Fundamentals of Immunohematology Lecture 1 Historical Perspectives: Pope Innocent VII 1492 Vein to vein transfusions Karl Landsteiner discovered ABO blood groups 1900 1915 anticoagulants-citrate-phosphate-dextrose WWII & American Red Cross Frequent transfusions /circulatory overload- need for components instead of whole blood This Photo by Unknown author is licensed under CC BY-SA. The Donation Process 1. Educational materials 2. Donor health history questionnaire 3. Abbreviated physical examination RBC Biology and Preservation RBC shape is a biconcave disc RBC composition- 52% protein, 40% lipid, and 8% carbohydrate Three areas of RBC biology are necessary for normal erythrocyte survival and function: 1. normal chemical composition and membrane structure. 2. Hemoglobin structure and function 3. RBC metabolism Defects in any of these will shorten the RBC survival time. This Photo by Unknown author is licensed under CC BY-SA. Normal length of survival of RBCs is 120 days in circulation. RBC metabolism – anaerobic glycolytic pathway, pentose phosphate pathway, methemoglobin reductase pathway, and the LueberingRapoport pathway. Hemoglobin's function is to transport O2 to the tissues and CO2 excretion. The amount of 2,3, DPG within RBCs has a significant effect on the affinity of hemoglobin for oxygen and therefore affects how well RBCs function post-transfusion. Oxygen dissociation curve- shift to the left, shift to the right Hemoglobin's function-transport O2 to tissues and CO2 excretion. Shift to the right-decrease in hemoglobinoxygen affinity. Increased levels of 2,3,DPG. Shift to the left- increases hemoglobinoxygen affinity. Decreased levels of 2,3,DPG. This Photo by Unknown author is licensed under CC BY-SA-NC. Anticoagulant solution CPDA-1 extends shelf life to 35 days at 1.0-6.0 C Additives can extend to 42 days from collection. RBC freezing for rare blood types and autologous units. Two concentrations acceptable: highconcentration glycerol 40% weight by volume, low-concentration glycerol 20% weight by volume. Frozen RBCs must be thawed and washed prior to transfusion. This Photo by Unknown author is licensed under CC BY-SA. Platelet Preservation: Platelets help to form the hemostatic plug Platelet apheresis minimum of 3.0 x 10 11th in platelets Gently agitated at 20-24C (room temperature) Spontaneous hemorrhage may occur when platelet count falls below 10,000. Stored at room temperature and have a 5 day shelf life. Two main reasons only 5 days- bacterial contamination at incubation of 22C more likely and loss of platelet quality during storage. Absence of platelet swirling is associated with loss of membrane integrity This Photo by Unknown author is licensed under CC BY-SA. Storage Lesion RBCs-loss of ATP which causes loss of membrane deformability and more fragile with age. Leads to release of free hemoglobin and K. Platelets- loss of platelet quality and viability due to loss of membrane integrity. A pH of <6.2 is associated with loss of platelet viability. This Photo by Unknown author is licensed under CC BY. • Basic Genetics • Gene: a segment of deoxyribonucleic acid that encodes for a particular protein • Trait: A genetically determined characteristic or condition • Gene locus: The specific location on chromosomes at which genes are arranged • Allele: Alternative forms of a gene • Chromosome: Gene-carrying structures that are visible during nuclear division in the nucleus of the cell • Each living cell has a characteristic set of chromosomes in the nucleus. These chromosomes are made up of nucleic acids and associated proteins that carry genes. • Each chromosome has 2 arms that are joined at a central constriction called the centromere. • The chromosomal arms are different lengths; the short arm (p) and the long arm (q). • Carrier: an individual who carries one gene for a recessive trait and one normal gene • Homologous chromosome: a pair of chromosomes in which males and females carry equivalent genes • Autosome: Any chromosome that is not a sex chromosome • Sex chromosome: Chromosome that determines gender Mendel’s laws of inheritance –law of independent segregation. Each gene is passed on to the next generation on its own. Mendel’s second law- law of independant assortment. Genes for different traits are inherited separately from each other. This Photo by Unknown author is licensed under CC BY-SA. This Photo by Unknown author is licensed under CC BY-NC-ND. Homozygous -identical alleles for a given locus or present on both chromosomes. (double dose) Heterozygous -different alleles are present at a given locus. Hemizygous -only a single copy of an allele is present instead of the customary two copies. Antigens that are encoded by alleles at the same locus are said to be antithetical. Inheritance of Genetic Traits A genetic trait is the observed expression of one or more genes. Autosomal Dominant Inheritance- trait is always expressed it appears in every generation and occurs in equal frequency in males and females. Autosomal Codominant Inheritance- when two different alleles are present (heterozygous) the products of both alleles are expressed. Autosomal recessive