Blood Bank Lecture 2 Fundamentals of Immunology

Primary lines of defense

Early evolutionary development

Non-specific

• - Natural. Present at birth

• - Immediately available

• - May be physical, biochemical, mechanical, or a combination of defense mechanisms

Mechanism does not alter on repeated exposed to any specific antigen

Innate or Natural Immunity Comparison

Supplements protection provided by innate immunity

Later evolutionary development - seen only in vertebrates

Specific

• - Specialized

• - Acquired by contact with a specific foreign substance

• - Initial contact with foreign substance triggers synthesis of specialized antibody proteins resulting in reactivity to that particular foreign substance

Memory

• - Response improves with each successive encounter with the same pathogen

• - Remembers the infectious agent and can prevent it from causing disease later

• - Immunity to withstand and resist subsequent exposure to the same foreign substance is acquired

Acquired/Adaptive Immunity

Internal Components:

Physical

• - Intact skin

• - Mucous membranes

• - Cilia

• - Cough reflexes

Biochemical

• - Secretions

  • - Sweat

  • - Tears

  • - Saliva

  • - Mucus

• - Very low pH of vag and stomach

Innate or Natural Immunity First Line of Defense

Internal Components:

Cellular

• - Phagocytic cells

  • - Macrophages-dendritic cells

  • - Monocytes

  • - PMNs Large granular leukocytes

  • - NK Cells

Humoral (Fluid) Biochemical

• - Complement-alternate pathway

• - Cytokines

  • - Interferons

  • - Interleukins

• - Acute inflammatory reaction

Innate or Natural Immunity Second Line of Defense

Internal Components

Cellular

• - Lymphocytes

  • - T cells

  • - TH

  • - TC

  • - T memory cells

• - B Cells

  • - B memory cells

  • - Plasma cells

Humoral

• - Antibodies

• - Complement-classic pathways

• - Cytokines

Acquired or Adaptive Immunity Third Line of Defense

Function of antibodies is to bind antigens

Binding is very specific: antibody reacts with only one epitope, or antigenic determinant, of an antigen

Binding inactivates the antigen and activates effector mechanisms that ultimately lead to the destruction of the antigen and the cell to which it is bound

Humoral Immunity

The laboratory study of antigen-antibody reactions - the basis of routine blood bank testing

Serology

Lag phase of 5-10 days, influence by the characteristics of the antigen and hosts immune system

IgM is produced first, IgG seen later

Primary immune response

Can detect antibodies within 1-3 days of exposure

Much higher concentration of antibodies that persist longer

Primary antibody is IgG

Secondary (anamnestic) immune response

Gamma (IgG)

Alpha (IgA)

Mu (IgM)

Delta (IgD)

Epsilon (IgE)

Heavy Chains

Kappa

Lambda

Light Chains

Constant and variable regions

Papain and pepsin digestion

• Disulfide bonds

Fc fragments binds complement or Fc receptors on other

Fab region binds antigen

IgM and IgA can exist in monomeric or polymeric

• IgM as pentamer

• IgA as dimer or trimer

IgG only exists in monomeric form

Characteristics of Immunoglobulins

IgG and IgM are the most significant for blood banking

Most clinically significant antibodies that react at body temperature are IgG

IgM antibodies are commonly encounter as naturally occurring antibodies and usually react best at room temperatures or colder

Immunoglobulins significant for blood banking

Heavy chain composition - Mu

Light chain composition - Kappa or Lambda

J Chain - Yes

Molecular Weight - 900,000

Valence - 10

Total serum concentration - 10

Serum half-life in days - 5

Crosses the placenta - No

Activation of classical pathways of complement - Yes; very efficient

Clearance of red cells - Intravascular

Detection in laboratory tests - Immediate spin

IgM

Heavy chain composition - Gamma

Light chain composition - Kappa or Lambda

J Chain - No

Molecular Weight - 150,000

Valence - 2

Total serum concentration - 70-75

Serum half-life in days - 23

Crosses the placenta - Yes

Activation of classical pathways of complement - Yes; not as efficient

Clearance of red cells - Extravascular

Detection in laboratory tests - Antiglobulin test

IgG

Activation when antibody (IgM, IgG1, or IgG3) binds to antigen

• - Activation by IgG requires many IgG molecules

• - Activation by IgM requires only 1 IgM molecule

Majority of blood group antibodies do not activate complement at body temperature

