LABORATORY ACTIVITY #8

DIRECT ANTIGLOBLIN TEST

I. Desired learning outcomes

The direct antiglobulin test is used to demonstrate that red cells have been coated (sensitized) with
antibodies in vivo. The test has many applications: to diagnose Hemolytic Disease of the Newborn (HDN), to
show whether fetal red cells are sensitized by maternal antibodies, in Autoimmune Hemolytic Anemia (AIHA),
and as an indication of incompatible transfusion or delayed hemolytic transfusion reaction.
A positive reaction in the DAT, however, can be confusing since such a result can be caused by variables
other than antigen-antibody sensitization. for example, a positive test can be caused by several disease
states (viral pneumonia, infectious hepatitis, infectious mononucleosis, favism, megaloblastic anemia,
reticulocytosis, acute intermittent porphyria, and most importantly, acquired hemolytic anemia.) Many drugs
too, are the cause of a positive DAT (e.g., alpha methyldopa, penicillin, caphalotin, Mesantoin, stibophen,
phenylhydrazine, quinine, and others).

After performing this activity, students should be able to:

a. perform the direct antiglobulin test;
b. enumerate the applications of the DAT, and
c. understand the significance of the DAT results.

II. Material
1. Test tubes 4. 2%-5% red cell suspension of the
2. Droppers patient
3. Centrifuge 5. NSS
6. Antihuman globulin
III. Procedures
1. Place one drop of 2%-5% red cell suspension of the patient into a test tube.
2. Wash the red cells four (4) times in large volumes of saline. Care should be taken to ensure adequate
removal of the supernatant saline after each wash.
3. Add one or two drops of anti-human globulin reagent.
4. Mix the red cells and reagents carefully and centrifuge at 3400 rpm for 15 to 30 seconds.
5. Gently resuspend the cell button and observe for any sign of agglutination. Check all negative reactions
microscopically.
6. Add IgG-sensitized red cells as control, centrifuge, and read. If a negative result is obtained, the test
result is invalid.

LABORATORY OUTPUT
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TITLE OF LABORATORY ACTIVITY: Lab. Act. 8 – Direct Antiglobulin Test (DAT) DATE SUBMITTED: 0

I. Observation/Results
Instructions: Paste pictures of how the agglutination reaction of red cells as they appear in the tubes.
Figure 1 Figure 2

Direct antiglobulin test (DAT) result in tube Direct antiglobulin test (DAT) result in slide

