Histotechnique

Histology is microscopic study of normal tissue of body

Term histology derived from greek word

histos-means tissue……logy –means study coined

by Mayer in 1819

Histopathology means sciences of studying

structural changes in human body by

diseases

Histotechnology is concerned with

processing and preparation of tissues in such

a manner that it enables a satisfactory study.

Histotechnique is that branch of biology

concerned with demonstration of minute

tissue structure in diseases.

Paraffin embedding technique

Parlodion embedding technique

Exfoliative cytology

Fine-needle aspiration cytology

Fine-needle aspiration biopsy

Ground section

Frozen section

Immunohistochemistry (or IHC)

Fluorescence technique

Tissue microarray

Molecular techniques

The lab should be well illuminated and well-ventilated.

Rules and Regulations governing formalin and

hydrocarbonds

such as xyleneand toluene.

Limits set by the Occupational Safety and Health Administration (OSHA) that should not be exceeded.

These limits should be revised and revived to reduced any mishap.

Check the sharpness of scalpel, scissors and

quality of other ones like ruler, probes weighing

machines.

Every instrument used in the laboratory should

meet electrical safety specifications and have

written instructions regarding its use.

Flammable materials may only be stored in

approved rooms and only in storage cabinets that

are designed for this purpose.

Fire safety procedures are to be posted.

Safety equipment including fire extinguishers,

fire blankets,

and fire alarms should be within easy access.

A shower and eyewash should be readily available.

No smoking, eating or movements in the labs

Use disposable gloves

Laboratory accidents must be documented and

investigated with incident reports and industrial

accident reports.

Specific hazards that you should know about

include:

Bouin's solution is made with picric acid. This acid is

only sold in the aqueous state. When it dries out, it

becomes explosive.

Tissue specimens received in the surgical

pathology laboratory have a request form

that lists the patient information and history

along with a description of the site of origin.

The specimens are accessioned by giving

them a number that will identify each

specimen for each patient.

Tissues removed from the body for diagnosis arrive in the Pathology Department and are examined by a pathologist, pathology assistant, or pathology resident.

Gross examination consists of describing the specimen and placing all or parts of it into a small plastic cassette which holds the tissue while it is being processed to a paraffin block. Initially, the cassettes are placed into a fixative

When a malignancy is suspected, then the specimen is often covered with ink in order to mark the margins of the specimen. Different colored inks can be used to identify different areas if needed. When sections are made and processed, the ink will mark the actual margin on the slide

Machine processing

Manual processing

1. FIXATION.

2. DECALCIFICATION (if required).

3. PROCESSING & EMBEDDING.

4. SECTIONING.

5. MOUNTING.

6. STAINING.

Definition –process by which constituents of

cells and tissues are fixed in a chemical so

that they will withstand treatment with

various reagent with minimum loss or

decomposition.

In simple words—it prevents autolysis of

tissue.

1- To prevent autolysis and bacterial attack.

2- To fix the tissues so they will not change

their volume and shape during processing.

3- To prepare tissue and leave it in a condition

which allow clear staining of sections.

4- To leave tissue as close as their living state

as possible, and no small molecules should be

lost.

Fixation is coming by reaction between the

fixative and protein which form a gel, so

keeping every thing as their in vivo relation to

each other.

Types of fixation

1-Immersion fixation

2-Perfusion fixation

3-Vapour fixation

4-Spray fixation

5-Freeze fixation

6-Microwave fixation

Types of fixatives

Aldehydes

Mercurials

Alcohols

Picrates

Oxidizing agents

Inhibition of autolysis

Hardening of tissue

Solidification of colloid material

Optical differentiation

Effects on staining

Loss of material during fixation

Tissue shrinkage

1-Buffer and pH

2-Temperature

3-Penetration capacity

4-Volume change

5-Agitation

6-Osmolarity of fixation solution

7-Concentration of fixation

8-Duration of fixation

Fixation is best carried out close to neutral pH, in the range of 6-8.

Hypoxia of tissues lowers the pH, so there must be buffering capacity in the fixative to prevent excessive acidity.

Acidity favors formation of formalin-hemepigment that appears as black, polarizable deposits in tissue.

Common buffers include phosphate, bicarbonate, cacodylate, and veronal.

Commercial formalin is buffered with phosphate at a pH of 7.

Penetration of tissues depends upon the diffusability of each individual fixative, which is a constant.

Formalin and alcohol penetrate the best, and glutaraldehyde the worst.

Mercurials and others are somewhere in between.

