6/21/2021 Coombs Test- Principle, Types, Procedure and Result Interpretation

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Coombs Test- Principle, Types, Procedure and Result Interpretation

Last updated: June 12, 2018 by Sagar Aryal

Coombs test is also known as antiglobulin test. The Coombs test tests for antibodies that may stick to the red blood cells
and cause red blood cells to die too early. It was discovered by Coombs, Mourant and Race in 1945. Coombs reagent is
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6/21/2021 Coombs Test- Principle, Types, Procedure and Result Interpretation

antihuman globulin. It is made by injecting human globulin into animals, which produce polyclonal antibodies specific for
human immunoglobulins and human complement system factors.

Principle of Coombs 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. 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.

Types of Coombs test

Direct Coombs Test (Direct Antiglobulin Test- DAT)

The direct Coombs test is used to detect antibodies (IgG or C3) that are stuck to the surface of red blood cells. Many
diseases and drugs can cause this. These antibodies sometimes destroy red blood cells and cause anemia.

This is the test that is done on the newborn’s blood sample, usually in the setting of a newborn with jaundice. The two
most commonly recognized forms of antibody-mediated hemolysis in newborns are Rh incompatibility and ABO
incompatibility.

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6/21/2021 Coombs Test- Principle, Types, Procedure and Result Interpretation

Procedure of Direct Coombs Test

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

Indirect Coombs Test (Indirect Antiglobulin Test- IAT)

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6/21/2021 Coombs Test- Principle, Types, Procedure and Result Interpretation

The indirect Coombs test looks for free-flowing antibodies against certain red blood cells. It is most often done to
determine if you may have a reaction to a blood transfusion.

This is the test that is done on the mother’s blood sample as part of her prenatal labs.  Frequently referred to as the
“antibody screen”, this test identifies a long list of minor antigens that could either cause problems in the newborns or
cause problems in the mother if transfusion is necessary.

Approximately 5% of patients have a positive IAT due to IgG antibodies, IgM antibodies, or both.

Procedure of Indirect Coombs Test

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

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6/21/2021 Coombs Test- Principle, Types, Procedure and Result Interpretation

4. In the test tube labeled as NC (Negative control), take 1 drop of normal saline.
5. Add one drop of 5 % saline suspension of the pooled ‘O’ Rho (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.
9. Keep for 5 minutes and then centrifuge at 1,500 RPM for one minute.
10. Resuspend the cells and examine macroscopically as well as microscopically.

Result Interpretation of Coombs Test

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).
This may be due to

Hemolytic anemia,
Chronic lymphocytic leukemia or similar disorder,
Erythroblastosis fetalis (hemolytic disease of the newborn),
Infectious mononucleosis,
Mycoplasmal infection,
Syphilis,
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6/21/2021 Coombs Test- Principle, Types, Procedure and Result Interpretation

Systemic lupus erythematosus and

Transfusion reaction, such as one due to improperly matched units of blood.

References:
1. Stanford School of Medicine
2. Institute For Transfusion Medicine
3. Johns Hopkins Lupus Center
4. University of Alberta
5. Madison College
6. Healthline
7. Medline Plus
8. Web MD
9. Lab Test Online
10. Patient
11. Cigna
12. Medscape
13. Healthlink BC
14. Laboratory Info
15. Wikipedia

 Haematology, Immunology, Laboratory

22 thoughts on “Coombs Test- Principle, Types, Procedure and Result Interpretation”

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6/21/2021 Coombs Test- Principle, Types, Procedure and Result Interpretation

Alfred
February 23, 2021 at 3:36 PM

why it is a need to incubate sample in doing the Indirect Coomb’s Test?

Reply

Madhu
October 13, 2020 at 1:13 PM

Nice explanation.But I have a doubt that why we r adding O pooled cells only ,we can use A or B also no ?

Reply

Muskhan
April 9, 2021 at 11:06 AM

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6/21/2021 Coombs Test- Principle, Types, Procedure and Result Interpretation

No we can’t since these A or B cells have antigens coated on their RBC surface hence may interfere the results
but for O positive cells , they don’t have any antigen except the Rh antigens which is important in the testing

Reply

Payal
February 12, 2020 at 2:03 PM

My indirect coombis test result are positive (grade 4+). Please help me.

Reply

Eddy
February 2, 2021 at 2:11 AM

Hallo Payal, since your indirect coomb’s test is positive, you need to find out what is reponsible as there are a
long list of antibodies – Anti D, C, E, Lewis, Duffy, etc – that could cause that. That helps you to prevent
transfusion problem, whenever you might need blood. And if you are still able to bear children, knowing the

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6/21/2021 Coombs Test- Principle, Types, Procedure and Result Interpretation

cause of your positive IAT will prevent the chances of having miscarriage or any post-natal complications,
because of medical intervention you would receive. hope that helps.

