Chapter 3 Fundamentals of Immunology PDF

 Chapter 3 Fundamentals of Immunology 47 Table 3–1 Comparison of the Major Mechanisms of the Immune System Innate or Natural Immunity Acquired...

 Chapter 3 Fundamentals of Immunology 47 Table 3–1 Comparison of the Major Mechanisms of the Immune System Innate or Natural Immunity Acquired or Adaptive Immunity Primary lines of defense Supplements protection provided by innate immunity Early evolutionary development Later evolutionary development—seen only in vertebrates Nonspecific Specific Natural—present at birth Specialized Immediately available Acquired by contact with a specific foreign substance May be physical, biochemical, mechanical, or a Initial contact with foreign substance triggers synthesis of specialized antibody proteins combination of defense mechanisms resulting in reactivity to that particular foreign substance Mechanism does not alter on repeated exposure to Memory any specific antigen 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 Table 3–2 Cellular and Humoral Components of the Immune System Innate or Natural Immunity Acquired or Adaptive Immunity First Line of Defense Second Line of Defense Third Line of Defense Internal Components Internal Components Internal Components Physical Cellular Cellular Intact skin Phagocytic cells Lymphocytes Mucous membranes Macrophages-dendritic cells T cells Cilia Monocytes TH Cough reflex PMNs: Large granular leukocytes TC Biochemical NK cells T memory cells Secretions Humoral (fluid)/Biochemical B cells Sweat Complement-alternate pathway B memory cells Tears Cytokines Plasma cells Saliva Interferons Humoral Mucus Interleukins Antibodies Very low pH of vagina and stomach Acute inflammatory reaction Complement-classic pathway Cytokines of innate defense is external, including skin and enzymes Importantly, it is the acquired immune response that present on the skin’s surface. The second line of innate de- responds more rapidly and efficiently against a repeat attack fense is internal and can recognize common invaders with a from a pathogen by establishing the memory response. The nonspecific response, such as phagocytosis that does not IS’s specificity also prevents the host from becoming attacked have to be primed. The last line of defense is the acquired and damaged during an immune response. The localized immune response, which needs time (days to weeks) and nature of an immune reaction also prevents systemic damage reorganization to mount an effective and specific reaction. throughout the host organism. The wide variety of potential 48 PART I Fundamental Concepts organisms and substances that can invade the host requires the macrophages in tissues). Phagocytic cells collaborate a vast array of means to recognize and remove them. The with molecules of the IS. Opsonins are factors that include acquired immune response must be capable of generating antibodies and complement components in plasma that coat a near-infinite level of specific responses to all the different pathogens and facilitate phagocytosis. When phagocytes complex organisms and substances that the host can ingest foreign cells and destroy them, they can become encounter over its lifetime. activated to release soluble polypeptide substances called cytokines that have numerous effects on other cells of the Innate and Acquired Immunity immune and vascular systems (Table 3–3 is a partial list of cytokines). There are a large number of different cytokines; The immune system has two major arms, innate and some with unique functions and others with overlapping acquired immunity, that work to prevent infection and dam- functions. Some work together, and some oppose the func- aged cells from destroying the host. The innate part of the tions of other cytokines. Many are secreted, and some are system is more primitive and does not function in a specific membrane receptors. Cytokines help to regulate the immune way; rather, it recognizes certain complex repeating patterns response in terms of specificity, intensity, and duration. present on common invading organisms. The innate arm can Another important component of the innate immune sys- function immediately to stop host organisms from being tem is the complement system. Complement has three infected. Therefore, innate immunity is the immediate (first) major roles in immunity: (1) the final lysis of abnormal and line of defense from invading pathogens. pathogenic cells via the binding of antibody, (2) opsoniza- The acquired immune response is more advanced and was tion and phagocytosis, and (3) mediation of inflammation. developed after vertebrates had evolved. It relies on the for- The proteins of the complement system are enzymes that are mation of specific antigen-antibody complexes and specific normally found in the plasma in a proenzyme inactive state. cellular responses. Acquired immunity allows for a specific Three ways the complement proteins can be activated are the response, and IS memory allows resistance to a pathogen that classic, alternative, and lectin pathways; all have essentially was previously encountered. the final result of cell lysis and inflammation. The classic pathway uses antigen-antibody binding and therefore is a Innate Immunity specific activator of complement. The alternative pathway activates complement by recognizing polysaccharides and There are two important features of innate immunity. First, liposaccharides found on the surfaces of bacteria and tumor the innate immune system is nonspecific. The same response cells; therefore, it uses nonspecific methods of activation. is used against invading organisms, no matter what the The lectin pathway is activated by mannose-binding proteins source is, as long as the innate IS can recognize them as non- bound to macrophages. self. Innate immunity is present at birth and does not have Inflammation is also a critical component of the innate to be learned