BB part 1 & 2 PDF Immunohematology Laboratory Guide

[MLS 416-LAB] Immunohematology - Laboratory P1: Basics of Immunology and Serology and Its Applications in Blood Banking, Part 1 Professor: Bea Angelli Laude Date: January 14, 2024 IMMUNOLOGY AND SEROLOGY...

[MLS 416-LAB] Immunohematology - Laboratory P1: Basics of Immunology and Serology and Its Applications in Blood Banking, Part 1 Professor: Bea Angelli Laude Date: January 14, 2024 IMMUNOLOGY AND SEROLOGY REVIEW: CELLS OF THE IMMUNE SYSTEM ➔ The body’s immune system is a compilation of integrated network of cells, tissues, organs, mechanical barriers, and secreted molecules THREE MAIN FUNCTIONS 1. Defense Prevents the entry of infectious agents, and in case they are able to gain entry, they are going to be eliminated Immune response has the ability to recognize self from non-self antigens → non-self should be eliminated right away Normally, the immune system would not attack the body’s antigens – if in case that this happens, it is already a defect referring to occurrence of autoimmune disorders in which the body is attacking its own antigens 2. Homeostasis These cells are mainly found in the blood; others The body maintains balance mainly because found in the lymphoid organs – most in the blood during an immune response, if the pathogenic agent and produced in the bone marrow is already eliminated, it also has its means of Coming from the same stem cell, but are going to stopping that particular response - it could not affect differentiate into two: the normal cells of the body ○ Can differentiate to become a lymphoid stem cell 3. Surveillance ○ Or a myeloid progenitor cell Immune response is also able to detect any MYELOID PROGENITOR CELL overgrowth of a particular cell population Rises the granulocytes If there is tumor formation or cancer formation, then Most common granulocytes: Polymorphonuclear it is signaling a defect in the body’s ability to cells (PMNs) → Neutrophil, eosinophil, basophil recognize any increased mitotic division / increased Mast cell, found in the tissues, and active for number of a particular cell population eliciting hypersensitivity reactions FOUR COMPONENTS Basophils are found in the blood, and its granules 1. Cells and tissues of the immune system contain histamine which initiates hypersensitivity a. Involves the cells, organs, and tissues of responses the immune system Eosinophils - granules contain the major basic b. Includes the skin, mucous-secreting protein, which is very essential in fighting off cells, enzyme producing cells → first parasites line of defense preventing any entry of invading pathogens in portals of entry of Neutrophil - most numerous granulocyte that is the body present and very active during any infection, particularly bacterial infection 2. Monocyte-Macrophage Cell System ○ Found in the peripheral blood while a. Functions as antigen-presenting cells migrating into tissues in times of infection via diapedesis when signaled by 3. T Lymphocytes (T cells) inflammatory mediators 4. B Lymphocytes (B cells) Monocyte - which will differentiate to become dendritic cell or macrophage Dendritic cells and macrophages - act as antigen-presenting cells, so they are going to capture antigens via phagocytosis of the foreign material, breaking them down and presenting them to the T cells ○ Main way of eliminating pathogens is mainly through phagocytosis and an antigenic remnant will be placed at the Astronomo, Kintanar | 1  plasma membrane/in its surfaces and it will B Lymphocyte Activation be presented to the T lymphocytes Since B lymphocytes have both IgM and IgD that are found LYMPHOID STEM CELLS in its surface any type of antigen going to bind with these Differentiate to become lymphocytes antibodies will lead to the activation of these B cells B cell, T cell, NK cells (Natural Killer Cells) upon contact with the antigen to become plasma cells and Natural Killer Cells → will release chemicals that lead to antibody secretion, or… kill infected cells, foreign cells, as well as tumor cells T helper cell is going to release chemical signals and will T cells are very important cells of the immune activate B cells to become plasma cells and will tend to system release antibodies ○ Mainly mature in the thymus, hence the name T IMMUNE RESPONSE / Types of Immunity ○ Expresses specific receptors, and is 1. Innate/ Nonspecific Immune Response involved in the cellular immunity Involves the body’s first line of defense ○ Has 2 very important Types → T helper Has a consistent response and T cytotoxic cell ○ Regardless of the amount/type of pathogen encountered by an individual, T helper Activates macrophages and B cells by the innate response has the same cell cytokine secretion magnitude and is governed by the same regulatory mechanism The cell where the dendritic cells and No