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Serology Review 2012

Serology Review 2012. By: Joanna Ellis, (MLS)ASCP. Overview. Study your notes and labs from the spring. Make sure you know the information related to the objectives.

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Serology Review 2012

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  1. Serology Review2012 By: Joanna Ellis, (MLS)ASCP

  2. Overview Study your notes and labs from the spring. Make sure you know the information related to the objectives. The certification exam will test over knowledge of immunology and serology, so need to know immunological theories as well as testing specific for conditions. The Serology final will also test over this. This review is BRIEF and covers the high points only.

  3. Overview Only a few select serological tests are performed in clinical laboratories. Due to this fact serology is performed on campus to give everyone equal experience. A kit test is a kit test. As an MLT you must be able to: Read and accurately follow directions of package insert. State principle, sample requirements, limitations of the procedure and causes of false positives and negatives. Accurately perform test according to package insert or written instructions. Report out results in required format.

  4. Traits of Immunogens (Antigens) • Foreignness • Ex. Plant protein more immungenic than animal protein • Size • >10,000 daltons • Complexity • Heterogeneity in building blocks that comprise the antigen • Proteins>polysaccharides>>lipids and nucleic acids

  5. Haptens • Small molecule that, by themselves, are NOT immunogenic. • When coupled to a high molecular weight protein, the hapten becomes the antigenic determinant (epitope) for the antigen .

  6. Definitions • Chemotaxis • the migration of cells to an inflammatory site following interaction with chemoattractants or chemokines. • Ex. C5a of the complement system • Opsonization • The process by which plasma proteins are attached to a foreign substance and prepare it for phagocytosis • Ex. IgG • Epitope (antigenic determinant) • Small part of the immunogen that is recognized by B or T cells. • Can be as few as 6 amino acids

  7. More Definitions • Antibody • Please tell me you can define antibody by now. • Heterophile antigen • Antigens that appear on the surface of tissues of several different species • Heterophile antibody • Antibodies that are generated in response to an antigen in one species, but are cross reactive with an antigen in another species. • Example: Epstein-Barr virus antibody detected in Monospot test

  8. Antibody Structure

  9. IgG

  10. Classes of Antibodies – Know Structure and Function • IgG, IgD, and IgE: all monomers • IgG: Gamma heavy chains • IgD: Delta (Δ or δ) heavy chains • IgE: Epsilon (Ε or ε) heavy chains • IgA: Alpha -monomer or dimer • IgM: Mu - pentamer

  11. Comparison of Antibodies

  12. Innate versus Acquired (Adaptive) Immunity

  13. Innate versus Adaptive Mechanisms

  14. Factors that determine Immune Response • Dose • High dose: Tolerance or immunological paralysis • Intermediate: Best response • Low dose: little stimulus OR induces tolerance • Route • Enteric administration : preferred route for intestinal parasite immunogens—frequently induces B cell tolerance rather than an immune response • Parenteral administration • Intradermal and subcutaneous routes stimulate the immune cells of lymph nodes , faster and more pronounced immune response. • IV administration stimulates immune cells in spleen first, slower and less effective • Adjuvants enhance the immune response • Often oil and water mixture • Aluminum potassium sulfate is the only one approved for human use

  15. Cellular versus Humoral Immunity

  16. Humoral Immunity Exposure to antigen causes production of specific antibodies by B cells. Life long Two types of responses – must know length of time for antibody production and antibody class produced Primary – first exposure, takes weeks to months, IgM then IgG, slow Secondary – second exposure to same antigen, IgG, fast – days to weeks

  17. Primary versus Secondary Response

  18. Complement Purpose Promote the inflammatory response by opsonization which enhances susceptibility of coated cells to phagocytosis. Alter biological membranes to cause direct cell lysis. Three pathways: Classical – activation caused by IgM (1 molecule minimum) or IgG (2 molecules minimum in close proximity) - know recognition, activation and membrane attack units. Alternative (properdin) - occurs independently of antibody, must know triggers Lectin - begins when mannan-binding protein (MBP) binds to the mannose groups of microbial carbohydrates. Must know the activation sequence of each Note: complement is destroyed by HEAT. If complement interferes with serological testing, it can be denatured by heating serum to 56C for 30 minutes.

  19. Classical Pathway of Complement Activation

  20. Specificity versus Sensitivity • Specificity: How true is the test result? • Ability of a test to correctly EXCLUDE individuals who do NOT have the given disease/condition • How likely is the test to detect the absence of a characteristic in someone without the characteristic? • Sensitivity: How small of an amount can be detected? • Ability of a test to correctly identify individuals who HAVE a given disease/condition • How likely is the test to detect the presence of a characteristic in someone with the characteristic?

  21. Principle of Serological Tests Cause a reaction between antigen and antibody to produce a DETECTABLE reaction. One must be UNKNOWN. Look for antigen OR antibody. An antibody MAY be the antigen in the test system, i.e., RF is an IgM class antibody directed against IgG. In this test latex particles coated with IgG antibody, looking for antigen which in this case is the IgM RF present. MANY different test systems developed.

  22. Factors affecting Ag/Ab Reactivity

  23. Basic Immunologic Procedures Measurement by Light Precipitation Electrophoretic Techniques Agglutination Reactions – still popular Labeled reactions – very popular Molecular techniques – gaining in popularity especially for pathogens.

  24. Nephelometry • Measures turbidity of sample by passing light thru it, amount of light scatter is measured. • Two types: • Endpoint – reaction goes to completion • Kinetic – light scatter measured at specific time. Reaction occurs at a steady rate and the timing of measurement can be done.

