Star Team

Receiving of Specimen
Check that the name, identification number of the form matches that of the bottles or the bags
Once every item received tallies with the request form, sign your name, stamp the time and write the date.
Sort the specimens out according to type, some specimens such as placenta require trimming, and must be put into a special box.


Scribing of gross description

Samples such as product of conception (POC), Breast biopsies and endometrial samplings have to go through this.

For POC, the information to be written down on the form are:
-The amount of sample used in milliliters
-If there is no more sample left after putting into a cassette, NR or No Reserve is written on the -form.
-The number of cassettes used is also written down

For breast biopsy, the information to be written down on the form are:
-The number of strips received
-The length, Breadth and width of strips
-The number of cassettes used is also written down
-The number of strips in each cassette is written down

Since the histopathology laboratory has to generate many blocks of tissue a day,
In order to differentiate them, different specimens have different colours.

-For paediatrics specimens, a yellow cassette is used
-For adult specimens, a green cassette is used
-For urgent specimens, a purple cassette is used
-For Post Mortem cases, a pink casstte is used.

Tissue processing

The cassettes containing the tissues are put into the Shandon Excelsior tissue processor,
It is an automated processor that removes the job of having someone to manually do the tissue processing process which takes about 18 hours. With the Shandon Excelsior tissue processor, the histopathology laboratory does not need to be a 24-hour laboratory, all the medical technologists have to do is to put in the cassettes at the end of the day and allow it to run overnight
Processes that the samples undergo during tissue processing:

Fixation
-A process that fixes the tissue samples in their current state so that the tissues can be studied
-Fixation is done with Formalin

Dehydration
-A process done for the removal of water
-Dehydration is done usually with alcohol, although other laboratories have been known to use alcoholic variants

Clearing
-Clearing agents used in the process must be miscible with both the dehydrating agent and the infiltration medium
-Clearing is done, to remove the dehydrating agents from the tissue and to prepare the tissue for addition of infiltrating medium
-Clearing is done with Xylene

Infiltration
-done with paraffin wax
-done to remove the clearing medium from the tissue and to prepare the tissue sample for embedding

Embedding

-The embedding process is done with the aid of the machine Sakura Tissue-Tek
-Prepare 3 different kinds of forceps for aiding of the tissue embedding process
-Soak them in in melted paraffin wax
-On the left panel of the tissue embedder, molds of different sizes are kept there and ready for use for tissue samples of different sizes
-The right panel is used to put the cassettes after they have been removed from the tissue processing machine
-Once the tissue has been removed from the cassette, it is placed into the the mold of an appropriate size
-Fill the mold with wax and use the forcep to press down the tissue, to ensure that the tissue sample is level. This ensures that shaving and sectioning would be easier
-Once the tissue is leveled, attach the base of the cassette to the top of the mold and fill up with more wax
-A piece of colour coded paper is embedded into the wax before it cools, this is done for tracebility purposes.
-The molds are then placed in ice to cool down
-Do not force the cassettes out of the mold, but instead wait for the paraffin wax to contract and become loose on its own
-In the cases where the cassette is still stuck on the mold after a period of time use abit of force but remember to place the tissue block back on the ice

Shaving

-The tissue blocks are shaved at 10 microns, to expose the full surface of the tissue to make sectioning easier
-It makes sectioning easier, as the person doing sectioning requires less shaving of the tissue block
-When shaving the tissue block, always check how much of the tissue has been exposed, as if too much is shaved off some parts of the tissue may be lost
-Some tissue samples such as fibroid are hard to cut as they contain calcium deposits, therefore they must be placed in softerner for 5 mins to soften the tissue again to make tissue sectioning easier
-After the tissue blocks have been softened, wash them under running water until the soapy feel is removed
-Dry the blocks before placing them on a cooler set at between –10 to –20 degrees, this makes sectioning easier

Sectioning

-Sectioning is done with either the leica or sakura manufactured rotary microtomes
-After the tissue blocks have been placed on the cooler for a period of time, they can be ready for cutting
-Set the microtome to cut at 4 microns
-After the appropriate section has been obtained, transfer it from the microtome to a water bath
-Fish the section onto a clean glass slide, before transferring it to another water bath
-The section is then fished back onto the same slide and left to dry off
-After the slides have been dried, they are put into racks to prepare for staining by baking in the oven at around 80°c for about 15 minutes

Routine H & E staining

-In the laboratory, routine Haematoxylin & Eosin ( H&E) staining is done on an automated machine the leica ST5010
-All we have to do is insert the rack of prepared slides (each rack can accommodate 30 slides) into the machine, ensure that the correct program is selected and then press load.

The machine has 16 different stations, and starts of with:
Xylene (removes wax)
Xylene
Absolute alcohol (hydration)
95% alcohol (hydration)
Haematoxylin (staining of the nucleus)
Haematoxylin
Ammonium (blueing)
Acid Alcohol
Eosin
95% alcohol
absolute alcohol
absolute alcohol
absolute alcohol
absolute alcohol
xylene
xylene

Once the process has been completed, the machine will sound alerting the user

Attaching of coverslip

Once the slides have been removed from the autostainer, they will be transferred into the Leica CV 5030, a fully automated glass coverslipper.
The machine individually applies Depex onto each glass slide before covering it with a cover slip.
Once the process is done, the machine will also alert the user that the process has been completed.

Slide sorting

The slides are then sorted according to case number, and compared to the original tissue blocks. If overlapping or folding of the tissue is observed, the tissue block would have to be recut to obtain a new slide.
Once the slides have been double checked and confirmed as okay, the appropriate labels are stuck on to them before the cases are dispatched to their respective pathologists to read the slides.

thanks

Randall

Tg02, 0503272G

# posted by Star team @ 3:04 PM