RPR Test: Principle, Procedure, Interpretations

The rapid plasma reagin (RPR) test is a macroscopic, nontreponemal flocculation card test used to screen for syphilis.  

Let’s first analyze the test name:

Rapid: It is a rapid test that can be done within a few minutes.

Plasma: The sample is plasma (or serum)

Reagin:  The term “reagin” means that this test does not look for antibodies against the actual bacterium, but rather for antibodies against substances released by cells when they are damaged by Treponema pallidum.

In some textbooks you can find reagin means an antibody to cardiolipin.

RPR test is one of the nonspecific tests (another non-specific test is VDRL Test) commonly used to screen the population for syphilis. The RPR test measures 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.

RPR Test card
RPR Test card

The anti-lipoidal antibodies are antibodies that are produced not only as a consequence of syphilis and other treponemal diseases but also in response to nontreponemal diseases of an acute and chronic nature in which tissue damage occurs.

Antigen used in RPR Test

RPR antigen contains cardiolipin, lecithin, cholesterol, 10% choline chloride, EDTA, charcoal, etc in a buffer.

The antigen is prepared from a modified Venereal Disease Research Laboratory (VDRL) antigen suspension containing choline chloride to eliminate the need to heat inactivate serum, ethylenediaminetetraacetic acid (EDTA) to enhance the stability of the suspension, and finely divided charcoal particles as a visualizing agent.

Positive Control: Prepared from human serum samples containing antibodies to Treponema pallidum. Serum is ready to use. Bring to room temp before use.

The test kits can be purchased from many commercial sources.

RPR Test Principle

RPR antigen is mixed with unheated or heated (to inactivate complement) serum or with unheated plasma on a plastic-coated card.

If antibodies are present, they combine with the lipid particles of the antigen, causing them to agglutinate. The charcoal particles coagglutinate with the antibodies and show up as black clumps against the white card (macroscopically visible flocculation-type precipitation if the patient’s sera contain reagin).

Without some other evidence for the diagnosis of syphilis, even a reactive nontreponemal test does not confirm T. pallidum infection and treponemal tests such as Treponema pallidum haemagglutination assay (TPHA) or Fluorescent Treponemal Antibody Absorption Test (FTA-Abs) must be done to confirm syphilis.

If antibodies are not present in the test serum, the test mixture is uniformly gray.


Bring RPR antigen suspension, positive control, negative control, and samples to room temperature (20–30°C) before use.

All reagents are ready for use as supplied. Gently mix the reagents before use; avoid foaming.

Qualitative RPR Test Procedure

  1. Label a RPR card with patient and control information being careful not to interfere with the test areas of the card.
  2. Using disposable serum dispensers or droppers, dispense one free-falling drop (0.05 ml) of serum or plasma sample onto a circle on the test card. When using droppers/dispensers, keep it in a vertical position to ensure accurate delivery.
  3. Repeat by adding one free-falling drop of positive control and negative control using a new dispenser for each sample.
  4. Spread the sample smoothly across the circle area using the paddle side of the dispenser as shown by instructor. Take care not to scratch the test area.
  5. After mixing the antigen solution by swirling, add one drop of the antigen suspension to each sample/control testing area.

    Note: hold the antigen container upside down directly over the test area such that the drop falls directly onto the center of the circle. DO NOT STIR OR SPREAD THE ANTIGEN.
  6. Place the card on an automatic rotator and cover to maintain humidity. Rotate at 100 ± 5 rpm for 8 minutes (7 minutes 50 seconds to 8 minutes 30 seconds). Following rotation, a brief hand rotation and tilting of the card (3–4 times) should be performed to aid in differentiating nonreactive from minimally reactive results.
  7. Immediately read results macroscopically in the “wet” state under a high-intensity light source.

Interpretation of RPR Test

  1. Non-reactive (NR) – smooth suspension, no clumping or slight roughness
  2. Reactive (R) – any degree of clumping

If the test is negative, but the physician still suspects the infection is present, the more specific treponemal tests  (FTA-ABS, MHA-TP, TPHA, etc.) should be performed.

