CLLS 3311Advanced Clinical

Immunohematology

Antigen and Antibody

Reactions

Demonstration of red cell antigen-antibody reactions is key to immunohematology.

AABB Technical Manual

• Red cell antigen-antibody reactions that we study and perform include Patient ABO grouping, Rh typing, Antibody screens and identifications, Crossmatching, etc.

• An awareness of how antigens and antibodies react is necessary to accurately perform the required testing including: quality control, trouble shooting, maintenance, etc.

Immune Response• Definition: Physiological mechanism to

fight disease or clear foreign substances.• Primary immune response: First

exposure of a foreign agent. IgM is the predominant antibody produced that attaches to and fights the foreign agent. In our case a foreign red blood cell antigen.

• Secondary immune response: Subsequent exposure of the same foreign agent. Rapid response in which IgG is the predominant antibody produced.

Antigen/Antibody Reactions

1. Antigen:

2. Antigenicity:

3. Epitopes:

1. Substance capable of eliciting an immune response when introduced into a immunocompetent individual to whom it is foreign.

2. Ability of an antigen to react with the products of an immune response

3. Antigenic determinants - structural site where the antibody combines with the antigen.

Characteristics of Antigens

1. Antigenicity of an Antigen is affected by its:

2. Foreign:

3. Size:

4. Most common forms of Blood Group Antigens:

1. Size, shape, rigidity, location, number of determinants (epitopes), dose, route of entry, and genetic factors.

2. Normally…don’t forget autoimmune processes

3. >10,000 daltons• Glycoproteins: HLA• Glycolipids: ABH, Le, Ii

and P antigens (Ag)• Proteins: Rh, M, N antigens

Characteristics of Immunoglobulins

(Antibodies)• RBC Immune Vs. Non-RBC Immune– RBC Immune Antibody: antibody that results

from exposure to foreign red cell antigen either by transfusion or pregnancy (anti-D, etc.)

– Non-RBC Immune Antibody: antibody that is present without any evidence of exposure to foreign red cell antigen (anti-A, anti-N, etc.)

• Clinically Significant Antibody: An antibody that decreases the red cell survival. Able to destroy red blood cells in vivo. (anti-A,B, Anti-Jka, etc.)

CharacteristicsCharacteristics of IgG Class Antibodies 1. Structure

2. Phase of Reactivity

3. Placenta

4. Complement (C’) Activation

5. Clinical Significance

1. Monomer

2. Warm: reacts best at 37oC

3. Can cross the placental barrier

4. Poor to good C’ activators. Two IgG required to activate C’ to completion.

5. Usually clinically significant RBC immune Antibody

Characteristics of IgM Class Antibodies1. Structure

2. Phase of reactivity

3. Placenta

4. Complement Activation

5. Clinical Significance

1. Pentamer

2. Cold: reacts best at 4-10oC

3. Cannot cross the placental barrier

4. GOOD (most) complement activators - one IgM can activate C’ to completion

5. “Usually” NOT clinically significant, Non RBC immune.

• We just stated that IgM antibodies are usually NOT clinically significant. There is a notable exception to this statement! What is it?

• ABO antibodies are VERY CLINICALLY SIGNIFICANT. Most transfusion fatalities are the result of an ABO incompatibility.

• Yet anti-M, anti-N, etc. are usually NOT clinically significant IgM antibodies.

IgG Antibody(Monomer Structure)

IgM Antibody(Pentamer Structure)

Images provided by textbook: Figure 14.3, page 97, KubyImmunology, 4th Edition, W.H. Freeman and Company

1. Cold reacting

2. Good complement activator

3. Pentamer

4. ABO, Ii, Lewis, MN, & P

1. Warm Reacting

2. Poor complement activators

3. Monomer

4. Rh, Kell, Kidd, Duffy, and Ss

IgG

Antibody’s

IgM Antibody’s

Separation of Red Blood Cells

• In vivo red blood cells are built in such a way as to keep their distance from each other. It is a good thing. Theories include:

• Waters of Hydration: Water bound by RBC membrane glycoproteins helps maintain the distance between rbc’s. (Water envelope)

• Electrostatic charges: Electron cloud surrounds RBC with a net negative charge called the zeta potential.

• To observe Ag/Ab reactions in vitro these forces need to be overcome to enable Ab’s to attach to their corresponding Ag.

In vitro (in the test tube) indicators of Ag/Ab Rxn’s

1. Antibody coating RBC without Agglutination

– Sensitization: Attachment of antibody to antigen that requires antihuman globulin to detect.

2. RBC Hemolysis– Immune mediated lysis of red blood cells.

3. Agglutination– Antibody mediated clumping of red blood cells

that express corresponding antigens on their surface.

Antibody Coating RBC Membrane without Agglutination

• Attachment of antibody to corresponding antigen on RBC membrane, ONLY.

• This reaction is NOT SEEN at the Immediate Spin (IS), Room Temperature (RT), or 37oC (LISS, low ionic strength solution) phases.

• This reaction requires the use of an Antihuman Globulin reagent in the Direct (DAT) or Indirect Antiglobulin Tests (IAT) to observe.

Hemolysis• When antibody (IgG or IgM) has

activated Complement to completion. • The complement (C’) cascade is the mechanism

that actually destroys the red blood cell. • Antibody (immunoglobulin) by itself

cannot tear a hole in the RBC membrane. It is the C’ that does that.

• Seen when supernatant is CLEAR and RED.• Not seen at the AHG phase! Why??

