Fundamentals of Immunology for Blood Bankers PDF

**CHAPTER 3** **Fundamentals of Immunology for Blood Bankers** **(IS) Immune System:** IS can be divided into two parts: a. b. The IS responds against damaged (senescent RBC or diseased cells (tumors) in the host body. Comparison of the major mechanisms of the immune system Table 3 -- 1 ![]...

**CHAPTER 3** **Fundamentals of Immunology for Blood Bankers** **(IS) Immune System:** IS can be divided into two parts: a. b. The IS responds against damaged (senescent RBC or diseased cells (tumors) in the host body. Comparison of the major mechanisms of the immune system Table 3 -- 1  **Cellular and Humoral Immunity** (IS) Component of two major components: 1. Cellular port is mediate by various cells of the IS "macrophages, T-cells and dendritic cells", lymphokines are communicate between cellular system also include cytokines and chemokines. 2. The Humoral port of the system are fluid port include *antibody* and *complement components* found in the plasma, saliva and other secretions. The function of the antibody is to bind to foreign molecules called antigen. Antigen-Antibody complex formation inactivates the antigen and elicit a number of complicated effect on mechanism the will ultimately result in the destruction of the antigen and the cell to which is bound. Antigen-Antibody reaction called serology. - **Innate Immunity and Acquired Immunity** 1. The innate system is less complicated and more primitive, primary lines of defense not specific where the natural presented at birth immediately available may by a. Physical barriers-like intact skin, mucosal, membrane, cilia and cough reflex. b. Biochemical secretions. sweat, tears, saliva, mucous, very low PH of vagina and stomach. Mechanism does not alter on repeated exposure to any specific antigen. 2. second line of defense:- a. Cellular Immunity -- Phagocytic cells include mononuclear cells (monocytes in plasma and macrophages in tissues), Polymorph nuclear cells (neutrophils, basophils and eosinophils) and Natural killer (NK) cells which can recognize host cells that have been infected with virus or bacteria or abnormal host cells "tumor" before acquired immune response can be activated and can secrete very potent cytokines "interferon". b. Humoral (fluid)/Biochemical Immunity, - **Cytokines** some have some function and other have specific function, some work together and some oppose the function of other cytokines and help to regulate the immune response in terms of specificity, intensity and duration. **Interferon** -- destroying virally infected cell and helps stop this rapid progression of disease, **Interleukins** act as signaling between leukocytes to become active and to divide to increase their numbers. - Complement -- alternate pathway by recognition of polysaccharides and liposaccharides on the surfaces of bacteria and tumor cells. c. Acute Inflammatory Reaction is imitated by any type of tissue damage which elicit an inflammatory response including:- i. Increase blood flow of damaged area. ii. Increase blood vessels permeable at that site to allow greater flow of cells. iii. Possible activation of heat-shock proteins. iv. Maintain the healing process at site of damaged tissue. v. In acute phase also liver produce proteins -- C reactive protein, fibrinogen and amyloid protein as highly inflammatory cytokines. - **Acquired or Adaptive Immunity** **Lymphocyte** 1. 2. 3. 4. 5. Different cell has markers which can distinguish between them called Cluster of Differentiation (CD). (IS) has adapted to be able to recognize many of different antigen with a specific antibody. The IS gets around this is by activate host cells that recognize the antigen and multiplying them to a great number that cells called Progenition which divided many times to make clone of genetic copy and all clones attack the same antigen. If antigen recognized by different cells, the cells make colonies and produce many different antibody for different epitops for antigen. Immune memory is also acquired when lymphocytes recognize antigen from previous encounters. Some of lymphocyte cell remain in the circulation to rapid immune response for repeated pathogen. - B-cells are responsible to produce antibody and can bind to antigen by antibody secreted from plasma cell "mature B-cell" or direct contact to pathogen where antibody membrane-bound. B-cells can remain in the host for providing life-long immunity and called Memory B-cells "which have antibody on their surfaces. "Opsonization" binding