Transfusion Medicine Quality Manual

Standard Operating Procedure for ABO Grouping

Tube Method
 Standard Operating Procedure for
ABO Grouping Tube Method
Provincial Blood
Coordinating Program

1.0 Policy Statements

1.1 Each recipient blood sample for compatibility testing shall be tested for
ABO group.

1.2 The results of the red cell and plasma test must agree. Current and previous
results should be compared to identify any ABO discrepancy.

1.3 All blood group discrepancy shall be resolved and the resolution
documented before issuing red cells. If transfusion is necessary before
resolving the ABO discrepancy the recipient should receive group O red
cells and AB plasma products.

1.4 Blood group testing for A, B and D antigens only is required for:
1.4.1 Infants less than 4 months of age; and
1.4.2 Confirmation of the ABO group on donor units;

2.0 Linkages
Patient Identification and Specimen Labeling. Available at:
http://www.health.gov.nl.ca/health/bloodservices/pdf/patient_id_and_specimen_la
beling.pdf

Determining Specimen Suitability. Available at:

http://www.health.gov.nl.ca/health/bloodservices/pdf/determining_specimen_suit
ability.pdf

Patient History Check. Available at:

http://www.health.gov.nl.ca/health/bloodservices/pdf/patient_history_check.pdf

Preparation of Red Cell Suspensions. Available at:

http://www.health.gov.nl.ca/health/bloodservices/pdf/preparation_of_red_cell_sus
pensions.pdf

Quality Control of Reagents and Antisera. Available at:

http://www.health.gov.nl.ca/health/bloodservices/pdf/quality_control_of_reagents
and_antisera_ver1.pdf

_______________________________________________________________________
This document may be incorporated into each Regional Policy/Procedure Manual.

NL2010.-008 –TMQ
Version: 3.0
Effective Date: 2015-04-03
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 Standard Operating Procedure for
ABO Grouping Tube Method
Provincial Blood
Coordinating Program

Resolving ABO & Rh Blood Group Discrepancies. Available at:

http://www.health.gov.nl.ca/health/bloodservices/pdf/resolving_abo_rh_blood_gr
oup_discrepancies.pdf

3.0 Scope
3.1 All Transfusion Medicine Laboratory Technologists who are responsible for
testing and reporting transfusion medicine samples.

4.0 General Information

4.1 ABO blood groups are determined by phenotyping the recipient’s red cells
for the presence or absence of A and B antigens on red cells and by testing
the recipient’s plasma for the presence or absence of anti-A and anti-B.

4.2 ABO typing and ABO compatibility testing is the foundation of

pretransfusion testing.

4.3 Transfusion of ABO incompatible blood can be associated with acute

intravascular hemolysis, renal failure and death.

4.4 Routine tests to determine the ABO group consists of testing red cells with
anti-A and anti-B (forward group) and testing the plasma with A1 and B red
cells (reverse group).

4.5 Hemolysis is interpreted as a positive result.

4.6 Pre-washing the red cells is not necessary, however, if a discrepancy is

detected the cells should be washed and the tests repeated.

4.7 Washed red cells may reduce false positive results associated with rouleaux
or autoantibodies.

4.8 Positive reactions in the forward group characteristically demonstrate a

grade three (3) to grade four (4) agglutination. Reactions in the reverse
grouping (serum and reagent red cells) are often weaker.

4.9 Reactions in the forward group that demonstrate less than grade 2 reactions
should be further investigated.

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NL2010.-008 –TMQ
Version: 3.0
Effective Date: 2015-04-03
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 Standard Operating Procedure for
ABO Grouping Tube Method
Provincial Blood
Coordinating Program

4.10 The serum tests may be incubated at room temperature for 5 to 15 minutes
to enhance weak reactions.

4.11 A mixed field reaction maybe detected if a recipient has been transfused
with ABO compatible red cells other than that of their own ABO group.

4.12 False positive or negative results can be caused by variables such as:
4.12.1 Improper technique
4.12.2 Contaminated materials
4.12.3 Omission of reagents or anti-sera
4.12.4 Delays in testing
4.12.5 Inadequate incubation time and temperature
4.12.6 Inappropriate centrifugation
4.12.7 Inappropriate or prolonged storage of red cells.

4.13 If performing the ABO grouping by automation follow manufacturer’s

instructions for operation and resulting.

