medical mircobiology [serology] (part I)

Hello all~ yanling here.. its my turn to blog this wk~!

I'm going to talk about serology as im in that department for 3 wks (again). This week is my last week. xD

I will talk about one test in a post. So there might be quite a few posts at a go. But at least questions can be directed to each test~! xDDecided to write about some of the manual tests done in serology, because I think they require more skills, especially in pipetting. xD

Venereal Disease Research Laboratory Test

Its is to check if the person has syphilis.

The first test done is to screen for presence of syphilis/ Treponoma pallidum Ab using RPR reagent which consist of carbon particles coated with syphilis Ag. [I had made a mistake here, SO SORRY~!] screen for presence of Ab that are specific to the substance that were released by the cells that are attacked by Treponoma pallidum which causes syphilis. Since its about Ag-Ab, so the principle of this test is agglutination.

So how the screening test is done? First, 50ul of serum is dropped onto a circle of RPR card (will upload picture soon). Then a drop of RPR reagent will be added to each circle then rotate for 8 mins at 100rpm.

Then 8 minutes later, one has to check for agglutination. Here comes the difficult part as for weak positive, the agglutination will not be very obvious so there are chances that one may mistaken with the RPR carbon. So everytime in doubt, got to ask the staff for help.

So for the reactive samples, they will be keep aside for further tests. Then for the non-reactive samples, they will be scan and reported as "non-reactive".

Now I'm going to talk abt the further tests for reactive samples. The next test is venereal disease titre, which to find how strong the syphilis Ab is. There will be 6 circles required, 50ul of saline is pipetted onto second to sixth circles. Then 50ul of reactive serum is added to 1st circle (neat). Then another 50ul of the same serum will be pipette to 2nd circle. Then a 50ul serial dilution is done from 2nd circle to 6th circle. Then the serum is spread to the entire circle from the highest dilution to the neat. Then 1 drop of RPR reagent is added to each circle and rotate for 8 mins at 100 rpm. Then titre of the serum will then be recorded b4 proceeding to the next test. The titre from neat to the 6th circle is as follows: 1:1 1:2 1:4 1:8 1: 16 and 1: 32. And if there are still strong agglutination at the 6th circle it will be recorded as >1:32.

The next test done is Treponoma pallidum Haemagglutination (TPHA), is a more specific test for syphilis as VDRL test can be reactive if there is present of malaria parasite.

The principle for TPHA is haemagglutination, which uses cells with the Ag to check for agglutination whereas for VDRL, it uses particle coated with the Ag for agglutination, and for this case is carbon particle.

The steps are a little complicated, but i will try to explain to the best i can. The test uses 96 u-shaped well. For each sample, 6 wells will be used. So the max number that can be done in a plate is 15 as one row will be used for control. xD

So before adding anything to the plate, the side of each row of 6 wells must be labelled with the last 3 number of the barcode no. or for control, it will be labelled as C. Then 190ul of sample diluent will be added to the 1st well of each sample or control. Next, leaving the 2nd well empty, add 25ul of sample diluent into 3rd to 6th wells. As for control, the sample diluent is added till the 4th well. This because the control will show agglutination from 3rd well onwards, so in order to save the diluent, it is done till the 4th well. After that, 10ul of control/serum is added to the 1st well and mix well. Then take 25ul from the 1st well to then 2nd well and the 3rd well. Then a 25ul serial dilution is done from 3rd well onwards.

After the serial dilution, one drop of unsensitised cells will be added to 2nd wells of each sample and control. And 1 drop of sensitised cells will be added to 3rd wells onwards. The unsensitised cells contains cells without any T. pallidum Ag, so the results will always be negative. Then as for sensitised cells, it contains the T. pallidum Ag, so if there is any presence of T. pallidum Ab in the patient's serum, there will be agglutination. Then the sides of the plate is tapped to ensure the contents are mixed. Its like how we do our ELISA during AIMM i think? Then it will be incubate for minimum of 45 mins at room temp.

So how the TPHA negative and positive results will look like? For negative results, it will appear as a compact button or small ring at the center of the well as the cells will settle. Then for positive results, there will be agglutination and so there will not be any button or small ring in the center of the well. The titre of the test will be then recorded and reported. The titre is from 3rd well onwards, 1:80, 1:160 and 1:320. If there is still no button or small ring, it will report as >1:320.

yaNLing xD