Blood group antigens (A antigen and B antigen) is present on the surface of red blood cells and antibody (Anti-A and Anti-B) is present in plasma. 

According to karl landsteiner law-

  • If an agglutinogen (antigen) is present on the surface of red blood cells of an individual, the corresponding agglutinin (antibody) must be absent in the plasma. 
  • If an agglutinogen (antigen) is absent on the surface of red blood cells of an individual, the corresponding agglutinin (antibody) must be present in the plasma.

ABO blood grouping can be performed by two methods-

  1. Slide method
  2. Tube method

Slide method: This is a simple technique, which is used for emergency ABO blood grouping particularly in outdoor camps where centrifuge is not available. This technique is less sensitive and not capable to detect weak antigens. It is not recommended as routine test. Drying of reaction can cause aggregation of cell and can give false positive result. This technique is used in forward typing (unknown antigen with known antibody).

Tube method: This technique is more sensitive then slide method. This is capable of detecting weak antigens and antibody. By this technique cell grouping (forward grouping) and serum grouping (reverse grouping) are performed separately.

Slide method

Forward blood grouping by slide method:

Requirements:-

  • Glass slide or ceramic tiles
  • Anti- A and Anti-B sera
  • Blood sample

Procedure:-

  • Test can be performed either on glass slides or on ceramic tiles at room temperature or lower.
  • Put one drop of Anti-A and Anti-B sera separately on two previously labeled slides.
  • Add 1 drop of whole blood of the test sample on each slide.
  • Mix the cell and reagent properly by using clean stick. Spread each mixture evenly on the slide.
  • Rock the slides in order to mix the cells and sera and leave at room temperature for 2 minutes.
  • Record the results.

Interpretation of results:-

If agglutination occurs, it is visible with naked eyes as dark reddish clumps of different sizes. If agglutination is minimal, it can be examined under the microscope. Agglutination (+) in one or both antisera is interpreted as follows:

Tube method:

Forward blood grouping (cell grouping) by tube method:

In this method 5% red cell suspension of donor/patient is tested against Anti- A and Anti-B and Anti-AB sera.

Requirements:-

  • Clean test tubes
  • Anti- A and Anti-B and Anti-AB sera
  • 5% red cell suspension of blood sample

Washing of red cells:- Fill 2/3rd of test tube with normal saline. Mix 0.5ml of red cells with normal saline in the test tube and centrifuge at 3000 rpm for 1 minutes. The supernatant is discarded. Refill the tube with same amount of normal saline and centrifuge again. Repeat the procedure three times and discard the supernatant every time. The remaining cells are washed cells.

Preparation of 5% red cell suspension:- Mix the washed cells and normal saline in one of the following ratio as per requirement:

0.1ml of cells + 1.9ml of normal saline

0.2ml of cells + 1.9ml of normal saline

0.5ml of cells + 1.9ml of normal saline

Centrifuge the mixture at 1000 rpm for 1 minute.

Procedure:

  • Take three test tubes and label them as A, B and AB.
  • Add 1 drop of Anti-A in the test tube labeled A, Anti-B in the test tube labeled B and Anti-AB in the test tube labeled AB.
  • Add 1 drop of 5% cell suspension in each tube.
  • Mix properly and incubate at room temperature for 5-10 minutes and the centrifuge at 1000rpm for 1 minute.
  • Check the agglutination. If no clump is seen by naked eyes, examine under the microscope for weak agglutination.
  • Record the results.

Interpretation of results:-

If agglutination occurs, it is visible with naked eyes as dark reddish clumps of different sizes. If agglutination is minimal, it can be examined under the microscope and interpreted as follows:

Reverse blood grouping (serum grouping) by tube method:

In this method the serum of donor/patient is tested against known cells of group A, B and O. These cells are either prepared in lab or can be acquired by the manufacturers.

Requirements:-

  • Clean test tubes
  • Serum sample
  • Known A, B and O cells

Procedure:

  • Take three test tubes and label them as A, B and O.
  • Put 2 drops of serum to be tested in each test tube.
  • Add 1 drop of A group cells to the tube labeled A, B group cells to the tube labeled B and O group cells to the tube labeled O.
  • Mix properly and incubate at room temperature for 5-10 minutes and the centrifuge at 1000rpm for 1 minute.
  • Check the agglutination. If no clump is seen by naked eyes, examine under the microscope for weak agglutination.
  • Record the results.

Interpretation of results:-

If agglutination occurs, it is visible with naked eyes as dark reddish clumps of different sizes. If agglutination is minimal, it can be examined under the microscope and interpreted as follows: