibodies are the best because they have more antigen-binding sites.
They can achieve binding of
two adjacent antigens by single IgM molecule. Only certain IgG subclasses
are capable of activating complement: IgG subclasses 1, 2, and 3. Of
these IgG subsets, IgG 3 is the best. IgG subclass 4 does not activate complement.
Number of antigen sites on red cell
The more antigen sites found on the red blood cell,
the more likely two adjacent ones are bound by antibody.
What does this mean in relationship to the ABO
The A and B antigens are in very high concentration on
the red blood cells. The natural-occurring, expected antibodies
(anti-A, anti-B, and anti-A,B) are IgM. ABO incompatibilities are the
most likely to result in intravascular transfusion reactions from the
activation of the classical pathway.
Possible Outcomes of Complement Activation in Blood
- Normal survival of the red blood cells can occur
but realize the other outcomes are more likely.
- Intravascular hemolysis (complement cascade goes to completion)
and the cells are lysed.
- Extravascular hemolysis where the complement cascade stops at C3b step.
Cells coated with C3b are removed from circulation via macrophages and
- "Damaged cells" (spherocytes and stroma fragments)
lead to decreased cell survival and possible activation of Hageman factor
that in turn leads to coagulation activation
- Disseminated Intravascular Coagulation (DIC) may also occur due to small clumps of
agglutinated cells in the blood stream, fibrinogen consumption, activation
of fibrinogen system and fibrin destruction.
- When leukocytes are exposed to various
antigen-antibody complexes they will also respond by secreting various
cytokines that will lead to: fever, a drop in blood pressure and
additional release of white blood cells from the bone marrow as well as a
number of other activities.
- Renal failure, which is the most common
complication of an untreated hemolytic transfusion reaction. This
occurs due to a combination of
Contraction of the blood
vessels in the kidney since it is one of the smooth muscles that responds
Toxic effects of free
- Temperature extremes (outside body)
- Hypotonic solutions (IV solutions)
- Mechanical (pumps, intravascular clots)
Immune-Mediated Cell Destruction (Hemolysis)
Causes of Immune Cell Destruction:
- Antibodies that bind complement
- Antibodies that do not bind complement
- Complement activation only
Types of Immune-Mediated Hemolysis
Intravascular hemolysis is complement mediated since
the hemolysis is due to activation of the complement pathway by the
classical pathway. Intravascular indicates the cell destruction takes
place within the blood vessels. This type of hemolysis can be
life-threatening due to both the possibility of anaphylaxis and renal
Extravascular Hemolysis may be antibody mediated or complement mediated.
The cell destruction takes place within RE system (spleen, primarily) and is
generally not life-threatening. The survival rate of the transfused
cells will be decreased.
Immune Response Following a Blood Transfusion Leading
- Donor red cells carrying foreign antigens die normally and are phagocytized by RE system.
- Foreign antigens processed and stimulate immune response.
- IgM antibody produced after several weeks, probably no cell
- Gradual decline of red cells results in continuous re-exposure of the
antigen to the immune system.
- Primary and secondary responses can overlap each other.
- IgG antibodies can be produced while IgM antibody productions is going
on, from the same blood transfusion (same "stimulating event").
- Low-titer IgM antibodies may not cause cell destruction, but
higher-titer IgG antibodies will eventually lead to extravascular hemolysis.
Intravascular Hemolysis (complement mediated) Summary
Mechanism of intravascular hemolysis
Complement is activated when two adjacent antigen sites are bound by
antibody. The activated complement rapidly proceeds through several
chemical changes to membrane attack complex. The membrane attack
complex bores a hole through red cell membrane, causing hemolysis. The
rate of complement activation and amount of complement activated determines
whether complement cascade goes to completion.
Signs of intravascular hemolysis
- Sudden drop in blood pressure due to the
anaphylactoxins: C5a and C3a
- Hemoglobinuria due to the lysis of of the red blood
cells. If more hemoglobulin is released that can be carried by
- Hemoglobinemia (plasma hemoglobulin levels) occurs
again due to the lysis of red blood cells in the blood vessels.
