this is a ppt on histotechniques,, all techniques from receiving samples to block making to sectioning to staining are discusses in detail..useful for postgraduate pathology students and lab technicians

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HISTO -TECHNIQUES

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OVERVIEW
• Definition
• Specimen Receiving
• Fixation
• Grossing
• Tissue processing
• Embedding
• Microtomy

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Definitions
• Histology – greek – histos + logia (A.F.J.K. MAYER 1819)
– Study of microscopic anatomy of cells and tissues
• Histopathology – Microscopic study of
diseased tissue
• Histotechnology – processing of tissues in
such a manner as to enable microscopy /
study of the tissue

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AIM
• Obtaining thin enough sections of the tissue
so as to enable light to pass through it

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SPECIMEN RECEIVING
• Documented policy defining the minimum
demographic details of the patient
• Spill kit – 5% hypochrorite, gauze piece, yellow
bag, gloves
• Sample transportation – covered containers,
in 10% buffered formalin, chutes
• Unique identification number
• Strict Sample rejection criteria

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“FIX AT A POINT IN TIME”
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TYPES OF FIXATION
• Immersion
• Perfusion – brain, spinal cord, lungs,
embalming
• Vapour
• Spray
• Microwave fixation/Stabilization
• Freeze drying

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MICROWAVE PRINCIPLE
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FREEZE DRYING
• Fresh tissue immersed in isopentane cooled by
liquid nitrogen at -160° to -180° - Quenching
• Tissue water removed under vacuum, absorbed
by phosphurous pentoxide – sublimation
• Put in embedding medium
• Little chemical alteration, no loss of glycogen
• Modification of this technique useful in enzyme
histochemistry

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• Standard histological processing abolishes the activity of most
enzymes
• Freeze drying is the optimal method of tissue preservation
maintaining tissue components in their native state. Conventionally,
however, the freeze dried tissue specimens are fixed and embedded
in wax after freeze drying.
• Glycol methacrylate resin has been widely used as an alternative
embedding medium to wax and the activities of a limited number
of enzymes have been shown in tissue fixed in aldehyde and
embedded in resin. Enzyme histochemistry performed on these
resin sections gives good results.

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PHYSICAL METHODS CHEMICAL METHODS
Heating
Microwaving
Freeze - drying
Aldehydes – Formal dehyde,
Glutaraldehyde
Alcohol – Methyl alcohol, ethyl alcohol
Methacarn
Glacial acetic acid
Bouin’s fluid
Osmium tetroxide
SIMPLE FIXATIVE COMPOUND FIXATIVE
Formal dehyde
Glutaryl dehyde
Ethyl alcohol
Zenker’s fluid
Bouin’s fluid

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Formulae
 Formal Saline
 40% Formaldehyde 100 ml
 NACL 9 gm
 Tap Water 900 ml
 10% Buffered Formalin
 40% Formaldehyde 10 ml
 Sodium Hydrogen phosphate 0.4gm
 Disodium Hydrogen phosphate 0.65gm
 Tap Water 90 ml
Buffered formalin prevents formation of pigment acid formaldehyde
hematin formed from hemoglobin at acidic pH.

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Formulae
Zenker’s fluid
 Distilled Water 1000 ml
 Hg Cl2 50 gms
 K-Dichromate 25 gms
 NaSO4 10 gms
• It provides excellent fixation of nuclear chromatin, connective
tissue fibres and some cytoplasmic features but does not
preserve delicate cytoplasmic organelles such as
mitochondria.
• Currently out of fashion because of the toxicity.

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Glutaraldehyde
• Used for electron microscopy with osmium
tetroxide
• Glutaraldehyde produces nuclear and
cytoplasmic shrinkage while osmium produces
swelling and balances it
• Formalin is not useful for EM because
methanol which is added to commercial
preparations has denaturing action on tissue
components.

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Factors affecting fixation
1. Hydrogen ion Concentration & Buffers
1. Satisfactory fixation occurs at pH between 6 and 8.
2. Buffers like phosphate, bicarbonate, s-collidine and
cacodylate are chosen.
2. Temperature
1. Higher temp causes faster fixation
2. Lower temperature preserves tissue better
3. A tissue of 4mm thickness will be adequately fixed in NBF
10 to 20 times the volume in about 8 hours at room
temperature. Fixation time is shortened by 25-40% if
temperature is raised to 40°c.
4. Faster fixation is obtained through agitation.

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6. Duration of Fixation ideal is 8 hrs
• over fixation causes hardening of tissue and loss of
antigens
7. Volume change – Tissue fixed in formaldehyde and
embedded in paraffin wax shrinks by 33%

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Fixation Artefacts
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Decalcification
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4. Ion exchange resins – polystyrene
5. Electrolytic method – Electrolytic solution of
HCl and formic acid used
6. Ultrasonic method
7. Surface decalcification – Achieved by
inverting paraffin block in 5% HCl for one
hour, top 30 micron is decalcified.

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Factors affecting rate of
decalcification
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Assesment of decalcification
• Needling, knifing, finger nailing
• X ray examination is the best
• Chemical method using ammonia

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Trimming
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Trimming

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Tissue Processing
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Tissue Processing
 Principle :
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
 Dehydration – to remove fixatives and water from tissue.
 Clearing – replacing dehydrating fluid with a fluid that is
totally miscible with dehydrating fluid and embedding
medium
 Impregnation – replacing clearing agent with the
embedding medium
 Embedding

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Dehydration
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Clearing
 Hydrocarbons which have refractive indices similar to
tissue proteins, end point noted by transparent
appearance of tissues.
 Criteria
 Miscible with both dehydrating and embeding agent
 Speedy removal of dehydrating agents
 Easy removal by paraffin
 Examples
 Xylene
 Toluene
 Chloroform
 Citrus fruit oils
 Cedar wood oil

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Impregnation
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Automated tissue processing
Processing schedule
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Important points
Bone, skin and CNS tissues require three
changes of wax
Two changes of wax suffice for other organs

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Embedding
 Requirements
 Embedding media
 Cold plate / ice plate
 Wax Dispenser
 L-molds / ice trays / paper blocks / tissue tek system
 Wooden blocks
 Forceps
 Spatula
 Typed labels

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Embedding

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Embedding

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Embedding media
 Water insoluble media
– Paraffin wax
– Carbowax
– Plastic embedding medium – Butyl methacrylate
– Acrylic
– Epoxy resin
– Epon
– Maraglas 655
– Polyester resin
Water soluble media
– Durcupan
– Aquon
– Glycol methacrylate

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Important points
• Size of the mould should be such that there is a
margin of 1-2 mm around the tissue
• Embed all bits of skin / cervical cone only few mm
apart in the same block with parallel epithelial
edges
• Endometrial samples and cell blocks are best kept
at centre of block and tamped to the bottom
• Stand mount – cyst walls, gall bladder walls,
ovarian wedges and skin sections – required edge
kept at the bottom

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Paraffin wax additives
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Special embedding techniques
• Double embedding
– Impregnated in celloidin, blocked in paraffin wax
– Used in making blocks of tissues with varying
consistency such as eyes where retina is easily
detached
• Tissue mat and Bioloid
– Thin walled and circular sections maintain their
original shape due to elasticity of the media

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Paraffin section cutting
 Requirements:
 Microtomes
 Disposable blades
 Water bath
 Fine forceps
 Small squirrel hair brush
 Slide rack
 Clean slides
 Ice tray
 Diamond pencil

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Microtomes
 Precision instruments that cut sections from the
paraffin blocks, thin enough for examination
under microscope.

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Sliding microtome
• Block is stationary, knife moves horizontally
against it
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Rocking microtome
• The knife is fixed, blok of tissue moves
through an arc and strikes against the knife

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Sledge Microtome
• Similar in function to sliding microtome

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Rotatoty Microtome
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Auto cut microtome

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Vibrating knife microtome
• Sections obtained without fixation /impregnation /freezing,
useful for histochemistry
• Sections are thick and not suitable for routine staining
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Ultracut microtome
• Used for cutting 500 to 600 A° thin sections for EM

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MICROTOME KNIFE
• Vicker’s Hardness number
– Important to use knives of gauranteed hardness
– VHN = 1.72p the desirable hardness is 400-900 VHS
D²
Where D is the diagonal between corners of rectangular indentation, P is standard load

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one side is flat, other is concave
used for cutting nitrocellulose blocks
originally designed for frozen sections,
now used for all microtomes
Used to section hard tissues such as
undecalcified bone

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Knife materials
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Sharpening of knives
• Manual
– Honing
• Coarse
• Fine
– Stropping
• Automatic

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Honing
• Honing is removing nicks and irregularities
from the knife edge
• Hones are sharpening stones
– Examples – Belgian Vein Black and Arkansas
• Lubricants used during the process to act as
coolants and flow away fine particles

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Honing
• Abrasive powders
– Diamond
– Carbodundum (silicon carbide)
– Aluminium oxide
– Iron oxide
– Ceric oxide
– Chromium oxide

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Stropping
• Polishing a fairly sharp knife
• Strops are made of hide from the rump of
horse

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Knife Sharpening machine

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Fine Cutting
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Sections

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Section adhesion
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Difficulties commonly encountered in
sectioning
 Failure of block to ribbon
 Too hard paraffin
 Knife
 Uneven Ribbons
 Irregular knife edge
 Knife not parallel to the block
 Impure paraffin
 Alternate thick and thin Ribbons
 Too soft wax
 Loose block or blade
 Faulty microtome

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Difficulties commonly encountered in
sectioning (Contd.)
 Tight coil of sections
 Blunt blade
 Too thick sections
 Adherence of sections to knife
 Dirty knife edge
 Dull knife

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Frozen sections & Cryostat
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Cryostat

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Cryostat
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Cryostat

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