Processing Flashcards
What are the steps in tissue processing?
- Fixation
- Decalcification (if required)
- Dehydration
- Clearing
- Infiltration
- Solidification
What factors affect processing?
- Agitation - increased solution mvmnt+ increase reagent flow into specimen
- Heat - increase temp = increase rate of penetration and fluid exchange
- Vacuum - removes reagent in exchange for next reagent (removes trapped air and good for dense/fatty tissue)
- Viscosity of reagents - smaller molecules = less flow resistances into tissue = lower viscosity and reagents permeate quicker
Fixation
- stabilizes and hardens tissue with minimal cellular distortion
-inadequate cross linking/coagulation = macro molecular rearrangement during subsequent steps ~ smudgy nuclei, nuclear bubbling etc
Decalcification
- removes mineralization of calcium in tissue (only performed on specimens with a large amount of calcium)
- 20:1 ratio of volume : tissue
What are the factors that determine the decalcifier?
- urgency
- size
- degree of mineralization - how hard is it?
- scope of investigation - can we put it in molecular studies?
Types of decalcifying techniques
- Acid : strong acids (HCl, nitric), weak acids (formic), ion-exchange resin, electrolytic methods
- Chelation : EDTA, preserves DNA/RNA for subsequent molecular studies
- acetic and picric acid will decal microcalcifications during fixation
Dehydration
- pulls free water from tissues
- most infiltrating media is immiscible with water
- if tissue is not fully dehydrated = clearing agent can’t penetrate = infiltrating media cannot penetrate/solidify tissue properly = under-processed tissue
- don’t want to overdehydrate by removing naturally bound h20 = micro chatter
What reagents are used in dehydration?
Alcohols, acetone and universal solvents
How do you select dehydration reagent?
- Agent selection: must be miscible with the reagent the tissue is in previously and following
- Tissue type: delicate tissue should start in lower levels of alcohol (30-50%) and normal in higher levels (70-80%)
- Fixation: non-aqueous fixative tissues should start in 100% (prevents water entering tissue thru osmosis/hypertonicity)
How do you store dehydrated tissue?
Can safely store in 70% alcohol without morphology changes
- anything lower, tissue will swell
-anything higher, tissue will shrink and become brittle
1: Properties of Ethanol
- clear, colorless and flammable
- not safe to consume as reagent due to addition of isopropanol and methanol
- miscible with water and may contain 1-2%
- used in gradations
- can add eosin/phloxine to 100% to dye tissues pink for better visibility in embedding and microtomy
- may cause autoflorescence
- Properties of Methanol
- aka wood alcohol
- evaporates quick, unpleasant odor, toxic
- rarely used as a reagent
- used in hematology and cytology as fixative - blood smears, cyto preps, Diff-quik staining
Poisonous thru skin; ingestion = blood stream = liver= formaldehyde + formic acid = blindness and death
- Properties of isopropanol
- retains moisture ~ 1% water in it
- doesn’t overharden tissue
- good substitute for ethanol for paraffin infiltration (cannot be used for celloidin technique, not miscible with nitrocellulose)
- cannot be used for some staining solutions = eosin
- mild irritation and toxicity if ingested
- Properties of butanol
- not common in lab
- requires longer dehydration time = acts slower
- has pronounced odor
- good for animal and plant work
Alcohol substitutes for dehydration
- Ethylene glycol - cellosolve
- not easily found
- rapid dehydrant
- not used in graded percentages - Propylene glycol ether (doesn’t fix tissue)
- pro-soft
-not used in graded %
- will not remove bound water = easier for microtomy
- will not fix tissue and cannot be used for staining, not in processing
- Properties of Acetone
- Rapid dehydrant
- but can cause excessive shrinking and hardening
- easily removed by clearing agents - Rapid evaporation ( watch volumes closely)
- Less expensive than alcohols
- Volatile
What are universal solvents?
They are reagents that dehydrate and clear (2 in 1)
- cannot be used with delicate tissue
Common reagents are: (all toxic)
A. Dioxane
B. Tertiary butanol
C. Tetrahydrofuran (THF)
Describe dioxane
- faster than ethanol
- less shrinkage than ethanol; leave in tissues for longer periods than ethanol
- if water is left in tissue = more shrinkage
-very toxic, pronounced odor, carcinogen
Describe tertiary butanol
- pronounced odor
- expensive
- may solidify at room temp
Describe tetrahydrofuran
- miscible with many solvents, water and paraffin
- acts rapidly w/o shrinkage or hardening
-least toxic of group but can cause dermatitis and conjunctivitis
What is a clearing agent?
- called that because it made tissue look translucent
-removes alcohol and sets tissue up for infiltration; must be done with proper dehydrant for adequate infiltration - clearing agents remove lipids
- don’t leave tissue in for too long or else microchatter will occur
How do you pick reagents for clearing?
