L03 - Fundamentals of Immunology PDF

9/20/2024 Chapter 3: Fundamentals of Immunology Preamble PowerPoints are a general overview and are provided to help students take notes over the video lecture ONLY. – PowerPoints DO NOT cover the details needed for the Unit exam Each student is responsible for READING the TEXTBOOK for de...

9/20/2024 Chapter 3: Fundamentals of Immunology Preamble PowerPoints are a general overview and are provided to help students take notes over the video lecture ONLY. – PowerPoints DO NOT cover the details needed for the Unit exam Each student is responsible for READING the TEXTBOOK for details to answer the UNIT OBJECTIVES Unit Objectives are your study guide (not this PowerPoint) Test questions cover the details of UNIT OBJECTIVES found only in your Textbook! 1  9/20/2024 Introduction Cellular defense mechanism –mediated by macrophages, T cells, and dendritic cells. Humoral mechanism –B cells produce specific antibodies –Complement binds to immunoglobulin molecules that have specific complement receptor sites. Introduction (cont’d) Detection of allo- and auto- antibodies –depends upon binding forces between antigens and antibodies, properties of the antibody itself, and individual host characteristics. –Antigen-antibody reactions are influenced by a number of factors, including distance, antigen- antibody ratio, pH, temperature, and immunoglobulin type 2  9/20/2024 Overview of the Immune System Innate or natural immunity Acquired or adaptive immunity Cellular and humoral immunity –Cellular defense mechanism: cells involved –Humoral defense mechanisms: antibodies and complement Overview of the Immune System (cont’d) Types of Antibodies –Autoantibodies: directed against self antigens –Alloantibodies: directed against non-self antigens Antigen: molecule found on the surface of foreign cells or on damaged internal cells 3  9/20/2024 Overview of the Immune System: Cells B cells – B memory cells – Plasma cells T cells – Th – turns on immune system – Tc - kills tumor cells and turns off immune system – T memory – remembers Antigen-presenting cells – Macrophages – Neutrophils – Dendritic cells – Some B cells Overview of the Immune System: Cells NK cells – Kill tumor cells Immune system organs – Primary lymphoid organs – Secondary lymphoid organs MHC Class I and Class II antigens – Cell membrane proteins that help T-cells recognize foreign antigens – MHC I helps T cytotoxic cells (CD8) – MHC II helps T helper cells (CD4) 4  9/20/2024 Overview of the Immune System: Cells (cont’d) Cell linage markers - specific cell receptors – Cluster Designation (CD) pluripotent stem cell -- CD 34 Granulocytes – CD11b, CD16, CD35 T cells – CD2, CD3, CD4, CD8 B cells – CD19, CD20, CD21, CD22, CD35 NK cells – CD16, CD56 Overview of the Immune System (cont’d) Importance of cytokines and immunoregulatory molecules – lymphokines – Monokines – See pages 49 and 50 for cytokine charts and functions Major Histocompatibility Complex (MHC) – Class I HLA molecules – Class II HLA molecules – Role of MHC Class I and Class II molecules 5  9/20/2024 Characteristics of Immunoglobulins  Heavy chains Gamma (IgG) Alpha (IgA) Mu (IgM) Delta (IgD) Epsilon (IgE) Characteristics of Immunoglobulins Light chains – Kappa – Lambda Basic immunoglobulin structure – Papain and pepsin digestion – Disulfide bonds – Constant and variable regions FC region binds complement Fab region binds antigen 6  9/20/2024 Characteristics of Immunoglobulins Immunoglobulins significant for blood banking – IgG, IgM, and IgA: most significant – Reaction temperatures – Naturally occurring antibodies – Commonly encountered IgM and IgG antibodies – IgG subclasses – Role of IgE – Role of IgD Significant Immunoglobulins for Blood Bank IgG – most significant –Reacts at body temperature IgM –Testing problems Mask reactivity of IgG’s –Found in ABO system Lewis system MNS system Ii system P system 7  9/20/2024 IgG Gamma heavy chain Reacts at 37oC Size – Sedimentation coefficient – 6.7 / Molecular weight, 150,000 Crosses placenta Fixes complement Found only in monomer form Opsonizes foreign material Transfusion reactions and HDN IgM Mu heavy chain Size –Sedimentation coefficient – 19 / Molecular weight, 900,000 Does not cross placenta Fixes compliment Found in pentamer form Opsonizes foreign material 8  9/20/2024 IgA Alpha heavy chain Size – Sedimentation coefficient 7-15 / Molecular weight 160,000 to 500,000 Found in body fluids Does