Direct Antiglobulin Test

Direct Antiglobulin Test – DAT (Direct Coombs)
PRINCIPLE
Normal human red blood cells in the presence of antibody directed toward an antigen they possess may
become sensitized, but fail to agglutinate due to the particular nature of the antigen and antibody involved.
Anti-Human Serum will react with the sensitized red cells. The Direct Antiglobulin Test (DAT) is used to
demonstrate in vivo coating of red cells with antibody or complement. It is used in investigating autoimmune
hemolytic anemia, drug-induced hemolysis, hemolytic disease of the newborn and alloimmune reactions to
recently transfused red cells.
● Note: Direct Antiglobulin Test is also known as Direct Coombs.
SPECIMEN
Use EDTA-anticoagulated whole blood. Test should be performed as soon as possible after collection, but for
best results within 48 hours. Store and keep specimens at 2-8 degrees C for at least 7 days.
REAGENTS
Polyspecific Anti-Human Globulin, Anti-IgG Serum, Anti-C3b,-C3d Serum
Complement Control Cells (check cells), IgG Control Cells (check cells)
Store all reagents at 2-8 degrees C when not in use. Do not use reagents beyond the expiration dates.
MATERIALS AND EQUIPMENT: Test tubes, glass slides, pipettes, physiologic saline, 37 C dribath, timer
centrifuge, optical aid.
TESTING REQUIREMENTS
For Cord Blood Testing:
● Anti-IgG (monospecific) Coombs
For Hemolytic Workup Testing and Transfusion Reaction Workup:

● Anti-IgG,-C3d (polyspecific) Coombs
● Anti-IgG (monospecific) Coombs (If poly pos)
● Anti-C3b, C3d Coombs (If poly pos)
Note: Red blood cells which agglutinate in the presence of polyspecific Anti-Human Globulin must be further
tested with anti-IgG and anti-C3b,-C3d to determine which component is causing the positive result.
(Monospecific DAT’s are performed in the same manner as the polyspecific DAT).
For Positive Antibody Workup:

● Anti-IgG (monospecific) Coombs
1
PROCEDURE
A crucial step in the DAT test procedure is the thorough washing of the test cells before adding Anti-Human
Globulin Reagent. Washing removes unbound human protein that would otherwise
neutralize the AHG.
Step Action
1 Prepare an approximate 3-4 % suspension in isotonic saline of the red blood cells to be
tested.
2 With a clean pipette, add one drop of the prepared cell suspension to a 10 x 75 test tube.
3 Wash the cells at least 3 times with saline, either manually or with a cell washer
NOTE When manually washing, carefully decant the saline between washes and resuspend the rbcs
thoroughly before adding the next volume of saline. Decant the saline completely and then
blot tubes following the last wash.
4 Add 2 drops of the appropriate AHG serum to the washed cell button and mix well.
5 Centrifuge for a time appropriate to the calibration of the centrifuge.
6 Resuspend the cells by gentle agitation and examine macroscopically for agglutination.
Examine negative tests with an optical aid.
7 Grade and record test results.
8 NOTE: If using polyspecific AHG or anti-C3d, incubate nonreactive tests at room temperature
for 5 minutes, then centrifuge, and read again.
9 a. Add one drop of IgG coated check cells (Coombscell-E) to any tests that show no
agglutination. (IgG and Poly AHG)
b. Add one drop of Complement check cells to the anti-C3b,-C3d tubes that show no
agglutination.
10 Mix, centrifuge and check for positive results.
Stability Of The Reaction

Following centrifugation, all tube tests should be read immediately and results interpreted without delay.
Time delays may cause a dissociation of the antigen-antibody complexes resulting to false negative or more
often weak positive reactions
INTERPRETATION OF TEST RESULTS

Agglutination: Indicates the presence of human IgG and/or complement on the red blood cells.
No agglutination: Indicates that there is no detectable IgG or complement components on the red blood cells,
subject to satisfactory control tests.
Invalid: If the Ortho Control Cells and complement check cells are not agglutinated, the negative DAT result is
considered invalid and the test must be repeated.
If a DAT result is positive for IGG and the Rh type is initially negative and the weak D is carried out, a positive
weak D result cannot be reported out as positive because of the positive DAT. The Rh type cannot be
determined and nursing services should be notified. A comment should be entered in the computer that the
Rh type could not be determined due to a positive DAT. If the DAT is positive for complement with a weak D
positive, the Rh type would be reported out as Rh positive.
2
REPORTING RESULTS
DAT results are recorded and entered in the LIS as an antibody.
● DCN = Direct Coombs Negative
● DCP = Direct Coombs Positive
● For all DCP results indicate if RBCs contain: IgG, Complement or both
○ After entering DCP, select COMMENT. Select F5, then F9
○ BBKDATC3 = “Rbcs coated with complement”
○ BBKDATIGG = “Rbcs coated with IgG”
○ BBKDIGGC3 = “Rbcs coated with IgG and complement”
● An additional comment may be added with the above, but is not required
○ BBKDATPNBL =
“This patient has not received blood recently. The clinical condition and/or drug therapy of the patient
may cause a positive direct coombs result”
● In case of a positive DAT (IgG) in a delayed transfusion reaction: Perform an elution.
● In case of a positive DAT (IgG) and patient received blood within 3 months: Perform an elution.
LIMITATIONS
As in all serological tests, such factors as contaminated materials, improper washing, improper incubation time
or temperature, improper centrifugation and improper examination for agglutination may give rise to false test
results. In addition:
1. False-negative results:
a. Failure to wash the red cells adequately is a major cause of false-negative antiglobulin tests. Since
globulins not bound to red cells will neutralize Anti-Human Serum.
b. If testing is interrupted or delayed. The entire washing process must be undertaken as quickly as
possible to minimize loss of bound antibody by elution from the cells.
c. Anti-Human Serum can lose activity following improper storage, bacterial contamination or
contamination with human serum. Store between 2-8C. Never freeze AHG reagent and do not
warm by placing in a 37C dribath or waterbath. The reagent can be neutralized very easily.
d. The sensitivity of antiglobulin test is greatly impaired if human protein is introduced into the test
system after washing the cells
e. Forgetting to add Anti-Human Serum during testing.
f. Under centrifugation provides suboptimal conditions for agglutination to occur.
g. The number of red blood cells present may influence reactivity so an insufficient number of red
cells may cause a false-negative result.
3
2. False-positive results:
a. Red cells may be agglutinated before they are washed. Red cells from patients with potent cold-
reactive autoantibodies may agglutinate in whole blood samples unless maintained at 37 degrees C.
b. Improperly cleansed glassware may be contaminated with dust, detergent or other matter that
causes red cells to aggregate.
c. Over centrifugation packs red cells so tightly that they cannot be dispersed completely.
d. Septicemia in a patient or bacterial contamination of stored specimens may cause a positive DAT.
REFERENCES
Roback, J. (2011). Technical manual (17th ed.). Bethesda, Md.: AABB
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Direct Antiglobulin Test – DAT (Direct Coombs)

For Hemolytic Workup Testing and Transfusion Reaction Workup:

For Positive Antibody Workup:

Stability Of The Reaction

INTERPRETATION OF TEST RESULTS

e. Forgetting to add Anti-Human Serum during testing.

f. Under centrifugation provides suboptimal conditions for agglutination to occur.

Roback, J. (2011). Technical manual (17th ed.). Bethesda, Md.: AABB

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