When can you use an antibody
to find another antibody?
The Antiglobulin Test
Basic Principle, Procedures, and Applications
The Acquired Immune
Response

From our Immunology Review we know:

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The body makes antibodies in response to foreign
antigen.
These antibodies coat the foreign object leading to:


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Clearance of the foreign antigen by the RES
Lysis of the foreign object via complement activation
IgG is the predominant antibody produced in most
responses

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Incomplete antibody
Usually not detected at room temperature/immediate spin phase
of testing
The Antiglobulin Test

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The purpose of the
antiglobulin test is to
detect cells that have
become coated with
antibodies &/or
complement.
The test is also known
as the Coombs test.
Anti-Human Globulin

The main reagent used in the
antiglobulin test is anti-human
globulin (AHG).
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
Also called Coombs serum.
Anti-human globulin (AHG) is an
IgG antibody directed against
human immunoglobulins or
complement components.
AHG Production
AHG Production

The globulins that AHG
may be directed against
include:
IgG
 IgM
 IgA
 C3

AHG Contents


Polyspecific AHG reagent
contains antibodies to
both IgG and C3.
Monospecific AHG
contains antibodies to
either IgG or C3.
AHG Action

AHG combines with
the Fc portion of a
sensitizing antibody.
This completes the
antigen-antibody
bridge, allowing
agglutination to
occur.
Y

When used to detect clinically significant
antibodies, AHG reagent MUST contain
anti-IgG.
Control
ANYTIME YOU ARE USING AHG
REAGENT, AND GET A
NEGATIVE TEST RESULT
(by tube)---
YOU MUST ADD
COOMBS CONTROL CELLS
AND GET POSITIVE RESULTS!!!
Coombs Control Cells


Rh positive cells
coated with anti-D
antibodies or cells
coated with the C3
portion of complement.
Coombs Control Cells
will react with the
antibody in the AHG
reagent.
D
D
D
D
D
D
Y
Coombs Control Cells will
prove that…
Coombs reagent was added.
 Coombs reagent was active.
 The wash step was adequate to
remove any unbound globulins.

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The most common error made
when performing the antiglobulin
test is inadequate washing.
Procedures

Direct Antiglobulin Test (DAT)
Detects antibody (or complement)
sensitizing red cells
 In vivo sensitization
 Uses patient’s cells

Procedures

Indirect Antiglobulin Test (IAT)
Antibody is free
 Uses incubation at 37oC to force red cell
sensitization in vitro.
 May be used to detect antigens or antibodies.

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Both DAT and IAT utilize Anti-Human
Globulin reagent (AHG).
The Direct Antiglobulin Test
Procedure
Steps to the DAT Procedure
(tube method)
1. One drop of patient’s red cells are washed with 0.9% NaCl
a minimum of 3 times to remove plasma that may contain
unbound antibodies.
2. AHG reagent is added.
3. Tube is centrifuged.
4. If IgG or C3 is coating the cells, agglutination will occur
(positive test).
This will depend on the type of AHG reagent used i.e. if C3 is coating
the cell, and monospecific anti-IgG AHG reagent is used, there will
be NO agglutination.
If neither is present there will be no agglutination (negative
test).
5. Each negative test is validated (controlled) through the
addition of Coombs Control Cells (also called check cells).
DAT Procedure
(tube method)
Pt
ID
√
Washed cell button
The Direct Antiglobulin Test
Applications
What causes a cell to
become coated with
antibodies in vivo?
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Hemolytic Transfusion Reaction
Hemolytic Disease of the Fetus and Newborn
Drugs
Disease
Indirect Antiglobulin Test
Procedure
Steps to the IAT Procedure
(tube method)
1.
2.
3.
4.
5.
6.
One or two drops of serum (may contain an antibody) are
added to a test tube.
One drop of red cells (antigen source) is added to the
tube.
The tube is incubated at 37oC. The length of incubation
is dependant on the medium.
Following incubation, the cells are washed with saline a
minimum of 3 times, to remove any unbound antibody.
Following the final wash, two drops of AHG reagent are
added to the dry cell button. The tube is centrifuged and
results are read. The tube may be read microscopically,
depending on the test medium.
Coombs control cells are added to each negative test.
The tubes are centrifuged and results read.
Indirect Antiglobulin Test
Tube Method
3
7
C
i
n
c
√
Indirect Antiglobulin Test
Antibodies sensitize the
red cells during incubation
at 37oC.
Following the wash step,
AHG reagent is added.
AHG reagent completes
the “bridge” between red
cells, allowing for visible
agglutination.
Indirect Antiglobulin Test
Applications
Indirect Antiglobulin Test

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Looking for in vitro cell sensitization.
Uses incubation at 37oC to allow antibody to
sensitize red cell.
Uses AHG reagent to complete the “bridging”
between red cells.

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Visible agglutination as a positive endpoint.
Enhancement reagents may be added during
incubation phase to increase sensitization and
agglutination.
Applications Using Patient’s
Serum

Antibody screen

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Detects antibodies in patient’s serum
Uses reagent red cells as a source of known
antigen
Antibody panel

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Identifies antibodies
Uses reagent red cells as a source of known
antigen
Applications Using Patient’s
Serum
 Antiglobulin
crossmatch
Determines patient’s compatibility with donor
 Uses donor red cells (antigens) and patient’s
serum (antibodies)
 Usually performed only when a patient has an
antibody or a history of antibodies

Applications Using Patient’s
Cells
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Antigen Typing
Weak D test
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Both use commercial
anti-serum which
contains antibodies,
versus the patient’s
cells (antigen).
Auto control – Patient’s
plasma vs. patient’s
cells

NOTE:
If the patient has a
positive DAT, the results
of any IAT using the
patient’s cells will be
invalid.

Cells are already coated
with antibody before the
incubation step!
Factors affecting the IAT
Serum/Cell ratio
 Incubation temperature
 pH
 Length of incubation
 Test environment (enhancement
media)

POTENTIATORS
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Some incomplete
antibodies will not react
in a saline environment.
Potentiators are
reagents that adjust the
test environment.

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Reduce the zeta potential
Promote agglutination
Enhance antibody uptake
22% Albumin
 Polyethylene glycol
(PEG) : a low ionic strength

medium. Removes water from the
test system, thereby concentrating
any antibody present.
LISS
 Enzymes

Sources of Error in the
Antiglobulin Test
Adequate wash
 Centrifugation
 Problems with reagents/saline
 Problems reading reactions
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