Immunohematology - 02 Fundamentals of Immunology and Immunohematology

• IMMUNOHEMATOLOGY FUNDAMENTALS OF IMMUNOLOGY & IMMUNOHEMATOLOGY OUTLINE • Introduction • Antigens o The Antigen o Formation of ABH RBC Antigens o Formation of ABH Soluble Antigens o Comparison of ABH Antigens on RBCs with ABH Soluble Substances o ABH Antigens in Diseases o Compound Antigens • Antibodies o The Antibody o Immunoglobulins o Antibody Detection o Antibody Identification o Resolving Difficult Antibody Identification Problem • The Complement System o Complement System o Classical Complement Pathway o Alternative Complement Pathway o Lectin Complement Pathway o Summary of the Complement System o Binding of Complement by RBC AB • Antigen-Antibody Reactions o The Binding Force of Antigen-Antibody Reaction o Different Types of Antigen and Antibody Reactions o Introduction to Immunohematology o RBC-Antibody Interaction • INTRODUCTION • • • The immune system is complicated, tightly controlled, and includes tissues, organs, cells, and biological mediators that coordinate to defend a host organism against intrusion by a foreign substance or abnormal cells of self-origin. In this module, we will be able to relate the concepts of immunology and immune-mediated diseases in the world of transfusion medicine. Immunity - process by which as host organism protects itself from attacks by both external and internal agents o Immune cells - substances that protect against pathogens Major Mechanisms of the Immune System o Innate Immunity ▪ Primary lines of defense ▪ Early development ▪ Non-specific • Naturally present at birth; immediately available • Has biochemical defense mechanisms • Mechanism does not alter on repeated exposure to any specific antigens o Adaptive Immunity ▪ Secondary and tertiary lines of defense ▪ Later development ▪ Supplements protection provided by innate immunity ▪ Specific, specialized • Acquired by contact of specific foreign substances ▪ Develops memory on exposure that improves response with each successive encounter with pathogen due to the components of response Cellular and Humoral Components of Immune System o First Line of Defense ▪ Physical component: skin, mucus membranes, cilia, cough reflexes ▪ Biochemical component: secretions (saliva, sweat, mucus, tears), low pH of vagina & gastric fluids (maintains equilibrium) o Second Line of Defense ▪ Cellular component: phagocytic cells (NK cells & macrophages) ▪ Humoral component: complement (alternative pathway), cytokines (interferons, interleukins), acute inflammatory rxn (acute phase reactants) o Third Line of Defense ▪ Cellular component: lymphocytes (T cells), B cells (memory B cell) & plasma cell ▪ Humoral component: complement (classical pathway), antibodies, cytokines Primary and Secondary Response Primary Secondary Stimulus 1st exposure to Subsequent antigens exposure to antigens Lag Phase Days to months Hours Type of IgM (initial); IgG Antibody IgG (after 2-3 weeks) Titer Slow rise → peak Rapid and high rise; → decline stay elevated longer ANTIGENS THE ANTIGEN • • • • Antigen o A substance recognized by the body as being foreign, which can cause an immune response o In blood banking, antigens usually, but not exclusively, found on the red blood cell membrane ▪ Antigens can be found on the surface Antithecal Antigens o Referring to antigens that are product of allelic genes (e.g., Kell [K] and Cellano [k]) Antigen Specificity o The affinity of an antibody and the antigen against which it is directed. o Antigen - Antibody specificity Blood Group Antigens o RBC antigens are very diverse in structure and composition and may be: o Proteins ▪ Rh (Rhesus) ▪ M ▪ N blood group substances o Glycolipids ▪ ABH ▪ Lewis ▪ Ii ▪ P blood group substance o Human leukocyte antigens (HLA) are glycoproteins FORMATION OF A, B, AND H RED BLOOD CELL ANTIGENS • • • • • • • • • • The formation of ABH antigens results from the interaction of genes at three separate loci (ABO, Hh, and Se). These genes do not actually code for the production of antigens but rather produce specific glycosyltransferases that add sugars to a basic precursor substance When the terminal galactose on the precursor substance is attached to the N-acetylglucosamine in a beta 1 → 4 linkage (Fig. 6–3), the precursor substance on erythrocytes is referred to as type 2. ABH antigens on the RBC are constructed on oligosaccharide chains of a type 2 precursor substance. A type 1 precursor substance refers to a beta 1 → 3 linkage between galactose and Nacetylglucosamine. H antigen o Precursor structure on which A & B antigens are made o Inheritance of the H gene results in formation of the H antigen. • FORMATION OF A, B, AND H SOLUBLE ANTIGENS • ABH antigens are integral parts of the membranes of RBCs, endothelial cells, platelets, lymphocytes, and epithelial cells. ABH-soluble antigens can also be found in all body secretions. Their presence is dependent on the ABO genes inherited and on the inheritance of another set of genes called Sese (secretor genes) that regulate their formation. Eighty percent of the random U.S. population are known as secretors because they have inherited a secretor gene (SeSe or Sese). The inheritance of a FUT 2 (Se) gene codes for the production of the transferase a-2-L-fucosyltransferase that modifies the type 1 precursor substance in secretions to form H substance. People who inherit the sese genotype are termed nonsecretors. COMPARISON OF