Immunohematology Basics PDF

IMMUNOHEMATOLOGY / ISBB / IH LESSON#1: BASICS OF IMMUNOSERO & ITS APPLICATION TO BLOOD BANKING PRELIMS | A.Y. 2023 - 2024 | SIR JASON “AB” CHUA IMMUNOLOGY AND SEROLOGY...

IMMUNOHEMATOLOGY / ISBB / IH LESSON#1: BASICS OF IMMUNOSERO & ITS APPLICATION TO BLOOD BANKING PRELIMS | A.Y. 2023 - 2024 | SIR JASON “AB” CHUA IMMUNOLOGY AND SEROLOGY REVIEW: CELLS OF THE IMMUNE SYSTEM The science of immunohematology embodies the blood group antigens and antibodies. Mainly the concepts of immunohematology is based on the field of Immunology mainly because it involves the immune responses to the transfusion of cellular elements. The body's immune system is a compilation of an integrated network of cells, tissues, organs, mechanical barriers and secreted molecules. 3 FUNCTIONS: Defense ○ Prevents the entry of infectious agents ○ if in case that they are able to gain entry then Immune responses of these are mainly found in the blood they're going to be eliminated whereas others are actually found in the lymphoid organs. ○ Recognized self vs. non-self Most of them are coming from the blood and they are produced in ○ anything that is going to be recognized as the bone marrow coming from the same stem cell but they are non-self or foreign must be eliminated right going to differentiate into 2: away 1. MYELOID PROGENITOR CELL➡ granulocytes ○ normally, the immune system is not going to Polymorphonuclear cells (PMN) -most common attack the body's antigens but if in case that Neutrophil this happens then therefore it is already a Most numerous and is very active defect and that is actually referring to the during any infection, particularly occurrence of autoimmune disorders in which bacterial infxn. the body now or immune system now is Found in the peripheral blood while attacking the body's own antigens migrating into tissues in times of Homeostasis infection via diapedesis when ○ maintains balance during an immune signaled by inflammatory response. If the pathogenic agent is already mediators. eliminated then it also has its means of Eosinophil stopping that particular response so it could Granules contain the major basic not affect the normal cells of the body. protein which is essential in Surveillance fighting parasites. ○ able to detect any overgrowth of a particular Basophil cell population. Found in blood ○ If there is any tumor or cancer formation then Granules contain histamine that that is signaling a defect in the body's ability initiates hypersensitivity responses to recognize any increased mitotic division or Mast cell increased number of a particular cell Found in tissues population. Active for eliciting hypersensitivity reactions 4 COMPONENTS: Monocyte Cells and tissues of the immune system ○ Dendritic cell and/or macrophages ○ First line of defense that prevents any entry Acts as antigen presenting cells of invading pathogen in portals of entry of the Capture antigens via body. phagocytosis(main way of Skin eliminating pathogens) and they Mucus secreting cells break them down. enzyme producing cells An antigenic remnant will be placed Monocyte-Macrophage Cell System at their plasma membrane or in its ○ Antigen presenting cells surfaces to be presented to T T Lymphocytes (T cells) lymphocytes. B Lymphocytes (B cells) 2. LYMPHOID STEM CELL ➡ Lymphocytes NK Cells: release chemicals (perforins & granzymes) to kill infected cells, foreign cells and tumor cells T Lymphocytes/Cells: very important cells in the immune system ○ Produced in the bone marrow and matures in the thymus (hence the name T cell) ISBB - LEC EMBOSCADO, MATUGAS, PALCO, TUGAY, APIL, DELFIN, LAMELA, INTING, VALDIVIA BSMLS 3I & 3J 1  ○ Expresses specific receptors ○ These cells/tissues/organs provide an unfriendly ○ Involved in the cellular immunity environment for most pathogens TWO CLASSES OF T CELLS ○ Includes phagocytic cells as well as chemical ○ T-helper cell: (CD4+) activate macrophages mediators to promote inflammatory responses and B cells via cytokine secretion; the cell Skin whom the macrophages and dendritic cells Mucosal linings will present its phagocytosed antigen to HCl of stomach Once it is being determined by the T Mucus-producing pseudostratified columnar helper cell, then it is going to elicit a epithelium of respiratory tract–which trap particular immune response foreign organisms ○ T-cytotoxic cell: (CD8+) releases chemicals Flushing action of the urine (perforins & granzymes) to kill infected cells, Unsaturated fatty acids on the skin foreign cells, as well as tumors Sweat B lymphocytes tears ○ Produced in the bone marrow and matures in Saliva the bone marrow which then differentiates to ○ In the event that the invading pathogens have plasma cell surpassed the first line of defense, then inflammation Plasma cell: activated by T-helper occurs cell; produces antibodies (humoral INFLAMMATION - second line of defense immunity) ○ Produce antibodies to block infections and REVIEW: INFLAMMATION eliminate extracellular pathogens Refers to the body's reaction to injury ○ Derived from stem cells in the bone marrow Results in 3 events: and is involved in humoral immunity ○ increased blood supply in the area Transforms to become plasma ○ increased capillary permeability cells and produces antibodies or ○ migration of leukocytes into surrounding tissues immunoglobulins which are The three events from above manifest symptoms including; important proteins involved in the ○ Pain (dolor) immune defense ○ Heat (calor) ○ Redness (rubor) 2 WAYS IN B LYMPHOCYTES ACTIVATION ○ Swelling (tumor) 1. Binding of an antigen to IgD and IgM on B cell surfaces Vasodilation - increase in diameter of blood