memory macrophages are going to present ○ Regarless of the type of pathogen that antigens. invaded the body → response is still the same Once it is being detected/ determined by Th cell, it is going to elicit a particular or Physical barriers of the body specific immune response. ○ Skin, mucosal linings Tissues and organs provide an T cytotoxic Releases chemicals to kill infected cells, unfriendly environment for most lymphocyte foreign cells, as well as tumor cells pathogens, so they include phagocytic cells and chemical B Lymphocyte mediators to promote Lymphocytes that mature in the bone marrow, inflammatory responses hence the name B lymphocyte ○ Other physiological factors that contribute Differentiates into plasma cell which is made to be first line of defense: possible by the activation of the T helper cell Hydrochloric acid in the stomach Once it becomes a plasma cell, will produce Pseudostratified ciliated columnar antibodies epithelium of the respiratory tract B cells produce antibodies to block infection and (produces mucus) eliminate extracellular pathogens Traps foreign organisms that goes in with the oxygen during inhalation Flushing action of urine Unsaturated fatty acids (skin) Sweat, tears, and saliva commensal normal flora Inflammation – Second line of defense ○ Refer to the events or body’s reaction to injury ○ Results in 3 specific events: Increased blood supply in the - Are derived from stem cells in the bone marrow area and are involved in humoral immunity, mainly Increased capillary permeability because they transform into plasma cells that will Migration of leukocytes produce a family of proteins known as ○ The 3 events → manifest symptoms which antibodies/immunoglobulins include: Pain, heat, redness, and swelling - Very important proteins involved in the Vasodilation → led to increased immune defense blood flow in the area → - Will become plasma cells first in order to produce Redness and Heat (increased antibodies temperature) Swelling is manifested by edema mainly due to increased Astronomo, Kintanar | 2  capillary permeability mother, if that Rh blood type of the mother and increase in fluid is incompatible with the baby accumulation in that Route of administration and dose – although not a particular area “physical nature” important for response Also due to the influx of ○ refers to the manner in which the antigen phagocytes (e.g. stimulus is introduced neutrophils, eosinophils, ○ could be introduced via intramuscular macrophages, NK cells) route, however in immunohematology, the Adaptive or specific immune response route of administration would be Immunologic memory and specificity develop in intravenous that is mainly by blood response to the antigen transfusion Can recognize self and non-self antigens Intravenous administration is Production of antibodies (humoral immunity) or the best routes for inducing an actions of T-cells (cell-mediated immunity). immune response ○ Produced by B-lymphocytes EPITOPES Activated to become plasma cells because of its recognition receptors, which are capable of discriminating many molecular configurations Interaction between both humoral and cell-mediated ANTIGENS Capable of provoking a specific immune response If antigen is able to produce an immune response → Immunogen ○ All immunogens are antigens ○ Not all antigens are considered as immunogens Recognized as foreign by the immune system there could be some instances Small regions of the molecule within the that an antigen lacks some immunogen factors that could lead to AKA antigenic determinants elicitation of immune response unique configurations which allows recognition by Capable of stimulating the formation of antibody corresponding antibody and the development of cell-mediated immunity ○ If antigen has multiple epitopes → a In the concept of blood banking → are referring to different antibody is produced from each of the antigens present on the RBC those epitopes Characteristics ○ The antibodies that is produced for Foreign nature different epitpoes tend to cross-react if ○ An immunogen must be identified as they have a common determinant non-self Allogeneic antigens and autologous antigens ○ the greater the difference from self = ALLOGENEIC greater the likelihood of eliciting an Allogeneic: It is from another individual but it is of immune response the same species Molecular size ○ In IHBB → Blood is coming from different ○ molecular antigens that has a molecular individual but (may have) the same blood weight of more than 10,000 daltons are type better immunogens AUTOLOGOUS Molecular complexity and composition Autologous: One’s own self antigen ○ Proteins are the best form of antigens Always tolerated by the immune response because because with its complexity, it is much it is recognized if they are self-antigens more effective in inducing