Processing & Embedding

Graded alcohols

A gradual increase in the percentage of ethanol in the dehydration step. These graded alcohols reduce the tissue shrinkage that occurs in the dehydration process.

Characteristics & functions of plastics in infiltration

ACRYLIC RESIN MISCIBLE W/WATER, infiltration after dehydration w/95% alcohol. GMA converted to a crystal-clear, hard solid by polymerization. Excellent support for VERY HARD TISSUES such as undecalcified bone. Glass knives are used for good thin sections. Cellular detail is well preserved & distortion minimal. Useful for kidney, bone marrow, & lymph node biopsy specimens. Embedding medium is not removed making staining difficult. Receptive to some enzyme techniques. GMA sections do not adhere well to glass slides. The catalyst benzoyl peroxide is a strong oxidizer; heat, shock, or contact w/other materials should be prevented b/c could result in fire or explosive decomposition.

QC of the tissue processor

Adequate reagent volume must be maintained, reagents need to be rotated or changed frequently & temperature must be monitored daily.

Factors influencing processing

Agitation, choice of fixative, pressure, reagent type & quality, temperature & time.

Agitation

Allows for greater exposure of fresh processing reagents around the tissue samples, this can greatly reduce the amount of time needed to process the tissue, (reduce by 30% or more).

Closed tissue processors: advantages & disadvantages

An advantage is the specimens can not dry out in the tissue chamber. A disadvantage is that not all types of reagents can be used on this processor, (mercury-dichromate or dichromate containing fixatives and chloroform).

Paraffin additives & functions

Beeswax (reduces crystal size & increases stickiness & adhesion), rubber (reduces brittleness, increases stickiness, & makes the formation of ribboning easier), other waxes (produce smooth texture & smaller crystal size), & plastics (increase hardness & support). They provide support for hard tissues & form ribbons at the microtome pushing the preceding section away from the knife edge w/each new section cut.

Characteristics & functions of methanol in dehydration

Blood smear fix, rarely used in dehydration, flammable, clear, overexposure can cause blindness or even death, PEL of 200 ppm.

Characteristics & functions of acetone in clearing

Boils off provided paraffin baths are kept at 58°C or higher, used like a universal solvent, shows more shrinkage than xylene.

Orienting large, dense specimens

Bone, prostate, uterus are commonly embedded flat but at an angle to prevent tissue from pulling out of the block, reduce vibration, and avoid compression during sectioning.

Characteristics & functions of water-soluble waxes in infiltration

CARBOWAX, are solid polyethylene glycols that are water soluble & used only for special projects. Infiltrate tissues directly from aqueous fixatives. Dehydration & clearing are unnecessary fat will not be dissolved & can be demonstrated on embedded tissue sections. Will not infiltrate tissues w/large amounts of fat. Tissues of the CNS require long periods of time for impregnation. Well-fixed tissue is suitable & usually 3 changes of wax for a total of 3 hours. Some enzymes remain active & these waxes will remain softer than embedded paraffin. FLOATING OUT of sections; sections dissolve on the usual flotation bath causing diffusion currents, disruption, & disintegration of tissue. The wax is hygroscopic.

Characteristics & functions of aviation gasoline in clearing

Carbon tertachloride, carbon bisulfide & aviation gasoline have been used for clearing but their associated hazards are so great that they are not used.

Characteristics & functions of essential oils in clearing

Clove, origanum, sandalwood, & cedarwood; expensive & volatile, if oil remains in tissue, microtomy is difficult. Removed by xylene or toluene before infiltration. Cedarwood is most common & will not cause further shrinkage, clear tissue after dehydration w/95% alcohol, can remain in cedarwood indefinitely. More steps associated due to removal with xylene or toluene increasing overall time of processing.

Infiltration

Displaces the clearant from the tissue to make room for the supporting media whose purpose is to hold the cells in their proper relationships to each other & prevent distortion of the tissue elements during microtomy.

Characteristics & functions of toluene in clearing

Doesn't harden as much as xylene, best of aromatic hydrocarbon clearing agents, greater tolerance for atmospheric water contamination than xylene. Uneven H & E & poor nuclear chromatin patterns was a result of incomplete clearing, when humidity is high, hygroscopic substances such as absolute ethanol absorb more water from atmosphere to reach equilibrium. Volatile, more than xylene & flammable, PEL of 50 ppm.

Resolutions of processing errors

Ensure no condensation in processor or mechanical problems, ensure a good schedule of reagent rotation is developed & maintained, process biopsy specimens separately from larger tissues, decrease time allowed in dehydrating solutions.

