Basic Principles of Immunology and Ag
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PREPARED BY: JOSEPHINE C. MILAN, RMT, MSMT WAGGAS AHMEDELAAS; M.Sc, MLT (Revised, Feb 2014)
Objective and Focus To recall basic concepts of Immunology and Antigen-
Antibody reactions as they apply to Blood Banking practice, such as: Immune response arising from blood group antigen stimulation Characteristics of antigens Describing clinically significant antibodies Enhancing Ag-Ab reactions
Topic Outline Definition and properties of Antigen, immunogen Definition and properties, types of antibodies The immune system response in relation to
Immunohematology or blood banking practice Nature/ principle of Ag-Ab reactions
ANTIGEN, IMMUNOGEN ANTIGEN (Ag)– substance recognized as foreign by the
body, which when introduced to a host , will stimulate an immune response. Immune response 1. activation of the cellular components of immune system 2. the production of specific antibodies Properties of antigen:
1. Specific reactivity – react with specific antibody 2. Immunogenicity- stimulate immune response
Blood Group Antigens Biochemically : Protein, large polysaccharide,
glycoproteins or glycolipids Immunogenic, antigenic Represent the blood group phenotype Determined by blood group genes Antigens are integral structural component of the RBC
BLOOD GROUP ANTIBODIES Gamma globulins / immunogloblins Produced in response to antigenic
stimulation. How? Exposure through pregnancy Exposure through transfusion Exposure through transplant Essentially of Ig M and IgG classes; some IgA Characterized with specific reaction with blood group antigen epitopes/ determinants
Antibodies: Produced and secreted by activated B lymphocytes
(plasma cells) Types: 1. Heteroantibodies (xenoantibodies) – produced in response to antigens from another species. 2. Alloantibodies / isoantibodies – produced in response to antigens from individuals of the same species; such as those involved in transfusion reactions 3. Autoantibodies – made in response to the body’s own antigens.
Ig CLASSES OF SOME BLOOD GROUP ANTIBODIES
IgM antibodies important in Blood banking includes those against : ABH, Ii, MN, Lewis, Lutheran, and P.
IgG antibodies important in Blood banking includes those against: Rh(D), Ss, Kell, Duffy, and Kidd.
IgA antibodies – Le, ABH subs
II. Antibody Classes IgM
75 – 85
160,000 & 400,000
9 - 11d
25 - 35d
56 C stable
Pass thru placenta
Ig G structure
Ig M structure
Secretory Ig A
Serologic properties of Abs: IgM Strong agglutinins (3-4+ agglutination) Reactive at cold temperature (4 0C to room temp) React to cells suspended in saline Fix/ activate the complement IgG Weak agglutinins, need AHG Reactive at warm temperature (37 oC) React to red cells in protein or potentiating medium Weakly activate the complement
ANTIBODY FUNCTIONS Primary function: to bind with antigen Secondary functions/ biological effector
Complement fixation/ activation
hemolysis of red cells Placental transfer ( Ig G) Bind on receptor sites on tissues and other cells
The Complement in Immunohematology Proteins normally produced in the body. Unstable, heat labile (56 oC); serum must be fresh When activated, they cause lysis of cell membrane
of antibody-coated RBCs. Complement is activated by the classic pathway : IgM or IgG3 antibodies bound to red cell antigens C1 component binds to the Fc portion of the Ab activate C4, C2, C3, C5, C6 + C7 + C8 + C9 lysis. Reason why: hemolysis of RC is a positive result.
IMMUNE RESPONSE Immune system – activated by the presence of
foreign antigen, or abnormal autoantigen. Cells involved : APCs (antigen presenting cells)= macrophages, dendritic cells, monocytes Lymphocytes = T helper, T cytotoxic, B/ plasma cells Natural killer, neutrophils, eosinophils, basophils, platelets
Plasma cells; Monocytes; Neutrophols; Platelets; Lymphocytes
IMMUNE RESPONSES in Immunohematology 1. antibody production IgM and IgG 2. hemolytic reaction mediated by Ab-Ag and
complement (cascade pathway) 3. inflammatory reaction tissue necrosis (organ transplantation) 4. allergic reaction mediated by IgE with basophils and mast cells
IMMUNE RESPONSE BY BLOOD GROUP ANTIGEN STIMULATION PREGNANCY – incompatible blood type of fetus
to that of mother (baby’s antigen foreign to the mother) E.g. Baby is D(+), mother is D(-) TRANSFUSION- incompatible blood type
between patient (recipient) and donor blood Tissue/ organ transplantation – incompatible
type between patient (recipient) and donor tissue
PRINCIPLES OF ANTIGEN- ANTIBODY REACTION
MANIFESTATIONS OF ANTIGEN – ANTIBODY REACTION 1. Red cell agglutination Clumping of red cells Various grades of strength of reaction (4+, 3+, 2+, 1+, +/-) 2. Hemolysis (red cell destructionHb) Red tinge of supernatant Minimal red cell button or none at all May be misread as negative
Grades of agglutination reaction
NON- SPECIFIC AGGREGATION OF RED CELLS; NOT (+) RESULT Rouleaux Formation
Stacked-coin formation Found in patients with Multiple Myeloma, Walderstrom’s Macroglobulinemia or Hyperviscosity Syndrome The red cells cluster and resembles a macroscopic agglutination)
NATURE OF Ag- Ab REACTIONS
A. Lock and Key principle specific reaction of antigen and antibody the antigenic determinant fit in a cleft / space formed by the combining site of the antibody (at the Fab region, formed by variable regions of light and heavy chains)
NATURE OF Ag – Ab REACTIONS B. Non-covalent Bonds
That hold the Ag in the antibody combining site Include hydrogen bonds, electrostatic bonds, Van der Waals forces and hydrophobic bonds Multiple bonding ensures the Ag bound to the Ab is tight and stable.
