Basic Principles of Immunology and Ag-Ab Reactions

PREPARED BY:JOSEPHINE C. MILAN, RMT, MSMTWAGGAS AHMEDELAAS; M.Sc, MLT

(Revised, Feb 2014)

Objective and FocusTo 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 antigensDescribing clinically significant antibodiesEnhancing Ag-Ab reactions

Topic OutlineDefinition and properties of Antigen,

immunogenDefinition and properties, types of antibodiesThe immune system response in relation to

Immunohematology or blood banking practiceNature/ principle of Ag-Ab reactions

ANTIGEN, IMMUNOGENANTIGEN (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 AntigensBiochemically : 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 membrane

BLOOD GROUP ANTIBODIESGamma globulins / immunogloblinsProduced in response to antigenic

stimulation. How?Exposure through pregnancyExposure through transfusionExposure 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 reactions3. 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 ClassesIgM IgG IgA IgD IgE

Other names Macro-globulin

- Secretory Ig - Reagin

% 5-10 75 – 85 5-15 0.001 0.0003

MW 900,000 150,000 160,000 & 400,000 180,000 190,000

Half-life 9 - 11d 25 - 35d 6-8d 2-3d 2d

56 C stable yes yes yes no no

Pass thru placenta

no yes no no No

valence 10 2 2,4 2 2

C-fixing yes IgG3 No no no

subclass 2 4 2 - -

form pentamer monomer dimer Monomer Monomer

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 salineFix/ activate the complement

IgGWeak agglutinins, need AHGReactive at warm temperature (37 oC)React to red cells in protein or potentiating

mediumWeakly activate the complement

ANTIBODY FUNCTIONSPrimary function: to bind with antigen

Secondary functions/ biological effector functions:Complement fixation/ activation hemolysis of red cellsPlacental transfer ( Ig G)Bind on receptor sites on tissues and

other cells

The Complement in ImmunohematologyProteins normally produced in the body.Unstable, heat labile (56 oC); serum must be

freshWhen 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 RESPONSEImmune system – activated by the presence of

foreign antigen, or abnormal autoantigen.

Cells involved :APCs (antigen presenting cells)= macrophages,

dendritic cells, monocytesLymphocytes = T helper, T cytotoxic, B/ plasma

cellsNatural killer, neutrophils, eosinophils, basophils,

platelets

Plasma cells; Monocytes; Neutrophols; Platelets; Lymphocytes

IMMUNE RESPONSES in Immunohematology

1. antibody production IgM and IgG2. 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 supernatantMinimal red cell button or none at allMay be misread as negative

Grades of agglutination reaction

NON- SPECIFIC AGGREGATION OF RED CELLS; NOT (+) RESULTRouleaux Formation

Stacked-coin formation Found in patients with Multiple Myeloma, Walderstrom’s Macroglobulinemia or Hyperviscosity Syndrome The red cells cluster and resembles amacroscopic agglutination)

NATURE OF Ag- Ab REACTIONS

A. Lock and Key principlespecific 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)

Ag-Ab binding

NATURE OF Ag – Ab REACTIONSB. 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 siteof the Ab and the epitope/ determinantof the Agin a reversible reaction).

2. SECONDARY STAGE OF RED CELL AGGLUTINATION : LATTICE FORMATION

Multiple erythrocytes with bound antibodies form alatticework through Ag--Ab bridgesformed between adjacenterythrocytes The lattice formed is thebasis of all visible agglutination reactions.

FACTORS AFFECTING THE PRIMARY STAGE OF REACTION

1. TemperatureThe 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 OCThis type of bond is normally associated

with protein antigen e.g. Rh, Kell, Duffy

FACTORS AFFECTING THE PRIMARY STAGE OF REACTION

2. Effect Of pHOptimum pH for most antigen-antibody

reactions = 6.5 to 7.5Some 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 IncubationTo allow antigen/antibody reactions

reach equilibrium 15 to 60 minutes

FACTORS AFFECTING THE PRIMARY STAGE OF REACTION

5. Effect Of Antigen-antibody RatioAmounts of antigen and antibody should

be in optimal proportions Optimum ratio is 80 parts antibody to 1

part antigen2 drops serum : 1 drop RCSExcess 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 POTANTIALThe 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 distance apart.

ZETA POTENTIAL

• IgG antibodies cannot causeagglutination when zeta potential exists.

• To overcome zeta potential techniques are needed to neutralize these charges.

FACTORS AFFECTING THE SECONDARY STAGE

2. Effect of CentrifugationThe 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 TypeMost efficient for agglutination reactions.The physical size of IgMThe 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 cross-linking 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.

Thanks