Immunologic laboratory-tests

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Immunologic laboratory-tests

  1. 1. Immunologic Laboratory Tests
  2. 2. • Agglutination reactions• DAT• IAT• Immunofluorescence• ELISA• Western blot• Flow cytometryImmunologic Lab Tests Outline
  3. 3. • What does it measure?• Where does the Ag-Ab interaction occur?• How is the Ag-Ab complex detected?Things to Remember About Each Test
  4. 4. • Agglutination reactionsImmunologic Lab Tests Outline
  5. 5. • Detection of Ag or Ab in patient specimen• Examples:• blood typing• testing for antibodies to infectious agents• testing for Hemophilus influenzae type B capsularantigen in CSFAgglutination Reactions: Purpose
  6. 6. • Use particles coated with Ag or Ab• Add patient’s serum (containing Ab or Ag)• See if particles clumpAgglutination Reactions: Method
  7. 7. Clumping = patient has the antibody (or antigen)Agglutination Reactions: Interpretation
  8. 8. negative positive
  9. 9. • Agglutination reactions• DATImmunologic Lab Tests Outline
  10. 10. • Detection of Ab (or C’) on patient’s red cells• Also called the direct Coombs Test• Performed in patients with hemolytic anemiaDAT: Purpose
  11. 11. • Use patient’s red cells (coated with Ab)• Add anti-human globulin (AHG) (Coombs reagent)• Look for agglutinationDAT: Method
  12. 12. patient red cells + AHG = agglutination
  13. 13. Clumping = patient red cells are coated withantibody and/or complementDAT: Interpretation
  14. 14. • Agglutination reactions• DAT• IATImmunologic Lab Tests Outline
  15. 15. • Detection of antibodies to red cell antigens• Also called the indirect Coombs Test• Performed as part of pre-transfusion testing• antibody screen• cross-matchIAT: Purpose
  16. 16. • Use patient serum (containing Ab)• Add donor RBCs (coated with Ag)• Add anti-human globulin (Coombs reagent)• Look for agglutinationIAT: Method
  17. 17. patientAb+ AHG = agglutinationdonorRBC=Ab-coateddonor RBC+
  18. 18. patient serum(without red cell Ab)AHGreagent RBC(with red cell Ag)patient serum(with red cell Ab)AHGreagent RBC(with red cell Ag)ANTIBODY SCREENINGno agglutination(negative test)agglutination(positive test)
  19. 19. Clumping = patient has an antibody to thedonor (or reagent) red cellsIAT: Interpretation
  20. 20. • Agglutination reactions• DAT• IAT• ImmunofluorescenceImmunologic Lab Tests Outline
  21. 21. • Detection of a specific antigen in a specimen• Examples:• detection of bacterial organisms• detection of antigen-antibody complexesImmunofluorescence: Purpose
  22. 22. • Fix specimen on slide• Add antibody specific forthe desired antigen• Look for fluorescence• Fix specimen on slide• Add antibody specific forthe desired antigen• Add second antibody• Look for fluorescenceDirect IndirectImmunofluorescence: Methods
  23. 23. Fluorescence = patient has the antigenImmunofluorescence: Interpretation
  24. 24. • Agglutination reactions• DAT• IAT• Immunofluorescence• ELISAImmunologic Lab Tests Outline
  25. 25. • Detection of antibodies in patient specimen• Examples:• home pregnancy tests• HIV tests• tests for some coagulation factors, cytokines,and autoantibodiesELISA: Purpose
  26. 26. • Add patient specimen to well coated with ligand• Add AHG with enzyme attached• Add substrate• Measure color changeELISA: Method
  27. 27. Color change = patient has the antibodyELISA: Interpretation
  28. 28. Sandwich immunoassay• detects antigen (not antibody)• coat well with antibody• rest is like ELISARadioimmunoassay• detects antibody or antigen• detector is a radioactive substance• otherwise like ELISA or sandwich immunoassayELISA: Variations
  29. 29. • Agglutination reactions• DAT• IAT• Immunofluorescence• ELISA• Western blotImmunologic Lab Tests Outline
  30. 30. • Detection of antibodies in patient specimen• Most common example: HIV testWestern Blot: Purpose
  31. 31. • Make a protein suspension of the target of theantibody you’re looking for (e.g., HIV)• Electrophorese the suspension onto a little gel strip• Apply the patient’s specimen (containing antibodies)to the strip• Add AHG that has an enzyme attached• Add substrate and look for bandsWestern Blot: Method
  32. 32. Bands on strip = patient has antibodies tocorresponding proteinsWestern Blot: Interpretation
  33. 33. Enough bands = patient is “positive”
  34. 34. • Agglutination reactions• DAT• IAT• Immunofluorescence• ELISA• Western blot• Flow cytometryImmunologic Lab Tests Outline
  35. 35. • Characterization of cell size, complexity, antigens• Examples:• diagnosis of leukemia and lymphoma• determination of CD4/CD8 counts in patientswith HIVFlow Cytometry: Purpose
  36. 36. Complicated! Combine size, complexity andantigen expression data to come up withmeaningful description of cells.Flow Cytometry: Interpretation

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