EFFECTS OF FIXATION

• X Autolysis/Putrefication CLASSIFICATION BASED ON:

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• X Change in shape/volume
• X Dessication/Shrinkage of
Tissue CHEMICAL AGENTS USE
1. Proteins →Paraformaldehyde →
• Clear Staining 1. Microanatomical Osmium Tetroxide
1. Cross-linking fixatives/Aldehyde
• Tissue-living state 2. Enxymes → Frozen sections→
- Formaldehyde, Glutaraldehyde - 10% formol saline
• Semifluid-Semisolid Chemical Fixatives
2. Protein denaturing/ Coagulants - 10% neutral buffered
• Optical differentiation 3. Lipids → Frozen sec.,Glut., Osmium
- Acetic acid, Methyl alcohol, Ethyl formalin
- Zenkers solution tetroxide → Alcoholic fixative, NBF
alcohol
- Bouin’s solution 4. Nucleic acids → Alcoholic fixatives →
3. Oxidizing agents
FACTORS AFFECTING - Rossman’s fluid Aldehyde
- Osmium tetroxide, potassium - Formol calcium
FIXATION 5. Mucopolysaccharides → Frozen
permanganate, potassium
sections → Chemical
1. Buffers & pH dichromate 2. Nuclear fixatives
6. Biogenic amines → Bouin’s solution,
2. Penetration 4. Other cross-linking agents
- glacial acetic a NBF
(Depth, D = K √t) - Carbodiimides
affinity for nuclear 7. Glycogen → Alcoholic fixatives →
3. Duration 5. Miscellaneous chromatin. Osmium tetroxide
4. Temperature - mercuric chloride, picric acid, non - - pH≤ 4.6
5. Concentration aldehyde – containing fixatives, dye - Flemming’s
6. Osmolality stuffs - Carnoy’s
- Newcomer’s
7. Volume - Clarke’s
8. Additives
Cytoplasmic fixatives
- glacial acetic a -
destroys mitochondria
COMMONLY USED IS 10% NBF and Golgi.
- pH ≥ 4.6.
ADVANTAGES: DISADVANTAGES: - Kelly flemmings
- Regauds’s fluid
1. Cheap, easy to prepare, rel. 1. Dermatitis
- Orth’s fluid
stable 2. Irritation
2. Allows subsequent appl. of 3. Asthma 3. Histochemical
most staining tech. without 4. Formation of - Formol saline 10%
Pigmets spl. Preliminary procedures - Absolute ethyl alcohol
3. Frozen sections- easily - Acetone
prepared
4. Staining for fat – easily
carried out
5. Penetrates tissues
reasonably
6. No excessive
#30 – MAGTANGOB, ILLONAH JANE D.
hardening / brittle
MT4 – HISTOPATH MODULE 4
 ACID
METHODS OF DECALCIFICATION:
DECALCIFICATION –
1. Acid decalcification common method
CRITERIA FOR GOOD CALCIUM-CONTAINING TISSUE: 1. Strong Acids:
2. Ion-exchange resin
DECALCIFICATION 3. Electrical ionization Hydrochloric
1. Bone
4. Chelating solution acid, Nitric acid
1. Complete removal of calcium 2. Tooth
5. Surface decalcification 2. Weak Acids:
2. Absence of damage to tissue cells 3. Pathological tissue
Formic acid,
or fibres such as in tuberculous lymph node,
Trichloroacetic
3. Non impairment of subsequent dystrophic calcification, and certain
acid
staining technique. tumours such as teratomas, etc.
CHELATING AGENT
4. Reasonable speed of
decalcification. Ethylenediaminetetra
TECHNIQUE acetic Acid (EDTA) 1. Aqueous nitric acid - 5%
-commonly used Advantages: Rapid in action, Good
1.Selection of tissue - Mode of action: nuclear stain
FACTORS AFFECTING RATE OF - 4-5 mm Binds with calcium of Disadvantages: Gives yellow color to
DECALCIFICATION 2.Fixation the hydroxyapatite the tissue
- Routine fixative: Formal saline ;
crystals to form a
1. Concentration of decalcifying Zenker’s: Bone Marrow (best) ; 15% 2. Nitric acid formaldehyde – 10%
non-ionized soluble
agent Formic Acid: Tooth ; Muller’s Fluid + 3% A: Rapid action (1–3 days), Less
complex
2. Temperature Formic Acid Formalin: Some fine prep. Of chance of tissue damage & swelling,
bones - Slow and gentle in
3. Agitation Long-time washing by water is not
3.Decalcification action
4. Suspension needed
4. Acid neutralization D: Not a very good nuclear stain
5. Thorough washing A: Morphological
preservation of tissue, 3. Hydrochloric acid – 8%
Suitable to do various A: Rapid action, Ideal decalcifying
other laboratory agent for the tooth
5. CHEMICAL TEST tests, Very good for D: Nuclear staining is not very good
END POINT OF DETERMINATION OF bone marrow
Chemical Solution DECALCIFICATION 4. Trichloroacetic acid - 5 g in 95 ml
trephine
Stock solution 1. Radiographic examination formal saline
biopsy
1. Ammonium hydroxide stock 2. Chemical test A: Good for small biopsies, Good
solution 3. Physical test D: Very slow process, nuclear stain
Ammonium hydroxide (28%) : 5 mL 4. Radiographic Examination Maintenance of Ph D: Not good for hard bony tissue,
Distilled water: 5mL 5. Chemical Test around 7 is Slower in action and takes 4–5 days
necessary., Thin tissue
2. Ammonium oxalate stock is needed 4. Formic acid - 5%
solution A: Decalcifying agent of choice in
Ammonium oxalate: 5 mL routine laboratory process
Distilled water: 95 mL D: Slow acting and takes many days
for decalcification
Removes free or unbound water
molecule of the tissue as the Common dehydrating agents: 1. Ethyl alcohol
– Ethyl alcohol A: Rapid and efficient dehydrating
supporting medium (paraffin) is not agent
miscible with – Methylated spirit
D: Needs license from the government, Inflammable, Hard and
water. – Methanol
brittle tissue if kept for long time
– Butyl alcohol
– Isopropyl alcohol
Sharp difference of concentration 2. Methanol
– Dehydrating agents other than
gradient of the dehydrating fluid may A: Equally effective as ethanol
alcohol: dioxane, ethylene glycol
damage the delicate tissue. D: Volatile, High cost
and acetone

