A Preface to

Histological and

Histopathological

Analysis

Tissue

ProcessingDr. Komal Parmar

PG- MS Anatomy, 1st year.

09.09.2016

Histology: Microscopic study of the normal

tissue.

Histopathology: Microscopic study of the

diseased tissue.

Histotechniques: Processing of the tissue

obtained from the body so as to render it

available for the microscopic analysis.

Histochemistry: Identification of chemical

components in biological cells and tissues.

Sources for tissue study in

Histology

Autopsy

Animal tissue

Biopsy

Cadavers

Receiving the Tissue Tissue removed from the surgical specimens or

dead bodies are liable to:

1. Autolysis

2. Pressure Injury

3. Osmotic Injury

4. Drying and desiccation

5. Microbial Breakdown

6. Excessive blood, mucus and contamination

Most of these problems are prevented by adequate

fixation.

Need for tissue fixation

Hardening and the prevention of putrefaction.

The weak molecular associations (H-bonds and salt linkages) are converted to stable condensations achieved by the action of stronger reactants leading to

Polymerization of tissue compounds

Denaturation and precipitation of proteins

Double bond cross linkages and cross saturation of fatty acids and lipids.

Concerns: Post

Fixation

Fixation(Aldehyde or

potassium dichromate in Water)

Microtomy

Embedding(Complex saturated

Hydrocarbons)

Dehydration(High concentration

lower alcohols replace water)

Clearing(Higher alcohols)

Tissues tend to fall apart and

separate on sectioning even after

the best fixation.

Embedding media like paraffin

and celloidin hold the tissue

together and make it possible to

make one cell layer thick section.

Paraffin does not mix with water

and mixes only with higher

alcohols that need to be used

before embedding (Antemedia

aka Clearing Agents)

While the clearing agents will mix

with paraffin, they will not mix with

the water (present in the fixative

as a solvent). Therefore a process

of Dehydration becomes

necessary wherein the alcohol

(polar organic solvent).

Tissue

procurement

Labeling and

Storage

Fixation

Dehydration

Clearing

Impregnation

Embedding

Section Cutting

Staining

Mounting

Protocols followed

in Tissue

Processing

Source, Specimen Identification

and Labeling

After receiving the

tissue specimens,

they undergo

careful Grossing

& Dissection,

and are thereafter

preserved in the

fixative solution.

Unfixed Tissue: Temporary

Mounts Direct Examination: Thin membranes

Smear and Squash Preparations: Friable

material

Freehand Sectioning

Frozen Sections

Fixation

o 2cm x 2 cm section of 2-3mm

thickness.

o Volume of fixative used= 10

times.

o Regularly change and agitate

the fixative

o Types of Fixatives

o Simple:o Ethyl Alcohol

o Formaldehyde

o Dilute Acetic Acid

o Picric Acid

o Chromic Acid

o Potassium Dichromate

o Mercuric Chloride

o Cadmium Chloride

o Acetone

o Osmium tetraoxide

FixativesGeneral Purpose Fixatives:

Ten Percent Formalin

Neutral Buffered Formalin

Zenker’s Fluid

Helly’s Fluid

Bouin’s Fluid

Carnoy’s Fluid

Alcohol- Formanlin

Plain Alcohol

Van de Grift’s Solution

Fixatives for Particular Purpose:

Aoyama’s Fluid: Golgi element.

Baker’s Calcium cadmium FormolSolution: Complex Lipids

Cobalt Calcium Formol Fixation: Complex Lipids

Gendre’s Fluid: Polysaccharides

Rossman’s Fluid: Polysaccharides.

Care During Fixation

Prepare ahead of time

Refrigerate

Placing the tissue in the bottle

Injecting and permeating the fixative through alternate approach.

Duration: Depends on the Tissue and the Fixative.

• Neutral Buffered Formalin fixes a block of tissue 1x2x0.4 cm in 4-6 hours.

• Time duration varies from 6-48 hours.

• Usually kept overnight.

Aftertreatment

Formalin requires no particular aftertreatment, and fixed tissues may be placed directly into the dehydrant. If using NBF, it is recommended that an alcoholconcentration of about 60% or so be used first.

If Picric acid is used, wash the tissue in 70% ethanol.

