tissue processing

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TISSUE PROCESSING Dr Neeta Chhabra Coordinator Dr Minu Bedi

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TISSUE PROCESSING Aim : To remove all extractable water from the tissue, replacing it with a solid medium firm enough to support the tissue and give it sufficient rigidity to enable sectioning without damage or distortion Types of processing Manual Automated

TISSUE PROCESSING: 

Stages of tissue processing Dehydration Clearing Impregnation & Embedding TISSUE PROCESSING

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DEHYDRATION Aim : To remove fixative and water from the tissue and replace them with dehydrating fluid Water Free Bound Types of Dehydration Dilution dehydration Chemical dehydration

DEHYDRATING AGENTS: 

CLASSIFICATION ALCOHOLS GLYCOL ETHERS ACETONE DIMETHOXYPROPANE PHENOL DEHYDRATING AGENTS Choice of dehydrant depends on Nature of task Processing method Embedding medium Economic factors

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Clear, colorless, inflammable, hydrophilic Act as dehydrants, Secondary coagulant fixatives

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Do not act as secondary fixatives

OTHER DEHYDRATING AGENTS: 

Acetone Clear, flammable, ketonic odor Fast , effective dehydrant Also act as secondary coagulant fixative Best for fatty specimens Toxicity low More Tissue shrinkage Dimethoxy propane Acidified dimethoxy propane Diethoxy propane Phenol, Aniline ,Beech wood creosote Used with solvents OTHER DEHYDRATING AGENTS

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TECHNIQUE OF DEHYDRATION Industrial Absolute Alcohol (99.24%) Transfer tissue to 1 st alcohol bath (70%) Volume of reagent : 20-50 times Graded series of alcohol 70%, 90%, Abs Alcohol I,II,III Conc of alcohol Time required Size & type of tissue Size of tissue Fixative used Tissue 1 mm thick - 30 minutes in each alcohol Blocks 5 mm thick- 90 minutes or longer in each change

ENTRY POINT OF TISSUE IN DEHYDRATING FLUID : 

Fixative Zenker’s fluid Washed→ 70% alcohol Helly’s fluid -do- Orth’s fluid -do- Flemming’s fluid -do- 10%Buffered Formalin 70% alcohol 10% Formal-saline 70% alcohol Bouin’s fluid 70% alcohol Formal sublimate 90% alcohol Susa fixative 95% alcohol Carnoy’s fluid 95% or Abs alcohol ENTRY POINT OF TISSUE IN DEHYDRATING FLUID

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Factors influencing rate of dehydration Agitation Heat Copper Sulphate DEHYDRATION Excessive dehydration- shrink ,hard and brittle Incomplete dehydration- prohibits infiltration of clearing agent and wax

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CLEARING Replacing the dehydrating fluid with a fluid miscible with both, the dehydrating fluid and the embedding medium and which may in turn be eliminated in the process of wax impregnation Hydrocarbons with refractive indices similar to protein Wax solvent & raises the refractive index of tissues Do not alter tissue reactivity Do not act as secondary fixatives

CLEARING AGENTS : 

Functions Dealcoholisation Facilitate penetration of paraffin wax Facilitate penetration of Canada balsam Make gross internal structure of embryos ,parasites and certain tissues more readily demonstrable to the naked eye . Some clearing agents also dehydrate Aniline oil –70% alcohol Oil of Bergamot- 90% alcohol Cedar wood oil- 95 % alcohol CLEARING AGENTS

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Volatile,toxic,inflammable.kept in containers with well fitted lids

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Odorless, inflammable, coagulate nitrocellulose

HYDROCARBONS : 

Petroleum solvents G entle ,non hardening Disadvantages Slow Do not render tissues transparent HYDROCARBONS Chlorinated hydrocarbons colorless, sweet odor, miscible with organic solvents & paraffin wax Carbon tetrachloride Cheap, Non inflammable Only little shrinkage Disadvantages Toxic & Slow

HYDROCARBONS : 

Chloroform Manual methods, Slower Advantages Tolerance, Used for specimens of CNS Non inflammable Disadvantages Does not effect Ref index V expensive, highly toxic HYDROCARBONS