Gradual dehydration is necessary. 3. Isopropyl alcohol

A: Relatively rapid action, Non-toxic, Minimal tissue
shrinkage
D: Not possible to use in celloidin technique
Too much time in the dehydrating
fluid: the tissue becomes hard and
brittle. 4. Dioxane
A: Rapid action, No shrinkage of tissue
D: Highly toxic gas is generated
Routine laboratory: 70, 90 and 100%

alcohol for 2 h each 5. Ethylene glycol

A: Rapid, No graded solution is needed, Tissue can be
kept in it for long time
D: Very expensive, Clearing agent is needed

6. Acetone
A: Rapid action, Cheaper than ethanol, Good for fatty
tissue processing
D: Quickly evaporates, Inflammable, Prolonged use may
cause shrinkage and brittleness of tissue.

AIMS OF CLEARING
– Removal of dehydrating agent
(e.g. alcohol) to facilitate
impregnation of paraffin wax
– To make the tissue clear and
improve the microscopic
examination
IDEAL CLEARING AGENT:
– Low viscosity and high
penetration rate
– Low melting point
– Miscible with both alcohol
and molten wax
– No tissue damage
– Less toxic
– Less inflammable
– Cheap
SELECTION OF APPROPRIATE CLEARING
AGENT:
1. Type of tissue
2. Type of processor
3. Processing condition (such as heat,
vacuum) safety factors and cost
1. Volume of clearing agent: 40
times the volume of the specimen
2. Total duration:
– Smaller biopsy: 1 h
– Larger tissue: Three changes in
xylene or toluene 60 min each
3. End point detection: Tissue
becomes transparent
4. Prolonged exposure to clearing
agent: The brittle and more friable
tissue
5. Different clearing agents: Xylene,
toluene, chloroform, amyl nitrate,
cedarwood oil and limonene
5. CLEARING AGENTS
Xylene
Tissue Shrinkage: Yes
Tissue Hardening: Yes
Inflammable: Yes
Harmful Effect: Irritant but less harmful
Cost: Cheap
Toluene
Tissue Shrinkage: Yes
Tissue Hardening: No
Inflammable: Yes
Harmful Effect: Irritant
Cost: Cheap
Chloroform
Tissue Shrinkage: Minimum
Tissue Hardening: No
Inflammable: No
Harmful Effect: Dangerous Toxic Gas
Cost: Very Expensive
Esters
Tissue Shrinkage: No
Tissue Hardening: No
Inflammable: Yes
Harmful Effect: Safe
Cost: High Cost
Cedarwood Oil – Expensive, Used in dense
tissue
Limonene – clear, difficult to remove from
tissue by paraffin wax