vessels that lead to ○ Since B lymphocytes have both IgM and IgD increased blood flow in the area. It may include the following that are found in its surface in which any type symptoms; of antigen that is going to bind with these ○ Redness (due to increased blood flow) antibodies →lead to the activation of B cells ○ Heat (due to increased temperature) upon in contact with antigen →become REMEMBER: If there’s an increased blood plasma cells → antibody secretion flow, there will also be an increased 2. Cytokine signaling of T-helper cells to B cells to temperature become plasma cells Swelling - manifested by edema ○ T helper cells will release chemical signals → ○ due to increased capillary permeability and increased activate B cells to become plasma cells fluid accumulation in the particular area →release antibodies Influx of phagocytes (neutrophils) - via diapedesis and exit from blood vessels towards the site of infection ACTIVATION OF B LYMPHOCYTES: ○ Accumulates in the injured area (1) binding to antigen → (2) chemical signaling → (3) becomes plasma cell ○ Apart from neutrophils, other WBC’s will also migrate to → (4) releases antibodies the infected area in the form of eosinophils, macrophages as well as NK cells Innate immunity has a consistent response regardless of the type or pathogen or the number of encounters with that pathogen. The innate response produces the same magnitude and is governed by the same regulatory mechanism. It also has no memory. 2. ADAPTIVE OR SPECIFIC IMMUNE RESPONSE Production of antibodies (humoral immunity) or actions of T-cell (cell mediated immunity). ○ Results from immunologic memory and specificity that develop in response to the antigen and involve the ability 2 TYPES OF IMMUNITY: to recognize self from non-self antigens. ○ Cells involved have the ability to recognize specific 1. INNATE OR NONSPECIFIC IMMUNE RESPONSE infectious agents in which repeated exposure results in Involves the body’s first line of defense against invading the production of a much more powerful response. pathogens ○ Product of adaptive response = production of antibodies Physical barriers Ab are highly specific to a particular agent ISBB - LEC EMBOSCADO, MATUGAS, PALCO, TUGAY, APIL, DELFIN, LAMELA, INTING, VALDIVIA BSMLS 3I & 3J 2  Ab are produced by B lymphocytes. B lymphocytes are then activated to become plasma cells due to its recognition receptors capable of discriminating many molecular configurations. Interaction between both humoral and cell-mediated. ○ Immunohematology is primarily concerned with the causes and effects of humoral immunity. Mainly because the administration of antigens via an intravenous route leads to the production of antibodies (specific reaction). ANTIGENS EPITOPES OR ANTIGENIC DETERMINANTS CHARACTERISTICS THAT MAKE AN ANTIGEN AN EFFECTIVE Small regions of the molecule within the immunogen IMMUNOGEN: Unique configurations which allows recognition by corresponding ○ Foreign nature antibody It is capable of producing a specific immune ○ If an antigen has multiple epitopes, then a different response. antibody is going to be produced from each of those Immunogen - an antigen capable of producing epitopes an immune response. Thus, there are tendencies wherein one “All immunogens are antigens but not all antigen elicits the production of multiple types antigens are immunogens.” of antibodies. The degree of foreignness: an immunogen Antibodies produced for different epitopes must be identified as non-self. Wherein the tend to cross-react if they have a common greater the difference from self antigens, determinant. produces a greater likelihood of eliciting an Recognized as foreign by the immune system immune response. Allogeneic antigens and autologous antigens ○ Molecular size ○ Allogeneic antigen Antigens that have a molecular weight of come from another individual but are of the more than 10,000 daltons are better same species. immunogens compared to those having a Recognized as foreign. MW < 10,000. ○ Autologous antigens ○ Molecular complexity and composition tolerated by the immune response Proteins are the best form of antigens d/t to recognized as self Ag. it’s complexity = much more effective in ○ NOTE: if in case the immune system could not recognize inducing immune response self-antigens, it is going to induce an immune response Carbohydrates (indicated as autoimmune) Lipids, EXAMPLE: Glycolipids- ABO group antigens which are In an allogeneic blood transfusion, they may have the same blood are also effective as immunogen. type but still it comes from a different individual of the same But, red cell antigens that are highly species. protein in nature (Rh Antigen) are In autologous transfusion, the blood is taken out of an individual very strong immunogens = and will be transfused back to the same individual produces high immune response ○ done mainly for rare blood types where their blood will (reason why it causes be donated in case of emergency but not for other miscarriage/death of baby in cases individuals, only for them. of Rh incompatibility are highly protein in nature (Rh antigens) are very strong immunogens. Thus, it has the ability to produce a high immune response leading to miscarriage or death of the baby in cases of incompatibility with the mother’s Rh and of the baby’s. ○ Route of administration and dose - Although not a “physical nature” important for response Refers to the manner wherein the antigen stimulus is introduced. Introduced via intramuscular route but often BLOOD ANTIGENS administered intravenously via blood Chemical structures embedded in or protruding from RBCs, WBCs, transfusion in blood banking or