ANTIGLOBULIN TEST (AHG) IMMUNOHEMATOLOGY LABORATORY

§ Because their single monomer structure

INTRODUCTION
is too small to directly agglutinate
§ Anti-human globulin test (aka Coombs’ Test) - is
sensitized RBCs
based on the principle that antihuman globulins
§ Adding AHG allows for the
obtained from immunized nonhuman species
hemagglutination of the sensitized
bind to human globulins such as IgG or
RBCs.
complement either free in serum or attached to
§ Crossmatching
antigens on red blood cells (RBCs).
The Antiglobulin Test – Principle
§ Anti-globulin – green color
§ Red cells coated with IgG antibodies DO NOT
§ Adding AHG reagent containing anti-IgG to
agglutinate directly when centrifuged. These
RBCs sensitized with IgG antibodies allows for
cells are said to be sensitized with IgG or
hemagglutination of these sensitized cells.
Complement.
§ An anti- antibody is used to observe the
§ In order for agglutination to occur, an additional
formation of an Ag/Ab complex that would
antibody which reacts with the Fc portion of the
otherwise go undetected
IgG antibody or with the C3b or C3d component
of the complement must be added to the system
HISTORY OF ANTIGLOBULIN TEST § This will form a “bridge” between the antibodies
§ 1945 – Coombs, Mourant and Race or complement coating the red cells, causing
§ Described the use of the antiglobulin hemagglutination.
test for the detection of weak and non -
agglutinating Rh antibodies in serum.
§ 1946 - Coombs
§ Described the use of AHG to detect in
vivo sensitization of the RBCs of
neonates suffering from hemolytic
disease of the fetus and newborn
(HDFN).
§ 1908 - Moreschi
§ Was the first to describe the principle of
antiglobulin test
Monospecific &
Monoclonal & Polyclonal
Polyspecific
THE ANTIGLOBULIN TEST
Demonstrates antibody
§ The Coombs’ test involved the injection of Demonstrates method of
range/scope of the
human serum (purified globulin: IgG or preparation
antibody and the reagent
complement) into rabbit to produce antihuman
Demonstrates antibody Demonstrates way of
serum/anti-sera
specificity manufacturing reagent
§ The AHG test detects the presence of
Demonstrates antibody Determines the purity of
nonagglutinating IgG antibodies attached to
activity if: anti-IgG or the antibodies in the
sensitized Red Blood Cells.
IgM - bind to corresponding antigen and directly anti-complement reagent
§
agglutinate RBCs suspended in saline.
§ ABO blood typing
§ IgG - “nonagglutinating”, or “incomplete
antibodies”

JJ LORESCO, RMT 1
ANTIGLOBULIN TEST (AHG) IMMUNOHEMATOLOGY LABORATORY

Polycloncal Antibodies Monoclonal Antibodies

ANTIGLOBULIN REAGENTS –
Mixture of antibodies
POLYSPECIFIC & MONOSPECIFIC
from different plasma cell
clones. The resulting Produced using
POLYSPECIFIC AHG MONOSPECIFIC AHG
polyclonal antibodies hybridoma technology;
recognize different derived from one clone
Contain antibody to
Contain only one antigenic determinants of plasma cells and
human IgG and to the
antibody specificity. (epitopes), or the same recognize a single
C3d of human
portion of the antigen epitope
complement.
but with different
Either anti-IgG or
Other anticomplement affinities
antibody to specific
antibodies, such as anti- Different plasma cells + Sample plasma cell +
components of
C3b, may also be Different epitopes Same epitope
complement, such as
present. Different plasma cells,
C3b or C3d Antibodies are pure,
different antibodies,
Anti-IgG – contain identical structurally and
Its use can facilitate different epitopes but
antibodies specific for immunologically, binds to
agglutination when RBCs binds to the same
the Fc fragment of the same area
have been sensitized with antigen
gamma heavy chain of
IgG or C3d or both.
the IgG molecule.
Anti-complement - such POLYCLONAL AHG PRODUCTION
as anti-C3b, -C3d § In vivo
reagents, are reactive § Conventional polyspecific antiglobulin reagents
Anti-IgG + Anti- against only the are produced by immunizing one colony of
complement designated complement rabbits with human immunoglobulin (IgG)
components and contain antigen and another colony with human C3
no activity against human antigen.
immunoglobulins § Both colonies of animals are hyperimmunized to
produce high-titer, high-avidity IgG antibodies.
§ Polyspecific antihuman globulin may
PREPARATION OF AHG REAGENTS
be manufactured by combining polyclonal anti-
§ The classic method of AHG production involves
IgG with either polyclonal or monoclonal
injecting human serum or purified globulin into
anticomplement components.
laboratory animals such as rabbits (polyclonal)
§ Blood specimens are drawn from the immunized
and mice (monoclonal).
animals, and if the antibody potency and
§ The human globulin behaves as foreign antigens,
specificity meet predetermined specifications,
the rabbit’s immune response is triggered and an
the animals are bled for a production batch of
antibody to human globulin is prepared
reagent
§ For example, human IgG injected into a rabbit
§ The rabbits are injected with pooled antigen;
results in anti-IgG production; human
either human IgG or human complement.
complement components injected into a rabbit
§ After a few weeks, the rabbits will be immunized
result in anticomplement
against the pooled antigens and its immune
system will produce antibodies against the
human IgG (anti-IgG) and human complement
components (anti-complement)
§ These antibodies can be harvested as antiserum

