Immunology Notes PDF

MTAP 100 - CLINICAL INTERNSHIP PROGRAM | SPC MLS IMMUNOSEROLOGY AND BLOOD BANKING 4TH YEAR - 1ST SEMESTER | A.Y. 2024-2025 | LECTURER: MS. JUDEA MARIE POLICARPIO...

MTAP 100 - CLINICAL INTERNSHIP PROGRAM | SPC MLS IMMUNOSEROLOGY AND BLOOD BANKING 4TH YEAR - 1ST SEMESTER | A.Y. 2024-2025 | LECTURER: MS. JUDEA MARIE POLICARPIO Tissue Monocyte / Histiocytes IMMUNOLOGY Liver Kupffer cells Immune System Integrated system of cells, substances, and Kidney Mesangial cells organs responsible for: Brain Microglial cells (1) destruction of foreign substances Bone Osteoclasts (antigens) Skin Langerhans cells (2) keeps the body safe from injury and Lungs Alveolar macrophage / Dust cells infectious agents Placenta Hoffbauer cells T Spleen Splenic macrophage Immunology The study concerned with the process (how?) by which all living organisms defend themselves RM against infection NK Cell: 3rd population of the lymphocyte family; assassin of the immune system Line of Defense 1st Line: “Barriers” (e.g. skin) ★ release Perforin (induce 2nd Line: “Soldiers” (e.g. neutrophil, monocytes) perforation) & Granzymes: also 3rd Line: “Back up” (e.g. immunocytes) produced by T cytotoxic cells Antigens substances that are considered foreign to a host Antigen Presenting Cell: connects innate and ★ not all antigens are enemy adaptive immunity Components of 1. Cellular Immunity A, Humoral Factors Acute Phase Reactants: ↑ in Inflammation Immunity 2. Humoral Immunity (Innate) ★ C-reactive Protein (CRP): most Cellular Immunity Cells that make up the immune system potent but a nonspecific indicator (e.g. WBC) (same w/ Serum Amyloid A) Humoral Immunity Non-cellular soluble substances that promote Categories of Immunity Innate Immunity immunity (e.g. proteins, antibodies) 1. Innate Immunity 2. Adaptive Immunity Natural or non-specific immunity L Adaptive Immunity Acquired or specific immunity UN ★ Neg. APRs = ↓ in Inflammation (e.g. Albumin); ↑ in Dehydration (Alb.) Cytokines: substances produced by cells in the immune system in general; from WBCs = “messages” ★ Interleukin 1: ↑ in Fever (Pyrogen); messenger to induce systemic Characteristics of a. Functions in early stages of host defenses in responses which include attacking BA Innate Immunity response to foreign agents foreign causes ★ 1st & 2nd line of defenses ⇒ Theoretically: best time for blood b. Magnitude (strength) or Kinetics (way) of the collection: 30-45mins BEFORE fever; innate immune response are unaltered by we need the “cause” to be alive subsequent encounters with the foreign agent Phagocytosis Discovered by Elie Metchnikoff ★ no memory CA Action of Neutrophil & Monocyte Components of 1. Anatomical or physical barriers ★ Steps: I-C-E-D: Innate Immunity 2. Resident Flora (Normal Microbiota) - Initiation 3. Cellular Factors - & Chemotaxis 4. Humoral Factors - ⑤ Engulfment Anatomical or Skin: largest barrier - ⑨ Degradation /Digestion physical barriers Cilia: filters antigens (RT) Characteristics of a. Adapts depending on the antigen that it N Mucus: traps antigens → sputum (RT) Adaptive Immunity encounters (strength adjusts) Gastric acid: ↑ production = ↓ pH (GIT) b. Appears following exposure to antigen (only Tears: defense of the brain to relieve tension appears after encounter) SO ★ “flushing mechanism” ★ Advantage: Specificity & Memory Earwax: prevents entry of organisms Components of 1. Cellular Factors - T cell & B cell ★ P. aeruginosa = Swimmer’s ear Adaptive Immunity 2. Humoral Factors - Cytokines & Antibodies Resident Flora Prevents multiplication of pathogens T cells Thymus-dependent; 60-80% of lymphocytes (Normal Oral: Viridans Strep. / Alpha-hemolytic Strep. Most predominant lymphocyte Microbiota) Intestine: Bacteroides / Gram(-) anaerobic bacilli JA Functions: Skin: Staph. epidermedis - Involved with cell-mediated immune ★ Blood culture contaminant response (Helper T cells) Vagina: Lactobacillus acidophilus - Regulate