STANDARD OPERATING PROCEDURE (SOP): ABO Blood Grouping

BLOOD CENTRE NAME

SCOPE  AND APPLICATION: 

 This Standard Operating Procedure provides the method to be followed to determine the correct ABO group of an individual and ensure the reliability of the result. This procedure describes the method of detection of ‘ABO’ antigens forward (red cell) and reverse (serum) grouping in  donors’s or patient’s serum by tube technique.

RESPONSIBILITY :

It is the responsibility of the technician in the red cell serology laboratory to perform the ABO grouping of donors and patients. If a discrepancy is encountered in cell and serum grouping, all tests should be repeated by the same technician using new reagent and anti A1and anti H lectins if required. It is the responsibility of all staff performing the ABO grouping to ensure that quality controlled reagents and proper cell concentrations are used and to report any discrepancies to medical officer in charge of the lab who will in turn resolve and record it.

REFERENCES :

• Model standard operating procedures for blood transfusion service, WHO, New Delhi 2002 
• Technical Manual of American Association of Blood Banks, 18^th edition,2004.

MATERIALS REQUIRED :

           Equipment:

• Refrigerator to store samples and reagents at 2- 6◦ C.
• Table top centrifuge.
•  Microscope.

            Specimen:

• Clotted and anticoagulated blood samples of donors/patients.
• Clotted blood sample of donors/patients.
• Test red cells suspended in native serum/plasma or saline.

            Reagents:

• Commercial blood grouping antisera Anti A,-B,-D,-AB ) and polyspecific AHG.
• Group A,B and O pooled  red cells.
•  0.9%  normal saline.
•  Distilled water.

        Glassware:

• Serum tubes.
•  Test tubes(10X75mm).
•  Pasteur pipettes.
•  Glass slides.

      Miscellaneous:

• Disposal bucket with Na hypochlorite 
• 2 glass beakers.
• Aluminium/plastic racks to hold sample tubes

PROCEDURE

Principle : Based  on the identification of the presence of A or B antigen on forward grouping and that of naturally occurring anti-A, anti-B(agglutinins) in the plasma/serum of the individual whose red cells lack the corresponding antigen in the reverse grouping. 

Forward grouping (cell grouping)

• Confirm the identification of blood sample by checking registration number/patients’s name 
• Prepare 2-5% cell suspension of test red cells by washing 3 times with normal saline
• Label 3 test tubes as anti-A,anti-B and anti-AB(optional)
• Add  two drops of anti-A, anti-B and anti-AB reagent in the appropriately labelled

tubes.

• Add one drop of a 2 – 5% red cell suspension to each of the test tubes
• Mix the contents of the tubes gently and   Centrifuge at 1000 rpm for 1 minute
• Gently re-suspend the cell button and examine macroscopically for agglutination
• And records the results immediately

            (Note: Always follow manufacturer’s instructions from package insert)

   Reverse  grouping( serum grouping)

• Confirm the identification of blood sample by checking registration number/patients’s name
• Separate the serum from the clotted blood serum
• Label 3 test tubes with donors number/patients name and tubes contents (Ac,Bc,Oc)
• Add 2 drops of test serum in all labelled areas.
• Add 1 drop of 5%  suspension of reagent A red cells to the tube labeled Ac.
• Add 1 drop of 5%  suspension of reagent B red cells to the tube labeled Bc
• Add 1 drop of 5%  suspension of reagent Ored cells to the tube labelled Oc
• Mix the contents of the tubes gently and Centrifuge at 1000 rpm for 1 minute
• Gently re-suspend the cell button and examine macroscopically for agglutination
• And records the results immediately

Results:

    Depending on presence (+) or absence (-) of agglutination, the test is described as positive or negative. 

INTERPRETATION :

ABO grouping: Interpret the test using the following table

• Any discrepancy between cell and serum typing tests should be resolved before an interpretation is recorded for the donor/ patient. 

DOCUMENTATION:

• Enter the results of donor grouping in the donor grouping register and computer. 
• Enter the results of patients grouping in the patient grouping register, blood group requisition form, serial case number register and computer.