Sunday, September 20, 2009

Trepanema pallidum Haemagglutination Assay

Hi everyone..

For this week i was again attached to the Microbiology department.
I was introduced to a new test called TPHA (Trepanema pallidum Haemagglutination Assay) .This is a confirmatory test used to confirm the presence of Trepanema pallidum(syphillis) in patient's blood sample.


This test is usually done when VDRL result is positive. Since VDRL is not a confirmatory test, this test therefore is important in determining whether the patient really has syphillis or instead due to other contributing factors and conditions which gave a false positive VDRL result.

A false positive VDRL can be encountered in infectious mononucleosis, lupus, hepatitis A, leprosy, malaria and, occasionally, pregnancy.

Hence TPHA test is a very important test to ensure the condition/disease is diagnosed correctly.

This test uses the Serodia-TPHA test kit. It takes 2 hours incubation after which result can be obtained. This test makes use of the 96-well microtiter plate. However for every individual test, only 4 wells (in vertical order) will be used.



These are the reagents used,available in the kit:

1) Unsensitized particles
2) Sensitized particles
3) Sample diluent


The following are the procedures:

1) Pipette 100 ul of sample diluent into the 1st (top most) well
2) Pipette 25ul each into the 2nd, 3rd and 4th wells
3) Dispense 25 ul of serum into the 1st well and pipette up and down to mix the sample diluent with the serum
4) After mixing the contents of the 1st well, pipette out 25ul of the mixture and serially dilute it into the 2nd, 3rd and 4th wells consecutively.
5) The 4th well therefore should have the lowest concentration of serum
6) Using the droppers provided in the kit, add 1 drop of unsensitized particles (Control) into the 3rd well.
7) Add 1 drop of sensitized particles into the 4th well
8)Gently tap the sides of the plate to mix the contents together.
9) Incubate at room temperature for 2 hours,away from any vibrations.
10) After two hours, read the result from the 4th well and report it.

Only the result from the 4th well should be taken into account.





This is an example of how the result may appear.

This picture only shows the 4th well of 4 separate tests.
The 4th well of Test no. 1 and 2 show positive results. For both cases, samples are positive for presence of Trepanema pallidum.
However for the 4th wells of Test no. 3 and 4, results are negative. Hence this shows that no Trepanema pallidum is present and the previous VDRL result is a false positive.


Final result will be reported as REACTIVE (positive) or NON-REACTIVE (negative).


Since this kit takes up to 2 hours for the result to be out, it is thus used only for routine specimens.

For urgent specimens, since the turnaround time is 1 hour, the lab makes use of a different TPHA kit which only takes up 15 min and also has a much simpler procedure.



Alright, that is all for now. Feel free to comment if you have any questions!

Thanks =D

Siti Hawa
TG02
GRP 10
Posted by at

13 comments:

  1. TG01 Group 1September 27, 2009 at 7:01 PM

    hi there!
    may i know who's this posting the entry?

    and what's VDRL?

    thanks!
    stella

    ReplyDelete
  2. emadtechsSeptember 27, 2009 at 8:20 PM

    Hi

    What is the purpose of the sensitized particles added in the 4th well?

    Liyana
    0703827F

    ReplyDelete
  3. TG01-Group 2September 28, 2009 at 1:24 AM

    You show us an image of 4 wells and state that test 1 and 2 is positive while 3 and 4 is negative. But how exactly do you differentiate between postive and negative result? What do we have to look for/at?

    Alvin

    ReplyDelete
  4. MedTech JrSeptember 28, 2009 at 8:22 AM

    Hi stella..
    oops, if forgot to add in my name..edited already..=)

    Sorry i wasnt very clear in the post..
    VDRL acutally stands for 'Venereal Disease Research Laboratory' Test. Its a common test that is done very frequently in the lab.

    VDRL is a serological screening test to detect the presence of syphillis antigen (trepanema pallidum)in serum.

    But unlike TPHA, its only a presumptive test and hence if VDRL screening result is positive, TPHA must be done as well in order to confirm presence of syphillis in a patient.

    Hope that answers ur qn..thanks!
    =)

    Siti
    tg02

    ReplyDelete
  5. MedTech JrSeptember 28, 2009 at 8:29 AM

    Hi Alvin,

    we have to visually differentiate the size and visibility of a ring that will be formed in the well after 2 hours of incubation.

    In the lab actually there is a chart showing the different sizes of the ring and what should be concluded (positive or negative).

    There are times when we would not be very confident of the final result. For that case, the test should be repeated again as most likely incubation was not done properly (eg. vibration of the plate while incubating).

    Thanks for the qn!
    =)

    Siti
    tg02

    ReplyDelete
  6. MedTech JrSeptember 28, 2009 at 8:35 AM

    Hi Liyana,

    sensitized particles are particles/cells (eg. RBC) that has been sensitized with Trepanema pallidum antigen.

    If it is exposed to a syphillitic serum in the 4th well, T.pallidum-sensitized particles will aggregate with the antibodies present in the patient's serum, forming distinctive patterns in the well - hence positive result.

    Thanks for the qn!

    Siti
    tg02

    ReplyDelete
  7. MichelleOctober 4, 2009 at 9:07 AM

    Hi Siti!
    May I know what's the other TPHA kit that your are using that takes only 15mins and the procedures?

    Thanks,
    Michelle

    ReplyDelete
  8. jenashling-shamunaOctober 14, 2009 at 7:29 AM

    Is this test affected by saliva as well? When I was attached to the serology lab, they made us wear mask everytime we did the test as they said that there are treponemal sp in the saliva that may cause false positives.

    MUNA
    0703791D

    ReplyDelete
  9. MedTech JrNovember 30, 2009 at 6:58 PM

    No in my laboratory we were not required to take those measures. Probably due to the principles of the tests are different hence resulting in different level of sensitivity.

    ReplyDelete
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    ReplyDelete
  11. amoebaOctober 14, 2020 at 7:35 AM

    This comment has been removed by the author.

    ReplyDelete
  12. amoebaOctober 14, 2020 at 7:36 AM

    Treponema pallidum hemagglutination Test abbreviated TPHA is a qualitative 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 read more www.labweeks.com/tpha-test/

    ReplyDelete
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    ReplyDelete

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