Immunology Key Concepts

Fundamentals of Immunology for Blood Bankers

• The immune system (IS) has two parts: defense against foreign entities and managing damaged cells
• It responds to damaged red blood cells and diseased cells in the host body

Comparison of Innate and Adaptive Immunity

• Innate immunity represents the primary defense line, naturally present at birth.
• It offers non-specific and immediate protection through physical, biochemical, and mechanical mechanisms
• Adaptive immunity supplements innate defenses and is acquired through contact with specific foreign substances
• It is specific to pathogens
• The initial contact with a foreign substance prompts the synthesis of specialized antibody proteins reactive to foreign bodies
• This creates immunological memory, which allows for improved responses upon subsequent encounters.

Cellular and Humoral Components of the Immune System

• Cellular immunity is mediated by macrophages, T-cells, and dendritic cells
• Lymphokines, cytokines, and chemokines facilitate communication within the cellular system
• Humoral immunity involves fluid-based elements like antibodies and complement components found in plasma, saliva, and other secretions
• Antibodies bind to antigens, forming a complex that inactivates the antigen and triggers mechanisms that destroy the antigen and the bound cell

Innate and Acquired Immunity

• IS prevents infection and damage
• Innate immunity is a simpler primary defense, nonspecific and present from birth

Second Line of Defense

• Cellular immunity involves phagocytic cells, including mononuclear cells, polymorph nuclear cells, and natural killer cells that detect infected or abnormal host cells
• Humoral immunity involves cytokines, which modulate immune responses in terms of specificity, intensity, and duration
• Interferon helps stop the rapid progression of disease by destroying virally infected cells
• Interleukins signal leukocytes to become active and increase their numbers

Complement Alternate Pathway

• The alternate pathway occurs by recognition of polysaccharides and liposaccharides on the surfaces of bacteria and tumor cells
• Acute inflammatory reactions are initiated by tissue damage, leading to increased blood flow, increased vessel permeability, activation of heat-shock proteins, and maintenance of healing

Acquired or Adaptive Immunity

• This depends on innate immunity, developing the ability to adapt and destroy complex pathogens
• The acquired IS employs antibodies with memory for defense against repeat pathogens

Lymphocytes

• T-lymphocytes mature in the thymus and produce cytokines to destroy infected host cells
• B-lymphocytes mature in bone marrow and secrete antibodies when stimulated
• NK cells protect against viruses
• Dendritic cells present in tissues are responsible for antigen presentation
  • Macrophages process antigens

Cluster of Differentiation

• Different cells possess markers called the Cluster of Differentiation (CD) for recognition.
• The IS activates host cells recognizing an antigen, multiplying them into a large number of cells
• The cells is called Progenition which divide many times to make clones of genetic copies that all attack the same antigen
• If the antigen is recognized by various cells, they form colonies and produce diverse antibodies
• Memory is acquired when lymphocytes recognize antigens from previous encounters; some remain in circulation for rapid immune response to repeated pathogens

B-Cells

• B-cells produce antibodies either through secretion from plasma cells or by direct contact with pathogens via membrane-bound antibodies
• B-cells can provide lifelong immunity and called Memory B-cells that "have antibody on their surfaces" called Opsonization making the cell lysis

T-Cells

• T-cells recognize antigens of host cells via the Major Histocompatibility Complex (MHC), which are produced by MHC genes
• MHC Class 1 genes code for HLA-A, HLA-B, while MHC Class 2 genes code for HLA-DR, HLA-DQ, and HLA-DC
• T-cells produce "cytokines" via TH1 and TH2 to stimulate B-cells, macrophages, and TC cells on antigen recognition or kill cells

Antigen Presenting Cells (APCs)

• APCs include macrophages, neutrophils, B-cells, and dendritic cells in the skin (Langerhans cells).
• Nervous tissue → glial cells and Lymph nodes, spleen, intestines, liver → “Kupffer cells”, thymus bone → osteoclasts
• Primary lymphoid organs are thymus and bone marrow, which are where cells differentiate and mature
• Secondary lymphoid organs are lymph nodes, spleen, and mucosa where cells interact with antigens

Immune Response Times

• The latency or window period lasts for days to months; the log phase reflects the duration of exposure to an immune response
• Primary antibodies are IgM but secondary responses are IgG, where antibodies are higher in quantity and avidity for antigens after cleared.