hematology. and platelets. Intravenous administration - Currently, 29 blood group systems exists, wherein there is 250 generally the best routes for unique red cell antigens that are recognized by the AABB inducing an immune response. (American Association of Blood Banks) ISBB - LEC EMBOSCADO, MATUGAS, PALCO, TUGAY, APIL, DELFIN, LAMELA, INTING, VALDIVIA BSMLS 3I & 3J 3  RBC antigens are determined by the inheritance of many blood that is why IgA is also group chains. found in the secretions. Every individual possesses a unique set of red cell antigens ○ While IgG, IgE, IgM, and through genetic inheritance. IgD are found in the 3 COMMON FORMS: serum. ○ Glycoproteins Light chains (2 types): combination of carbohydrates and proteins ○ Kappa HLA system ○ lambda ○ Glycolipids ○ Biochemical properties combination of proteins and lipids Variable region ABH/ABO, Lewis, Li and P blood group concerns in binding with the antigen systems Constant region ○ Proteins uniqueness of antibody class blood group antigens that can highly induced function such as activation of an immune response complement or the attachment to considered the most effective immunogen certain cells Rh, M, N blood group systems IgE and IgD → attached to certain cells ○ IgE → surface of basophil and mast cells ○ IgD → surface of B lymphocytes IgG → mainly found in the serum IgM → surface of B cells IgA → secretions ○ Serological activity Hinge region composed of disulfide bonds Impart the flexibility of the molecule during its attachment to their ANTIBODIES antigens counterpart of antigens Can be acted upon by certain enzymes called Specific to a particular antigen papain and pepsin Produced by plasma cells which are your activated B-cells and Papain → yields the antibody into 3 they are going to produce proteins in the form of antibodies. fragments (2 Fabs & 1 Fc). 5 types of antibodies: [Pa1 - pa2 - in3] ○ IgG, IgA, IgM, IgE, IgD ○ Fragments for antigen CHARACTERISTICS: binding (Fab) → site for ○ Size antigen binding and gives made up of monomeric units in which some its antigenicity which are having more than one units produce agglutination Monomer: IgG, IgE, IgD (smallest) reactions IgA Pepsin → 2 fragments (1 Fab & 1 Dimer (secretions) Fc) [Pep1 - sin2] Monomer (serum) ○ Cutting is below the hinge IgM (largest) region. Fab is still intact Pentamer: 5 units ○ Regardless if there is no ○ Biologic Function Fc region it is still Heavy chains capable of binding to impart unique features to the antigens. different immunoglobulin classes which permit them to function biologically heavy chains determine what type of antibody or what class of antibody they are. ○ so if the heavy chain is containing the gamma form it is an IgG. NOTE: ○ If the heavy chain is an IgG & IgM alpha form it is unique considered of high clinical significance in the study of because the alpha heavy immunohematology. chain enables the b/c these forms are produced if there is introduction of red cells whose antibody to be residing in antigens are absent or are not the same w/ the antigens of the recipient the mucosal lining and ISBB - LEC EMBOSCADO, MATUGAS, PALCO, TUGAY, APIL, DELFIN, LAMELA, INTING, VALDIVIA BSMLS 3I & 3J 4  IMMUNOGLOBULIN MU (IgM) MOST CLINICALLY SIGNIFICANT antibody in blood banking Considered to be of high clinical significance among the five types because: of immunoglobulins ○ It is Reactivity at 37°C ( body temperature ) Largest of all the antibody molecules ○ It can activate complement system ○ Consists of five basic units/pentamers in which it is ○ Indirect agglutination and hemolysis. joined together by a structural component mainly Which means IgG agglutination is not readily composed of disulfide bonds located in the middle visible ( disadvantage). Unlike IgM, known as chain. agglutination is very visible due to its large ○ Restricted entirely in the intravascular space and is able size to activate the classical pathway of complement with Indirect agglutination - means that there is a great efficiency. need for some reagent ( Anti-human globulin ) ○ Accounts for about 5 to 10% of the immunoglobulin in order for the agglutination reactions to be pool visible. Fixes complement 4 subclasses ( result of minor variations in the gamma heavy First antibody to be produced chains, so the subclasses has slight differences on their amino ○ Part of the primary immune response to an infecting acid ) organisms ○ IgG1 Does not cross the placenta ○ IgG2 ○ Due to absence of receptors ○ IgG3 Saline agglutinins ○ IgG4 ○ Capable of agglutinating antigens presents on rbcs suspended in saline if they are processed at room ANTIBODIES temperature (eg. 22-24°C, ) or @4°C (aka Cold reacting Particularly IgM and IgG are highly significant in the study of Antibodies) immunohematology because it is associated with the following : Activate complement very efficiently Has caused hemolytic transfusion reactions (HTRs) ○ Much more efficient compared to IgG since it only ○ If a particular recipient of has received a packed red call requires 1 IgM to initiate the classical pathway. or blood component , in which it has antigen that is absent from its system , therefore its body will produce IMMUNOGLOBULIN GAMMA ( IgG) antibody against of such antigen Most abundant in the plasma ○ First encounter with the non-self antigens would ○ Comprises 80% of the total immunoglobulin population undergo sensitization phase( slow progress of antibody ○ Ability to cross the placenta - it has Fc receptors in the production ) placenta which allows its crossing during pregnancy. ○ 2nd time of encounter with the same antigen, there will ○ Advantage: be faster production of the antibodies ( since B cell has Able to protect the baby from infection memory) particularly if the mother has an infection that ○ Since there is faster production of antibodies → there occur during pregnancy could be immediate transfusion reaction → complement ○ Disadvantage: activation → hemolysis ( fatal for the blood recipient) If IgG is produced against certain red cell Implicated in Hemolytic disease of the fetus and newborn antigen (HDFN) Complement activation not as efficient compared to IgM because of its smaller structure and requires 2 molecules of IgG for it to activate the classical pathway of the complement EXAMPLE: If the baby has an antigen that is absent in the mother, it will lead to the desensitization of the mother’s antigen and in turn it will produce an antibody → If the antibody is in IgG form, it could cross the placenta and attach to the red cell of the baby and will activate the complement which causes hemolysis. ○ Involves RH blood group system ○ Occurs when a baby is RH antigen positive and the mother is RH antigen negative ( mother lacks RH Predominant antibody produced in the secondary response antigen) ISBB - LEC EMBOSCADO, MATUGAS, PALCO, TUGAY, APIL, DELFIN, LAMELA, INTING, VALDIVIA BSMLS 3I & 3J 5  Most likely , the baby inherited the gene that Avidity - strength of the bond between antigen and antibody. produces RH antigens from the father COMPLEMENT ○ On the first pregnancy with an RH + baby would be safe the complement system is a group of serum proteins that have a Since it will be the first encounter of the number of biologic rules related to antigenic clearance, cell lysis mother with said antigen as well as vasodilation Baby may survive , but a possibility would normally circulate in an inactive or pro-enzyme state appear YELLOW, because of the degradation ○ during activation they are converted into active enzyme of RBC→ that lead to hemolysis of some that enhance the immunologic process RBC→ resulting in the presence of bilirubin in FUNCTIONS OF THE COMPLEMENT SYSTEM the circulation of the baby Opsonization Feto-maternal circulation - a particular term in Anaphylaxis pregnancy where the baby’s blood will actually Lysis go to the circulation of the mother → leading Chemotaxis to sensitization ○ On the second pregnancy with an RH + baby would be OPSONIZATION not safe Able to clear immune complexes through this process since the immune system of the mother has Covering of the foreign material with your complement proteins memory , during the feto-maternal circulation enhances phagocytosis ,there will be faster production of antibodies Promote release of enzymes from neutrophils (IgG) against RH antigen of the baby→ leading to hemolysis of the baby’s blood ANAPHYLAXIS Since the IgG is produced → it could cross the There is increased smooth muscle contraction and it induces placenta → attach to RBC of baby → activates inflammation complement→ leads to hemolysis → death of the baby LYSIS ANTIGEN-ANTIBODY COMPLEX Manner of killing foreign antigens by membrane lysis The binding of antigens to antibodies is based on noncovalent ○ A result of the transfusion reaction is if a red cell that is forces : transfused to the patient is containing an antigen that is NONCOVALENT ORIGIN absent on his system. Therefore, antibodies will be FORCES produced and activate complement that could lead to Ionic forces that is ELECTROSTATIC based on the lysis of transfused red cells. FORCES Attraction between opposite charges CHEMOTAXIS Attraction due to Referring to the recruitment of platelets and phagocytes in which Hydrogen shared it will induce the immune response between 2 electronegative HYDROGEN PATHWAYS FOR COMPLEMENT ACTIVATION (negative charges) BONDS atoms particularly Nitrogen and ,Oxygen Fluctuations/attract ions between 2 electron clouds VAN DER WAALS around molecules FORCES oppositely polarized neighboring atoms Hydrophobic groups interact unfavorably with water and tend to pack together to Classical Pathway exclude water MB-Lectin Pathway molecules. The Alternative Pathway HYDROPHOBIC attraction also CASCADE OF THE CLASSICAL COMPLEMENT SYSTEM FORCES involves van der Main antibody directed mechanism for triggering complement Waals forces. activation Weak bonds that is formed as a result NOTE: not all antibodies are able to activate the complement of the exclusion of CAN ACTIVATE COMPLEMENT CANNOT ACTIVATE water in IgM IgG4 antigen-antibody IgG1 IgA complex IgG2 IgE Two terms often use in describing Antigen-antibody complex: IgG3 IgD Affinity - the tendency that an epitope has for combining with the antigen binding site of antibody molecule. ISBB - LEC EMBOSCADO, MATUGAS, PALCO, TUGAY, APIL, DELFIN, LAMELA, INTING, VALDIVIA BSMLS 3I & 3J 6  the classical complement pathway is going to start by an antigen and antibody interaction so if the FC portion of your antibody has attached to an antigen then it is going to be recognized by the first complement protein and that is your C1 ○ It has three subunits you have C1q, C1r and C1s so collectively they are your C1qrs This is going to cleave now your second complement protein so you have your C4 complement in which it will cleave to have your C4b and C4a so mawala C4a and what is retain would be your C4b Next is the activation of your C2 complement in which it will be cleaved to have C2a and C2b so what is going to be retained will be your C2a so you have here now the combination of your C4b2a which is now going to be termed as your C3 convertase next C4b2a will now cleave your C3 so your C3b will now join with your C4b2a3b which is now termed as your C5 convertase The C5 complement protein which will cleave to C5a and