an immune ○ If in case the immune system cannot response recognize such self-antigens, it is going to ○ Carbohydrates and lipids → next to the induce an immune response that will be proteins indicated as autoimmune ○ ABO antigens are made of glycolipids in which it is an effective immunogen ○ However, Red cell antigens that are highly protein in nature, particularly the Rh antigens, are very strong immunogens → Ability to produce high immune response → could lead to misscarriage of the Astronomo, Kintanar | 3  Characteristics Size ○ antibodies are made up of monomeric units in which some are considered to be having more than one units ○ Smallest: IgG, IgE, IgD They are monomers ○ IgA could be a dimer or a monomer Monomeric form: If it is in the In IHBB → Autologous tranfusion: An individual will serum be taken out with blood and it is transfued back to Dimeric form: if they are found in the same individual the secretions done mainly if one has a very rare blood type ○ Largest: IgM ○ They are going to have their blood donated made up of five units of but it is intended only for them – NOT antibodies (pentamer) intended for another individual Biologic function Commonly indicated for individuals with rate ○ Due to the presence of their heavy chains blood types their heavy chains are going to impart unique features to the BLOOD ANTIGENS different immunoglobulin classes which permit them to function biologically their heavy chains determines what type of antibody or class they belong to EX: If heavy chain has the gamma form → IgG In IgA, the alpha heavy chain is unique because it enables the antibody to reside in the mucosal linings, thus found in the secretions The rest of the antibodies are mainly found in the serum ○ There are two types of light chains: Kappa and Lambda Biochemical properties ○ Variable region which is concerned Chemical structures embedded in or protruding mainly in binding with the antigen from RBCs, WBCs, and platelets ○ Constant region which imports the Currently, there are 29 blood group systems uniqueness of the antibody class function There are 250 unique red cell antigens that are such as activation of the complement or recognized by the American Association of Blood the attachment to certain cells Banks (AABB) IgE attaches to surfaces of Are determined by the inheritance of many blood basophils and mast cells group genes IgD in the surface of B Every individual possesses a unique set of Red Cell lymphocytes antigens through genetic inheritance IgG is mainly found in the serum 3 Common forms IgM is found in the surface of B Glycoproteins - HLA system cells Glycolipids - ABH, Lewis, Ii, and P blood group IgA is found in the secretions systems. ○ Hinge region of antibodies are made up of Proteins - Rh, M, N blood group systems disulfide bonds and they are going to ANTIBODIES impart the flexibility of the molecule during are specific to a particular antigen its attachment to their antigens are produced by the plasma cells, which are the ○ Somehow, these antibodies can be acted activated B cells, and are going to produce proteins upon by certain enzymes. in the form of antibodies Serological activity Five types of antibodies: ○ Gamma, Alpha, Mu, Epsilon, Delta Astronomo, Kintanar | 4  number of antibody class is the PAPAIN AND PEPSIN ENZYME Immunoglobulin gamma. Papain → yields antibody into three fragments Fixes complement Two FAbs, 1 Fc First antibody to be produced ○ Part of the primary immune response to an infecting organism Does not cross the placenta ○ Does not have any receptors for the placenta Saline agglutinins ○ Due to its large structure, is actually capable of visible agglutinations of cells Pepsin → yields antibody into two fragments that are suspended in saline One FAb, 1 Fc ○ Capable of agglutinating antigens present Regardless of having an Fc or not, it is still capable of binding to antigens and could still on the RBCs that is suspended in saline IF produce agglutination reactions. they are going to be processed at room temperature (22-24 deg. C or lower); and it could still react at very low temperatures ○ Also known as Cold reacting antibodies Activate complement very efficiently ○ Considered to be much more efficient than IgG in complement activation, mainly because it requires ONE immunoglobulin Mu to initiate the classical complement pathway In the study of hematology, we are going to look at the antibodies or study of antibodies that are of high clinical significance; and namely that is IgG and IgM because these are the antibodies that could be produced if there is introduction of red cells whose antigens are absent– not the same with the recipient Immunoglobulin MU (IgM) Immunoglobulin GAMMA (IgG) Most abundant in the plasma ○ Comprises 80% of the total Immunoglobulin population Largest of all the antibody