It is processing and preparation of the tissue of body in such a manner as to satisfactory study of the tissues can be done.

Handling of SpecimenSpecimen should be transported in glass, plastic or metal container or in a plastic bag in 10% formalin. If formalin is not available at hand, place the specimen in refrigerator at 4oC to slow down autolysis. fresh material is needed for the following purpose:1. Frozen section2. Immunocytochemistry3. Cytological examination4. Microbiological sampling before histopathology5. Chromosome analysis6. Research purpose7. Museum display

Basic stepsPreparation of the tissuesProcessing of the tissuesPreparation of the sectionsStainingMounting

SpecimenHistology lab

Registration

Fixation

Grossing

Labelling

Dehydration

Clearing

Infiltration & impregnation

Embedding Trimming

Section cutting

Deparaffinization

Hydration

Staining

Dehydration

Clearing

Mounting

Fixation - AIMPrevent putrefaction & autolysisPreservation of cells & tissue constituentsHardening of soft tissuesConversion of semifluid consistency of cell to

an irreversible semisolid consistencyAlteration of refractive indices to varying

degree which enables unstained components to be seen easily.

Ideal fixativesCheap & easily availableStable & easy to handleFix quicklyMinimal loss of tissueEven penetration

Reagents employed as fixativesFormaldehydeMercuric chloridePotassium dichromatePicric acidEthyl alcoholGlutaraldehydeOsmium tetraoxide

Decalcification

A process to remove calcium from bone and other mineralized hard tissue in order to facilitate the process of cutting thin section .Stages:-I. Selection of tissueII. FixationIII. DecalcificationIV. Acid neutralizationV. Washing

Processing of tissue Embed the tissue in a solid medium.Firm enough to support the tissue and

enable thin sections to be cut.Soft enough not to damage the knife of

tissue.

There are two kinds of tissue processors:1- Moving tissue type 2- Moving fluid type.

Histokenette Automatic Tissue Processor

Tissue Cassettes

Tissue Processor With Vacuum And Fume Control

Vacuum Tissue Processor

Linear Tissue Processor With Touch Screen

PROCESSING SCHEDULEAutomated processing schedule1-Overnight schedule2-short processing schedule

Manual processing schedule

Over night schedule1- 10% formaline.2- 70% alcohol3- 95% alcohol4- 100% alcohol5- 100% alcohol6- 100% alcohol7- 100% alcohol8- 100% alcohol 9- xylene10- xylene11- wax12- wax

0 hrs½ hrs.½ hrs½ hrs1 hrs1 hrs1 hrs½ hrs1 hrs2 hrs2 x1/2 hrs4 hrs

Schedule for processing for small biopsy or for urgent work1- 10% formaline.2- 95% alcohol3- 95% alcohol4- 100% alcohol5- 100% alcohol6- 100% alcohol7- 100% alcohol8- 100%

alcohol/ethylene9- xylene10- xylene11- wax12- wax

20 min vacuum heat5 min y

450c5 min5 min5 min5 min5 min5 min5 min5 min5 min5 min

Rapid technique for thin slices of tissue

1- carnoys fluid- 45 min2- 100% alcohol x 6 15 min each3-xylene 10 min4- xylene 15 min5- wax 20 min6- wax 45 min

Manual processing schedule

Manual processing schedule for blockes

1- 70% alcohol2- 95% alcohol3- 95% alcohol4- 100% alcohol5- 100% alcohol6- 100% alcohol7- 100% alcohol8- xylene9- xylene10- wax with vacuume11- wax with vacuume12- wax with vacuume

0900 hrs to 1000 hrs1000 hrs to 1100 hrs1100 hrs to 1300 hrs1300 hrs to 1430 hrs1430 to 1600 hrs1600 to 1730 hrsovernight0900 to 1000 hrs 1000 hrs to 1130 hrs1130 hrs to 1230 hrs 1230 hrs to 1400 hrs1400hrs to 1600 hrs

½ hrs½ hrs1 hrs1 hrs1 hrs½ hrs1 hrs2 hrs2 x1/2 hrs4 hrs

Processing of tissueImportant steps of tissue processing by

paraffin wax technique.a)Dehydration.b)Clearing.c)Infiltration.d)Embedding.

Factors influencing the rate of processing-

a)Heat ( increase the rate of penetration, but limited to 45 degree)

b)Agitation (tissue lies on the base of the container ,rate of exchange of fluid is much less)

c)Viscosity ( quickness of impregnation due to lower viscosity of paraffin in fluid state )

d)Vacuum ( little increase dehydration and clearing but reduces the impregnation time)

DehydrationI. Water is completely removed from the

fixed tissues. II. Tissue blocks are placed in cassettes

with the identification number. III . Passed through increasing

concentration of alcohol with changes in each concentration.