or acquired. Second, it does not need modifi- IS and is familiar to most people when they have a minor cations to function and is not altered with repeated exposure wound that has redness and warmth at the abrasion site. to the same antigen. Because innate immunity functions so Inflammation is initiated by any type of tissue damage, well as a first line of defense, it was maintained in the IS of whether it be to the skin or to an internal organ. Burns, vertebrates during evolution. infections, fractures, necrosis, and superficial wounds all The innate IS is comprised of physical and biochemical elicit an inflammatory response that is characterized by an barriers as well as numerous cells. Physical barriers in- increase in blood flow to the wounded area, increased blood clude intact skin, mucous membranes, cilia lining the mu- vessel permeability at the site to allow for greater flow of cous membranes, and cough reflexes. Biochemical barriers cells, a mobilization of phagocytic cells into the site, and of the innate system include bactericidal enzymes such as a possible activation of acute phase and stress response pro- lysozyme and RNases, fatty acids, sweat, digestive enzymes teins at the site of tissue damage. Eventually the wound is in saliva, stomach acid, and low pH. Innate immune cells repaired, new tissue grows in place of the damaged tissue, include phagocytic leukocytes and natural killer (NK) cells. and inflammation is stopped. Uncontrolled inflammation Phagocytic cells of different types are found in most tissues can result in unwanted damage to healthy tissues; therefore, and organs of individuals, including the brain, liver, intes- the regulation of inflammation is tightly controlled and tines, lungs, and kidneys. Phagocytes such as circulating requires signals to effectively turn it on and off. monocytes in the blood and peripheral macrophages can move between vessel walls. Phagocytes recognize complex Acquired Immunity molecular structures on the surface of invading cells or in the secretions and fluids of the host body. They remove the Acquired immunity is the other major arm of the host’s IS invading organisms by engulfing and digesting them with and is the most highly evolved. It is also the most specific vesicle enzymes. and allows the IS to have memory of pathogens it has Two major cells that can use phagocytosis to remove encountered previously. The acquired system is present only pathogens are the polymorphonuclear cells (which include in vertebrates. The term acquired refers to the fact that the neutrophils, basophils, and eosinophils) and the mono- immunity is acquired via specific contact with a pathogen or nuclear cells (which include the monocytes in plasma and aberrant cell. The term adaptive refers to the ability to adapt  Chapter 3 Fundamentals of Immunology 49 Table 3–3 Various Cytokines and Their Functions Cytokine Source Stimulatory Function IL-1 (IL-1α, IL-1β) Mf, monocytes Induction of proinflammatory proteins, enhances production of other cytokines IL-2 T Proliferation of activated T and B cells, activates NK cells IL-3 T, NK, MC Hematopoietic growth factor IL-4 T, MC, NK Proliferation of activated B, T, MC, induces B to produce IgE IL-5 T, MC Differentiation/proliferation of eosinophils, activated B, induces B to produce IgA IL-6 T, Monocytes, BM Stroma Differentiation of myeloid stem cells, convert B to plasma cells IL-7 BM and thymic stroma Production/differentiation of B, T IL-8 MF, Monocytes Chemotaxis/activation of neutrophils IL-9 T Proliferation of T and MC IL-10 T, B, Mf, monocytes Downregulates MHC class II and cytokine production by monocytes, MF IL-11 BM stroma Differentiation of pro-B and megakaryocytes IL-12 B, Mf, monocytes Enhances NK and CD8+T cytotoxicity IL-13 T, MC Upregulates MHC class II, induces B switch to IgG and IgE IL-14 T, B, and T lineage lymphomas Proliferation of activated B IL-15 T, B, NK, Mf, monocytes Proliferation of T, NK, activated B IL-16 T Induces MHC class II, chemoattractant for CD4+T IL-17 T Proinflammatory-stimulates cytokine production IL-18 Mf Induces T to produce IFN, enhances NK cytotoxicity IL-19 Monocytes Modulates T helper activity IL-20 Monocytes Regulation of inflammatory responses IL-21 T Regulation of hematopoiesis, activates B, differentiation of NK IL-22 T Inhibits IL-4 production IL-23 Dendritic cells Induces IFNγ production by T IL-24 T, Mf, monocytes Induction of TNF, IL-1, IL-6 IL-25 T, Mf, MC Induction of IL-4, IL-5, IL-13 Colony Stimulating Factors GM-CSF T, Mf, fibroblasts, MC, endothelium Growth of granulocyte and Mf colonies G-CSF Fibroblasts, endothelium Growth of neutrophil progenitors M-CSF Fibroblasts, endothelium, epithelium Growth of monocyte progenitors Steel Factor BM stroma Enhances cell division Tumor Necrosis Factors TNFα Mf, T Tumor cytotoxicity, promotes cytokine secretion, activates Mf, antiviral TNFβ T Tumor cytotoxicity, promotes phagocytosis by neutrophils and Mf, lymphoid organ development, antiviral Continued 50 PART I Fundamental Concepts Table 3–3 Cytokines and Their Functions—cont’d Cytokine Source Stimulatory Function Interferons IFNα Leukocytes Enhances MHC class II, antiviral IFNβ Fibroblasts Antiviral, enhances MHC class II IFNγ T, NK Inhibits T helper, induces B to IgG2, antagonizes IL-4 Other TGFβ T, B, Mf, MC Proinflammatory, induces B to produce IgA, promotes tissue repair LIF Thymic epithelium, BM stroma Induces acute phase proteins Adapted from Delves, PJ, Martin, SJ, Burton, DR, Roitt, IM: Roitt’s Essential Immunology, 13th ed. Wiley-Blackwell, 2017 APC = antigen-presenting cells; BM = bone marrow; CSIF = cytokine synthesis inhibitory factor, FC = immunoglobulin Fc receptor for IgE; G-CSF = granulocyte colony-stimulating factor; GM-CSF = granulocyte monocyte/macrophage colony-stimulating factor; IFN = interferon; IL = interleukin; LIF = leukocyte inhibitory factor; MC = mast cell; M-CSF = monocyte