  25. Precipitation • Involves combining soluble antigen with soluble antibody to produce visible, INSOLUBLE complexes. • Relative concentrations of antigen and antibody must be equal

  26. Liquid Precipitation

  27. Gel Precipitation: Radial Immunodiffusion (RID)

  28. Double Gel Diffusion : Ouchterlony

  29. Capsular Precipitation • Swelling of the capsule surrounding a bacterium as a result of interaction with anticapsular antibody, consequently the capsule becomes more refractile and conspicuous.

  30. Electrophoresis • Process of separating proteins in a mixture utilizing their different net electrical charges • Size and shape can cause frictional drag • Types • Moving Boundary • Disc • Capillary zone • Immunoelectrophoresis

  31. Rocket Immunoelectrophoresis • Adaptation of radial immunodiffusion (RID). • Antibody incorporated (mixed) into the gel. • Antigen added to wells. • Apply electrical current and antigen will move forward and will bind to antigen. • Dissolution and reformation occurs. • Height of precipitin band related to concentration of antigen. • Much faster than RID

  32. Hybridoma • Where we get our monoclonal antibodies • A cell hybrid produced in vitro by the fusion of a lymphocyte that produces antibodies and a myeloma tumor cell. It proliferates into clones that produce a continuous supply of a specific antibody.

  33. Agglutination Occurs in two stages: Sensitization – cannot be seen Lattice formation – visible Antigen or antibody can be coated onto or an integral part of a carrier particle: Latex particles Red blood cells--HEMAGGLUTINATION Charcoal Bacteria Agglutination indicates presence of substance being tested for.

  34. Labeled Reactions One of the reactants labeled with a tag: I125 – measure radioactivity Enzyme – color or intensity of color measured. Measure intensity of light emitted as a result of reaction. Fluorescence Review lecture guide for specifics.

  35. Molecular Rapidly exploding field. Polymerase Chain Reaction (PCR) allows replication of genetic material specific to an infectious agent or malignancy. Probe is prepared which has target sequence. Use thermocycler to cause DNA to denature (separate) then cool to cause annealing to probe. Amplify the specific target. Becoming EXTREMELY popular. Review lecture notes for specifics.

  36. C-Reactive Protein • Non-specific protein that appears in serum as a response to inflammatory conditions • Involved in opsonization and complement fixation • Latex agglutination available • Coated with Anti-CRP • Conditions elevated • Bacterial infection • Viral infection • Active rheumatic fever • Active rheumatoid arthritis • TB infections • Malignancies • Following surgeries

  37. C-Reactive Protein • ESR also used to gauge inflammation. • C-RP has the following advantages over the ESR: • Rises quickly DURING inflammation. • Decreases quickly once inflammation resolved. • Not affected by anemia or abnormal serum proteins.

  38. Syphilis • Caused by spirochete Treponema pallidum • Early: chancre: 90% seropositive within 3 weeks • Secondary: 6-8 weeks 100% seropositive • Latent: seropositive, symptoms absent • Tertiary: Years later, new lesions any body part

  39. Syphilis Lab Tests • Direct Microscopic • Darkfield • Fluorescent Antibody • Nontreponemal: detect antibody to cardiolipin called reagin • VDRL: flocculation, prone to false positives • Antigen composed of cardiolipin, cholesterol, lecithin • RPR: modified VDRL with charcoal particles, more sensitive • Trust: similar to RPR • Treponemal: detect antibody to the spirochete or spirochete itself • FTA-ABS: confirmatory test that uses slides fixed with Nichols strain of T. pallidum that will react with patient antibody • EIA: not as sensitive as FTA • DNA probe

  40. Syphilis Testing REMEMBER if a non-treponemal antibody screening test is positive MUST do specific treponemal antibody test. RPR CANNOT be performed on CSF or cord blood. VDRL can be performed on CSF. Review lecture notes for causes of false positive and false negative reagin tests.

  41. Syphilis Testing VDRL Must heat serum to 56C for 30 minutes prior to testing to inactivate complement which can cause a false positive. Antigen must be prepared daily. Test read microscopically. RPR Modified commercially prepared antigen attached to charcoal. Serum does not have to be heated. Plasma can be used. Read macroscopically.

  42. Lyme Disease • Causative agent: Borrelia burgdorferi • Transmitted by Ixodes scapularis • Bull’s eye rash

  43. Lyme Disease Stages • Stage 1 • Flu-like symptoms • Rash at bite (maybe) • Fatigue • Headache • Stage 2 • Paralysis of face/neurologic problems • Carditis/arrythmia • Meningits • Stage 3 • Chronic joint and muscle pain • Restless sleep

  44. Lyme Disease Laboratory Testing • Immunofluorescence assay (IFA) • B. burgdorferi antigen on slide • Cross-reactivity possible • Enzyme immunoassay (EIA) • Sonicated B. burgdorferi • Western Blot most common confirmatory test • PCR

  45. Group A Streptococcus

  46. Streptococcal Infections • Causative agent Streptococcus pyogenes. • Organism found only in man. • Leading cause of oropharyngitis which may lead to serious complications (sequelae) • Rheumatic fever • Acute glomerulonephritis • Culture and rapid screening tests detect early infection. • Two major sites of infection • Upper respiratory tract - oropharyngitis • Skin - Pyoderma or Impetigo

  47. Group A Lab Tests • Culture • Rapid Test • Detects antibodies to carbohydrate specific to Group A • PCR – DNA probes becoming popular in place of serology and culture • ASO • Antistreptolysin O detected suggests recent infection • Anti-Dnase B

  48. Cold Agglutinins • Transient Ab that appears in patients with Mycoplasma pneumoniae • Anti-I specificity • Reacts preferentially at 4C, agglutination reversed if heated at 37C. • Must run RBC control • Fourfold or more rise in titer is significant

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