ANTI GLOBULIN TEST

(COOMB’S TEST)
Presenter – Dr.Ishita Bansal
Moderator – Dr.Vaibhav Goyal
ANTIGLOBULIN TEST ( COOMBS’ TEST )
• Coombs, Mourant and Race first described the antiglobulin test (also called Coombs’
test) in 1945.
• Antiglobulin test is used to detect either in-vivo sensitization of red cells by antibodies
or to detect red cell antibodies present in plasma after sensitizing them in-vitro onto
red cells. The test helps in differentiating immune causes of hemolysis from non-
immune causes and in identifying clinically significant red cell alloantibodies capable of
causing red cell hemolysis.
Antiglobulin test is based on the following principle:
• Antibody molecules (IgG) and complement components are globulins.
• Antihuman globulin (AHG) is an antibody against human globulin molecules
that reacts with them either bound to the RBCs or present free in the
serum.
• Washed red cells coated with human globulins (IgG and complement) are
thus agglutinated by AHG,resulting in visible agglutination.
ANTIHUMAN GLOBULIN
• Anti-human globulin represents antibodies against Fc portion of human
immunoglobulins and complement components. Anti-human globulins are
prepared either by classic method or by hybridoma technology.
• The most commonly used AHG is directed towards IgG and C3d. It is called
polyspecific if both anti-IgG and anti-C3d are present and monospecific if only
one component, i.e., either anti-IgG or anti-C3d, is present
• AHG forms the cross-links between red cells sensitized with IgG and/or C3d,
bridging the gaps between red cells and helps in a lattice formation
• Antiglobulin test is of two types:
1. Direct coombs test – It is used to detect in vivo sensitization
( coating) of red cells with immune antibody ( IgG ) or the
complement generally C3d.
• Applications:-
- Diagnosis of hemolytic disease of new born (HDFN)
- Diagnosis of auto immune haemolytic anemia (AIHA)
- Investigation of drug induced red cell sensitization
- Investigation of haemolytic transfusion reaction
 Direct Antiglobulin test
Clinical Application In vivo Sensitization

HDFN Maternal antibody coating fetal RBCs

HTR Recipient antibody coating donor RBCs

AIHA Autoantibody coating individual’s RBCs

2. Indirect coombs test – It is used to detect incomplete
antibodies and complement binding antibodies in the serum
after coating red cells in vitro
• Applications:-
-Compatibility testing
-Screening and identification of unexpected antibodies in serum
Control cells for AHG Tests
Preparation of IgG coated positive control cells
Materials –
• Test tubes
• Group O Rh (D) positive cells
• Anti – D serum
• Anti human globulin reagent
• Saline

Method –
1.Wash the O Rh(D) , To wash the red cells, fill two-third of the test tube with Normal
saline , thoroughly mix the red cells with it followed by centrifugation by giving a
hard spin and then decant the supernatant saline. Repeat this at least three times.
2.Take 0.5ml of washed & packed cells in test tube
3.Add 2-3 drops of anti D
• Note: Select the dilution of Anti-D (usually 1:4 dilution ) which only coats
the red cells but does not agglutinate them at 37 C.
• 4.Mix and incubate at 37 C for 30 minutes . If there is agglutination, repeat
the procedure using more diluted anti-D
• 5.Wash the cells 3 times and then make 5% suspension in saline by taking
950mcl of saline and 50mcl of red cells
• 6.Take one volume(2 drops) of 5% cell suspension and 2 volume of AHG
reagent . Mix gently and centrifuge the tube . There should be a 2+ reaction
• 7.These sensitized cells can be stored at 4 C for 48 hours in Alsever’s
solution
Material required for DCT
Materials required
• Patient Sample (EDTA Vial)
• AHG reagent-Polyspecific (anti-IgG & C3d)
• Sensitized red cells / Check cells
• Normal saline (0.9%)
• Glass tubes
• Pasture pipette
• Tabletop centrifuge
Material required for DCT

• Patient Sample (EDTA Vial)

AHG reagent-Polyspecific (anti-IgG & C3d)
LISS
Pasture pipette and tips
Tabletop centrifuge
Centrifugation for hard spin
Procedure for DCT :