molecules, Ability to cross the placenta ○ Consists of five basic units which is also ○ Because it has Fc receptors in the known as a pentamer in which it is joined placenta, it allows its crossing during together by a structural component, pregnancy composed mainly of disulfide bonds in the ○ ADV: able to protect the baby from middle known as J CHAIN infection, particularly if the mother has an ○ Restricted mostly entirely in the infection that occured during pregnancy intravascular space mainly because of its ○ DADV: If the IgG is produced AGAINST a large size, but it is able to activate the certain red cell antigen classical pathway of complement with If the baby has an antigen that is great efficiency. absent in the mother, it will lead to ○ Accounts for about 5-10% of the the sensitization of the mother Immunoglobulin Pool whereas the highest Astronomo, Kintanar | 5  against that certain antigen and Hemolytic Disease of the Fetus and Newborn will produce antibodies If the antibodies are in IgG form, it will cross the placenta and attach to the red cell of the baby and will activate complement, leading to hemolysis IgG has the ability to activate the complement system, however it is NOT as efficient as compared to IgM ○ Because of its small structure, it will require 2 Molecules of IgG to activate the classical pathway of complement Predominant antibody produced in the secondary response. MOST CLINICALLY SIGNIFICANT in blood Implicated in Hemolytic disease of the fetus and banking. newborn (HDFN) ○ Reactivity at 37°C (body temperature), Caused / in which a particular blood group is complement activation, indirect involved, which is the Rh blood group system agglutination, and hemolysis. Occurs if the baby has the Rh antigen which the ○ DADV: Agglutination is not as visible as mother is lacking IgM Most probably, the baby has inherited the gene that ○ You need reagents to have visible produces Rh antigens from the father agglutination reaction with IgG → using FIRST PREGNANCY – SAFE. This is the first antihuman globulin encounter of the mother with the said antigen. Baby Effect: Hemolysis will survive and the baby will appear yellow due to Four subclasses: IgG1, IgG2, IgG3, and IgG4 the degradation of red cells that led to the ○ As a result of minor variations in the hemolysis of some red cells therefore bilirubin gamma heavy chain might be present in the circulation of the baby ○ Slight amino acid differences - however, it ○ During the pregnancy – fetomaternal affects its activity circulation – at a particular term in the Antibodies pregnancy Has caused hemolytic transfusion reactions (HTRs) ○ The baby’s blood will go to the circulation ○ If a particular recipient of a blood has of the mother which tends to lead to received a red cell/ packed red cell/ blood SENSITIZATION component in which it has an antigen that ○ Because the first pregnancy is the first is absent from the system of the recipient, encounter, the baby survives, but the ○ The recipient will produce an antibody mother is sensitized with the Rh antigen. against such antigens SECOND PREGNANCY – UNSAFE. Since the Steps in Antibody Formation (From Hemolytic mother’s immune system has memory, then Transfusion Reactions) fetomaternal circulation, the mother has 1. SENSITIZATION – slow progress of antibody encountered the same antigen for the second time, production there is faster immune response - production of 2. FASTER PRODUCTION OF ANTIBODIES – antibody which could lead to hemolysis happens the second time the patient encounters the ○ Antibody produced → IgG same antigen ○ Has receptors in the placenta, and could a. Because the B cells tend to have memory8 cross the placenta and lead to attachment b. Immediate transfusion reaction which to the red cells of the baby, activated could lead to complement activation, complements so that could lead to hemolysis which could be fatal to the blood hemolysis and eventual deeath recipient ANTIGEN-ANTIBODY COMPLEX The binding of antigens to antibodies is based on noncovalent forces: Astronomo, Kintanar | 6  FUNCTIONS OF COMPLEMENT SYSTEM 1. Opsonization a. Able to clear immune complexes b. Opsonization – covering of foreign material with complement proteins enhances phagocytosis; also they are going to promote release of enzymes from the neutrophils 2. Anaphylaxis a. There is increased smooth muscle contraction and induces inflammation 3. Lysis a. Manner of killing foreign antigens by 1. Electrostatic Forces – ionic forces based on the membrane lysis attraction between two molecules on the basis of b. This is going to be the result of the opposite charges transfusion reaction if in case the red cell 2. Hydrogen bonds – Attraction between two transfused to the patient is containig an negatively charged groups, namely Nitrogen and antigen that is absent on their