ANTIHUMAN

GLOBULIN (AHG)
TEST
BLOOD BANKING PROCEDURES

ANTIHUMAN GLOBULIN TEST (COOMBS’ TEST)

Principle: A technique for detecting cell-bound immunoglobulin. It

is used to detect incomplete antibodies (IgG).
•  The antihuman globulin test, which is also
referred to as the Coombs’ test

•  AGT is based on the principle that

antihuman globulins (AHGs) obtained from
immunized nonhuman species bind to
human globulins such as IgG or
complement, either free in serum or
attached to antigens on red blood cells
(RBCs).
 IgM IgG
Natural Immune
Complete Incomplete
Agglutinating Coating/Sensitizing
Cold-Reacting Warm-reacting
Saline-reactive Albumin/AHG-reactive
Ex. ABO antibody Ex. Rh antibody
Complement binding Complement binding
(more potent)
HISTORY OF AGT

•  1945: Coombs and associates described the use of the

antiglobulin test for the detection of weak and
nonagglutinating Rh antibodies in serum.

•  1946: Coombs and coworkers described the use of AHG to

detect in vivo sensitization of the RBCs (later called the
direct antiglobulin test [DAT]) of babies suffering from
hemolytic disease of the newborn (HDN). Although the test
was initially of great value in the investigation of Rh HDN,
its versatility for detecting other IgG blood group antibodies
soon became evident.
HISTORY OF AGT
•  The first of the Kell blood group system antibodies
and the associated antigen were reported only weeks
after Coombs had described the test.

•  Although Coombs and associates were instrumental

in introducing the antiglobulin test to blood group
serology, the principle of the test had in fact been
described by Moreschi in 1908
HISTORY OF AGT

•  1947: Coombs and Mourant demonstrated that the

antibody activity that detected Rh antibodies was
associated with the anti–gamma globulin fraction in
the reagent.

•  1951: Dacie presented the first indication that there

might be another antibody activity present that
influenced the final reaction. He observed that
different reaction patterns were obtained when
dilutions of AHG were used to test cells sensitized
with warm as compared with cold antibodies.
HISTORY OF AGT

•  1957: Dacie and coworkers published data showing

that the reactivity of AHG to cells sensitized with warm
antibodies resulted from anti–gamma globulin activity,
whereas anti–nongamma globulin activity was
responsible for the activity of cells sensitized by cold
antibodies. The nongamma globulin component was
shown to be beta globulin and had specificity for
complement
AHG reagents (Commercially Prepared)
1. Polyspecific AHG Reagents – consists of a pool of rabbit
anti-human IgG and mouse monoclonal anti-C3b and anti-C3d.
- Also referred to as Broad Spectrum Coombs Reagent.

2. Monospecific AHG Reagents – contains only one antibody specificity.

Either: a. Anti-IgG
b.Anti-C3b or C3d
•  Polyspecific AHG reagents contain antibody to
human IgG and to the C3d component of human
complement
•  Other anticomplement antibodies, such as anti-
C3b, anti-C4b, and anti-C4d, may also be
present. Therefore, its use can facilitate
agglutination when RBCs have been sensitized
with IgG or C3d or both.
•  Commercially prepared polyspecific AHG contains
little, if any, activity against IgA and IgM heavy
chains. However, the polyspecific mixture may
contain antibody activity to kappa and lambda light
chains common to all immunoglobulin classes, thus
reacting with IgA or IgM molecules.
ü  Monospecific AHG reagents contain only one antibody
specificity: either anti-IgG or antibody to specific
complement components such as C3b or C3d (i.e.,
anticomplement)

Anti-IgG reagents contain antibodies specific for the FC

fragment of the gamma heavy chain of the IgG
molecule.

If not labeled “gamma heavy chain–specific,” anti-IgG may

contain anti–light chain specificity and may therefore
react with cells sensitized with IgM, IgA, and IgG.
Monoclonal AHG reagents
•  Anticomplement reagents, such as anti-
C3b, anti-C3d reagents, are reactive
against only the designated complement
components and contain no activity
against human immunoglobulins.
Monospecific anticomplement reagents
are often a blend of monoclonal anti-
C3b and monoclonal anti-C3d
Stages of Antigen-Antibody Interaction
The first stage is sensitization. Sensitization occurs when
antibodies react with antigens on the cells and coat the cells.