TERPENES : 

Cedar wood oil Advantages Least hardening effect, makes tissue transparent Used as clearing agent for dense tissue Complete dehydration not essential before clearing Disadvantages Expensive ,Slow, more time Tissue has to be soaked in benzene or toluene Several extra changes of paraffin wax required. Contamination with acetic acid→ Partial solidification (add 0.5ml xylene to each 40ml cedar wood oil) TERPENES

ESTERS : 

Esters Xylene substitute Butyl acetate Amyl acetate Methyl benzoate Advantages Low toxicity, Least hardening Used as nitrocellulose solvents in double embedding techniques Disadvantages Expensive ,Slow, more time Tissue has to be soaked in benzene or toluene Several extra changes of paraffin wax required ESTERS

NEWER CLEARING AGENTS: 

Volatile oil in citrus peels-contains limoline Long chain aliphatic hydrocarbons( Clearite ) Less hazardous but not good on poorly fixed and dehydrated tissues NEWER CLEARING AGENTS

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CLEARING Choice of a clearing agent depends upon Type of tissues to be processed,& type of processing Processor system to be used Processing conditions- temp, vacuum and pressure Safety factors Cost and convenience Speedy removal of dehydrating agent Ease of removal by molten paraffin wax Minimal tissue damage

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TECHNIQUE OF CLEARING Tissue is lightly blotted Volume of clearing agent 25- 50 times Xylene , Benzene,Toulene One change ( 30- 60 min) Chloroform/ Carbon tetrachloride Overnight Cedar wood oil Till sinks

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IMPREGNATION 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 Purpose To remove clearing agent from tissues for them to be completely permeated by paraffin wax which is subsequently allowed to harden to produce a block from which sections may be cut.

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EMBEDDING OR CASTING Is the process by which tissues are surrounded by a medium such as agar, gelatin, or wax which when solidified will provide sufficient external support during sectioning Types of Impregnation / Embedding media Ribboning Media Water Soluble Gelatin, Gum, Polyethylene glycol Soap ,Paraffin Non ribboning media Water Insoluble Sugar and gum solution Paraffin wax Nitrocellulose

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EMBEDDING TECHNIQUES Paraffin Embedding Cellulose Nitrate Embedding Double Embedding Ester wax Embedding Water soluble wax Embedding Synthetic Resin Embedding Gelatin Embedding Freeze drying Freeze substitution

WAX IMPREGNATION: 

Paraffin wax - Melting point from 39°C to 68°C (54 °C preferred) Provides support for hard tissue Easy ribboning of tissues Hard wax – Melting point 60 °C Soft wax – Melting point 45 °C Lower melting point waxes hardened by 10-20 % of bees wax Advantage Easier cutting of sections without tissue being exposed to higher temp WAX IMPREGNATION

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MODIFIED PARAFFIN WAXES Properties of paraffin wax are improved for histological purposes by the addition of some substances They are called Plasticizers Substances added Bees wax Rubber Ceresin Plastic polymers Diethyl glycol distearate Advantages Improve ribboning Increase hardness Decrease melting point Improve adhesion between specimen and wax

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Bees wax Does not burn with smoky flame,imparts hardness Candle wax Reduces tendency of paraffin wax to soften in hot atmospheric conditions Ester wax Soluble in alcohol Clearing not required Harder blocks can be made Carbowax Polyethylene glycol ,water-soluble Fixed tissues can be directly transferred to embedding media . OTHER WAXES

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WAX IMPREGNATION OVENS/INCUBATORS Temp 50°C to 60°C Electrical Gas heated TECHNIQUE Tissue blotted lightly Transfer to filtered molten paraffin wax. Volume 25 -50 times Temp 56°C Fragile specimens are transferred to graded mixtures of clearing agent and wax

WAX IMPREGNATION: 

Duration of impregnation & No of changes of wax Size & consistency of tissue Clearing agent used Use of Vacuum embedding oven WAX IMPREGNATION

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VACUUM IMPREGNATION Principle By reducing pressure during wax impregnation air bubbles and clearing agent are more rapidly removed leading to rapid impregnation Indications Tissue required urgently Tissue from CNS Lung, Bone, Lymph glands, fatty tissue

