TPHA Test - About, Principle, Procedure & Test results

Treponema pallidum hemagglutination abbreviated TPHA is a qualitative and semiquantitative test used for the detection of specific antibodies to Treponema pallidum in human serum by hemagglutination. TPHA test is used in the diagnosis of syphilis

TPHA Test - About, Principle, Procedure &  Test results

Treponema pallidum hemagglutination Test abbreviated TPHA is a qualitative and semi-quantitative test used for the detection of specific antibodies to Treponema pallidum in human serum by hemagglutination test. TPHA test is used in the diagnosis of syphilis

Principle of Treponema Pallidum Hemagglutination Test

TPHA test is based on Hemagglutination. Stabilized fowl/chicken erythrocytes are sensitized with an antigenic extract of Treponema pallidum (Nichols strain). These sensitized erythrocytes will agglutinate with Treponema pallidum antibodies if present in the patient’s serum. The cells form a characteristic pattern of cells in the bottom of a micro titration plate well. In the absence of antibody they form a compact button in the well. Non-specific reactions are detected by the unsensitized control reagent. Non-pathogenic Treponemal antibodies are absorbed by an extract of Reiter’s treponemes included in the diluent solution.

TPHA Test Performance specifications

  • Cross reactions can occur with other species of Treponema.
    • TPHA test cannot discriminate between antibody due to pallidum infection and antibody due to infection with other pathogenic treponemes, i.e, T. carateum and T. pertenue
    • Also, false positive reactions can occur with samples from patients with infectious mononucleosis, autoimmune diseases, leprosy and drug addiction.
    • Use of samples other than serum or CSF have not been validated in this test.
    • False positive reactions may occasionally occur if plasma is used as specimen.

    Sample Collection

    Serum  is the appropriate sample for TPHA Test.

    • Collect 2 mL of venous blood in a plain red topped vacutainer tube
    • Allow the tube to stand for 30 minutes and separate the serum by centrifugation at 2500–3000 rpm for 5–10
    • Take note not to use lysed or grossly lipemic or contaminated serum.
    • It is advisable to process the specimen on the same day. If analysis is not done on the same day of collection, separate the serum and store it at 2–8 °C for up to 5 days or at –20°C for longer periods.

    Required Reagents/consumables

    Reagents

    Description

    Antigen reagent suspension

    Suspension of sensitized fowl/chicken erythrocytes.

    Control reagent suspension

    Suspension of unsensitized chicken erythrocytes.

    Diluent solution         

    Phosphate buffered saline solution containing soluble components of Reiter Treponemal strain and stabilizing agents.

    Positive control serum         

    Immune rabbit serum. Prediluted to 1:20. See exact titer on the vial label. A variation of titer of ± one doubling dilution is accepted.

    Negative control serum

    Normal rabbit serum. Prediluted to 1:20

     

    Required Equipment for TPHA Test

    • Micro pipettes/cell droppers
    • Microtiter plates are required.

    Procedure for Qualitative TPHA Test

    Bring all the reagents and specimens to room temperature before use.

    • Make sure to thoroughly re-suspend or mix the antigen and control reagents suspensions well before use
    • Dispense diluent into the micro titration plates as follows:

    In well 1

    25 μL (1 drop)

    In well 2

    100 μL (4 drops)

    In well 3

    25 μL (1 drop)

    In well 4

    25 μL (1 drop)

    • Dispense 25 μL (1 drop) of each sample into a well in row 1
    • Using a micro pipette, mix well 1 and transfer 25 μL volume to well 2. Mix and transfer 25 μL to well 3. Mix and discard 25 μL from well 3. Transfer a 25 μL from well 2 to well 4, mix and discard 25 μL from well 4.
    • Add three drops (75 μL) of test cells to well 4 using the attached cell dropper and three drops (75 μL) of control cells to well 3 using the attached cell dropper.
    • This results in final serum dilutions in well 3 and 4 to 1/80
    • Mix the contents of the wells by tapping the sides of the plate or by using a plate shaker for at least 30 seconds.
    • Cover and let stand for 45–60 minutes.
    • Examine for agglutination patterns.

    TPHA Test results interpretation

    Agglutination pattern

    Description

    4+

     

    Smooth mat of agglutinated cells covering the entire bottom of the

    well, sometimes with folded edges

    3+

     

    Smooth mat of agglutinated cells partially covering the bottom of the well.

    2+

    Smooth mat of agglutinated cells surrounded by a ring of cells.

    1+

    Smooth mat of agglutinated cells surrounded by a heavy ring of cells.

    ±

    Button of cells with a small central opening

    Negative

    Button of cells with or without a very small hole in the center.

     

    Positive and negative results

    4+ to 1+

    Considered as positive

    ±              

    Considered as Borderline

    No agglutination

    Considered as a negative test result

    • Dilute the test serum ¼ with control cells (25 μL of test serum + 75 μL of
      control cells) and allow to stand at room temperature for 45–60 minutes.
    • Centrifuge the sample for 5 minutes at 1000 rpm (25 μL of 1:4 diluted
      serum + 100 μL of diluent)
    • After centrifugation dilute the supernatant 1/5 in diluent. Test this dilution directly. Add 25 μL of diluted treated serum to two cells. Add 75 μL of control wells to well 1 and 75 μL of test cells to well 2
    • A positive result indicates the presence of antibodies to T. pallidum, resulting from a past or present infection. All positive test samples should be tested with further dilutions of the sample to get end titer.
    • A negative result indicates the absence of antibodies to T. pallidum
    • A borderline may correspond to a low level of antibodies in early stages of syphilis or to residual antibodies in treated syphilis.

    TPHA test semi-quantitative estimation

    • 25 μL of the diluent is added to 5 wells in a row

    • 25 μL from the second well of the sample showing reactive result is added to 25 μL of diluent in the first well, mixed and transferred to the second well and like-wise proceeded till the fifth well.
    • 25 μL is discarded from the fifth well.
    • 75 μL of the test cells are added to all the five wells of the diluted sample
    • Mix the contents of the wells by tapping the sides of the plate or by using a plate shaker for at least 30 seconds.
    • Cover and let stand for 45-60 minutes
    • Examine for agglutination patterns.
    • The titre of the sample is the reciprocal of the last dilution that shows agglutination.

    TPHA test quality control procedure

    • Make sure to include the positive and negative controls in each series of samples taking into account that the controls are prediluted to 1:20.
    • Add 25 μL of each control directly into wells 3 and 4.
    • Do not add any diluent solution.

    Precautions          

    • Handle all samples as potentially infectious.
    • Handle all reagents with care and avoid contact with eye, mouth and skin
    • Do not perform mouth pipetting.
    • Discard used reagents and sample as per disposal procedure

    Potential sources of variability

    • False positive reactions may occur if plasma is used as specimen
    • Specific antibodies may persist for a long period of time, even after successful treatment of the disease. In order to assess the response to treatment, the use of Reagin test (RPR test) is recommended
    • Any agglutination seen in the control well indicates the presence of non-specific agglutinins.

    References

    Sankara Nethralaya’s, TPHA test, Manual of Medical Laboratory Techniques page 357

    What's Your Reaction?

    like
    0
    dislike
    0
    love
    0
    funny
    0
    angry
    0
    sad
    0
    wow
    0