C5b, your C5b will now be attaching to your cell membrane (it is actually rapidly inactivated but unless it is going to be bound with your C6) Next C5b is going to form the beginning of your MAC or your membrane attack complex so C5b will join with C6, C7, C8, and C9 to become C5b6789 and this is now your membrane attack complex which will now induce cell lysis [END] ISBB - LEC EMBOSCADO, MATUGAS, PALCO, TUGAY, APIL, DELFIN, LAMELA, INTING, VALDIVIA BSMLS 3I & 3J 7  IMMUNOHEMATOLOGY / ISBB / IH LESSON#1.2: BASICS OF IMMUNOSERO & ITS APPLICATION TO BLOOD BANKING PART 2 PRELIMS | A.Y. 2023 - 2024 | SIR JASON “AB” CHUA FACTORS AFFECTING THE REACTIONS BETWEEN ANTIGENS AND The optimum reaction of antigen and ANTIBODIES antibody happens in this phase Equal amount of antigen and antibody 2. Concentration of Antigens 1. Nature of Antibodies and Antibodies 3. Effect of Surface 4. Use of Reaction Media Charge 5. pH 6. Temperature 7. Incubation time 8. Washing 9. Centrifugation 10. Chemical Reduction 11. Antiglobulin Testing 12. Cell Dosage 3. EFFECTS OF SURFACE CHARGE 13. Location of the antigen Sialic acid ○ Components found in red blood cell membrane 1. NATURE OF ANTIBODIES ○ responsible for the negative charge of the cell Reaction of Antigen-Antibody depends on the class of antibody membrane. ○ 5 antibodies (IgG, IgM, IgA, IgD, IgE) ○ If you suspend the rbc(-) in a saline environment(+) , they ○ Also depends on the structure of the antibody will attract since their charges are opposite. Ie. If IgG is monomer; has 2 heavy and 2 light Cationic cloud chains, and is connected by covalent bonds ○ Numerous cations (+) are attracted to the anion(-) IgM is pentameric, since it is big, it has surface of red blood cells. numerous antigen binding site → more ○ responsible for the distance of 1 RBC to another. visible reaction Zeta potential: ○ repulsion force from 1 RBC to another. RECALL: from part 1 AFFINITY ○ created because of the ionic cloud of cations that ○ the tendency that an epitope has for combining with cause the force repulsion between red cells. the antigen binding site of antibody molecules. AVIDITY ○ strength of the bond between antigen and antibody. IgM (Cold reacting) ○ Monomer ○ not reactive at body temperature (22-24°C or lower) IgG (Warm reacting) ○ Pentamer ○ reactive in body temperature ○ can cross the placenta 4. USE OF REACTION MEDIA 2. CONCENTRATION OF ANTIGEN AND ANTIBODIES 22% albumin MNEMONICS: GAMDE (highest to lowest conc.) Low Ionic Strength Solution (LISS) ○ IgG (80%) ○ 22% albumin and LISS has the same reaction, both ○ IgA (13) potentiators (enhancers) for a reaction to happen. ○ IgM (6%) They decrease zeta potential (act as a buffer) ○ IgD (1%) hence faster agglutination ○ IgE ( system > human body but genotypically, they are heterozygous – A parent as AO and the B parent as BO ○ If gene combination takes place to determine the genotype of the offspring, there could be a possibility that offspring could have; AB, AO, BO or OO (OO as homozygous while rest is heterozygous) ○ Recall: phenotype is based on antigens present on the red cells and is the byproduct of genetic make up If parents have A and B genes, both antigens Nitrogenous bases (Adenine, Thymine, Guanine, Cytosine) will be expressed. Offspring will be Type AB ○ 3 consecutive Nitrogenous bases constitutes a codon that carries a particular amino acid. PUNNETT SQUARE Amino acids linked by peptide bonds forming a protein. Punnett square is a very important tool wherein it is responsible GENOTYPE & PHENOTYPE for illustrating the probability of genotypes from known or inferred PHENOTYPE genotypes ○ Outward expression of genes. A B ○ Product of genotype. O AO BO ○ Ex. antigen of red cells (if blood O AO BO type A -> A antigens found in red cells) Punnett Square Illustration Explanation: GENOTYPE ○ Parent no. 1 (homozygous O) ○ Responsible for expression of genes. ○ Parent no. 2 (heterozygous A & B) but both of the genes are considered DOMINANT Sample scenario: ○ Simply combine O & A = AO; O & B = BO An individual has B antigen which means a blood type of B, the Genotype present: phenotype will be B since B is the antigen present on the red cell ○ AO ○ The genotype from the above scenario will be either ○ BO homozygous (BB) or heterozygous (BO) – a Percentage: combination of one dominant gene and one recessive ○ AO → ¹⁄ or 50% gene. ○ BO → ¹⁄ or 50% ISBB - LEC EMBOSCADO, MATUGAS, PALCO, TUGAY, APIL, DELFIN, LAMELA, INTING, VALDIVIA BSMLS 3I & 3J 1  Phenotype: organ rejection, but due to the ○ 50% Blood type A complexity of HLA gene, finding a ○ 50% Blood type B match for organ transplantation is quite difficult; even for the case of A B twins. A AA AB B AB BB INHERITANCE PATTERNS Blood group antigens are inherited by the following patterns: Genotype present: ○ CODOMINANT ○ AA If you have inherited two dominant genes, ○ AB although different, both of their gene products ○ BB will be expressed Percentage: ○ DOMINANT ○ AA → ¹⁄ or 25% If dominant ○ AB → ¹⁄ or 50% gene/double dose, ○ BB → ¹⁄ or 25% more expressed Phenotype: If paired with a ○ 25% Blood Type A recessive gene, it will ○ 50% Blood Type AB still be expressed. ○ 25% Blood Type B ○ RECESSIVE Expressed only if inherited homozygously GENES, ALLELES AND POLYMORPHISM In ABO, Genes are located in specific loci on a particular chromosome. ○ A & B are considered to be dominant, while ○ chromosome 9, houses ABO ○ O is considered to be recessive. blood group In the presence of a dominant and recessive gene, the dominant ABO blood gene is still able to express its gene product (i.e antigen) group/gene, has If you have AO and BO, still, you have A and B antigens on