The rapid plasma reagin (RPR) 18-mm circle card test is a macroscopic,
nontreponemal flocculation card test used to screen for syphilis (Creighton,
1990). The antigen is prepared from a modified Venereal Disease Research Laboratory
(VDRL) antigen suspension containing choline chloride to eliminate the need
for heat inactivation of serum, ethylenediaminetetraacetic acid (EDTA) to enhance
the stability of the suspension, and finely divided charcoal particles as a
visualizing agent. For 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 antibodies to lipoidal material released from damaged host cells as
well as to lipoprotein-like material, and possibly cardiolipin released from
the treponemes. If antibodies are present, they combine with the lipid particles
of the antigen, causing them to agglutinate or clump. 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 nontreponemal
test does not confirm T. pallidum infection.
Specimen Collection
Serum—Collect whole blood into a clean,
dry tube without an anticoagulant.
Plasma—Collect blood in a tube containing
EDTA as an anticoagulant. Completely fill the tube or collect blood until the
vacuum in the collection tube has been exhausted.
Label each specimen with patient identifier, and date.
Procedure
Qualitative Test
To prepare antigen for testing, attach the hub of the dispensing
needle to the fitting on the plastic dispensing bottle. Shake the antigen ampule
to resuspend the particles. Open the ampule. Squeeze the dispensing bottle
to collapse it. Insert the needle into the ampule and withdraw all the antigen
suspension into the dispensing bottle.
Place serum or plasma onto a 18-mm circle of the RPR test
card, using a disposable Dispenstir or a safety pipetting device.
Using the inverted Dispenstir (closed end) or flat toothpicks,
spread the serum or plasma to fill the entire circle. Do not spread the specimen
beyond the confines of the circle.
Gently shake the antigen dispensing bottle to resuspend the
particles.
Holding the dispensing bottle and needle in a vertical position,
dispense several drops to clear the needle of air. Then add antigen suspension
to each circle containing serum or plasma. Do not mix.
Place the card on the mechanical rotator under a humidifying
cover. Rotate the card.
Immediately remove the card from the rotator; briefly rotate
and tilt the card by hand to aid in differentiating nonreactive from minimally
reactive results.
Perform the quantitative test on serum specimens showing any
degree of reactivity (clumping) or "roughness."
Reading and Reporting of Qualitative Results
Read the test reactions in the "wet" state under
a high-intensity incandescent lamp. Read the test without magnification.
Report the results as follows:
Reading
Report
Characteristic
clumping ranging from marked and intense (reactive) to slight but definite
(minimally to moderately) reactive
Reactive (R)
Slight
roughness or no clumping
Nonreactive (N)
Note: Only two reports with the RPR card test
are possible: Reactive, no matter how much clumping, or Nonreactive.
Interpretation of Results
The RPR card test is an aid in the diagnosis of syphilis.
Clinicians combine the RPR card 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 card test is commonly unrelated to Treponema pallidum infection.
The predictive value of a reactive RPR card 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.
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 card 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.
A nonreactive RPR card test without clinical evidence of syphilis
may suggest no current infection or an effectively treated infection. A nonreactive
RPR card test with clinical evidence of syphilis can be seen in early primary
syphilis; in secondary syphilis, as a result of the prozone reaction (see test
limitations); and in some cases of late syphilis. A nonreactive RPR card test
result does not rule out an incubating syphilis infection.
When the quantitative RPR card test is performed on patients
with syphilis, a fourfold rise in titer on a repeat specimen may suggest an
infection, a reinfection, or a treatment failure; a fourfold decrease in titer
in early syphilis usually indicates adequate syphilis therapy.
All reactive qualitative RPR card tests should be diluted
to an endpoint and the endpoint titer reported.
Sources of Error
If the temperatures of the sera, reagents, or testing area
are less than 23%C (73%F), test reactivity decreases; if temperatures are greater
than 29%C (85%F), test reactivity increases.
If the speed of the mechanical rotator is too fast or too
slow, improper antigen-antibody interaction will cause unpredictable test results.
If the time of rotation is too long test reactivity may be
increased, or if too short test reactivity may be decreased.
If the card is excessively rotated and tilted (to-and fro
motions) by hand after removal from the rotator, a false- reactive result may
occur.
If lighting produces a glare on the card, the reactions may
be obscured.
If the antigen is outdated or not adequately tested for standard
reactivity, the results may be unpredictable.
If the serum is unevenly spread in the circle, the antigen
and antibody may not mix properly.
If hemolyzed, contaminated, or improperly collected serum
or plasma samples are tested, the reaction may be masked.
If the moistened humidifying cover is not used to cover tests
as they are being rotated, proper humidity will not be maintained, and test
components may dry on card, and false reactive results may occur.
Test Limitations
The RPR card test cannot be used to test spinal fluids.
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.
The RPR card test may be reactive in persons from countries
where yaws, pinta or nonvenereal syphilis is endemic. Generally, residual titers
from these infections will be no higher than 1:4.
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.
Nontreponemal test titers of persons who have been treated
in latent or late stages of syphilis or who have become reinfected do not decrease
as rapidly as do those of the persons in the early stages of their first infection.
In fact, these individuals may remain "serofast," retaining a low-level
reactive titer for life.
Centers for Disease Control and Prevention
1600 Clifton Rd, Atlanta, GA
30333, USA
800-CDC-INFO (800-232-4636) TTY: (888) 232-6348, 24 Hours/Every Day cdcinfo@cdc.gov
Program Operations Guidelines for STD Prevention
