When can you use an antibody to find another antibody?

1. When can you use an antibody to find another antibody?

2. Immunohematology The Antiglobulin Test Basic Principle, Procedures, and Applications

3. Objectives • Discuss the preparation, contents and use of Anti-Human Globulin reagent (AHG). • Explain the principle of the antiglobulin reaction. • Name the control system for the antiglobulin test. • Interpret both positive and negative “check-cell” results.

4. Objectives • Differentiate between the direct and indirect antiglobulin procedures. • Discuss applications of the indirect and direct antiglobulin tests. • Evaluate the Direct Antiglobulin Test in the following situations: • HTR • HDFN • Hemolytic Anemias • Drugs

5. Objectives • Evaluate the significance of a positive Direct Antiglobulin Test in the following situations: • Antigen typing • Weak D testing • Autocontrol • Evaluate the Indirect Antiglobulin Test in the following situations: • Antibody detection • Antibody identification • Antiglobulin crossmatch • Antigen typing

6. Objectives • List at least four factors affecting the antigen-antibody reactions in the indirect antiglobulin test. • Discuss the action of potentiators. • Assess sources of error affecting the antiglobulin test.

7. The Acquired Immune Response • From our Immunology Review we know: • The body makes antibodies in response to foreign antigen. • These antibodies coat the foreign object leading to: • Clearance of the foreign antigen by the RES • Lysis of the foreign object via complement activation • IgG is the predominant antibody produced in most responses • Incomplete antibody • Usually not detected at room temperature/immediate spin phase of testing

8. The Antiglobulin Test • The purpose of the antiglobulin test is to detect cells that have become coated with antibodies &/or complement. • The test is also known as the Coombs test. Y Y Y

9. Anti-Human Globulin • The main reagent used in the antiglobulin test is anti-human globulin (AHG). • Also called Coombs serum. • Anti-human globulin (AHG) is an IgG antibody directed against human immunoglobulins or complement components.

10. AHG Production

11. AHG Production • The globulins that AHG may be directed against include: • IgG • IgM • IgA • C3

12. AHG Contents • Polyspecific AHG reagent contains antibodies to both IgG and C3. • Monospecific AHG contains antibodies to either IgG or C3.

13. AHG Action • AHG combines with the Fc portion of a sensitizing antibody. • This completes the antigen-antibody bridge, allowing agglutination to occur. Y Y Y

14. When used to detect clinically significant antibodies, AHG reagent MUST contain anti-IgG.

15. ANYTIME YOU ARE USING AHG REAGENT, AND GET A NEGATIVE TEST RESULT (by tube)--- YOU MUST ADD COOMBS CONTROL CELLS AND GET POSITIVE RESULTS!!! Control

16. Coombs Control Cells • Rh positive cells coated with anti-D antibodies or cells coated with the C3 portion of complement. • Coombs Control Cells will react with the antibody in the AHG reagent. Y D Y D D Y Y Y Y D D D Y

17. Coombs Control Cells will prove that… • Coombs reagent was added. • Coombs reagent was active. • The wash step was adequate to remove any unbound globulins. • The most common error made when performing the antiglobulin test is inadequate washing.

18. Procedures • Direct Antiglobulin Test (DAT) • Detects antibody (or complement) sensitizing red cells • In vivo sensitization • Uses patient’s cells

19. Procedures • Indirect Antiglobulin Test (IAT) • Antibody is free • Uses incubation at 37oC to force red cell sensitization in vitro. • May be used to detect antigens or antibodies. • Both DAT and IAT utilize Anti-Human Globulin reagent (AHG).

20. The Direct Antiglobulin Test Procedure

21. Steps to the DAT Procedure (tube method) 1. One drop of patient’s red cells are washed with 0.9% NaCl a minimum of 3 times to remove plasma that may contain unbound antibodies. 2. AHG reagent is added. 3. Tube is centrifuged. 4. If IgG or C3 is coating the cells, agglutination will occur (positive test). This will depend on the type of AHG reagent used i.e. if C3 is coating the cell, and monospecific anti-IgG AHG reagent is used, there will be NO agglutination. If neither is present there will be no agglutination (negative test). 5. Each negative test is validated (controlled) through the addition of Coombs Control Cells (also called check cells).

22. DAT Procedure (tube method) Pt ID √ Washed cell button

23. The Direct Antiglobulin Test Applications

24. What causes a cell to become coated with antibodies in vivo? • Hemolytic Transfusion Reaction • Hemolytic Disease of the Fetus and Newborn • Drugs • Disease

25. Hemolytic Transfusion Reaction • The patient has an antibody in the plasma that is directed against an antigen on the donor red cells. • The patient’s antibody coats the donor cells. Y Y Y Y Y Y Y Y Y

26. Hemolytic Transfusion Reaction • In an acute (immediate) hemolytic transfusion reaction, complement is activated. • The donor cells are lysed (intravascular hemolysis). • The DAT may be positive due to IgG or complement. • The DAT may be negative if all the donor cells are rapidly destroyed. Y Y Y Y Y Y Y Y Y

27. Hemolytic Transfusion Reaction • In a delayed hemolytic transfusion reaction, the antibody coated cells are removed via phagocytosis. • (extravascular lysis) • A drop in hemoglobin usually occurs 2-10 days following transfusion. • The DAT is usually positive due to IgG. Y Y Y

28. Hemolytic Transfusion Reaction • Other types of transfusion reactions share the symptoms of an acute HTR, including: • Febrile, nonhemolytic • Bacterial • TRALI • A positive DAT establishes the diagnosis of an acute hemolytic transfusion reaction.