system Oxygen for a hydrogen atom c. Antibodies will be produced and these Abs 3. Van der Waals forces - attractions between two could activate complements leading to electron clouds of one atom and the protons within lysis of transfused red cells another atom’s nucleus 4. Chemotaxis 4. Hydrophobic forces - they are weak bonds formed a. Recruitment of platelets and phagocytes as a result of the exclusion of water from the which induces the immune response antigen and antibody complex Three Pathways 1. Classical Complement Pathway AFFINITY AVIDITY 2. Alternative Pathway Tendency that an epitope Strength of the bond 3. Mannose-binding lectin (MBL) Pathway has for combining with the between the antigen and antigen binding site of an the antibody antibody molecule COMPLEMENT ANTIBODIES THAT CAN ACTIVATE COMPLEMENT IgM IgG1 IgG2 IgG3 → IgG4, IgA, IgE, and IgD CANNOT activate complement pathway CLASSICAL COMPLEMENT PATHWAY 1. Starts with antigen-antibody reaction a. If the Fc portion of the antibody has attached to an antigen, it will be recognized by the first complement protein → C1 2. C1 and its three subunits → C1q, C1r, C1s a. Collectively, they are the C1qrs 3. Cleaves the second complement protein → C4 complement Complement system is a group of serum proteins 4. C4 will be cleaved to have C4a and C4b that have a number of biologic roles related to 5. C4a will be removed and C4b will be retained antigenic clearance, cell lysis, as well as 6. Activation of C2 complement in which it will be vasodilation cleaved to have C2a and C2b Normally circulate in an inactive or pro-enzyme 7. C2a will be retained state 8. New combination: C4b2a → C3 convertase During activation, they are converted into active 9. C4b2a cleaves C3 → C3b will now join C4b2a → enzyme that enhances the immunologic process; C4b2a3b 10. C4b2a3b → C5 convertase Astronomo, Kintanar | 7 11. C5 complement protein will be cleaved to C5a and C5b 12. C5b will be attached to cell membrane and it is rapidly inactivated, but unless it is going to be bound with C6 a. Forms the beginning of the MAC (Membrane Attack Complex) 13. C5b is joined by C6, C7, C8, and C9 a. C5b6789 → Membrane Attack Complex b. Induces cell lysis Astronomo, Kintanar | 8  [MLS 416-LEC] Immunohematology - Lecture Basics of Immunology and Serology and Its Applications in Blood Banking, Part 2 Professor: Bea Angelli Laude, RMT Date: January 19, 2024 → Why can IgG cause hemolysis even though it is a small ○ You cannot produce antibodies right away antibody? – it is POSSIBLE only if it is the second Due to complement; the membrane attack time you have encountered the antigen complex causes now the hemolysis. ○ Because B cells have memory 13 FACTORS AFFECTING REACTIONS BETWEEN In Prozone… ANTIGENS AND ANTIBODIES If fever has persisted for a long time 1. Nature of Antibodies Possible that the patient has taken antibiotics IgM and IgG are the most important antibodies in ○ Antigen has been lost, antibodies are more the blood banking concepts prominent. ○ Not so much on IgA as it focuses on secretion – but somehow, there is complication in Px if they do not have IgA This is why Red Cell Suspension is important. – because they are able to produce an If RBC Suspension is 5%, with the amount of antibody that is in the form of Anti-IgA that antibody present in the antisera / in the sample could lead to transfusion reactions. of the patient, it will give you the right Focus on blood groups, antigens and antibodies concentration for Antigen-Antibody → it is usually IgM and IgG interactions. If Ab produced against a certain blood group Basically, due to Red Cell Suspension, the antigen will ALWAYS BE IN TWO FORMS ONLY. antigen-antibody ratio is equal ○ IgM and IgG It will yield good, visible, agglutination reactions. Hence, this justifies why we are focusing on IgM and IgG. 3. Effects of Surface Charge Why red cells normally do not attach to each other? 2. Concentration of Antigens and Antibodies They have negative charges → Zeta potential What causes the zeta potential? Why is this diagram important? It is mainly dependent on the presence of antigen on a particular antibody (And the other way around). → Tendency is when we have false results, it could be false positive, or false negative - In a red blood cell, the negative charges are at the In the diagram above, it is ALWAYS FALSE surface brought about by SIALIC ACID RESIDUES NEGATIVE whether it is prozone or postzone. (negative in charge) from the Glycophorins. Take note of the concentration of the - Negative charges attract positive charges → in the Antigen-Antibody present in the sample environment of the cell, all positive charges go to ○ If the concentration is not good, you end the negative charge of the surface. up with postzone or prozone → result is - If you have another red cell with the same