Good morning

- By Dr Shruthi B S

Tissue

Processing

References • Theory and Practice of histological

techniques, 6th Ed - Bancroft.• Cellular pathology technique, 4th Edn-

Culling• Theory & practice of Histotechnology,

2nd Edn – Sheehan• AFIP laboratory methods in

Histotechnology• Histopathology Techniques: Tissue

Processing and Staining, Dr. S.I Talukder

• Internet

Presentation outline• Introduction• Specimen handling• Tissue processing• Factors affecting tissue

processing• Stages of tissue processing• Methods of tissue

processing• Conclusion

Introduction • The most commonly used

method of examining tissue microscopically is by sectioning

• Fixation of the tissue stabilizes the structure and stabilised tissues must be adequately supported by infiltrating and embedding in support medium before they can be sectioned for microscopical examination

Specimen handling

• Correct labelling & identification are the first essentials of processing

• Emphasis should be given to1. A foolproof system of labelling2. Ensuring the correct label

remains with the specimen throughout

3. Constant vigilance

Labelling of tissues(a)Handwritten in soft lead pencil or

waterproof ink (b)A numbered card label generated by

computer-printer(c)Automated embossing systems that

permanently etch or emboss tissue cassettes and slides and produce serial numbers

(d)Tissue- Tek system – where tissue identity is written on cassette & retained as permanent record

Tissue Tek System

• The cassettes consisted a small perforated plastic container with metal or plastic lid

• Edges of the cassettes are roughened enabling them to be marked in pencil

• As the cassette forms a part of final wax block, the id number stays with the specimen from the time its put in and thus reduces the margin of error

Tissue processing• Definition It refers to any treatment of

the tissue necessary to impregnate them with a solid medium to facilitate production of sections for microscopy

Principle of tissue processing

• Fick's Law: The rate of solution diffusion through tissues is proportional to the concentration gradient as a multiple of temperature dependant constants for specific substances.

• Tissue processing is concerned with the diffusion process results from the thermodynamic tendency of processing reagents to equalise concentrations inside and outside blocks of tissue and thus stabilize it

Factors affecting the rate of processing

Tissue processin

g

Specimen size

Heat

Agitation

Viscosity

Vaccum

1. Specimen size:• Thicker the specimen, longer will be the

impregnation• Ideally, specimen thickness has to be 3-

4mm• If thicker slices are unavoidable,

processing schedule has to be prolonged 2. Heat:

• Increases the rate of penetration & fluid exchange• Temperature is limited to 45ºC• Disadvantages:

– Hardens tissue, increases brittleness, shrinkage

– Interferes with staining

3. Agitation:• The greater the surface area of the tissue in

contact with processing fluid, more effective the fluid interchange.

• Agitation ensures the effective interchange between fluid within the tissue & fluid outside

• Commercial tissue processors has automatic agitation with either vertical or rotatory movements of specimen container or by drawing & refilling the containers

4. Viscosity: • Higher the viscosity of processing fluid,

slower the rate of penetration.• Clearing agent like cedarwood oil &

molten wax are of high viscosity and require longer immersion times to achive impregnation.

• Heating reduces viscosity but this is avoided when using cederwood oil as this causes tissue hardening

5. Vaccum:• Vacuum reduces the time necessary

for impregnation by one-half particularly dense and fatty tissues

• Helps in removal of trapped air from tissues

• Assists in the removal of clearing agents because of increased volatility

• Some Automatic processing machines incorporated vacuum at all stages of processing cycle

Stages of processing

Dehydration

Clearing

Impregnation

Embedding

Removal of water and fixative from the tissue

Removal of dehydrating solutions Permeating the tissue with a supporting mediumOrienting the tissue sample in a supporting medium

Dehydration • First stage of processing concerned with

the removal of unbound water and aqueous fixatives from the tissue components

• Specimens are processed in a graded series of reagents of increasing concentration to prevent its distortion

• Excessive dehydration- hard, brittle & shrunken tissue

• Incomplete dehydration- inhibit the penetration of clearing agents

Duration of dehydration

• Duration of dehydration should be kept to the minimum consistent with the tissues being processed.

• Tissues may be held and stored indefinitely in 70% ethanol without harm.

