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.
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)
- Test Cell suspensions: Preserved RBCs treated with tannic acid and coated with T. pallidum antigen.
- Control cell suspension: Preserved RBCs (without immobilized T. pallidum antigen)
- Buffer: Phosphate buffered saline solution containing adsorbers (used to remove possible cross-reacting heterophile antibodies).
- 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.
- 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
- Add 10μL of patient’s serum in the first well (say well A).
- Add 190 μL of diluent (provided by the manufacture).
- 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
- Add 75μL of “control cells” to well B and 75 μL of “test cells” to well C.
- Add 25μL of diluted serum on each B and C well.
- Shake the plate gently to mix the contents thoroughly.
- Cover the plate and protect to direct sunlight, heat and any source of vibration.
- Incubate 45-60 minutes at room temperature.
- 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.
- 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.
- 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.
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).
- 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.
- Microhemagglutination Assay for Treponema pallidum (MHA-TP): It is another confirmatory test to detect treponemal antibodies. This test is used much less commonly now.