antibody-Antigen makes lysis in the cell by binding complement to antibody. Pathogen can be destroyed intra-or extravascular. - T-cells -- one of the major and important function of (IS) is recognize antigens of the host cell where can be recognized Major histocompatibility Complex (MHC) cell membrane-protein which produce by MHC genes *MH* that determine the humor leukocyte antigen (HLA) on cells. MHC Class 1 genes code for the HLA-A, HLA-B and HLA-C while MHC Class 2 genes code HLA-DR, HLA-DQ and HLA-DC. **The Functional of T-Cells as follow:** 1. Produce immune mediating substance "cytokines" by TH1 or TH2 when recognize antigen", along with MHC Class II CD4 to stimulate B-cells and macrophages and TC cells. 2. Killing cells that contain foreign antigen which interact with MHC Class I and by receive help from TH cells then destroy tumor cells infected cells and grafts tissue. **Antigen Presenting Cells (APCs)** **the antigen presenting cells consist of macrophages** - neutrophils -- some B-cells **dendritic cell** present in the skin "Langerhans cells", **Nervous tissue** → glial cells **Lymph nodes**, spleen, intestines, liver → "Kupffer cells", thymus bone → osteoclasts; all of them can phagocytize the foreign antigen. **Immune System Organs:** 1. Primary lymphoid organs are thymus and bone marrow where cells differentiate and mature. 2. Secondary lymphoid organs are lymph nodes and spleen and mucosa -- associated tissue where cells interact with each other and antigen. **Immune Maturation** The immune responses usually take days to months. Latency or window period; antigen not detect and antibody not produce. "Log phase time of exposure to immune response occur. T and B cells are very active to initiate the primary response to the antigen. Primary antibodies are IgM. Secondary responses are IgG. After antigen is cleared, Memory cells are stored in immune organs of the host. This mechanism is all (IS) to be rapid and strong response to repeated antigen where IgG antibodies high quantitative and higher avidity for antigen. **Cell Lineage and Markers** Hematopoietic stem cells, "Pluripotent hematopoietic progenitors" or "CD34 -- positive cells". **Myeloid Path-way** monocytes → macrophages "mononuclear phagocytic system (MPS) polymorph nuclear cells (PMNs) neutrophils, eosinophils and basophils. Also erythrocytes and platelets. **Lymphoid Lineage,** T cells which divided to T(H) T Helper TH~1~ and TH~2~, B cells and NK cells. FC receptor and complement receptor (CD16) CR1 are used by phagocytes during opsonization. PMNs have FC receptors (CD16) CR1 (CD35), C5a receptors and CR3 (CD11b). **Eosinophils** play critical role in allergic reaction and inflammation in parasitic infection. **Basophils and mast cells** possess high-affinity FC immunoglobulin and allergic reaction and can cause the release of localized histamine. **B-cells** possess complement receptor CD35 and CD21, FE receptors for IgG and CD19, CD20 and CD21 markers. **NK cells** "third population" referred to as large granular lymphocytes and possess CD56, CD16 and able to lyses virally infected cells and tumor cells directly in a process known as antibody-dependent cell mediated cytotoxicity (ADCC) throw FC receptor. **Cytokines and Immunoregulatory Molecules** Cytokines are soluble protein or peptide molecules that function as powerful mediators of the immune response. **Cytokines function**: 1. 2. 3. **The major classes of cytokines include**:- Interleukin (IL), Interferon (IFN), Tumor necrosis, Factor (TNF) and colony stimulating factor (CSF). Cytokine act by binding to specific target cell receptors. Chemokines are attractant molecules that interact between cells, immunoglobulin and complement protein. **Basic Immune System Genetics** Not all transfusion recipients make antibodies to alloantigen on transfused RBCs depend on individual if he high responders or low responders. During early development and differentiation lymphocyte germ line DNA can move to another position on the chromosome. A rearrangement is necessary to bring together sets of gene that result in complete new protein arrangement. B-cell form specific antibody and also can make a different isotype for same antigen specificity. Isotype switching requires further DNA arrangement in mature B-cells. Isotype like antibodies react at the different temperature and phases. The genetic recombination events that required to produce mature B and T cells that secret antibodies and have T cell antigen receptors (TCR) respectively with defined specificity are under control of the RAG-1 and RAG-2 genes. MHC Class I are found on all nucleated cells except trophoblast and sperm and play a key role in (T Cell) function. MHC Class II found on APCs. **Immune Suppression** Can cause by:- 1. 