inheritance expressed only in a person who is homozygous for the allele Ann has inherited the recessive allele from both parents. Sex linked inheritance a trait that is sex linked is one that is encoded by a gene located on the X or Y chromosome. In females the inheritance of X linked genes can be dominant or recessive. Males are hemizygous for genes on the X or Y chromosome because there is only one chromosome present. Thus, an X linked trait that is recessive in females is expressed by all males who carry the gene for the trait. With X linked inheritance that trade is never transmitted from father to son. Sex linked dominant inheritance- males pass to all daughters, who then all express the trait. Heterozygous females pass the trait to 50% of their children. Homozygous females will pass the trait to all of their children. Ex:Xga Sex linked recessive inheritance is carried but not expressed by a heterozygous female. A male inherits the trait from his mother who is usually a carrier. An unaffected male transmits the trait to all of his daughters, who in turn transmit the trait to half of their sons. The prevalence of the expression of an X linked recessive trait is much higher in males than in females. ex: XK in McLeod phenotype. Click to add text This Photo by Unknown author is licensed under CC BY-SA-NC. Fundamentals of Immunology The majority of blood bank testing is focused on the prevention, detection, and identification of blood group antibodies and on the typing of RBC antigens. Antigen characteristics and host factors have an impact on the immune response. The detection of alloantibodies or autoantibodies in routine testing procedures is one of the utmost importance in providing compatible blood to patients. This detection is dependent upon several factors, including binding forces between antigens and antibodies, properties of the antibody itself, and individual host characteristics. Antigen-antibody reactions are influenced by a number of factors, including distance, antigen-antibody ratio, pH, temperature, and immunoglobulin type. Innate immune response consist of physical barriers, biochemical effectors and, an immune cells. The first step of the innate defenses external including skin and enzymes presence on the skin's surface. The second line of innate defenses internal an can recognize common invaders with a nonspecific response. Cellular immunity is mediated by various IS cells such as macrophages, T cells, and dendritic cells. Humoral immunity consists of fluid parts of the IS, such as antibodies and complement components found in plasma, saliva, and other secretions. Antibodies are also called immunoglobulins, immune because of their function and globulin because they are a type of globular soluble protein. They are found in the gamma globulin portion of the plasma or serum. The function of the antibody is to bond afford molecules called antigens. Only one antibody reacts with one antigen, or one part of a complex antigen. This Photo by Unknown author is licensed under CC BY-SA. Innate or natural immunity is the immediate line of immune defense and is present at birth. 1st line (innate)- skin, mucous membranes, cilia, cough reflex, secretions, low ph of vag and stomach. 2nd line (innate)-phagocytic cells (macrophages, monocytes, PMNsgranulocytes, NK cells, complement- alternate pathway, cytokinesinterferons and interleukins, acute inflammatory reaction. Acquired or Adaptive immunity- specific, evolved immune system The acquired IS uses antibodies as specific immune effectors and has memory. There are two major arms of the acquired system: Humoral- mediated by B cells and antibody production Cellular – mediated by T cells and lymphokines This Photo by Unknown author is licensed under CC BY-SA. •T lymphocytes matures in the thymus gland and is responsible for making cytokines and destroying virally infected host cells •T cell mediated immunity is involved in response against fungal and viral infections, intracellular parasites, tissue grafts, and tumors •B lymphocytes matures in the bone marrowand when stimulated by an antigen, evolve into plasma cells that secrete antibody •Memory B cells have antibody on their surface This Photo by Unknown author is licensed under CC BY-SA. T and B cells communicate with each other and are both necessary for antibody production. B cells undergo gene rearrangement in order to have the correct antibody made so that they can react with the correct antigen. T cells also have receptors that undergo gene rearrangement. The receptors of the cell membranes of T&B lymphocytes allow them to recognize foreign substances. Self vs non-self This Photo by Unknown author is licensed under CC BY-SA. Mature B cells are called plasma cells and they bind to antigens in a specific manner. The images are usually insoluble form in plasma. Memory B cells have antibody on their surfaces. Plasma cells are antibody factories that make large amounts of one specific type of antibody in a soluble form that remains in the circulation in the plasma, body secretions and the lymphatics. Binding of energy and by antibody brings about opsonization which aids in