Fixation of Fresh

tissue
Prepared by: Ms. Roselin Tsauses
Anatomical Pathology 2A (ANP611S)
February 2022
Objectives
• Understand the importance of timely specimen transfer to the
laboratory.
• Understand the need for accurate specimen reception and patient
details supplied with samples.
• Understand the principles of specimen fixation and factors affecting
fixation.
• Understand the importance of specimen decalcification and how this
can be achieved.
 Pre-learning activity
• Take the pre-learning quiz in class.
Terms
• Autolysis –Self digestion within the cell; refers to the destruction of
a cell through the action of its own enzymes. When an organism dies,
one of the processes that is triggered is cellular destruction by these
internal enzymes.
• Putrefaction –cellular degradation due to attack by microorganism;
This process, prevalent in moist climates, is associated with green
discoloration of the body; gas production with associated bloating;
skin slippage; and a foul odor.
• Hypoxia of tissues lowers the pH; a condition in which the body or a
region of the body is deprived of adequate oxygen supply at the tissue
level.
• Buffered - Although the pigment can be removed from sections with
saturated aqueous picric acid before staining, it is preferable to avoid
its formation in the first place. For this reason and
because formaldehyde reacts most effectively at about a neutral pH,
10% formalin solutions are usually buffered to pH 6.8 – 7.2.
Introduction
• Histopathology is a complex process involving multiple steps,
from the acquisition of tissues to diagnosis.
• Fixation forms part of the five (5) steps of tissue processing.
• Fixation refers to the preservation of biological tissues from
decay due to autolysis or putrefaction.
• It terminates any ongoing biochemical reactions and may also
increase the treated tissues' mechanical strength or stability.
• Fixation consists of two steps:
• cessation of normal life functions in the tissue (killing)
• stabilization of the structure of the tissue (preservation).
• The goal of fixation is to preserve structure as faithfully as
possible compared to the living state.
Steps to tissue processing

• Fixation
• Dehydration
• Clearing
• Infiltration
• Embedding
Order of Histo-technique steps in
histology
• Collection of specimen
• Fixation
• Grossing
• Dehydration
• Clearing
• Infiltration/ impregnation
• Embedding
• Microtomy
• Staining
• Mounting
• Microscopy
Cell and tissue preservation

• Most routine specimens received in the laboratory are suitable for

formalin fixation.
• When tissue is removed from the body, it dies due to the absence of
oxygen and food, and the build up of waste material. The DNA,
proteins, sugars, and fats start to deteriorate, initially through the build
up of waste products and uncontrolled release of proteases. This
process is known as autolysis.
• Autolyzed cells stained with haematoxylin and eosin (H&E) show
enhanced cytoplasmic staining or eosinophilia (i.e., more pink) and
reduced nuclear staining (less purple) due to the break-down of RNA
molecules in the cytoplasm and the breakdown of DNA molecules in
the nuclei.
• Later, bacterial or fungal contamination can cause further destruction
of cells and tissues, which is a process known as putrefaction.
• If the shape and structure of the cells deteriorate, then diagnostic
analysis becomes impossible.
Cell and tissue preservation…

• Therefore, the first part of the histopathology process involves

making sure the cells do not deteriorate.
• Prevention of cellular deterioration is achieved by fixation or
freezing.
• The nature of preservation in histopathology is to keep cells in
as life-like a state as possible.
• The freezing of histopathology samples occurs only in a
minority of cases where the tests required are incompatible with
the chemicals used in fixation.
• Examples of tissues that should be frozen on receipt are
muscle biopsies, specimens for immunofluorescence and
suspected rare tumors where molecular studies may be of
interest.
Cell and tissue preservation…

• Hence, fixation aims to preserve cells and tissue constituents in

as close to a life-like state as possible.
• It is concerned with the stabilization of proteins within the tissue
and arresting the effects of autolysis and bacterial
decomposition.
• Note: Cell preservation is only part of the fixation effect.
• Fixatives provide additional benefits for the histologist as they
allow the tissues to undergo further preparative procedures
without change and make the cellular components more easily
stained by dyes.
• Note: The appearance of the tissue after fixation is artefactual.
• The stabilization of proteins is required to prevent their diffusion
during subsequent processing, but this also changes the
structure and appearance of these proteins.
Cell and tissue preservation…

• Information is available when the nucleus is examined.

