Microtomy =D

Hi everybody,

Sorry for the very late posting. This would be the final post of the whole 20 weeks! Haha..I would like to talk about Microtomy as it the the last area that i'm posted to.

Subject Title: Lab Technique

Topic: Microtomy

Principle: The principle of microtomy is to sectioned tissue specimens into thin sections so that they can viewed and diagnose under microscope.

Below is a picture of the rotary microtome used for microtomy.



Materials Required

Alcohol Floatation bath 1
Soft Pencil 1
Rotary Microtome 1
Microscopic glass slides 1
Disposable microtome blade 1
Cryoplate 1
Warm floatation bath 1

Steps involved:

1) The block is required to shave first before cutting. The paraffin wax block is secured to the block holder of the microtome and adjusted it to ensure that it clears the knife. THe block holder screws are also readjusted to place the block parallel to the knife. During shaving, while the machine is manually advanced, the block is repeatedly sectioned at 20 microns thickness per slice. Shaving is stopped when the entire surface of the tissue is exposed. The block is then removed from the holder.

2) All blocks should be shaved so that dense and hard tissue can be identified more rapidly and separated from the rest. Those blocks will then pre-treated before sectioning. By doing so, it can prevent nicks and scorelines to the blade. It can also ensure that all staples or sutures are removed from the blocks before sectioning as pathologists might forget to remove them during trimming. Hard bone can also be softened first in 10% commercial fabric softener for 5 minutes, so that the tissue can be sectioned more easily.

3) After the blocks are softened, they are placed face down on the cryoplate to chill the block to allow fast sections.

4) The blocks are secured and adjusted to ensure that it is parallel and clears the knife. It is sectioned at 3-4 microns using the handwheel by allowing the block to advance automatically or manually.

5) The section is then lowered onto the floatation bath. If the tissue has difficulty in spreading, it is placed on alcohol floatation bath first to increase the surface tension before transferring onto the warm floatation bath. The section is then transferred onto a glass slide where the corresponding biopsy number is written on the frosted end of the glass slide.

6) The glass slides are separated according to their respective stains such as H&E, special stains and unstained slides.

In our lab, different biopsies are sectioned at different thickness and different number of slides are required. Below is some examples of different types of tissue biopsies and the thickness and number of slides that are required.



I will stop here for now. Please feel free to ask questions =D
Enjoy your holidays =D

Lok Pui
(0704138G)

Processing :)

Hi people, this is my last post for SIP. Hope you guys are doing fine for your project. :) I have been posted to the trimming room for this month and will be talking about processing for this post.

In the trimming room, pathologists will trimmed large biopsy specimens or specimens that are too big for the technicians to pass. Some of the large specimens are breast, colon, uterus etc. Basically, my job is to assist the pathologists. In the morning before they arrive, my job is to distribute the specimens and write cassettes. Normally for the large specimens around 10-12 cassettes are needed while for those very small 1-2 is enough.

Specimen example colon is labeled with eg.A, B or C etc, according to the alphabet that is written on the form. In other words, if the pathologists uses 14 cassettes for the colon specimen, the cassettes will be written until eg. A14. During the labelling of the cassettes, one have to remain alert and make sure that the correct biopsy number is written, otherwise wrong diagnosis will be given. Following that, i need to make sure that there are enough trimming blades and scalpel, paper towels and paint, so that they do not run out during trimming.

After the trimming is completed for the specimens, the cassettes have to be put into a rack which is soak in 10% neutral buffered formalin. The purpose of this is to fix the specimen. After that, the cassette rack will be placed into the automated tissue processor for processing. There are basically three main steps for processing, which are dehydration, clearing followed last by infiltration.


Dehydration

Principle: It is to remove the water contain inside the tissues so that wax can infiltrate as wax is not miscible with water. (Ethanol is used in this case)


Clearing

Principle: It is to remove the alcohol from the previous step using an agent that is misicble with both the wax and alcohol and for clearing purpose. (Xylene is used)

Infiltration

Principle: It is to replace the clearing agent with embedding medium (Paraffin wax is used)


Zi Shuang
0703383J

Tetraplex PCR

Dear all,

last 10-14 working days left. Hang in there!
Anyway i have mentioned about Real Time PCR and the Molecular probes for Real Time PCR. So lets talk old school, Conventional PCR.

Tetraplex is a panel developed by the scientific officers of the lab. Heard that it is a request from doctors thus resulting in the test being developed.
The panel consist of 4 viruses,
1. Cytomegalovirus (CMV)
2. Varicella – Zoster Virus (VZV)
3. Toxoplasma Gondii (Toxo)
4. Herpes simplex virus (HSV)

Specimen received are either CSF or Aqueous eye fluid (no preference of which eye).

The specimen will go through a manual DNA extraction process to get the DNA.

The difference from what we learn in school as compared to this is that in school, there is only 1 time PCR (1round PCR). However, for Tetraplex, this is a 2 round PCR.
As told by the med techs, the 2nd round is to improve the sensitivity.
Also, for PCR, each patient will have samples, 1 normal sample and another is a spike in.
Spike in is to add positive control to the sample. This is for ruling out any false positive.