negative just false negative charge and you have attracted positive charges → they have the same charge. - They will repel. Prozone More antibodies present vs. antigens - The distance from one red cell to another red cell is the ZETA POTENTIAL. Postzone More antigens compared to antibodies Small amount of antibodies produced (E.g. Px on their 1st day of fever) In Postzone… This is why the doctor asks “How many days have you been experiencing fever?” The body needs ample time to generate antibodies Astronomo, Budo | 1  Zeta Potential Antihuman Globulin (Anti-IgG) This is why we’re going to add ANTIHUMAN GLOBULIN (AHG). Example… Is the reason why IgM can give visible agglutination while You have an antigen that is attached w/ IgG. From IgG cannot this, there is no visible agglutination yet. IgG 25 nm; approximate distance between one distance cannot cover the entire span of the zeta potential of to another the RBC → cannot connect IgM has 5 monomeric units (pentamer) → can AHG bridges the zeta potential span that the IgG actually span the distance between the zeta cannot cover, because AHG is attracted to the Fc potential (35 nm span) portion of the antibody Red Cells w/ antigen, and their antibody is IgM → it WILL react and clumping will occur Characteristic of AHG: Reactive to human antibodies, particularly IgG HOW? → It is attracted to the Fc portion of the IgG therefore AHG attaches to it, resulting in an agglutination reaction. Now, IgG is too small, it can ONLY SENSITIZE but CANNOT give you agglutination reaction. AHG is ONLY important for IgG and its detection. Why not for IgM? ○ The Fc for IgM is found in the center, failing the agglutination reaction. ○ IgG on the other hand has a Free Fragment for crystallization (Fc) portion where it is attracted → in which it is considered the antigen for AHG. Because of the surface charges, now we know the difference between IgG and IgM. When we do antigen-antibody mixing, we require incubation time. ○ Incubation time takes 30 minutes - 1 hour. If we are going to utilize AHG in performing blood typing, it will take too long and become counterproductive ○ We now require reaction media 4. Use of Reaction Media Function: Reduces incubation time by enhancing the antigen-antibody binding. Low Ionic Strength Solution (LISS) Commonly used in the lab as it is very cheap Has the reduced amount of incubation Reduces incubation time to 10 minutes only Astronomo, Budo | 2  With antigen-antibody binding, the FAb will attract 6. Temperature ions. 4 deg. C → IgM ○ Usually, we are to use NSS, so ions would ○ IgM tends to have a wider range (4 deg. C be in the form of sodium and chloride, - 22-24 deg. C (room temperature)) which could also hinder the interaction Body temp → IgG between the antigen and antibody ○ The environment needs to be low ionic – 7. Incubation Time therefore LISS is needed If saline alone (NSS), incubation time would take ○ The lesser ion component there is, there is 30-60 minutes increased antigen-antibody binding. With albumin, PEG, or LISS → 10-15 minutes only ○ Common → LISS, very safe, very cheap, 22% Albumin (Bovine Albumin) and reduces time significantly. 22-30%, with 22% being the most common Comes from cows 8. Washing Acts as a buffer Before RBC suspension, washing is very important Albumin is in the plasma, extraction from cows for removal of any impurities would be hazardous → some bacteria and ○ Only RBC should be present microorganisms would be transmitted to humans, ○ If other substances are present, there will posing a risk be false agglutination (false negative), Function of Albumin: Notice the ionic cloud neutralizing the antibody with an antigen present on the zeta potential, bovine albumin will not of the red cell disperse the positive charges. Must be thorough ○ After dispersion, the distance between the Must be rapid two cells will be reduced ○ But not too fast as this will lead to hemolysis and defeats the purpose of Polyethylene glycol (PEG) washing Disperses water molecules. There is increased ○ Refers to the washing that separates the uptake of antigens and antibodies. red cell from the NSS (diluent) Mechanism: Dehydration If it is not separated rapidly, it will lead to dilution. Enzymes ○ When you do testing, specifically AHG Are said to be proteolytic testing, it involves washing – if you are ○ Lyses protein unable to wash or separate rapidly, there Function: Breaks down protein present in the will still separation and dilution → FALSE surface of the red cell. Either NEGATIVE 1. To reduce zeta potential a. Because sialic acid residues 9. Centrifugation coming from the glycophorins to 5-10 seconds reduce the distance between red Purpose: RBC clotting cells ○ No need for 5 minutes 2. For antibodies to detect small antigens ○ 1 minute is way too much near the RBC membrane a. It is very