Week 13 Sharing of experiences in histopathological laboratory

In tissue processing room

• Whole mount preparation
  -Completely cross-sections of a pathological specimen
  -Usually too large for the ordinary cassette and require individual handling prior to machine processing
  -Example: whole mount sections of prostate

Observations of prostate trimming by pathologists
-Material: Prostate cutting tool
-Whole mount is necessary as tumour is located in prostate and can only be seen under microscope.
-For whole mount processing, the timing of fixation will be extended for 41/2
hours (total 211/2 hours) to ensure tissue is thoroughly fixed.
-The dehydration steps will also be extended to ensure proper dehydration due to the size of the tissue.
-Other tissues will not be processed together with whole mount prostate as over fixation and dehydration of smaller pieces will cause the tissues to be harder.


Microtomy

1. Microtome safety (Safe Operating Procedure)
2. Handle blades very carefully when installing or removing. Follow the manufacturer’s guidelines explicitly.
3. Tungsten-Carbide knives can put through your shoes, if dropped. Be careful where your feet are positioned when installing or removing blades.
4. Store blades in a covered container that has guides to hold the blades rigid.
5. Never leave blades on countertops. Lacerations can occur when reaching across the countertop and inadvertently contacting on unprotected table.
6. When setting up the microtome, position the sample first, and then put in the blade. Never the other way around.
7. When applying the brake, ensure that it is tight. Most accidents occur when the brake slips and the operator’s hand is drawn into the blade.
8. When leaving the microtome, even for a short time, ensure that the blade guard is in place.
9. When preparing a paraffin sample for the microtome, remember to clamp the sample down tight. The movement is allowed by a loose clamp increase your risks of cut.
10. Use forceps or brushes during retrieving and transferring slides and ribbons from the blade, thereby keeping your hands free from the moving parts of the microtome.

Common faults encountered in microtomy and remedies

Causes / Remedies

Ribbon fails to form
1. Paraffin too hard / 1. Use softer wax i.e. lower melting
2. Blade too blunt point / 2. Change to a new blade edge
3. The tilt is too great / 3. Tilt the blade less

Crooked ribbons
1. Edge of block is trimmed not parallel to blade. / 1. Re-trim the block.
2. Irregularities in blade edge / 2. Try another part of the blade

Sections compressed, wrinkled and jammed together
1. Blade too blunt / 1. Change to a new blade edge.
2. Paraffin block is warm / 2. Cool the block on cryoplate
3. Blade tilt is too slight, therefore / 3. Increase the tilt
the cutting facet rubs over the block
4. Blade edge gummed with wax / 4. Wipe blade with xylene

Sections crumble and specimens fall out, mushy and soft
1. Incomplete dehydration / 1. Re-hydrate
2. Improper embedding / 2. Re-embed

Tissues turn hard and cut like stone
1. After clearing, the specimen was / 1. Nothing can be done except repeat
accidentally left to dry in air with fresh specimen
2. Paraffin bath is too hot and burns the tissue / 2. Decrease the temperature of parrafin
bath

Split ribbon or lengthwise scratches in ribbon
1. Nicks in the blade / 1. Use a different part of the blade.
2. The tilt of the knife is too great / 2. Decrease the tilt
3. Blade edge dirty / 3. Wipe blade edge with xylene
4. Hard particles (calcified materials) in the block / 4. Do surface decalcification
5. Crystal from fixative e.g. Mercuric chloride / 5. Treat tissues to remove crystals

Undulations in the surface of the section
1. The blade is not tightened / 1. Tighten all screws
2. The title of the blade is too great / 2. Decrease tilt to prevent vibration
3. Tissue too hard e.g. cervix, fibroid / 3. Soften tissue in phenol or mollifex
4. Blade edge is dull or rounded / 4. Change to new blade edge

Sections of varying thickness and skipping of sections
1. Blade not tilted enough to clear facet, / 1. Adjust the tilt
or too much and tissue is compressed until
the inevitable expansion gives a thick section
2. The blade and specimen holder not tightened / 2. Tighten all screws
3. Tissue too hard e.g. cervix, fibroid / 3. Soften tissue in phenol or mollifex
4.Cutting stroke is not regular or interrupted / 4. Maintain regular cutting stroke

Sections full of fine lines running across
1. Blade edge consist of fine serration / 1. Change to a new blade edge
2. Cutting edge is dirty / 2. Clean blade edge

Sections full of folds
1. Water bath is too hot or too cold / 1. Adjust temperature to 50 degree
Celsius
2.Blade tilt is too slight so cutting facet / 2. Increase the hilt
rubs over the block.
3. Paraffin block is warm / 3. Cool block on cryoplate

Alright i shall stop here then. :)

Ang Xiao Si Sharon
TG02
0503219H