Cell Lineage and Markers

• Hematopoietic stem cells are "Pluripotent hematopoietic progenitors or CD34+ cells
• Myeloid Pathway: monocytes to macrophages in tissues and blood – Lymphoid Lineage: T cells (TH1, TH2), B cells, NK cells are included.
• FC receptor and complement receptor (CD16) CR1 are used by phagocytes during opsonization
• PMNs possess FC receptors (CD16) CR1 (CD35).
• Eosinophils are critical in allergic reactions and for inflammation in parasitic infection
• Basophils and mast cells possess high-affinity FC immunoglobulin, releasing histamine during allergic reactions B-cells possess complement receptor CD35 and CD21, FE receptors for IgG, plus CD19, CD20, and CD21 markers

NK Cells

• NK cells, also known as third population possess CD56 and CD16, can lyse infected cells and tumor cells through antibody-dependent cell-mediated cytotoxicity (ADCC) Cytokines some have some function and others opposing function.
• Cytokines regulate immune response with the specificity, intensity and duration
• Interferon destroys infected cells, where Interleukins signal leukocytes to become active

Cytokines

• These are soluble protein or peptide molecules mediating immune responses by lymphocytes or macrophages
• The major classes of cytokines include Interleukins (IL), Interferon (IFN), Tumor necrosis Factor (TNF), and colony stimulating factor (CSF).
• Cytokine act by binding to specific target cell receptors, also called “Chemokines" which are attractant molecules that interact between cells, immunoglobulin and complement protein.

Immune System Genetics

• During lymphocyte development, gene rearrangements form new protein arrangements, enabling B-cells to produce specific antibodies
• Isotype switching requires DNA arrangement to make antibodies react
• RAG-1 and gene RAG-2 regulate genetic recombination and antibodies produced

MHC Markers

• MHC Class I markers are on nucleated cells except for trophoblast and sperm and play a key role in (T Cell) function
• MHC Class II markers are located on APCs (Antigen-presenting cell).

Immune Suppression

• Caused by cytokines, little immunoglobulin, loss of T and B cells, immune stimulation, organ failure, drugs like corticosteroids, diseases like AIDS, genetic lesions causing a decrease in immune responsiveness
• Immunocompromised patients require irradiated blood products for transfusion to defend against donor lymphocyte attacks

Immunoglobulin Characteristics

• Antibodies are known as complex proteins produced by plasma cells
• They bind to antigens neutralizing toxic substances, classified by the structure of their heavy chain for IgG, IgA, IgE, IgD, and IgM.
• The basic Ig structure features two identical light chains and two heavychains linked by covalent disulfide bonds

Immunoglobulins

• The heavy chains are linked by disulfide linkages in the hinge region
• The enzyme papain splits the antibody at the hinge region, with the Fc section responsible for complex fixation
• Fab portions attach to antigens to the molecule
• IgG, IgM, IgA signify important blood banking antibodies where IgG reacts best at 37°C

ABO Antibodies

• IgM antibodies, ABO respond to intestinal flora, pollen, Lewis, I; P and MNS-Produce IgM which react best at (22-24°C)
• IgM is exists in both monomeric and polymeric form as (pentamers) containing a J “joining chain, B-2“
• IgG antibodies are effective with causing HDN which reacts with mercaptoethanol (2-ME) or dithiothreitol (DTT) that can cleavage covalent bonds sulfhydryl

IgG Variants

• Classes have different constant regions and disulfide bonds where HDN is associated with IgG antibodies that are capable of crossing the placenta from mother to infant

IgM, IgA, IgE, IgD

• IgA and IgM exist as monomers and polymers
• IgA is found mucosally
• Anti-IgA can trigger anaphylaxis.

IgE

• This important monomeric in allergic reactions, its FC portion attaches to mast cells and facilitate histamine release

IgD

• This primarily matures B cells and antibody production that are bound membrane.

Ig Variants

• Isotype, Allotype, Idiotype are variants that have Antibody inherited variations

Ig FC Receptors

• B-cells, and neutrophils have receptors to IgG₁ and IgG3 bind at CH3 domain of the Fc

Complement System

• Complement functions include; primary is direct lysis of cell and bacteria, secondary is facilitate phagocytosis
• The components are C1 to C9, factor “B”/D/P, Activation need Ca²+ and Mg2+
• The inhibitors proteins:- C1 inhibitor and Factor H/I (anaphylatoxin inactivator) membrane attack complex (MAC) inhibitor and C3 nephritic Factor act in process

Complement Activation

• Complement is typically activated via classical pathway by antibodies, or alternative pathways by bacteria or Lectin pathways by MBL

Membrane Attack Complex

• Cell membrane death occurs when the transmembrane structures create a pore; antibody-mediated killing occurs through complement binding
• C1 molecules require two adjacent Fc regions; IgG antibodies typically don't bind due to low antigen levels.
• Anti-Lewis IgM can activate complement, though is limited because of its limited optimal reactivity temperature

Antigen Characteristics

• Antigens are anything can form and react with antibodies by initiating an IS response

Molecular Size

• Increasing in size of molecule weight larger than 10,000 Daltons, stimulate the most rigorous IS response
• Less soluble antigens are less elicit an immunes responses.

