Rapid Plasma Reagin (RPR) Card Test - UNMC

University of Nebraska Medical Center
For student use only
Clinical Laboratory Science program CLS 418/CLS 419
Method: Slide flocculation
Rapid Plasma Reagin (RPR) Card Test
Principle:
The RPR (rapid plasma reagin) 18 mm Circle Card Test is a nontreponemal serological test for
syphilis that detects reagin, an IgM or IgG immunoglobulin present in the plasma or serum of patients
with syphilis and occasionally present in the serum or plasma of individuals with other acute or
chronic conditions. The antigen consists of cardiolipin mixed with carbon particles (aids in
visualization), cholesterol (provides absorption centers), and lecithin (aids in reproducibility). If
antibody is present in the patient specimen, it binds to the cardiolipin antigen resulting in flocculation.
The carbon particles become trapped in the flocculation and appear agglutinated or as black clumps
against a white background on the plastic-coated test card. The agglutination can be seen
macroscopically. Specimens that are nonreactive, that is, do not contain reagin, have a uniform lightgray
color and even particle distribution (no clumping).
Sample Preparation:
Either clear unhemolyzed serum (specimen of choice) or plasma from EDTA anticoagulated blood
may be used for this procedure. Cerebrospinal fluid is not acceptable. Collect blood by venipuncture
using aseptic technique. Centrifuge the specimen at a force sufficient to sediment cellular elements.
Keep the serum or plasma in the original collection tube. Heat inactivation is not necessary for this
procedure.
Materials:
1. Test antigen suspension: 0.003% cardiolipin, 0.020-0.022% lecithin, 0.09% cholesterol, EDTA
(0.0125 mol/L), Na2HPO4 (0.01 mol/L), 0.1% thimerosal (preservative), 0.02% charcoal, 10%
choline chloride (w/v), and distilled water. Refrigerate (2-8ºC) to store. Unopened ampules have
a shelf life of 12 months from date of manufacture. Once opened, antigen in the dispensing bottle
may be used for approximately 3 months, or until the expiration date. A new lot of antigen
suspension should be compared with an antigen suspension of known reactivity before placed
into routine use. Antigen suspensions should be well mixed and at room temperature when used.
2. Diagnostic test cards: specially prepared plastic-coated cards designed for use with the RPR
card antigen, 18 mm diameter test circles.
3. Dispenstirs, 0.05 ml per drop.
4. Needles, 20 gauge without bevel.
5. Stirrers.
6. Plastic dispensing bottle.
7. Pipet or syringe, 1 ml.
8. Rotator, 100 rpm, circumscribing a circle 2 cm in diameter with an automated timer and a cover
containing a moistened sponge or blotter.
9. Controls: known reactive and weakly reactive sera prepared from pooled rabbit sera, and
nonreactive pooled human sera. These need to be stored at 4ºC and may be used until the
expiration date on the label.
Quality Control:
1. Testing accuracy of delivery needle: It is of primary importance that the proper amounts of
reagents be used. For this reason, the needle should be checked each day. Place the needle on
a 1 ml pipet. Fill the pipet with well-mixed antigen suspension, and holding in a vertical position,
count the number of drops delivered in 0.5 ml. A needle is considered satisfactory if 30 +/- 1
drops are obtained in 0.5 ml. The needle not meeting this specification should be replaced with
another needle that does meet this specification.
2. Testing temperature: Controls, antigen suspension, test cards, and test specimens should be at
room temperature for use.

3. Rotator speed: Rotator must be checked daily for a proper rotation speed of 100 +/- 2 rpm.
4. Controls: Test control sera of graded reactivity daily. Always include nonreactive, weakly
reactive and reactive controls. In testing patient samples, use only those antigen suspensions
that have given the appropriate reactions with the controls.
Procedure:
1. Hold a Dispenstir device between the thumb and forefinger near the sealed end. Squeeze and
do not release until open end is below surface of the specimen. Hold specimen tube vertically to
minimize stirring up of cellular elements when suing original blood tube. Release finger pressure
to draw up the sample. Hold Dispenstir in a vertical position over the card test area (18 mm
circle). Do not touch the surface of the card. Squeeze Dispenstir, allowing one drop (0.05 ml) of
serum to fall onto card.
2. Spread serum with inverted Dispenstir (closed end) or a stirrer (broad end) to fill the entire circle.
Discard Dispenstir.
3. Hydrate the lyophilized controls with a drop of distilled water using a Dispenstir. Spread each
control with its own inverted Dispenstir or a stirrer to fill the entire circle. Discard Dispenstir.
4. Attach the needle to the tapered fitting on the dispensing bottle that contains the antigen
suspension. Gently shake antigen-dispensing bottle before use. Hold in a vertical position, and
dispense several drops into bottle cap to make sure the needle passage is clear. Place 1 freefalling
drop onto each test or control area. Return test droplets from the bottle cap to the
dispensing bottle. Do not mix antigen and specimen. Mixing is accomplished during rotation.
5. Place card on rotator and cover with a humidifier cover.
6. Rotate 8 minutes at 100 rpm.
7. Immediately following rotation, briefly rotate by hand with tilting of the card (3-4 to-and-fro
motions). Read macroscopically in the “wet” state under a high-intensity incandescent lamp or
strong daylight.
8. Report as follows:
Reactive –clumping, ranging from slight but definitive to marked and intense
Nonreactive – slight roughness or no clumping
NOTE: Specimens giving any degree of clumping should be subjected to further serologic
study. The RPR test may be falsely positive in a number of situations. Any reactive serum
should be tested by a treponemal specific test.
9. Upon completion of tests, remove the needle from the dispensing bottle and rinse with distilled or
deionized water. Do not wipe the needle, as this may remove the silicon coating and affect
accuracy of delivery. Recap dispensing bottle and store in refrigerator.
Reference Range:
Normal healthy individuals should be nonreactive.
References:
1. Manual of tests for syphilis, PHS Publication No. 411, 1969.
2. Stevens, C.D. (1996): Clinical Immunology and Serology, F.A. Davis, pages 246-247.
3. Macro-Vue RPR 18-mm circle Card Test (Brewer Diagnostic Kit) package insert, Becton
Dickinson and Company, Cockneysville, MD.