Transfusion Medicine Quality Manual Standard Operation Procedure for
Direct Antiglobulin Test
Provincial Blood
Coordinating Program
_______________________________________________________________________
This document may be incorporated into each Regional Policy/Procedure Manual.
NL09-005-TMQ
Version: 4.0
Effective Date: 2016-02-15
Page 2 of 13
Standard Operation Procedure for
Direct Antiglobulin Test
1.0 Policy Statements
1.1 A direct antigloulin test (DAT) shall be performed for investigation of:
1.1.1 Hemolytic disease of the newborn;
1.1.2 Autoimmune hemolytic anemia;
1.1.3 Hemolytic transfusion reactions;
1.1.4 Sensitization caused by drugs; and
1.1.5 Complications during pretransfusion and/or compatibility testing.
1.2 A DAT is required if an auto control is not completed in the antibody screen
and:
1.2.1 Antibody identification is required and an auto control cannot be
completed (e.g., limited volume of plasma);
1.2.2 Antigen typing of the patient cells is required.
1.3 A control reagent (e.g., Saline or 6% albumin) is required when
agglutination is observed with all antisera tested.
1.4 Tests shall be read immediately after centrifugation. Delay may cause bound
IgG to dissociate from the red cells causing false negative results.
1.5 The antiglobulin reagent shall contain antibodies to IgG and C3d component
of complement.
Exception: Cord blood testing that may be performed with a
monospecific anti-IgG reagent.
1.6 If a DAT performed on a clotted specimen identifies complement on the red
cell surface, the result shall be verified using an EDTA sample.
1.7 An elution shall be performed on red cells from patients who have a positive
DAT and:
1.7.1 Have received a transfusion or red cells or platelets in the past three
(3) months;
1.7.2 Have been pregnant in the past three (3) months; or
1.7.3 The patients history is questionable or unavailable.
Provincial Blood
Coordinating Program
_______________________________________________________________________
This document may be incorporated into each Regional Policy/Procedure Manual.
NL09-005-TMQ
Version: 4.0
Effective Date: 2016-02-15
Page 3 of 13
Standard Operation Procedure for
Direct Antiglobulin Test
2.0 Linkages
Determining Specimen Suitability. Available at:
http://www.health.gov.nl.ca/health/bloodservices/pdf/determining_specimen_suitability.p
df
Guidelines for Investigation of Adverse Transfusion Reactions. Available at:
http://www.health.gov.nl.ca/health/bloodservices/pdf/Guidelines_for_Investigation.pdf
Patient History Check. Available at:
http://www.health.gov.nl.ca/health/bloodservices/pdf/patient_history_check.pdf
Patient Identification and Specimen Labeling. Available at:
http://www.health.gov.nl.ca/health/bloodservices/pdf/patient_id_and_specimen_labeling.
Preparation of Red Cell Suspensions. Available at:
http://www.health.gov.nl.ca/health/bloodservices/pdf/preparation_of_red_cell_suspensio
ns.pdf
Quality Control of Reagents and Antisera. Available at:
http://www.health.gov.nl.ca/health/bloodservices/pdf/quality_control_of_reagents_and_a
ntisera_ver1.pdf
3.0 Scope
3.1 All Transfusion Medicine Laboratory Technologists
4.0 General Information
4.1 The DAT can determine if red cells have been coated in vivo with
immunoglobulin, complement, or both.
4.2 The interpretation of a positive DAT result should take into consideration:
4.2.1 The patient’s recent drug, pregnancy and transfusion history;
4.2.2 Clinical data; and
4.2.3 Results of other laboratory tests.
Provincial Blood
Coordinating Program
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NL09-005-TMQ
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Effective Date: 2016-02-15
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Standard Operation Procedure for
Direct Antiglobulin Test
4.3 A positive control may be due to strong cold agglutinin in the recipient’s
serum/plasma. Washing of the recipient red cells with 37°C saline and
repeating DAT may correct this issue.
4.4 Mixed field reaction may indicate a transfusion reaction.
4.5 Weak anti-C3d reactions may be enhanced if tubes containing polyspecific
AHG/red cells or anti-C3d/red cells are incubated for five (5) minutes at
room temperature (RT) after initial reading of the test.