False-positive results

As reagin antibodies are also produced by patients with other infectious and non-infectious conditions, false-positive results are likely to occur.

Patients with the following infectious diseases may produce reagin antibodies;

  • Chancroid
  • Chickenpox
  • Hepatitis
  • Infectious mononucelosis
  • Leprosy
  • Leptospirosis
  • Lymphogranuloma venereum (LGV)
  • Malaria
  • Measles
  • Ricekttsial disease
  • Trypanosomiasis and
  • Tuberculosis

Non-infectious conditions

  • Autoimmune disorders (rheumatoid disease)
  • Drug addiction
  • Other factors (old age, pregnancy, recent immunization

Uses of non-treponemal tests

As antibodies to Treponema pallidum may persist indefinitely regardless of the disease state or prior therapy, treponemal tests (FTA-ABS, TPHA or TPPA) fail to differentiate between active and past syphilis infection but non-treponemal tests (such as VDRL and RPR) may help to differentiate between active and past syphilis infection.

Nontreponemal antibodies are generally detected by the rapid plasma reagin (RPR) assay, which is typically positive during current infection and negative following treatment or during late/latent forms of syphilis.


  1. Phaosavasdi, S., Snidvongs, W., Thasanapradit, P., Ungthavorn, P., Bhongsvej, S., Jongpiputvanich, S., Vejjajiva, S., Udomsantisuk, P., Chitwarakorn, A., & Panikabutra, K. (1989). Rapid Plasma Reagin test (RPR) compared to Venereal Diseases Research Laboratory test (VDRL) for the diagnosis of syphilis in pregnancy. Journal of the Medical Association of Thailand = Chotmaihet thangphaet, 72(4), 202–206.
  2. Walker A. N. (1971). Rapid plasma reagin (RPR) card test. A screening method for treponemal disease. The British journal of venereal diseases, 47(4), 259–262. https://doi.org/10.1136/sti.47.4.259

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.

8 thoughts on “RPR Test: Principle, Procedure, Interpretations

  1. i worked in a hospital laboratory approximately 1979, I was calibrating the drops of reagent for the RPR test using a mouth pipetting hose…. (dumb I know). Well i sucked some of the charcoal antigen into my mouth, of course I spit it out and did not think to much about it. Our hospital required several test once a year and one of the test was an RPR, I was actually performing the test for the evening, one was slightly positive, i looked at the patient and to my amazement it was MY sample. Long story short it was sent to state for confirmation and it came back negative for syphilis. When I had children I got a letter from the state health department notifying me that i had a positive RPR. Years later i donated blood, got the same letter from health department … yes I’m a slow learner. Is this positive screening result possibly from ingesting the charcoal antigen ???

    1. Positive or reactive RPR may caused from biological false positive (both infectious and non-infectious condition as above even your pregnancy). Did your RPR result showed the regain titer? If the titer showed below 1:8, it should be false positive. I think the lab from health department may confirmed the specific treponemal test and the result was non reactive that was meant that you were not get syphilis disease. So I think (less possible) your blood can be found a low reagin or low anti-cardiolipin from the RPR antigen not from the charcoal. Don’t worry for the RPR result.

  2. I ve done test rpr 1:4 and tpha positive and same day I ve done next test to another lab its negative m in confuse. Again tested negative. I ve consulted with doctor and take penicillin injection after 6 month later I test blood 1st lab which I ve done previous rpr 1:4 result gave me negative result and 2nd lab which gave me negative result give me positive result. I asked him why this they said me it’s may be result of cross reaction of yr medication please tell me what to do I m in confusion

  3. sir i am munir ahmed I have to take a medical for foreign from afip Rawalpindi my result of VDRL was” positive” and TPHA was’ NEGATIVE’ then i consult with Doctor and took Medicine for one Month after that I have to got Test of VDRL/RPR from many Laboratories Such as AFIP (Cmh Rawalpindi ) Shoukat khanam Lab Chughati Lab Zeenat Lab in all above labs my test of VDRL /RPR is” NEGATIVE ” Sir when I carry out test in AKUH Lab my Result is” Positive” pl z guide me which Lab Result is accurate

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