not cross placenta Does not activate complement Found in both monomer and dimer forms Has secretory component IgE Epsilon heavy chain Size: – Sedimentation coefficient – 8 / Molecular weight 196,000 Does not cross placenta Does not activate complement Found only in monomer form Associated with hypersensitivity 9  9/20/2024 IgD Delta heavy chain Size – Sedimentation coefficient 7 / Molecular weight 160.000 Does not cross placenta Does not activate complement Found only in monomer form Characteristics of Immunoglobulins Immunoglobulin variation – Isotype – antibody variants present in all members of a species – Allotype – antibody variant in a constant region that does not occur in all members of a species – Idiotype – antibody variant in variable region and is specific for each antibody molecule Immunoglobulin Fc receptors – IgG subclasses involved IgG1 and IgG3 can attach to phagocytic receptors Allows for removal of incompatible RBCS – Cells involved Macrophages and monocytes 10  9/20/2024 Complement System Biological roles – Direct lysis of cells, bacteria and viruses Mediation of inflammation – Split fragments cause increased vascular permeability, smooth muscle contractions, chemotaxis, migrations and adherence Circulating and cell membrane proteins – Inactive forms as proenzymes except factor D – Once activated they in a cascade of events Activation pathways – Classical – Alternative – Lectin – Membrane attack complex – Binding of complement by RBC antibodies Can destroy RBC’s needed in transfusion Complement System  Classical Complement Pathway Activation when antibody binds to antigen Activation of components Fragments with anaphylatoxin activity Membrane attack complex formation Cell lysis 11  9/20/2024 Complement System Alternative complement pathway Activation of alternative pathway – Surface contacts with complex molecules Important protein factors – Factor D – Factor B – Factor P – C3 Complement System Lectin Complement Pathway – Activation Attachment of Mannose Binding Lectin to microbes – Elements common with classical pathway 12  9/20/2024 Complement System Membrane Attack Complex (MAC) –Initiation of MAC in the classical pathway –Initiation of MAC in the alternative pathway –Complement mediated cell destruction Complement System Binding of complement by RBC antibodies –Activation by IgG Binds less efficiently due to small size 2 molecules are needed in close proximity to bind –Activation by IgM Can activate complement –IgG Rh antibodies Do not usually bind complement –IgM Lewis antibodies Can bind complement but rarely cause HTR –ABO antibodies Can activate complement and cause HTR 13  9/20/2024 Characteristics of Antigens Initiate formation of and reactions to antibodies Antigen characteristics influencing immune response: – size, – complexity, – conformation, – charge, – accessibility, – solubility, – digestibility, – chemical composition Different blood group antigens differ in their immunogenicity Characteristics of Blood Group Antibodies Polyclonal antibodies Monoclonal antibodies Naturally occurring antibodies – Produced without transfusion, injection, pregnancy – IgM, RT or lower, activate complement, may be hemolytic at 37°C –ABH, Hh, Ii, Lewis, MN, P 14  9/20/2024 Characteristics of Blood Group Antibodies Immune antibodies –Transfusion or pregnancy –IgG, 37°C –Require AHG for detection –Rh, Kell, Duffy, Kidd, Ss Characteristics of Blood Group Antibodies Unexpected antibodies – All antibodies directed against red cell antigens except for the ABO group are considered UNEXPECTED. Naturally occurring ABO antibodies –Isoagglutinins Unexpected antibodies –Detection techniques –Importance in pretransfusion testing 15  9/20/2024 Characteristics of Blood Group Antibodies Alloantibodies and autoantibodies Production of alloantibodies – Produced after exposure to genetically different (non- self antigens Production of autoantibodies – Produced in response to SELF antigens – Cold auto or Warm auto antibodies –Associated with autoimmune disease –Special transfusion techniques needed Characteristics of Antigen-Antibody Reactions Intermolecular binding forces Antibody properties –Affinity – First attraction –Avidity - Strength of Binding Antibody specificity –Specific reaction –Cross-reaction –No reaction 16  9/20/2024 Characteristics of Antigen-Antibody Reactions Host factors influencing immune response – Age, Health, Immune status Duffy system and malaria – Those who