ABO GROUPING, ABO GROUPING METHODS

ABO GROUPING, Preparation of Red Cell Suspensions, Reaction of Antisera with Red Cells,  ABO GROUPING METHODS

ABO GROUPING

Determination of ABO group is necessary both for the donor as well as the recipient, preliminary to blood transfusion. The blood group may be determined by

1. Detecting the antigens on the red cells If an individual has A antigen on his red cells, he is said to belong to group A, those having B antigen on red cells belong to group B, those having both the antigens belong to group AB and those who have neither A nor B antigen on their red cells belong to group O. This process of grouping is known as cell grouping.

2. Detecting antibodies in the serum According to Landsteiner's rule, corresponding antigens and antibodies cannot co-exist in the same person's blood. For example, persons who are blood group A cannot have antibodies to antigen A (anti-A) in their blood, and group B persons can not have anti-B in their blood. However, for ABO system, natural antibodies are present in the blood of every individual. Therefore, group A person will not have anti-A in his serum, but will possess antiB because B antigen is absent on his red cells. This process of grouping is known as serum grouping.

This can be summarized thus:

BLOOD GROUP	ANTOGEN ON RED CELLS	ANTIBODIES IN SERUM
A	A	ANTI-B
B	B	ANTI-A
AB	AB	NONE
O	NONE	ANTI-A, ANTI-B

ABO Grouping Sera Selection and preparation ABO grouping sera are usually obtained from selected donors whose antibody levels are suitable for use as a laboratory reagent. A satisfactory antiserum must have: 

1. Specificity It must be specific for the antigen to be detected, under the test conditions. The specificity of the antiserum can be established by testing equal volumes of serum against 2-5% suspensions of red cells of ABO groups. Agglutination should occur only with appropriate cells. 

2. Sufficient titre A serum to be used for grouping should have an anti-A titre of 512 and anti-B titre of 256 when titrated against A and B cells respectively.

To find the titre, prepare serial doubling dilutions of the antiserum (e.g. 1:2 to 1:1024) in saline and add cells of the appropriate group. The titre is the reciprocal of the highest dilution showing agglutination. 

3. Avidity It is the power of antibody to agglutinate quickly and strongly. A suitable serum should produce complete agglutination in 30 seconds when mixed with a 2-5% suspension of appropriate cells. A vidity of the antiserum to A antigens is essential to detect weak subgroups of A.

The antisera obtained from the above sources should be purified to remove unwanted fractions, distributed into small amounts under aseptic conditions, labelled and stored at 4°C. 

Lectins Extracts of seeds of some plants contain certain substances that can agglutinate red cells. In most plants, the extracts do not seem to exhibit antigen specificity and bring about agglutination irrespective of the blood group antigen. However, there are two extracts from seeds which can be used in blood grouping.

1. Dolichos bifloris (commonly known as horse gram): If diluted appropriately, this extract reacts specifically with A, antigen and does not react with A.

2. Ulex europaeus (common gorse): This reacts specifically with H-substance and agglutinates A2, A B and Ocells more strongly than A, B or B cells.

Preparation of Red Cell Suspensions

Suspensions of washed red cells having known and unknown antigens on their surface are required for various tests in the laboratory. The washing of cells is necessary to remove plasma, which may interfere with the reaction of the cells. In addition, blood group substances in plasma may neutralise the antiserum leading to false negative results. 

It is also necessary to use a red cell suspension of appropriate strength for optimum reaction with antibody molecules. The red cells are suspended in an isotonic solution of sodium chloride. (0.15 mol/L or 8.5 g/L), pH adjusted to 7.0.

Technique

(1) Place 0.2 to 0.5 ml of blood in a test tube.

(2) Fill the tube with saline to 1 cm from the top.

(3) Centrifuge at 200 g for 1-2 minutes to obtain packed red cells.

(4) Remove the supernatant by pouring off the saline in one quick, continuous movement or by using a pasteur pipette.

(5) Tap the tube to re-suspend the cells in the residual fluid. This constitutes one wash.

(6) Repeat the procedure with the same cells at least twice. The last wash should show clear, colourless supernatant with no signs of haemolysis.

(7) To make a 2% cell suspension, add 1 volume of packed red cells to 49 volumes of saline (e.g. 0.1 ml cells and 4.9 ml saline).

(8) To make a 5% cell suspension, add 1 volume of packed cells to 19 volumes of saline.

