Histological Techniques: Perform Fixation and Tissue Processing

Topic 2: Perform
Fixation & Tissue
Processing
Histological Techniques
Part 1

Learning Outcome
1) Describe the aims of fixation.
2) Describe the properties of an ideal fixative.
3) Describe the classification of fixation methods.
4) Explain the factors that affect fixation.
5) Describe the aims of tissue processing.
6) Explain the steps involved in tissue processing.
7) Explain the selection criteria of a suitable dehydrating and clearing agent.
8) Describe the types of tissue processor.
9) Describe the factors influencing the rate of tissue processing and processing
schedule.
10) Perform the following:
✓ fixation of tissues
✓ tissue processing with the use of the automatic tissue processor (ATP)

Histology Laboratory Workflow
Biopsy
Source: www. //i.ytimg.com/vi/JDbPgtb2j8Y/maxresdefault.jpg

Fixation
• an attempt to preserve cells and tissue constituents in a life-like
condition.
• must render cells/tissues to withstand any subsequent treatment with
various reagents with minimum loss, significant distortion, or
decomposition.

Aims of Fixation
One
To prevent autolysis
(enzyme attack) and
putrefaction
(bacterial attack).
Two
To preserve cells and
tissue constituents in
a life-like condition.
Three
To prevent distortion
(change in shape or
volume) of tissues
during subsequent
procedures.
To avoid decomposition
due to deprivation of
oxygen, accumulation of
carbon dioxide and
metabolites.
Four

Autolysis
a) autolysis means “self-destruction”.
b) caused by action of intracellular enzymes.
c) when a tissue is removed from the body or cut from its blood supply:
➢ it is deprived of oxygen and essential metabolites and the cell begins to die.
➢ the cell and its organelle membranes rupture .
➢ enzymes are released causing the breakdown of protein and eventual
liquefaction of cells.
d) Autolysis changes:
➢ independent of any bacterial action.
➢ retarded by cold; greatly accelerated by keeping at 37oC.
➢ affects highly specialized cells of complex organs (e.g. liver, pancreas, brain
and kidney) more rapidly.
➢ cell nuclei show condensation (pyknosis), fragmentation (karyorrhexis), or
lysis and eventual disappearance (karyolysis).
Self Digestion

Parts of the Normal Cell
Cytoplasm
Medium for chemical
reaction
Nucleus
Store the cell’s DNA
Membrane
Barrier to protect the cell
from its surroundings
Mitochondria
Generate energy

Normal Cell
Pyknosis
Autolysis Changes
Karyorrhexis
Normal nucleus
Condensation of
nucleus
Fragmentation of
nucleus
Karyolysis
Disappearance of
nucleus

Putrefaction
a) a process of bacterial decomposition.
b) caused by bacterial invasion:
➢ by multiplying bacteria in the diseased tissues at time of
death as in septicaemia (blood poisoning).
➢ by bacteria normally present in the body such as the
non-pathogenic organisms from the alimentary tract.
➢ microorganisms secrete enzymes to breakdown proteins,
lipids and carbohydrates into smaller units to provide
nutrients for themselves.

Fixative
•A substance which preserve the shape, structure, relationship and
chemical constituents of tissues of cells, after removal from the body.
•must be able to achieve all the aims of fixation.
•Common fixatives used: Formalin/Formaldehyde, Glutaraldehyde and
Osmium Tetroxide

Ideal Fixative
● preserve tissue volume without shrinking, or
swelling or distortion.
● preserve tissues in the natural state and fix
all components (protein, CHO, fat ).
● penetrate the tissue and cells rapidly and
evenly.
● inhibit bacterial decay and autolysis
● harden the tissue and render it insensitive
to subsequent treatment.
● allow tissue to be stored for long periods
of time.
● permit the restoration of natural colour for
photography and mounting as museum
specimens
● simple to prepare and economical in use.
● non-toxic and non-allergenic .
Properties 1 Properties 2
To date, no ideal fixative has been found yet.