5.0 Process
5.1 Quality Control

5.1.1 All reagents shall be used and controlled according to the

manufacturer’s written instructions.
5.1.2 All anti-sera must be visually inspected for contamination such as
discoloration, cloudiness, turbidity and/or particulate matter.
5.1.3 All reagent red cells must be visually inspected for hemolysis and/or
discoloration.
5.1.4 The results of the visual inspection, reagent lot number, expiry date,
date of the inspection and the individual performing the inspection
must be documented.
5.1.5 The expiry date should be checked on each reagent used. Do not use
reagents beyond expiry date.

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NL2010.-008 –TMQ
Version: 3.0
Effective Date: 2015-04-03
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 Standard Operating Procedure for
ABO Grouping Tube Method
Provincial Blood
Coordinating Program

5.1.6 A control consisting of 6-8% bovine serum albumin or a diluent

control may be used with the recipient’s red cell suspension.
5.1.7 The reactivity of blood grouping reagents shall be confirmed each
day of use by control tests with known antigen positive and negative
red cells. Positive control cells should be selected to represent weak
expression of the specific antigen.

5.2 Procedure

5.2.1 Centrifuge specimen according to facility policy.

5.2.2 Determine specimen acceptability.
5.2.3 Perform a patient history check.
5.2.4 Ensure patient information on the sample corresponds with the
patient information on the worksheet.
5.2.4.1 Forward group:
5.2.4.1.1 Place 1 drop of anti-A in a clean, labeled test tube;
5.2.4.1.2 Place 1 drop of anti-B in a separate, clean, labeled
tube;
5.2.4.1.3 Add 1 drop of a two (2)% to five (5)% patient cell
suspension to each tube;
5.2.4.1.4 Mix the contents of the tubes gently;
5.2.4.1.5 Centrifuge for the calibrated spin time;
5.2.4.1.6 Gently resuspend the cell buttons, and examine them
for agglutination; and
5.2.4.1.7 Read, interpret, and record the test results.
5.2.4.2 Reverse group
5.2.4.2.1 Add 2 or 3 drops each of serum or plasma to two
clean, labeled test tubes;
5.2.4.2.2 Add 1 drop of A1 reagent red cells to the tube
labeled A1;
5.2.4.2.3 Add 1 drop of B reagent red cells to the tube
labeled B;
5.2.4.2.4 Gently mix the contents of the tubes; then centrifuge
for the calibrated spin time;

_______________________________________________________________________
This document may be incorporated into each Regional Policy/Procedure Manual.

NL2010.-008 –TMQ
Version: 3.0
Effective Date: 2015-04-03
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 Standard Operating Procedure for
ABO Grouping Tube Method
Provincial Blood
Coordinating Program

5.2.4.2.5 Examine the serum overlying the red cell buttons for
evidence of hemolysis. Gently resuspend the cell
buttons, and examine them for agglutination;
5.2.4.2.6 Examine the serum overlying the red cell buttons for
evidence of hemolysis. Gently resuspend the cell
buttons, and examine them for agglutination; and
5.2.4.2.7 Read, interpret, and record test results.

5.3 Guidelines (NA)

5.4 Materials

5.4.1 Reagents
5.4.1.1 Anti-A anti-sera
5.4.1.2 Anti-B anti-sera
5.4.1.3 A1 reagent red cells
5.4.1.4 B reagent red cells
5.4.1.5 Isotonic saline
5.4.2 Supplies
5.4.2.1 Test tubes (10x75mm)
5.4.2.2 Transfer pipettes
5.4.2.3 Test tube rack
5.4.3 Equipment
5.4.3.1 Serological centrifuge
5.4.4 Specimen
5.4.4.1 Blood sample collected in EDTA anticoagulant; or
5.4.4.2 Red cells from clotted samples (SST tubes with gel
separator should not be used); or
5.4.4.3 Venous or capillary blood sample from neonates. Cord
blood must not be used for pre-transfusion testing.

_______________________________________________________________________
This document may be incorporated into each Regional Policy/Procedure Manual.