Characteristics & functions of isopropanol in dehydration

Eosin is insoluble so not a good substitute for ethanol for staining. Does not harden or shrink like ethanol, contains 1% water always, can not be used in celloidin, PEL of 400 ppm.

Processing for electron microscopy

Epoxy resins which require dehydration & unless miscible w/ethanol, they require use of a transitional fluid (similar to clearing agent in paraffin processing, propylene oxide). Common epoxy resins are Araldite, Epon & Spurr & are cut with a diamond knife to 60-90 nm.

Dehydration: Repeated dilution

Exposing the tissue to multiple changes of the same, fresh reagent which over time removes all the water.

Characteristics & functions of benzene in clearing

Fast acting & does not overharden, but it hardens muscle, tendon, & uterus more than toluene. Evaporates fast from bath & volume is difficult to maintain. Carcinogen, primarily affecting blood & bone marrow, PEL of 10 ppm.

Routine tissue processing steps & reagents

Fixation using 10% NBF, dehydration using graded alcohols; 70%, 80%, 95%, 95%, clearing using xylene, & the last step is infiltration of paraffin.

Choice of fixative

Fixing the tissue prior to processing can reduce steps in processing.

Special techniques & temperature

For IHC techniques that can be done on paraffin-embedded tissue, paraffins with lower melting points have become popular b/c heat may inactivate the antigens.

Orienting epithelial surfaces

Gastrointestinal tissue, skin, bladder, must be embedded on edge so the epithelium can be seen in relationship to the underlying connective tissue and with the epithelium facing the same direction in the mold.

Processing for enzyme histochemistry

Glycol methacrylate (GMA) is used which is on acrylic resin that is miscible w/water but is usually performed after dehydration of tissue w/95% ethanol. GMA is converted to a crystal-clear hard solid by polymerization.

Characteristics & functions of butanol in dehydration

Good for dehydrating plant & animal material, causes less shrinkage & hardening than ethanol, excellent dehydrant but takes a long time, PEL of 100 ppm.

Paraffin MP's

If the MP is too high the paraffin becomes harder & provides more support but ribboning becomes difficult, if the MP is too low the wax is softer & provides less support but ribboning is easier. Paraffin should be matched to the hardness of the tissue, the temperature at which sectioning is done, & the stain to be used.

Time

Inadequate time can result in under processed tissue.

Temperature

Increased temperature can decrease processing time but excessive exposure to heat can compromise tissue morphology.

Orienting inked specimens

Inked side of specimen should be facing up when embedding

Role of eosin in tissue processing

It is added to the alcohols on closed tissue processors to dye the tissue a light pink. Eosin can be added to ethanol but NOT ISOPROPANOL. The reasoning for this is due to the fact that small biopsy specimens are often difficult to identify at embedding station.

Tissue size & processing

Larger tissue size increases the amount of time necessary to process the tissue.

Characteristics & functions of chloroform in clearing

Leaves tissue less brittle than xylene, penetrates slowly, readily absorbs atmospheric pressure, dessicate connective tissue, better for uterus, muscle, & tendon. Volatile but not flammable or combustible. Heating chloroform may cause formation of phosgene, a toxic gas. PEL of 50 ppm, does not make tissue transparent.

Characteristics & functions of universal solvents: dioxane

Less shrinkage during dehydration than ethanol, faster dehydrant than ethanol, frequently contains water & if left can cause 50% shrinkage during infiltration. Expensive, reclaimed by 18-22 hours w/anhydrous calcium chloride or calcium oxide. Flammable & suspected carcinogen, PEL of 100 ppm.

Substitutes for ethanol

Methanol, isopropanol, butanol & acetone.

Poorly processed tissue at microtomy

Microchatter, thick & thin sections.

Characteristics & functions of universal solvents: tetrahydrofuran

Miscible w/lower alcohols, water, ether, chloroform, acetone, benzene, toluene, xylene, & melted paraffin. Rapid without causing excessive shrinkage or hardening & is the best of the universal solvents. Conjunctivitis & dermatitis are results of overexposure. Volatile w/a flash point of -14.5°C & the lower the explosive limit in air is 11.8%, PEL of 200 ppm.

Characteristics & functions of paraffin in infiltration

Most popular, short time serial sections are easily obtained, & routine & most special staining can be done easily. Mixture of hydrocarbons produced by cracking of petroleum. Additives such as beeswax, other waxes, rubber, & plastics, enhance the ability of paraffin compounds to provide support for hard tissues. As melting point increases the paraffin becomes harder & provides better support for hard tissues, vice versa. MP decreases, thin sections become more difficult to obtain but ribboning becomes easier. Lowering MP's have become popular b/c heat may inactivate the antigens. MP of paraffin for routine work is 55°C-58°C. Aided by vacuum but vacuum & heat on small specimens results in overhardening. Overnight processing for large & fatty tissues.

