Antiglobulin

test

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 Antiglobulin test
• Also known as Anti Human Globulin test
(AHG) or coombs test

• Test was first described by Coomb’s et

al in 1945

• It is very important in detecting

clinically significant unexpected
antibodies either in-vivo or in-vitro
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Principle of test
Red blood cells
coated with
IgG coated incomplete IgG
red blood antibodies or
cells
complement do
not readily
agglutinate.
These cells are
said to be
sensitized with
Complement
coated red blood IgG or
cells complement

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• For agglutination to occur, an
additional antibody that reacts with
the FC portion of the IgG or the C3b
or C3d must be added to system to
bridge the antibodies or the C3b or
C3d forming agglutination

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 Anti human globulin
reagent production
• It is made by injecting rabbits, sheep or
goat with purified human IgG or C3.

• The animal is bled after a specified period

and the reagent purified by absorbing
unwanted antibodies

• The reagent may be polyspecific or

monospecific 5
 Anti human globulin reagent
production

• Polyspecific Anti-human Globulin:

contains a blend of Anti-IgG & Anti-
C3b, -C3d and sometimes C4

• Monospecific reagents: contains

Anti-IgG alone or Anti-C3b,-C3d alone

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Antiglobulin test and
interpretation

Whether the cells have

been coated, or
sensitized, in vivo or in
vitro the final
interpretation is based
on the following:
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Positive antiglobulin test
Incomplete Antibodies
attach to the red cells

•Wash cells 3 to 4 times to remove any unattached antibodies

•Only antibodies attached with the cell remain

Add antihuman globulin

Visible agglutination in

test tube 8
Negative antiglobulin
test
Antibodies are
not attached to
antigens during
incubation

Wash cells three to four times to remove any unattached

antibodies

Add anti human globulin

No visible agglutination
therefore a negative test

Check cells
Add coombs control check cells (CCC) agglutinated whilst
original test cells
remained
unagglutinated9
Agglutination following addition of CCC verifies negative result
 Preparation of coombs
control check cells (CCC)
• Positive Control :
Sensitized 0 Rh (D) positive cells

• Negative Control :
Sensitised 0 Rh (D) negative cells
Unsensitised 0 Rh (D) positive cells

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• Take 0.5 ml of 5-6 times washed and packed
0 Rh (D) +ve red cells in a test tube.

• Add 2-3 drops of IgG anti-D (select a dilution

(titer 1:4) of anti-D which coats the red cells
but does not agglutinate them at 37°C).

• Mix and incubate at 37°C for 30 minutes. If

there is agglutination, repeat the procedure
using more diluted anti-D.

• Wash 3-4 times and make 5% suspension in

saline for use.
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• Perform a Direct antiglobulin test
which should give a 2+ reaction. If
no agglutination occurs, repeat the
test by using less diluted anti-D
serum.

• 0 Rh(D) negative sensitized red cells

are also prepared by treating 0 Rh(D)
negative cells in the same manner.
The preparation should give a
negative direct antiglobulin test
(DAT) 12
 False negative and
positive results in
antiglobulin test
• False-negative and false-positive
reactions can occur in antigen-antibody
reactions in the following ways.

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• False negatives results may be
obtained when there is:

1. Inadequate cell washing

2. Delay in adding antiglobulin
reagent after the washing step
3. Presence of small fibrin clots
among the cells
4. Inactive, or forgotten
antiglobulin reagent
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Inadequate washing

Inadequate cell washing will

lead to unbound antibody
remaining in the red cell
suspension that are available
to neutralize the AHG
(Coombs serum) so it will not
react with red cells bound
with antibody 15
Delay in adding AHG
Delay in adding Coombs serum
after washing step will lead to
antibody eluting off, detaching
from the cell while cells are sitting
in saline. Now free antibody
present in the saline neutralizes
the AHG or Coombs reagent so it
will not be able to react with the
cells bound with antibody. 16
 Presence of Small
fibrin clot among cells
• Small fibrin clot among the
cells that were not washed
away will have immunoglobulin
and complement present. The
antibodies and complement in
the fibrin clot neutralizes AHG
leading to a negative test.
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 False positive results
• Using improper sample (clotted cells
instead of EDTA for Direct
Antiglobulin Test, DAT)
• Spontaneous agglutination (cells
heavily coated with IgM)
• Non-specific agglutination ("sticky
cells")

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 Types of Antiglobulin
test
• There are two types:
a. Direct antiglobulin test
(DAT)
b. Indirect antiglobulin test
(IAT)

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 DAT
• Direct antiglobulin test is used to detect in-vivo
sensitization (coating) of red cells with immune
antibody (IgG) or the complement component
(C3b or C3d) in :

1. Diagnosis of haemolytic disease of the newborn

(HDN)
2. Diagnosis of autoimmune haemolytic anaemia
(AIHA)
3. Investigation of haemolytic transfusion reaction
(HTR)
4. Investigation of drug induced red cell
sensitization
eg. Penicillins, phenacetins, quinidine 20
21
Procedure

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• Prepare a 5% suspension of isotonic saline of
the the RBCs to be tested

• With clean Pasteur pipette, add a drop of the

prepared cell suspension to a small tube

• Wash three ties with normal saline to remove

all the traces of serum

• Decant completely after the last wash

• Add 2 drops of antihuman serum

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• Incubate at at 37oC for 5-10 mins

• Mix well and centrifuge at one

minute at 1500 RPM

• Resuspend the cells by gentle

agitation and examine
macroscopically and or
microscopically for agglutination

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 Result interpretation
• Negative results:
• No clumping of cells (no agglutination)
• This means you have no antibodies to red cells

• Positive results:
• Clumping agglutination of the red cells during a
DAT means that you have antibodies on the red
cells or have a condition that causes
destruction of red cells y your immune system

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 IAT
• The purpose of the indirect antiglobulin test is to
detect In vitro sensitization of red cells. This is done
when sensitization does not lead to direct
agglutination
Uses
• Weak D's in donor bloods and pregnant females of
individuals who type D (-) at room temperature when
doing ABO and Rh typing

• Screening and identification of unexpected (irregular)

antibodies in serum.
• Compatibility testing

• Detection of red cell antigens using specific antibodies

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reacting only in antiglobulin test (K, Fya, Fyb, Jkb, etc.)
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PROCEDURE

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 Factors affecting sensitivity of
IAT
Temperature
Optimal temperature : 37°C.
Incubation at higher or lower temperature may give false results.

Serum Cell ratio

Increasing the ratio of serum to cells increases the antibody coating.
Commonly used ratio in saline suspension is 2:1 but in LISS suspending
cells, use equal volume of serum and 2% cell suspension.

Incubation time
Saline, Albumin or enzyme technique : 45-60 minutes
LISS suspended cells - Routine 15 minutes
Emergency : 5 minutes

Suspension medium
The sensitivity of IAT can be increased with addition of 22% bovine 29
albumin, enzyme or by using LISS suspended cells.
 Mechanism of
enhancement
• LISS: reduces the ionic strength of the
reaction medium thereby enhancing
uptake of antibodies by the red cells

• Proteolytic enzymes: cleave

sialoglycoprotein from red cells thereby
reducing net charge between red cells

• Albumin: reduces the repulsion charges

between red cells
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