A, B, AND H ANTIGENS ON RBCS WITH A, B, AND H SOLUBLE SUBSTANCES • • • • • The term secretor refers only to secretion of A, B, and H soluble antigens in body fluids. The demonstration of A, B, and H substances in saliva is evidence of the inheritance of an A gene, B gene, H gene, and Se The glycoprotein-soluble substances (or antigens) normally found in the saliva of secretors . Both ABH red blood cell antigens and ABH-soluble substances are formed due to the attachment of an immunodominant sugar to an oligosaccharide chain. • • • COMPOUND ANTIGENS • • • • Various disease states seem to alter RBC antigens, resulting in either progressively weaker reactions or additional acquired pseudoantigens, which can be seen during forward grouping. Leukemia, chromosome 9 translocations, and any hemolytic disease that induces stress hematopoiesis (e.g., thalassemia) have been shown to depress antigen strength. Two or more antigens that interact and are recognized as a single antigen by an antibody f (ce) It is expressed on the RBC when both c and e are present on the same haplotype. Previously referred to as cis products to indicate that the antigens were on the same haplotype ANTIBODIES THE ANTIBODY • • • • • • • • • • ABH ANTIGENS IN DISEASE • Hodgkin’s disease also has been reported to weaken or depress ABH RBC antigens, resulting in variable reactions during forward grouping similar to those found in leukemia. The weakening of the antigen tends to follow the course of the disease. The antigen strength increases again as the patient enters remission. A lack of detectable ABO antigens can occur in patients with carcinoma of the stomach or pancreas. The patient’s red blood cell antigens have not been changed, but the serum contains excessive amounts of blood group–specific soluble substances (BGSS) that may neutralize the antisera utilized in the forward grouping. • Also known as immunoglobulins/gamma globulins Secreted by named as plasma cell Bind foreign molecules known as antigen Five immunoglobulins: IgG, IgA, IgM, IgD, IgE Primary antibodies are IgM and secondary antibodies (anamnestic) are IgG Detection of antibodies directed against red blood cell (RBC) antigens is critical in pretransfusion compatibility testing. It is one of the principal tools for investigating potential hemolytic transfusion reactions and immune hemolytic anemias. In addition, it aids in detecting and monitoring patients who are at risk of delivering infants with hemolytic disease of the fetus and newborn (HDFN). Immune alloantibodies - produced in response to RBC stimulation through transfusion, transplantation, or pregnancy. Other unexpected antibodies may be “naturally occurring” (i.e., produced with- out RBC stimulation). Naturally occurring alloantibodies - form as a result of exposure to environmental sources, such as pollen, fungus, and bacteria, which have structures similar to some RBC antigens. Passively acquired antibodies - antibodies produced in one individual and then transmitted to another individual via plasma-containing blood components or derivatives such as intravenous immunoglobulin (IVIG). Autoantibodies - antibodies directed against antigens expressed on one’s own RBCs and generally react with all RBCs tested. Presence of naturally occurring alloantibodies, passively acquired antibodies, and autoantibodies may complicate the detection and identification of clinically significant, immune alloantibodies. Clinically significant alloantibodies - are those that cause decreased survival of RBCs possessing the target antigen. These antibodies are typically IgG antibodies that react at 37°C and/or at the antihuman globulin (AHG) phase of the indirect antiglobulin test (IAT). • IMMUNOGLOBULINS Class H chain IgG gamma L Chain Subunits mg/mL kappa/ H2L2 6-13 lambda kappa/ (H2L2)5 0.5-3 lambda Notes Transferred across placenta 1st antibodies to appear after immunization Much higher concentrations in secretions Function uncertain IgM mu IgA alpha kappa/ lambda (H2L2)2 0.6-3 IgD delta H2L2 <0.14 IgE epsilon kappa/ lambda kappa/ lambda H2L2 <0.0004 Binds to basophils and mast cells sensitizing them for certain reactions • • • IgG - smallest antibody; monomer o Only immunoglobulin able to cross placenta o Most clinically significant ▪ May cause HDFN o Small incompatibility may cause hemolytic transfusion reaction (HTR) & autoimmune hemolytic anemia IgM - first to appear o Pentamer; five times bigger than IgG IgA - secretory antibody; dimer • • • • • • COMPARISON OF IgM AND IgG IgM IgG To observe for hemolysis, the tube is carefully removed from the centrifuge so as not to dislodge the RBC button. The supernatant is observed for pink or red discoloration. To observe for agglutination, the tube is gently tilted or rolled to dislodge the cell button. The tubes are then washed with 0.9% saline a minimum of three times to remove all antibodies that remain unbound. AHG reagent, also known as Coombs’ serum, is added to each tube. The tubes are centrifuged and examined for hemolysis, if applicable, and agglutination. Different reagents used in tube method: RBC reagents, Enhancement reagents, AHG reagents A cell with homozygous antigen expression is from an individual who inherited only one allele at a given genetic locus. Therefore, the cell surface has a “double dose” of that antigen. A cell with heterozygous antigen expression is from an individual who inherited two different alleles at a given genetic