NL2010.-008 –TMQ
Version: 3.0
Effective Date: 2015-04-03
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 Standard Operating Procedure for
ABO Grouping Tube Method
Provincial Blood
Coordinating Program

6.0 Acronyms
EDTA Ethylenediamine tetracetic acid
(anticoagulant)
SST Serum-separating tubes

7.0 Definitions
rouleaux The stacking up of red blood cells caused
by extra or abnormal proteins in the blood
that decrease the normal distance red cells
maintain between each other.
compatibility testing Testing to determine if the donor's blood is
compatible with the blood of an intended
recipient.
ABO discrepancy The result of red cell tests disagree with
that of the serum tests.
mixed field The presence of two populations of red
cells.

8.0 Records Management

8.1 The recipient transfusion data file in the Transfusion Medicine Laboratory
shall be retained indefinitely.

8.2 All transfusion records in the recipient’s medical chart shall be retained in
accordance with health care facility policy.

8.3 Quality control of blood components, blood products, reagents and

equipment shall be retained for 5 years.

8.4 Date and time of specimen collection and phlebotomist’s identification shall
be retained for 1 year.

8.5 Request form for serologic tests shall be retained for one month or as per
facility policy.

_______________________________________________________________________
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NL2010.-008 –TMQ
Version: 3.0
Effective Date: 2015-04-03
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 Standard Operating Procedure for
ABO Grouping Tube Method
Provincial Blood
Coordinating Program

8.6 Documentation of staff training and competency must be kept for a

minimum of ten years.

9.0 Key Words

Forward blood group, reverse blood group, ABO group

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NL2010.-008 –TMQ
Version: 3.0
Effective Date: 2015-04-03
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 Standard Operating Procedure for
ABO Grouping Tube Method
Provincial Blood
Coordinating Program

10.0 Supporting Documents

10.1 Process Flow/Algorithm
Determine
specimen
suitability Label Test Tubes Add RBC suspension
NL2010.012 A/A, A/B, A1, B to tubes labeled A/A
and A/B
No
Is specimen Collect new
suitable? specimen
Mix contents of tubes
Yes
Add plasma to
tubes labeled
Centrifuge A1 and B Centrifuge tubes
specimen

Remove tubes from

Check centrfuge
specimen Prepare RBC
suitability after suspension
centrifugation NL2012-033
Verify recipient’s
identification on
tubes, specimens and
No worksheet match
Is specimen Collect new
suitable? specimen
Add appropriate
antisera to labeled Re-suspend cell
Yes tubes A/A, A/B button

Perform
patient history
check Read macroscopically
NL2010.013
Add A1 and B cells to
labeled tubes
Check name A1 and B
Grade and record
on specimen
matches
worksheet

Interpret results and

compare to previous
reports

Yes
Resolve Discrepancy
ABO Discrepancy
before reporting

No

Report Results

_______________________________________________________________________
This document may be incorporated into each Regional Policy/Procedure Manual.

NL2010.-008 –TMQ
Version: 3.0
Effective Date: 2015-04-03
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 Standard Operating Procedure for
ABO Grouping Tube Method
Provincial Blood
Coordinating Program

10.2 Tables/Charts

Interpreting ABO Test Results

Forward Grouping (Cells) Reverse Grouping (Plasma) Interpretation

Anti-A Anti- B A1 cells B cells ABO Group
Neg Neg Pos Pos O
Pos Neg Neg Pos A
Neg Pos Pos Neg B
Pos Pos Neg Neg AB

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NL2010.-008 –TMQ
Version: 3.0
Effective Date: 2015-04-03
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 Standard Operating Procedure for
ABO Grouping Tube Method
Provincial Blood
Coordinating Program

References

Cooling L. ABO, H, and Lewis blood groups and structurally related antigens. In: Fung
M, Grossman BJ, Hillyer CD, Westhoff CM, eds. Technical manual. 18th edition.
Bethesda, MD: AABB, 2014:291-315.

Immucor Gamma. (2012) Blood Grouping Reagent: Package Insert.

Immucor Gamma. (2012) Reagent Red Cells; Package Insert.

American Association of Blood Banks, (2011). Standards for Blood Banks and
Transfusion Services, (27th ed.). Bethesda, MD: Author.

Canadian Standards Association, (2010). CAN/CSA-Z902-10, Blood and Blood

Components. Mississauga, ON: Author.

Canadian Society for Transfusion Medicine, (2011). Standards for Hospital

Transfusion Services, Version 3.0. Ottawa, ON: Author.

_______________________________________________________________________
This document may be incorporated into each Regional Policy/Procedure Manual.

NL2010.-008 –TMQ
Version: 3.0
Effective Date: 2015-04-03
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