Useful Notes on Coombs’ Test

The Coombs’ test was first described in 1945 by Cambridge immunologists Robin Coombs (after whom it is named), Arthur Mourant and Rob Race.

Historically, it was done in test tubes. Today, it is commonly done using microarray and gel technology.

The two Coombs tests are:

i. Direct Coombs test (also known as direct antiglobulin test or DAT).

ii. Indirect Coombs test (also known as indirect antiglobulin test or IAT).

Direct Coombs’ Test (DCT):

The direct Coombs’ test is used to detect autoantibodies on the surface of red blood cells. Many diseases and drugs (quinidine, methyldopa, and procainamide) can lead to production of these antibodies. These antibodies sometimes destroy red blood cells and cause anaemia. This test is sometimes performed to diagnose the cause of anaemia or jaundice.

Principle:

The direct Coombs’ test is a test of whether IgG antibodies are already attached directly to the patients red cells. The test is based on the fact that, serum containing anti-human IgG, is added to patients red cell. The anti-human anti-bodies are produced by immunizing laboratory animals (e.g. Rabbit, sheep, goat, horse, donkey and chicken) with human serum.

The mixture is observed for agglutination. If human IgG antibody has already attached to the patient’s red cells in vivo (in the bloodstream), then the addition of anti-human IgG will cause the cells to agglutinate. This is a positive direct anti-human globulin test (DAT) or a positive direct Coombs test.

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Requirements:

Test tubes centrifuge.

Reagents:

Anti-human globulin (AHG) reagent.

Presensitized red cells (Coombs’ control cells).

Saline.

Procedure:

1. Wash the red cells suspected of being sensitized, 3 to 4 times in large volume of saline. Complete removal of free globulin is important.

2. Add two drops of anti-human globulin serum to the sedimented cells.

3. Mix well and centrifuge at 1500 rpm for one minute.

4. Examine agglutination by holding against a lighted background and tapping the bottom of the tube.

5. If the agglutination is not seen, leave the tube at room temperature for 10 min. then re-centrifuge and read. A weaker reacting antibody will show delayed reaction. Consider this as positive.

6. If haemagglutination is not seen in step 5, add one drop of presensitized red blood cells (5% suspension is saline). This should result in haemagglutination of pre-sensitized cells indicating that the AHG is reactive and the result is valid.

Interpretation:

Haemagglutination of red cells with the addition of AHG (positive) indicates that the cells are sensitized inside the body.

Positive Coombs’ test means patient have antibody that acts against his own RBC. This may be due to:

i. Autoimmune haemolytic anaemia without another underlying cause.

ii. Drug-induced haemolytic anaemia (many drugs have been associated with this complication).

iii. Erythroblastosis fetalis (haemolytic disease of the newborn).

iv. Infectious mononucleosis.

v. Mycoplasmal infection.

vi. Syphilis.

vii. Chronic lymphocytic leukaemia or other lympho proliferative disorder.

viii. Systemic lupus erythematosus or another rheumatologic condition.

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

Indirect Coombs’ Test (ICT):

The indirect test looks for anti-RBC antibodies that flow freely in blood serum. The indirect Coombs’ test is only rarely used to diagnose a medical condition. More frequently, it is used to determine a reaction.

Principle:

The indirect Coombs’ test determines whether there are free IgG antibodies in the patient’s serum, as opposed to being on the red cells. The test is called indirect because, in this test the red cells are first sensitized in laboratory and then detected by direct Coombs’ test.

When a person’s serum is suspected to be having some ‘incomplete antibodies’, such serum is mixed with cell- suspension of cells known to have antigen corresponding to the suspected antibody. After incubation at a temperature appropriate for the suspected antibody for an appropriate time the cells are washed and direct Coombs’ test is performed.

Requirements and Reagents:

Same as that of direct Coombs’ test.

Procedure:

1. Prepare a 4% saline suspension of the test cells.

2. Add two drops of cell suspension to a small test tube.

3. Add two drops of antiserum to the cell suspension.

4. Incubate in a water bath at 37°C for 30 min.

5. Remove the tube from the water bath and wash 3 to 4 times with large volume of saline. Decant completely after last washing.

6. Add immediately two drops of anti human globulin (AHG) and mix well.

7. Centrifuge at 1500 rpm for one min.

8. Examine for haemagglutination.

9. In case of negative haemagglutination, add presensitized reagent cells or Coombs’ control cells to test the reactivity of AHG. Agglutination must be seen with the addition of Coombs’ control cells.

Positive indirect Coombs’ test means patient have antibodies that the body views as foreign. This may be due to:

i. Erythroblastosis foetalis haemolytic disease.

ii. Incompatible blood match (when used in blood banks).

iii. Autoimmune or drug-induced haemolytic anaemia.

Precautions while performing the test:

iv. Proportion of serum to cells: 1 drop of 2.5% cells + 2 drops of serum.

v. Incubation temperature: optimum reacting temperature for antibody is 37^JC

vi. Incubation time: 30-40 min. for 1 drop of 5% cells with two drops of serum.

vii. Cell wash: It is must to prevent neutralization of AHG by free antibody in serum.

