When can you use an antibody to find another antibody

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

2. 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. The antiglobulin test was first described in 1908, but wasn�t applied to human testing until 1945 by Coombs, Mourant, and Race. Prior to this, only directly agglutinating (IgM) antibodies could be detected. The antiglobulin test (AGT) soon lead to the detection and definition of many previously unrecognized red cell antigen systems, the first of which was the Kell system. The antiglobulin test was first described in 1908, but wasn�t applied to human testing until 1945 by Coombs, Mourant, and Race. Prior to this, only directly agglutinating (IgM) antibodies could be detected. The antiglobulin test (AGT) soon lead to the detection and definition of many previously unrecognized red cell antigen systems, the first of which was the Kell system.

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 Traditionally, anti-human globulin was produced by injecting human globulins into rabbits. The rabbits produced antibodies to the human globulins (anti-human globulin). These antibodies were harvested and purified. They are the active ingredient in the AHG reagent. This is an example of a heterophile or xenophile antibody. Today, monoclonal techniques are used to produce most of the AHG reagent available. Traditionally, anti-human globulin was produced by injecting human globulins into rabbits. The rabbits produced antibodies to the human globulins (anti-human globulin). These antibodies were harvested and purified. They are the active ingredient in the AHG reagent. This is an example of a heterophile or xenophile antibody. Today, monoclonal techniques are used to produce most of the AHG reagent available.

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. In other words, polyspecific Coombs serum contains anti-IgG and anti-C3 from a non-human source. Polyspecific AHG is also called polyvalent or broad spectrum AHG. Anti-IgM and anti-IgA AHG are also available, but not routinely used. In other words, polyspecific Coombs serum contains anti-IgG and anti-C3 from a non-human source. Polyspecific AHG is also called polyvalent or broad spectrum AHG. Anti-IgM and anti-IgA AHG are also available, but not routinely used.

13. AHG Action AHG combines with the Fc portion of a sensitizing antibody. This completes the antigen-antibody bridge, allowing agglutination to occur. The specificity of the antibodies in the AHG reagent are to either the Fc portion of an immunoglobulin molecule (usually IgG) or C3. The specificity of the antibodies in the AHG reagent are to either the Fc portion of an immunoglobulin molecule (usually IgG) or C3.

14. When used to detect clinically significant antibodies, AHG reagent MUST contain anti-IgG. Both monospecific anti-IgG and polyspecific AHG reagent would be acceptable for this purpose.Both monospecific anti-IgG and polyspecific AHG reagent would be acceptable for this purpose.

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!!! A negative test with Coombs Control cells invalidates the antiglobulin test. The test would need to be completely repeated.A negative test with Coombs Control cells invalidates the antiglobulin test. The test would need to be completely repeated.

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.

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. Many labs use AHG reagent that has been tinted green to help assure the tech that AHG reagent has been added. The most common error made when performing the antiglobulin test is inadequate washing. Any unbound antibodies may neutralize the AHG reagent once it has been added to the test tube. As few as 1:4000 unbound globulins can neutralize AHG. Many labs use AHG reagent that has been tinted green to help assure the tech that AHG reagent has been added. The most common error made when performing the antiglobulin test is inadequate washing. Any unbound antibodies may neutralize the AHG reagent once it has been added to the test tube. As few as 1:4000 unbound globulins can neutralize AHG.

18. Procedures Direct Antiglobulin Test (DAT) Detects antibody (or complement) sensitizing red cells In vivo sensitization Uses patient�s cells AKA Direct Coombs Test. Red cells that became coated with antibodies in the body are detected by the DAT. AKA Direct Coombs Test. Red cells that became coated with antibodies in the body are detected by the DAT.

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). AKA Indirect Coombs Test. In the IAT procedure, the red cells become coated with antibody during the incubation step of the procedure. The IAT uses the patient�s red cells when looking for antigens on those cells. Reagent red cells are used when looking for antibodies in the patient�s plasma. AKA Indirect Coombs Test. In the IAT procedure, the red cells become coated with antibody during the incubation step of the procedure. The IAT uses the patient�s red cells when looking for antigens on those cells. Reagent red cells are used when looking for antibodies in the patient�s plasma.