4.6 A DAT should be performed on every patient in whom the presence of
hemolysis has been established to distinguish immune from nonimmune
hemolytic anemia.
4.7 False positive test results due to in vitro coating of red cells with
complement may occur if testing is performed on a clotted specimen.
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 The expiry date shall be checked on each reagent used. Do not use
reagents beyond expiry date.
5.1.3 The addition of IgG control cells should demonstrate agglutination
or the test is invalid and shall be repeated.
5.1.4 The addition of complement control cells should demonstrate
agglutination or the test is invalid and shall be repeated.
5.2 Procedure
5.2.1 Procedure for Testing with Polyspecific Antiglobulin Reagent
(AHG)
5.2.1.1 Determine specimen suitability.
5.2.1.2 Centrifuge specimen (speed and time as recommended by
manufacturer’s directions).
5.2.1.3 Check patient history.
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Coordinating Program
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NL09-005-TMQ
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Effective Date: 2016-02-15
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Standard Operation Procedure for
Direct Antiglobulin Test
5.2.1.4 Wash an aliquot of red blood cells to be tested at least once
with isotonic saline. Prepare a 2%–5% suspension of red
cells in isotonic saline.
5.2.1.5 Label test tube with patient identifier and ‘AHG.’
5.2.1.6 Dispense 1–2 drops of red cells into labelled tube.
5.2.1.7 Wash three (3) times with isotonic saline. Completely
decant saline after each wash and re-suspend cells prior to
the addition of saline for subsequent washes.
5.2.1.8 Completely decant saline after final wash, blot to remove
any residual saline in order to obtain a ‘dry’ red cell button.
5.2.1.9 Add two (2) drops (or volume indicated as per
manufacturer’s directions) of polyspecific AHG to labelled
tube.
5.2.1.10 Mix and centrifuge (speed and time as per manufacturer’s
directions).
5.2.1.11 Immediately after centrifugation re-suspend red blood
cells and examine macroscopically for agglutination. If
negative macroscopically, read microscopically.
5.2.1.12 Grade and record the results.
5.2.1.13 If test is negative, incubate test at room temperature for 5–
10 minutes. If test is positive, perform DAT with
monospecific reagents, anti-IgG and anti-C3d, to determine
which molecules are present.
5.2.1.14 After incubation mix and centrifuge, re-suspend the red
cells. Read macroscopically and microscopically.
5.2.1.15 Grade and record results:
If test is positive, perform DAT with monospecific
reagents, anti-IgG and anti-C3d, to determine which
globulins are present.
If test is negative add one (1) drop (or volume indicated as
per manufacturer’s directions) of IgG coated red cells to
both tubes and centrifuge.
5.2.1.16 Examine the tube for macroscopic agglutination.
Provincial Blood
Coordinating Program
_______________________________________________________________________
This document may be incorporated into each Regional Policy/Procedure Manual.
NL09-005-TMQ
Version: 4.0
Effective Date: 2016-02-15
Page 6 of 13
Standard Operation Procedure for
Direct Antiglobulin Test
5.2.1.17 Grade and record results:
If positive, test is valid. DAT is negative.
If negative, test is not valid. Procedure must be repeated.
5.2.2 Procedure for Testing with Monospecific Anti-IgG and Anti-
C3d
5.2.2.1 Perform steps 5.2.1.1 to 5.2.1.4 as in procedure 5.2.1.
5.2.2.2 Label two (2) test tubes: one with patient identifier and
‘IgG’ and one with patient identifier and ‘C3d.’
5.2.2.3 Dispense 1–2 drops of red cell suspension into each
labelled tube.
5.2.2.4 Wash three (3) times with isotonic saline. Completely
decant saline after each wash and re-suspend cells prior to
the addition of saline for subsequent washes.
5.2.2.5 Completely decant saline after final wash, blot to remove
any residual saline in order to obtain a ‘dry’ red cell button.
5.2.2.6 Add two (2) drops (or volume as indicated as per
manufacturer’s directions) of Anti-IgG to tube labelled
‘IgG’ and add two (2) drops of anti- C3d to the tube
labelled ‘C3d.’
