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IMPORTANCE OF IN VITRO LABORATORY METHODS IN ALLERGOLOGY

IMPORTANCE OF IN VITRO LABORATORY METHODS IN ALLERGOLOGY. Dr. Sándor Sipka. http://rimm.dote.hu. Johansson et al. Hypersensitivity: hyperreactive reaction of the organisen elicited by a trigger of enviroment. Allergic hypersensitivity (immunologic mechanism defined or strongly suspected).

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IMPORTANCE OF IN VITRO LABORATORY METHODS IN ALLERGOLOGY

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  1. IMPORTANCE OF IN VITRO LABORATORY METHODS IN ALLERGOLOGY Dr. Sándor Sipka http://rimm.dote.hu

  2. Johansson et al. Hypersensitivity: hyperreactive reaction of the organisen elicited by a trigger of enviroment Allergic hypersensitivity (immunologic mechanism defined or strongly suspected) Nonallergic hypersensitivity (immunologic mechanism excluded) IgE-mediated Not IgE-mediated T cell:e.g.,contact dermatitis, celiac Nonatopic Atopic Insect sting Eosinophil:e.g., gastroenteropathy Helminths IgG-mediated:e.g.allergic alveolitis Drugs Other Other

  3. Allergy: a pathologic state in certain group of (atopic) patients responding with inflammatory reactionson certain types of antigens (allergens) being otherwise neutral for a greater part of the population. Backround: a diathesis of polygenic type (inherited pathologic state) with: - increased production of allergen specific IgE - raised production of total IgE stimulated by IL-4 - overproduction of inflammatory cells -increased sensitivity of tissues to certain types of mediator substances

  4. Types of allergic reactions: Type I. reaction: IgE mediated allergic reaction Type II. reaction: cytotoxic reaction induced by IgG and IgM Type III. reaction: tissue damages caused by immunocomplexes Type IV. reaction: delayed type hypersensitivity induced by Th1 lymphocytes

  5. Type I. allergic reaction The reaction is mediated by allegen specific IgE The reaction is of immediate type ( the symtoms of inflammation appear within 4 hours afterthe allergen challange) The symptomes are elicited by mediator substances released from mast cells, basophils,eosinophils, macrophages or platelets. Mediators of mast cells/basophils eosinophils macrophages platelets histamine, triptase ECP proteases serotonine PGD2, LTC4 MBP PGD2, PGE2 histamine PAF ROS TxA2, LTB4 TxA2 IL-1, IL-4, IL-5 LTC4, PAF LTC4, PAF ROS TNFa, IFNgIL-5 IL-1, TNF ROS

  6. Types of allergens: Drugs (penicilline, etc) Inhalative allergens: pollens (ragweed, mugwort, etc) animal epithelium (cat, dog,etc) mites fungi (mucor, aspergillus, etc) textile/cotton Insects bee, wasp Nutritive allergens: milk, egg, soybeen, etc. Atopy: pathologic hypersensitivity to allergic reactions. It is a diathesis. Anaphylaxy: a lifethretening state when enormously high amouts of the inflammatorymediators get into the circulation, skin, lung andgastrintestinal truct a.) IgE mediated b.) not IgE mediated forms (mediated by complement and other factors)

  7. Laboratory diagnosis: Serum total IgE (nephelometry, turbidimetry) Allergen specific IgE (RAST, ELISA, FIA, dot-blot) Activity markers: increased levels of eosinophil cationic protein (ECP)and tryptase Blood film: eosinophylia

  8. FIRST ALLERGEN STIMULUS SECOND ALLERGEN STIMULUS

  9. Type II. allergic reaction Mechanism: cytolytic and cytotoxic reactions induced by IgG and IgM, causing tissue damages: -complement mediated cytolysis (classic pathway) -stimulation of PMN, Eo cells and monocytes/macrophages by activated C3 -IgG bindig to effector cells: killer cells, PMN, Eo cells and monocytes/macrophages Allergens: drugs: chinine, furosemide, gold salt, indomethacine, sulphonamides,salicylate, chloramphenicole Laboratory diagnosis: measurement of complement activity demonstration of the activation of PMN, Eo, monocytes/macrophages ADCC

  10. Type III. allergic reaction Mechanism: tissue damages caused by immunocomplexes sedimentation of IC in circulation sedimentation of IC in tissues Allergens: drugs, antibiotics, benzotiazine, hidantoine, bacteria: streptococcus, etc viruses: hepatitis B,C, etc. Laboratory diagnosis: Measurement of IC level in serum Measurement of complement factor activity in serum Histology: microscopic IC verification

