[PATHO] Immunologic Diagnosis.pdf

PATHOLOGY 09/01/2024. MOD 4: IMMUNOLOGIC DIAGNOSIS Ma. Patrici...

PATHOLOGY 09/01/2024. MOD 4: IMMUNOLOGIC DIAGNOSIS Ma. Patricia Lourena A. de Guzman, MD, DPSP Trans Group/s: 7B I. DEFINITION OF TERMS A. AFFINITY A. ANTIGEN Any substance that can represent antigenic sites (epitopes) to produce corresponding antibodies from small molecules such as haptens, hormones, protein, glycoproteins, glycolipids and artificial chemical compounds. Should have at least one epitope. B. ANTIBODIES Produced in response to antigenic stimulation 1. IMMUNOGLOBULINS Refers to antibodies in the context of biological Affinity. function Have a heavy chain with a kappa (k) or lambda (g) Strength of a single antigen-antibody reaction light chain Each IgG antigen binding site typically has high There a five isotypes: affinity for its target. ○ IgG Initial force of attraction that an antibody has for a ○ IgM specific antigenic epitope or determinant ○ IgA How well the antibody fits to the shape of the antigen ○ IgD will determine stability of the bond ○ IgE Perfect lock and key fit will have the strongest affinity 2. CATEGORIES OF ANTIBODIES IN LABORATORY B. AVIDITY MEDICINE 1.1 Antibodies as Analytes IgG IgM IgA 1.2 Antibodies as Reactants Prepared from antiserum obtained through animal immunization with purified antigen II. ANTIGEN-ANTIBODY BINDING Depends on two characteristics: ○ Affinity ○ Avidity Avidity. Sum of all attractive forces between an antigen and an antibody It is the force that stabilizes the antigen-antibody reaction keeping the two together. IgM typically has low affinity antigen-binding sites, but there are ten of them, so avidity is high. Overall strength of binding antigen and antibody Antigen-Antibody Binding. Pathology - Mod # Topic Title 1 of 6 The use of trans, practice questions, and evals ratio must be used discreetly and social media/public exposure of the aforementioned shall be strictly prohibited. C. PRECIPITATION CURVE Occurs when there is excess antibody present. There is so much antibody that all antigen sites are quoted and a reaction cannot occur that results in false negative reactions. 3. POSTZONE PHENOMENON Precipitation Curve. Dependent on the amount of antigen and antibody present in the test system The x-axis represents the amount of antigen present. The y-axis represents the amount of antibody present. Prozone Phenomenon. 1. ZONE OF EQUIVALENCE Occurs when excess antigen is present. There is so much antigen that antibody molecules bind in such a way that a lattice network cannot occur. This also causes a false negative reaction. III. CLASSES OF IMMUNOASSAYS A. PRECIPITATION IMMUNOASSAY Simplest method for antigen and antibodies to react with each other without involving the detection of any labels Zone of Equivalence. Where antigen and antibody are present in optimal proportions to create a precipitation reaction. Sufficient antibodies can bind to antigens molecules resulting in lattice formation. 2. PROZONE PHENOMENON Precipitation. This involves the combination of soluble antibody with soluble antigen to produce insoluble complexes or precipitate that may be visible to the naked eye The resulting antigen-antibody complex may be detected: 1. QUALITATIVE By the naked eye By visual inspection As a precipitant 2. QUANTITATIVE By a detector Prozone Phenomenon. Pathology - Mod 4 🏠 Immunologic Diagnosis 2 of 6 The use of trans, practice questions, and evals ratio must be used discreetly and social media/public exposure of the aforementioned shall be strictly prohibited. B. PARTICLE AGGLUTINATION IMMUNOASSAY 1. ADVANTAGES AND DISADVANTAGES OF ENZYME Uses inert particles as labels IMMUNOASSAY Antigen or antibodies attached to particles such as red blood cells, latex, or metal solution react with the analyte Advantages Disadvantages in the specimen Large particles show significant agglutination patterns Sensitive assays can be Enzyme reactions very that may be visible to the naked eye developed by the sensitive to temperature amplification effect of COMMON TESTS USING AGGLUTINATION enzymes Direct Antiglobulin Indirect Antiglobulin Relatively cheap and Some samples may have (Coombs) Test (Coombs) Test plenty labels with long natural inhibitors shelf life Multiple simultaneous Measurement of enzyme assays can be developed activity can be more complex Equipment can be Enzyme activity may be inexpensive and is widely affected by plasma available constituents No radiation hazards Some assays available at occur during labeling or the present time are not as disposal of wastes sensitive as radioimmunoassays EIAs for large protein molecules require complex immunochemical Antibodies that bind to RBCs cause visible large particles of reagents agglutination that are visible. 