ABO blood grouping

Cell and Serum Testing

The procedure describe the method of detection of ABO antigens on the red cells and reciprocal antibodies in the serum (Land Steiner’s Law) It provides guidance for the use of blood grouping reagents (anti-sera and standard red cells) in order to detect week variants, acquired antigens in the Bombay (OH) blood group, and irregular red cell antibodies. It is the responsibility of the laboratory technician or supervisor in the red cell serology lab to perform the ABO grouping of donors and patients. If any discrepancy is encountered in cell and serum grouping, all tests should be repeated by the same technician using anti-Ai and anti-H lectin when required. If the discrepancy persists, the sample should be referred to an advanced red cell serology laboratory for further workup. Documentation of results is the responsibility proof of the staff performing the test.

Basic Techniques for ABO groups

1. Slide test
2. Tube method
3. Microplate method (not done)
4. Gel technology

Slide Test Method of Blood Grouping

Principle:

For detection of type of agglutinogen present on the surface of Rest Blood Cell and type of Agglutinin present in the test Seru

Materials

Materials required as follows.

Procedure

1. Take a clean glass slide and divide it into two halves with a glass-marking pencil.

2. Place 1 drop of anti-A in the first part and, anti-B in the second part of the glass slide.

3. Place one drop of anti-AB on another slide.

4. Add 1 drop of the 30% suspension (in saline) of the cell to be tested to each part of the slide.

5. Mix the reagents and the red cells with a clean plastic or glass stick separately.

6. Gently rotate the slide continuously for 2 minutes.

7. Read, interpret and record the results.

Forward Grouping			Group Interpretation	Reverse Grouping
Anti-A	Anti-B	Anti-AB		A1 Cell	B Cell
+ + + +	–	+ + + +	A	–	+ + + +
–	+ + + +	+ + + +	B	+ + + +	–
+ + + +	+ + + +	+ + + +	AB	–	–
–	–	–	0	+ + + +	+ + + +

All negative results must be seen under a microscope.

Interpretation

• Strong agglutination of red cells against anti-sera (ABO typing reagents) should be interpreted as positive results.
• Agglutination of anti-A reagent suggests the A group.
• Agglutination of anti-B reagent suggests the B group.
• Agglutination of both anti-A and anti-B reagents suggests the AB group.
• No agglutination of anti-A and anti-B reagents suggests the O group.

Procedure

1. Put one drop of anti-A reagent in a clean, labeled test tube.
2. Put one drop of anti-B reagent in a clean, labeled test tube.
3. Put one drop of anti-A, B reagent in a clean, labeled test tube.
4. Add one drop of 5% cell suspension of the red cells (to be tested) to each test tube.
5. Mix the contents of the tube gently and centrifuge, then according to the reagent manufacturer’s direction (1 minute at 1000 RPM).
6. Gently re-suspend the cell buttons and examine for agglutination.
7. Read, interpret, and record the test results.

Interpretation

1. Agglutination or hemolysis of tested cells represents positive result.
2. No agglutination represents a negative test result.

TESTING SERUM FOR DETERMINATION OF ABO TYPE

Principles

Detection of agglutinin like anti-A and anti-B present in the serum tested by known corresponding antigen present in the red cells of the respective blood group.

Materials

1. 75 X 10 mm plastic or glass tubes

2. Test serum

3. Freshly prepared 5% suspensions of A, B & O cells.

4. Pasture pipette

5. Normal saline in clean glass beaker

6. Table lamp

7. Sticker labels

8. Cotton

9. Waste beaker

10. 0.1% Sodium hypochlorite

Procedure

1. Label three clean test tubs as ‘A’, ‘B’, ‘O’.

2. Add 1 drop of serum to each tube.

3. Add one drop of 5% suspension of ‘A’ cell to the tube labeled ‘A’.

4. Add one drop of 5% suspension of ‘B’ cell to the tube labeled ‘B’.

5. Add one drop of 5% suspension of ‘O’ cell to the tube labeled ‘O’.

6. Mix the contents of the tube gently and centrifuge at 1000 RPM for 1 minute.

7. Examine for any evidence of hemolysis or agglutination.

8. Read, interpret, and record the test result.

Interpretation

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