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IMMUNOLOGY

IMMUNOLOGY. LANGE CHAPTER 64 AND 65 DR SAMUEL AGUAZIM. Major uses of serologic (antibody-based) tests. Major uses of serologic (antibody-based) tests. I. Diagnosis of infectious diseases . II. Diagnosis of autoimmune diseases III. Determination of blood type and HLA type.

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IMMUNOLOGY

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  1. IMMUNOLOGY LANGE CHAPTER 64 AND 65 DR SAMUEL AGUAZIM

  2. Major uses of serologic (antibody-based) tests. • Major uses of serologic (antibody-based) tests. • I. Diagnosis of infectious diseases. • II. Diagnosis of autoimmune diseases • III. Determination of blood type and HLA type

  3. Antigen/Antibody reaction in the Laboratory • In this test, the antigen is particulate (e.g., bacteria and red blood cells)' or is an inert particle (latex heads)coated with an antigen. Antibody, because it is divalent or multivalent, cross-links the antigenically multivalent • particles and forms a latticework, and clumping (agglutination) can be seen.

  4. TESTS FOR QUALITATIVE AND QUANTITATIVE TESTS FOR ANTIGENS AND ANTIBODIES • IMMUNODIFFUSION: measures antigen-antibody complexes through the formation of precipitates. The most common method used is double immunodiffusion(ouchterlony radial immunodiffusion technique. It is also known as DID (double immunodiffusion). • ELECTROPHORESIS: method of separating proteins in an electric field. Zone electrophoresis separates proteins based on the surface charge. The method can quantitate serum proteins and immunoglobulins. • It is useful in the detection of multiple myeloma, Waldenstrommacroglobulinemia and hypergammaglobulenimia.. It can also detect the reduced or total absence of immunoglobulins in diseases.

  5. Agglutination • DIRECT TYPE AB + AG COMPLEX • INDIRECT TYPE (PASSIVE) AG + RBC,INERT PARTICLE  AB Ag+AB LATEX/ HEMA AGGLUTINATON IgM BETTER SEEN THAN IgG

  6. Latex agglutination

  7. Agglutination • HEMAGGLUTINATION - BLOOD TYPING -PRESENCE OF ANTIGENS ON THE SURFACE OF RBC -CORRESPONDS TO THE BLOOD TYPE

  8. Your Blood Type is the ANTIGEN • Inherited antigen from parent • Sugar – Protein

  9. Hemagglutination

  10. Hemagglutination • Serum + ANTI A AND ANTI B antiglobulin separately • (+) AGGLUTINATION: anti-A antibody • (-) AGGLUTINATION : anti-B antibody A antigen (+) B antigen(-) • blood type A

  11. Coombs regeant • USES ANTIHUMAN GLOBULIN • ANTIBODY SPECIFIC FOR HUMAN IG AND COMPLEMENT FACTORS • COOMBS TEST MEASURES AB WHICH ARE TOO LOW TO AGGLUTINATE BY THEMSELVES • Useful in blood typing, in hemolytic disease of the newborn, and autoimmune hemolytic anemias

  12. Direct Coombs test detects serum proteins adherent to RBCs taken from the patient

  13. Indirect Coombs test detects incomplete antibodies in the serum, which is then incubated with RBCs. The antibody-coated cells are then agglutinated by a Coombs antiglobulin serum

  14. Coombs test • DIRECT: SERUM PROTEINS ADHERE TO THE PXS RBC AUTOIMMUNE DISEASES DRUG-INDUCED DISEASES • INDIRECT: DETECTS INCOMPLETE AB IN THE SERUM; INCUBATED WITH RBC HEMOLYTIC DISEASE BLOOD TRANSFUSIONS

  15. IMMUNOFUORESCENCE • DETECTS AB OR AG IN TISSUES OR FLUIDS • DERMATOLOGIC DISEASES, SLE • DIRECT: AB BOUNDED TO TISSUE ANTIHUMAN IG (FLUOR) • INDIRECT : FREE AB IN SERUM ,PLASMA OVERLAY WITH TISSUE ANTIHUMAN IG(FLUOR) These are good in the detection of SLE, pemphigus, bullouspemphigoid, and Henoch-Schonleinvasculitis

  16. Complement fixation • MEASURES THE LEVELS OF AG AND AB BY THE CONSUMPTION OF COMPLEMENT • AG-AB-COMPLEMENT COMPLEX • RBC ADDED, BIND TO UNFIXED COMPLEMENT • This is useful for HBsAg determination, antiplatelet antibodies, Ig , and DNA.

  17. Compliment fixation

  18. Polymerase chain reaction • IDENTIFY THE PRESENCE OF DNA: CSF, BLOOD, TISSUES • ADD SYNTHETIC DNA PRIMER • COMPLEMENTARY TO THE DNA • REPLICATION OF THE DNA FRAGMENTS • AMPLIFICATION OF THE MINUTE DNA • Each cycle : 1-3 minutes

  19. Polymerase chain reaction

  20. Polymerase chain reaction • This important in the detection of low level bacterial infections or rapid changes in transcription at the single cell level, • as well as the detection of a specific individual's DNA in forensic science . • It can also be used in DNA sequencing, screening for genetic disorders, site specific mutation of DNA, or cloning or sub-cloning of DNAs.