Classification of Fixation Methods
01 Chemical properties or methods used
02
03
Action upon the cell and tissue constituents
Number of fixative chemicals used in a fixing
solution

Chemical Properties
or Methods Used
Classification of Fixation Methods
1) Physical agents: Microwave,
heat.
2) Coagulants: acetic acid, methyl
alcohol, ethyl alcohol.
3) Non-coagulants: formaldehyde
Glutaraldehyde, acrolein, picric
acid, mercuric chloride
Action Upon Cell
1) Micro-anatomical Fixatives
• preservation of the various layers of
tissues and cells in relation to one
another so that general structure can
be studied.
• 10% formol-saline
2) Cytological Fixatives (nuclear or
cytoplasmic)
• preservation of the constituent
elements of the cells.
• nuclear fixatives : Flemming’s fluid
• cytoplasmic fixatives: Flemming’s fluid
without acetic acid (chromic acid,
osmium tetroxide, glacial acetic acid).
Number of
Fixatives
1) Simple fixatives (only one
chemical): Formaldehydes,
mercuric chloride, osmium
tetroxide, picric acid
2) Compound fixatives (2 or
more chemicals): Bouin’s
fluid, Helly’s fluid,
Heidenhain’s Susa, Carnoy’s
Fluid, Zenker’s fluid
Protein stabiliser

Duration of
fixation
Temperature Size and
thickness
of tissues
➢ Volume ratio
Concentration
of fixatives
Agitation Buffers and pH
01 02 03 04
05 06
Factors affecting Fixation
08
07
Osmolality
of fixatives
Additives

• Moderate heat.
• Accentuates fixation
Temperature
Room
temperature
• To Increases, the rate
of fixation increases.
• For surgical and post
mortem specimens.
0 to 4 oC
• Ideal temperature.
• Especially for EM
(Electron Microscopy)
& Histochemistry.
37 to 56oC
60oC
• Rapid fixation for
very urgent biopsy
specimen.
Note: High temperature also increases the rate
of autolysis and diffusion of cellular elements

Size and thickness of tissues
a) Thickness of the tissue (for routine processing),
section should be < 3mm thick.
b) Large specimens must be sliced or opened:
● Uterus: should be opened
● Spleen: should be sliced thinly
● Segments of colon: should be opened
c) Volume ratio
● Tissue volume : Fixative volume
● Fixative volume 15 to 20 times > tissue volume
● Poor fixation staining problems
F F
X


Tissue volume : Fixative volume
Source: live.staticflickr.com/5229/5641048109_d8a16d9438_b.jpg

Duration of fixation
a) Tissue should be placed in fixative immediately after
surgical removal.
b) Adequate fixation is needed so that the tissue will not be
distorted by the subsequent processing steps.
c) Tissue that is not well fixed does not process well, and will
not stain well
d) For electron microscopy, tissues are fixed for 3 hours.
e) Formalin:
• Tissues are usually fixed for at least 6 to 8 hours, ok up to
24 hours.
• Reacts rapidly with tissue components.
• Prolonged fixation can cause shrinkage and hardening of
tissues.

03
Above 10% tend to cause increased hardening and shrinkage
Below 70% do not remove excess water from tissues efficiently
02
Concentration of Ethanol
01
Concentration of Fixative
Solubility of the fixatives.
Effectiveness
Concentration of Formalin

a) Fixation accelerated by agitation.
b) by the mechanical tissue processors.
Agitation

Buffers and pH
a) Fixatives occurs best between p 6 to 8.
b) Gastric mucosa is best fixed at pH 5.5.
c) pH can be adjusted using buffers:
• Phosphate, veronal acetate, bicarbonate and tris.
a) The effect of pH on fixation with formaldehyde is dependent on the targets.
• Acidic:
➢ Affect the NH2 group to become NH3
+ & become unreactive to
formaldehyde.
➢ Affect the COO- and lose the charge to become COOH.
➢ Will produce brown-black, insoluble, crystalline, birefringent pigment with
haemogloin breakdown products.

