SOP-S-003 — Syphilis Rapid Qualitative Treponemal Antibody and RPR Screening Procedure SOP

1.0 Purpose

This procedure covers syphilis screening at Tarangnan Community Hospital using rapid qualitative treponemal antibody immunoassay cassettes and Rapid Plasma Reagin (RPR) nontreponemal card flocculation tests, including quality control and confirmatory referral.

2.0 Scope

This procedure applies to all antenatal, pre-employment, inpatient, and outpatient syphilis screening by Medical Technologists in the Clinical Laboratory. It covers specimen preparation, rapid treponemal lateral-flow testing, RPR card flocculation, prozone effect identification, quantitative titration, and referral pathways to Samar Provincial Hospital. General serological operations are governed by Serology Section Workflow Quality Control and Result Reporting Sop.

3.0 Definitions

• Treponema pallidum: The causative spirochete bacterium responsible for human syphilis.
• Treponemal Test: Diagnostic assay detecting specific antibodies directed against Treponema pallidum cellular proteins (such as rapid immunochromatographic cassette or TP-PA).
• Rapid Plasma Reagin (RPR): Macroscopic carbon flocculation card assay detecting non-specific reagin (anti-cardiolipin) antibodies released during active spirochetal tissue damage.
• Prozone Phenomenon: False-negative or deceptively weak flocculation in neat serum resulting from extreme antibody excess, which resolves upon specimen dilution.
• Flocculation: Rapid, macroscopic clumping of carbon-antigen immune complexes visible on a standardized test card.

4.0 Responsibility

4.1 Hospital Director

• Approves this procedure and ensures access to referral networks for confirmatory testing.

4.2 Pathologist

• Reviews this procedure and advises on syphilis testing algorithms and discordant results.

4.3 Chief Medical Technologist / Laboratory Quality Manager

• Maintains version control, reviews internal QC records and rotator logs, and ensures technologist competency.

4.4 Medical Technologists

• Verify specimen identity and enforce environmental controls (especially ambient room temperature for RPR).
• Run treponemal rapid cassettes and RPR card flocculation strictly following this procedure.
• Identify prozone patterns, perform serial dilutions when indicated, and route reactive specimens for referral.

4.5 Laboratory Aides

• Clean test card rotators, log daily ambient room temperatures, and assist in transporting sealed referral packs.
• Shall not perform immunoassay runs, pipet sera, interpret card flocculation, or authorize laboratory results.

5.0 Policy / Procedure

5.1 Diagnostic Principles and Testing Algorithms

The syphilis diagnostic pathway integrates rapid treponemal antibody screening, RPR card flocculation, serial dilution titration, and confirmatory referral (see Figure 1).

Figure 1 — Syphilis rapid treponemal screening, RPR qualitative card, and serial titration flow

• Initiate syphilis screening per diagnostic testing algorithm, differentiating treponemal and nontreponemal utility: - Treponemal Rapid Cassette: highly sensitive screening marker indicating exposure to Treponema pallidum. Treponemal antibodies typically persist for life even following curative therapy. - RPR Flocculation Card: nontreponemal marker correlating with active disease, spirochetal burden, and response to treatment. RPR titers decline following successful antimicrobial therapy.
• Follow the sequential testing algorithm: all reactive rapid treponemal screening results must be evaluated with an RPR card test to determine titer activity, and confirmed through referral testing when clinically discordant or unconfirmed.

5.2 Specimen Requirements and Environmental Controls

• For specimen collection, draw venous blood into plain red-top or gold-top SST tubes. Centrifuge at 1,000–1,200 × g for 10 minutes to obtain clear serum. Unheated serum is used for both assays.
• Verify specimen quality: avoid gross hemolysis, chyle, or microbial growth. Do not use specimens showing active bacterial turbidity.
• Maintain ambient room temperature strictly between 23 °C and 29 °C (73–85 °F) during RPR card testing: - Temperatures below 23 °C decrease reaction sensitivity, producing false-negative results. - Temperatures above 29 °C accelerate evaporation and cause non-specific agglutination, producing false-positive results. - Log bench temperature before performing any RPR run.

5.3 Method A: Rapid Treponemal Antibody Immunoassay Cassette

• Bring the foil pouch to room temperature (15–30 °C). Open the pouch and place the cassette horizontally on a clean, dry surface.
• Dispense 10 µL serum and buffer onto Treponemal Rapid Cassette; read at 15 to 20 minutes: - Using the supplied micro-dropper or calibrated micropipette, dispense exactly 10 µL (or 1 drop) of serum, plasma, or whole blood into the round sample well (S). - Immediately add 2 to 3 drops (approximately 80–100 µL) of manufacturer assay buffer into the buffer well.
• Start the digital timer immediately. Read the test visually at 15 to 20 minutes. Do not interpret results after 20 minutes.
• Interpret visual findings: - Control line C and test line T reactive: two distinct colored lines appear: one at the control region (C) and one at the test region (T). - Non-Reactive: only one colored line appears at the control region (C). - Invalid: the control line (C) fails to appear. Discard cassette and retest immediately using a fresh device.

