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Immunologic laboratory-tests
- 2. • Agglutination reactions • DAT • IAT • Immunofluorescence • ELISA • Western blot • Flow cytometry Immunologic Lab Tests Outline
- 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. • Agglutination reactions Immunologic Lab Tests Outline
- 5. • Detection of Ag or Ab in patient specimen • Examples: • blood typing • testing for antibodies to infectious agents • testing for Hemophilus influenzae type B capsular antigen in CSF Agglutination Reactions: Purpose
- 6. • Use particles coated with Ag or Ab • Add patient’s serum (containing Ab or Ag) • See if particles clump Agglutination Reactions: Method
- 9. Clumping = patient has the antibody (or antigen) Agglutination Reactions: Interpretation
- 12. • Agglutination reactions • DAT Immunologic Lab Tests Outline
- 13. • Detection of Ab (or C’) on patient’s red cells • Also called the direct Coombs Test • Performed in patients with hemolytic anemia DAT: Purpose
- 14. • Use patient’s red cells (coated with Ab) • Add anti-human globulin (AHG) (Coombs reagent) • Look for agglutination DAT: Method
- 15. patient red cells + AHG = agglutination
- 16. Clumping = patient red cells are coated with antibody and/or complement DAT: Interpretation
- 17. • Agglutination reactions • DAT • IAT Immunologic Lab Tests Outline
- 18. • Detection of antibodies to red cell antigens • Also called the indirect Coombs Test • Performed as part of pre-transfusion testing • antibody screen • cross-match IAT: Purpose
- 19. • Use patient serum (containing Ab) • Add donor RBCs (coated with Ag) • Add anti-human globulin (Coombs reagent) • Look for agglutination IAT: Method
- 20. patient Ab + AHG = agglutination donor RBC = Ab-coated donor RBC +
- 21. patient serum (without red cell Ab) AHG reagent RBC (with red cell Ag) patient serum (with red cell Ab) AHG reagent RBC (with red cell Ag) ANTIBODY SCREENING no agglutination (negative test) agglutination (positive test)
- 22. Clumping = patient has an antibody to the donor (or reagent) red cells IAT: Interpretation
- 23. • Agglutination reactions • DAT • IAT • Immunofluorescence Immunologic Lab Tests Outline
- 24. • Detection of a specific antigen in a specimen • Examples: • detection of bacterial organisms • detection of antigen-antibody complexes Immunofluorescence: Purpose
- 25. • Fix specimen on slide • Add antibody specific for the desired antigen • Look for fluorescence • Fix specimen on slide • Add antibody specific for the desired antigen • Add second antibody • Look for fluorescence Direct Indirect Immunofluorescence: Methods
- 31. Fluorescence = patient has the antigen Immunofluorescence: Interpretation
- 32. • Agglutination reactions • DAT • IAT • Immunofluorescence • ELISA Immunologic Lab Tests Outline
- 33. • Detection of antibodies in patient specimen • Examples: • home pregnancy tests • HIV tests • tests for some coagulation factors, cytokines, and autoantibodies ELISA: Purpose
- 34. • Add patient specimen to well coated with ligand • Add AHG with enzyme attached • Add substrate • Measure color change ELISA: Method
- 36. Color change = patient has the antibody ELISA: Interpretation
- 37. 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 ELISA: Variations
- 39. • Agglutination reactions • DAT • IAT • Immunofluorescence • ELISA • Western blot Immunologic Lab Tests Outline
- 40. • Detection of antibodies in patient specimen • Most common example: HIV test Western Blot: Purpose
- 41. • 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 Western Blot: Method
- 44. Bands on strip = patient has antibodies to corresponding proteins Western Blot: Interpretation
- 45. Enough bands = patient is “positive”
- 46. • Agglutination reactions • DAT • IAT • Immunofluorescence • ELISA • Western blot • Flow cytometry Immunologic Lab Tests Outline
- 47. • Characterization of cell size, complexity, antigens • Examples: • diagnosis of leukemia and lymphoma • determination of CD4/CD8 counts in patients with HIV Flow Cytometry: Purpose
- 59. Complicated! Combine size, complexity and antigen expression data to come up with meaningful description of cells. Flow Cytometry: Interpretation