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

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

3. 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

4. The Antiglobulin Test Y Y Y
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
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.

5. 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.

6. 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.

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

8. 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.

9. 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
The specificity of the antibodies in the AHG reagent are to either the Fc portion of an immunoglobulin molecule (usually IgG) or C3.

10. 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.

11. YOU MUST ADD COOMBS CONTROL CELLS AND GET POSITIVE RESULTS!!!
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.

12. Coombs Control Cells Y Y Y Y Y Y Y
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

13. 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.

14. 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.

15. 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.

16. The Direct Antiglobulin Test
Procedure

17. 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.

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

19. The Direct Antiglobulin Test
Applications

20. 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.

21. Indirect Antiglobulin Test
Procedure

22. Steps to the IAT Procedure (tube method)
One or two drops of serum (may contain an 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.

23. Indirect Antiglobulin Test Tube Method
37C inc √

24. 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.

25. Indirect Antiglobulin Test
Applications

26. 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.

27. 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

28. 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.

29. 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!

30. 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 min inc. Potentiators may be added to decrease the incubation time (anywhere from 10 – 30 minutes).

31. 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.

32. 22% Albumin
Polyethylene glycol (PEG) : a low ionic strength medium. Removes water from the test system, thereby concentrating any antibody present.
LISS
Enzymes

33. 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)