Laboratory Procedure Manual
Analyte: Syphilis
Matrix: Serum
Method: Rapid Plasma Reagin Circle Card
Test
Method No.:
Revised:
as performed by: Division of HIV, STD, and TB Laboratory Research
National Center for Infectious Diseases
Contact: Dr. Vicki Pope
Important Information for Users
NCHSTP, CDC periodically refines these laboratory methods. It is the responsibility of the
user to contact the person listed on the title page of each write-up before using the analytical
method to find out whether any changes have been made and what revisions, if any, have
been incorporated.
Rapid Plasma Reagin in Serum
NHANES 2003–2004
Public Release Data Set Information
This document details the Lab Protocol for NHANES 2003–2004 data.
A tabular list of the released analytes follows:
Lab
Number
Analyte SAS Label
l36_c LBDSY3 Syphilis
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1. SUMMARY OF TEST PRINCIPLE AND CLINICAL RELEVANCE
The rapid plasma reagin (RPR) 18-mm circle card test is a macroscopic, nontreponemal flocculation card
test used to screen for syphilis (1–4). The antigen is prepared from a modified Venereal Disease Research
Laboratory (VDRL) antigen suspension containing choline chloride to eliminate the need to heat-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 released from damaged host cells as well as to lipoprotein-like material, and
possibly cardiolipin released from the treponemes (5, 6). The anti-lipoidal antibodies are antibodies that are
produced not only as a consequence of syphilis and other treponemal diseases, but also in response to
nontreponemal diseases of an acute and chronic nature in which tissue damage occurs (7). 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. The test can be purchased in kit form or in component
parts from many commercial sources. Without some other evidence for the diagnosis of syphilis, a reactive
nontreponemal test does not confirm T. pallidum infection.
2. SAFETY PRECAUTIONS
The risk of infection due to an occupational exposure to blood depends upon the prevalence of blood-borne
pathogens in the population supplying the blood specimens, the probability of infection given a particular
type of exposure to a blood-borne pathogen, and the frequency of exposures (9, 10).
T. pallidum is present in circulating blood during primary and secondary syphilis. The minimum number
(LD50) of T. pallidum organisms needed to infect by subcutaneous injection is 23 (11). The concentration of
T. pallidum in patients' blood during early syphilis, however, has not been determined. The ability of blood
inoculated with T. pallidum to infect animals is reduced by refrigerated storage (12, 13). Although multiple
instances of transmission of T. pallidum due to transfusion of an infected donor's blood were reported prior
to the introduction of penicillin for treatment of syphilis and of refrigeration for blood storage (12).
Subsequent reports have been rare (12, 13). Infection of a health care or laboratory worker following
exposure to T. pallidum-infected blood has, apparently, not been reported.
Authoritative sources focus attention on infection with hepatitis B virus (HBV), hepatitis C virus (HCV), and
human immunodeficiency virus (HIV) as the principal concerns associated with exposure to blood (10, 15–
18). The prevalence of these infections varies greatly among patient populations tested for T. pallidum
infection. HBV infection is most common. HBV viremia is indicated by tests for HBV surface antigen
(HBsAg) in serum. Prevalence of anti-HBsAg, from published studies of patients in hospitals and emergency
rooms cited in a recent review, ranged from 0.9 to 6% (9, 19–22). Unlike initial HBV infection, in which only
a minority of individuals continues to be viremic, initial HCV and HIV infections lead to persistent viremia in
most individuals. Consequently, serum antibody to HCV and HIV are indicators of potential infectiousness.
Seroprevalences of antibody to HCV in studies of patients in hospitals and emergency rooms cited in a
recent review ranged from 2 to 18% (18, 21-24). HIV prevalence ranged from 0.1 to 5.6% in patients
enrolled in a national hospital surveillance system (9, 25). All three infections are more common among
patients at increased risk for syphilis, especially patients with a history of illegal drug use. For example,
seroprevalences of antibody to HCV were 10% among non-drug-using attendees at sexually transmitted
diseases clinics and 60% among injection-drug users (26–28).
Infections with HBV (27, 29) and HIV (17, 30–32) can occur with skin and mucus membrane exposures to
blood; however, needle-stick and percutaneous injury with blood-coated sharp objects are the principal
sources of laboratory-associated acquisition of these agents. The risk of infection following exposure to
blood from an infected patient is greatest for HBV, except for exposed individuals who are immune due to
prior HBV infection or vaccination. The risk is highest if the source individual is HBSAG-positive (27, 33–35)
and is positive for envelope (E) antigen. A vaccine to prevent HBV infection has been available since 1982
and is strongly recommended for health care workers with potential exposures to blood or other body fluids
(33, 36, 37). Individuals with anti-HBV antibody from vaccination or prior infection are considered to be
immune to HBV infection.
