Procedure: Venereal Disease Research Laboratory (VDRL) test for Syphillis
Posted by: Azhar Hamdan (0503269C)
Introduction
Syphilis is a disease caused by the spirochaete organism Treponema pallidum. There are two types of serological tests that are useful for the diagnosis of primary, secondary and tertiary stages of syphilis.- Non-treponemal tests: Such as the Venereal Disease Research Laboratory (VDRL) test and the Rapid Plasma Reagin (RPR) test, which may be used as screening tests.
- Treponemal tests: Such as the (TPPA) test and the LIA-Sphilis (Line Immuno Assay) IgM and LIA-Syphilis IgG tests. The LIA-Syphilis test is to be used as a confirmatory test depending on clinical situations.
The most common test requested would be the Venereal Disease Research Laboratory (VDRL) test (which is a non-treponemal test) and when this test turns out to be positive, we would then proceed with Treponema Pallidum Particle Agglutination (TPPA) test (which is a treponemal test).
VDRL is a non-treponemal test. Unlike TPPA (a treponemal test) which is based on the agglutination of gel particles sensitized with T. pallidum antigen by the patient’s antibodies, VDRL measures anti-lipid IgG and IgM antibodies called reagin directed agains substance in the mitochondrial membrane which are formed by the host in response to lipid from the treponemal surface.
Most of you would be quite familiar with TPPA, which is very similar to the Treponema Pallidum Haemagglutination (TPHA) tests we have done in school thus I would be focusing on the VDRL test only
VDRL Test using a Patient’s Serum Sample
Principle
The Venereal Disease Research Laboratory (VDRL) test is a slide microflocculation test for syphilis. It measures anti-lipid IgG and IgM antibodies called reagin directed against substances in the mitochondrial membrane which are formed by the host in response to lipid from the treponemal surface. The antigen which is suspended in buffered saline is composed of cardiolipin, cholesterol and lecithin and forms clumps when combined with lipoidal antibodies in serum from syphilitic patients.
If the serum is reactive, flocculation occurs due to the antigen reacting with the antibody. These complexes can be seen under the microscope. A reaction reported as weakly reactive shows small clumps, while if there are no clumps seen this is reported as nonreactive.
The antilipoidal antibodies are antibodies that are not only produced as a consequence of syphilis and other treponemal diseases, but also may be produced in response to non-treponemal diseases of an acute and chronic nature in which tissue damage occurs. Without some other evidence for the diagnosis of syphilis, a reactive non-treponemal test does not confirm T. pallidum infection.
Specimen Requirements
Heat serum obtained from centrifugation of clotted blood (5ml) at 56°C in a water bath for 30 minutes before testing. Cool down to room temperature. Examine the specimen after it is removed from the water bath. Re-centrifuge if debris is seen. Test sera within four hours after original heating. If there is a delay in testing reheat the sera at 56°C for 10 minutes. Refrigerate the sera at 2 to 8°C if there is a delay in doing the test. Avoid freezing and thawing of specimen repeatedly.
Equipment
- Light microscope
- Rotating machine adjustible to 180 rpm circumscribing a ¾ inch diameter circle.
- Slide holders for 5x7.5 cm microscope slides.
- Nondisposable calibrated needles without bevels, 18 gauge needle- for testing sera.
- Slides 5x7.5 cm with ceramic rings approximately 14mm in diameter
- 2 ml syringe
Reagents
IMMUTREP® VDRL Antigen Ref OD011 from Omega Diagnostics Ltd is used. The antigen suspension is a colourless alcoholic solution containing 0.03% cardiolipin, 0.9% cholesterol and 0.2% lecithin. Antigens are stored in the dark at room temperature.
The VDRL antigen suspension has to be prepared following the manual included in the kit prior to use. This preparation is done daily as the antigen is not stable. Quality controls are tested before the antigen is used for patient samples.
Qualitative Test
- Transfer 50 μL of serum into one ring of a ceramic-ringed slide.
- Gently resuspend the VDRL antigen suspension.
- Holding the VDRL antigen suspension dispensing needle and syringe in a vertical position, dispense several drops to clear the needle of air. Then add exactly 1 free-falling drop (17μL) of antigen suspension to each circle containing serum.
- Place the slide on the mechanical rotator. Rotate the slide for 4 minutes at 180 ± 2 rpm
- Immediately after rotating the slide, remove it from the rotator and read the test results.
Quantitative Test
- Perform quantitative tests on all serum specimens that produce reactive, weakly reactive or “rough” non-reactive results in the qualitative VDRL test to their end-point titres.
- Dilute Reactive sample from 1:1 to 1:32 (e.g., use 6 circles, number 1 to 6 for dilutions 1:1, 1:2, 1:4, 1:8, 1:16 and 1:32 respectively) and dilute Weakly reactive and rough Nonreactive sample from 1:1 to 1:4 by 2-fold dilution.
- Add 50 μL of 0.9% saline in each of the circles except number 1 circle (1:1 dilution).
- Transfer 50μL of serum in circle 1 and (to?) 2
- Mix the saline and the serum in circle 2 by drawing the mixture up and down 7 to 8 times, transfer 50 μL diluted serum from circle 2 to circle 3 and mix thoroughly.
- Repeat step 4 until the last circle, mix thoroughly and discard the last 50μL mixture from the last circle.
- Gently resuspend the antigen suspension.
- Following the way described in “Qualitative test” add 1 free-falling drop of antigen suspension to each circle.
- Rotate the slide for 4 minutes at 180 ± 2 rpm and read the test.
- If the highest dilution (1:32) is reactive, repeat the sample by higher dilution until the end-point.
Reading of test results
The results are read microscopically using 10X occular and a 10X objective.
Qualitative tests are read as follows:
Qualitative Method Report
Medium or large clumps Reactive (R)
Small clumps Weakly reactive (W)
No clumping or very slight roughness Non-reactive (NR)
Quantitative tests: The end-point titre is the last dilution that produces a reactive result.
Interpretation of Test Results
The VDRL test is an aid in the diagnosis of syphilis. A reactive VDRL may be unrelated to T. pallidum infection. The predictive value of a reactive VDRL in the serological diagnosis of syphilis is increasd when combined with a reactive treponemal test.
A reactive VDRL test may indicate past or present infection with a pathogenic treponeme. False-positive reactions can result from laboratory error as well as from serum antibodies that are unrelated to syphilis infection (1-2% of normal population, transiently post-immunization or febrile disease or during pregnancy)
A non-reactive VDRL test and no clinical evidence of syphilis may indicate no current infection or an effectively treated infection. A non-reactive VDRL test with clinical evidence of syphilis as a result of the prozone reaction, and in some cases of late syphilis, a fourfold rise in titre on a repeat specimen may indicate an infection, a reinfection, or a treatment failure; a fourfold derease in titre in early syphilis usually indicates adequate syphilis therapy.
All reactive qualitative VDRL tests should be diluted to an endpoint, and the endpoint titre should be reported. Unusually high VDRL titre can be seen with concurrent HIV-1 infection. Unusually high false-positive titres may be seen in some patients with lymhomas.
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