COLEGIO SAN AGUSTIN-BACOLOD

Medical Technology Program MLS 116 LABORATORY ACTIVITY SHEET

LABORATORY ACTIVITY #9
INDIRECT ANTIGLOBLIN TEST

I. Desired learning outcomes

The indirect antiglobulin test is used to demonstrate the presence of free antibody in the patient’s
serum. This is achieved by incubation of the serum with red cells of various antigenic make-ups at 37 C,
allowing sensitization of the red cells to occur (i.e., sensitization in vitro). The addition of antiglobulin reagent
(once free antibody has been diluted out by washing red cells) provides the bridge between sensitized red
cells by employing an antibody produced in a rabbit, which reacts with the antibody or complement attached
to the red cells. The rabbit antihuman globulin reacts with the sensitized red cells and produces agglutinates.
If either antigen or antibody is missing from the mixture, the red cells will fail to agglutinate, and the test is
interpreted as invalid.

The indirect antiglobulin test is the most widely used serological test in modern blood banking.
Although its most important application is in the compatibility testing, it is also used in antibody
identification, in hemolytic disease of the newborn (HDN), in cases of autoimmune hemolytic anemia (AIHA),
in the investigation of adverse reactions to transfusion and in practically all other investigations undertaken
by the blood bank.

After performing this activity, students should be able to:

a. perform the indirect antiglobulin test;
b. enumerate the applications of the IAT; and
c. understand the significance of the IAT results.

II. Material
1. Test tubes 5. 2%-5% red cell suspension of the
2. Droppers patient
3. Centrifuge 6. serum of the patient
4. Water bath 7. Antihuman globulin
III. Procedures
1. Place 2-3 drops of the serum under test in a test tube.
2. Add one drop of 5% suspension of the test cells (donor’s red cells or screening cells).
3. Mix well.
4. Incubate at 37 C for 15-30 minutes or longer.
5. Immediately upon removal from the incubator, centrifuge for 15 seconds at 3400 rpm. Examine for
agglutination or hemolysis.
6. Wash the red cells 3-4X in large amounts of saline. Decant each was as completely as possible.
7. Add one or two drops of antihuman globulin.
8. Mix well.
9. Centrifuge at 3400 rpm for 15 seconds.
10. Examine for agglutination, using an optical aid. Check all negative reactions microscopically.
11. Optional: Add one drop of known sensitized red cells to all tests giving negative results. Centrifuge at
 COLEGIO SAN AGUSTIN-BACOLOD
Medical Technology Program MLS 116 LABORATORY ACTIVITY SHEET

3400 rpm for 15 seconds. If no agglutination is seen, the test is invalid.

Interpretation:

As soon as centrifugation is complete, the contents of the tube(s) should be examined for hemolysis or
agglutination. The appearance of free hemoglobin that was not present in the original sample must be
considered as positive reaction. If any of the two reactions occurs, then an antibody sensitization in vitro is
demonstrated.
 COLEGIO SAN AGUSTIN-BACOLOD
Medical Technology Program MLS 116 LABORATORY ACTIVITY SHEET

LABORATORY OUTPUT

NAME OF STUDENT: ________________________________________ DATE PERFORMED: ___________________

TITLE OF LABORATORY ACTIVITY: ______________________________ DATE SUBMITTED: ____________________

IV. Observation/Results
Instructions: Paste pictures of the result of the test as it appears in the tube.

V. Discussion
 COLEGIO SAN AGUSTIN-BACOLOD
Medical Technology Program MLS 116 LABORATORY ACTIVITY SHEET

VI. Guide Questions

1. What are the applications of IAT?
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2. What does a positive IAT signify?
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3. What is the difference of IAT and DAT?
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 COLEGIO SAN AGUSTIN-BACOLOD
Medical Technology Program MLS 116 LABORATORY ACTIVITY SHEET

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VII. Conclusion
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VIII. Reference/s