DHA Standard Operating Procedures Template

Standard Operating Procedures Title: Direct Antiglobulin Test (DAT), Using Polyspecific
Ownership: Effective Date: 04/10/2014 Code: DHA/PGD/COM/TS/017
Pathology & Genetics Department. Revision Due Date:04/10/2017 Type:
Revision No: 03 Technical
First Edition Date:04/10/2008

Applies to:
Rashid Hospital Dubai Hospital Latifa Hospital Hatta Hospital PHC
1.0 Purpose:
To perform Direct Antiglobulin test (DAT) -Direct Coombs' test (DCT)-, using IgG+C3d polyspecific anti
human globulin reagent (AHG), which recognizes IgG antibodies, complement component as well as the IgM
antibodies on surface of red blood cells, since IgM always fixes complement in vivo.

2.0 Principle:
The direct antiglobulin test is based on the principle of haemagglutination. The addition of IgG+C3d
polyspecific AHG serum induces agglutination of the red blood cells sensitized in vivo. It determine if red
blood cells have been coated in vivo with immunoglobulin, complement or both. The bound antibodies could be
autoantibodies, as in AIHA, or alloantibodies-as in HDN, transfusion reaction, and drug induced Hemolytic
complications.

3.0 Special Safety Precautions:

3.1 Refer to Lab Safety Manual & apply the universal safety precautions- as indicated.
3.2 All reagents should be treated as potentially infectious.
3.3 Wear protective lab gown & gloves throughout the procedure.
3.4 Special protective measures, conditions for clinical waste disposal & disinfection should be followed
in accordance to Lab Infection Control Guidelines.

SOP No Effective Date Revision Due date Revision No First Edition Date
DHA/PGD/COM/TS/017 05/10/2014 04/10/2017 03 04/10/2008

Pages 1 of 8 Corporate Excellence Department ‐ Form No (CED‐SOP‐Template 001 E)

 DHA Standard Operating Procedures Template
4.0 Specimen:
Aseptically and freshly drawn sample, with or without anticoagulant. If not immediately tested, samples can be
stored between 2 - 8ºC & must be examined within 48 hours. Blood specimens exhibiting gross haemolysis or
contamination should not be used. Donor blood stored in citrate anticoagulant may be tested until the expiry
date of the donation.
At the time of testing, centrifuge the sample at 1200g for 3 minutes.

5.0 Reagents /Supplies:

5.1 Polyspecific AHG (IgG+C3d).
5.2 Coombs Control –IgG &/or complement -sensitized RBCs.
5.3 Isotonic saline solution (0.9% NaCl).
5.4 10 or 12 X 75 mm test tubes.
5.5 Test tube rack.
5.6 Pipettes.

6.0 Equipment:
6.1 Calibrated Centrifuge.
6.2 Cell washer

7.0 Quality Control:

7.1 Validate all negative reactions by adding IgG &/or complement- sensitized red blood cells.
7.2 The reagents must be stored between 2 – 8°C, inspected & tested each day of use. Make sure the reagent
is stored in the refrigerator between uses.
7.3 Don't use reagent beyond expiry dates & don't use damaged or leaking reagents.
7.4 Standardization of equipments used is a must.
7.5 Follow the reagent manufacturer's QC instructions.

SOP No Effective Date Revision Due date Revision No First Edition Date
DHA/PGD/COM/TS/017 05/10/2014 04/10/2017 03 04/10/2008

Pages 2 of 8 Corporate Excellence Department ‐ Form No (CED‐SOP‐Template 001 E)

 DHA Standard Operating Procedures Template
8.0 Procedure:
8.1 Check name & ID number of the samples to be tested.
8.2 Allow the reagents to reach room temperature (18 -25 ºC).
8.3 Label the tube with the patient name or health card number or accession number or donor
number, or as appropriate.
8.4 Prepare 2%-5% patient red cell suspension by adding 20 uL of packed cells to 0.5 ml (500ul) isotonic
saline.
8.5 Label another tube as in step 8.3 and dispense 1 drop of a 2%-5% of patient red cell suspension.
8.6 Wash the tube three times with saline. Completely decant the final wash.
8.7 Immediately add two drops of AHG reagent.
8.8 Gently shake the tubes to homogenize the mixture.
8.9 Centrifuge at 3400 rpm for 20 seconds or as appropriate.
8.10 Gently shake the tubes so as to detach the red blood cells pellet.
8.11 Examine the cells macroscopically for agglutination, and then grade the strength of reaction-as
Attachment 3: Grading Tube Agglutination Reactions.
8.12 Confirm the validity of negative test by adding one drop of check cells or IgG sensitized cells.
8.13 Read the reaction immediately & record the findings in Transfusion Services daily registers, as
appropriate and Sunquest (laboratory system).