Osmolality
a) Important for ultrastructural studies.
b) Hypertonic solution cell shrinkage.
c) Hypotonic solution swollen cell.
d) Slightly hypertonic solution: 400 to 450 mOsm for EM work
e) Isotonic solution: 340 mOsm.
Cell shrinkage
(Hypertonic)
Swollen cell
(Hypotonic)
Happy cell
(Isotonic)

Additives for fixatives
Electrolytes
Non-electrolytes
• Sucrose
• Dextrant
• Detergent
• Calcium chloride
• Potassium thiocyanate
• Ammonium sulfate
• Potassium dihydrogen phosphate
➢ Can be added during fixation.
➢ To improve the cell morphology of the fixed tissues.

Application for fixatives
➢ volume of fixative should be approximately 15 to 20 times the
volume of tissue.
➢ tissue must be put in a suitably sized container .
➢ most fixatives cause expansion of tissue during fixation.
➢ if a container is too small, tissue will be squashed .
Conditions
Why?

Application for fixatives
Source: www.leicabiosystems.com Source: www.istock.com

Heart
Intestine
• open up the middle to allow easy
access of fixative to mucosa.
• specimen may be pinned to a
board, mucosal surface upward.
Lungs
• Inflate with fixative
through a cannula into
main bronchi.
Brain Solid Organs
• spleen, kidney, liver.
• should be sliced and paper placed
between slices to draw in fixative.
• fill chambers with cotton
wool to draw in fixative
01 02 03
05
Requirements to ensure effective and
even fixation
• perfuse it via the middle
cerebral arteries.
04
Penetration of Fixative

Quote of the Day
Source: www.redbubble.com/shop/medical+laboratory+professionals

Topic 2: Perform
Fixation & Tissue
Processing
Histological Techniques
Part 2

Grossing Station
Source: www.thermofisherscientific.com

Examination of Specimen
Source: https://www.researchgate.net/

Tissue Processing
• Fixed human tissues are soft and need support to cut thin sections.
• Sections : 3 – 5 m thick.
• Tissue must first undergo preparatory treatment (tissue processing) before
being sectioned .
• After processing → tissue block for microtomy (section-cutting) .
• Embedding medium (wax) is used to provide support for microtomy .

Tissue Blocks
Source: cpb-us-w2.wpmucdn.com/sites.gsu.edu/dist/3/1174/files/2015/02/paraffin-block-27vno1c.jpg

Tissue Cassettes
Source: www.sks-science.com/lab-supply-p-9687.html

Aims of Tissue Processing
One
To remove water from
the tissue and
impregnate with
another medium (solid)
to provide support.
Two
Three Four
firm enough to support
the tissue and give it
the rigidity to enable
thin sections to be cut.
soft enough to enable the
knife to cut sections with
little or no damage to the
knife or tissue
Source: www.123rf.com
Paraffin Wax
Tissue
Tissue
cassette

Steps Involved in Tissue Processing
• Replace clearing
agent with
embedding medium
• Replace dehydrating
fluid which is miscible
with dehydrating fluid
and embedding medium.
• Removal of fixative and
water from tissue.
• To replace them with
dehydrating fluid.
02 03
Dehydration Clearing Infiltration
01

1)First stage of tissue processing.
2)To remove fixative and water from tissue, replacing them with dehydrating fluid.
3)Essential as paraffin wax will not penetrate tissues in the presence of water.
4)Dehydrating reagents are hydrophilic.
- strong polar group with hydrogen bonds to remove water from tissues.
5) Accomplished by using graded alcohols.
- First into 70% -> 95% -> 100% alcohol.
- Direct transfer to absolute (100%) alcohol) lead to distortion of tissues.
- due to diffusion currents with excessive concentration gradients.
6) Delicate tissues, start with 30% alcohol.
7) Direct transfer to absolute alcohol can lead to distortion of tissues.
8) Excessive dehydration cause the tissue to become hard, brittle and shrunken.
9) Incomplete dehydration will impair the penetration of the clearing agents into tissues.
- Tissue will become too soft and cannot infiltrate into the tissues.
Dehydration