AIMS: To provide support to the tissue.
PRINCIPLE:
Clearing agent is removed by the process
of diffusion, and the tissue space is now
infiltrated with the embedding media.
IDEAL IMPREGNATING MEDIUM:
• Miscible with clearing agent
• Liquid in higher temperature and solid
in room temperature
• Homogenous and stable
• Non-toxic and cheap
• Transparent
• Fit for sectioning the tissue
TIME DURATION AND THE NUMBER OF
CHANGES OF EMBEDDING MEDIUM:
• Size of tissue: Large versus small.
• Type of tissue: Hard versus soft.
• The type of clearing agent: Cedarwood
oil takes longer time.
• Type of processing: Vacuum
processing accelerates.
DIFFERENT EMBEDDING MEDIUM:
1.Paraffin wax
2. Dimethyl sulphoxide
1. PARAFFIN WAX
A: Tissue block can be stored for long
duration, Non-toxic, Cheap, Safe
D: It may cause tissue shrinkage and
hardening in case of prolonged
impregnation, Paraffin wax takes long
duration for the impregnation of the
bone and eye
A -a completely closed processor. Here
ADDITIVES AND MODIFICATION OF PARAFFIN WAX
1. To increase hardness: addition of stearic acid
2. Reduction of melting point: addition of
phenanthrene.
3. Improving adhesiveness with tissue and wax:
addition of 0.5% of ceresin
2. DIMETHYL SULPHOXIDE (DMSO)
- Addition of small amount of DMSO in
paraffin wax reduces the infiltration time of
the wax and removes the residual clearing
agent.
-It produces a homogenous matrix and better
support.
TISSUE PROCESSING METHOD
1. Manual Method – small lab, few
tissue numbers.
2. Automated Tissue Processor –
widely used
TYPES OF AUTOMATED TISSUE
PROCESSOR
1.Tissue transfer processor
-bucket of tissue is transferred from
one carousel to other after a
specified time.
2. Fluid transfer processor
the tissue is kept in the container,
and the container is periodically filled
with particular fluid.
ADVANTAGE : Vacuum pressure
makes the system faster and
efficient, No chance for tissue drying.
MICROWAVE PROCESSING
- reduces time processing, suitable for
small number of delicate tissues.
Microwave Oven has:
1. System to control the temperature
2. System to control the time duration
of particular temperature
3. Proper exhaust to remove the toxic
gas

