ANTIGLOBULIN TEST (COOMBS TEST) Direct Antihuman Globulin Test (DAT, Direct Coombs' Test) , Indirect Antihuman Globulin Test (IAT, Indirect Coombs' Test)

ANTIGLOBULIN TEST (COOMBS TEST) 

Direct Anti human Globulin Test (DAT, Direct Coombs' Test) 

Coombs reagent

Indirect Anti human Globulin Test (IAT, Indirect Coombs' Test), Coombs reagent

 

ANTIGLOBULIN TECHNIQUES - Antiglobulin techniques are used for the detection of incomplete univalent antibodies which cannot be detected by the other methods described above. These antibodies react with the red cells, but the reaction is not observable as agglutination or haemolysis.

ANTIGLOBULIN TEST (COOMBS TEST)

Antiglobulintest are used for the detection of incomplete univalent antibodies which cannot be detected by the other methods described above. These antibodies react with the red cells, but the reaction is not observable as agglutination or haemolysis.

Such red cells are called sensitised red cells. 

The antibody binds with one antigen site on one red cell, but is unable to reach the adjacent red cell with the other binding site because the distance between two cells is greater than the size of the antibody molecule. Thus, bridging and subsequent agglutination of red cells does not occur. However, in vivo, these incomplete antibodies are capable of bringing about severe reactions.

To demonstrate the presence of these antibodies in vitro, a reagent was developed by Coombs which is an antibody to human globulin. This antiglobulin antibody is IgM in nature and can agglutinate the incomplete antibody coating the adjacent red cells.

Coombs reagent:

The antihuman globulin (AHG) reagent is prepared by inoculating laboratory animals, usually rabbits, with human serum (globulin) or purified globulin. The animals produce an antibody to this human globulin, AHG. The serum of the animal is purified till it reacts specifically only with human globulin. This AHG is monospecific. Some AHG reagents can combine both with globulin and with complement. These are called polyspecific AHG.

The monospecific AHG are produced by injecting purified fractions of human serum into rabbits where as the polyspecific reagent can be prepared by pooling the required monospecific reagents together.

The antiglobulin techniques can be used for cell typing with incomplete antibodies, for antibody screening before transfusion, for antibody detection in post-transfusion reactions and other reactions such as the haemolytic disease of the newborn (HDN), haemolytic anaemias etc.

ANTI HUMAN GLOBULIN TESTS (Coombs' Tests):- There are two types of antiglobulin tests:

i) Direct test is used to detect in vivo sensitisation of red cells by an incomplete antibody.

(ii) Indirect test detects the presence of incomplete antibody in the patient's serum.

(A)Direct Antihuman Globulin Test (DAT, Direct Coombs' Test) :- DAT is performed on red cells which are suspected of being coated with an antibody in vivo as in haemolytic disease of the new-born (HDN) and auto immune haemolytic anaemia. These sensitised red cells are washed and mixed with the AHG which binds the incomplete antibody on the red cell surface and brings about agglutination of red cells.

Note:-It is essential to remove all traces of serum globulins by thoroughly washing the cells.

Specimen:- Whole blood or 5% washed red cell suspension in saline.

Reagents:-

1. Monospecific or polyspecific AHG serum,

2. O Rh-positive red cells.

3. Anti-D serum incomplete antibody, IgG) 

Technique:- Preparation of IgG coated red cells

1. Wash O Rh-positive red cells three times in saline.

 2. To the packed red cells, add equal volume of anti-D serum.

3. Incubate at 37°C for 30 minutes.

4. Wash the red cells four times in saline, and prepare a 5% suspension.

5. Take one volume of the 5% cell suspensionand add two volumes of AHG to it.

6. Centrifuge at 1000 g for 15-20 seconds andexamine for agglutination.

-Strong agglutination with AHG indicates that thecells are properly coated with IgG. The IgG coated red cells can be stored at 4°C for 48 hours.

Direct Coombs' Test

Prepare a 5% suspension of patient's red cells.

