**Your Laboratory Name**

**Country xxxxx**

**Phone (xx) xxx-xxxx FAX (xxx) xxx-xxxx**

+----------------+-----------------------------------------------------+
| ## Procedure   | ### Rapid Plasma Reagin Test                        |
+----------------+-----------------------------------------------------+

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  **Prepared By**         **Date Adopted**       **Supersedes Procedure
                                                 \#**

                                                 
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  **Review Date**         **Revision Date**      **Signature**

                                                 

                                                 

                                                 

                                                 

                                                 

                                                 

                                                 

                                                 
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  **Distributed To**                             **Number of Copies**

                                                 

                                                 

                                                 

                                                 
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# Principle: The RPR card antigen suspension is a cardio-particle cardiolipin antigen which detests "reagin", an antibody-like substance present in sera from syphilitic persons, and occasionally in serums of persons with other acute or chronic conditions.

When a specimen contains antibody, flocculation occurs with coagulation
of the carbon particles of the RPR Card antigen, which appear as black
clumps against the white background of the plastic-coated card. This
coagglutination can be read macroscopically. By contrast, non-reactive
specimens appear to have an even light gray color.

> The RPR Card test serves as a non-treponemal screening test for
> syphilis, and serves as a means of monitoring the course of treatment,
> relapse or reinfection.

# Scope / Field of Application: The RPR is a presumptive screening test for Syphilis, which is a venereal disease, caused by spirochete Treponema pallidum. 

# Definitions and Acronyms:

-   RPR (Rapid Plasma Reagin)

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-   VDRL (Venereal Disease Research Laboratory)

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-   MLS (Medical Laboratory Scientist)

-   N/A (does not apply)

-   SOP (Standard Operating Procedure)

-   RPM (Revolutions per minute)

-   TPHA (Trponema pallidum haemagglutination test)

-   QC (Quality Control)

# Responsibility: Only Medical Trained personnel under the Supervision of the MLS

# Specimen: Serum and plasma

## Type:

## Use unheated serum collected in a 16 X 100 vacutainer tube with no anticoagulants and allow to clot. Collect a minimum of 3 to a maximum of 8 ml. of whole blood.

-   Do not use hemolyzed, contaminated or lipemic sera.

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-   Samples rejected due to hemolysis or otherwise unsatisfactory
    conditions must be posted on the Laboratory requisition form and
    returned to the provider.

## Handling Conditions: 

## Specimens must be refrigerated at 2-8°C.

-   Separate serum sample from blood by centrifugation and store at
    2-8°C .

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-   Samples stored at 4°C for up to 72 hours or -20°C are satisfactory.

> Handle all specimens as potentially infectious. Follow all special
> precautions according to Universal Precautions.

# Patient Prep: No special patient preparation is required.

# Equipment and Materials Required:

## Equipment: 

-   50 υl eppendorf and yellow tips

-   rotator 100 rpm and humidifying cover

-   high intensity lamp

-   centrifuge

-   refrigerator

-   time clock

-   test tube rack

## Materials:

-   RPR card antigen suspension\*

-   RPR test cards\*

-   Dispensing bottle\*

-   Dispensing needle-20 gauge, without bevel\*

-   Dispenstirs-0.05/drop\*

-   Controls\*

-   Internal positive and negative controls

-   10% bleach

## Preparation:

-   10% bleach; Use household bleach and dilute 1:10

> \*Supplied by manufacturer.

## Performance Parameters: N/A

# Storage Requirements:

## Container: N/A

## Temperature: 

-   The carbon antigen is stored between 2-8°C in the original glass
    bottle. **Do not freeze.**

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-   The test cards must be stored at room temperature.

## Stability: Do not use reagents beyond manufacturers expiration date. 

## Labeling: Once a bottle is opened date and initial it.

# Calibration:

## Standard Prep: (need info. On how internal standards are prepared)

## List of Standards Used:

-   Controls provided by manufacturer

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-   Internal laboratory controls

## Directions for Preparation: N/A

## Special Instructions: Be sure all reagents are at room temperature before testing.

## Degree of Accuracy: Results of reactive control must be reactive and the negative must be negative.

## Storage Requirements:

### ***Container:***

-   Aliquot internal controls into cryogenic vials.

### ***Temperature:***

-   Manufacturer's controls must be stored at 4°C.

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-   Internal Controls must be aliquoted and stored at -20°C.

### ***Stability:*** 

### The controls must not be used beyond their expiration date.

### ***Labeling:*** 

### The internal control must have the date prepared,date expired and the initials of the person who prepared.

## Calibration Procedure: (information required)

# Quality Control:

## Materials:

-   Rotator

-   Antigen suspension

-   Antigen dispensing needle

-   Controls

-   Thermometer

## Instructions: 

-   The testing area temperature must be 23°-29°C

-   Rotator: To check rotator speed, hold a pen next to the rotating
    platform and count the number of taps per minute. Recommended speed
    is 95-110 rpm. If not functioning properly notify the Supervisor.

-   Allow antigen suspension and controls to come to room temperature
    before proceeding.

-   Check the accuracy of the antigen dispensing needle. Count the
    number of drops delivered from 0.5 ml. of antigen suspension drawn
    up in a 2 ml. pipette. Needles should deliver 30 drops ± 1 drops per
    0.5ml.

