Podcast
Questions and Answers
What are the three major steps of tissue processing?
What are the three major steps of tissue processing?
What is the most common agent used in dehydration?
What is the most common agent used in dehydration?
Ethanol
Water mixes well with infiltrating or embedding media.
Water mixes well with infiltrating or embedding media.
False
What is the purpose of dehydration in the context of staining?
What is the purpose of dehydration in the context of staining?
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What are the main reasons for using a graded concentration of alcohol for dehydration?
What are the main reasons for using a graded concentration of alcohol for dehydration?
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What are some advantages of using toluene as a clearing agent?
What are some advantages of using toluene as a clearing agent?
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Choose the most appropriate option for the disadvantages of using benzene as a clearing agent.
Choose the most appropriate option for the disadvantages of using benzene as a clearing agent.
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What is the primary purpose of infiltration in tissue processing?
What is the primary purpose of infiltration in tissue processing?
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Which of the following infiltration media is considered the most common?
Which of the following infiltration media is considered the most common?
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Carbowax is a type of celloidin infiltration medium.
Carbowax is a type of celloidin infiltration medium.
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What is the main difference between an open and a closed tissue processor?
What is the main difference between an open and a closed tissue processor?
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Microwave processors are preferred for urgent tissue processing because they utilize a standard, closed system.
Microwave processors are preferred for urgent tissue processing because they utilize a standard, closed system.
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Explain why the first station of each reagent in tissue processing is the most contaminated.
Explain why the first station of each reagent in tissue processing is the most contaminated.
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What is the general approach to changing reagents in a tissue processor and how is it called?
What is the general approach to changing reagents in a tissue processor and how is it called?
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The first station is the most contaminated, but can still be safely re-used.
The first station is the most contaminated, but can still be safely re-used.
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What contaminant is most likely to appear in a xylene (clearing agent) station?
What contaminant is most likely to appear in a xylene (clearing agent) station?
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How can you tell if paraffin is contaminated with a clearing agent?
How can you tell if paraffin is contaminated with a clearing agent?
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Study Notes
Tissue Processing I & II
- Tissue processing aims to remove water from tissues and replace them with a medium that solidifies, allowing thin sections to be cut.
- The process involves several steps: collection, accessioning, grossing, processing, embedding, microtomy, staining, assigning, reporting, and finally analysis by a pathologist.
The Path of a Tissue Specimen
- Tissue specimens journey from collection (doctor's office, clinic, or hospital) to the pathology lab.
- Key steps in the lab include accessioning, grossing, processing, embedding, microtomy, staining, and assigning a report to the pathologist.
Steps to Tissue Processing
- Fixation must be complete before processing begins. If fixation isn't complete, add more fixation time to the schedule.
- Dehydration, clearing, infiltration, and embedding are the subsequent steps.
Processing (Detailed Steps)
- Dehydration removes water from the tissue.
- Clearing removes alcohol from the tissue.
- Infiltration infiltrates the tissue with paraffin wax.
Dehydration
- Dehydration removes water from tissue using hydrophilic reagents that attract water from the tissue and repeats the dilution of aqueous tissue fluids.
- Ethanol is commonly used as a dehydrating agent, frequently increasing in concentration gradually (e.g., 50%, 60%, 70%, 80%, 90%, 100%).
- Gradual increases in concentration prevent tissue damage (e.g., shrinkage, distortion).
- Dehydration is an intermediate step between aqueous and non-aqueous media (e.g., paraffin).
- Dehydrating agents must be miscible with water (from fixatives) and clearing agents (e.g., alcohol mixes with both water and xylene).
- Dehydration is performed after staining in routine staining, because water doesn't mix with resinous mounting media.
- After cutting sections, hydration is needed for aqueous staining.
After Embedding/Cutting/Drying
- Deparaffinize slides (remove wax) using xylene.
- Hydrate with a gradient of alcohol concentrations (e.g., 100% to 70%).
- Stain the tissue.
- Dehydrate with a gradient of alcohol concentrations (e.g., 70% to 100%).
- Clear using xylene.
- Mount using resinous mounting material.
