NAME OF THE LAB……………..

Name Position Signature Date

Prepared by

Reviewed by

Approved by

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1. Purpose.
i. For different reasons, some patients do not have adequate quantity of
blood in the body and are treated using blood transfusions. The
transfused blood has to fulfil compatibility requirements to ovoid post-
transfusion reaction dues to incompatibility within ABO and Rhesus
systems and which can lead to death.
ii. This procedure provides instructions for performing ABO and Rhesus (D) blood
grouping using tube method.

2. Principle.
i. This method is based on immunophenotyping principle. Known antibodies (Antisera)
are used to detect unknown antigens on the red cells surface that determines the blood
groups of patient/blood donor, this is known as forward or cells grouping. Also
known A, B and O cells are used to detect unknown antibodies in the patient/donor
serum; this is known as backward or serum blood grouping. Known Anti-D is used
to detect the presence of D antigen on red cells surface which determine the Rhesus
group of an individual, either as Rh (D) Positive or Rh (D) Negative.

3. Scope
i. This procedure is used in Blood Transfusion section of ………….. Hospital
Laboratory when performing ABO & Rhesus blood group typing for donors and
patients.

4. Responsibility
 Laboratory manager and section head.
Ensure that all users have been trained, understand content of this
SOP and is documented.
 Section head and all laboratory staff.
Ensure that all necessary materials are available and of good
quality.
 Section head and all laboratory staff.
Ensure the quality of the samples.
 Section head and all laboratory staff.
Ensure proper transport of sample.
 All laboratory staff.
To determine ABO and Rhesus blood groups.
 Section head and all laboratory staff.
Ensure the verification and reporting of results.

5. Performance Characteristics.
i. Refer to the method verification report.

6. Type of Sample.
i. Whole blood collected in EDTA tube.

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7. Patient Preparation.
i. No special patient preparation required.

8. Type of sample, container and additives.

i. 2 to 5mls of whole blood in EDTA tube for forward typing.
ii. 2 to 5mls of clotted blood sample in plain tube for backward typing.

9. Required Equipment and Reagents.

Reagent Supplies Equipment

 ABO grouping test kits:  Test tubes,  Centrifuge,

 Anti –A  Test tube rack,  Refrigerator,
 Anti-B  Marker pen,  Timers,
 Anti-D (saline, IgM)  Beakers, ,  37˚C water bath.
 Incomplete anti-D (IgG)  Pasteur pipettes,
 Low Ionic Strength Solution (LISS)
 Anti-Human Globulin Serum (AHG)
 Physiological Saline
 Anti sera should be kept at 2◦C – 8◦C
10. Environmental and Safety Controls.
i. Personnel Protective Equipment must be worn at all times and samples must be
treated as potentially infectious.
ii. Room and refrigerator temperatures are monitored twice daily.
iii. All blood samples and reagents must be considered as
potentially infectious. Their handling requires the use of
appropriate PPE.
iv. For best results sample should be at less than 2days of transport
at room temperature or 7days at 4°C.

11. Calibration (Metrological traceability).

i. Ensure that calibration of the ancillary equipment (centrifuge, water
bath,timer, thermometers and Refridgerator is done annually).

12. Procedural Steps.

A. Sample preparation.

i. Arrange test tubes in the rack.

ii. To obtain serum, centrifuge the blood in the plain tube at 3000rpm for 3minutes and
extract the serum into a micro tube, clearly labeled with patient unique sample number.

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iii. Wash red cells by suspending the cells in physiological saline in the ratio of 1 to 9
respectively.

iv. Centrifuge the tube at 3000rpm for 1 minute.

v. Discard the supernatant and add physiological saline in the same ratio as in (iii) above
and centrifuge again.

vi. Repeat step (v) above for the third wash.

vii. After the third wash, discard supernatant and remain with cells.

viii. To prepare 3% cell suspension add 3 drops of washed cells to 97 drops of physiological
saline in a test tube.

ix. Clearly label the test tube with the patient’s unique identifier number.

B. Forward/Cell Grouping.

a) For each patient/donor, label 4 test tubes as Anti-A (A), Anti-B (B), Anti-AB (AB) and
anti-D (D).

b) Label the 4 tubes with patient ID.

c) Put one drop of 3% cell suspension of A, B, AB, & O Rh (D) Positive into each of the
tubes above.

d) Add one drop of anti-A, Anti-B, Anti-AB and Anti-D into the respective tubes above.

e) Spin all the tubes at 1000rpm for 1 minute

f) Observe for agglutination.

