STANDARD OPERATING PROCEDURE FOR ABO GROUPING TUBE METHOD

Standard Operating Procedure for ABO Grouping Tube Method

Provincial Blood Coordinating Program

TITLE: STANDARD OPERATING PROCEDURE FOR ABO GROUPING TUBE METHOD

1.0 Principle
1.1 1.2 To determine the ABO group in human blood. ABO blood groups are determined by phenotyping the recipients red cells for the presence or absence of A and B antigens and by testing the recipients plasma for the presence or absence of anti-A and anti-B.

2.0 Scope and Related Policies

2.1 The facility shall develop and maintain operating procedures for each activity that affects the safety of recipients. The facilitys SOP manual shall be available to all staff at all times to cover the activities they perform. Quality control shall be carried out as specified in an operating procedure. Testing for ABO blood groups must be preformed on properly collected and labelled blood samples from the recipients. (NL2010.014 Patient Identification and Specimen Labeling). 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). The results of the red cell and plasma test should agree. Current and previous results should be compared to identify any ABO discrepancy. The 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. Only the forward grouping is required for: 2.7.1 Infants less than 4 months of age 2.7.2 Confirmation of the ABO group on donor units

2.2

2.3 2.4

2.5

2.6

2.7

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This document may be incorporated into each Regional Policy/Procedure Manual.
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Standard Operating Procedure for ABO Grouping Tube Method

Provincial Blood Coordinating Program

3.0 Specimens
3.1 3.2 Blood sample collected in EDTA anticoagulant. Red cells from clotted samples (SST tubes with gel separator should not be used). 3.3 Venous or capillary blood sample from neonates. Note: Cord blood must not be used for pre-transfusion testing

4.0 Materials
Reagents: Anti-A anti-sera Anti-B anti-sera A1 reagent red cells B reagent red cells Isotonic saline Supplies: Test tubes (10x75mm) Transfer pipettes Test tube rack Equipment: Serological centrifuge

5.0 Quality Control

5.1 All reagents shall be used and controlled according to the manufacturers written instructions. All anti-sera must be visually inspected for contamination such as discoloration, cloudiness, turbidity and/or particulate matter. All reagent red cells must be visually inspected for hemolysis and/or discoloration.

5.2

5.3

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Standard Operating Procedure for ABO Grouping Tube Method

Provincial Blood Coordinating Program

5.4

The results of the visual inspection, reagent lot number, expiry date, date of the inspection and the individual performing the inspection must be documented. The expiry date should be checked on each reagent used. Do not use reagents beyond expiry date. A control consisting of 6-8% bovine serum albumin or a diluent control may be used with the recipients red cell suspension. 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.

5.5

5.6

5.7

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This document may be incorporated into each Regional Policy/Procedure Manual.
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Standard Operating Procedure for ABO Grouping Tube Method

Provincial Blood Coordinating Program

6.0 Process Flowchart

Determine specimen suitability NL2010.012

Label Test Tubes A/A, A/B, A1, B No

Add RBC suspension to tubes labeled A/A and A/B

Is specimen suitable? Yes Centrifuge specimen

Collect new specimen Mix contents of tubes Add plasma to tubes labeled A1 and B

Centrifuge tubes

Check specimen suitability after centrifugation

Remove tubes from centrfuge Prepare RBC suspension NL2012-033 Verify recipients identification on tubes, specimens and worksheet match Collect new specimen Add appropriate antisera to labeled tubes A/A, A/B Re-suspend cell button

No Is specimen suitable?

Yes Perform patient history check NL2010.013

Read macroscopically Add A1 and B cells to labeled tubes A1 and B

Check name on specimen matches worksheet

Grade and record

Interpret results and compare to previous reports

Yes ABO Discrepancy

Resolve Discrepancy before reporting

No Report Results

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This document may be incorporated into each Regional Policy/Procedure Manual.
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Standard Operating Procedure for ABO Grouping Tube Method

Provincial Blood Coordinating Program

7.0 Procedure
7.1 Determine specimen suitability. (See NL2010.012 Determining Specimen Suitability). Centrifuge specimen (Speed and time as recommended by manufacturers directions). Check specimen for abnormal appearance after centrifuging (e.g. hemolysis). Perform a patient history check. (See NL2010-013 Patient History Check). Ensure patient information on the sample corresponds with the patient information on the worksheet. Label tubes with the recipients identifier, anti-sera reagent (e.g. A/A, A/B) and reverse grouping cells (e.g. A1 and B). Add 2 drops of plasma to the tubes labelled A1 and B. Prepare a 3- 5 % recipient red cell suspension.(See NL2012-033 Preparation of Red Cell Suspensions) Add the appropriate anti-sera to the tubes according to manufacturers instructions. 7.9.1 Anti-A to the tube labelled A/A 7.9.2 Anti-B to the tube labelled A/B

7.2

7.3

7.4 7.5

7.6

7.7 7.8

7.9

7.10 Add 1 drop of A1 cells to the tube labelled A1. 7.11 Add 1 drop of B cells to the tube labelled B. 7.12 Add 1 drop of the recipients red cell suspension to the tubes labelled A/A, A/B. 7.13 Mix the contents of the tubes. 7.14 Centrifuge the tubes (speed and time as recommended by manufacturers directions). _______________________________________________________________________
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Standard Operating Procedure for ABO Grouping Tube Method

Provincial Blood Coordinating Program

7.15 Remove the tubes from the centrifuge. 7.16 Verify that the recipients identification on the tubes, specimens and worksheet correspond. If more than one recipient is being tested, read and record results on one recipient at a time. 7.17 Re-suspend the red cell button. 7.18 Read macroscopically for agglutination. 7.19 Grade and record test results. 7.20 Interpret results and compare to previous reports. 7.21 Any ABO discrepancy should be resolved before reporting.