• The size, shape, texture, and staining properties of the nucleus alter
in differing pathological conditions.
• Note: Not all specimens received in the laboratory are suitable for
fixation, because subsequent tests required on some specimens are
not compatible with fixation. Therefore, it is important to know what
tests will be required before the sample is taken.
• Hence, some samples (usually less than the majority) may be
received fresh or in Michel’s transport medium.
• Michel’s transport medium is not a fixative, but a solution of salts plus
an antibacterial agent, which acts to sustain biopsies in an osmotically
balanced solution during transportation to the laboratory.
• It is mainly used with skin and mucosal samples for investigation of
inflammatory disorders.
Cell and tissue preservation…

• Specimens not suitable for fixation are:

• Muscle biopsies for enzyme histochemistry.
• Skin/ mucosal biopsies for investigation of inflammatory skin
conditions.
Principles & mechanisms of
fixation
• Principles:
• There are a number of features that make a fixative ideal (to be discussed
later in the presentation).
• In addition to the preserving agent, often salts or buffers are included.
• The salts are added to adjust the osmolality of the solution, which ideally
should be similar to the fluid within the tissue being fixed.
• This ensures that cells do not shrink or swell.
• Mechanisms:
• Fixation is a chemical process by which the constituent molecules within
the cells can be manipulated in several ways.
• Cross-linking (or additive) fixatives act by binding amino acids within
proteins to adjust their three-dimensional (3D) structure, preventing
autolysis and putrefaction.
• Formalin fixation is not suitable when handling specimens for enzyme
histochemistry as the cross-linking acts to denature many enzymes.
• Precipitating or (non-additive) fixatives act by removing water from the
cellular matrix, which acts to disrupt the 3D (tertiary) protein structure,
thereby precipitating the protein.
Principles & mechanisms of
fixation…
• The use of fixation results in significant changes to the tissue
structure and components.
• These changes are beneficial in terms of tissue preservation.
however, it is important to recognize that some tissue components
are extremely labile and so would be readily inactivated by the use of
these chemicals.
• Well-fixed cells should show:
• Good nuclear detail
• Not swollen or shrunken
• Integrity of the general architecture is not disturbed
• Cellular proteins are well preserved for future tests.
Principles & mechanisms of
fixation…
• Poorly fixed cells have poor nuclear detail and may be swollen or
shrunken.
• The general architecture of the piece of tissue may be distorted, and
cellular proteins may be lost.
• Fatty tissue is susceptible to poor fixation as the aqueous reagents do
not penetrate fat well.
Types of fixation
• Involves physical or chemical methods.
• Physical methods (not commonly used in the histology lab):
• Heating
• Microwaving
• Freeze-drying
• Chemical fixation (mostly used in the histology lab):
• Liquid fixatives
Types of fixation
Chemical fixation
• Makes use of organic or non-organic solutions to maintain adequate
morphological preservation.
• Chemical fixatives are members of three major categories:
• Coagulant
• Cross-linking
• Compound fixatives
Coagulant fixatives
• Organic and non-organic solutions may coagulate proteins, making
them insoluble.
• Cellular architecture is mostly maintained by lipoproteins and fibrous
proteins e. g. collagen.
• Coagulating such proteins maintains tissue histomorphology at light
microscopic level.
• Coagulating proteins:
The change in the structure of protein (from a liquid form to solid or a
thicker liquid) brought about by heat, mechanical action or acids.
Enzymes may also cause protein coagulation e. g. cheese making.
• Coagulant fixatives result in cytoplasmic flocculation (formation of
loose aggregations or soft flakes) and poor preservation of the
mitochondria, therefore not useful.
Compound fixatives
• Formaldehyde-based fixatives are used in order to ensure
reproducible histomorphometric patterns.
• Other agents may be added to formaldehyde to produce specific
effects that are not possible with formaldehyde alone.
• Example: dehydrant ethanol to produce alcoholic formalin
• Third combination preserves molecules like glycogen and results in
less shrinkage and hardening than pure dehydrants.
• Compound fixatives are useful for specific tissues e. g. alcoholic
formalin for fixation of some fatty tissues, e .g. breast.
 Features of an ideal
fixative
• Penetrates tissue rapidly and evenly:
• The solution diffuses through the tissue quickly.
• Kills cells quickly and evenly with little distortion:
• This stops the build-up of harmful metabolites that disrupt the cellular appearance.
• Not swell or shrink cells of the tissue:
• Maintains the normal shape and size of the cell in relation to the size in situ. This allows for
meaningful diagnostic and prognostic measurements.
• Give good optical differentiation
• Help with good staining
• Harden tissue – easier grossing
• Prevent autolysis and putrefaction:
• Stops cellular functions immediately, preventing self-destruction and microbial damage.
• Prevent desiccation and drying of tissue:
• Keeps the cells hydrated.
• Support long-term tissue storage giving excellent microtomy of paraffin blocks
• Minimizing the loss or enzymatic destruction of cellular and extracellular
molecules
 What affects the speed and
effectiveness of fixation?
• Temperature of fixative:
• The hotter the fixative, the quicker the fixation.
• Concentration of fixative:
• The stronger the fixative, the more rapidly fixation takes place.
• Volume of fixative:
• The larger the volume of the fluid to specimen ratio, the more rapidly fixation will
take place. It is recommended that there is 20 times as much fixative as
specimen, although this is not always possible.
• Duration of the fixation:
• The longer the tissues are in fixative, the more complete the fixative will be.
• Density/ size of the tissue and penetration of the fixative:
• Large specimens take longer to fix than small specimens. The penetration rate is
the rate at which the chemicals permeate the tissue. This may be only four or five
millimetres a day; consequently, large resections need slicing open before the
processes of autolysis destroys the detail within the tissue.
Speed and effectiveness of
fixation…
• pH and buffers:
• The addition of buffer does slow the fixation process slightly, bit it does
ensure that the chemicals are at the correct pH. Acidic formalin fixatives react
with tissues and can create pigments. Ph: Ideal pH required is 6-8
• Osmolarity:
• Fixatives should be the same osmolarity as the cells they are fixing. This will
help to prevent shrinking or swelling.
Fixative –general use
Action of Fixatives
Why buffered formalin?

