Red Cell Antigens & Immunohematology

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

Red Cell Antigens

• Most common forms of blood group antigens: glycoproteins (HLA), glycolipids (ABH, Lewis, li and P), and proteins (Rh, M, N)

Characteristics of RBC Antigens

• Single-pass transmembrane proteins span the lipid bilayer once
• Multipass polypeptide chains cross multiple times
• Anchored antigens do not traverse

Immunogenicity

• Defined as the ability to trigger the immune system
• Most immunogenic blood group antigen: Rh (D)

Relative Immunogenicity

• D (Rh): 50%
• K (Kell): 5%
• c (hr'): 2.05%
• E (rh"): 1.69%
• k (Kell): 1.50%
• e (hr"): 0.56%
• Fy (Duffy): 0.23%
• C (rh'): 0.11%
• Jka (Kidd): 0.07%
• S (MNSS): 0.04%
• Jkb (Kidd): 0.03%
• s (MNSS): 0.03%

Immunoglobulins/Antibodies

• RBC Immune Antibody: found in the serum of individuals previously exposed to RBC antigens by transfusion, injection, or pregnancy
• Examples of blood group systems include Rh, Kell, Duffy, Kidd, and Ss, usually IgG
• Non-RBC Immune Antibody or Naturally-occurring: Found in the serum of individuals who have NEVER BEEN PREVIOUSLY EXPOSED to RBC antigens by transfusion, injection, or pregnancy
• Examples of blood group systems: ABH, Hh, li, Lewis, MN, and P, usually IgM
• Probably produced in response to substances in the environment that resemble RBC antigens such as pollen grains
• Clinically significant antibody: An antibody that decreases red cell survival and is able to destroy red blood cells in vivo (anti-A/B, anti-Jka) is reactive at 37°C
• Alloantibody is an antibody produced in response to foreign antigens
• Autoantibody is an antibody produced in response to self-antigen and may be warm or cold reacting
• Newborns don't have antibodies

Characteristics of IgG Class Antibodies

• Monomer structure and warm phase of reactivity
• IgG can cross the placental barrier except IgG2
• IgG has poor to good complement (C') activators, where two IgG are required to activate C' to completion except IgG4
• IgG has usually clinically significant RBC immune antibody
• Rh, Kell, Kidd, Duffy, Lub and Ssu are blood group systems

Characteristics of IgM Class Antibodies

• Pentamer structure, can be dissociated by sulfhydryl reducing reagents
• Cold phase of reactivity, best at 4-10°C and cannot cross the placental barrier
• A good (most) complement activator where one IgM can activate C' to completion
• "Usually" NOT clinically significant, non-RBC immune antibody
• Blood Group Systems included are ABO, li, Lewis MN, P, and Lu
• NOTE: IgM → 3Ms: MALAKI, MALAMIG, MAGALING

IgA

• About 30% of anti-A and anti-B antibodies are of the IgA class
• Anti-IgA antibodies can cause severe anaphylaxis if IgA are transfused in plasma products to patients who are deficient in IgA
• Individuals who are IgA deficient must be given washed components
• IgA can increase the effect of IgG induced RBC hemolysis and related to SECRETIONS

IgE

• The FC portion of the IgE molecule attaches to basophils and mast cells and facilitates HISTAMINE release when an allergen binds to the Fab portion of the molecule and cross-links with a second molecule on the cell surface
• Presence of IgE antibodies may cause urticaria

Separation of Red Blood Cells

• In vivo red blood cells are built in such a way as to keep their distance from each other
• Waters of hydration: water bound by RBC membrane glycoproteins helps maintain the distance between RBCs (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 Indicators of Ag/Ab Reactions

• Antibody coats RBC without agglutination
• Requires use of an antihuman globulin reagent in the Direct (DAT) or Indirect Antiglobulin Tests (IAT) to observe

RBC Hemolysis

• Complement mediated lysis of red blood cells where when antibody (IgG or IgM) has activated complement to completion, i.e. MAC(C5b-9) is formed
• Antibody (immunoglobulin) by itself cannot tear a hole in the RBC membrane, but Cl does that Seen when supernatant is CLEAR and RED

Complement

• Cl molecules bind with two adjacent Ig Fc regions
• A pentameric IgM molecule provides two Fc regions side by side, thereby binding complement
• A monomeric IgG molecule binds Clq less efficiently, and two IgG molecules are needed in close proximity to bind complement
• Antibodies against the Rh antigens usually do not bind complement due to the low level of Rh antigens on RBC surfaces

Agglutination

• Antibody mediated clumping of red blood cells that express corresponding antigens on their surface
• Two stage process: Sensitization (attachment of antibody to antigen on the RBC membrane and Lattice formation (formation of bridges between the sensitized red cells to form the lattice that constitutes agglutination

Stages of Agglutination

• Antibody molecules attach to their corresponding antigenic site (epitope) on the red blood cell membrane where there is no visible clumping

Attachment

• Attachment between antigen and antibody is dependent on spatial complementarity (Lock and Key concept) and on weak non-specific intermolecular binding forces:
• Electrostatic force/attraction: attraction between a (-) and (+) pole
• Hydrogen bonds, Hydrophobic force, Van der Waals force/London dispersion force

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
• 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 (4-10°C), warm (37°C)
• PH affects electrostatic bonds
• Incubation Time takes 1-2 hours, use of enhancement media reduces incubation
• Ionic strength: Low ionic strength increases the rate of Ag-Ab association

Stage 2 of Agglutination

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

Factors Affecting Stage 2 of Agglutination

• Size of the Immunoglobulin: IgG and IgM
• Number of binding sites: IgG and IgM Location and Number of Antigenic Determinants: A, B, M and N antigens and Kidd

Centrifugation

• Brings Ab's and Ag's into close proximity
• Net negative charge surrounding RBC

Waters of Hydration

• Acts as an insulating bubble around RBC
• Water molecules tightly bound to hydrophilic macromolecules on the red cell surface
• Protein Media, LISS, Macromolecule additives, Polycations, Enzymes, Albumin, AHG

Reading and Interpreting Ag/Ab Reactions

• Strong reaction (4+) indicates strong reaction
• A weak reaction (-/+) indicates weak reaction
• A reaction with 0 indicates there is no agglutinate

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

Other

• Rouleaux are increased proteins can cause RBCs to clump and stack (false positive)
• Mixed Field Agglutination are result of the Presence of two cell populations
• Always observe the supernatant of the test tube after centrifugation, where clear and red hemolysis is Indicated

Samples

• Serum preferred for complement studies
• EDTA chelates divalent ions and prevents complement activation
• Sodium heparin on the other hand prevents cleavage of C4
• Plasma is preferred for DAT and elution studies, since fibrin clot cause false positive reactions
• Plasma is comparable to serum and used routinely instead of the blood component

Explore red cell antigen-antibody reactions in immunohematology. Learn about RBC antigen characteristics, including glycoproteins and proteins like Rh and ABH. Understand immunogenicity and the role of immune antibodies in blood group systems.