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The risk of HCV infection due to needle stick exposure to blood from an individual with antibody to HCV was
10% in one study (27, 38, 39), but HCV does not appear to survive long in serum held at room temperature
(27, 40). A vaccine is not yet available to immunize against HCV infection. Repeated infection with HCV
seems to be possible in spite of detectable serum anti-HCV antibody, although the significance of
reinfection is unknown (26, 41, 42).
The risk of infection with HIV after a single needle-stick exposure to blood from a patient known to be
infected with HIV is approximately 0.3% (9). The risks following mucous membrane or skin exposures to
HIV-infected blood average approximately 0.1% and <0.1%, respectively (17, 30, 32, 43). The lower rate of
transmission for HIV than for HBV or HCV probably reflects a lower concentration of HIV in the blood of
infected persons. A vaccine is not available to immunize against HIV infection. The frequency and
significance of repeated exposures of individuals with prior anti-HIV antibody is unknown.
3. COMPUTERIZATION; DATA SYSTEM MANAGEMENT
A. Each shipment of specimens received from the NHANES III mobile unit contains a corresponding
transmittal sheet and an ASCII data file (KOUTPUT.TXT) on a 3 ½ “ high density floppy diskette. The
data file, containing the specimen ID, collection date, and type of sample (i.e., whole blood, serum,
plasma) is checked against the information on the transmittal sheet and specimen label prior to the
assay.
B. After the data is calculated and the final values are approved by the reviewing supervisor for release,
all results are entered onto the NHANES diskettes by using the program provided by National Center
for Health Statistics (NCHS).
C. After the results are entered on diskettes, back up copies are made and stored in locked areas.
D. The original diskettes containing analytical results are mailed to NCHS.
4. SPECIMEN COLLECTION, STORAGE, AND HANDLING PROCEDURES; CRITERIA FOR SPECIMEN
REJECTION
A. No special instruction such as special diet or fasting is necessary.
B. Fresh serum samples are the specimens of choice for the RPR. Serum specimens may be collected
using regular red-top or serum separator Vacutainers. Specimens are allowed to clot at room temp and
centrifuged.
C. Transfer serum to 2-mL polypropylene screw-capped vials. Freeze at <–20°C.
D. Each week, batches of frozen serum samples are placed in a Styrofoam-insulated shipping container
with dry ice and sent to the laboratory by an overnight courier.
E. Serum specimens are stable up to 72 hours at 4–8°C. For longer periods, store the serum at <–20°C in
glass or plastic vials, as long as the vials are tightly sealed to prevent desiccation of the sample.
F. Excessively hemolyzed, contaminated, or lipemic sera may give aberrant results and should not be
used. A specimen is too hemolyzed for testing when printed material cannot be read through it. Heat-
inactivated sera may be used (56°C for 30 minutes). Excessive inactivation time or temperature may
increase nonspecific background activity which could result in equivocal results.
G. The optimal amount of serum is 0.5 to 1.0 mL. Specimen volumes of less than 0.4 mL are not
acceptable.
H. Avoid repeated freeze-thawing cycles, which may compromise specimen integrity.
Specimens should generally arrive frozen.
Residual samples are frozen at <–20°C.
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5. PROCEDURES FOR MICROSCOPIC EXAMINATIONS; CRITERIA FOR REJECTION OF
INADEQUATELY PREPARED SLIDES
Not applicable for this procedure
6. EQUIPMENT AND INSTRUMENTATION, MATERIALS, REAGENT PREPARATION, CALIBRATORS
(STANDARDS), AND CONTROLS
A. Instrumentation
(1) Micropipettes to deliver 50 μL.
(2) Mechanical rotator, fixed-speed or adjustable to 100 ±2 rpm, circumscribing a circle 3/4-inch in
diameter in a horizontal plane.
(3) High-intensity incandescent lamp.
B. Other Materials
(1) Disposable, calibrated 20-gauge needle without bevel, silicone treated, to deliver 17 μL per drop.
(2) Plastic antigen dispensing bottle, I dram.
(3) Plastic-coated RPR cards, with 10 circles, each approximately 18 mm in diameter. Store cards at
room temperature.
(4) Dispensters, a disposable (plastic) dispensing-stirring device that delivers 50 μL.