Determination of D^u:

Indirect Coombs test is applied in D^u testing. D^u factor is variant of D antigen present on the red cells of individuals of D^u blood type.

Principle:

Cells with D^u antigen are sensitized with anti-D by incubating at 37°C for 30 min. This results in the adsorption of anti-D on the surface of the cell without producing haemagglutination (sensitization). The presence of reacted antibody on the surface of D^u cells is recognized by using antihuman globulin (AHG) which reacts with the coated antibody and brings about haemagglutination. If the cells do not show agglutination, even after anti-D treatment and AHG reaction, the cells are truly Rh- negative.

Additional Requirement:

1. Antihuman serum (Coombs’ serum)

2. Bovine albumin, 22% (control)

3. Incubator or water bath (37°C)

Procedure:

1. Prepare 5% suspension of red blood cells in isotonic saline.

2. Label one tube as ‘T’ and add in it one drop of anti-D serum (test).

3. Label second tube as ‘C’ and add in it one drop of 22% bovine albumin reagent (control).

4. By using a Pasteur pipette, add one drop of the cell suspension to each of the test tubes, mix well.

5. Incubate the test and the control for at least 15 minutes at 37°C.

6. After incubation, wash the cells in each tube three times with fresh normal saline (for each washing add test tube full of normal saline).

7. Decant the tubes completely after the last washing.

8. To each tube, add two drops of antihuman serum (containing anti-IgG).

9. Mix the contents of the tube gently and centrifuge at 1500 RPM for 1 minute.

10. Resuspend the cells by gentle agitation and examine macroscopically and confirm the results microscopically.

Interpretation:

Agglutination indicates Rh-positive cells and absence of agglutination is Rh- negative cells.

Antibody Titration:

The titre of an antibody is the reciprocal of the highest dilution that causes agglutination of the corresponding antigen cells. Thus the titration value gives the quantitative value of the antibody and the relative strength of the antigens in the cells.

But it is not the total strength of the antibody in the serum. It indicates only the amount of antibody which is dissociated and is free in solution. The titre of an antibody is usually determined by testing the serial two fold dilution of the serum’ against appropriate red cells.

To obtain exact titration results, maintain meticulous pipetting, optimum incubation time and temperature. Use freshly drawn and prepared red cell suspension and evaluate entire series of the tubes.

Specimen:

Serum (antibody) to be titred.

Reagents:

1. Red blood cells that express the antigen (s) corresponding to the antibody specificity (ies) in a 5% saline suspension. Uniformity of cell suspension is very important to ensure compatibility results.

2. Normal saline (dilutions may be made with 6% albumin if desired)

Procedure (Double Dilution):

1. Label a row of test tubes, according to the serum dilution, usually 1:1 through 1:512.

2. Add 0.1 ml of saline to all tubes except the first tube.

3. Add 0.1 ml of serum to tubes 1 and 2 (dilution 1:1 and 1:2).

4. Mix the contents of the tube 2 with a clean pipette and then transfer 0.1 ml of the mixture to tube 3 (1:4 dilution).

5. Continue the same technique, through all dilutions and remove 0.1 ml from dilution tube with dilution of 1:512 and discard or save for further dilution if required.

6. Add 0.1ml of 5% saline suspension of appropriate red cells to each tube.

7. Incubate at the appropriate temperature according to the antibody being tested. In case of anti-A and anti-B, incubate at room temperature for 30-45 minutes.

8. Gently dislodge the red cells and observe macroscopically for agglutination. The agglutination titre is recorded.

Results are expressed as the reciprocal of the highest serum dilution that causes macroscopic agglutination.

Thus a serum which gives visible agglutination at 1:256 is said to have titre of 256. In describing the titre of the serum, it is usual to ignore the diluting effect of the cell suspension.

Note:

i. For IgG antibody titre add 0.1 ml of bovine albumin 22% or papain cystein after step 5.

ii. Incubate at 37°C for 30-40 min.

iii. Observe for the agglutination.

iv. IgG antibody titre can also be done by IAT.

Avidity:

Speed and strength of agglutination is termed as avidity. The test is done by mixing two drop of anti-serum with one drop of 40-50% cell suspension on a slide and rocking gently at room temperature.

The time for clearly visible reaction (+1) and then for strong reaction (+4) reaction to occur are recorded with the help of stop watch.

Grading of Agglutination Reaction:

i. +4 single clump of agglutination with no free cells

ii. +3 three or four individual clumps with few free cells

iii. +2 many fairly large clumps with many free cells

iv. +1 fine granular appearance visually, but definite small clumps (10-15 cells) per low power field

v. +W 2 to 3 cells sticking together per low power field, uneven distribution. Visually no agglutination

vi. – All cells are free

vii. H haemolysis (partial or total) must be interpreted as positive.