Frozen Sections

FROZEN SECTIONS
FROZEN SECTIONS
• Uses
▫ Rapid pathologic diagnosis during surgery
▫ Diagnostic and research enzyme histochemistry
▫ Diagnostic and research demonstration of soluble
substances such as lipids and carbohydrates
▫ Immunoflourescent and immunocytochemical
staining
▫ Some specialized silver stains, particularly in
neuropathology
Two methods of preparing frozen section
▫ Cold knife procedure

▫ Cryostat procedure (cold microtome)
Cold knife procedure
• Tissue blocks can be frozen by adapting a

conventional freezing microtome gas supply of
carbon dioxide gas from CO2 cylinder, or by using
a specially made piece of equipment known as
cryostat.
• Almost any microtome can be utilized for the
purpose, provided means are made available for
freezing and maintaining the specimen and the
knife at low temperatures, usually by utilizing the
carbon dioxide technique.
Cryostat procedure
• Uses cryostat, an apparatus used in fresh tissue
microtomy, consisting of an insulated
microtome housed in an electrically driven
refrigerated chamber maintained at
temperature near -20C, where microtome knife,
specimen and atmosphere are kept at the same
temperature.
• The optimum working temperature is -18 to
-20C.
Cryostat procedure
• The commonly used methods of freezing include
▫ Liquid nitrogen
▫ Isopentane cooled by liquid nitrogen
▫ Carbon dioxide gas
▫ Aerosol sprays
Liquid nitrogen
▫ Used in histochemistry and during intra-operative procedures
and the most rapid of the commonly available freezing agents.
▫ Main disadvantage is that soft tissue is liable to crack due to
rapid expansion of the ice within the tissue, producing ice
crystals or freeze artifacts.
▫ It also overcools urgent biopsy blocks, causing damage to both
block and blade if sectioning is done at minus 70C or below.
▫ It causes a vapor phase to form around the tissue, acting as an
insulator that causes uneven cooling of tissue, particularly of
muscle biopsies, making diagnostic interpretation difficult.
Isopentane
• Isopentane is liquid at room temp. A pyrex
glass beaker is usually suspended in a flask of
liquid nitrogen until half liquid and half solid
stage is reached. The beaker is removed from the
liquid nitrogen when small crystals start forming
on the side of the beaker (approximately -170C),
and the tissue to be frozen (affixed on a cork
disc, aluminum foil or cryostat chuck) is dropped
into cooled liquid isopentane
Aerosol spray
• The use of aerosol spray has become increasingly
popular in recent years, and is adequatefor
freezing small pieces of tissue except muscle.
Quick freezing spray can of fluorinated
hydrocarbons have a distinct advantage of
freezing blocks of any tissue type rapidly.
considerations
• Fresh frozen tissue requires that the tissue be
maintained in the frozen solid state during
cutting of section, thereby supporting and
protecting the tissue from damage and distortion
by the knife during the process of cutting.
• The microtome knife needs to be chilled and
maintained at low temperature to prevent
complete melting of the tissue, thereby forming
a sticky, distorted mass along the knife edge.
considerations
• When the tissue is too cold, on the other hand,
resistance to cutting is increased, so that the tissue
becomes brittle and is broken down into fragments
upon cutting.
• The success of fresh tissue sectioning therefore,
depends to a large extent on the temperature, both of
the knife and the tissue.
• Certain tissues such as fat or mucin, and hard or dense
structures in a soft matrix require much lower
temperatures to impart a suitable consistency for
cutting
MOUNTING OF TISSUE BLOCK
• Synthetic water soluble glycols and resins are generally
used as mounting media for tissue blocks that need to be
sectioned on a cryostat.
• It is marketed in a convenient 8 oz plastic dispensers in
three temperature ranges, depending on the tissue being
cut.
▫ -5Cto -15C- brain, lymph nodes, liver, spleen, uterine
curettings, soft cellular tumors
▫ -15C to -25C- non=fatty breast tissue, ovary, prostate,
tongue and GI tract
▫ -35C for fatty breast and omental tissue
• The cryostat is usually set at -18 to -20C.
• The tissue block should be 2-4mm thick in order
to minimize the risk of the knife hitting the
metal tissue block holder.
• Small fragments of tissue, such as curettings or
brain biopsies, are placed on a thick base of
freezing medium.
• The blocks are then surrounded and covered with
an additional matrix.
• The frozen tissue is mounted on the microtome.
Both the microtome knife and the tissue block are
left in the cryostat for 15-30 minutes at -20C, to
ensure that they are cooled to correct temperature.
• Sections between 5-10 micra are then cut slowly
and steadily, removed from the knife with a camel
hair brush, attached directly to slides of cover-glass
at room temperature, air dried, fixed and stained.
MOUNTING CRYOSTAT SECTIONS
• To mount cryostat sections after cutting, one edge