Reply

Kent
November 2, 2018 at 5:20 PM

POSITIVE CONTROL- use antibody-coated cells, which are commercially available or maybe prepared in the
laboratory

NEGATIVE CONTROL – known non-sensitized cells serve as negative control

RMT from PH

Reply

Jen
April 3, 2017 at 3:04 AM

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6/21/2021 Coombs Test- Principle, Types, Procedure and Result Interpretation

Great presentation. I used to work in the blood bank as a medical technologist performing DAT and IDAT testing.
This presentation is simple and to the point. This was a good review for me.

Reply

sandy
November 1, 2016 at 11:21 AM

sir, actually in coombs test there is a negative result means? pls clear my doubt.

Reply

Muka Robert
April 8, 2019 at 7:55 PM

@Sandy. A negative result means that the complement was not fixed to prevent agglutination.

It equally implies that the antibodies (Ab) were not sensitized.

Reply

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6/21/2021 Coombs Test- Principle, Types, Procedure and Result Interpretation

Eddy
February 2, 2021 at 2:15 AM

provided the test was done acccurately, negative result simply means you do not have any foreign immune
antibody on your cells, that could cause immune reaction (agglutination reaction). It could also be that the titre
value of the antibody is too low to be detected. This is usually the case with people who had received anti-D
prophylaxis in the past.

Reply

sundararaju
September 2, 2016 at 9:16 AM

ICT:Three tubes T,NC,PC In tube T:pt serum+O pooled cells. Tube NC:O Dneg cells+ anti D antisera 1/5 diluted.
Tube PC:O Dpos cells+ anti D antisera 1/5 diluted.

Reply

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6/21/2021 Coombs Test- Principle, Types, Procedure and Result Interpretation

kamau
August 24, 2016 at 12:48 PM

in ict there was error in procedure.no human serum was used.so which blood do we use to test for cell
sensitization in vitro.but good notes in DCT.

Reply

Ajitha Augustine
August 4, 2016 at 7:18 AM

Can u tell me that whether this test is altered by steroid therapy

Reply

Eddy
February 2, 2021 at 2:20 AM

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6/21/2021 Coombs Test- Principle, Types, Procedure and Result Interpretation

as far as i could remember, there has been no study to establish this. but since steroid therapy relates to immune
system, one can not completely rule out the effect on the ICT especially.

Reply

Devinder Singh Bandral

May 5, 2016 at 12:12 AM

There is not clearly verify that how to make negative and positive controls

Reply

Devinder Singh Bandral

May 5, 2016 at 12:09 AM

Bt there is no clearly verify of negative control

Reply

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6/21/2021 Coombs Test- Principle, Types, Procedure and Result Interpretation

kamaa
March 30, 2016 at 4:14 PM

i didn’t recognise the third tube.

Reply

Usman
December 27, 2015 at 3:25 PM

Very fantastic descriptions of reaction bt error in ICT procedure step 2&3 instead

1 Test tube label T serum

2 Test tube label PC Anti D

3Test tube label NC saline

Reply

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6/21/2021 Coombs Test- Principle, Types, Procedure and Result Interpretation

Eddy
February 2, 2021 at 2:21 AM

to save yourself this hurdle, just buy a commercially prepared ICT test kits.

Reply

H M Saxena
October 6, 2015 at 3:35 PM

We have developed a new Superagglutination test for serodiagnosis of infectious diseases. It differs from
conventional plate/slide agglutination tests (PAT/SAT) by three additional steps: prior staining of serum antibody
by adding a dye and addition of diluted biotinylated antiglobulin and avidin in sequence after mixing the antigen
with the test serum. The new steps circumvent the problems of false positive and false negative results of
PAT/SAT. In serodiagnosis of brucellosis, Superagglutination test had higher positive predictive value and
specificity than Rose Bengal Plate Test (RBPT) and Standard Tube Agglutination Test (STAT) and higher negative
predictive value and sensitivity than RBPT, STAT, ELISA and Complement Fixation Test (CFT).

Read the full paper at:

http://www.sciencedirect.com/science/article/pii/S2215016115000424

doi:10.1016/j.mex.2015.08.001

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6/21/2021 Coombs Test- Principle, Types, Procedure and Result Interpretation

Reply

hao wang
July 31, 2018 at 12:29 PM

is there a kit for super agglutination test

Reply

Seth Malovany MT AMT

June 29, 2015 at 1:44 AM

It has been several years since I have worked in BB is there now a completely automated way to do a Coombs
test? A more general question can a cross match NOW be done by machine….or is it still the familiar labor
intensive procedure I am familiar with?

With Respect

Seth Malovany MT AMT

Reply

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6/21/2021 Coombs Test- Principle, Types, Procedure and Result Interpretation

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