2. 3. 4. 5. 6. 7. 8. Immunosuppressed and immunocompromised patient must receive irradiated blood products so the donor lymphocytes do not engraft and attack the recipients IS and destroy it. **Characteristics of Immunoglobulin** Also called antibody is a complex protein produced by plasma cells where bind to antigen or neutralize toxic substances in the circulation. Ig are classified according to the molecular structure of their heavy chain to IgG, IgA, IgE, IgD, IgM. Characteristic of serum immunoglobulin show in Table 3-2. Table 3-2 Characteristic IgA IgD IgE IgG IgM --------------------------- --------- ------- --------- ------- ------ Heavy Chain Type Alpha Delta Epsilon Gamma Mu Sedimentation coefficient 7-15 7 8 6.7 19 Molecular weight (kD) 160-500 180 196 150 900 Biological half life days 5.8 2.8 2.3 21 5.1 Placental transfer no no no yes No Complement fixation no no no \+ +++ Agglutination in saline \+ no no ± ++++ kD=Kilodaltons A. **Immunoglobulin (IG) Structure** The basic Ig structural unit is composed of four polypeptide chains:- - two identical light chains - two identical heavy chains - covalent disulfide bonding holds both the light and heavy chains together. - The two heavy chains interconnected by disulfide linkages in the hinge region of the molecule. All heavy chain consists of constant region and variable region. The enzyme papain splits the antibody at hinge region. Fc portion responsible for complex fixation as well as monocyte binding by Fc receptors on cells and placental transfer (IgG only). Fab portions responsible for binding antigen. B. **Immunoglobulin Significant for Blood Banking** IgG, IgM and IgA have significance for transfusion. Most clinically significant antibodies react at 37°C like IgG. ABO antibodies are IgM encountered as naturally occurring. They produce due to response to antigen-like intestinal flora and pollen grains, also the Lewis, I; P and MNS-Produce IgM which react best at (22-24°C). IgM exists in both monomeric and polymeric form as (pentamers) containing a J (joining) chain. *B-2- mercaptoethanol (2-ME)* or *dithiothreitol (DTT)* can cleavage covalent bonds **J chain** by sulfhydryl reducing reagent. IgG antibodies are significant because can cause (HDN). Biological properties of IgG subclasses in the Table 3-3 Table 3-3 Characteristic IgG~1~ IgG~2~ IgG~3~ IgG~4~ ------------------------------------------------------------------------------------------------------------ -------- -------- -------- -------- Proportion of total serum IgG (%) 65-70 23-28 4-7 3-4 Complement fixation classical pathway ++ \+ +++ \- Binding to microphage Fc receptors +++ ++ +++ ± Ability to cross placenta ++ ± ++ \+ Dominant antibody activities Anti -- RH ++ \- \+ ± Anti -- Kell \+ \- \- \- Anti - Duffy \+ \- \- \- Anti - platelet \- \- \+ \- Biological half life (days) 21 21 7-8 21 (-)= absent; (±)= weak or negative; (+)= slight reactive; (++)= moderate reactive; (+++)= strong reactive. IgG subclass have different in constant regions of the gamma heavy chain and the number of disulfide bonds between the two heavy chains in the hinge region. HDN has been most often associated with IgG antibody. The IgM, IgA, exists in two main forms, a monomer and polymer forms. IgA is usually found in mucosal tissues. Anti-IgA can cause potentially fatal anaphylaxis. IgE is monomeric form important in allergic reaction. Fc portion of IgE attaches to basophils and mast cells and facilitate histamine release. IgD have function that deal primarily with maturation of B cells into plasma cells which bound to the membrane. IgD necessary for regulatory roles during B-cell differentiation and antibody production. C. **Ig Variation** Types of Antibody-inherited Variation 1. Isotype -- refers to variant in all members of species "IgG, IgM, IgA, IgE, IgD. 2. Allotype -- variation is present in constant region. 