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1. PURPOSE: To prepare cell suspension for blood ABO blood grouping

This procedure applies to all testing that requires red cell suspension preparation.
It is the responsibility of Medical Technologist (Lab) in the red cell serology
laboratory performing a given test to prepare the appropriate red cell suspension.
Every morning, the technologist must prepare A, B& O red cell suspension for the
routine use.
WHO (2002).Model standard operating procedures for blood transfusion service;
New Delhi. Mark E. Brecher (eds). Technical Manual of the American Association of
Blood Banks; 15th Edition; Bethesda, Maryland; AABB 2005; p 727.
5.1. Equipment: Calibrated Centrifuge
5.2. Reagent: 0.9% saline P a g e 19
5.3. Specimen: Anti-coagulated blood sample of donor, Donor unit segment
5.4. Glassware: Test tubes, Pasteur pipette
5.5. Miscellaneous: Disposal box
2 plastic beakers
Racks to hold sample tubes
6.1 Principle:
The ratio of serum to red cells may affect the sensitivity of agglutination tests.
A 5% red cell suspension is a common reagent in many serological procedures.
The suspension need not be exactly 5%; an approximation achieves the appropriate
serum-to-cell ratio for most test procedures.
6.2 Pooled Cell Suspension:
1. Label tubes with A, B & O groups.
2. Place 1 drop of red cells each from 3 of A group sample tubes into the A labelled
3. Place 1 drop of red cells each from 3 of B group sample tubes into the B labelled
4. Place 1 drop of red cells each from 3 of O group sample tubes into the O labelled
5. Fill the tube full with 0.9% saline to re-suspend the cells. Centrifuge the tubes
for at least 1 minute at 1000 rpm. To make 5% red cell suspension, add 1 drop of
RBC to 19 drops of saline.
6. Test the pooled cells prepared by adding the antisera (anti-A, B, AB & D) in use.

6.3 Limitations: Haemolysis of red cells from improper washing may result in false

Reverse Grouping Procedure

1. Label 2 test tubes with specimen identifying information (patients name or
initials or blood donors number). Label one tube a and one tube b.
2. Place 2 drops of the specimens serum or plasma into each tube.
3. Add one drop of A1 RBC reagent to the a tube and one drop of B RBC reagent
to the b tube.
4. Gently shake each tube to mix the contents, and then centrifuge tubes for 15-20
seconds on high speed (3500 rpm).
5. Examine for hemolysis, then gently resuspend the RBC button, and read for
6. Immediately grade the strength of the reactions and record the results on the
appropriate worksheet for this test.
7. Reporting Results Forward Grouping Unknown Cells Tested Against Reverse
Grouping Unknown Serum