Tissue processing

Definition Tissue processing is defined as the process of preparing the tissue by embedding it in a solid medium that is firm enough to support it and give sufficient rigidity to enable thin sections to be cut and yet soft enough to enable the knife to cut the sections with little damage to the knife or the tissue Stages of tissue processing 1. Collection, labelling & fixation of specimen 2. Dehydration 3. Clearing 4. Impregnation 5. Embedding 6. Trimming & sectioning Labelling of tissue .Correct labelling is essential for: Methods of labelling tissue White card I. II. III. Writing in soft lead pencil, india ink, typewritten, printed or stencilled Card accompanies specimen in tissue carrier Labelling should withstand the fluids used

Tissue tek system The tissue identity is written on the cassetes & retained as permanent record during processing, sectioning & storage. Automated number embossing machines Numbers can be embossed onto cassete by embossing machine which can be programmed manually or by computer Bar code All the relevant informations are stored in the bar codes & are generated by clinical or medical record staff. It is sticked on request form & specimen jar.

Completion of fixation before processing It is desirable to complete fixation of tissue before processing begins.If small fragments of tissues are inadequately fixed then it may be fixed completely by dehydrating alcohol.Dehydration by alcohol should be avoided as this may alter staining characteristics.

Post fixation treatment If tissues are fixed with chrome oxide fixatives then they should be washed in running tap water for 24 hours to avoid formation of chrome oxide pigment.Tissues fixed with picric acid forms water soluble picrates hence tissue blocks should be placed directly into 70% ethanol.Tissues fixed in high alcohol fixatives should be placed directly into 100% ethanol.

Factors influencing rate of processing Specimen size Ideally tissue slices or biopsy specimen should not be thicker than 3-4mm . This will permit satisfactory processing overnight on automatic tissue processors or in two working days if manually processed.Thicker the tissue longer will impregnation take.

Agitation It enhances the interchange of fluids between the tissue and its surrounding medium. Surface area of tissue Efficient agitation reduces overall processing time by upto 30%.

Method Agitation Rate of agitation

 It should not be too slow so that it is ineffective.It should not be too violent causing damage to small fragments & fragile tissue fragments. Heat Increases rate of penetration, hence increase interchange of processing fluids. Disadvantages Hardens tissue, increases brittleness & causes shrinkage Viscosity Fluids with higher viscosity requires longer time to achieve impregnation. Vacuum Enclosed processing machines consists of vacuum systems. It is effective during impregnation stage.Reduced pressure helps in removing trapped air from within tissues hence bringing intimate contact with processing fluids.Clearing agents are removed more rapidly because of increased volatility.Tissues requiring vacuum impregnation are - lungs, heart muscles, spleen, decalcified bone, skin and brain. Dehydration This is the first step in the processing of fixed tissues.In this the aqueous fixative fluids are removed from the tissues and are replaced with dehydrating fluids.pExamples ethanol, methanol, acetone etc.

Steps for dehydration 1. Washing out the fixative 2. Immersing the tissue in dehydrating fluid. Ex : ethanol. Tissues are placed in following order:Alcohol replaces water by diffusion. Dehydrating fluids Ethanol c2h5oh denatured alcohol (industrial methylated spirit) Methanol Propan-2-ol, isopropyl alcohol ch3chohch3

acetone ch3coch3 additives to dehydrating agents Ethanol (c2h5oh)

It is a clear, colourless; flammable liquid with pleasant odour.It is hydrophilic and miscible in water & many organic solvents.It is expensive but still used commonly because it ensures total dehydration Denatured alcohol (industrial methylated spirit) .It contains ethanol to which 1 % methanol has been added which renders the fluid unfit for consumption & thus attracts a much lower excise duty.It has same physical properties as ethanol & for tissue processing it is used in the same way as ethanol. Methanol

It is clear, colourless and flammable fluid which is highly toxic.It is miscible with water, ethanol & most organic solvents.It is rarely used but can be a substitute for ethanol. Propan-2-ol, isopropyl alcohol ch3chohch3

