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Enhancing Media for IAT

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10. Enhancing Media for IAT

The following enhancing media may be used to increase the sensitivity of the indirect antiglobulin test

1. Albumin
2. Enzyme
3. LISS (Low ionic strength saline) solution

Albumin

Albumin has no effect on sensitization of the antigen by the antibody. It enhances agglutination by reducing the repulsion charges between red cells.

Procedure

1. First three steps are same as in IAT.

2. At step 4, incubate mixture for 15-20 minutes at 37°C.

3. Add 1 drop of 22% bovine albumin along the side of the tube.

4. Incubate further for 15-20 minutes.

5. Spin at 1000 rpm for 1 minute and examine for agglutination.

6. Proceed as at step 6 of the IAT and continue till step 10.

Enzymes.

Proteolytic enzymes also enhance agglutination by cleaving sialoglycoproteins from the red cell surface and thereby reducing the net charge between red cells.

One stage method for enzyme

Reagent

1% solution of papain-cystein
01 and OIl reagent cells

Procedure

1. Add 1 drop to test serum in each of the tubes labelled 01 & Oil

2. Add I drop of papain-cystein solution to each of the tubes.

3. Add 1 drop of 2-5% cell suspension of 01 cells to the tube labelled Cl and I drop of Oil cells to the tube labelled 011.

4. Mix and incubate at 37°C for 45 minutes.

5. Spin at 1000 rpm for 1 minute, examine for agglutination and record the results.

6. Proceed to the antiglobulin test from step 6 onwards in the IAT.

Two stage method for enzyme

Reagent

0.1% solution of papain-cystein

Procedure

1. Place 1 drop of washed 5% 01 and Oil cells in labelled test tubes.

2. Add 2 drops of papain-cystein solution to each of the tubes.

3. Mix and incubate at 37°C for the duration which has been determined by the enzyme standardization procedure. This is usually around 15 minutes

4. Wash 3 times with saline and decant last wash completely on filter paper.

5. Add 1-2 drops of serum, mix and incubate at 37°C for 15-30 minutes.

6. Spin at 1000 rpm for 1 minute. Look for haemolysis or agglutination and record the results.

7. Proceed onwards as from step 6 in the IAT.

Note: A similarly treated autologous control should be used.

LISS (Low ionic strength saline) solution

LISS reduced the ionic strength of the reaction medium and thereby enhances antibody uptake by the red cell antigens. This helps in increasing the sensitivity of the test.

Reagents

01 & OIl reagent cells in LISS (Low ionic strength saline) solution.

Preparation of 0 cells in LISS

Pool known 0 Rh (D) positive cells from 2 donors each, in each of the 2 tubes labelled 01 & Oil. Wash twice in normal saline and once in LISS. Make 2-5% cell suspension in LISS.

Procedure

Procedure is identical to TAT except that the incubation period is reduced to 15 minutes at step 5. In an emergency even 5 minute incubation will be sufficient.

Note: It is imperative to put positive and negative controls, as LISS may give few false-positive reactions.

Washing of red cells

While performing direct or indirect antiglobulin test it is mandatory to make sure that pipette, saline, test tubes, etc. are serum-free and absolutely clean.

Always wash the cells throughly after incubation (sensitization) to remove all traces of human serum except that coating the red cells. Presence of the smallest amount of human globulin can neutralize the antihuman globulin reagent and give a false-negative result.

Checking negative results

Confirm a negative direct and indirect antiglobulin test to rule out ineffective or neutralized anti-human globulin reagent.

To do this, add one volume of presensitized well washed cells to all negative tests. If the negative reaction is true-negative, the antiglobulin reagent will be active and will agglutinate these cells. If the cells are not agglutinated, the test must be repeated.

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