Standard Operating Procedure: Syphilis Serology (VDRL / RPR)

1. Purpose and Scope

To perform nontreponemal serologic screening for syphilis (VDRL and RPR) and to quantitate reactive titers for monitoring disease activity and treatment response, with a protocol for confirmation by treponemal tests.

2. Specimen Requirements

• Type: Serum (plain tube or SST); fresh, non-hemolyzed.
• Volume: Minimum 1.0 mL.
• Interference: Lipemia and hemolysis interfere with flocculation reading; grossly contaminated sera cause false reactions.
• Stability: Serum stable 3-5 days at 2-8°C; freeze if testing is delayed beyond the validated window.
• Timing: RPR uses warm serum; do not heat inactivate because heat destroys some antibodies and destabilizes the reagent.

3. Safety and Precautions

• Standard PPE (gloves, lab coat). Serum and syphilis reagents are potentially infectious.
• Dispose of pipettes and slides as biohazard; do not mouth-pipette.
• RPR/VDRL reagents contain cardiolipin and are alcohol-based; keep away from open flames.

4. Equipment and Reagents

• VDRL flocculation slide or RPR card kit with RPR antigen (carbonized cardiolipin-lectinin-cholesterol).
• Rotation platform (rotator) at the specified speed and serum dilution pipettes.
• Syringe or dropper delivering the correct antigen drop size.
• Positive (weakly reactive) and negative controls provided with the kit.

5. Step-by-Step Procedure

1. Bring serum and RPR card/antigen to room temperature; run weakly reactive positive and negative controls before patient samples.
2. Place the required amount of serum (or dilution) on the card ring.
3. Add one drop of antigen, mix with the applicator, and rotate at the specified speed for the validated time.
4. Read under the card lamp for clumping: record as nonreactive, weak reactive, or reactive.
5. Titrate reactive sera by twofold dilution (1:2, 1:4, 1:8 ... 1:64 and higher) and report the endpoint titer.
6. For VDRL, use the flocculation slide method with fresh serum; titrate reactive samples similarly.
7. Consult the algorithm for treponemal confirmation (TP-PA/FTA-ABS/EIA) on any reactive result before reporting a positive diagnosis.

6. Quality Control

• The weakly reactive control must give a weak positive pattern in the expected low-titer range; the negative control must remain nonreactive.
• Check antigen uniformity (a flocculent, slightly clumped preparation is discarded).
• Participate in EQA for syphilis serology; validate batch of cards and rotator timing.

7. Decision limits

The interpretive thresholds below are clinical decision limits (there is no canonical reference range for serologic reagents).

• Reactive screen: any reactive (or weak reactive) nontreponemal result must be confirmed with a treponemal test (TP-PA, FTA-ABS, or EIA/CMIA) — a reactive screen alone is never reported as syphilis.
• Biological false positives (BFP): nnon-reactive treponemal test with a reactive nontreponemal screen occurs in pregnancy, autoimmune disease, intravenous drug use, infectious mononucleosis, malaria, and advanced age.
• Titer monitoring: a change of fourfold (two dilutions) between serial titers is considered clinically significant; a falling titer indicates adequate treatment response, a rising titer suggests progression or reinfection.
• Prozone effect: very high antibody titers (secondary syphilis) can produce a false-negative screen; dilute the serum and repeat when clinical suspicion is high.
• Primary syphilis: the nontreponemal screen may be nonreactive in early primary disease because antibody has not yet appeared.