colony-stimulating factor; MHC = major histocompatibility complex; Mf = macrophage; NK = natural killer cells; PGE2 = prostaglandin E2; T = T lymphocyte; TGF = transforming growth factor; TNF = tumor necrosis factor to and destroy new complex pathogens, although it must globulin because they are a type of globular soluble protein. first react to them through complex recognition processes. They are found in the gamma globulin portion of plasma Acquired immunity is specific in recognition of new or serum when it is separated by fractionation or elec- pathogens and has specific responses, depending on the type trophoresis. The function of the antibody is to bind to for- of pathogen it encounters. eign molecules called antigens. Most antigens are found on The acquired IS uses antibodies as specific immune the surface of foreign cells or on damaged self-cells. effectors. Antigen specificity and uniqueness determine A key feature of antigen-antibody reactions is their speci- the particular antibody that will bind to it. The antigen- ficity. Only one antibody reacts with one antigen, or one part antibody complex is a three-dimensional interaction, which (an epitope or antigenic determinant) of a complex anti- helps to prevent the binding and induction of an immune gen. An immune reaction against an antigen stimulates the response by nonspecific antigens. For example, antibodies production of antibodies that will match the epitope of against one blood group antigen do not react against an- the antigen. The binding reaction of antigen and antibody other blood group antigen. An antigen that an antibody is has often been called a lock and key mechanism, referring made against is sometimes referred to as its antithetical to its specific conformation. Antigen-antibody complex antigen. Antibodies do not always remain in plasma at lev- formation inactivates the antigen and elicits a number of els observable with serologic testing, and if antigen-positive complicated effector mechanisms that will ultimately result RBC units are transfused in a sensitized patient, the second in the destruction of the antigen and the cell to which it is antibody response against the transfused cell antigens can bound. The laboratory study of antigen-antibody reactions be more vigorous, resulting in intravascular RBC hemoly- is called serology and has been the basis of blood bank tech- sis. Due to this second (memory) response, obtaining the nology for many years. Antibody screening methods such medical history of a patient who requires a transfusion is as the indirect antibody test and crossmatching techniques absolutely critical. rely on the detection of antigen-antibody complexes by screening for antibodies in the plasma or sera. The direct Cellular and Humoral Immunity antiglobulin (Coombs) test relies on the detection of anti- bodies (and complement) bound to the surface of RBCs. The two major components of the vertebrate adaptive IS are cellular and humoral immunity. Cellular immunity is medi- Cells and Organs of the ated by various IS cells, such as macrophages, T cells, and Immune System dendritic cells. Lymphokines are powerful molecules that in- clude cytokines and chemokines. These effector molecules The different types of cells within the immune system can play a critical role in cellular immunity by activating and de- be distinguished by the membrane markers they possess. activating different cells, and allowing cells to communicate These are referred to as clusters of differentiation (CD) throughout the host body. markers and are detected by immunotyping methods to Humoral immunity consists of the fluid parts of the identify cells of the immune system. These cell surface mole- IS, such as antibodies and complement components found cules specify cellular definitions and functions, including in plasma, saliva, and other secretions. One of the most maturation levels and lineage specificity. important parts of humoral immunity is the antibody, Lymphocytes, which are critical for acquired immunity, which is produced by B cells. Antibodies are also called are divided into two major types, the T lymphocyte (T cell) immunoglobulins—immune because of their function, and and the B lymphocyte (B cell). The T lymphocyte matures 60 PART I Fundamental Concepts CLASSIC PATHWAY ALTERNATIVE PATHWAY C4b, 2C MEMBRANE ATTACK MECHANISM C5b67 C5b6789 Figure 3–5. Schematic diagram illustrating the sequential activation of the complement CELL LYSIS system via the classical and alternative pathways. C3. In this pathway, complement component factor D is C3b encounters normal cells, it is rapidly eliminated through analogous to C1s in the classical pathway; factor B is analo- the combined interactions of factors H and I. The accumu- gous to C2; and the cleavage product C3b is analogous lation of C3b on microbial cell surfaces is associated with to C4. Also, factor C3bBbP is analogous to C4b2a, and attachment of C3b to factor B. The complex of C3b and fac- C3b2BbP is analogous to C4b2a3b in the classical pathway. tor B is then acted on by factor D. As a result of this action, Activation of the alternative pathway requires that a C3b factor B is cleaved, yielding a cleavage product known as Bb. molecule be bound to the surface of a target cell. Small The C3bBb complex is stabilized by the presence of prop- amounts of C3b are generated continuously, owing to the erdin (P), yielding C3bBbP. This complex cleaves C3 into spontaneous hydrolytic cleavage of the C3 molecule. When additional C3a and C3b. C3b, therefore, acts as a positive feedback mechanism for driving the alternative pathway. The C3b2BbP complex acts as a C5 convertase and initiates the IgG Antibody later steps of complement activation (see Fig. 3–5). Lectin Complement Pathway Lysed Target Cell The third pathway of activation, the lectin pathway, is acti- vated by the attachment of MBL to microbes. The subsequent Red Cell