5.2.2.7 Mix and centrifuge (speed and time as per manufacturer’s
directions).
5.2.2.8 Immediately after centrifugation re-suspend red blood cells
and examine macroscopically for agglutination. If negative
macroscopically, read microscopically.
5.2.2.9 IgG
5.2.2.9.1 If negative:
Add one (1) drop (or volume indicated in
manufacturer’s directions) of IgG coated red cells.
Centrifuge, examine macroscopically, grade and
record results.
5.2.2.9.2 If positive:
Report as positive, IgG coating red cells.
Provincial Blood
Coordinating Program
_______________________________________________________________________
This document may be incorporated into each Regional Policy/Procedure Manual.
NL09-005-TMQ
Version: 4.0
Effective Date: 2016-02-15
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Standard Operation Procedure for
Direct Antiglobulin Test
5.2.2.10 C3d
5.2.2.10.1 If negative:
Incubate at room temperature for five (5)
minutes, and follow steps 5.2.1.14 to 5.2.1.15. If
test is then negative add one (1) drop (or volume
indicated in manufacturer’s directions) of
complement coated red cells. Incubate test at
room temperature for 5–10 minutes. Centrifuge,
read and record results.
5.2.2.10.2 If positive:
Report as positive, complement coating red cells.
5.2.2.11 If C3d and IgG are both positive, perform control if not
previously completed.
5.3 Guidelines (NA)
5.4 Materials
5.4.1 Reagents:
5.4.1.1 Polyspecific Anti-Human Globulin (AHG)
5.4.1.2 Anti-IgG (Monospecific)
5.4.1.3 IgG sensitized cells
5.4.1.4 Anti-C3d (Monospecific)
5.4.1.5 C3d coated cells
5.4.1.6 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
5.4.2.4 Microscope slides
Provincial Blood
Coordinating Program
_______________________________________________________________________
This document may be incorporated into each Regional Policy/Procedure Manual.
NL09-005-TMQ
Version: 4.0
Effective Date: 2016-02-15
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Standard Operation Procedure for
Direct Antiglobulin Test
5.4.3 Equipment:
5.4.3.1 Serological centrifuge
5.4.3.2 Cell washer
5.4.3.3 Interval timer
5.4.3.4 Microscope
5.4.4 Specimen:
5.4.4.1 Blood sample collected in EDTA anticoagulant.
5.4.4.2 Cord blood sample (clotted), if test is positive with anti-
C3d, the test must be repeated with an EDTA specimen.
6.0 Acronyms
DAT Direct anti-globulin test
EDTA Ethylenediaminetetraacetic acid
RBC Red blood cells
RT Room temperature
7.0 Definitions
Elution A technique that dissociates antibodies from sensitized red cells
In vitro Outside the living body
In vivo In living body of plant or animal
Macroscopically Large enough to be observed by the naked eye
Microscopically Observed with the use of a microscope
8.0 Records Management
8.1 The recipient transfusion data file in the transfusion service laboratory shall
be retained for 50 years.
8.2 All transfusion records in the recipient’s medical chart shall be retained in
accordance with health care facility’s retention policy for medical records.
Provincial Blood
Coordinating Program
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NL09-005-TMQ
Version: 4.0
Effective Date: 2016-02-15
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Standard Operation Procedure for
Direct Antiglobulin Test
8.3 Quality control of blood components, blood products, reagents and
equipment shall be retained for five (5) years.
8.4 Date and time of specimen collection and phlebotomist’s identification shall
be retained for one (1) year.
8.5 Request form for serologic tests shall be retained for one (1) month.
8.6 Documentation of staff training and competency must be kept for a
minimum of 10 years.
9.0 Key Words
Direct anti-globulin test, Coombs test, complement
Provincial Blood
Coordinating Program
_______________________________________________________________________
This document may be incorporated into each Regional Policy/Procedure Manual.