After the second round, it will under go a gel electrophoresis.
Gene ruler, the 4 different controls, "spike in" samples and actual samples are added to separate well but on the same slab of gel.

After electrophersis, the gel will be put into the chemimager and we will check for the corresponding base pairs to check if it is pos/neg.

Upon receive a POS, the ministry has to be notified.

Thats all for this post. I'm sorry that i cant go into the details (Supervisor dont approve)...

Cheers.
Tiong han
Tg01

Rotavirus detection

Time really flies, 5 months of attachment is ending in a couple of weeks! Back to my post, i am going to talk about a small, easy-to-use and rapid kit that is used for the detection of rotavirus.

Rotavirus is a major cause of diarrheal illness among infants and childrens. As the name implies, it is a virus and can be classified into Group A-E based on their antigenic group on VP6. Group A is known to be the major cause of human rotavirus disease. The symptoms includes vomiting, watery diarrhoea and fever and may even leads to death due to severe dehydration.



[Rotavirus kit]
Test procedure:

1) Pipette 1 ml of the Rotavirus Extraction buffer into a test-tube.
2) Add around 100 ul or 50mg of stool sample into the test-tube.
3) Mix the mixture in the test-tube using the pipette
4) Let the stool sample to settle for around 3 minutes
5) Pipette 4 drops near the top part of the mixture into a round opening on the Rotavirus kit
3) Read the result on the kit after 5 minutes.


Interpretation:
A blue control band will appear on the test kit.
A positive result will be indicated by a red test band along with the blue control band.
A negative result will only shows the blue control band.
If there is no blue control band, the test will be repeated with a new kit.



Signing off,
Yong Herng
0702243G

HbA1c test

Hey (:

Subject Title: Clinical Chemistry
Topic: HbA1c test

Haemoglobin A1c (HbA1c) Test using Bio-Rad D-10

Note: I will just be focusing on the main procedure of running the samples. I will talk about the Calibration next week.


Principle

This test is based on ion- exchange high-performance liquid chromatography. Samples are automatically diluted on the D-10, injected into the analytical flow path, and applied to the analytical cartridge. The D-10 provides a programmed buffer gradient of increasing ionic strength to the cartridge, where the haemoglobins are separated based on their ionic interactions with the cartridge material. The separated haemoglobins then pass through the flow cell of the filter photometer, where changes in the absorbance at 415 nm are measured.

An accurate index of the mean blood glucose concentration maybe established by the measurement of haemoglobin A1c every 2 to 3 months. HbA1c is formed in 2 steps by non-enzymatic glycation of HbA. The first step is the formation of an unstable aldimine (pre- A1c), a reversible reaction between the carbonyl group of glucose and the N –Terminal valine of the b-chain of haemoglobin. During red blood cell circulation, some of the pre- A1c is converted to form a stable ketoamine, HbA1c. The level of HbA1c is proportional to both the average glucose concentration and the life span of the red blood cell in the circulation. The measurement of HbA1c has therefore been accepted as the clinical management of diabetes.


Materials

1.Bio-Rad D-10 Machine
2.D-10- Haemoglobin A1cRecorder Pack
a) Elution Buffer 1
b) Elution Buffer 2
c) Wash/Diluent Solution
d) Analytical Cartridge
e) Floppy Diskette
f) Calibrator/Diluent Set
g) Whole Blood Primer
h) Samples Vials
I) Thermal Paper
3.Sample Vial Adapter, 10 x 1.5mL
4.Lymphochek Diabetes Bi-level Control, 6 x 0.5 mL
5.Pipettes, 5mL, 0.5mL, 1mL, 7mL
6.Deionised water
7.Disposable gloves



Method

1.Start up the machine if it is in “Sleep” mode.
2.Collect EDTA.
3.Check the amount of blood present.
4.If there is less than about 2 mL of blood in the tube, the sample will be pre-diluted.
5.To pre-dilute, pipette 1500 mL of Wash/Diluent Solution into a vial, followed by 5 mL of the whole blood sample. Use a sample vial adapter for 1.5 mL vials.
6.Invert the tubes about 6 times.
7.Load the EDTA tube(s) into D-10 sample rack and place the rack in D-10. Must ensure that the sample barcodes are facing towards the back of the instrument.
8.Wait for the sample’s laboratory request number to appear on the screen (if they are not pre-diluted) before pressing “Start”.
9.When more than 1 tube is tested, press “Edit” and then, press “Done” for all the tubes and finally, press “Start”.