C. Backward/Serum Grouping.

i. For each patient/donor, label 3 test tubes as A cells, B cells and AB cells.

ii. Put one drop of 3% cell suspension of A, B and AB into each of the tubes above
respectively,

iii. Add one drop of patient/donor serum into each of the tubes above.

iv. Spin tubes at 1000rpm for 1minute

v. Observe for agglutination or haemolysis

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D. Procedure for Weak Rh (D)

If the above Anti-D reaction is negative, confirm weak Rh (D) as follows:
i. Add two drops of LISS

ii. Incubate at 37˚C for 15 minutes or at room temperature for 1hour

iii. Spin for 1000rpm for 1 minute.

iv. Observe for agglutination macroscopically and microscopically or for haemolysis.

v. If agglutination or haemolysis is observed at this stage, report result as Rh (D) Positive

and the procedure ends here.

vi. If there is still no agglutination, proceed as follows:

vii. Wash contents of the tube 3 times with physiological saline

viii. Discard supernatant after third wash

ix. Add one drop Anti-Human Globulin Serum (AGS)

x. Spin for 1000rpm for 1 minute

xi. Observe for agglutination (macroscopically and microscopically) or haemolysis

xii. If agglutination is observed at this stage, report results as Rh (Du) Positive

xiii. If reaction is still negative, add one drop of O sensitised cells to the tube

xiv. Spin for 1000rpm for 1minute

xv. Observe for agglutination (macroscopically and microscopically) or haemolysis

xvi. Presence of agglutination or haemolysis indicates a valid negative result.

xvii. Abscence of agglutination or haemolysis means the test is invalid, therefore it has to be
repeated.

NB: For purposes of transfusion, patients with Rh(D u) Positive can be given rhesus negative
blood

13. Quality Control Procedures.

a) Controls are done once in a week.

Controlling of anti-sera
b) Commercial anti-sera is quality controlled by reacting them with known cell
suspension of A,B, AB,O, Rh( D) Positive and Rh (D) Negative.

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c) Arrange 6 tubes labeled A, B, AB, O and D positive and D negative.

d) Put one drop of 3-5% cell suspension of A, B, AB, O, Rh (D) Positive and Rh (D)
Negative cells to the corresponding tubes above

e) Add one drop Anti-A, Anti-B, Anti-AB and Anti-D into corresponding tubes.

Controlling of O sensitised cells

f) Label two tubes as O sensitized cells and O unsensitised cells.

g) Put one drop of corresponding of 3-5% cell suspension in the two tubes above.

h) In the two tubes, add one drop of AHG each.

Controlling of Low Ionic Strength Solution

i) Label three tubes as LISS, Saline and Distilled water respectively and put one drop of
O sensitised cells in each of the tubes.

j) Add one drop of LISS, Saline and distilled water to the respective tubes above.

k) Add one drop AHG to the tubes labelled LISS and Saline Only

Common step
l) Centrifuge all tubes above at light speed (1000rpm for 1 minute).

m) Agglutination or haemolysis indicates positive reactions.

n) Expected reactions are shown in table below

o) Record results on ABO & Rh blood Group Quality Control

Table 1: Interpretation of QC results

Commercia Known cell suspensions

l Anti-sera
A cells B cells AB cells O cells Rh D Pos Rh D Neg. O sensitized Unsensitis
Cells Cells cells cells

Anti-A + - + -

Anti-B - + + -

Ant-AB + + + -

Anti-D + -

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AGS + -

LISS +++

Saline + to ++

Distilled Haemolysis
water

+ Agglutination
- No agglutination

NB: Strength of agglutination is graded from 1+ (separate agglutinates) to 3+ (one solid

agglutinate)

14. Interferences or cross reaction.

i. Clotted blood sample may lead to incorrect results during
Blood Grouping.
ii. Avoid using haemolysed samples as this may lead to false positive results.
iii. Patients who have had recent multiple transfusions may develop Allo-
antibodies that can interfere with antigen – antibody reactions.

15. Principle of procedure for calculating results.

i. Not applicable.

16. Biological reference intervals or clinical decision values.

i. Not applicable.

17. Reportable interval of examination results.

i. Blood group A, B, AB, O with Rhesus D factor Positive or
Negative.

18. Instruction for determining quantitative results.

i. Not applicable.

19. Alert/critical values.

i. Rhesus D negative to pregnancy woman.

20. Laboratory clinical results interpretation.

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Patient/Donor Cell Grouping Patient/ Donor Serum Blood group

Grouping
Anti-A Anti-B Anti-D A-cells B-cells
+ - + - + A Rh(D)Pos
- + + + - B Rh(D)Pos
+ + + - - AB Rh(D)Pos
- - + + + O Rh(D)Pos

21. Potential source of variation.

i. Clotted and hemolysed sample.

22. Definitions
i. Blood: Biological liquid containing cells, minerals, proteins
and water that circulates in vessels to bring nutrients and
protect our body against infections and order disorders.
ii. Blood groups: Classification of blood into groups according
to the presence or absence of certain erythrocytes surface
antigens and the presence or absence of specific antibodies to
these erythrocyte antigens
iii. ABO system: Blood groups characterized by the presence or
absence of Ag A, Ag B, Ag A and B on erythrocytes or the
presence or absence in the serum/plasma of specific
antibodies directed to different erythrocytes.
iv. Rhesus system: Blood groups characterized by the presence
or absence of Ag D on the erythrocyte surface.

23. Supporting documents

i. Communication Log.
ii. Haematology Request Form.
iii. Quality control log.

24. References.
i. Sample collection manual

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ii. Controlled external documents from BTS and Ministry of

Health as indicated in the master list.

Amendment Records Log:

Amendment Version Reviewed by Next review date

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User acknowledgement: I acknowledge that I have read, understand and

agree to follow this procedure.
Date Name Signature

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