standard operating procedure for the direct antiglobulin test

STANDARD OPERATING PROCEDURE FOR THE
DIRECT ANTIGLOBULIN TEST

Provincial Blood
Coordinating Program
Standard Operating Procedure For
Performing The Direct Antiglobulin
Test.
TITLE: Standard Operating Procedure for Performing the
Direct Antiglobulin Test
1.0 Principle
To detect in vivo sensitization of red blood cells (RBC) and to determine which
protein is coating red cells.
A 3% saline suspension of the red blood cells to be tested is washed with saline.
Antiglobulin reagents, polyspecific (anti-IgG and C3) and/or monospecific (anti-
IgG and anti-C3) antisera, are added to the washed red cell button, mixed,
centrifuged and then examined microscopically.
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.
2.2 The facility’s SOP manual shall be available to all staff at all times to cover
the activities they perform.
2.3 Quality control shall be carried out as specified in an operating procedure.
2.4 To provide work instruction for consistent testing, interpretation and
reporting of the Direct Antiglobulin Test (DAT).
2.5 The Direct Antiglobulin Test (DAT) may be performed for investigation of
• Hemolytic disease of the newborn
• Autoimmune hemolytic anemia
• Transfusion reactions
• Sensitization caused by drugs
2.6 A DAT is required if an auto control is not done in the antibody screen and:
• Antibody identification is required and an auto control cannot be done
(e.g. limited volume of plasma)
• Antigen typing of the patient cells is required
2.7 The antiglobulin reagent shall contain antibodies to IgG and C3d component
of complement. The only exception is cord blood testing that may be
performed with a monospecific anti-IgG reagent.
_______________________________________________________________________
This document may be incorporated into each Regional Policy/Procedure Manual.
NL09-005
Version: 2.0
Effective Date: 2011-02-14
Page 2 of 12

Provincial Blood
Coordinating Program
Standard Operating Procedure For
Performing The Direct Antiglobulin
Test.
2.8 If a direct antiglobulin test performed on a clotted specimen identifies
complement on the red cell surface, the result shall be verified using an
EDTA sample.
3.0 Specimens
3.1 Blood sample collected in EDTA anticoagulant
3.2 Clotted blood sample – Cells from a clotted specimen may be used.
However, if the test is positive with anti-C3, it must be repeated with an
EDTA specimen.
4.0 Materials
4.1 Polyspecific Antihuman globulin (AHG) reagent containing Anti-IgG and
Anti-C3
4.2 Monospecific Anti-IgG reagent
4.3 Monospecific Anti-C3 reagent (anti -C3b, -C3d)
4.4 IgG coated red cells (Coombs control)
4.5 C3 coated red cells
4.6 0.9% Normal saline
4.7 Serologic centrifuge
4.8 Cell washer
4.9 Microscope
4.10 Test tubes
4.11 Transfer pipettes
Note: A control reagent is required when agglutination is observed with all antisera
tested.
5.0 Quality Control
5.1 Test polyspecific and anti-IgG reagents against IgG coated cells, C3 coated
cells and unsensitized cells at time of use or once daily as applicable.
_______________________________________________________________________
This document may be incorporated into each Regional Policy/Procedure Manual.
NL09-005
Version: 2.0
Effective Date: 2011-02-14
Page 3 of 12

Provincial Blood
Coordinating Program
6.0 Process Flowchart
Standard Operating Procedure For
Performing The Direct Antiglobulin
Test.
6.1 Process Flow – Testing with Polyspecific AHG
_______________________________________________________________________
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NL09-005
Version: 2.0
Effective Date: 2011-02-14
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Provincial Blood
Coordinating Program
Standard Operating Procedure For
Performing The Direct Antiglobulin
Test.
6.2 Process Flow– Testing with monospecific reagents
_______________________________________________________________________
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NL09-005
Version: 2.0
Effective Date: 2011-02-14
Page 5 of 12

