Rapid Plasma
Reagin(RPR) Test
Ram Prashad Ojha
Introduction
The RPR test is a nontreponemal testing
procedure for the serologic detection of
syphilis.
Principle of RPR
The RPR Card antigen suspension is a carbon
particle containing cardiolipin antigen that
detects reagin.
Reagin is an antibody like substance present in
serum or plasma from individuals with syphilis.
The reagin binds to the test antigen which
consists of cardiolipin-lecithin coated particles
that cause macroscopic flocculation.
Cont…
When a specimen such as serum or plasma
contains antibody, flocculation occurs with
the resulting aggregation of the carbon
particles.
The flocculation appears as black clumps
against the white background of the plastic
coated card.
Cont…
Antibodies associated with syphilis begin to
appear in the blood 4 to 6 weeks after
infection.
Nontreponemal tests determine the presence
of reagin.
Reagin is a nontreponemal autoantibody
directed against cardiolipin antigens.
Materials for RPR
RPR Test Cards
RPR Controls(Reactive and Non-Reactive)
RPR Antigen
◦ (cardiolipin-lecithin-cholesterol,choline
chloride,EDTA & carbon particle)
Dispenstirs
Rotator
Reactions for Controls
The following reactions should be observed to
compare against the test results:
Reactive control - characteristic strong
clumping.
Reactive moderate control - moderate
clumping.
Non-reactive control - smooth, grayish
appearance of unclumped particles
Specimen Collection
Unheated serum- centrifuge for
sedimentation of cellular elements, serum
may be frozen until time of testing.
Unheated Plasma - specimen should be
collected with an anticoagulant such as EDTA
or heparin, plasma must be stored at 2°C to
8°C. Plasma must be tested within in 24 hrs
of collection.
Theaddition of choline chloride,(block
inhibitors in serum,eliminate the heat
inactivation & allow testing of plasma)
EDTA(stabilizethe antigen &allow it to be
used for upto 6month at 4-10◦c)
Procedure for Test
1. Label rings on test card with numbers of
samples to be tested
2. Use Dispenstir to draw up serum sample.
3. Hold Dispenstir in a perpendicular position
directly over the test circle to which the
specimen is to be delivered.
4. Squeeze Dispenstir to allow 1 drop to fall on
to each circle.
5. Spread the specimen in the confines of the
circle.
6. Reconstitute the antigen bottle, by shaking.
Holding the bottle in a straight vertical position,
place one “free falling” drop on each test area.
7. Rotate card for 8 minutes on a mechanical
rotator at 100 rpm.
8.After rotating mechanically, the test card
should be rotated manually by hand 3 to four
rotations and then read immediately
macroscopically in the “wet” state under a high
intensity lamp.
The test results should be reported as
reactive (even if minimally reactive) or non-
reactive.
All test results that are reactive should be
quantitated.
Causes of Negative test result
1. The patient does not have syphilis.
2. The infection is too recent for antibodies to
be produced. (Repeated tests should be
administered at 1 week, 1 month, and 3
month intervals to establish presence or
absence of disease).
3. The syphilis is latent or inactive
4. Faulty immunodefense mechanism
5. Faulty lab techniques
A positive reaction is not conclusive for
syphilis. Several conditions produce biologic
false positive results for syphilis. (False
positive means that the test revealed a
positive reaction when it was actually
negative).
False positives may reveal the presence of
other serious diseases.
Non-syphilitic Conditions Giving
Biologic False-Positive Results
Malaria
Leprosy
Relapsing fever
Infectious Mononucleosis
Atypical pneumonia
Viral pneumonia
Lupus erythematosus
Measles
pregnancy
Lymphoma
Tuberculosis
Connetive tissue disease
Endocarditis
Drug abuse
Contamination
Resolving False Positive RPR Tests
False positive RPR tests may be resolved by
testing the patient’s serum with a specific
treponemal antigen test.
Conformation of result
Diagnosis of syphilis requires correlation of
geographical area or country, patient history, physical
findings, and results of syphilis antibody tests.
T. pallidum is diagnosed when both the screening and
confirmatory test are reactive.
Treponemal tests are more specific than non-
treponemal tests
Treponemal tests confirm syphilis when a reactive or
positive non-treponemal result is obtained.
Confirmatory Tests for Syphilis
FTA-ABS (Fluorescent Antibody Absorbed
test)
TPHA (T.pallidum Haemaggultination Assay)
EIA (Enzyme Immunoassay)
Treponemal tests are used to confirm reactive
non-treponemal procedures.
A positive FTA-ABS test almost always
remains positive and therefore is not
recommended for monitoring therapy.
Theory of Antigen-Antibody Reaction
It is theorized that reagin is an antibody
against tissue lipids.
Lipids are presumed to be liberated or free
from body tissue in the normal course of
activity.
As a result of being free in the body, these
lipids may induce antibody formation.
Interfering Factors
Alcohol decreases reaction intensity in tests
and therefore should be avoided for a least
24 hrs before blood is drawn.
Avoid drawing the blood sample immediately
after the patient has eaten.
Clinical Information
Sexual partners of patients with syphilis
should be evaluated for the disease.
After treatment, patients with early stage
syphilis should be tested at 3 month intervals
for one year to monitor declining reactivity of
the syphilis.
Summary
RPR (Rapid Plasma Reagin) is Non specific (screening) test
for syphilis and detect reagin antibody present in serum.
It is modification of VDRL in which cardiolipin Ag is absorb
on finely divided carbon particles, which are suspended in
choline chloride which blocks inhibitory factor in serum,
thus eliminating the need of heating serum sample before
testing.
The Ag gets clump and flocculated with serum containing
Reagin antibody.
Report:
No clump- Non reactive
Clump - Reactive with titer (eg: Reactive, titer1:8)
Thank You…….