important b/c some of 10. Chemical Reduction the antigens housing in very high Sometimes, IgM would serve as an interference proteins – cannot be detected in ○ When we do testing of red cell and the surface of the red cells antibodies, it is done at room temperature ○ And IgM is reactive at room temperature Bromelin Solution: 0.5% ○ IgM should be broken down or we should Reactive at pH 5.5 get IgM through absorption so IgG would To utilize this enzyme, you have be left to acidify the environment If we cannot remove IgM through absorption, we (solution) break it ○ We break IgM into monomeric units through the use of Dithiothreitol, Ficin and Conc.: 0.1% β-mercaptoethanol (IgM) and/or ZZAP Trypsin Reactive at pH 7.3 (IgG) - seldomly used Most often the RBCs would interact with the antibodies at a ZZAP is a combination of dithiothreitol and an neutral pH enzyme mostly bromelin Commonly utilized enzyme ○ will reduce the disulfide bonds in the hinge region of the IgG Papain Conc.: 0.1% ○ This extends the Fab portion of our IgG so Reactive at pH 6.5 that it further spreads; this wider span An acidic environment would allows IgG to attach from one red cell to lead to hemolysis. another ○ This is mainly possible if potentiators are 5. pH added. There are antibodies that tend to react at a more ○ Zeta potential reduced from potentiators + acidified pH IgG hinge region reduced = higher ○ pH 6.5 ⇒ Anti-M (for confirmation) propensity to attach to red cells The rest are neutral Astronomo, Budo | 3  11. Antiglobulin Testing The sample used is (1) Red Cell or (2) Detection of non-agglutinating Ab, especially IgG1 Plasma and IgG3 or complement components (C3d) affixed to RBCs in vivo or in vitro; it has 2 types → DAT & Note: When dealing with antiglobulin testing, the antibody IAT involved is always IgG. PRINCIPLE OF DAT & IAT If what you want to find out is a certain ANTIGEN what will serve as your sample is the RED CELL and what will serve as your reagent is PLASMA If what you want to find out is a certain ANTIBODY what will serve as your sample is the PLASMA and what will serve as your reagent is RED CELL Polyspecific AHG Abs against human IgG Abs against human C3d (C3b breaks down to C3c and C3d) Direct Agglutination Test Diagram Acts as a Screening test When the reaction is positive, you do not know whether it reacted to IgG or C3d because it contains both antibodies. You first need to find out if which of the 2 is sensitized in the RBC Advantage: may detect complement-dependent Abs on RBCs (anti-Jka ) Disadvantage: more nuisance positives Indirect Agglutination Test Diagram Monospecific AHG **More comprehensive diagram of DAT and IAT on the Appendix Abs to human IgG only or human C3d only (both in separate bottles) Fewer nuisance positives; may miss an important Direct Detection of sensitization of Ab antiglobulin RBC is (coated with Abs and/or Acts as Confirmatory test test (DAT) complement components) in Vivo It is NOT RECOMMENDED to directly test for monospecific - Red cell in vivo has IgG already AHG, because in cases that the actual RBC is Polyspecific attached to its cell AHG negative, for sure it will not have a positive reaction on - Directly concerned with the Monospecific AHG, therefore it would just be a waste of sensitization of your antigen materials. with IgG Coombs Check Cells How do you detect if it has been Content: IgG coated red cells sensitized by IgG? Purpose: To confirm negative result in AHG AHG is added and is attracted (+) Reaction: Visible agglutination (The sample is to the Fc portion and truly negative) automatically there will be You only add coomb’s check cells if the results are agglutination due to NEGATIVE pre-sensitization of IgG to the red cell surface Why negative?… ➔ Because if you added coomb’s check cells when The sample used is (1) Red Cell and not the result is positive it would be useless since the the Plasma AHG already binded to the sensitized red cell the person has. Indirect Detection of sensitization of ➔ if you added coomb’s check cells and there was no antiglobulin RBCs in Vitro reaction observed, there are 3 meanings: test (IAT) - No antibody has attached yet to 1. Expired AHG (unreactive AHG) he red cell surface; it will be us 2. No washing happened who will facilitate the a. After incubation procedure, we sensitization of our red cell with need to do washing, in order for antibody the unbound antibodies to be eliminated It has 2 phases: b. This will now eliminate the 1. Sensitization (Red cell + IgG possibility of a false positive antibody) reaction of the IgG coated red 2. Addition of AHG → Will result to cells to the unbound antibodies agglutination 3. No AHG was added Astronomo, Budo | 4  12. Cell Dosage Causes of FALSE POSITIVE REACTIONS in The number of antigens is dependent on the genes that you agglutination have