Note. Direct antiglobulin test (DAT) result Note. Direct antiglobulin test (DAT) result when examined
when examined macroscopically. No microscopically. No agglutination reaction or a negative
visible agglutination reaction or a negative result observed which corresponds to the macroscopic
reation observed but only just a smooth examination of tube, thereby indicating absence of
suspension. detectable antibodies bound to red blood cells.
 II. Discussion
The Direct Coombs test, also known as DAT, is conducted in the Blood Bank to identify if red blood cells are
covered with immunoglobulin, complement, or both within the body (Rodrigue, 2022) and it is commonly requested
when looking for hemolysis, as the presence of bound immunoglobulin and/or complement can result in the breakdown
of RBCs.
As guided by the American Association of Blood Banks, to conduct the Direct Coombs Test (DAT), one needs to
start by labeling a test tube with the lab number and the patient's name (Miller, 2005). Next, one should add 1-2 drops
of a 3 to 5% suspension of the patient's red blood cells into the labeled tube. Then, add 1-2 drops of antihuman globulin
reagent (AHG) to the same tube. Gently mix the contents of the tube and centrifuge it at 2500 rpm for 20 seconds. After
centrifugation, carefully resuspend the contents of the tube and observe for any agglutination. A positive test result is
indicated by agglutination, signifying that the patient's DAT is positive, while no agglutination indicates a negative test
result. It's important to note that the DAT is used to identify in-vivo sensitization of red blood cells, whereas the in-vitro
sensitization is identified through the indirect antiglobulin test (IAT). To confirm a positive DAT result, a control tube is
run in parallel with the DAT test tube. One should label the control tube accordingly and follow the same procedure as
the DAT test tube, but instead of AHG, add 1-2 drops of 6% albumin. Again, observe for agglutination in the control tube.
If agglutination occurs in the control tube, it indicates a negative result for DAT, suggesting in-vivo sensitization of red
blood cells. If no agglutination is observed in the control tube, and agglutination is seen only in the DAT test tube, it
confirms a true positive result for DAT. In the case of a negative DAT result, one should use a positive control to validate
the test. After performing the DAT test as described earlier, if no agglutination is observed, add 1-2 drops of 5%
sensitized cells (check cells) to the tube. Gently mix and centrifuge the tube again, then observe for agglutination. If
agglutination occurs after the addition of sensitized cells, it confirms the correctness of the negative DAT result.
However, if no agglutination is observed even after adding sensitized cells, it indicates that the negative DAT result is not
valid, and the test must be repeated.
Zantek et al. (2012) Enumerated various clinical applications of DAT in identifying the causes of hemolysis.
Firstly, it aids in distinguishing between immune and nonimmune etiologies of hemolysis, which is essential for
appropriate management. Common clinical scenarios where the DAT proves beneficial include autoimmune hemolytic
anemia (AIHA), transfusion-related hemolysis, hemolytic disease of the fetus/newborn (HDFN), drug-induced hemolytic
anemia, passenger lymphocyte syndrome, and cases of DAT-negative hemolytic anemia. For instance, in AIHA, the DAT
helps differentiate between warm and cold autoimmune hemolytic anemia by detecting IgG or IgM antibodies on red
blood cells. Similarly, in transfusion-related reactions, the DAT can indicate acute or delayed hemolytic reactions,
providing crucial information for patient care. Additionally, the DAT assists in diagnosing HDFN by detecting maternal
IgG antibodies bound to fetal red blood cells.
As pointed out by Theis and Hashmi (2022), the significance of direct antiglobulin test (DAT) results lies in their
clinical utility in diagnosing conditions characterized by autoantibody-induced red blood cell (RBC) hemolysis. Typically,
DAT involves using a polyspecific reagent containing IgG and complement C3, aiding in the detection of circulating
antibodies potentially causing RBC destruction. Positive results necessitate careful analysis within the clinical context to
ensure accurate diagnosis. By initially screening with a polyspecific reagent before confirming with monospecific or
quantitative analysis, healthcare costs and laboratory time can be minimized. Additionally, quantitative DAT testing may
identify less common antibody subtypes, aiding in the diagnosis of autoimmune hemolytic anemia (AIHA) even in the
absence of positive DAT results. Clinical significance areas include AIHA classification, alloimmune-mediated hemolytic
transfusion reactions (AHTR), ABO blood group typing for transfusions and stem cell transplants, and hemolytic disease
of the fetus and newborn (HDFN), particularly in cases of ABO incompatibility.

III. Guide Questions

 1. What are the applications of DAT?
The direct antiglobulin test (DAT), also known as the direct Coombs test, is employed to identify immunoglobulin
and/or complement on the surface of red blood cells (RBCs) (Lin, 2018). This test holds significant value in various clinical
scenarios, including autoimmune hemolytic anemia (AIHA), drug-induced immune hemolysis, hemolytic disease of the
newborn, investigation of hemolytic transfusion reactions, and passenger lymphocyte syndrome. AIHA, characterized by
autoantibodies against the patient's RBCs, can be diagnosed and classified using DAT, revealing patterns of reactivity
indicative of warm or cold AIHA. Drug-induced immune hemolytic anemia, caused by more than 150 drugs, can also be
identified through DAT, distinguishing between drug-independent and drug-dependent mechanisms. Hemolytic disease
of the newborn, resulting from maternal alloantibodies binding to fetal RBC antigens, is detected through positive DAT in
the newborn and positive indirect antiglobulin test in the mother. Investigation of hemolytic transfusion reactions and
passenger lymphocyte syndrome are other applications where DAT plays a crucial role. Despite its utility, the
interpretation of DAT results requires consideration of other various factors such as the patient's clinical history and the
limitations of the test itself.

2. Should a result with an agglutination reaction on the control tube be considered for reading? Explain.
No, a result with an agglutination reaction on the control tube should not be considered for reading, as it
functions as a positive control containing IgG-sensitized red cells, according to the procedure of Karp (2021). IgG-coated
RBCs are included in all negative tests with anti-IgG or polyspecific AHG, while complement-coated RBCs are added to all
negative tests with anti-C3. The tests are repeated if IgG-coated RBCs and/or complement-coated RBCs show no
reactivity. According to ALBAcyte®’s (2019) package insert, their IgG Sensitized Red Blood Cells serve to confirm the
validity of negative antiglobulin tests by demonstrating the anti-IgG activity of the anti-human globulin (AHG) reagent
used in the test. The resultant agglutination indicates both the presence and the activity of the anti-human globulin,
verifying the effectiveness of the test and ensuring its accuracy.

