Download The Antiglobulin Test

When can you use an antibody
to find another antibody?
Immunohematology
The Antiglobulin Test
Basic Principle, Procedures, and Applications
Objectives
Discuss the preparation, contents and use of
Anti-Human Globulin reagent (AHG).
 Explain the principle of the antiglobulin
reaction.
 Name the control system for the antiglobulin
test.
 Interpret both positive and negative “checkcell” results.

Objectives
Differentiate between the direct and indirect
antiglobulin procedures.
 Discuss applications of the indirect and
direct antiglobulin tests.
 Evaluate the Direct Antiglobulin Test in the
following situations:





HTR
HDFN
Hemolytic Anemias
Drugs
Objectives
Evaluate the significance of a positive Direct
Antiglobulin Test in the following situations:




Antigen typing
Weak D testing
Autocontrol
Evaluate the Indirect Antiglobulin Test in the
following situations:





Antibody detection
Antibody identification
Antiglobulin crossmatch
Antigen typing
Objectives



List at least four factors affecting the antigenantibody reactions in the indirect antiglobulin test.
Discuss the action of potentiators.
Assess sources of error affecting the antiglobulin
test.
The Acquired Immune
Response

From our Immunology Review we know:


The body makes antibodies in response to foreign
antigen.
These antibodies coat the foreign object leading to:



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


Incomplete antibody
Usually not detected at room temperature/immediate spin phase
of testing
The Antiglobulin Test


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).


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.


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.


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?




Hemolytic Transfusion Reaction
Hemolytic Disease of the Fetus and Newborn
Drugs
Disease
Hemolytic Transfusion
Reaction
The patient has an
antibody in the
plasma that is
directed against an
antigen on the
donor red cells.
 The patient’s
antibody coats the
donor cells.

Y
Y
Y
Y
Hemolytic Transfusion
Reaction



In an acute (immediate)
hemolytic transfusion
reaction, complement is
activated.
The donor cells are lysed
(intravascular hemolysis).
The DAT may be positive
due to IgG or
complement.

The DAT may be negative if
all the donor cells are
rapidly destroyed.
Y
Hemolytic Transfusion
Reaction

In a delayed hemolytic
transfusion reaction, the
antibody coated cells are
removed via phagocytosis.



(extravascular lysis)
A drop in hemoglobin
usually occurs 2-10 days
following transfusion.
The DAT is usually positive
due to IgG.
Y
Hemolytic Transfusion
Reaction

Other types of transfusion reactions share
the symptoms of an acute HTR, including:
Febrile, nonhemolytic
 Bacterial
 TRALI


A positive DAT establishes the diagnosis of
an acute hemolytic transfusion reaction.
Hemolytic Disease of the Fetus
and Newborn
(HDFN)
HDFN



Mother must have been
stimulated to make an IgG
antibody.
Antibody must cross the
placenta.
Fetus must be antigen
positive.
Fetal cells become coated
with maternal antibody &
are cleared by the fetal
RES.
Y

HDFN
In HDFN, the infant’s cells are coated with
maternal IgG antibody, resulting in a
positive DAT in the infant.
 The DAT is THE diagnostic test for HDFN.


If the DAT is negative, the infant is not suffering
from HDFN.
Drug-induced Hemolytic
Anemia
Mechanisms

Drug Adsorption




Drug attaches to RBC.
Antibody directed at drug only
DAT positive due to IgG
Immune Complex



Antibody directed at a “neoantigen” having
determinants on both the drug and the red cell
membrane.
Antibody activates complement
DAT positive due to complement
Mechanisms

Induction of autoimmunity
Drug modifies red cell membrane resulting in
production of auto antibody.
 DAT positive due to IgG and occasionally
complement.


Membrane Modification
Nonspecific adsorption of plasma proteins (Nonimmunologic )
 DAT will be positive due to whatever has “stuck”
to the membrane – IgG, complement, IgM, IgA.

Disease
Autoimmune Hemolytic Anemia
Diseases

Various diseases may cause autoantibody to coat
the patient’s red cells.

Cold Hemagglutinin Disease


Warm Auto Immune Hemolytic Anemia


DAT positive due to complement
DAT positive due to IgG and sometimes also due to complement
Paroxysmal Cold Hemoglobinuria

DAT positive due to complement
Indirect Antiglobulin Test
Procedure
Steps to the IAT Procedure
(tube method)
1.
2.
3.
4.
5.
6.
One or two drops of plasma or anti-serum containing
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



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.


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



Detects antibodies in patient’s serum
Uses reagent red cells as a source of known
antigen
Antibody panel


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


Antigen Typing
Weak D test


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


Some incomplete
antibodies will not react
in a saline environment.
Potentiators are
reagents that adjust the
test environment.



Reduce the zeta potential
Promote agglutination
Enhance antibody uptake
Zeta Potential
RBCs have a negative surface charge, and
attract cations such as Na+.
 The electrical potential between the red cell
surface and the outer ionic cloud
surrounding it is the zeta potential.

Zeta Potential

By reducing the
ionic cloud, the
potentiator allows
red cells to come
closer together.
-----------

This promotes the formation of antibody bridges
between cells which we call Agglutination.
22% Albumin
High molecular weight protein
 Reduces the zeta potential by
dispersing some of the cations
surrounding each negatively charged
red cell.
 Increases the dielectric constant,
defined as a measure of ability to
dissipate a charge.

LISS
 Low
Ionic Strength Solution
 Made
of NaCl, glycine and albumin
 Creates a low ionic environment
 Lowers
the zeta potential
 Promotes antibody uptake by the red
cells
PEG
Polyethylene glycol in a low ionic
strength medium.
 Removes water from the test system,
thereby concentrating any antibody
present.
 Antibody uptake is also increased.

PEG


PEG can cause cellular aggregation, therefore,
tests using PEG can not be centrifuged and
evaluated following a 37oC incubation. Testing
should proceed immediately to the wash phase,
with a minimum of 4 washes performed.
PEG is not the potentiator of choice when the
patient has elevated proteins, such as in multiple
myeloma. In these cases, LISS is the preferred
enhancement.
Polybrene
A positive polymer
 Will aggregate normal RBCs.



Sodium citrate is then added to the test
system. If an antibody is not present, the
aggregates will disperse. If an antibody is
present, the agglutination will persist.
Polybrene is currently used mainly to
resolve ABO discrepancies due to
polyagglutination.
Enzymes

Ficin, papain, bromelin and trypsin are
commonly used in blood banking
Proteolytic substances
 Modifies the red cell membrane by removing
sialic acid residues, thereby reducing surface
charge.
 Splits polypeptide chains, which further
exposes some antigens.
 May enhance the hemolytic activity of some
complement dependant antibodies.

POTENTIATORS
Enzymes diminish or destroy M,N, S,
Fy, Xga.
 Enzymes enhance Rh, P, Le, Jk and I.
 Enzymes enhance the hemolytic activity
of some complement binding antibodies
such as Lea, Jka, Vel, PP1Pk, ABO.

Sources of Error in the
Antiglobulin Test
Adequate wash
 Centrifugation
 Problems with reagents/saline
 Problems reading reactions

The End