LABORATORY ACTIVITY #9

INDIRECT ANTIGLOBLIN TEST

I. Desired learning outcomes
The indirect antiglobulin test is used to demonstrate the presence of free antibodies 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 an antiglobulin reagent (once the 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 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 transfusion
reactions, and 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 complete 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
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 a positive reaction. If any of the two reactions occur, then an antibody sensitization in vitro is
demonstrated.
LABORATORY OUTPUT
002024
TITLE OF LABORATORY ACTIVITY: Lab. Act. 9 – Indirect Antiglobulin Test (IAT) DATE SUBMITTED: 00

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

Figure 1 Figure 2

Indirect antiglobulin test (IAT) result in tube Indirect antiglobulin test (IAT) result in
slide

Note. Indirect antiglobulin test (IAT) Note. Indirect antiglobulin test (IAT) results when
results when examined macroscopically. examined microscopically. No agglutination reaction
No visible agglutination reaction or a nor hemolysis—meaning, a negative result—was
negative reaction was observed but only observed which corresponds to the macroscopic
just a smooth suspension. However, there examination of the tube, thereby indicating the
is still the need to check microscopically absence of clinically significant antibodies in the
for a reaction. patient’s plasma or an antibody sensitization in vitro
has taken place.

II. Discussion
The indirect antiglobulin test (IAT), also known as the indirect Coombs test, is employed for identifying IgG
antibodies directed against red blood cells (RBCs) in the plasma of a patient (Gerber, 2024).
 To perform the indirect antiglobulin test (IAT), typically, monospecific anti-IgG is employed in modern labs
(Letendre, 2019a). This procedure primarily aims to detect in vitro sensitization, yet it can also identify in vivo
sensitization if the red cells utilized are pre-sensitized with IgG. Initially, 2 to 4 drops of serum, preferably 4, are
incubated at 37°C for 15 to 60 minutes, along with 1 drop of 3 to 5% red cells. Should the serum contain a specific
antibody for an antigen on the red cells, the antibody will sensitize the cells without causing agglutination if it's IgG.
Subsequently, the test is visually inspected for agglutination and hemolysis post-incubation, constituting the saline 37°C
phase of the AHG test, although microscopic examination at this stage is rare. Following this, thorough washing of the
red cells is conducted 3 to 4 times to eliminate all unbound protein, which is crucial for preventing false negatives by
neutralizing the AHG serum. Effective decanting during washing is vital to remove residual saline, which could otherwise
dilute the AHG serum and lead to false negatives. Once the last wash is complete, 2 drops of AHG serum are added,
followed by centrifugation and macroscopic inspection solely for agglutination, as the serum is removed to avoid
interference with hemolysis detection. In case of a negative result, a microscopic examination is performed, and if still
negative, IgG-sensitized cells are introduced for further testing.

One application of the Indirect Antiglobulin Test (IAT) is varied and significant, but primarily, it serves to detect
and identify antibodies, utilizing antihuman globulin (AHG) reagent to detect in vitro sensitization of red cells—making it
a vital tool for RBC phenotyping and crossmatch screening for blood transfusion (Alba, 2024; Theis & Hashmi, 2024).
Letendre (2019b) further specified that this test method is instrumental in detecting IgG antibodies in systems such as
Rh, Kell, Kidd, and Duffy, with additional capability to detect IgM antibodies binding C3, like anti-Lea, anti-Leb, autoanti-I,
and anti-P1, if clotted specimens and polyspecific AHG are employed. Its utility extends to crossmatching, antibody
detection and identification, and antigen typing in various clinical scenarios, offering comprehensive insights into in vitro
sensitization.

Understanding the significance of the Indirect Antiglobulin Test (IAT) results is crucial for determining
compatibility and potential risks. A negative result indicates the absence of antibodies to donor red blood cells, ensuring
safe blood transfusion and alleviating concerns regarding hemolytic disease in unborn babies (Haldeman-Englert et al.,
2024; Rath, 2021). Conversely, a positive result signals potential incompatibility between your blood and the donor's,
necessitating careful consideration by healthcare providers during blood transfusion. For individuals requiring frequent
transfusions, multiple antibodies may develop, complicating blood selection. During pregnancy, a positive result
suggests the need for protective measures for the baby, prompting further testing to identify specific antibodies and
inform appropriate medical interventions.

III. Guide Questions

1. What are the applications of IAT?
One main application of the Indirect Antiglobulin Test (IAT) is to detect and identify antibodies, employing
antihuman globulin (AHG) reagent to discern in vitro sensitization of red cells—which also makes it essential for RBC
phenotyping and crossmatch screening for blood transfusion (Alba, 2024; Theis & Hashmi, 2024). Letendre (2019b)
elaborated on its utility, highlighting its efficacy in detecting IgG antibodies across systems like Rh, Kell, Kidd, and Duffy,
with added capability to identify IgM antibodies binding C3, such as anti-Lea, anti-Leb, autoanti-I, and anti-P1, when
utilizing clotted specimens and polyspecific AHG. This method extends its usefulness to crossmatching, antibody
detection and identification, and antigen typing across diverse clinical contexts, providing comprehensive insights into in
vitro sensitization.

