CHAPTER 10: FROZEN SECTIONS
 Tissues that are frozen too hard will chip into fragments when cut.
Frozen Section
- normally used when rapid diagnosis is required
- especially recommended when lipids and nervous tissue elements
are to be demonstrate
Uses:
1. Rapid pathologic diagnosis during surgery
2. Diagnostic and research enzyme histochemistry
3. Diagnostic and research demonstration of soluble substances
such as lipids and carbohydrates
4. Immunofluorescent and immunocytochemical staining
5. Some specialized silver stains, particularly in neuropathology
Preparing tissue for freezing:
- Tissue for freezing should be frozen or fixed as promptly as
possible after cessation of circulation to avoid morphological
distortions and damage due to:
1. Tissue drying artifact
2. Autolysis - The destruction of tissues or cells by the action of
substances, such as enzymes, that are produced within the
organism. Also called self-digestion.
3. Putrefaction - Decomposition by microorganisms.
WHY SNAP FREEZE OR FIX AND CRYOPROTECT FOR FREEZING?
- Slow freezing can cause distortion of tissue due to ice crystal
formation that replaces the architecture with a “Swiss Cheese”
effect.
- The object is to freeze so rapidly that water does not have time to
form crystals and remains in a vitreous form that does not expand
when solidified.
- Freezing artifact in sections of Brain, Spleen and Skeletal Muscle
Requirements:
- Tissue is fresh, completely unfixed or have been previously
treated briskly with formalin not requiring embedding instead
frozen and cut in a freezing microtome
Methods of preparing frozen sections:
1. CoId Knife procedure
2. Cryostat procedure (Cold Microtome)
COLD KNIFE PROCEDURE
- uses carbon dioxide gas from a CO2 cylinder, or by using a
cryostat.
- almost any microtome can be utilized provided there is a means
of freezing or maintaining the specimen and the knife at low
temperatures using carbon dioxide technique.
- a piece of filter paper soaked with gum syrup is placed on the
microtome stage then frozen by applying short bursts of CO2
- then the selected piece of tissue approximately 3-5 mm thick, is
oriented on the stage, applied with a few drops of gum syrup and
frozen solid with several intermittent burst of CO2 each for 1-2
seconds duration, every 5 seconds intervals.
- tissue should be frozen and firm enough for sectioning.
- tissue is then lifted up to the knife manually and trimmed until the
surface is flat.
- surface is then warmed with the finger until the hard frozen tissue
starts to thaw and becomes visible to the naked eye.
 This is the Dew Line, the point at which sections may then
be cut at 10 micra thickness.
Note:
 Sections do not form ribbons but rather stick to the knife blade
and should be removed with a camel hair brush or finger
moistened with water.
 Transfer section to a dish of distilled water to separate, and
picked up individually for mounting and staining.
 Use water dish in a dark or black background, in order to see the
sections which are usually colorless or very light in color.
Problems:
REMEDY: softened by warming slightly with the ball of the finger
or thumb.
 Tissues that have not been sufficiently frozen will cut thick and
crumble, and the block may come away from the stage.
REMEDY: more bursts of CO2 gas should then be given to refreeze
the block.
Temperature:
- Whether used in a cold environment or not, a different
temperature between the tissue and the knife is usually
employed.
- KNIFE SHOULD BE COLDER THAN THE TISSUE
o Knife = - 40° to - 60°C
o Tissue = - 5° to - 10°C
o Environment = 0° to - 10°C
Success of the procedure:
1. ambient temperature
2. humidity
 It is very hard to cut sections in a hot or humid surrounding.
 Facility of cutting and quality of sections are always improved in a cold
environment.
CRYOSTAT PROCEDURE
Cryostat
- a chamber consisting of an insulated microtome housed in an
electrically driven chamber maintained at temperatures near
“ -20°C ”
- Optimum working temperature = -18 to -20°C
- majority of the sections can be cut in isothermic conditions:
 the temperature for sectioning can be accurately established
and controlled
More commonly used methods of freezing include:
1) Liquid nitrogen
- used in histochemistry and intro-operative procedures
- most rapid of the commonly available freezing agents.
Disadvantage
1. soft tissue is liable to crack due to the rapid expansion of the ice
within the tissue, producing ice crystals or freeze artifacts.
2. overcools urgent biopsy blocks, causing damage to both block and
blade if sectioning is done at - 70°C or below.
Note:
 Tissue snap-frozen in liquid nitrogen must be allowed to
equilibrate to cryostat chamber temperature before sectioning is
attempted.
 Temperature for non-fatty unfixed tissues = -10°C to -25°C
Problems:
 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.
REMEDY: freezing the tissue in Isopentane, O.C.T., or Freon 2.2
that has a high thermal conductivity.
2) Isopentane (cooled by liquid nitrogen)
- liquid at room temperature
3) Aerosol sprays
- adequate for freezing small pieces of tissue except muscle.
- used for freezing tissues rapidly using quick-freezing spray cans of
fluorinated hydrocarbons (e.g., Cryokwik)
4) Carbon dioxide gas
 Success of fresh tissue sectioning depend on the extent on the
temperature, both of the tissue and of the knife.
 Fat or mucin, and hard or dense structures in a soft matrix: require
lower temperatures to impart a suitable consistency for cutting.