your red three different cells → typed as type A or type B; but forms (A gene, B If you have received O gene from both parents, since both are gene, O gene) = recessive, they can only be expressed if inherited homozygously. alleles If heterozygous is paired with a dominant gene, it will always be Alleles may exist for each locus the product of the dominant gene that will be expressed ○ Alleles are variations of a particular gene If you have inherited both dominant genes, whether it is A or B, Aa ○ A chromosome may have multiple locus/ loci (plural), or BB. homozygous for the dominant genes, or heterozygous for but not all loci can have the ABO gene (only 1 loci the dominant genes, still, antigens will be expressed houses a specific gene) SUMMARY: Dominant + recessive = antigen Chromosome 1 has a loci that contains Rh gene SILENT, AMORPH, NULL GENES Chromosome 17 - Diego gene (from Diego Genes that do not code for any product. blood group system) CAUSES: Allelic genes → ANTITHETICAL ○ Amorphic gene inherited from both parents ○ Only one form of the gene could be present on a ○ Action of suppressor genes particular locus ○ Ie. ABO blood group/gene, has three different forms (A Sample scenario: gene, B gene, O gene), but only one allele can be present There will be instances where despite inheriting a dominant gene, the inheritance of a suppressor gene will inhibit the on a particular locus = most blood groups are having expression of the products of a gene, regardless of inheriting genes that are said to be “antithetical” dominant genes = product of genes will not be expressed Polymorphic genes ○ Dominant gene: Lu a/Lu b ○ More than one allele that is contained in a particular loci ○ Suppressor gene: : In(Lu) at a particular chromosome Ie. Rh blood group system; has 5 different ○ Examples of null phenotypes alleles (D, C, c, E, e) O Rh antigens could be a combination The amorph phenotype of the ABO of these alleles, but only three are blood group system present Rh null ○ DCe, DcE, Dce, DCE or Ce, Rh blood group etc Does not allow the expression of Ie. HLA/Human Leukocyte Antigen (more DCcEe antigens complex than Rh due to excessive number of Lu (a-b-) possible combinations) From the inheritance of both the Reason why HLA typing is recessive genes of the Lutheran important in organ transplantation blood group aka “tissue typing” ○ Absence of Lu a and Lu b It is important that the donor and the recipient must match to avoid ISBB - LEC EMBOSCADO, MATUGAS, PALCO, TUGAY, APIL, DELFIN, LAMELA, INTING, VALDIVIA BSMLS 3I & 3J 2  Could also be from the presence of Both blood group genes, though coming from a suppressor gene. Despite having separate chromosomes, are expressed dominant genes which could separately. possibly express an antigen, the suppressor gene inhibits the CHROMOSOMAL ASSIGNMENT expression of these antigens, BLOOD GROUP SYSTEM CHROMOSOME Rh 1 Amorph and Suppressor Genes in Blood Groups (1p36-p34) -short arm BLOOD GROUP RESULTING Duffy AMORPH GENE SUPPRESSOR GENE 1 SYSTEM PHENOTYPE (1q22-q23) - long arm ABO O O MNS 4 H h Bombay Chido/Rodgers 6 Kell 7 Kell K0 Kellnull ABO 9 Lutheran Lu In (Lu) Lu(a-b-) Though Rh and Duffy come from the same chromosome, they are Kidd Jk In (Jk) Jk(a-b-) located in different Loci. The same goes with Lewis, LW, Lutheran Duffy Fy Fy(a-b-) and Hh. “O” as the amorph gene will have the phenotype “O” if you are Chido/Rodgers is a miscellaneous blood group. homozygous for the amorph gene. CHROMOSOMAL ASSIGNMENT The dominant gene for “H” would be H, but if you have inherited the BLOOD GROUP SYSTEM CHROMOSOME recessive “h” gene, then there will be no H antigens present on the Diego 17 red cell. Thus , it will be classified as the bombay phenotype (hh). Kidd 18 2 blood groups that have suppressor genes: Lewis 19 ○ Lutheran and Kidd LW 19 ○ Recall: despite having dominant genes, the presence of Lutheran 19 suppressor genes successfully inhibits the antigens. Hh 19 For Duffy, a double dose of the Fy (Fy/Fy) gene would have the P 22 Fy(a-b-) phenotype, Currently, there are almost 40 blood groups. Most of them are ○ (-) indicating the absence of the particular antigen. considered miscellaneous, which means they are only found in a particular group of people. Whereas ABO is considered a major MENDELLIAN PRINCIPLES blood group since 99% of the population in the world contains Gregor Mendel - Father of Genetics either A,B or the lack of either A/B antigen. INDEPENDENT SEGREGATION HETEROZYGOSITY vs. HOMOZYGOSITY Each parent has a pair of gene for a particular trait and either of It is important in blood banking because it leads to a dosage effect which can be transmitted to the next generation. that pertains to the number of antigens on the red cell, and this is ○ Punnett square: demonstrates the law of independent significant serologically. segregation A B B AB AB B AB AB INDEPENDENT ASSORTMENT Blood group antigens are inherited from different chromosomes, but are expressed separately without the interference of one blood group gene coming from a different chromosome with the expression of the antigen(s) coming from another chromosome. Genogram: illustrates the law of independent assortment. Example: If you have the phenotype Jk (a+ B-), it means that you have the Phenotype “A” indicates the presence of A antigen, hence the presence of Jka antigens on your red cell and you do not contain genotype is “AO” or homozygous. Jkb antigen. ○ Recall: ABO blood group originates from chromosome 9. ○ This means you have a double dose of the Jka antigen ○ Recall: Kell blood group, where K and k antigens are present on your red cell pertaining only to the kidd present, come