DHA Standard Operating Procedures Template
Standard Operating Procedures Title: Indirect Antiglobulin Test (IAT), LISS (Tube Method)
Ownership: Effective Date: 04/10/2014 Code: DHA/PGD/COM/TS/014
Pathology & Genetics Department. Revision Due Date:04/10/2017 Type:
Revision No: 03 Technical
First Edition Date: 01/08/2008
Applies to:
Rashid Hospital Dubai Hospital Latifa Hospital Hatta Hospital PHC
1.Purpose:
To perform Indirect Antiglobulin test (IAT) - also known as Indirect Coombs' test (ICT), or Antibody Screen
(AS), for detecting the presence or absence of immune red cells antibodies in patient's serum, using IgG+C3d
polyspecific anti-humanglobulin reagent (AHG) that recognizes IgG antibodies and IgM antibodies as well as
complement proteins (c3d).
2.0 Principle:
The Indirect Antiglobulin test is based on the principle of haemagglutination. The addition of polyspecific AHG
(IgG+C3d) reagent induces agglutination of the red blood cells sensitized in vitro. It detects the presence of
immune antibodies (against donor/reagent red cells) present in patient's serum. Specific antibody will attach to
the corresponding antigen on the surface of red blood cells. Patient may develop immune antibodies, when
exposed to red cell antigens by transfusion and/or pregnancy. In vitro reaction will take place as follows; RBCs
exposed to IgG antibodies, antibodies will bind to the RBCs carrying the corresponding antigen (sensitization),
and after washing, IgG+C3d AHG will be added inducing agglutination of sensitized RBCs.
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.
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DHA Standard Operating Procedures Template
3.4 Special protective measures, conditions for clinical waste disposal & disinfection should be followed in
accordance to Lab Infection Control Guidelines.
4.0 Specimen:
Aseptically and freshly drawn blood sample, collected in anticoagulant (EDTA, heparin, CPD-A or citrate) or
without anticoagulant. It should be tested as soon as possible after collection. If testing will be delayed or the
sample will be used for future testing, plasma or serum should be separated from the red cells and to be stored at
2-8 ºC to be examined within 48 hours. Blood specimens exhibiting gross haemolysis or contamination should
not be used. Clotted sample to be used must be cleared by centrifugation at 1500g for 10 minutes or by
acceptable alternative g force and time.
5.0 Reagents /Supplies:
5.1 Polyspecific AHG (IgG+C3d).
5.2 Antibody Screening Cells, commercially prepared (e.g. Ortho C1 & C2 or BioRad CI, CII, CIII).
5.3 Coombs Control (IgG-coated RBC).
5.4 Isotonic Saline Solution (0.9% NaCl).
5.5 LISS
5.6 10 or 12 X 75 mm test tubes.
5.7 Test tube rack.
5.8 Pipettes
6.0 Equipment:
6.1 Cell washer/Centrifuge.
6.2 Water bath, at 37 ± 2 °C.
7.0 Quality Control:
7.1 Validate all negative reactions by adding check cells (IgG sensitized red blood cells). If this gives
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DHA Standard Operating Procedures Template
positive reaction, the activity of AHG is confirmed, and the negative reaction prior to the addition of the
sensitized red cells is validated.
7.2 Standardization of equipments used is a must.
7.3 The reagents must be stored between 2 – 8 °C. It's also advisable to minimize the reagent time outside
refrigerator between uses.
7.4 Don't use reagent beyond expiry dates & don't use damaged or leaking reagents.
7.5 Follow the reagent manufacturer's QC instructions.
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 tubes to be used with patient ID or name or accession number and if the test will used
more cells as of antibody screening, the corresponding cell number can be used for labeling the tube
(depending on number of red cell vials in the antibody screening kit: can be two or three tubes)
8.4 Add 2 drops of serum/plasma to each of the properly labeled test tubes.
8.5 Add 2 drops of LISS.
8.6 Add one drop of reagent red cells to each tube
8.7 Mix thoroughly and incubate at 37 °C for 15 minutes.
8.8 Centrifuge and observe for hemolysis or agglutination. Grade and record (INC) result– as in
Attachment 3.