  21. ENZYME LINKED IMMUNOSORBENT ASSAY (ELISA) • ONE OF THE MOST SENSITIVE METHODS OF DETECTING SPECIFIC AG, AB • AG-AB COMPLEX, FREE AB WASHED AWAY • ENZYME ADDED, BIND TO AG-AB COMPLEX • SPECTROPHOTOMETRY

  22. Enzyme linked immunosorbent assay

  23. Western blot • MODIFIED ELISA • ANTIGENS ARE ELECTROPHORESED, SEPARATED • BLOT STEP: SEPARATED AG TRANSFERRED TO NITROCELLULOSE • SERUM AB ADDED TO THE NITROCELLULOSE • AG-AB COMPLEXES,EXCESS AB ARE WASHED OFF • ANTI-ANTIBODIES ADDED • RADIOISOTOPES ADDED

  24. Northern blot

  25. Mantoux tuberculin skin test (TST) • standard method of determining whether a person is infected with Mycobacterium tuberculosis • Purified protein derivative

  26. Mantoux tuberculin skin test • intradermal injection • one tenth of a milliliter (mL) of tuberculin; 5 tuberculin units. • insert the needle bevel slowly at a 5° to 15° angle, to below 3 mm of the skin surface • wheal should be 6mm to 10mm in diameter • Read after 48-72 hours for induration • Size of the wheal determines exposure to MTB

  27. Patch Test

  28. Hypersensitivity 1) Hypersensitivity reactions occur when the immune responds in such a way to cause moreharm than good to the body. 2) Hypersensitivity reactions must involve inflammation. Hence IgA, which does not cause inflammation, is never implicated, all other classes of antibody and the cell-mediated Immune system maybe. 3) Hypersensitivity reactions are classified into 4 groups depending on which branch of the immune system is involved

  29. Hypersensitivity An immunologic reaction which produces tissue damage on re-exposure to antigen. • Type I (IgE-mediated) • Type II (Fc and complement-mediated) • Type III (Immune complex-mediated) • Type IV (Delayed-type hypersensitivity)

  30. TYPE 1 HYPERSENSITIVITY DISEASES Type I Hypersensitivity Diseases: • Allergic rhinoconjunctivitis (hay fever) • Asthma • Eczema (atopic dermatitis) • Acute urticaria • Anaphylaxis

  31. TYPE 1 HYPERSENSITIVITY DISEASES • Type hypersensitivity reactions involve IgE antibodies • IgE antibodies have two heavy (epsilon) chains and two light chains • Little IgE antibody is found inserum, it functions by binding to mast cells through a high affinity receptor • Mast cells are filled with granules which contain inflammatory mediators such as histamine. There are two variants but these are sufficiently similar we need not bother about the differences • When antigen binds to the bound IgE antibody at at least two sites then calcium enters the cell. This results in two responses

  32. The Immediate Reaction • Mast cell granules contain a range of inflammatory mediators. Most important is histamine, which causes dilation of blood vessels and increases blood vessel permeability (but causes constriction of bronchial smooth muscle). The result is the four classic symptoms of inflammation (redness, swelling, heat and pain - in Latin rubor, tumor, calor, dolor) • In addition the granules contain chemotactic factors, especially for eosinophils, and activating factors (e.g. for platelets) • In addition to their direct effects factors in the mast cell granules activate the alternative pathway for complement fixation and the kinin system

  33. Mast Cell Mediators • Preformed – Vasoactive amines: Histamine – Neutral proteases: Tryptase, Chymase – Acid hydrolases: Hexoseaminidase – Proteoglycans: Heparin, Chondroitin sulfate • Newly formed – Eicosanoids: PGD2, LTC4 – Cytokines: TNF, IL-4, IL-5, IL-

  34. Histamine • Produced almost exclusively by basophils & mast cells (3-8 pg/cell) • Immediate pharmacologic effects: • Pruritus (H1) • Vascular permeability/vasodilatation (H1) • Smooth muscle contraction (H1) • Gastric acid secretion (H2)

  35. Type I hypersensitivity – sensitization to an inhaled allergen or bee sting • Antigens (red dots) from inhaled pollen are ingested and presented by macrophages to T cells. Activated T cells produce cytokines leading to the production of IgE, which binds to receptors on mast cells and causes the release of histamine, which is responsible for allergy symptoms. Onset is usually within minutes of contact with antigen.  

  36. result

  37. Acute Anaphylaxis Shock: very low blood pressure due to systemic arteriolar dilation • Bradycardia and decreased respiratory rate • Examples: Bee sting, penicillin allergy • Treated with epinephrine i.v., high dose fluids, vasopressor agents

  38. Type II Hypersensitivity • Antibodies • complement activation • anti-68kDa protein antibody • SLE like reaction, Goodpasture’s

  39. Type II Hypersensitivity Reactions: Mechanisms of Tissue Damage • Complement-mediated cytolysis • Antibody-dependent cell-mediated cytotoxicity (ADCC)

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