INDIRECT ANTIGLOBULIN TEST

BSMT 3A - GROUP 7
INTRODUCTION
■ Indirect antiglobulin test is used to assess in vitro sensitization of the
RBCs. It tests the presence of antibodies in the patient’s serum by
reacting it with RBCs with known antigen. A positive or negative
agglutination is then observed and noted.
■ If the samples are negative, the addition of AHG is done. AHG acts as
a potentiator to enhance agglutination reactions and in order to have
a better macroscopic visualization of the otherwise, weak
agglutination reactions.
■ Significant in testing for antibody titration, antibody detection,
crossmatching, RBC antigen phenotyping and antibody investigation.
DISCUSSION
Indirect Antiglobulin Test (IAT), is used for the detection of in vivo
sensitization of the red blood cells. The recipient’s serum which contains
antibodies is added with the donor’s blood sample and will form an
antibody-antigen complex. AHG is then added to the solution and will link
the antibodies attached on the surface of the red cells, causing
agglutination.
This is used in the following situations:
 Detection of incomplete (nonagglutinating) antibodies to potential donor
RBCs (compatibility testing) or to screening cells (antibody screen) in
serum
 Determination of RBC phenotype using known antisera (e.g., Kell typing,
weak D testing)
 Titration of incomplete antibodies
PROCEDURE AND
RESULTS
 Place two drops of the serum to be tested in a properly labeled
test tube.
 Add two drops of 2-5% RCS with known antigens.
 Incubate for one hour at 37°C.
 A similarly prepared test tube should be incubated at 4°C to test
for cold reacting antibodies.
 Spin down and then examine for agglutination. If no agglutination
is observed, continue.
 Rapidly wash the cells three times in normal saline.
PROCEDURE AND
RESULTS
Completely decant the final wash supernatant.
Add two drops of AHG to the cells.
Mix and spin down.
Re-suspend lightly, then examine macroscopically
and microscopically.
Interpret the results.
(+) IAT= presence of agglutination
(-) IAT= no agglutination
GUIDE QUESTIONS
1. What is the principle of the test?
The IAT detects in vitro sensitization of RBCs.
The recipient’s serum which contains antibodies
is added with the donor’s blood sample and will
form an antibody-antigen complex. AHG is then
added to the solution and will link the antibodies
attached on the surface of the red cells, causing
agglutination.
INDIRECT ANTIGLOBULIN
TEST
GUIDE QUESTIONS
2. What are the causes of a false reaction?
POST-LAB CONFERENCE
1. How is quality control done in IAT?
Two types of quality control RBCs are normally used to
standardize the antisera and to confirm true-negative anti-
globulin reaction: those that are coated with IgG and others
that are coated with C3b and/or C3d.
To sensitize RBCs with IgG, Rh antibodies are usually used.
RBCs coated with C3b are prepared by incubation of whole
blood in low ionic strength saline (LISS) or with human anti-
Lea or anti-I. While C3d-coated RBCs are prepared by
incubating C3b-coated cells with fresh serum or trypsin to
split C3b→C3d. IgG or complement-coated control cells
should give a 1+ to 2+ reaction.
POST-LAB CONFERENCE
2. Compare and contrast DAT with IAT. Tabulate your answers
and present it to the class.
DAT IAT
PRINCIPLE In vivo sensitization of RBCs In vitro sensitization of RBCs
SPECIMEN Red cell suspension Serum
REAGENT Anti-human globulin Reagent
CONTROL O check cells Cells with known Antigen

37°C and 4°C

INCUBATION n/a
(different tubes)

Positive = Presence of agglutination

INTERPRETATION OF RESULTS
Negative = No agglutination

SOURCES OF ERROR EDTA isn’t use in collection of samples Sample for DAT is used

Cell suspension either too weak or heavy Improper use of Polycation Enhancement Reagent

AHG Reagent isn’t added Inadequate incubation periods

Updates
Paper-based assay for red blood cell antigen typing by the
indirect antiglobulin test
N. Yeow, et.al., May 2016;
https://rd.springer.com/article/10.1007/s00216-016-9617-6

Abstract

A rapid and simple paper-based elution assay for red blood

cell antigen typing by the indirect antiglobulin test (IAT) was
established. This allows to type blood using IgG antibodies
for the important blood groups in which IgM antibodies do
not exist. Red blood cells incubated with
IgG anti-D were washed with saline and spotted onto the paper assay
pre-treated with anti-IgG. The blood spot was eluted with an elution
buffer solution in a chromatography tank. Positive samples were
identified by the agglutinated and fixed red blood cells on the original
spotting area, while red blood cells from negative samples completely
eluted away from the spot of origin. Optimum concentrations for both
anti-IgG and anti-D were identified to eliminate the washing step after
the incubation phase. Based on the no-washing procedure, the
critical variables were investigated to establish the optimal conditions
for the paper-based assay. Two hundred ten donor blood samples
were tested in optimal conditions for the paper test with anti-D and
anti-Kell. Positive and negative samples were clearly distinguished.
This assay opens up new applications of the IAT on paper including
antibody detection and blood donor-recipient crossmatching and
extends its uses into non-blood typing applications with IgG antibody-
based diagnostics.
REFERENCES
■ Modern Blood Banking and Transfusion Practices by Denise
M. Harmening, Sixth Edition
■ Henry's Clinical Diagnosis and Management by Laboratory
Methods by Richard A. McPherson, Matthew R. Pincus, 22nd
Edition