Antigenic Determinant Site reactions are the same as those of the classical pathway. Remember that all three methods of activation lead to a final common pathway for complement activation and membrane Effector Cell (Monocyte) attack complex (MAC) formation. C3b Receptor Membrane Attack Complex The final step of complement activation is the formation Fc Receptor of the MAC, which is composed of the terminal components of the complement sequence. There are two ways in which the MAC is initiated. In either classical or alternative path- Figure 3–6. Schematic representation of the mechanism of antibody-dependent cell-mediated cytotoxicity (ADCC). Note the role of effector cell surface receptors ways, the formation of C5 convertase is necessary. In the for the FC fragment of IgG. activation of the classical pathway, the MAC is initiated by  Chapter 3 Fundamentals of Immunology 61 the enzymatic activity of component C4b2a3b on C5. In the antigen is more commonly used in blood banking because alternative pathway, it is C3b2Bb that has the ability to cleave the primary testing is for the detection of antibodies to blood C5. The next step in either pathway is the same. Component group antigens. The immune reaction to any immunogen, C5 is split into the fragments C5a (a potent anaphylatoxin) including antigens, is determined by the host response and and C5b; the C5b fragment attaches to cell membranes and by several biochemical and physical characteristics of the im- can continue the complement cascade by initiating the mem- munogen. Properties such as size, complexity, conformation, brane attachment of C6, C7, and C8. After the attachment charge, accessibility, solubility, digestibility, and biochemical of C9 to the C5b678 complex, a small transmembrane chan- composition influence the amount and type of immune nel or pore is formed in the lipid bilayer of the cell mem- response (Box 3–2). brane, and this allows for osmotic lysis and subsequent death Molecules that are too small cannot stimulate antibody pro- of the cell. duction. Immunogens having a molecular weight (MW) less than 10,000 D, for example, are called haptens and usually do Binding of Complement by RBC Antibodies not elicit an immune response on their own; however, coupled with a carrier protein having a MW greater than 10,000 D, they Disruption in the activation of either complement pathway can produce a reaction. Antibodies and cellular responses are can result in damage to the host’s cells. The formation of an- very specific for an antigen’s physical conformation as opposed tibodies with complement capacity that can bring about the to its linear sequence. Overall charge is important, as antibody destruction of RBCs are of particular concern to transfusion response is also formed to the net charge of a molecule, medicine specialists. In order to initiate activation, C1 mole- whether it is positive, negative, or neutral. As an antigen must cules bind with two adjacent Ig FC regions. A pentameric be seen by the IS, the accessibility of epitopes influences the IgM molecule provides two FC regions side by side, thereby immune response. Also, antigenic substances that are less binding complement. However, a monomeric IgG molecule soluble are less likely to elicit an immune response. The bio- binds C1q less efficiently because two IgG molecules are chemical composition of the stimulus plays a role in immune needed in close proximity to bind complement. Therefore, stimulation. Remember that RBC antigens are very diverse in many molecules of IgG are required to be bound to a single structure and composition and may be proteins (such as the target in order for complement activation to occur. For Rh, M, and N blood group substances) or glycolipids (such as example, as many as 800 IgG anti-A molecules may need to the ABH, Lewis, Ii, and P blood group substances). Further- attach to one adult group A RBC in order to bind a single C1 more, human leukocyte antigens are glycoproteins. Because of molecule, so there is little complement activation by IgG these differences in structure, conformation, and molecular anti-A immunoglobulins.4 nature, not all blood group substances are equally immuno- Antibodies against the Rh antigens usually do not bind genic in vivo (Table 3–7). complement due to the low level of Rh antigens on RBC sur- faces. Antibodies to the Lewis blood group system are gener- Characteristics of Blood Group ally IgM, and as such can activate complement but rarely cause Antibodies hemolytic transfusion reactions due to their low optimal reactivity temperature. Therefore, in blood banking, with the There are many different and important characteristics of exception of the ABO system, only a few antibodies activate blood group antibodies, such as whether they are polyclonal the complement sequence to induce complement-mediated or monoclonal, naturally occurring or immune, and allo- intravascular hemolysis. However, extravascular hemolysis antibodies or autoantibodies. usually occurs as a result of antibody coating of RBCs, and the split products of complement activation can stimulate the Polyclonal and Monoclonal Antibodies reticuloendothelial system and cause anaphylatoxic effects. Antibody-coated RBCs, either self or nonself, are removed In laboratory testing there are two types of antibody (reagent by cells of the mononuclear phagocyte system and by the antibodies are called antisera) that are available for use; they cells lining the hepatic and splenic sinusoids. These phago- cytic cells are able to clear antibody-coated RBCs because they have cell surface receptors for complement CR1 (C3b) BOX 3–2 and Ig FC receptors (see Fig. 3–6). IgM-coated RBCs are not Characteristics of Antigens: Properties That Influence eliminated through FC receptor–mediated phagocytosis, but if erythrocytes are coated with IgG and complement, they Immune Response will be cleared rapidly from circulation