3. Idiotype -- variation is present in antigen-binding specificity. D. **Ig Fc Receptors** Macrophages and monocytes have receptors to IgG~1~ and IgG~3~ which bind at CH3 domain of the Fc portion. Also, other phagocyte NK, neutrophils and mature B-cells. **Complement System** Complement is a complex group that have a multitude of function within the immune response. **Primary role:** - direct lysis of cell - direct lysis of bacteria - direct lysis of involve of viruses - assisting with opsonization to facilitate phagocytosis. **Another role:** - Production of peptide fragment split products to stimulate inflammation and immune response. - The complement component circulated in inactive form except factor D. **The complement component**:- 1. C1 to C9 2. Serum protein "factor B", factor D, factor P (properdin) 3. Activation pathways require Ca²+ and Mg²+ 4. Inhibitor proteins:- C1 inhibitor (C1INH) Factor H, factor I, C4-binding protein (C4BP), anaphylatoxin inactivator, anaphylatoxin inhibitor, membrane attack complex (MAC) inhibitor and C3 nephritic factor (NF). The complement activated by:- 1. Classical complement pathway -- activated by the binding of an antigen with IgM, IgG~1~ or IgG~3~ antibody. 2. The alternative pathway is activated by high molecular weight "polysaccharides and lipopolysaccharides found on the surfaces of target cells ". Bacteria, fungi, parasites and tumor cells. 3. The Lectin pathway is activated by attachment of plasma Mannose-Binding Lectin (MBL) which activates proteins of the classical pathway. Figure 3-1 **Membrane Attack Complex** After the attachment of C9 to C5b678 complex, a small transmembrane channel or pore is formed in the cell membrane which had to osmotic lysis and subsequent death of the cell. Binding of complement by RBC Antibodies. - C1 molecules bind with two adjacent Ig Fc regions. - C1 need two IgG and one IgM - IgG antibodies against the Rh antigen usually do not bind complement due to the low level of Rh antigen on RBC surfaces. Anti-Lewis blood group IgM and can activate complement but rarely cause HTR due to low optimal reactivity temperature. IgM coated RBC are not eliminated through Fc receptor-mediated phagocytosis. RBCs are coated with only C3b not cleared but only sequestered temporarily. **Characteristics of Antigens:** Antigen "can initiate formation of and react with antibody. Immunogen "can initiate an immune response immunogenic. Increase size of antigen molecular weight (MW) more than 10,000 Daltons. MW less than 10,000 Daltons called haptons. Hapton can stimulate IS, if this protein antigen MW \> 10,000 Daltons. More chemically complex produces more rigorous immune response. Change is important to react antibody to antigen. Accessibility of epitops influences. Less soluble antigens are less elicit an immune responses. Chemical composition of antigen which can stimulate IS. **Characteristics of Blood Group Antibodies** 1. **Polyclonal Antibodies** -- produced by B-cell which produces antibodies against one antigen for different epitope. - Polyclonal reagent antisera produced by animal or human can give confusing test results. - Individual sera "Ig can recognize nonspecific epitops" cross-reacting. 2. **Monoclonal Antibodies** -- produced by B-cell by hybridoma technology which produce antibodies for specific single epitope. - Monoclonal antibodies are preferred in testing because they are highly specific well-characterized and uniformly reactive. 3. **Naturally occurring**:- - The RBC antibodies are considered naturally occurring when they are found in the serum of individuals who have never been previously exposed to RBC antigen by transfusion, injection or pregnancy. Most of antibodies are IgM. 4. **Immune Antibodies** - The RBC antibodies considered immune when found in the serum of individual who have been transfused or pregnant. Most of Antibodies are IgG. 5. **Unexpected Antibodies** - Anti-A and anti-B antibodies are routinely detected in human serum and useful to confirm blood group. - All other antibodies directed against RBC antigen are considered unexpected. 6. **Alloantibodies** are produced after exposure to non self antigen. 7. **Autoantibodies** are produced in response to self antigen. - Some autoantibodies do not have detectable specificity and are referred to as pan-or polyagglutinins. **Characteristics of antigen-antibody Reaction** Intermolecular Binding Forces c. d. e. f. g. **Antibody Properties:** 1. Antibody affinity -- strength of a single antigen-antibody bond attractive and repulsive forces. 2. Avidity used to express the binding strength of a multivalent antigen with antisera produced in an immunized individual. 3. Specificity is related to antibody relative avidity for antigen. a. b. c. 4. Valence of an antibody. **Host Factors**:- 1. Nutritional status -- malnutrition lead to reduction in TH cells → low affinity of antibody. 