VDRL Procedure and Interpretation | Arlington Scientific

Preparation of the VDRL & Synthetic VDRL Antigen Suspension

The VDRL ANTIGEN SUSPENSION must be prepared fresh each day.
The temperature of the VDRL BUFFERED SALINE, VDRL ANTIGEN and equipment should be between 23 and 29°C before preparing the suspension. All glassware, pipets and equipment must be dry.
With a 1 ml serological pipet, deliver 0.4 ml of VDRL BUFFERED SALINE to the bottom of a 30 ml round, glass stoppered bottle with flat inner bottom.
Pipet 0.5 ml of VDRL ANTIGEN gradually into the VDRL BUFFERED SALINE while continuously but gently rotating the bottle on a flat surface. Add the Pipet 0.5 ml of VDRL ANTIGEN gradually into the VDRL BUFFERED SALINE while continuously but gently rotating the bottle on a flat surface. Add the VDRL ANTIGEN drop by drop at a rate allowing approximately 6 seconds for 0.5 ml of VDRL ANTIGEN. Keep the pipet tip in the upper third of the bottle. Do not splash the BUFFERED SALINE onto the pipet. The proper rotation speed is obtained when the center of the bottle circumscribes a 2-inch (5 cm) diameter circle approximately three (3) times per second. Expel the last drop of VDRL ANTIGEN from the pipet without touching the pipet to the BUFFERED SALINE, and continue rotation of the bottle for 10 seconds.
Add 4.1 ml of VDRL BUFFERED SALINE from a 5 ml pipet.
Cap the bottle and shake it from bottom to top and back approximately 30 times in 10 seconds. The VDRL ANTIGEN SUSPENSION is ready for use and is usable for 8 hours.
Mix the VDRL ANTIGEN SUSPENSION by gently swirling it each time it is used. Do not mix the SUSPENSION by forcing it back and forth through the syringe and needle since this may cause breakdown of particles and loss of reactivity.
To achieve reliable and reproducible test results, the VDRL ANTIGEN SUSPENSION, controls and test specimens must be at 23-29°C when the tests are performed.

Preparation of the Samples

Each serum sample must be heat-inactivated for 30 minutes at 56°C prior to testing.
If heat-inactivation occurs more than four (4) hours prior to testing, reheat the serum for an additional 10 minutes at 56°C.

Assay Protocol - Qualitative

Using a micropipettor, pipet 50 ml of serum into one 14 mm test circle.
Gently resuspend the VDRL ANTIGEN SUSPENSION.
Draw a sufficient volume of VDRL ANTIGEN SUSPENSION into the needle and glass syringe assembly. Dispense several drops into the 30 ml SUSPENSION bottle to make sure the passage is clear.
Holding the VDRL ANTIGEN SUSPENSION dispensing needle and syringe in a vertical position, dispense exactly one (1) free-falling drop of ANTIGEN SUSPENSION into each circle containing serum.
Immediately after rotating the slide, remove it from the rotator and read the test microscopically, using 100x magnification. Record the results.

Assay Protocol - Semiquantitative

To quantitate serum samples for determination of endpoint, dilutions can be prepared directly on the glass slide.
Using a micropipettor, dispense 50 μl of saline into the circles numbered 2-5. Do not spread.
Dispense 50 μl of serum onto circles 1 and 2 of the glass slide.
Mix the saline and the serum in circle 2 by drawing the mixture up and down in the pipet 5 or 6 times. Avoid any bubble formation.
Transfer 50 μl from circle 2 to circle 3 and mix as in step (4) above. Repeat this serial dilution procedure to circle 5 and discard 50 μl from the last circle. Circles 1 through 5 represent a dilution series as follows:

Circle

Dilution

1:1

1:2

1:4

1:8

1:16

Gently resuspend the VDRL ANTIGEN SUSPENSION in the 30-ml bottle, and draw sufficient volume into the syringe and needle assembly.

Holding the VDRL ANTIGEN SUSPENSION dispensing needle and syringe in vertical position, dispense several drops into the 30-ml bottle to clear the needle of air. Then add exactly one (1) free-falling drop of ANTIGEN SUSPENSION to each circle.

Interpretation

Reactive – Any degree of agglutination within the test area as compared to the nonreactive control.

Nonreactive – Smooth or finely granular suspension with no visible agglutination.