One way to get around this problem is sectioning the tissues thinly (2 to 3 mm).

Penetration into a thin section will occur more rapidly than for a thick section

The volume of fixative is important.

There should be a 10:1 ratio of fixative to tissue.

Obviously, we often get away with less than this,

but may not get ideal fixation.

One way to partially solve the problem is to

change the fixative at intervals to avoid

exhaustion of the fixative.

Agitation of the specimen in the fixative will also

enhance fixation.

Increasing the temperature, as with all chemical reactions, will increase the speed of fixation, as long as you don't cook the tissue.

Hot formalin will fix tissues faster, and this is often the first step on an automated tissue processor.

Concentration of fixative should be adjusted down

to the lowest level possible, because you will expend

less money for the fixative.

Formalin is best at 10%;

Glutaraldehyde is generally made up at 0.25% to

4%.

Too high a concentration may adversely affect the

tissues and produce artefact similar to excessive

heat.

Also very important is time interval from of removal of tissues to fixation.

The faster you can get the tissue and fix it, the better.

Artefact will be introduced by drying, so if tissue is left out, please keep it moist with saline.

The longer you wait, the more cellular organelles will be lost and the more nuclear shrinkage and artefactual clumping will occur.

Penetrate cells or tissue rapidly

Preserve cellular structure before

cell can react to produce

structural artifacts

Not cause autofluorescence, and

act as an antifade reagent.

Coagulating Fixatives

Crosslinking Fixatives

Fix specimens by rapidly changing hydration state of

cellular components

Proteins are either coagulated or extracted

Preserve antigen recognition often.

DISADVANTAGE

Advantages

Disadvantages

• Cause significant shrinkage of specimens.

• Difficult to do accurate 3D confocal images.

• Can shrink cells to 50% size (height).

• Commercial preparations of formaldehyde contain

methanol as a stabilizing agent.

Glutaraldehyde

Formaldehyde

Ethelene glycol-bis-succinimidyl succinate (EGS)

Form covalent crosslinks that are determined by

the active groups of each compound

Simple fixative

Eg-Formaldehyde,Glutaraldehyde,Ethyl alcohol

Compound fixative

Eg-Carnoys fluid,Zenker’s fluid,Bouins fluid

According to action upon cell and tissue

1-Micro-anatomical fixative

To preserve microscopic structure of tissues.

Eg-Formal-saline,Buffered neutral formalin,Zenker’s fluid

2-Cytological fixative

To preserve intracellular structure.

Eg-Carnoy’s fluid,Clarks fluid,Flemings fluid

3-Histochemical fixative(freezing-drying

technique)

Eg-Buffered neutral formalin,Cold acetone

According to action

1-Physical methods ------heating

------microwaving

-------freeze drying

2-Chemical methods(biochemical approach)

Tolerant fixative---eg-formalin

Non tolerant—eg carnoy’s fixative

MOST COMMONLY USED FIXATIVE -----

1-10%formalin

2-10%formal saline

3-10%buffered formalin

The Process of removing calcium salts from the tissue and making them suitable for sectioning.

Some tissues contain calcium deposits which are extremely firm and extremely firm and which will not section properly with paraffin with paraffin embedding owing to the difference in densities between densities between calcium and paraffin.

Bone specimens are the most likely type here, but other tissues may but other tissues may contain calcified areas as well. well.

This calcium must be removed prior to embedding to allow sectioning. embedding to allow sectioning.

A variety of agents or techniques have been used to decalcify tissue and to decalcify tissue and none of them work perfectly. perfectly. Mineral acids, organic acids, EDTA, and

electrolysis have all been used.

Specimens should be decalcified in hydrochloric acid/formic acid working solution 20 times their volume.

Change to fresh solution each day until decalcification is complete. It may take 24 hours up to days or months depending

on size of the specimens.

Once the decalcification is complete, rinse specimens in water briefly and transfer to ammonia solution to neutralize acids left in specimens for 30 minutes.

Wash specimens in running tap water thoroughly up to 24 hours.

Routine paraffin embedding.