29. Hemolytic Disease of the Fetus and Newborn (HDFN)

30. HDFN • Mother must have been stimulated to make an IgG antibody. • Antibody must cross the placenta. • Fetus must be antigen positive. • Fetal cells become coated with maternal antibody & are cleared by the fetal RES. Y Y Y Y

31. HDFN • In HDFN, the infant’s cells are coated with maternal IgG antibody, resulting in a positive DAT in the infant. • The DAT is THE diagnostic test for HDFN. • If the DAT is negative, the infant is not suffering from HDFN.

32. Drug-induced Hemolytic Anemia

33. Mechanisms • Drug Adsorption • Drug attaches to RBC. • Antibody directed at drug only • DAT positive due to IgG • Immune Complex • Antibody directed at a “neoantigen” having determinants on both the drug and the red cell membrane. • Antibody activates complement • DAT positive due to complement

34. Mechanisms • Induction of autoimmunity • Drug modifies red cell membrane resulting in production of auto antibody. • DAT positive due to IgG and occasionally complement. • Membrane Modification • Nonspecific adsorption of plasma proteins (Non-immunologic ) • DAT will be positive due to whatever has “stuck” to the membrane – IgG, complement, IgM, IgA.

35. Disease Autoimmune Hemolytic Anemia

36. Diseases • Various diseases may cause autoantibody to coat the patient’s red cells. • Cold Hemagglutinin Disease • DAT positive due to complement • Warm Auto Immune Hemolytic Anemia • DAT positive due to IgG and sometimes also due to complement • Paroxysmal Cold Hemoglobinuria • DAT positive due to complement

37. Indirect Antiglobulin Test Procedure

38. Steps to the IAT Procedure(tube method) • One or two drops of plasma or anti-serum containing antibody are added to a test tube. • One drop of red cells (antigen source) is added to the tube. • The tube is incubated at 37oC. The length of incubation is dependant on the medium. • Following incubation, the cells are washed with saline a minimum of 3 times, to remove any unbound antibody. • Following the final wash, two drops of AHG reagent are added to the dry cell button. The tube is centrifuged and results are read. The tube may be read microscopically, depending on the test medium. • Coombs control cells are added to each negative test. The tubes are centrifuged and results read.

39. Indirect Antiglobulin Test Tube Method 37C inc √

40. Indirect Antiglobulin Test • Antibodies sensitize the red cells during incubation at 37oC. • Following the wash step, AHG reagent is added. • AHG reagent completes the “bridge” between red cells, allowing for visible agglutination.

41. Indirect Antiglobulin Test Applications

42. Indirect Antiglobulin Test • Looking for in vitro cell sensitization. • Uses incubation at 37oC to allow antibody to sensitize red cell. • Uses AHG reagent to complete the “bridging” between red cells. • Visible agglutination as a positive endpoint. • Enhancement reagents may be added during incubation phase to increase sensitization and agglutination.

43. Applications Using Patient’s Serum • Antibody screen • Detects antibodies in patient’s serum • Uses reagent red cells as a source of known antigen • Antibody panel • Identifies antibodies • Uses reagent red cells as a source of known antigen

44. Applications Using Patient’s Serum • Antiglobulin crossmatch • Determines patient’s compatibility with donor • Uses donor red cells (antigens) and patient’s serum (antibodies) • Usually performed only when a patient has an antibody or a history of antibodies

45. Antigen Typing Weak D test Both use commercial anti-serum which contains antibodies, versus the patient’s cells (antigen). Auto control – Patient’s plasma vs. patient’s cells NOTE: If the patient has a positive DAT, the results of any IAT using the patient’s cells will be invalid. Cells are already coated with antibody before the incubation step! Applications Using Patient’s Cells

46. Factors affecting the IAT • Serum/Cell ratio • Incubation temperature • pH • Length of incubation • Test environment (enhancement media)

47. POTENTIATORS • Some incomplete antibodies will not react in a saline environment. • Potentiators are reagents that adjust the test environment. • Reduce the zeta potential • Promote agglutination • Enhance antibody uptake

48. Zeta Potential • RBCs have a negative surface charge, and attract cations such as Na+. • The electrical potential between the red cell surface and the outer ionic cloud surrounding it is the zeta potential.

49. Zeta Potential • By reducing the ionic cloud, the potentiator allows red cells to come closer together. - - - - - - - - - - - - - - ++++++++++++++ - - - - - - - - - - - ++++++++++++++ - - - - - - - - - - - - - - -

50. This promotes the formation of antibody bridges between cells which we call Agglutination.