(9) To make a 10% cell suspension, add 1 volume of packed cells to 9 volumes of saline.

Reaction of Antisera with Red Cells

The reaction between the antiserum and red cells can occur in various ways: 

Agglutination If the red cells and the antiserum contain corresponding antigen and antibody, they will react to bring about agglutination (clumping) of the red cells. If either antigen or antibody is absent, there will be no agglutination.

Haemolysis Sometimes, the antigen-antibody reaction may result in haemolysis due to activation of the complement system. Complement, if present in the antiserum, can bind to the antigen-antibody complex, and lyse the red cells. Complement can be easily inactivated by heating at 56°C for 30 minutes.

Rouleaux formation A high concentration of globulin in patient's serum can hold the red cells together to appear like a stack of coins. This is rouleaux formation and can be mistaken for agglutination.

Note

The speed and strength of antigen-antibody reactions are affected by many factors. It is necessary to use optimum concentrations of antigen and antibody. Similarly, the physical conditions, such as the pH and temperature, should be maintained at an optimal level for the test. Most blood group antibodies show optimum activity at pH 6.5-7.5 and at 37°C.

ABO GROUPING METHODS

Method 1: The Tile Method

Reagents

1. ABO grouping anti-sera: Anti-A, anti-B and Anti-AB.

2. ABO control cells (10% suspension): Az cells, B cells, O cells. 

Specimen

1. 10% suspension of patient's cells in saline.

2. Patient's serum.

Technique

(1) Mark a clean white tile with 16 wells as shown in Fig.

ANTI –A	ANTI-B	ANTI A+ ANTI B	PATIENT”S SERUM
PATIENT CELLS	PATIENT CELLS	PATIENT CELLS	PATIENT CELLS
A2 CELLS	A2 CELLS	A2 CELLS	A2 CELLS
B CELLS	B CELLS	B CELLS	B CELLS
O CELLS	O CELLS	O CELLS	B CELLS
ABO GROUPING -CELLS

(2) Place one drop of anti-A serum in all the wells of row 1, one drop of anti-B serum in row 2, one drop of anti-AB serum in row 3 and one drop of patient's serum in row 4.

(3) Place one drop of 10% suspension of patient's cells in all the wells of the horizontal row 1, 10 % A cells in row 2,10 % B cells in row 3 and 10% O cells in row 4.

(4) Mix the cells and the serum in each well using a separate applicator stick.

(5) Allow to stand at room temperature for five minutes.

(6) Rock the tile gently and read the results in the control and test wells macroscopically.

Results for the reactions of the control and the test serum, refer to Table

Method 2: The Tube Method

Reagents

1. Control blood grouping sera : anti-A, anti-B, anti-AB

2. Control red cells: 2% suspension of A cells, B cells and O cells.

Specimen 1.2% suspension of patient's red cells 2. Patient's serum

Technique

(1) Arrange and label six tubes from one to six in a rack.

(2) Add 1 drop of anti-A serum in tube 1, one drop of anti-B serum in tube 2 and one drop of antiAB serum in tube 3.

(3) Add one drop each of 2% red cell suspension of patient's cells in tubes 1, 2 and 3.

(4) Add two drops of patient's serum in each of the tubes 4,5 and 6.

(5) To tube 4, add 1 drop of 2% suspension of group A red cells, in tube 5, add 1 drop of 2% group B cells and in tube 6, add 1 drop of 2% group O cells.

(6) Mix the contents of all the tubes by shaking the rack

(7) Allow the tubes to stand at room temperature for two hours. Alternatively, centrifuge the tubes at 200g for 2-3 minutes after five minutes incubation.

(8) After incubation or centrifugation, re-suspend the cells by tapping the tubes.

(9) Read the results microscopically. 

Caution:- It is important to rinse the pasteur pipette used in transferring the cells on to the slides after each transfer.
 

Results:- The findings in the first three tubes with known antisera must agree with those in tubes 4,5 and 6 with known red cell antigens. The reactions of the ABO groups are as shown in Fig. 8.4. The patient's blood group should be interpreted accordingly.

ABO GROUPING: REACTION OF CELLS WITH KNOWN ANTISERA
GROUP	ANTI-A	ANTI-B	ANTI-A+B(GROUP O SERUM)
A	+	-	+
B	-	+	+
AB	+	+	+
O	-	-	-