1 –Acid decalcification

2- Ion exchange resins

3-Electrical ionization

4-Chelating methods

1)Aqueous nitric acid(clayden ,1952)

-nitric acid—5-10ml

-distilled water—100ml

2)Nitric acid –formaldehyde(recommended for urgent

biopsies)

---nitric acid—10ml

----formalin—5-10 ml(added to prevent tissue swelling)

-----distilled water—100ml

3)Gooding and Stewarts fluid(1932)

Formic acid---5ml

Formalin-----5ml

Distilled water----90ml

4)Trichloroacetic acid

5)Von Ebners fluid

-Sodium chloride---50ml

-HCl—15ml(added daily 0.5% until

decalcification)

-Distilled water---100ml

Perenyl’s fluid—

10%nitric acid-----40ml

absolute alcohol---30ml

chromic acid(0.5%)—30ml

Excellent cytological preservation are possible-

--

Chemical test cannot be carried out---x-ray

should be used

Nitric and

Hydrochloric acids

rapid

damage cellular morphology,

so are not recommended for delicate tissues such as bone

marrow.

Acetic and Formic acid are better suited to bone marrow, since they are not as harsh.

However, they act more slowly on dense cortical bone.

Formic acid in a 10% concentration is the best all-around decalcifier.

Some commercial solutions are available that combine formic acid with formalin to fix and decalcify tissues at the same time.

EDTA can remove calcium and is not harsh (it is not an acid) but it penetrates tissue poorly and works slowly and is expensive in large amounts.

Electrolysis has been tried in experimental situations where calcium had to be removed with the least tissue damage. It is slow and not suited for routine daily

use.

Most used is EDTA which as ability to bind

calcium forming non-ionized soluble complex

EDTA works best on cancerous bone

Agent of choice for electron microscopy

EDTA solution(hilleman/lee)

----EDTA disodium salt---5.5 g

----Distilled water-----90ml

-----Formalin------10ml

Concentration of active reagent

Temperature

Agitation

Density of bone

X-ray (the most accurate way)

Chemical testing (accurate)

Physical testing (less accurate and

potentially damage of specimen)

Insert a pipette into the decalcifying solution containing the specimen.

Withdraw approximately 5 ml of the hydrochloric acid/formic acid decalcification solution from under the specimen and place it in a test tube.

Add approximately 10 ml of the ammonium hydroxide/ammonium oxalate working solution, mix well and let stand overnight.

Decalcification is complete when no precipitate is observed on two consecutive days of testing. Repeat this test every two or three days.

The Physical tests include bending the specimen or inserting a pin, razor, or scalpel directly into the tissue.

The disadvantage of inserting a pin, razor, or scalpel is the introduction of tears and pinhole artifacts.

Slightly bending the specimen is safer and less disruptive but will not conclusively determine if all calcium salts have been removed.

After checking for rigidity, wash thoroughly prior to processing.

Once the tissue has been fixed, it must be processed into a form in which it can be made into thin microscopic sections.

The usual way this is done is with paraffin.

Tissues embedded in paraffin, which is similar in density to tissue, can be sectioned at anywhere from 3 to 10 microns, usually 6-8 routinely.

The technique of getting fixed tissue into paraffin is called tissue processing

TISSUE PROCESSING

The aim of tissue processing is to embed the tissue in a solid

medium firm enough to support the tissue and give it sufficient

rigidity to enable thin sections to be cut , and yet soft enough not

to damage the knife or tissue.

Stages of processing:1- Dehydration.

2- Clearing.

3- Embedding.

Wet fixed tissues (in aqueous solutions) cannot be directly infiltrated with paraffin.

First, the water from the tissues must be removed by dehydration.

This is usually done with a series of alcohols, say 70% to 95% to 100%.

Sometimes the first step is a mixture of formalin and alcohol.

Other dehydrants can be used, but have major disadvantages. Acetone is very fast, but a fire hazard, so is safe only

for small, hand-processed sets of tissues.

Dioxane can be used without clearing, but has toxic fumes

Alcohols –

1)ethanol

2)methanol

3)isopropanol

Normal and tertiary butanols

glycol-ethers

Ethoxyethanol,polyethylene glycols

Other dehydrants-----1)acetone

2)phenol,beechwood

cresolate and aniline

Ethanol

-clear,colorless flammable

-hydrophillic

Advantages -------non toxic,reliable

Disadvantage------expensive,tissue shrinkage

Supplied as 99.85%ethanol

Anhydrous cupric sulphate added to final

ethanol scavanges any water present.

Duration of dehydration should be kept to

the minimum consistent with the tissues

being processed. Tissue blocks 1 mm thick

should receive up to 30 minutes in each

alcohol, blocks 5 mm thick require up to 90

minutes or longer in each change. Tissues

may be held and stored indefinitely in 70%

ethanol without harm

Removal of the dehydrant with a substance that will be miscible with the embedding medium (paraffin).

The commonest clearing agent is xylene.

