The antigiobulin test popularly known as the Coomb's test was described by Coomb.

Mourant & Race

in 1945 for detecting non agglutinating (incomplete) antibodies in serum.

PRINCIPLE
of the complement either
Red blood cells coated with incomplete antibody (lgG) or C3 component
or in-vitro as in indirect test (LAT),
will be aggiutinated
in-vivo as in direct antiglobulin test (DAT)
to the IgG antibodies coating
the cells. The
by the antihuman globulin (AHG) reagent binding the
antihuman antibody combines with the Fc portion
of the antibody molecules which is present on
two Fab regions
and not with the red cell antigen itself. The
red cells (sensitizing immunoglobulin), the gap
of two sensitizing antibodies (Figure 6.1) bridging
of the AHG molecule attach to Fe portion attached
visible agglutination. Cells that have no
between adjacent antibody coated cells to produce
immunoglobulins are not agglutinated.

Y Antigiobalun
Molecules Y igG
Molecules

Figure 6.1: Principie of antiglobulan

test

125
Antiglobulin Test
The
antiglobulin test popularly known
in 1945 tor
detecting agglutinating the Coomb's test described
non
as
was
(incomplete) antibodies in serum.by Coomb, Mourant & Race
PRINCIPLE
Red blood cells
coated with
in-vivo as in direct incomplete antibody (IgG)
by the antihuman antiglobulin test(DAT) or in-vitro
as in
C3 or
component of the complement either
antihuman antibodyglobulin (AHG) reagent indirect test (IAT), will
be
combines with the
binding to the
IgG antibodies agglutinated
red cells Fc
portion of the coating the cells. The
of the AHG
(sensitizing immunoglobulin), and not with the antibody
red cell
molecules which is
present on the
molecule attach to Fc antigen itself. The two Fab
portion of two regions
between adjacent
antibody coated cells to producesensitizing
visible
antibodies (Figure 6.1)
bridging the gap
immunoglobulins are not
agglutinated. agglutination. Cells that have no attached

Antiglobulin
Molecules

Figure 6. i: Principle
Y
of
lgG
Molecules

antiglobulin test

125
 ANTI HUMAN GLOBULIN REaGENTS (AHG)
wo
types ot anti human globulin reagents are available (Table 6.1):
Polyspeaitic AHG reagent
Monospecitic AHGi reagent
oypecite AHG reagent usually contains Anti lgG and Anti C3d but may also contain Anti C3b
a d Anti CA t is prepared by injecting human globulin into animals, which produce polyclonal
antibodies specitie tor human immunoglobulins and human complement system factors.

Monospeciie AHG reagent contains either of these i.e., Anti IgG, IgM, IgA or complement components.

Table 61: Types of antihuman globulin reagents

Type Source Content
Polyspecifc Polyclonal (Rabbit) Anti lgG, anti C3d, may contain other
anti complement components and
other anti immunoglobulin antibodies
Rabbit polyclonal+ murine Blend of rabbit polyclonal
monoclonal blend anti lgG and murine monoclonal anti
C3b and anti C3d
Murine monoclonal
Monoclonal anti lgG, anti C3b, and
anti C3d.
Monospecific Anti lgG Rabbit polyclonal
Contains anti lgG with no anti
complement activity
Monoclonal lgG
Murine monoclonal anti
Anti complement Rabbit polyclonal
lgG
Anti C3d, anti C3b, anti C4b, anti
Murine monoclonal C4d
Anti C3d or anti C3b and anti
C3d

APPLICATIONS OF ANTIGLOBULIN TEST

Direct Antiglobulin Test (DAT)
Direct test
t is used to detect in vivo
sensitization
generally C3d, as in:(coating) red cells with immune
the complement component of
antibody (IgG) or
Diagnosis of haemolytic disease of new born
(HDN)
Diagnosis of auto immune haemolytic anemia
(AIHA)
investigation of drug induced red cell
sensitization
Investigation of haemolytic transfusion reaction