Reasons for Ascending Concentration in Dehydration
- 70-95-100% Alcohol prevents tissue shrinkage, hardening, and distortion from turbulence.
- The different specific gravities of water and alcohol prevent tissue layers from separating during dehydration.
Delicate Tissues
- Delicate tissues may require lower initial alcohol concentrations (e.g., 35%, 50%, 60%, 75%, 95%, 100%) to avoid damage.
- Manual processing or specialized processor programs are used.
Microwave Dehydration
- Microwave dehydration can reduce the time needed for dehydration but may lead to tissue distortion.
- Only one step is needed.
Closed Processors
- Closed processors typically start at approximately 60% alcohol concentration.
Common Dehydrating Reagents
- Ethyl alcohol is the most common dehydrating agent.
- Other common agents include isopropanol, acetone, methyl alcohol, and denatured alcohol.
Ethyl Alcohol
- Advantages: non-toxic, mixes well, slight shrinkage, good for delicate tissues (eyes, embryos), fast-acting, reliable.
- Disadvantages: cost, prolonged exposure can cause shrinkage and hardening, record-keeping and theft prevention issues, some dye extraction issues.
Safety - Alcohols
- Alcohols are flammable.
- Ethyl, methyl, and 2-propanol are potential drinking hazards.
- Violent reactions can occur with oxidizing agents like silver nitrate for concentrations >24%.
Alcohol Safety Concerns
- Alcohols are flammable and can cause violent reactions with oxidizing agents.
- 24%+ concentrations require special disposal.
Isopropanol
- Advantages: good penetration, less shrinkage, affordable.
- Disadvantages: not very good for celloidin embedding. Eosin is insoluble in 2-propanol.
Acetone
- Advantages: fast, inexpensive, does not remove dyes from stained sections.
- Disadvantages: needs more volume, flammable, low flash point, excessive shrinkage, volatile, evaporates, and melts coverslips.
Methyl Alcohol
- Works well but may cause blindness or death
Denatured Alcohol
- Works well, but not subject to alcohol taxation. Security measures are not needed. Record keeping is avoided.
Dehydration - Verification
- Adding alcohol to xylene-water mixture turns white.
- Checking specific gravity determines the water content. (Desiccation with calcium or copper sulfate is an option, though not always used)
Added to Dehydrant
- Phenol, mollifex (glycerin/alcohol mixture), or dish detergent (DI water) helps maintain tissue flexibility for optimal sectioning.
- Eosin helps with embedding.
Common Clearing Reagents
- Xylene.
- Toluene.
- Benzene.
- Chloroform.
- Acetone.
- Essential Oils (like cedarwood)
- Xylene substitutes (e.g., limonene, clearite)
Universal Solvents
- Advantages: speed.
- Disadvantages: toxicity, cost, and unpleasant odor.
- Dioxane, tertiary butanol, and tetrahydrofuran are examples.
Clearing (Closed Processor)
- Clearing, also known as dealcoholization, removes the alcohol from the tissue and replaces it with a clearing agent.
Clearing Agents
- Clearing agents alter the refractive index of tissue.
- Leaving clearing agents in contact with tissue too long can cause issues (e.g., incomplete clearing, paraffin infiltration problems, resulting in mushy blocks).
Criteria for Choosing Clearing Agents
- Speed of dehydrant removal, ease of removal by molten wax, and minimal tissue damage, flammability, toxicity and cost are important factors.
Xylene Advantages
- Speed; clear end result, good for celloidin, does not remove aniline dyes.
Xylene Disadvantages
- Toxic, makes tissues hard, hazardous waste, solvent recycling, and licensed waste disposal are needed.
Xylene Safety
- Ventilation, gloves, and flammable precautions are necessary. Automation of coverslipping reduces exposure.
Toluene
- Advantages: less hardening of tissue, especially nerve and brain tissues, clear endpoint, speed, considered best by many.
- Disadvantages: toxicity, headache, dizziness, dermatitis, and lung injury.
Benzene Advantages
- Speed, clear endpoint, hardens better than xylene, leaves paraffin readily available, and less shrinkage.
