So for me, I'm doing the serology testing in the micro laboratory such as Venereal Disease Research Laboratory (VDRL) test and Treponema Pallidum Haemagglutination (TPHA) test which are syphilis tests that test for the presence of the antibody that is produced in the presence of Treponema Pallidum. Although both of the tests are syphilis test, VDRL is non-specific as the antibody produced with Treponema Pallidum infection that it tests for can also be found when the patient is not suffering for syphilis but other diseases such as hepatitis A and malaria. TPHA is used as a confirmation tests (unless the doctor ordered the test) for syphilis. TPHA uses cells sensitized with Treponema Pallidum specific antigen, hence any agglutination observed would mean a positive that patient is tested positive for syphilis.
Steps involved for VDRL:
1. A drop of serum is taken and spread evenly on the test card.
2. A drop of reagin is added to the serum. (Remove the first few drops to remove the bubbles trapped to prevent uneven volume of reagin added)
3. Test card is placed on an electronic rotator for 8 minutes with approximately two rotations per minute.
4. Test result is observed and recorded.
If agglutination or reaction is observed, proceed to serial dilution to determine the titre:
1. 50µl of saline is placed on 4 different test areas on the test card.
2. 50µl of serum is added to the first test area labelled 1:2. Mix well by pipetting up and down.
3. 50µl of serum+saline mixture is transfered from the first test area to the next labelled 1:4. Mix well by pipetting up and down. Continue till the fourth well and discard the remaining 50µl of mixture.
4. A drop of reagin is added to the different test areas.
5. Test card is placed on an electronic rotator for 8 minutes with approximately two rotations per minute.
6. Test result is observed and recorded.
The titre is taken as the lowest dilution with reaction (agglutination). Once the titre is obtained. TPHA will be performed to confirm the result if the patient is doing a package (a series of test). If the patient is not doing a package, the confirmation will only be done if the doctor order for the test.
Since VDRL is testing for the non-specific antibody for Treponema Pallidum, it is not specific and cannot be used clinically to determine whether patient is suffering form syphilis without a confirmation test.
Hui Juan
TG01
0702012F
Hi Hui Juan ^^ Jess here
ReplyDeleteJust wanna double check with u, so as long as there is agglutination formed (for both VDRL and TPHA), we can conclude that the patient has Syphilis? I mean is it reliable enough just by naked-eye observation? ehehhehe
And may I know what is the purpose of determining the titre? lolz
yay yay!
Vo Thu Hong Anh [Jess]
0705364H
Hello!!
ReplyDeleteActually, the test procedures that we always can get online requires us to observe the agglutination under the microscope. But in our laboratory, we determine that VDRL is negative when there is a tail-like structure form. The reagin contains microparticulate carbon which will settle on the card after standing for 1 minute. So when the test card is rotated manually, a tail-like structure will be form.
Similarly, after standing for 1 minute, if an agglutination occurs, it will be very obvious as the microparticulate carbon will agglutinate instead of forming the tail-like structure. Yes, as TPHA is a test which test for antibody specific to Treponema Pallidum, if TPHA is positive, we can conclude that patient has Syphilis. XD
Oh, and the purpose of determining the titre is to give the doctor an idea of how serious the patient is, because the higher the titre, the stronger the infection. VDRL can also be used as a monitoring test for the treatment of Syphilis, which in many case, the neat reaction will be reactive. Hence, the titre is required to check whether the treatment is appropriate for the patient. By the way, once TPHA is positive, it will be positive for life.
Hope my explanation is clear!!XD
Hui Juan
0702012F
wow! thanks a lot for the explanation. I'm much clearer about the tests now ^^
ReplyDeletebtw, is it possible for u to put up some pix of the test results? [showing tail-like structure and agglutination] ehhe, coz I just wanna see how they'are different from each other, lolz
Vo Thu Hong Anh [Jess]
0705364h
haha. i will try to get a camera and get it down. my manager told us to show him the pictures 1st. XD
ReplyDeleteHui Juan
0702012F
Hey Hui Juan!
ReplyDeleteHow's SIP?
You mentioned that as long as VDRL and TPHA gives an agglutination, it's positive yea? So even a 1+ grade agglutination is positive?
If so~ is there any chances that a patient sample could be diagnosed wrongly, like due to false positive/negative results?
Is there a control utilised in this test to ensure that the results you get later when you run the patient sample are accurate and reliable?
Sherman (TG02)
Group 10
Hi Sherman!!
ReplyDeleteHaha, SIP is fun!! Yah, for VDRL, agglutination observed is considered positive even if it is a 1+ agglutination. As long as there is agglutination, we will run the test with diluted serum to get the titre. For TPHA, the test is done on a microtitre plate and the reaction is observed by the appearance of the sediments at the bottom of the plate.
Since we are observing the VDRL reaction manually, and testing for non-Treponema Pallidum specific antibodies present, there is a high chance for false positive result. False negative result is hard to get as the 'tail-like' structure is only possible to be observed in VDRL negative serum. TPHA is done for all the VDRL reactive cases after the titre is determined, and act as a confirmation test for Syphilis disease. If TPHA is negative for the patient with positive VDRL, this either indicate that it is a false negative test or the patient is suffering from some other diseases such as infectious mononucleosis, hapatitis A or malaria which induces the production of the non-Treponema Pallidum specific antibodies.
The positive and negative controls are run every day before the patients' serum are run to ensure that the reagin is working normally. Once the positive and negative controls are run, the results obtained by running VDRL with the same reagin is considered accurate and reliable.
i will try to upload some pictures for better illustration of what i mention in the content. Hope i answered your questions! XD
Hui Juan
0702012F