Philippines
ABO Grouping
SOP Approvals
Title Name Signature Date
Technical Authority
Lab Director
Quality Assurance
ABO Grouping
Location: Philippines Document Code: PHI/QMS/HEM/015
Issue No: 01 Revision No: 00 Effective date: 01/06/2023
Document Review/Amendment Details:
Review Review/Amendment Details Authorized By Effective Date
No.
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ABO Grouping
Location: Philippines Document Code: PHI/QMS/HEM/015
Issue No: 01 Revision No: 00 Effective date: 01/06/2023
1. Scope & application
The scope of this document is to introduce laboratory staff to the identification of ABO
grouping and provide them with technical guidance in doing so.
2. Responsibility
Lab Personnel are responsible for complying with this SOP.
Quality Manager is responsible for ensuring that this procedure is instituted and followed
and that all technical staff have been trained in this procedure.
Lab Director is responsible for Implementation of Procedure..
3. Purpose of the examination
For studying the identification of ABO &Rh group.
4. Principle and method of the procedure used
Spin Tube method
ABO & Rh grouping tests are based on the principle of agglutination reaction. The red cell
surface is normally negatively charged and the force of this charge keeps the individual cells
apart. Agglutination is brought about by the linked of the red cells by the antibodies
bindings the antigen sites on one red cell to the antigen sites on another IgM molecules
being larger in size are able to link the red cells and bring about agglutination in a saline
medium. IgG antibodies, being smaller in size, are unable to bring about the linkage
between the red cells. IgG antibodies, however, bring about the agglutination of the red
cells, thereby allowing closer contact.
Red cells show presence or absence of blood group antigens (A,B, A&B) on their surface and
accordingly an individual’s group is A,B,AB, or O (denotes absence of A & B). There are
naturally occurring antibodies anti- A and anti-B in the plasma/ serum of individuals whose
red cells lack the corresponding antigen.
Blood Group Red cell Antigen Serum Antibodies
A A Anti B
B B Anti A
AB A+B None
O Neither A nor B present Anti A, Anti B
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ABO Grouping
Location: Philippines Document Code: PHI/QMS/HEM/015
Issue No: 01 Revision No: 00 Effective date: 01/06/2023
5. Performance characteristics
a. AMR/Linearity: NA
b. Precision CV%: NA
c. Measurement of Uncertainty (scope CV x 1.96): NA
6. Sample type, vacutainer, and additives
EDTA vacutainer
7. Patient preparation
No special patient preparation is required.
8. Environmental and safety controls
a. Room temp: 22-28°C & Humidity 30 – 80%
b. Wear personal protective equipment such as gloves and lab coats or aprons while
working in the lab.
c. Dispose of contaminated materials according to the laboratory’s Biomedical waste
disposal policies.
d. Keep your work area clean and disinfected.
9. Required equipment and reagents
Leishman stain & Buffer, slide and microscope
10. Stability & storage
The reagent is stable, unopened, and up to the stated expiry date when stored at 2-28°C..
11. Procedural steps
Slide Method
o The slide test may be performed on glass slides or ceramic opaque tiles.
o Take 2 glass slide and divide in to 2 parts by drawing a line in the middle by a
glass marking pencil.
o Label parts- A, B on one slide and similarly divided 2nd slide and label as D
o Put one drop each of Anti- A serum in part labelled A, Anti-B serum in part
labelled B, Anti- D Serum on part labelled D
o Add one drop of patient’s blood to each compartment.
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ABO Grouping
Location: Philippines Document Code: PHI/QMS/HEM/015
Issue No: 01 Revision No: 00 Effective date: 01/06/2023
o Mix the cells and reagents using a clean stick separately for each mixture spread
evenly on slide.
o Rotate the slide and leave the test for 2 min. at room temperature and look for
agglutination.
o Record the results.
