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.
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.
light-colored antibody molecule represents the anti-globulin reagent that
binds with the Fc portion of the IgG antibody attached to the red blood
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
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
There are two types of antiglobulin tests:
- Direct Antiglobulin Test (DAT)
antibodies or complement coating patient's cells in vivo.
- Indirect Antiglobulin
Test (IAT) - Uses a 37oC 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.
Production Methods of Anti-Human globulin (AHG or
- 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
Polyspecific Anti-human Globulin: blend of Anti-IgG
& Anti-C3b, -C3d
Monospecific reagents: Anti-IgG alone or
Note: Reagent does not contain antibodies to IgM.
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
- Only antibody attached to the cells remain
Add Anti-Human Globulin
Visible Agglutination in the test
Grade the reaction strength
Summary of the reaction:
- Antigen-antibody reaction, which can take place
either in vivo or in vitro
- Cells coated with IgG antibody and/or complement
- Cells washed 3-4X to remove unbound or free
antibody or complement
- The only antibody or complement left is attached
to red cells
- AHG (Coombs serum) added
- Antibodies in Coombs serum react with antibodies
or complement on red cells, causing agglutination
- If no agglutination add Coombs control reagent cells*
*Coombs control reagent cells will be discussed under
False Negative Reactions.
Negative Antiglobulin Test
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.
- NO antigen-antibody reaction occurred.
- No attachment of antibody or complement to
- Cells washed three to four times = all
plasma or serum antibodies was washed away.
- Anti-human globulin, Coombs, serum added, which
would react with antibody-coated cells if present.
- But no agglutination, because no antibodies or
complement on red cells for the anti-human globulin, Coombs, serum to react with
- Must add Coombs Control Check Cells to negative
- 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
False Positives and Negatives
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
- 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
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,
- Spontaneous agglutination (cells heavily coated
- Non-specific agglutination ("sticky
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
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
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.
- Add 1 drop of patient cells from EDTA tube
- Wash these drops of blood 3-4X to remove
plasma antibodies and make a 3% cell suspension.
- Add a drop of 3% cell suspension to a clean, labeled
- Add drop of Polyspecific AHG (Coombs serum) to
- 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.
- We want to make the test as sensitive as possible,
so allow all negatives to incubate 5 minutes to enhance complement
- Read all negatives microscopically to detect weak
- False pos. possible if red top tube used to
- 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.
- 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)
- Recent transfusion history of both red cell and
- Other lab values that may indicate red cell
destruction (hematocrit, bilirubin, LDH)
Clinical Causes of Positive DAT
- Normal patient with unexplainable reasons for a
- 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.
- 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
- 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.
- 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.
- 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
- 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.
- 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
- 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..
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
- Incubate cells with serum at 37oC for the
recommended time. (Usually 15 to 30 minutes.)
- After incubation wash the
cells three to four times.
- Add AHG, Coombs reagent, centrifuge and
read for agglutination.
- If the test is negative, add Coombs Control
Check Cells to check for false negatives.
Screening Serum for Unexpected Antibodies Procedure
- 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
- Screening Cells have known antigens for the common
clinically significant antibodies.
(attach screening cell sheet)
- If there is agglutination after Coombs step with
either (or both) Screening Cells, patient has an unexpected antibody.
- 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
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.
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+.
|Should be at least
||Should be Negative
||Should be Negative
||May be Positive (2+)
OBJECTIVES - ANTIGLOBULIN TESTING
- Explain the principle of the antiglobulin test.
- Explain the difference between the direct antiglobulin test and the indirect antiglobulin test.
- Explain how Coombs reagents are produced.
- Explain the role of Coombs control cells in antiglobulin testing.
- Explain the mode of action of the Coombs control
- Name five reasons for a false negative antiglobulin test.
- Name two reasons for a false positive antiglobulin test
- Explain why a lavender top tube is best for DAT
- Describe the differences in the procedure between
testing for IgG on the cells and testing for complement on the cells.
- Name two reasons for a false positive DAT, and
explain why each would produce a false positive result.
- List six clinical situations in which the DAT
would be positive, and explain why each would cause the positive DAT
- List the patient information that should be
obtained if a positive DAT result is obtained on his or her sample
- Describe the basic procedure for indirect antiglobulin testing
- List three applications of the indirect antiglobulin test to detect red cell antigens
- List three applications of the indirect antiglobulin test to detect serum antibodies
- State the controls used in antigen typing and
explain the purpose of each
- Correctly perform and interpret the DAT, using
good washing technique.
- Use monospecific reagents appropriately, and
correctly interpret results.
- Use Coombs control cells appropriately.
- Correctly perform and interpret serum antibody
- Correctly perform and interpret red cell antigen typings.
- Correctly select and use controls when performing
red cell antigen typing.