It is miscible with water, ethanol & most organic solvents.Many of the processing methods for use in microwave oven recommend this agent Acetone ch3coch3

It is clear, colourless and flammable fluid miscible with water; ethanol & most organic solvents.It is rapid in action but poor in penetration & causes brittleness in tissues if use is prolonged.Only used when urgent reports are required.Most lipids are removed from tissues with this agent. Additives to dehydrating agents Phenol - phenol acts as a softening agent for hard tissues like nails, tendon, dense fibrous tissue & keratin masses (4% phenol added to 95% ethanol)Glycerol (mollifex)- hard tissue can also be immersed in a glycerol & alcohol mixture. Additives to dehydrating agents Anhydrous copper sulphate - it acts as both a dehydrating agent and an indicator of the water content of the last bath of 100% ethanol . Anhydrous copper sulphate removes water from the alcohol as it in turn removes it from the tissue. Anhydrous copper sulphate which is white in colour turns blue on exposure to water. This blue coloured cus04 indicates that alcohol is contaminated with water and should be replaced with fresh dehydrating alcohol.This additive cannot be used in the pump-action enclosed processors . Clearing This process relates to the appearance of tissue after it is chosen to remove dehydrating agent.Most of the clearing agents have refractive index similar to protein.When dehydrating agent is entirely replaced by clearing agent it appears translucent . The step following dehydration is called "clearing" and consists of replacing the dehydrant with a substance that will be miscible with the embedding medium (paraffin). The term "clearing" comes from the fact that the clearing agents often have the same refractive index as proteins. As a result, when the tissue is completely infiltrated with the clearing agent, it becomes translucent. This change in appearance is often used as an indication of the effectiveness or completeness of the clearing process. The most common clearing agent is Xylene. Xylene is reasonably cost effective and works well for short-term clearing of small tissue blocks. Long-term immersion of tissue in xylene results in tissue distortions. Toluene is better at preserving tissue structure and is more tolerant of small amounts of water left behind in the tissues than xylene. However, toluene is more expensive than xylene and more toxic, so toluene is less commonly used. Chloroform has been used in some applications, but it is a severe health hazard, acts slowly and may lead to sectioning difficulties. Methyl salicylate is safe and effective, though rarely used due to cost. Orange oil based clearing agents, such as National Diagnostics Histo-Clear, offer the best clearing action with the lowest hazard rating of all xylene alternatives. Histo-Clear is excellent for preserving fine tissue structure, and can often be used in place of xylene with no alteration of protocol. Because orange oils can break down to produce compounds which will interfere with staining procedures, it is important to use a product, such as Histo-Clear, which has been rigorously purified and then stabilized.

Most tissue processing is done using automated machines that carry out the steps automatically. Tissues coming off a tissue processor are in a plastic box ready for the embedding stage. Examples of clearing agents:Xylene, toluene, chloroform, paraffin, cedar wood oil, citrus fruit oil . Criteria for choosing clearing agent Speedy removal of dehydrating agent

Clearing agents, Xylene/toluene,Chloroform,Paraffin,Methyl benzoate & methyl salicylate,Cedar wood & clove oil, 1,1,1 trichlorethane & petrol, Citrus fruit oil Xylene / toluene It is most widely used.Alcohol is replaced due to difference in refractive index & tissue becomes clearer. Advantage:Rapid in action.Tissue becomes clearer so end point can be determined as alcohol is replaced. Disadvantage: Highly inflammable.Damaging to tissue on prolonged immersion.Side effect of vapour inhalation.Toluene is less damaging to tissues on prolonged immersion Chloroform Commonest reagent used by manual methods.Advantages: slower in action than xylene / toluene. tissues can be left for prolonged time,noninflammable.Disadvantages:longer immersion is required for complete penetration & replacement of alcohol, tissue placed in chloroform does not become translucent. The end point of Clearing cannot be determined.it is highly toxic (phosgene gas is released). Paraffin Can be used as clearing agent.The time of immersion is similar to chloroform. Advantages: it is less inflammable,it is cheap.Disadvantages :Variability of action from batch to batch,Clearing agents . Methyl benzoate and methyl salicylate These are slow acting clearing agents and commonly used in double Embedding techniques. Advantage :Minimal distortion of tissues.Disadvantage:strong penetrating odour ,Clearing agents. Citrus fruit oil These are extracted from orange & lemon rinds and commercially available as clearing agents. Advantages: it is non-toxic & miscible in water, thus avoiding the cost of disposal. Disadvantages: Strong odour,Small mineral (calcium or copper) deposits in tissues are dissolved out of tissue blocks,if used to clear sections prior to mounting, dyes are leached out