-   Include all he quality control data on a quality control sheet in
    the RPR QC notebook. Include lot numbers and expiration dates. All
    reagents must be dated and initialed by the analyst when opened.

-   Do not mix reagents from different lots.

## Frequency: 

## The quality control checks must be performed daily.

-   Test the needle for antigen delivery:

> -daily
>
> -each time a new needle is used
>
> -if control patterns cannot be reproduced
>
> -when a drop of antigen does not drop clearly from the tip

## Acceptable Limits: 

## The Reactive Control must show characteristic strong clumping. The Non-reactive control must show the smoothe grayish appearance of unclumped particles. 

## Corrective Action:

-   If the established reactivity pattern is not obtained, delay routine
    testing until the optimal reactivity is obtained by rechecking
    antigen suspension, test procedures, room temperature, and equipment
    (including use of rotator with humidifying cover).

# Procedure:

## Accessioning: (need info)

## Sample Preparation: (need info)

## Centrifugation: (need info)

## Criteria for rejection of samples:

-   Samples that are hemolyzed, lipemic or quantity not sufficient.

## Detailed Stepwise Procedure:

## Prepare a daily in-house worksheet.

1.  Remove reagents and samples from the refrigerator and allow to
    attain room temperature (23° -29°C).

2.  Antigen preparation: When opening a new ampule , vigorously shake
    for 10-15 seconds to re-suspend antigen, siphon into antigen
    dispensing bottle. To resuspend from previously filed dispensing
    bottle, shake bottle for 10-15 seconds vigorously. Attach dispensing
    needle to bottle.

3.  Label with specimen number an appropriate number of 10 well cards
    for qualitative test.

4.  Place 0.05ml (50υl) of unheated serum on the 18 mm circle of the
    test card, using a dispenstir (hold vertically, do not touch card
    surface).

5.  Spread serum with inverted dispenstir (pipstir) closed end to fill
    the entire circle. Repeat for each specimen to be tested.

6.  Gently re-suspend antigen by inverting dispensing bottle several
    times prior to each use. Hold the bottle in a vertical position and
    dispense approximately 5 drops into the dispensing bottle cap, then
    place one free falling drop of antigen onto each test area of the
    card.

7.  Place the card on the rotator and cover with the humidifier cover.

8.  Rotate 8 minutes at 100 rpm

9.  Read macroscopically, under a high intensity light. A brief rotating
    and tilting of the card (3-4 circular motions) must be done to aid
    in differentiating non-reactive from minimal -- moderate reactive
    results.

10. Report results as follows:

> Small-large clumps Reactive R
>
> No clumping,slight roughness Non-reactive NR

12\. Remove all reactive samples for TPHA confirmation testing.

## Glassware: N/A

## Measurements from instruments: N/A

# Safety: 

# Follow universal precautions, as all samples are considered potentially infectious. Gloves and a face shield must be worn when performing this test.

# Calculations: N/A

## Instructions: N/A

## Equations: N/A

## Computer Validation Protocol: N/A

## Examples: N/A

# Reporting Results:

## Reference intervals: Non-reactive to Reactive

## Range Values: N/A

## Demographic Values: N/A

## Confidence Limits: N/A

## Procedure for Abnormal Results: N/A

## Reporting format: Non-reactive or reactive

# Documentation: All test results are recorded in the laboratory log book. All QC is recorded in the RPR Serology QC book.

# Procedural Notes:

## Reason for Special Precaution: 

> Risk of exposure to HIV, Hepatitis etc.

## Possible sources of errors:

-   Antigen has expired

-   Needle not delivering the proper drop

-   Temperature too high or too low on the room

-   Reagents are not at room temperature

-   Rotator is not at correct rpm

## Helpful hints:

-   Be sure the samples in the rack match the order of the worksheet

-   Have another technologist check your rack order and results after
    you post them to the final laboratory report

## Clinical situations that may influence test:

-   Biological false positive reactions have been reported in disease
    such as infectious mononucleosis, leprosy, malaria, lupus
    erythematosus, vacinia, and viral pneumonia. In pregnancy, several
    reports indicated the occurrence of false positive reactions.
    Narcotic addiction and autoimmune diseases also may give false
    positive reactions. Penta, yaws, bejel, and other related trponemal
    diseases produce positive reactions in this test but should not be
    considered false.

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-   The presence of even low concentrations of positively charged
    chemicals such as quaternary ammonium compounds may produce false
    positive reactions.

## Clinical applications:

-   It is a presumptive screening test for syphilis a non-reactive is
    indicative of the absence of disease but a reactive must be
    confirmed by TPHA.

## Turn around time:

-   Completed within 24 hours of receipt.

# Limitations of Procedure

## Reportable range: N/A

## Interfering Substances: Refer to section 16.4

# Reference Procedures: N/A

# References:

## Manufacturer Product Literature: Biotec package insert

## Textbooks: [American Society for Microbiology]{.underline}

## Standard Publications: Manual of Tests for Syphilis , PHS #411

## Written Personal Communications: N/A

## Research: N/A

# Revision History: N/A

# Appendix

## Manufacturer Product Instructions: Biotec

## Flow Diagrams: N/A

## Card Indexing: N/A

#