the direct killing of the pathogens by cell lysis. This Photo by Unknown author is licensed under CC BY-SA. T cell-mediated immunity is involved in the response against fungal and viral infections, intracellular parasites, tissue grafts, and tumors. T cell receptors do not recognize foreign antigen on their own they require help in the form of cell membrane proteins known as the major histocompatibility complex molecules (MHC). This Photo by Unknown author is licensed under CC BY-SA. Two major classes of MHC genes and antigens: MHC class I – on nucleated cells, code for HLA-A, HLA-B, HLA-C antigens MHC class II- on most antigen presenting cells, code for HLA-DR, HLA-DQ, HLA-DC This Photo by Unknown author is licensed under CC BY. It usually takes days to months from exposure to form an initial antibody. This is called the lag phase or preseroconversion window. It is during this time the antibody cannot be detected. The primary antibodies are IgM antibodies. After the antigen is cleared memory cells are stored in the immune organs of the host when the same antigen is encountered again the memory cells are activated and produce a stronger and more rapid response. IgG antibodies are formed during the secondary response intermating great quantities. Secondary antibodies can usually be measured within one to two days. Immune Maturation • Takes days to months for immune response to make initial antibody • The lag phase-latency, preseroconversion or window period • After antigen is cleared, memory cells are stored • When the same antigen is encountered again the immune response is quicker and stronger. This is called the secondary (memory) response. Immunoglobulin Structure • An antibody is a tetramer of two identical heavy chains and two identical light chains • Each heavy and light chain contain a variable region- which varies from antibody to antibody and binds antigen- and a constant region • Human antibodies consist of two light chain families and any given antibody has either two identical kappa or two identical lambda light chains The Fab fragment consists of the heavy-and the light- chain variable regions, the lightchain constant region, and one heavy-chain constant region domain. The Fab fragment binds antigen but does not activate effector mechanisms. The Fc fragment, which consists only of heavy-chain constant regions, activates effector mechanisms, which allows for the destruction of the antibody target. The Fc constant regions differ based on antibody isotype and subclass This Photo by Unknown author is licensed under CC BY-SA. There are five different antibody isotypes which are determined by the constant region of the heavy chain. These include IgM, IgG, IgE, IgA, and IgD. Antibody isotypes can differ in the number of antigen-binding sites per molecule The number of antigen-binding sites per antibody affects the binding avidity for the antigen. This Photo by Unknown author is licensed under CC BY-SA. IgM antibodies are capable of activating complement by changing their structure after antigen binding; they are known to cause hemolysis during transfusion reactions IgM antibodies are capable of activating complement by changing their structure after antigen binding; they are known to cause hemolysis during transfusion reactions. This Photo by Unknown author is licensed under CC BY-SA-NC. IgG antibodies are divided into four subclasses: IgG1, IgG2, IgG3 and IgG4. Each subclass has a different constant region and interacts differently with Fc receptors on phagocytes. Some are capable of complement activation. IgG1 and IgG3 are the most effective in activating complement. This Photo by Unknown author is licensed under CC BY-SA. Complement system- has 3 major roles: final lysis of abnormal and pathogenic cells via the binding of antibody, opsonization and phagocytosis, and mediation of inflammation. Classical pathway and Alternate pathway of Complement system This Photo by Unknown author is licensed under CC BY-SA. 1. Classical Pathway is initiated when antibody binds to antigen. C1 binds to Fc fragment of IgM, IgG1 or IgG3 subclass antibody. Complement 2. Alternative pathway is activated by surface contact with complex molecules and artificial surfaces. This Photo by Unknown author is licensed under CC BY-SA. 3. Lectin pathway is activated by attachment to MBL to microbes. All three methods of activation lead to a final common pathway for complement activation and membrane attack complex (MAC) formation. •Lymphokines are powerful molecules that include cytokines and chemokines •Antibodies are also called immunoglobulins and are found in the gamma globulin portion of the plasma or serum •The function of the antibody is to bind to foreign molecules called antigens. Only one antibody reacts with one antigen or one part of a complex antigen. Lock and key mechanism. • Alloantibody- antibody produced in an individual against red blood cell antigens of another individual • Autoantibody- antibody produced against the individuals own red blood cell antigens • Antigen and antibody reactions are influenced by a number of factors, including distance, antigen-antibody ratio, pH, temperature, and