Coombs’ Test

Coombs’ Test Direct

Sample for Coombs’ Test

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 coomb’s test direct (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 an Rh-negative mother when the baby is Rh-positive and in the case of erythroblastosis fetalis.
2. T0 diagnoses autoimmune hemolytic anemias.
3. To diagnose blood transfusion reactions.
4. To diagnose drug-induced hemolytic anemia.
5. It also detects C3 on patients’ RBCs.

Precautions for Coomb’s test

1. Remember that some drugs give false positive coombs direct tests, like cephalosporin, chlorpromazine, ampicillin, captopril, indomethacin, isoniazid (INH), streptomycin, tetracycline, sulphonamide, penicillin.

Pathophysiology of the Coomb’s test

1. Coombs’ direct test will detect coated RBCs with antibodies.

RBC coated with Antibody

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 to 2 days in the second exposure (Secondary immune response).

Immune response, primary and secondary

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

RBC coated with Antibody in direct Coombs’ test

5. Such antibodies can also develop due to drugs like methyldopa and penicillins (Non-blood grouping antigens).
   1. Also, autoantibodies can attach to RBC.

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).

Coombs’ serum (Antibody):

1. It is prepared in the rabbit against the human antibodies IgM and IgG.
2. Rabbit produces anti-human (IgM and IgG) antibodies called Coombs’ serum.

Coombs’ Test: Coombs’ serum formation

The procedure of Coomb’s direct test

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

Coombs’ direct test

Coomb’s direct

Result of reading Coomb’s direct test:

1. It is normally negative; there is no agglutination.
2. Positive direct Coomb’s show agglutination immediately after centrifugation, indicating that RBCs are coated with antibodies.
   1. This test is read positive when the clumping is on a trace scale, trace to 4⁺.

All the reactions are reported as follows:

False-positive results are seen in the following conditions:

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.
5. It may be seen in m multiple myeloma and Waldenstrom’s macroglobulinemia.

The positive Coomb’s direct test is seen in:

1. Autoimmune hemolytic anemia.
   1. Warm reactive autoantibody.
   2. Cold reactive autoantibody.
   3. Cold haemagglutinin disease is seen in Lymphoma and pneumonia.
   4. Paroxysmal cold hemoglobinuria.
   5. Systemic lupus erythematosus.
2. Drug-induced hemolytic anemia, e.g., penicillin, quinidine, Cephalosporin, and α-methyldopa.
   1. α-methyldopa hemolytic reaction occurs in <30% of the patient with therapy, but only <1% shows hemolysis; rarely in the first 6 months of the treatment.
   2. If it is not found within 12 months of the treatment, then less likely to see the reaction.
   3. A reversal takes place from weeks to months after stopping the drugs.
3. Hemolytic anemia: Transfusion reaction to incompatible RBC and hemolytic anemia of the newborn.
   1. Erythroblastosis fetalis.
   2. Incompatible blood transfusion.
   3. Delayed hemolytic blood transfusion reaction.
4. Malignant diseases:
   1. Lymphoma.
   2. Chronic lymphocytic leukemia.
   3. Acute and chronic leukemias.
5. Infections :
   1. Infectious mononucleosis.
   2. Mycoplasma pneumoniae
6. Weak positive tests are seen in:
   1. Renal diseases.
   2. Rheumatoid arthritis.
   3. Inflammatory bowel disease.
   4. Epithelial malignancy.
   5. Weak positive tests usually have no clinical significance.

Coomb’s direct negative results are seen in the following conditions:

1. Hemolytic anemia is caused by the intrinsic defect in the RBC, e.g., G6PD deficiency hemoglobinopathies.
2. Hemolytic anemias due to weak or smaller amounts of IgG bound to RBCs.

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