ASO (Anti-streptococcal O)Test: Introduction, Principle, Procedure, Normal Range, Result- Interpretation, and Keynotes

Introduction of ASO Test

ASO stands for Antistreptococcal O and it is an antibody targeted against streptolysin O, a toxic enzyme produced by group A Streptococcus bacterium. Antistreptococcal O and anti -DNase are 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 test measures the amount of ASO in the blood.  The toxin of S. pyogenes (hemolysin) is of further two types and they are streptolysin O and streptolysin S. Streptolysin O is oxygen and heat-labile having a molecular weight of 60,000.

It is Hemolytic in a reduced condition and immunogenic. It lyses  RBC by binding to the cholesterol of the cell membrane and is toxic to platelets and cardiac tissue. Streptolysin S is oxygen stable and non-immunogenic and it shows leucocidin action in addition to hemolytic properties. Features of Antistreptococcal O: ASO appears in sera of humans following streptococcal infection. It is an antibody produced by the host body against the toxin streptolysin O produced by groups A, C, and G of beta-hemolytic streptococci. 

Streptolysin O

Streptolysin O toxin is heat-labile, oxygen-liable, and highly antigenic, therefore patients with these infections produce specific antibodies. The “O” in the name stands for oxygen-labile; the other related toxin is oxygen-stable streptolysin-S. The main function of streptolysin O is to cause hemolysis. The test helps direct antimicrobial treatment and is used to assist in the diagnosis of scarlet fever, rheumatic fever, and Post-streptococcal glomerulonephritis (PSGN). 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.

Only a fourfold or greater rise in titer on successive serum samples taken 10–14 days apart should be considered indicative of recent infection. The rise in ASO indicates concurrent, past, or present infection. Antistreptococcal O begins to rise after 1-3 weeks of infection and peak level in  3-5 months while falls after 6 months. Antistreptococcal O produced against Streptolysin O co-reacts with human antigens (normally collagen) and hence attacks the cellular membrane of the heart, joints, skin, kidney, etc. This test is mainly used in the diagnosis of acute rheumatic fever, acute glomerulonephritis, and other post-streptococcal diseases. It is elevated in 80% of 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 titers (200 IU or higher).
2. ASO tube test is a hemolysis inhibition test that is used to determine ASO antibody titer 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 an 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 fine flocculation (agglutination) within 2 minutes.

Result- Interpretation of ASO Test

A negative reaction shows no agglutination. A positive result will be obtained at an antistreptococcal O serum concentration of 200 IU/ml or more and a negative result will be obtained at an ASO concentration below 200 IU/ml. If the flocculation appears within 2 minutes, the serum should be titrated with the anti-streptolysin O tube test.

Semi-Quantitative Methods

• 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)
• Do the same as above mention using each diluted serum.
• The serum ASO concentration can then be calculated approximately by multiplying the dilution factor.
• If the agglutination titer appears at 1/4 the approximate serum ASO concentration is 4Χ200= 800 IU/ml

ASO Tube Test (Neutralization Test)

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

Materials Required for ASO Tube 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 of  ASO Tube Test

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 1.5 ml of buffer to control tube 7 and 1 ml of buffer to control tube 8.
10. Add 0.5ml of reduced streptolysin O to all test tubes, except control tube 7.
11. Mix and refrigerate at 4ºC for two hours to allow the antibody-antigen reaction to take place.
12. 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.
13. Centrifuge the tubes at 1000 g for 2 minutes and observe for hemolysis.

Result -Interpretation of ASO Tube Test

Control tube 7 should show no hemolysis and control tube 8 should be completely hemolyzed. The ASO titer is determined as the highest dilution showing no sign of hemolysis. If there is hemolysis in all tubes, report the result as “ASO titer 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 titer”.

The normal value of the ASO test

• Adult<200 units and children <400 units (Todd units)

Keynotes on ASO Assay

• The false negatives rate is 20-30%. If a false negative is suspected, then an anti-DNase B titer should be sought.
• False positives can result from liver disease and tuberculosis.

Further Readings

• http://atlasmedical.com/upload/productFiles/201002/ASO%20latex%20Package%20Insert.pdf
• https://www.childrensmn.org/references/lab/chemistry/aso-(antistreptolysin-o).pdf
• https://www.linear.es/wp-content/uploads/2018/03/2340005-ing-Rev.-04.pdf
• http://atlas-medical.com/upload/productFiles/201002/ASO%20latex%20Package%20Insert.pdf
• https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3357621/
• https://www.sciencedirect.com/science/article/pii/S1201971208017153