The second stage of the reaction is agglutination.

Agglutination occurs when antibodies on coated cells form
cross-linkages between cells resulting in visible clumping.
 TYPES OF AHG PROCEDURES
1. DIRECT AHG TEST (DAT)
- Detects in vivo sensitization of red cells with IgG
and/or complement.
- Useful in the ff. situations:
- investigation of transfusion reactions (e.g.HTR)
- diagnosis of HDN
- diagnosis of autoimmune and drug-induced
hemolytic anemias

***Cells used for DAT should be collected into either EDTA or

citrates containing anticoagulant to minimize the possibility of in
vitro attachment of complement components.
DAT
•  The direct antihuman globulin test (DAT) is
needed to demonstrate antibodies in the event of
in vivo erythrocyte sensitization.
•  Thus, antibodies or complement components
already fixed to the patient's erythrocytes are
detected.
•  Following a triple washing process with the
sensitised cells, the AHG serum is added.
Importance of washing in DAT
(1) Presence of other unbound Abs
Washing phase (2)
Addition of AHG (3)
IgG bridge formation (4)
Agglutination (5)
 Evaluation of a positive
DAT
•  AABB Technical Manual: “A positive DAT alone is
not diagnostic”
•  Significant positive result requires knowledge :
1. Patient’s diagnosis
2. Recent drug intake
3. Pregnancy
4. Transfusion history
5. Info on the presence of acquired or unexplained
hemolytic anemia
2. INDIRECT AHG TEST (IAT)
- A two step procedure (sensitization and agglutination)
that determines in vitro sensitization of red cells

- Useful in the ff. situations:

- Detection of incomplete antibodies in compatibility
testing or to screening cells in antibody screen
- Identification of antigen specificity, using a panel
of red cells
- Determination of red cell phenotype using known
antisera (e.g. Du testing, Kell typing)
- Titration of incomplete antibodies
 FACTORS AFFECTING THE AHG TEST

1. Ratio of serum to cells.

Minimum ratio 40:1 = 2 drops serum and 1 drop of 5%v/v cell
suspension
2. Temperature- Optimal: 37ºC
3. Incubation Time – In saline suspension: 30-120 minutes
LISS/PEG suspension: 10-15 minutes
4. Reaction medium (albumin, LISS and PEG)
ü  60-minute saline test = 30-minute albumin technique
22% Albumin – 2 drops 22% albumin + 2 drops serum + 1 drop 3-5%
cell suspension
ü  LISS- is said to reduce the zeta potential between RBCs thus increasing
the rate of antibody uptake on the cell
- also increases sensitivity and shortens incubation times from 30-60
minutes to 10-15 minutes
 LISS – 2 drops 3% RBC suspension in LISS + 2 drops
serum

ü  PEG: removes water surrounding the RBCs to effectively concentrate

the antibodies

5. Washing of cells – minimum of three times

6. Saline for washing –should be fresh and buffered to a
pH of 7.2-7.4
7. Addition of AHG reagents should be added to washed cells
immediately after washing.
8. Centrifugation- 1000 rcf for 15-20 seconds,
SOURCES OF ERROR IN THE AHG TECHNIQUE

False-Positive Results
1. autoagglutinable cells
2. bacterial contamination or other contamination in cells or saline
3. cells with a POSITIVE DAT used for IAT
4. overcentrifugation and overreading
5. polyagglutinable cells
6. dirty glasswares
7. saline contaminated with silica or heavy metals
False Negative Results

1. Inadequate or improper washing of cells (most

common cause)
2. AHG reagent nonreactive owing to deterioration or
neutralization
3. AHG reagent not added
4. serum not added in the indirect test
5. serum is nonreactive owing to deterioration of
complement
6. inadequate incubation conditions
7. Postzone and Prozone (cell suspension either too
weak or too heavy)
8. Undercentrifugation
9. Poor reading technique
Patient Cells + Donor Serum

Unbound
Immunoglobulins
Unwashed cells + AHG reagent
(Anti-human IgG)

False negative reaction

AHG is neutralized by the
unbound immunoglobulins
Washed cells + AHG reagent

True positive reaction

n  Modified and automated AHG tests
techniques :
1.low-iconic polybrene technique,
2. enzyme-linked antiglobulin test,
3. solid phase technology, and
4. gel test.