2. What does a positive IAT signify?

According to Case-Lo (2022), a positive Indirect Antiglobulin Test (IAT) signifies the presence of antibodies in the
bloodstream, which have the potential to trigger an immune response against foreign red blood cells. This outcome is
particularly significant in prenatal screening during pregnancy, where it indicates an incompatibility between the
birthing parent's blood type and the baby's, posing serious risks for both during pregnancy or birth if blood mixing
occurs. Moreover, in pre-transfusion testing, a positive IAT indicates an incompatibility between the donor and recipient
blood types, emphasizing the importance of ensuring compatibility to prevent complications during transfusion.

3. What is the difference between IAT and DAT?

As Chaffin (2022) has differentiated, the primary difference between the Indirect Antiglobulin Test (IAT) and the
Direct Antiglobulin Test (DAT) lies in the method of interaction between antibodies or complement proteins and red
blood cells (RBCs). In the case of the IAT, which is commonly used in pre-transfusion testing and antibody detection, the
interaction occurs in the laboratory setting. Here, patient serum or plasma is mixed with RBCs from a donor blood
sample with a known antigen profile, followed by incubation and subsequent washing to remove unbound antibodies,
before the addition of the AHG reagent. Conversely, the DAT, employed in transfusion reaction workup and hemolytic
anemia investigation, evaluates whether such interaction has occurred within the patient's body. In this test, RBCs are
extracted from the patient, washed, and directly mixed with the AHG reagent, without any additional laboratory-
induced interactions. Despite these distinctions, both tests share the common step of assessing visible agglutination
resulting from the binding of antiglobulin reagents to antibodies or complement proteins already attached to RBCs.

IV. Conclusion
The objectives were sufficiently achieved as I performed the indirect antiglobulin test, enumerated the applications
of the IAT, and implicated the significance of the IAT results. The negative indirect antiglobulin test (IAT) results signify
no visible agglutination; thus, I have learned that IAT is crucial for blood transfusion safety, indicating the absence of IgG
antibodies against red blood cells (RBCs) as assessed in the laboratory and highlighting its significance in prenatal and
pre-transfusion screenings, contrasting with the Direct Antiglobulin Test (DAT) which evaluates interaction within the
patient's body. A clarified misconception is that a macroscopic negative result in the Indirect Antiglobulin Test (IAT) does
not guarantee negativity; microscopic examination is also necessary to confirm the absence of detectable antibodies
bound to red blood cells.

V. Reference/s
Alba, M. (2024). Indirect antiglobulin test (IAT). Indirect Antiglobulin Test (IAT) - LabCE.com, Laboratory Continuing
Education. https://www.labce.com/spg1867617_indirect_antiglobulin_test_iat.aspx

Case-Lo, C. (2022, February 4). Coombs test: Purpose, procedure, and results. Healthline.
https://www.healthline.com/health/coombs-test

Chaffin, J. (2022, March 20). What's in a name? ending direct/indirect confusion. Blood Bank Guy.
https://www.bbguy.org/2016/04/27/whats-name-ending-directindirect-confusion/
Gerber, G. F. (2024). Image: indirect antiglobulin (Indirect Coombs) test. MSD Manual Professional Edition.
https://www.msdmanuals.com/professional/multimedia/image/indirect-antiglobulin-indirect-coombs-test?
mredirectid=2487

Haldeman-Englert, C., Turley Jr, R., & Novick, T. (2024). Indirect Antiglobulin. In Health Encyclopedia. University of
Rochester Medical Center. Retrieved from https://www.urmc.rochester.edu/encyclopedia/content.aspx?
contenttypeid=167&contentid=indirect_antiglobulin

Letendre, P. (2019a). Antiglobulin Test. University of Alberta. Dept. of Laboratory Medicine & Pathology.
https://sites.ualberta.ca/~pletendr/tm-modules/methods/70met-iat.html

Letendre, P. (2019b). Uses of the Indirect Antiglobulin Test. University of Alberta. Dept. of Laboratory Medicine &
Pathology. https://sites.ualberta.ca/~pletendr/tm-modules/methods/70met-uses.html

Rath, L. (2021). Coombs test: Purpose, procedure, and results explained. WebMD. https://www.webmd.com/a-to-z-
guides/antibody-coombs-test

Theis, S. R., & Hashmi, M. F. (2024). Coombs Test. In StatPearls [Internet]. Treasure Island, FL: StatPearls Publishing.
Retrieved from [URL]