8.0 Reporting / Interpretation

8.1 Interpreting ABO Test Results Reverse Grouping (Plasma) A1 cells B cells Pos Pos Neg Pos Pos Neg Neg Neg Interpretation ABO Group O A B AB

Forward Grouping (Cells) Anti-A Anti- B Neg Neg Pos Neg Neg Pos Pos Pos 8.2

Anti-sera (A/A and A/B) and recipient cells (forward group) should react at grade 3 or stronger. A1 and B reagent red cells (reverse group) should react at grade 2 or stronger with the recipients plasma. Hemolysis is considered a positive reaction in the reverse group. If available compare previous ABO group.

8.3

8.4 8.5

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This document may be incorporated into each Regional Policy/Procedure Manual.
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Standard Operating Procedure for ABO Grouping Tube Method

Provincial Blood Coordinating Program

9.0 Procedural Notes

9.1 Pre-washing the red cells is not necessary, however, if a discrepancy is detected the cells should be washed and the tests repeated. Washed red cells may reduce false positive results associated with rouleaux or autoantibodies. Positive reactions characteristically demonstrate a grade 3 to grade 4 agglutination by reagent ABO antibodies. Reactions in the reverse grouping (serum and reagent red cells) are often weaker. Reactions that demonstrate less than grade 2 reactions should be further investigated to ensure no discrepancy exits. Incubation for 5-60 minutes at 18-30C may be necessary to enhance the reactivity of the blood group reagents with some of the weak subgroups of A and B. 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. False positive or negative results can be caused by variables such as: 9.7.1 Improper technique 9.7.2 Contaminated materials 9.7.3 Omission of reagents or anti-sera 9.7.4 Delays in testing 9.7.5 Inadequate incubation time and temperature 9.7.6 Inappropriate centrifugation 9.7.7 Inappropriate or prolonged storage of red cells If performing the ABO grouping by automation follow manufacturers instructions for operation and resulting.

9.2

9.3

9.4

9.5

9.6

9.7

9.8

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Standard Operating Procedure for ABO Grouping Tube Method

Provincial Blood Coordinating Program

10.0 Records Management

10.1 The recipient transfusion data file in the Transfusion Medicine Laboratory shall be retained indefinitely. 10.2 All transfusion records in the recipients medical chart shall be retained in accordance with health care facility policy. 10.3 Quality control of blood components, blood products, reagents and equipment shall be retained for 5 years. 10.4 Date and time of specimen collection and phlebotomists identification shall be retained for 1 year. 10.5 Request form for serologic tests shall be retained for one month. 10.6 Documentation of staff training and competency must be kept for a minimum of ten years

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This document may be incorporated into each Regional Policy/Procedure Manual.
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Standard Operating Procedure for ABO Grouping Tube Method

Provincial Blood Coordinating Program

11.0 References
11.1 Canadian Standards Association. Blood and blood components Z902-10. Mississauga (ON): Canadian Standards Association; 2010. 11.2 Canadian Standards for Transfusion Medicine. CSTM standards for hospital transfusion services Version 3.0. Ottawa: Canadian Society for Transfusion Medicine; 2011. 11.3 Canadian Standards for Transfusion Medicine. Guidelines for serologic quality control in the transfusion medicine laboratory, 2nd ed. Saskatoon, SK: Canadian Standards for Transfusion Medicine; 1998. 11.4 Dominion Biologicals Limited. Blood grouping reagents NovacloneTM AntiA; Anti-B; Anti-A,B murine monoclonal blend manufacturers instruction. Dartmouth (NS): Dominion Biologicals Limited; 2011. 11.5 Immucor Inc. Reagent red blood cells for ABO serum grouping manufacturers instructions. Norcross,(GA) USA: Immucor Gamma; 2010. 11.6 Ortho-Clinical Diagnostics, Inc. Blood grouping reagents Anti-A, Anti-B, Anti-A,B murine monoclonal blend BioClone manufacturers instructions. Raritan (NJ): Ortho-Clinical Diagnostics, Inc.; 2004. 11.7 Ortho-Clinical Diagnostics, Inc. Reagent red blood cells Affimagen manufacturers instructions. Raritan (NJ): Ortho-Clinical Diagnostics, Inc.; 2010. 11.8 Manitoba Provincial Blood Coordinating Office. Manitoba transfusion quality manual for blood banks Version 2.0. Winnipeg (MB).Manitoba Provincial Blood Programs Coordinating Office; 2007. 11.9 Roback, J., Grossman, B., Harris, T & Hillier, C. Technical manual 17th ed. Bethesda, Maryland: AABB; 2011. 11.10 Transfusion Ontario Programs Ottawa Office. Ontario regional blood coordinating network standard work instruction manual. Ottawa (ON): Transfusion Ontario Programs Ottawa Office; 2009.

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