HISTOTECHNIQUES

PART -1

Introduction

• 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 given a number that will identify each specimen for each patient.

• Tissues arrive in the Pathology Department and are examined.

• Gross examination consists of describing the specimen and placing all or parts of it into a small plastic cassette which after fixation will be processed to a paraffin block.

INTRODUCTION• Histo techniques deals with the

preparation of tissues for microscopic examination.

• Submission of total or selective part of the tissue for examination into a series of processes,

1.Fixation 2.Dehydration tissue 3.Clearing processing 4.Embedding 5.Cutting 6.Staining.

• Fixation : It is a process to preserve tissues permanently in as life-like state as possible.

• Dehydration: Gradual removal of water from the tissue using ascending grades of ethyl alcohol to prevent tissue shrinking.

• Clearing: Replacement of alcohol in tissue by clearing fluid like xylene, benzene, or acetone

• Embedding: Tissues are impregnated in paraffin – with in a capsule.

• Cutting: Paraffin blocks are cut by microtome using metal knife, into thin sections ~ 3-4µ.

• Staining : Sections are spread on the glass slide coated with egg-albumin and taken for staining.

• Mounting : Stained slides are dried and mounted with a cover slip via mounting media.

Did you observe the order of processing?

• Why “fixation” first? • As soon as the tissue is removed – deprivation of

oxygen and metabolites.• Autolysis – in those tissues which have maximum

metabolism.• To preserve as nearly as possible the natural state of

the tissue cells and check these processes of decomposition with a minimum of delay.

• Natural color and appearance and consistency changes with minimum delay in fixation – best to view in the fresh state.

Other reasons…• Cells contain – colloidal solution of salts,

proteins, CHO, lipids, enzymes and organic acids.

• Were the cells not “fixed” – many of them would be lost.

• Autolysed tissues loose their staining affinities.

• Fine intracellular organelles are lost.

Definition

Fixation is a process by which the constituents of the cells and therefore of the tissues, are fixed in a physical and partly also in a chemical state, so that they withstand subsequent treatment with various reagents with a minimum loss, less significant distortion or decomposition….

Mode of action and effects on tissues…

• Fixatives act by denaturing / precipitating the proteins.

• This forms a meshwork which holds the other cell constituents together.

• Produce some tissue hardening which helps in cutting.

• But this hardening is reinforced by the alcohols used in dehydration.

• Micro-organisms are also fixed and killed –prevents putrefaction.

• Mordant – to enhance the staining process.

Ideal fixative

• Cheap, stable, safe to handle.• Penetrate tissues easily ,rapidly, isotonic• Cause minimum loss of properties• Preserve tissues in their natural state• Avoid excessive hardness of tissues• Non toxic and non allergic.• There is no perfect fixative, though

formaldehyde comes the closest.

Types of fixatives

• There are five major groups of fixatives, classified according to mechanism of action:– Aldehydes – Mercurials– Alcohols – Oxidizing agents – Picrates

1.Aldehydes

Formaldehyde (formalin) Glutaraldehyde• Tissue is fixed by cross-linkages/polymerization formed

in the proteins forming methylene bridges, particularly between lysine residues.

• This cross-linkage does not harm the structure of proteins greatly, so that antigenicity is not lost.

• Therefore, formaldehyde is good for immunohistochemical techniques.

• However, they cause some alteration which may interfere with the staining, which can be reversed by washing in water.

Formalin - penetrates tissue well, but is relatively slow.

- The standard solution is 10% neutral buffered formalin in water. - A buffer prevents acidity that would promote autolysis and prevents precipitation of formol - heme pigment in the tissues.

Glutaraldehyde - causes deformation of alpha- helix

structure in proteins so is not good for immunoperoxidase staining. - However, it fixes very quickly so is good for electron microscopy. - It penetrates very poorly, but gives best overall cytoplasmic and nuclear detail. - The standard solution is a 2%

buffered glutaraldehyde.

Formalin • Commercial formaldehyde (formalin) – saturated solution

of formaldehyde gas in water (40%gas by weight) – regarded as 100% formalin.

• A mixture of 10ml formalin with 90ml water or saline is known as 10%formalin.

