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.


  1. Kashyap, B., Goyal, N., Gupta, N., Singh, N. P., & Kumar, V. (2018). Evaluation of Treponema pallidum Hemagglutination Assay among Varying Titers of the Venereal Disease Research Laboratory Test. Indian journal of dermatology, 63(6), 479–483. 
  2. Gupta, K., Bhardwaj, A., Dash, S., & Kaur, I. R. (2018). Role of Treponema pallidum hemagglutination assay for diagnosis of syphilis in low titers of VDRL-reactive sera: A prospective study from a large tertiary care center of East Delhi. Journal of family medicine and primary care, 7(6), 1594–1595.

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.

36 thoughts on “TPHA: Principle, Procedure, Results, Interpretations

  1. hey! can you please explain why the hemagglutinated cells DONT form a button?
    thanks! 😀

    1. Dear Marko thank you for your query. The settling patterns of free RBCs and agglutinated RBCs are different. Free RBCs roll down the sides of the V or U bottom well and settle as a button. Agglutinated RBCS do not roll down like free RBCs to form button but instead are held in the lattice structure so settle as a diffuse film.

    1. Bandy, there is no problem. This test detects antibodies against Treponema pallidum, a bacterium that causes syphilis. Negative result means you are less likely to have syphilis. Please consult with your physician.

  2. Actually my prblm is coming pus from my uretra and it is paining and burning I went to doctor he treated me he took my blood and urine sample and the tpha test is negative so is this any prblm I m asking
    y pus come from uretra?

    1. Dear Bandy TPHA is done for Syphilis, non-reactive results means, you do not have Syphilis, but you might have other infection. Please refer with physician, they will send appropriate test, diagnose the infection and prescribe medicine for the treatment.

  3. Thanx sir I went to doctor now my result of urine was all okay but lecocytes was positive and blood sugar 94 and tpha once I checked it is nehative.I was scared I was with said bladder infection with bacteria but thanks for u r valuable information sir.sorry for asking sir once again there in prblm right

  4. Hi Tankweschar, I work in the lab, currently I am on the serology manual bench. I have always felt confident interpreting results from tests, however I do have one question, how do I determine the difference between a borderline positive and a positive result for a TPHA test. This will maybe just serve as a confirmation of an answer I already know, but thanks in advance.

  5. I have a pregnant patient with TPHA POSITIVE with 1:1280 dilution. is it a significant titre

  6. Good evening:)

    What does it mean if VDRL is negative, TPHA negative but FTA positive( 1/150) ?
    Does that mean syphilis?

    1. Dear Ewine,
      thank you for your query.
      FTA-ABS is a confirmatory test for Syphilis. Both FTA-ABS and THPA are treponemal tests, once positive these test may give positive results throughout life. Getting negative TPHA and positive FTA-ABS happens rarely, but we can not rule out such results. Some research have shown that in people having both VDRL and TPHA negative, very few (less than 5%) may come positive in FTA-ABS. Regarding the status of syphilis, such tests results should be interpreted by the attending physician taking care of both history and physical/clincal examination.

  7. Thanks for a beautiful post.
    Please can you kindly include pictures of the microtiter wells for a positive and negative TPHA test? Visuals are an indispensable learning tool. Thanks

  8. hello sir my tpha positive and i want to ivf treatment but i dont know tpha positive what effected in my ivf treatment pls sir

  9. My cousin first blood test for syphilis is 1.36, in her 2nd test 2.38 and in her 3rd test it is 0.36 all negative. Is it normal?

  10. Dear Sir,
    I checked my blood for VDRL, RPR and TPHA. And the results came back: VDRL-Non-reactive, TPHA-Negative and Treponema(IgG/IgM)- 0.8 (Reference range- <1.1 index). The fact I'm worried about is that although Treponema (IgG/IgM) is lower than the cut off index , 0.8 is closer to 1.1. If I don't have syphilis , my titre should be lower than 0.8 index. Am I right, Sir? I'm confused with the result. Do I need to test again? Please kind give your expert opinion, Sir.
    With respect,

  11. Hello sir,
    Just a small inquiry, is the TPHA test done only on fasting in the morning? I thought it could be done any time.

  12. respected sir i had dan TPHA Test last weak results shows 471.5 please tell what this value mean and what is normal value and what i use medicence

  13. Hello Dear
    Am very happy to get your help
    My wife is pregnancy and TPHA positive and Hemagglution negative and disappointed.

  14. My Tpha value 160. So i take pancline injection 24 lack 4 my value 160 what i treatment

  15. My TPHA result is non reactive gray zone.COV 0.212 TITER 0.191HCV non reactive what is that means.what should I do?thanks

  16. Hi sir.. Recently ive tested for tpha titre after secondary infection, now reading shows vdrl negative and tpha positive titre 1:1280, even i got treated with penincilin 4 doses the tpha titre will go down or remain same? Why the tpha titre always remain same? Even after get treated?

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