 MOUNTING OF TISSUE BLOCK
- Mounting media: Synthetic water-soluble glycols and resins
- Example: O.C.T. (Optimal Cutting Temperature compound);
marketed in convenient 8 oz. plastic dispensers in three
temperature ranges:
o - 5 to - 15°C = brain, lymph nodes, liver, spleen, uterine
curetting, soft cellular tumors
o -15 to - 25°C = non-fatty breast tissue, ovary, prostate,
tongue and GI tract
o - 35°C = fatty breast and omental tissue.
- Thickness: 2-4 mm to minimize the risk of the knife hitting the
metal tissue block holder.
- Small tissue, such as curettings or brain biopsies, are placed on a
thick base of O.C.T. compound. The blocks are then surrounded
and covered with an additional matrix of O.C.T. compound, and
frozen by liquid nitrogen.
- Both the microtome knife and the tissue block are left in the
cryostat for 15-30 minutes at -20°C, to ensure that they are
cooled to the correct temperature.
- Cutting: sections 5 - 10 micra are cut slowly and steadily, removed
from the knife with a camel hair brush, attached directly to slides
of cover-glasses at room temperature, air-dried, fixed (optional)
and stained.
 MOUNTING CRYOSTAT SECTIONS
CARE OF THE MICROTOME:
- cryostat should be left on at all times, since it will require several
hours to reach operating temperature from a room temperature
start.
- it takes at least one hour for a knife to come down to operating
temperature, so a spare knife should always be kept inside the
cryostat cabinet.
- to ensure that the sections will cut smoothly and freely onto the
knife surface, the knife as well as the undersurface and edge of
the anti-roll plate must be kept scrupulously clean and dry.
 Cleaning agent: Soft tissue paper (dry or moistened with
absolute alcohol)
- cryostat should be defrosted during the weekend, including
cleaning and oiling of microtome with special low-temperature
oil.
 SPECIAL PROCESSING TECHNIQUES
- frozen section is the most ideal and preferred means of
preserving tissues in order to avoid complete or partial loss of
enzymes due to chemical fixation.
The methods if chemical fixation of tissue blocks is to be avoided:
1. Freeze-drying
2. Freeze substitution
Common principle:
- rapidly preserve the tissue block by freezing (quenching), to
produce instant cessation of cellular activity thereby preventing
chemical alteration of tissue constituents and displacement of
cellular tissue components.
- Freezing agent: liquid nitrogen
 Agents that have a higher conductivity: Isopentane,
pentane,propane, and dichloro-difluoromethane
Freeze-Drying
- rapid freezing (quenching) at -160°C
- subfollowed by removing ice water molecules (dessication) by a
physical process of transferring the still frozen tissue block in a
vacuum at a higher temperature, e.g. -40°C (sublimation) without
the use of any chemical fixative.
- restricted to specialized or research laboratories.
Method:
1. 2 mm thick is plunged into Isopentane or propane-isopentane
mixture chilled to -160°C to -180°C with liquid nitrogen.
2. Solidification in 2-3 seconds, to prevent the formation of large ice
crystals, autolysis and putrefaction.
3. Transfer frozen tissue into a high vacuum drying apparatus
maintained at a temperature of -30°C to -40°C
4. Dessication by sublimation and dehydrated of the tissue within
24-48 hours.
5. Once drying is completed, the tissue is removed, fixed and
embedded
 Infiltration and impregnation are done in a vacuum embedding oven.
 Sectioning of the tissue is in a routine manner and specific staining is
applied.
Disadvantages: time-consuming and expensive
 Drying is the most consuming part of the process, as tissues
contain 70-80% water by weight, that has to be removed without
damage to the tissue.
 Freeze-dried materials are difficult to section than ordinary
paraffin blocks.
 The tissue is brittle and inadequately supported due to the
relative short period for wax impregnation.
 Water must be avoided and warm alcohol, acetone, mercury are
preferred.
Good features:
 It produces minimum tissue shrinkage, and allows tissues to be
processed in a fresh state, allowing minimal chemical change on
the cells, especially on the protein components, and less
displacement of tissue and cell constituents, avoids destruction of
enzymes, and loss of tissue constituents
 used to demonstrate hydrolytic enzymes, mucous substances,
glycogen and protein
 may also be used for special studies, including:
1. Immunocytochemistry
2. Fluorescent antibody studies of polypeptide and polypeptide
hormones
3. Autoradiography
4. Microspectrofluorimetry of autofluorescent substances
5. Formaldehyde -induced fluorescence of biogenic amines
6. Scanning electron microscopy
Freeze-Substitution:
- similar to freeze-drying except instead of being dehydrated in an
expensive vacuum drying apparatus, it is fixed in Rossman's
formula or in 1% Acetone and dehydrated in absolute alcohol.
- alternative method from rapid freezing of tissue to -160°C in
isopentane supercooled in liquid nitrogen.
- Cryostat sections are cut 8-10 um
- Transferred to water free acetone, and cooled to -70°C for 12
hours.
- Sections are floated onto coverslips or slides and allowed to dry
for subsequent histochemical staining.
Advantage: more economic and less time-consuming
Overall cryostat sections advantage:
- simple, quick and least labor- intensive for producing frozen
sections, intraoperative and rapid diagnosis of surgical specimen.
PandaMT13
(Lotho, Katrina L.)