From patient to pathologist, preparing tissue specimens for histological examination requires care, skill and sound procedures. This guide provides practical advice on best-practice techniques and simple ways to avoid common errors.
Tips for better processing and embedding are highlighted in this week's guide. We hope each step provides a valuable reminder of good histology practice and also helps with troubleshooting when unacceptable results do occur.
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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.
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).
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.
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.
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.
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.
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.
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.
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.
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.
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.
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This reference document is presented as a service to health care professionals by Leica Biosystems and has been compiled from available literature. Although every effort has been made to report faithfully the information, Leia Biosystems cannot be held responsible for the correctness. This document is not intended to be, and should not be construed as medical advice. For any use, the product information guides, inserts and operation manuals of the various drugs and devices should be consulted. Leica Biosystems and the editors disclaim any liability arising directly or indirectly from the use of drugs, devices, techniques or procedures described in this reference document.
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