  11. Type IV. allergic reaction Mechanism: „delayed type” hypersensitivity induced by the cytokines of Th1 cells.The symptoms appear within 12-24 hours after the allergen challange. Forms: a.) Contact sensitivity Hapten-carreir complexes processed by Langerhans cells to Th1 lymphocytes:cytokine release antigens: nickel, gutta percha, oils, Hg salts, stains, drugs, cosmetics

  12. b.) Tuberculin-type reaction Mononuclear (monocyte-lymphocyte) cell infiltration at the site of antigenpenetration Antigen: bacterial proteins, chemicals (circorium, berillium) The same mechanism is involved in the rejection of transplants Laboratory diagnosis   Histology (mononuclear cell infiltration) Lymphoblast transformation induced by the antigen Measurement of cytokine production

  13. Questionnaire The most frequent allergens in your clinical pratctice • Country: City: • Place of work: 3/a.Universitiy Department 3/b. Hospital 3/c. Private practice • Position: • Number of your allergic patients/year: 5/a.<1000 5/b. 1000-5000 5/c> 5000

  14. Questionnaire The most frequent allergens in your clinical pratctice

  15. The main methods used for diagnosis of allergy Type I. reaction:in vivo: cutaneous Prick test in vitro: serum total IgE allergen specific IgE allergen specific IgG produced mainly in infants against milk, egg, soybeen, etc mast cell/basophil activation products: histamine, tryptase, leukotrine eosinophil activation products: ECP Type III. reaction:in vitro: allergen-IgG/IgM complex measurement (ELISA, Ouchterlony) Type IV. reaction:in vivo: epicutaneous skin test (contact allergens) in vitro: lymphocyte proliferation assay (for drugs)

  16. Methods used for the determination of allergen specific IgE Types of methods Allergens used: purified natural extracts molecules produced by recombinant technique mix of allergens Anti-IgE immunoassays: radioimmunoassay (RIA) enzyme linked immunosorbent assay (ELISA) fluorescence immunoassay (FIA) chemiluminescence immunoassay (CHLIA) Values of measurements: 1. kU/l 2. Spec. IgE positivity class: 0-6 Criteria of use: sensitivity specificity positive predictive value negative predictive value 

  17. SPECIFIC IgE POSITIVITY CLASSES

  18. Other forms of in vitro tests for allergy Basophil activation tests: Measurement of histamine release Measurement of leukotriene release Measurement of CD63 expression Lymphocyte activation tests:  T cell proliferation assay cytokine release nitroblue tetrazolium (NBT) test Quantitative PCR for IL-4 Chip technique with recombinant allergens

  19. Indications for using of allergen specific IgE measurements Patients with any types of skin diseases Lack of allergens used for skin tests Diversity of the result of skin test and the anamnesis At the beginning and the end of specific immunotherapy When the skin test may provoke anaphylaxis (e.g penicilline) At infants  At patients when the physical or mental conditions exclude the skin test taking : antihistamines, benzodiazepines or corticosteroid, etc. The value of a negative specific IgE test: The clinician can exclude the allergic pathomechanism (testing for food allergy)

  20. The problems with allergen specific IgE determinations CROSS REACTIVITY BETWEEN ALLERGENS: e.g. ragweed: water melone, banana, cucumber peanut: chestnut, soya bean, pea The lack of chemically well defined allergens The in vitro tests are more expensive than the skin tests The possibility for misusing during the ordering of these tests. Lack of widely accepted and used international standards for the tests. The international external quality controls did not come into general use. (NEQUAST, Pharmacia, QualiCont)

  21. The clinical value of allergenspecific IgG measurements This antibody is not in a direct relation to allergy. The production of anti-milk, -egg, -soybeen,- tomato IgG is possible by an increased intestinal permeability, by an inflammation, mainly in infants. Therefore, it reflects a state of anincreased enteral permeability for food antigens. The association of specific IgG with IgE, however, may predict the prolonged persistance of a hypersensitivity to milk, for example.

  22. The fundamental in vitro laboratory tests of allergy Serum total IgE Serum allergen specific IgE Verifies the allergen ( children) Early definition of allergen (chicken egg, bovine milk) Can be used for series measurements ( in a population) Following up the specific immunotherapy Markers of activity: serum ECP and tryptase levels   Free radical production (chemiluminescence) of   peripheral phagocytes

  23. I. Occurence of allergens in children with food allergy less than 1 year old in Debrecen

  24. II. Occurence of allergens in 1-6 years old children with food allergy in Debrecen (Hungary)

  25. III. Occurence of allergens in children with inhalative allergy less than 1 year old in Debrecen

  26. IV. Occurence of allergens in 1-6 years old children with inhalative allergy in DebrecenI.

  27. V. Occurence of allergens in 1-6 years old children with inhalative allergy in DebrecenII.

  28. 3rd Department of Internal Medicine Regional Immunology Laboratory

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