Toluene works well, and is more tolerant of small amounts of water left in the tissues, but is 3 times more expensive than xylene.

Chloroform used to be used, but is a health hazard, and is slow.

Methyl salicylate is rarely used because it is expensive, but it smells nice (it is oil of wintergreen).

Replacing the dehydrating fluid with a fluid that is totally miscible with both the dehydrating fluid and the embedding medium.

Choice of a clearing agent depends upon the following:

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

- The processor system to be used.

- Intended processing conditions such as temperature, vacuum and pressure.

- Safety factors.

- Cost and convenience.

- Speedy removal of dehydrating agent .

- Ease of removal by molten paraffin wax .

- Minimal tissue damage .

Chloroform – tolerant, no effect on RI

Xylene, Benzene, toluene – rapid, intolerant, flammable, affects RI

Esters—n butyl acetate----xylene substitute

cedar wood oil – tolerant, expensive

Some clearing agents:

- Xylene.

- Toluene.

- Chloroform.

- Benzene.

- Petrol.

When xylene has completely replaced the

alcohol in the tissue, the specimen is ready to be

infiltrated with paraffin.

It is removed from the xylene and placed in a

dish of embedding paraffin, and the dish is put in

a constant temperature of about 60 °C

The exact temperature depend upon melting

point of the paraffin used.

During the course of several hours the specimen

is changed to two or three successive dishes of

paraffin so that all xylene in tissue is replaced by

paraffin

Finally, the tissue is infiltrated with the embedding agent, almost always paraffin.

Paraffins can be purchased that differ in melting point, for various hardnesses, depending upon the way the histotechnologistlikes them and upon the climate (warm vs. cold).

Wax hardness (viscosity) depends upon the molecular weight of the components and the ambient temperature.

High molecular weight mixtures melt at higher temperatures than waxes comprised of lower molecular weight fractions.

Paraffin wax is traditionally marketed by its melting points which range from 39°C to 68°C.

1802------lce gelatin glycerin

1879------Nitrocellulose

1881-------Paraffin

1950-------Acrylics(L R White)

Paraffin wax embedding

Water soluble waxes embedding

Celloidin embedding

Double embedding

Gelatin embedding

Ester wax embedding

There are four main mould systems and associated embedding protocols presently in use :1- Traditional methods using paper boats.2- Leuckart or Dimmock embedding irons or metal containers.3- the Peel-a-way system using disposable plastic moulds and4- Systems using embedding rings or cassette-bases which become an integral part of the block and serve as the block holder in the microtome.

Tissue processing

Embedding moulds:

(A) paper boat;

(B) metal bot mould;

(C) Dimmock embedding

mould;

(D) Peel-a-way disposable

mould;

(E) base mould used with

embedding ring

( F) Cassette

General Embedding Procedure1- Open the tissue cassette, check against worksheet entry to ensure the correct number of tissue pieces are present.

2- Select the mould, there should be sufficient room for the tissue with allowance for at least a 2 mm surrounding margin of wax.

3- Fill the mould with paraffin wax.

4 Using warm forceps select the tissue, taking care that it does not cool in the air; at the same time, correct orientation of tissue in a mould is the most important step in embedding. Incorrect placement of tissues may result in diagnostically important tissue elements being missed or damaged during microtomy.

5- Insert the identifying label or place the labeled embedding ring or cassette base onto the mould.

6- Cool the block on the cold plate, or carefully submerge it under water when a thin skin has formed over the wax surface.

7- Remove the block from the mould

.8- Cross check block, label and worksheet.

Turn on the water bath and check that the temperature is 35-37ºC.

Use fresh deionized water (DEPC treated water must be used if in situ hybridization will be performed on the sections).

Blocks to be sectioned are placed face down on an ice block or heat sink for 10 minutes.

Place a fresh blade on the microtome.

Insert the block into the microtome chuck so the wax block faces the blade and is aligned in the vertical plane. Set the dial to cut 4-10 µM sections.

The blade should be angled 4-6º.

Face the block by cutting it down to the desired tissue plane and discard the paraffin ribbon.

If the block is ribboning well then cut another four sections and pick them up with forceps or a fine paint brush and float them on the surface of the 37ºC water bath.

Float the sections onto the surface of clean glass slides.

If the block is not ribboning well then place it back on the ice block to cool off firm up the wax.

If the specimens fragment when placed on the water bath then it may be too hot.

Place the slides with paraffin sections in a 65°C oven for 20 minutes (so the wax just starts to melt) to bond the tissue to the glass.