2. Hormones -- Hormone receptor found on various immunologic cells "can enhance and suppress immune response". 3. Genetic inheritance of MHC. 4. Age immune response decrease with age increase. 5. Individual race may be factor in susceptibility to certain disease. 6. Sex 7. Physical activity 8. Environmental exposure 9. Healthy **Tolerance** -- is defined as the lack of an immune response or an active immunosuppressive response. Detection of Ig in the Blood Sample: - Serum → viable complex. - EDTA "Ethylene-diaminetetraacetic-acid - Sodium heparin - Plasma samples are preferred for DAT and elution studies because they lack fibrin strands which cause false-positive. - Complement activity remain 48H at 4°C or frozen at - 50°C. - For deactivate complement in the serum, heating serum at 56°C for 30min. which destroys C1 and C2 and damages C4. - Factor B of the alternate pathway is inactivated by heating at 50°C for 20min. - Complement system can be activated during storage in the blood bag, by alternate pathway through contact of plasma C3 to plastic surface of blood bag and may cause hemolysis. **Traditional Laboratory Methods** 1. 2. 3. 4. 5. **Factors that influence Agglutination Reaction** 1. **Centrifugation** -- cause decrease reaction time by increase the gravitational forces on the reactants and bringing reactants closer together. 2. **Antigen-Antibody Reaction** h. Equivalence: Antigen-Antibody reaction that is necessary for agglutination reaction to occur. i. Prozone: Excess of unbound immunoglobulin. j. Postzone: Excess of antigen. 3. **pH** -- Antigen-Antibody reaction occur at range of PH 6.5 -- 7.5. 4. **Temperature** -- IgM Antibody react at \< 22°C; IgG Antibody react at 37°C. 5. **Ig Type** -- IgM antibodies react and agglutinate RBCs suspended in 85% - 90% saline media. IgM molecule can bind 10 separate antigen sites while IgG can bind only 2 separate antigens. 6. **Enhancement Media** -- accomplish this by reducing the zeta potential of RBC membrane. "negative charge surrounding RBC repels the RBC from each other due to sialic acid on the surface of RBCs. a. **protein media** Colloidal substances, colloids are a type of clear solution that contains particles permanently suspended in solution include Albumin, polyethylene glycol (PEG), polybrene, polyvinylpyrrolidone (PVP) and protamine. These substance work by increasing the dielectric constant, which then reduce the zeta potential of RBC. b. **Low ionic strength solution (LISS)** LISS contain 2% sodium chloride, they decrease the ionic strength of reaction medium which reduce the zeta potential. Increase rate of antibody uptake during sensitization and decrease incubation time (5-15min). c. **Polyethylene -- Glycol (PEG) and Polybrene** Polyethylene -- Glycol (PEG) and Polybrene are macromolecule additives used with LISS to bring sensitized RBC closer to each other to facilitate antibody cross-linking and agglutination reaction. d. **PEG** PEG produce very specific reaction with reduction in false positive or nonspecific reaction also useful for detection of weak antibodies. e. **Proteolytic Enzymes** Proteolytic Enzymes are protein molecules that function by altering reaction condition and bring about changes in other molecules without being changed themselves. used in the detection and identification, enzyme include: ficin, papain, trypsin, bromelin. treating cell by release of sialic acid from the membrane then, decrease zeta potential. Enzyme remove hydrophilic glycoproteins from the membrane causing the membrane to become more hydrophobic, which would allow RBC to come closer together. Enzyme enhance Rh, Kidd, P, Lewis and I antigen. Enzyme denature Fy(a,b), MNSs. f. **AHG Reagent** AHG determine if RBCs are coated with antibody or complement or both. AHG allow for agglutination to occur by cross-linking the antibodies on the RBCs. g. **Chemical Reduction of IgG and IgM Molecules** - **DTT** (Dithiothreitol) and β-2-mercaptoethanol (2-ME) break the J (joining) of IgM and leave IgG. - **ZZAP**, consists of thiol reagent plus proteolytic enzyme causes the dissociation of IgG molecules from the surface of sensitized RBCs and alters the surface antigen of the RBC. **Monoclonal versus Polyclonal Reagent** - Polyclonal reagents are directed against multiple epitops for one antigen. - Monoclonal reagents are directed against specific epitops made by "hybridoma technology". Monoclonal reagents are produced from immortal clones maintained in vitro, no batch variation exist nearly unlimited