Sunday, June 29, 2008

Week 1 SIP (Ernest)

Hello

I am posted to Histopathology department of a hospital. (Hospital name is confidential). This department involves everything we're learnt in Histological Technique, taught to us by Dr. Khin in year 2. My classmates who are in the same department as me are Ying chee and Athirah.

The whole process starts from obtaining the tissue, storing it in formalin, "passing" it into cassettes, stored in formalin again, processed, embedding, microtomy, staining, and finally sorting and sending out the glass slides. Is it confusing? I will go through it over the next few weeks. I haven't touched on everything yet in the lab.

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Ok, the first week, we have done fishing, labelling, heating, and staining.

Fishing is when the block is cut into 4um thin pieces, and placed on 40-50deg Celcius water bath. The slice of sample is then "fished" out using a glass slide. The first few times, it was hard to scoop up the tissue. But after 2 days of practising, we can easily fish it up easily, maybe even blind-folded. Haha.

Next the glass slide is labelled using a pencil. We need to write the biopsy number, batch number, year, and medical technologist's initials. It is important to label as it will be used for sorting, according to the doctor's request.

Heating the glass slide on a hot plate to remove the wax and to dry the specimen. The glass slide must be removed of all water first, otherwise the tissue will smudge when heated. To remove all the water on the slide first, we must place the slide only partially on the hot plate. So that the slide won't be too hot. The temperature of the hot plate is above 60 deg celcius, I'm not sure what is the exact temperature.

Then the glass slides are stained using an automated staining machine. However, this machine can only stain tissues with Haematoxylin and Eosin. If you want to stain with other types, you have to use other machines, eg to stain PAS, Perl's stain etc. The slide holder can hold 30 slides, and the machine can hold about 5+ holders. That means it can stain over 150 slides at a time! It can also mount a glass cover slip for you automatically. Very amazing machine, it saves the time of the med techs by staining alot of glass slides at any point of time.

After it is stained, it is sorted according to the batch number and biopsy number and sent to the doctor's in charge for microscopy. The glass slide is attached to the doctor's form.
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We have also assisted pathologists in trimming organs. Most of tissues or organs have tumours. The organs/tissues we have seen so far are breast, rectum, iluem, liver, uterus, prostate, and pancreas. We stand beside the pathologists while they cut. We assist them by labelling, giving them cassettes and showing them the patient's forms. Very interesting to see them dictate what they see.

Dictate means saying out the observation into a micophone. For example, the pathologist will say "Cyst measuring 6cm by 2cm by 3 cm is located at the upper quadrant of the right lobe". It is recorded into a computer, then sent to LIS (Lab information system - we learnt this in LMQA). An admin staff will then download the dictation and type it out and attach it to the patient's form.
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Histopathology department is rather fast paced as we handle about 500-700 slides in a day. Some are urgent while some are routine. Thus eventhough there are quite a number of staff, we need to work very fast.

By Ernest Lim
TG01 0606330I
5.45pm
29 June 2008 (Sunday)

13 comments:

Fluid collectors said...

Hello

Wow! What an amazing staining machine you mentioned~

Anyway, I would like to know if there is any difference in the process for different specimens.

Thanks

LeeJin
TG02

group1 said...

Hi!

I miss those days back in HTECH lab.

Can you tell me the reason for fishing at 50-60 degree? Is it to melt to wax? I've kind of forgotten already.

And you have also mention about labelling the slides. what is batch number? how do you assign the numbers or where did you get it from?

Last question! How many glass slides of the organ specimen do the lab usually make? And you mention sending the glass slides out, but to where?

Thanks!

Chew Yu Mei
TG01

Fluid collectors said...

Hello

Were the organs fixed in formalin as well?

How do they trim the organ? How they determine which part to take?

Thanks!

ShiHui
TG02

hellomedtech said...

Hi Ernest,

Would like to know..how long does the automated staining machine take to stain 1 batch of slides?

Thanks.

Nur Farhana
0604834B

THE CODEC 5 said...

Hi!

Your lab seems fun.

By the way, what is a biopsy number? And how do you guys trim the organs? Trim them as in cutting them or ..?

Oh .. and how does the automated staining machine stain the glass slides?

Thanks =)

Lyn
TG02

tg01 group 2 said...

