T. S.

MISRA MEDICAL COLLEGE

DEPARTMENT OF PATHOLOGY
Histopathological And Histochemical

Techniques In Processing Of A

Tissue In Routine Biopsy

Presented by :

1. Sourabh Kundu
Moderated by : 2.Tripti Sharma

Dr.(Prof) MONIKA GUPTA 3. Trisha Singh

4. Utkarsh Jaiswal

5. Vartika Singh
INTRODUCTION
Histology, the study of tissue had been done for century in order to detect and observe
normal structure of tissue. However, there are several reasons that might alter tissue
from its original structure. Several processes, such as fixation, processing, embedding,
cutting and staining had been done to reduce the limitation of tissue observation. In this
study, ileum, skin, and appendix were experimental by using neutral buffered
formaldehyde as fixative and stained with Van Gieson stain, Haematoxylin and eosin,
Masson's Trichrome stain and Periodic Acid Schiff.

Histology' term is derived from Greek: Histos means tissue while Logio means study of
or knowledge. By refer to these terms, it actual refer to study of tissues for living
organism. Nowadays, histology is used loosely to include all subdivision of microscopic
anatomy. The correlation between structure and function provide the evidence that
show the histology is an intriguing and readily understandable subject. In the study of
histology, preparation of tissue for microscopic viewing is an important consideration.
This is because cell and tissue cannot be studied to advantage unless they are well
prepared for microscopic examination. There are two subdivisions in whole method of
tissue preparation: method involving direct viewing of living cell and method employed
with dead cell (fixed and stained). In this practical session, fixed and stained
preparation was applied. Ileum, thick skin, and appendix were selected.However,
different tissues that stacked together are hard to be recognized and differentiated, as
most of them appear as colourless compound. This study provided with a full
processes which allow the observation of tissue become easier.

"Tissue processing" describes the steps required to take an animal or human tissue
from fixation to the state where it is completely infiltrated with a suitable histological
wax and can be embedded ready for section cutting on the microtome.

Tissue processing can be performed manually (hand processing), but where multiple
specimens must be dealt with, it is more convenient and much more efficient to use an
automated tissue processing machine (a “tissue processor”). These devices have been
available since the 1940’s1 and have slowly evolved to be safer in use, handle larger
specimen numbers, process more quickly, and to produce better quality outcomes.
There are two main types of processors: the tissue-transfer (or “dip and dunk”)
machines where specimens are transferred from container to container to be processed,
and the fluid-transfer (or “enclosed”) types where specimens are held in a single
process chamber or retort and fluids are pumped in and out as required. Most modern
fluid-transfer processors employ raised temperatures, effective fluid circulation and
incorporate vacuum/pressure cycles to enhance processing and reduce processing
times

TECHNIQUES :

Fixation

Fixation is done as the first step to preserve the tissue substrate and render protein
structure and other tissue components insoluble in all reagents exposed later in either
processing or staining process. This step must be done as soon as possible as most
cells contain lysosome which will carry out cell autolysis and release digestive enzymes
to break down cell components after the cells have died. Besides that, extracellular
microorganism, mostly bacteria, will take opportunities and break down the dead cell
through putrefaction for nutrient absorption. By fixation, autolysis and putrefaction can
be halted and tissue substrates can be preserved.

Aims and Effects of fixation

If a fresh tissue in kept as such at room, temperature it will become

liquefied with a foul odour mainly due to action of bacteria i.e. putrefaction

and autolysis so the first and fore most aim of fixation is

1.To preserve the tissue in as If like manner as possible.

2.To prevent postmortem changes like autolysis and putrefaction Autolysis is the lysis
or dissolution of cells by enzymatic action probably as a result of rupture of lysosomes.

Putrefaction The breakdown of tissue by bacterial action often with formation of gas.

3. Preservation of chemical compounds and microanatomic constituents so that further

histochemistry is possible

4. Hardening :the hardening effect of fixatives allows easy manipulation of soft tissue
like brain, intestines etc.

5. Solidification: Converts the normal semifluid consistency of cells (gel) to an

irreversible semisolid consistency (solid).

6. Optical differentiation - it alters to varying degrees the refractive indices of the

various components of cells and tissues so that unstained components are 7.Effects of
staining certain fixatives like more easily visualized than when unfixed.formaldehyde
intensifies the staining character of tissue especially with haematoxylin.

Properties of fixatives
1.Coagulation and precipitation as described above

2.Penetration Fixation is done by immersing the tissue in luid containing the fixative.
Faster a fixative can penetrate the tissue better it s penetration power depends upon the
molecular weight e.g. formalin fixes faster than osimic acid.

3. Solubility of fixatives

All fixatives should be soluble in a suitable solvent, preferably in water so that adequate
concentrations can be prepared.

4.Concentration It is important that the concentration of fixative is isotonic or

hypotonic

Commonly used Fixatives

• 10% Formalin Solution.

• Buffered Neutral Formalin Solution,

• Formalin Alcohol. (glyogen, good cytoplasmic fixation)

• Zenker Fluid. (bone marrow aspirates)

• Bouin's Fluid. (embryological specimens, Purkinje cells)

• Acetone. (Rabies)

• Ethyl Alcohol. (glycogen, pigments, amyloid, hylaine, elastic fibers and bacteria)

◦ Glacial acetic acid. (rapid fixation, swelling of cells)

• Potassium Dichromate. (cytoplasm, not nucleoproteins)

Composition of Formalin:-

 Formalin Solution (10%, unbuffered)

 Formaldehyde (37-40%)-10 ml

 Distilled water - 90 ml
Composition of Zenkers Fluid:-

 Distilled water: 100 ml

 Mercuric chloride: 5 g

 Potassium dichromate: 2.5 g

 Sodium sulphate: 1 g

 5 ml glacial acetic acid

Trimming of tissue

Tissue, enclosed in tissue cassette and placed in basket of automatic tissue processor.

Washing of the Tissue

• Washing of tissue under running tape water overnight (12 hours) remove excessive
Fixative Solution.

Tissue Processing
The tissue processing is the heart of any tissue section which wlill be cut adequately
only if the tissue is properly preserved and processed. The study of this topic is to
understand the coarse and fine details of tissue processing so that excellent sections
are obtained.

Steps in tissue processing :

1.Fixation

2.Dehydration

3.Clearing - with a substance which is totally miscible with both the dehydrating agent
which precedes it, and embedding agent which follows it.
4.Embedding

All these 4 processes depend upon complete impregnation of the tissue by the agent
like paraffin wax being used

Dehydration
Dehydration is simply the removal of water from aqueous-fixed tissue. Since most
fixatives are aqueous, this step is necessary to prepare the tissue for embedding in non-
aqueous media like paraffin.

Alcohols are most commonly used in the laboratory for tissue dehydration, since they
are miscible with aqueous fixatives like 10% formalin. In this step, the alcohol
penetrates tissue quickly and the water is replaced with alcohol. Since alcohols act
rapidly and may shrink and harden tissue too much, care must be taken when
calculating the amount of time needed in the dehydration step. This step is performed
at room temperature. Ethyl alcohol and isopropyl alcohol are used most often, with
methanol and butanol being used to some degree in special techniques.

•The purpose of dehydration to remove fixative and water from the tissue and replace
them with dehydrating fluid.

•There are a variety of compounds many of which are alcohols. Several are hydrophilic
so attract water from tissue.
Types of dehydrating agents:

1. Ethanol

2. Methanol

3. Acetone

To minimize tissue distortion from diffusion currents, delicate specimens are

dehydrated in a graded ethanol series from water through 10%-20%-50%-95%-100%
ethanol.

Ethyl alcohol 70% 2 hour

Ethyl alcohol 80% 1 hour

Ethyl alcohol 95% 1 hour

Ethyl alcohol (Absolute 1) .5 hour

Ethyl alcohol (Absolute 2) .5 hour

‣ To increase the process of dehydration, the tissue blocks should be agitated either
mechanically in an automatic tissue processor or by shaking the container periodically.'

• The volume of alcohol should be at least 50 times more than the tissue placed for
dehydration.

Clearing:
It involves removal of dehydrating agent and replacement by some fluid which is
miscible with dehydrating agent and embedding medium.

The process is called as clearing because in addition of removal of alcohol, the tissues
are rendered translucent because refractive index of clearing agents is approximately
equal to that of tissue proteins.

⁃ Choice of a clearing agent dopends upon the following

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

  The processor system to be used.

 Extended processing conditions such as tomperature vacuum and pressure

Safety factors.

 Cost and convenience.

 Speedy removal of dehydrating agent.

 Ease of removal by molten paraffin wax.

 Minimal tissue damage

• Like ethanol, xylene should also be kept in tightly stoppered bottle to prevent the
evaporation.

Equal parts of Absolute alcohol +Xylene .5 hour

Xylene I 1 hour

Xylene ll 1 hour

• If xylene is not available then benzene may be used for 3 hours as its action of clearing
is slower than xylene.

‣ On complete clearing, the tissue becomes transparent hen they should be transferred
in paraffin wax fo impregnation.

Name different clearing agents :

 Benzene

 Xylene

 Toluene

 Choloroform

 Petroleum ether

 Oil of wintergreen

 Cedar wood oil

  Carbon tetrachloride

 Clove oil

 Dioxane

 Aniline oil

Impregnation -
It is the complete removal of clearing reagents by substitution of paraffin or any such
similar media.

Impregnation with wax Impregnation with paraffin wax takes place in an oven heated to
56-60°C depending upon the melting point of the wax in use.

Frequent check of the temperature of paraffin baths is required since temperature 5°C
above the melting point of the paraffin will cause tissue shrinkage and hardening.

Properties of paraffin wax

1. Easy to prepare large number of tissue blocks in comparatively short time.

2. Minimum supervision is required

3.It is cheaper than other impregnating media

4. During staining there is very little difficulty than other media.

Points to be remembered during use of paraffin wax

1. It should be free from dust, grit and other foreign matter.

2.It should not contain water, which causes it to crystallize and turn it white

Technique of impregnation:
The tissue is transferred from clearing agent to molten paraffin wax. The amount of
wax should be 25-50 times the volume of tissue. The tissue must be submitted to 3
changes in wax. The temperature of the wax bath should be 2-3°C above the melting
point of wax.
Time of impregnation

Depends on the following 3 factors

1. The size and type of tissue

2.The clearing agent employed

3. The use of vacuum embedding oven.

 Size and type of tissue: The thicker the tissue the longer will be the time
required for wax to penetrate to the centre in addition a thick tissue has more of
clearing agent so more changes of wax are necessary to remove it. If even small
amounts of clearing agents remains with the wax this will cause crystallization
and produce crumbling of the sections during cutting. The type of tissue is also
important since bone, skin, CNS needs twice as long as soft tissue like liver or
kindly. Tissue like muscle and fibrous tissue tends to overharden and become
brittle in wax bath so the time for impregnation must be kept to a minimum. The
reduction of time can be achieved by using vacuum embedding medium.

 Clearing agent employed Some clearing agents are more rapidly and easily
cleared than other e.g. Xylene, benzene and toluene are easiest to remove, and
one change of wax is normally sufficient; whereas for chloroform and carbon
tetrachloride 2-3 changes are needed.

Embedding -
Now that the specimen is thoroughly infiltrated with wax, it must be formed into a
"block" which can be clamped into a microtome for section cutting. This step is carried
out using an "embedding centre" where a mold is filled with molten wax and the
specimen placed into it. The specimen is very carefully orientated in the mold because
its placement will determine the "plane of section", an important consideration in both
diagnostic and research histology. A cassette is placed on top of the mold, topped up
with more wax, and the whole thing is placed on a cold plate to solidify. When this is
completed, the block with its attached cassette can be removed from the mold and is
ready for microtomy. It should be noted that, if tissue processing is properly carried out,
the wax blocks containing the tissue specimens are very stable and represent an
important source of archival material.
Types of moulds

a) Leuckhart's L pieces - These are two 'L' which are resting metal - usually brass, which
are resting on a flat metal or glass plate.

b) Compound embedding units - consists of square shaped brass or metal plates in a

series of interlocking plates.

c) Others like plastic embedding blocks (tissue Tek system)

Techniques of casting

1. Molten paraffin wax which is heated at a temperature 2-3° above the melting point is
poured into the mould to an adequate depth so as to cover the thickest tissue block.

2. The wax touching the mould will quickly form a thin semi solid layers, Now introduce
the tissue with a prewarmed forceps to prevent the wax to stick to it. The tissue is
pressed in this semisolid wax to orient it at the bottom of mould in a correct plane.

3. Fix the label in position by pressing one edge against solidifying wax usually sides of
the mould are preferred.

4. As soon as a film of solid wax is formed on the surface, the whole block with mould
are submerged in cold water at 20°C. If this is not done there will be crystallization of
wax, using ice water to do initial cooling will also cause the block
MICROTOMY AND SECTION CUTTING
Microtomy or section cutting is the technique of making the very thin slices of tissue
specimens for the microscopic examination. process by which processed tissue, most
commonly a paraffin embedded tissue, is trimmed and cut into uniformly thin slices or
"sections" to facilitate studies under the microscope

Principle : a spring-balanced teeth or pawl is brought into contact with, and turns a
ratchet feed wheel connected to a micrometer screw, which is in turn rotated, moving
the tissue block at a predetermined distance towards the knife for cutting sections at
uniform thickness.

EQUIPMENTS HISTOPATHOLOGY

 Microtome

 Oil stone and strope for sharpening knife

 Pair of needles with wooden handles

 Diamond marker or glass marker

 Flat dish of water at 45°C to 50°C according to melting point of wax

 Slides coated with albumin glycerin mixture or celloidin or collodion

 Metal handling scalpel

 Microtome knife
TYPES OF MICROTOME

1. Rocking microtome

2. Rotary microtome

3. Base sledge microtome

4. Rotary rocking microtome

5. Sliding microtome

6. ultramicrotome

Methods

 Take a tissue block, trim it and fix it to the microtome.

 Take care to fix microtome knife properly.

 Mark the slides with serial number of blocks and arrange them on a rack in order
in which fixed and then they have to be cut.

 The block is fixed in such a way that it should edge. parallel in relation to the
microtome knife

 Adjust to thickness indicator to 12 microns and cut sections until the whole
surface of the object is in contact with the knife edge.

 Set the indicator to the desired section thickness usually 7 microns. Sections
come in ribbon.

 Sometimes sections may be irregular, then it may need to be checked once again.
Float this ribbon of sections on a dish of water, heated to be just warm to the
hand with the help of a pair of needles.

 Take the individual sections which float on to the slides.

 Stand the slide dry and transfer to oven at 50°C to 55°C for 2 hours and then
keep it in a incubator at 37°C overnight.

 Slides can be smeared with egg albumin or any other adhesive agent to fix the
section on to the slide.

 The slide is stained with H&E and arranged in slide tray and kept horizontally.
ERROR OF SECTION CUTTING

 Ribbon is not formed properly if wax is too hard.

 Ribbon is curved- upper and lower surface, block is not parallel

 Knife is not properly placed - if edge is not very sharp then use another segment
of knife or proper edge.

 Section are fragmented - wax is too soft

 Sections coming out may be thick or thin - knife improperly fixed or block is not
properly fixed.

 Width of the section less than that of block - sections will not float, that is due to
knife defect.

 Decalcification is not complete - then soak in 4% aqueous solution of phenol for

1 to 3 days.

STAINING :
The Hematoxylin and Eosin stain (H&E) is the most widely used histological stain
because : comparative simplicity Ability to demonstrate clearly an enormous number of
different tissue structures. Hematoxylin stains cell nuclei blue black → shows good
intranuclear detail. Eosin stains cell cytoplasm and most connective tissue fibers in
varying shades and intensities of pink, orange, and red.

Haematoxylin and Eosin stain:

Haematoxylin is a natural dye extracted from the core wood of Haematoxylin cam-
pachianum tree. It is available as brown powder. This powder by oxidation made in to
colouring agent - haematin. This process is called ripening. It takes several days or
weeks. It can be or accelerated by addition of oxidizing agent as mercuric oxide, sodium
iodide, potassium permanganate. This dye with combination of heavy metals becomes
a powerful nuclear stain. The commonly used metals are - alluminium, iron, tungsten,
lithium and are known as flakes. Aluminium flakes are most used.
Methods :

1.Treat with xylol to remove wax after warming the slides

2.Dip in alcohol to remove xylol (2-3 changes)

3. Rinse in water

4. Stain with alum haematoxylin 5 - 10 minutes, then rinse in water

5. Differentiate in acid - alcohol (1% concentrated hydrochloric acid in 70% alcohol)

6. Rinse in water

7. Dip in ammonia water - 2 to 3 ml of ammonia solution in 1000 ml of tap water orin

saturated lithium carbonate till sections are bright blue or wash in tap water till the
sections becomes blue. This is called as bluing.

8. Wash in water for few minutes.

9. Stain with eosin for 5 minutes or more, wash in running tap water till eosin is
differentiated. Then dehydrate in alcohol (70%, 80% and 90%). Then dip in xylol to
remove alcohol- 2 to 3 changes, a then dip in xylol to clear it.

10. Mount it with DPX or Canada balsam. Keep horizontally till it gets fixed. SAMPLE
FOOTER TEXT

MOUNTING

In histology or a pathology laboratory, mounting is the last procedure in the series that
ends with a permanent histological preparation on the table. well after the tissue
processing and staining. Viz.. (1) fixing, (2) paraffin embedding, (3) sectioning. (4)
staining, (5) dehydrating, and (6) clearing operations.
The mounting medium is the solution in which the specimen is embedded, generally
under a cover glass. It may be liquid, gum or resinous, soluble in water. alcohol or other
solvents and be sealed from the external atmosphere by non-soluble ringing media.The
main purpose of mounting media is to physically protect the specimen; the mounting
medium bonds specimen, slide and coverslip together with a cleardurable film. The
medium is important for the image formation as it affects the specimen's rendition

DISCUSSION :
Histopathology is the study of tissue to look for disease. Pathologists perform
histopathology in a lab. They examine tissue under a microscope and develop a report
of their findings. Histopathology reports can include descriptions of the tissue,
diagnosis, and prognosis. In addition to evaluating the shape and structure of cells,
pathologists may also use other techniques to assess and diagnose.

Routine (conventional) processing requires 2–3 working days before a diagnosis is

delivered to the patient. The rapid processing schedules that are available currently
require a minimum of 16–48 h for completion.

Diagnosis pathology is largely dependent on formalin-fixed paraffin-embedded tissue

sections. This study presents a rapid processing technique (RaPT) [Table 1] and is
compared with the routine processing technique (RoPT). The properties of tissues
processed by rapid technique were comparable with routinely processed tissues, with
reference to ease of sectioning, yield of good ribbons, good staining, permanency of
stain (6–12 months observation) and satisfactory staining quality. These properties
were observed in both soft tissues and hard tissues processed by these techniques.
Furthermore, decalcified tissues were subjected to both the techniques to observe and
compare. However, no statistical comparison was made for hard tissues as the sample
size for hard tissues was less.

Histology techniques are among the gold-standard techniques for the morphological
evaluation of biological specimens. From academic to clinical labs, several types of
traditional histology protocols are used every day. One of the most important
applications of histology techniques is their application in patient diagnosis. When
evaluated by experienced pathologists, histological images may provide information on
disease staging and progression. However, the application of standard histologic
techniques is limited to the study of dead, fixed samples, and the study of living
samples is out of scope.

H&E stain contain haemotoxylin and eosin. Haemotoxylin is basic stain. It will stain
basophilic component, such as nuclei as dark blue . Meanwhile, eosin, which is acidic
stain, will stain acidophilic substrate as pinky red in different degree. For instance,
cytoplasm which fulfilled with granular mass is stained pink; collagen and muscle is
stained pink and erythrocyte (red blood cell) is stained intensely red.

PAS stain contains periodic acid and Schiff reagent. Periodic acid can oxidize the vicinal
diol (glycol part) in the glycol-sugar and produce two aldehyde groups at two free tips of
each monosaccharide ring while splitting the bond between the two carbons. On the
other hand, Schiff's reagent consists of para-saniline solution (Basic fuchsin), which
was decolourised by the sulphurous acid by adding extra sulphurous group to the
central carbon of dye. For the reaction with aldehydes in the tissue, the alkyl sulphonic
derivative of the dye is formed and restores the quinoid chromophobic group to give a
magenta colour on the tissue. At the end of reaction, basement membrane and brush
border (microvilli) of ileum stain pink and the goblet cell stain magenta red. The staining
mechanism for Masson Trichrome stain is mainly based on molecular size theory. For
instance, erythrocyte is stained red by the smallest molecular size dye, Ponceau 2R
(MW-480); Acid fuchsin, which has intermediate molecular size (MW-586), stains
muscle and cytoplasm pink; Collagen, which is more permeable, stained green by the
light green, which has large molecular size.