SOFT TISSUE PROCESSING

Contents Definition Factors influencing rate of processing Stages Specimen handling Paraffin wax embedding Orientation Automated tissue processing Microwave processing Ultrasound tissue processing references

The tissue processing is designed to remove all extractable water from the tissue , replacing it with a support medium that provide sufficient rigidity to enable sectioning of the tissue without parenchymal damage or distortion.

For microscopic examination , tissue specimens must be thin enough to permit the passage of transmitted light and for detailed , morphological examination no more than one cell in thickness. To achieve this end ,tissue slices need to be approx 4-6um in thickness.

Factors influencing rate of processing

agitation Heat Viscosity Vacuum Specimen size ultrasonic

Stages of tissue processing

Fixation Dehydration Clearing Infiltrating embedding

Fixation

Preserving cells and tissue component with minimal distortion is the most important aim of processing tissue sample.

It stabilizes proteins and render the cell and the components resistant to further autolysis by inactivating lysosomal enzymes.

Dehydration

Removal of “free “ unbound water and aqueous fixatives from the tissue components.

Hydrophilic Excessive dehydration hard and brittle Incomplete dehydration impair

penetration of clearing reagent Use of copper sulphate

Dehydration is effected as follows: Dilution dehydrationChemical dehydration- acidified dimethoxypropane or diethoxypropane,

Dehydrating fluids

ETHANOLClear , colorless, flammmable liquid.Miscible with water and other organic solvents

Graded conc 70 % ethanol in water 95 % 100 %Delicate tissue –processing starts 30 %

INDUSTRIAL METHYLATED SPIRIT(DENATURED ALCOHOL)Physical properties same as ethanolEthanol+ methanol(about 1%)+ isopropyl alcohol or combination of alcohol

Methanol

Clear ,colorless and flammable fluid Miscible with water , ethanol and most organic

solvents Highly toxic Can be substituted for ethanol

Propanol ,isopropyl alcohol

Used in microwave processing Doesn’t cause over hardening and shrinkage of

tissue

Butyl alcohol, butanol

Used primarly for plant and animal histology Slow dehydrant causing less shrinkage and

hardening of tissue

Acetone

Rapid in action but poor penetration and cause brittleness in tissue ,if its use in prolonged

It remove lipid from the tissue during processing

Additive

Phenol softening agent for hard tissues such as tendon , nail and dense fibrous tissue and keratin masses.

Phenol (4%) + 95% ethanol Glycerol/ alcohol mixture

Universal solution

No longer used It dehydrate and clear tissue during tissue

processing Dioxane, tertiarty butanol and tetrahydrofuran Not recommended due to hardening properties

Clearing It is an intermediary between the dehydration and infiltration

solutions It should be miscible with both solution Mostly hydrocarbons with refractive index similar to protein CRITERIA Rapid penetration of tissue Rapid removal of dehydrating agents Ease of removal by melted paraffin wax Minimal tissue damage Low flammability Low cost

Boiling point of clearing agents gives an indication of its speed of replacement by melted parffin wax

xylene

Flammable , colorless liquid with characteristic petroleum or aromatic odor

Suitable for less than 5 mm thick (2-4hrs) Overexposure- hardening Recyclable

Toluene

Similar properties to xylene Less damaging More flammable and volatile than xylene

Chloroform

Slower than xylene Cause less brittleness Tissue do not become translucent Non flammable but highly toxic produce phosgene

gas when heated Commonly used in processing of CNS

Xylene substitute

Aliphatic hydrocarbons that exist in short and long chained forms

Differ in number of C atoms in the carbon chain Amyl acetate, methyl benzoate and methyl

salicylate are chiefly used as nitrocellulose solvents in double embedding techniques.

Citrus fruit oil- limonene reagentsNon toxic and miscible in waterDisadvantage- can cause sensitization and have strong pungent odor that may cause headacheMineral deposits such as Cu and Ca may dissolve and leach from tissueExtremely oily and cant be recycledGRASClearene, Hemo-De and Histo-Clear

CPN 30 AND InhibisolLess toxic than xylene , toluene and chloroformMore volatileVapours more uncomfortable than xyleneMaxwell (1978)

CEDARWOOD OILLeast hardening effectsUsed for hard tissue 5-7mm thick (2-5 days) has a role in forensic histopathology in processing the hardened skin margins of electrical burns and bullet woundsFormation of crystalline cedrol in cedarwood oil can be overcome by the addition of 1 ml xylene or toluene to 80 ml cedarwood oil

Embedding is the process by which tissues are surrounded by a medium such as agar, gelatine, or wax which when solidified will provide sufficient external support during sectioning

Infiltration is the saturation of tissue cavities and cells by a supporting substance which is generally, but not always, the medium in which they are finally

Double embedding is the process by which tissues are first embedded or fully infiltrated with a supporting medium such as agar or nitrocellulose, then infiltrated a second time with wax in which they are also embedded embedded

Investment generally refers to the practice of embedding wax infiltrated tissues in another wax, such as Piccolyte-paraffin wax, modified to provide improved tissue support and sectioning qualities.

