Tissue Processing Flashcards
What is histology?
The study of the structure of cells and tissues microscopically that demonstrates disease processes and the effects on tissues.
Histology is essential for understanding how diseases affect tissue structure.
What is pathology?
The study of diseased tissues and conditions as well as the process of diseases.
Pathology plays a critical role in diagnosis and treatment.
List the key tissue preparation techniques.
- Fixation
- Accession
- Grossing
- Processing (dehydrate, clear, infiltrate)
- Embedding
- Paraffin sectioning
- Frozen sectioning
- Staining
Each technique is crucial for preparing tissue samples for microscopic examination.
What is the purpose of fixation in tissue processing?
To maintain tissues in as life-like state as possible and harden tissues.
Fixation is a critical first step in preserving tissue morphology.
What is accession in tissue processing?
The verification of samples and requisition; entry into LIS (Laboratory Information System) where a unique histology number is assigned.
Accessioning ensures accurate tracking and management of specimens.
Define dehydration in the context of tissue processing.
The removal of ‘free’ water from tissues, which is not bound to tissue components, through diffusion.
Dehydration is essential for allowing paraffin to infiltrate the tissue.
What are common dehydrating agents used in tissue processing?
- Ethanol
- Isopropyl alcohol
- Acetone
Each agent has specific properties affecting the dehydration process.
What is the main disadvantage of using ethanol as a dehydrating agent?
Alcohol fumes may ignite.
Safety precautions must be taken when using flammable agents.
What happens if dehydration is incomplete?
The clearing agent will not act properly, yielding soft, mushy blocks.
Proper dehydration is crucial for successful tissue processing.
What is the purpose of clearing in tissue processing?
To remove alcohol from tissue and prepare it for infiltration with wax.
Clearing is essential for ensuring that tissues can accept the embedding medium.
List the consequences of improper clearing.
- ‘Moth eaten’ appearance in tissue
- Sections crumble and tend to tear out of the block
Improper clearing can severely affect the quality of tissue sections.
What are some common clearing agents?
- Xylene
- Toluene
- Benzene substitutes
- Cedarwood Oil
- Chloroform
- Carbon
- Xylene substitutes
Clearing agents have different effects and applications in tissue processing.
What is a universal solvent in tissue processing?
Reagents that perform both dehydrating and clearing steps but are not suitable for delicate tissues.
Universal solvents can lead to tissue distortion.
What factors affect the duration of dehydration?
- Fixative used
- Size of tissue
- Type of tissue (density)
- Amount of tissue
These factors influence how long tissues need to be dehydrated.
True or False: Xylene is miscible with water.
False.
Xylene is insoluble in water, which is important for its role as a clearing agent.
What is the effect of excessive clearing on tissue?
It can cause further denaturation of tissue proteins, making microtomy difficult.
Balance in clearing time is crucial for maintaining tissue integrity.
What is the main advantage of using toluene as a clearing agent?
Tissues may be left up to 12 hours without excessive hardening.
Toluene is preferred for certain delicate tissues due to its slower action.
Fill in the blank: The process of _______ is used to remove alcohol or wax from tissue.
[clearing]
What can happen if tissues remain in high concentrations of dehydrating agents for extended periods?
It interferes with staining and can lead to over-dehydration, resulting in microscopic chatter at tissue edges.
Monitoring exposure times is critical for optimal tissue processing.
What is a major disadvantage of toluene as a clearing agent?
Clears only from absolute alcohol; tissue must be totally dehydrated
Toluene fumes are toxic and it is flammable.
How does toluene affect tissue compared to xylene?
Does not harden as extensively as xylene; clears tissue rapidly
There is a change in refractive index as a result of the clearing process.
What are the advantages of benzene as a clearing agent?
Rapid; tissue rendered less brittle than toluene or xylene; easily removed from wax
Benzene hardens muscle, tendon, and uterus more than toluene.
What are the disadvantages of benzene?
Flammable; very volatile; carcinogenic; extremely toxic; implicated in aplastic anemia
Toxic by inhalation and skin absorption.
What are the properties of cedarwood oil as a clearing agent?