(5) Humidifying cover.
(6) 1-mL and 5-mL serologic pipettes
(7) Test-tube to hold 2-mL and 5-mL volumes
(8) Latex gloves, safety glasses, and protective clothing.
(9) Discard container and disinfectant.
C. Reagent Preparation
Each Serodia TP-PA kit contains enough reagents to test 92 samples and the controls. Reagents
should be mixed gently to avoid possible deterioration of the antigen-carrier complex. Reagents are
stable until the expiration date printed on the label. All reagents should be stored at 4–8°C.
(1) RPR antigen suspension.
Stabilized combination of 0.003% cardiolipin, 0.020-0.022% lecithin, 0.09% cholesterol, 10%
choline chloride, 0.0125M EDTA, 0.01875% charcoal, 0.01M Na2HP04, 0.01M KH2P04, 0.1%
thimerosal in distilled water (1). The antigen suspension is packaged in ampules. Store unopened
ampules at 2 to 8°C; do not store the antigen in bright sunlight or in temperatures above 29°C; do
not freeze. An unopened ampule of antigen is stable up to the expiration date.
(2) Control serum samples.
Liquid reactive (R), minimally reactive (Rm), and nonreactive (N) control serum specimens of
graded reactivity. If quantitative tests are to be performed, a control serum that can be titered to at
least a 1:4 dilution should be used. Store control cards or serum samples according to the
manufacturer’s directions
(3) 0.9% Saline.
Prepare a 2% solution of saline, by diluting 0.9 grams of sodium chloride in 100 mL of distilled
water.
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(4) Diluent (liquid).
Aqueous solution of 2% normal human serum made by diluting human serum 1:50 in 0.9% NaCl.
Used to dilute serum specimens at dilutions of 1:32 and above.
D. Preparation of Control Serum Samples
(1) Positive Control Serum
Prepared from human serum samples containing antibodies to T. pallidum. Serum is ready to use.
Bring to room temp before use.
(2) Reactive Minimal Control Serum
Prepared from human serum samples containing antibodies to T. pallidum. Serum should be
nonreactive at 1:2 dilution, but reactive at 1:1 dilution. Serum is ready to use. Bring to room temp
before use.
(3) Nonreactive Control Serum
Prepared from human serum samples free of T. pallidum antibodies. Serum is ready to use. Bring
to room temp before use.
7. CALIBRATION AND CALIBRATION VERIFICATION PROCEDURES
A. Working Standards
The reactive control is used to determine level of reactivity of the test for lot to lot comparison and as
an indication of whether reagents are deteriorating. Reactive control should have a titer of 1:8 ± 1
doubling dilution.
B. Pipettors and Tips
With the pipettors currently available, the measurement of small serum volumes is routine. Most
manufacturers include in the specifications of the pipettors the accuracy for frequently used microliter
volumes. Daily use may affect pipettors, making them lose their initial accuracy. The differences in
disposable tips from sources other than the pipette manufacturer are probably the most common error.
For budgetary reasons, a less expensive brand of pipette tips may be substituted for those of the
manufacturer. Although the less expensive brand may be satisfactory, the laboratory should verify the
accuracy of the substitute pipette tips in their system. Commercial kits to check the accuracy are
available. Also, manufacturers provide procedures for checking the accuracy of their equipment.
Historically, the gravimetric or spectrophotometric procedures, which use the weight of water or
absorbance of a substance at a given wavelength, have been the most accepted methods used to
calibrate pipettors. These procedures should not be used instead of those specified by the
manufacturer nor do they substitute for an annual verification and repair by a company qualified to do
this.
C. Needles
(1) Check the calibrated needle each time a new needle is used, when needle has been dropped or
wiped, or when the control pattern is not met to ensure the delivery of the correct volume of
antigen suspension (60 drops ± 2 drops per mL; 17 μL per drop).
(2) Place the needle on a 1-mL syringe or on a 2-mL pipette. Fill the syringe or pipette with RPR
antigen suspension. Holding the syringe or pipette in a vertical position, count the number of drops
delivered in 0.5 mL. The needle is correctly calibrated if 30 drops + 1 drop is delivered in 0.5 mL.
(3) Replace the needle if it does not meet this specification. Be sure to test the calibration of the
replacement needle.
D. Rotator
(1) Speed. For rotators without a digital readout, the speed can be estimated by counting the number
of rotations made per minute. To count the rotations place your finger next to the rotator and count
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the number of times the rotator touches your finger in 15 seconds. If the rotator is properly
adjusted, the count should be 25. The rotator’s speed should be calibrated each day it is used.