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TECHNIQUE OF VACUUM IMPREGNATION

VACUUM IMPREGNATION : 

Expulsion and readmission of air should be slow to avoid rupture of delicate tissues If ATP is used ,tissue is transferred to V.E.O from second wax bath.- kept for 30mts for final change Advantages Evaporation of clearing agent & impregnation with paraffin wax occurs more quickly & completely Removes trapped air in specimens and air contained in blocks of lung tissue Reduces the duration of contact of tissue with molten wax, prevents the tissue from becoming too brittle VACUUM IMPREGNATION

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TYPES OF MOULD Leuckhart ΄ s 2 L shaped pieces of metal in various lengths (depth 1.5-2 cm) Paper or Aluminium foil boxes Watch glasses ,Glass or Metal Petri dishes Plastic ice cube trays and plaster of Paris boxes Metal or glass moulds lightly smeared with glycerin to prevent sticking of wax The most common types of moulds used are:

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TECHNIQUE OF EMBEDDING

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ORIENTATION OF TISSUE IN THE BLOCK Elongated tissues - diagonally across the block Tubular and walled specimens - embedded so as to provide transverse sections showing all tissue layers Tissues with an epithelial surface - embedded to provide sections in a plane at right angles to the surface Multiple tissue pieces- aligned across the long axis of the mould

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PRECAUTIONS WHILE EMBEDDING IN WAX No dust particles must be present The wax is clear of clearing agent Immediately after tissue embedding, the wax must be rapidly cooled to reduce the wax crystal size

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PARAFFIN WAX EMBEDDING MANUAL PROCESS Procedure Time Fix the tissue Wash in running water if reqd 70 % alcohol ( during day) 3-8h 90 % alcohol (overnight ) 16h Absolute alcohol I 2h Absolute alcohol II 3h Absolute alcohol III 3h Toluene (overnight ) 16h Paraffin wax I 1h Paraffin wax II 1h Paraffin wax III 1h Embed in fresh filtered paraffin

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RAPID MANUAL PROCESS METHOD When section required urgently Curetting / Small pieces of biopsies( not >3mm) Fix in Carnoy’s fluid 30-60min Absolute alcohol I 30 min Absolute alcohol II 30 min Absolute alcohol III 30 min Xylene or Toluene 15-30min Paraffin wax I (Vacuum oven) 20 min Paraffin wax II ˝ 20 min Paraffin wax III ˝ 20 min Embed in fresh filtered paraffin wax

PARAFFIN WAX EMBEDDING: 

Advantages Reasonable speed of tissue processing Good consistency for serial sectioning Wide range of thickness of section possible Ribboning of sections is possible. Blocks are durable and can be stored indefinitely Disadvantages Tissue becomes hardened and shrunken Difficult to cut sections of cortical bone Not suitable for large pieces of brain PARAFFIN WAX EMBEDDING

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CELLOIDIN EMBEDDING Cellulose nitrate (Soluble gum Cotton or Pyroxylin ) Used for exceptionally hard tissues because of its rubbery consistency. Celloidin wool damped with N Butyl Alcohol Solution Dissolved in equal amounts of Absolute alcohol & Ether. Kept in well stoppered jars NECOL ( Necoloidin ) Similar, Cheaper

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CELLOIDIN EMBEDDING TECHNIQUE Thoroughly fixed & dehydrated tissue (5-8mm) Abs Alcohol & Ether mixture 24 h 2% Celloidin in Abs Alcohol& Ether 5-7 days 4% Celloidin in Abs Alcohol& Ether 5-7 days 8% Celloidin in Abs Alcohol& Ether 3-4 days Embed

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Embed in a mould containing 10% Celloidin Position tissue with surface to be cut UPWARDS Pour more celloidin up to depth of 30-40 mm leaving a margin of 10 mm on each side Transfer to a glass dish with well fitting lid Keep ether in small dish alongside. Leave for 1-3 hrs Turn the tissue with surface to be cut facing DOWNWARDS Ether vapor for further 1-3 hrs Replace Ether with chloroform Leave for 12-24 h or until Celloidin of hard rubbery consistency Store in 70% alcohol CELLOIDIN BLOCKS