JJ LORESCO, RMT 2
ANTIGLOBULIN TEST (AHG) IMMUNOHEMATOLOGY LABORATORY

§ Antiserum drawn from an animal will contain § This allows the production of a consistently pure
antibodies from multiple clones of B cells, with and uncontaminated AHG reagent.
each B cell responding to a specific epitope on § All antibodies produced by a clone of cells
the antigen. recognize a single epitope present on an antigen

MONOCLONAL AHG PRODUCTION

§ Monoclonal AHG production and Hybridoma
technology were introduced by Kohler and APPLICATIONS OF ANTIGLOBULIN
Milstein TEST
§ In vitro tissue culture DIRECT ANTIGLOBULIN TEST (DAT)
§ Monoclonal antibody production begins with the
§ Detects in vivo sensitization of RBCs with IgG or
immunization of laboratory animals, usually mice,
complement components.
with purified human globulin
§ Performed directly by testing the sample of
§ After a suitable immune response, mouse spleen
washed RBCs with AHG reagent
cells containing antibody-secreting lymphocytes
§ Clinical conditions that can result in in vivo
(plasma cells: Anti-IgG and anti-C3d) are fused
coating of RBCs with antibody or complement
with myeloma cells (cells that reproduces or
are the following: (+) results:
multiplies infinitely)
§ Hemolytic disease of fetus and newborn
§ The resulting hybridomas (lymphocyte–myeloma
§ Hemolytic transfusion reactions
hybrid cells that produces monoclonal
§ Autoimmune and drug-induced
antibodies) are screened for antibodies with the
hemolytic anemia
required specificity and affinity.
§ (+) DAT is an important indicator of potential
§ The antibody-secreting clones may then be
immune-mediated red cell destruction in the
propagated in tissue culture or by inoculation
body.
into mice, in which case the antibody is collected
§ As a consequence of IgG or
as ascites
complement attachment to red cells,
§ Those producing the desired mAb are grown in
macrophages are signaled to clear then using
tissue culture; the culture medium is harvested
the mononuclear phagocytic system, particularly
periodically and mAbs are purified from the
in the spleen.
medium.
§ This can signal immune destruction of red cells &
§ All antibody molecules produced by a clone of
often leads to anemia.
hybridoma cells are identical in terms of antibody
§ DO NOT need incubation step
structure and antigen specificty

JJ LORESCO, RMT 3
ANTIGLOBULIN TEST (AHG) IMMUNOHEMATOLOGY LABORATORY

§ A one stage procedure § Antibody Identification (Patient serum +

§ RBC Washing: to remove unbound or free Panel cells)
antibodies in the reaction and to avoid AHG § RBC phenotyping (Patient RBC + Panel
neutralization antibodies)
§ False negative: inadequate washing § Antibody Titration (Patient serum +
Panel cells)