high titers of antibodies. The IS immortal clones can be kept growing in vitro culture for years without loss of production. Monoclonal can react strongly with the small quantities of antigen. **Disadvantages of Monoclonal** - over specificity which lead to false-positive. - complement may not be fixed in the antigen-antibody reaction which lead to false-negative result. These advantages may resolve by blend of different monoclonal reagent or blend with polyclonal reagent. **New and Nontraditional Laboratory Method** 1. **Flow Cytometery:-** Fluorescence-assisted cell sorting (FACs) and flow cytometery makes use of antibodies that are tagged with a fluorescent dye. Fc can be used to obtain quantitative and qualitative date on cell population and to sort different cell population. The principle of flow cytometery is based on the scattering of light. Application: 1. 2. 3. 4. 2. **Solid-Phase Adherence Test:-** These tests use antigen-antibody reaction and adherence to a solid phase support system as opposed to hemagglutination in solution to determine if a reaction is positive or negative. **3. Gel Test** The gel test uses a type of gel matrix instead of saline to separate positive and negative reaction. - Used for cell antigen detection and identification - Serum antibody detection and identification - Cross-matching **4. RBC Affinity Column Test (ACT)** ACT cards have an immunoreactive gel with mixture of the high affinity Fc receptor binding reagent, protein G and protein A. \- Protein G bind to all subclasses of IgG. \- Protein A binds to IgG~1~, IgG~2~, IgG~4~ \- Protein A and G place of AHG. **Disease Important in Blood Bank Serologic Testing** 1. Immunodeficiency Box 3-1. 2. Hypersensitivity -- allergy is an inflammatory response to a foreign antigen and can be cell or antibody-mediated or both. **Type I reaction** "anaphylaxis or immediate hypersensitivity" involve histamine, Ig-A deficient, Urticarial reaction "transfusion of plasma product or certain food allergens. **Type II** involve IgG, IgM, complement, phagocytes and proleolytic enzyme and cause HDN, HTR and AIHA. **Type III** involve IgG, IgM, complement phagocytes, proleolytic enzyme plus tissue damage like a drug induce hemolytic reaction. **Type IV** reaction -- involve only T-cell mediated response and their cytokines such as graft versus host, for Immunocompromised patient must receive irradiated blood products. 3. Monoclonal And Polyclonal Gammopathies -- increase concentration of serum proteins can cause nonspecific aggregation like multiple myeloma patient. 4. Autoimmune Disease, autoantibodies are produced against the host's own cells and tissues. 5. Hemolytic Disease of the newborn HDN can result when the maternal IS produces an antibody directed at an antigen present on fetal cells. IgG~1~, IgG~3~, IgG~4~ capable of crossing the placenta. Severe HDN is most often with IgG1.  - The immune system is very old and complex: - It interacts with other host systems and maintains the host equilibrium. - The two major roles for the IS are: - Protection from pathogens and foreign substances - Removal of abnormal and damaged host cells - The two major branches of the IS are: - Innate, or natural; the nonspecific primitive IS - Acquired, or adaptive; the specific, evolved IS - The two major arms of the acquired IS are: - Humoral, mediated by B cells and antibody production - Cellular, mediated by T cells and lymphokines - The basic mechanisms used by the IS are: - Recognition of self and nonself organisms, cells and tissues - Removal of unwanted organisms, cells, and tissues (self/nonself) - Repair of damaged host tissues - The acquired IS demonstrates: - Diversity and uniqueness: - Individual B and T cells have vast arrays of unique membrane molecules - These molecules can have configurations to match nearly any antigen in the environment. - Each individual lymphocyte has one unique receptor per cell that recognizes one epitope. - Recognition: - Lymphocytes can differentiate self molecules from nonself molecules - Specificity: - Antibodies and T cell receptors recognize and react only with the antigen that matches and fits their specific configuration. - Memory: - Selected T and B cells can remain dormant and later respond more rigorously upon second exposure of a previously recognized antigen - Tolerance: - Immune responses against the host are either removed or down regulated. - There are three types of lymphocytes: T cells, B cells and NK cells. - T cells (or lymphocytes) have the