Benzene Disadvantages
- High volatility, proven carcinogen.
Chloroform
- Advantages: less hardening than xylene, not flammable, good for hard tissues, good penetration, works for celloidin.
- Disadvantages: absorbs water, slow, volatile, damages vacuum pump membranes, and disposal problems.
Chloroform Disadvantages
- Violent reaction with acetone, no clear endpoint, and smelly.
Acetone
- Advantages: boils off at 58°C, less wax contamination and changing, dehydrates and clears tissue.
- Disadvantages: shrinkage, best graded with xylene (needs careful handling), volatile, flammable, low flash point, and may interfere with automatic coverslipping.
Essential Oils
- Advantages: quick clearing, less hardening than others, low evaporation.
- Disadvantages: expensive, must be removed with xylene, has a strong smell, oil residue can make cutting difficult.
Limonene
- Advantages: hardens tissues less than xylene, citrus odor, more paraffin changing.
- Disadvantages: greasy, same waste disposal concerns as xylene, potential mounting issues.
Aliphatic Hydrocarbons (Alkanes)
- Advantages: low in toxicity, combustible, gentle on tissue.
- Disadvantages: intolerant to water, not compatible with all mounting media, requires more processor/staining stations, and changes to lab routines may be necessary.
Xylene Substitute (Clearite)
- Advantages: low in toxicity compared to other options, gentle on tissue.
- Disadvantages: intolerant to water, potential water contamination via humidity, not compatible with all mounting media (coverslipping), and requires more processor stations.
Infiltration
- Infiltration uses a supporting medium to hold cells and intercellular structures in proper relationships during sectioning.
- Important in supporting cells for proper relationships for thin sectioning.
Common Infiltration Medias
- Paraffin wax (most common), water-soluble wax, celloidin, plastics, epoxy resin.
Paraffin
- Advantages: cheap, easy to handle, relatively easy section production, and quicker processing times.
- Other advantages: allows serial sectioning, many staining procedures, safe to handle, and can be stored directly after cutting.
Paraffin Additives
- Beeswax increases stickiness.
- Rubber reduces brittleness.
- Other waxes provide a smoother texture and smaller crystals.
- Plastic adds hardness and support.
Proprietary Paraffin
- Advantages: increased melting temperature for hard wax, thinner sectioning, and ribboning is difficult.
- Disadvantages: decreased temperatures yield softer wax, better for ribboning but harder to get thin sections and less tissue support.
Paraffin (Tissue Processor)
- Using a short processing time and proper temperature monitoring.
- Avoid any exposure between the clearing agent and paraffin (contaminating the wax).
Paraffin under Pressure
- Speeds up processing.
- Removes residual air.
- Might damage small biopsies.
Tissue Processors
- Open processors (e.g., "dip-and-dunk"): require longer time, more hazardous, and more prone to error if steps are skipped in the processing steps (dipping into the wrong substance, etc).
- Closed processors (e.g., fluid transfer): most common, uses solution pumping into/out of the retort (section holder) in a specific order. Vacuum and heat are often applied.
Microwave Processor
- Quickens the processing time but requires proper fixation prior to the commencement of the process. Isopropyl alcohol is a good substitute for the clearing reagent.
Manual/Hand Processing
- Reserved for high-priority specimens or urgent procedures, or high-risk specimens (e.g., Prons).
Processor Protocols
- Processors are typically programmed with tissue/embedding readiness in mind.
- Protocols use different times based on different tissue/lab requirements. Includes whether pressure and/or heat is applied.
- Protocols should be followed.
Processing - Sample Protocol
- Table of different stations, reagents, times, heat settings, and vacuum/pressure cycles for a fluid-transfer processor.
Routine for Changing Reagents
- The first station of each reagent is typically the most contaminated. Discard the first station and rotate the others. Fresh reagents are typically added to the final stage of the procedure.
Potential Contaminants
- Alcohol stations can be contaminated from water coming from the tissue/air.
- Clearing stations become contaminated with alcohol or water.
- Paraffin stations are possibly contaminated with clearing agent or alcohol.
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