Department: Health

Programme: Medical Laboratory Sciences

Tissue
Processing
Prepared by: Ms. Roselin Tsauses
Anatomical Pathology 2A (ANP611S)
March 2022
Quiz on previous lectures

• Define fixation.
• Fixation refers to the preservation of biological tissues from decay due to
autolysis or putrefaction.
• Name the chemical that is used to fix tissue?
• 10% Neutral Buffered Formalin (NBF)
• What do we mean by buffered?
• To lessen or moderate the impact of something. E.g., 10% formalin
solutions are usually buffered to pH 6.8 – 7.2
• Why is formalin buffered?
• To avoid the formation of formalin pigments.
• Name any two (2) tips for better grossing.
• Avoid Specimen Trauma
• Avoid Overloading Cassettes
Pre-learning activity

• Name the five stages of tissue processing:

• Fixation
• Dehydration
• Clearing
• Infiltration
• Embedding
Contents
• Concepts and definitions
• Steps to tissue processing:
• Fixation
• Dehydration
• Clearing
• Infiltration
• Embedding
• Automated and manual tissue processing
• Microwave processing
• Illustrative examples of the different steps
• Summary
Learning objectives

• Demonstrate practical understanding of the stages involved in tissue

processing for microscopic examination.
• Discuss principles and practices to tissue processing.
• Discuss chemicals, equipment/ instruments used during each stage of
tissue processing.
Introduction

• In previous lectures, we looked at specimen collection, reception,

fixation and dissection.
• In this lecture, we will briefly revise fixation and look in more detail at
the laboratory processes which follow specimen dissection as part of
tissue processing.
• Tissue processing involves preparing tissues so that they are
infiltrated with a supporting medium.
• The supporting medium is usually paraffin wax.
Concepts and definitions

• Fixation:
• Prevents autolysis and stabilizes tissue to maintain cellular structure.
• Dehydration:
• Removes water and unbound fixative from the tissue.
• Clearing:
• Displaces dehydrating solutions, making the tissue components
receptive to the infiltrating medium.
• Infiltration:
• Permeates the tissue with a support medium.
• Embedding:
• Orientation of the tissue sample in a support medium to create a tissue
“block” suitable for sectioning.
Revision: Fixation…

• Preserving cells and tissue components with minimal distortion is the

most important aim of processing tissue samples.
• Fixation does the following:
• Stabilizes proteins, rendering the cell and its components resistant to further
autolysis by inactivating lysosomal enzymes.
• Changes the tissues’ receptiveness to further processing.
• Fixation must finish before subsequent steps in the processing
schedule are initiated.
• If fixation is not complete prior to processing, stations should be
designated on the processor for this purpose.
• If the tissue is inadequately fixed, the subsequent dehydration
solutions may complete the process, possibly altering the staining
characteristics of the tissue.
Fixation cont.

• The size and type of specimen in the tissue cassette determines the
time required for complete fixation and processing.
• The tissue should be dissected to 2 – 4 mm in thickness.
• Care must be taken not to overfill the cassette, as this would impede
the flow of reagents around the tissue.
• If possible, larger and smaller pieces of tissue should be separated
and processed using different schedules.
• The most commonly used reagent for the fixation of histological
specimens is:
• 10% NBF
• The usual 10% formalin used in fixation of tissues is a 10% solution of
formalin; i. e. it contains about 4% weight to volume of
formaldehyde.
Dehydration

• The first stage of processing is the removal of “free "unbound water

and aqueous fixatives from the tissue components.
• Dehydration should be accomplished slowly.
• If the concentration gradient is excessive, diffusion currents across
the cell membranes may increase the possibility of cell distortion.
• Specimens are therefore processed through a graded series of
reagents of increasing concentration.
• Excessive dehydration may cause the tissue to become hard, brittle
and shrunken.
• Incomplete dehydration will impair the penetration of the clearing
reagents into the tissue, leaving the specimen soft and non-receptive
to infiltration.
Dehydration cont.

• There are numerous dehydrating agents:

• Ethanol
• Ethanol acetone
• Methanol
• Isopropyl
• Glycol
• Denatured alcohols
• Graded concentrations of ethanol are used for dehydration.
• The tissue is immersed in 70% ethanol, followed by 95% and 100%
solutions.
• Ethanol ensures total dehydration, making it the reagent of choice for
processing.
• For delicate tissue it is recommended that the processing starts in
30% ethanol.
Dehydration cont.
Clearing

• Clearing reagents act as an intermediary between the dehydration

and infiltration solutions.
• They should be miscible (forming a homogeneous mixture when
added together) with both solutions.
• When the dehydrating agent has been entirely replaced by most of
these solvents, the tissue has a translucent appearance: hence the
term clearing agent.
• Criteria for choosing a suitable clearing agent are:
• Rapid penetration of tissues
• Rapid removal of dehydrating agent
• Ease of removal by melted paraffin wax
• Minimal tissue damage
• Low flammability
• Low toxicity
• Low cost
Clearing cont.

• Xylene is used as the preferred clearing agent in Histology.

