TISSUE ACCESIONING

• New specimens are registered in a book and

are assigned a number
• Specimens usually come in formalin fixative
• Specimen are macroscopically examined
– Described and weighed
– Suspicious portions are selected and cut
 Tissue processing
• Handling of tissue specimens until it is ready for staining.
• Includes:
– Fixation
– Dehydration
– Clearing
– Infiltration
– Embedding
– Blocking
– Trimming
– Sectioning
 Fixation-AIMS
1. To prevent decomposition
• Decomposition may be due to O2 and
essential metabolite deprivation, deprivation
of blood supply and accumulation of by-
product of metabolism.
• The fixative denature or precipitate proteins
which form sponge or meshwork that tend to
hold other cells constituents.
2. Prevents cellular autolysis
By two mechanisms
a. The fixative inactivates the lysosomal
enzymes.
b.The fixative chemically alters the cell
components so that they become resistant to
enzyme degradation.
3. Prevents putrefaction
• Fixation seems to protect the tissue from
microbial damage.
4. Fixation protects the tissue from damage
during subsquent preparative operations e.g.
dedydration, embedding, sectioning and
mounting.
5. Insolubilisation of tissue
• Fixation renders insoluble certain tissue
components that woud otherwise leach out
during subsquent handling.e.g lipids.
• However, no one fixative is capable of
insolubilising all tissue components.
• Therefore, the success of examining a particular
component lies in the proper selection of fixative
to use.
 Factors affecting fixation
1. Buffering and pH.
• Fixation is best carried out close to neutral pH.
• Hypoxia of tissue lowers the tissue pH, as such,
there should be a buffering capacity to prevent
excess acidity.
• Acidity favors formation of formalin, lead to
formation of black pigment in tissue.
• Common buffers; phosphates, bicarbonates,
cocodylates and veronal.
• The buffer must not react with the fixative, inhibit
enzymes or react with incubation medium.
2. Penetration
• The depth of tissue penetrated is directly proportional
to the square root of the time
• Fixation depends not only on the diffusion of the fixative
into the tissue but also on the rate at which it reacts
with tissue components.
• Formalin and alcohol penetrates the best, and
formaldehyde, the worst.
• Penetration into a thin section will occur more rapidily
than for a thick section.
3.Temperature
• Fixation of most surgical specimens is at room
temperature.
• For electron microscopy and some
histochemistry, the temperature range is 0-4 oC
• As a rule, cold temperatures retards fixation and
mderate heat accerates fixation but also hastens
autolytic changes and enzyme destruction
4. Concentration of fixative
To be adjusted down to the lowest level for
economic reasons.
Formalin works best 10% while gluteraldehyde
is generally made up 0.25-4%.
Too high concentration may adversely affect the
tissues and produce artifacts apart frm being
too expensive.
5. Volume of fixative
There should be a 10-20:1 ratio of fixative to
tissue volume.
 FACTORS RETARDING FIXATION
1. Size and thickness of tissue specimen
2. Presence of mucus-preventing complete
penetration of fixative. Excess mucus can be
washed away with normal saline.
3. Presence of fat
4. Presence of blood
5. Cold temperature
PRINCIPLES AND PRECUATIONS IN HANDLING
AND FIXATION OF SPECIMENS IN GENERAL
• Both autopsy and surgical material should fixed as
soon as possible.
• All tissues should be properly labelled and
identified.
• Avoid put refrigerated tissues at 0oC to avoid ice
fromations
• Tissues shuld not be more than 5mm thick.
• Required time of fixation should not be exceeded
toavoid hardening and britleness of tissue.
• There must be adquate supply of fixative at all times
• Avoid drying of specimens to avoid shrinkage and
distortion of cellular details.
• Solid organs should be injected with as well as
emersed in enough fixative to ensure enough
fixation.
• Hollow organs should be packed with cotton soaked
in fixative or completely opened before being
emersed in adequate fixative.
• Air-filled organs like lungs float on the fixative.
To avoid this, the organ may be covered with
several layers of guaze to keep it
undersurface.
• Dense tissues are poorly penetrated hence
require longer fixation.
 CHARACTERISTIS OF A GOOD
FIXATIVE
• Must be cheap
• Must be stable
• Safe to handle
• Must kill the cell quickly producing minimum distortion of
cell constituents
• Must inhibit autolysis and bacterial decomposition
• Must produce minimum shrinkage
• Must penetrate tissue rapidily and evenly
• Must harden the tissue making cutting of sections easier
but prevent overhardening.
• Must be isotonic
must preserve tissue volume
Must be non-allergic or non-toxic.