Vacuum infiltration is the impregnation of tissues by a molten medium under reduced pressure

Infiltrating and embedding reagents

Paraffin wax – mixture of long chained HC Melting point 47-64 C To promote desirable ribboning during microtomy,

paraffin wax of suitable hardness at room temp should be used

compatible

Paraffin wax additives it contain plasticizer or other resins Ex – beeswax Rubber Ceresin Plastic polymer Diethylene glycol disterate Higher m.p than paraffin increase hardness: add stearic acid decrease melting point: add spermaceti or

phenanthrene

Piccolyte 115, a thermoplastic terpene resin added at the rate of 5%-10% to the infiltrating wax,or to the final investing paraffin wax to improve tissue support for thin sectioning and facilitate flattening and expansion of sections on the waterbath

Plastic polymers such as polyethylene wax, added to improve adhesion, hardness and plasticity.

Dimethyl sulphoxide (DMSO) reduces infiltration times and facilitates thin sectioning. DMSO scavenges residual transition solvent and alters tissue permeability by substituting for or removing bound water thus improving infiltration

Alternative embeddding media

Processing reagents remove or destroy tissue components that are object of investigation. Ex lipids

Sections are required to be thinner, e.g. lymph nodes

The use of heat may adversely affect tissues or enzymes

The infiltrating medium is not sufficiently hard to support the tissue

Resin

Used for EM

Ultrathin sections for higher resolution and also for undecalcified bone

Agar alone does not provide sufficient support for

sectioning cohesive agent for small friable pieces of tissue

after fixation

Agar Embedding Medium Agar-Paraffin Wax Double Embedding For

Fragments, Biopsies And Friable Specimen Agar-Paraffin Wax Double Embedding For Bone

Marrow Aspirates And Cell Suspensions Using The Collodion Bag Technique

AGAR - ESTER WAX DOUBLE INFILTRATION

Gelatin

Gough Wentworth technique and in frozen section Rarely used

Celloidin

celloidin has rubbery consistency allowing a continous shearing type of cutting which is beneficial for delicate tissue

Dehydration – impregnation with LVN in solution – solvent will evaporate –produce block

Water soluble waxes

Eliminating the necessity of dehydration and clearing

Thus avoiding shrinkage

Synthetic resins

Used for ultra thin sections for EM 0.5-2 micrometer Glass knife used Methacrylate and epoxy resins Also used for bone and teeth

Specimen handling

Specimens should not be tightly packed into processing cassettes or containers, but should have sufficient free space to facilitate fluid exchange.

Small specimens and tissue fragments are processed in fine mesh containers, wrapped in lens tissue, sandwiched between sponge biopsy pads or more safely, double embedded in agar-paraffin wax

Specimens are generally identified by a numbering system that is not bleached by subsequent fluid and solvent treatment

Tissue marking

India ink Silver nitrate artist’s grade pigments Particulate pigments Alcian blue Eosin, erythrosin, and rose bengal

Paraffin wax embedding

Orientation

It is important for demonstration of proper morphology

Products(marking system)- tattoo dyes , biopsy bags , sponges and papers

Time of impregnation

Size and type of tissue Clearing agent employed Use of vacuum embedding oven

Leuckhart ‘s pieces

Ice trays

Paper boats

Embedding cassettes

Automated tissue processing

The basic principle of tissue processing requires the exchange of fluids using a series of solutions for a predetermined length of time in a controlled environment

12 stage cycle

TISSUE-TRANSFER PROCESSORS

These processors are characterised by the transfer of tissues, contained within a basket, through a series of stationary reagents arranged in-line or in a circular carousel plan. 9-10 reagent and 2-3 wax positions

of 30-110 cassettes vertical oscillation or rotary motion

FLUID-TRANSFER PROCESSORS

In fluid-transfer units processing fluids are pumped to and from a retort in which the tissues remain stationary

10-12 reagent temperatures adjustable between 30-45°C, 3-4

paraffin wax stations with variable temperature settings between 48-68°C, and vacuum-pressure options for each station