Tube Method –
o Select 3 clean test tubes and labels them A, B and D
o Place two drops each of Anti-A serum in the tube labelled A, Anti-B serum in the
tube labelled B and Anti –D in tube labelled D
o Place 1-2 drops of the patient’s whole blood in test tube marked with the
patient’s name / lab I.D and add physiological saline to fill the tube. Shake the
contents well to resuspend all of the red cells. Examine the cell suspension and
remove any particles of fibrin that may be present.
o Centrifuge to pack the red cells and remove supernatant saline. Repeat this cell
wash 3 times and make an approximate 5 % suspension of red cells.
o Add two drops of the once washed 5 % suspension to each of the test tubes
labelled A, B & D and shake to mix the contents. Incubates tubes A, B& D at
room temperature for 5-10 minutes.
o Centrifuge the tubes at low speed (1000-1500 rpm) for one minute.
o Remove the tubes from the centrifuge and with a good source of the light in the
background agitate the tubes just sufficiently to remove the cells from the
bottom of the tubes, and observe for clumping of cells.
o Record agglutination or absence of agglutination.
o All negative results must be examined under microscope
Reverse Grouping
o Test Tube method (Serum grouping)
o Use a similar tube technique to test patient’s / donor’s serum against 5 % saline
suspensions of [pooled 2-3 red cells samples of group A cells, B cells and O cells
o Label three tubes A cell, B cells and O cells.
o Place two drop of the test serum in each tube.
o Add two drops of A cells to the tube labelled A, two drop of B cells to tube
labelled B and two drop of O cells to tube labelled O.
o Mix the contents of each tube by gentle shaking and centrifuge at low speed
(1000-1500 rpm) for 1 min. after 5-10 min. incubation at room
temperature
o Observe the supernatant fluid for the presence of haemolysis against well lighted
background.
o Gently disperse cell button and see for agglutination
o All negative results must be examined under microscope.
o Record the results immediately in format.
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ABO Grouping
Location: Philippines Document Code: PHI/QMS/HEM/015
Issue No: 01 Revision No: 00 Effective date: 01/06/2023
Preparation of pooled cells
o Label three tubes as A, B, O.
o Add 1 ml each of red blood cells from atleast three specimen of known group to
the respective tubes i.e., three specimen of group A, group B and group O, to the
respective tube.
o Suspend the cells in 5-7 ml. of normal saline.
o Centrifuge the tubes at 1500-2000 rpm for 5 mins.
o Decant the supernatant saline.
o Repeat steps c, d & at least three more times.
o Suspend the cells in the normal saline to prepare a 2-5% suspension of the
pooled red cells
Anti –A Anti –B Anti –D Blood Group A Cells B Cells O Cells
+ - + A Pos - + -
+ - - A Neg - + -
- + + B Pos + - -
- + - B Neg + - -
+ + + AB Pos - - -
+ + - AB Neg - - -
- - + O Pos + + -
- - - O Neg + + -
12. Calibration procedures (metrological traceability)
Not applicable
13. Quality control procedures
Inter pathologist comparison is done Quarterly and verified by the Lab Director.
External Quality Assurance.
14. Interference
a. Manufacturer’s instruction to be followed for storage.
b. Daily check reagents for appearance – should be no precipitate, particles or gel
formation by visual inspection
15. Calculation of values
Not Applicable.
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ABO Grouping
Location: Philippines Document Code: PHI/QMS/HEM/015
Issue No: 01 Revision No: 00 Effective date: 01/06/2023
16. Biological reference intervals
ABO-
Agglutination in A-A group
Agglutination in B-B group
Agglutination in A&B- AB group
No Agglutination in AB - O group
Rh-
Agglutination – Positive
No Agglutination - Negative
17. Alert/critical values
NA
18. Sample Dilution
NA.
19. Clinical reportable range
ABO-
Agglutination in A-A group
Agglutination in B-B group
Agglutination in A&B- AB group
No Agglutination in AB - O group
Rh-
Agglutination – Positive
No Agglutination – Negative
20. Laboratory clinical interpretation
Based on the results, health care providers can determine which type of blood can safely
receive:
type A blood, can only receive types A and O blood.
type B blood, can only receive types B and O blood.
type AB blood, can receive types A, B, AB, and O blood.
type O blood, can only receive type O blood.
Rh+, can receive Rh+ or Rh- blood.
If Rh-, can only receive Rh- blood.
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ABO Grouping
Location: Philippines Document Code: PHI/QMS/HEM/015
Issue No: 01 Revision No: 00 Effective date: 01/06/2023
Type O blood can be given to anyone with any blood type. That is why people with type O
blood are called universal blood donors.
21. Potential source of variation
Not Applicable
22. References
Wiliams Haematology.
Read & Understood
Following staff of PHI SANTE has read & understood this document w.e.f. 01-06-2023
Date Name
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ABO Grouping
Location: Philippines Document Code: PHI/QMS/HEM/015
Issue No: 01 Revision No: 00 Effective date: 01/06/2023
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