Provincial Blood
Coordinating Program
Standard Operating Procedure For
Performing The Direct Antiglobulin
Test.
7.0 Procedure
7.1 Procedure for Testing with Polyspecific AHG
7.1.1 Check specimen suitability. Ensure patient information on
specimen matches requisition. (See NL2010-012 Determining
Specimen Suitability)
7.1.2 Check patient history. (See NL2010-013 Patient History
Check)
7.1.3 Wash an aliquot of red blood cells to be tested at least once
with normal saline. Prepare a red cell suspension in normal
saline (concentration as indicated in manufacturer’s directions)
7.1.4 Label 2 test tubes: one with patient identifier and AHG (eg. JD-
AHG) and one with patient identifier and control (eg. JD-
CTRL).
Perform the remaining steps without interruption.
7.1.5 Dispense an aliquot (volume as indicated in manufacturer’s
directions) of patient’s red cell suspension into each tube.
7.1.6 Wash 4 times with saline. Completely decant saline after each
wash and resuspend cells prior to the addition of saline for
subsequent washes.
7.1.7 Completely decant saline after final wash, blot to remove any
residual saline in order to obtain a ‘dry’ red cell button.
7.1.8 Immediately add 2 drops (or volume indicated in
manufacturer’s directions) of polyspecific AHG to tube
labelled “AHG” and add 2 drops of saline to the tube labelled
“CTRL”
7.1.9 Mix and centrifuge (speed and time as recommended by
manufacturer’s directions) .
7.1.10 Immediately after centrifugation re-suspend red blood cells and
examine macroscopically for agglutination. If negative
macroscopically, read microscopically.
7.1.11 Grade and record the results.
7.1.12 If negative, incubate test at room temperature for 5 minutes
7.1.13 After incubation mix and centrifuge, re-suspend the red cells,
read macroscopically and microscopically.
7.1.14 Grade and record results.
7.1.15 If test is negative add 1 drop (or volume indicated in
manufacturer’s directions) of IgG coated red cells to the tube
labelled “AHG”and centrifuge.
7.1.16 Examine the tube for macroscopic agglutination.
7.1.17 Grade and record results.
_______________________________________________________________________
This document may be incorporated into each Regional Policy/Procedure Manual.
NL09-005
Version: 2.0
Effective Date: 2011-02-14
Page 6 of 12

Provincial Blood
Coordinating Program
Standard Operating Procedure For
Performing The Direct Antiglobulin
Test.
NOTE: If agglutination (must be at least 2+) following the addition of IgG
coated red cells is not detected the test is invalid and must be
repeated.
If test with polyspecific AHG is positive, sample must be tested
with monospecific Anti-IgG and Anti-C3.
7.2 Procedure for Testing with Monospecific Anti-IgG and Anti C3d
7.2.1 Check specimen suitability. Ensure patient information on
specimen matches requisition. (See NL2010-012 Determining
Specimen Suitability)
7.2.2 Check patient history. (See NL2010-013 Patient History
Check)
7.2.3 Label 3 test tubes: one with patient identifier and IgG, one with
patient identifier and C3 and one with patient identifier and
control (CTRL).
Perform the remaining steps without interruption
7.2.4 Dispense an aliquot (volume as indicated in manufacturer’s
directions) of previously prepared patient red cell suspension
into each tube and wash 4 times with saline. Completely decant
saline after each wash and resuspend cells prior to the addition
of saline for subsequent washes.
7.2.5 Completely decant saline after final wash, blot to remove any
residual saline in order to obtain a ‘dry’ red cell button.
7.2.6 Immediately add 2 drops (or volume indicated in
manufacturer’s directions) of IgG to the tube labelled “IgG”, 2
drops of C3 (or volume indicated in manufacturer’s directions)
to the tube labelled “C3” and 2 drops of saline to the tube
labelled “CTRL”.
7.2.7 Mix and centrifuge (speed and time as recommended by
manufacturer’s directions) .
7.2.8 Immediately after centrifugation re-suspend red blood cells and
examine macroscopically for agglutination. If negative
macroscopically, examine the cells microscopically.
7.2.9 Grade and record the results.
_______________________________________________________________________
This document may be incorporated into each Regional Policy/Procedure Manual.
NL09-005
Version: 2.0
Effective Date: 2011-02-14
Page 7 of 12