- Speed at which it removes dehydrate
- Ease of inliftration
- Flammability
- Toxicity
- Cost
Need to be disposed of by hauling away
Refractive index
Tissues with different refractive indices (RI) will have light bend and it will appear hits
Tissues with similar RIs will appear clear/translucent
Name the clearing reagents:
- aromatics hydrocarbons : benzene, xylene, toluene
- chloroform
- essential oils
- limonene
- aliphatic hydrocarbons
- universal solvents (dual role of dehydration and clearing)
Properties of xylene
- mixed isomer, hydrocarbon
- clears quickly to endpoint
- time in reagent needs to be controlled, can easily harden tissue
- toxic (neurotoxin that can cause a headache, dizziness, lack of coordination, mental confusion
OSHA PEL = 100 ppm, STEL = 150 ppm
Properties of toluene
- Aromatic hydrocarbon, similar to xylene
- clears to translucency and has safety risks
- will NOT overharden tissue (tissue can stay overnight; recommended for *brains, muscle and tendon *
- smaller molecule than xylene so evaporates quicker = more toxic fumes
OSHA PEL= 50ppm
Properties of Benzene
- aromatic hydrocarbon, similar to xylene
- clears to translucency, serious safety risks
- carcinogen of bone marrow, SHA PEL = 10 ppm
- tissue hardening is in between toluene and xylene
- evaporates rapidly - do not need to change paraffin as frequently
Properties of chloroform
- chlorinated hydrocarbon
- penetrates slow but far (needs more time than xylene)
- causes less brittleness than aromatic hydrocarbons (good for uterus, muscle, tendon)
- rapidly evaporates, use in a well ventilated area; toxic= toxic gas, phosgene and is a known carcinogen
- different RI than tissue, will it create translucency
Essential oils
- very slow to clear - used for special research or long term storage
- can go from 95% alcohol to essential oil ( must remove with xylene or toluene before using for microtomy)
- very strong odor, use in well ventilated ares
E.g:- cedarwood
- clove
- sandalwood
- wintergreen
Limonene
- terpene is marketed as a xylene substitute
- by product of citrus (generally regarded as safe; techs can become sensitized to citrusy smell)
- safer than aromatic hydrocarbons
- compared to xylene: slower to clear by 1.5 times, hardens tissues must change paraffin more frequently (guess its bigger molecule)
- similar to xylene - cannot dump down the drain, must be hauled away
Aliphatic hydrocarbons (alkanes)
- xylene substitutes
- proprietary chemicals - alkanes (short chain hydrocarbon (between paraffin and octane)
- safety : non irritating, non sensitizing, low inhalation toxicity
- compared to xylene:
- take 2x as long
- not tolerant of water (rotate alcohols more)
- not miscible with all mounting media ( not recommended for automated coverslippers
- disposal = waste hauler/ recycling
5. Infiltration
Exchanging clearant fluid with fluid media that will be used for embedding and subsequently support upon solidification
Types of infiltrating media (must know)
- paraffin
- water soluble waxes
- polyethylene glycol (PEG)
- celloidin
- plastics — used for electron microscopy and hard tissue
- glycol methacrylate
- epoxy resins
Paraffin
- Most common
- excellent for large output and diff types of tissue
- infiltrates with aid of vacuum (increase infiltration speed)
- commercially bought and has additives
Paraffin additives
A. Beeswax - reduces crystal size and increases stickiness
B. Rubber - reduces brittleness, increases stickiness and ease of sectioning
C. Bayberry wax
Hardness increased by Ceres in, stearic acid, diethylene glycol diesterate, microcrystalline waves, plastic
- each paraffin will vary amount of additives to vary cutting properties (every company is different)
Paraffin properties
- high melting point = harder paraffin; better support for harder tissues and thinner sectors
- more difficult to ribbon
- lower melting point = softer paraffin; less support for harder tissues
~ easier to ribbon
Routine work done with paraffin is usually between 55 - 58 degrees C
- too much time in paraffin - will cause shrinkage and hardening
- paraffin should be kept 2-4 degrees C above melting point
~ in processor, if it goes above, tissue will harden
~ in embedder - cutting properties of paraffin will change
Advantages of paraffin
- cheap
- easy to use
-ribbons well - non toxic
- thin sections ~ 2um (one cell layer)
- easily stored
- works well on automated processors
Disadvantages of paraffin
- requires heat
- removes water
- shrinkage, hardening, distortion (microchatter)
- inactivates enzymes and some antigens that will be required further down the line in IHC f.e
- temp must be controlled
- removes water
- can’t be used for electron microscopy because the sections aren’t small enough
- messy
Polyethelene glycol (PEG) (not used routinely)
- aka carbowax
- water soluble wax
- can go from fixative into graded percentages of alcohol
- have to heat it to melt
- can go form any percentage alcohol into PEG (from 95% don’t have to worry about shrinkage)
- can cut moderately think sections (3-4um)
- doesn’t ribbon as well as paraffin
How to float PEG in waterbath
PEG is water soluble so add:
- Potassium dichromate and gelatin to waterbath
- Diethylene glycol, formaldehyde and carbowax to waterbath
- Place directly on slide and then into oven
How to store PEG blocks
- hygroscopic blocks
