Immunology and Serology

Immunology and Serology

  • The body’s immune system is an integrated network of cells, tissues, organs, mechanical barriers, and secreted molecules.

  • Three main functions:

    • Defense: Prevents entry of infectious agents and eliminates those that gain entry. Recognizes self from non-self antigens, eliminating non-self. Autoimmune disorders occur when the body attacks its own antigens.

    • Homeostasis: Maintains balance, stopping the immune response after eliminating pathogenic agents.

    • Surveillance: Detects overgrowth of particular cell populations, such as tumor or cancer formation, signaling a defect in the body’s ability to recognize increased mitotic division.

Components of the Immune System

  • Four components:

    • Cells and tissues of the immune system: Cells, organs, and tissues, including the skin, mucous-secreting cells, and enzyme-producing cells, serve as the first line of defense.

    • Monocyte-Macrophage Cell System: Functions as antigen-presenting cells.

    • T Lymphocytes (T cells)

    • B Lymphocytes (B cells)

Cells of the Immune System

  • Found mainly in the blood and lymphoid organs, produced in the bone marrow.

  • Differentiate from the same stem cell into:

    • Lymphoid stem cell

    • Myeloid progenitor cell

Myeloid Progenitor Cell

  • Gives rise to granulocytes:

    • Polymorphonuclear cells (PMNs): Neutrophils, eosinophils, basophils.

    • Mast cells: Found in tissues, active in eliciting hypersensitivity reactions.

    • Basophils: Found in the blood, contain histamine, which initiates hypersensitivity responses.

    • Eosinophils: Granules contain major basic protein, essential in fighting off parasites.

    • Neutrophils: Most numerous granulocyte, active during bacterial infection. Migrate into tissues via diapedesis when signaled by inflammatory mediators.

    • Monocyte: Differentiates into dendritic cells or macrophages.

    • Dendritic cells and macrophages: Act as antigen-presenting cells, capturing antigens via phagocytosis, breaking them down, and presenting them to T cells. The main way of eliminating pathogens is through phagocytosis.

Lymphoid Stem Cells

  • Differentiate into lymphocytes: B cells, T cells, NK cells (Natural Killer Cells).

    • Natural Killer Cells: Release chemicals that kill infected cells, foreign cells, and tumor cells.

    • T cells: Mature in the thymus, involved in cellular immunity, express specific receptors.

      • T helper cell: Activates macrophages and B cells by cytokine secretion. Presents antigens.

      • T cytotoxic lymphocyte: Releases chemicals to kill infected cells, foreign cells, and tumor cells.

    • B Lymphocytes:

      • Mature in the bone marrow.

      • Differentiate into plasma cells, made possible by the activation of the T helper cell.

      • Plasma cells produce antibodies to block infection and eliminate extracellular pathogens.

      • Involved in humoral immunity. Transform into plasma cells, which produce antibodies/immunoglobulins.

      • B Lymphocyte Activation:

        • Antigen binding: Any antigen binding with IgM and IgD found on the surface of B lymphocytes leads to activation upon contact.

        • T helper cell activation: T helper cells release chemical signals to activate B cells, leading to antibody secretion.

Types of Immunity

  • Two types:

Innate/Nonspecific Immune Response

  • Involves the body’s first line of defense.

  • Has a consistent response regardless of the amount/type of pathogen encountered, governed by the same regulatory mechanism.

  • No memory.

  • Physical barriers:

    • Skin, mucosal linings: Provide an unfriendly environment for pathogens.

    • Phagocytic cells and chemical mediators promote inflammatory responses.

    • Physiological factors:

      • Hydrochloric acid in the stomach.

      • Pseudostratified ciliated columnar epithelium of the respiratory tract (produces mucus).

      • Flushing action of urine.

      • Unsaturated fatty acids (skin).

      • Sweat, tears, and saliva.

      • Commensal normal flora.

Inflammation – Second Line of Defense

  • Body’s reaction to injury.

  • Results in:

    • Increased blood supply in the area.

    • Increased capillary permeability.

    • Migration of leukocytes.