AIMS
1. To give support of the tissue
2. To prevent distortion of the tissue
during cutting
3. To preserve the tissue for archival
use
FACTORS AFFECTING:
1. Type of tissue: The density of the
tissue and the embedding medium
should be close otherwise tissue
may not be sectioned properly, and
tissue will be deformed.
2. Type of microtome
3. Type of microscope
DIFFERENT TYPES OF MOULD
USED FOR BLOCK
1. Leuckhard Embedding Moulds -
have two arms, one arm of the L
is longer than the other arm
2. Stainless Still Mould - flat and
well-polished that helps to
remove liquid paraffin. The
mould can be covered by a
plastic ring.
3. Plastic Mould - made of
chemical-resistant plastic
TISSUE MARKING
PURPOSES
EMBEDDING MEDIUM 1. To identify the resection plane or outer margin of the tissue
1. Paraffin Wax
2. To help in embedding the tissue
2. Epoxy Resin - used in
3. Any area of interest to identify such as the area of transitional
electron microscopy as it
zone in cone biopsy of cervix.
provides better resolution
and greater details of tissue.
3. Acrylic medium - provides a
hard and clear block INKS USED IN TISSUE MARKING
4. Agar gel - helps in cohesion
1. India ink: This is the most commonly used marker in the
of friable and fragmented
routine surgical pathology laboratory. It takes 15 min time to
tissue particularly in cytology
mark the tissue.
sample and also endometrial
2. Silver nitrate: This is also a good marker. It produces brown-
curetting and small
black colour.
endoscopic biopsies.
3. Rose Bengal: For surgical margin stain, 1% e Bengal dye is
5. Gelatin - used in small friable
used. It stains within 5 min provides pink stain.
tissues and frozen section
containing friable and
necrotic tissue.
6. Celloidin medium - was
mainly used for embedding
hard tissue, not used
anymore.
THINGS NEEDED FOR EMBEDDING
1. Molten paraffin wax
2. Mould with cover
3. Metal plate (cold plate) to work
TISSUE -TEK SYSTEM
-commercially available
1. Dispenser of liquid paraffin in a
constant
temperature
2. A metal plate to make the tissue
block
3. Cold plate

TYPES OF MICROTOME
• Rotary microtome
• Rocking microtome
• Base sledge microtome
• Sliding microtome
• Cryomicrotome
• Ultramicrotome
• Laser microtome
ROTARY MICROTOME
Advantages:
1. Good-quality 2–3-μm-thin section is
possible.
2. Heavy and stable automated rotary
microtome reduces health hazard and
gives the best-quality section.
3. Good tissue ribbon production.
4. Easy-to-cut various types of tissue:
firm, fragile, small biopsy, etc.
Disadvantages:
1. Expensive.
2. Unsuitable to cut large block.
3. Knife faces up and so may be
dangerous to the technical staff
ULTRAMICROTOME - used to cut ultrathin
sections for transmission electron microscopy.
Sections are cut between 40and 100
nanomicron thickness with the help of glass
knife or diamond knife.
LASER MICROTOME - laser beam is used to cut
the biological section without any processing or
embedding the material.
TYPES OF KNIVES BASED ON SHAPE:
Plano concave (Profile A):
– Very sharp knife and is used for cutting soft
tissues
Biconcave (Profile B):
– Used for rocking microtome
ROCKING MICROTOME – Vibrates during cutting
Advantages: Wedge (Profile C):
1. Thin section – Most commonly used
2. Easy to operate Tool edge (Profile D):
3. Low-cost instrument and reliable – Used for hard tissue
Disadvantages:
1. Tissue is curved and the microtome does
not provide flat section. TYPES BASED ON DISPOSABILITY
2. As the microtome is of light weight so
Permanent
vibration may occur.
Disposable
– Low-profile blade: To cut small
biopsy
BASE SLEDGE MICROTOME – High-profile blade: To cut firm to
Advantages: hard tissue
1. Hard tissue can be cut.
2. Large tissue sample can be cut.
3. The best microtome for ophthalmology MATERIALS USED IN KNIFE:
and large neuropathology section. Conventional knife: Steel
Disadvantages: Diamond knife: Made of diamond and used to
1. Difficult to get thin section. cut epoxy resin blocks
2. Large slides are required.
CRYOMICROTOME
SLIDING MICROTOME
Advantages:
Advantages:
1. To get rapid section for rapid diagnosis
1. Large sections can be cut.
2. Mainly used for celloidin-embedded tissue. 2. To study nerve biopsy
3. Simpler design and easy maintenance. 3. To study enzyme histochemistry
Disadvantages:
4. Brain sections can be cut better by this type of
microtome. 1. It needs continuous supervision to
Disadvantages: maintain the temperature.
1. The knife may glide in case of hard tissue and 2. Freezing artefact is often seen.
may jump. 3. Very expensive instrument.
4. Fixed tissue is very difficult to cut.
2. Long knives are difficult to sharpen