TPHA: principle, procedure, results and interpretations

Treponema pallidum Hemagglutination Assay (TPHA) is a treponemal test for the serologic diagnosis of syphilis, a sexually transmitted infection caused by a 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 1960’s.  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-quantative detection of Anti-treponemal antibodies.

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 in room temperature (25 – 30º C).  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 

Results Test Cells Control Cells
Strongly Reactive Full cell pattern covering the bottom of the well. No agglutination tight button
Weakly Reactive Cell pattern covers approx. 1/3 of well bottom No agglutination tight button
Indeterminate (Equivocal) Cell pattern shows a distinctly open centre No agglutination tight button
Nonreactive Cells 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:

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 molecule. Some studies has 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.