Coombs’ Test – Part 1- Coombs’ Direct, Direct-Anti-globulin test
- The blood is collected in EDTA.
- Separate RBC immediately to prevent the absorption of the complement to RBC.
- Avoid clotted blood if possible. In the case of clotted blood, keep the blood at room temp. 37 °C, until the cells are separated.
- Can store samples at 4 °C for one week.
Purpose of the test (Indications)
- To diagnose the hemolytic anemia of the newborn.
- 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.
- T0 diagnose autoimmune hemolytic anemias.
- To diagnose blood transfusion reactions.
- To diagnose drug-induced hemolytic anemia.
- 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.
- Coombs’ test will detect coated RBCs with antibodies.
- Most of the antibodies are due to ABO and Rh antigens.
- 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).
- Once there is a reaction, then these antibodies are coated to RBC.
- Such antibodies can also develop due to drugs like methyldopa and penicillins (Non-blood grouping antigens).
- Also, autoantibodies can attach to RBC.
- Signs and symptoms of blood transfusion reaction are:
- Fainting and dizziness.
- Fever and chills.
- Back pain and pain in the flanks.
- Hematuria (blood in the urine).
- Coombs’ serum (antibody) is prepared in the rabbit against the human antibodies IgM and IgG.
- Rabbit produces anti-human (IgM and IgG) antibodies, which are called Coombs’ serum.
- Wash the patient’s RBC three times with saline.
- Decant the supernatant saline.
- Now make 2 to 5 % of these RBCs suspension in saline.
- Now add Coombs serum and centrifuge.
- Look for the agglutination (clumping of RBC).
- It is normally negative; there is no agglutination.
- Positive direct Coombs show agglutination immediately after centrifugation, indicating that RBCs are coated with antibodies.
- This test is read positive when the clumping on a scale of a trace to +4.
- False-positive results are seen in:
- Insufficient washing of the RBCs.
- Over Centrifugation of the test.
- Contaminated reagents.
- If the washed RBCs are left for a longer period of time.
The positive test is seen in:
- Autoimmune hemolytic anemia.
- Warm reactive autoantibody.
- Cold reactive autoantibody.
- Cold haemagglutinin disease is seen in Lymphoma, pneumonia.
- Paroxysmal cold hemoglobinuria.
- Systemic lupus erythematosus.
- Drug-induced hemolytic anemia, e.g, penicillin, quinidine, Cephalosporin, and α-methyldopa.
- Hemolytic anemia: Transfusion reaction to incompatible RBC and hemolytic anemia of the newborn.
- Erythroblastosis fetalis.
- Incompatible blood transfusion.
- Malignant diseases:
- Chronic lymphocytic leukemia.
- Infections :
- Infectious mononucleosis.
- Mycoplasma pneumoniae