Week 3. 17 more weeks to go.

Hey all. How are you guys doing?


I was attached to a private laboratory, together with Sharon, Lloyd, Maya and Ivan. It's CRAZY, I tell you. Working hours are a bit odd, but I guess we're all getting used to it already. Most of us worked from 1 pm to 9 pm daily. Each of us were posted to different departments but we still get to see a lot of one another as the departments are all located closely together. For the first 2 weeks of SIP, I was attached to the Serology department, which is a semi-automated lab where some of the tests are done manually while others are automated.


Samples are usually received at the lab around 1 pm and I usually have to wait for the specimen reception staffs to do the labelling and sorting out of the samples. My busy hours usually starts around 1.30 pm till break time which is around 5 pm or 6 pm. Samples usually come in hundreds up to thousands.

Most of the tests done in the Serology department are testing for presence of the different types of Sexually-Transmitted diseases. The most common and first test done upon receiving a sample is the Venereal disease (VD) test, which is done manually. However, I won't be talking about VD test as Farhana from another group has already talked about it in week 1. The procedures done in my department and hers are more or less the same. So, I'll explain about another test which I have carried out a few times by myself, but under the supervision of one of my Filipino colleagues.

Test:
Mycoplasma Pneumoniae Antibodies test (MPA)

Principle of test:
Agglutination of sensitised gelatin particles in the presence of Mycoplasma Pneumoniae antibodies in human serum.

Purpose of test:
An in-vitro diagnostic test to detect anti-Mycoplasma Pneumoniae antibodies in human serum. Mycoplasma Pneumoniae is the microorganism responsible for causing Mycoplasma Pneumonia, which is an infection of the lungs.

Materials:

- "U" shaped microwells plate
- 100 µl pipette
- 25 µl pipette
- 2 droppers (one for sensitised particles and the other for unsensitised particles)
- human serum
- Serodia®-Myco II test kit produced by Fujirebio Inc.

(Photo taken in lab. Permission given by supervisor.)

Contents of test kit:

- sample diluent (ready-to-use) - for diluting specimens (human serum) and reconstituting sensitised and unsensitised particles
- sensitised particles (lyophilized form) - gelatin particles coated with antigen specific to mycoplasma pneumoniae antibodies
- unsensitised particles (lyophilized form) - gelatin particles not coated with any antigen
- positive control (ready-to-use) - contains mycoplasma pneumoniae antibody positive rabbit serum diluted with sample diluent with dilution 1:10

Methods:

1. Label the sides of each row of the microplate with either 'C' for control (first row) or the last 3 digit of the patient's barcode number e.g. '319'. Every time we carry out a test, we have to do controls, thus labelling the first row of the microplate 'C'. Each sample takes up 6 wells; 1st well - neat (1:1), 2nd well - 1:20, 3rd well - 1:40, 4th well - 1:80, 5th well - 1:160, 6th well - 1:320. These are dilution factors or better known as titre.

2. Add 100 µl of sample diluent to the 1st wells of every row.

3. Add 25 µl of sample diluent from the 2nd to 6th wells of every row.

4. Add 25µl of positive control into the 1st well of the 1st row ('C'). Pipette up and down to mix the positive control and sample diluent together.

5. Obtain 25µl of solution from the 1st well and add it into the 2nd well and mix again. From the 2nd well, obtain another 25 µl of solution and add it into the 3rd well. Do this serial dilution up till the 6th well. Remember to discard the final 25 µl of solution from the last well to obtain the desired dilution.

6. Repeat steps 4 & 5 for every other patient's serum, but instead of adding positive control to the 1st well, add the respective patient's serum.

7. Add 1500 µl of sample diluent into the lyophilised sensitised particles and 500 µl of sample diluent into the lyophilised unsensitised particles. The amount of sample diluent to be added is written at the side of the bottles. This step is done to prepare the sensitised and unsensitised particles as they come in lyophilised form and are not ready to use. Shake the solutions gently to obtain homogenous solutions.

8. Add 1 drop of unsensitised particles to the 2nd wells of each row.

9. Add 1 drop of sensitised particles from the 3rd wells to the 6th wells of each row.

10. Incubate the wells for 3 hours.

Results:

The principle of the test is visible agglutination between sensitised particles, coated with antigen, in the presence of mycoplasma pneumoniae antibodies. Therefore, positive result will show agglutination while negative result will show no sign of agglutination.

A positive result will show a definite large ring with agglutinated particles spread within the ring. This type of result is read as (+). A more obvious positive result, read as (++), will show a uniform agglutination of agglutinated particles spread out evenly to cover the bottom of the well. A negative result will show a definite button at the centre of the well with a smooth round margin, and will be read as (-). Sometimes, the result is indeterminate and is read as (±). A small ring with smooth outer margin will be observed. If most of the results are indeterminate, I was taught to compare the compactness of the ring. The least compact ring will be reported as positive.

As the 2nd wells are added with unsensitised particles, a negative result is expected to be observed. Sensitised particles are added from the 3rd well to the 6th well of the positive control, 'C' row. Therefore, a positive result is expected to be observed in the 4 wells. The positive control is expected to give a 1:320 end point titre.

Positive result in patient's serum depends on the presence or absence of mycoplasma pneumoniae antibodies. The titre at which the positive results end is recorded. For example, a uniform agglutination is observed in the 4th well where the titre is 1:80 and a ring is observed in the next well, 5th well, where the titre is 1:160. The result is then recorded as '1:80'. A negative result in patient's serum will show no sign of agglutination and a compact ring or button is observed at the bottom of the well. The result is then recorded as 'NEG'.

Usually, we record down the titre at which the positive results end. After recording down the titre, we have to write our initial down so that we know who is responsible for doing and recording of the results.

(Photo taken in the lab. Permission given by supervisor in charge.)

This is a visual interpretation of some examples of how a positive or a negative result should look like. This chart is given by the supplier as a guide when reading test results. The interpretation of each well is written on the top right hand side of the well. Although the chart shows results till the last or the 12th well, we usually only do up till the 6th well in the lab. So, for example, the results for the first row shall be recorded as 1:40.

That's all I have to share with you guys this time. Hope to hear from you guys soon.
Enjoy C:

Nur Azeimah
0607060A
TG 02