8.9 Wash the red blood cells 3 times with isotonic saline solution & completely decant the saline after
last wash.
8.10 Add 2 drops of AHG reagent to each dry cell button.
8.11 Mix thoroughly and centrifuge all tubes for 20 seconds at 1000rcf or for a suitable alternative g
force and time.
8.12 Gently resuspend cell button and read macroscopically or microscopically if desired for
agglutination. Grade the reaction– as in Attachment 3- and record the result.
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DHA Standard Operating Procedures Template
8.13 12. Confirm the validity of negative test results by adding IgG-coated cells (check cells).
9.0 Calculations: NA
10.0 Expected values:
AS IS INC AHG CC Interpretation
cells
C1 0 0 0 + Negative
C2 0 0 0 +
C3 0 0 0 +
Auto 0 0 0 +
IS: Immediate Spin Phase
INC: Incubation at 37 °C phase
AHG: Antihuman Globulin phase
CC: Coombs Control Cells
C1 & C2: reagent red cells
Auto: Autocontrol.
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 from the test
serum. Further testing has to be done for identification of the antibody/ies detected. For titration-
Refer to related P&Ps.
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. No further testing required.
11.3. Record your results in Blood bank records & computer.
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DHA Standard Operating Procedures Template
12.0 Procedure Notes:
12.1. False negative may be observed if RBCs are not sufficiently washed.
12.2. It's necessary to validate the negative reactions with IgG &/or complement sensitized red cells.
13.0 Method limitations:
13.1 Only qualified personnel should perform the testing procedure.
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 & expiry date must be observed.
13.5 Strict compliance with procedures for use is required.
14.0 Tools/Attachments Form:
14.1 Attachment 1: Grading Tube Agglutination Reactions.
15.0 References:
15.1 Manufacturer’s instructions leaflet.
15.2 AABB Technical Manual, 16th Edition, 2008
16. Revision History
New issue Part revision Complete revision
SOP No Effective Date Revision Due date Revision No First Edition Date
DHA/PGD/COM/TS/014 04/10/2014 04/10/2017 03 01/08/2008
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Date Status Change Reference Section
01/08/2008 Deleted New
Modified
Added
3.2: added
01/08/2010 Deleted
4.0: 5 ml removed.
Modified
8.3: detailed. AABB Technical
Added
8.6, 8.8, 8.11: reference attachment added. Manual,16TH Edition
14.1: Attachment added.
15.2: Reference edition changed.
AWH changed to Latifa
Deleted Title changed, LISS added
18/09/2012 Modified 2.0 word will attach added and last sentence
Added was rephrased
4.0 word Hatta hospital is included AABB Technical Manual
aseptically and sentence 3-5 added 16th edition
5.2 commercially prepared and Biorad added Manufacturer’s
5.8 and 6.1 added instructions leaflet
8.3 added
8.9 edited
8.11 added
8.12 added
8.13 check cells added
13.5 added
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DHA/PGD/COM/TS/014 04/10/2014 04/10/2017 03 01/08/2008
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DHA Standard Operating Procedures Template
Title edited(Tube method added) AABB Technical Manual
01/10/2014 Deleted 1,2 edited 16th edition
Modified 5.2 CI, CII, CIII added Manufacturer’s
Added 6.1 centrifuge added instructions leaflet
7.1 edited. Check cells added.
8.8 attachment 3 changed to 1.
8.9 4 changed to 3.
11.1 edited.
New template used
Search words: Standard Operating Procedures
17. Search words: Standard Operating Procedures.
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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.
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DHA/PGD/COM/TS/014 04/10/2014 04/10/2017 03 01/08/2008
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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
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