ASO-BAR

Intended use

During infection, group A streptococci produce a range of enzymes (including streptolysin O and streptodornase B) that contribute to their pathogenicity.

Streptolysin causes erythrocyte lysis and liberation of hemoglobin. These different molecules, in particular streptolysin O, are immunogenic. The human body responds to infection via the formation of specific antibodies targeted against these toxins.

The quantitative determination of anti-streptolysin O antibodies in patient serum, allows the confirmation of an active or recent streptococcal A infection.

The ASO-BAR test is important in the management of patients with acute articular rheumatism characterized by elevated ASO levels.

Principle

The detection of anti-streptolysin O antibodies is based upon the neutralization of the streptolysin O hemolytic activity by antibodies present in the test serum.

The antigen-antibody immunological complexes that are formed are revealed by the addition of a suspension of sheep erythrocytes. The inhibition of streptolysin activity by anti-streptolysin O antibodies leads to a reduction in erythrocyte lysis.

Either:

1) The erythrocytes remain intact due to the concentration of antibodies being greater than the concentration of streptolysin. Reading of the test is carried out before sedimentation occurs with the medium remaining cloudy.

2) The erythrocytes are lysed, releasing hemoglobin due to the concentration of antibodies being less than the concentration of streptolysin. The cloudy medium becomes limpid.
The reaction is carried out in wells 1 to 8 of the tray. These wells contain increasing concentrations of streptolysin in order to enable the determination of the serum antibody concentration.

ASO-BAR profile:

The antibody titre corresponds to the last well of the tray that is not lysed. For each row of tests, reagent and test controls are included in order to verify the correct operation of the test:

• A negative control (well 9, labelled C-) containing streptolysin, but no serum in order to verify that streptolysin mediated erythrocyte lysis occurs in the absence of antistreptolysin antibodies.
• A positive control (well 10, labelled C+) containing serum but no streptolysin in order to verify that lysis does not occur in the presence of anti-streptolysin antibodies and erythrocytes. This well also acts as a test control for the reading of the results.

Simple protocol

Easy-read and easy-to-interpret results

The C+ control well facilitates reading and interpretation in the absence of lysis.
Absence of hemolysis (cloudy medium)
The absence of hemolysis correlates to there being a sufficient concentration of anti-streptolysin antibodies in the test serum in order to neutralize the streptolysin in the wells.
Presence of hemolysis (limpid medium)
The presence of hemolysis correlates to there being an insufficient concentration of anti-streptolysin antibodies in the test serum to neutralize the streptolysin in the wells.

The titre of anti-streptolysin antibodies in the serum, in U/ml, corresponds to the LAST WELL THAT DOES NOT DEMONSTRATE HEMOLYSIS. A titer greater than 200 U/mL is considered to be pathological.

Reagents and material

• DIL:
  • 5 mL vial of diluant containing sodium barbital
  • Ref. 04125 Quantity: 2
• R1:
  • Vial of lyophilized reducer containing dithioerythritol.
  • To be reconstituted with 600 μL of sheep erythrocytes (R2).
  • Ref. 04125 Quantity: 12
• R2:
  • 5 mL vial of sheep erythrocytes. Reagents available separately
  • Ref. 04905 Quantity: 2
• ASO-BAR tray:
  • Dividible tray of 2 x 10 wells (2 tests), individually wrapped in aluminum sachets. The trays contain increasing concentrations of dehydrated streptolysin in the “Test” wells. The control C- well contains streptolysin and the control C+ well is empty.
  • Ref. 04125 Quantity: 6

Stability and storage

• Before opening:
  • The reagents stored at 2-8 °C are stable until the expiry date.
• After opening:
  • The DIL reagent is stable for 3 months at 2-8 °C. The reconstituted R1 reagent should only be used once. The R2 reagent is stable at 2-8 °C until the expiry date. The R2 reagent is also available separately (Ref. 04905).The unused half of the ASO-BAR tray is stable for 2 months at 2-8 °C, provided that it is stored hermetically in its original packaging.

Performance

• EM) and the ASO-BAR (Technique Biologique), routinely used by the reference laboratory, and between the ASOBAR (IM) and the ASL-Kit (Biomérieux). 120 serum specimens were tested. The qualitative results that were obtained are summarized in the following table:
  

  	ASO-BAR (EM)* versus ASO-BAR (TB)**	ASO-BAR (EM) versus ASL-Kit
  Negative serums (n=)	80	86
  Positive serums (n=)	40	34
  Agreement	97,5%	95%
  Diagnostic sensibility	95,2%	97,1%
  Diagnostic specificity	98,7%	94,5%
  PPV	97,5%	87,1%
  NPV	97,5%	98,8%

• The titres obtained with the different techniques were compared. The results are summarized in the following table:
  

  	ASO-BAR (EM)* versus ASO-BAR (TB)**	ASO-BAR (EM) versus ASL-Kit
  Same titre	62,5%	52,9%
  Titre within 1/2 dilution	87,5%	91,1%
  Titre within 1 dilution	95%	97%

  * ASO-BAR (EM): New ASO-BAR test manufactured by ELITech MICROBIO.
  ** ASO-BAR (TB): Old ASO-BAR test manufactured by Technique Biologique.

Material required but not provided

• Automatic pipette(s) appropriate to the volumes measured
• Incubator calibrated to 37 °C
• Contaminated waste container