the production of antibodies Cellular Factors Basophil: Allergic reaction by B cells (Suppressor T cells) (Innate) Eosinophil: Parasitic infection - May act directly with the antigen or ell Neutrophil: Bacterial infection / Acute infection infected cell (Cytotoxic/Cytolytic T cell) ★ acts as a Phagocyte B cells Thymus-independent; 20-30% of lymphocytes Monocyte: Chronic infection; arrive late to clear Function: produces Antibodies up the infected area (via phagocytosis) Archer of IS = provide long-range attack ★ Scavenger cell BY: KYLE GENE J. CALATRAVA | 1 |  2 Primary 1. Bone Marrow → B cell maturation Endogenous Ag Antigens that are derived from within the body’s Lymphoid Tissues ★ Birds: Bursa of Fabricius own cells; may also be sub-fragments or 2. Thymus → T cell maturation INSIDE THE compounds or antigenic products that are ★ Site: Thorax ⇒ Atrophy as we age BODY produced by the person’s own body 5 Secondary Antigen-trapping sites: Tonsils, Spleen, Peyer’s → autoantigen, alloantigen, heteroantigen Lymphoid Tissues Patches, Appendix, Lymph nodes Autoantigen/ Refers to the person’s own antigens Types of Acquired According to HOW the immunity was acquired: Autologous Ag Do not normally evoke an immune response Immunity - Natural: contracted the dx. OIUNSA Ex: Thyroglobulin, DNA, Corneal tissue, etc. /transplacental transfer Significance: Autoimmune Diseases - Artificial: injected/administered Autoimmune Conditions in which damage to organs or tissues ★ Clue: “Anti-” or “Ig”) Disease results from the presence of autoantibody or T According to WHAT IS RECEIVED UNS/ autoreactive cells SELF ATTACK SELF - Active: antigens Type 1 Diabetes Anti-beta cells: destroys beta cells = ↓ Insulin RM - Passive: antibodies Mellitus Natural Active Natural disease process Pernicious Anemia Anti-parietal cell Abs: destroys parietal cells = ↓ Natural Passive Transplacental Transfer of Antibodies (IgG) HCl & IF = Malabs. of Vit. 12 = Impaired DNA * Colostrum (IgA): breastfeeding Synthesis = Megaloblastic Anemia ★ Clutching: direct feeding via baby’s ★ BAO & MAO = can end up to 0 mouth to the breast Hashimoto’s Anti-microsomal (Anti-thyroid peroxidase): Artificial Active HBV Vaccine: prevention/pre-exposure Disease (Primary impaired iodination in thyroid hormone prod. A, ★ Before: Formalin-killed Ag/Virus Hypothyroidism) Anti-thyroglobulin Abs Rabies Vaccine: live-attenuated rabies virus; Grave’s Disease Anti-TSH receptor Abs (Thyroid-stimulating administered to dogs/cats (primary reservoir) (Primary D Hormone Immunoglobulin): attaches to ★ discovered by Louis Pasteur Hyperthyroidism) receptors = uncontrolled Thyroid Hormone prod. Artificial Passive Tetanus Toxoid: Modified from toxin → nontoxic HBIg: HBV abs; after injury/post-exposure Anti-rabies: abs to rabies virus ★ administered w/in 24 hrs after bitten Shots vary depending on: L - Distance from the head UN Multiple Sclerosis Anti-myelin sheath Abs Goodpasture Syndrome Al Systemic Lupus ★ CSF: Oligoclonal bands; suggestive Anti-glomerular basement membrane (Kidneys) Anti-alveolar basement membrane (Lungs) Anti-dsDNA; Anti-Smith: uses dsDNA from - Severity Erythematosus ★Crithidia luciliae as the reagent Anti-tetanus: anti-toxin ★ (+) Butterfly Rash (face) # BA RhoGAM: Rho (Anti-D) Gamma-globulin ★ (+) LE cell: neutrophil with ingested nuclear body/nucleus VS Tarte cell Antigens Originated from “antibody generators” (differentiated): Monocyte with Serve as the target of antibodies ingested nuclear body/nucleus Can be polysaccharides, proteins, lipids, nucleic acids, etc. “anything under the sun” Rheumatoid Rheumatoid factor (IgM): Antibodies to the Fc CA arthritis portion of IgG an IgM that targets 1g6 Epitope Antigenic determinants; specific regions of * ★ RF LAT: uses IgG-coated latex antigens that are recognized by the IS particles to detect Rheumatoid Factor Monovalent/ Antigen that has a single epitope Myasthenia gravis Anti-acetylcholine