TPHA and FTA-ABS

Mr. Parag Chauhan

Introduction

The TPHA and FTA-ABS tests are confirmatory tests for syphilis after a non-treponemal test (such as VDRL) shows a positive result. These tests detect antibodies specific to Treponema pallidum bacteria. Thus, they are highly specific and can differentiate syphilis from other infections or diseases that may give false-positive results in non-treponemal tests.

  • TPHA (Treponema Pallidum Hemagglutination Assay): The TPHA test detects antibodies produced against Treponema pallidum using sensitized red blood cells (RBCs) that agglutinate when they bind to these antibodies.
  • FTA-ABS (Fluorescent Treponemal Antibody Absorption Test): The FTA-ABS test uses a fluorescent-labeled anti-human antibody to detect specific treponemal antibodies in the patient’s serum.

Both tests are used for confirmatory testing in cases of suspected syphilis. They can help diagnose latent and tertiary syphilis, where non-treponemal tests (like VDRL) may not be reliable.

 

Principle

  1. TPHA Test:

    • The TPHA test is based on the principle of hemagglutination. It uses red blood cells (RBCs) coated with Treponema pallidum antigen. Agglutination occurs when the patient’s serum, containing antibodies against Treponema pallidum, is added to the RBCs.
    • Positive reaction: The antibodies present in the patient’s serum will bind to the treponemal antigens on the RBCs, causing visible agglutination.
    • Negative reaction: No agglutination occurs if no antibodies against Treponema pallidum are present.

  2. FTA-ABS Test:

    • The FTA-ABS test is based on the principle of immunofluorescence. The patient’s serum is incubated with T. pallidum antigen fixed to a microscope slide.
    • If treponemal antibodies are present in the serum, they will bind to the antigen. The slide is then treated with a fluorescent-labeled anti-human antibody, which binds to the antibody-antigen complex.
    • Positive reaction: The complex fluoresces under a UV microscope.
    • Negative reaction: No fluorescence indicates the presence of treponemal antibodies.

 

Requirements

  1. Sample:

    • Serum: The TPHA and FTA-ABS tests are performed on serum samples. Blood should be collected in a sterile tube, allowed to clot, and then centrifuged to separate the serum.
    • Cerebrospinal Fluid (CSF): In cases of suspected neurosyphilis, CSF may be used for FTA-ABS testing.

  2. Reagents & materials

    • TPHA Reagent: This includes red blood cells (RBCs) sensitized with Treponema pallidum antigens.
    • Fluorescent-labeled Anti-human Globulin: For the FTA-ABS test to bind to the antigen-antibody complexes.
    • T. pallidum Antigen: The antigen used in the FTA-ABS test is derived from Treponema pallidum bacteria.
    • Positive and Negative Controls: Used to validate test results.
    • Microscope: For examining the FTA-ABS test (fluorescent microscope).
    • Buffer Solutions: For dilution and washing of samples in both tests.

 

Procedure

  1. TPHA Test Procedure

  1. Prepare the Sample:
    • Collect serum from the patient after blood collection.
    • If necessary, dilute the serum with saline or buffer.
  2. Add Serum to Reagent:
    • Place a small drop (e.g., 50 µL) of patient serum onto a well or glass slide.
    • Add a drop of the TPHA reagent (RBCs coated with T. pallidum antigen) to the serum.
  3. Mix:
    • Gently mix the serum and reagent.
  4. Incubation:
    • Incubate the slide or well at room temperature for about 30 minutes.
  5. Examine:
    • After incubation, observe for agglutination (clumping of RBCs). Visible agglutination indicates a positive result.
    • If there is no agglutination, the result is negative.

  1. FTA-ABS Test Procedure

  1. Prepare the Sample:
    • Obtain serum from the patient. If the sample is not immediately processed, store it at 4°C.
  2. Coat the Slide:
    • Place a few drops of T. pallidum antigen on a glass slide and allow it to air dry. This ensures that the antigen binds to the slide surface.
  3. Add Patient’s Serum:
    • Add a small amount (e.g., 50 µL) of patient serum to the slide with the antigen.
  4. Incubate:
    • Incubate the slide at 37°C for 30 minutes to allow antibodies in the serum to bind to the antigen.
  5. Washing:
    • After incubation, wash the slide thoroughly with phosphate-buffered saline (PBS) to remove unbound proteins.
  6. Add Fluorescent Antibody:
    • Add fluorescent-labeled anti-human globulin to the slide. This antibody will bind to any human (treponemal) antibodies already bound to the T. pallidum antigen.
  7. Examine:
    • After incubation, examine the slide under a fluorescent microscope. If treponemal antibodies are present, the antigen-antibody complex will fluoresce.
    • Positive result: The fluorescence will indicate the presence of treponemal antibodies.
    • Negative result: No fluorescence indicates the absence of treponemal antibodies.

 

Results

  1. TPHA Test Results:

    • Positive: Agglutination (clumping) of the RBCs coated with T. pallidum antigens.
    • Negative: No agglutination.
    • Titer: The test can be performed in serial dilutions to determine the titer of antibodies, which reflects the severity of the infection.

  2. FTA-ABS Test Results:

    • Positive: Fluorescence under a UV microscope indicates the presence of treponemal antibodies specific to T. pallidum.
    • Negative: No fluorescence indicates the presence of specific antibodies.

 

Clinical Significance

  1. Diagnosis of Syphilis:
    • Confirmatory test for syphilis after a positive result in a non-treponemal test like VDRL.
    • The TPHA and FTA-ABS tests detect antibodies specific to Treponema pallidum, making them highly specific for diagnosing syphilis.
  2. Detection of Latent Syphilis:
    • Both tests are useful for diagnosing latent syphilis, where non-treponemal tests like VDRL may be negative or show low titers.
  3. Monitoring Treatment:
    • After syphilis treatment, FTA-ABS and TPHA antibodies typically remain positive for life, even if the patient is successfully treated. Therefore, these tests are useless for monitoring treatment success or serological cure.
  4. Neurosyphilis Diagnosis:
    • FTA-ABS can be performed on cerebrospinal fluid (CSF) to detect neurosyphilis (a complication of syphilis affecting the nervous system).
  5. False Positive Results:
    • The TPHA and FTA-ABS tests are more specific than non-treponemal tests. However, they can still show false-positive results in conditions like lupus, autoimmune diseases, or other treponemal infections (e.g., yaws, pinta).
    • Confirmatory results should always be interpreted in the context of clinical symptoms and history.

 

Limitations

  1. False Positives:
    • Other treponemal infections (such as yaws or pinta) may cause false-positive results.
    • Autoimmune disorders or other chronic diseases may also cause false-positive reactions.
  2. Persistence of Antibodies:
    • Both tests may remain positive for years or even a lifetime after treating the infection, especially for TPHA.
  3. Sensitivity and Specificity:
    • TPHA and FTA-ABS have high specificity for syphilis, but sensitivity may be lower in the primary stage of syphilis or early latent syphilis.

 

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