Coombs’ Test:- Part 2 – Coombs’ Indirect test, Anti-globulin indirect test

Coombs’ Test Indirect

Sample

• This is done on mother serum.

Indication

1. To diagnose the presence of antibodies in the pregnant mother serum.
2. Detection of certain weak antigens in RBCs like D^u.
   1. To find weak antibodies or incomplete types of antibodies like found in Duffy or Kidd blood groups.
   2. Detection of incomplete antibodies in serum, before pretransfusion screening or may do titration of the antibody.
   3. Detection of cold agglutinins autoantibodies.
3. To diagnose autoimmune hemolytic anemia.
4. To find the red cell sensitization by drugs.

Precaution of Coombs’ test indirect

1. This should be done on fresh serum.
2. Some of the drugs that give false-positive results are cephalosporin, antiarrhythmic, insulin, methyldopa, Dilantin, sulphonamides, and tetracycline.
3. The false-positive or negative results are seen due to:
   1. Clerical mistakes.
   2. Technical errors like not washing RBCs, failure to add reagents.
   3. Contamination by 5% or 10% glucose in water but not glucose in saline from intravenous tubing.
4. Poor quality of commercial coombs’ reagents.

Pathophysiology

1. The indirect Coombs test detects the circulating antibodies in the serum, these are the free antibodies.
   

   RBC coated with Antibody in direct Coombs’ test

2. The major purpose of this test is to detect if the recipient or the patient has serum antibodies other than the ABO / Rh system to RBC before receiving the blood transfusion.
3. This is the test for screening purposes in routine blood transfusion.
4. This test can diagnose other agglutinins like cold agglutinins which are usually associated with Mycoplasmal infection.
5. Circulating antibodies against RBC may be seen in the Rh-negative mother who is carrying an Rh-positive fetus.
   1. There is a mixing of the feto-maternal blood during the first delivery.
   2. Fetal blood Rh-Positive when goes into mother circulation will sensitize the mother and there is the formation of IgG type of antibodies.
   3. These antibodies can cross the placental barrier and enter fetal circulation.
   4. In the second pregnancy, there is a Type II cytotoxic reaction that will destroy the fetal RBCs and give rise to hemolytic anemia.
      

      Sensitization of the Rh-negative mother

6. Coombs’ serum is prepared by injecting IgG immunoglobulin (antibody) to the rabbit, then from his serum anti-IgG antibody is separated, which is called Coombs serum.
   

   Coombs’ serum formation

The procedure of  coombs’ indirect test

1. This test is done in the serum.
2. Add recipient serum (patients)  to donor RBC  (known blood O) in a test tube.
3. Incubate and then wash the RBC three times with saline.
4. Now add Coombs’ serum and again centrifuge, then see for agglutination.
5. If the test is positive then you will see clumping or agglutination.
   

   Coombs’ indirect test

Normal

• Negative and there is no agglutination.

A positive coombs’ indirect test indicates:

1. Incompatible blood transfusion.
2. Mother has anti-Rh antibodies.
   1. Erythroblastosis fetalis.
3. Acquired hemolytic anemia.
4. due to drugs and cold antibodies.

 

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