3. Session Objectives
Define the terms Direct Antiglobulin (Coombs)
Test and Indirect Antiglobulin (Coombs) Test
State the purpose of the Direct Antiglobulin
Test
Describe the principle of the Direct
Antiglobulin Test
Describe the steps in the Direct Antiglobulin
Test
Mention the purpose of the Indirect
Antiglobulin Test
4. Describe the principle of the Indirect
Antiglobulin Test
Describe the steps in the Indirect Antiglobulin
Test
Differentiate between the Direct Antiglobulin
and Indirect Antiglobulin Test
5. Direct Antiglobulin (Coombs) Test: a test that
determines the absence or presence of
antibodies coating the patient’s red blood
cells
Indirect Antiglobulin (Coombs) Test: a test
that determines the absence or presence of
antibodies in the patient’s serum
Coombs: the name of the man credited with
inventing antihuman globulin for the purpose
of detecting IgG antibodies attached to red
blood cells
6. Purpose of the Direct Antiglobulin Test
The Direct Antiglobulin Test (abbreviated as
DAT) is used to detect invivo sensitization
(coating) of the patient’s red blood cells with
antibodies.
The DAT is used in the investigation of the
following:
HDFN: IgG maternal antibodies cross the
placenta and coat the foetal red blood cells
that have the corresponding antigen
7. Transfusion reactions: the patient’s
antibodies (can be IgM or IgG) coat the
transfused red blood cells that have the
corresponding antigen
Autoimmune haemolytic anaemia: IgG
and/or IgM antibodies coat the patient’s red
blood cells, shortening the survival of the red
blood cells, leading to anaemia
8. Principle of the Direct Antiglobulin
Test
When the patient’s red blood cells are coated
with antibody (globulin), agglutination may
not occur until antihuman globulin is added
(the cells are sensitized, but not agglutinated)
The antihuman globulin forms a bridge
between sensitized red blood cells to produce
visible agglutination.
The term “Direct” refers to the fact that no
incubation is required.
10. Steps in the Direct Antiglobulin Test
Direct Antiglobulin Test
Step Action
1. Label two test tubes – one for Test and another for Control.
2. Add 2 drops of patient’s red cells into the test tube marked ‘Test’ and 2
drops of Sensitized cells into the tube marked ‘Control’.
3. Wash patient red cells 3 times
4. Add one drop of Anti human Globulin serum into both tubes.
5. Mix well both tubes
6. Centrifuge both tubes gently at 1000 rpm for 30 seconds
7. Re-suspend the cell buttons and examine for agglutination.
Agglutination –means POSITIVE
No agglutination – means NEGATIVE*
*To confirm negativity, add one drop of Sensitized cells and repeat step 6
and 7. If there is agglutination, indicates a true Negative result.
11. Purpose of the Indirect Antiglobulin Test
• The Indirect Antiglobulin Test (abbreviated as IAT) is
used to detect in vitro sensitization (coating) of
reagent red blood cells with antibodies that are in
the patient’s serum or plasma.
The IAT is used in the following situations:
• Full crossmatch
• Antibody screen
• Antibody identification
• phenotyping/detection of red cell antigens
12. Principle of the Indirect Antiglobulin Test
• Review of the two stages of antigen-
antibody reactions
Sensitization: antibody attaches to the
antigen
Lattice formation
13. The patient’s serum is incubated with reagent
red blood cells. If antibodies (globulins) are
present, they will bind to their corresponding
antigen on the reagent red blood cells. All
excess plasma is removed by thoroughly
washing with saline.
Antihuman globulin is added and forms a
bridge between sensitized red blood cells to
produce visible agglutination.
The term “Indirect” refers to the fact that
incubation and the addition of AHG are
required.
15. Steps of Indirect Antiglobulin Test
Step Action: Antibody Screening Procedure
1. Prepare 50% cells suspension of O cells by adding 5 drops of washed O
cells and 5 drops of saline into the tubes.
2. Arrange two test tubes, one marked test (T) and another marked control
(C).
3. Add 4 drops of patient serum in tube marked T and 4 drops of Incomplete
Anti-D into the tube marked C
4. Add one drop of 50% of washed O cells into both tubes
5. Incubate both tubes at 37oC for 30 minutes
6. Wash both tubes three times by using saline and remove the supernatant
7. Add one drop of AHG in to both tubes
8. Spin immediately and observe for agglutination
Agglutination –means POSITIVE
No agglutination – means NEGATIVE*
*To confirm negativity, add one drop of Sensitized cells and repeat step 8. If
there is agglutination, indicates a true Negative result.
16. Compare the Direct Antiglobulin and
Indirect Antiglobulin Tests
Direct Indirect
Detects in-vivo sensitization Detects in-vitro sensitization
AHG added to well washed cells Serum and red cells incubated at
37º
Wash to remove unbound
globulins
Add AHG
Positive test indicates HDFN,
haemolytic anaemia, transfusion
reaction
Used for full cross match,
antibody screen or adverse and
antibody identification
17. Key Points
The DAT is used to detect in vivo sensitization of the
patient’s red blood cells with antibodies. The DAT
requires no incubation.
The DAT is used to investigate HDFN, transfusion
reactions and autoimmune haemolytic anaemia.
The IAT is used to detect in vitro sensitization of
reagent red blood cells with antibodies that are in the
patient’s serum or plasma.
The IAT requires incubation to allow antibodies to
sensitize the reagent RBCs.
The IAT is used in the following situations: full
crossmatch, antibody screen, antibody identification.
Both the DAT and the IAT use antihuman globulin to
produce visible agglutination.
18. References:
• F.J. Baker, R.E. Silverton (2001). Introduction to Medical
Laboratory Technology, Oxford University Press; 7th Edition
• Monica Cheesbrough (1981). Medical Laboratory Manual
for Tropical Countries Volume I, Butterworth & Co.
(Publishers) Ltd; 2nd Edition
• Monica Cheesbrough (2006) District Laboratory Practice in
Tropical Countries Part 1 & 2 Cambridge University Press,
2nd Edition
• World Health Organization (2002). Safe Blood and Blood
Products, Module 3: Blood Group Serology, World Health
Organization.
• W.J. Lockyer (1982). Essentials of ABO-Rh Grouping and
Compatibility Testing, Wright-PSG.
• D.M. Harmening, (2005) Modern Blood Banking and
Transfusion Practices, F.A Davis, 5th Edition.