(2) Time. The rotator’s timer should be checked against another laboratory timer or stop watch. The
rotator’s timer should be within ± 15 seconds of the set time.
8. PROCEDURE OPERATING INSTRUCTIONS; CALCULATIONS; INTERPRETATION OF RESULTS
A. Preliminaries
(1) Bring all reagents and serum samples to room temp before beginning test.
(2) The reactive control should be titered every day that samples are tested. The reactive minimal and
negative controls should also be run each day testing is done.
(3) 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.
B. Sample Preparation
All samples are initially tested undiluted.
C. Operation of Assay Procedure
Qualitative Test Procedure
(1) Place 50 μL of serum or plasma onto an 18-mm circle of the RPR test card, using a disposable
Dispenstir or a safety pipetting device.
(2) 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.
(3) Gently shake the antigen dispensing bottle to resuspend the particles.
(4) Holding the dispensing bottle and needle in a vertical position, dispense several drops to clear the
needle of air. Then add exactly 1 free-falling drop (17 μL) of antigen suspension to each circle
containing serum or plasma. Do not mix (2, 3).
(5) Place the card on the mechanical rotator under a humidifying cover. Rotate the card for 8 minutes
at 100 ± 2 rpm.
(6) Immediately remove the card from the rotator; briefly rotate and tilt the card by hand (three or four
to-and-fro motions) to aid in differentiating nonreactive from minimally reactive results.
(7) Perform the quantitative test on serum specimens showing any degree of reactivity (clumping) or
“roughness.”
Quantitative Test (3)
(1) Dilute to an endpoint titer all serum specimens with rough nonreactive results in the qualitative
test. Test each specimen undiluted (1:1), and in 1:2, 1:4, 1:8, and 1:16 dilutions (see Fig. 1).
(2) Place 50 μL of 0.9% saline in circles numbered 2 through 5. Do not spread the saline.
(3) Using a safety pipette device, place 50 μL of serum in circle 1 and 50 μL of serum in circle 2 (see
Fig. 1).
(4) Mix the saline and the serum in circle 2 by drawing the mixture up and down in a safety pipette
eight times. Avoid forming bubbles.
(5) Transfer 50 μL from circle 2 (1:2) to circle 3, and mix.
(6) Transfer 50 μL from circle 3 (1:4) to circle 4, and mix.
(7) Transfer 50 μL from circle 4 (1:8) to circle 5 (1:16), mix, and then discard the last 50 μL.
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(8) Using the broad end of a clean Dispenstir, spread the serum dilution to fill the entire surface of
circle 5, the highest dilution (1:16). Using the same Dispenstir, repeat for circle 4(1:8), 3(1:4),
2(1:2), and 1 (undiluted).
(9) Gently shake the dispensing bottle to resuspend the antigen particles.
(10) Holding the antigen dispensing bottle in a vertical position, dispense 1 or 2 drops to clear the
needle of air. Then add exactly 1 free-falling drop (17 μL) of antigen suspension in each circle. DO
NOT MIX.
(11) Place the card on the rotator under the humidifying cover and rotate the card for 8 minutes at 100
± 2 rpm.
(12) Immediately remove the card from the rotator; briefly rotate and tilt the card by hand (three or four
to-and-fro motions) to aid in differentiating nonreactive from minimally reactive results.
Figure 1. Diagram of card for quantitative test
*Begin dilutions in 2% normal human serum
(13) If the highest dilution tested (1:16) is reactive, continue as follows:
(a) Prepare a 1:50 dilution of nonreactive serum in 0.9% saline to be used for making 1:32
and higher dilutions of the specimen to be tested.
(b) Prepare a 1:16 dilution of the test specimen by adding 0.1ml of serum to 1.5ml of 0.9%
saline. Mix thoroughly.
(c) Place 50 μL of the 1:50 nonreactive serum diluent in circles 2 through 5 of an RPR card.
(d) Using a safety pipetting device with disposable tip, place 50 μL of the 1:16 dilution of the
test specimen in circle 1 and 50 μL in circle 2.
(e) Using the same pipette and tip, make serial twofold dilutions. Complete test as
described in steps 4 through 13 (see “Quantitative Test”). Use a clean tip for each
specimen tested. Prepare higher dilutions if necessary in 1:50 nonreactive serum
diluent.
