Terry Kotrla, MS, MT(ASCP)BB

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Unit 3 Immunology and Complement

• Part 2
• Terry Kotrla, MS, MT(ASCP)BB

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Overview of Immunity
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Immunoglobulins

• Humans produce specific proteins or
  immunoglobulins which can be differentiated on
  the basis of
• Size
• Biologic function
• Biochemical properties
• Serological activity

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Basic Structure of Immunoglobulins

• An antibody digested by papain yields three
  fragments
• Two Fab which consist of antigen binding site,
  can sensitize.
• One Fc, whic is the region that determines
  biological properties of the Ig.

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Basic Structure of Immunoglobulins

• An antibody digested by pepsin yields two
  fragments
• One Fab2 which consist of 2 antigen binding sites
  joined together, able to agglutinate.
• One Fc,the region that determines biological
  properties of the Ig.

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Immunoglobulin Classes
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IgM Class
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IgM

• Largest of all the antibody molecules, consists
  of five of the basic units (pentamer) mu heavy
  chains joined together by a structure known as
  J-chain.
• Accounts for about 5-10 of the immunoglobulin
  pool.
• Restricted almost entirely to the intravascular
  space due to its large size.
• Fixes complement, much more efficient than IgG in
  the activation of complement and agglutination.
• First antibody to be produced and is of greatest
  importance in the first few days of a primary
  immune response to an infecting organism.
• Does not cross the placenta.
• Many blood group antibodies that are capable of
  agglutinating antigen positive RBCs suspended in
  saline in tests performed at 22 C are IgM causing
  visible agglutination, ie, ABO antibodies.
• IgM antibodies are potent agglutinators that
  activate complement very efficiently.

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IgG

• Most abundant of the immunoglobulins in the
  plasma
• One basic structural unit, i.e. Y-shaped molecule
  having 2 light chains and 2 Gamma heavy chains.
• Produced in response to a wide variety of
  antigens, including bacteria, viruses and RBC and
  WBC allo-antigens.
• Coats organisms to enhance phagocytosis by
  neutrophils and macrophages.
• Through its ability to cross the placenta,
  maternal IgG provides the major line of defense
  against infection for the first few weeks of a
  baby's life.
• It is the predominant antibody produced in the
  secondary response.
• The serologic behavior and characteristics of IgG
  antibodies make them one of the most clinically
  significant in blood banking.
• Most blood group antigens capable of eliciting
  an immune response result in the production of
  IgG antibodies.
• These antibodies are detected by serologic test
  procedures based on their behavior
  characteristics, such as reactivity at 37 C,
  complement activation, indirect agglutination and
  hemolysis.
• Much of routine blood banking involves serologic
  test procedures designed to detect and identify
  IgG antibodies.
• Four subclasses which differ in their heavy chain
  composition and in some of their characteristics
  such as biologic activities. IgG1, IgG2, IgG3
  and IgG4.

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IgA

• Found in saliva, tears, colostrum breast milk and
  in nasal, bronchial and intestinal secretions.
• IgA present in large quantities in colostrum and
  breast milk, is transferred across the gut mucosa
  in the neonate and plays an important role in
  protecting the neonate from infection.
• Produced in high concentrations by lymphoid
  tissues lining the gastrointestinal, respiratory
  and genitourinary tracts.
• Plays an important role in protection against
  respiratory, urinary tract and bowel infections
  and preventing absorption of potential antigens
  in the food we eat.
• Represents 10 to 15 of the total circulatory
  immunoglobulin pool.
• In plasma IgA may exist as a single basic
  structural unit or as two or three basic units
  joined together.
• The IgA present in secretions exists as two basic
  units (a dimer) attached to another molecule know
  as secretory component.
• 1) This substance is produced by the cells lining
  the mucous membranes.
• 2) It is thought to protect the IgA in secretions
  from destruction by digestive enzymes.
• IgA does not cross the placenta and does not bind
  complement.
• For blood banking, an IgA deficient individual
  may produce anti-IgA which can cause severe,
  life-threatening anaphylactic reactions during
  transfusion. Once identified these individuals
  must be transfused with blood and components
  which lack IgA.

