The document discusses the Coombs test, also known as the antiglobulin test. It is used to detect antibodies attached to red blood cells, such as the Rh antibody. There are two types of Coombs tests - direct and indirect. The direct test detects antibodies directly attached to red blood cells, while the indirect test detects antibodies in the patient's serum that could cause red blood cell agglutination. The document provides detailed procedures for performing both the direct and indirect Coombs test in a laboratory setting. It also lists conditions where a positive Coombs test could indicate, such as hemolytic anemia.

1. By: Nityanand Upadhyay
Associate Professor
Department of MLT
Integral University, Lucknow

2. ⦁ This is also known as Anti-Globulin Test.
⦁ Antiglobulin serum (Coombs’Serum) was
discovered by Coombs etal in 1945.
Coomb’s test is an Investigation done for
identification of anti-D antibody or Rh
antibody.
The Rh- antibody is not naturally occurring
antibody

3. ⦁ The antiglobulin test can be used to detect
red cells sensitized with IgG alloantibodies,
IgG autoantibodies or components.

4. 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.

5. 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.

6. ⦁ Two Types
1. Direct Antiglobulin Test/ Direct
Coomb’s (DCT).
2. Indirect Antiglobulin Test/ Indirect
Coomb’s Test (ICT).

7. 1. This test is performed to detect anti-D
antibody attached with RBC,s to get
sensitized .
2. The anti-D attached with RBC’s are found
in certain clinical conditions.
3. The DCT is performed in -

8. 1.HDN/ Erythroblastosis.
2. Transfusion Reaction.
3. Drugs Induced sensitized RBC.
4. Autoimmune Haemolytic Anaemia

10. ⦁ Test Tubes (10x75mm)
⦁ Centrifuge
⦁ NS
⦁ Micropipettes
⦁ Tips
⦁ Incubator

11. Specimen: 2 Ml. Blood drawn into EDTA is
preferred but oxalated, or clotted, citrated whole
blood may be used (specimen need n.ot be fasting
sample).
Reagent: AHG (Anti Human Globulin)/Coomb’s.

12. 1. Prepare a 5 % suspension in isotonic saline of
the red blood cells to be tested.
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.

13. 4. Decant completely after the last washing
5. Add two drops of Antihuman serum
(AHG).
6. Incubate at 37 Centigrade for 5-10 min.
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.

14. Looking for Agglutination
Reaction

15. ⦁ Negative Result:
No clumping of cells (no agglutination).
This means you have no antibodies to red blood
cells.
⦁ Positive Result:
Clumping (agglutination) of the blood cells during a
direct Coombs test means that you have antibodies
on the red blood cells and that you may have a
condition that causes the destruction of red blood
cells by your immune system (hemolysis).

16. ⦁ Hemolytic anemia,
⦁ Chronic lymphocytic leukemia or similar disorder,
⦁ Erythroblastosis fetalis (hemolytic disease of the
newborn),
⦁ Infectious mononucleosis,
⦁ Mycoplasmal infection,
⦁ Syphilis,
⦁ Systemic lupus erythematosus and
⦁ Transfusion reaction, such as one due to
improperly matched units of blood.

17. The ICT is used to detect presence of Rh-
antibody in patient serum.
e.g.
1. The Rh-ve women married with
Rh+ve husband.
2.The Rh-ve person received
Rh+ve Blood.

18. 3. Rh-ve mother carry Rh+ve
featus.
4. Abortion of Rh+ve featus .

20. ⦁ Test Tubes (10x75mm)
⦁ Centrifuge
⦁ NS
⦁ Micropipettes
⦁ Tips
⦁ Incubator

21. Specimen: Serum (need not be fasting)
Reagents: 1. Antihuman serum
2. Anti-D serum
Additional Requirements: Coombs control cells
A. Make a pooled ‘O’ Rho (D) positive cells from at
least three different ‘O’ positive blood samples.
B. Wash these cells three times in normal saline
(these cells should be completely free from serum
with no free antibodies).

22. ⦁ Label three test tubes as T (test serum) PC
(Positive control) and NC (negative control).
⦁ In the tube labeled as T (Test), take 2 drops of
test serum.
⦁ In the test tube labeled as PC (Positive control),
take 1 drop of anti D serum.
⦁ In the test tube labeled as NC (Negative control),
take 1 drop of normal saline.

23. ⦁ Add one drop of 5 % saline suspension of the
pooled ‘O’ Rho (D) positive cells in each tube.
⦁ Incubate all the three tubes for one hour at
37°C.
⦁ 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).

24. ⦁ 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.
⦁ Resuspend the cells and examine
macroscopically as well as
microscopically.

25. ⦁ Look for agglutination reaction
1. PC Tube- Agglutination (Test
correctly performed).
No agglutination(Coombs serum
may not be proper. Repeat the
test again.)

26. 2. Negative control (NC)- It should show no
agglutination, since saline does not contain Anti-D
or any other antibodies.
3. Test (Serum) (T).
a. Agglutination (and if PC results are correct)-
Patients serum contains AntiD.
b. No Agglutination- Negative.

27. Thanks…………..