2. MECHANISM PARTICLE AGGLUTINATION TESTS Enzyme-antibody conjugates to deliver a detectable substrate to the site of an antigen Hemagglutination Simple, easy to perform The substrate may be colorless molecule converted to No special equipment colored end product after an enzyme activation needed Treponema pallidum antibody test, Hepatitis B and C antibody test, HIV 1, HIV 2, Thyroglobulin Gelatin Particle Uses special gelatin particles Agglutination (instead of RBCs) with highly hydrophilic surface that prevents non-specific binding of materials present in a specimen Latex Uses latex beads as labels Agglutination hCG in home pregnancy kits Enzyme Immunoassay Mechanism. Particle Counting Uses optical cell counting to D. FLUORESCENT IMMUNOASSAY Immunoassay assess the decrease in number of Uses fluorophores as labels unagglutinated particles AFTER an Require optimal wavelength light energy to produce immune reaction detectable emission light 1. COMPOUNDS FREQUENTLY USED AS LABELS C. ENZYME IMMUNOASSAY Quantitative immunoassay that uses enzymes as labels Enzymes can amplify signal, depending on the Emits green color, has high turnover of enzyme catalytic activity Fluorescein intensity and good e.g., Horseradish Peroxidase, Alkaline phosphatase, photostability B-galactosidase, Glucose oxidase, Urease, Catalase Tetramethylrhodamine Emits red light Algae, Porphyrins, and Chlorophylls Pathology - Mod 4 🏠 Immunologic Diagnosis 3 of 6 The use of trans, practice questions, and evals ratio must be used discreetly and social media/public exposure of the aforementioned shall be strictly prohibited. 2. ADVANTAGES AND DISADVANTAGES OF FLUORESCENT IMMUNOASSAY Advantages Disadvantages Methods fairly simple Fluorescent compounds are sensitive to environmental changes No hazardous reagent Labels are less stable compared to enzymes Increased sensitivity over Bilirubin or hemoglobin can radiolabeled and enzyme absorb emission energy reactions Expensive Addition of chemiluminescent substrate. E. CHEMILUMINESCENT ASSAY Uses chemiluminescent compounds as labels IV. COMMON IMMUNOLOGIC TESTS Labels include chemically synthesized molecules or natural products (e.g., Luminol derivatives, Acridinium A. ANTINUCLEAR ANTIBODY (ANA) TEST esters, Nitrophenyl oxalate derivatives) Used to diagnose Systemic lupus erythematosus (SLE) Requires chemical energy to generate emission of light and other immune diseases SLE” systemic autoimmune disease that can affect 1. ADVANTAGES AND DISADVANTAGES practically every organ and tissue ○ Systemic, MSK, cutaneous, hematologic, renal, ADVANTAGES AND DISADVANTAGES OF lungs, heart, CNS CHEMILUMINESCENT ASSAY Almost 100% of SLE patients have a positive test A negative ANA is strong evidence against SLE Advantages Disadvantages After a positive screening test, ANA specificity may be determined to a certain specific antigen to which the Reactive stability of label False results may be ANA reacts: obtained if there is lack of ○ Anti-dsDNA in 50-70% patients precision in addition of ○ Anti-Sm (Anti-smith antibody): extremely specific hydrogen peroxide (almost diagnostic), in 30% Indirect immunofluorescent test (IFA): method of Speed of detection Need dedicated choice for screening of ANA instrumentation In ANA, the higher the titer, the more antibodies are present More sensitive compared ANA patterns are also reported. to radio immunoassay and enzyme immunoassay 1. COMMON PATTERNS OF ANA Common patterns include: Low cost 1. Homogeneous — SLE 2. Speckled — SLE, mixed connective tissue disease, 2. MECHANISM scleroderma, Sjogren’s syndrome 3. Centromere — CREST syndrome The antigen-antibody complex is attached to an 4. Nucleolar — scleroderma, CREST syndrome enzyme. The chemiluminescent substrate is added, chemical 1.1 Homogenous reaction occurs and light is emitted. The entire nucleus is stained with ANA Most common pattern Can be seen in any autoimmune disease, including SLE Can result from antibodies, DNA, and histones Antigen-antibody complex of chemiluminescent assay. Homogeneous pattern. Pathology - Mod 4 🏠 Immunologic Diagnosis 4 of 6 The use of trans, practice questions, and evals ratio must be used discreetly and social media/public exposure of the aforementioned shall be strictly prohibited.  