Agglutination ReactionsTwo Stage Process:

Stage 1• Sensitization: attachment of Antibody to

Antigen on the RBC membrane. TM

Stage 2• Lattice formation: formation of bridges

between the sensitized red cells to form the lattice that constitutes agglutination. TM

This represents what occurs during stage one of agglutination: Sensitization

Antibody molecules attach to their corresponding Antigenic site (epitope) on the red blood cell membrane. There is no visible clumping.

Stage 1

This represents what occurs during stage 2 of agglutination: Lattice Formation

Antibody molecules crosslink RBCs forming a lattice that results in visible clumping or agglutination.

Stage 2

Stage 1 of Agglutination: Sensitization

• Attachment of antibody to antigen• Attachment between Antigen and Antibody

is dependent on spatial (spacial) complementarity (Lock and Key concept) and on weak non-specific intermolecular forces including:– Electrostatic forces, Hydrogen Bonds,

Hydrophobic forces, and van der Waals forces. (Does NOT involve covalent bonding.)

Weak Non-specific Intermolecular Forces

Electrostatic forces• Attraction of oppositely charged or ionized molecules• Force of attraction is inversely proportional to

square of the distance between charges: closer you get the stronger the attachment of Ag to Ab.

Hydrogen bonds• Weak, reversible hydrogen bridges between

hydrophilic groups which are stronger at lower temperatures.

Weak Non-specific Intermolecular Forces

Hydrophobic forces• Water hating groups (valine, leucine) come into

close contact and exclude water between them, resulting in lower free energy system disrupting (breaking up) the water envelope.

• Disruption of water envelope is the result of hydrophobic forces: requires close proximity

Weak Non-specific Intermolecular Forces

van der Waals bonds

• Temporary disruption of electrons in one molecule effectively forms a dipole which causes the formation of a dipole in another molecule, the two dipoles then exhibit an attraction for each other: Requires close proximity.

LAW OF MASS ACTION

• Due to weakness of these forces Ag/Ab complexes obey the Law of Mass Action which is:

Antigen and antibody complexing is REVERSIBLE (bilateral).

• Equilibrium is the point at which the number of bonds being formed equals the number of bonds being broken.

Factors Affecting Stage 1 of Agglutination

Temperature• Cold (4-10oC): Exothermic reactions occur when

H+ bonds are made with carbohydrates at LOW temperatures. – ABO, P1, Lewis antibodies bond better at low

temperatures due to carbohydrate nature of antigens

• Warm (37oC): Entropy driven reactions associated with hydrophobic bonding to proteins.– Rh antibodies bond better at 37oC due to protein nature

of Rh antigens.

Factors Affecting Stage 1 of Agglutination

pH• Changes in pH can affect electrostatic bonds.• Optimal range is physiologic pH• Some Ab’s like lowered pH particularly anti-M

Incubation Time • The time needed to reach equilibrium• Saline systems: 30-60 minutes at 37oC• Enhancement media can reduce incubation time

significantly: LISS (low ionic strength solution) is 10-15 minutes

Factors Affecting Stage 1 of Agglutination

Ionic Strength• In normal saline, Na+ and Cl- ions cluster around

and partially neutralize opposite charges on Ag and Ab molecules, which hinders (blocks) the association of Ab with Ag.

• If you lower the ionic strength of the test system then you increase rate of Ag-Ab association.

• Using LISS (low ionic strength solution) decreases incubation time.

Stage 2 of Agglutination:Lattice Formation

• Lattice formation: formation of bridges between the sensitized red cells to form the lattice that results in agglutination (clumping) of red blood cells.

Concentration of Ag and Ab also affects agglutination reactions, both the first and second stages.

Factors Affecting Stage 2 of Agglutination

Size of the Immunoglobulin• IgG: Monomer, takes two to activate C’, etc.

• IgM: Pentamer, takes one to activate C’, etc.

Number of binding sites• IgG: Two binding sites (anti-D, anti-Jka, etc.)

• IgM: Ten binding sites (anti-A,B, anti-I, etc.)

Factors Affecting Stage 2 of Agglutination

Location and Number of Antigenic Determinants

• A, B, M and N antigens: 600,000 to >1,000,000 antigen sites per RBC

• Kidd: approximately 10-20,000 Ag sites/RBC

Centrifugation• Brings Ab’s and Ag’s into close proximity• Undercentrifugation: may result in false negative• Overcentrifugation: may result in false positive

Factors Affecting Stage 2 of Agglutination

Zeta Potential• Net negative charge surrounding RBC• Now considered a minor player

Waters of Hydration• Acts as an insulating bubble around RBC• Water molecules tightly bound to

hydrophilic macromolecules on the red cell surface.

Reading and Interpreting Ag/Ab Reactions

Agglutination• Grade reaction strength: Neg to 4+• Harmening Color Plate 2.

Hemolysis• ALWAYS observe the supernatant of the test tube

after centrifugation. If the supernatant is clear and red Hemolysis is indicated. THIS IS A POSITIVE REACTION! And indicates a Nasty antibody.

Reading and Interpreting Ag/Ab Reactions

Rouleaux• Increased proteins can cause RBCs to

clump and stack - false positive

Mixed Field Agglutination• Presence of two cell populations: such as

Group O cells in a Group A patient. Anti-A will only agglutinate A cells resulting in a mixture of clumping cells and free cells.

Potentiators

• We have not covered the topic of potentiators. This is found on pages 60-62 in Harmening and is Objective #10.

• We also did not cover Solid Phase and Gel testing techniques. This is found on page 62-63 in Harmening and is Objective #6.