Histology - Microtomy

HELLOS.

been experiencing some problems with blogger.. can’t make my postings. but anyway, i’m back to histopathalogy routine lab.. after a month at cytology and a month at processing area of histo lab.. so far what have been shared by the others on histology is staining, processing/trimming, embedding.. i shall touch on microtomy then~ =)

Microtomy, otherwise also known as sectioning, is as we all know from HTECH, the sectioning of tissue blocks using a microtome. After tissues are processed and embedded into paraffin blocks, it is then sent to the microtomy section for sectioning. Each tissue section produced can range from 2-25 microns.

Tissue blocks are first shaved/rough-cut, before it is being sectioned for microscopic viewing. Rough-cutting/Shaving is done at 20 microns per rotation/slice, until the entire surface of the tissue has been exposed. With rough-cutting/shaving, dense and hard tissues which may cause nicks and blunts to the microtome blade can be easily identified, as well as sutures and staples. If tissues are dense and hard, it is will then be pre-treated with RDO (decalcifier) and sectioned last (after all the other tissue blocks have been sectioned), as they might still cause nicks and blunts to the blade. Sutures and staples will be removed if identified. Nicks and blunts on the blade will cause score lines and poor sectioning of the tissue block, which may affect the diagnosis when evaluated by the pathologists.

After blocks are being shaved, it is then placed in 10% fabric softener solution for 5 minutes, and washed in water before placing them on cryoplate for chilling. Chilling of blocks is actually to facilitate fast sectioning, and renders tissue blocks hard for thin sections (normally 3-4 microns) to be produced.

The microtomes are those rotary microtomes, whereby the rotary movement is converted into up and down movement for sectioning, and a forward movement to advance the block for continuous sectioning.

Tissue blocks which are chilled, will be clamped onto the microtome, whereby thickness of the sectioning will be set at 3-4 microns, and section into even thickness and long ribbon of sections. These tissue sections will then be transferred onto the illuminated floatation warm water bath (set at 48 degree Celsius +/- 4 degree Celsius). Tissue sections can also be placed into 1% alcohol floatation bath before transferring to the warm water bath, which serves as an alternative when folds on the tissues are difficult to get rid of. Alcohol have a low vapour pressure which will increase surface tension of the sections, thus aid in the spreading of the tissue sections, so that it can be transferred flat, monolayer onto the microscopic glass slides.

Transferring of the tissue sections onto microscopic glass slides is done by ‘fishing’, whereby the desired sections will be ‘fished’ out of the floatation bath using the glass slides, and subsequently labelled with technican’s (who sectioned the block) initial, accession number (of the specimen), batch number, and block number.

Slides will then be placed on the hot plate (with tissue section facing upwards) for 3 minutes to ensure that the sections will not float off during staining. After which, it will be sent for staining (either H&E by the autostainer, or special stains such as PAS, GMS etc.)

Common faults which may be encountered during sectioning:

1. Ribbons fail to form, which can be due to

• paraffin wax too hard
• blade too blunt
• tilt/angle of blade is too great.

2. Sections compresed, wrinkled or jammed, which can be due to

• Blade too blunt
• Tissue block is still warm
• tile of blade and block is too slight

3. Split ribbon or scratches (score lines) in ribbons, which can be due to

• nicks in the blade
• tilt/angle of blade is too great.
• tissue is too hard
• blade edge is blunt (dull/rounded)

4. Sections full of folds, which can be due to

• floatation bath is too cold/warm
• tissue block is still warm

           Automated Microtome which we use in the lab!

               Controller for the automated microtome (close up)

Cryoplate

Illuminated floatation water bath

Manual Microtome(similar to the one we have in sch!)

Pictures Credit to Lab, taken with permission.

Cheers,

Ang Yu Hui Jacelyn
0702632A