126
 Antiglobulin Test

Indirect Antiglobulin Test (IAT) antibodies

detect the presence of incomplete antibodies and complement binding
IAT test is uscd to
theserum after coating red cells in vitro in:
in
Compatibility testing
identification of unexpected antibodies in serum
Screening and test such as
antibodies reacting only in antiglobulin
Detection of red cell antigens using specific
K, Jk'. Jk* etc.
Fy', Fy,

AHG TESTS
CONTROL CELLS FOR and are added to
Coomb's control cells
Sensitized O Rh (D) positive
cells are used as

Positive control:
reaction to test the efficacy
of AHG reagent.
a negative from attaching to
membrane-

unbound globulins and may prevent it of AHG

preferentially to before addition
Since AHG reacts free of unbound proteins it
be washed result. Therefore,
bound globulins,
the red cells must c a u s e a false-negative
neutralize AHG and
control cells.
these unbound globulins
may
s e r u m , as addition of Coomb's
of AHG with the
check the efficacy
is imperative to
CONTROL
CELLS
POSITIVE
COATED
PREPARATION OF IgG
Materials

75 x 12 mm test tubes
(D) positive cells
D
Rh Polyclonal anti
Group O & IgM
(monoclonal) o r

-IgG type
or blend of IgG
Anti -D serum

Anti human
globulin reagent

Saline
saline.
Method times in
cells three
positive
Wash the O Rh (D) tube.
1. cells in test
washed & packed
Take 0.5ml
of
2. (monoclonal)
D blend of IgG & gM anti D
3. Add 2-3 drops of anti D in c a s e
polyclonal
anti D or

does not agglutinate them

at 37C.

the dilution of Anti coats the

red cells but
Note: Select only more
which procedure using
1:4 dilution) agglutination, repeat the
is
is used (usually minutes. If there
for 30
incubate at 37°C
4. Mix and
diluted anti -D. suspension
in saline.
make 5% cenirifuge
times and
then
AHG reagent.
Mix gently and
cells 4 v o l u m e s of
5. Wash the and 2
suspension
volume of
5% cell
6. Take o n e 2+
reaction.
should be a
There
the tube. for 48 hours.
stored at 4°C
be
sensitized cells can

These
7.

127
 Medicine
Practice of Transfusion
Principles&

DIRECT ANTIGLOBULIN TEST

Materials
test tubes
l12
75 x
mm

Test red cells

(AHG) reagent
Antihuman globulin
coated)
cells (lgG
Positive control Serum
should be
test tube.
clean prelabelled vitro uptake
Method in a in
be tested EDTA to
prevent
to in
suspension sample
% cell
I drop of 2-4 collect the blood
Place more than 24 hours
old) or

resh (not

of complement. final wash

completely.
decant the
times with saline &
Wash the cells 3
2
3. Add 1-2 drop of AHG reagent.
1 minute.
centrifuge at 1000 rpm for
4. Mix and
the results.
shake the tubes,
read and record
5. Gently
control cells.
is negative, add 1 drop of
result
agglutination is
seen
If the test
6.
minute and look for agglutination. If
no
for 1
Mix and centrifuge at 1000 rpm
the test procedure.
the result is invalid. Repeat

INDIRECT ANTIGLOBULIN TEST

Materials

75 x 12 mm test tubes

Test Serum
cells
Reagent Opositive red blood
Antiglobulin reagent
coated red cells)
Positive control (IgG

Method
1.Take 2 drops of serum to be tested in a prelabelled tube (sample should be fresh for detecting
complement binding antibodies, otherwise fresh AB serum should be added to it).

2 Add one drop uf 2-4% suspension of reagent O cels.

3. Incubate at 37"C for 45-60 minutes.

4. Look for haemolysis or

agglutination. Agglutination or
haemolysis at this stage indicates presence
of saline reacting antibody
128
 Antiglobulin Test

5. 1f no hacmolysis or agglutination is seen, wash the sample three to four times in saline and decant
the last wash completely.
.Add -2 drops of AHG reagent to the washed cells and mix.

7. Centrituge the tubes at 1000 rpm for I minute.

8. Gently shake the tubes and look for agglutination.

9. Record the result
10. If the test is negative, add I drop of control cells (IgG coated cells)
11. Mix and centrifuge at 1000 rpm for I minule.
is
is seen, the test is invalid and
whole test prOcedure
12. Look tor agglutination. If no agglutination
repeated.

