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 1960s. TPHA is a good primary screening
test for syphilis at all stages beyond the early primary stage.

Principle:
TPHA test is a passive hemagglutination assay based on
hemagglutination of erythrocytes sensitized with T. pallidum antigen by
antibodies found in the patients 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)

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

A:Dilution of serum sample

1. Add 10L of patients serum in the first well (say well A).
2. Add 190 L 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 75L of control cells to well B and 75 L of test cells to well C.
2. Add 25L 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

Full cell pattern covering the No agglutination

Strongly Reactive bottom of the well. tight button

Cell pattern covers approx. 1/3 of No agglutination

Weakly Reactive well bottom tight button

Indeterminate Cell pattern shows a distinctly No agglutination

(Equivocal) open centre tight button

Cells settled to a compact bottom, No agglutination

Nonreactive typically with a small clear center. 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 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.