THE ANTIGLOBULIN

TEST
(coomb’s
direct test)
& indirect
GROUP 1
 THE ANTIGLOBULIN
TEST
Antiglobulin serum (Coombs’Serum) was discovered by Coombs
et.al. in 1945.
The antiglobulin test can be used to detect red cells sensitized with
IgG alloantibodies, IgG autoantibodies or complement components.
Sensitization of red cells can occur in vivo or vitro. The use of AHG
serum to detect sensitization of red cells in vitro is a two stage
technique known as indirect antiglobulin test (IAT). The sensitization
of red cells in vivo is detected by one stage technique the direct
antiglobulin test (DAT).
 principle
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.
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.
 ANTI-HUMAN GLOBULIN (AHG)
REAGENT
Preparation
• Anti-human globulin reagent is produced by immunizing
rabbits, goats or sheep with human serum or purified
add type antigen.
• Animals are bled after a specified period and the
reagent is purified by absorbing unwanted antibodies.
 types of AHG reagent
Polyspecific Antiglobulin Reagent
Contains antibodies to human IgG, C3 and C4 components
of the complement.
Monospecific Antiglobulin Reagent
 May be against any one of the human IgM, IgD, IgA or
complement component C3 or C4.
 Monoclonal AHG is now available against IgG or
complement components.
 DIRECT ANTIGLOBULIN TEST
(DAT)
• The direct antiglobulin test (DAT) detects sensitized red cells
with IgG and/or complement components C3b and C3d in
vivo.
• In vivo coating of red cells with IgG and/or complement may
occur in any immune mechanism is attacking the patient's
own RBC's.
• This mechanism could be autoimmunity, alloimmunity or a
drug-induced immune-mediated mechanism.
DIRECT ANTIGLOBULIN TEST
(DAT)
 blood sample
 it should be as fresh as possible not more than
24 hours old, otherwise,
 the sample should be taken in EDTA
(1.5 mg EDTA for I ml of blood)
 procedure of DAT
1. Prepare a 5% suspension in isotonic saline of the RBCs to be tested.
2. With clean pipette, add 1 drop of the prepared cell suspension to a
small tube.
3. Wash three times with NSS to remove all the traces of serum.
4. Decant completely after the last washing.
5. Add two drops of Anti-human serum;
6. Mix well and centrifuge for 1 minute at 1500 rpm.
7. Resuspend the cells by gentle agitation and examine macroscopically
and microscopically for agglutination.
 INDIRECT ANTIGLOBULIN TEST
Indications
(IAT)
The IAT is done to determine the presence of sensitization of
red cells with IgG and/or complement in vitro in the following
conditions.
1. Compatibility testing.
2. Screening and detection of unexpected antibodies in serum.
3. Determination of red cells phenotype K, Lea, Fya Fyb, Jka, Jkb
and sub-group of Rh etc by using known sera.
INDIRECT ANTIGLOBULIN TEST
(IAT)
 procedure of IAT
1. Label 3 test tubes as T (test serum), PC (positive control), and NC
(negative control).
2. In the test tube labelled as T = take two drops of test serum
3. In the test tube labelled as PC = take one drop of anti-D serum
4. In the test tube labelled as NC = take one drop of NSS
5. Add one drop of 5% saline suspension of the pooled “O” Rho (D)
positive cells in each test tube.
6. Incubate all the three tubes for one hour at 37⁰C.
7. Wash the cells three times in NSS to remove excess serum with no free
antibodies
 procedure of IAT
8. Add two drops of Coombs serum (Antihuman serum) to each tube
9. Keep for 5 minutes and then centrifuge at 1500 rpm for 1 minute
10. Resuspend the cells and examine macroscopically and microscopically.
 interpretation
NEGATIVE RESULT (-)
no clumping of cells (no agglutination)
YOU HAVE NO ANTIBODIES TO RBCs

POSITIVE RESULT (+)

clumping of blood cells (agglutination)
YOU HAVE ANTIBODIES ON THE RBCs and that you may have a
condition that causes the destruction of RBCs by your immune system
(hemolysis). This may be due to certain diseases.
 sources of error in AHG tests
FALSE NEGATIVE RESULTS
General DAT & IAT
Failure to wash red blood cells adequately, since globulins not bound to
rbcs will neutralize the AHG reagent.
Interpretation of test: the washing process and the addition of AHG
reagent must be undertaken as quickly as possible to minimize loss of
bound antibodies by elution.
Improper storage, bacterial contamination and contamination with
human serum will impair the AHG reagent activity.
 sources of error in AHG tests
FALSE NEGATIVE RESULTS
General DAT & IAT
Not adding the AHG reagent
Improper centrifugation
 Number of cells present in the test: -too many cells give weak reactions
-too few cells will impair the reading of the agglutination
 sources of error in AHG tests
FALSE POSITIVE RESULTS
General DAT & IAT
In specimens containing potent cold-reactive antibodies agglutination
may occur before adding the AHG reagent.
Dirty glassware may cause clumping of cells.
Over centifugation
 antigen-antibody ratio
The optimum ratio is 80 parts antibody to 1 part antigen. There are specific terms
for variations in this ratio.

Prozone - antibody excess:

Antibodies saturating all antigen sites; no antibodies forming cross-
linkages between cells; no agglutination
Zone of equivalence:
Antibodies and antigens present in optimum ratio, agglutination
formed
Zone of antigen excess (Post-zone):
Too many antigens - any agglutination is hidden by masses of
unagglutinated antigens
 false negative results
DAT
All samples negative at the AHG phase should be incubated at room
temperature for 5 minutes to achieve maximal sensitivity needed for
complement detection.
IAT
• Serum and/or rbcs lose reactivity if improperly stored.
• Plasma used instead of serum can lead to failure to detect antibodies depending on
presence of active complement (anti-Jka, -Jkb)
• Temperature and incubation time affect attachment of antibody or complement to
cells.
• An optimal proportion of serum to cells should be achieved: usually 2-3 drops
serum to one drop of 5% cell suspension.
 false positive results
DAT
• A positive DAT from a clotted sample should be repeated on an EDTA
sample
• Samples collected from infusion lines may have complement present
on the cells.
IAT
• Cells with a positive DAT will give a positive result in any indirect antiglobulin
procedure.
 COOMB’S CELLS
• To show that test cells were properly washed and that no neutralization or reagent
deterioration has occurred, antibody-coated cells are used as a positive indicator.

• In a negative antiglobulin test the anti-human globulin should remain active and this
can be demonstrated by the addition of IgG sensitized cells.

• Agglutination of the IgG sensitized cells after mixing and centrifuging confirms that
the anti-human globulin was added to the test, that the test cells were properly
washed and all free globulin molecules were removed and that the anti-human
globulin was active.

• Failure of the IgG sensitized cells to agglutinate indicates that the original negative
antiglobulin test result is not valid and testing must be repeated.
 preparation of coomb’s cells
Preparing Coombs control cells is very easy. To about 10 drops of washed O Positive
red cells add 5-6 drops of anti-D antisera. Incubate at 37C for 15 minutes. Wash 4
times then prepare a 3 to 5% cell suspension.
To verify reaction, add two drops of AHG into test tube and one drop of newly
prepared Coombs cells. Centrifuge on High for 15 seconds,,,should get 1 -2 +
reaction.