Coombs Test

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History
The antiglobulin test was first described in 1908 by Moreschi.
Introduced to human testing by Coombs’, Mourant and Race
in 1945. Purpose
• To detect IgG antibodies (incomplete antibodies) in the
serum.
• To detect RBCs coated with antibodies.
Importance of Antiglobulin Test (Coombs’ Test)
• Red cells coated with antibodies (IgG) will not agglutinate
and these antibodies are too small to span the distance
between the RBCs which are normally repelled by negative
surface charges (zeta potential).
• Cells coated with only complement will not agglutinate.
• Anti-human globulin reagent solves this problem, when
added to IgG/complement sensitized red cells the additional
antibodies are able to bridge the gap between the red cells.
Agglutination indicates presence of antibody/complement
or both and information about strength of agglutination can
be obtained. Grade the agglutination from 1+ to 4+, 4+ is
the strong reaction.
Antiglobulin reagents
• Poly-specific Coombs’ serum can recognise multiple epitopes
on one antigen, and this contains anti-IgG and anti-C3
antibodies.
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67
• Mono-specific Coombs’ serum can detect only one epitope
of antigen, specific against heavy chains of IgG, IgM and
IgA or can detect only C3b or C3d.
Quality Control of Anti-globulin Reagents
ISBT/ICSH freeze dried reference reagent is used to evaluate
the following:
• Specificity
• Potency of IgG
• Potency of anti-complement antibodies against C3b/
C3d present at controlled levels. No anti-C4d should be
present.
• Free from false positive tests (stored blood cells uptake C3d,
further augmented incubation with fresh serum).
Fig. 8.1: Role of AHG in Coombs’ test (direct antiglobulin test)

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Essentials of Blood Banking and Transfusion Medicine
COOMBS’ TEST
• Direct
• Indirect
Direct Test Performed Directly on the Red Cells
Indirect test name is a misnomer. In this test the patient’s serum
is tested for red cells antibodies.
Direct Coombs’ Test/Direct Antiglobulin Test (DAT)
• This test is performed to detect anti-IgG antibody or other
antibodies attached to the red cell surface within the blood
stream.
• DAT is carried out in the following circumstances:
– Transfusion reactions
– Non-ABO antibodies
– Drug-induced red cells sensitization
– For example, pencillin, quinidine, α-methyldopa, cephalo-
sporins
– Autoimmune haemolytic anaemia
– Paroxysmal cold haemoglobinuria (PCH) for presence of
IgG/C3
– Haemolytic disease of newborn
Precautions
• EDTA blood is preferred
Requirements
• Test tubes (10 × 75 mm)
• Pasteur pipettes
• Incubator
• Centrifuge
• AHG reagent
• Positive control cells (IgG coated)
Procedure
1. Prepare a 5% cell suspension in isotonic saline warmed at
37°C of the red blood cells to be tested. Warm saline removes
cold antibodies and non-specifically bound proteins.

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Coombs’ Test
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2. With clean pasture pipette add one drop of the prepared
cell suspension to a small tube.
3. Wash three times with normal saline to remove all the
traces of serum.
4. Decant completely after the last washing.
5. Add two drops of anti-human globulin serum.
6. Mix well and centrifuge for one minute at 1500 RPM.
7. Resuspend the cells by gentle agitation and examine
macroscopically and microscopically for agglutination.
8. With negative reaction further incubated at room tempera-
ture, centrifuged and read again. This additional incuba-
tion promotes positive reaction with the presence of
complement.
9. If negative, positive control cells (IgG coated) are added
to the test tube. Agglutination confirms AHG is active.
10. Initial testing is done with poly-specific AHG and later
can be confirmed with mono-specific AHG.
Indirect Coombs’ Test (Indirect Antiglobulin Test)
This test is performed to detect the presence of Rh-antibodies
or IgG antibodies produced in patients serum in case of the
following:
To check whether an Rh-negative patient has developed
a. Rh-antibodies
b. Anti-Rh antibodies may be produced in the blood of any
Rh-negative person by exposure to D antigen by:
i. Transfusion of Rh positive blood
ii. Pregnancy, if infant is Rh positive (when father is Rh-
positive).
iii. Abortion of Rh-positive fetus.
c. Rare blood groups which are similar to Rh blood group.
Rare blood groups which are similar to Rh blood group are
mentioned below:
• MNSs—anti-S and anti-sare IgG class as a result of
pregnancy or transfusion.
• Antibodies to Lutheran antigens are IgG.