Results and Interpretation

1. Go to “data” and click “details’’
2. The graph must be above 0.02. If below 0.02, repeat test
3. F must not be more than 3%
4. Total area: 1- 4 million (cannot be more than 5 million)
5. P3 cannot be more than 3
6. Cannot have “unknown” below A1c, if not reject the test
7. The overall results must not be more than 10, otherwise, rerun the test and record “R:” in the worksheet.
8. If got variant window, record “variant window” in worksheet and then
- select sample with variant window
- choose “selected samples”
- Press ‘’Print”
9. At the end of the day, go to “data”, daily summary and press ‘‘Print”
10. If the total area is low (lesser than 1 million), do Manual run (dilution). If total area is very low, take more of sample and vice versa.
a. Take the diluent bottle (ensure that lot number on the bottle is the same with main diluent bottle located behind of the machine)
b. Take the patient’s sample and pipette out 5ul to 1500ul of diluent in the sample vial
c. Invert a few times to mix it
d. Manually key in sample number and run HbA1c test again


Samples from patients with haemolytic anaemia will exhibit decreased glycated haemoglobin values due to shortened lifespan of the red blood cells.

Do not report results if the peak shape of the graph is abnormal.

Expected Value Range

Factors such as duration of diabetes, adherence to therapy, and the age of the patient should also be considered in assessing the degree of blood glucose control.

Histopathology (Processing)

Hi! This will be my second last post =D. I have been posted back to Histopathology lab since the last six weeks. For the four weeks, i went to processing and then went to main lab to do embedding for the next two weeks. As Zi Shuang have posted on embedding, I will talk about processing for this post. For processing, we used two types of tissue processor machine which are Leica and Peloris. For the morning batch of the trimmed tissues, they will be put into the Leica tissue processor. The program used will be extended 16 hours tissue processing whereby it is extended to 7.30am the following morning. For the afternoon batch, tissue sections will be put in the Peloris tissue processor. The program used will be the extended 9.5 hours Xylene free tissue processing whereby it is extended to 8.30am the following day. Thus, for this post I will be emphasizing on the Leica tissue processor.

Subject Title: Lab Technique

Topic: Tissue Processing

Below is a picture of the Leica tissue processor:


The cassettes rack used for this rack is shown below:



Principle: The principle of tissue processing is to remove the extractable water from tissue specimens and replace it with a medium that solidifies to allow sectioning. It consists of 3 stages which are dehydration, clearingand infiltrating. The purpose of dehydrating is to remove water from the tissue using graded alcohols from a lower to a higher concentration. Clearing is to remove alcohol from the tissue with a solvent that is miscible with paraffin wax such as xylene. Infiltrating is to infiltrate the tissue with paraffin wax to allow sectioning of tissues.

Steps involved:



I have observed that sometimes the tissue sections tend to sunk down or bulge up after they are processed and embedded. If the tissue sections are sunk down, it is normally due to the lack of infiltration. However, if the tissue sections are bulged up, it is normally due to the lack of dehydration.

I also observed that the cassette racks and the machine must undergo quick clean to wash off the reagents used for tissue processing. This quick clean process will take about 30 minutes. After this cleaning session, new cassette racks can then be loaded.

Some precautions to take note is that gloves should be wore at all times when you are at the trimming room. This is because you will be dealing with formalin when you are placing the tissue cassette in the formalin container for fixation before they are being processed. As formalin is carcinogenic, gloves can protect your hands from having contact with the formalin. Rinse with water immediately after you had accidentally splashed with formalin. Try to wear mask with shield to protect your eyes as formalin will cause eye irritation which will make you tear.

I will post until here for now =). If you have any questions, feel free to ask me =D.

References:

Picture of Tissue processor
www.dotmed.com/images/listingpics/370518.jpg

Lok Pui
0704138G

cytology (gynae)

Hi everyone, this is my third post. In this post, i am going to talk about processing of the gynaecology specimens in cytology lab.

Basically, the gynae specimens come in two forms, Thin Prep vials or conventional smears. The thin prep vials contain preservcyt solution which is basically a methanol-based medium. The purpose is to preserve the cells to allow proper evaluation under microscope.


Collection of specimen

1)Specimen is collected with a spatula or a cytobrush
2)It is rinsed immediately in the presercyt solution
3)The vial will be capped tightly and labeled with the patient information before being set to the lab

In the lab

1)Once specimen is received, the patient information on the form must be double check with that on the vial and thin prep number is assigned to both of them(eg.0983)
2)The vials are then taken into the processing room to be processed by the Thin Prep 2000
3)Thin prep slides are labeled with the thin prep number and the patient's ID
4)The slide is first inserted into the slide holder
5)This is then followed by putting in the fixative vial which contains 95% ethanol
6)Specimen vial is inserted next followed by slotting in the filter
7)Press program '4'to process the gynae specimens

After processing

1)Once processing is done, the slide holder will drop the slides into the fixative vial
2)The slides in the fixative vial will be left aside for around 15 minutes to adequately fix the cells before staining
3)The stain used is papanicolaou stain which is bascially carried out by the machine
4)Slides are then manually mounted using Depex

Things to take note

1)The slides must be adequately fixed before undergoing staining so as to prevent cells from dropping during that process
2)The specimen vial must always be placed into the Thinprep 2000 before the filter to prevent the filter membrane from being scratched
3)At the end of the day, make sure that grease is applied onto the black rings of the filter cap so as to reduce friction

That is the end of my post, which is generally an overview of how processing of thinprep specimens are carried out. Questions are welcome. :)

zi shuang
0703383J