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

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: 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

10. negativepositive

11. Agglutination reactions DAT Immunologic Lab Tests Outline

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

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

14. patient red cells+AHG=agglutination

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

16. Agglutination reactions DAT IAT Immunologic Lab Tests Outline

17. 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

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

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

20. 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)

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

22. Agglutination reactions DAT IAT Immunofluorescence Immunologic Lab Tests Outline

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

24. 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 DirectIndirect Immunofluorescence: Methods

30. Fluorescence = patient has the antigen Immunofluorescence: Interpretation

31. Agglutination reactions DAT IAT Immunofluorescence ELISA Immunologic Lab Tests Outline

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

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

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

36. 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

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

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

40. 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

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

44. Enough bands = patient is “positive”

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

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

58. Complicated! Combine size, complexity and antigen expression data to come up with meaningful description of cells. Flow Cytometry: Interpretation