Anti Human Globulin Test (Coomb’s Test)

Coomb’s test or Anti Human Globulin test is very useful in recognized weak immunologic reactions. It is widely used in the identification of Du, Compatibility testing, Antibody screening and identification of Sensitized red cells. Anti Human Globulin is made by injecting human globulin in rabbit and purifying the Anti human globulin (AHG) produced by the rabbit. It is commercially available. There are two types of Coomb’s test :

  1. Direct Coomb’s Test (DCT) or Direct Anti Human Globulin Test (DAT)
  2. Indirect Coomb’s Test (ICT) or Indirect Anti Human Globulin Test (IAT)
Anti Human Globulin Test (Coomb’s Test) : http://medicoinfo.org/anti-human-globulin-test-coombs-test/

Direct Coomb’s Test (DCT) or Direct Anti Human Globulin Test (DAT)

Direct Coomb’s Test (DCT) or Direct Anti Human Globulin Test (DAT) is performed to detect anti-D antibody or other antibodies attached to the Red Blood Cell surface with in the blood stream. This occurs in the following circumstances :

  1. When there is a ‘Rh’ Positive baby in the womb of a sensitized ‘Rh’ Negative woman; the antibodies produced in the mother serum, cross the placenta and after entering the baby’s blood stream. These antibodies will attach to the baby’s ‘Rh’ positive red blood cells. These coated (or Sensitized) cells are clumped and removed from the circulation, causing haemolytic anaemia (Haemolytic Disease of the Newborn; Erythroblastosis foetalis).
  2. Transfusion Reaction.
  3. Drug induced red cell sensitization.
  4. Auto-Immune Haemolytic Anaemia.

Principle :

Direct Coomb’s Test or Direct Anti Human Globulin Test detects “Sensitized red cells”; where the red blood cells get coated with IgG or globin (anti-D) but do not agglutinate. When the sensitized red blood cells come in contact with Anti Human Globulin reagent they agglutinate.

Direct Anti Human Globulin Test (DAT) : http://medicoinfo.org/anti-human-globulin-test-coombs-test/

Method :

Two methods are used for determination of Direct Coomb’s test (DCT) or Direct Anti Human Globulin Test (DAT).

  1. Tube method        
  2. Gel method

In the developing country Tube method is commonly performed. But Special circumstances Gel method is performed. In the Laboratory We performed Tube method.

Requirements :

  1. Clean and dry grass free glass test tube.
  2. Pasteur pipette.
  3. Centrifuge machine.
  4. Timer & Marker pen
  5. Microscope

Reagent :

  1. Anti Human Globulin (AHG)
  2. Coomb’s Control Cells (5% ‘O’ Cell Suspension)
  3. Normal Saline (0.85% NaCl Solution)

Specimen :

Blood drawn into EDTA anticoagulated vial is preferred but oxalated or clotted, citrated whole blood may be used ( the specimen need not to be  fasting sample).

Procedure :

  1. All the reagents and sample to be brought to the room temperature before performing the test.
  2. Prepared 5% suspension in isotonic solution of the red cells of patient to be tested.
  3. Taken a clean and dry glass test tube. Added 1 drop of the pre-paired Red cell suspension to the test tube by a clean Pasteur pipette.
  4. Washed the cell suspension three times with normal saline to remove all the trace of serum. Decant completely after the last washing.
  5. Added two drops of Anti Human Globulin (AHG).
  6. Mixed well and centrifuge for 1 minute at 1500 RPM.
  7. Resuspended the cells by gentle agitation and examine macroscopically or microscopically for agglutination.

Interpretation :

  1. Agglutination        :     Direct Coomb’s test Positive (Patient serum contain Anti-D)
  2. No Agglutination   :    Direct Coomb’s test Negative (Patient serum does not contain Anti-D)

Special Note :

  1. The sensitivity of the test can be increased by incubation at room temperature for 5 to 10 minutes and by recentrifugation.
  2. All Negative result is cross-checked by adding 1 drop of Coomb’s Control Cells that agglutinate in contact with AHG reagent.