by monocytes and Size macrophages. If RBCs are coated with only C3b, they may Complexity not be cleared but only sequestered temporarily. Conformation Charge Characteristics of Antigens Accessibility Solubility The immune response is initiated by the presentation of an Digestibility antigen (initiates formation of and reacts with an antibody) Chemical composition or immunogen (initiates an immune response). The term 62 PART I Fundamental Concepts Table 3–7 Relative Immunogenicity of from the cell culture contains antibody from a single type of B cell, clonally expanded, and therefore has the same variable Different Blood Group Antigens region and a single epitope specificity. This results in a mono- Blood Blood clonal antibody suspension. Monoclonal antibodies are Group Group Immunogenicity preferred in testing because they are highly specific, well Antigen System (%)* characterized, and uniformly reactive. Most reagents used D (Rho) Rh 50 today are monoclonal in nature or are a blend of monoclonal antisera. K Kell 5 c (hr’) Rh 2.05 Naturally Occurring and Immune Antibodies E (rh’’) Rh 1.69 There are two types of antibodies that concern blood bank- ing: one is naturally occurring and the other is immune. k Kell 1.50 Both are produced in reaction to encountered antigens. RBC e (hr’’) Rh 0.56 antibodies are considered naturally occurring when they are found in the serum of individuals who have never been pre- Fya Duffy 0.23 viously exposed to RBC antigens by transfusion, injection, C (rh’) Rh 0.11 or pregnancy. These antibodies are probably produced in response to substances in the environment that resemble Jka Kidd 0.07 RBC antigens such as pollen grains and bacteria membranes. S MNSs 0.04 The common occurrence of naturally occurring antibodies suggests that their antigens are widely found in nature and Jkb Kidd 0.03 have a repetitive complex pattern. Most naturally occurring s MNSs 0.03 antibodies are IgM cold agglutinins, which react best at room temperature or lower, activate complement, and may be he- Adapted from Kaushansky, K, et al: Williams Hematology, 8th ed. McGraw-Hill molytic when active at 37°C. In blood banking, the common Professional, New York, 2010. *Percentage of transfusion recipients lacking the blood group antigen (in the first naturally occurring antibodies react with antigens of the ABH, column) who are likely to be sensitized to a single transfusion of red blood cells Hh, Ii, Lewis, MN, and P blood group systems. Some naturally containing that antigen. occurring antibodies found in normal serum are manufactured without a known environmental stimulus.10 In contrast to nat- ural antibodies, RBC antibodies are considered immune when are manufactured differently and have different properties. found in the serum of individuals who have been transfused An antigen usually consists of numerous epitopes, and it is or who are pregnant. These antigens have a molecular makeup the epitopes, not the entire antigen, that a B cell is stimulated that is unique to human RBCs. Most immune RBC antibodies to produce antibody against. Therefore, these different epi- are IgG antibodies that react best at 37°C and require the use topes on a single antigen induce the proliferation of a variety of antihuman globulin sera (Coombs’ sera) for detection. The of B-cell clones, resulting in a heterogeneous population of most common immune antibodies encountered in testing serum antibodies. These antibodies are referred to as poly- include those that react with the Rh, Kell, Duffy, Kidd, and Ss clonal or serum antibodies and are produced in response to blood group systems.10 a single antigen with more than one epitope. In vivo, the polyclonal nature of antibodies improves the Unexpected Antibodies immune response with respect to quality and quantity. Anti- bodies against more than one epitope are needed to give Naturally occurring anti-A and anti-B antibodies are routinely immunity against an entire antigen, such as a pathogen. detected in human serum and depend on the blood type of However, this diversity is not optimal in the laboratory, and the individual. Blood group A has anti-B; blood group B has in vitro reagents produced by animals or humans can give anti-A; blood group O has both and anti-A, B. Blood group confusing test results. Consistency and reliability are needed AB has neither antibody present in their serum. These natu- in laboratory testing, and polyclonal sera can vary in anti- rally occurring antibodies, or isoagglutinins, are significant body concentration from person to person and animal to and useful in blood typing. They are easily detected by use of animal. Sera from the same animal can also vary somewhat, A and B reagent RBCs with a direct agglutination technique. depending on the animal’s overall condition. In normal, healthy individuals, anti-A and anti-B are generally Individual sera also differ in the serologic properties of the only RBC antibodies expected to be found in a serum the antibody molecules they contain, the epitopes they rec- sample. People with an IS that does not function normally ognize, and the presence of additional nonspecific or cross- may not have the expected naturally occurring antibodies. reacting antibodies. One way to avoid this problem is to use All other antibodies directed against RBC antigens are con- monoclonal antibodies produced by isolating individual B sidered unexpected and must be detected and identified cells from a polyclonal population and propagating them in before blood can be safely transfused, no matter their reaction cell culture with hybridoma technology. The supernatant strength or profile.  