• Formalin is cheap, easy to make, keeps well on the shelf, and is easily
transported.
• It is an aqueous acidic solution that contains a concentration of
dissolved formaldehyde gas.
• Acid formalin causes formalin pigments, a brown residue seen in red
blood cells.
• Pigments are not seen when pH of the solution is neutralized, so
buffering agents are added in most circumstances.
• 10% formalin solution is the commonly used fixative in routine
laboratories.
Formalin: Chemical structure

• The chemical formula of

formaldehyde is CH2O: it has
one carbon (C) atom, two
hydrogen (H) atoms and one
oxygen (O) atom.
• Note the double bond between
the carbon and the oxygen,
which gives the highly reactive
aldehyde functional group.
 Saline
10% Buffered
Formaldehyde
1. Saline solution:
9.0 gm NaCl diluted in 1000ml DDW

2. 10% Buffered Formaldehyde (formalin):

• Sodium dihydrogen Phosphate - 80gm (10gm) 4.0gm
• diSod hydrogen Phosphate – 130gm(16.25gm) 6.5gm
• Formaldehyde – 2000L (250ml) 100ml
• DDW – 18L (2250ml) 900ml
• pH reading – 7.2-7.4
Formalin fixation

• Fixation is divided up into two parts:

• The penetration rate ( the time it takes the fixative to reach the innermost
parts of the tissue).
• The reaction time for the tissue to be fully fixed.
• The thicket the tissue, the longer the fixation process will take.
• Formalin is a protein fixative, but fixation of DNA is not ideal with this
agent.
• As formalin enters the cell, the proteins are fixed first and any
carbohydrate present is ‘pushed’ to the unfixed part of the cell, giving
rise to the characteristic flow pattern that is usually seen
microscopically (we will cover this under staining).
Formalin fixation: common errors
and pitfalls
• Inadequate length of fixation
• Tissue not sliced before fixation
• Faulty batches of solution
• Incorrect pH of solution
Health and safety: Formalin

• Colourless liquid
• Causes burns
• Very toxic by inhalation, ingestion, and through skin absorption
• Readily absorbed through the skin
• Possible human carcinogen
• May cause allergic reactions
• Causes watery eyes at levels over 20 ppm
• First aid measures:
• Inhalation: remove to fresh air, seek medical assistance if necessary.
• Eye contact: rinse well with running water, seek medical advice if necessary.
• Skin contact: rinse affected area, seek medical advice if necessary.
Decalcification

• After tissues have been fixed, they are then ready for the next stage of
laboratory investigation.
• Some tissues cut easily with a knife and require no further pre-
treatment.
• Some tissue structures (e. g. bone and teeth) are extremely hard due
to the presence of calcium phosphate salt (hydroxyapatite).
• This mineral is essential in life for these structures to perform their
role, but unless this mineral is removed, then the production of thin
sections for microscopy will be problematic.
• The method of achieving this is called decalcification as it is primarily
calcium salts that are removed. However, during the process,
materials other than calcium will be removed, so a more accurate
term to apply would be deminerelization. Calcium crystals may also
be present in other tissues as part of a pathological process.
Decalcification…

