9. Antiglobulin Test
-
Upload
carinajonglee -
Category
Documents
-
view
222 -
download
1
description
Transcript of 9. Antiglobulin Test
-
HEM 2133
Immunohaematology IImmunohaematology I
Lesson 9: Antihuman Globulin
Reaction
-
History of Antiglobulin Testing
Discovered by Coombs, Mourant and Race in 1945
Found that red blood cells (RBC) can become sensitized without visible agglutinationsensitized without visible agglutination
Preceded by two important discoveries by Race and Wiener
Race found what he believed to be anti-D components in some patients sera that contained detectable anti-D
-
These anti-D components seemed to combine
with D-positive red cells but did not cause
direct agglutination of those D-positive red
cells incomplete antibodiescells incomplete antibodies
Coombs visualized red cells coated with
human, incomplete antibodies being linked
together by other antibodies that were
directed against human antibodies
-
This simple hypothesis was then tested by
Coombs, Mourant and Race as the principle of
the antiglobulin test
The routine application of the antiglobulin test The routine application of the antiglobulin test
to immunohematology have allowed major
discoveries in human blood groups, immune
and acquired hemolytic anemias and
antiglobulin reagents
-
Principle of the Antiglobulin Test
The antiglobulin test depends on the following
basic premises:
Antibodies are globulins
Animals injected with human globulins Animals injected with human globulins
produce anti-human globulins. These anti-
human antibodies include antibodies to
complement components and IgG
-
The anti-human antibodies bind to the Fc
portion of sensitizing antibodies and form
bridges between antibody-coated red cells,
resulting in visible agglutinationresulting in visible agglutination
Anti-human antibodies will bind to human
antibodies that are both bound to red cells
(sensitizing) and unbound. Anti-human
antibodies will react first with any unbound
human antibodies
-
Depending on the amount of unbound human
antibody present for binding with the anti-
human globulins (AHG), AHGs may not be
available to form bridges between sensitized available to form bridges between sensitized
red cells
-
Principle of the Antiglobulin Test
IgM pentameric (large structure) when
bound to RBCs antigens, agglutination will
occur (usually at room temperature)
-
Principle of the Antiglobulin Test
When IgG antibodies attach to their
appropriate red cell antigens may fail to
cause agglutination
Will remain firmly bound even when the cells Will remain firmly bound even when the cells
are thoroughly washed in saline
These bound antibody may be detected by the
addition of anti-human globulin (AHG) act as
a bridge molecule to help IgG to agglutinate
RBCs
-
Principles of the Antiglobulin Test
-
Principles of the Antiglobulin Test
Injecting an animal with human globulin (IgG)
and complement proteins
This stimulates the animal to produce
antibodies to these foreign proteinsantibodies to these foreign proteins
AHG will react with human globulin molecules
whether bound to RBCs or free in serum
AHG will agglutinate with washed RBCs coated
with human globulin
-
Principles of the Antiglobulin Test
If the red cells in the test are sensitized with
IgG or complement, the AHG reagent will
crosslink and cause agglutination
Anti-IgG attaches to the Fc portion of the IgG
molecule that is bound to the red cellmolecule that is bound to the red cell
Anti-C3 attaches to C3 molecules bound to the
red cell
Agglutination = positive antiglobulin test
No agglutination = negative antiglobulin test
-
Immunoglobulin
-
Positive Antiglobulin Test
-
Negative Antiglobulin Test
-
Reagents
1. Monoclonal (single clone of cells-specificity)
2. Polyclonal (human source: mixture of cells-
contains multiple antibodies)
Assist in identifying antigen present on Assist in identifying antigen present on
patients RBC that may cause and reaction in
vivo
-
Polyspecific AHG
Abs to human IgG and Abs to human C3d (anti-
C3d to detect Ab bound with complements
and those rare Abs)and those rare Abs)
Advantage is that polyspecific AHG may detect
complement-dependent Abs on RBCs
-
Monospecific AHG
Abs to human IgG or human C3d only
May miss an important antibody
-
The AHG serum that is routinely used by most
labs for pretransfusion IATs is monospecific
anti-IgG
When doing DATs to detect in vivo When doing DATs to detect in vivo
sensitization with IgG or C3, blood banks must
use polyspecific AHG containing anti-IgG, anti-
C3b and anti-C3d
-
Zeta Potential
An expression of the difference in electrostatic
potential at the surface of the red cell and the
ionic cloud of positive cations that are
attracted to the negative charges on the attracted to the negative charges on the
surface
The net negative charge surrounding red cells
is part of the force that repels red cells from
each other
-
Reduce the zeta potential facilitate the RBCs
to agglutinate by IgG molecules
zeta potential zeta potential
Agglutination
-
Enhancement Media
Low Ionic Strength Solution (LISS)
Decrease the ionic strength of a reaction medium
Thus, reduce the zeta potential
Leads to an increase in the attraction between
positively charged Ab molecules and negatively positively charged Ab molecules and negatively
charged red cells
Increase the rate of antibody uptake incubation
time of 5-15 mins; instead of 30-60 mins
-
Albumin
Enhances antibody detection test by reducing
the zeta potential
Allow Ab-sensitized cells to become closer Allow Ab-sensitized cells to become closer
together
Do not promote the antibody uptake stage of
agglutination
-
Enzymes
Can enhance or suppress the reaction of
certain blood group antigens and antibodies
There are certain enzymes can be used:
Ficin (Fish)Ficin (Fish)
Trypsin (pig stomach)
Papain (papaya)
Bromelin (pineapple)
-
Action of Enzyme
The treatment of red cells with enzyme
results in the release of sialic acid from the
membrane of RBC
Subsequent decrease in the negative charge Subsequent decrease in the negative charge
of the cells which lead to reduction in the
zeta potential
-
The use of proteolytic enzymes in in vitro test
systems facilitates antibody identification
Blood group antigen-antibody reactions are
differently affected by enzyme treatmentdifferently affected by enzyme treatment
Enzymes cleave certain proteins from the
surface of the red blood cells, destroying some
antigens while enhancing others
-
Abs reactivity which can be enhanced by
enzyme treatment are Rh, Kidd, P1, Lewis and
I Ags
Ags that can be destroyed or depressed by Ags that can be destroyed or depressed by
enzyme treatment are Fya, Fyb, M, N and S
-
Reagent Uses
Bovine serum albumin
(BSA)
Rh antibodies enhanced
at 37C
Low ionic strength saline
(LISS)
Increases antibody
uptake, decreases
incubation time, cost-incubation time, cost-
efficient
Proteolytic enzymes
(papain, ficin, trypsin,
bromelin)
Kidd, Rh, Lewis
antibodies enhanced,
generally warm and cold
autoantibodies
enhanced