Cat. No. S168-0
Sterling Diagnostics, Inc.
For In Vitro Diagnostic Use
Sterling Heights, MI 48312 U.S.A.
RPR CARD TEST
INTENDED USE
The Sterling RPR Card Test for Syphilis is a qualitative and semiquantitative non-treponemal flocculation test for the determination of reagin
antibodies in human serum. These materials are intended to be acquired and
used only by health professionals for in vitro diagnostic use.
Prior to testing, centrifuge samples at a force sufficient to sediment
cellular elements.
Samples that are to be sent out for RPR testing should be placed on ice
packs and treated like any other biohazardous material capable of
transmitting infection.
SUMMARY AND PRICIPLE
MATERIALS PROVIDED
Treponema pallidum, the etiological agent of syphilis, induces the
production of at least two types of antibodies in human infection, antitreponemal antibodies that can be detected by FTA-ABS1 antigen, and antitreponemal antibodies (reagin) that can be detected by RPR antigen 2. The
RPR test is a macroscopic non-treponemal flocculation test to be used for the
detection of reagin. The micro-particulate carbon RPR antigen enhances the
visual discrimination between reactive and non-reactive results.
REAGENTS
1.
2.
3.
4.
RPR Carbon Antigen
RPR Reactive Control
RPR Weak Reactive Control
RPR Non-Reactive Control
Store all reagents at 2-8°C in an upright position. Do not freeze. After
use, immediately return to refrigeration. All reagents and controls are stable
until their indicated expiration date when properly stored.
All reagents contain 0.1% Sodium Azide as a preservative and are ready
to use as supplied. Gently mix the reagents before use and avoid foaming.
Vigorously agitate the RPR Antigen Reagent for 10-15 seconds before each
use to ensure homogeneity.
RPR Carbon Antigen, RPR Reactive Control, RPR Weak Reactive Control,
RPR Non-Reactive Control, 3 ml Dropping Bottle, 20 gauge Dispensing
Needle (60 drops/ml), 10-Well RPR Test Cards, 0.05 ml Disposable Stirrer
Pipets
ADDITIONAL MATERIALS REQUIRED
Pipet capable of dispensing 50 µl (0.05 ml), Saline (0.85-0.9% Sodium
Chloride Solution), Mechanical Rotator set at 100 ± 2 RPM with Humidity
Cover
PROCEDURE
All reagents and serum samples must attain room temperature (2025°C) prior to use.
QUALITATIVE CARD TEST
1.
INDICATORS OF DETERIORATION
1. Turbidity or precipitation in serum controls is indicative of deterioration
and the component should not be used.
2. Bacterial contamination of reagents or specimens may cause false
positive agglutination.
3. Sterling Diagnostics cannot guarantee the stability or performance of
reagents, which have been transferred from their original containers,
stored improperly or contaminated during use.
2.
3.
4.
Precautions:
Reagents containing sodium azide may combine with copper and lead
plumbing to form highly explosive metal azides. Dispose of reagents by
flushing with large amounts of water to prevent azide buildup.
All materials of human source have been tested by FDA cleared assays
for hepatitis B surface antigen (HbsAg) and antibodies to HIV and found to
be non-reactive. However, all materials of human derivation should still be
considered a potential biohazard capable of transmitting infection and
handled accordingly.
5.
6.
Using the stirrer pipette, dispense one drop (0.05 ml) of serum or plasma
onto a separate circle on the test card. Use a fresh stirrer pipette for
each sample. When using the stirrer pipette, keep it in a vertical
position to ensure accurate delivery. Repeat using the reactive, weak
reactive and non-reactive control sera. Note the location of each sample
by the number located above and to the left of each circle.
Using the flat end of the stirrer pipette spread the sample over the entire
area of the test circle.
Attach the needle to the dropping bottle. Mix the RPR Carbon Antigen
suspension well. Squeeze the dropping bottle and draw a sufficient
volume of the antigen suspension into the bottle. Dispense several
drops into the dispensing bottle cap to make sure the needle passage is
clear.
Dispense one (1) drop of the RPR Carbon Antigen suspension onto each
sample while holding the bottle in a vertical position. DO NOT MIX
the sample and the antigen. Aspirate the pre-dropped antigen from the
bottle cap.
Place the card on an automatic rotator and cover to maintain humidity.
Rotate at 100 ± 2 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 non-reactive from minimally reactive results.
Read results macroscopically in the "wet" state under a high intensity
incandescent light source.
RESULTS
SPECIMEN COLLECTION AND PREPARATION
The test may be performed on heated or unheated serum samples free
from bacterial contamination and hemolysis, or plasma samples collected in
EDTA anticoagulant. Specimens can be drawn by venipuncture or
convenient finger stick method.
Keep samples stored at 2-8°C in the original tubes and test them within
48 hours. Samples stored longer than 48 hours should not be used in the
assay because false reactive results may occur.
A reactive result is indicated by the presence of large aggregates in the
center or periphery of the test circle.
Minimally reactive results are indicated by the presence of small or fine
aggregates.
A non-reactive result will give a smooth grey appearance within the
circle.
SEMI-QUANTITATIVE CARD TEST
LIMITATIONS
1. Using the stirrer pipette, dispense one drop (0.05 ml) of serum or plasma
sample onto circle 1 on the test card.