DehydrationDehydrating agents 1-Alcohols- ethenol, methanol,

isopropanol,Polyethylene glycols (PEG) , diaxone

2-Other dehydrantsAcetone , Tetrahydrofuran , 2,2

dimethoxypropane Phenol,

Clearing

1-. It is the process in which water from cell & tissues is removed & is replaced by a fluid in which wax is soluble

2- .Most commonly used agent is xylene.3-. Xylene is miscible in both paraffin wax & alcohol.4-. Replaces alcohol & make room for paraffin.5- Other clearing agent – toluene , benzene ,

chloroform & cedar wood oil.

Xylene or toluene or benzene : Advantages – Cheap and rapid in action, can

be used for almost all tissue, used for both paraffin and celloidin embedding, benzene has less hardening effect then xylene.

Disadvantages –a)Make the tissue brittle if kept in the fluid for

a longer period.b)Excessive shrinkage for delicate tissue.c)May cause dermatitisd)Benzene is more inflammable and toxic and

known to be carcinogenic.

Infiltration & Impregnation

Infiltration – xylene is eliminated from the tissue by diffusion in the surrounding melting wax.

Impregnation – wax diffuses in the tissue by replacing the xylene.

It maintain the intra cellular structure during the section cutting on microtome.

EmbeddingI.Casting or blocking.II. Infiltrated & impregnated tissue is places in

warm liquid which forms a firm block after cooling.

III. Enables the tissue to be cut on a microtome.

IV. Most commonly used material is paraffin.V. Leuckhard embedding box ( consisting of

two L shaped pieces of heavy metallic material brass) arranged on a glass plate.

Ideally an infiltrating and embedding medium should be:

• soluble in processing fluids • suitable for sectioning and ribboning • molten between 30°C and 60°C • translucent or transparent; colorless • stable • homogeneous • capable of flattening after ribboning • non-toxic • odorless • easy to handle • inexpensive

Paraffin Melting point Feature

Commonly used

54 c

Hard wax 60 c Hard fibrous tissue

Soft wax 45 c Fetal & areolar tissue

Celloidin media ( nitrocellulose )1.Used when it is desired to avoid the use of

heat in CNS2.It is rubbery material.3.Give better support to tissue like skin, sclera

& subcutaneous tissues.

Gelatin media – for friable tissues like lung.

Resin , Agar

Used for cutting paraffin tissue sections of uniform thickness. A knob on the machine is used to adjust the thickness of section.

A knife is fixed in a clamp . The tissue block is drawn across the knife edge, the top and bottom of the block should be parallel and horizontal and at least 1 mm of paraffin should be present on all side of tissue.

Then ribbon of sections is transferred to warm water.

MICROTOME

MICROTOMERotary microtome – for paraffin embedding.Rocking microtome – for paraffin

embedding.Sliding microtome – for celloidin embeddingFreezing microtome – for frozen sectionCold ( cryostat) – for frozen sectionUltramicrotome – for electron microscopeLaser microtome-for contact free slicing

Trimming of the paraffin block.Attach the block to the microtome.Cutting of the section.Fix the section on the slides. adhesives – starch paste albumin ( glycerol +

white egg + distilled

water)

STAININGDeparaffinized (2 jars of xylene – each 2 min)

2 jars of alcohol – each for 2 min.

Rehydrated (90% alcohol – 1min f/b 70% alcohol for 1 min)

Rinsed in water

Stained in harris haematoxylene for 2 – 5min

Washing in running water till sections turn blue

Differentiation (1% acid alcoholic solution for 10 sec)Dip in 0.5% hydrochloric acid, nuclear aaper dark purple.( bluing) Rinse in water for 10-15 min

Counterstain (1% aqueous solution of eosin for 1-3 min)

Rinse in tap water

Dehydrate

Clearing in xylene

Mounting ( DPX / Canada balsam)

Frozen sectionMethod of sectioning of tissue after

making the tissue hard by rapid freezing.Advantages – rapidly prepared, Minimum shrinkage of tissue, Every method of staining is allowed

especially suitable for immunochemistry and almost obligatory for enzyme study

Essential technique for demonstration of certain lipid and enzymes.

Disadvantages – sections are thick and sometime difficult to interpret properly,

not always possible to maintain the structural element in their natural position .

it is practically impossible to obtain and correlate serial section

Methods Freezing microtome with carbon dioxide

which is popular method.Freezing microtome with thermoelectric

molecules.Use of refrigerated microtome ( cryostat).10 % formal saline is most common fixative

used, fresh unfixed tissue may be used.Tissue thickness should not exceed 3 mm.

AdvancesUse of different type of resins as alternative

to wax.Large block preparation and large sections

for study of whole pathological region of tissue.

Specimen radiography or thin section ultrasonography on freshly removed specimen to be sure that the whole pathology has been removed.

Microwaves for quick fixation, processing.

Microwave processing of tissueTissue is cut on small pieces ---- tissue

block are placed in isotonic saline and subjected to microwave irradiation for 120 sec. (full power )- ( 50 % power) dehydration by 70 % alchohal for 4 min, 100 % alchohal for 5 min, chloroform for 5 min then impregnation in wax for 5 min---- embedding- section cutting.

THANK YOU

PRESENTED BY – Dr. Narmada Prasad Tiwari