• Xylene is a flammable, colourless liquid with a characteristic
petroleum odor, which is miscible with most organic solvents and
paraffin wax.
• It is suitable for clearing blocks that are less than 5 mm in thickness
and rapidly replaces alcohol from the tissue.
Clearing cont.
Infiltrating and embedding

• Paraffin wax is the most popular infiltration and embedding medium.

• Paraffin wax permeates the tissue in liquid form and solidifies rapidly
when cooled.
• The tissue is impregnated with the medium, forming a matrix and
preventing distortion of the tissue structure during microtomy.
• It has a wide range of melting points, which is important for use in
different climatic regions.
• Paraffin wax is compatible with most routine and special stains, as
well as immunohistochemistry protocols.
Infiltration
Infiltration cont.
Embedding

• Involves the enclosing of properly processed, correctly orientated

specimens in a support medium that provides external support
during microtomy.
• The embedding media must fill the matrix within the tissue,
supporting cellular components.
• The medium should provide elasticity, resisting section distortion
while facilitating sectioning.
Embedding workstation

• Embedding workstation with cold plate.

Embedding workstation cont.
 Tissue
embedding
cassettes
Metal moulds
Embedding cont.
Embedding cont.
Wax blocks with different tissues
Automated tissue processing

• The basic principle for tissue processing requires the exchange of

fluids using a series of solutions for a predetermined length of time in
a controlled environment.
Automated tissue processing
Manual tissue processing

• Manual tissue processing has stopped in most laboratories.

• There are circumstances requiring tissue samples to be processed
manually:
• Power failure
• Equipment malfunction
• Large tissue samples requiring more time than can be allocated on an
automated processor
• Small biopsies, such as transplant specimens needing a rapid diagnosis
Manual
tissue
process
ing
Manual tissue processing cont.
Processing schedule - overnight
Microwave tissue processing

• The microwave shortens the processing time from hours to minutes.

• The microwave exposure stimulates the diffusion of the solutions
into the tissue by increasing the internal heat of the specimen, thus
accelerating the reaction.
• Tissues are manually transferred from container to container of the
reagent.
• Most laboratory microwave ovens contain precise temperature
controls, timers and fume extraction systems.
• The processing time depends on the thickness and density of the
specimen.
• Disadvantage: The system is labour intensive, because the solutions
are manually manipulated.
Microwave Processing schedule –
Rapid histopathologic diagnosis
Microwave Processor
Order of Histo-technique steps in
histology

• Collection of specimen
• Fixation
• Grossing
• Dehydration
• Clearing
• Infiltration/ impregnation
• Embedding
• Microtomy
• Staining
• Mounting
• Microscopy
 Histo-technique Steps

Collection of Specimen Removal of fresh tissue

Cut to appropriate size - trimmed

Fixing
Decalcifying
Washing/post-fixing
Processing
Dehydrating
Infiltration & Embedding
Clearing

Sectioning of Tissue
Remove paraffin wax

Biological Staining Rehydrating

Clearing
Mounting

Microscopy Observation
 Summary:
Alcohol Alcohol
Formaldehyde 90% 96%
Fixing Alcohol Alcohol
70% 96%

Post fixing Alcohol

(water) 100%

Polymere Alcohol
Wax 100%

Polymere
Alcohol
Wax
100%
100%
Wax:Xylene Alc:Xyl
1:1 1:1
Xylene Xylene
100% 100%
Embedding
Summary

• The key aspects you are looking to optimize are:

• Overall morphology
• Nuclear detail
• Tissue integrity
 What can go wrong?
Plan before removing tissue – know what you want to
do beforehand.
Collection of Specimen
Fix asap to keep tissue in same state

Fixing Post-wash= rinse excess fixative from tissue- if its not

replaced by clearing agent, affect embedding, sectioning &
staining.
Processing
Clearing= bridging step- alcohol must be extracted for
Infiltration & Embedding wax to penetrate tissue, if not fully extracted, affect
sectioning & staining

Sectioning of Tissue Infiltration= gradual extraction of clearing agent from

tissue without distorting/changing tissue

Biological Staining
Embedded material must be orientated properly &
levelled on microtome block
Mounting
Ensure proper staining with stain & counterstain

Microscopy Observation Careful mounting to avoid bubbles

Quiz 1

• Opens: Thursday, the 10th of March 2022 @ 23:00 pm

• Closes: Thursday, the 17th of March 2022 @ 23:00 pm
• Covers: lectures 1 – 4
• Assessment weight: 5%
• No extensions will be given.
Summary

• In summary, have a look at the following video on YouTube:

• Histology Techniques and Equipment
• Link: https://www.youtube.com/watch?v=4DJm4NLECQs
Next lecture

• During our next lecture, we will have a look at microtomy.

References

• John D. Bancroft, Christopher Layton and S.Kim Suvarna, 2013,

Bancroft’s Theory and Practice of Histological Techniques, 7thEdition,
Elsevier, China
• J.A. Kiernan,2015, Histological and Histochemical Methods,
5thEdition, Scion, UK