1.

PURPOSE

1.1 Describing the ABO and Rhesus Blood Groupings test procedure.

2. SCOPE

2.1 The SOP applies to all technologists/technicians.

3. PRINCIPLE

3.1 ABO Grouping

The ABO bloodgroup of an individual isdetermined by the agglutination reactions

of the individual’s red cells with known anti-sera [colourcoded Anti –A (blue),
Anti-B (yellow) or Anti-AB (colourless)].This is known as forward grouping or cell
typing. The results of theforward grouping is then confirmed by adding the patients
own serum /plasma to known red cell antigens, a process known as reverse
grouping or serum typing.

3.2 Rhesus Grouping

The Rhesus factor is determined by the ability of red cell antigens to react OR the
lack of reaction, with a known Anti-Rh Sera, commonly known as anti-D.

4. EQUIPMENTREQUIRED

4.1 Sterile glass test tubes (10x75mm or12x75 mm)

4.2 Clean glass tubes (10x75mmor12x75mm)
4.3 Metal,Perspex or wooden test tube racks.
4.4 Perspextiles with shallow wells (where available and used in a laboratory)
4.5 Transfer pipettes –Pasteur pipettes (plastic or glass)
4.6 Glass slides
4.7 Waterbath at 37ºC
4.8 Centrifuge with variable speed of1,000 RPM up.
4.9 A microscope with a10x magnification (dry lens).

5. REAGENTS

5.1 Isotonic Saline (0.9%NaCl)

5.2 Anti-A –monoclonal IgM blend
5.3 Anti-B –monoclonal IgM blend
5.4 Anti AB–monoclonal IgM blend
5.5 Known Red Blood cells Type A,B and O
5.6 Anti HumanGlobulin Reagent (AHG)
5.7 Anti-Rh sera (anti D)

6. SPECIMENS REQUIRED

6.1 The choice of the blood collecting tube is the plain (red), EDTA (purple) or
heparin (green) tube.
 6.2 Suitability of samples

Haemolysed samples are not suitable. Samples are stable for up to 7 days at 2 – 8 degrees.

7. PREPARATION

7.1 Test Sample Preparation

7.1.1 Check that the Lab numbers and names on the work sheets
correspond to the name and number on the specimen tubes.
7.1.2 Remove from the sample serum /plasma as required for reverse
grouping.
7.1.3 Prepare the patient’s 2% to10% red cell suspension.

7.2 Preparinga Red Cell Suspension

7.2.1 Make a ±5% red cell suspension using thepatient red cells and
isotonic saline.
(Add1 volume red cells to ±20 volumes of Saline).
A cell suspension concentration between 2% and 10% is
acceptable.

7. TESTPROCEDURE

7.1 ForwardGrouping

7.1.1 Add 1 drop of A,B,AB, D antisera to the corresponding tubes/tiles.

7.1.2 Add 1 drop of the patient cell suspension to the tubes/tiles.

7.2 Reverse Grouping

7.2.1 Add 1 drop of the patient serum to marked tubes.tiles.

7.2.2 Add1 drop of corresponding known cells to the tubes/tiles

7.3 Incubation Period and Agglutination Check

7.3.1 Rock the tiles for 1 – 2 minutes or centrifuge tubes at 1000 rpm for 1-2
minutes or 2000-3000 rpm for 20sec to 1 min, gently resuspend the red cell
button and observefor agglutination.
7.3.2 Any agglutination reaction observed is regarded as a positive reaction.
7.3.3 When in doubt, examine the reactions microscopically.
7.3.4 The forward grouping results confirms the reverse grouping results and visa
versa and this is an internal control validation for the reagents used.
 8. FURTHER INVESTIGATION OF RHESUS NEGATIVE RESULTS

8. Incubation at 37ºC

8.1.1 Incubate the red cell suspension and Anti-D sera mixture at
37ºC for 15 to 30 minutes.
8.1.2 Centrifuge at 1000rpm for1-2 minutes or 2000-3000 rpm for 20sec to 1 min,
gently resuspend the red cell button and read microscopically for
agglutination.
8.1.3 If agglutination is present, report patient results as positive,if no
agglutination(negative) continue to do coombs test.

8.2 Coombs

8.2.1 Wash the cells at least 3 times with isotonic saline. After last wash
decant supernatant saline completely.
8.2.2 Add 1 or 2drops of AHG (Anti-HumanGlobulin)
8.2.3 Centrifuge at 1000 rpm for1-2 minutes or 2000-3000 rpm for 20 sec to 1 min,
gently resuspend the red cell button and read microscopically for
agglutination.
8.2.4 A positive agglutination result indicates the presence of Rh0 variant (DU)
and must be reported as DUpositive
8.2.5 Treat patient with Rh0 Varient (DU) as Rh positive donors and Rh
negative receivers.

9. REFERENCES