Coombs’ Test – Part 1- Coombs’ Direct, Direct-Anti-globulin test

Sample

1. The blood is collected in EDTA.
2. Separate RBC immediately to prevent the absorption of the complement to RBC.
3. Avoid clotted blood if possible. In the case of clotted blood, keep the blood at room temp. 37 °C, until the cells are separated.
4. Can store samples at 4 °C for one week.

Purpose of the test (Indications)

1. To diagnose the hemolytic anemia of the newborn.
   1. This test is done on the cells (RBC), especially in the case of a newborn in the Rh-negative mother when the baby is Rh-positive and in the case of erythroblastosis fetalis.
2. T0  diagnose autoimmune hemolytic anemias.
3. To diagnose blood transfusion reactions.
4. To diagnose drug-induced hemolytic anemia.

Precautions

1. Keep in mind that some of the drugs give false positive coombs direct tests, like cephalosporin, chlorpromazine, ampicillin, captopril, indomethacin, isoniazid (INH), streptomycin, tetracycline, sulphonamide, and penicillin.

Pathophysiology

1. Coombs’ test will detect coated RBCs with antibodies.
2. Most of the antibodies are due to ABO and Rh antigens.
3. Antibodies appear within 7 to 10 days of primary exposure (Primary immune response) and 1 o 2 days in the second exposure (Secondary immune response).

4. Once there is a reaction, then these antibodies are coated to RBC.

5. Such antibodies can also develop due to drugs like methyldopa and penicillins (Non-blood grouping antigens).
   1. Also, autoantibodies can attach to RBC.
6. Signs and symptoms of blood transfusion reaction are:
   1. Fainting and dizziness.
   2. Fever and chills.
   3. Rash.
   4. Back pain and pain in the flanks.
   5. Hematuria (blood in the urine).
7. Coombs’ serum (antibody) is prepared in the rabbit against the human antibodies IgM and IgG.
   1. Rabbit produces anti-human (IgM and IgG) antibodies, which are called Coombs’ serum.

Procedure

1. Wash the patient’s RBC three times with saline.
2. Decant the supernatant saline.
3. Now make 2 to 5 % of these RBCs suspension in saline.
4. Now add Coombs serum and centrifuge.
5. Look for the agglutination (clumping of RBC).
   

   Principle of Coombs’ direct test

Result

1. It is normally negative; there is no agglutination.
2. Positive direct Coombs show agglutination immediately after centrifugation, indicating that RBCs are coated with antibodies.
   1. This test is read positive when the clumping on a scale of a trace to +4.
3. False-positive results are seen in:
   1. Insufficient washing of the RBCs.
   2. Over Centrifugation of the test.
   3. Contaminated reagents.
   4. If the washed RBCs are left for a longer period of time.

The positive test is seen in:

1. Autoimmune hemolytic anemia.
   1. Warm reactive autoantibody.
   2. Cold reactive autoantibody.
   3. Cold haemagglutinin disease is seen in Lymphoma, pneumonia.
   4. Paroxysmal cold hemoglobinuria.
   5. Systemic lupus erythematosus.
2. Drug-induced hemolytic anemia, e.g, penicillin, quinidine, Cephalosporin, and α-methyldopa.
3. Hemolytic anemia: Transfusion reaction to incompatible RBC and hemolytic anemia of the newborn.
   1. Erythroblastosis fetalis.
   2. Incompatible blood transfusion.
4. Malignant diseases:
   1. Lymphoma.
   2. Chronic lymphocytic leukemia.
5. Infections :
   1. Infectious mononucleosis.
   2. Mycoplasma pneumoniae

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