Step 20 - Use an Appropriate Schedule
An appropriate schedule is chosen for the tissue type and size.
An inappropriate schedule is chosen. For example, a very long schedule for a small endoscopic biopsy or a very short schedule for a large, fatty breast specimen.
This micrograph of a small area of subcutaneous tissue from a large, fatty specimen shows the effects of under-processing. The fibro-fatty tissue is poorly supported and therefore fragmented while the epithelial tissue of the glands shows a lack of nuclear definition and peculiar staining due to retained solvent (H&E).
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This endoscopic biopsy has been over-processed and has become very brittle. As a consequence many fine cracks are visible through out the section. Poor microtomy technique will exacerbate the problem (H&E).
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Step 21 - Provide Additional Fixation
For optimal processing and good morphology tissue should be well fixed before processing. Where specimens are incompletely fixed additional formalin fixation is provided in the processing schedule.
Incompletely fixed specimens go directly into alcohol producing zonal fixation (formal in fixation for the outside of the specimen, alcohol fixation for deeper areas).
This micrograph shows the effects of zonal fixation on a section of a marrow aspirate (H&E). In the upper left portion the red cells are intact where as in the lower part they are hemolyzed.
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This micrograph shows a low power view of liver stained with a trichrome stain. The staining result in the outer zone of the specimen is different to that of the inner.
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Step 22 - Maintain Reagent Quality
Processing reagents are replaced strictly according to established guidelines (ideally using are agent management system in an advanced tissue processor such as Leica Biosystem’s PELORIS).
Guide lines for there placement of processing reagents are ignored, meaning that ineffective, contaminated or diluted reagents are used (e.g “out-of-threshold” warnings from the PELORIS reagent management system are ignored).This can cause poor processing quality.
In this section – from a large skin specimen – the poor preservation of the dense collagen is due to inadequate processing. In this case we believe it was due to the use of heavily contaminated reagent swell “out-of-threshold”.
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Step 23 - Use High Quality Wax
High quality wax is used for infiltration and especially for embedding (blocking out) to ensure high quality blocks that are easy to cut.
Cheap, poor quality wax from little-known sources is used for infiltration and embedding. Poor quality wax produces blocks that are difficult to cut.
A ribbon of sections was slowly cut from this block while the block was cold. The sections show considerable compression despite the low temperature used. Here the poor quality wax failed to properly support the tissue.
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Step 24 - Avoid Hazardous Reagent
Where possible, xylene-free protocols are used (such as those available when using Leica Biosystems’ PELORIS). This provides a safer laboratory environment without compromising processing quality.
No consideration is given to the health effects of xylene use. The possibility of using alternatives has not been considered.
Xylene-free processing can improve laboratory safety while maintaining quality.
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Step 25 - Orientate Specimens Carefully
Specimens are carefully orientated. Competent grossing ensures flat surfaces on most specimens. Staff performing embedding have ready access to each specimen description and are appropriately trained.
Orientation is incorrect. This can result in loss of tissue as re-embedding is required. Some poorly prepared specimens require extensive trimming on the microtome to obtain a full-face section.
This endoscopic biopsy has been orientated incorrectly and shows only the superficial level of the mucosa.
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Step 26 - Choose an Appropriate Mold
A mold of suitable size is always chosen for each specimen.
The same mold size is used for every specimen. Often the tissue touches the edge of the mold.
The mold used for this specimen was too small. The specimen is in contact with the edges of the block and may therefore be difficult to section.
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Molds of different sizes are available for a variety of specimen sizes.
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Step 27 - Handle Specimens Gently
Specimens are handled gently during embedding.
Specimens are handled forcefully during embedding to make them lie flat in the mold. Some tissue can be fractured by this process.
An H&E stained section of spleen which was fractured during embedding in an attempt to make the specimen lie flat on the base of the mold.
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Step 28 - Avoid Excessive Heat
Before handling tissue, forceps are heated to the point where the wax just melts.
Forceps are heated well beyond the melting point of wax. This can cause local heat damage and a change in morphology in the area close to the contact point.
This micrograph shows the surface of a section of liver (H&E). Extreme local damage (making the tissue almost unrecognizable) has been caused by the application of heat to the tissue during embedding.
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Step 29 - Check Temperatures Regularly
The temperature of the embedding center hot plate and wax reservoir is regularly checked.
The temperature of the embedding center hot plate is never checked. Even at this stage of processing specimens can be damaged by excessive local heat.
This lymph node was damaged by over-heating of the embedding center hot plate. Note the shriveled, pyknotic nuclei and extensive cracking. Cracking like this can also be caused by flotation on a water bath that is too warm, or by drying on a hot plate without sufficient draining.
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Step 30 - Do Not Over-fill Molds
Molds are filled to an optimum level and do not overflow.
Molds are over-filled, requiring scraping of the back and edges of the cassette prior to microtomy. Over-filled blocks may sit unevenly in the microtome chuck causing instability that may lead to the tissue becoming damaged during microtomy.
Priorities are important when delivering specimens to the laboratory. This is particularly so when frozen sections are involved.
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