CELLOIDIN EMBEDDING : 

Advantages Clearing not required Less tissue shrinkage (absence of heat) Facilitates preparation of sections of brain Improved cutting qualities Superior cohesion of tissue layers . e.g. Eye Disadvantages Highly inflammable Processing very slow Difficult to cut thin sections Non ribboning medium Blocks & sections must be stored in 70 % alcohol Sectioning requires a heavy microtome CELLOIDIN EMBEDDING

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LOW VISCOSITY NITROCELLULOSE Advantages Low viscosity- higher concentration can be used Thinner sections can be cut Greater water tolerance Disadvantages Highly explosive Sections can crack during handling and staining Difficult to stain because of alcohol solubility Solution: 5%, 10%, 20% 1% Tricresylphosphate or 0.5% Oleum ricini used as plasticiser

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DRY CELLOIDIN EMBEDDING Overcomes the disadvantages of standard method

DOUBLE EMBEDDING: 

Combines the advantages of Paraffin & Celloidin Useful for Brain Bone Muscle Large pieces of dense fibrous tissue TECHNIQUE Thoroughly dehydrate Transfer to Abs alcohol & Ether (equal parts) 1 or 2% Celloidin at room temp 2-3 days Draw off excess Celloidin Transfer to chloroform 24 h Impregnate in molten paraffin wax Embed DOUBLE EMBEDDING

DOUBLE EMBEDDING: 

Peterfi’s Double embedding method Thoroughly dehydrate Transfer to 1%Celloidin Methyl benzoate mixture. 24h (3changes) Tissue is transparent – complete impregnation Transfer to Toluene 8h (3changes) If 3 changes for Celloidin – 3 changes of Toluene required Impregnate with several changes of paraffin wax Embed DOUBLE EMBEDDING

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ESTER WAX EMBEDDING Ester wax Mixture of Diethylene glycol distearate Glyceryl monostearate 300 Polyethylene glycol Much harder than paraffin wax (lower MP 46°C- 48°C ) Compressible like celloidin Used for heat labile tissues. Advantages Thin sections are more easily cut (1 μ ) Ribboning is easy

TECHNIQUE ESTER WAX EMBEDDING: 

70% Cellosolve 8h 90% Cellosolve Overnight Pure Cellosolve I 2 ½ h Pure Cellosolve II 2 ½ h Pure Cellosolve III 3 h Cellosolve & Ester wax (50:50 at 37°C) Overnight Ester wax in oven (48°C-50°C,3 changes) 1h Embed in molten wax. Rapidly cooled , not submerged. Cut slowly ,sharp knife. Microtome Sledge type. TECHNIQUE ESTER WAX EMBEDDING

WATER SOLUBLE WAXES: 

For demonstration of lipids & to preserve enzymatic activity in plants To overcome tissue shrinkage, damage &distortion inherent in paraffin wax technique Advantages Dehydration and clearing not reqd Less tissue shrinkage (4.7%) Disadvatages Floating of section can cause fragmentation Tissues with large amount of fat are not penetrated AC labs are required due to hygroscopic nature . WATER SOLUBLE WAXES Solid Polyethylene glycols

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TECHNIQUE WATER SOLUBLE WAXES Wash tissue & treat with acetone if fat present 50% PEG 900 in d/w 10- 15 min 4 changes of molten PEG 900 (28°C-30°C) 45 min each Mix of 1: 1 PEG 900 & Nonex 63 B (39°C) 30-40 min Mix of 1: 3 PEG 900 & Nonex 63 B (39°C) 15min 3 changes of Nonex 63B (39°C) 30-40 min each Embed in paper / paraffin wax boat Blocks solidified at room temp/refrigerator Not immersed in water. Protected by coating with paraffin wax.

