Life's Blood

CLASS NOTES

IMMUNE HEMOLYSIS & COMPLEMENT

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 phagocytosis (opsonization)
  • 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: 

  • Complement components are identified by C and their number: C1, C2, C3 etc
  • Complement products resulting from the splitting of these proteins during the activation process are followed by a lower case letter: C3a, C3b, C1q
  • If complement complexes develop that have enzymatic activity are written with a bar above the top: C5b678

Complement controls various biological process:

  1. Cell destruction through lysis
  2. Cell destruction through opsonization (enhanced phagocytosis)  especially with C3b
  3. Chemotaxis via certain split products acting as chemical signals to the phagocytic cells
  4. 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 turn:
  • Increases vascular permeability
  • Smooth muscle contraction to preserve blood for vital organs
  • 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

  1. Antigen-Antibody reaction occurs
  2. Complement senses adjacent Fc receptors
  3. Complement is activated and forms C1qrs complex
  4. C1qrs activates C4
  5. Activated C4 breaks down to C4a and C4b
  6. C4b activates C2 to form C4b2a complex
  7. C4b2a complex also known as C3 convertase
  8. In presence of Ca+, C4b2a activates C3
  9. Activated C3 breaks down to C3a and C3b
  10. C3b attaches to rbc membrane - is sensed by RE system and removed from circulation (extravascular hemolysis)
  11. Activated C3a activates many C5 molecules that leads to the development of the Membrane Attack Complex.
  12. Each activated C5 activates many more C6 and C7 molecules
  13. C6 and C7 conversion proceed rapidly to C8 and C9 activation
  14. Activated C8 activates many more C9 molecules
  15. 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 System?

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 Banking

  • 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 neutrophils
     
  • "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
  1. Hypotension

  2. Contraction of the blood vessels in the kidney since it is one of the smooth muscles that responds in anaphylaxis.

  3. Intravascular clots

  4. Toxic effects of free hemoglobin

Non-Immune Hemolysis

Physical

  • Temperature extremes (outside body)
  • Hypotonic solutions (IV solutions)
  • Mechanical (pumps, intravascular clots)

Chemical- toxins

Immune-Mediated Cell Destruction (Hemolysis)

Causes of Immune Cell Destruction:

  1. Antibodies that bind complement
  2. Antibodies that do not bind complement
  3. Complement activation only

Types of Immune-Mediated Hemolysis

Intravascular 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 failure.

Extravascular Hemolysis

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 to Alloimmunization

  1. Donor red cells carrying foreign antigens die normally and are phagocytized by RE system.
  2. Foreign antigens processed and stimulate immune response.
  3. IgM antibody produced after several weeks, probably no cell destruction.
  4. Gradual decline of red cells results in continuous re-exposure of the antigen to the immune system.
  5. Primary and secondary responses can overlap each other.
  6. IgG antibodies can be produced while IgM antibody productions is going on, from the same blood transfusion (same "stimulating event").
  7. 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 albumin. 
  • 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 a
  • IgG antibodies = anti-A,B, auto anti-P, anti-D, anti-Kidd, anti-Kell, etc.

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 destruction

Extravascular hemolysis processes are caused by IgG antibodies:

  • Rh antibodies (anti-D, anti-C, anti-c, anti-E, and anti-e)
  • Anti-Kell (anti-K, anti-k, anti-Kpa, anti-Jsa etc.)
  • Anti-Kidd (anit-Jka  and anti-Jkb)
  • Anti-Duffy (anit-Fya  and anti-Fyb)
  • Anti-S
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 extravascular hemolysis.

OBJECTIVES - IMMUNE HEMOLYSIS & COMPLEMENT

  1. Briefly explain what complement is
  2. List the effects complement activation can have on the body, in addition to cell destruction
  3. Explain what is required for complement to be activated in the classical pathway
  4. Explain how the alternate pathway differs from the classical pathway in the complement cascade
  5. Outline the steps of the complement cascade in intravascular cell destruction
  6. Explain the role of complement in extravascular cell destruction, including the roles of C3b and C3d
  7. Explain what C3 convertase is
  8. Explain what the membrane attack complex is
  9. List two factors that help determine if complement will be activated during an antigen-antibody reaction
  10. Explain why IgM is a better complement activator than IgG
  11. List four possible results of complement activation regarding red cell survival
  12. List at least four causes of red cell hemolysis, other than immune-mediated
  13. List the three main causes of immune-mediated hemolysis
  14. Describe the mechanism of intravascular hemolysis
  15. Describe the signs and physical symptoms of intravascular hemolysis
  16. List the IgM antibodies that typically cause intravascular hemolysis
  17. List the IgG antibodies that typically cause intravascular hemolysis
  18. Describe the mechanism for antibody-mediated extravascular hemolysis
  19. List the symptoms of extravascular hemolysis
  20. List pertinent laboratory findings in extravascular hemolysis
  21. Describe the mechanism for complement-mediated extravascular hemolysis
  22. List three causes of complement-mediated extravascular hemolysis
  23. 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 involved.
 

Clinical Microbiology Syllabus