TPHA: Principle, Procedure, Results, Interpretations

Treponema pallidum Hemagglutination Assay (TPHA) is a treponemal test for the serologic diagnosis of syphilis, a sexually transmitted infection caused by spirochetes, Treponema pallidum. Based on the principle of passive haemagglutination, this test detects anti-treponemal antibodies (IgG and IgM antibodies) in serum or CSF. TPHA  has been used as a confirmatory test for the diagnosis of Treponema pallidum infection since the mid-1960s. TPHA is a good primary screening test for syphilis at all stages beyond the early primary stage.


TPHA test is a passive hemagglutination assay based on hemagglutination of erythrocytes sensitized with T. pallidum antigen by antibodies found in the patient’s serum or plasma.  It is used for both qualitative and semi-quantitative detection of anti-treponemal antibodies.

TPHA Test Principle
TPHA Test Principle

The test sample is diluted in absorbing diluent to remove possible cross-reacting heterophile antibody and to remove, block, or absorb potentially cross-reacting, nonpathogenic treponemal antibodies. Sera containing antibodies to T. pallidum react with erythrocytes (chicken or avian) sensitized with sonicated T. pallidum, Nichols strain (the antigen), to form a smooth mat of agglutinated cells in the microtiter tray well. If antibodies are not present the cells settle to the bottom of the tray well, forming a compact button of unagglutinated cells.

Reagents (supplied by manufacturers)

  1. Test cell suspensions: Preserved  RBCs treated with tannic acid and coated with T. pallidum antigen.
  2. Control cell suspension: Preserved RBCs (without immobilized T. pallidum antigen)
  3. Buffer: Phosphate buffered saline solution containing adsorbers (used to remove possible cross-reacting heterophile antibodies).
  4. Positive Control serum: Human serum containing antibodies against T. pallidum. Ready for use. This will give an equivalent titer of 1/640 to 1/2560 with the quantitative test.
  5. Negative Control serum:  Human serum free of antibodies against T. pallidum


Before performing the test procedure, bring the sample, diluent, control, and test cells at room temperature (25 – 30ºC). For each qualitative test, a test card with three wells is needed.

A:Dilution of serum sample 

  1. Add 10μL of patient’s serum in the first well (say well A).
  2. Add 190 μL of diluent (provided by the manufacturer).
  3. Mix the content well using a micropipette; we will use this diluted serum later.

B: Testing of serum sample for the presence of specific antibodies 

  1. Add 75μL of “control cells” to well B and 75 μL of “test cells” to well C.
  2. Add 25μL of diluted serum on each B and C well.
  3. Shake the plate gently to mix the contents thoroughly.
  4. Cover the plate and protect to direct sunlight, heat and any source of vibration.
  5. Incubate 45-60 minutes at room temperature.
  6. Read the test results and interpret.

Positive control and negative control should be run along with the test serum (see quality control section below). 

Results and Interpretation 

ResultsTest CellsControl Cells
Strongly ReactiveFull cell pattern covering the bottom of the well.No agglutination tight button
Weakly ReactiveCell pattern covers approx. 1/3 of well bottomNo agglutination tight button
Indeterminate (Equivocal)Cell pattern shows a distinctly open centreNo agglutination tight button
NonreactiveCells settled to a compact bottom, typically with a small clear center.No agglutination tight button

If the controls (positive control and negative control) do not give the expected result, all assays performed in that batch are invalid and must be tested again.

  1. Reactive (R): Reactive results may indicate an active, past, or successfully treated infection. A diagnosis should be made with a careful history of the patient and a physical examination as well as pertinent laboratory results.
  2. Indeterminate: indeterminate results are confirmed with the MHATP and FTA-ABS test tests.

False Positive results: Although TPHA test is highly specific, false-positive results have been known to occur in patients suffering from leprosy, infectious mononucleosis, and connective tissue disorders. For confirmation, FTA-ABS test should be used.

Quality Control

Positive and negative control are included in the test kit  for the quality control. Control should be recommended in the following cases:

  • At least once a run
  • At least once within 24 hours
  • When changing vial of reagent.

If the control is not showing expected results; the test is invalid (whatever be the test results).

Similar Tests

  1. Treponema pallidum Particle Assay (TP-PA) is another treponemal test. It uses gelatin particles as carrier molecules. Some studies have reported that TPPA has higher sensitivity than the TPHA in detecting cases of primary stage syphilis.
  2. Microhemagglutination Assay for Treponema pallidum (MHA-TP): It is another confirmatory test to detect treponemal antibodies.This test is used much less commonly now.
  3. Treponema pallidum immobilization (TPI) test
    This test measures the presence of antibodies against Treponema pallidum in the patient’s serum.
    T. pallidum (Nichols strain) grown in rabbit testes is used as the antigen. If the patient’s serum contains antibodies against treponema, antibody and complement immobilizes the living treponemes. The test results are read by dark-field microscopy. Failure to immobilize the treponema strain suggests the absence of antibody in the patient’s serum, thus not infected with syphilis.
    TPI test is technically difficult, expensive, time-consuming so rarely performed nowadays.

Acharya Tankeshwar

Hello, thank you for visiting my blog. I am Tankeshwar Acharya. Blogging is my passion. As an asst. professor, I am teaching microbiology and immunology to medical and nursing students at PAHS, Nepal. I have been working as a microbiologist at Patan hospital for more than 10 years.

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