~ store in plastic bag with small amounts of desiccant (to absorb water molecules)
Celloidin
- nitrocellulose compounds used from embedding
~ most common = parlodion - process:
- from 95% EtOH tissue is placed in a 1:1 of ether and ethanol
- infilatrated though graded% of celloidin dissolved in the 1:1 ether and ethanol
- starts at 2% and embedded in 12%
- celloidin hardens thru evaporation of ether ethanol solution to the consistency of gelatinous mix, or gum drop then hardened using **chloroform* (good for brain tissue)
- sections held in 80% ETOH until stain and then mounted on glass slides
Advantages
- no heat required (shrinkage and hardening are minimal
- good for brain sections
Disadvantages
- takes a long time (weeks to months)
- cannot cut thin sections or serial sections
- requires explosive reagents
- anhydrous ether
- nitrocellulose
Resins and Plastics
- hardest of the embedding media
- support hard tissues and cut very thin
- types :
- glycol methacrylates
- can support cutting un-decalcified whole bone or ceramic implants (pathos study this to see surrounding infection etc)
- epoxy resins
- cut very thin sections for electron microscopy (600-800 angstroms)
- glycol methacrylates
SOLIDIFICATION
Process of transitioning from a liquid to a solid state
- crystallization (paraffin and carbowax)
- evaporation (celloidin)
- polymerization (resins and plastics)
A. Crystallization
Paraffin and carbowax
- media is solid at room temp - heated to turn to a liquid
- embedding media is called rapidly to create a solid structure with a small crystal size
- small enough for light microscopy
- too large for electron microscopy
- if cooling is not done fast enough - will result in large crystal size
b. Evaporation
Celloidin
- cannot use heat - would prematurely harden media
- takes a long time — days to weeks to months depending on size of block (ether ethanol solution has to evaporate )
C. Polymerization
Resins and plastics
- except celloidin
- liquids are combined together to react and create solid matrix
- electro microscopy — epoxy resins
- heat used to speed up solidification
- histology -glycol methacrylates
- exothermic rxn -heat is generated during polymerization
- usually done on ice to prevent tissue from overheating
Microwave processing
- more commonly used for small tissue biopsies
- can be used for large specimens
- utilizes heat to speed up processing
Fixation —> isopropanol —> paraffin
automated processors and schedules
- can add heat, agitation and vacuum to decrease processing times
- Custom programs can be created for different tissues
- small, short runs for biopsies
- standard runs for larger tissue
- extended runs for fatty tissue - breast and brain
- can use the delays to end process at specific times/days (will hold in fixative until time to start)
- containment of fluids and fumes
- some will even calculate management of reagents so you can replace
- open system = carousel and rotates for replacements
- closed system - tubing sucks agent from retort reagents
Tissue processor QC
- Reagent volumes should be maintained to cover sections in retort chamber
- schedules created on an indiv. Lab basis
- higher vol labs done on reg. basis
- smaller vol labs done after certain # of runs (tissue vol.)
- charts should be maintained to keep tract on how often the machine is run ad how often it is cleaned (hot water flushes)
- solutions should be rotated not dumped; first solution dumped and replaced with new reagent; last 2 pushed forward
- temp of processor, paraffin pots and embedding centers kept (can aft how paraffin ribbons form; can destroy additives if not monitored)
- low vol labs should replace paraffin after a week if not used
- buffering salt precipitate can be removed with cute acetic acid or thru preventative measures, eg how water flashes
Tissue processing schedules
Need minimal time for xylene and paraffin for the following (to prevent over hardening)
- hard tissue
- muscle
- fibrous tissue
- scar tissue
- spleen
- bloody tissue
- small biopsies
- delicate tissue
- animal tissue
Note difference in processing schedules of normal tissue vs biopsies
Biopsies:
- do not have to be fixed/fixed for too long
- less alcohol gradations
- less time spent in graded alcohol solutions
- less time spent in other solutions compared to other tissues;
Biopsies process for 2-3 hrs while normal tissues go 6-8 hrs
Underprocessed tissue causes
- wrong time in processing solutions
- wrong schedule chosen — biopsy vs normal tissue
- mix of routine and biopsy cassettes eg placenta in GI biopsies
- xylene substitute using xylene processing times
- xylene substitutes need 1.5 - 2 times longer
- solutions place in wrong place in processor
- switching of 95% and 100%
- solutions not changed often enough
- carry over from previous solutions; H2O presence can dilute graded alcohols — 100 to 95, 95 to 90 etc
- paraffin contains too much xylene - prevents crystallization
Correcting the prob- fixing underprocessed tissue
- need to go backwards the reprocess
What’s left in the tissue? - xylene - need a fresh change of paraffin
- alcohol - go into paraffin and xylene
-water - go into paraffin, xylene and absolute alcohol - several methods; where is it decided?
- embedding, microtomy or have pathology decide
- reprocessing will look better than original specimen but not as good as getting it right the first time
