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Antiglobulin Test

You are here : Home/ Blood Bank Zone/ Red Cell Serology/ 9. Antiglobulin Test

9. Antiglobulin Test

Antiglobulin test is one of the most important serological tests done in a routine blood transfusion laboratory. It utilizes the anti-human globulin (AHG) reagent to bring about agglutination of red cells coated with immunoglobulin or complement component, which do not show any agglutination in saline.

Principle

Red cells which are coated with incomplete (IgG) antibodies show agglutination on addition of anti-human globulin (AHG or Coombs; reagent). The coating can occur either in vivo or in vitro following incubation with serum containing the antibody.

The majority of incomplete antibodies are IgG which attach to the red cell membrane by he Fab portion. The two arm of IgG molecule are unable to bridge the gap between red cells which are separated from each other because of the negative charge on their surface. While this results in sensitization of the cells, agglutination is not seen as the RBCs do not form lattice.

Addition of AHG reagent results in the Fab portion of the AHG molecule combining with the Fc portion of two adjacent IgG molecules, thereby bridging the gap between the red cells and causing agglutination.

There are two types of antiglobulin tests

a. Direct

b. Indirect

Direct antiglobulin test (DAT/DCT)
Direct antiglobulin test is used to detect in-vivo sensitization (coating) of red cells with immune antibody (IgO) or the complement component (C3d or C3c) in

Diagnosis of haemolytic disease of the newborn (l-IDN)
Diagnosis of autoimmune haemolytic anemia (All-IA)
Investigation of haemolytic transfusion reaction
Investigation of drug induced red cell sensitization

Indirect antiglobulin test (IAT/ICT)
This test is used to detect the presence of incomplete antibodies and complement-binding antibodies in the serum after coating on to the red cell in-vitro in.

Screening and identification of unexpected (irregular) antibodies in serum.
Compatibility testing
Detection of red cell antigens using specific antibodies reacting only in antiglobulin test (K, Fya, Fyb, Jkb, etc.)
Investigation of haemolytic disease of the newborn.

Anti-Human Globulin (AHG) Reagent
Anti-human globulin reagent is produced by immunizing rabbits, goats or sheep with human serum or purified add type antigen. Animals are bled after a specified period and the reagent is purified by absorbing unwanted antibodies. Anti-human globulin reagent can be polyspecific and monospecific.

Polyspecific antiglobulin reagent contains antibodies to human IgG, C3 and C4 components of the complement. Monospecific antiglobulin reagent may be against any one of the human IgM, IgO, IgA or complement component C3 or C4.
Monoclonal AHG is now available against IgG or complement components.

Direct Antiglobulin Test (DAT)
Sample collection
Blood sample for DAT should be collected in EDTA (Na2 or K2) to prevent in-vitro uptake of complement.

Reagents
Anti-human globulin reagent (AHG)
Positive Control : Sensitized 0 Rh (D) positive cells
Negative Control : Sensitized 0 Rh (D) negative cells Unsensitized 0 Rh (D) positive cells

Preparation of 0 Rh (D) positive sensitized red cells

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 (titre 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.
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).

Procedure (DAT)

Place 1 drop of 2-5% suspension of red cells in a clean labeled test tube.
Wash the red cells 3-4 times with saline and decant the final wash completely.
Add 1-2 drops of Al-IG reagent.
Mix and centrifuge at 1000 rpm for 1 minute.
Shake the tube gently to dislodge the cell button and read the results using a concave mirror.
If result is negative, incubate the test for further 5 minutes at room temperature, centrifuge and look for agglutination and record the results.
Add 1 drop of 5% IgG-sensitized red cells to the negative test. Look for agglutination, if a negative result is obtained the test result is invalid and the test should be repeated.
Appropriate controls are put with the test.

Interpretation
Agglutination of red cells indicates a positive DAI Controls tubes should be read before final interpretation. A positive reaction after immediate spin indicates presence of IgG coating antibodies. Reactions due to IgG become weaker after incubation. A positive reaction after a 5 minute incubation indicates coating by complement component.

Indirect Antiglobulin Test (IAT)

Sample collection
Blood sample for IAT should be collected in plain labeled test tube.

Reagents
Anti-human globulin reagent. Reagent 0 cells, commercially available or prepared in the laboratory (01 arid OIl)

Preparation of reagent 0 cells

Obtain poled 0 Rh (D) positive cells in 2 test tubes 0 I and 0 II. In each tube, cells from 2 donors are taken.
Wash e times with normal saline.
Prepare a 5% suspension for use.

Reagent cells should preferably be homozygous (double dose) for antigens and must carry the main important antigens e.g. Rh, Kell, Kidd, Duffy, MNS and Lewis.
Controls Same as for DAT.

Procedure (IAT)

Centrifuge the tubes at 3000 rpm for 5 minutes to separate the serum.
Add 2 drops of serum in each of the tubes labeled 01 and 011 (sample should be fresh for detecting complement - binding antibodies otherwise fresh AB serum should be added to it).
Add 1 drop of 5% suspension of 01 cells to tube labeled 01 and I drop of Oil cells to tube labeled 011.
Mix and incubate both tubes at 37�C for 45-60 minutes.
Spin at 1000 rpm for 1 minute and examine for agglutination. Record the results. Agglutination will not occur if incomplete antibodies are present.
Wash the cells in each of the tube 3-4 times with warm saline. Decant the saline completely after the last wash over a filter paper.
Add 1-2 drops of AI-IG reagent to each tube. Centrifuge immediately and look for centrifugation.
If negative, incubate at room temperature (22�-24�C) for further 5 minutes.
Re-centrifuge and look for agglutination.
Confirm negative test by adding a drop of IgG sensitized 0 Rh (D) positive cells. Agglutination should be seen.

Note : Autocontrol must be kept with IAT.

Interpretation
Agglutination in one or both the tubes indicates presence of unexpected antibody in the test serum. If no agglutination occurs, use enhancing techniques. Results of control tubes should be considered before final interpretation.

Factors Affecting the 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 albumin, enzyme or by using LISS suspended cells.

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