Chapter 3 Fundamentals of Immunology 63 The reactivity of unexpected antibodies is highly varied and antigen; this antibody amino acid sequence cannot be changed unpredictable, as they may be either isotype IgM or IgG; rarely, without altering its specificity. The extent of the reciprocal both may be present in the same sample. These antibodies relationship, also called the fit between the antigen and its may be able to hemolyze, agglutinate, or sensitize RBCs. Some binding site on the antibody, is often referred to as a lock and antibodies require special reagents to enhance their reactivity key mechanism. Factors influencing antigen-antibody reac- and detection, especially if more than one antibody occurs in tions include intermolecular binding forces, antibody proper- the sample. Due to the enormous polymorphism of the human ties, host factors, and tolerance. population, a diversity of RBC alleles and antigens exist, re- quiring a variety of standardized immunologic techniques and Intermolecular Binding Forces reagents for their detection and identification. The in vitro analysis of unexpected antibodies involves Intermolecular binding forces such as hydrogen bonding, the use of antibody screening procedures to optimize anti- electrostatic forces, van der Waals forces, and hydrophobic gen-antibody reactions. The majority of these procedures bonds are all involved in antigen-antibody binding reactions. include reacting unknown serum from a donor or patient Stronger covalent bonds are not involved in this reaction, sample with known reagent cells at various amounts of time, although they are important for the intramolecular confor- temperature, and media. All routine blood bank testing re- mation of the antibody molecule. Hydrogen bonds result quires the use of samples for both expected and unexpected from weak dipole forces between hydrogen atoms bonded antibodies. to oxygen or nitrogen atoms in which there is incomplete transfer of the electronic energy to the more electronegative Alloantibodies and Autoantibodies oxygen or nitrogen. When two atoms with dipoles come close to each other, there is a weak attraction between them. Antibodies can be either alloreactive or autoreactive. Allo- Although hydrogen bonds are singularly weak, many of antibodies are produced after exposure to genetically differ- them together can be strong. They are found in complex ent, or nonself, antigens, such as different RBC antigens after molecules throughout the human body. transfusion. Transfused components may elicit the formation Electrostatic forces result from weak charges on atoms in of alloantibodies against antigens (red blood cell, white cell, molecules that have either a positive or negative overall and platelets) not present in the recipient. A potentially charge (like charges repel and unlike charges attract). This serious problem for blood bankers is transfused patients who is seen in the formation of salt bridges. Van der Waals forces have alloantibodies that are no longer detectable in the are a type of weak interaction between atoms in larger mole- patients’ plasma or serum. If these individuals are transfused cules. Hydrophobic (water-avoiding) bonds result from the with the immunizing antigen again, they will make a overlap of hydrophobic amino acids in proteins. The hydro- stronger immune response against those RBC antigens, phobic amino acids bury themselves together to avoid water which can cause severe and possibly fatal transfusion reac- and salts in solution. The repulsion of these amino acids to tions. These recipients will then have a positive autocontrol prevent contact with water and aqueous solutions can be or direct antiglobulin test (DAT), also referred to as the very strong collectively. direct Coombs’ test. In addition, there are hydrophilic (water-loving) bonds Autoantibodies on the other hand, are produced in that allow for the overlap of amino acids that are attracted to response to self-antigens. They can cause reactions in the water. Hydrophobic and hydrophilic bonds repel each other, recipient if they have a specificity that is common to the and these repulsive forces also play a role in the formation of transfused blood. Some autoantibodies do not have a the antigen-antibody bond. All of these bonds affect the total detectable specificity. Autoantibodies can react at different conformation and strength of antigen-antibody reactions and temperatures, and both cold and warm autoantibodies may IS molecules. be present. Patients with autoantibodies frequently have autoimmune diseases and may require considerable num- Antibody Properties bers of blood products and special techniques to find com- patible units. Autoantibodies can be removed from RBCs Just as antigen properties influence the amount and type of by special adsorption and elution techniques and then immune response, antibody properties influence the strength tested against reagent RBCs. Therefore, in order to trans- and characteristics of the immune response. Antibody affinity fuse blood safely, the identity of antibodies should be is often defined as the strength of a single antigen-antibody determined and recorded. bond produced by the summation of attractive and repulsive forces. In other words, affinity is the strength of the interac- Characteristics of Antigen-Antibody tion between the antigen and the antibody’s binding site at Reactions one individual site. Avidity is used to express the binding strength of a multivalent antigen with antisera produced in There are many complex properties of antigen and antibody an immunized individual. Avidity, therefore, is a measure of reactions that influence serologic and other testing methods the functional affinity of an antiserum for the whole antigen involving antibodies. The antigen-binding site of the antibody and is sometimes referred to as a combination of affinities. molecule is uniquely structured to recognize a corresponding Avidity can be important in blood banking because high-titer, 68 PART I Fundamental Concepts Table 3–8 Serologic Systems Used in Traditional Laboratory Methods for Red Blood Cell Antibody Detection Ig Class Purpose and Tests That Use Reaction Commonly Mechanism of Serologic Type of Antibodies Phase Detected Reaction System Commonly Detected Immediate spin IgM IgM antibodies react best at ABO reverse testing Expected ABO alloantibodies cold temperatures IgM is an agglutinating anti- Cross-match Unexpected cold-reacting body that has the ability to alloantibodies or autoantibodies easily bridge the distance between red blood cells. 