Upload
drsyed-asif
View
95
Download
1
Tags:
Embed Size (px)
Citation preview
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