Treat mercury fixative containing sections with iodine mixture (Lugol’s Iodine + Sodium thiosulphate.

On using Zenker or Helley’s fluid the tissue needs to be washed for 10-15 hrs under tap water to get rid of excess chromium.

Decalcification

Immerse tissue cassette in 10% formic acid

with a stir bar overnight in a fume hood.

Rinse in running water for 30-60 minutes (the

smell should be gone).

Other decalcifying agents: Nitric Acid

Hydrochloric Acid

Picric Acid

Acetic Acid

Citric Acid

Dehydration

50% alcohol 90% Alcohol Absolute Alcohol Absolute Alcohol

Embed

Clearing Agents: Xylene, Toluene, Cederwood Oil,

Methyl Benzoate, Cederwood oil.

Also known as Antemedia.

The toxic effects of xylene include acute neurotoxicity, cardiac and kidney injury, cancer, blood dyscrasias, skin diseases, gastrointestinal disturbances, musculoskeletal system disorders, fetotoxicity and so on.

Embedding Media

Various mixture of the paraffin hydrocarbons have

melting points in the range of 35ºC to 65ºC.

The most commonly used paraffin wax has the mean

melting point near 55ºC.

Other media used for embedding are Celloidin,

Carbowax (Polyethylene glycol), synthetic epoxy

resins etc.

Step 1

• Impregnation• Wax bath for 6-8 hrs

Step 2

• Embedding• Transferring the tissue to

wax mould.

Infiltration Tissue is removed from

the clearing agent and is

placed in successive baths

of melted pure paraffin at a

set temperature slightly

above the melting point.

Successive changing

reduces clearing agent

retention.

Vaccum Embedding

Impregnated tissue

shifted to the mould

filled with molten

wax and is allowed

to cool and solidify.

Types of Moulds:

Leuckhart’s

Moulds- L shaped

brass pieces with

adjustable opposite

vertices.

Glass or Metal petri

dishes

Watch glass

Block Preparation

Tissue Sectioning

Microtome

Front View

-Utilize safety features properly.

-Clamps and Brakes

-Camel brush- Soft and coarse

-Ice blocks

-Warm water

-Hot plate and oven

Trimming: The goal of properly trimming a

block is to conservatively expose the tissue

do to a level where a representative section

can be obtained.

Trimming is usually done at thicknesses

between 10 and 30 μm.

Consider Factors Affecting Section Thickness.

Floating Out The Sections Adhesive Solution: Mayer Glycerine-egg albumin

Glycerine-Egg-Albumin: GEA

Egg white: 1 part

Glycerin: 1 part

Thymol:1 crystal

The adhesive mixture used for routine purposes is a dilution of the above. 15ml of water with 3 drops of GEA.

Flattening:-Water Bath

-Individual Slide flattening on hot plate

Drain excess water from beneath the section before drying. This is vital if slides are dried flat on a hot plate.

Slides can be stored in racks in an upright position, then dried in an oven.

Generally drying temperatures should not exceed 65 ˚C.

Excessive heat can cause droplets of water underneath a section to boil and this will cause damage.

Dry sections for between 10 and 30 minutes.

Some delicate specimens will produce best results when dried at 37 ˚C for a longer time (several hours to overnight).

Clean and maintain the Microtome thoroughly.

Automated Tissue Processor:

Histokinette Reduction of work load.

Constant agitation and mixing of the components.

Dehydration to remove all water & infiltration of paraffin

Permits the tissue to be cut into sections for slide staining

Working Tissues are inserted in stainless capsules/ uni-cassettes

Identified & placed into wire mesh baskets that are mounted on hood for processing

Movable hood raises, transports & lowers the baskets through series often via glass beakers containing processing agents & two stainless steel pots containing hot paraffin

Tissue is sequentially submerged & oscillated in a fixative dehydrated cleansing agent and finally immersed in paraffin

Programmable 12 stage timing sequence, having 24 hours calibrated disc

Delay action of 24 hours

Precision gear mechanism for trouble frees working

Safety device for tissue protection

Thermostatically controlled wax bath

Each Station with capacity of 1 Liter/2 Liter

Automatic agitations allow the effective processing of tissues

Assure utmost convenience in operation

Recognizing the common faults

in Slide Processing