Provincial Blood
Coordinating Program
Standard Operating Procedure For
Performing The Direct Antiglobulin
Test.
IgG
C3
Positive
Report
result
Report
result
Negative
• add 1 drop IgG coated red cells
• mix and centrifuge
• read macroscopically
• grade and record results
• report result
• incubate “C3” and “CTRL”
tubes at room temperature 5 minutes
• mix and centrifuge
• read macroscopically and microscopically
• grade and record result
▪ if still negative, add 1 drop C3 coated
red cells
▪ mix and centrifuge
▪ read macroscopically
▪ grade and record results
▪ report result
NOTE: If agglutination following the addition of the appropriate check
cells is not detected the test is invalid and must be repeated
8.0 Reporting / Interpretation
8.1 Interpretation using Polyspecific AHG reagents:
Polyspecific Control Test Result Interpretation
AHG
Negative Negative Negative
Positive Negative Positive. Test with monospecific reagents.
Positive Positive Unable to report. See note
_______________________________________________________________________
This document may be incorporated into each Regional Policy/Procedure Manual.
NL09-005
Version: 2.0
Effective Date: 2011-02-14
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Provincial Blood
Coordinating Program
Standard Operating Procedure For
Performing The Direct Antiglobulin
Test.
8.2 Interpretation using Monospecific antiglobulin reagents:
Anti-IgG Anti-
Control Test Result Interpretation
Complement
Negative Negative Negative Negative
Positive Positive Negative Positive – IgG and Complement coating
cells
Positive Negative Negative Positive – IgG coating cells
Negative Positive Negative Positive – Complement coating cells
Negative TNP Negative Neonate specimens only: Negative
Positive TNP Negative Neonate specimens only: Positive – IgG
coating cells
Positive Positive or Positive Unable to report See note
TNP
*TNP - Test not performed
9.0 Procedural Notes
9.1 Invalid tests
9.1.1 The addition of IgG control cells should demonstrate a 2+
reaction or the test is invalid and shall be repeated.
9.1.2 The addition of complement control cells should demonstrate a
1+ reaction or the test is invalid and shall be repeated.
9.2 False Negative test
9.2.1 Tests should be read immediately after centrifugation. Delay
may cause bound IgG to dissociate from the red cells causing
false negative results.
9.3 False Positive test
9.3.1 False positive test results due to in vitro coating with
complement may occur if testing is performed on a clotted
specimen.
9.4 Positive Control
9.4.1 A positive control may be due to strong cold agglutinin in the
recipient’s serum/plasma. Wash recipient cells with 37°C and
repeat DAT.
9.5 Mixed Field Reaction
9.5.1 Mixed Field reaction may indicate a transfusion reaction.
9.6 Weak Anti-complement Reactions
9.6.1 Weak anti-complement reactions may be enhanced if tubes
containing polyspecific AHG /red cells or anti-complement /
red cells are incubated for 5 minutes at room temperature after
initial reading of the test. Centrifuge and read again.
_______________________________________________________________________
This document may be incorporated into each Regional Policy/Procedure Manual.
NL09-005
Version: 2.0
Effective Date: 2011-02-14
Page 9 of 12