Orienting loose material

Multiple specimens must be arranged in an organized pattern horizontally & in a line.

Orienting multiple pieces

Multiple specimens must be arranged in an organized pattern horizontally & in a line.

Characteristics & functions of aliphatic hydrocarbons in clearing

Newest class, alkanes, low in reactivity & toxicity, light weight (short chain), aliphatic hydrocarbons used in histotechnology b/c they penetrate faster, remove fat more effectively, intolerant to water & incompatible with some mounting media. Difficult in high humidity b/c atmospheric moisture absorbed in alcohol preceding clearing agent, less aggressive than xylene. PEL of 300 ppm.

Characteristics & functions of celloidin

Nitrocellulose compound use for embedding, rarely used except in research & neuropathology, paraloidin is most frequently used, any fixative can be used. Dehydration w/95% & absolute ethanol, treatment of equal parts of absolute & ether. Infiltration begins w/a 2% solution & carried through graded solutions up to 12%-14% celloidin (considered thick celloidin). Embedded in 12%-14% & celloidin is allowed to thicken by slow evaporation until it has the consistency of a gum drop. Hardened w/chloroform & cut either wet or dry. Wet, the block, knife & sections are kept wet w/80% alcohol. Dry, infiltrated by cedarwood before sectioning & then may be cut dry. Both methods, the cut sections must be kept wet w/80% alcohol until stained. Hand stained & mounted on slide just before clearing w/xylene. Heat not required & shrinkage & hardening are minimal. Takes weeks or even months. Anhydrous ether & nitrocellulose reagents are EXPLOSIVE.

Characteristics & functions of 30% sucrose in infiltration

Obtaining frozen sections from formalin-fixed, unprocessed tissue infiltrate w/this before it is frozen, cryoprotectant & a high-quality frozen section can be obtained, staining is easily performed w/H & E & fat stains, dried well before adhesion.

Characteristics & functions of xylene in clearing

Overhardens, especially in fibrous, muscular, CNS, or cartilaginous tissues. Rapid displacement of alcohol & miscible w/paraffin, intolerant to any water left in tissue, xylene turns cloudy in presence of water. Flammable, is a neurotoxin, headaches, dizziness, lack of coordination, mental confusion & fatigue are short-term, damage to CNS in repeated exposure, is also a defatting agent, PEL of 100 ppm.

Universal Solvent

Perform both dehydrating & clearing functions & avoid the need for both the dehydrating & clearing reagents, (Dioxane, tertiary butanol, tetrahydrofuran).

Poorly processed tissue at microscopy

Poor nuclear staining.

Reagent type & quality

Poor reagent selection can lead to loss of tissue components and/or unwanted artifacts and reagent quality may result in poorly processed tissue.

Pressure

Pressure pushes the processing reagents into the tissue allowing for faster movement of chemicals deep into the tissue sample.

Characteristics & functions of ethanol in dehydration

Reliable & fast-acting, mixes with water, best to start with 60% ethanol & then increase, if phosphate-buffered formalin is used for fixation, salts will precipitate if alcohol solution is greater than 70%. Causes excessive shrinkage & hardening, PEL of 1,000 ppm. Denature methanol &/or isopropanol.

Processing

Removes all of the free water from the sample and replaces it with a media that will provide support for the tissue structures.

Clearing

Removes the dehydrant from the tissue in preparation for infiltration.

Characteristics & functions of epoxy resins in infiltration

Require dehydration, & unless miscible w/ethanol, they also require a transitional fluid. Final embedding mixture contain epoxy resins, hardeners, & catalysts; polymerization is done at approx. 60°C. Used when EM or ultrastructural examination is desired. Diamond knife for EM & glass knife for light microscopy.

Characteristics & functions of agar & gelatin in infiltration

Single block of friable tissue or multiple fragments. Washed over night, impregnated for 24 hours w/12.5% solution of gelatin at 37°C & impregnated again for 24 hours w/a 25% solution at 37°C. Block will harden in fridge then further in 5% formalin for 24 hours.

Poorly processed tissue at embedding

Soft mushy tissue or hard, brittle tissue.

Characteristics & functions of universal solvents: tertiary butanol

Solidify at room temperature, expensive, half tertiary butanol & half paraffin used for initial paraffin infiltration, PEL of 100 ppm.

Hygroscopic

Taken up & retained under some conditions of humidity & temperature, take in water.

