Treponema Pallidum Hemagglutination Assay CH.U

TPHA is abbreviation of treponema pallidum hemagglutination assay to 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 is a good primary screening test for syphilis at all stages beyond the early primary stage.

1. Treponema Pallidum Hemagglutination Assay Prepared by: Hawdam Latif Abdulla Charmo University

2. Overviews What is TPHA? Principle Of Treponema Pallidum Hemagglutination Assay Procedure Results and Interpretation

3. What Is TPHA? TPHA is abbreviation of treponema pallidum hemagglutination assay to 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 is a good primary screening test for syphilis at all stages beyond the early primary stage.

4. Principle Of Treponema Pallidum Hemagglutination Assay 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 . 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.

5. Principle Of Treponema Pallidum Hemagglutination Assay 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. Negative Control serum: Human serum free of antibodies against T. pallidum.

6. Procedure 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.

7. Procedure 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.

8. 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 Cell pattern shows a distinctly open center No agglutination tight button Nonreactive Cells settled to a compact bottom, typically with a small clear center. No agglutination tight button

9. Any Questions?