37°C incubation IgG IgG antibodies react best at Antibody screening/ warm temperatures. identification Autocontrol Antibody screening/ identification No visible agglutination Cross-match (if needed) commonly seen. IgG is sensitizing antibody Autocontrol with fewer antigen-binding sites than IgM and cannot undergo the second stage of agglutination, lattice formation. Complement may be bound during reactivity, which may or may not result in visible hemolysis. Antiglobulin test IgG Antihuman globulin (AHG) Antibody screening/ Unexpected warm-reacting has specificity for the identification alloantibodies or autoantibodies FC portion of the heavy chain of the human IgG molecule or complement components. Cross-match (if needed) Autocontrol AHG acts as a bridge cross- Direct antiglobulin test linking red blood cells sensi- (DAT) tized with IgG antibody or complement. Commercially available enhancement media reduces the zeta neutral and go into solution because of their microscopic potential and allows the more positively charged antibodies size. There are several colloidal solutions currently utilized to get closer to the negatively charged RBCs and therefore in blood bank testing to enhance agglutination reactions. increases RBC agglutination by IgG molecules. Colloids include albumin, polyethylene glycol (PEG), poly- brene, polyvinylpyrrolidone (PVP), and protamine. These Protein Media substances work by increasing the dielectric constant (a measure of electrical conductivity), which then reduces Colloidal substances, or colloids, are a clear solution that the zeta potential of the RBC. contains particles permanently suspended in solution. Col- loidal particles are usually large moieties like proteins as Low Ionic Strength Solution Media compared with the more familiar crystalloids, which usually have small, highly soluble molecules, such as glucose, that Low ionic strength solutions (LISS), or low salt media, are easily dialyzed. The colloidal solutes can be charged or generally contain 0.2% sodium chloride. They decrease 72 PART I Fundamental Concepts known. Some of the more well-known immunodeficiencies Type III reactions, like type II, involve phagocytes and IgG are listed in Box 3–4.17 Immunodeficiencies can influence and IgM and complement. Type III reactions result in tissue blood bank test results and transfusion decisions, such as damage from the formation of immune complexes of when there is a false-negative reverse grouping for ABO due antigen-antibody aggregates, complement, and phagocytes to low immunoglobulin levels. and are therefore very serious. Penicillin and other drug- induced antibodies can lead to hemolytic reactions through Hypersensitivity type III hypersensitivity. The type IV reaction involves only T cell–mediated responses and their cytokines and can be Hypersensitivity is an inflammatory response to a foreign fatal if untreated. The most important type IV reaction is antigen and can be cell- or antibody-mediated or both. There graft-versus-host, of which there is also more than one type. are four different types of hypersensitive reactions, and the Immunocompromised and immunosuppressed patients symptoms and treatment required for each are different. All must receive irradiated blood products so that T lympho- four types can be caused by blood product transfusions and cytes do not engraft and attack the host tissues. Type IV may be the first sign of a transfusion reaction. Type I reac- reactions are a problem for bone marrow transplant and stem tion, also called anaphylaxis or immediate hypersensitivity, cell transfusion recipients as well. (Refer to Chapter 17, involves histamine release by mast cells or basophils with “Adverse Effects of Blood Transfusion.”) surface IgE antibody. It can occur in IgA-deficient individuals who receive plasma products containing IgA. Urticarial re- Monoclonal and Polyclonal Gammopathies actions (skin rashes) may also result from transfusion of cer- tain food allergens or drugs in plasma products. A type II Plasma cell neoplasms result in proliferation of abnormal im- reaction can involve IgG or IgM antibody with complement, munoglobulin from either a single B-cell clone (monoclonal phagocytes, and proteolytic enzymes. HDN or transfusion gammopathies) or multiple clones (polyclonal gammo- reactions caused by blood group antibodies and autoimmune pathies) and may be a specific isotype or involve just light hemolytic reactions are all type II reactions. or heavy chain molecules. Increased serum viscosity is a BOX 3–4 Classification of Immunodeficiency Disorders Antibody (B Cell) Immunodeficiency Disorders Cellular immunodeficiency with abnormal immunoglobulin synthesis (Nezelof syndrome) X-linked hypogammaglobulinemia (congenital hypogammaglobulinemia) Immunodeficiency with ataxia-telangiectasia Transient hypogammaglobulinemia of infancy Immunodeficiency with eczema and thrombocytopenia (Wiskott-Aldrich syndrome) Common, variable, unclassifiable immunodeficiency (acquired by hypogammaglobulinemia) Immunodeficiency with thymoma Immunodeficiency with hyper-IgM Immunodeficiency with short-limbed dwarfism Selective IgA deficiency Immunodeficiency with adenosine deaminase deficiency Selective IgM deficiency Immunodeficiency with nucleoside phosphorylase deficiency Selective deficiency of IgG subclasses Biotin-dependent multiple carboxylase deficiency Secondary B-cell immunodeficiency associated with drugs, Graft-versus-host disease protein-losing states AIDS X-linked lymphoproliferative disease Phagocytic