Indirect Coomb’s Test (ICT) or Indirect Anti Human Globulin Test (IAT)

Indirect Coomb’s Test (ICT) or Indirect Anti Human Globulin Test (IAT) is very important test in Immunohaematology. This test is performed to detect presence of ‘Rh’ antibodies or other antibodies in the patient serum in case of following :

  1. To check whether an ‘Rh’ Negative woman (married to ‘Rh’ Positive husband) has developed Anti-Rh antibody.
  2. Anti-D may be produced in the blood of any ‘Rh’ Negative persons by exposure  to D antigen by :
    • Transfusion of ‘Rh’ Positive blood.
    • Pregnancy, if the infant is ‘Rh’ positive (if husband is ‘Rh’ Positive)
    • Abortion of ‘Rh’ Positive foetus.

Principle :

Indirect Coomb’s test or Indirect Anti Human Globin Test detects Anti-D antibody if present in the patient serum. Known ‘O’ Positive red cells are sensitized by reacting with patient serum (contain anti-D) afterwards these ‘O’ Positive added with Anti Human Globulin reagent. When the sensitized red cells come in contact with Anti Human Globulin (AHG) they agglutinate. If patient serum have anti-D hence after sensitization ‘O’ Positive cells agglutinate with AHG. If patient serum does not have anti-D then ‘O’ Positive cells not agglutinate with AHG.

Indirect Anti Human Globulin Test (IAT) : http://medicoinfo.org/anti-human-globulin-test-coombs-test/

Method :

Two methods are used for determination of Indirect Coomb’s test (ICT) or Indirect Anti Human Globulin Test (IAT). These are as follows__

  1. Tube method        
  2. Gel method

In the developing country Tube method is commonly performed. But Special circumstances Gel method is performed. In the Laboratory We performed Tube method.

Requirements :

  1. Clean and dry grass free glass test tube (10 x 75 mm).
  2. Pasteur pipette.
  3. Centrifuge machine.
  4. Microscope
  5. Incubator
  6. Timer & Marker pen

Reagent :

  1. Anti Human Globulin (AHG)
  2. Anti-D Serum (IgG Nature)
  3. Coomb’s Control Cells (5% ‘O’ Cell Suspension)
  4. Normal Saline (0.85% NaCl Solution)

Preparation of Coomb’s Control Cells :

Make pooled ‘O’ ‘Rho(D) Positive cells from at least three different ‘O’ Positive blood samples. Washed these cells three times in normal saline (the cells should be completely free from serum with No-free antibodies). Make 5% saline suspension of these cells.

  1. Mixed 5 drops of well mixed sedimented red blood cells with 2 ml of normal saline.
  2. Centrifuge at 1500 RPM for 1 to 2 minutes. Discarded supernatant.
  3. Again added 2 ml of normal saline and mixed well. Centrifuge the tube at 1500RPM for 1 to 2 minutes. Discarded the Supernatant. (this procedure done Three times).
  4. Then added 4 ml of normal saline with the sedimented red blood cells and mixed well. (Approximately 5% RBCs suspension)

Specimen :

Fresh Serum Specimen (the specimen need not to be fasting sample).

Procedure :

  1. Labelled three test tube as ‘T’ (Test serum), ‘PC’ (Positive Control), ‘NC’ (Negative Control).
  2. Added 2 drops of patient serum in the tube labelled as ‘T’.
  3. Added 1 drop of Anti-D in the ‘PC’ labelled test tube and 1 drop of normal saline in the ‘NC’ labelled test tube.
  4. Added 1 drop of 5% saline suspension of the polled ‘O’ ‘Rho(D) Positive cells in each tubes.
  5. Incubated all three test tubes for 45 minutes at 37°C temperature.
  6. Washed the cells three times in normal saline to remove excess serum with No-free antibody. (In the case of inadequate washing of the red cells; Negative result may be obtained).
  7. Added 2 drops of Coomb’s serum (AHG reagent) to each tubes. Keep for 5 minutes and then centrifuged at 1500 RPM for 1 minute.
  8. Resuspended the cells and examine macroscopically as well as microscopically.

Interpretation :

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