TCR, which is usually associated with the CD3 complex. T cells require APCs to respond to antigens. There are two well-characterized subpopulations of T cells distinguished by CD markers, T helper (T~H~, CD4-positive) and T cytotoxic (Tc, CD8-positive) lymphocytes. In laboratory testing, T cells can be distinguished from B cells by their ability to bind sheep erythrocytes (called the CD2 marker) as well as specific markers - T~H~ lymphocytes: - Have CD4 markers on their cell membranes - Provide B cell help to stimulate the immune response - Release lymphokines when stimulated - Recognize antigens in association with MHC class II molecules - T~C~ lymphocytes: - Have the CD8 marker on their membranes - Can eliminate specific target cells without the help of antibody (cytotoxicity). - Recognize antigens in association with MHC class I molecules - B lymphocytes (or cells) make up about 5 to 15 percent of circulating lymphocytes and are characterized by their membrane-bound antibodies (or immunoglobulins). Membrane-bound antibodies are manufactured by B cells and inserted into their cell membranes where they act as antigen receptors. - Stimulated B cells differentiate into plasma cells to secrete humoral immunoglobulin. - B cells receive T cell help for antibody production and for immunologic memory - A single B cell clone manufactures Ig of a single specificity for a specific antigen for its entire cell lifetime. - NK cells: - Also called large granular lymphocytes; play a role in host resistance to tumors and viral infections - The primary, or orginal, immune response occurs after the first exposure to an antigen. The secondary, or anamnestic, immune response happens after a second exposure with the same specific antigen. - Complement consists of a large group of different enzymatic proteins (convertases/esterases) that circulate in an inactive proenzyme form. Once the cascade is started, they activate each other in a sequence to form products that are involved in optimizing phagocytosis and cell lysis. Complement can be activated through three pathways: - The classical pathway is initiated by antigen-antibody complexes and requires C1q for activation to proceed. - The alternative pathway is activated by certain macromolecules on the cell walls of bacteria, fungi, parasites, and tumor cells and requires C3b, serum factors B, D, properdin, and initiating factor - The lectin pathway is activated by binding of MBL to microbes - All three pathways meet at a common point in the cascade and result in fprmation of the membrane attack complex to remove unwanted cells. - There are three different types of complement receptors on cells, CR1 on RBCs and platelets, CR2 on B cells, and CR3 on myeloid cells - There are five classes (or isotypes) of immunoglobulins, all of which have a basic four-chain protein structure, consisting of two identical light and two identical heavy chains. Disulfide (covalent) bonds link each light chain to a heavy chain and the two heavy chains to each other. - Antibody molecules are isotypic (based on heavy chain subtype), allotypic (based on one heavy chain mutation), or idiotypic (based on hypervariable and variable regions of light and heavy chains) and are reflected in the Ig sequences. - The N-terminal domains of both heavy and light chains are the variable regions that make up the antigen-binding site of the antibody and vary according to the antibody specificity (the CDR or complementary determining regions) and contain the Fab portion of the Ig molecule. The rest is the C terminal domains of heavy chains that contain the constant (C) and Fc regions that bind the antibody to cell membrane receptors. - An immune reaction is determined by host response as well as various characteristics of foreign substances. Many factors such as size, complexity, conformation, charge, accessibility, and biochemical composition influence the amount and type of immune response. Blood group antigens may be proteins, glycolipids, or glycoproteins. Not all blood group substances are equally immunogenic in vivo. Generally, molecules of a certain size and complexity as well as significant doses are necessary for a good immune response. Blood group antibodies may be characterized by such factors as epitope and variable region diversity (monoclonal or polyclonal), mode of sensitization (naturally occurring or immune), expected or unexpected presence in routine serum samples, isotype class (IgM or IgG or; rarely IgA), activity (warm or cold reactive or both