No agglutination is a negative test result and indicates
the absence of human IgG or components of complement
In all phases of the compatibility test, if no agglutination or
hemolysis is observed, then the patient and the donor may
be considered compatible. If hemolysis or agglutination
at any point till the completion of the antiglobulin phase
is observed, the patient and the donor are considered
1. If plasma is used in the indirect antiglobulin test,
the complement-dependent antibodies may not be
detected due to the absence of calcium.
2. To all negative test results, after the antiglobulin test
phase, one drop of Coomb’s control cells should be
added. If Coomb’s control cells do not agglutinate then
the compatibility test must be repeated.
3. In the indirect antiglobulin test procedure an auto
control tube (individual’s cells in his own serum)
4. Red blood cells showing a positive direct antiglobulin
test cannot be used for the indirect antiglobulin test.
preferably on a daily basis using Coomb’s control cells
6. All glassware used in the test should be scrupulously
clean dry and free from contamination with human
7. Contaminated bovine serum albumin, saline or
glassware may inactivate anti-human globulin reagent.
8. Use of various drugs and certain diseases (such as
megaloblastic anemia) are known to be associated
with a positive direct antiglobulin test.
hemolytic disease of the newborn, especially due to
ABO incompatibility may give false negative results.
10. Eryclone Anti-human globulin reagent does not
contain anti-C4 and is free from anti-T activity.
11. As undercentrifugation or overcentrifugation could
lead to erroneous results, it is recommended that each
laboratory calibrate its own equipment and determine
the time required for achieving the desired results.
PREPARING COOMB’S CONTROL CELLS Agtrol®
(Courtesy: Tulip, Starter Pack)
Anti-human globulin reagent is used in blood group
serology for performing compatibility testing, antibody
screening, antibody detection and detection of Du red
cell type. Usage of Coomb’s control cells is advocated for
functional validation of anti-human globulin reagent and
procedural validation of tests employing anti-human
Agtrol starter pack for preparing Coomb’s control cells
1. Ready-to-use, standardized-prediluted anti-D (IgG)
2. Red blood cell preserving solution for serological
Each batch of reagent undergoes rigorous quality
control at various stages of manufacture for its performance
Store the reagent at 2–8°C. Do not freeze.
The shelf life of the reagent is as per the expiry date
mentioned on the reagent vial label.
Human ‘O’ Rho(D) positive cells in presence of Agtrol
prediluted monoclonal reagent do not agglutinate but are
sensitized with IgG antibodies. After processing, these
sensitized red blood cells are resuspended in red blood
cell preserving solution for long-term storage and use.
When anti-human globulin reagent is added to these
sensitized cells the incomplete anti-D (IgG) antibodies
are agglutinated by the anti-human IgG component. The
agglutination reaction validates the serological activity of
1. In vitro diagnostic reagent for laboratory and
professional use only. Not for medicinal use.
2. Agtrol anti-D reagent is not from human source, hence,
contamination due to HBsAg and HIV is practically
3. Agtrol anti-D reagent and red cell preserving solution
contains 0.1% sodium azide as preservative. Avoid
contact with skin and mucosa. On disposal flush with
4. Extreme turbidity in both Agtrol anti-D IgG and red
blood cell preserving solution reagent may indicate
microbial contamination. Such reagent must be
No special preparation is required prior to sample collection
by approved techniques. Samples should be stored at 2–8°C
if not tested immediately. Do not use hemolyzed samples.
Anticoagulated blood using various anticoagulants should
be tested within the below mentioned time period:
¾ Sodium citrate or sodium oxalate: 14 days
¾ Clotted whole blood should be used within 14 days.
Additional Collection and Storage
Test tubes (12 × 75 mm), Pasteur pipettes, isotonic saline/
isotonic buffered saline (available from Tulip:Osmosol),
anti-human globulin reagent (Available from Tulip:
Eryclone anti-human globulin reagent), Rho(D) positive
red blood cells, incubator at 37°C, laboratory centrifuge,
Bring all the reagents to room temperature (25–30°C)
Preparation and Validation of Coomb’s Control
Preparation of 5% Coomb’s control cell suspension:
1. Collect fresh O Rho(D) positive red blood cells
preferably with citrate as an anticoagulant.
2. Wash 1 mL of freshly collected O Rho(D) positive red
blood cells with isotonic saline at least three times.
(IgG) reagent and gently resuspend the red blood cells.
4. Incubate the mixture at 37°C for 15 minutes.
5. After incubation, wash the sensitized red blood cells
thoroughly at least 4 to 5 times with isotonic saline.
Blood Banking (Immunohematology) 359
6. Decant the supernatant thoroughly after the last wash.
Resuspend the cell button gently with about 1–2 mL of
Agtrol red blood cell preserving solution. The complete
resuspended red cells should be added back to the
balance Agtrol red blood cell preserving solution in the
dropper vial. A stabilized suspension of 5% Coomb’s
control cells is thus, obtained. Label appropriately with
7. Store the Coomb’s control cells at 2–8°C. Use within
Validation of Prepared 5% Coomb’s Control Cell
1. Add one drop of Coomb’s control cells into a test tube.
2. Add two drops of anti-human globulin reagent and
3. Centrifuge for 1 minute at 1000 rpm or 20 seconds at
4. Very gently, resuspend the cell button and observe for
agglutination macroscopically.
Validation of Anti-human Globulin Reagent
1. Add one drop of Coomb’s control cells into a test tube.
2. Add two drops of anti-human globulin reagent and
3. Centrifuge for 1 minute at 1000 rpm or 20 seconds at
4. Very gently, resuspend the cell button and observe for
agglutination macroscopically.
Confirmation of Negative Antiglobulin Test Reactions
1. Add one drop of Coomb’s control cells to the samples
negative during direct or indirect antiglobulin test.
2. Centrifuge for 1 minute at 1000 rpm or 20 seconds at
3. Very gently, resuspend the cell button and observe for
agglutination macroscopically.
Agglutination reaction indicates that the anti-human
globulin reagent is functional and the test is valid. No
agglutination indicates that the anti-human globulin
reagent does not have sufficient activity and the test is
1. As undercentrifugation or overcentrifugation could
lead to erroneous results, it is recommended that
each laboratory calibrates its own equipment and
determine the time required for achieving the desired
2. Erroneous results may also occur due to improper red
blood cell concentration, improper temperature while
3. Store the Coomb’s control cells at 2–8°C with cap tightly
4. Do not contaminate the prepared Coomb’s control cell
suspension as it may subsequently effect the stability.
5. Glassware used to retrieve the Agtrol reagent and
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