100-300 cassettes tidal action

Steedman (1960) Paraffin + celloidin Harder M.p 46-68 1-2 micrometer

Ester wax

technique

Transfer into mixture of clearing agents (3-6 hrs) 3 changes of wax (3-6 hrs each) 68-78 degree Heavy microtome Ralwax histoplast

Water soluble waxes

Carbowax No need to be dehydrated and cleared before

infiltration Less shrinkage Miles and linder (1952)

Technique Wash tissue 50 % polyethylene glycol 900 in distilled

water (10-15 mins) Four changes of molten polyethylene glycol

at 28-30 degree (45 mins) Polyethylene + nonex 63B at 39 degree (30-

40 mins) 3 parts of nonex+ 1 part polyethylene

(15mins) 3 changes of nonex at 39 degree (30-45

mins each)

Gelatin

Frozen sections of friable or partially necrotic tissue

Immeresed in 10 % formalin In phospholipid and enzyme studies tissues may

be infiltrated and embedded in gelatine

Technique

Tissue is fixed Washed for 6-12 hrs 10% gelatin in 1% phenol (24 hrs ) 20% gelatin (12 hrs at 37 degree ) Embed in 20% gelatin

Celloidin

Cox (1983)

Preparation

Difficult to dissolve than LVN Once dissolved equal amount of ether is added Thin solution – 5% LVN or 2% celloidin in

ethanol/ether Medium solution- 10 % LVN or 4% celloidin Thick solution – 20% LVN or 8% celloidin

Processing

70% ethanol 2 changes 24 hrs each 95% ethanol 2 changes 48 hrs each Absolute alcohol 2 changes over 2-5 days Thin 3-5 days Medium 5-7 days Thick 5-7 days

Casting and blocking Follwing impregnation in thick solution 8% celloidin Mould 1 quarter inches in depth L pieces cant be used Glass petri dishes 2 inches in depth with loose fitting

ground glass lids Blocks hardedned by evaporation Rubbery consistency is enough Chloroform vapours Block is fixed to vulcanite or hardwood

Dry celloidin

Overcome the disadvantage of storing in 70% alcohol

Gilson ‘s mixture (chloroform+cedarwood oil) Transparent and exposed to air Air tight bottle

Double embedding Tissue is first impregnated in celloidin

subsequently blocked in paraffin waxPeterfi ‘s procedure Dehydration Celloidin methylbenzoate mixture (2-3 days) Benzene 2-3 changes each 6 hrs Paraffin embedding

Resin embedding

Undecalcified bone 0.5- 1.5 micrometer Methyl + butyl methacrylate Newer- 2 hydroxythyl methacrylate and glycol

methacrylate 2 phases JB -4 , LR White

Method Resin solution A monomer HEMA 80 ml 2 Butoxythanol 8ml Bezoyl peroxide 1g Resin solution B – activator Polyethylene glycol 400 15 parts N-N dimethylaniline 1 part Fix into 10% neutrl formal saline

70% ethanol 1 hr 90% ethanol 1 hr 100% ethanol 3 changes 30 mins Solution A monomer 2 hrs Fresh monomer overnight Embed Solution A 42 parts Solution B 1 part Mix well

Sodium carboxy methyl cellulose (CMC)

Frozen tissues are transferred from coolant directly into 5% CMC, briefly placed under vacuum to remove trapped air, then frozen to a solid block for sectioning.

Polyvinyl alcohol (PVA)

Cross-linking PVA with glutaraldehyde provides a final hydrophobic block containing some 10% water, with improved sectioning characteristics and good preservation of lipids, proteins and carbohydrates

Microwave processing

precise temperature control and timer, and an interlocked fume extraction system to preclude accidental solvent vapour ignition. Agitation is provided by an air-nitrogen system

The microwave oven shortens the processing time from hours to minutes.

diffusion of the solutions into the tissue by increasing the internal heat of the specimen

ethanol, isopropanol and proprietary mixtures of alcohol, and paraffin

Xylene and formalin are not used in this process, which eliminates toxic fumes and carcinogens.

Disadvantage--temperatures must be maintained between 70 and 85°C, and the size of tissue sample is critical (2 mm).

Ultrasound-stimulated processing

The most important effect of ultrasound at frequencies of 100 kHz-1 MHz is agitation.

Processing is performed in reagent containers suspended in a detergent solution within the transducer tank of an ultrasonic cleaner operated at 50 watts

REFERENCES

Bancroft’s Theory and Practice of Histological Techniques

7th edition Culling’s Tissue processing by Leigh Winsor