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Immunological investigations

Immunological investigations. Dr. Cismaru Cosmin Andrei Cosmin.Cismaru@umfcluj.ro. Medical investigations. Laboratory tests – important in establishing a diagnosis and conducting treatment strategy Applicability: - diagnosing or excluding a disease among symptomatic persons

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Immunological investigations

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  1. Immunological investigations Dr. CismaruCosmin Andrei Cosmin.Cismaru@umfcluj.ro

  2. Medical investigations • Laboratory tests – important in establishing a diagnosis and conducting treatment strategy • Applicability: - diagnosing or excluding a disease among symptomatic persons - diagnosing a disease among asymptomatic persons (screening) Evaluating a therapeutic response

  3. Characteristics of medical tests • Accuracy • Cost • Interfering factors • Precision • Normal values • Sensitivity • Specificity Sensitivity: The probability of having a positive test when you are sick is the power of a disease test Specificity: The probability of having the negative test when you are healthy, or the proportion of those with a negative test among thehealthy

  4. Laboratory investigations • Hematology: HG with leucocitary formula (complete blood count) • Biochemistry: liver tests, kidney, thyroid, metabolic. • Imunology-serology: Ab, auto-Ab, tumor markers • Microbiology: hemoculture, coproculture, urine culture. • Imagery • US • X-ray • CT-scan • MRI • Scintigraphy • Paraclinical investigations • Spirometry • EKG

  5. Laboratory investigations • Hematologice: HLG cu formula leucocitara • Biochimie: testehepatice, renale • Imunology-serology: anticorpi, auto-anticorpi, markeritumorali • Microbiologie • Imagistica • Ecografie • Radiografie • Tomografie • Scintigrafie • Investigatiiparaclinice • Spirometrie • EKG

  6. SEROLOGY AND IMMUNOHISTOCHEMISTRY

  7. Ab detection methods • Immunoprecipitation • Detects Ab in solutions • Quantitative evaluation of the presence of Ab • End-point is the visualization of Antigen-antibody precipitates • Complement fixation • It is based on fixation / activation of the complement that follows the binding of the complement system components to the immune complexes

  8. Neutralization • Capacity of specific antibodies to neutralize various toxins / pathogens • Rarely used for diagnostic purposes • For the detection of post-vaccine antibody synthesis • Agglutination • Simple and fast test • Capable of detecting small antibody concentrations • Detection of anti-viral antibodies • It uses latex-attached antigenic particles • Direct or indirect method

  9. Immunofluorescece • Requires microscope with uv lamp • Direct or indirect method • ImmunoenzymaticTechnique (ELISA) • The most sensitive • Usually the indirect method is the most used and it depends on the use of an anti-human conjugate (IgG or IgM) • Conjugated antibody - an anti-human Abthat has an attached enzyme (HRP) at the Fc portion, the enzyme that is capable of catalysing the conversion of a substrate to a colored product, which will then be read with a spectrophotometer • Radio-immune technique • High sensitivity • Increased costs, risk of irradiation

  10. 1. Precipitation reactions • When certain molecules of antigen (Ag) interact with antibodies (Ac), they will precipitate out of solution • Indications: • Detection of serum antigens / antibodies

  11. a. Reactions in liquid medium b. Reactions in gel: • Immunoelectrophoresis • Immunofixation • Western blotting • Radial immune diffusion

  12. a. Reactions in liquid medium • Laser nephelometry - based on the property of immune complexes to deviate laser light • Indications: • - quantification of serum proteins: Ig, C3, CRP, ceruloplasmin.

  13. b. Reactions in gel a. Immunlectrophoresis b. Imunofixarea c. Western blot d. Imunodifuziaradiala

  14. Immunlectrophoresisallows the evaluation of the gammaglogulins, the three major classes of immunoglobulins. • M (IgM), • G (IgG), • A (IgA).

  15. a. Imunoelectroforeza b. Immunofixation (SDS-PAGE) c. Western blot d. Imunodifuziaradiala

  16. The serum proteins will be separated on SDS based on molecular weight (Sodium dodecyl sulfate polyacrylamide gel).

  17. a. Imunoelectroforeza b. Imunofixarea (SDS-PAGE) c. Western blot d. Imunodifuzia radiala

  18. Transfer Membrane After Blotting Gel after blotting Transfer Membrane SDS Gel Electro- blotting Gel-bound proteins will be transferred to a nitrocellulose membrane by applying an electrical charge.

  19. 1. Blocking 2. 1st Antibody 3. 2nd Antibody 4. Detectection Protein X Nitrocellulose membrane

  20. a. Imunoelectroforeza b. Imunofixarea (SDS-PAGE) c. Western blot d. Radial immunodiffusion

  21. Radial immunodiffusion(Mancini) • Quantitative dosing of the quantity of anantigen in a sample. The diameter of the circle increases with time as the antigen diffuses into the medium, reacts with the antibody, and forms insoluble precipitin complexes.[he antigen is quantitated by measuring the diameter of the precipitin circle and comparing it with the diameters of precipitin circles formed by known quantities or concentrations of the antigen 1 1 2 2 3 3

  22. Applications • Quantitative determination of certain proteins: • - immunoglobulins, • - complement, • - alpha-1 antitrypsin

  23. 2. Agglutination reactions • Highlighting the presence of particles (bacteria, blood cells, cells) in suspension (serum) IgM + Eritrocite aglutinare

  24. Antibody measurement in patients serum • Serial Dilutions (1: 2) • Mixing with known Ag. • The occurrence of precipitate if Abis present. • Abtiter Blood typing: yes/no example of agglutination.

