VDRL Test: Introduction, Principle, Procedure, Result-Interpretation,Limitations, and Keynotes

Introduction of VDRL Test

VDRL stands for Veneral disease research laboratory. The laboratory was renamed the Treponemal Pathogenesis and Immunology Branch of the United States Public Health Service. The Venereal Disease Research Laboratory (VDRL) test is one of two variations of micro flocculation procedures used for serological testing of syphilis. Flocculation testing is based on antibody detection with the interaction of soluble antigen with an antibody that results in a precipitate formation of fine particles. The VDRL can be used for qualitative and quantitative measurements and is recommended when a patient suspected of having syphilis has a negative dark field microscopy result.

Principle of VDRL Test

In a VDRL procedure, the patient’s serum is heat-inactivated and mixed with a buffered saline suspension of VDRL antigen containing cardiolipin, lecithin, and cholesterol that binds with reagin (an antibody-like protein). A combination of reagin and VDRL antigen forms microscopic clumping called flocculation.
VDRL Antigen is a nontreponemal antigen composed of cardiolipin cholesterol and lecithin. The nontreponemal tests measures anti-lipid antibodies, which are formed by the host in response to lipids released from damaged host cells early in infection with Treponema pallidum, and lipid-like material from the treponemal cell surface. During syphilis infection, an antibody-like substance called reagin can be detected in the patient’s serum or cerebrospinal fluid (CSF).

The VDRL tests measure immunoglobulin M (IgM) and immunoglobulin G (IgG) antibodies to lipoidal material released from damaged host cells as well as to lipoprotein-like material, and possibly cardiolipin released from the treponemes. 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 nontreponemal diseases of an acute and chronic nature in which tissue damage occurs. Without some other evidence for the diagnosis of syphilis, a reactive nontreponemal test does not confirm T. pallidum infection.

Requirements for the VDRL Test

• VDRL antigen:- A colorless alcohol solution containing 0.03% cardiolipin, 0.9% cholesterol, and sufficient purified 0.21% + 0.01% lecithin.
• VDRL-Buffered Saline:- pH 6.0 ±0.1 (1.0% NaCl).
• Control serum samples:- Reactive (R), weakly reactive (W), and nonreactive (N) sera in dehydrated or liquid form are used as controls in the test.
• 10 % saline
• VDRL slide:- 2 x 3 inches, with 12 paraffin or ceramic rings approximately 14 mm in diameter
• Non-disposable calibrated needles without bevel.
• Serum test: 18-gauge b.Spinal fluid test: 21-or 22-gauge
• Water bath
• Mechanical rotator adjustable to 180 ±2 rpm
• Microscope

Specimens for VDRL Test

Serum:-An acceptable serum specimen should not contain particulate matter that would interfere with reading test results. Serum specimens that are excessively hemolyzed, grossly contaminated with bacteria, chylous, or otherwise extremely turbid are unsatisfactory for testing. On the day of testing, heat the serum in a 56°C water bath for 30 minutes. Remove the serum from the water bath and examine for debris. Re-centrifuge all serum specimens containing particulate debris. Reheat serum at 56°C for 10 minutes if testing is delayed more than 4 hours. Spinal fluid:-An acceptable CSF specimen should not contain particulate matter that would interfere with reading test results. CSF specimens with even traces of blood are unsuitable for testing. Do not heat spinal fluid before testing. Specimens must be at room temperature, 23 -29°C (73 -85°F) when tested.

VDRL Test Procedure

Preparation of fresh antigen daily:-Pipette 0.4ml of VDRL-buffered saline to the bottom of a round, 30-ml, glass-stoppered bottle with a flat inner-bottom surface. Add 0.5 ml of VDRL antigen suspension directly onto the saline, while rotating the bottle continuously but gently on a flat surface. Put antigen drop by drop at a rate allowing approximately 6 seconds for each 0.5ml of antigen. Add 4.1 ml of buffered saline. Cap the bottle and shake it from bottom to top and back approximately 30 times in 10 seconds. The antigen suspension is ready for use and may be used during that day (8 hours). Mix the VDRL antigen suspension by gently swirling it each time it is used.

Qualitative VDRL Test (serum)
(Slide flocculation test)

VDRL antigen suspension, controls, and test specimens must be at room temperature, 23⁰C-29⁰C. Place 50 μl of serum into one ring of paraffin or ceramic-ringed slide using a pipetting device. Gently resuspend the VDRL antigen suspension. Holding the VDRL antigen suspension dispensing needle and syringe in a vertical position, add exactly 1 free-falling drop (17 μl) 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.

Reading and Reporting Results

Read slides microscopically, using 10X oculars and a 10X objective.
Reading Report-

Observations	Test Report
Medium or large clumps	Reactive (R)
Small clumps	Weakly reactive (W)
No clumping	Non-reactive (N)

Quantitative VDRL Test (serum)

Dilute serum samples to 1:8 dilution. Place 50 μl of 0.9% saline in each circle from 2. Do not spread saline. Using a safety pipette device, place 50 μl of serum in circle 1 and 50 μl of serum in a circle. Mix the saline and the serum in circle 2 by drawing the mixture up and down in the safety pipette eight times. Transfer 50 μl from circle 2 (1:2) to circle 3, and mix. Transfer 50 μl from circle 3 to circle 4, mix, and then discard the last 50 μl. Gently resuspend the antigen suspension. Holding the VDRL antigen suspension dispensing needle and syringe in a vertical position, exactly 1 free-falling drop (17 μl) of antigen suspension to each circle. Place the slide on the mechanical rotator.