2. Using a stirrer pipette (or other accurate volumetric pipette capable of
delivering 0.05 ml), dispense 1 drop of saline onto the circles to be
numbered 2 to 5. DO NOT SPREAD.
3. Using an accurate volumetric pipette, dispense 0.05 ml of the test sample
onto circle 2. Insert the tip of the pipette into the resulting mixture and
mix them by carefully drawing the sample up and down the pipette 5 or
6 times. Avoid any bubble formation.
4. Transfer 0,05 ml of the mixture in circle 2 to circle 3 and mix as above.
Repeat this serial dilution procedure to circle 5 and discard 0.05 ml
from this last circle. Circles 1 to 5 now represent a dilution series as
follows:
Circle:
Dilution:
1
1:1
2
1:2
3
1:4
4
1:8
5
1:16
5. Using the flat end of the stirrer pipette spread the diluted samples over the
entire area of the test circles, starting at circle 5 (highest dilution).
Repeat this spreading procedure in circles 4, 3, 2, and 1.
6. Dispense 1 drop of RPR Carbon Antigen suspension from the dropping
bottle onto each circle. DO NOT MIX.
7. Place the card onto the automatic rotator and rotate for 8 minutes. Read
results immediately,
RESULTS
1. Biological false positive reactions occur occasionally with the carbon
antigen. Such reactions sometimes occur in samples from individuals
with a history of drug abuse, or with diseases such as Lupus
Erythematosus, Mononucleosis, Leprosy, Viral Pneumonia, and after
Smallpox vaccinations.
2. Reactive RPR test samples should be substantiated using a confirmation
test 3.
3. Reagents and controls should not be used after their indicated date of
expiration. Reagents should not be frozen.
4. Contaminated, lipemic, or grossly hemolyzed sera should not be used
because of the possibility of nonspecific reactions.
5. Temperature of the reagents is crucial to test outcome.
6. Reaction times longer than specified might cause false positive results
due to a drying effect.
7. In accordance with diagnostic methods, a final diagnosis should not be
made on the result of a single test, but should be based on a correlation
of test results with other clinical findings.
PERFORMANCE CHARACTERISTICS
The Sterling RPR Card Test was evaluated for equivalence in its
reactivity pattern against the CDC Reference RPR Card Antigen Suspension.
A total of 1209 samples were tested by RPR Card Test in comparison
with the Hynson, Westcott abd Dunning (HWD) product. The following
results were obtained. Among the 9 samples found to be non-reactive by the
RPR Card Test, 7 were also confirmed negative by the FTA-ABS test. There
was a 99.2% overall agreement between the two products.
The highest dilution in which visible agglutination occurs is the end
point. The titer is the reciprocal of this dilution.
A reactive result is indicated by the presence of large aggregates in the
center or periphery of the test circle.
Minimally reactive results are indicated by the presence of small or fine
aggregates.
A non-reactive result will give a smooth grey appearance within the
circle.
HWD
RPR Test
Reactive
Non-Reactive
In another evaluation, a total of 332 clinical specimens were tested by
RPR Card Test in parallel with the VDRL Slide Test. There was an overall
agreement of 99.4%.
Titers Greater Than 1:16
1. Prepare a 1:50 dilution of non-reactive serum in saline. This is to be used
for making 1:32 and higher dilutions of samples to be titered. Dispense
0.05 ml of this solution onto circles labeled 2 through 5.
2. Prepare a 1:16 dilution of test sample by adding 0.1 ml of serum to 1.5 ml
of saline. Mix thoroughly. Dispense 0.05 ml of this diluted sample onto
circles 1 and 2.
3. Mix the solution on circle 2 by drawing the solution up and down 5 or 6
times into the tip of a volumetric pipette. Avoid any bubble
formation.
4. Transfer 0.05 ml of the mixture to the next circle (3) and mix as above.
Continue the serial dilution through circle 5 and discard 0.05 ml from
the last circle after mixing. Circles 1 to 5 represent a dilution series as
follows:
Circle:
Dilution:
1
1:16
2
1:32
3
1:64
4
1:128
5
1:256
5. Proceed with the test as described in Steps 5-7 under the Qualitative Card
Test procedure.
6. Continue with additional dilutions as required until an endpoint is
reached.
QUALITY CONTROL
1. Reactive and non-reactive controls should be included in each run of test
samples.
2. If control samples do not elicit the expected response, the assay should be
considered invalid and the assay should be repeated.
3. If the repeat assay does not elicit the expected results for the control
samples, discontinue use of the kit and contact Sterling Diagnostics
Technical Services Department at 1-800-637-2661.
RPR Card Test
Reactive
Non-Reactive
462
9
1
737
VDRL SLIDE
TEST
Reactive
Non-Reactive
RPR Card Test
Reactive
Non-Reactive
10
0
2
320
The two specimens, which were shown to be positive also by the RPR
Card Test, also gave positive results when tested by the TPHA (Treponema
Pallidum Hemagglutination) confirmation test.
BIBLIOGRAPHY
1. Hunter, E.F. et al, An Improved FTA Test for Syphilis, The Absorption
Procedure (FTA-ABS). Public Health Reports, 79, 410-412, 1964.
2. Larsen, S.A. et al, Clin. Microbiol., 17, 341-345, 1983.
3. Manual of Tests for Syphilis, Public Health Service Publication No. 411,
1969.
4. Fischer, G.S. et al, ibid, 27, 188-189, 1989.
Lit. No. S168 (Rev 5/31/94)