  25. ABO blood groups

  26. Blood group AB0 and Rh testing • Reactives • Anti-A, Anti-B and Anti-AB solution of monoclonal antibodies (hybridometechnqiue)

  27. Indications • Establishment of blood groups ABO, Rh • Demonstration of antibodies to certain bacteria • Detection of rheumatoid factor

  28. 3. Complement Fixation • indications • - Diagnosis of viral and bacterial infections Human serum Complement Positive R Antigen (viral, bacterian) Negativa R

  29. 4. Reactions with marked reagents • The immunofluorescence reaction • Radio-immunological analysis • Immunoenzymatic analysis

  30. a. The immunofluorescence reaction • Direct- Antigen detection (biopsy) + Ag Ag • Indirect - detection of antibodies (serum) + + Ag Ag Ag

  31. Indications • Detection of antinuclear antibodies (ANA) LES LES, Sdr.Sj, Sclerodermie Miozita LES, BMTC, Sdr. Raynaud, Psoriazis Sdr. CREST Hepatita cronica, Vasculita, Trombocitopenie Sclerodermie, Miozita, CBP, Sdr. Sj Sclerodermie

  32. b. Radioimmunological tests Detection of IgEspecific to a suspected / known antigen - total / specific IgE count 1 kUI / l = 2.4 μg / l Anti-IgE-radiolabel Antigen IgE wash Patient serum

  33. b. Radioimmunoassay (RIA) Anti-IgE-radiolabelled • Direct method Antigen Patient serum IgE wash • Indirect method Antigen IgE Patient serum wash

  34. RAST (radio-alergo-sorbent test) - detection of IgE specific to a suspected / known Ag - quantification of total / specific IgE Indications: Quantitative determination of different antigens:bacterial, viral, tumor, hormoneMedicines (digital, theophylline, cortisone)Determination of allergens in allergic diseases

  35. c. Immunoenzymatic analysis • ELISA, RIST

  36. ELISA - classical- quantification of Ab HRP Ser uman Ac anti IgG-HRP HRP - sandwich- quantification of antigen HRP Antigen Ac anti Ag-HRP HRP

  37. Indications: • Quantitative determination of some important diagnostic proteins: alpha-fetoprotein, carcinoembryonicantigen, HBs antigen, etc. • Quantitative determination of bacterial, viral, fungal, parasitic or appropriate antigens in serum of patients for diagnostic purposes • Evidence and titration of autoantibodies as markers of autoimmune diseases

  38. 5. Flowcitometry Fluid cells susp Procedure: A cell suspension is passed through a special detector, which allows multiparametric (physico-chemical) detection of thousands of cells in a relatively short time Laser Light 488nm Light detectors FL1 FITC Computer

  39. Flow-citometry (FCM) • Main surface markers

  40. 6. Investigations in hypersensitivity • in vivo - skin tests - Challenge test: oral, nasal, bronchial - Hot, cold test • In vitro - total IgE - specific IgE (RAST, ELISA) - mediators determinations: triptase

  41. Indications: • Diagnosis of Allergic Diseases: • - drug allergy / food allergy • contact dermatitis / atopic • - allergic asthma • Triptase- diagnostic and forensic value • - VN: 1-10ng / ml (> 20ng / ml- mastocytosis, anaphylaxis)

  42. 7. Detection of immune complexes • Physical methods:- gel filtration,- precipitation,- ultracentrifugationDetecting cryoglobulinsradiolabelled C1q

  43. Indications • Estimating the severity of chronic infections: • Viral Hepatitis B, C • Bacterial endocarditis • parasitosis • Estimating the severity of autoimmune disorders: • Systemic lupus erythematosus • Rheumatoid arthritis

  44. 8. Investigations in autoimmune diseases • Specific organ autoantibodies • ELISA, IFI → Abanti-Thyroglobulin (Hashimoto's thyroiditis) • ELISA, RIA → Abanti-Acetyl Choline Receptor (MG - miastenia • gravis) • IFI → AbAnti Mitochondria (Primary Biliary Cirrhosis) • Non-specific organ autoantibodies • IF → ANA (LES, SdrSj, Polymositis, Dermatomyositis)

  45. 9. Investigation of immunodeficiencies • Humoral immunity (Lymphocytes B, Antibodies) - Full HLG → leukocyte formula - Radial gel immuno-diffusion → Ig dosing - Flow cytometry with Ac anti-CD19 → LB separation • Cellular Immunity (Lymphocyte T) - Full HLG → leukocyte formula - Flow cytometry → Population tracking of T lymphocytes (LTh, LTc,)

  46. Evaluation of lymphocytes • Lymphocyte populations: LB, LT, NK Subpopulations: LTHelper, LT cytotoxic • Quantitative assessment - Flow cytometry • Qualitative / functional evaluation - Activation tests - Proliferation tests - Differentiation tests - The leukocyte adhesion inhibition test

  47. Lymphocyte activation test • NBT Reduction Test • Determination of the percentage of granulocytes producing intracellular superoxide anion due to the activation of cellular oxidases • The optical microscope determines the percentage of cells that contain blue intracellular formazan deposits • Blast transformation test of lymphocytes • Determine the number of blast cells formed in response to in vitro stimulation with a specific allergen

  48. Indications • Diagnosis of immunodeficiencies primary secondary • Diagnosis of late-onset hypersensitivity • Evaluation of post-vaccination immune response

  49. MoAb humanized chimeric (have fragments from the mouse) recombinant generically called by suffix:       "Mab" - Mo Ab

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