  • Manifests as pain, heat, redness, and swelling:

    • Vasodilation leads to increased blood flow in the area, causing redness and heat.

    • Swelling is manifested by edema due to increased capillary permeability and fluid accumulation. Also due to the influx of phagocytes (e.g., neutrophils, eosinophils, macrophages, NK cells).

Adaptive or Specific Immune Response

  • Immunologic memory and specificity develop in response to the antigen.

  • Can recognize self and non-self antigens.

  • Production of antibodies (humoral immunity) or actions of T-cells (cell-mediated immunity).

    • Produced by B-lymphocytes, activated to become plasma cells because of their recognition receptors, which are capable of discriminating many molecular configurations.

    • Interaction between both humoral and cell-mediated.

Antigens

  • Capable of provoking a specific immune response.

  • If the antigen can produce an immune response → Immunogen

    • All immunogens are antigens.

    • Not all antigens are considered as immunogens.

  • Capable of stimulating the formation of antibody and the development of cell-mediated immunity.

  • In blood banking, refers to the antigens present on the RBC.

Characteristics

  • Foreign nature: An immunogen must be identified as non-self. The greater the difference from self = greater the likelihood of eliciting an immune response.

  • Molecular size: Molecular antigens with a molecular weight of more than 10,000 daltons are better immunogens.

  • Molecular complexity and composition:

    • Proteins are the best form of antigens because with its complexity, it is much more effective in inducing an immune response.

    • Carbohydrates and lipids are next to the proteins.

    • ABO antigens are made of glycolipids, which are effective immunogens.

    • Red cell antigens that are highly protein in nature, particularly the Rh antigens, are very strong immunogens

  • Route of administration and dose:

    • Intramuscular route.

    • Intravenous administration (blood transfusion the best route for inducing any response)

Epitopes

  • Recognized as foreign by the immune system.

  • Small regions of the molecule within the immunogen. AKA antigenic determinants.

  • Unique configurations allow recognition by corresponding antibody.

    • If the antigen has multiple epitopes, a different antibody is produced from each of those epitopes.

    • The antibodies produced for different epitopes tend to cross-react if they have a common determinant.

  • Allogeneic antigens: From another individual but of the same species.

    • In IHBB: Blood from a different individual but the same blood type.

  • Autologous antigens: One’s own self-antigen. Always tolerated by the immune response because it is recognized if they are self-antigens. If the immune system cannot recognize such self-antigens, it is going to induce an immune response that will be indicated as autoimmune.

    • In IHBB: Autologous transfusion: An individual will have their blood taken out and transfused back to themselves. Done if someone has a very rare blood type. Intended only for them, NOT another individual.

Blood Antigens

  • Chemical structures embedded in or protruding from RBCs, WBCs, and platelets.

  • Currently, there are 29 blood group systems.

  • There are 250 unique red cell antigens that are recognized by the American Association of Blood Banks (AABB).

  • Determined by the inheritance of many blood group genes.

  • Every individual possesses a unique set of Red Cell antigens through genetic inheritance

Three Common Forms

  • Glycoproteins - HLA system

  • Glycolipids - ABH, Lewis, Ii, and P blood group systems

  • Proteins - Rh, M, N blood group systems

Antibodies

  • Specific to a particular antigen.

  • Produced by the plasma cells, which are the activated B cells, and produce proteins in the form of antibodies.

  • Five types of antibodies: Gamma, Alpha, Mu, Epsilon, Delta

Characteristics

  • Size

    • Smallest: IgG, IgE, IgD (Monomers)

    • IgA: Could be a dimer or a monomer. Monomeric form = serum; Dimeric form = secretions.

    • Largest: IgM (Pentamer made up of five units of antibodies)

  • Biologic function: heavy chains impart unique features to the different immunoglobulin classes, determining antibody type or class.

    • EX: Gamma heavy chain → IgG. Alpha heavy chain in IgA allows residing in mucosal linings, in secretions.

    • Kappa and Lambda light chains.

  • Biochemical properties: Variable region binds with the antigen. Constant region imports the antibody class function (e.g., complement activation or attachment to certain cells).