from chromosome 7. antigens ISBB - LEC EMBOSCADO, MATUGAS, PALCO, TUGAY, APIL, DELFIN, LAMELA, INTING, VALDIVIA BSMLS 3I & 3J 3  ○ However, if you are going to test for Jka antigen, you are cannot have both M and N there; you could only have just going to add a particular antibody, and that is anti-Jka one of them. That’s why I placed a slash in between. antibodies, and because all of the kidd antigens only ○ But in another loci that is very near to your M or N gene have Jka on their red cell→ there would be numerous is your big S and small s gene (Ss). antibodies that would attach to the red cell, giving you a So that’s why these two, although separated three-plus reaction. because they are different genes, somehow, ○ Note: adding anti-Jkb to Jk(a+b-) = no reaction due to their close proximity in the No Jkb antigens for the anti-Jkb ab to attach chromosome, they are usually inherited as a unit, which is termed LINKED GENES. Considered to be one blood group (MNSs) MS, Ms, NS, Ns These are different genes, so each antigen is going to be coded by the different genes. But because of Whereas if you are heterozygous to the JK antigens (a+ B+), you their close proximity on the both have Jka and Jkb antigens; your genotype could be Jka and chromosome, they are inherited as Jkb. a unit. The genetic combination of ○ This means that the kidd antigens on your red cell are these linked genes is known as both Jka and Jkb. HAPLOTYPES. ○ If you added anti-Jka, but since it is a mixed population of Jka and Jkb antigens, it is going to give a lesser NOTE: hemagglutination reaction(1+/2+). In the Philippines: ○ Rh+: 99% HETEROZYGOUS ○ Rh-: 1% If you have a mixed population of antigen, you are considered to be heterozygous LINKED GENES ○ Example: Genes that are in close proximity on the same chromosome, If you have M on one chromosome and N on making them likely to be inherited together. another, you will have a two-cell antigen ○ MNSs population because you are both positive for M and N HAPLOTYPE HOMOZYGOUS Genetic combination of linked genes If you have a double dose of the antigen, you are considered to be ○ MS, Ms, NS, Ns homozygous ○ Example: LINKAGE DISEQUILIBRIUM If one parent contains the M gene and the In some communities, geneticists would tend to compute in the other parent also contains it, your red cell will population how many percent are the N+, N-, A, B Rh+, Rh- but due contain a lot of M genes because both parents to the presence of linked genes they have what we call linkage have given you those specific chromosomes; disequilibrium. therefore, you are homozygous. ○ For example, if they are not inherited as a unit, the M and LINKAGE, HAPLOTYPES AND CROSSING OVER S are separate. Exceptions to the law of independent assortment M - 17% in the population There is an instances the loci of your genes are actually located in S - 12% in the population a very close proximity ○ But because they are inherited as a unit, there is an increase in the frequency in the people that have M. Because they are haplotype, which is a unit, it will be MS - 24 %. there will be an increase because they can be inherited as a unit. CROSSING OVER happens in cell division where there is an exchange in the genetic material from sister chromatids Example: ○ This is a centromere of your chromosome (refer to the ○ During cell division there will be an exchange of genetic drawing above). Let’s say that in this particular locus you material(prophase 1). have the N/M genes, but it’s antithetical since you ○ In the picture: the blue portion goes to the violet portion and the violet portion goes to the blue portion ISBB - LEC EMBOSCADO, MATUGAS, PALCO, TUGAY, APIL, DELFIN, LAMELA, INTING, VALDIVIA BSMLS 3I & 3J 4  ○ Because of the occurrence of crossing over, each EXAMPLE 2 individual is going to be different. A patient has an anti-Fya, Anti-Jkb and Anti-K, how many units that is why no individuals are alike even if they should be tested to find 2 units of the appropriate phenotype? are twins ○ Remember that the patient has antibody against Fya ○ During crossing over, there will be new sets of genes that antigen , Jkb antigen and K antigen would be transferred from one chromosome to another. ○ Now, what type of red cell do we need to transfuse to POPULATION GENETICS the patient ? red cell to be transfuse must lack or devoid COMBINED PHENOTYPIC CALCULATIONS Fya antigen , Jkb antigen and K antigen Determines the frequency of a particular phenotype of a population, to find a donor unit of RBCs with certain antigen POPULATION FREQUENCIES characteristics (Fya -) Provides an estimate of the number of units that may need to be Fya 68% 32% or 0.32 tested to find the unit with desired antigens 100% - 68 % = 32 % (Jkb-) Jkb 74% 26% or 0.26 EXAMPLE 1 100% - 74 % = 26 % A patient produced antibodies such as anti-C- anti-E and anti-S. (K-) RBC units negative for the antigens, C, E and S would be required K 9% 91% or 0.91 for transfusion. 100% - 9 % = 91 % ○ So if this individual is in need of a blood if the patient is anemic = needs packed RBC SOLUTION: The patient has an antibody, so the red cell ○ 0.32 x 0.26 x 0.91= 0.076 or 8% that you will donate should not have C, E, and 8% of the population are negative for Fya S antigens. antigen , Jkb antigen and K antigen. ○ If you gave a red cell that has C-E-S, it could lead to How many units should be tested to find 2 units of the hemolytic transfusion reaction. appropriate phenotype? That is why we need to find a blood unit ○ solve for x: whose red cells do not contain the C, E, and S ⁄ = 2/x antigens. 