Life's Blood

ANTIGLOBULIN TESTING

The antiglobulin test, which is also referred to as the anti-human globulin test (AHG) or the Coombs test, is the cornerstone of detecting clinically significant unexpected antibodies that have coated cells either in vivo or in vitro.  For a historical perspective, see "The Discovery of the Anti-Globulin Test" written by A. E. Mourant pages 180 to 183 Vox Sang. 45: 180-83 (1983)

Principle of Antiglobulin Test

Red cells coated with complement or IgG antibodies do not agglutinate directly when centrifuged.  These cells are said to be sensitized with IgG or complement.

IgG-coated red blood cells

Complement-coated red blood cells

In order for agglutination to occur an additional antibody, which reacts with the Fc portion of the IgG antibody, or with the C3b or C3d component of complement, must be added to the system.

This will form a "bridge" between the antibodies or complement coating the red cells, causing agglutination.

The light-colored antibody molecule represents the anti-globulin reagent that binds with the Fc portion of the IgG antibody attached to the red blood cells.

The light-colored antibody molecule represents the anti-globulin reagent that binds with the complement attached to the red blood cells.

Traditionally rabbits were immunized with human gamma globulin to  make this antibody to IgG or C3d.

Types of Antiglobulin Tests

The original work done by Coombs and Mourant was detecting those antibodies, especially in the Rh system, that would cause hemolytic disease of the newborn, which we now classify as the Indirect Antiglobulin Test.  

There are two types of antiglobulin tests:

• Direct Antiglobulin Test (DAT) - Detects antibodies or complement coating patient's cells in vivo.
• Indirect Antiglobulin Test (IAT) - Uses a 37^oC incubation step so antibodies in serum can react with antigens on cells in vitro,  After washing the cells antiglobulin reagent is used to detect antibody coating of cells.

Reagents

Production Methods of Anti-Human globulin (AHG or Coombs) Reagent

• May be made by injecting rabbits with purified human IgG or C3, then harvesting the antibodies produced by the rabbit.
• Monoclonal technology may be used to make monoclonal antiglobulin reagent

Specificity types

Polyspecific Anti-human Globulin: blend of Anti-IgG & Anti-C3b, -C3d

Monospecific reagents: Anti-IgG alone or Anti-C3b,-C3d alone

Note:  Reagent does not contain antibodies to IgM.  Information about IgM coating of cells comes from the presence of C3 coating the cells since IgM is a strong complement activator.

Interpretation of Antiglobulin Tests

Whether the cells have been coated, or sensitized, in vivo or in vitro the final interpretation is based on the following

Positive Antiglobulin Test

	(Wash Bottle Image)	Wash cells three times to remove unbound antibody Only antibody attached to the cells remain
	(Anti-globulin Image)	Add Anti-Human Globulin
	Visible Agglutination in the test tube:  Grade the reaction strength

Summary of the reaction:

1.  Antigen-antibody reaction, which can take place either in vivo or in vitro
2. Cells coated with IgG antibody and/or complement
3. Cells washed 3-4X to remove unbound or free antibody or complement
4. The only antibody or complement left is attached to red cells
5. AHG (Coombs serum) added
6. Antibodies in Coombs serum react with antibodies or complement on red cells, causing agglutination
7. If no agglutination add Coombs control reagent cells* (CCC).

*Coombs control reagent cells will be discussed under False Negative Reactions.

Negative Antiglobulin Test

	Antibodies are not attached to the antigens during incubation.
Wash the cells 3 times to remove any unattached antibodies.
	Add Anti-human globulin
No visible agglutination and therefore a negative test
	Add Coombs Control Check Cells	Check cells agglutinated and original test cells remain unagglutinated.

Coombs Control Agglutinated by Anti-Human Globulin

Coombs Control Check Cells tell you if you did the test properly when you have a negative test.