IAT: PROCEDURE

TASK PURPOSE

INDIRECT ANTIGLOBULIN TEST (IAT) Incubate RBCs with Allows time for antibody
§ The IAT is performed to determine in vitro antisera molecule attachments to
sensitization of RBCs. RBC antigen
§ It is a two-stage procedure (thus, “indirect”). Perform a minimum of Removes free globulin
§ Incubation stage (facilitates sensitization) three saline washes molecules
§ Antiglobulin phase (adds antiglobulin Add antiglobulin reagent Forms RBC agglutinates
reagent to demonstrates agglutination) (RBC Ag + Ab + anti-IgG)
§ Ab first must combine w/red cell antigens in vitro Centrifuge Accelerates agglutination
through the incubation step, after w/c is washed by bringing cells closer
to remove any unbound proteins. together
§ Following the washing, AHG reagnet is added in Examine for agglutination Interprets test as positive
the 2nd stage. or negative
§ Any agglutination in the 2nd stage is a (+) IAT, it Grade agglutination Determine strength of
means a specific reaction between an Ab in the reactions reaction
serum & an antigen present on the red cells. Add antibody-coated Checks for neutralization
§ Incubate at 37 C RBCs to negative of anti-sera by free
§ Applications: reactions globulin molecules
§ Crossmatching (Patient serum + Donor (Coombs’ control cells are
RBC) D-positive RBCs coated
§ Determine if the with anti-D)
recipient/patient is compatible
with the donor Coombs check cells: Type “O” (Rh positive) RBCs coated
§ Major crossmatch: PS + DR with anti-D
- To determine if the patient § Used if IAT (-)
has antibodies against the § Differentiate true negatives from false
antigens present on donor negatives
RBC § True negative: agglutination would occur
- Agglutination (+) = after adding Coombs check cells to IAT (-)
Incompatible § False negative: absence of agglutination after
- Without Agglutination (-) adding Coombs check cells; AHG has been
= compatible neutralized by interfering antibodies
§ Minor crossmatch: PR + DS
§ Antibody screening (Patient serum +
Screen cells)

JJ LORESCO, RMT 4
ANTIGLOBULIN TEST (AHG) IMMUNOHEMATOLOGY LABORATORY

FACTORS AFFECTING THE COMMON SOURCES OF ERROR

ANTIGLOBULIN TEST
1. Ratio of Serum to Cells (Serum-to-Cell-Ratio) False Negatives Possible Explanation /
§ 2:1 ratio Mechanism
§ 2 drops of serum to 1 drop of 5% RCS Failure to wash red cells Unbound human serum
2. Reaction Medium (speeds up antigen antibody adequately during the test globulins neutralize the
reaction by shortening the incubation time) procedure before the AHG reagent
§ Albumin Medium (Obsolete) addition of AHG reagent
§ Allow antibody-coated cells to come into Testing is interrupted or Bound IgG or
closer contact with each other so that delayed; AHG reagent is nit complement molecules
agglutination occurs. added immediately after may detach from the
§ Increases cell-to-cell contact washing coated red cells
§ Low Ionic Strength Solution (most commonly Failure to identify W+ Technical error in
used) reactions testing
§ Enhance antibody uptake and allow Loss of reagent activity Improper reagent
incubation times to be decreased (10-15 storage bacterial
mins) contamination
§ Reduces the zeta potential surrounding or contamination
the RBCs. w/human serum
§ Polyethylene Glycol (10-15 mins) Failure to add AHG reagent Technical error in
§ Increased antibody uptake by removing testing
water molecules around the RBCs (the Improper centrifugation: Conditions for
water of hydration theory), thereby undercentrifugation promoting
increasing antibody concentration. agglutination are
3. Incubation Temperature not optimal
§ Optimal temperature is 37 C Inappropriate red cell Concentration of red
4. Incubation Times concentrations: red cell cells influences the rxn
§ In Saline: may vary from 30 minutes – 2 hours suspensions fall outside the
§ In LISS or PEG: may vary from 10- 15 mins optimal 25%
5. Washing of RBCs
§ RBCs must be saline-washed a minimum of 3x False Positives Possible Explanation /
before adding AHG reagent. Mechanism
§ Washing the RBCs removes free unbound serum Red cells are agglutinated Potent cold
globulins before washing step & reactive Ab or
6. Saline for Washing addition of AHG reagent patient origin.
§ Ideally, the saline used for washing should be Use of dirty glass ware Particles or contaminants.
fresh (suggested open expiration of 30 days) and Improper centrifugation: Red cell button packed so
buffered to a pH of 7.2 – 7.4 Overcentrifugation tightly on centrifugation
7. Addition of AHG Reagent that nonspecific clumping
§ AHG should be added to the cells immediately
cannot be dispersed.
after washing to minimize the chance of antibody
eluting from the cell and subsequently
neutralizing the AHG reagent.
8. Centrifugation for Reading
§ 500 RCFs for 15-20 seconds

JJ LORESCO, RMT 5