STAGES OF RED CELL AGGLUTINATION 1. PRIMARY STAGE ( ANTIBODY SENSITIZATION) Involves binding of the paratope /combining site of the Ab and the epitope/ determinant of the Ag in a reversible reaction).
2. SECONDARY STAGE OF RED CELL AGGLUTINATION : LATTICE FORMATION
Multiple erythrocytes with bound antibodies form a latticework through Ag--Ab bridges formed between adjacent erythrocytes The lattice formed is the basis of all visible agglutination reactions.
FACTORS AFFECTING THE PRIMARY STAGE OF REACTION 1. Temperature The nature of the bonds determines if the reaction occurs better at colder or warmer temperature. With
hydrogen bonds- they are exothermic, reaction occurs better at colder temperature Associated with carbohydrates antigen e.g. ABH, Lewis, P1, I)
FACTORS AFFECTING THE PRIMARY STAGE OF REACTION If
with hydrophobic bonding, the reaction is optimally reactive at body temperature, 37 OC This type of bond is normally associated with protein antigen e.g. Rh, Kell, Duffy
FACTORS AFFECTING THE PRIMARY STAGE OF REACTION 2. Effect Of pH Optimum
pH for most antigen-antibody reactions = 6.5 to 7.5 Some antigens show stronger reactivity at lower pH e.g. anti-M, and anti-D at pH 6.5 – 7.0
FACTORS AFFECTING THE PRIMARY STAGE OF REACTION 3. Ionic Strength
refers to charge concentration of the suspending (reaction) medium.
decreasing the ionic strength of the suspending medium reduces the interfering effects of the electrostatic barrier… THUS, better attraction between antigen and antibody.
FACTORS AFFECTING THE PRIMARY STAGE OF REACTION Low Ionic Strength Saline (LISS) Consists of 0.2 % NaCl Bring about increased rate of antibody uptake for the Ag during sensitization shortens incubation period of 15 to 5 minutes.
FACTORS AFFECTING THE PRIMARY STAGE OF REACTION 4. Time of Incubation To
allow antigen/antibody reactions reach equilibrium 15 to 60 minutes
FACTORS AFFECTING THE PRIMARY STAGE OF REACTION 5. Effect Of Antigen-antibody Ratio Amounts
of antigen and antibody should be in optimal proportions Optimum ratio is 80 parts antibody to 1 part antigen 2 drops serum : 1 drop RCS Excess in either antigen or antibody results to non occurrence of lattice formation leading to a false-negative result.
FACTORS AFFECTING THE PRIMARY STAGE OF REACTION
FACTORS AFFECTING THE SECONDARY STAGE 1. ZETA POTANTIAL The surface of red cells carry a negative charge . In saline, red cells will attract positively charged Na+, and an ionic cloud will form around each cell. Thus the cells repel each other and stay a certain
• IgG antibodies
cannot cause agglutination when zeta potential exists. • To overcome
zeta potential techniques are needed to neutralize these charges.
FACTORS AFFECTING THE SECONDARY STAGE 2. Effect of Centrifugation The antibody - sensitized red cells are subjected to high gravitational force to overcome the natural repulsive effect of the red cells to one another Closer
physical proximity (nearness) increases antigen-antibody bridging
FACTORS AFFECTING THE SECONDARY STAGE 3. Effect of Immunoglobulin Type Most efficient for agglutination reactions. The The
physical size of IgM
number of antigen binding sites of IgM molecules (valence 10) compared with IgG (valence 2), increases the chances of random antibody-antigen collisions, leading to a greater chance of effective red cell cross-linking.
FACTORS AFFECTING THE SECONDARY STAGE
ENHANCEMENT MEDIA FOR Ag- Ab REACTIONS 1. ALBUMIN:
1. Reduce the charge density (dielectric constant) of the red cell suspending medium. 2. Reduce net repulsive force between red cells 3. It replaces the water hydration surrounding the red cell
Therefore: IgG antibody able to span the gap between individual positive red cells to produce agglutination.
ENHANCEMENT MEDIA FOR Ag- Ab REACTIONS 2. ENZYMES
The proteolytic enzymes: 1) papain (papaya) 2) ficin (figs) 3) bromelin (pineapples) 4) trypsin (lining of a hog's stomach) Enzyme treatment is known to increase the avidity of both IgM & IgG Abs. They reduce Zeta potential allowing cells to come closer. They also remove proteins adjacent to antigens, allowing Abs to have better access to Ags.
3. ANTIHUMAN GLOBULIN (AHG) Produces
a “bridging effect” by cross linking the antibody-sensitized red cells.
4. POLYETHYLENE GLYCOL: Water- soluble polymer used with AHG to bring sensitized cells close together and facilitate crosslinking and enhancement of agglutination reaction. Does not produce non-specific reactions. 5. POLYBRENE (hexadimethrine bromide) Positively charged macromolecule which cause non-specific aggregation of sensitized red cells with IgG antibody after incubation with LISS.