VDRL Test: Principle, Procedure, Results

The venereal disease research laboratory (VDRL) test is a nonspecific microflocculation test for the screening of syphilis. It uses antigen-containing cardiolipin, lecithin, and cholesterol and measures both IgG and IgM antibodies to lipoidal and lipoproteins released as a result of damage to host cells and also to cardiolipin released from treponemes. VDRL test is easy to perform and inexpensive, so it is commonly used in the screening of the population for syphilis. Without some other evidence for the diagnosis of syphilis, a reactive nontreponemal test does not confirm Treponema pallidum infection.


  1. Serum (plasma can not be used)
  2. Cerebrospinal fluid (CSF)

Acceptable CSF and serum specimens should not contain particulate matter that would interfere with reading test results. Serum samples that are excessively hemolyzed, contaminated, and turbid are not suitable for testing.

Principle of VDRL Test

VDRL Test Kits
VDRL Test Kits

Non-treponemal antigen (cardiolipin-cholesterol-lecithin) is used to detect the presence of “reagin antibodies” (IgM and IgG antibodies to lipoidal material released from damaged host cells as well as to lipoprotein-like material,   and possibly cardiolipin released from the treponemes) in the patient’s serum.

When the heat-inactivated serum (to destroy complement) of a patient reacts with freshly prepared non-treponemal antigen, a flocculation reaction (Ag-Ab complex are suspended) occurs. The flocculation can be observed by using a microscope with a 10x objective and 10x eyepiece.

Reactive VDRL test serum can be quantitated to obtain the titre of “reagin antibodies” by using the serial double dilution method.

Procedure of VDRL Test

The sample for the VDRL test is fresh serum or plasma. Before starting the process, make sure all the antigens suspensions, controls, and samples are at room temperature. 

There are two ways to perform the VDRL test: Qualitative and Quantitative.

Qualitative Method

  1. Dispense a drop of negative control, positive control, and 50 μl of the test sample into the separate designated test circles. 
  2. Then, using the mixing stick provided, spread all the dispensed substances over the entire area of the test circle. 
  3. After that, dispense a drop of antigen to each of the test samples. Avoid stirring. 
  4. Rotate the test card using an automatic rotator for 8 minutes at 100 rpm or a mechanical rotator for 4 minutes at 160-180 rpm. 
  5. Read the result. A false positive result may appear if the test is read after 8 minutes if it is rotated in the automatic rotator and 4 minutes if it is rotated in the mechanical rotator.

Quantitative Methods

  1. Using a volumetric pipette, dispense 50 μl of 0.85% saline onto the first five circles of the test card. 
  2. Likewise, with the help of the volumetric pipette, dispense 50 μl of the test sample on the first circle and label it 1. Place the tip of the pipette into the drop and mix by filling and discharging the pipette at least six times. Avoid bubble formation. 
  3. Withdraw 50 μl from the first circle and transfer it to the second circle, labeling it 2. Repeat the mixing procedure. 
  4. Repeat steps 2-3 from second to third, third to fourth, and fourth to fifth, labeling them as 3, 4, and 5. 
  5. Using the provided mixing sticks, spread the suspension in the 5th circle to the entire circle area. Repeat the same mixing step with separate sticks in the remaining circles. 
  6. Then, dispense a drop of antigen in all the five circles. Avoid stirring. 
  7. Now rotate the test card like in qualitative methods. 
  8. Read the result as soon as possible, i.e., before 8 minutes.

Result and Interpretation of VDRL test

  • Reactive: medium or large clumps
  • Weakly reactive: small clumps
  • Nonreactive: no clumping or very slight roughness
  • Report titers in terms of the highest dilution that produces a reactive (not weakly reactive) result

VDRL test is positive in most cases of primary syphilis and is almost always positive in secondary syphilis. The titer of reagin antibodies decreases with effective treatment, so the VDRL test can be used to determine the treatment response of syphilis.

You may be interested in these tests: 

TPHA: Principle, Procedure, Results and Interpretations 
RPR Test: Princple, Procedure and Interpretations 
FTA-Abs test: Princple, Procedure, Results and Interpretation

Limitation of the Test

A. False positive VDRL test  result 

  1. Reagin antibodies may be produced in response to nontreponemal diseases of an acute and chronic nature in which tissue damage occurs such as:
    • Leprosy
    • Hepatitis B
    • Infectious Mononucleosis
    • Various autoimmune diseases
  2. VDRL may be reactive in persons from areas where yaws are endemic. As a rule, residual titers from these infections will be <1:8.
  3. Nontreponemal test titers of persons treated in latent or late stages of syphilis or who have become reinfected do not decrease as rapidly as do those from persons in the early stages of their first infection. In fact, these persons may remain “serofast,” retaining a low -level reactive titer for life.

B. False negative VDRL test

It can be seen because of the prozone phenomenon (no flocculation due to antibody excess). In that case test serum has to be diluted further to obtain a zone of equivalence (where maximum flocculation of Ag-Ab occurs).

Further Reading and References

  1. ASM: Veneral Disease Research Laboratory (VDRL) slide Test.
  2. Yetty Fakile, Weiping Cao, Mayur Shukla, and Allan Pillay, 2023. 13.5 Laboratory Diagnosis of Syphilis, Clinical Microbiology Procedures Handbook, 5th Edition. ASM Press, Washington, DC. doi: 10.1128/9781683670438.CMPH.ch13.5 

Acharya Tankeshwar

Hello, thank you for visiting my blog. I am Tankeshwar Acharya. Blogging is my passion. As an asst. professor, I am teaching microbiology and immunology to medical and nursing students at PAHS, Nepal. I have been working as a microbiologist at Patan hospital for more than 10 years.

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