(14) After completing the day’s tests, remove the needle from the antigen dispensing bottle. Rinse
needle in distilled water, and air dry. Do not wipe needle (wiping removes the silicone coating). A
satisfactory needle may be retained as a spare for replacement of an unsatisfactory needle.
(15) Recap the plastic dispensing bottle containing the antigen suspension and refrigerate at 2–8°C.
Do not freeze the antigen. Antigen stored in the dispensing bottle will retain its reactivity for 3
months or until the expiration date, whichever is sooner.
D. Interpretation of results
Qualitative Test
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NHANES 2003–2004
(1) Read the test reactions in the “wet” state under a high-intensity incandescent lamp. Read the test
without magnification.
(2) Report the results as given in Table 1.
Table 1. Reporting qualitative results
Reading Report
Characteristic clumping ranging
from marked and intense (reactive)
to Reactive (R) slight but definitely
(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.
Quantitative Test
(1) Read the test reaction in a “wet” state under a high-intensity incandescent lamp as for the
qualitative test.
(2) Report the results in terms of the highest dilution that has given a reactive result, including a
minimally reactive result, as shown in Table 2.
Table2. Reporting quantitative results
Serum Dilutions
Undiluted (1:1)
1:2 1:4 1:8 1:16
Report
Rm N N N N Reactive, undiluted 1:1, or R 1
R R N N N Reactive, 1:2 dilution, or R 2
R R R N N Reactive, 1:4 dilution, or R 4
R R R Rm N Reactive, 1:8 dilution, or R 8
R, reactive; Rm, minimally reactive; N, nonreactive.
E. Recording of Data. Quality control data: record lot number of kit, date of testing, and titer of reactive
control serum.
(1) The titer of the reactive control should be 1:8 ± 1 doubling dilution.
The nonreactive control should have a uniform appearance with no clumping.
(2) Analytical results
Results should be recorded as reactive or nonreactive. If reactive, should be followed by a number
indicating the titer or the serum.
F. Replacement and Periodic Maintenance of Key Components
All pipettors should be checked, repaired, and recalibrated at least yearly.
G. Calculations
Not applicable to this procedure.
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H. Special Method Notes
(1) 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.
(2) If the time of rotation is too long test reactivity may be increased, or if too short test reactivity may
be decreased.
(3) 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.
(4) If lighting produces a glare on the card, the reactions may be obscured.
(5) If the antigen is outdated or not adequately tested for standard reactivity, the results may be
inaccurate.
(6) If the serum is unevenly spread in the circle, the antigen and antibody may not mix properly.
(7) If hemolyzed, contaminated, or improperly collected serum or plasma specimens are tested, the
reaction may be masked.
(8) 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 giving rise to false reactive
results.
9. REPORTABLE RANGE OF RESULTS
Results are reported as Reactive, Nonreactive, or Inconclusive.
10. QUALITY CONTROL (QC) PROCEDURES
A. Evaluation of RPR kits is the responsibility of the user. Reagents evaluated as described here must
produce results comparable to those obtained with reference reagents. All glassware used must be
free of contamination, and distilled water used as diluent must be pure.
B. Evaluation Procedure
Test 10 individual serum samples of predetermined reactivity on each of 2 days. The recommended
distribution is three reactive serum samples, three minimally reactive serum samples, and four
nonreactive serum samples. If necessary, prepare reactive serum samples of various levels of
reactivity by diluting reactive samples with nonreactive serum samples. These pooled samples may be
substituted for some of the individual serum samples.
C. Testing
The RPR reagents from the new and the reference lots are tested on 2 days by using reactive and
nonreactive control serum samples from the new kit and the reference kit and 10 individual serum
samples.
(1) Assemble the 10 individual serum samples described above in B.
(2) Perform the tests on reactive control, nonreactive control and individual serum specimens. Test all
serum specimens in parallel, using new and reference (old) reagents.
(3) Read and record test results.
(4) Compare the results obtained with reference and new reagents. Determine whether new RPR
reagents meet the criteria of acceptability.
(5) If results between reagent lots are discordant, additional testing may be necessary.
(6) If the new kit gives the established reactivity patterns for known controls other than the
manufacturer supplied controls, further testing can continue.
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D. Daily Control
(1) Temperatures of refrigerators must be recorded daily.
(2) At each routine test run, check expiration date on kit.
(3) Test kit reactivity with control serum specimens of graded reactivity (reactive, minimal reactive,
and nonreactive controls). Use only if results fall within ±1 doubling dilution of the titer of the
reactive control.
