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Laboratory Activity No. 6

MLSIMHE23
AHG Testing

Objectives

At the end of this exercise, you are expected to:
1. Differentiate the two methods of AHG tests based on their principle and applications.
2. Perform direct and indirect antiglobulin tests with accuracy.
3. Interpret the results of DAT and IAT correctly.

Introduction

The antihuman globulin test is one of the most useful and most universally applied tests
employed in blood banks. The Direct Antihuman Globulin Test (DAT) is used to demonstrate in vivo
coating of red cells with antibodies or complement, in particular IgG and C3d (in vivo sensitization). On
the other hand, Indirect Antihuman gloulin Test (IAT) detects the presence of unexpected antibodies in
the patient’s serum that are able to coat antigens in type “O” red cells in vitro (in vitro sensitization).

Materials Needed

Test tubes (wasserman) Marking pen Water bath
Centrifuge Pasteur pipettes Forceps
Test tube rack Gum label
Gloves Nescofilm

Reagents/Samples

Anticoagulated blood samples
Antihuman-globulin sera
Normal Saline Solution (0.85-0.9% NaCl) in wash bottle
22% Bovine Serum Albumin (22% BSA)
Coombs’ check cells

A. Procedure – DAT
1. Prepare a wassermann test tube and label it as U.
2. Deliver 2 drops of well-mixed anticoagulated blood into the tube.
3. Wash the anticoagulated blood three times with NSS.
4. Decant completely the supernatant after the last washing.
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 5. Add 2 drops of AHG sera to the tube and mix well.
6. Centrifuge for 15 seconds at 3,400 rpm.
7. Gently dislodge the cell button and examine for hemolysis or agglutination.
8. Grade the reaction and record the result.
9. If agglutination is not observed, add 1 drop of Coombs’ check cells to the tube and centrifuge for
15 seconds at 3,400 rpm.
10. Gently dislodge the cell button and examine the reaction. Record the result.
Note: Coombs’ check cells are also added to confirm if AHG was previously added. If AHG was
not added, no agglutination will be observed after the addition of check cells. It is suggested
that the procedure should be repeated.


Interpretation:
With agglutination = DAT positive
No agglutination = DAT negative

DAT positive reaction indicates that the red cells have been sensitized in vivo. IgG or
complement bound to red cells initiate complement activation which eventually results to hemolysis
that happens intravascularly. This is expected from patients sufferring from hemolytic conditions:
Hemolytic Disease of the Newborn (HDN), Drug-Induced Hemolytic Anemia (DIHA), Autoimmune
Hemolytic Anemia (AIHA), Hemolytic Transfusion Reaction (HTR). Usually, further procedures can be
done to remove the bound antibodies (elution) and identify them subsequently (antibody screening and
identification through panel cells).

DAT negative reaction indicates that the red cells are not sensitized in vivo either by IgG or
complement. This is expected in healthy normal patients.



B. Procedure - IAT

1. Prepare two tubes and label them “U” for unknown and “NC” for negative control and follow
the table below.

Content Unknown Negative Control
Unknown Serum 2 drops -
2-5% “O” Red Cells 2 drops 2 drops
22% Bovine Serum Albumin - 2 drops

2. Mix gently and cover with Nescofilm. Incubate at 37°C water bath for 15 minutes.
3. At the end of incubation, centrifuge for 15 seconds at 3,400 rpm. Examine for agglutination.
4. If no agglutination is observed, wash the unknown tube three times with NSS. Decant
completely the supernatant after the last washing.
5. Add 2 drops of AHG sera to the tube and mix well.
6. Centrifuge for 15 seconds at 3,400 rpm.
7. Gently dislodge the cell button and examine for hemolysis or agglutination.
8. Grade the reaction and record the result.
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 9. If agglutination is not observed, add 1 drop of Coombs’ check cells to the tube and centrifuge for
15 seconds at 3,400 rpm.
10. Gently dislodge the cell button and examine the reaction. Record the result.
Note: Indirect Antihuman Globulin Test is a procedure to determine in vitro sensitization of red cells. It
uses Type “O” red cell suspension because it primarily detects antibodies other than the naturally
occurring anti-A and anti-B. This test is usefull in detecting weak variants of D antigens, red cell
phenotyping, in antibody screening and identification and in minor crossmatching.




























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 Laboratory Activity No. 6
MLSIMHE23
AHG Testing

Observation

Draw and color your results. Label the tubes completely then interpret and grade the reactions.
Determine the DAT and IAT result by shading the box. If performed, record the result of the addition of
the check cells.

A. Direct Antihuman Globulin Test (DAT)
Unknown:
Interpretation

DAT Positive
U
DAT Negative




Reaction Grade: __________

B. Indirect Antihuman Globulin Test (IAT)
Unknown:

Interpretation

IAT Positive
U NC
IAT Negative

Reaction Grade: __________ __________

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Student’s Signature Over Printed Name Instructor’s Signature Over Printed Name

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 Laboratory Activity No. 6
MLSIMHE23
AHG Testing

Post Laboratory Questions

1. What is Coombs’ reagent? How is it prepared?
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2. Enumerate and discuss some uses of direct and indirect Coombs test.
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3. What are the possible causes of false positive and false negative results in AHG tests?

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Name: ___________________________________________________

Section/Cluster: __________________ Date Submitted:____________

Instructor: _________________________________________________