Division of Blood Transfusion Services: Ministry of Health and Family Welfare

Division of
Blood Transfusion Services
Ministry of Health and Family Welfare

Antiglobulin Test
Teaching Aims
• To understand the principle of AGT
• To learn the techniques of AGT
• To know about the AHG reagent

Antihuman Globulin (AHG)
 Antihuman: antibodies against human antigens
 Globulin: all antibody molecules are globulins
 Antihuman Globulin is antibody directed against the Fc portion

of human antibodies and/or complement components.
Anti-globulin Test
 Introduction by Coombs in 1945
 Detects incomplete antibodies
 Principle of AGT
• Antibodies and complement components are
globulins.
• Animals injected with human globulins produce
anti-human globulin (AHG).
• AHG forms bridges between antibody coated red
cells.
Antihuman Globulin Reagents
 Polyspecific
• Contains both, anti-IgG and anti-C3d (complement)
 Monospecific
• Contains only one specificity, either anti-IgG or anti-

C3d
Preparation of AHG reagent
Direct Antiglobulin Test
Detects in vivo sensitization of red cells
+ =
Coombs Reagent
Patient’s RBCs
(Antiglobulin)
Method of DAT
Wash test red cells 3-6 times with saline
Decant supernatant saline from the last wash
Make 5% suspension of washed red cells
Add one drop of 5% washed cells to a labeled tube
Add one drop of AHG reagent
Mix well and centrifuge at 1000 RPM x 1 min
Note the results

Applications of DAT
 Diagnosis of HDN in newborn
 Diagnosis of autoimmune hemolytic anemia
 Diagnosis of drug induced immune hemolytic anemia
 Investigation of hemolytic transfusion reaction

Indirect Antiglobulin Test
Detects free antibodies in the serum
+ =
Step 1 Patient’s Pooled O RhD +
Serum RBCs
Step 2 + =
Coombs Reagent
(Antiglobulin)
Method of IAT
Add 2 drop of test serum to a test tube
Add 1 drop of 5% suspension of pooled O + red cells
Incubate at 37oC for 60 minutes
Wash the red cells 3-6 times with normal saline
Add 1 drop of AHG reagent to red cell button
Look for agglutination
Negative results should be confirmed

by adding 1 drop of check cells
Applications of IAT
 Detection and identification of unexpected antibodies in
the serum
 Cross matching
 Typing of minor red cell antigens such as Duffy, Kell, Kidd
 Detection of weak D (earlier Du test)
 Titration of antibodies
• Anti-D in maternal serum in HDN
Factors affecting AGT
 Temperature - 37oC
 Ratio of serum to cells – 2:1
 Incubation time- 60 min
 Reaction medium –Saline/ LISS
 Washing of cells – 3 to 6 times
 Addition of AHG reagent – remember to add
 Centrifugation for results – speed & time
 Quality of AHG reagent – QC of reagent
Sources of errors: false negative results in AGT
 Inadequate washing of red cells.

 Test is interrupted or delayed.
 Problems with AHG reagent
• Bacterial contamination of reagent
• Improper storage.
• Failure to add AHG reagent.
• Decreased reactivity of AHG reagent.
Sources of errors: false positive results in AGT
 Autoagglutination or polyagglutination of red blood cells.
 Improper washing of glassware.
 Over centrifugation.
 Presence of other antibodies in the AHG reagent.
 Contaminated reagents
 Saline contaminated by heavy metals or colloidal silica.

Preparation of Check Cells
 Perform doubling dilution of commercially available

monoclonal anti-D (IgG)
 Select the highest dilution which gives +2 reaction with ORhD

positive red cells
 For eg. For dilution of 1:64, add 630 µL of NS to 10 µL of

undiluted anti-D
 Take one volume of diluted anti-D to which add equal vol of

pooled O RhD positive red cells
Preparation of Check Cells (contd…)
 Incubate at 370C for 45 min
 Wash 3 times with NS
 Add 1 drop of AHG to 1 drop of 5% suspension of washed red

cells
 Check cells can be stored in Alsever’s solution for 1 week at

40 C
Validation of negative AGT
Add IgG sensitized red cells (check cells) to

negative coombs test
Agglutination present Agglutination absent
Valid Test Invalid test

Points to remember…
a) Even 10 mL of AHG can be neutralized by a tiny amount of

free serum protein.
b) A technologist can forget to add AHG to a test tube.
c) A couple of drops of residual saline can dilute the AHG
reagent below detectable levels.
d) Normally people do NOT produce unexpected antibodies.
Therefore the test should normally be negative.
What are reagent red cell panels?
 Red cell suspensions used in tests employing the principles of

hemagglutination and hemolysis for the detection and
identification of blood group antibodies.
Commercial
 Sources Regular donors
In-house
Staff members
Applications of Reagent Red Cells
 Reverse ABO grouping
 Antibody screening
 Antibody identification
 Antibody titration
 Allogenic adsorption
 Control of AHG technique

Reagent cells for antibody screen of donors
 Pooled reagent screening cells used only for testing samples

from blood donors
 Group O red cells

• Naturally occurring anti-A or anti-B do not interfere with
detection of unexpected antibodies
 As a minimum the following antigens should be expressed:

D; C; c; E; e; K.
Reagent cells for antibody screen of patients
 Un-pooled cells from a minimum of two donors must be used
 One reagent red cell should be R2R2; other R1R1.
 Must express K;k;Fya;Fyb;Jka;Jkb;S;s;M;N;P1;Lea and Leb.
 Homozygous expression of some of these antigens is essential

for reliable detection of weak antibodies.
Antibody Screening
 An anti-gram listing the antigen makeup of each cell provided
with each lot of screening cells issued from a manufacture.
 Lot number on the screening cells must match with the lot
number printed on the anti-gram because antigen make up
will vary with each lot.
 The “ideal” screening cells have red cells with homozygous

expression of as many antigens as possible.
Three cell screening panel
Rh Kell Duffy Kidd Lewis P MNS

D C E c e K k Fy a Fyb Jka Jkb Lea Leb p M N S s
I + + 0 0 + 0 + 0 + 0 + 0 + 0 0 + 0 +
II + 0 + + 0 + + + 0 + + + 0 + + 0 + +
III 0 0 0 + + 0 + + 0 + 0 0 + + + + + 0
All circled antigens are in homozygous state

Antibody Screening in Test Tubes
Take 3 test tubes and mark them as 1,2 and 3
Add 2 drop test serum in all tubes
Add 1 drop of screening cells in respective tubes
Add 2 drops of LISS to test tubes
Incubate at 37ºC for 15 min- 30 min
Centrifuge at 1000 rpm for 1 min.
Wash cells 3 times with saline
Add 2 drops of AHG reagent
Centrifuge at 1000 rpm for 1 min.
Examine for Agglutination
Antibody identification
What is the importance of identification of antibody?
 To determine specificity of antibody
 To provide antigen negative blood for transfusion
 To determine clinical significance of antibody
 To investigate hemolytic disease of newborn
 To investigate transfusion reactions

Potentiators
 Used in antibody screening and identification to enhance
antigen-antibody reaction
 Low-ionic strength solution (LISS)
 Bovine serum albumin (BSA)
 Polyethylene glycol (PEG)
 Proteolytic enzymes
• Papain
• Ficin
• Bromelin
Potentiators in Red Cell Serology
Potentiator Action Detects

22% Reduces zeta potential IgG antibodies
albumin between red cells
LISS Low ionic strength IgG antibodies
environment increases
antibody uptake by red cells
Enzymes Destroys some red cell Destroys Fya, Fyb,
antigens and enhances other MNS.
red cell antigens Enhances Rh, Kidd,
P, Lewis antibodies
PEG Macromolecules reduce IgG antibodies
distance between two red cells
Learning Outcomes
• You will now understand the principal of AGT
• You will be able to carry out AGT
• You must have known about the reagent in use

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Division of Blood Transfusion Services: Ministry of Health and Family Welfare

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Division of

Blood Transfusion Services

Ministry of Health and Family Welfare

• To understand the principle of AGT

• To learn the techniques of AGT

• To know about the AHG reagent

 Antihuman: antibodies against human antigens

 Globulin: all antibody molecules are globulins

 Antihuman Globulin is antibody directed against the Fc portion

 Detects incomplete antibodies

• Contains both, anti-IgG and anti-C3d (complement)

• Contains only one specificity, either anti-IgG or anti-

Detects in vivo sensitization of red cells

Wash test red cells 3-6 times with saline

Decant supernatant saline from the last wash

Make 5% suspension of washed red cells

Add one drop of 5% washed cells to a labeled tube

Add one drop of AHG reagent

Mix well and centrifuge at 1000 RPM x 1 min

Note the results

 Diagnosis of HDN in newborn

 Diagnosis of autoimmune hemolytic anemia

 Diagnosis of drug induced immune hemolytic anemia

 Investigation of hemolytic transfusion reaction

Detects free antibodies in the serum

Add 1 drop of 5% suspension of pooled O + red cells

Incubate at 37oC for 60 minutes

Wash the red cells 3-6 times with normal saline

Add 1 drop of AHG reagent to red cell button

Look for agglutination

Negative results should be confirmed

 Typing of minor red cell antigens such as Duffy, Kell, Kidd

 Detection of weak D (earlier Du test)

 Inadequate washing of red cells.

 Autoagglutination or polyagglutination of red blood cells.

 Improper washing of glassware.

 Presence of other antibodies in the AHG reagent.

 Saline contaminated by heavy metals or colloidal silica.

 Perform doubling dilution of commercially available

 Select the highest dilution which gives +2 reaction with ORhD

 For eg. For dilution of 1:64, add 630 µL of NS to 10 µL of

 Take one volume of diluted anti-D to which add equal vol of

 Incubate at 370C for 45 min

 Wash 3 times with NS

 Add 1 drop of AHG to 1 drop of 5% suspension of washed red

 Check cells can be stored in Alsever’s solution for 1 week at

Add IgG sensitized red cells (check cells) to

Agglutination present Agglutination absent

Valid Test Invalid test

a) Even 10 mL of AHG can be neutralized by a tiny amount of

 Red cell suspensions used in tests employing the principles of

 Reverse ABO grouping

 Control of AHG technique

 Pooled reagent screening cells used only for testing samples

 Group O red cells

 As a minimum the following antigens should be expressed:

 Un-pooled cells from a minimum of two donors must be used

 One reagent red cell should be R2R2; other R1R1.

 Must express K;k;Fya;Fyb;Jka;Jkb;S;s;M;N;P1;Lea and Leb.