receptor Univalent Antigen Sjogren syndrome Anti-SSa and Anti-SSb: ↓ tears & ↓ saliva Multivalent Antigen Antigen with more than one identical epitope ★ Anti-lacrimal gland Abs (tears) & ↑ Immunogen Antigens capable of stimulating a host’s IS Anti-salivary gland Abs (saliva) N ★ All immunogens are antigens but Primary biliary Anti-mitochondrial Abs NOT ALL antigens are immunogens cirrhosis Haptens Small molecules that are not immunogenic by SO Chronic active Anti-smooth muscle Abs themselves but can be immunogenic when hepatitis coupled to a carrier Alloantigen/ Antigens that are found in another member of Carrier Macromolecular substances to which a hapten Allogenic Ags the host’s species that are capable of eliciting OR immunogen is coupled to produce an immune response immune response Ex. A and B Ags, HLAs same species JA ⇒ Hapten + Carrier = Immunogen Significance: Tissue Transplantation and Blood ⇒ Immunogen + Carrier = ↑ Immunogenicity Transfusion Ex. RBC (blood groups), bacteria, charcoal (RPR), latex (LATs), beads, bentonite Human Leukocyte Major Histocompatibility Complex molecules Antigen (HLA) “Leukocyte” = where HLA was first found Soluble Ag Particulate Ag Antigen that does not have a carrier Antigen that is attached to a carrier INOcarriera Coded by MHC genes located in the short arm of Chromosome 6 Exogenous Ag Antigens that enter the body or system and start Polymorphic & Codominant " FROM circulating in the body fluids ★ 1:100,000: an individual’s MHC OUTSIDE BODY → bacteria, viruses, fungi, pollens, drugs, " type is about as unique as fingerprint pollutants Often used as a molecular basis for T cells to BY: KYLE GENE J. CALATRAVA | 2 |  caused by discriminate self from non-self Cold Agglutinin Rx.: Primary Atypical/WalkingPneumonia Mycoplasma - Ex. Bacteria (No HLA) = nonself Test Dtx: Cold Agglutinin abs pneumoniae Classes of HLA HLA Class I - A, B, C Rgt: RBCs at cold temp. - Found in almost all nucleated cells Spp.: Humans ★ (+) in PLT(no nucleus) but from Paul Bunnell Test Rx.: Infectious Mononucleosis Megakaryocytes (multinucleated) IM Dtx: Heterophile abs HLA Class II - DP, DQ, DR Sheep Rgt: Sheep RBCs - Found in APCs (dendritic cell, Spp.: Sheep macrophage, b-cell) APC-AntigenPresentinga ★ Monospot (Rapid): Horse RBCs ★ Most potent: Dendritic cell Immunization Method of generating protection against HLA Class III infectious disease by administering Ag T - Secreted proteins (minor group) ★ Old methods: Insufflation- Importance of 1. Antigen Presentation: Respiratory; Variolation - Skin RM HLA/MHC - HLA Class I = CD8+ T cells 1 8 - Factors affecting 1. Foreignness molecules - HLA Class II = CD4+ T cells 2 4 - Immunogenicity 2. Chemical Composition 2. Predisposition to diseases: 3. Molecular Size ★ HLA-B27 = Ankylosing spondylitis 4. Molecular Complexity or Diversity 3. Anthropologic Marker: 5. Susceptibility to Antigen Processing and ★ to determine genetic history (race) Presentation 4. Paternity Exclusion testing: babies' HLA must 6. Genetic Composition be seen in EITHER of the parents’ HLAs 7. Route and Dosage A, 5. Organ Transplantation: closest HLA (donor) 8. Addition of Adjuvants as possible: Foreignness Ag must be considered non-self - Host Versus Graft Disease (HVGD)/ Graft Rejection: immune response of Chemical Comp. Proteins > Polysaccharides > Lipids and NAs - the host to the graft Immunocompetent Graft Versus Host Disease (GVHD): graft attacks the host esp. In Immunocompromised px ★ Most Immunogenic: Bone Marrow ★ Least Immunogenic: Cornea L UN Molecular Size Molecular Complexity or Diversity Bigger molecules are more immunogenic - Heterogeneity in the building blocks that comprise the antigen ↑ Hetero = ↑ Immuno - Immunogen itself must be diverse rather than being composed of a single nucleic acid or 6. Transfusion: Ex. Single Donor Plt (SDP) is monosaccharide better than Random Donor Plt (RDP) ✓ Pro-Val-Thr-Tyr-Leu BA - SDP: from platelet apheresis = 1 - Molecules with repeating units are human source = lesser HLA types non-complex and are less immunogenic - RDP: pooled PLTs from routine WB X Val-Val-Val-Val-Val donations = more HLA types - Conformation of the molecular structure - Px. produce multiple Abs contributes to the immunogenicity CA against different HLAs = Susceptibility to - Large, insoluble macromolecules generally are HLA Alloimmunization Antigen more immunogenic than small, soluble ones - 1st trans. = Processing and - Macromolecules that cannot be degraded and sensitization Presentation presented with MHC molecules are poor - 2nd trans.: immunogens destruction of PLT Genetic Inherited factors such as HLAs by HLA Abs = N Composition Platelet Refractoriness Route and Dosage 1. Amount of Antigen Exposed to: - An insufficient dose will not stimulate SO Heteroantigen/ Antigen of a species different from that of the an immune response Heterologous or host, such as other animals, plants, or 2. Number of Exposure: Xenogenic Ags microorganisms Diff species ↓ - A single dose of most experimental. Heterophile -Exist in (totally) unrelated plants or animals but hence the immunogens will not induce a strong pressence antigens are either identical or closely related structure of busters Kay huga mutate response = Sensitization only! unrelated but identical -Ab to one will cross-react with Ag of the other any all - Repeated administration over weeks is JA identical Ags in the cells of different species usually required for repeated exposure -Ags shared by different species = Boosters! → since Ab may ↓ Cross-reacting 1. Weil-Felix reaction for Typhus Fever 3. Mode of Transmission: Microbial Ags 2. Cold Agglutinin Test in Primary Atypical - Oral: Mouth Examples Pneumia - Intradermal (ID): into the skin 3. Paul Bunnell Test - Subcutaneous (SC): beneath the skin Weil Felix Rx.: Typhus fever - Intravenous (IV): into a vein Dtx: Rickettsia abs - Intraperitoneal (IP): into the peri. cavity Rgt: OX-2 & OX- 19 (vulgaris), OX-K (mirabilis) - Intramuscuolar (IM): into a muscle Spp.: Proteus ★ Most common; best to introduce in the line of defenses BY: KYLE GENE J. CALATRAVA | 3 |  Addition of - included in the Ag preparations of vaccines to Best in agglutination and complement activation Adjuvants enhance immunogenicity Primary response Ab - Actions: IgG Secondary/Anamnestic response Ab → Prolong antigen persistence; Most predominant Ab in the serum enhances co-stimulatory signals Ab with the longest half-life → Increases local inflammatory Only Ab that can cross the placenta response Has 4 subclasses; differ on the number and → Stimulate nonspecific proliferation arrangement of their disulfide bonds of lymphocytes IgE Reaginic Ab Ex. Alum precipitate, Squaline, MF-59, Freund’s complete adjuvant IgA 2 subclasses; monomeric (IgA1) & dimeric (IgA2) T Antibodies Immunoglobulins/proteins found in the gamma IgA1 Most predominant IgA in the blood regions during electrophoresis; part of the IgA2 Most predominant IgA in the secretions Humoral component of adaptive immunity w/ secretory component RM Abs are 1. Antigen recognition - Enables it to be transported safely into responsible for 2. Opsonization the secretions 3. Complement activation IgM & IgD Surface of the B cells Paratope Antibody determinants; specific regions of IgM & IgA2 With J-chain "Antiqu binding site" antibodies that recognize and bind to Ag IgM & IgG Activate complement Structure of Ab ✓ pair of Light Chains IgM & IgE Ab with CH4 12 A, ✓ pair of Heavy Chains ↳ who discovered * Gerald Edelman Complement by discovered Set of 14-20 serum proteins; found in BETA * Rodney Porter ✓ each chain has Variable & Constant regions "JULES BORDET " during serum protein electrophoresis Types of Light Kappa, Lambda (either never both) : B ratio Naming system of 1. Numbers (e.g. C1 to C9) Chains Complement 2. Symbols (Factor B, Factor D) Types of Heavy Chains Amino Terminal End ⑧ Carboxy