Dysfunction Cellular (T Cell) Immunodeficiency Disorders Chronic granulomatous disease Congenital thymic aplasia (DiGeorge syndrome) Glucose-6-phosphate dehydrogenase deficiency Chronic mucocutaneous candidiasis (with or without endocrinopathy) Myeloperoxidase deficiency T-cell deficiency associated with purine nucleoside phosphorylase Chediak-Higashi syndrome deficiency Job’s syndrome T-cell deficiency associated with absent membrane glycoprotein Tuftsin deficiency T-cell deficiency associated with absent class I or II MHC antigens or Lazy leukocyte syndrome both (base lymphocyte syndrome) Elevated IgE, defective chemotaxis, and recurrent infections Combined Antibody-Mediated (B Cell) and Cell- Mediated (T Cell) Immunodeficiency Disorders Severe combined immunodeficiency disease (autosomal recessive, X-linked, sporadic) Ammann, AJ: Mechanisms of immunodeficiency. In Stites, DP, Terr, AI, and Parslow, TG (eds): Basic and Clinical Immunology, 8th ed. Appleton & Lange, Norwalk, 1994, with permission.  Chapter 3 Fundamentals of Immunology 73 result of these diseases and can interfere with testing. The antibodies and may require exchange transfusion. Antigens increased concentrations of serum proteins can cause of the ABO, Rh, and other blood group systems such as Kell nonspecific aggregation (as opposed to agglutination) of have been shown to cause HDFN. (Refer to Chapter 20, erythrocytes called rouleaux, which is seen as a stacking of “Hemolytic Disease of the Fetus and Newborn [HDFN].”) RBCs. It often occurs in multiple-myeloma patients. If rouleaux is suspected, saline replacement technique may Blood Product Transfusions and the be needed to distinguish true cell agglutination from non- Immune System specific aggregation. Rouleaux primarily causes problems in the ABO reverse grouping, antibody screening, and compat- The immune system may undergo transient depression fol- ibility testing procedures. However, excess immunoglobulin lowing the administration of blood and blood products. coating red blood cells can cause spontaneous aggregation This is referred to as transfusion-related immunomodula- to occur as well. tion (TRIM). With a weakened immune system, there is an increased risk of an individual developing infections or Autoimmune Disease certain cancers. The exact mechanism(s) causing TRIM are not known and have not been fully elucidated, but three Autoantibodies are produced against the host’s own cells and major mechanisms seem to have emerged: clonal deletion, tissues. It is unknown why this loss of tolerance to self- induction of anergy, and immune suppression. In clonal antigens occurs, but there are many possible explanations deletion, alloreactive lymphocytes are inactivated and such as aberrant antigen presentation, failure to obtain clonal removed, thus preventing a potential graft rejection (as in deletion, anti-idiotypic network breakdown, and cross- kidney organ). With induction of anergy, there is an unre- reactivity between self and nonself antigens. Autoimmune sponsiveness of the immune system, most likely due to hemolytic anemias are an important problem in testing and a failure of T cells to respond. In immune suppression, transfusion. They may produce antibodies that cause responding cells appear to be inhibited by some sort of RBC destruction and anemia and result in antibody- or cellular mechanism or cytokine. complement-sensitized RBCs. The direct antiglobulin test Immune suppression can be due to a number of factors. should be done to detect sensitized RBCs and determine if Certain cytokines, including interleukins and some growth the cells are coated with antibody or complement. Special factors, can decrease immune responsiveness. Medically in- procedures such as elution or chemical treatment to remove duced suppression of some IS components is critical at times antibody from cells may be required to prepare RBCs so the IS does not become overactivated and attack host cells. for antigen typing. Serum autoantibodies may interfere Immature T and B cells that recognize host cells, and poten- with testing for clinically significant alloantibodies. Special tially destroy them, must be removed before they can deve- reagents and procedures to denature immunoglobulins lop into mature lymphocytes. Specific suppressor cells may be required to remove autoantibodies from serum so have recently been isolated that have unique CD profiles they do not interfere with the testing. (Refer to Chapter 21, and play a role in modulating lymphocyte function. Immune “Autoimmune Hemolytic Anemias.”) suppression can also occur when there is too little im- munoglobulin made or when too many T and B cells are lost Hemolytic Disease of the Newborn through severe infection, extreme immune stimulation, or IS organ failure. Hemolytic disease of the fetus and newborn (HDFN) can re- It is believed that some constituents of cellular blood sult when the maternal IS produces an antibody directed at products may be responsible for TRIM. These constituents an antigen present on fetal cells but absent from maternal include allogeneic mononuclear cells, chemicals released by cells. The mother is exposed to fetal RBCs as a result of allogenic mononuclear cells as they age, and soluble HLA fetomaternal transfer of cells during pregnancy or childbirth. peptides that circulate in plasma. The decision to transfuse Maternal memory cells can cause a stronger response during blood products is approached with caution, and if transfu- a second pregnancy if the fetus is positive for the sensitizing sion is required, the patient’s immune status must be taken antigens. IgG1, IgG3, and IgG4 are capable of crossing the into consideration. The use of leukoreduction filters may placenta and attaching to fetal RBCs, whereas IgG2 and IgM help to reduce the risk for TRIM. (Refer to Chapter 16, are not. Severe HDFN is most often associated with IgG1 “Transfusion Therapy.”)