Cedar wood oil and clove oil Very slow acting clearing agents.Not recommended for routine use.For delicate tissues they cause little brittleness hence good sectioning.They can be used in humid conditions also .Combined dehydrating clearing agents Dioxane 1:4 diethylene dioxide Slower than ethanol as dehydrating agent.The process of clearing &, impregnation is faster.Disadvantage:It is highly toxic.Advantage:Little damage to the tissues,Tolerates little amount of water.Disadvantage :It is slow acting Impregnation It is intracellular and extracellular deposition of an embedding agent in tissues so that tissues acquires a firm consistency, enabling an easy cutting later on by microtome knife.Paraffin wax is the most satisfactory embedding material till date. It should throughly penetrate tissue in fluid form & solidify with little damage to tissue.Other embedding agents are plastics, synthetic resins. Paraffin wax It is a mixture of straight chain hydrocarbons produced from vacuum distilled crude oil.It is the most popular embedding medium because:It is cheap.It can be easily handled.Section production provides lesser difficulties.It can be used in different climatic conditions due to its wide range of.Melting points (40- 70c) Paraffin wax The higher the melting point of paraffin wax the harder it will be at any given temperature.The plastic point of paraffin wax governs its natureThe plastic point is the temperature at which the crystalline rearrangement takes place during solidification and at which the behavior characteristics of the wax change.It is generally 10c below the melting point of the wax.

Types of paraffin wax Depending upon its melting point the types of paraffin wax are: I) soft paraffin wax It has a low melting point (about 45c).It is most suitable for soft, friable tissue.Used for impregnation Ii) hard paraffin wax

It has a high melting point (about 60c).It is used for harder tissues like dense fibrous tissue.Used for embedding Paraffin wax additives These are utilized to modify the consistency of wax along with its melting.Point. They alter the crystalline structure of the wax Role of additives is to: Increase hardness in order to cut thinner sections or to cut sections at ambient temperatures.Increase hardness in order to give necessary support in sectioning harder tissues, increase the stickiness/plasticity of wax to aid the production of serial sections of ribbons.

The various additives used are: I) beeswax, ceresin, rubber, dental wax, diethylene glycol distearate Ii) microcrystalline waxes These are produced from petroleum distillation.They have much finer crystalline structure but a high range of melting points (62-88 c).Adding 5% of these waxes to paraffin wax produce a harder medium Iii) dimethyl sulfoxide (dmso)- commercial paraffin wax with added resins of.Various types imparts hardness & plasticity to paraffin wax. Disadvantages of additives I) many additives have a higher melting point than the paraffin wax and thus make the tissues more brittle Ii) many additives are not pure so consistency is difficult to achieve if these mixtures are made in the laboratory Embedding Its the process when the tissue is finally transferred from last wax bath to a mould filled with molten paraffin wax, inverting the tissue to free all surfaces of air bubbles & orienting the intended cutting surface so that it faces the base of the mould. Blocks or moulds used in embedding 1. Leukharts l pieces 2. Ice trays

3. Paper boats 4. Embedding cassetes 5. Glass petri dishes 6. Metal petri dishes 7. Watch glass 8. Test tubes Leukharts l pieces

it consists of two l -shaped pieces of metal which is laid on a metal or a glass plate to form an oblong.By adjusting l-pieces relative shape & size of mould can be modified according to size & number of tissues.When wax has set, l-pieces can be easily removed & used again. Advantages :Various sizes of l-pieces are available and enabling almost any piece of tissue to be blocked by this method.Disadvantages :It is time consuming.There is wastage of paraffin wax.The identification tags comes out easily

