INTRODUCTION : Rapid plasma reagin is commonly abbreviated as RPR. RPR test is a macroscopic, nontreponemal, flocculation test. It is a non-specific screening test for diagnosis of syphilis. Syphilis is a venereal disease caused by the spirochete Treponema pallidum.  After acquiring infection, the host forms antibodies to Treponema pallidum, in addition, the host also forms Non Treponemal antilipoidal antibodies in response to the lipoidal material released from the damaged host cell during treponemal invasion. These antibodies are traditionally referred to as ‘Reagins.’

PRINCIPLE OF RPR TEST

RPR test is performed in a card of 18 mm circle with white background.  The antigen is prepared from a modified Venereal Disease Research Laboratory (VDRL) antigen suspension containing choline chloride to eliminate the need of heat to inactivate serum, ethylenediaminetetraacetic acid (EDTA) to enhance the stability of the suspension, and finely divided charcoal particles as a visualizing agent.

In the test, the RPR antigen is mixed with unheated or heated serum or with unheated plasma on a plastic-coated card. The RPR test measures IgM and IgG antibodies to lipoidal material. 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. If antibodies are not present, the test mixture is uniformly gray. Without some other evidence for the diagnosis of syphilis, a reactive RPR test does not confirm T. pallidum infection. To confirm syphilis, treponemal tests such as Treponema pallidum Heamagglutination Assay (TPHA), Microhemagglutination assay for antibodies to T. pallidum (MHA-TP) or Fluorescent Treponemal Antibody Absorption test (FTA-Abs) should be performed.

CONTROL

  1. For reliable and reproducible test results, the RPR Card antigen suspension, controls, and test specimens must be at room temperature (23° – 29°C; 73° – 85°F), when tests are performed.
  2. At each routine test run, check the expiration date on the ampule.
  3. Determine antigen suspension reactivity with control cards or control serum specimens of graded reactivity (reactive, minimally reactive, and nonreactive)

 PROCEDURE FOR QUALITATIVE RPR TEST:

  1. Label a RPR card with patient number 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 (50µl) 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. 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.

 

OBSERVATAION

Reactive – any sign of clumping ranging from marked and intense (reactive) to Reactive (R) slight but definite reactive no matter how much clumping

Nonreactive – Slight roughness or no clumping

 

INTERPRETATION OF RESULTS

  1. The RPR test is a screening test for syphilis. Clinicians combine the RPR test with results of other serologic tests, darkfield examinations, clinical signs and symptoms, and risk factors in arriving at a syphilis diagnosis. Without some other support for the diagnosis of syphilis, a reactive RPR test is commonly unrelated to T. pallidum infection. The predictive value of a reactive RPR test in a serologic diagnosis of syphilis is increased when combined with a reactive treponemal test, such as the fluorescent treponemal antibody absorption (FTA-ABS) test or the microhemagglutination assay for antibodies to T. pallidum (MHA-TP).
  2. A reactive RPR card test may suggest past or present infection with a pathogenic treponeme; however, it may also be a false-positive reaction. False-positive reactions can result from laboratory error as well as serum antibodies unrelated to syphilis infection. Technical errors are detected by a nonreactive RPR test with a second serum specimen. False-positive RPR card tests from infections with nontreponemal diseases or other disease conditions are identified by an accompanying nonreactive treponemal test.
  3. A nonreactive RPR test without clinical evidence of syphilis may suggest no current infection or an effectively treated infection. A nonreactive RPR test with clinical evidence of syphilis can be seen in early primary syphilis; in secondary syphilis, as a result of the prozone reaction; and in some cases of late syphilis. A nonreactive RPR test result does not rule out an incubating syphilis infection.
  4. All reactive qualitative RPR tests should be diluted to an endpoint and the endpoint titer reported. Unusually high false-positive titers may also be seen in patients with lymphomas and concurrent HIV-1 infection with syphilis.

LIMITATIONS OF RPR TEST

  1. The RPR card test cannot be used to test spinal fluids.
  2. A prozone reaction may be encountered occasionally. In a prozone reaction, complete or partial inhibition of reactivity occurs with undiluted serum (maximum reactivity is obtained only with diluted serum). The prozone phenomenon may be so pronounced that only a rough reading is produced in the qualitative test by a serum that will be strongly reactive when diluted. All test specimens producing any degree of roughness or reactivity with the RPR card test antigen in the qualitative test should be retested by using the quantitative procedure. In addition, a specimen should be tested for the prozone phenomenon when the clinician suspects syphilis, even if qualitative RPR test is nonreactive.
  3. The RPR card test may be reactive in persons from areas where yaws, pinta or nonvenereal syphilis is endemic. Generally, residual titers from these infections will be <1:8.
  4. Biological false-positive (BFP) reactions occur occasionally with cardiolipin antigens, mainly in specimens from persons who abuse drugs; who have diseases such as lupus erythematosus, mononucleosis, malaria, leprosy, or viral pneumonia; or who have recently been vaccinated.

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