Tissue Processing

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GOOD AFTERNOON

TISSUE PROCESSING

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-A histotechnique.............

INTRODUCTIONDEFINITIONAIMPRINCIPLESTEPS CONCLUSION

CONTENTS

INTRODUCTION

• History of tissue processing

• Histology

• Histopathology

• Histotechnologist

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• SECTIONING

•EMBEDING

ALEXANDER BUTLEROV, 1859

MICROTOME

GROSS EXAMINATION

SPECIMEN ACCESSIONING

•REQUEST FORM

• NUMBER

•COLOUR

•CONSISTENCY

•TEXTURE

SPECIMEN LABELLING

• Should be insoluble • Should not contaminate• Should not penetrate • Should not react • Clearly identifiable• Eg:India ink, silver nitrate, alcian blue

SOFT LEAD PENCIL

DEFINITIONThe tissue must undergo preparatory

treatment before being sectioned, entailing to impregnation of specimen with an embedding medium to provide support and suitable consistency for microtome is tissue processing.

FORMALIN ALCOHOLS XYLENE PARAFIN 10% WAX

PREPARATORY TREATMENT

FIXATION DEHYDRATION CLEARING IMPREGNATION

To remove water from tissue and replace with a medium that solidifies to allow the thin sections of the tissue to be cut.

AIM

FIXATION DEHYDRATION

4-6mmHCT-2-3mmLC-10-20mm

PRINCIPLE

“FICK’S LAW”

REAGENTS REAGENTS

TISSUE FLUID TISSUE FLUID

cold heat=

TEMP GRADIENTSTABILIZE POROUS TISSUE

THERMODYNAMIC TENDENCY OF PROCESSING REAGENTS

FICK’S LAW- The rate of diffusion of solutions through tissue is proportional to the concentration gradient of various tissue processing reagents.

FACTORS EFFCTING THE RATE OF TISSUE PROCESSING(TP)

Vacuum-

Fixation-

Viscosity-

Agitation-

Heat-(45oc

) (TP)TPf Highly flammable

r

V-E-OReduced pressure 15 inches of hg

Removes air trapped, doesn’t make hard tissueMolten parafin

wax

Temp-hardens, shrinkage

CEDAR WOOD OIL, MOLTEN WAX

STEPS1.FIXATION-

2.DEHYDRATION-

3.CLEARING

4.IMPREGNATION

5.EMBEDDING

Mini-6hrs, max-12hrs to 24hrs,25times AIM, washed in running h2o

types

ACTION, ARTIFACTS,FACTORS, TECHNIQUE

DEHYDRATIONRemoval of unbound water & aqueous fixatives from tissue components is called Dehydration.

DEHYDRATING AGENTSEthanol- fast acting, electron microscopy specimen, clear colourless, flamable liquidIndustrial Methylated Spirit-methonol 1%+IPA, Same physical property as ethanol

Methanol- subsituted for ethanol,toxic, inflammablePropan-2-01,Isopropylealcohol- Microwave processing schedule,Butyl alcohol- plant and animal histology, slow dehydrantAcetone-Rapid in action with poor penetration, clear colorless & Inflammable liquid,Removes lipids & causes brittleness of the tissue

Universal Solvents-Tertiary butanol, Tetra hydro furan, Dioxane, dehydrate+clear, not for delicate tissues.

No shrinkage & hardness of the tissue

TECHNIQUE

70% 80%50% 90% 100% 100%

THE GRADUAL INCREASING CONCENTRATIONS OF ALCOHOL

2- 4 hrs

7-8hrs

•In celloidin embedding after dehydration ,tissue is transferred to a mixture of equal parts of absolute alcohol & ether.

USE OF COPPER SULPHATE IN FINAL ALCOHOLS

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white white

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BLUE BLUE

CLEARING•Intermediate step

•Refractive index

•Boiling point

•Short chain aliphatic hydrocarbons

CRITERIA FOR CHOOSING CLEARING AGENTS

Rapid removal of dehydrating agent

Ease of removal by melting paraffin

Minimal tissue damageToxicity

Flammability Time Cost

viscocity Stor

age

Disposal

CRITERIA FOR CHOOSING CLEARING AGENTS

TECHNIQUE OF CLEARINGVolume of clearing agent should be 50-100 times the volume of tissue.

In chloroform or carbon tetrachloride tissues are left over night for clearing .

In Xylene,benzene,toluene tissues are given one change after 30 to 60 minutes.

Technique for cedar wood oil is different.

CEDAR WOOD OILTECHNIQUE:-

Cedar wood oil poured into specimen jar & same quantity of absolute alcohol superimposed on it .

The specimen is placed into alcohol , it floats at the interface of two fluids.

As tissue is cleared it sinks into cedar wood oil.

The alcohol is removed & specimen is transferred to fresh cedar wood oil

Cedar wood oil

Absolute alcohol

Cedar wood oil

In processing for electron microscopy clearing is done in 1,2 epoxy propane,2 changes 15 min in each.