• If stored in cold environment – formalin becomes turbid – due to the formation of paraformaldehyde.

• This can be removed by filtration or by adding 11-16% ethanol.

• Formation of formic acid due to oxidation causes interference in staining techniques.

• This is prevented by using formal saline with calcium carbonate.

• Amount of fixative should be approx 10-20 times the volume of the specimen.

• 4mm thick sections of the tissues will be adequately fixed with the above amount in 8hrs at room temp & 4hrs with agitation.

• Complete fixation requires 12-24 hrs at room temperature.

Advantages Disadvantages

• Cheap, easy to prepare, stable.

• Penetrates tissues easily.

• No excessive hardening/brittleness.

• Tissue enzyme studies can be done.

• Irritant – dermatitis.• If exposure is >1ppm

conc for 30min – reduction in ventilating capacity, asthma in allergic individuals.

• Forms artifactual pigment – acid formaldehyde hematin.

Removal of formalin pigment

1.Kardasewitsch’s method.

70%ethyl alcohol,28%ammonia

2.lillie’s method.

acetone, hydrogen.per,28% ammonia

3.Picric acid method.

saturated alcoholic sol of picric acid

Routine formalin fixatives

• 10% formol saline – water

sodium chloride

formalin• 10% buffered neutral formalin – water

NaH2PO4

Na2HPO4

formalin

2.Mercurials • Mercurials fix tissue by an unknown mechanism. and

they contain mercuric chloride Zenker's fixative. • These fixatives penetrate relatively poorly and cause

some tissue hardness, but are fast and give excellent nuclear detail.

• Mercury deposition on the tissues must be removed.• Best application is for fixation of hematopoietic and

reticuloendothelial tissues. • Since they contain mercury, they must be disposed of

carefully.

3.Alcohols • Alcohols, including methyl alcohol (methanol)

and ethyl alcohol (ethanol ), are protein denaturants and are not used routinely for tissues because they cause too much brittleness and hardness.

• However, they are very good for cytological smears because they act quickly and give good nuclear detail.

• Contraindicated when lipids are to be demonstrated.

• Carnoy’s fixative – for glycogen.

4.Oxidizing agents

• Oxidizing agents include permanganate fixatives (potassium permanganate), dichromate fixatives (potassium dichromate), and osmium tetroxide.

• They cross-link proteins, but cause extensive denaturation.

5.Picric acid fixatives

• Picrates include fixatives with picric acid. Bouin's solution – 1.2% sat.picric acid formalin glaceal acetic acid • It does almost as well as mercurials with nuclear

detail but does not cause as much hardness. • Picric acid is an explosion hazard in dry form. • As a solution, it stains everything it touches

yellow, including skin.• It is a best fixative for glycogen demo.

Summary • Formalin is used for all routine surgical pathology and autopsy

tissues when an H and E slide is to be produced.

• Glutaraldehyde is recommended for fixation of tissues for electron microscopy.

• Zenker‘s (Mercuric) fixatives are recommended for reticulo-endothelial tissues including lymph nodes, spleen, thymus , and bone marrow.

• Zenker's fixes nuclei very well and gives good detail. • However, the mercury deposits must be removed

(dezenkerized) before staining or black deposits will result in the sections.

• Bouin's (Picric acid) solution is recommended for fixation of testis, GI tract, and endocrine tissue

• Alcohols, specifically ethanol, are used primarily for cytologic smears. Ethanol (95%) is fast and cheap.

Factors effecting fixation

There are a number of factors that will affect the fixation process:– Buffering - Neutral pH(6-7), phosphate, bicarbonate.– Penetration - Formalin and alcohol penetrate the best. Glutaraldehyde the worst. – Volume - 10:1 ratio of fixative to tissue– Temperature - Increase– Concentration - Formalin is best at 10% Glutaraldehyde at 0.25% to 4%.

Tissue processing

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

• The main steps in this process are

dehydration and clearing.

Dehydration

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

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

• A reagent which can easily react with water so that it can easily penetrate into the tissues.

• Ethyl alcohol – graded, beginning with 70%.• Other option – isopropyl alcohol.• Amount at each stage should not be <10 times

the volume of tissue to be dehydrated.

