1. The specimen is allowed sufficient time for it to be fully fixed with 10% formol calcium (at least 24 hours after removal from the body) before processing is commenced. Specimens which are labelled with tetracycline (noted on request form) for subsequent demonstration with fluorescence, must be fixed only in 70% alcohol and not formol calcium, for at least 24 hours after removal from the body.
2. If the specimen is too large, it should be trimmed/cut so that adequate size block(s) are taken from the part(s) required for examination. Surplus tissue not required for examination should be cut off. Should cutting/trimming be necessary, then advice must be sought from an experienced MLSO.
3. Large specimens especially those which require cutting/trimming may require longer fixation than 24 hours before processing is commenced.
The following processing schedule is suitable for biopsy size specimens which are processed in a 10ml glass vial.
1. 70% Alcohol 2 hours
2. 90% Alcohol 2 hours
3. 100% Ethanol x 2 2 hours each
4. Ethanol/methyl methacrylate monomer (1:1) overnight
5. Infiltrating solution A x 2 4 hours each
6. Infiltrating/embedding solution B 23 hours
1. Specimens must only be processed under an operational fume hood.
2. Specimens are agitated continuously on a roller mixer for all steps.
3. Waste solutions containing alcohol can be discarded down the sink.
4. Any waste solution containing methyl methacrylate must be discarded after being allowed to evaporate off according to waste-dosposal policies.
INFILTRATING SOLUTION A (STORE AT 4°C)
--Methyl methacrylate monomer (stabilised) 70ml
--Dibutyl phthalate 30ml
--Benzoyl peroxide (previously dried) 2g
Make up solution under a fume hood in a darkened bottle. Benzoyl peroxide must be dissolved before use by agitating on a roller mixer.
INFILTRATING/EMBEDDING OF SOLUTION B (STORE AT 4°C)
--Infiltrating solution A 100ml
--Poly methyl methacrylatelow mol wt beads 30g
Make up mixture under a fume hood in a darkened bottle, mixing on a roller mixer until beads have fully dissolved (approximately 4 days)
DRYING BENZOYL PEROXIDE
Benzoyl peroxide is supplied damped with water as it is potentially explosive in its dry form. However, it is essential that only dried benzoyl peroxide is used in the solutions A and B. It is dried as follows:
1. Spoon out approximately 10g of benzoyl peroxide into a filter paper bent in the shape of a cone on a beaker.
2. Dry in a 37°C oven overnight. Use only the oven in the laboratory, and place a sign to warn staff.
3. Excess dried benzoyl peroxide is stored on the shelf in the container provided away from direct heat.
1. Allow specimen in resin embedding solution B to stand for 1 hour.
2. Orientate the label using a wooden cocktail stick, so that it is bent at right angles across the specimen.
3. Secure lid on vial/container.
4. Partially immerse in pre-heated 60°C water inside a staining dish. Ensure lid is replaced on dish.
5. Polymerise resin overnight at 60°C in the oven. Use only the 60°C oven in the resin laboratory.
RELEASING BLOCK FROM GLASS CONTAINER
1. Place glass container (minus lid if possible) in freezing compartment of the refrigerator for 15 minutes.
2. Wrapping the container well in paper towels, break the container by hitting gently with the hammer provided. Wear eye protection.
3. Discard both waste paper and broken glass in the glass bin, and ensure no glass is lying on the bench.
4. Carefully wash block(s) under a running tap.
1. Clamp block in specially designed holder on Reichert-Jung Autocut so that the bone is vertical to the knife edge.
2. Using the trimming tungsten carbide knife, trim into block until a representative area is reached, lubricating both knife and block with 30% alcohol with the aid of an artist's brush.
3. Remove the trimming knife and replace it with the cutting tungsten carbide knife and cut sections at 6µ, lubricating both block and knife as previously described. Sections that require fluorescence to examine tetracycline labelling should be cut at 12µ and kept separate from other sections. Sections can be picked up using a pair of fine forceps.
4. Cut at least 6 sections per routine case so that spares are available should they be required.
5. Store sections in 30% alcohol (or 70% alcohol for tetracycline-labelled tissue). Use a separate labelled small specimen pot for sections from each block.
NOTE: No responsibility is assumed by The University of Nottingham or the Queens Medical Centre NHS Trust for any injury and/or damage to persons or property as a matter of products liability, negligence or
otherwise, or from any use or operation of any methods, products, instructions or ideas contained in the material herein. It is the users responsibilty to ensure that all procedures are carried out according to appropriate Health and Safety requirements.
© Copyright 1997 University of Nottingham Medical School Division of Histopathology. This page was last built on Mon, May 5, 1997
with Frontier. Thanks for looking in. Comments to James.Lowe@nottingham.ac.uk