Terminal Gamma, Alpha, Mu, Delta, Epsilon "GAMDE" Where the variable region is located (V = A) amino -> variable "AU" Where the constant region is located (C = C) L UN proteins Function of Complements 3. Fragments: small case letters (“a” & “b”) ↑ phagocytosis by serving opsonins (C4b, C3b) ★ coating → enhance phagocytosis ↑ inflammatory response as anaphylatoxins (C3a, C4a, C5a) = clue: w/ small “a” ★ induce localized vasodilation End carboxy -> constant Pepsin Hydrolyze Ab into 2 frag. (1=pep, 2=sin) ↑ directed movement of WBCs as chemotactic agents (C3a, C5a) BA Breaks in the Fc portion (partial) PEP-SIN(2) ↑ clearance of immune complex and cell lysis ★ Fc’ + F(ab)2 - prime (‘) = degraded ★ cell lysis = ending of ALL pathways - 2 = divalent (2 Ag-binding sites) General concepts ✓ plays a role in cytolytic destruction Papain Hydrolyze Ab into 3 frag. (1=pa, 2=pa, 3=in) of Complements ✓ activity is destroyed by heating sera to 56degC for 30mins; inactivated serum is used CA Breaks in the Hinge region PA - PA - IN(3) ★ Fc + Fab + Fab within 4 hours, if exceeded, re-inactivate at - Fc = complete 56degC for 10 mins - Fab = monovalent (1 Ag-binding site) ★ VDRL & FTA-Abs ✓ IgM & IgG are the only Ig that react with Fab involves 1 light chain + ½ of heavy chain complement Fc involves ½ of the heavy chain ✓ Some Abs can activate but some can’t = IgG4 Hinge region Located between CH1 or CH2 ✓ Most potent = IgM N ★ Flexible d/t Proline (AA) ✓ Most active IgG = IgG3 CH2 (IgG) Complement binding site ✓ Binding sites = CH2 or CH3 SO CH3 (IgM) Pathways of 1. Classic Pathway Fc region WBC binding site Complement → Ag-Ab complex Variation in Ab 1. Molecular Weight IgM-heaviest Activation (w/ 2. Alternative Pathway/Properdin Pathway 2. Heavy chain subclasses 1g6-lightest triggers) → Activating surface (Ex. LPS) 3. Percent of total Ig 3. Mannan-Binding Lectin Pathway 4. Serum half-life: time it takes to reach half of # → Mannan/Mannose JA 5. Complement Fixation Stages of 1. Recognition Complement 2. Activation IgG IgM IgA IgD IgE Activation 3. Membrane Attack Complex MW 150,000 900,000 160,000 180,000 190,000 Exclusive - C1q HCS 4 0 2 0 0 Components to - C1r % of Total Ig 70-75 10 10-15 Smallpox MASP-1 similar to C1r: MBL-associated serine Strength of Binding Affinity: refers to the association constant protease-1 MASP-2 Regulatory Proteins similar to C1s: splits C4 & C2: MBL-associated serine protease-2 - C1 Inhibitor: inactivates C1 by dissociating C1r and C1s from C1; inactivates MASP-2 - Factor I: cleaves C3b and C4b L UN Precipitation between Ab and a univalent Ag Avidity: refers to the measure of the overall binding between Ag-binding sites and multivalent Ag the interaction of an Ab with a Soluble Ag; this - Factor H: cofactor to Factor I to inactivate C3b; NO CARRIER occurs since multivalent Ags can cross-link Abs prevents binding of B to C3b to form immune complexes BA - C4-Binding Protein: acts as cofactor with ★ soluble = no carrier Factor I to inactivate C4b Agglutination the interaction of an Ab with a Particulate Ag - S protein (vitronectin): prevents attachment of w/ CARRIER ★ particulate = with carrier C5b67 complex (early phase of MAC) to cell ★ more common in the laboratory membrane Stages of 1. Sensitization or Coating: refers to the - Decay-accelerating factor (DAF): dissociates CA Agglutination combination of Ab with a single antigenic C3 convertase (CD55) determinant on the surface of a cell without Hereditary C1 Inhibitor deficiency agglutination Angioneurotic Edema (HANE) 2. Lattice Formation: formation of large SLE-like C4 and C2 deficiency aggregates and visible clumping Syndrome; ★ most common Non-agglutinating antibodies that can perform sensitization but N Immune Complex or Incomplete Abs NOT the lattice formation Disorder ★ usually IgG; monomeric Neisseria infection/ C5 to C8 (MAC) deficiency NO visible agglutination = false (-) SO Gonococcal Antihuman Remedy to