Clearing (dealcoholization)

• The next step is called "clearing" and consists of removal of the dehydrant with a substance that will be miscible with the embedding medium (paraffin).

• The commonest clearing agent is xylene.

• Others :Toluene - expensive than xylene.

Chloroform - health hazard & slow.

Methyl salicylate - expensive.

• Refractive index of the clearing agents is equal to that of the tissues so that they are rendered transparent.

• Amount – not <10 times the volume of the tissue.

• Applications: before wax impregnation.

before mounting the slides.

for making tissues

transparent.

Automated tissue processor

Graded alcohols

In our lab…

• Microm automated tissue processor• Contains 12 bottles• Each adjusted for one hour.• 0ne – 10% formalin• 6 –isopropyl alcohol in graded percentages.• 3 –xylene• 2 -paraffin

Impregnation and embedding

• It is a process that involves impregnation of the tissues with a medium that will fill all the natural cavities and spaces of the tissues..even of the cells.

• That will set into a sufficiently firm consistency to allow cutting.

• Formation of a block of the embedding medium around the specimen – for easier handling fixing it to the holder on microtome and cutting.

• Volume of impregnating medium – 25 times the volume of the tissue.

• Two types of embedding : paraffin wax embedding celloidin embedding• Paraffin is routinely used. Tissues

dehydrated and cleared

impregnated with paraffin wax by immersion in successive molten wax baths. embedded in wax block• For firmness of the medium – many combinations were

in use• Introduction of paraplast.

Advantages

• It is a mixture of highly purified paraffin wax and synthetic plastic polymers.

• Maintains the melting point.• It gives uniform blocks, regardless of the rate of

setting.• Sections may be cut without cooling on the block

face.• Does not crack.

Carbowax

• Does not need dehydration and clearing – soluble in water – lipids/neutral fats are not removed and processing time is reduced.

• It is highly hygroscopic – blocks must not come in contact with water.

• Water soluble – sections cannot be floated on water – needs other solutions.

• Tendency to crumble while cutting.• However, can be used for enzyme histochemical

studies.

Impregnation and embedding

Blocking out the molds

• Leuckhart’s embedding irons

• Waxed paper cups

• Tissue Tek system

Procedure Wax used is kept molten at 2 deg.cel higher than its MP.

The capsules are opened and the number noted.

Suitable mold is chosen and is filled with wax within a few mm of its top.

Tissue is picked out of its cassette by a blunt forceps that was preheated.

Tissue is placed on the molten wax with the cutting surface facing down and kept pressed for some time.

When the wax becomes partly solid, they removed from the steel base and are cooled in the refrigerator – prevents crystallization of the wax.

Gives blocks of uniform, smooth and solid consistency.

At least 2mm of wax should surround the tissue.

A small paper tag bearing the tissue no. must be attached.

Dimensions – 5mm – depth 2mm - surrounding tissue 4mm - surface

Leuckhart’s moulds : L-molds L- shaped pieces of brass base – 1/8th inch 3/2 inches square good initial setting of blocks but too slow for busy labs.

Tissue Tek system• Stainless steel base molds in which the tissue block

is embedded.• Tissue is placed in the steel base with the required

plane of cutting and then filled with wax.• Another plastic mold is placed on top of the steel

base which is filled with tissue and wax.• The number of the specimen is then fixed to the side

of the block.• The metal bases are pre coated with an alcoholic

substance so that the wax do not adhere to them.• The assembly is then allowed to set and then

removed from the steel base and cooled in a refrigerator

Different tissue teks

• Mark I & II tissue tek systems Five stainless steel molds – 7x7 15x15 24x24 30x24 37x24• With 5mm depth.• Peel-a-way tissue tek – disposable plastic walls –needs no rough

cutting.• TIMS tissue tek – capsules are used through out - contains 3 compartments. - Required to pen the lower lid in order to cut – complete in-built system

In our lab…

• Embedding unit – contains 2 parts

EC 350-1 – paraffin in molten state (67

deg.cel)

EC 350-2 – cooling chamber

• Microm company

• Uses tissue tek system of embedding.

• Blocks are ready for tissue /section cutting……

to be continued in PART-II