locus. Gel Technology- a process that detects RBC antigenantibody reactions by means of a chamber filled with polyacrylamide gel. Solid-Phase Technology- may be used for performing antiglobulin tests. Several different techniques have been reported using either test tubes or microplates.With the availability of microplate readers, this technology can be fully automated. Direct and indirect tests can be performed. INTERPRETATION OF RESULTS • Heavy chain Light chain J chain Valence Structure Detection Optimum temperature Completeness • Mu Kappa or Lambda Present 10 Pentamer Immediate spin 25C / room temperature Complete Gamma Kappa or Lambda Absent 2 Monomer Antiglobulin test Warm-reacting antibody (37C) Incomplete • ANTIBODY DETECTION • • • • • • Tube Method - traditional testing method used to detect clinically significant antibodies is the IAT performed in a test tube. In the test tube method, the patient’s serum is mixed with reagent RBCs that have known antigen content This test may include an immediate spin (IS) phase to detect antibodies reacting at room temperature. The test must include a 37°C incubation phase; during this phase, IgG antibodies, if present in the patient’s serum, will sensitize any reagent RBCs that possess the target antigen. Enhancement reagent may be added to the test tube prior to incubation at 37°C to increase the degree of sensitization. The tubes may be centrifuged and observed for hemolysis and agglutination following this incubation phase; tubes only need to be observed for hemolysis when fresh serum and reagent RBCs that do not contain EDTA are used because complement is necessary for hemolysis to occur. A positive test result at any phase of testing indicates the need for antibody identification studies. Evaluation of antibody screen results, including the autologous control if tested at this time, and patient history (i.e., age, sex, race, diagnosis, pregnancy and transfusion history, current medications, and intravenous solutions) can provide clues and give direction for identification and resolution of the antibody or antibodies present. In complex cases, patient history can be particularly valuable. The investigator should consider the following: o Phases the reaction occurred. o If the autologous control is negative or positive- The autologous control is the patient’s RBCs tested against the patient’s serum in the same manner as the antibody screen. o If more than one screen sample reacted. o If hemolysis or mixed-field agglutination is present. o If the cells truly agglutinated, or if rouleaux is present. LIMITATIONS • • • The screen will not detect antibodies when the antibody titer has dropped below the level of sensitivity for the screening method employed. The screen also cannot detect antibodies directed against low-prevalence antigens that are not present on any of the RBCs in the screen cell set. Antibodies showing dosage may not be detected if none of the screen cells have homozygous expression of the target antigen. ANTIBODY IDENTIFICATION • • • • • • • • • • In general, reviewing patient history and testing an antibody identification panel, along with an auto control or DAT (if not previously per- formed), comprises initial, additional testing/review. Patient History. Information concerning the patient’s age, sex, race, diagnosis, transfusion and pregnancy history, medications, and intravenous solutions may provide valuable clues in antibody identification studies, particularly in complex cases. Knowing the patient’s race can be beneficial, as some antibodies are associated with a particular race. Information regarding recent transfusions is also important when antigen-typing the patient’s RBCs. An antibody identification panel is a collection of 11 to 20 group O reagent RBCs, with various antigen expression. The pattern of antigen expression should be diverse so that it will be possible to distinguish one antibody from another and should include cells with homozygous expression of Rh, Duffy, Kidd, and MNSs antigens. When interpreting panel results, the first step is to exclude antibodies that could not be responsible for the reactivity seen. Evaluation of Panel Results. After each negatively reacting cell has been evaluated, the remaining antigens that were not excluded should be examined to see if the pattern of reactivity matches a pattern of antigen-positive cells (inclusion technique). Additional techniques for resolving antibody identification: Selected Cell Panels, Enzymes, Neutralization, Adsorption, Alteration in pH. Commercial reagents for adsorption: Human platelet concentrate, Rabbit erythrocyte stroma. RBC adsorption- Autoantibodies are commonly removed using RBC adsorption techniques so that any underlying, clinically significant antibodies can be detected. Autoadsorption. The simplest method for removing autoantibodies is adsorption using the patient’s own RBCs (autologous adsorption). Allogeneic Adsorption. When the volume of autologous RBCs is insufficient or when a patient has been recently transfused (i.e., within the last 3 months), phenotypically matched or differential adsorptions may be employed instead of autoad- sorption to remove autoantibodies from patient serum; autologous adsorption is preferred over allogeneic adsorption for the removal of autoantibodies, since allogeneic RBCs have the potential to adsorb an antibody to a high-prevalence antigen. Phenotypically Matched Adsorption. If the patient’s phenotype is known or can be determined (i.e., not transfused within the last 3 months and DAT-negative RBCs are available), then phenotypically matched RBCs can be used for adsorption in place of autologous cells. Differential Adsorption. Differential adsorption can be performed when a patient’s phenotype is unknown and performing antigen-typing is not an option (i.e., positive DAT or transfused within the last 3 months). DAT AND ELUTION TECHNIQUES • DAT detects cells that were sensitized with antibody in vivo. The patient’s cells are washed thoroughly to remove any unbound antibody, and then AHG reagent is added. If IgG antibody or complement is fixed to the cell, agglutination will be observed. If neither is present, no • • • agglutination will be observed. Coombs’ control cells are added to validate the negative test. Elution techniques are used to release, concentrate, and purify antibodies. The methods used to remove the antibody change the thermodynamics of the environment, change the attractive forces between antigen and antibody, or change the structure of the RBC surface. Temperature- Heat may be used to remove antibody when washed, coated RBCs are suspended in an equal volume of saline or albumin. Heat method performed at 45C, allows for antibody removal while leaving the RBC intact. Elution performed at 56C is a total elution method, allowing for antibody identification.30 The Lui freeze method also produces a total elution. In this method, washed, coated RBCs suspended in saline or albumin are frozen at 18C or colder until solid. The mixture is then thawed rapidly, causing the RBCs to burst, freeing the bound antibody. Temperature-dependent elutions are best at detecting IgG antibodies directed against antigens of the ABO system. pH- The most common and relatively quick and easy method for total elution in order to detect non-ABO antibodies is acid elution. Washed antibody-coated cells are mixed with a glycine acid solution at a pH of 3.0. The supernatant is harvested, and the pH is neutralized so that antibody identification testing can take place. Organic Solvents- Organic solvents have been used in total elution methods, among them dichloromethane, xylene, and ether. These solvents act on the lipids in the RBC membrane to reduce surface tension and lead to the reversal of the van der Waal forces that hold antigens and antibodies together. These procedures are timeconsuming, and the chemicals pose several health and safety hazards as they are carcinogenic and flammable. The most critical step in preparing any eluate is the original washing, which is used to remove unbound immunoglobulins. ANTIBODY TITRATION • An antibody titration can help determine antibody concentration levels. Twofold serial dilutions of serum containing an antibody are prepared and tested against a suspension of RBCs that possesses the target antigen. The titer level is the reciprocal of the greatest dilution in which agglutination is observed. Performing a titer is one way to confirm the presence of antibodies belonging to the group collectively known as HTLA (high titer, low avidity). These high-frequency antibodies are observed at the AHG phase of testing with weakly positive reactions. PROVIDING A COMPATIBLE BLOOD PRODUCTS • Difficulty in providing compatible blood products is determined by the frequency of the antigen in the population and by the clinical significance of the antibody. If the antibody does not cause decreased survival of antigen positive RBCs, then use of random blood products that are crossmatch-compatible is acceptable. Knowing the incidence of the antigen in the population is helpful when determining the number of units that must be antigen-typed to find a sufficient number to fill the crossmatch request. The number of units requested is divided by the frequency of antigen-negative individuals. RESOLVING DIFFICULT ANTIBODY IDENTIFICATION PROBLEM • • • • • THE COMPLEMENT SYSTEM COMPLEMENT SYSTEM Case One: Multiple Antibodies o Suspect multiple antibodies when all or most of the screen and panel cells are positive but reactions are at different strengths or in different phases and the autocontrol is negative. Resolution may include performing a selected cell panel to exclude certain specificities. Several sets of cells may need to be tested in order to narrow the list of possible antibodies Case Two: Antibody to a High Frequency Antigen o High-frequency antigens are those that are present in almost all individuals (98 percent or more). Suspect an antibody to a high-frequency antigen when all or most screen and panel cells are positive, with reactions in the same phase and same strength, and a negative autocontrol. Resolution would be testing of additional cells that lack the antigen would be most useful. Case Three: Antibody to a Low Frequency Antigen o Low-frequency antigens are present in less than 10 percent of the population. Antibodies to these antigens are uncommon because exposure to the antigen is rare. Resolution would be testing with additional antigen-positive cells will be required to confirm the specificity. Case Four: Cold-Reacting Autoantibodies o These antibodies are usually IgM and of no clinical significance. They are troublesome in that they may interfere with the detection of significant antibodies, resulting in prolonged workups and delayed transfusions. Cold-reacting autoantibodies may be suspected when the screening