do not inherit the Duffy Blood system antigens (Fya or Fyb) are resistant to malarial invasion. Immune tolerance effects – Lack of an immune response or an active immunosuppressive response. Detection of RBC Antigen-Antibody Reactions Blood samples required – Serum – Plasma (EDTA) Different tests may require different samples – Serum Ensures adequate amounts of complement are available – Plasma (EDTA) – MOST COMMON EDTA binds calcium and will obstruct compliment activation – Plasma (Sodium Heparin) Inhibits the cleavage of C4 and obstructs compliment activation 17  9/20/2024 Traditional Laboratory Methods Hemagglutination – Major technique used in Blood Banking – Red cell agglutination reactions Precipitation – Soluble antigen + soluble antibody to create an insoluble antigen-antibody complex Agglutination Inhibition – Non-agglutination is a positive test Hemolysis – Antigen-Antibody reaction has occurred, and complement has been fixed and RBC lysis has occurred. ELISA (EIA), IF, WB Red Cell Agglutination Reactions Sensitization – Antigen binding to the antibody occurs Lattice formation – Multiple antigen-antibody bridges between RBC antigens and antibodies is formed. This allows for agglutination to form 18  9/20/2024 Factors Influencing Agglutination Reactions Centrifugation – Decreases reaction time by increasing the gravitational forces on the reactants and brings them closer together. Zeta potential – RBC’s natural repulsive effect Antigen-antibody ratio – Prozone – Equivalence – Postzone Factors Influencing Agglutination Reactions Effects of pH – Ideal for Ag/Ab complexes is 6.5 – 7.5 Temperature – IgM react best at 22 C or below and are seen at the Immediate Spin Phase of testing – IgG react best at 37 C require incubation and Anti-Human Globulin reagent to be seen Immunoglobulin type – IgM antibodies of importance are: ABH, Ii, MN Lewis, Lutheran and P – IgG antibodies of importance are: Ss, Kell (Kk, Jsa, Jsb, Kpa, Kpb) Rh (DCcEe), Luthern (Lub), Duffy (Fya, Fyb) and Kidd (Jka, Jkb) 19  9/20/2024 Factors Influencing Agglutination Reactions Different techniques for IgG and IgM – IGM – Immediate spin reactions; ABO testing – IgG – 37 C Incubation; Antibody screening Crossmatch testing – IgG – AHG reagent; Antibody screening, Antibody Identification and Crossmatch testing Enhancement media – especially for IgG antibodies – Also known as Potentiators – AHG – Cross-links sensitized cells – LISS – Causes RBC’s to take up antibody more rapidly – Enzymes – Reduce surface charge; can destroy or enhance different RBC antigens Protein media – 22% Albumin –Adjusts the zeta potential between RBC’s – PEG – Increases test sensitivity by causing closer proximity of RBC Factors Influencing Agglutination Reactions (3 of 3) Zeta potential, sialic acid in red cells Low Ionic Strength Media (LISS) Polyethylene Glycol (PEG) and polybrene Proteolytic enzymes Antihuman Globulin (AHG) reagents Chemical reduction of IgG and IgM 20  9/20/2024 Monoclonal versus Polyclonal Reagents Differences in manufacturing techniques Disadvantages of monoclonal reagents – Overspecificity – Complement may not be fixed in antigen-antibody reaction – Oversensitivity Nontraditional Laboratory Methods Flow Cytometry – Used to obtain quantitative and qualitative data on cell populations to sort different cell populations – In Blood Bank it is used to: Quantify fetomaternal hemorrhage Study transfused cells Distinguish heterozygous and homozygous antigen expression 21  9/20/2024 Diseases Important in Blood Bank Serologic Testing Immunodeficiency Hypersensitivity Monoclonal and polyclonal gammopathies Autoimmune disease Hemolytic Disease of the Newborn (HDN) Blood Product Transfusions and the Immune System Transfusion-Related Immunomodulation (TRIM) – Immune system may undergo transient depression following a blood transfusion – Causes an increased risk of developing an infection – Cells, cytokines involved in TRIM Alloreactive lymphocytes are inactivated and removed Immune system unresponsive due to failure of T cells to respond Cells are inhibited by cytokines – Effects of leukoreduction Transfusion of Leukoreduced packed cells has decreased the risk or TRIM 22  9/20/2024 Postamble READ the TEXTBOOK for the details to answer the UNIT OBJECTIVES. USE THE UNIT OBJECTIVES AS A STUDY GUIDE All test questions come from detailed material found in the TEXTBOOK (Not this PowerPoint) and relate back to the Unit Objectives 23