Note: Auto control should be kept with the IAT

SENSITIVITY OF IAT
FACTORS AFFECTING THE

Temperature can reduce sensitivity

at lower temperature
for IAT test is 37°C. Incubation
Optimum temperature
red blood cells.
while higher temperature damages

Serum:cell ratio of
used ratio is 2 drops
sensitivity. Commonly
to red cells,
increases
ratio of serum volumes of cell and
Increasing the In low ionic strength
saline (LISS) equal
serum 1& of 2-4 % cell suspension.
drop
serum are used.

Incubation time
45-60 minutes
albumin o r enzymes
technique
a) For saline,
10-15 minutes
cells
b) For LISS suspended
Suspending medium
enzyme or LISS.
addition of 22% bovine albumin,
be increased with
of IAT can
The sensitivity
ANTIGLOBULIN TEST
INDIRECT
STRENGTH SALINE
LOW IONIC
(LISS/IAT) the red cells and
increases the rate and degree of antibody uptake by
IAT generally
The use of LISS in
time considerably.
reduces the incubation

Materials
12 test tubes
75 x mm

saline (LISS)
Low ionic strength

129
 Test serum

Reagent red cells

Antiglobulin reagent
Positive control (lgti coated cells)

Methods
1. Wash the red cells twice in normal saline.
Wash these cells once in LISS.
.Make 2-4% reagent red cell suspension in LISS.
4. Take equal volume of serum & LISS suspended cells in a test tube.
Incubate for 15 minutes at 37C
( for 5 minutes in emergency).
Remove the tube and look for haemolysis /agglutination if present. Record the result.
If no
agglutination is seen, wash the cells 3 times in saline.
Add 1-2 drops of AHG serum to the washed red cells.
9.
Centrifuge at 1000 rpm for 1 minute.
10. Gently
shake the tube and look for
agglutination using optical aid.
11. Record the result.
12. If test results are negative, add one drop of control
IgG coated cells. Mix and
centrifuge at 1000
rpm for 1 minute. Look for agglutination. If no
agglutination is seen, the test is invalid and whole
test procedure is repeated.
Note: LISS suspended cells and serum should be brought to room
temperature before use.

SOURCES OF ERROR IN ANTIGLOBULIN TEST

Problems in antiglobulin test can arise either due to false
such, all efforts must be made to avoid any negative results or false positive results. As
possible source of error for
achieving accurate and
results. precise
False Negative Results
Inadequate washing of cells
Delay or interruption of washing procedure
Failure to add AHG
reagents
Under centrifugation
Too heavy or weak red cell
suspension
Prozone effect

Insufficient incubation
AHG reagent that has lost
potency
Presence of small fibrin clots
among ceils

130
False Positive Results
AHG
Agglutination of red cells before adding reagent

Over Centrifugation
Dirty glass ware
container
from glass ware and from metal
Leaching of silica and metallic ions in saline
AHG reagent containing antihuman species antibody
Cold auto antibodies in sample
Test, DAT)
EDTA for Direct Antiglobulin
(clotted cells instead of
Using improper sample
(cells heavily coated with IgM)
Spontaneous agglutination

Non-specific agglutination ("sticky cells")

EXERCISE
ASSESSMENT AND REVIEW
SELF
globulin test is performedto detect
Q1. Anti human

1. Complete antibodies

2. Incomplete antibodies

test?
What is the principle of Coomb's
Q2. available?
human globulin reagents
3. What are the types of anti
Q
control cells?
Q4. What is the role of Coomb's
direct antiglobulin test indicates.
Q5. A positive

Antibody in
serunm
1.

on red cell
2. Antibody
for IAT test is
temperature
Q6. Optimum
1. 25C

2. 4°C
monitor the
3. 37C reactions in AHG test is a way to
cells to all negative
Addition of IgG coated
07.
results.
to false
after incubation in IAT may give rise
cells
Inadequate washing ofred
Q8. used to enhance IAT:
media which may be
suspension
two important
Q9. List IAT are
affect the sensitivity of
factors which
Q 10. Three important

Q11.List three uses of JAT.