VDRL & TPHA

Done By: Janice Yeh
Department: Serology
Section: VDRL and TPHA
*Pictures: Taken with the permission from the Head of Department. No patient information or ID is revealed.


Abbreviations:
VDRL- Venereal Disease Research Laboratory (Venereal Disease refers to diseases that are transmitted by sexual intercourse)
RPR - Rapid Plasma Reagin
TPHA - Treponoma Pallidum Haemagglutination



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3 Parts to test for Syphilis:
1) RPR: a non-specific treponemal test: Screening test
2) RPR titer: to find out how infectious the disease is or to monitor progress of treatment in syphilis positive patients
3) TPHA: a specific treponemal test: Confirmatory test


The VDRL test is a screening test for syphilis. It measures antibodies, that can be produced by Treponema pallidum, the bacteria that causes syphilis.
The test is similar to the newer rapid plasma reagin (RPR) test. RPR is a screening test and if a positive result is obtained, a further confirmatory test is conducted, which is TPHA.


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(1) RAPID PLASMA REAGIN TEST


RPR is known as a non-treponemal test. It detects for antibodies in the patient that are against substances that a released from cells that are damaged by the bacterium. Unlike RPR, TPHA is more specific as it detects antibodies against the actual bacterium. Hence, we cannot based on the results of RPR alone as there could be many false positive reasons.


Procedure: Samples are collected from trays as such or from the Sample Receival (SR) department. Samples are spun down in a centrifuge to obtain seperation between the serum and the RBCs.

Procedure: Use a dropper to obtain a small amount of serum (~10microlitres) as shown in the picture. Ensure that only serum is obtained.
Purpose:
Why obtain serum only:
(1)Our main aim is to detect for antibodies present in the cell. Antibodies are only present in serum.
(2)Obtaining the serum only is important. Because if it is mixed with the blood to do the test, it will be harder to spot for agglutination (will be explained).

Procedure: An immuno RPR test card is used. The serum is dropped with the circle and smeared to fill the circle. The circle is a gauge of how much serum is needed. Then a drop of reagent is added. The reagent contains carbon particles coated with a mixture of lipid materials which will bind to the antibodies present in the patient's serum.
Purpose:
Why must we ensure that there is enough serum:
(1) As the samples will not be read immediately, we need to ensure we do not take to little serum as they may dry up after the next step, which is the mixing part.

Procedure: The card is then placed on a automated shaker for 8 minutes. The card should avoid being shifted around or touched.
Purpose:

Why should we avoid disturbing the card:

(1) As the samples are mixing, movement may cause cross contamination of samples as the circles are very close to each other.

Procedure: After 8 minutes, the card is taken off the shaker and swirled alittle to see clearly for agglutination.
Results: Figure 1 - Positive with grey agglutination
Figure 2 - Negative with a button of non-aggregated carbon particles in the centre of
the circle http://www.bmb.leeds.ac.uk/mbiology/ug/ugteach/icu8/images/std/vdrl.jpg
on the left: negative
on the right: positive
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(2) TITER MEASUREMENT


Titer is a measure of concentration, that employs serial dilution of the serum. Titer corresponds to the highest dilution factor that still yields a positive reading. The purpose of measuring the titer of the sample is find out how serious is disease or for doctors to monitor the progress of the treatment. Procedures: Aliquot 50ul of patient serum onto the first circle with a pipette and 50ul of saline onto circles 2 to 6. Then serial dilution is conducted by transferring 50ul of the sample from the tube to the 2nd, mix well and transfer 50ul of the mixed sample to the next circle. The first circle is purely 50ul of sample without having to mix with saline. Discard the last 50ul of mixture from the 6th circle. Then place one drop of reagent (same one used in RPR) onto the circle. Place the card on the automated shaker for 8 mins before results are read.
Results: The last circle that contains agglutination is the titer of the sample. The titer from the first to the last circle is 1:1, 1:2, 1:4, 1:8, 1:16, 1:32.


Example, if there is agglutination present from the 1st circle to the 4th circle, it means that the sample has a titer of 1:8.


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(3)TREPONOMA PALLIDUM HAEMAGGLUTINATION

TPHA screening kit uses preserved avian erythrocytes coated with antigens of T.pallidum which will bind with specific antibody present in the patient's serum or plasma. The cells are suspened in a medium containing components to eliminate non-specific reactions such as reactions caused by other bacterium.

Procedures:
1st Roll: is always for the controls. Takes up 4 wells.
Sample Roll: takes up 6 wells.

1) WELL #1: for both Control and Patient Sample (PT): 190ul of diluent + 10ul of serum
2) WELL #2: No diluent + 25ul of diluted sample from well 1
3) WELL #3-4 (for control) : 25ul of diluent
WELL #3-6 (patient sample): 25ul of diluent
4) Transfer 25ul of mixed sample from well #1 to well #3. Mix well and transfer to the next well. 5) Discard the last 25ul of mixed sample.

Procedures:
1) Add one drop of cells to the wells:

(a) Add Control Cells (Unsensitized) to Well#2
(b) Add Test cells (Sensitised) to Well #3-4 (for control), and Well #3-6 (Patient Sample)
2) Mix the microplate well by gently tapping on the corners of the plate. This is to ensure that sample or reagents along the side of the well will sink to the bottom of the well and be well mixed.

3) Incubate the well in room temperature for 45 minutes before reading the the results.

Results: After 45 minutes, check the results. The results in the RED CIRCLE are positive/reactive. It means that patient is syphilis positive. It is denoted by large rins with peripheral agglutination. Negative results are denoted by concentrated buttons at the bottom of the wells.

That's all, I hope it's clear enough! :D