Ice trays

Domestic ice trays made of plastic can also be used as moulds. Advantage :Each tissue is blocked into individual compartment saving on the labour involved in separate blocks. Disadvantage :The compartments are of uniform size. Small pieces of tissue need to have a large excess of wax to be removed before microtomy.They tend to wear out with use. Paper boats They make cheap & convenient alternative.Ordinary paper can be used but they are best prepared from glossy paper. Advantage :Paper needs to be removed only from the cutting surfaces & upper edges.Blocked tissue can be stored with the identifying number written on the projecting tag.Wax block can be clamped directly into the microtome vice without the need of mounting on wooden block or special holders .Embedding cassette/tissue tek system .A plastic cassette which is provided with either a metal snap on lid or an integral plastic one holds the

tissue during processing.A roughned surface is provided for writing the referece number.Embedding is performed in a specially made base mould and casstte is inverted over the tissue and filled with wax.After solidification of wax the cassette can be clamped into microtome.Embedding cassette/tissue tek system After sectioning the block can be stored with the cassette.Moulds are available in a variety of sizes and cassettes in a variety of depths & colours. Advantage:ease of use,Less paraffin wax is used,Speed ,Tissue & holder are firmly attached,Blocks are filled immediately after sectioning,Permanent identification Glass petri dishes

Convinient embedding moulds if previously smeared with glycerin.Used to embed several pieces of tissues at one time .When wax has set , it is turned out of dish & is then cut allowing a margin of about inch all around each piece of tissue Metal petri dishes

Commercially available & is made up of aluminium.Are strong & have long life.Final block of wax is easily removed due to its contraction during setting Watch glass

Used as mould for small pieces of tissues.Glass is smeared with glycerin for easy removal of wax block Test tubes

Used for small fragments , ex- bone marrowThe fragments will collect & concentrate at the tip of the tube without the use of forcepsDisadvantage is it is often necessary to break the tube to remove the block

Technique of embedding metal mould should be sprayed or smeared lightly with mould release fluid.The mould is filled with appropriate paraffin wax.Molten paraffin wax is dispensed into mould to a depth more than adequate to cover the thickest tissue block.When a thin film of semisolid wax is formed on base of mould, the tissue is introduced rapidly to this wax.Ensure that no air bubbles are trapped.Now the tissue with the surface to be sectioned is placed down against the base of mould.Now allow the wax in mould to solidify sufficiently to hold tissue in place.The block should be immediately trasferred to a refrigerated surface.Once a skin forms on the surface the block should be immersed in trough of cold water.On completion of solidification the block is removed from mould. Precautions to be taken during paraffin wax embedding The wax to be used must not contain traces of clearing agent.No dust particles should be present.Immediately after tissue embedding, the wax must be rapidly cooled to reduce wax crystal size

Orientation of tissues

Epithelium skin or mucosa covered tissue must be cut at right angles to the surface so that full thickness of epithelium is visualized.

Muscle biopsies Muscle tissue should be sectioned so that both the longitudinal & tranverse sections are obtained and embedded accordingly Tubes Tubular tissues should be sectioned transversely so that the lumen is clearly visible.Orientation of specimen to the blade .The longest axis of the specimen should be parallel to the blade.Hard tissues like bone or teeth should be embedded in an oblique orientation so that blade meets a corner of specimen first. Majority of histopathology departments now use machines to process tissue blocks .This has reduced the processing time as compared to manual methods by atleast 24 hours Automated tissue processor

Types of processing machines Carousal type It has the facility for 12 separate stages in processing.Minimum time at any stage in old carousal type machine is 30 min.New carousal type can be programmed electronically & minimum time per stage is 15 min .Types of processing machines

Enclosed pump fluid type This type also has facility for 12 separate stages in processing.Tissue cassettes are held in static reaction chamber & processing fluids are pumped in & out .