- Decreased haptoglobin since haptoglobin can also
carry hemoglobin. There are limited amounts of haptoglobulin
produced so once it is used, it will be removed by the liver and the
haptoglobulin levels decreased.
- No rise in hematocrit following blood transfusion
since the donor blood is destroyed.
- Anaphylactic shock, death may result if large
amounts of complement activated
Antibodies that bind complement (classical pathway)
- IgM antibodies = anti-A, anti-B, anti-I, anti-Lewis
- IgG antibodies = anti-A,B, auto anti-P, anti-D, anti-Kidd, anti-Kell,
Extravascular Hemolysis - Antibody Mediated
Mechanism of antibody-mediated hemolysis:
Antibodies attach to antigens on red cell membrane and are sensed by
phagocytes in RE system. The the antibody-coated red cells are
ingested by the phagocytic cells and destroyed at a rate faster than normal
cell destruction. RE system plucks antibody off cells, leaving damaged
membrane. Cell becomes a spherocyte with shorter lifespan.
Antibodies that do not bind complement but promote extravascular cell
Extravascular hemolysis processes are caused by IgG antibodies:
- Rh antibodies (anti-D, anti-C, anti-c, anti-E, and
- Anti-Kell (anti-K, anti-k, anti-Kpa,
- Anti-Kidd (anit-Jka and anti-Jkb)
- Anti-Duffy (anit-Fya and anti-Fyb)
Signs of extravascular hemolysis:
Signs of extravascular hemolysis includes a falling hematocrit,
increased bilirubin (unconjugated), increased LD, and an abnormal peripheral smear (polychromasia, spherocytes, fragments)
Extravascular Hemolysis - Complement Mediated
In extravascular hemolysis that is complement mediated
the breakdown products of activated complement attach to red cell membrane
(primarily C4b and C3b and C3d). Presence of C3b coated cells are
sensed by phagocytes in RE system. Complement-coated cells are ingested and
destroyed at a rate faster than normal cell destruction.
Causes of Complement-Mediated Extravascular Hemolysis:
Any of the IgM or IgG antibodies that are capable of
activating complement can cause this type of hemolysis. Besides the
classical pathway the alternate pathway of complement activation can also
occur due to the presence of various components like cell walls of bacteria
and yeast, dialysis membranes, dextran, and some tumor cells. Certain
drugs can also activate complement and can lead to a complement-mediated
OBJECTIVES - IMMUNE HEMOLYSIS & COMPLEMENT
- Briefly explain what complement is
- List the effects complement activation can have on the body, in
addition to cell destruction
- Explain what is required for complement to be activated in the
- Explain how the alternate pathway differs from the classical pathway
in the complement cascade
- Outline the steps of the complement cascade in intravascular cell
- Explain the role of complement in extravascular cell destruction,
including the roles of C3b and C3d
- Explain what C3 convertase is
- Explain what the membrane attack complex is
- List two factors that help determine if complement will be activated
during an antigen-antibody reaction
- Explain why IgM is a better complement activator than IgG
- List four possible results of complement activation regarding red
- List at least four causes of red cell hemolysis, other than
- List the three main causes of immune-mediated hemolysis
- Describe the mechanism of intravascular hemolysis
- Describe the signs and physical symptoms of intravascular hemolysis
- List the IgM antibodies that typically cause intravascular hemolysis
- List the IgG antibodies that typically cause intravascular hemolysis
- Describe the mechanism for antibody-mediated extravascular hemolysis
- List the symptoms of extravascular hemolysis
- List pertinent laboratory findings in extravascular hemolysis
- Describe the mechanism for complement-mediated extravascular
- List three causes of complement-mediated extravascular hemolysis
- Outline the steps in an immune response and
subsequent cell destruction following a blood transfusion, including types
of white cells involved in each step, and the approximate length of time