Eco-friendly; causes minimal shrinkage; can clear from 90% alcohol; excellent for fragile tissues
Tissues may be left for prolonged periods without damage.
What is a disadvantage of cedarwood oil?
Very slow penetrating; difficult to remove from wax; very expensive
Must be removed with hydrocarbon prior to paraffin impregnation.
What is a key advantage of chloroform as a clearing agent?
Clears readily from 95% ethanol; not flammable or combustible; useful for large tissue specimens
Causes very little shrinkage and is less brittle than xylene.
What is a significant disadvantage of chloroform?
Difficult to determine the endpoint of clearing; toxic; must be used in a well-ventilated room
Formerly used as an anesthetic.
What are the advantages of carbon tetrachloride?
Properties similar to chloroform; cheaper; very tolerant
Clears tissues up to 5.0 mm in 8 to 15 hours.
What are the disadvantages of carbon tetrachloride?
Very toxic; difficult to remove from wax; endpoint determination is difficult
Considered narcotic, may cause tissue damage.
What is a key characteristic of aliphatic hydrocarbons as xylene alternatives?
Low in reactivity and toxicity; derived from paraffin and petroleum jelly
Can be used on all tissue processors and automated stainers.
What are terpenes?
Naturally occurring hydrocarbons derived from plants; primary constituents of essential oils
Known for their strong aroma and must be used in well-ventilated areas.
What is the purpose of infiltration in tissue processing?
Replacement of clearing agent with wax; allows for tissue hardening suitable for cutting thin sections
Maintains proper relationship of cellular structures to prevent distortion.
What factors affect the duration of infiltration?
Size of tissue; type of tissue; type of clearing agent; type of processing; type of infiltration method; volume of tissue
Vacuum infiltration can reduce time significantly.
What are the properties of paraffin wax as an embedding medium?
Must be free from dust; melts easily; does not harm tissue; inert mixture of hydrocarbons
Preferred melting point is 54°C to 60°C.
What are the advantages of paraffin infiltration?
Same paraffin can be used for infiltration and embedding; process many tissue blocks quickly; easy to obtain serial sections
Tissue blocks can be stored indefinitely once embedded.
What is a disadvantage of paraffin infiltration?
Tissue may undergo distortion due to shrinkage; sectioning difficult at higher temperatures
Overheated paraffin can result in cracked blocks.
What is celloidin?
Generic term for nitrocellulose embedding compound; supports hard or fragile tissues
Used for embedding whole organs and specimens with large cavities.
What is a disadvantage of using celloidin?
Difficult to obtain serial sections and thin sections; rarely used except in research
No clearing agent is required and no heat is used.
What is the initial infiltration solution concentration for tissues using celloidin?
2% solution for 1 week
This is the first step before transferring to higher concentrations.
What is the final concentration of celloidin used for tissue embedding?
12-14%
This concentration is referred to as thick celloidin.
What is the purpose of adding chloroform to the celloidin mold?
Causes the mold to harden
This allows for the cutting of blocks wet or dry.
What are the disadvantages of using celloidin?
- Very slow (4 to 8 weeks)
- Tedious staining
- Sections typically thicker (12 to 15 um)
- Difficult to cut sections smaller than 10 um
- Ribbons cannot be cut
- Blocks must be stored in 80% alcohol
- Explosive due to ether and nitrocellulose
- Highly flammable
- Produces toxic nitrogen oxide when burned
- Rarely used in surgical pathology
These factors limit the practicality of using celloidin.
What is ‘double embedding’ in histology?
Infiltration with both celloidin and paraffin wax
This technique combines the advantages of both methods.
What are the components of water-soluble waxes typically used in histology?
Solid polyethylene glycols
These waxes are graded according to their molecular weight.
List the advantages of polyethylene glycol over paraffin wax.
- Minimal shrinkage
- Easier cutting of thinner sections
- Lipid demonstration
- Enzyme demonstration
- Softer texture than paraffin embedded sections
However, it requires chilling like paraffin.
What are the disadvantages of polyethylene waxes?