5.4 Method B: RPR (Rapid Plasma Reagin) Card Flocculation Procedure

• Prepare required RPR equipment and reagents: - RPR 18 mm card with clear circular testing fields - RPR carbon antigen suspension (refrigerated at 2–8 °C; bring to room temperature before testing) - Calibrated dispensing needle and plastic bottle (verified to deliver 60 drops ± 2 drops per mL; 17 µL per drop) - Mechanical rotator operating at a constant 100 ± 2 rpm with humidifying cover - High-intensity incandescent or halogen reading light
• Dispense 50 µL serum and 1 drop carbon antigen onto 18 mm circle of RPR card: - Dispense exactly 50 µL of unheated serum into an 18 mm circle of the RPR test card using a calibrated pipette. - Spread the serum drop with a clean spreader tip to completely fill the 18 mm circle without scratching the card or crossing the perimeter line. - Invert the antigen dropper bottle gently several times to ensure uniform carbon suspension. - Holding the dropper bottle completely vertical, allow exactly 1 free-falling drop (17 µL) of carbon antigen suspension to fall directly into the center of the serum pool. Do not restir. - Rotate for exactly 8 minutes at 100 rpm under humidified cover: place the card on the mechanical rotator, cover with the humidified cover containing a damp sponge, and rotate for exactly 8 minutes at 100 rpm. - Remove card immediately upon timer completion, gently tilt the card manually by hand 3 to 4 times to facilitate flocculation observation, and read macroscopically under direct high-intensity light.
• RPR card qualitative result reactive or weakly reactive: - Reactive (R): large or medium black carbon aggregates clumped in the center or periphery against a clear background. - Weakly Reactive (W): finely dispersed, small but definite carbon flakes throughout the circle. - Nonreactive (N): smooth, uniform grey suspension with no visible clumps or aggregates.
• Screen for prozone effect in neat serum: - If a neat serum displays a rough, grainy, or atypically non-clumped appearance, or if strong clinical suspicion of secondary syphilis exists, suspect prozone phenomenon. - Prepare a 1:4 and 1:16 dilution of the patient serum in 0.9% sterile saline and retest. If the diluted specimen shows distinct flocculation while neat serum was non-reactive, report as reactive with prozone noted.
• Perform two-fold serial dilutions to determine endpoint titer: - For every reactive or weakly reactive qualitative RPR, perform two-fold serial dilutions to determine endpoint titer: neat (1:1), 1:2, 1:4, 1:8, 1:16, 1:32, etc. - Test each dilution per the standard card procedure. - Report the highest serum dilution producing a clear reactive result (for example, "RPR Reactive, Titer 1:16").

5.5 Quality Control and Test Limitations

• Treponemal rapid cassette QC: the appearance of the procedural C line on each device is mandatory. Retest any device lacking a C line.
• RPR card testing QC: - Test commercial Reactive, Weakly Reactive, and Nonreactive control sera daily with every batch of patient cards. - Verify antigen dispensing needle calibration weekly: aspirate 1.0 mL distilled water into a syringe and verify that the needle dispenses 60 ± 2 free-falling drops per mL. - Do not release patient results if daily controls fail expected reactivity.
• Test limitations and biological false positives: - Biological false-positive RPR reactions occur in viral infections, autoimmune connective tissue disorders, malaria, leprosy, and normal pregnancy. - Treponemal antibodies cannot differentiate between venereal syphilis (T. pallidum) and endemic non-venereal treponematoses (yaws, pinta).

5.6 Result Reporting and Confirmatory Referral Protocol

• Standardized result reporting: - Report Treponemal Cassette as Non-Reactive or "Reactive for Treponema pallidum Antibodies." - Report RPR as Nonreactive, or Report RPR as Reactive with documented endpoint titer (such as "Titer 1:4"). - Result reporting and confirmatory referral complete upon technical validation and dispatch.
• Refer reactive serum to Samar Provincial Hospital for confirmatory TP-PA or FTA-ABS: - Refer reactive serum to Samar Provincial Hospital for confirmatory TP-PA or FTA-ABS: patients with reactive treponemal screening or reactive RPR card findings shall have at least 1.0 mL serum referred to Samar Provincial Hospital (SPH) or the National Reference Laboratory (NRL) for confirmatory Treponema pallidum Particle Agglutination (TP-PA) or Fluorescent Treponemal Antibody Absorption (FTA-ABS) testing. - Ship referred specimens under refrigerated triple-packaging conditions per Referral Laboratory Policy. - Maintain strict confidentiality per Confidentiality Policy. Notify attending obstetrician/clinician immediately for antenatal cases, and submit statutory infectious disease notifications in compliance with RA 11332.

6.0 Records

7.0 References

• DOH Clinical Laboratory QMS Tool, Section 3 — Service Delivery
• DOH Administrative Order No. 2021-0037, Annex B1 — Technical Standards for Clinical Laboratories
• ISO 15189:2022, Section 7.3 — Examination Processes
• WHO Guidelines for the Treatment of Treponema pallidum (Syphilis)

Companion policies:

• Serology Section Workflow Quality Control and Result Reporting Sop — Serology section general workflow, QC, and result reporting
• Referral Laboratory Policy — Specimen referral, packaging, and transmission procedures
• Laboratory Retention Schedule Policy — Master retention schedule for serological records and referral documents