B. RHEUMATOID FACTOR 1.2 Speckled An autoantibody that binds a variety of antigenic Fine and coarse ANA staining seen throughout the determinants of the Fc portion of IgG antibodies nucleus ○ Autoantibodies may be of IgM, IgG, IgA or IgE More commonly associated with antibodies to classes extractable nuclear antigens NOT specific for rheumatoid arthritis Associated with SLE, Sjogren’s syndrome, systemic ○ Often also seen in cases of chronic infections and sclerosis, polymyositis, and rheumatoid arthritis other inflammatory diseases Speckled pattern. 1.3 Centromere ANA staining is seen along the chromosomes It can be associated with: ○ Limited Systemic Sclerosis ○ Primary biliary cirrhosis ○ Other autoimmune diseases like Raynaud’s Latex Agglutination Test. phenomenon [HANDOUT] LATEX AGGLUTINATION TEST Test Parameter Positive test Latex beads agglutinate Negative test NO agglutination Semi-quantitative determination may be done using serial dilutions Centromere pattern. C. C-REACTIVE PROTEIN 1.4 Nucleolar General scavenger molecule and nonspecific The ANA staining is seen around the nucleolus inside acute-phase reactant protein nucleus It is produced by the liver and its presence indicate an It can be associated with: ongoing inflammatory process ○ Systemic sclerosis Highly sensitive acute phase reactant ○ Scleroderma Rises strikingly whenever there is tissue necrosis ○ CREST syndrome Sometimes used as a rapid test to differentiate a Calcinosis bacterial infection from a viral infection Raynaud's Phenomenon ○ Bacterial infection have HIGH CRP Esophageal Dysmotility ○ Viral infection have LOW CRP Sclerodactyly Used by rheumatologists to monitor progression or Telangiectasia remission of autoimmune disorders When latex particles coated with human anti-CRP are mixed with a patient’s serum containing C-reactive proteins, this results in visible agglutination within 2 minutes. ○ Positive test HAVE agglutination of latex particles ○ Negative test HAVE NO agglutination Nucleolar Pattern. Pathology - Mod 4 🏠 Immunologic Diagnosis 5 of 6 The use of trans, practice questions, and evals ratio must be used discreetly and social media/public exposure of the aforementioned shall be strictly prohibited.  CRP/Latex Agglutination Test. Case of Anaplastic Large Cell Lymphoma (ALCL) D. IMMUNOHISTOCHEMICAL STAINING highlighting the neoplastic lymphocytes. Used to check certain antigen markers in tissue samples 3. HER-2-NEU Antibodies linked to an enzyme or a fluorescent dye Used as a prognostic marker in breast carcinoma and become activated after it binds to an antigen. Breast carcinoma that stains for this marker expresses a The resulting antibody-enzyme conjugate enhances protein called the human epidermal growth factor microscopy receptor-2 (HER-2) In diagnostic surgical pathology, formalin-fixed and HER-2-NEU: promotes the growth of cancer cells paraffin-embedded tissues utilize these stains to visualize cell types: ○ HER-2 positive breast cancers tend to be more ○ Confirm cancer cell type aggressive than other types ○ Determine the possible origin of metastatic ○ Treatments that specifically target HER-2 are very cancer of unknown primary effective that even if the tumor is aggressive, a ○ Prognostic markers positive HER-2-NEU stain is a good prognostic factor in breast carcinoma. COMMON IMMUNOSTAIN IN SURGICAL PATHOLOGY Cytokeratin (CK) Leukocyte Common Antigen (LCA) HER-2-NEU 1. CYTOKERATIN (CK) Used to differentiate epithelial malignancies (stain positive) as opposed to non-epithelial malignancies (stain negative) Example of Breast Carcinoma. Tissue Section from a Thyroid with Papillary Carcinoma showing cytokeratin. 2. LEUKOCYTE COMMON ANTIGEN (LCA) Commonly used marker of hematopoietic cells EXCEPT erythrocytes and platelets Pathology - Mod 4 🏠 Immunologic Diagnosis 6 of 6 The use of trans, practice questions, and evals ratio must be used discreetly and social media/public exposure of the aforementioned shall be strictly prohibited.