1. Identify the patient sample by checking and matching patient details from the
request form and sample
2. Centrifuge the sample by hard spin (3000 rpm for 2-3 min) to separate red cells
from plasma.
3. Take a clean test tube and label with a patient identifier (name or hospital
number).
4. Transfer patient's packed red cells to the labelled test tube and wash them at
least three times.
5. To wash the red cells, fill two-third of the test tube with Normal saline , thoroughly
mix the red cells with it followed by centrifugation by giving a hard spin and then
decant the supernatant saline. Repeat this at least three times.
6.Add 990 mcL of LISS to test tube and then add 10mcL of patient’s
washed cells in test tube ( 1% cell suspension )
7.For control ‘0’ cell suspension is made by adding 990 mcL of LISS to
10 mcL of O+ red cells
8. Then the control ‘0’ cells is added (50mcL) to one card of LISS gel card
9.For positive control Anti-D of that blood group(i.e O) is added and one is
negative control in this normal saline is added.
10.For DCT we add 50 mcL of patients suspension made above in one
gel card.
11.Then the card is centrifuged at 1000rpm for 10 min
12.Remove the card and grade and record the reaction
Interpretation of DCT

1. If agglutination is observed , DAT is recorded as positive. Report the result as

positive with grading in brackets along with the technique of testing. e.g.
• DAT – Positive (3+) by gel card technique
 Validation of Negative AGT
Add IgG sensitized red cells
(check cells ) to negative
coombs test

Agglutination present Agglutination absent

Valid test Invalid test

Indirect Coomb’s test
• Materials required
• Test serum/plasma
• Glass tubes and Test tube racks
• Pasteur pipettes and tips
• LISS (optional)
• AHG reagent-Polyspecific (anti-IgG & C3d)
• Pooled ‘O’ cells
• Sensitized red cells / Check cells
• Tabletop centrifuge
• Serologic Incubator
Serologic Incubator
Procedure for ICT
1. Identify the patient sample by checking and matching patient's details
from the request form and sample.
2. Centrifuge the sample by hard spin (3000rpm for 2-3 min) to separate
red cells from plasma.
3. Take a clean test tube and label with a patient identifier (name or
hospital number) .
4. Add 10 mcL of O+ RBCs to 990 mcL of Normal saline and then wash this
three times making 1% ‘O’ red cell suspension
5.Now add this 50 mcL of O red cells suspension and 25 mcL of
patient’s plasma in gel card
6.Incubate the card at 37 C in serological incubator for 15 min . Now
centrifuge the card at 1000 rpm for 10 min.
7. Remove the card and grade and record the reaction

→ In case of cross matching instead of O pooled rbcs donor’s RBC are

used to see if donor and recipient are compatible
Interpretation of ICT
1. If agglutination is not observed and agglutination is observed after the addition
of check cells,then report the result as negative along with the technique of testing.
e.g.
• IAT – Negative by gel card technique
2. If agglutination is observed , it is suggestive of clinically significant IgG antibodies.
Report the result as positive along with the technique of testing. e.g.
• IAT – Positive by gel card technique using a pool of ‘O’ cells
3. If agglutination is not observed at step 9 as well as after addition of check cells
the test is considered invalid and need to be repeated.
Tasks and purposes
TASK PURPOSE

Incubate RBCs with antisera Allows time for antibody molecule attachment to RBC
antigen
Perform a minimum of 3 saline washes It removes free globulin molecules

Add antiglobulin reagent It forms RBC agglutinates ( RBC Ag + Ab + anti –IgG )

Centrifuge Accelerates agglutination by bringing cells closer

together
Examine for agglutination Interprets test as positive or negative

Grade agglutination reactions Determines strength of reaction

Add antibody coated RBCs to negative reaction Checks for neutralization of antisera by free globulin
molecules (Coombs control cells are D – positive RBCs
coated with anti – D )
 Factors affecting sensitivity of ICT
Temperature :
Optimum temperature for IAT test is 37 C . Incubation at lower temperature can
reduce sensitivity while higher temperature damages red blood cells

Serum:cell ratio :
Increasing the ratio of serum to red cells , increases sensitivity

Incubation time :
a) For saline , albumin or enzymes technique 45-60 min
b) For LISS suspended cells 10-15 min

Suspending medium :
The sensitivity of IAT can be increased with addition of 22% bovine albumin ,
enzyme or LISS
SOURCES OF ERROR IN COOMBS’ TEST
False positive results
1. Auto-agglutinable cells
2. Bacterial contamination of cells or saline used in washing
3. Cells with a positive DAT used for IAT
4. Dirty glassware
5. Over centrifugation or over-reading
6. Polyagglutinable cells
7. Preservative dependent antibodies in LISS reagents
8. Contaminating antibodies in the antihuman globulin reagent
• False negative results :
1. Inadequate washing of cells
2. Delay or interruption of washing produre
3. Failure to add AHG reagents
4. Under centrifugation
5. Too heavy or weak red cell suspension
6. Insufficient incubation
7. AHG reagents that has lost potency
8. Presence of small fibrin clots among cells
THANKYOU