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). Patient�s red cells are at a 2-5% cell suspension in saline. Patient�s red cells are at a 2-5% cell suspension in saline.

22. DAT Procedure (tube method)

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 When investigating a positive DAT, it is important to obtain a thorough history of the patient�s transfusions, transplants, pregnancies, medications and diagnosis.When investigating a positive DAT, it is important to obtain a thorough history of the patient�s transfusions, transplants, pregnancies, medications and diagnosis.

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.

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.

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.

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. The three serologic categories of HDFN are: ABO; Rh (which refers to anti-D only); �Other� (all other IgG antibodies including the other Rh antibodies). ABO HDFN occurs in Group O mothers who have naturally occurring anti-A, anti-B, and anti-AB that are IgG antibodies. Rh and �other� HDFN requires that the mother have a previous pregnancy, transfusion or transplant that stimulated IgG antibody production. IgG is the only antibody class that can cross the placenta. The fetus must have inherited the target antigen from the father, and can only be heterozygous for the antigen. (Mother could not have passed along the gene for the antigen�She must be antigen negative in order to see the antigen as foreign and form the antibody.)The three serologic categories of HDFN are: ABO; Rh (which refers to anti-D only); �Other� (all other IgG antibodies including the other Rh antibodies). ABO HDFN occurs in Group O mothers who have naturally occurring anti-A, anti-B, and anti-AB that are IgG antibodies. Rh and �other� HDFN requires that the mother have a previous pregnancy, transfusion or transplant that stimulated IgG antibody production. IgG is the only antibody class that can cross the placenta. The fetus must have inherited the target antigen from the father, and can only be heterozygous for the antigen. (Mother could not have passed along the gene for the antigen�She must be antigen negative in order to see the antigen as foreign and form the antibody.)

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 Autoantibody � the mechanism that recognizes foreign antigen goes �haywire�. The body sees self antigens as foreign and produces antibodies to the self antigens. May be primary (idiopathic) or secondary to other diseases such as CLL, lupus, viral infections, etc. PCH is associated with anti-P, CHD is associated with anti-I, and WAIHA is associated with anti-Rh antibody, most often anti-e.Autoantibody � the mechanism that recognizes foreign antigen goes �haywire�. The body sees self antigens as foreign and produces antibodies to the self antigens. May be primary (idiopathic) or secondary to other diseases such as CLL, lupus, viral infections, etc. PCH is associated with anti-P, CHD is associated with anti-I, and WAIHA is associated with anti-Rh antibody, most often anti-e.

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. Red cells are at a 2-5% cell suspension in saline.Red cells are at a 2-5% cell suspension in saline.

39. Indirect Antiglobulin Test Tube Method

40. Indirect Antiglobulin Test

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 The antiglobulin crossmatch (AGXM) gives added assurance that the antibodies in the patient�s serum will not react with antigens on the donor red cells.The antiglobulin crossmatch (AGXM) gives added assurance that the antibodies in the patient�s serum will not react with antigens on the donor red cells.

45. Applications Using Patient�s Cells 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!

46. Factors affecting the IAT Serum/Cell ratio Incubation temperature pH Length of incubation Test environment (enhancement media) Serum/cell ratio is most often 2 drops serum to 1 drop cells. Too weak of a cell suspension (too few antigens) = prozone. Too heavy of a cell suspension (too many antigens) = postzone Temperature 36-38oC (Needs to be at �body temperature� to induce sensitization) Neutral pH (6.5 to 7.5) Incubation time will depend on test environment. Incubate for too short or too long of time for the medium being used and agglutinates will either not have formed or will have formed and be falling apart. Saline environment 45-60 min inc. Potentiators may be added to decrease the incubation time (anywhere from 10 � 30 minutes).Serum/cell ratio is most often 2 drops serum to 1 drop cells. Too weak of a cell suspension (too few antigens) = prozone. Too heavy of a cell suspension (too many antigens) = postzone Temperature 36-38oC (Needs to be at �body temperature� to induce sensitization) Neutral pH (6.5 to 7.5) Incubation time will depend on test environment. Incubate for too short or too long of time for the medium being used and agglutinates will either not have formed or will have formed and be falling apart. Saline environment 45-60 min inc. Potentiators may be added to decrease the incubation time (anywhere from 10 � 30 minutes).