Provincial Blood
Coordinating Program
Standard Operating Procedure For
Performing The Direct Antiglobulin
Test.
10.0 Records Management
10.1 The recipient transfusion data file in the transfusion service laboratory shall
be retained indefinitely.
10.2 All transfusion records in the recipient’s medical chart, shall be retained in
accordance with health care facility’s retention policy for medical records.
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 phlebotomist’s 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.
10.7 Refer to CSTM Standards, Appendix A and CSA Standards, Table 4 for
additional record retention requirements.
_______________________________________________________________________
This document may be incorporated into each Regional Policy/Procedure Manual.
NL09-005
Version: 2.0
Effective Date: 2011-02-14
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Provincial Blood
Coordinating Program
Standard Operating Procedure For
Performing The Direct Antiglobulin
Test.
11.0 References
11.1 American Association of Blood Banks. Standards for blood banks and
transfusion services, 26 th ed, Bethesda, Maryland: American Association of
Blood Banks; 2009.
11.2 Becher M. Technical manual. 15 th ed. Bethesda, Maryland:
American Association of Blood Banks; 2005.
11.3 Canadian Standards Association. Blood and blood components Z902-10.
Mississauga (ON): Canadian Standards Association; 2010.
11.4 Canadian Standards for Transfusion Medicine. CSTM standards for hospital
transfusion services Version 2.0. Ottawa: Canadian Society for Transfusion
Medicine; 2007.
11.5 Dominion Biologicals Limited. Anti-Human Globulin Novaclone TM
Anti-C3d monoclonal blend manufactures’ instructions. Dartmouth (NS):
Dominion Biologicals Limited; 2007.
11.6 Dominion Biologicals Limited. Anti-Human Globulin Novaclone TM
Anti-IgG, –C3d polyspecific manufactures’ instructions. Dartmouth (NS):
Dominion Biologicals Limited; 2008.
11.7 Dominion Biologicals Limited. Anti-Human Globulin Novaclone TM
Anti- IgG monoclonal blend manufactures’ instructions. Dartmouth (NS):
Dominion Biologicals Limited; 2008.
11.8 Immucor Inc. Checkcell® (Weak) antiglobulin control IgG-coated pooled
red blood cells manufacture’s instructions. Norcross,(GA) USA: Immucor
Gamma; 2007
11.9 Immucor Inc. Complement control cells manufacture’s instructions.
Norcross,(GA) USA: Immucor Gamma; 2005.
11.10 Ortho-Clinical Diagnostics, Inc. Anti-human globulin Anti-C3d
monoclonal blend manufactures’ instructions. Raritan (NJ): Ortho-Clinical
Diagnostics, Inc.; 1999.
_______________________________________________________________________
This document may be incorporated into each Regional Policy/Procedure Manual.
NL09-005
Version: 2.0
Effective Date: 2011-02-14
Page 11 of 12

Provincial Blood
Coordinating Program
Standard Operating Procedure For
Performing The Direct Antiglobulin
Test.
11.11 Ortho-Clinical Diagnostics, Inc. Anti-human globulin BioClone® Anti-
IgG, -C3d polyspecific manufactures’ instructions. Raritan (NJ): Ortho-
Clinical Diagnostics, Inc.; 2005.
11.12 Ortho-Clinical Diagnostics, Inc. Anti-human globulin BioClone® Anti-
IgG manufactures’ instructions. Raritan (NJ): Ortho-Clinical Diagnostics,
Inc.; 2005.
11.13 Ortho-Clinical Diagnostics, Inc. Reagent red blood cells Ortho® Coombs
Control manufacture’s instructions. Raritan (NJ): Ortho-Clinical
Diagnostics, Inc.; 2007
11.14 Manitoba Provincial Blood Coordinating Office. Manitoba transfusion
quality manual for blood banks Version 2.0. Winnipeg (MB).Manitoba
Provincial Blood Programs Coordinating Office; 2007.
11.15 Transfusion Ontario Programs Ottawa Office. Ontario regional blood
coordinating network standard work instruction manual. Ottawa (ON):
Transfusion Ontario Programs Ottawa Office; 2009.
11.16 TraQ Program of the British Columbia Provincial Blood Coordinating
Office. Technical resource manual for hospital transfusion services, 2 nd
edition. British Columbia: British Columbia Provincial Blood
Coordinating Office; 2005.
_______________________________________________________________________
This document may be incorporated into each Regional Policy/Procedure Manual.
NL09-005
Version: 2.0
Effective Date: 2011-02-14
Page 12 of 12