    • IgE attaches to surfaces of basophils and mast cells.

    • IgD in the surface of B lymphocytes.

    • IgG is mainly found in the serum.

    • IgM is found in the surface of B cells.

    • IgA is found in the secretions.

    • Hinge region of antibodies contain disulfide bonds to impart flexibility during their attachment to antigens.

  • Serological activity

Papain and Pepsin Enzyme

  • Papain → yields antibody into three fragments: Two FAbs, 1 Fc

  • Pepsin → yields antibody into two fragments: One FAb, 1 Fc

  • Regardless of having an Fc or not, it is still capable of binding to antigens and could still produce agglutination reactions.

  • In hematology, clinically significant antibodies are IgG and IgM because these are the antibodies that could be produced if there is the introduction of red cells whose antigens are absent.

Immunoglobulin MU (IgM)

  • Largest of all antibody molecules.

    • Consists of five basic units (pentamer) joined by a structural component (J CHAIN) composed of disulfide bonds in the middle.

    • Restricted mostly entirely in the intravascular space because of its large size, but it is able to activate the classical pathway of complement with great efficiency.

    • Accounts for about 5-10% of the Immunoglobulin Pool.

  • Fixes complement.

  • First antibody to be produced during the primary immune response to an infecting organism.

  • Does not cross the placenta.

  • Saline agglutinins

    • Capable of visible agglutinations of cells that are suspended in saline.

    • Capable of agglutinating antigens present on the RBCs that is suspended in saline IF they are going to be processed at room temperature or lower; and it could still react at very low temperatures.

    • Also known as Cold reacting antibodies.

  • Activate complement very efficiently

    • Considered to be much more efficient than IgG in complement activation because it requires only one immunoglobulin Mu to initiate the classical complement pathway.

Immunoglobulin GAMMA (IgG)

  • Most abundant in the plasma.

    • Comprises 80% of the total Immunoglobulin population.

  • Ability to cross the placenta

    • Fc receptors in the placenta, allowing it to cross during pregnancy.

    • ADV: Able to protect the baby from infection, particularly if the mother has an infection that occurred during pregnancy.

    • DADV: If the IgG is produced AGAINST a certain red cell antigen

      • If the baby has an antigen that is absent in the mother, it will lead to the sensitization of the mother against that certain antigen and will produce antibodies

      • If the antibodies are in IgG form, it will cross the placenta and attach to the red cells of the baby and will activate complement, leading to hemolysis.

  • IgG has the ability to activate the complement system; however, it is not as efficient as compared to IgM.

    • Because of its small structure, it will require 2 Molecules of IgG to activate the classical pathway of complement.

  • Predominant antibody produced in the secondary response.

  • MOST CLINICALLY SIGNIFICANT in blood banking.

    • Reactivity at 37°C (body temperature), complement activation, indirect agglutination, and hemolysis.

      • DADV: Agglutination is not as visible as IgM

      • You need reagents to have visible agglutination reaction with IgG → using antihuman globulin

        • Effect: Hemolysis.

  • Four subclasses: IgG1, IgG2, IgG3, and IgG4

    • Result of minor variations in the gamma heavy chain, slight amino acid differences, affecting its activity.

Antibodies

  • Has caused hemolytic transfusion reactions (HTRs).

    • If a recipient of a blood transfusion has an antigen that is absent from the system of the recipient.

    • The recipient will produce an antibody against such antigens

Steps in Antibody Formation (From Hemolytic Transfusion Reactions)

  • SENSITIZATION – the slow progress of antibody production

  • FASTER PRODUCTION OF ANTIBODIES – Happens the second time the patient encounters the same antigen

    • Because the B cells tend to have memory

    • Immediate transfusion reaction which could lead to complement activation, hemolysis which could be fatal to the blood recipient.

Hemolytic Disease of the Fetus and Newborn

  • Implicated in Hemolytic disease of the fetus and newborn (HDFN).

  • A particular blood group that is involved, which is the Rh blood group system.