1)Second stage of tissue processing.
2)To remove alcohol and make the tissue receptive to infiltration medium. fixative and
water from tissue, replacing them with dehydrating fluid.
3)To treat tissues with a reagent which is miscible with both dehydrating and embedding
medium.
4)Factors affecting selection of clearing agents:
i) Flammable liquids:
- Boiling point gives an indicative of its speed of replacement by melted paraffin wax.
- Low boiling point more readily replaced by melted paraffin wax.
ii) Viscosity:
- Affect speed of penetration. High viscosity; lower rate of fluid penetration.
- Prolonged exposure tissue become brittle.
iii) Cost:
- expensive due to chemical waste disposal’s charges.
Clearing

1)Third stage of tissue processing.
2)After dehydration & clearing, tissues must be infiltrated (impregnated) with embedding
medium (wax).
3)Transfer tissues from clearing agent into molten wax at 60oC.
4)Clearing agent is eliminated from tissues by diffusion into wax, which in turn diffuses into
tissues to replace it.
5)Support tissues internally and externally.
- to hold the cells and intracellular structures in their proper relationship.
6) Most common embedding medium: Paraffin Wax.
- Cheap
- Easy to handle.
- Allow section with few problems
- Non-toxic.
Infiltration

Selection Criteria of Suitable Agent
1. Rapid penetration.
2. Speedy removal of dehydrating agent.
3. Ease of removal by molten.
4. Minimal tissue damage.
5. Low flammability, toxicity and cost.
6. Common clearing agents:
i. Xylene
ii. Toluene
iii. Chloroform.
Dehydration Clearing
1. Able to remove water.
2. Common dehydrating agents:
i. Ethanol
ii. Methanol
iii. Acetone
iv. Commercial industrial
methylated spirit
v. Propan-2-ol
vi. isopropyl alcohol

Common Clearing Agents
No. Clearing Agents Advantages Disadvantages
1. Xylene
(commonly used in
routine histology lab)
• Cheap.
• Rapid in action.
• Renders tissue transparent.
• Readily eliminated in paraffin
oven.
• Highly flammable.
• Prolonged immersion causes
tissues to be brittle.
• Does not dissolve well in water.
2. Toluene • Has similar properties to xylene
• Less toxic.
• Less damaging on prolonged
immersion of tissues.
• Highly flammable.
• More volatile than xylene.
3. Chloroform
(commonly used for
nervous tissues)
• Non-flammable.
• Very gentle in tissues, minimal
shrinkage or hardening of tissues.
• Expensive
• Slower in action than xylene or
toluene.
• Difficult to assess end-point as
tissues are not rendered
transparent.
• Vapour is anaesthetic and toxic.

Types of Automated Tissue Processors (ATP)
Enclosed Carousel Microwave
Source: www.leica.com Source: www.thermofisher.com Source: BioWave Pro+ tissue processing system (agarscientific.com)

Carousel ATP
• This traditional design consists of 12 reagent containers arranged in a circle.
• The containers are for fixatives, dehydrating, clearing agents and the last two
containers are reserved to melt wax.
• Tissues are contained in metal baskets and are suspended in the first container.
• It is then mechanically moved to the following container based on a pre-set timer .
• At the end of the cycle, the tissues should be in the last wax bath.
• Modern machines are now equipped with an electronic timer for greater
programming flexibility.
• Safety devices are also incorporated to provide protection for tissues and personnel.

Enclosed ATP
• Tissue cassettes and 12 reagent containers are arranged in a closed system.
• Fluids are pumped in and out as required.
• Incorporates the use of a microprocessor.
• Many different schedules can be programmed and stored for different types of
tissues.
• Vacuum and temperature may be applied to any or all the stages, thus speeds up
the processing time.
• A large number of tissues can be processed at a time.
• No fumes are vented from the machine making the work environment safer.