• Tissue such as breast may be X-rayed prior to being

processed.
• The identification of this calcium has diagnostic significance and
therefore it is important that it remains in the tissue.
• Bone samples are not suitable for decalcification when they are
to be studies for suspected disturbances to calcium metabolism
(e. g. osteomalacia in adults or rickets in children).
• In these conditions, the amount of mineralized to non-
mineralized bone is reduced.
• In these cases, the sample must be treated by embedding the
mineralized bone into a hard embedding medium such as a
plastic resin.
• This will allow suitable quality sections to be produced and
stained.
Factors affecting the rate of
decalcification
• Whichever decalcification method is used, there must be a balance
between speed of decalcification and preservation of cellular
morphology.
• The speedier the rate of decalcification, the greater the rate of
destruction (maceration) of cellular morphology.
• The choice of decalcifying method and chemical will be determined
by the type of specimen investigated.
• Factors:
• Concentration of the acid used in the decalcifying agent.
• Agitation of the tissue.
• Saturation of calcium ions in the decalcifying solution.
• Note: Specimens must not be decalcified before fixation is complete,
as the decalcifying agent will cause excessive maceration.
Decalcifying agents

• Note: decalcifying agents are categorized as follows:

• Acid decalcifiers
• Chelating agents
• Acids: Acid decalcifying agents act by reacting with the calcium in the tissue to
form soluble salts.
• Nitric acid: very fast acting but produces the most tissue damage. Suitable for the controlled
decalcification of very hard bones such as the skull.
• Hydrochloric acid: Fast acting but produces less tissue damage than nitric acid.
• Formic acid: Much slower than nitric or HCL acid and so its effect is more gentle. It is
probably the most popular choice of decalcification fluid for most diagnostic histopathology
applications.
• Chelating agents: These offer alternatives to the use of acids. They act as calcium
sponges, continuously mopping up the free ionic calcium that is always present
around mineralized bone.
• Chelating agents offer a greater approach and may be the decalcifying agent of
choice for tissue likely to require immunocytochemistry.
• Ethylenediaminetetraacetic acid (EDTA): Main chelating agent used. Calcium
removal is very slow and as a result, there is minimal tissue damage. EDTA is the
decalcifying agent of choice if immunocytochemistry is likely on the specimen.
Practical demonstrations
Fixation
Unfixed Heart
Fixed Heart
Heart
Unfixed brain
Fixed brain
Brain cut in half
Unfixed uterus
Fixed uterus
Opened
Uterus
Cervix
cone
biopsy
Cervix
cone
biopsy
Cervix
cone
biopsy -
inked
Cervix
cone
biopsy
Grossing
cancerous
breast
lump
Colored
ink for
orientation
Normal
appendix
Inflamed
appendix
Sectioned
appendix
Colon
Colon
sections
- stained
Skin/mole
specimen
Skin/mole
specimen
- inked
Skin/mole
specimen
Prostate
“chips”
Stained
prostate
“chips”
In summary
• The key objective of fixation is to stabilize the protein framework of
cells to achieve consistency and reliability in subsequent
demonstration techniques.
• In the field of anatomy, fixation is the preservation of
biological tissues from decay due to autolysis or putrefaction.
• It terminates any ongoing biochemical reactions and may also
increase the treated tissues' mechanical strength or stability.
Test your knowledge

• What changes do cells and tissues undergo wen they are removed
from the body?
• What are the principles of fixation?
• What might be the implications of a poorly fixed specimen for
accurate diagnosis?
• What does formalin actually fix?
• What are the key hazards associated with formalin?
• What factors affect the speed of fixation?
• What methods are used for decalcification?
Next lecture
• We will cover grossing, which refers to the gross examination
and inspection of pathology specimens with the bare eye to
obtain diagnostic information, while being processed for further
microscopic examination.
Tasks
• Lecture 1, which is an introductory lecture will be uploaded on
Thursday, the 25th of March 2021.
• This lecture need to be attended to during the ANP611S time
slot on Friday, the 26th of March 2021 from 08:30 am to 10:30
am.
• A quiz will be uploaded to be attempted at the end of the
lecture.
 References
• Pathcare Academy Notes
• 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
• Shambayati,B, 2011, Cytopathology, Fundamentals of biomedical
Science series, Oxford University Press, Oxford
• Keebler, CM and Somrak, TM, The Manual of Cytotechnology,
7thEdition, American Society of Clinical Pathologist, Hong Kong.
• Guy Orchard and Brian Nation,(2012) Histopathology, Fundamentals of
biomedical Science series, Oxford University Press, New York
• Cook,DJ, (2006), Cellular Pathology: An Introduction To Techniques and
Applications, 2ndEdition, Scion Publishing Ltd, UK.