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IgA Structure

• The dimeric IgA molecule.
• 1 H-chain,
• 2 L-chain,
• 3 J-chain,
• 4 secretory component

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IgE

• Trace plasma protein (only about 0.004) in the
  plasma of non-parasitized individuals.
• Major importance mediating some types of allergic
  reactions and is generally responsible for an
  individual's immunity to invading parasites.
• Fc region binds strongly to a receptor on mast
  cells and basophils and, when antigen is bound it
  causes the basophil (or mast cell) to release
  histamines and heparin from these cells,
  resulting in allergic symptoms.
• Clinical effects of IgE mediated reactions
  include increased vascular permeability, skin
  rashes, respiratory tract constriction
  (wheezing), and increased secretions from
  epithelium (watery eyes, runny nose).
• Not much else is known about its biologic role.
• IgE does not fix complement and does not cross
  the placenta.
• No blood group antibodies have been reported to
  belong to this class.

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IgE
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IgD

• Accounts for less than 1 of the total
  immunoglobulin pool.
• This is primarily a cell membrane immunoglobulin
  found on the surface of B lymphocytes.
• IgD does not fix complement and does not cross
  the placenta.
• Little is known about the function of this class
  of antibody.
• No blood group antibodies have been reported to
  belong to this class.

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Clinical Significance of Blood Group Antibodies

• A blood group antibody is considered clinically
  significant if it has been associated with the
  following
• Has caused hemolytic transfusion reactions
  (destruction of transfused red cells) or
• Implicated in Hemolytic disease of the fetus and
  newborn (HDFN) (destruction of fetal cells)

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Blood Group Antigens

• At least 30 blood groups with over 600 antigens.
• Individuals may produce antibodies to blood group
  antigens they do not possess when exposed to
  blood through transfusion or pregnancy.
• Second exposure may result in immune hemolysis of
  red blood cells.

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Transfusion Reaction

• Term used to describe an unfavorable response by
  a recipient to the infusion of blood or blood
  products and include the following
• In-vivo hemolysis (either immediate or delayed)
• Decreased survival of transfused cells
• Anaphylaxis
• Graft-versus-host disease
• Post-transfusion purpura
• Alloimmunization
• Sepsis due to bacterial contaminated components,
• Disease transmission.
• Will be discussed in detail later

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Severity

• Depends on a number of factors, including the
  characteristics of the antibody class involved.
• Antibodies to the ABO system antigens are
  predominantly IgM, cause complement activation
  and intravascular hemolysis.
• Other RBC antigens induce formation of IgG class
  antibodies which may cause accelerated RBC
  destruction extravascularly.
• Symptoms of response to incompatible ABO
  transfusion may include fever, low back pain,
  nausea and vomiting, circulatory shock, anemia,
  jaundice, and kidney failure which may ultimately
  result in death.
• Primary immune response may be asymptomatic due
  to slow destruction of RBCs.
• Secondary response symptomatic due to memory B
  cells and rapid antibody production.

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Antibody Mediated Hemolysis

• Hemolysis can be intravascular or extravascular.
• INTRAVASCULAR Antibodies destroy the red cells
  IN THE CIRCULATION. Due to of IgM and activation
  of complement with destruction of RBCs, VERY BAD,
  will see RED serum/plasma.
• EXTRAVASCULAR hemolysis is due to RBCs being
  coated with IgG and destroyed OUTSIDE the
  circulation in the RES system. If it occurs
  slowly may not be detectable.

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Transfusion Reactions

• Screening donor blood for disease markers
  significantly decreased transfusion transmitted
  diseases.
• Reactions to donor WBCs and platelets relatively
  common but usually not severe.
• ABO reactions severe and PREVENTABLE by following
  protocols.
• Other blood group antibodies may or may not be
  detectable.
• It is YOUR duty to provide serologically
  compatible blood and blood components for
  transfusion.

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Complement

• Spend quality time on your notes from Serology.

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Complement

• Integral part of the immune system.
• Three pathways
• Classical
• Alternative or properdin
• Lectin
• Three primary functions
• Lysis of antibody coated cells, such as bacteria
  and RBCs.
• Mediation of opsonization, preparation of foreign
  cells for phagocytosis.
• Generation of peptide fragments that regulate
  features of the inflammatory and immune response.