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GELATIN EMBEDDING Frozen sections of friable, Necrotic tissue Multiple small fragments Technique Wash in running water 24 h 12.5% gelatin in 1% phenol (37°C) 24 h 25% gelatin in 1% phenol (37°C) 24 h Embed in 25% gelatin Set in cold room or refrigerator Trim leaving margin of 3mm & little on surface

GELATIN EMBEDDING: 

Fixative should be thoroughly washed to prevent premature hardening or fixation of the gelatin. Tissue should not be > 2-3mm in size Immersed in 10 % formalin for 12- 24 hrs to harden Disadvantages Gelatin holds the stain giving an indifferent background. Sections difficult to manipulate as they have a tendency to crease & fold over themselves GELATIN EMBEDDING

AGAR EMBEDDING: 

Friable tissues to be cut on freezing microtome TECHNIQUE Place tissue in 5ml of 10% formalin .Boil Replace formalin with 2% sterile agar in water. Boil for 1min Pour off Agar. Remove tissue and place on stage of freezing microtome. Freeze & cut sections Advantage Agar not stained with H&E AGAR EMBEDDING

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SYNTHETIC RESIN EMBEDDING Used for EM (0.005-0.1 μ ) Acrylic resins : Ethyl , Methyl, Butyl Methacrylate Epoxy resins : Araldite Polyester resins Advantages Exceptionally hard material( undecalcified bone) can be sectioned. Tissue structure is not destroyed Thin sections can be cut Disadvantages Require special reagents for dehydration and clearing Special microtome for sectioning

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FREEZE DRYING Histochemical investigations After freeze drying tissue can be embedded in Paraffin wax Polyethylene glycol Ester wax Synthetic resin Steps Immediate freezing (Quenching) Drying Chamber (Vacuum 0.01mm Hg) Temp- 30°C to-60°C Ice is removed by sublimation. Water vapor absorbed by drying agent –Phosphorus pentoxide . Impregnated with impregnating medium under reduced pressure

FREEZE DRYING: 

Embedding in paraffin wax VEO containing molten wax at routine pressure Tissue impregnated on sinking to the bottom of wax bath Thoroughly degassed embedding medium placed at the bottom of drying tube. When drying is complete temp of tube is brought to room temp and tube is heated slowly until wax melts. Dried tissue sinks & is impregnated. FREEZE DRYING

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FREEZE SUBSTITUTION Done at low temp in liquid dehydrating agents-alcohol & acetones No expensive equipment required Steps Quenching Simultaneous fixation & removal of ice from tissue by immersion in absolute alcohol or acetone at-60°C-70°C for several days Gradually raise temp & complete the embedding process at appropriate temp .Embedded in Polyester/ Esterwax Advantages : P revents autolysis & putrefaction Prevents diffusion and dissolution of tissue ingredients Prevents ice crystal formation in quenched tissues

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AUTOMATIC TISSUE PROCESSOR Time required for processing tissue is reduced if tissue is Suspended in fluid Continuously agitated Moved from one reagent to another when desirable . AUTOMATIC TISSUE PROCESSORS Tissue transfer processors Fluid transfer processors

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AUTOMATIC TISSUE PROCESSOR Automation consists of an instrument that moves the tissues/ fluid on a preset time scale. A T P while removing all the water from a tissue sample and replacing it with paraffin wax, does so with optimum speed to minimize damage to the tissue caused by dehydration and shrinkage

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AUTOMATIC TISSUE PROCESSOR Temp is maintained around 60°C Timings controlled by a timer which can be adjusted in respect to hours and minutes Tissue blocks not > 5mm with label are placed in tissue cassettes. Different jars containing reagents. Arranged in a sequence Tissue moved from one jar to another by a mechanical device.

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AUTOMATIC TISSUE PROCESSOR Advantages Time for processing of tissues is greatly reduced. Because of continuous agitation ,the time the tissue remains in various agents is greatly reduced

ROUTINE AUTOMATIC PROCESS ( BLOCKS 3-5MM) : 

Formal Sublimate 4 h 90% alcohol 2 h Abs alcohol I 1 h Abs alcohol II 1 h Abs alcohol III 2 h Abs alcohol IV 2 h Toluene I 1½ h Toluene II 2½ h Wax bath I 3 h Wax bath 3 h 22 h ROUTINE AUTOMATIC PROCESS ( BLOCKS 3-5MM) REAGENT TIME

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THANK YOU