  • Occurs if the baby has the Rh antigen, which the mother is lacking.

  • Most probably, the baby has inherited the gene that produces Rh antigens from the father.

    • FIRST PREGNANCY – SAFE. This is the first encounter of the mother with the said antigen; baby will survive and the baby will appear yellow due to the degradation of red cells that led to the hemolysis of some red cells therefore bilirubin might be present in the circulation of the baby

      • During pregnancy – fetomaternal circulation – at a particular term in the pregnancy

      • The baby’s blood will go to the circulation of the mother, which tends to lead to SENSITIZATION

      • Because the first pregnancy is the first encounter, the baby survives, but the mother is sensitized with the Rh antigen.

    • SECOND PREGNANCY – UNSAFE. Since the mother’s immune system has memory, then fetomaternal circulation, the mother has encountered the same antigen for the second time, there is faster immune response - production of antibody which could lead to hemolysis

      • Antibody produced → IgG

      • Has receptors in the placenta, and could cross the placenta and lead to attachment to the red cells of the baby, activated complements so that could lead to hemolysis and eventual death

Antigen-Antibody Complex

  • The binding of antigens to antibodies is based on noncovalent forces:

    • Electrostatic Forces: ionic forces based on the attraction between two molecules on the basis of opposite charges

    • Hydrogen bonds: Attraction between two negatively charged groups, namely Nitrogen and Oxygen for a hydrogen atom

    • Van der Waals forces: attractions between two electron clouds of one atom and the protons within another atom’s nucleus

    • Hydrophobic forces: These are weak bonds formed as a result of the exclusion of water from the antigen and antibody complex.

Affinity

  • Tendency that an epitope has for combining with the antigen binding site of an antibody molecule

Avidity

  • Strength of the bond between the antigen and the antibody

Complement

  • The complement system is a group of serum proteins with a number of biologic roles related to antigenic clearance, cell lysis, as well as vasodilation.

  • Normally circulate in an inactive or pro-enzyme state.

  • During activation, they are converted into active enzyme that enhances the immunologic process

Functions of Complement System

  • Opsonization:

    • Able to clear immune complexes

    • Covering of foreign material with complement proteins enhances phagocytosis, promoting the release of enzymes from the neutrophils.

  • Anaphylaxis:

    • Increased smooth muscle contraction and induces inflammation.

  • Lysis:

    • Foreign antigens are killed by membrane lysis.

    • This will be the result of the transfusion reaction if the red cell transfused to the patient is containing an antigen that is absent on their system

    • Antibodies will be produced and these Abs could activate complements leading to lysis of transfused red cells.

  • Chemotaxis:

    • Recruitment of platelets and phagocytes, which induces the immune response.

Three Pathways

  • Classical Complement Pathway

  • Alternative Pathway

  • Mannose-binding lectin (MBL) Pathway

Antibodies that can Activate Complement

  • IgM

  • IgG1

  • IgG2

  • IgG3 → IgG4, IgA, IgE, and IgD CANNOT activate complement pathway

Classical Complement Pathway

  • Starts with antigen-antibody reaction

    • If the Fc portion of the antibody has attached to an antigen, it will be recognized by the first complement protein → C1

  • C1 and its three subunits → C1q, C1r, C1s

    • Collectively, they are the C1qrs

  • Cleaves the second complement protein → C4 complement

  • C4 will be cleaved to have C4a and C4b

  • C4a will be removed and C4b will be retained

  • Activation of C2 complement in which it will be cleaved to have C2a and C2b

  • C2a will be retained

  • New combination: C4b2a → C3 convertase

  • C4b2a cleaves C3 → C3b will now join C4b2a → C4b2a3b

  • C4b2a3b → C5 convertase

  • C5 complement protein will be cleaved to C5a and C5b

  • C5b will be attached to cell membrane; unless it is going to be bound with C6 it is rapidly inactivated.

    • Forms the beginning of the MAC (Membrane Attack Complex)

  • C5b is joined by C6, C7, C8, and C9

    • C5b6789 → Membrane Attack Complex

    • Induces cell lysis

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