cells, panel cells, and the autocontrol are all positive at the immediate spin phase and get weaker or disappear with incubation at 37C. Case Five: Warm-Reacting Autoantibodies o Warm-reacting autoantibodies are some of the most challenging to work up. Warm autoantibodies are uncommon, but they may occur secondary to chronic lymphocytic leukemia, lymphoma, systemic lupus erythematosus, and other autoimmune diseases or as a result of drug therapy. Partial elution using gentle heat or chemical methods is used to produce an antibody-free cell. ZZAP is one chemical that works particularly well for this. It is composed of DTT and papain, resulting in the destruction of enzymesensitive antigens and denaturing Kell, LW, Dombrock, Knops, and Cromer antigens. The cells used for homologous or differential adsorption are usually treated with enzymes or with ZZAP to enhance certain antigens and facilitate autoantibody removal. These treatment and adsorbing steps are time-consuming. An alternative method, in which PEG is used to enhance antibody removal, cutting the processing time approximately in half while still providing for adequate autoantibody removal. • • • • • • • • • • • • • Complement - a complex group of over 20 circulating and cell membrane proteins that have a multitude of functions within the immune response. Primary Roles: o Direct lysis of cells, bacteria and enveloped viruses (membrane attack complex - MAC) o Assisting with opsonization to facilitate phagocytosis o Production of peptide fragment split products, which play roles in Other roles: o Production of peptide fragment split products which play roles in inflammatory responses: ▪ Increased vascular permeability ▪ Smooth muscle contraction ▪ Chemotaxis ▪ Migration ▪ Adherence Complement proteins are activated in a cascade of events o Three main pathways ▪ Classical Complement Pathway ▪ Alternative Complement Pathway ▪ Lectin Complement Pathway Complement components are sequentially numbered C1 through C9 Four unique serum proteins : o Factor D o Factor B o Factor P (properdin) o IF ( Initiating Factor) The complement system is able to modulate its own reactions by inhibitory proteins such as o C1 inhibitor (C1INH) o Factor H, Factor I o C4-binding protein (C4BP) o Anaphylatoxin inactivator o Anaphylatoxin inhibitor o Membrane attack complex (MAC) inhibitor o C3 nephritic factor (NF) Chemotaxins : C5a , C5b , C6 , C7 Immune adherence : C3b Kinin Activator : C2b Anaphylatoxins: C3a, C4a, C5b Opsonins: C3b, C4b , C5b For virus neutralization: C4b, C1 CLASSICAL COMPLEMENT PATHWAY • • • • • • • • • • • • • • • • • • • • • • • • Activated by Antigen- Antibody binding Requires IgM or IgG for activation IgG antibody depends on concentration of cell surface antigen and antigen clustering IgM molecule is necessary to activate complement because it is large and has Fc monomers close to each other. C1 component is a complex composed of three subunits : C1q, C1r, C1s Stabilized by Calcium ions . C1q - for catalyzing C1r C1r - generate to activate C1s C1s - a serine-type protease Acts on C2 and C4 to form C4b2a C4b2a uses component C3 as natural substrate By products from the activation includes C3a C4b – binds to the cell surface C4a – stays in the medium; modest anaphylatoxin activity C3b - attaches to the microbial surface Note: complement fragments that have b type bound to membranes, whereas a fragments often have anaphylatoxic activity Human RBCs have CR1 receptors for c4d and C3b and some cleaved products will attach to the RBC membrane. Fragments degrades to C4d and C3d Factor H (binds C3b) Factor I (degrades both C4b and C3b) C3b binds to C4b2a complex > C4b2a3b complex which functions as C5 convertase. C5 convertase – acts on C5 to produce : o C5a – strong stimulator of anaphylatoxins o C5b – binds to cell membrane and recruits C6, C7, C8 and C9 When C5b and C6, C7, C8 and C9 bound , the MAC (membrane attack complex) forms causing lysis of the foreign cells . ALTERNATIVE COMPLEMENT PATHWAY • • • • • Activated by surface contacts with complex molecules and artificial surfaces such as dialysis membranes and dextran polymers Allows activation of complement without acquired immunity Four important proteins in this pathway: o Factor D – analogous to C1s in the classical pathway o Factor B – analogous to C2 o Properdin o C3 ▪ C3b – analogous to C4 ▪ Factor C3bBbp – analogous to C4b2a ▪ C3b2BbP – analogous to C4b2a3b ▪ Activation requires C3b to be bound to the surface of a target cell ▪ C3b encounters normal cells Rapidly eliminated through the combined interaction of factor H and factor I o Accumulation of C3b on microbial surfaces Attachment of C3b to Factor B > Acted by Factor D = cleaved Factor B (Bb) • • • • • • • C3bBb complex Stabilized by Properdin (P) > C3bBbP C3bBbP complex Cleaves C3 into C3a and C3b C3b Acts as a positive feedback mechanism C3b2BbP complex – act as C5 convertase and initiates the later steps of complement activation. LECTIN COMPLEMENT PATHWAY • • • • • • Activated by the attachment of MBL (Mannan/Mannosebinding Lectin) to microbes. Reactions are the same as those of the Classical Pathway Activates C4 and C2 C1 is not involved in activation of lectin pathway Effectors : o MBP/MBL o MASP-MBP- associated serine protease The three pathways of activation leads to a final common pathway for complement activation and membrane attack complex (MAC) formation. FINAL STEP OF COMPLEMENT ACTIVATION: FORMATION OF MAC / MEMBRANE ATTACK SYSTEM • • • • • Classical pathway- MAC is initiated by the enzymatic activity of C4b2a3b on C5 Alternative pathway- C3b2Bb has the ability to cleave C5 MAC- compose of terminal components of complement sequence C5 split into C5a a potent anaphylatoxin and C5b which is attach to cell membrane of C6 C7 and C8 Osmotic lysis and Death of Cell – by the small transmembrane channel that formed by the attachment of C9 to C5678 SUMMARY OF THE COMPLEMENT SYSTEM Classical Immune complexes (IgG or IgM) Recognition C1q , C1r , unit C1s C3 convertase C4b2a C5 convertase C4b2a3b MAC C5b6789 End Result CELL LYSIS Activating Substance Alternative Lectin LPS (bacterial Mannose capsule ) IgA groups on microbial cell C3, Factor B MBP, MASPand Factor D 1, MASP-2 C3bBb C4b2a C3bBb3b C4b2a3b BINDING OF COMPLEMENT BY RBC AB • • • • • • Antibodies with complement capacity destruction of RBC C1 bind with 2 adjacent Ig Fc regions IgM: pentameric, provides 2 Fc region so its possible for complement binding. IgG: monomeric, 2 or more IgG are needed in order to bind complement IgG- anti-A Immunoglobulin o Little complement activation o 800 IgG anti-A may need to attach to one adult group A RBC Ab and Rh Ag : do not bind complement because of low level of Rh Ag in RBC • • • • • Ab to lewis blood group = IgM, can activate complement and cause hemolytic transfusion Intravascular Hemolysis- Induces when Ab activate complement sequence Extravascular Hemolysis- occurs when Ab coats RBC Ab-coated RBCs : removed by cell of mononuclear phagocytic system Phagocytic cell: can clear Ab coated RBC thru its cell surface receptor for CR1 and Ig Fc receptor but IgM coated RBC cant remove by phagocytic cell because of its Fc receptor mediated phagocytosis. ANTIGEN-ANTIBODY REACTIONS • There are many complex properties of antigen and antibody reactions that influence serologic and other testing methods involving antibodies. The antigen-binding site of the antibody molecule is uniquely designed to recognize a corresponding antigen; this antibody amino acid sequence cannot be changed without altering its specificity. THE BINDING FORCE OF ANTIGEN-ANTIBODY REACTION • • • The binding force between antigen and antibody is due to three factors. They are closeness between antigen and antibody, non-covalent bonds or intermolecular forces, and affinity of the antibody. When antigen and antibody are closely fitted, the strength of binding is great. When they are apart binding strength is low. The bonds that hold the antigen to the antibody combining site are all non-covalent in nature. These include hydrogen bonds, electrostatic bonds, Van der Waals forces, and hydrophobic bonds. Antibody affinity is the strength of the reaction between a single antigenic determinant and a single combining site on the antibody. The chief application of antigen-antibody reactions is to determine blood groups for transfusion, to study serological ascertainment of exposure to infectious agents, to develop immunoassays for the quantification of various substances, to detect the presence or absence of protein in serum, and to determine the characteristics of certain immunodeficiency diseases. DIFFERENT TYPES OF ANTIGEN AND ANTIBODY REACTIONS • The reaction between soluble antigen and antibody leads to visible precipitate formation, which is called precipitin reaction. Antibodies that bring about precipitate formation on reacting with antigens are called as precipitins. • Whenever a particulate antigen interacts with its antibody, it would result in clumping or agglutination of the particulate antigen, which is called agglutination reaction. The antibody involved in bringing about agglutination reaction is called agglutinin. o Factors That Influence Agglutination Reactions: ▪ Centrifugation - Centrifugation is an effective way to enhance agglutination reactions because it decreases reaction time by increasing the gravitational forces on the reactants and bringing reactants closer together. ▪ Antigen-Antibody Ratio - Antigen and antibody have optimal concentrations; in ideal reactive conditions, an equivalent amount of antigen and antibody binds. Any deviation from this decreases the efficiency of the reaction and a loss of the zone of equivalence between antigen and antibody ratio that is necessary for agglutination reactions to occur. An excess of unbound immunoglobulin leads to a prozone effect, and a surplus of antigen leads to a postzone effect. ▪ Effect of pH - The ideal pH of a test system for antigen-antibody reactions ranges between 6.5 and 7.5, which is similar to the pH of normal plasma or serum. Exceptions include some antiM and some Pr(Sp1) group antibodies that show stronger reactivity below pH 6.5.13 Acidification of the test serum may aid in distinguishing anti-M and anti-Pr(Sp1) antibodies from other antibodies. ▪ Temperature - Different isotypes of antibodies may exhibit optimal reactivity at different temperatures. IgM antibodies usually react optimally at ambient temperatures or below 22°C at the immediate spin (IS) phase of testing, whereas IgG antibodies usually require 37°C incubation and react optimally at the antihuman globulin (AHG) phase of testing. Because clinically significant antibodies may be in both these temperature ranges, it is important to do testing with a range of temperatures ▪ Immunoglobulin Type - IgM antibodies are generally capable of agglutinating RBCs suspended in a 