Non-agglutinating or Incomplete Abs Infection Globulin (AHG) / ★ Green-colored reagent Severe Recurrent C3 deficiency Coombs Reagent / Infection/ ★ most severe: C3 is the central Antiglobulin Glomerulonephritis pivotal point of all pathways Factors that affect 1. Antigen-Antibody Ratio JA Ag-Ab Interaction 2. pH Classical Alternative MBL 3. Centrifugation/Rotations Trigger Ag-Ab complex Activating Surface Mannan/Mannose 4. Addition of Colloids and Enhancement Media Recognition C1qrs C3b MBL + MASP 1 + 5. Length of Incubation MASP 2 6. Number of Available Antigens on the Carrier C3 convertase C4b2a C3Bb C4b2a 7. Location of the Antigen C5 convertase C4b2a3b C3Bb3b C4b2a3b 8. Roleaux Formation vs True Agglutination MAC C5b6789 9. Type of Ab and the Temperature Reaction Endpoint Cell Lysis Antigen-Antibody Zone of Equivalence: ideal reactive condition Ratio (2:1) where the number of multivalent sites of antigen and antibody is approximately equal (Ab = Ag) BY: KYLE GENE J. CALATRAVA | 5 | CLASSICAL calcium-holds the C1ars C4b29-C3 convertase of classical of Classical C462936 - C5 convertase Pathway ALTERNATIVE / Properdin C3b- opsonin C3bBb - properdin C3Bb3b - C5 convertase of alt. Pathway Me Me  ★ Harmening (Latest): 6-8x Prozone: Antibody excess ★ Turgeon (Old): 5x - Case: peak of infection - Choose an answer - Cause: false negative depending on the choices - Remedy: serial dilution (1-digit - 5x; range - 6-8x) Type of Ab and the IgM: cold-reactive; room temp. & below Postzone: Antigen excess Temperature IgG: warm-reactive; body temp. - Case: window period Reaction - Cause: false negative Reporting 1. Quantitative: in terms of concentration - Remedy: wait for the px to produce Ab Serological Test → mg/dL, mg/L, ng/dL, ug/dL pH (6.5-7.5) Optimum pH for antigen-antibody binding Results 2. Qualitative: presence or absence of Ab/Ag T ★ Anti-M: Ab in blood banking that is → pos/neg; reactive/non-reactive enhanced by acidic pH (6.5) 3. Semi-quantitative: in terms of titer RM Centrifugation/ Enhances the physical contact btw Ag & Ab Titer the reciprocal of the highest dilution of the Rotations → Manual Rot: ASO LAT = 2 mins biological sample that remains in a positive → Rotators: result → depends on the principle RPR = 100 RPM for 8mins How to answer (1) Know the PRINCIPLE VDRL = 180 RPM for 4mins “determine the (2) Always check the CONTROLS → Immediate Spin/Saline phase: 20 secs titer” sample (3) Focus on POSITIVES ★ > time = false positive d/t drying out problems? (4) See HIGHEST DILUTION ★ Calibrated: every 3 months A, → a negative rx.must be see ★ Disinfected: weekly Types of 1. Direct Agglutination Addition of Colloids 22% Albumin: Agglutination Tests 2. Indirect or Passive Agglutination and Enhancement ↑ dielectric constant = ↓ zeta potential 3. Reverse Passive Agglutination Media Polyethylene Glycol (PEG): # brings RBC closer together and concentrates Abs by removing water molecules from the testing sample Low Ionic Strength Solution (LISS): ↑ ionic cloud = ↓ zeta potential L UN ⑪ Direct Agglutination Agglutination 4. Agglutination Inhibition 5. AHG-mediated Agglutination use: Ag → naturally present in carrier dtx: Ab in the serum Examples of Direct Widal Test Weil-Felix Length of A certain amount of contact time must occur for Incubation Ab to bind the Ag Forward/Direct/Front/Cell ABO Typing Reverse/Indirect/Back/Serum ABO Typing BA 22% Albumin → 30-60 mins PEG —> 10-30 mins Rh typing LISS → 10-15 mins Direct Agg. Test Use (Rgt) Dtx #of Available Ags Lattice formation becomes FASTER if more Widal Text Bacterial Ag (O,H,K/Vi) Salmonella Ab on the Carrier antigens are present in the surface of cell/carrier Weil-Felix Proteus spp. Ag Rickettsia Ab CA Location of the Binding occurs faster if the antigens are (OX-2/19, OX-K) Antigen ACCESSIBLE to the antibody’s reach Forward/Direct/Front/ Anti-sera (Anti-A,B) RBC Ag Roleaux Formation Rouleaux formation (pseudoagglutination) may Cell ABO Typing