immunoglobulin type The immune response is initiated by the presentation of an antigen (initiates formation of and reacts with an antibody) or immunogen (initiates an immune response). Properties such as size, complexity, conformation, charge, accessibility solubility, digestibility, and biochemical composition influence the amount and type of immune response. RBC antigens are diverse in structure and composition and may be proteins (such as Rh, M, and N blood group substances) or glycolipids (such as ABH, Lewis, Ii, and P blood group substances). Human leukocyte antigens (HLAs) are glycoproteins. Most naturally occurring antibodies are IgM cold agglutinins. These react best at room temperature or lower, activate complement, and may be hemolytic when active at 37C. Ex: ABO, Hh, Ii, Lewis, MN, and P blood group systems Most RBC antibodies are IgG that react best at 37C and require the use of antihuman globulin sera for detection. The most common and clinically significant of these are Rh, Kell, Duffy, Kidd, and Ss blood group systems. Naturally occurring anti-A and anti-B antibodies are routinely detected in human serum and depend on the blood type of the individual. These naturally occurring antibodies or isoagglutinins are significant and are useful in blood typing. This Photo by Unknown author is licensed under CC BY-SA. All other antibodies directed against RBC antigens are considered unexpected and must be detected and identified before blood can be safely transfused, no matter their reaction strength or profile. Antibodies can be either alloreactive or autoreactive. Alloantibodies are produced after exposure to genetically different (non-self) antigens, such as RBC antigens from transfusion. Titers of alloantibodies may fall and be undetectable. If these indivduals are transfused with the immunizing antigen again they will make a stronger immune response which can cause a severe or possibly fatal transfusion reaction. This is why the previous history of any patient is a critical part of the testing. Intermolecular binding forces- hydrogen bonding, electrostatic forces, van der Waals forces, and hydrophobic bonds are involved in antigen-antibody binding reactions. Affinity-strength of a single antigen-antibody bond produced by attractive and repulsive forces. Avidity- binding strength of a multivalent antigen with antisera produced from an immunized individual. High-titier, low-avidity antibodes exhibit low antigen binding capacity but still show reactivity at high serum dilutions. This Photo by Unknown author is licensed under CC BY-SA. Blood samples 4 testing The anticoagulant EDTA (ethylenediaminetetraacetic acid) [ethylenediamine-tetra-acetic-acid] at a ratio of 2mg to 1ml, is most commonly used. EDTA inhibits complement activation by binding calcium and to a lesser extent magnesium. Another anticoagulant that is sometimes uses sodium heparin which inhibits the cleavage of C4. Plasma samples are often preferred over serum for testing because they lack fibrin strands which can cause false positives. Serum can be used for testing. Centrifugation- enhance agglutination reactions because it decreases reaction time by increasing gravitational forces on the reactants and bringing reactants closer together. High speed centrifugation is the most efficient method used in blood banking. Antigen-antibody ratio: Prozone-excess unbound immunoglobulin Postzone- surplus antigen Both prozone and postzone can yield a false negative result. PH- between 6.5 and 7.5 Temperature-IgM antibodies usually react optimally at ambient temperature or below 22C at the immediate spin (IS) phase, IgG usually require 37C incubation and react optimally at the antihuman globulin (AHG) phase. Enhancement media reduces the zeta potential of RBC membranes. The net negative charge surrounding RBCs is part of the force that repels RBCs from each other and is due to sialic acid molecules on the surface of the RBCs. Albumin 22% and PEG are colloids that increase the dielectric constant which then reduces the zeta potential of the RBC. Low ionic strength solutions (LISS) decreases the ionic strength of a reaction medium which reduces the zeta potential. Enzymes are protein molecules that function by altering reaction conditions and bringing about changes in other molecules without being changed themselves. Ex: ficin (from fig plants), papain (from papaya), trypsin (from pig stomach) and bromelin (from pineapple). Enzymes enhance Rh, Kidd, P1, Lewis, and I antigens. Enzymes destroy Fya, Fyb, M, N, and S antigens. Dithiothreitol (DTT) and beta-2-mercaptoethanol (2-ME) are thiolreducing agents that break the disulfide bonds of the J chain of the IgM molecule but leave the IgG molecule intact. ZZAP is a thiol reagent plus a proteolytic enzyme. It causes dissociation of IgG molecules from the surface of sensitized RBCs and alters the surface antigens of RBCs. Flow cytometry makes use of antibodies that are tagged with a fluorescent dye. Fluorescence occurs when the compound absorbs light energy of one wavelength and emits light of a different wavelength. End of Lecture 1 Read Chapters 1-4 and complete quiz.