1. NO antigen-antibody reaction occurred.
2. No attachment of antibody or complement to red cells
3. Cells washed three to four times  = all plasma or serum antibodies was washed away.
4. Anti-human globulin, Coombs, serum added, which would react with antibody-coated cells if present.
5. But no agglutination, because no antibodies or complement on red cells for the anti-human globulin, Coombs, serum to react with
6. Must add Coombs Control Check Cells to negative reactions

• CCC are cells coated with IgG antibody
• Will react with antibodies in Coombs serum still "floating around" in the tube.
• Agglutination will now result
• Agglutination following addition of CCC verifies negative result

False Positives and Negatives

 False-Negative Reactions

False-negative reactions can occur when antigen-antibody reactions have occurred but WASHING IS INADEQUATE and free antibody remains when the anti-human globulin is added.

• Anti-human globulin (Coombs) antibody prefers to react first with free antibody and then with antibody-coated cells
• If the free antibody has already reacted with the anti-human globulin, no free Coombs serum to react with Coombs Control Check Cells (CCC)
   

False negatives that are detected by negative Coombs control cells includes

• inadequate cell washing
• delay in adding antiglobulin reagent after the washing step
• presence of small fibrin clots among the cells
• inactive, or forgotten, antiglobulin reagent

Inadequate cell washing will lead to unbound antibody remaining in the red cell suspension that are available to neutralize the AHG (Coombs serum) so it will not react with red cells bound with antibody.

Delay in adding Coombs serum after washing step will lead to antibody eluting off, detaching from, cell while cells are sitting in saline.  Now free antibody present in the saline neutralizes the AHG, Coombs, serum so it will not be able to react with the cells bound with antibody.

Small fibrin clot among the cells that were not washed away will have immunoglobulins and complement present.  The antibodies and complement in the fibrin clot neutralizes AHG, Coombs, serum leading to a negative test.

Inactive AHG (Coombs serum) or the failure to add AHG (Coombs serum) will also be detected by a negative reaction when adding Coombs Control Check Cells.

There are also false negatives NOT detected by negative Coombs Control Cells that include:

• Too heavy cell suspension
• Delay during cell washing procedure, which can lead to antibody eluting off cells while they are sitting in saline and then the antibody is washed away during the remaining washes
• Improper centrifugation can either lead to lost of cells during the washing or the need to shake too hard during resuspension.

False positives

False positive reactions can also occurred when performing this test.  These would not be detected by the use of Coombs Control Check Cells.  Reasons for a false positive reaction could be the following:

• Using improper sample (clotted cells instead of EDTA for Direct Antiglobulin Test, DAT)
• Spontaneous agglutination (cells heavily coated with IgM)
• Non-specific agglutination ("sticky cells")

All of these reactions would be the result of cells appearing to agglutinate, or actually agglutinating.  Using a clotted tube for the DAT may allow complement to become activated in the test tube since calcium ions are free to be part of the complement cascade.

Direct Antiglobulin Testing

Principle

The Direct Antiglobulin Test detects in vivo coating of patient cells - either IgG antibodies, complement, or both.  Within the patient's blood stream antibodies attach to their specific antigens on the red blood cells.  This happens in Hemolytic Disease of the Newborn (HDN), in transfusion reactions, and in autoimmune hemolytic anemia.  Certain drugs are also known to activate complement and it can also coat the cells in vivo. 

When the blood is drawn the antibodies and/or complement have already attached to the red cells.  Those red cells from the EDTA tube will be washed 3 or more times and a 3% cell suspension is made. A drop of cell suspension and the anti-human globulin are mixed in a tube and then centrifuged.  If agglutination occurs, it indicates the patient has a positive Direct Antiglobulin Test due to antibody coating the cells in vivo.   If IgM antibodies involved, DAT will be identified by complement binding since the polyspecific antisera has both anti-IgG and anti-C3.  The meaning of a positive DAT is found under Clinical Causes of a Positive DAT.

Technique

1. Add 1  drop of patient cells from EDTA tube to tube
2. Wash these drops of blood 3-4X to remove plasma antibodies and make a 3% cell suspension.
3. Add a drop of 3% cell suspension to a clean, labeled tube.
4. Add drop of Polyspecific AHG (Coombs serum) to the tube.
5. If test is positive with polyspecific reagent, set up again using monospecific reagents to see if it is antibody or complement or both coating the cells.
6. We want to make the test as sensitive as possible, so allow all negatives to incubate 5 minutes to enhance complement coating.
7. Read all negatives microscopically to detect weak coating.
8. False pos. possible if red top tube used to collect sample.