- Solubility leading to ‘floating out’ of sections
- Requires aqueous mountant for mounting
- Hygroscopic storage; best sealed with desiccant
These factors limit routine use.
What types of plastics are used for infiltration and impregnation?
- Glycol Methacrylate
- Epoxy Resins
- Agar
- Gelatin
- 30% Sucrose
Each plastic differs in the hardness of the final product.
What is the main application of glycol methacrylate?
Embedding hard tissues like bone
It allows for cutting tissues into very thin sections (1 to 2 microns).
What is the polymerization process for epoxy resins?
Requires a hardener and is done at 60ºC
This results in a hard block suitable for electron microscopy.
What is the primary use of agar and gelatin in histology?
To produce a single block of friable tissue or multiple fragments
They are also used in frozen sections.
What is the role of 30% sucrose in tissue processing?
Cryoprotectant for preparing frozen sections
It results in high-quality frozen sections from formalin-fixed tissue.
What are the requirements for a good automated processor?
- Closed system
- Computerized with digital readout
- Alarm system for mechanical issues
- LIS connectivity
- Ventilation to minimize toxic vapor exposure
- Features like agitation and spinning movements
These ensure effective tissue processing.
What is the significance of monitoring the pH of zinc formalin?
Prevents precipitation in tissue
Maintaining pH below 7.0 is crucial to avoid processing issues.
What is a common artifact seen with over-dehydration in tissue processing?
Chatter artifact
It appears as a washboard appearance on stained tissue.
What is the purpose of embedding in histology?
To orient and support tissue for sectioning
Embedding may also be referred to as casting or blocking.
What is the most popular infiltrating and embedding medium?
Paraffin
It is typically used at temperatures just above the melting point.
What is the critical step during tissue embedding?
Specimen orientation
Proper orientation is essential for quality sectioning.
What is the most critical step in the embedding process?
Tissue orientation
Proper orientation is essential for morphology demonstration and proper sectioning.
What are the purposes of embedding?
- To orient and support tissue in a mold
- To enable attachment to a microtome or cutting device
- Also referred to as casting or blocking
What should be used to fix tissues for lipid demonstration?
Osmium tetroxide or fresh tissue cut using cryostat
Xylene and other solvents will dissolve fats of the tissues.
How should elongated tissues be placed in the embedding mold?
Diagonally across the block
What is the purpose of tissue inking?
To inform the embedding technologist of orientation position
India ink or tattoo ink may be used for inking.
What should be ensured about the margin of embedding medium around the tissue?
It will assure support of the tissue and facilitate ribboning.
What is the recommended way to embed tubular structures?
Provide transverse sections showing all tissue layers and the lumen.
What is the effect of applying light pressure during tissue orientation?
It helps in orienting and stabilizing the specimen.
What is the consequence of embedding multiple specimens at one time?
Only one cassette of tissue is to be embedded at one time.
What happens if the embedding forceps are not wiped between tissue samples?
Increases the chance of tissue contamination and misdiagnosis.
What should be done to blocks after embedding?
Cool rapidly to reduce paraffin crystal size.
What are the advantages of using an embedding system like Tissue Tek?
- Ease of use
- Speed
- Tissue and holder firmly attached
- Blocks can be filed immediately after sectioning
- Permanent identification
What is the ideal temperature range for the paraffin dispenser?
2°C to 4°C above the melting point of wax.
What is the purpose of the refrigerated plate at -5°C in the embedding center?
To quickly cool the embedded specimens and reduce large ice crystal formation.
What is the procedure for embedding tissue in a mold?
- Select appropriate size mold
- Fill warmed mold with molten paraffin
- Use warmed forceps
- Place tissue flat against base
- Press tissue lightly with tamper
- Cover tissue with wax and add labeled cassette
- Cool on cold plate
What is a potential error when the longest side of tissue is closest to the cutting edge?
It may cause the section to compress softer tissues, producing rough sections.
What is the effect of improper cooling on the tissue block?
It can lead to a ‘dish effect’ or cracking of the tissue block.
What should be done if tissue is improperly embedded?
Melt the tissue block in a heated chamber and start over.
True or False: The smaller the specimen, the easier it is to orient.
False