NL09-005-TMQ
Version: 4.0
Effective Date: 2016-02-15
Page 10 of 13
Standard Operation Procedure for
Direct Antiglobulin Test
10.0 Supporting Documents
10.1 Process Flow/Algorithm
Process Flow – Testing with Polyspecific AHG
Determine specimen suitability
Centrifuge specimen
Check patient history
Wash an aliquot of RBC once and prepare RBC suspension
Label test tube with ‘AHG’ and patient identifier
Dispense 1– 2 drops of RBC suspension into labelled tubes
Wash 3 times with saline
Add 1– 2 drops of AHG to labelled tube
Mix and centrifuge
Immediately re-suspend red cells and examine macroscopically and
microscopically
Preform DAT with monospecific reagents
Incubate at room temperature for 5 minutes
Mix and centrifugeRe-suspend red cells and
examine macroscopically and microscopically
Add one drop of IgG coated red cells to the tube
Examine macroscopically
Report test as Negative
Test invalid. Repeat
POS
POS
POS
NEG
NEGNEG
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Coordinating Program
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Effective Date: 2016-02-15
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Standard Operation Procedure for
Direct Antiglobulin Test
Process Flow– Testing with Monospecific Anti-IgG and Anti-
C3d
Determine specimen suitability
Centrifuge specimen
Check patient history
Wash an aliquot of RBC once and prepare RBC suspension
Label test tubes ‘C3d’ and ‘IgG’ with
patient identifier
Dispense1– 2 drops of RBC suspension into labelled tubes
Wash 3 times with saline
Add 2 drops of Anti-IgG to ‘IgG’ tube and 2 drops of Anti-C3d
to ‘C3d’ tube.
Mix and centrifuge
Immediately re-suspend and examine macroscopically and
microscopically
IgG Results C3d Results
Add IgG coated red cells (Check Cells)
Incubate at RT for 5 minutesReport as Positive
for IgG
Centrifuge and re-suspend
Report as Positive for C3d
Read macroscopically
Report test as Negative
Test Invalid. Repeat
Centrifuge and re-suspend
Read macroscopically
and microscopically
Report as Positive for C3d
Add C3d coated red cells (complement
control cells)
Centrifuge and re-suspend
Read macroscopically
Report test as Negative
NEG POS NEG POS
NEG
POS
NEG
POS
POS
NEG
Provincial Blood
Coordinating Program
_______________________________________________________________________
This document may be incorporated into each Regional Policy/Procedure Manual.
NL09-005-TMQ
Version: 4.0
Effective Date: 2016-02-15
Page 12 of 13
Standard Operation Procedure for
Direct Antiglobulin Test
10.2 Tables/Charts
Interpretation using Polyspecific AHG reagents:
Polyspecific
AHG
Control Test Result Interpretation
Negative Negative Negative
Positive Negative Positive. Test with monospecific reagents.
Positive Positive Test invalid.
Interpretation using Monospecific antiglobulin reagents:
Anti-IgG Anti-C3d Control Test Result Interpretation
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
Positive Positive Positive Test invalid.
Provincial Blood
Coordinating Program
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NL09-005-TMQ
Version: 4.0
Effective Date: 2016-02-15
Page 13 of 13
Standard Operation Procedure for
Direct Antiglobulin Test
References
AABB Standards for Blood Banks and Transfusion Services, 29th
edition pg 88.
Canadian Standards Association (2015). Blood and blood components, Z902- 15.
Toronto (ON): Author.
Canadian Society for Transfusion Medicine. (2011). CSTM Standards for Hospital
Transfusion Service. Version 3. Ottawa: Author.
Dominion Biologicals. (2011). Anti-Human Globulin NOVACLONE™ Anti-C3d:
Instructions for use. Dartmouth, NS: Author.
Dominion Biologicals. (2011). Anti-Human Globulin NOVACLONE™ Anti-IgG, -C3d
Polyspecific: Instructions for use. Dartmouth, NS: Author.
Dominion Biologicals. (2011). Anti-Human Globulin NOVACLONE™ Anti-IgG:
Instructions for use. Dartmouth, NS: Author.
Immucor. (2011). CHECKCELL®: Instructions for use. Norcross, GA: Author.
Immucor. (2010). Complement Control Cells. Instructions for use. Norcross, GA: Author.