Orienting cystic structures

The edges of the dome are properly embedded down in the mold creating a concave appearance to the completed tissue block, flat side down.

Processing reagent maintenance

The frequency that the reagents must be rotated or changed is dictated by the number of cassettes processed or the use of a hydrometer to measure the water content within the alcohols on the processor. If this is not properly maintained & rotated & changed, this can result in poorly processed tissue.

Tissue size & constituents

The greater the size of the samples the longer it takes to process them. Calcified tissue may require additional time on the processor to manage the decal. solution.

Viscosity

The lower the viscosity of the reagents means the molecules are smaller & therefore can enter the tissue more freely.

Dehydration: Hydrophilic properties

The reagents possess strong polar groups that attract water & remove it from the tissue.

Temperature "rule of thumb" for the tissue processor

The reagents should be kept at 45°C & the paraffin should be set at 60°C.

Dehydration

The removal of water, a process necessary to prepare the tissue for embedding in a nonaqueous medium such as paraffin, celloidin, & some plastics.

Closed tissue processors: function & maintenance

The tissue is stationary & fluids are pumped in & out of the pressurized chamber holding the tissue. Diffusion of various substances into & out of stabilized porous tissues. Routine reagent rotation &/or change cycle determined by usage and number of cassettes.

Microwave processor

The waves of NRG generated from the microwaves create friction that heats things up much like a traditional processor only faster. It uses 3 reagents only, ethanol, isopropanol & paraffin taking approx. 45 minutes. Fix tissue for 30 min., place on the agitator or mechanical stirrer, rinse cassettes with water to eliminate possibility of salt precipitation, place cassettes into microwave leaving top rack empty to allow for evaporation. Place rack in plastic container & fill w/100% ethanol to rinse off water then fill w/fresh 100% ethanol. Place container in microwave, microwave at 67°C for 5 min., remove & empty & fill with isopropanol, put back in microwave at 74°C for 3 min., empty & fill w/paraffin at 60°C, agitate rack to remove excess isopropanol, dump paraffin in vat & refill w/paraffin. Place back in microwave at 65°C for 2 min., during this, open door & agitate several times, then increase temp. to 84°C for 5 min.

Embedding orientation: flat

These tissues are consistent throughout their histologic structure (e.g. liver, spleen). The orientation is intended to see the most surface area of the specimen. Orient these such that they are evenly pressed (gently for those that are delicate) to the bottom of the embedding mold.

Embedding orientation: on end

These tissues will have a lumen in the center of a long tube (e.g. arteries, vas deferens). Orient these such that you see the center of the tube (lumen) with the concentric layers around it.

Embedding orientation: on edge

These tissues will have multiple layers to them running in close to parallel to each other (e.g. small intestine, skin). Orient these such that you can see each of the distinctive layers within the sample.

Runback procedure

Tissue is reprocessed beginning by melting the paraffin from the tissue & working backwards through xylene & absolute. This should be applied when the tissue is underprocessed & tissue may smell of xylene.

Embedding in same plane

Tissue must be embedded in the same plane meaning tissue should be pressed against the bottom of the mold to ensure that all of the tissue is represented on the slide.

Tissue & temperature

Tissues can shrink & become brittle at higher temperatures & can negatively the staining quality.

Importance of cooling rapidly in embedding

To obtain the smallest paraffin crystal size possible, b/c the crystal size affects sectioning quality. Small crystalline structure will allow the wax to fit closely to the embedded tissue.

Substitutes for xylene

Toluene, benzene, chloroform, acetone, essential oils, limonene reagents, aliphatic hydrocarbons, & universal solvents.

Cause of processing errors

Underprocessed or overprocessed tissue, overdehydration, too much heat or improper QC of processor.

Vacuum

Vacuum removes air bubbles from porous tissue components allowing the processing reagents to move in & out of the tissue with less resistance. This speeds up reagent penetration & reduces the amount of time to process the tissue. Average vacuum is 15 mmHg

Characteristics & functions of acetone in dehydration

Very flammable w/ a flash point of -17°C, its PEL is 1,000 ppm. Rapid-acting & less expensive, causes excessive shrinkage, if exposed to air it will absorb water. Volatile & difficult to maintain proper levels.

Substitutes for paraffin

Water-soluble waxes, celloidin, plastics (Glycol Methacrylate/GMA & epoxy resins), agar & gelatin, & 30% sucrose.

Characteristics & functions of limonene reagents in clearing

Xylene substitute, have an overpowering citrus odor, harden less than xylene but more paraffin contamination. Repeated exposure predisposes individuals to allergic reactions, not water-soluble.