Slides can be stored overnight at room temperature

A microtome is a mechanical

instrument used to cut biological

specimens into very thin segments for

microscopic examination. Most

microtome use a steel blade and are

used to prepare sections of animal or

plant tissues for histology.

STEEL KNIVES

NON-CORROSIVE KNIVES FOR CRYOSTATS

DISPOSABLE BLADES

GLASS KNIVES

DIAMOND KNIVES

The embedding process must be reversedin order to get the paraffin wax out of the tissue and allow water soluble dyes to penetrate the sections.

Therefore, before any staining can be done, the slides are "deparaffinized" by running them through xylenes (or substitutes) to alcohols to water.

There are no stains that can be done on tissues containing paraffin.

The staining process makes use of a

variety of dyes that have been chosen for

their ability to stain various cellular

components of tissue.

The routine stain is that of hematoxylin

and eosion (H and E).

Other stains are referred to as "special

stains" because they are employed in

specific situations according to the

diagnostic need.

Slides being stained on automated Stainer

Frozen sections are stained by hand, because

this is faster for one or a few individual sections.

The stain is a "progressive" stain in which the

section is left in contact with the stain until the

desired tint is achieved.

The stained section on the slide must be covered with a thin piece plastic or glass to protect the tissue from being scratched, to provide better optical quality for viewing under the microscope, and to preserve the tissue section for years to come.

The stained slide must go through the reverse process that it went through from paraffin section to water.

The stained slide is taken through a series of alcohol solutions to remove the water, then through clearing agents to a point at which a permanent resinous substance beneath the glass coverslip, or a plastic film, can be placed over the section.

Exfoliative Cytology – In this method, cells are collected

after they have been either spontaneously shed by the body

("spontaneous exfoliation") or manually scraped/brushed off

of a surface in the body ("mechanical exfoliation"). An

example of spontaneous exfoliation is when cells of

the pleural cavity or peritoneal cavity are shed into the

pleural or peritoneal fluid. This fluid can be collected via

various methods for examination. Examples of mechanical

exfoliation include Pap smears, where cells are scraped from

the cervix with a cervical spatula, or bronchial brushings,

where a bronchoscope is inserted into the trachea and used to

evaluate a visible lesion by brushing cells from its surface

and subjecting them to cytopathologic analysis

Suitable for hard structure like bone and

teeth.

Similar to paraffin embedding technique and

the only difference is infiltration of

embedding is done in parlodion instade of

paraffin.

It is a diagnostic procedure used to investigate superficial (just under the skin) lumps or masses. In this technique, a thin, hollow needle is inserted into the mass for sampling of cells that, after being stained, will be examined under a microscope. There could be cytology exam of aspirate (cell specimen evaluation, FNAC) or histological (biopsy - tissue specimen evaluation, FNAB). Fine-needle aspiration biopsies are very safe, minor surgical procedures. Often, a major surgical (excisional or open) biopsy can be avoided by performing a needle aspiration biopsy instead. Now a day, this procedure is widely used in the diagnosis of cancer.

Micrograph of a needle aspiration biopsy

specimen of a salivary gland showing adenoid

cystic carcinoma.

It is the process of detecting antigens (e.g., proteins) in cells of a tissue section by exploiting the principle of antibodies binding specifically to antigens in biological tissues.

IHC takes its name from the roots "immuno," in reference to antibodies used in the procedure, and "histo," meaning tissue (compare to immunocytochemistry).

Immunohistochemical staining is widely used in the diagnosis of abnormal cells such as those found in cancerous tumors. Specific molecular markers are characteristic of particular cellular events such as proliferation or cell death (apoptosis).

Immunohistochemistry labels individual proteins,

such as TH (green) in the axons of

sympathetic autonomic neurons

It consist of paraffin blocks in which up to 1000,separate tissue cores are assembled in array fashion to allow multiplex histological analysis.

In the tissue microarray technique, a hollow needle is used to remove tissue cores as small as 0.6 mm in diameter from regions of interest in paraffin-embedded tissues such as clinical biopsies or tumor samples. These tissue cores are then inserted in a recipient paraffin block in a precisely spaced, array pattern.

Sections from this block are cut using a microtome, mounted on a microscope slide and then analyzed by any method of standard histological analysis. Each microarray block can be cut into 100 – 500 sections, which can be subjected to independent tests. Tests commonly employed in tissue microarray include immunohistochemistry, and fluorescent in situ hybridization. Tissue microarrays are particularly useful in analysis of cancer samples