15mins 15mins

ELECTRON MICROSCOPY

1,2 epoxy propane1,2 epoxy propane

CLEARING AGENTS•Chloroform

•Toluene , Xylene , Benzene

•Carbon Tetrachloride

•Cedar Wood oil

•Methyl Benzoate , Methyl Salicylate

•Citrus Fruit Oil

CHLOROFORM•Most common reagent used in processing of CNS specimens.

•DIS ADV:- Slower in action, Highly toxic. Does not effect refractive index of tissue.

End point of clearing can not be determined.

Expensive compared to other clearing agents.

Tissue can be left in this with out any damage for longer time, non-flammable.

•ADV:-

CARBON TETRACHLORIDE

•Similar action as chloroform.

•Much cheaper.

•Non inflamable.

•Is toxic.

IMPREGNATION

•Impregnation with paraffin wax is carried out in a oven heated to 54-56 degree.

•The temperature depends up on the melting point of wax used for impregnation.

•Types of impregnation oven: Electric heated oven. Vacuum embedding oven. Gas heated oven.

•Permeating the tissue with a supporting medium is called Impregnation.

FACTORS EFFECTING IMPREGNATION

•Size & type of tissue.

•Clearing agent employed.

•Use of vacuum embedding oven.

IMPREGNATION OVEN

TECHNIQUE OF IMPREGNATION

•After clearing the tissue is transferred to oven maintained at 56 degree temperature.

•Volume of wax should be 25-50 times the volume of tissue.

•Wax must be changed at least once during the process.

PARAFFIN WAX

•Should be free from dust & foreign particles.

•Should not contain water.

•Melting point should be 54degrees.

•Soft wax(45 dg) for foetal & areolar tissue.

•Hard wax (60 dg) for hard & fibrous tissue

PARAPLAST

•Mixture of purified paraffin & plastic polymers.

•Greater elasticity than paraffin .

•Wrinkle free serial sections can be cut.

•Does not need ice application while sectioning

ESTER WAX

•From clearing agent transfer to mixture of clearing agent & wax.

CELLOIDIN EMBEDDING

2% celloidin:- 5-7 dayS.

4% celloidin:-5-7 days.

8% celloidin:-2-3 days.

L.V.N

•5% L.V.N:- 3-5days.

•10%L.V.N:-2-3days.

•20%L.V.N:-1 day.

DOUBLE EMBEDDING

Tissue is impregnated in a mixture of celloidin& methyle benzoate.

ARTIFACTS

•In adequate impregnation leads to loss of architectural details.

AUTOMATIC TISSUE PROCESSOR

•All the before mentioned procedures upto the impregnation step can be done automatically in a single, unmanned instrument , which is the Automated Tissue processor.

AUTOMATIC TISSUE PROCESSOR

•Transfer tissue both by day & night.

•Reduce time in each fluid by continual agitation.

•The 24hr clock is provided.

•Allows rapid processing.

•Racks to carry 25 cassettes.

•Eliminates human error.

•Tissue containers: Has 24 containers subdivided into 2 Compartments.

•Beakers &Wax baths: Provided with 10 beakers for reagents & 2 wax baths maintained at 56 degree temp. .

•Stirring Mechanism: One arm supports stirring mechanism.

• Time Mechanism: Consists of 1hr,24hr or 7-day electric Clock which has a time disc.

•Disc revolves once in 24hrs.

MICOROWAVE PROCESSING

•Invented by percy spencer in 1945.

•Based on principle, that heat peaks up diffusion of liquids in & out of tissues.

•Steps:- Dehydration. Clearing. Impregnation.

PROCESSING SCHEDULES -Thin sections less than 1mm -- 25 min. -For medium sections 1-2mm -- 60 min. -For thick sections 2-5mm --165 min.

•Isopropanol is used as clearing agents in microwave processing instead of xylene.

PROCESSING SCHEDULE FOR MICROWAVE PROCESSING•Dehydration with ethyl alcohol at 67 d-centigrade for 5 min.•Clearing with isopropyl alcohol at 74 d-centigrade for 3 min. •Impregnation with paraffin at 65 d-centigrade for 2 min.•Followed by 80 d-centigrade for 5 min.

• Tissue processing is a very much critical step that needs to be monitored with utmost care.

• Since it takes longer time to process the tissue, any mistakes alters the tissues requiring repetition.

• The tissues should not be under processed or over processed that may hamper the tissue details.

• It is essential that the tissues are processed with proper techniques and rendered them for subsequent steps.

CONCLUSION

•To get sections of diagnostic value, processing of tissue should be done with appropriate reagents & embedded in suitable embedding medium depending upon type of tissue.

Thank You!!!

REFERENCES

•Hand book of histopathologic techniques: C.F.A. CULLING.

•Theory & practice of histological techniques: JHON.D.BANCROT•Internet source, W.W.W.histotechniques.com, youtube.com