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Immunologic Laboratory Tests

Immunologic Laboratory Tests. Kristine Krafts, M.D. Immunologic Lab Tests Outline. Agglutination reactions DAT IAT Immunofluorescence ELISA Western blot Flow cytometry. Things to Remember About Each Test. What does it measure? Where does the Ag-Ab interaction occur?

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Immunologic Laboratory Tests

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  1. Immunologic Laboratory Tests Kristine Krafts, M.D.

  2. Immunologic Lab Tests Outline • Agglutination reactions • DAT • IAT • Immunofluorescence • ELISA • Western blot • Flow cytometry

  3. Things to Remember About Each Test • What does it measure? • Where does the Ag-Ab interaction occur? • How is the Ag-Ab complex detected?

  4. Immunologic Lab Tests Outline • Agglutination reactions

  5. Agglutination Reactions: Purpose • Detection of Ag or Ab in patient specimen • Examples: • testing for antibodies to infectious agents • testing for Hemophilus influenzae type B capsular antigen in CSF

  6. Agglutination Reactions: Method • Use particles coated with Ag or Ab • Add patient’s serum (containing Ab or Ag) • See if particles clump

  7. Agglutination Reactions: Interpretation Clumping = patient has the antibody (or antigen)

  8. negative positive

  9. Immunologic Lab Tests Outline • Agglutination reactions • DAT

  10. DAT: Purpose • Detection of Ab (or complement) on patient’s red cells • Also called the direct Coombs Test • Performed in patients with hemolytic anemia

  11. DAT: Method • Use patient’s red cells (coated with Ab) • Add anti-human globulin (AHG) (Coombs reagent) • Look for agglutination

  12. patient red cells + AHG = agglutination

  13. DAT: Interpretation Clumping = patient red cells are coated with antibody and/or complement

  14. Immunologic Lab Tests Outline • Agglutination reactions • DAT • IAT

  15. IAT: Purpose • Detection of antibodies to red cell antigens • Also called the indirect Coombs Test • Performed as part of pre-transfusion testing • antibody screen • cross-match

  16. IAT: Method • Use patient serum (containing Ab) • Add donor RBCs (coated with Ag) • Add anti-human globulin (Coombs reagent) • Look for agglutination

  17. patient Ab donor RBC Ab-coated donor RBC + = + AHG agglutination =

  18. ANTIBODY SCREENING patient serum (without red cell Ab) patient serum (with red cell Ab) reagent RBC (with red cell Ag) reagent RBC (with red cell Ag) AHG AHG no agglutination (negative test) agglutination (positive test)

  19. IAT: Interpretation Clumping = patient has an antibody to the donor (or reagent) red cells

  20. Immunologic Lab Tests Outline • Agglutination reactions • DAT • IAT • Immunofluorescence

  21. Immunofluorescence: Purpose • Detection of a specific antigen or antibody in a histologic specimen • Examples: • detection of bacterial organisms • detection of antigen-antibody complexes

  22. Immunofluorescence: Methods • Fix specimen on slide • Add antibodyspecific for the desired antigen • Look for fluorescence Direct Indirect • Fix specimen on slide • Add antibody specific for the desired antigen • Add second antibody • Look for fluorescence

  23. Immunofluorescence: Interpretation Fluorescence = patient has the antigen

  24. Immunologic Lab Tests Outline • Agglutination reactions • DAT • IAT • Immunofluorescence • ELISA

  25. ELISA: Purpose • Detection of antibodies in patient specimen • Examples: • home pregnancy tests • HIV tests • tests for some coagulation factors, cytokines, and autoantibodies

  26. ELISA: Method • Add patient specimen to well coated with ligand • Add AHG with enzyme attached • Add substrate • Measure color change

  27. ELISA: Interpretation Color change = patient has the antibody

  28. ELISA: Variations Sandwich immunoassay • detects antigen (not antibody) • coat well with antibody • rest is like ELISA Radioimmunoassay • detects antibody or antigen • detector is a radioactive substance • otherwise like ELISA or sandwich immunoassay

  29. Immunologic Lab Tests Outline • Agglutination reactions • DAT • IAT • Immunofluorescence • ELISA • Western blot

  30. Western Blot: Purpose • Detection of antibodies in patient specimen • Most common example: HIV test

  31. Western Blot: Method • Make a protein suspension of the target of the antibody 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 bands

  32. Western Blot: Interpretation Bands on strip = patient has antibodies to corresponding proteins

  33. Enough bands = patient is “positive”

  34. Immunologic Lab Tests Outline • Agglutination reactions • DAT • IAT • Immunofluorescence • ELISA • Western blot • Flow cytometry

  35. Flow Cytometry: Purpose • Characterization of cell size, complexity, antigens • Examples: • diagnosis of leukemia and lymphoma • determination of CD4/CD8 counts in patients with HIV

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