Anti-Streptolysin O (ASO) test

Introduction

1. The Anti-Streptolysin O (ASO) test is a vital diagnostic tool for measuring antibodies produced against Streptolysin O (SLO), a cytotoxic enzyme secreted by Streptococcus pyogenes.
2. These antibodies indicate exposure to or infection by Group A Streptococcus.
3. Elevated ASO titers are associated with post-streptococcal complications such as:

• Rheumatic fever (affecting the heart, joints, and nervous system).
• Post-streptococcal glomerulonephritis (inflammation of the kidneys).

Key Points:

• ASO titers begin to rise 1–3 weeks post-infection, peak around 3–6 weeks, and decline over months.
• ASO levels are expressed in Todd units (International Units, IU/mL).

 

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Principle

The ASO test relies on the ability of anti-SLO antibodies in the patient’s serum to neutralize the hemolytic activity of the SLO enzyme:

1. Neutralization of Hemolysis:
   • SLO can lyse red blood cells (RBCs).
   • When a serum containing anti-SLO antibodies is mixed with SLO, the antibodies neutralize the enzyme, preventing RBC lysis.
2. Qualitative Test: Determines the presence of anti-SLO antibodies.
3. Quantitative Test: Determines the highest serum dilution (titer) that inhibits hemolysis completely.

 

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Requirements

1. Sample

• Collect venous blood in a plain (clot) tube.
• Allow clotting at room temperature and centrifuge at 2,500–3,000 rpm for 10 minutes to separate serum.
• Avoid hemolysis or contamination.
• Use fresh serum or store at 2–8°C (up to 7 days). For longer storage, freeze at −20°C.

2. Reagents

1. Streptolysin O (SLO):
   • A lyophilized preparation reconstituted per manufacturer instructions.
   • Concentration must be standardized (e.g., causing complete hemolysis of a defined RBC volume).
2. Sensitized RBCs:
   • Sheep red blood cells (1%–2%) washed and suspended in isotonic saline.
3. Diluent:
   • Normal saline or phosphate-buffered saline (PBS).
4. Controls:
   • Positive Control: Serum with known anti-SLO concentration.
   • Negative Control: Normal saline or serum without ASO antibodies.

3. Equipment

• Test tubes or microtiter plates for serial dilution.
• Micropipettes and disposable tips.
• The incubator or water bath is maintained at 37°C.
• Timer or stopwatch.

 

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Procedure

Qualitative ASO Test (Screening)

1. Mix a fixed amount of serum (e.g., 0.5 mL) with a standardized amount of SLO in a test tube.
2. Incubate the mixture at 37°C for 15–30 minutes to allow antibody-antigen interaction.
3. Add an equal volume of sensitized sheep RBC suspension to the tube.
4. Incubate the tubes at 37°C for 1 hour.
5. Observe for hemolysis:
   • No hemolysis: Positive (presence of ASO antibodies).
   • Complete hemolysis: Negative (absence of ASO antibodies).

 

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Quantitative ASO Test (Titration)

1. Prepare Serial Dilutions:
   • Label tubes sequentially as 1:100, 1:200, 1:400, etc.
   • Add 1.9 mL of saline to the first tube and 1 mL to the remaining tubes.
   • Add 0.1 mL of serum to the first tube and mix thoroughly.
   • Transfer 1 mL from the first tube to the second, continuing serial dilution. Discard 1 mL from the last tube.
2. Add Streptolysin O:
   • Add an equal volume of standardized SLO to each tube.
3. Incubate:
   • Mix and incubate tubes at 37°C for 15–30 minutes.
4. Add Sensitized RBCs:
   • Add one drop of RBC suspension (equal volume to SLO-serum mixture) to each tube.
5. Second Incubation:
   • Incubate at 37°C for 1 hour.
6. Observation:
   • Look for hemolysis in each tube.
   • The highest dilution showing no hemolysis is the ASO titer.

 

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Results

Normal ASO Ranges

• • Adults: Up to 200 IU/mL.
  • Children (5–12 years): Up to 250 IU/mL.

Significant Titers

• • A single elevated ASO titer is not conclusive of active disease. Instead:
    • Rising Titers: A fourfold increase in ASO titer over 2–3 weeks is significant.

Reporting

• • Results are expressed in Todd units or IU/mL.
    • 1 Todd unit = the amount of antibody that neutralizes 0.01 mL of SLO.

 

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Clinical Significance

1. Post-Streptococcal Sequelae:
   • Elevated ASO titers support the diagnosis of:
     • Rheumatic fever: Associated with carditis, arthritis, and Sydenham chorea.
     • Post-streptococcal glomerulonephritis: Inflammation of the kidney.
2. Epidemiological Studies:
   • Monitoring streptococcal infections in communities.
3. Disease Monitoring:
   • Serial ASO testing helps assess the course of disease and treatment effectiveness.

 

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Limitations

1. False Negatives:
   • Not all streptococcal infections produce significant ASO levels (e.g., skin infections like impetigo).
2. False Positives:
   • Elevated ASO titers may persist after the resolution of infection.
3. Non-Specificity:
   • ASO cannot distinguish between recent and remote infections.
   • Results must be interpreted alongside clinical findings and other investigations.

 

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Precautions

1. Reagents and Controls:
   • Always use fresh or properly stored reagents. Validate test accuracy with controls.
2. Temperature Control:
   • Maintain consistent incubation temperatures (37°C) for reliable results.
3. Interpretation:
   • Always interpret ASO titers in the clinical context; elevated titers alone are not diagnostic of disease.