Polyclonal Vs Monoclonal

• Polyclonal antibodies produce with different epitopes with confusing data, while Monoclonal specific and uniform when produced produce single epitopes

Antibody Classifications

• Natural: Antibodies found naturally occur and are found in individuals who have had no previous exposures to transfusions and pregnancy, commonly called IgM
• Immune: Antibodies only found in individuals that have had previous exposure to blood or those that have had a pregnancy, commonly IgG
• Unexpected: A and B antibodies, all other directed is unexpected

Alloantibodies vs Autoantibodies

• Allo-: Alloantibodies produce from those only exposed to nonself antigens
• Auto-: Autoantibodies produce upon onself, and have detectable specificities, otherwise referred as pan- polyagglutinins

Intermolecular Binding Forces

• Hydrogen/ electrostatic bonds help with the charges with molecules Van der Waal forces appear when molecule is a weak interaction between atoms, while bond that is hydrophobic results in over lap of amino acids called the protein

Antibody Factors

• Affinity, Avidity, and Specificity all interact with the attractive-repulsive force single and multivalent with how relative its antigen is
• Reactions include specific reaction or no reaction when there is similar epitope or not to the epitope

Nutritional Factors (Host)

• Nutritional status with malnutrition decreasing Th cells with lower ability to bind
• Hormones affect binding and can suppress or increase immune responses

Host Factors

• Genetic inheritance of MHC
• Age declines response levels

Potential to Increase Response

• Race, Sex, Physical Activity, Overall health

Tolerance

• This is defined as an inadequate response or inability to achieve immune response

Blood Sample Readings

• Serum, edta, and sodium heparin are used to see viable complex, while plasma is preferred for DAT and elution studies because they lack fibrin
• Complement maintains activity for 48 hours at 4 degrees Celsius, or -50 degrees Celsius when frozen
• At 56°C for 30min, serum is deactivated by heat, or inactivation of factor B occurs at 50°C after 20 min

Traditional Lab Methods

• Hemagglutination/hemolysis: Detects Ag-Ab reactions directly by the rupturing of the RBCs that is being tested

• RIA/ELISA (or EIA), IF techniques: These immunologic methods focus on radiolabels by enzyme or antigen-antibody.

Main Effects of Tests:

• Centrifugation increases gravity and time while Antigen/Antibodies make sure you have all the right reactants with them in order to not cause error

• You NEED to have an equalivalent/prozone/ and post Zone for things not to get misinterpreted.

• When pH is between 6.5 and 7.5 and Temperature is 22°C (IgM) 37/C for (IgG) with a saline media from 85%- 90% can show accurate results.

• Protein and enhancements* increase in order to reduce charge where sodium chloride in a low ionic strength reduce zeta potential (5-15minutes of sensitization and incubation)

Additional factors to be wary of for the tests:

Polythylene can add Glycol and macromolecule additives that help with the reaction/crosslink the antigents when read.

PEG produces reliable reactions and is well for detection.

Enzymes react with condition alteration and change other molecules without being able to be altered with things like " Ficin and Papain"-

AhG determine if cells are coated with antibodies. AHG allows for the link on red bloood cells. DTT reduces with its enzyme and cause the antigens on the surface of antigens.

• Reagents made in polyclonal means there are a lot of changes to happen that causes error (epitopes are increased) by the cell as opposed to
• Monoclonal which gives an exact replica

New lab methods:

1. Flow- use Antibodies for antigens and cell sorting.
2. Solid: a system used to oppose hemoglobin to see positive vs negatives
3. Gel: a type of gel used for identification.

• Affinity AcT* and proteins are added to card for reactions

1. "G" proteins for all types of IgG
2. A place where AhG
3. Protein "A" and IgG

Immunoglobulin disease:

• Autoimmune: antibodies produced from own cells and/or tissue and is from the immune system
• Monocolonal gammopathes will cause aggregatons of aggulitation
• High doeses are given to the mother for fetal cells and Igg levels can move through the mother.

Summarized chapter notes:

• The equilibrium of human is dependent on the immune system.

• Damage is caused by removal and recognition.
• TH, Tc, B are types of cells that have CD molecules -Lymphocytes can determine from self and Non-self -Antibodies attach and react if they correlate.

·Memory exists because they stay stagnant untile second exposures

·Immunoglobins are determined by disuffide.and other types of chains the result in goodimmune

·Epitopes are variable.

Explore key immunology concepts. Investigate antibody variations, complement pathways, and the membrane attack complex. Learn about antigen complexity, antibody types, and the advantages of monoclonal antibodies in diagnostics.