ABO antibodies DO bind complement and induce intravascular hemolysis

Classical Complement Pathway

Activation occurs by surface contacts with complex molecules and artificial substances with repeating units

Allows complement to be activated without acquired immunity

Alternative complement pathway

Activation occurs when mannose-binding lectins (MBL) bind to microbes and then follows classical pathway

Lectin complement pathway

Though majority of blood group antibodies do not activate the classical complement pathway and result in intravascular hemolysis, clinically significant blood group antibodies can result in extravascular hemolysis via reticuloendothelial (mononuclear phagocytic) system and other anaphylatoxic effects

Complement System

Antigens are large molecular weight proteins and polysaccharides that may initiate formation of and react with antibodies

• Can be located on the surfaces of cell membranes or as an integral portion of the cell membrane

Characteristics of antigen

Degree to which an antigen is capable of eliciting an immune response

Immunogenicity

Size

Complexity

Conformation

Charge

Accessibility

Solubility

Digestibility

Chemical composition

Antigen characteristics influencing their immunogenicity

Blood group antigens differ in their immunogenicity

Transfused red cells express antigens that may be recognized as foreign to the patient

The exposure to these foreign antigens may cause an immunogenic response resulting in production of corresponding antibodies

RBC antigens

Blood group antibodies are present in a patients serum/plasma

• Polyclonal or monoclonal

• Naturally occurring or immune

• Alloantibodies or autoantibodies

Characteristics of blood group antigens

Secreted by different B cell lineages

They are a collection of immunoglobulin molecules that react against a specific antigen, each identifying a different epitope

Polyclonal antibodies

Made by identical B cells that are all clones of a unique parent cell using hybridoma technology

They have monovalent affinity, in that they bind to the same epitope

Monoclonal antibodies

Which of the following would likely be more optimal for laboratory testing

Polyclonal antisera

Monoclonal antisera

Monoclonal antisera

Produced without transfusion, injection, or pregnancy

IgM, RT or lower, activate complement, may be hemolytic at 37C

Visible agglutination in saline

ABO, Hh, Ii, Lewis, MN, P

Naturally occuring antibodiesA

Acquired through transfusion or pregnancy

IgG, 37C

Require AHG testing for detection

Rh, Kell, Duffy, Kidd, Ss

Immune antibodies

A red cell antibody is considered clinically significant if it can cause accelerated destruction of transfused donor red cells. Which of the following is FALSE concerning clinically significant red cell antibodies

Most clinically significant antibodies are IgM

Directed at non-self antigens after exposure/transfusion

Alloantibodies

Directed at self-antigens

Can have a specificity common to transfused blood or no detectable specificity

Autoantibodies

Most clinically significant antibodies react at body temp 37C, are IgG, and can cause immune destruction of transfused red cells possessing the antigen

IgG can cross the placenta, weakly cause complement fixation, and mark red cells for phagocytosis

The destruction of red cells can cause transfusion reactions, anemia, and Hemolytic Disease of the Fetus and Newborn (HDFN)

IgM antibodies react best at room temp 20-22C or lower and are usually not implicated in the destruction of transfused red cells (they are usually not clinically significant)

The antibodies to ABO antigens are an important EXCEPTION to this generalization

• A transfusion of the wrong ABO group antigens would effectively activate the classical complement system, initiate intravascular hemolysis of the transfused cells, and cause a severe hemolytic transfusion reaction

RBC antibodies

Antigen-Antibody ratio

pH

Temperature

Immunoglobulin type

Incubation time

Ionic strength

Distance between cells

Factors that influence antigen-antibody reactions

Detection of blood group antibodies depends on characteristics of antigen-antibody reactions

These reactions depend upon binding forces between antigens and antibodies, properties of the antibody itself, and individual host characteristics

Antigen-antibody reactions

Can occur in vivo or in vitro

The binding of an antigen and antibody is a reversible process

When the immune complex has been generated, non-covalent attractive forces, including electrostatic forces (ionic bonding), hydrogen bonding, hydrophobic bonding, and van der Waals forces hold the complex together

Intermolecular binding forces

Strength of a single antigen-antibody bond

Affinity

A measure of functional affinity

Avidity

Specific reaction, cross-reaction, no reaction

Specificity

Number of antigen/antibody binding sites

Valency

Antigen-antibody reactions in vitro are detected by visible agglutination of red cells or evidence of hemolysis at the end of the testing

