Histology

1. INTRODUCTION

Histology is the microscopic study of normal tissues while histopathology is the study of

diseased tissues. The techniques required for histological or histopathological microscopic

studies are termed Histotechniques (Histological Techniques or Microtechinques). Infact it is the

study of procedures or stages to reach the final stained slide of the specimen for

microscopic examination. The persons responsible for performing these procedures are

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These techniques, employed in a histopathology laboratory, help a histo-pathologist to

determine whether a patient has malignancy or some other disease process. The

microscopic examination involves following steps:

ƒ Documentation:

Every specimen is given the histopathology [HP] number which is pen down at top corner

of the request slip, in a specified record register and on the top of container.

ƒ Fixation:

It is a process by which the constituents of cells are fixed in a physical and partly also in a

compound state, so they will endure resulting treatment with different reagents with

a minimum of loss, or decomposition. This is accomplished by presenting the tissue to compound

compounds called fixatives.

ƒ Gross Examination:

The specimen is examined grossly by noting anatomical features of it. Representative

sections are selected for further procedures while small biopsies are selected as a whole.

ƒ Decalcification:

In case of calcified tissue (bone), calcium salts are removed from the bony tissue. It is done

before gross examination.

ƒ Tissue Processing:

It involves a number of stages in which selected portions of tissue are passed through a

number of chemicals in a sequence. In this stage tissues are impregnated with a setting

medium to facilitate its fine sectioning. Following four stages are involved:

i. Completion of Fixation 
ii. Dehydration
iii. Dealcoholization or Clearing

iv. Impregnation or Infiltration

ƒ Embedding:

In this step all processed tissues are blocked out in a solidifying medium (,.e.g. paraffin

wax) which is used in the impregnation stage.

ƒ Sectioning:

Paraffin blocks of tissues are then sectioned by means of a device termed as Microtome.

The sections which are produced are capable of transmitting light and are at a micron level

thickness.

ƒ Staining:

Tissue sections are picked onto slides and staining is performed in order to examine

different tissue components under the microscope.

ƒ Mounting:

Cover slip is applied on each section using appropriate medium to obtain permanent

preparation for microscope

2. FIXATION

As soon as the tissue is removed from the body, cells begin to undergo changes, which

result in their breakdown and ultimate destruction. These are referred to as post-mortem

changes, which might be either putrefactive or autolytic in nature. These changes are

prevented by decreasing the temperature or using chemicals called fixatives. So Fixation is

a process by which a tissue is safe-guarded against post-mortem changes.

x Putrefaction is due to the invasion of the tissue by bacteria, which generally disseminate

from the alimentary tract and spread quickly into the surrounding organs causing

decomposition.

x Autolysis is due to the action of enzymes from dead cells. This phenomenon mainly

occurs in the CNS and the endocrine system.

 

2.1 Rationale of Fixation:

There are number of reasons for which fixation is the first step in histotechniques. Most

cells consist of an outer complex membrane containing the fluid protoplasm, which is a

true, colloidal, mixed solution of salts, proteins, carbohydrates, lipids, organic acid and

enzymes. If the cells are not fixed, many of these substances would be lost by simple

solution, dialysis, osmotic swelling and rupture of the cells in the processing that must

precede the cutting and staining of sections. On microscopic examination, autolyzed tissues

show changes in the nuclei while the cytoplasm becomes cloudy and loses its staining

affinities. Fine intracellular structures such as mitochondria are lost in a very short period if

material is left unfixed.

2.2 Mechanism of Fixation:

Most fixatives act by denaturing or precipitating proteins. A fixative when acts on a protein

component of tissue, cross links them to form a sponge or meshwork due to which

structural proteins are stabilized and most of other free molecules, e.g. lipids, mucins etc.

are trapped inside meshes and so preserved. How much fixing liquid ought to be

approximately 10-20 times the volume of the specimen, except when osmium tetra oxide is

used.

2.3 Effects of Fixation:

2.3.1 Most fixatives produce some tissue hardening, which help in cutting of sections, but

this hardening effect will be reinforced by the action of alcohols used during the

process of dehydration.

2.3.2 Certain fixatives act as mordents for certain stains, e.g. after the use of mercuric

fixatives the staining of tissue constituents with many dyes is enhanced.

2.3.3 Fixatives usually increase the optical differentiation of cell and tissue components.

2.3.4 Fixatives render the cells insensitive to hypo and hypertonic solutions used

subsequent to fixation.

2.3.5 Micro-organisms are composed of proteins and they will also be fixed (killed)

preventing putrefactive changes in tissues.

2.4 Properties of an Idea Fixative:

A decent fixative ought to be equipped for satisfying the accompanying necessities:

2.4.1 It must penetrate the tissue rapidly and evenly. 

2.4.2 It must be simple to prepare and economical to use.

2.4.3 It should impart sufficient hardness to facilitate sectioning. 

2.4.4 It must inhibit bacterial decay and autolysis.

2.4.5 It must give good optical differentiation.

2.4.6 It must be non-irritant, non-toxic and non-corrosive.

2.4.7 It should not cause shrinkage, swelling or other distortion. 

2.4.8 It should allow tissue to be stored for long periods of time.

2.4.9 It should permit the restoration of natural colour for photography and mounting as

museum specimens.

2.5 Classification of Fixatives

 

There are three classifications of fixatives as below:

2.5.1 Classification I:

Initially fixatives were classified as simple and compound fixatives.

2.5.1.1Simple Fixatives:

These fixatives comprise a single chemical substance, e.g. formalin, ethanol etc

2.5.1.2Compound Fixatives:

These may be described as the product of two or more simple fixatives mixed together in

order to obtain the combined effect of their individual actions upon the tissue components.

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(formaldehyde + mercuric chloride) etc.

2.5.2 Classification II:

Fixatives are also grouped according to their action upon the cell and tissue constituents. 

2.5.2.1Microanatomical Fixatives:

These are those fixatives which preserve the tissue in a manner which permits the general

microscopical study of the tissue structures and allows the various layers of tissues to retain

their former relationship with each other.

2.5.2.2Cytological Fixatives:

These are employed for their specific action upon a specific part of the cell structure. They

are subdivided into nuclear and cytoplasmic fixatives depending upon which of the cell

inclusions they act.

ƒ Fixatives having glacial acetic acid or pH of 4.6 or less are termed nuclear fixatives.

ƒ Fixatives having pH above 4.6 or lacking glacial acetic acid are termed cytoplasmic

fixatives.

2.5.3 Classification III:

The most recent classification includes:

2.5.3.1Aldehydes:

These are organic compounds having functional group ±CHO. Fixation properties of

aldehydes are due to reactions of aldehydic group, e.g. formaldehyde, glutaraldehyde etc.

2.5.3.2Oxidizing Agents:

These bring about fixation through oxidation reduction reactions, e.g. chromic acid, 

osmium tetraoxide and potassium dichromate etc.

2.5.3.3Fixatives of Unknown Mechanism:

This group includes certain fixatives whose mode of action in fixation process is still not

clear, e.g. mercuric chloride, picric acid.

2.5.3.4Protein Denaturing Agents:

These fixatives bring about precipitation of structural proteins and more over denature

lysosomal enzymes in the course of their fixing actions, e.g. ethanol, methanol, glacial

acetic acid.

2.5.3.5Physical Methods of Fixation:

In this group of fixatives, fixation is done by means of heat and microwave energy. Heat

and microwave energy enhances oscillation energy of polar molecules as higher as 2650 Hz

at which proteins in tissues are precipitated and so fixed. (Microwave energy is heat energy

used in a control chamber).