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Importance in Blood Banking

• Two major areas
• Some antigen-antibody complexes cause sufficient
  quantities of complement to be bound to RBCs to
  complete activation cycle, causing hemolysis.
• Antigen-antibody complexes initiate complement
  binding in such a way that allows demonstration
  of the existence of such complexes by the use of
  serologic techniques.
• Fresh serum necessary to detect complement
  mediated in-vitro reactions.

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The Classic Pathway

• Eleven components involved, numbered C1 to C9.
• Complement cascade requires presence of cations,
  both calcium and magnesium.
• Activation of the classic pathway almost always
  initiated by immunoglobulin.
• Requires only 1 molecule of IgM (has 5 Fc).
• Requires 2 molecules of IgG (has 1 Fc).

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Two IgG, One IgM
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The Classic Pathway

• Recognition Phase - Recognition unit
  C1q,C1r,C1s.
• Activation Phase -Activation Unit
  C4b,C2b,C3b,C5b
• Attack Phase Attack Unit C5b,C6,C7,C8 and C9
• Classic pathway C1,C4,C2,C3,C5,C6,C7,C8,C9
• Must go to completion for hemolysis to occur.
  The next two slides are to assist you in your
  studies.

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Classical Pathway
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(No Transcript)
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Alternative (Properdin) Pathway

• Proteins in the alternative pathway perform
  activities similar to those in the classic
  pathway but are usually non-antibody triggered.
• Any one of a variety of substances can initiate
  complement activation including
• bacterial polysaccharides and lipopolysaccharides,
  
• endotoxins,
• cobra venom,
• trypsin like enzymes,
• aggregates of IgA and IgG4 that do not activate
  C1.
• C1, C4 and C2 do not participate.
• Alternative pathway C3,C5,C6,C7,C8,C9

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Alternative Pathway
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Lectin Pathway

• Activation begins when mannan-binding protein
  (MBP) binds to the mannose groups of microbial
  carbohydrates.
• Two more lectin pathway proteins called MASP1 and
  MASP2 (equivalent to C1r and C1s of the classical
  pathway) now bind to the MBP.
• This forms an enzyme similar to C1 of the
  classical complement pathway that is able to
  cleave C4 and C2 to form C4bC2a, the C3
  convertase capable of enzymatically splitting
  hundreds of molecules of C3 into C3a and C3b.
• The beneficial results are the same as in the
  classical complement pathway above
• trigger inflammation (C5agtC3agtc4a)
• chemotactically attract phagocytes to the
  infection site (C5a)
• promote the attachment of antigens to phagocytes
  via enhanced attachment or opsonization
  (C3bgtC4b)
• serves as a second signal for the activation of
  naive B-lymphocytes (C3d)
• cause lysis of gram-negative bacteria and human
  cells displaying foreign epitopes (MAC)
• and remove harmful immune complexes from the body
  (C3bgtC4b).

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Lectin Pathway - FYI

• Overview of the lectin complement pathway. In
  humans, MBL and ficolin that are lectins form
  complexes with MASPs (MASP-1,MASP-2 and MASP-3)
  and sMAP. Note that MBL consists of several sizes
  of oligomers and that the composition of MASPs
  and sMAP of each MBL oligomer has not been fully
  elucidated. Once the complexes bind to
  carbohydrates on the surfaces of microbes,
  activated MASPs cleave C4, C2 and C3.

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Activation of Pathways
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Order of Activation of 3 Pathways
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Regulation of Complement

• Activation of complement cascade results in
  complex series of molecular event with potent
  biologic consequences.
• Modulating mechanisms are necessary to regulate
  complement activation and control production of
  biologically active split products.
• First mechanism is spontaneous decay of activated
  components.
• Second mechanism involves specific control
  proteins that modulate the activity of certain
  complement components at critical activation
  steps.
• C1 inhibitor blocks activities of C1r and C1s.
• Other factors inhibit activation of other
  complement components.
• A number of proteins act to control the membrane
  attack unit.
• Bottom line, gotta turn it off!

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References

• http//en.wikipedia.org/wiki/Antibody
• Complement http//www.medicine.uiowa.edu/martinl
  ab/complement.html