Tissue Processing
1 2 3 4 5 6 7 8 9 10 12
11
Reagent
Containers
Formalin Alcohol Xylene
70%
Paraffin
95% 100%
Source: www. researchgate.net/figure/Schematic-presentation-of-sectioning-fixed-and-paraffin-embedded-tissue-16_fig2_344313790

Microwave ATP
• Shortens processing time.
• Do not use clearing agents because high temperature facilitates evaporation
of the alcohols from the tissues.
• Cannot be used for tissue sample more than 2mm.
• labour intensive because need to manually input the solutions.

Time Taken for Tissue Processing (Routine)
1 2 3 4 5 6 7 8 9 10 12
11
Reagent
Containers
70%
Paraffin
95% 100%
2 h 2 h 1 h 1 h 1 h 2 h 1 h 1.5 h 2 h 3 h
2 h 1.5h
Total time: 20 hours

Time Taken for Tissue Processing (Short Schedule)
1 2 3 4 5 6 7 8 9 10 12
11
Reagent
Containers
70%
Paraffin
95% 100%
30m 15m 15m 15m 15m 15m 45m
15m
Total time: 4 hours 15 mins
15m 15m
15m 45m

Time Taken for Tissue Processing (Rapid)
1 2 3 4 5 6 7 8 9 10 12
11
Reagent
Containers
Formalin (600C) Fresh acetone Xylene Paraffin
20m 20m 10m 30m
20m
Total time: 3 hours 10 mins
20m 10m 60m
At each step, agitate frequently in transfer of fluids

Factors Influencing Tissue Processing
● Agitation
● Heat
● Viscosity
● Vacuum
● Size of the Tissue
● Number of tissue cassettes
● Types of Reagent Used
● Types of Tissue Processor
● Whether Vacuum or Raised
Temperature is Being Used
Rate of Procesing Processing Schedule

• Property of resistance to the flow of fluid that is
dependent on the size of the molecules in the solution.
-Bigger molecules -> more viscous
• Most fluids used in dehydration & clearing have similar
viscosities.
• Paraffin wax: low viscosity in the fluid state enhance
impregnation quicker.
Agitation
• Improves the rate of processing .
- too slow -> ineffective
- too violent -> damage to soft, friable tissues
• Difficult with manual processing & time consuming.
• Automated Tissue Processor incorporates agitation.
• Efficient agitation reduce processing time by 30%.
Heat
• Increases the rate of penetration & fluid exchange.
• Many fluids used are flammable & heating may
increase the fire hazard.
• Used only when urgent reports are required.
• Higher Temperature:
-> Adversely affect staining & immunocytochemistry.
-> Cause tissue shrinkage, hardening or embrittlement.
• Improve and speed up rate of impregnation (using
pressure).
• Remove any air bubbles trapped within the tissue.
• Processing fluids more intimate contact with parts of
the tissue (especially with dense & fatty tissues)
• Produce good ribbons of sections.
• Vacuum should not exceed 50.79kPa.
Factors Influencing the Rate of Tissue Processing
Vacuum
Viscosity

Good Ribbons of Tissues
Source: www.leica-microsystems.com/uploads/pics/microtomy.png
Source: www. mayo.edu/research/core-resources/pathology-research-core/services/tissue-sectioning

Improper Impregnation
• Inadequate wax impregnation drying and shrinkage of embedded tissue block.
• Inadequate support by wax crumbling of sections
Source: www.sciencephoto.com/media/1145655/view/cutting-tissue-section-with-rotary-microtome
Source: www.123rf.com

Quote of the Day
Source: www.res.cloudinary.com/teepublic/image/private/s--8i37cC-Q--
/t_Preview/b_rgb:191919,c_limit,f_auto,h_630,q_90,w_630/v1600417633/production/designs/14140168_0.jpg

Histological Techniques: Perform Fixation and Tissue Processing

Recommended

Recommended

More Related Content

What's hot

What's hot (20)

Similar to Histological Techniques: Perform Fixation and Tissue Processing

Similar to Histological Techniques: Perform Fixation and Tissue Processing (20)

Recently uploaded

Recently uploaded (20)

Histological Techniques: Perform Fixation and Tissue Processing