11. REMEDIAL ACTION IF CALIBRATION OR QC SYSTEMS FAIL TO MEET ACCEPTABLE CRITERIA
If the titer of the reactive control is more than ±1 doubling dilution pattern of agglutination for the
unsensitized particles is other than, the test must be repeated.
If the controls are still out of compliance when repeated, a new kit should be used.
12. LIMITATIONS OF METHOD; INTERFERING SUBSTANCES AND CONDITIONS
Serum that is excessively lipemic, hemolyzed, or contaminated may interfere with the reaction.
Serum that has been repeatedly frozen and thawed may be falsely negative in the test.
Serum or reagents that have not reached room temperature before performing the test may cause false
negative reactions.
Improperly diluting the serum samples will cause erroneous results. If the sample is diluted too much, it may
be falsely negative. If not diluted enough, a false-positive result may occur.
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, but the qualitative RPR is nonreactive.
13. REFERENCE RANGES (NORMAL VALUES)
Not applicable to this procedure.
14. CRITICAL CALL RESULTS (PANIC VALUES)
Not applicable to this procedure.
15. SPECIMEN STORAGE AND HANDLING DURING TESTING
Specimens must be at room temp (18–25°C) during preparation and testing. Otherwise, store the serum at
<–20°C. If the sample is going to be retested within 24 hours, store at 4–8°C to avoid a freeze-thaw cycle.
16. ALTERNATE METHODS FOR PERFORMING TEST OR STORING SPECIMENS IF TEST SYSTEM FAILS
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17. TEST RESULT REPORTING SYSTEM; PROTOCOL FOR REPORTING CRITICAL CALLS (IF
APPLICABLE)
Not applicable to this procedure.
18. TRANSFER OR REFERRAL OF SPECIMENS; PROCEDURES FOR SPECIMEN ACCOUNTABILITY AND
TRACKING
We recommend that records, including QA/QC dat, be retained for 2 years beyond the duration of the
survey. Only numerical identifiers (e.g., NCHS ID numbers) should be used.
For the NHANES III study, residual samples are stored at <–20°C for 1 year after analysis, then returned to
the NCHS serum repository at Rockville MD.
19. SUMMARY STATISTICS AND QC GRAPHS
Qualitative assays are qualitative assays with a positive, negative or borderline/indeterminate result. The
absorbance or reactivity values of specimens are compared with a cutoff value that is a ratio of the negative
control mean and the positive control mean. Since the controls are read as cutoff values, plots of these
values are not generated for quality control purposes.
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REFERENCES
1. Portnoy J, Brewer JH, Harris A. Rapid plasma reagin card test for syphilis and other treponematoses.
Public Health Rep 1962;77:645-52.
2. Portnoy J. Modifications of the rapid plasma reagin (RPR) card test for syphilis, for use in large scale
testing. Am J Clin Pathol 1963;40:473-9.
3. Portnoy J. A note on the performance of modifications of the rapid plasma reagin (RPR) card test for
syphilis, for use in large scale testing. Public Hlth Lab 1965;23:43.
4. RPR Macro-Vue Card Test - Procedures Manual. Hynson Westcott and Dunning: Baltimore, MD 1977.
5. Matthews HM, Yang TK, Jenkin HM. Unique lipid composition of Treponema pallidum (Nichols virulent
strain). Infect Immun 1979;24:713-9.
6. Belisle JT, Brandt ME et al. Fatty acids of Treponema pallidum and Borrelia burgdorferi lipoproteins. J
Bacteriol 1994;176:2151-7.
7. Catterall, RD. Presidential address to the M.S.S.V.D.: Systemic disease and the biological false-
positive reaction. Br J Vener Dis 1972;48:1-12.
8. Larsen SA, Pettit DE, Perryman MW, Hambie EA, Mullally R, Whittington W. EDTA-treated plasma in
the rapid plasma reagin card test and the toluidine red unheated serum test for serodiagnosis of
syphilis. J Clin Microbiol 1983;17:341-5.
9. Chamberland ME, Ciesielski CA, Howard RJ, Fry DE, Bell DM. Occupational risk of infection with
human immunodeficiency virus. Surgical Clin N Amer 1995;75:1057-70.
10. Short LJ, Bell DM. Risk of occupational infection with blood-borne pathogens in operating and delivery
room settings. Amer J Infect Control 1993;21:343-50.
11. Magneson HJ, Thomas EW, Olansky S, Kaplan BL, DeMello L, Cutler JC. Inoculation syphilis in human
volunteers. Med 1956;35:33-82.
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