Advantages Safety Potentially hazardous fluids are housed separately from reaction chamber hence reducing risk of fire & spillage.Machine stops & sounds an alarm if there is any electronic fault.Each fault has an alarm number which is displayed on a vdu

CapacityBy using a single reaction chamber it is possible to provide facilities to process tissues with the application of heat & vacuum at all stages . Flexibility Microprocessor control & memory permit a great flexibility & variety of processing schedules.It is possible to process tissue in short time Automated processing schedule Most laboratories use an automated processors with an overnight schedule of approximately 1618 hours duration. Overnight schedule The 1st container with 10% formalin is used in conjunction with the delay mechanism for weekend & holiday times.Short processing schedule, this is for small biopsies or urgent work.Urgency is the reason for rapid processing.Recently excised endoscopic biopsies & needle biopsies can be adequately processed in 2-5 hrs using heat (37-45 c) & vacuum Alternative embedding media When paraffin wax cannot be used or it is unstable medium for the type of section required, are when: Processing agents remove or destroy tissue components that are to be investigated,section are required to be thinner,use of heat may adversely affect the tissue and when the impregnation medium is not sufficiently hard to support the tissue . Various alternative embedding media are : Water soluble waxes

These waxes are solid polyethylene glycol.They do not require tissue to be dehydrated.The degree of shrinkage of tissue is less due to avoidance of dehydrating & clearing agents Resins Resin is much harder material than paraffin wax. They have been used to cut hard material such as decalcified bone and to cut thin sections (0.5 - 1.5 m) Two groups of resins are used : Acrylic resins and epoxy resins Acrylic resins These are monomers of acrylic & mehthacrylic acids.2-hydroxy ethyl metha acrylate (hema) is now widely used.To be effective the resin must have two phases: a liquid phase to infiltrate the tissue and a solid phase to support the tissue during microtomy. Agar Agar gel alone does not provide sufficient support for sectioning of tissues.Mainly used as cohesive agent for small friable pieces of tissue after fixation.The fragments are embedded in molten agar and when solidified & trimmed, processed to paraffin wax Gelatin It may be used:When frozen sections of friable or partially necrotic tissue or number of small fragments are required.It finds its main use in the production of sections of whole organs in the gough - wentworth technique and in frozen sectioning Celloidin & lvn (low viscosity nitrocellulose) Celloidin forms less viscous fluid which penetrates tissue more easily & gives Classical embedding medium for cns tissues.It was popular medium for large tissue blocks of brain. Lvn (low viscosity nitrocellulose) It is an explosive when dry & is kept damp with butanol.Shrinkage is less.Tough tissues not made any harder.Used for embedding bone & teeth, cns & eyes. Double embedding This procedure was adapted to deal with hard tissues.Used in cases where morphological appearance of tissues is desired to be maintained for morphometric studies.Ex: lung Artifacts in tissue processing Inadequate fixation before processing:If tissue fragments are inadequately fixed then fixation may be completed by dehydrating alcohols but should be avoided as it affects staining

characteristics. Post fixation treatment is necessary while using certain fixatives like chrome fixatives, picric acid fixatives & carnoys fluid. Prolonged exposure to clearing agent make the tissue brittle & more difficult to section. Drying oven or hot plate:Excess heat will cause splitting or cracking of the sections.Surface decalcification affects the staining properties of tissue. Fluffy white spots appear on the block due to impurities of clearing agent left in the block or when block is allowed to cool slowly.

sDEPARTMENT OF ORAL AND MAXILLOFACIAL PATHOLOGY

D.J College of Dental Sciences and Research, Modinagar (U.P)

EGE OF D E OLL NT .C J A D.

IENCES AN D SC

EAR ES R
CH, MODIN AG A

COL L EGE
st d.

99 S ince 1

SEMINAR- Tissue Processing

SUBMITTED BY- Dr. Aanchal Puri

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