vs True be confused with true agglutination Reverse/Indirect/Back/ Known cells (A,B) RBC Ab Agglutination + Add NSS: Serum ABO Typing - Clumping is dispersed = Rh typing Anti-sera (Anti-D) D Ag ROULEAUX FORMATION N - Clumping remains intact = Indirect or Passive use: Ag → artificially attached to a carrier TRUE AGGLUTINATION ② Agglutination Causes of Rouleaux: Examples of ★ ASO (Anti-Streptolysin O) LAT SO 1. Monoclonal Gammopathy Indirect or Passive use: ASO ag → Latex (Arti. Carrier) - Multiple Myeloma (↑IgG) Agglutination dtx: Anti-ASO - Waldenstrom’s Macroglobulinemia (↑IgM) T. pallidum Particle Agg. (TP-PA) T. pallidum Hemagglutination (TPHA) 2. Plasma or Volume Expanders Microhemaggulutination T. pallidum (MHA-TP) - Colloids such as Dextran JA Hemagg. treponemal test for Syphillis (HATTS) ↑ protein = ↑ rouleaux = interfere BB Reverse Passive use: Ab → artificially attached to a carrier ↑ BV = Tachycardia = Cardiac Arrest ③ Agglutination ★ C-reactive Protein (CRP): ★ Blood Volume NV: use: Anti-CRP → Latex - M: 5-7 Liters dtx: CRP (Ag) - F: 4-6 Liters Agglutination (1) neutralization of antigen by addition of ★ Colloids = contain protein (alb, dextran) vs Inhibition soluble reagent Ab Crystalloid = contain salt (NSS) # (+) - Absence of agglutination (2) indicator phase; addition of antigen-coated -) presence of agghstination particles to bind free reagent Ab 3. Wharton’s Jelly in Cord Blood specimen: - - Remedy: wash with NSS before using (+) ABSENCE of Agglutination BY: KYLE GENE J. CALATRAVA | 6 |  (-) Agglutination High-Resolution Electrophoresis: → show up to 12 bands ★ Semi-quantitative Pregnancy Testing One-stage → electrophoresis only SID ⑤AHG-mediated use: AHG/Coombs Rgt Electrophoresis - single immuno diffission Agglutination for: Non-agglutinating/Incomplete Abs (IgG) Rocket Electrophoresis: Direct Antiglobulin ★ dtx. IN VIVO sensitization "INSIDE" C [SID + Electric current] test (DAT) Qualitative: Yes/No or (+/-) ★ Height = Concentration Ag Ab DAT (+): move from well in the gel 1. AIHA: Autoimmune Hemolytic Anemia (+) Precipitin Rockets/Bullets → Autoantibodies T DID 2. HTR: Hemolytic Transfusion Reaction Counterimmunoelectrophoresis: Immuno Double- diffusion → Anti-Kell [DID + Electric current] RM 3. HDN: Hemolytic Disease of the Newborn ★ Counter = Ag & Ab move on → Rh Incompatibility opposite sides Indirect ★ dtx. IN VITRO sensitization "OUTSIDE" Ag (t) Ab H Antiglobulin Test happens in test tube container well toward anode (+) toward cathode (-) - , , Bovine Serum ★ Rh control → not used anymore (+) Precipitin Line Albumin (BSA) ✓ NO agglutination = Valid → Albumin = naturally present in our Two-stage → electrophoresis + 2nd method A, blood; if it agg., then we’re dead now Electrophoresis Precipitation interaction of antibody with soluble Ag "no carrier" Classic Immunoelectrophoresis Types of 1. Precipitation by Light Measurement [Electrophoresis + Passive ID] Precipitation Tests 2. Passive Immunodiffusion Precipitation by 3. Electrophoresis Nephelomentry: Light Measurement → measures light that has been SCATTERED Turbidimetry: L → measures the light that has been BLOCKED UN Ag run via electrophoresis (+) Precipitin Arc Immunofixation Electrophoresis [Electrophoresis + Passive ID] Ab trough ★ Multiple Myeloma: Bence Jones Passive Single Immunodiffusion (S.I.D): GEN Protein identification BA Immunodiffusion → only Ag diffuses; Ab in the gel - Kappa or Lamda? Double Immunodiffusion (D.I.D): Ag Ab → both Ag and Ab diffuse run via electrophoresis overlain in gel (+) Precipitin Bands Single Single Linear Immunodiffusion Immunodiffusion (+) Precipitin Line CA Crossed Immunoelectrophoresis Single Radial Immunodiffusion [Electrophoresis + Electrophoresis] (+) Precipitin Ring Ag Ab Single Radial Endpoint Method (Mancini): run via electrophoresis in the second gel Immunodiffusion ★ measured after 24hrs ⇒ IgG (+) Precipitin Rockets ★ measured after 50-72hrs ⇒ IgM N Kinetic (Fahey-Mckelvey) ★ measured after 18hrs; w/deadline SO “finished or not finished” Double both Ag and Ab diffuse Immunodiffusion - Ouchterlony Double Immunodiffusion ★ best example under DID JA single spurr Double spur Electrophoresis Electrophoresis for Separation of Substances One-stage Electrophoresis Two-stage Electrophoresis Electrophoresis for Zone Electrophoresis: Separation of → show up to 5 bands Substances BY: KYLE GENE J. CALATRAVA | 7 |  Labeled interaction of antigen, antibody, and label - Real-Time (Homogeneous, Immunoassays Kinetic) PCR Labels act as a marker to detect Ag-Ab reaction - Rapid-Cycle PCR Characteristics of ✓ must not alter the reactivity of the molecule - Digital PCR a good label ✓ remain stable for the shelf life of the reagent - Transcription-Based Amplification - Strand-Displacement Amplification Labeled Enzymes: ALP, beta-galactosidase, - Loop-Mediated Amplification immunoassays horseradish peroxidase - Helicase-Dependent Amplification can be classified 4. Target Amplification according to the Fluorescent dyes: Fluorescein isothiocyanate 5. Unamplified type of label used: (FITC), phycoerythrin - Northern Blot (RNA) T - Southern Blot (DNA) Radioisotopes: 131I, 125I, 3H Polymerase Chain DNA target amplification technique Labeled species may be an Ag or Ab to which label is attached Reaction RM Solid phase media such as the well or microplate, to which PCR Reagents Water _ an Ag or Ab may be attached to Buffer Heterogeneous requires a step to physically separate from the Magnesium chloride w/separation unbound analyte ★ Nucleotides: building blocks ★ Forward & Reverse Primers: starting point ★ Taq polymerase: solidifies extension Homogenous no separation step is necessary because ★ Thermus aquaticus (Taq): a A, No separation enzyme activity diminishes when the binding of heat-stable organism which is the Ag and Ab occurs source of the enzyme used in PCR Purpose of provides a way to separate bound & free PCR Steps (1) Denaturation (95oC): Separation Step Methods used in the Separation Step reactants physical means: decanting, centrifugation, or filtration, followed by a washing step to remove any remaining unbound analyte solid-phase vehicle - polystyrene test tubes, L UN "DAE" → separates the double-stranded DNA (2) Annealing (55oC): → primers attached to each starting point (3) Extension/Elongation/Synthesis (72oC): microtiter plates, glass or polystyrene beads, → nucleotides are added to form new copies magnetic beads, and cellulose membranes ★ Optimum cycles: 30x BA Detection Step counting radioactivity using gamma (solid scintillation) counters for radioimmunoassays Complement- interaction of antigen, antibody, & complement label related Tests Depends on used change in absorbance in a substrate is Complement Detects for antibodies, esp IgM measured via spectrophotometry for enzyme Fixation Test ★ (+) result: NO HEMOLYSIS ★ (-) result: HEMOLYSIS CA immunoassays fluorescence can be determined via Steps: fluorometers, fluorescent microscopes, flow - + reagent Ag to serum cytometers, and spectrofluorometers - + complement (★ Guinea Pig) Examples of Direct Fluorescent Staining for T. pallidum - + sensitized/indicator RBC (★Sheep) Labeled → dtx. for T. pallidum on the spx - observe for hemolysis/absence N Immunoassays → rgt: anti-T. pallidum Ab attached to Treponema ★ treponemal test of reference fluorescent label pallidum detects for anti-Treponema pallidum antibody immobilization → Spx: serum SO Fluorescent Treponemal Antibody-Absorption → Rgt: suspension of live motile T.pallidum → dtx. for anti-T. pallidum on the spx "GUINEA from experimental rabbits (testicles) + guinea → rgt: dead T. pallidum (Nichols PIG" pig complement strain), fluorescent-labeled AHG Nucleic Acid Tests dtx. for DNA or RNA ★ Interpretation: - Positive: >50% immobilized JA Classifications of 1. Probe Amplification Method Nucleic Acid Tests - Cleavage/Invader Technology - Negative: