Histotechniques

PREVIEW ONLY
To download full presentation
follow this link
https://www.scribd.com/doc/200310129/Histotechniqueshttps://www.scribd.com/doc/200310129/Histotechniques
HISTO -TECHNIQUES

PREVIEW ONLY
follow this link
https://www.scribd.com/doc/200310129/Histotechniques
OVERVIEW
• Definition
• Specimen Receiving
• Fixation
• Grossing
• Tissue processing
• Embedding
• Microtomy

PREVIEW ONLY
follow this link
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

PREVIEW ONLY
follow this link
AIM
• Obtaining thin enough sections of the tissue
so as to enable light to pass through it

PREVIEW ONLY
follow this link
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

PREVIEW ONLY
follow this link
This slide is not
available in this
preview

PREVIEW ONLY
follow this link

PREVIEW ONLY
follow this link
“FIX AT A POINT IN TIME”
This slide is not
available in this
preview

PREVIEW ONLY
follow this link
TYPES OF FIXATION
• Immersion
• Perfusion – brain, spinal cord, lungs,
embalming
• Vapour
• Spray
• Microwave fixation/Stabilization
• Freeze drying

PREVIEW ONLY
follow this link
MICROWAVE PRINCIPLE
This slide is not
available in this
preview

PREVIEW ONLY
follow this link
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

PREVIEW ONLY
follow this link
• 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.

PREVIEW ONLY
follow this link
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

PREVIEW ONLY
follow this link
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.

PREVIEW ONLY
follow this link
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.

PREVIEW ONLY
follow this link
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.

PREVIEW ONLY
follow this link
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.

PREVIEW ONLY
follow this link
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%

PREVIEW ONLY
follow this link
Fixation Artefacts
This slide is not
available in this
preview

PREVIEW ONLY
follow this link
Decalcification
This slide is not
available in this
preview

PREVIEW ONLY
follow this link
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.

PREVIEW ONLY
follow this link
Factors affecting rate of
decalcification
This slide is not
available in this
preview

PREVIEW ONLY
follow this link
Assesment of decalcification
• Needling, knifing, finger nailing
• X ray examination is the best
• Chemical method using ammonia

PREVIEW ONLY
follow this link
Trimming
This slide is not
available in this
preview

PREVIEW ONLY
follow this link
Trimming

PREVIEW ONLY
follow this link
Tissue Processing
This slide is not
available in this
preview

PREVIEW ONLY
follow this link
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

PREVIEW ONLY
follow this link
Dehydration
This slide is not
available in this
preview

PREVIEW ONLY
follow this link
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

PREVIEW ONLY
follow this link
Impregnation
• This slide is not available in
this preview

PREVIEW ONLY
follow this link
Automated tissue processing
Processing schedule
This slide is not
available in this
preview

PREVIEW ONLY
follow this link
Important points
Bone, skin and CNS tissues require three
changes of wax
Two changes of wax suffice for other organs

PREVIEW ONLY
follow this link
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

PREVIEW ONLY
follow this link
Embedding

PREVIEW ONLY
follow this link
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

PREVIEW ONLY
follow this link
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

PREVIEW ONLY
follow this link
Paraffin wax additives
This slide is not
available in this
preview

PREVIEW ONLY
follow this link
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

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

PREVIEW ONLY
follow this link
Microtomes
 Precision instruments that cut sections from the
paraffin blocks, thin enough for examination
under microscope.

PREVIEW ONLY
follow this link
Sliding microtome
• Block is stationary, knife moves horizontally
against it
This slide is not
available in this
preview

PREVIEW ONLY
follow this link
Rocking microtome
• The knife is fixed, blok of tissue moves
through an arc and strikes against the knife

PREVIEW ONLY
follow this link
Sledge Microtome
• Similar in function to sliding microtome

PREVIEW ONLY
follow this link
Rotatoty Microtome
This slide is not
available in this
preview

PREVIEW ONLY
follow this link
Auto cut microtome

PREVIEW ONLY
follow this link
Vibrating knife microtome
• Sections obtained without fixation /impregnation /freezing,
useful for histochemistry
• Sections are thick and not suitable for routine staining
This slide is not
available in this
preview

PREVIEW ONLY
follow this link
Ultracut microtome
• Used for cutting 500 to 600 A° thin sections for EM

PREVIEW ONLY
follow this link
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

PREVIEW ONLY
follow this link
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

PREVIEW ONLY
follow this link
Knife materials
This slide is not
available in this
preview

PREVIEW ONLY
follow this link
Sharpening of knives
• Manual
– Honing
• Coarse
• Fine
– Stropping
• Automatic

PREVIEW ONLY
follow this link
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

PREVIEW ONLY
follow this link
Honing
• Abrasive powders
– Diamond
– Carbodundum (silicon carbide)
– Aluminium oxide
– Iron oxide
– Ceric oxide
– Chromium oxide

PREVIEW ONLY
follow this link
To watch stropping and honing, search for the same on you tube… many videos are available
• This slide is not available in
this preview

PREVIEW ONLY
follow this link
Stropping
• Polishing a fairly sharp knife
• Strops are made of hide from the rump of
horse

PREVIEW ONLY
follow this link
Knife Sharpening machine

PREVIEW ONLY
follow this link
Fine Cutting
This slide is not
available in this
preview

PREVIEW ONLY
follow this link
Sections

PREVIEW ONLY
follow this link
Section adhesion
This slide is not
available in this
preview

PREVIEW ONLY
follow this link
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

PREVIEW ONLY
follow this link
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

PREVIEW ONLY
follow this link
Frozen sections & Cryostat
This slide is not
available in this
preview

PREVIEW ONLY
follow this link
Cryostat

PREVIEW ONLY
follow this link
Cryostat
This slide is not
available in this
preview

Histotechniques

Recomendados

Recomendados

Mais conteúdo relacionado

Mais procurados

Mais procurados (20)

Semelhante a Histotechniques

Semelhante a Histotechniques (20)

Mais de Ashish Jawarkar

Mais de Ashish Jawarkar (20)

Último

Último (20)

Histotechniques