• In-vitro complement coating frequently happens when sample clots or cools down due to weak cold-acting auto-antibodies like anti-I
• Prevent by using lavender top tube to tie up Ca+ and Mg+ ions and prevent complement activation in vitro.

8. Whenever positive DAT is obtained, obtain the following information on the patient:

• Diagnosis (particularly autoimmune hemolytic anemia, hemolytic disease of the newborn and transfusion reactions)
• Medications
• Recent transfusion history of both red cell and plasma components
• Other lab values that may indicate red cell destruction (hematocrit, bilirubin, LDH)

Clinical Causes of Positive DAT

1. Normal patient with unexplainable reasons for a positive DAT
    
2. Transfusion reaction work-ups require that a DAT be performed on the post-transfusion specimen since the patient's antibodies and/or complement may coat the transfused donor cells.  These reactions are usually a weak positive or mixed field agglutination since you are testing a mixed population of patient and donor cells.

3. Warm-acting Autoimmune disease, can lead to patient antibodies coating their own cells.  This  results in a strong positive result.  A cold-acting autoimmune hemolytic anemia would be due to IgM antibodies that in turn activate complement. The complement-coated cells would then be detected by the antiglobulin reagent.

4. Hemolytic disease of the newborn is due to the mother's IgG antibodies crossing the placenta and coating the antigens on the fetal red blood cells.  Cord blood collected at the time of birth would be tested, but may need to followed up by a heel stick of EDTA blood.  The reaction is usually a strong positive.
    
5. Complement on the red cells may be the result of antigen-antibody reactions which may t involve red cells.  Complement can also be activated if immune complexes are present in the plasma and the activated complement attaches to the red cells.  Complement can also become activated by the C3 by-pass mechanism and the lectin activation process.  Again once the activation of complement occurs in the blood stream, it can become attached to the red cells.
    
6. Passive transfer of antibody from donor units of plasma or platelets may attach to the patient's red cells since recipients are given ABO compatible blood but  other unexpected red cell antibodies may not have been detected.  These antibodies in donor plasma can coat antigens on patient cells when group AB, A, or B receive group O plasma products (and possibly platelets)
    
7. ABO mismatched transplants of particularly bone marrow can occur if an universal "O" donor bone marrow is given to an A, B, or AB recipient.  "Passenger lymphocytes" from group O donor organ make antibody to group AB, A, or B recipient cells and these in turn can activate complement.  It is also more common for "O" individuals to make an IgG anti-A,B, which would also contribute to a positive DAT.
    
8. Sensitization of red cells due to medications like penicillin and cephalosporins that usually involves non-specific coating of red cells.  Other drugs like tetracyclines, antihistamines and sulphonamides cause the development  of immune complexes that are capable of activating complement.  Some drugs, like ibuproten, levodopa and methyldopa, are also known to cause autoimmunity.  If a patient has a positive DAT, drug-induced problems should be considered.

Indirect Antiglobulin Testing

The indirect antiglobulin test is one of the most important and commonly used techniques in immunohematology.  It is used to commonly for the detection of:

• Weak D's in donor bloods and pregnant females of individuals who type D (-) at room temperature when doing ABO and Rh typing.
• The presence or absence of antigens on a person cells from particularly the Kell, Kidd, and Duffy Blood Group systems.
• Unexpected, clinically significant antibodies in the patient's serum during the antibody screening procedure and the antibody identification procedure..

Principle

The purpose of the indirect antiglobulin test is to detect In vitro sensitization of red cells.  This is done when sensitization does not lead to direct agglutination.  This occurs when there are too few antigens on the red cell, too few antibodies in the serum and those antibodies are in the IgG class.