Rotate the slide for 4 minutes at 180 ±2 rpm. Immediately after rotation, read the test. If the highest dilution tested (1:8) is reactive, continue as follows: Prepare a 1:8 dilution of the test specimen in a test tube. Add 0.1 ml of serum to 0.7 ml of 0.9% saline. Mix thoroughly. Place 50 μl of 0.9% saline into paraffin rings 2, 3, and 4. Prepare additional serial dilutions for strongly reactive specimens. Add 50 μl of the 1:8 dilution of the test specimen to paraffin rings 1 and 2. Prepare serial two-fold dilutions beginning with ring 2. Complete the test. After completing the day’s tests, discard the antigen suspension and clean dispensing needle and syringe by rinsing with water, alcohol, and acetone, in that order. Remove the needle from the syringe after cleaning.

Reading and Reporting of Results

Read the results microscopically using 10X oculars and a 10X objective for the qualitative test. Report titers as the highest dilution that gives a reactive (not weakly reactive) result.

Test Procedure for Cerebrospinal fluid (VDRL-CSF)

Preparing the Sensitized Antigen Suspension:-Prepare the VDRL antigen suspension as described for the VDRL slide tests on serum. Add one part of 10% saline to one part of VDRL antigen suspension. Mix by gently rotating the bottle or inverting the tube. Allow the mixture to stand for at least 5 minutes.
The sensitized VDRL-CSF antigen suspension is good for only 2 hours after preparation.

Qualitative test

Holding the VDRL-CSF sensitized antigen suspension dispensing needle (21-or 22-gauge) and syringe in a vertical position, dispense exactly 1 free-falling drop (10 μl) of sensitized antigen suspension to each slide concavity that contains 50 μl spinal fluid. Rotate the slide for 8 minutes at 180 ±2 rpm. Read under a microscope.

Report the results as follows

Reading Report
Definite clumping of any degree- Reactive (R)
No clumping or very slight roughness- Nonreactive (N)

Quantitative Test

Test each specimen undiluted and in 1:2, 1:4, and 1:8 dilutions or more.

Interpretation of result VDRL Test

• Non-reactive VDRL – with clinical evidence may indicate early primary syphilis, a prozone reaction in secondary or late syphilis. Nonreactive VDRL – with no clinical evidence may indicate no current infection or an effectively treated infection.
• Reactive VDRL:- may indicate past or present infection with a pathogenic treponeme; however, the result may also be a false-positive reaction. Quantitative VDRL – detects changes in reagin titer. Serum samples displaying a fourfold increase in titer(1:4 -1:16) on a repeated sample may indicate an infection, reinfection, or treatment failure. A fourfold decrease(1:8-1:2) during treatment indicates adequate therapy. All reactive qualitative VDRL tests should be quantitated to an endpoint, and the endpoint titer should be reported. Unusually high VDRL titers may be seen with concurrent HIV-1 infection. Unusually high false-positive titers may be seen in serum from some patients with lymphomas.

Cerebrospinal Fluid(CSF)

A reactive VDRL test on CSF, free of blood or other contaminants, usually suggests past or present syphilis infection of the central nervous system. A biological false-positive VDRL test result for syphilis is rare in spinal fluid. A nonreactive VDRL test on CSF may indicate that the patient does not have neurosyphilis. However, a negative result may occur in some serum from neurosyphilis patients.

Limitations of VDRL tests

1. Tedious test procedure
2. VDRL antigen suspension for the VDRL test on serum must be prepared fresh each day of testing.
3. The VDRL-sensitized antigen suspension for the VDRL-CSF test is good for only 2 hours after preparation.
4. The VDRL-CSF test should be performed only when the patient’s serum treponemal test result is reactive.
5. Plasma cannot be used for the VDRL assay.
6. A microscope is required.
7. VDRL test is replaced by the RPR test.

Keynotes on VDRL Assay

1. Serum heat inactivation is mandatory to proceed VDRL test.
2. VDRL is a cumbersome assay.
3. VDRL is a flocculation-based assay that is a special type of precipitation test.
4. It is a non-specific test used for screening syphilis caused by the bacteria, Treponema pallidum.
5. TPHA is a confirmatory test for syphilis.
6. Biological false-positive (BFP) reactions occur occasionally. Non-syphilitic conditions giving biologic false-positive results in non-treponemal tests like-

Infectious origin	Non-infectious origin
Malaria	Pregnancy
Leprosy	Advanced cancer
Tuberculosis	Multiple myeloma
Scarlet fever	Connective tissue diseases (e.g. rheumatoid arthritis, lupus)
Trypanosomiasis	Multiple blood transfusion
pneumococcal pneumonia
Bacterial endocarditis
Mycoplasma pneumonia
Measles
Infectious mononucleosis
Mumps
Viral hepatitis
Chickenpox
HIV
Vaccination
Viral hepatitis
Pneumococcal pneumonia

Further Readings

• https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3312652/
• https://www.himedialabs.com/TD/HTI021.pdf
• https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3312652/
• https://www.himedialabs.com/TD/HTI021.pdf
• http://whqlibdoc.who.int/hq/2006/TDR_SDI_06.1_eng.pdf
• https://sti.bmj.com/content/27/2/52.full.pdf