2 To one volume (e.g. 0.1 ml) of the red cell suspension, add two volumes of AHG.

3. Mix gently and centrifuge at 1000g for 1520 seconds.

4. Read the tubes microscopically for agglutination.

5. Use the IgG coated cells as positive control and repeat steps 1-4. 

Result:- Agglutination of patient's red cells with AHG indicates a positive direct Coombs' test.

Indirect Antihuman Globulin Test (IAT, Indirect Coombs' Test):-This test is useful for the detection of antibodies in patient's serum that may have formed as a result of sensitisation by transfusion or pregnancy. In autoimmune haemolytic anaemia, the patient mayhave free circulating antibodies in addition to those sensitising the red cells. 

The IAT technique can also be used for the detection of antigens e.g. for cell typing, using a known incomplete antibody and AHG serum.

The indirect Coombs' test is performed in four stages as follows:

1. Incubation of control red cells and test serum (for antibody detection) at 37°C: This step will result in sensitisation of red cells because antibodies in serum will bind to the antigen sites on red cells.

2. Washing of the red cells to remove excess of free antibodies in the suspension: This step should ensure that there are no free reacting antibodies or interfering proteins in the suspension to give false results.

3. Addition of antihuman globulin serum: A positive test will be indicated by agglutination in this step.

4. Checking of negative results with sensitised red cells: If the test is negative, add sensitised red cells to the test. The presence of agglutination indicates that the AHG is active and is not neutralised by any other antigen in the preparation

.

Note:-This technique can be adapted for the detection of antigen on red cells by using a known positive antiserum.

Reagents

1.5% washed red cell suspension in saline. For example, O-Rh positive cells for the detection of anti-Rh antibodies.

2. Patient's serum

3. Antihuman globulin serum.

4. IgG coated red cells prepared as for the direct Coomb's test.

Technique

1. Mix one volume of 5% cell suspension with 3-4 volumes of patient's serum.

2. Incubate at 37°C for 45-60 minutes.

3. Centrifuge at 1000 g for 15-20 seconds, and examine for haemolysis or agglutination. If agglutination is observed, do not proceed further. The agglutination at this stage cannot be due to IgG. If partial haemolysis is observed, record and proceed further.

4. Wash the cells at least three times, taking care to remove as much supernatant as possible after each wash. Resuspend the cells in saline by tapping before centrifugation.

5. Resuspend the cells by tapping and add two drops of antihuman globulin serum.

6. Mix and centrifuge the tubes at 1000 g for 15-20 seconds.

7. Read the tubes macroscopically and microscopically for agglutination.

 -Record results.

8. If there is no agglutination in step 7, leave the tubes at room temperature for 5 minutes, recentrifuge and examine for agglutination.

9. If the test is still negative, add 1 drop of IgG coated red cells, centrifuge and examine again.

10. It is necessary to run a positive control, using known antibody in parallel with the test.

Note:-

 For the detection of antigens on red cells, follow the same technique using known positive antiserum.

Results:- A positive reaction in step 8 indicates that the negative reaction in step 7 is valid; and the test should be recorded as positive indirect Coombs test.

A positive reaction in step 9 indicates a negative indirect Coombs' test.

A negative reaction in steps 7, 8 and 9 indicates that the test is invalid and must be repeated.

Applications of Antihuman Globulin Tests (Coombs' Tests)
Direct Coomb's test is positive in:	Indirect Coombs' test is used for the detection of:
1. Haemolytic disease of the new-born(HDN). 2. Autoimmune haemolytic anaemia. 3. Recipients of incompatible blood transfusion. 4. Drug induced haemolytic anaemia e.g. due to penicillin, methyldopa, caphalothin.	1. Certain blood factors e.g. some blood group antigens of systems such as Duffy, Kell Kidd. 2. Antibodies to the above blood factors. 3. Incompatibility in the donor's and recipient's blood samples. 4. Antibodies which are destroyed by proteolytic enzymes. 5. Complement fixing antibodies on the red cells.