Summary of the Indirect Antiglobulin Technique

1. Incubate cells with serum at 37^oC for the recommended time. (Usually 15 to 30 minutes.) 
   
2. After incubation wash the cells three to four times.  
   
3. Add AHG, Coombs reagent, centrifuge and read for agglutination.  
   
4. If the test is negative, add Coombs Control Check Cells to check for false negatives.

Uses:

Screening Serum for Unexpected Antibodies Procedure

1. Involves patient serum plus reagent red cells (Screening Cells) Duet I and II ® (attach photo of screening cells)

• The patient's serum potentially has unknown antibody.
   
• Screening Cells have known antigens for the common clinically significant antibodies. (attach screening cell sheet)

2. If there is agglutination after Coombs step with either (or both) Screening Cells, patient has an unexpected antibody.
    
3. If antibody screen positive, must do additional tests to specifically identify antibody

The uses for antibody screen are:

• Testing donor plasma to make sure no unexpected antibodies will be transfused to the recipient.
• Testing recipient serum before transfusion to make sure patient has no unexpected antibodies to react with donor cells.
• Testing maternal serum to make sure pregnant mother has no antibodies to react with fetal cells causing hemolytic disease of the newborn.

Red Cell Antigen Typing

Red cell antigen typing involves patient cells plus reagent antiserum.  The patient's cells are the unknown antigen and the reagent antiserum is the known antibody.  The antiglobulin technique is used for antigen typing for a weak D and a number of other clinically significant antibodies like the Kell, Kidd, and Duffy antibodies.  If there is agglutination after the addition of anti-human globulin, or Coombs step, patient cells had that specific antigen.

The specific procedure varies depending on what antigen is being tested for, and what brand of antiserum is being used.  Remember you must always read  and follow directions in product insert carefully

Uses for red cell antigen typing are:

• Typing donors for antigen if patient has antibody.  You would want units that are negative for that antigen.
• Verifying that patient is negative for antigen if he/she has made the antibody.
• Typing patient to see what antigens he/she lacks so can predict what antibodies he/she is capable of making if they seem to be particularly likely to make additional antibodies.

Controls

When performing red cell antigen testing always run known positive and negative controls.  This will verify that antiserum is acting properly and helps you interpret your test results.  The positive control should be heterozygous for the antigen to ensure antiserum is capable of detecting weaker antigens.  For example, when performing antigen typing for K, you would want a cell that is K+ and k+. 

POSITIVE CONTROL	NEGATIVE CONTROL	PATIENT CONTROL	PATIENT TEST
Heterozygous Positive cells	Cells without Antigen	Patient Cells	Patient cells
+	+	+	+
Reagent Antiserum	Reagent Antiserum	Rh control	Reagent. Antiserum
Should be at least 2+	Should be Negative	Should be Negative	May be Positive (2+) Negative or Mixed Field

OBJECTIVES - ANTIGLOBULIN TESTING

1. Explain the principle of the antiglobulin test.
2. Explain the difference between the direct antiglobulin test and the indirect antiglobulin test.
3. Explain how Coombs reagents are produced.
4. Explain the role of Coombs control cells in antiglobulin testing.
5. Explain the mode of action of the Coombs control cells.
6. Name five reasons for a false negative antiglobulin test.
7. Name two reasons for a false positive antiglobulin test
8. Explain why a lavender top tube is best for DAT testing.
9. Describe the differences in the procedure between testing for IgG on the cells and testing for complement on the cells.
10. Name two reasons for a false positive DAT, and explain why each would produce a false positive result.
11. List six clinical situations in which the DAT would be positive, and explain why each would cause the positive DAT
12. List the patient information that should be obtained if a positive DAT result is obtained on his or her sample
13. Describe the basic procedure for indirect antiglobulin testing
14. List three applications of the indirect antiglobulin test to detect red cell antigens
15. List three applications of the indirect antiglobulin test to detect serum antibodies
16. State the controls used in antigen typing and explain the purpose of each

Performance objectives:

1. Correctly perform and interpret the DAT, using good washing technique.
2. Use monospecific reagents appropriately, and correctly interpret results.
3. Use Coombs control cells appropriately.
4. Correctly perform and interpret serum antibody screens.
5. Correctly perform and interpret red cell antigen typings.
6. Correctly select and use controls when performing red cell antigen typing.

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