3. What does a POSITIVE DAT signify?

As signified by Nedelcu (2013), a positive Direct Antiglobulin Test (DAT) indicates the presence of antibodies or
complement fractions bound to red blood cells (RBCs) in vivo, leading to RBC agglutination upon the addition of AHG or
Coombs' serum. This signifies decreased RBC survival or destruction, often associated with conditions causing hemolysis.
Positive DAT results are observed in various scenarios, including hemolytic transfusion reactions, hemolytic disease of
the fetus and newborn (HDFN), autoimmune hemolytic anemia, drug-induced hemolytic anemia, and passenger
lymphocyte syndrome. Hypergammaglobulinemia can also yield positive DAT results due to nonspecific immunoglobulin
adsorption onto RBCs. Despite its utility, a negative DAT does not rule out these conditions entirely. Detection limits of
the test may miss antibodies of certain immunoglobulin subclasses. In cases of positive DAT following RBC exposure via
transfusion or pregnancy, detachment of antibodies from patient RBCs via RBC eluate can aid in identifying RBC antigen
specificity. Correct interpretation hinges on knowledge of reagent types and reaction conditions, as certain factors like
polyclonal reagents can yield false positives.

4. What are the drugs that may affect the DAT?

Among the most common drug culprits that affect DAT are penicillin and its derivatives, cephalosporins (such as
cefotetan and ceftriaxone), methyldopa, β-lactamase inhibitors, and quinidine, as suspected in the case of Sarkar et al.
(2013). These drugs can induce positive DAT results through various mechanisms, including the production of
autoantibodies (drug-independent) or the formation of immune complexes (drug-dependent). Additionally, some drugs
may lead to non-immunologic protein adsorption onto red blood cells (RBCs), contributing to positive DAT findings.

IV. Conclusion
The objectives were sufficiently achieved as I performed the direct antiglobulin test, enumerated its
applications, and understood the significance of the results. I have learned about the Direct Coombs test, or DAT, which
identifies immunoglobulin or complement on red blood cells (RBCs), crucial for investigating hemolysis, with applications
including diagnosing autoimmune hemolytic anemia, drug-induced immune hemolysis, hemolytic disease of the fetus
and newborn, transfusion reactions, and passenger lymphocyte syndrome, while positive results may indicate antibodies
or complement bound to RBCs, suggesting decreased survival or destruction, often associated with conditions causing
hemolysis, including hemolytic transfusion reactions and autoimmune hemolytic anemia, with drugs like penicillin,
cephalosporins, methyldopa, and quinidine potentially affecting DAT results. A misconception I have clarified about is
that a macroscopic non-reactive or no agglutination result in the direct antiglobulin test might appear negative, yet
further microscopic examination is required to confirm its negativeness.

V. Reference/s
ALBAcyte®. (2019). IgG Sensitized Red Blood Cells II: ALBAcyte® for the control of the indirect and direct antiglobulin test
[Package Insert]. Retrieved from https://www.fda.gov/media/120185/download

Karp, J. K. (2021, June 26). Direct antiglobulin testing. Overview, Clinical Indications/Applications, Test Performance.
https://emedicine.medscape.com/article/1731264-overview#a6

Lin, J. S. (2018). Clinical applications of direct antiglobulin test. Blood Heart Circ, 2, 1-5.

Miller, W. V. (Ed.). (2005). The Technical Manual of the American Association of Blood Banks (15th ed.). American
Association of Blood Banks.

Nedelcu, E. (2013). Pre-analytical issues and interferences in transfusion medicine tests. In Accurate Results in the
Clinical Laboratory (pp. 273-294). Elsevier.

Rodrigue, B. (2022). Direct antiglobulin test (DAT). The Blood Project.

https://www.thebloodproject.com/wp-content/uploads/2022/06/DAT2.pdf

Sarkar, R. S., Philip, J., Mallhi, R. S., & Jain, N. (2013). Drug-induced immune hemolytic anemia (Direct Antiglobulin Test
positive). medical journal armed forces india, 69(2), 190-192.

Theis, S. R., & Hashmi, M. F. (2022). Coombs Test. In StatPearls. StatPearls Publishing.
https://www.ncbi.nlm.nih.gov/books/NBK547707/

Zantek, N. D., Koepsell, S. A., Tharp Jr, D. R., & Cohn, C. S. (2012). The direct antiglobulin test: a critical step in the
evaluation of hemolysis. American journal of hematology, 87(7), 707-709.