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 Also called enhancement media.Also called enhancement media.

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.

51. 22% Albumin High molecular weight protein Reduces the zeta potential by dispersing some of the cations surrounding each negatively charged red cell. Increases the dielectric constant, defined as a measure of ability to dissipate a charge.

52. LISS Low Ionic Strength Solution Made of NaCl, glycine and albumin Creates a low ionic environment Lowers the zeta potential Promotes antibody uptake by the red cells LISS lowers the zeta potential to promote agglutination, just as albumin does. However, LISS also works to decrease the shielding effect around antibodies and antigens, which increases the amount of antibody that sensitizes the red cell. This increases the likelihood of agglutination. LISS is the most commonly used enhancement media at this time. Not only can it be used with the tube method, but also with Gel and Solid Phase Adherence methods.LISS lowers the zeta potential to promote agglutination, just as albumin does. However, LISS also works to decrease the shielding effect around antibodies and antigens, which increases the amount of antibody that sensitizes the red cell. This increases the likelihood of agglutination. LISS is the most commonly used enhancement media at this time. Not only can it be used with the tube method, but also with Gel and Solid Phase Adherence methods.

53. PEG Polyethylene glycol in a low ionic strength medium. Removes water from the test system, thereby concentrating any antibody present. Antibody uptake is also increased. PEG is LISS with the addition of polyethylene glycol, so it has all the advantages of LISS. By concentrating the antibodies, PEG improves the chances of antibody encountering antigen. PEG is LISS with the addition of polyethylene glycol, so it has all the advantages of LISS. By concentrating the antibodies, PEG improves the chances of antibody encountering antigen.

54. PEG PEG can cause cellular aggregation, therefore, tests using PEG can not be centrifuged and evaluated following a 37oC incubation. Testing should proceed immediately to the wash phase, with a minimum of 4 washes performed. PEG is not the potentiator of choice when the patient has elevated proteins, such as in multiple myeloma. In these cases, LISS is the preferred enhancement.

55. Polybrene A positive polymer Will aggregate normal RBCs. Sodium citrate is then added to the test system. If an antibody is not present, the aggregates will disperse. If an antibody is present, the agglutination will persist. Polybrene is currently used mainly to resolve ABO discrepancies due to polyagglutination.

56. Enzymes Ficin, papain, bromelin and trypsin are commonly used in blood banking Proteolytic substances Modifies the red cell membrane by removing sialic acid residues, thereby reducing surface charge. Splits polypeptide chains, which further exposes some antigens. May enhance the hemolytic activity of some complement dependant antibodies.

57. Enzymes Enzymes diminish or destroy M,N, S, Fy, Xga. Enzymes enhance Rh, P, Le, Jk and I. Enzymes enhance the hemolytic activity of some complement binding antibodies such as Lea, Jka, Vel, PP1Pk, ABO.

58. Sources of Error in the Antiglobulin Test Adequate wash Centrifugation Problems with reagents/saline Problems reading reactions Wash � a minimum of 3 times, to remove unbound antibodies that could neutralize AHG reagent. Most common error in the AGT is inadequate wash. Centrifugation- throws cells together in closer contact, allowing agglutination to occur more readily. Too slow or too short of time, not enough contact. Too fast or too long of time, mechanical packing of cells (not agglutination due to antigen-antibody interaction) Reagents/saline � Neutralization, bacterial contamination Reading reactions � Debris often confused with microscopic positive reactions. If complement is present, may see hemolysis (positive reaction) Wash � a minimum of 3 times, to remove unbound antibodies that could neutralize AHG reagent. Most common error in the AGT is inadequate wash. Centrifugation- throws cells together in closer contact, allowing agglutination to occur more readily. Too slow or too short of time, not enough contact. Too fast or too long of time, mechanical packing of cells (not agglutination due to antigen-antibody interaction) Reagents/saline � Neutralization, bacterial contamination Reading reactions � Debris often confused with microscopic positive reactions. If complement is present, may see hemolysis (positive reaction)

59. The End