9.0 Calculations: N/A

10.0 Expected Values/Results:

*Reaction DAT Result

0 Negative
± to 4+ Positive

*Test & controls reaction pattern

SOP No Effective Date Revision Due date Revision No First Edition Date
DHA/PGD/COM/TS/017 05/10/2014 04/10/2017 03 04/10/2008

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 DHA Standard Operating Procedures Template
11.0 Interpreting/ Reporting Results:
11.1. If there is agglutination, the reaction is positive. The agglutination of red blood cells in the presence of
AHG indicates that cells have been sensitized by IgG &/or complement components.
11.2. If there is no agglutination observed the reaction is negative, indicating that IgG &/or complement
components have not been detected on surface of RBCs.
11.3. Confirm the validity of negative test by adding IgG &/or complement- sensitized red blood cells.
11.4. Check the previous record of the patient if any and verify your results.
11.5. Enter the results in blood bank records & computer.

12.0 Procedure Notes:

12.1 Positive DAT result requires parallel use of monospecific Anti-IgG & Anti-C3d reagent sera.
12.2 The agglutination reactions must be read immediately after centrifuging & resuspending.
12.3 False negatives may be observed if red blood cells are not sufficiently washed.
12.4 In HDN, the DAT may be negative, particularly in ABO incompatibility.

13.0 Method limitations:

13.1 Only qualified personnel should perform the test.
13.2 It is imperative to use the calibrated dropper provided in the reagent vial to dispense the reagent drop.
13.3 It is imperative to work with clean supplies and non-contaminated reagents- bacterial or other
contamination.
13.4 Storage conditions must be observed.
13.5 Expiry date and reagent lot number must be verified & documented in reagent’s daily QC sheet.

14.0 Tools/Attachments Forms:

14.1 Attachment 1: Grading Tube Agglutination Reactions.

15.0 References:

SOP No Effective Date Revision Due date Revision No First Edition Date
DHA/PGD/COM/TS/017 05/10/2014 04/10/2017 03 04/10/2008

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 DHA Standard Operating Procedures Template
15.1 Manufacturer’s instructions leaflet
15.2 AABB Technical Manual, 16th Edition, 2008.

16. Revision History

New issue Part revision Complete revision

Date Status Change Reference Section

04/10/2008 Deleted New
Modified
Added
14/09/2010 Deleted 4.0: 5 ml removed.
Modified 3.2, 8.2, 11.3, 11.4, 13.5: added points.
Added 8.9: Reference attachment added.
14.0: Attachment added.
15.2: Reference edition changed.
20/09/2012 Deleted Hatta hospital is included
Modified AWH changed to Latifa AABB Technical Manual
Added 2.0 last sentence added 16th edition
4.0 aseptically and sentence 3 and 4 added Manufacturer’s
6.2 added instructions leaflet
8.3 to 8.6, 8.12 added
8.13 Transfusion Services daily registers
and Sunquest added

30/09/2014 Deleted AABB Technical Manual

Modified 16th edition
SOP No Effective Date Revision Due date Revision No First Edition Date
DHA/PGD/COM/TS/017 05/10/2014 04/10/2017 03 04/10/2008

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 DHA Standard Operating Procedures Template
Added 1. complement component added. Manufacturer’s
2. Edited . instructions leaflet
8.4 four times removed.
8.9 as appropriate added.
New template used
Search words: Standard Operating
Procedures

17. Search words: Standard Operating Procedures

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DHA/PGD/COM/TS/017 05/10/2014 04/10/2017 03 04/10/2008

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 DHA Standard Operating Procedures Template

Attachment 1
GRADING TUBE AGGLUTINATION REACTIONS

 The purpose of grading reactions is to allow comparison of reaction strengths.

 This is beneficial in detecting multiple antibody specificities or antibodies exhibiting
dosage.
 Grading agglutination reactions gives an indication of the relative amount of antigen
or antibody present.
 All tubes tests should be graded.
 The technique used in the resuspension of the cells will affect the grading of the
reaction.

Interpretation of Agglutination Reactions

 4+ = one solid agglutinates.

 3+ = several large agglutinates.
 2+ = medium-size agglutinates; clear background.
 1+ = small agglutinates; cloudy background.
 1+W = very small agglutinates, cloudy background.
 ± or w+ = Barely visible agglutination; cloudy background.
 0 = negative, no agglutination.
 MF = mixed field. Mixture of agglutinated & unagglutinated red.
 H = Complete hemolysis (a positive reaction).
 PH = Partial hemolysis, some red cells remain.

SOP No Effective Date Revision Due date Revision No First Edition Date
DHA/PGD/COM/TS/017 05/10/2014 04/10/2017 03 04/10/2008

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 DHA Standard Operating Procedures Template

Refer to the below illustrations for comparison.

References:

 AABB Technical Manual, 16th Edition, 2008.

http://faculty.matcmadison.edu/mljensen/BloodBank/lectures/Basic_Laboratory_techniques&Re
agents.htm

SOP No Effective Date Revision Due date Revision No First Edition Date
DHA/PGD/COM/TS/017 05/10/2014 04/10/2017 03 04/10/2008

Pages 8 of 8 Corporate Excellence Department ‐ Form No (CED‐SOP‐Template 001 E)