 Types of fixatives
• A. ACCORDING TO COMPOSITION
1.Simple fixative-made up of only one
component substance e.g aldehydes,
formaldehyde, acetone, acetic acid
B. ACCORDING TO ACTION
1. Microanatomic fixative-permit microscopic
study of tissue substances without altering
structural patterns e.g 10% formal saline
• 2. cytological fixatives
• 3. Nuclear fixatives-e.g. Bouin’s fluid, Carroy’s
fluid, Flemming’s fluid.
4. Cytoplasmic fixatives e.g. Helley’s fluid
C. Histochemical fixatives
Those that preserve tissue chemicals e.g. 10%
formalin, absolute ethyl alcohol, acetone
 EMBEDDING
• This is the fixation of tissue specimen in a firm
medium inoder to keep it intact during the
cutting into thin sections.
 CHARACTERISTICS OF A GOOD
EMBEDDING MEDIUM
• Easily convertible from liquid to solid
• Should have the capacity to penetrate tissue
organelles when in liquid state
• Should not react with cell components or destroy
cell anatomy
• Should not change tissue volume
• Should have good cutting qualities
• Should be removed easily from the tissue
• Should be cheap
 EMBEDDING MEDIA
1. Is paraffin/paraplast mixture
• Could be substituted by, paraplast, embeddol,
bioloid, and carbowax; all with their
advantages and disadvantages.
2. Celloidin
Used to embed hard tissue
3. Gelatin
Used in histochemical and enzyme study
 EMBEDDING MOULDS
• Used for tissue blocking
• Blocking is casting of tissue blocks in an
embedding medium so that sections can be
cut with a microtome.
• Blocking is done for support and easy handling
of a tissue.
 TYPES
• Leukhart’s embedding molding
• Compound embedding unit
• Plastic embedding rings
• Disposable embedding units
 Factors affecting duration of
embedding
• Size of specimen
• Density of specimen
• Nature of specimen
 SECTIONING
• Is a process of cutting tissue into uniformly
thin slices or sections with the aid of a
microtome to facilitate study under the
microscope.
 Microtome
• Is a machine or instrument designed for actual
cutting of tissue sections.
 Kind of microtomes
1. Sliding microtome
• For cutting celloidin embedded sections
• Has concave knife
• Two models exist
– Base-sledge (old model)-movable block i.e. Block
of tissue goes to knife while knife is stationary
– Standard sliding(new model)- movable knife i.e
block is stationary while knife is moved backwards
or forward.
2. Standard Rotary microtome
• For cutting paraffin-embedded sections
• Knife is biconcave
3. Rustproof Rotary microtome
• For cryostat sectioning
4. Clinical freezing microtome
• For cutting fresh and fixed unembedded tissue that
has been frozen with CO2 and other refrigerants
Uses
• When rapid diagnosis is required
• When histological demonstration of fat is
needed.
• When certain neurological structures are to
be stained
• When senstive tissue constituents to be
studied are damaged or destroyed by heat.
5. Rocking mcrotome
Used for cutting serial sections of large blocks of
paraffin embedded sections
6. Ultra-thin microtome
Used for cutting sections for electron
microscopyn (0.5µm)
 NOTE
• Type of microtome to be used depends on
– Type of work
– Nature of tissue preparation
– Embedding medium used
PREPARATION OF THE BLOCK AND
EQUIPMENT
• The top and bottom sides of tissue are
trimmed until perfectly level and all sides are
parallel almost to the edge of the tissue.
• Tissue block is trimmed away until the entire
tissue space has been partly exposed.
• The block is then allowed to harden for proper
cutting by facing them down in ice cold or
refrigerator for 5-10 minutes.
• The block is then placed in the microtome for
cutting
• Series of glass slides are numbered corresponding to
those on tissue blocks.
• The slides are cleaned with a thin cloth and smeared
with a very thin coat of adhessive
• Examples of adhessives
– Meyer’s egg albumin
– Dried abumin
– Gelatin
– Starch paste
– plasma
 Floating out bath
• Sections are removed in ribbons to allow easy
location of serial sections.
• Sections are then floated out in a water bath set at
about 45-50oC which is approximately 6-10oC lower
than melting point of the embedding medium.
• This is done to flatten the sections before mounting
them onto the slides.
• Another way of flattening is by placing the sections
on a glass slide and flood them with 20% alcohol.
 Sections fixation onto the slide
• May be done in one of several ways;
– In wax oven at 56-60oC for 2hrs
– In an incubator for overnight
– On a hotplate at 45-55oC for 30-45 mnutes,
– By carefully holding the slide, section upward
above bunsen burner, until the wax just melts.
 STAINING
• Processing of imparting of or formation of
color in different tissues and cells.
 PURPOSE OF STAINING
• To render different tissue constituents more
visible through variations in color
• To promote easier optical differentiation for
identification of cells and tissue components.
• To display affinities of tissues and cells for most
dyes
• Physical characteristics and structural
relationships are better studied and evaluated.
The Hematoxylin and eosin
staining procedure