8x = 200 ( cross multiplication) RBC units that are negative for C, E, and S 8x/ 8=200/8 (divide both sides by 8 to get the antigens would be required for transfusion. x) The % of donors negative for the individual antigen is expressed x= 25 as a decimal point and then multiplied. Why solve x? To determine how many may be POPULATION FREQUENCIES required to be tested to find 2 units ( C- ) negative for the three antigens. C+ 68% 32% or 0.32 ANSWER: 100% - 68 % = 32 % ○ x= 25 ( E- ) ○ Therefore, out of 100 blood bags, I need to test 25 E+ 29% 71% or 0.71 blood bags to get 2 units that are negative for Fya 100% - 29% = 71% antigen , Jkb antigen and K antigen. ( S- ) S+ 32% 68% or 0.68 so that the patient's transfusion will be safe 100% - 32 % = 68% and there will be no hemolytic transfusion ○ based on the AABB manual, the portion of the population reaction because the RBC does not contain that contains the C+ antigen is 68%. these antigens. We are looking for the portion of the population that has no C antigen. GENE FREQUENCIES ○ So if 68% is C+ antigen, 32% is C- antigen. HARDY-WEINBERG EQUATIONS E + (29%) = E - (71%) S + (32%) = S - (68%) Hardy - english mathematician | Weinberg -german physician 2 2 ○ Next is to convert them to decimal. FORMULA : p + 2pq + q = 1 C- (32%) = 0.32 p+q=1 E- (71%) = 0.71 WHEREIN: S- (68%) = 0.68 ○ p = frequency of the dominant allele in a population SOLUTION: homozygous ○ (C-) 0.32 x (E-) 0.71 x (S-) 0.68 = 0.154 or 15% ○ q = frequency of the recessive allele in a population. homozygous ANSWER: ○ pq= combination of dominant and recessive alleles ○ 15 % of the population has a probability of being heterozygous negative for all 3 antigens. ○ 1 out of 10 RBC units are likely to be negative for combined antigens. EXAMPLE 1 This will give as the idea that 15 % of these p = allele A blood bags are negative for C , E and S q = allele a PROBLEM: If the frequency of p is 0.3, what is the value of q? ISBB - LEC EMBOSCADO, MATUGAS, PALCO, TUGAY, APIL, DELFIN, LAMELA, INTING, VALDIVIA BSMLS 3I & 3J 5  SOLUTION: Obligatory gene - A and O ○ Acc to the formula Remember that p + q= 1 Since the child's blood type is B, the genetic ○ p+q=1 marker is absent in both mother and father so ○ 0.3 + x =1 that is direct exclusion. ○ x=1-0.3 ( transpose) Therefore, the child is not from ○ x= 0.7 father “A”. CHECKING: ○ p+q=1 INDIRECT EXCLUSION ○ 0.3+0.7 = 1 Only one of the parents contains the genetic marker on the child. ANSWER: Could be a possibility that the absence of the particular antigen or ○ 0.7 is the value of the recessive gene gene from the father or the expression of the antigen from the FOLLOW UP PROBLEM: What proportion of the population is AA, father is mainly due to the presence of the suppressor gene. Aa and aa? ○ AA - homozygous dominant ○ Example using the kidd blood group system: ○ aa- homozygous recessive ○ Ae- heterozygous MOTHER FATHER CHILD SOLUTION: PHENOTYPE Jk (a+b-) Jk (a-b+) Jk (a+b-) ○ Formula : p2 + 2pq + q2 = 1 GENOTYPE Jka/Jka Jkb/Jkb Jka/Jka ○ Given : Mother: p=0.3 Phenotype: Jk (a+b-) q= 0.7 Genotype: Jka/Jka pq = 0.3 x 0.7 = 0. 21 Father: ○ p2 + 2pq + q2 = 1 Phenotype: Jk (a-b+) ○ (0.3)2 + 2 (0.21) + (0.7)2 = 1 ( substitute the values) Genotype: Jkb/Jkb ○ 0.09 + 0.42+ 0.49 =1 Obligatory gene: Jkb ○ 1=1 Child: ANSWER: Phenotype: Jk (a+b-) ○ AA= p2 or 0.09 Genotype: Jka/Jka ○ Aa= 2pq or 0.42 Since the father is homozygous to Jkb, ○ aa= q2 or 0.49 therefore it should have been passed to the ○ The answer is shown like this , in order to determine the child. proportion of the population that is homozygous In this case, it is indirect exclusion because dominant, homozygous recessive, and heterozygous. the absence of Jka on the father could be due This equation is usually applicable only to determine Gene to the presence of the suppressor gene. frequencies in communities wherein residents are actually It is termed as indirect exclusion because this permanent and no migration has occurred. alone cannot disprove paternity, mainly In urban areas this is not applicable because people come and go because of the possibility of the father's inheritance of the suppressor gene that has RELATIONSHIP TESTING suppressed the expression. No longer applicable because we have higher methods in determining paternity which are much more accurate, such as DNA testing [END] PATERNITY TESTING ○ DNA testing Making use of polymer chain reaction, molecular studies, and molecular genetics to prove and disprove paternity. ○ Blood typing Used for screening tests for paternity testing. Not a confirmatory test 2 types of paternity testing: ○ Direct Exclusion and Indirect Exclusion Both are dependent on the Obligatory gene Gene that is expected to be passed on to the baby coming from the father. Maternity is always assumed DIRECT EXCLUSION Genetic marker of the child is lacking from both parents ○ example: MOTHER FATHER CHILD PHENOTYPE ”O” ”A” ”B” GENOTYPE OO AA or AO BO ISBB - LEC EMBOSCADO, MATUGAS, PALCO, TUGAY, APIL, DELFIN, LAMELA, INTING, VALDIVIA BSMLS 3I & 3J 6  IMMUNOHEMATOLOGY / ISBB / IH LESSON#3: HH & ABO BLOOD GROUP SYSTEM PRELIMS | A.Y. 2023 - 2024 | SIR JASON “AB” CHUA Hh AND BLOOD GROUP SYSTEM Cartwright (Yt) YT 011 Blood group antigens are part of the red cell membrane Xg XG 012 ○ Expression depends on the inheritance of specific blood Scianna SC 013 Dombrock DO 014 group gene Colton CO 015 ○ Classified to be as carbohydrates linked to either lipids Landsteiner-Wiener LW 016 (termed as glycolipids) or proteins (termed as Chido/Rodgers CH/RG 017 glycoproteins) Hh H 018 Exposure to such antigens through blood transfusion / pregnancy Kx XK 019 can lead to the production of antibodies towards the antigen that Gerbich GE 020 the recipient is lacking