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HEM 2133

Immunohaematology I
Lesson 9: Antihuman Globulin

History of Antiglobulin Testing

Discovered by Coombs, Mourant and Race in
Found that red blood cells (RBC) can become
sensitized 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 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
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
produce anti-human globulins. These antihuman 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 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 antihuman globulins (AHG), AHGs may not be
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
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

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 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 cell
Anti-C3 attaches to C3 molecules bound to the
red cell
Agglutination = positive antiglobulin test
No agglutination = negative antiglobulin test


Positive Antiglobulin Test

Negative Antiglobulin Test

1. Monoclonal (single clone of cells-specificity)
2. Polyclonal (human source: mixture of cellscontains multiple antibodies)
Assist in identifying antigen present on
patients RBC that may cause and reaction in

Polyspecific AHG
Abs to human IgG and Abs to human C3d (antiC3d to detect Ab bound with complements
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
When doing DATs to detect in vivo
sensitization with IgG or C3, blood banks must
use polyspecific AHG containing anti-IgG, antiC3b 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
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

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
charged red cells
Increase the rate of antibody uptake incubation
time of 5-15 mins; instead of 30-60 mins

Enhances antibody detection test by reducing
the zeta potential
Allow Ab-sensitized cells to become closer
Do not promote the antibody uptake stage of

Can enhance or suppress the reaction of
certain blood group antigens and antibodies
There are certain enzymes can be used:
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
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 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
enzyme treatment are Fya, Fyb, M, N and S

Bovine serum albumin

Rh antibodies enhanced
at 37C

Low ionic strength saline Increases antibody

uptake, decreases
incubation time, costefficient
Proteolytic enzymes
Kidd, Rh, Lewis
(papain, ficin, trypsin,
antibodies enhanced,
generally warm and cold