Histopathology Lab

Nadiah Sukma TG02


Helo0o..

for today's post, i'll be sharing with u what ive learnt in the main lab. basically it is a continuation of my first post.

Once the tissue are processed, they will have to be embedded, sectioned and stained. and all this takes place in the main lab.


Embedding

After the completion of the processing cycle, the tissues are removed from the tissue processing machines to the blocking room for embedding.
Processed tissues are placed in molten paraffin (56 C melting point) such that after the paraffin cools, the tissue and paraffin wax will form a block of sufficient consistency to cut thin sections.
Embedding can done by:

1. Filling a mould of suitable size with molten wax
2. Orientating the specimen in the mould to ensure it is being cut in the right plane
3. Cooling the mass to promote solidification

Orientation of tissue specimen:

1. Tissue specimens must be embedded flat to ensure that a complete section is obtained.
2. Tissues must be placed such that the resistance to the knife flows from lower to greater amount. this is to achieve smooth sections as the knife cuts through the soft to the hard part of the specimen.
3. There should be adequate paraffin wax surrounding the tissue to provide maximum cutting support. (must ensure there is no air bubbles which will results to holes in the block)
4. Tubular structures such as veins and arteries and fallopian tubes must be embedded vertically in the mould such that the knife cuts across the lumen.
5. Tissues with epithelial surfaces such as skin, intestine, gallbladder and uterus must be positioned such that all tissue layers are cut across.
6. Multiple specimens should be placed side by side, close to each other so that all pieces can be sectioned.
7. Rectangular tissues should be placed parallel to each other with their long axis perpendicular to the plane of section.
8. Small bisected cyst (half of a sphere shape) must be embedded with the cut surface down and must ensure that no air bubbles are trapped in the praffin.
9. Muscle biopsies in 2 pieces should be embedded with one piece in a longitudinal and the other in vertical position.

Shaving

After solid blocks are formed and cooled, they are sent for shaving also known as rough cutting before they are ready for sectioning. Shaving at 20 microns is to removed the paraffin wax and to expose the entire surface of the tissue including the margins.

*If entire surface could not be exposed due to un-flat embedding, the blocks must be sent for re-blocks.

Once shaved, the blocks are then soaked in 10% fabric softener for 5 mins. This is to smoothen the tissue surface to ease sectioning to obtain very thin ribbons.But using a commercial softener is not essential. it is also possible to just soak them in cold water for 5 mins.

*Tiny biopsy tissues and fats should NOT be soaked.

Microtomy

After shaving and soaking, all blocks must be kept cool at all times , hence they are placed on a cooler before being sectioned.

Sectioning using rotatory microtome is to produce 4 microns (one cell thick) thick of section to be fixed on microscopic glass slide for easy examination under the microscope.

Things needed:

1. Rotatory microtome
2. Warm floatation water bath (48 +- 4C)
3. 1% alcohol cold floatation bath (95% alcohol 5ml and SRW 500ml)
4. Microscopic glass slides
5. Forceps, brush and pencil.

Sections are cut using an undamaged part of the knife. The motion must be continuous to produce even thickness sections and long ribbon of sections. Interrupted motion or change of speed during cutting will result in uneven thickness which can be seen after staining under the microscope.

The long ribbon of large tissue specimens produced are then transferred to the 1% alcohol foatation bath first before transferring to the warm bath for fishing. The ribbon is let to float in the alcohol solution to expand the tissue to prevent folds (or to unfold the tissue when the folds are difficult to get rid using forceps, brush). This is because alcohol having low vapour pressure will increase the surface tension of the tissue when transferring to the heated floatation bath. Once the section has spread sufficiently, they are then transfered on to the glass slides and sent for staining (H&E or Special stains).

OKIES thats all for now..

my next post will be on staining...

Thanks! have a happy happy day...