Specimen Time For whole organ 24-48 hrs in each

For delicate tissue & cytological research

2-4 hrs in each according to size

For post mortem tissue of not > 7mm

70,90,100% (3 changes) for 1-2 hrs in each

Dehydrating fluids• Dehydrating fluids should be

water miscible• Choice of a dehydrant is

determined by the nature of the task, the embedding medium, processing method, and economic factors

• Dehydrants differ in their capacity to cause tissue shrinkage

Concentration of dehydrants

• The dehydrant concentration at which processing is initiated depends largely upon the fixative employed

70%Specimen Concentration of dehydrant

Embryonic tissue in aqueous fixative

10%-20%-50%-95%-100%

Tissue in carnoy’s fluid 100%

Most specimens in aqueous fixative

70%-80%-100%

Use of copper sulphate in final alcohol:

• A layer (1.2 – 5mm thick) of Anhydrous copper sulphate added to the final absolute ethanol on a tissue processor scavenges any water present

• The salt is self-indicating: white when anhydrous, blue when hydrated

• This action not only speeds the dehydration process of tissue but also prolongs the life of alcohol

Ethanol • Clear, Hydrophilic, miscible with

water and organic solvents• Rapid, efficient, widely applicable

& most commonly used dehydrant in histology

• Ensures total dehydration, making it as choice for the processing of electron microscopic specimens

• The volume of alcohol should be 50-100 times that of tissue

Advantages • Nontoxic• Miscible in all

proportions of water• Little shrinkage • Fast acting• Reliable

Disadvantages • Expensive• Longer period may

cause shrinkage• May have prohibitive

taxes• Extracts methylene

blue from sections

Methanol

• It is a good ethanol substitute but rarely used for routine processing because of its volatility, flammability and toxicity

• It is miscible with water, ethanol & organic solvents

• In microwave processing it tends to harden tissues more than ethanol

Isopropyl alcohol

• Miscible with water, ethanol and most organic solvents

• Often used in microwave processing schedules

• Does not cause overhardening or shrinkage of the tissue

Acetone • Clear, colourless, flammable

fluid miscible with water, ethanol and most solvents.• Has characteristic pungent

odour• Rapid in action, with poor

penetration & Causes brittleness of tissue if use is prolonged.• Removes lipids from the tissue

during processing

Additives

• Phenol- (4%) softening agent for hard tissues like tendon, nail, keratin masses

• Hard tissue can be mixed even in alcohol-glycerol mixture

Clearing • Clearing is the transition step

between dehydration and infiltration with the embedding medium

• Dehydrants are immiscible with paraffin wax, and a solvent miscible with both the dehydrant and the embedding medium is used to facilitate the transition between dehydration and infiltration

Why is it named so??

• The term clearing arises because some solvents have high refractive indices approaching that of dehydrated fixed tissue protein and, on immersion, anhydrous tissues are rendered transparent or clear

Criteria

• The type of tissues to be processed, and the type of processing to be undertaken

• Rapidity of removing dehydrating agent

• Ease of removal by infiltrating agent

• Minimum tissue damage• Toxicity & inflammability• Cost and convenience

Clearing agentsXylene & Toulene:• Flammable, colourless, miscible with

most of the organic solvents & paraffin

• Rapid in action, 30-60 min for small pieces of tissue and about 2-4 hours for 5mm thick specimens

• Toulene has same properties as xylene but less damaging to tissue and more volatile

Advantages • Clears quickly• End point is easily

determined• Used for clearing in

staining sequence• Recyclable

Disadvantages • Flammable• Hardens the tissue• Toxic to skin &

various systems of body

• Not miscible with water

Advantages • Penetrates well• Makes tissue less

brittle• Thicker tissue

>1mm can be processed

• Non flammable

Disadvantages • Highly toxic due to

phosphogene gas• End point cannot

be determined• Used in well

ventilated room

Chloroform:•Commonest agent in routene use by manual methods because of its tolerance•Often used when processing specimens of CNS

Advantages • Rapid clearing• Least hardening &

shrinkage• Less toxic• Excellent for tissues

such as skin, uterus, muscle & tendon

Disadvantages • Must be removed with

xylene before impregnation

• Expensive• Remanants may cause

difficulty in sectioning

Cedar wood oil:• Best reagent for research• Tissues can be left for longer period even for months without damage

Methyl benzoate and methyl salicylate:• Slow acting and can be used when

double embedding techniques are required

• Moderate speed of action• Minimal distortion of tissue

Citrous fruit oils:• They are extracted from orange and

lemon• Nontoxic and miscible with water, so

they can be discharged through ordinary waste pipes thus avoiding the cost of disposal

Universal solvents• Some fluids are capable of acting as

dehydrating agents and at the same time they are miscible with molten paraffin wax, thus avoiding intermediate clearing stageDioxane, 1:4 diethlene dioxide

- highly toxic therefore not used

Ethylene glycol monoethyl ether (cellosolve)

-less toxic, slow action & requires further investigation

Recycling of processing fluids

• Distillation equipment is used• Advantages include

Reduced costRapid, efficientEliminates the risk of disposal of

toxic chemicals

Impregnation/Infiltration

• 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 embedded

Ideal requisites • Soluble in processing fluids• Suitable for sectioning and

ribboning• Molten between 30°C and 60°C• Translucent or transparent;

colourless• Stable, non toxic, odorless• Homogeneous• Capable of flattening after

ribboning• Easy to handle• Inexpensive

Paraffin wax• Most popular due to ease with which

large number of tissue blocks may be processed in comparatively short times

• Staining presents fewer difficulties than other media

• Cheap, easy handling, inexpensive, wide range of melting point & provides quality section

• It can be used in different climatic regions

• Volume of wax should be about 25-30 times the volume of tissues

Properties • It is a mixture of hydrocarbons

produced by sweating and pressing the residue to vacuum distilled crude oil.