inherited Causes Remedy Heterozygous production - Has less dosage; less reactive ○ AO → A antigen Over Centrifugation Regulate speed and time; ○ AB → A and B antigens 10-20 secs only ○ BO → B antigen Contaminated glassware Keep supplies covered and Homozygous production - Has more dosage; stored in a clean place more reactive ○ AA → A antigen Autoagglutination Use control and saline ○ BB → B antigen Cross-reactivity Use purified reagents and 13. Location of the antigen monoclonal Antibodies Vital for protection Presence of heterophile Use pre-absorbed serum We have antigens that are embedded in our red cell antibodies membrane. One of them is our Rh antigen whereas you have glycophorins which houses your MNSs Delayed reading slide test Read results immediately Sometimes, if you are going to add anti-Rh and you have a Too heavy cell suspension Prepare red cell lot of glycophorins in your surface, it is hard for the antibody suspension properly; 2-5% to reach the antigen concentration So with the use of proteolytic enzymes, the glycophorins will be removed and the antibody now can reach the antigen on Causes of FALSE NEGATIVE REACTIONS in the surface. agglutination AGGLUTINATION REACTIONS Causes Remedy Considerations: Hemolysis - most often not used Under Centrifugation Regulate speed and time ○ Needs to be considered because when RBC bursts/ dies, we now have no antigen Inadequate red cell Wash red cells properly ○ A good sign in immuno because a washing complement was activated. ○ Reasons: Over centrifugation; Wrong Reagents not active Proper storage of reagents diluent Rouleaux formation Delays in carrying out the Once a procedure has ○ Is caused by increased proteins in the procedure begun, follow through until plasma (CRP) → Due to infection or the end without delay inflammation → results to decreased zeta potential → RBCs will now clump/stack like coins Insufficient incubation time Observe proper time and ○ Reason: cells were not washed prior to and temperature temperature making red cell suspension ○ Solution: If it occurs in vivo = wash and Too light red cell Prepare red cell the Rouleaux formation is solved suspension suspension properly Sample problem ○ In vivo sensitization or presence of cold Special Techniques in Blood Banking reacting (IgM) antibodies Enzyme Treatment ○ Or the patient has autoantibodies Can be done in the patient’s sample or could be Reagents used utilized as the procedure to make your reagent red ○ Try to check the lot numbers and the cells expiration dates before performing the Enzymes remove very long antibodies such as your procedure daffy A and daffy B and MNS ○ Always make sure to have the reagents in ROOM TEMP before using it. Elution of Antibodies from the surface of RBCs At refrigerated temp, your Disassociating Abs from RBC membranes antibodies are When we centrifuge, antibodies attached to RBCs non-reactive/inactive will dissociate and be displaced into the supernatant Equipment used ○ Avoid putting the centrifuge in the same ○ Make sure pipette and centrifuges are table as the red cell suspension is placed, calibrated because it causes the table to shake and Technical Problems eluate the suspension ○ Question of skill, skipped or omitted some Supernatant with antibodies is called the Eluent steps ○ Eluent can be used for antibody screening or testing for the detection of antibodies. Astronomo, Budo | 5  Adsorption polypeptide chains and exposing or destroying Removal of Ab from serum by combining serum antigens with appropriate RBCs ○ E.g. papain, bromelin, ficin, trypsin ○ If there is an unwanted antibody it will be adsorb using a RED CELL that contains a ENHANCED DECREASED UNAFFECTED particular antigen * ** *** ○ So what will be left is the antibody of interest Useful if the patient has both autoantibody and an ABO-related MNS system Kell antibody against the antigen of another individual -ABO/H Duffy system systems system Neutralization - Lewis Composed of system These are taller disulfide bonds Uses soluble Ag to inhibit reactivity of some Abs in - I system antigens on the because the testing - P1PK/GLO RBCs antigen contains ○ Just like adsorption, but with the use of a B a lot of cysteine SOLUBLE SUBSTANCE Rh system Some are even amino acids ○ The unwanted antibody will form a Kidd attached to the precipitate with antigen system sialic acid Sensitive with residues the use of beta Saline Replacement Technique mercaptoethan Removing the serum and replacing with saline ol or dilutes the interfering proteins so that rouleaux dithiothreitol formation is prevented NOTE: *Increased protection from enzyme | **decreased Enzyme Treatment protection from enzyme | ***no effect from enzyme Proteolytic substances Modifies RBC membrane by reducing sialic acid residues reducing surface charge, splitting APPENDIX Astronomo, Budo | 6