of the glass slide is rested on the knife surface,
about 1 inch beyond the section, and the other end
is lowered gently until it is about ½ to 1mm thick
from the knife face.
• The section will automatically transfer from the
cold knife to the relatively warm slide.
• It should be noted that cryostats cut only
individual sections, and do not form ribbons, as in
paraffin blocks.
FREEZING PREVIOULY FIXED TISSUE
• Cryostat sections of fresh, unfixed tissue usually

attached easily to the slide, even without adhesives,
and will preserve enzymes and other substances
that may be studied by histochemical techniques.
• Sections of formalin-fixed tissue, however, may not
adhere to the slide, and will fall off or detached
during staining. To enhance attachment of frozen
fixed sections, clean slides should be coated with
albumin or chrome glycerin jelly.
FREEZING PREVIOULY FIXED TISSUE
• The tissue block may be immersed in boiling 10%

buffered formalin for 1 to 2 minutes before
freezing and sectioning for rapid surgical
diagnosis.
• Special fixatives, such as 10% formol calcium at
4C may be used in histochemistry and for lipid
demonstration.
• Tissue that have been fixed or stored in alcohol
should be washed in water for 12-24 hours before
sectioning, since alcohol inhibits freezing.
CARE OF THE MICROTOME
• It takes at least one hour for knife to come down to
operating temperature, so that a spare knife should
always be kept inside the cryostat cabinet.
• To ensure that the sections will cut smoothly and
freely onto the knife and anti roll plate must be kept
scrupulously clean and dry.
• Soft tissue paper, either dry or moistened with
absolute alcohol, may be used for cleaning the knife
and anti-roll plate.
• The cryostat should be defrosted and clean after use
SPECIAL PROCESSING TECHNIQUE
• Frozen section is deemed to be the most ideal and preferred means

of preserving tissues in order to avoid complete or partial loss of
enzymes consequent to chemical fixation.
• In addition to fresh frozen sections, there are two methods that
may be resorted to, if chemical fixation of tissue blocks is to be
avoided, namely:
▫ Freeze drying
▫ Freeze substitution
• These techniques have the common principle of rapidly preserving
the tissue block by freezing, to produce instant cessation of cellular
activity thereby preventing chemical alteration of tissue
constituents and displacement of cellular tissue components.
FREEZE DRYING
• A special way of preserving tissues by rapid

freezing of fresh tissue at -160C and subsequently
removing ice water molecules by physical process
of transferring the still frozen tissue block in a
vacuum at a higher temperature, without the use
of any chemical fixatives.
• Not used in routine surgical laboratories, and is
restricted to specialized or research laboratories.
• Time consuming and expensive.
FREEZE DRYING
• Produces minimum tissue shrinkage, and allows tissues to be processed
in a fresh state, thereby allowing minimal chemical change on the cells,
most especially on the protein components, less displacement of tissue
and cell constituents.
• In addition to demonstrating hydrolytic enzymes, mucous substances,
glycogen and proteins, freeze drying may be used for special studies,
including
▫ Immunohistochemistry
▫ Fluorescent antibody studies of polypeptide and polypeptide hormones
▫ Autoradiography
▫ Microspectrofluorimetry of autoflourescent substances
▫ Formaldehyde induced fluorescence of biogenic amines
▫ Scanning electron microscopy
FREEZE SUBSTITUTION
• Similar to freeze drying in preparing and preserving

tissue blocks for subsequent sectioning because both
involve the rapid freezing of tissues and subsequent
infiltration and embedding of the frozen tissue block in
paraffin or celloidin.
• The only variation is that the frozen tissue, instead of
being subjected to dehydration in an expensive vacuum
drying apparatus, is fixed in Rossman’s formula or in
1% acetone and dehydrated in absolute alcohol .
• Infiltration and embedding is then carried out in the
same way as in paraffin sections.
• Overall, cryostat section provide the simplest,
quickest and least labor intensive method for
producing frozen sections, and are routinely
used for intraoperative and rapid diagnosis of
surgical specimen.
End….

Frozen Sections

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Frozen Sections

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FROZEN SECTIONS

▫ Cold knife procedure

• Tissue blocks can be frozen by adapting a

• To mount cryostat sections after cutting, one edge

• Cryostat sections of fresh, unfixed tissue usually

• The tissue block may be immersed in boiling 10%

• Frozen section is deemed to be the most ideal and preferred means

• A special way of preserving tissues by rapid

• Similar to freeze drying in preparing and preserving

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