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Essentials of Blood Banking and Transfusion Medicine
• Antibodies to Kell system antigens are IgG
• Duffy antibodies are almost exclusively IgG
• Antibodies to the Kidd antigens are almost exclusively IgG.
Requirements
• Test tubes (10 × 75 mm)
• Pasteur pipettes
• Incubator
• Centrifuge
Specimen: Serum
Reagents
• Anti-human globulin serum
• Anti-D serum
Additional Requirements
• Coombs’ control cells (‘O’ Rh (D) positive cells).
• Make a pooled ‘O’ Rh (D) positive cells from at least three
different ‘O’ positive blood samples.
• Wash these cells three times in normal saline (these cells
should be completely free from serum with no free anti-
bodies). Make 5% saline suspension of these cells.
Procedure
1. Label three test tubes as ‘T” (test serum), PC (positive control)
and NC (negative control).
2. In the tube labeled as ‘T’, add two drops of serum
3. In the tube ‘NC’, add one drop of saline
4. In the tube PC, add positive serum.
5. Add one drop of 5 % saline suspension of the pooled ‘O’ Rh
(D) positive cells in each tube.
6. Incubate all the three tubes for one hour at 37°C.
7. Wash the cells three times in normal saline to remove excess
serum with no free antibodies, (in the case of inadequate
washings of the red cells, negative results may be obtained).
8. Add two drops of Coombs’ serum (anti-human serum) to each
tube. Keep for 5 minutes and then centrifuge at 1,500 RPM
for one minute.

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9. Re-suspend the cells and examine macroscopically as well
as microscopically.
Test interpretation
Test
Observations
Conclusions
Positive
Agglutination
Correctly performed test
Control (PC)
procedure.
No agglutination
Coombs’ serum may not be
proper, repeat the test again
Negative
It should show no agglutination, since saline does not
Control (NC)
contain anti-D or any other antibodies.
Test serum (T)
Agglutination
Patient’s serum contains
anti-D
If PC results are correct,
Patient’s serum does not
no agglutination
contain anti-D
• Poly-specific AHG sera can detect
– Anti-IgG, anti-C3 antibodies
• Mono-specific AHG can detect only IgG/ IgM / IgA/C3
False positive test
• Clotted blood at cold temp. or room temp.
• Hospitalized patients can have anti-C3d
• If clotted sample, complement can bound to the red cells
and test can give positive test.
False negative test
• Failure to wash the red cells properly, AHG neutralized by
the immunoglobulins or complement.
• Excessive agitation may breakup agglutinates at reading
stage.
• Use of impotent antisera, such that weakly sensitised cells
not detected.
• Use of antisera lacking the antibody corresponding to the
subclass of immunoglobulin responsible for sensitisation.
• Not adding AHG.
• Improper centrifugation.
• Improper storage, bacterial contamination, and contamina-
tion with human serum will impair AHG reagent activity.

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Essentials of Blood Banking and Transfusion Medicine
• Presence of an antibody which can be readily eluted during
washing.
New technology for detection of antibodies by antiglobulin
test
1. Solid phase red cell adherence method: Sensitized red cells
are immobilized on solid matrix.
2. Column agglutination technique (matrix gel method):
Microtubes contain dextran acrylamide gel particles or Gel
bead and AHG to which the donor cells are added. After
centrifugation; agglutinated red cells are trapped at the top.
Non-agglutinated red cells will be at the bottom.
Fig. 8.2: Coombs’ test matrix gel method
Thus, Coombs’ test detects incomplete antibodies (IgG) and
C3 or both in the serum or on the red cell surface.