• Melting point range is between 40-70°c, normally for routine use 54-58°c is satisfactory

• Higher the melting point of wax, harder the wax at any given temperature

• Plastic point governs the behaviour of the wax. Plastic point [at which crystalline rearrangement occurs] is 10°c below the melting point.

Paraffin wax additives

• Plasticizers or other resin additives to provide desired hardness

• Substances added to paraffin are bees wax, rubber, ceresin, plastic polymers

• Additives with higher melting point than paraffin can make the tissue brittle

Time of impregnation

• Duration & number of changes depends on:

• Size & type of the tissue• The clearing agent employed• Use of vaccum-embedding

oven

Size and type of tissue:

Size:• Thicker the tissue, longer will be the

time required for wax to penetrate• Thick tissue will carry more of the

clearing agents, & hence requires more change of wax to remove it.

Type:• For dense tissue impregnation time is

twice that of soft tissues (liver and kidney)

E.g: Bone, skin, central nervous system.

The clearing agents employed:

• Some clearing agents are replaced faster than others.

• Xylene, chloroform, toluene and food oils require two changes of wax. While cedarwood oil requires several changes

Vacuum impregnation:• Normal paraffin wax impregnation with

two changes of wax takes over a period of 4 hours. By use of vacuum impregnation this time may be halved.

• This procedure not only speeds up impregnation but also rapidly remove air bubbles and clearing agents from the tissue

• The degree of vacuum should not exceed 500 mm of mercury

• Tissues benefited from this are lungs, muscles, spleen, decalcified bone, skin and central nervous system tissue.

Methods of processing

Manual tissue processing

Automated tissue processing

Manual tissue processing

Manual tissue processing has now largely been superseded by automatic tissue processing but there are occasion when it may be necessary to adopt this procedure.

These circumstances are– power failure or breakdown of a tissue processor– requirement for a non-standard processing

schedule like:• rapid processing of an urgent specimen • delicate material • very large or thick tissue blocks • hard, dense tissues (nitrocellulose

methods)

Advantages • Flexibility of reagent selection,

conditions and schedule design to provide optimum processing for small batches of tissues.

• Exposure of tissues to the deleterious effects of some reagents can be carefully monitored and regulated through observation and precise timing

• Use of fluids on grounds of inflammability, volatility or cost would be unacceptable in automated techniques.

Manual processing schedules

Automated tissue processing

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

• Automated machines are most widely used to overcome the possibilities of human error and forgetfulness leading to changing of fluids at inconvenient times

Types

Tissue-transfer processors

Fluid-transfer processors

Microwave tissue processors

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

• The rotatory or carousel is the most common model of automatic tissue processor

• Fluid agitation is achieved by vertical oscillation or rotary motion of the tissue basket

Fluid-transfer Processors

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

• Depending upon the model these machines can process 100-300 cassettes at any one time

• Agitation is achieved by tidal action

Processing Schedules

Microwave processors• Rapid manual microwave-stimulated paraffin

wax processing gives excellent results compared to tissues processed by longer automated non-microwave methods

• Processing is undertaken in a dedicated microwave oven which is fitted with precise temperature control and timer, and an interlocked fume extraction system to preclude accidental solvent vapour ignition.

• Toxic and flammable solvent vapours generated during processing cannot always be adequately vented from these ovens and present an ignition hazard if the electrical system is unprotected

Rapid Automated Processing

• The enclosed system machine have the facility to use vacuum and heating at all stage which can greatly increase the speed of processing.

• Spleen, muscle, skin, decalcified tissue and tissues containing blood clot to become unduly hardened if high temperature are used

• This can be minimised by using limited temperature during fixation, dehydration and clearing

Techniques for increasing speed of processing:• The use of warm (40-50C) 45º C for

30 min, fixative to ensure that fixation is complete before commencing dehydration.

• Dehydration commencing at 95% alcohol stage

• The use of a fast acting clearing agents

• The use of a vacuum infiltration at all wax stage.

• Agitation at all stage even during fixation.

• The minimum of time at all the stage

Conclusion

Producing quality slides for diagnosis is not an accident rather it essentially requires skills that are developed by continued practice & experience

To be conscious that you are ignorant is a great step to knowledge

- Sir Benzamin Disreli