0.85% to 0.90% saline medium. The IgM antibody is 160 Å larger than an IgG molecule and approximately 750 times as efficient as IgG in agglutination reactions. This allows it to easily bridge the distance between two RBCs. • The IgM molecule has the potential to bind 10 separate antigen sites; however, an IgG molecule has only 2 binding sites per molecule, which implies that an IgG molecule would have to bind 2 RBCs with only 1 binding site on each cell. IgM molecules rarely bind 10 sites, owing to the size and the spacing of antigens in relation to the size and configuration of the IgM molecule. ▪ Enhancement Media - One of the key ways to enhance the detection of IgG antibodies is to increase their reactivity. Many of the commercially available enhancement media accomplish this by reducing the zeta potential of RBC membranes. The net negative charge o surrounding RBCs (and most other human cells) in a cationic media is part of the force that repels RBCs from each other and is due to sialic acid molecules on the surface of RBCs. Most acids have a negative charge, and the large concentration of these molecules on RBCs creates a “zone” of negative charge around the RBC. This zone is protective and keeps RBCs from adhering to each other in the peripheral blood. A potential is created because of the ionic cloud of cations (positively charged ions) that are attracted to the zone of negative charges on the RBC membrane. Grading Agglutination Reaction • • • • 2 WAYS TO DETECT ANTIGEN-ANTIBODY INTERACTION • • • • o Opsonisation or enhanced attachment is the process by which a pathogen is marked of ingestion and destruction by a phagocyte. Opsonisation involves the binding of an opsonin i.e., antibody, to a receptor on the pathogen’s cell membrane. After opsonin binds to the membrane, phagocytes are attracted to the pathogen. So, opsonisation is a process in which pathogens are coated with a substance called an opsonin, marking the pathogen out for destruction by the immune system. This results in a much more efficient phagocytosis. The neutralization reactions are the reactions of antigenantibody that involve the elimination of harmful effects of bacterial exotoxins or a virus by specific antibodies. These neutralizing substances i.e., antibodies are known as antitoxins. This specific antibody is produced by a host cell in response to a bacterial exotoxin or corresponding toxoid (inactivated toxin). INTRODUCTION TO IMMUNOHEMATOLOGY • • • Immunohematology- branch of science that deals with the study of RBC antigens and its corresponding antibodies in response to transfusion medicine Transfusion Medicine- Branch of Medicine that deals with blood transfusion Blood Banking- process of donor selection, blood colelction, preparation, and storage RBC-ANTIBODY INTERACTION • • • • • • • In the laboratory, BB procedure are performed to detect antigen-antibody reaction by means of agglutination RBC antigen could react to specifically to an antibody Red cell antigen antibody reaction is always specific in nature When we test for antigen, RBC/RCS are used as samples Antibodies are to be tested use serum sample Antigens are to be tested use red cells/RCS When antibody is unknown, the antigen is used as the reagent/known sample. When antigen is unknown, the typing serum/antibody is used as the reagent One of these 2 reactants must be known and that is the reagent will be used Antigens are also known as agglutinogens because these antigens are agglutinated/clumped by antibodies Antibodies are also known as agglutinins They are capable of agglutinating antigens Hemolysis o Cell destruction o When there’s cell destruction, this indicates that o It occurs due to the following: ▪ Complement protein activation ▪ Strong agglutination reaction ▪ Physical manifestation of hemolytic reaction: the supernatant fluid appears clear red, indication that hemoglobin is released from RBC • Hematuria - intact cells under microscope • Hemoglobinuria - lysed RBCs Hemagglutination o Cell clumping o Most common serologic reaction seen when there is Ag-Ab interaction o It is due to lattice formation (cross bridging of antibodies adjacent to the antigen o In laboratory, there are 2 antibodies of clinical interest that are able to clump RBC: IgM and IgG o 2 steps in detecting serologic reactions (agglutination) ▪ Sensitization - ag is specific to ab, ag recognizes a particular ab to clump ▪ Lattice formation - RBC is bound to series of abs, manifested by agglutination rxn o IgM ▪ Pentameric antibody ▪ Biggest antibody ▪ Can readily cross bridge with other antibodies thus, most efficient antibody o IgG ▪ Monomeric antibody ▪ Smallest antibody ▪ Cannot readily cross bridge with other IgG (incomplete) ▪ For IgG to create Lattice formation, there must be a bridging antibody known antihuman globulin (AHG) ▪ Can cross placenta (IgG1, IgG3) ▪ Clinical significance • Hemolytic transfusion reaction, HDN, autoimmune hemolytic anemia o Factors affecting agglutination of RBC ▪ Antibody length • Expressed in Angstroms unit (A) • IgM - 1000A (longest) o Allow IgM to be efficient • IgG - 250A (shortest) • Longer antibody, faster agglutination ▪ Zeta potential • Aka electric repulsion/clouding between RBC • It could delay agglutination of RBC • Increase Zeta potential, RBC repel each other. • ▪ ▪ ▪ ▪ Decrease Zeta potential, RBCs can interact readily facilitating agglutination • Potentiators - reagents that could lower zeta