ASO test

ASO stands for Antistreptococcal O. Antistreptolysin O (ASO) is an antibody targeted against streptolysin O, a toxic enzyme produced by group A Streptococcus bacterium. ASO and anti -DNase Bare the most common of several antibodies that are produced by the body’s immune system in response to a strep infection with group A Streptococcus. This ASO test measures the amount of ASO in the blood.

History

Toxin of S. pyogenes (hemolysin) is of further two types and they are streptolysin O and streptolysin S.

Differences between those shown below:

Streptolysin O

Oxygen and heat labile

Mol. wt. 60,000

Hemolytic in reduced condition

Immunogenic

Lyse RBC by binding to cholesterol of cell membrane

Toxic to platelets and cardiac tissue

Streptolysin S

Oxygen stable

Non immunogenic

Leucocidin action in addition to hemolytic property

Introduction

1.  ASO appears in sera of human following streptococcal infection.

2. It is antibody produce by host body against toxin streptolysin O produced by group A, C and G of beta hemolytic streptococci

3. Streptolysin O toxin is heat labile, oxygen liable and highly antigenic, therefore patients with these infections produce specific antibodies.

4. The “O” in the name stands for oxygen-labile; the other related toxin being oxygen-stable streptolysin-S. The main function of streptolysin O is to cause hemolysis

5. The ASO test helps direct antimicrobial treatment and is used to assist in the diagnosis of scarlet fever, rheumatic fever, and Post-streptococcal glomerulonephritis (PSGN).

6. The anti-streptolysin O test is based on the fact that patients with Streptococcus pyogenes (group A streptococcal) infections develop antibodies that inhibit the hemolytic activity of streptolysin O.

7. Only a fourfold or greater rise in titre on successive serum samples taken 10–14 days apart should be considered indicative of recent infection.

8. Rise in ASO indicates concurrent, past or present infection.

9. ASO begins to rise after 1-3 weeks of infection

peak- 3-5 months

Falls- 6 months

10. ASO produced against Streptolysin O co-reacts with human antigens (normally collagen) and hence attack cellular membrane of heart, joints, skin, kidney etc.

11. This test is mainly used in the diagnosis of acute rheumatic fever, acute glomerulonephritis and other post-streptococcal diseases.

12. Elevate in 80% acute or chronic rheumatic fever

Two types of commercial anti- streptolysin O test kits

1. ASO latex slide agglutination test is used to screen sera to identify those with raised ASO titres (200 IU or higher).

2. ASO tube test is a hemolysis inhibition test that is used to determine ASO antibody titre in serum samples that are positive in the ASO latex slide agglutination test.

ASO latex slide agglutination

Principle

Polystyrene latex particle coated with purified and stabilized streptolysin O. when the latex suspension is mixed with serum containing elevated level of ASO antibodies on a slide, clear agglutination is seen within 2 minutes.

 Materials required for ASO test

Disposable cards, with 6 wells each

Disposable dropper

Positive control serum

Sensitized latex reagent (with streptolysin O)

Applicator sticks

Procedure of ASO test

1. Dilute the serum 1: 20.

2. Place 1 drop of the serum solution in a well on the disposable card.

3.Use a new dropper to add 1 drop of sensitized latex reagent.

4. Use an applicator stick to mix the two drops and spread them over the entire well.

5. Examine for agglutination within 2 minutes.

6. A positive reaction appears as a fine flocculation (agglutination) within 2 minutes.

7. A negative reaction shows no agglutination.

9. Positive result will be obtained at an ASO serum concentration of 200 IU/ml or more and negative result will be obtained at ASO concentration below 200 IU/ml

10. If the flocculation appears within 2 minutes, the serum should be titrated with the anti-streptolysin O tube test.

Semi-Quantitative methods

1. using isotonic saline prepare serial dilution of the patient’s serum (1/2,1/4,1/8,1/16,1/32,1/64 so on)

2. Do same as above mention using each diluted serum.

3. The serum ASO concentration can then be calculated approximately by the multiplying the dilution factor.

4.  If the agglutination titre appears at 1/4 the approximate serum ASO concentration is 4Χ200= 800 IU/ml

ASO tube test (neutralization test)

Based on ability of ASO antibody to prevent lysis of erythrocytes by streptolysin O.

Materials required for ASO test

Positive control serum

Reduced streptolysin O antigen (dried preparation)

Sheep red blood cells

Standard anti-streptolysin O antibody (dried preparation)

Streptolysin O buffer

Distilled water

Pipettes

Test-tubes

Water-bath

Procedure

1. Reconstitute the reduced streptolysin O antigen (dried preparation) with the appropriate volume of distilled water to give a potency of 2 IU equivalent per ml.

2. The solution should be used within 6 hours of reconstitution as it does not contain preservatives.

3. Reconstitute the standard anti-streptolysin O antibody (dried preparation, 20 IU/bottle) with 10 ml of streptolysin O buffer. The solution can be stored for six months at 4ºC provided it does not become contaminated.

4. Dilute 20 ml of the streptolysin O buffer (25 Χ  concentrated solution) with 480 ml of distilled water before use.

5. Wash and centrifuge 1 ml of sheep red blood cells three times in streptolysin O buffer and pipette the supernatant fluid off.  Add streptolysin O buffer to give an 8% cell suspension.

6. Make a 1: 10 dilution of patient serum in a test-tube (0.1 ml serum + 0.9 ml streptolysin O buffer).

7. Prepare 2 master dilutions from the 1 : 10 dilution as shown in the table below:

8. Rearrange the tubes in rising dilutions: 1: 50, 1: 200, 1: 300, 1: 400, 1: 600, 1: 800.

9. Add 1.5ml of buffer to control tube 7 and 1ml of buffer to control tube 8.

10. Add 0.5ml of reduced streptolysin O to all test-tubes, except control tube 7.

10. Mix and refrigerate at 4ºC for two hours to allow the antibody–antigen reaction to take place.

11. Add 0.5 ml of the 8% cell suspension to each tube, including control tubes 7 and 8, mix and incubate in a water-bath at 37ºC for 30 minutes.

12. Centrifuge the tubes at 1000 g for 2 minutes and observe for hemolysis.

Control tube 7 should show no hemolysis and control tube 8 should be completely hemolyzed.

The ASO titre is determined as the highest dilution showing no sign of

hemolysis:

If there is hemolysis in all tubes, report the result as “ASO titre less

than 200 IU”.

If there is no hemolysis in the tubes with a higher serum dilution,

report the result as “ASO reactive with the titre”.

Normal value of ASO test

1. Adult<200 units and children <400 units (Todd units)

2. The false negatives rate is 20-30%. If a false negative is suspected, then an anti-DNase B titre should be sought.

3. False positives can result from liver disease and tuberculosis.

Note: For visualization aid:

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