Sunday, 8 July 2007


Hi all,
I am posted to a private clinical lab for SIP. Of which, my first 3 weeks is spent at the serology department before moving on to other departments of the laboratories.

Serology is basically a branch of immunology that deals with testing of patient's serum.
During this 2 weeks, I was mainly assigned to do the VDRL testing and Human ASOT because the other tests would require the usage of the LIS, which attachment students are not allowed to access.

This is the summary of the VDRL testing.

Principle of the test

RPR measures IgG and IgM produced in response to lipoidal material released from damaged host cells and also the lipoprotein released from Treponema pallidum. Thus, the antibodies detected are not specific for T. pallidum, which is the causative agent for syphilis.

Qualitative test
1. Using a Pasteur straw, place 100 µl of test serum into a circle of the test card.
2. Use the flatten tip of the straw to spread the serum evenly over the circle area.
3. Shake a plastic dropper containing the carbon antigen provided in the test kit to evenly mix the carbon.
4. Invert the bottle, holding it vertically to dispense a drop of the antigen. Each drop is approximately 0.4 µl.
5. Place the test card on an automatic rotator and rotate at 100 rpm (rounds per minute) for 8 minutes.
6. Read and interpret the results.
Semi-quantitative test
This test is performed if the result for the qualitative test is positive. This is to determine the antibody titre to aid the doctor in the treatment of the disease because the antibodies tend to disappear after successful treatment.To confirm the diagnosis, TPHA (T. Pallidum Haemagglutination) is performed.

The steps are the same as the qualitative test but instead, dilutions of the patient's serum of up to 1:32 is made using saline solution.

This is the Summary of TPHA testing.

Principle of the test

Gelatin particles, sensitised by purified T. Pallidum, agglutinate in the presence of antibodies against T.pallidum in human serum. A purple colored button would be developed after 1 hour of incubation at room temperature. This is compared with the positive control for interpretation of the results.

1. Label the test plate with the last 3 digits of the patient's ID and "C" for the positive control.

2.Prepare serial dilution of the patient specimen as follows:

Well no Diluent Specimen Test cells

1 190 10 75 for all wells
2 100 100 (carried over)
3 100 100 (carried over)
4 100 100 (carried over)
5 100 100 (carried over)
6 100 100 (carried over)

Final dilution
well 1 1 : 20
well 2 1 : 40
well 3 1 : 80
well 4 1 : 160
well 5 1 : 320
well 6 1 : 640

3. Mix the content by gently tapping the tray and incubate for 1hour at room temperature.

This is the summary of the Human ASOT

This test is basically antigen- antibody reaction of latex particles coated with stabilised streptolysin O as antigen against anti-streptolysin O (ASO) antibodies of patient's serum. This antibody is produced in response to group A Streptococci bacteial infections such as rheumatic fever or glomerulonephritis.

The method is basically the same as RPR. Instead of using a white background card, a black or dark background card is used. Instead of carbon antigen in the RPR, latex particles are used (caracterised as a milky colored solution). Instead of rotating for 8 mins in the RPR, 2 mins of rotation is needed or else it would give a false positive result.

I have also learnt how to use the Bio-Rad coda for EBV, HSV-1, HSV-2 and chlamydia IgG/IgM testing and was allowed to attend the training for the usage of their newly purchased equipment, the Bio-rad Evolis, which has an extensive list of tests it is able to perform.

The senior I was attached to also taught me how use the Serodia-Myco II test kit to test for anti-mycoplasma pneumoniae antibodies although I have yet to perform the test.


Yeng Ting


first6weeks said...

wads the purpose of adding carbon antigen?its specific for the bacteria to be detected in VDRL testing?
and why the duration of rotation for RPR and Human ASOT is different?
June, TG02

ALsubs said...

Hi Yeng Ting...

just wondering... what is the purpose of the serial dilution of the paitent's serum in TPHA testing?


Anonymous said...

To June:

1.The antigen is made of carbon particles coated with lipid antigens specific for the bacteria detection. The purpose of using carbon is to be able to visualise the agglutination easily on a white background card. If a sample is lipemic, the test can still done but if there is a positive, the sample, like any other positive samples, undergo the confirmatory test : TPHA.

2. Firstly, the difference in rotation time is because the protocol says so. :p Secondly, the the protocol says that for Human ASOT, if rotated for more than 2 minutes, it would give a false positive result due to the drying effect. The drying effect does not affect the RPR method unless the serum is totally dried.

Hope I'd answered your question.
Yeng Ting

Debra said...

Hey, what compound produces the purple color in the TPHA test? Any significance or is it just because it happens to be stained that colour?

- Debra, TG02

J.A.M.M.Y.S said...


I was wondering how does the purple coloration develop? some form of enzymatic reaction or sumtin else? thanx!!

shahirah, TG01

Anonymous said...

To cass:

The serial dilution is to obtain the antibody titre. =)

Hope that helped.

Yeng Ting

ALsubs said...

Hi Yeng Ting, I am going to be posted to the very same serology lab soon! So I better clear the slightest doubts I have aha. Just wondering how do you read and interpret the results as? And do you go about submitting the results?

Thanks :)


Anonymous said...

To sasi,

For RPR testing,agglutination=positive. Do antibody titre using NSS solution. 100 microliteres of serum to 100 microliters of NSS and carry over 100 microliters to make a 2 times, 4 times, 8 times, 16 times and 32 times dilution. If strong agglutination is seen for 32 times, report as more than 1:32. (use a marker and write the titre on a blank spot of the bar code and place the tube on the rack labelled for TPHA only).

For human ASOT,
agglutination=positive. INform the seniors and they will key in the results into the LIS.

For TPHA, compare the results with the positive control just as I have written on my second post and the reporting is what I have written for the first post about the respective dilutions and tell the seniors to key in the results. They would have to verify if your interpretation is the same as theirs. If there are any discrebancies, they would refer to the HOD.

Alternatively, if you have any doubts, the SOPs are always there and the seniors will always be there to help you.

Hope that this helps. Happy working with the serology department. They rox! =)

Yeng Ting

ALsubs said...
This comment has been removed by the author.
ALsubs said...

hi yeng ting..

Just want to find out why do you mean by this??

Well no Diluent Specimen Test cells

1 190 10 75 for all wells
2 100 100 (carried over)
3 100 100 (carried over)

Final dilution
well 1 1 : 20
well 2 1 : 40

Can you explain to me ?? I dun really quite understand.