potential thus, promoting agglutination, lower incubation period, increased test sensitivity Position and number of antigen sites • If the antigens of the RBCs are positioned on the outer part of the cell membrane, there is a faster agglutination reaction • If the antigen are positioned at the inner part of the cell membrane, there is a delay/increase in agglutination • Higher number of antigenic sites has higher possibility of faster & stronger agglutination • Example o Antigen A1 = 0.81 - 1.17x106 o Antigen A6 = 0.24 - 0.29x106 o Antigen B = 6.75x106 o Antigen D = 9x106 o Antigen Kell = 3.5 - 6.1x106 Length of Incubation Time • The length of incubation is based on the Zeta potential level • If zeta potential is increased, there is an increased electric repulsion resulting to longer incubation time • If zeta potential if decreased. Electric repulsion is lowered, RBC can interact more readily, shorter incubation time • Electric potential level is directly proportional to the length of incubation time • How will we know if there’s lower zeta potential? When we add potentiator, this can readily lower the electric repulsion which will shorten incubation time, promote agglutination rxn, increase test sensitivity Reaction Temperature • Based on the time of ab that reacts with the ag • Ab is IgM, reaction temperature is room temperature (20-24C) o Optimal temperature: Ref temp (4C) o Example: typing sera • Ab is IgG, reaction temperature is body temp (37C) (warm reacting antibodiess) o Example: anti-D typing reagent (viewing box, incubation is needed to enhance reaction of IgG) • IgG is seen in the patient’s sample, these IgGs are considered as clinically significant because the temperature for reaction is body temperature pH • Ab reacts as a plasma at plasma Ph (7.357.45; neutral) • Some Ab that reacts at lower pH such as Anti M (6.0-6.5) (optimal for IgM activity) • If you are to test a patient serum for Anti M, what are the requirements to see optimum reaction of anti M. The serum must undergo Acidification process (acidosis) to lower the pH thus optimizing IgM activity ▪ o o Concentration of Ag and Ab • In BB lab, reaction would occur when there is Point of Equivalence o Point of Equivalence - when optimal concentration of Ag reacts with optimal concentration of ab. If either ag or ab has excessive concentration, reaction will not occur (false negative) o Ab & ag should be equal • When either Ag or Ab is excessive, there will be Zonal Effect/Phenomenon that will occur o Zonal effect: excess antibody/antigen ▪ Prozone - Ab is excessive • How to counter act this phenomenon? Dilute the serum by serial dilution ▪ Postzone - Ag is excessive • How to counteract this phenomenon? Dilute RBC by adding diluent by RCS; by adding conc. of NSS o When there is Postzone or Prozone effect, what is the effect on the result Ag-Ab reaction? False negative rxn Grading Agglutination Reactions ▪ Grade macroscopically & microscopically ▪ When there is no macroscopic agglutination, confirm using microscope and look agglutination ▪ Grade based on • Size of RBC • Color of supernatant Grade Reaction based on the Appearance Grade % Ag Score Description of Appearance 4+ 100% 12 One solid clump, no free cells, clear supernatant 3+ 75% 10 Several large clumps, clear supernatant 2+ 50% 8 Many medium-sized clumps, cloudy red supernatant 1+ 25% 5 Numerous small clumps, cloudy red supernatant +/<25% 3 Numerous very small clumps easily dispersed, cloudy red supernatant + <25% 1 Appears negative macroscopically, but agglutination visible microscopically 0 0 0 Negative, no agglutination seen macroscopically H 0 Complete hemolysis, no intact RBCs remaining PH 0 Partial hemolysis, some RBCs still intact but hemolysis visible in supernatant mf 0 Mixed field-mixtures of agglutinated and agglutinated cells ▪ ▪ ▪ ▪ Clear red serum Cloudy red supernatant Do not vigorously shake the sample Mixed field should be confirmed microscopically • • • Zeta Potential Theory o Repulsion of RBCs o RBCs have sialic acid molecule on their surfaces ▪ Present specifically in the glycophorins of the cell membrane of RBCs ▪ Sialic acid - negatively charged; makes RBCs net negative ▪ In saline, cations will be attracted and form repulsion causing delay in agglutination o Add potentiators to lower zeta potential ▪ Enzymes, albumin, bovine albumin, LISS ▪ Promotion agglutination o Ionic charges on the surface of red cells keep them separated ▪ Negative charges attract positive charges which in turn repel positive charges from other cells o IgG cannot bridge the space between red cells because it is too small as a molecule o IgM is large enough to bridge space between RBC o Lectins o Antibody like substances or agents obtained or derived from plant sources specifically seed extracts that could detect specific antigen Specificity Plant Source Anti-A1 Dolichos biflorus Anti-B Banderas simplicifolia (Griffonia simplicifolia) Anti-H Ulex europaeus Anti-M *Iberis amara (most common source) Iberis umbellate Iberis semperivens Maclura aurantiaca Anti-N Bauhinia variegata Bauhinia bonatian Bauhinia purpura *Vicia graminea (most common source) Prolectins o Same with lectins but it is derived from animal sources most commonly from snail o Detect antigen due to antibody specificity Specificity Snail Source Anti-A Helix pomania Helix aspera Cepaea nemoralis Anti-A1 Euphradaperionphala spp Bradybaemafructicum spp Anti-B Salmo iriderus Anti-H Eel o