Hi

>Replying to Leejin

Yes different tissues need different processing techniques. eg. bone needs to be decalcified.

There are 8 steps to tissue preparation to view the specimen under the microscope.

1. Fixation
2. Decalcification (only for bones)
3. Dehydration
4. Clearing
5. Impregnation
6. Embedding
7. Cutting
8. Staining

To answer your question, tissues such as prostate have to be processed as a whole, thus the whole processing time takes longer, about 14.5 hours in xylene-free processor. While breast and colon may take up to 11 hours. There are quicker methods which take 9hours but this is used only in soft tissues.

There are also alot of types of stain such as H&E, PAS, TB stain etc.

Thanks
Ernest

tg01 group 2 said...

Hi

>Replying to Yumei

Not 50 to 60 degrees. The ideal temperature is 50 degrees but range from 40 to 55.

The melting point of wax is 58deg. The water bath is set to about 50deg to decrease the surface tension of water. Few drops of alcohol may be added too. The lower the surface tension, the easier it is to fish the specimen and the specimen will be better spread out more nicely on the glass slide.

The specimens comes in batches. As there are hundreds of patient samples being examined by my hospital, they are split into 4 batches for easy management and also traceability. eg. Batch 3a has 20 organs and is cut by Doctor Tan while batch 3b has 35 organs and is cut by Doctor Lim.

Sorry I do not understand your last question. The number of slides depends on the size of the organ. For example, a breast can have up to 30 slides. After the slides are stained, they are sorted and given to a pathologist to be examined, usually to confirm malignancy of the tissue.

Thanks
Ernest

tg01 group 2 said...

Hi

>replying to shihui

All specimens are fixed in formalin immediately UNLESS it is needed to be examined "freshly", then it will have to be frozen and cut. I have not been attached to the frozen section department yet so i do not know the SOP behind it.

Trimming is done usually to source out the malignant area. The pathologist know which part to take simply by looking at the tissue! They are trained in locating tumours. The histologist simply process the tissue. Trimming is done by either with a very sharp knife or a scaple.

Thanks
Ernest

tg01 group 2 said...

Hi

>Replying to Farhana

Instead of telling you the whole staining time, let me break it down for you.

Xylene 2 mins

Xylene 2 mins

Abs alcohol 1 min

95% alcohol 1 min

70% alcohol 1 min

Water 1 min

Haematoxylin 4 mins

Haematoxylin 3 mins

Water 1/2 min

0.5% acid alcohol 2 dips

Water 1 min

Lithium carbonate 2 mins

Water 2 mins

Water 3 mins

Eosin 1/2 min

70% alcohol 1 min

95% alcohol 1 min

4 rounds of abs alcohol each for 1 min

3 rounds of xylene each for 1 min

Mount cover slip

tg01 group 2 said...

Hi

>Replying to Lyn

A biopsy number is a 5 digit number given to a patient. eg. 15123. This number will be used on the specimen, it is labelled on the glass slide.

As mentioned in my reply to shihui, the pathologist trims the organs, not the histologist. It is done using a very sharp blade or scaple. Trimming is cutting out the desired tissue so that it is small enough to be processed. usually 4cm x 3cm x 2cm or less.

Refer to my reply to farhana on how the automated staining machine
works. Haematoxylin stains the nucleus blue-purple, while eosin stains the cytoplasm pink-orange.

There are other types of special stains such as alcian blue, Giemsa stain, Congo red, Gram staining being used in the lab. However these are quite rare.

Example: Some tissues may be infected with acid-fast microorganisms. Thus, we use a commercial TB reagent. TB comes from the word tubecule bacillus. A type of bacteria.

Thanks
Ernest.

Fluid collectors said...

hello Ernest. what is the principle of haematoxylin and eosin stain?
Yuxuan

MadTechs said...

Hey, Ernest ^.^

(referring to your reply to farhana)

lithium carbonate?
may i know what is it for?

Merci!


Nor Liyana
0607927A
Group8

tg01 group 2 said...

Hi

Replying to Liyana

Lithium carbonate is an alkali. It is a fast way to "blue" the specimen.

Note that it DOES NOT stain the specimen, it just makes the haematoxylin stain MORE blue.

Thanks
Ernest