• No agglutination means no reaction

Two stages of hemagglutination reaction

• Sensitization: Binding of antibody and antigen

• Lattice Formation: Cross-linking of antibody-coated RBCs resulting in visible agglutination of cells

Hemagglutination

The probability of antigen-antibody interaction relies upon the effect of the ratio of the antibody & antigen concentrations

Antigen-antibody ratio

Postzone effect in an antigen-antibody reaction would likely result in a

False-negative result

Recall heterozygous genotypes can lead to weaker phenotype expression, less antigen being present on RBCs can lead to weaker antigen-antibody reactions

Test system can be manipulated in the lab to overcome the effects of excessive antigen or antibody

Dosage effect

Antigen-antibody ratio

Dosage effect

pH

Temperature

Incubation Time

Ionic Strength

Factors that influence sensitization

Optimal is 7.0

pH

Generally acts by increasing reaction rate, but different Ig isotypes also have different optimal reactivity temperatures

IgM: immediate spin phase at room temperature

IgG: Antihuman globulin (AHG) phase with 37C incubation

Temperature

Allowing adequate time to attain equilibrium; varies by test procedure

Incubation time

Na+ and Cl- ions in isotonic environment (saline) are attracted to oppositely charged groups on antigen/antibodies, hindering their interaction with each other; lowering increases antibody uptake

Ionic strength

Distance between cells

• Zeta potential

• Centrifugation

• Zone of equivalence

Factors influencing lattice formation

Red cells possess a net negative charge on the cell surface in a saline suspension

Cations from the saline environment are attracted to those negative charges and form a stable cationic cloud around each cell, resulting in a force of repulsion between any similarly charged molecules

Because of this, the red cells remain at a distance from each other, proportional to the zeta potential

IgM is larger and has pentameter shape with multivalent properties; thus agglutination can be seen without enhancement media

IgG is smaller and cannot always overcome the zeta potential but enhancement media can help form macroscopically visible agglutination

Zeta potential

Helps facilitate lattice formation by forcing the red cells closer together in the test environment

Centrifuges are calibrated to the optimal speed and time for the best visible reaction (may vary by test)

Centrifugation

Again, optimal antibody to antigen ratio is very important for both stages of agglutination

Washing is another procedural step to remove unbound antibody that we do not want to detect

Zone of equivalence

What can enhance hemagglutination reactions

Increasing the incubation time

To help us detect IgG in our patients, we use potentiators or enhancement media to enhance the reactivity of IgG

Most enhancement media works to reduce the zeta potential and allow the more positively charged antibodies to get closer to the negatively charged red cells

Factors influencing agglutination

Low Ionic Strength Solution

Polyethylene Glycol (PEG)

Proteolytic Enzymes

Antihuman Globulin (AHG)

Potentiators

Decreases the ionic strength of test system

Reduces zeta potential

Increases antibody uptake during the sensitization stage and helps lower the needed incubation time

Low Ionic Strength Solution LISS

Concentrates test system by removing water molecules

Brings sensitized red cells closer together, helping to form those crosslinks for lattice formation

Is considered to be more effective than albumin and LISS for detection of weak antibodies

Polyethylene Glycol (PEG)

Ficin, Papain, Trypsin, Bromelin

Modifies various blood group antigens, which can be especially useful when IDing multiple antibodies

ENHANCES reactivity of Rh, Kidd, P1, Lewis, and I blood group antigens by removing hydrophilic glycoproteins from RBC membrane

DESTROYS reactivity of Fya, Fyb, M, N, and S blood group antigens

Proteolytic Enzymes

Reveals if red cells are coated with antibody and/or complement proteins

If the antibodies present in serum/plasma do not cause red cell agglutination but only sensitize (bind) them, AHG reagent allows for visible agglutination to occur by cross-linking the antibodies coating the RBCs

Antihuman Globulin (AHG)

Potentiators Chart

In routine blood banking, red cell agglutination is graded qualitatively

Testing can be performed in test tubes, microplate wells, or in microtubes filled with gel particles

Defined grading system in place to help standardize the subjectivity uses a 0 to 4+ scale

ANY Agglutination or hemolysis in the supernatant is considered a positive result

Grading hemagglutination

4+ reaction

3+ reaction

2+ reaction

1+ reaction

Hemolysis

Negative reaction