MODULE 3 eliminating most types of cancer before it
EXERCISE 5 has spread to lymph nodes or distant
FROZEN SECTION PROCESSING
Frozen section processing employs a different
method of processing anatomical samples
compared to routine processing that uses
formalin-fixation, paraffin-embedding.
While it is still possible to freeze tissues fixated by
formalin and embedded in paraffin, current
technologies have advanced to employ the use of
a cryostat for frozen section processing.
Two methods of routine frozen section
processing:
1. Cold-knife procedure
2. Cryotomy
INDICATIONS FOR FROZEN SECTION
PROCESSING
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 neuropathy.
 Traditional/routine formalin-paraffin
tissue processing often removes or
affects certain constituents such as
enzymes, lipids, & carbohydrates.
SURGICAL REMOVAL OF TUMORS
 Frozen section processing is especially
recommended when lipids and nervous
tissue elements are to be demonstrated.
 Frozen section processing is useful for
the diagnosis / evaluation of tumors.
 Surgery is a traditional form of cancer
treatment. It is the most effective in
sites (metastasized).
 If a tumor appears to have metastasized,
a sample of the suspected metastasis is
sent for cryosection to confirm its identity.
 This will help the surgeon decide whether
there is any point in continuing the
operation.
 Aggressive or radical surgery is involved
in the excision or resection of the tumor
tissue. It often involves removal of the
tumor itself including the margin, which is
healthy tissue surrounding the tumor.
 The degree of removal may depend
based on diagnostic findings. Usually,
aggressive surgery is performed only if
there is a chance to cure the patient.
 If the tests indicate a high chance for
metastasis, or the tumor has spread
across a large portion of the organ, a may
conservative (palliative) surgical
approach may be considered including
other cancer treatments.
 Debulking removes part, but not all of the
tumor. This is indicated if the tumor is too
large and removal may cause more
damage, or involve removal of the organ.
Debulking tries to remove majority of the
cancerous tissue, and
chemotherapy/radiation therapy will be
indicated to remove the rest/hinder
metastasis.
 Tissue must reach histopathology
laboratory immediately, often directly
from the operating room.
 To avoid tissue being dried it should be
kept in saline.
 The size of the tissue should be
small/thin, so that good smooth sections
can be obtained and freezing is quick.
 Thickness of the tissue should be about
3mm to 4mm.
 The tissue can directly be taken to
cryostat, or can be fixed with 10%
formalin or formol –alcohol
COLD KNIFE PROCEDURE
  Almost any microtome can be utilized for
this purpose, provided that means are
made available for freezing and
maintaining the specimen and the knife
at low temperatures, usually by utilizing
the carbon dioxide technique.
 But mainly, the cold-knife procedure is
employed using a freezing microtome.
 A freezing microtome is an apparatus
specifically designed for cutting frozen
sections.
 It takes the principle of the cold knife
procedure and allows control of the
microtome knife, the introduction of CO2
gas, and has an automatic feed
mechanism.
 This device is clamped onto a desk or
table. A rubber hose is attached from the
CO2 gas cylinder onto the freezing
attachment or stage where the specimen
is placed.
 The CO2 freezing attachment is provided
with a hard rubber non-conducting plate
between the corrugated surface to which
the object is frozen and the rest of the
apparatus, which prevents the
conduction of heat from the other parts of
the apparatus to the specimen; thus
saving time and CO2.
 Thickness of fresh tissue from which
frozen sections are to be cut must not be
thicker than 4-5 mm.
1. Place a drop of water on the block holder under
the tissue in the freezing stage to aid in attaching
it securely.
2. The tissue is held flat on the stage and carbon
dioxide is released in short bursts. (Frozen
section must appear white & firm for the
recommended consistency).
3. Adjust height of the freezing stage to bring the
face of the block a hair’s width below the
microtome knife.
4. Move the knife into the tissue block slowly;
cutting sections at short intervals until the tissue
softens and produces satisfactory samples. (Cut
is made through unfrozen tissue just above the
“frozen line”. Knife must be wiped clean and dry
while cutting.
5. With the dampened tip of the little finger, lift the
sections from the knife.
You may also use a camel brush if sections were
able to ribbon
6. Float sections on a wide dish of distilled water
(sections are colorless or very light in color)
Mount the section onto an albuminized slide
Temporary stains – Rapid Eyedropper Staining
Method
1. Float the frozen section onto a
clean glass slide
2. Place a drop or two of toluidine
blue directly on the tissue using
an eyedropper
3. Leave stain on about 10 seconds
4. Rinse gently with water from eye
dropper to remove excess stain
(keep slide in a fixed horizontal
position during staining &
rinsing)
5. Drain the slide. Add cover slip
with an aqueous mountant. Slide
is ready for examination.
Permanent stain – H&E (as in routine method)
ADVANTAGES/DISADVANTAGES
ADVANTAGES:
• Can be employed with any microtome
(requiring only CO2 gas)
• Rapid and inexpensive
• If the tissue is too hard, you can gently
warm it with your finger. If the tissue
block is too soft, you can immediately
refreeze it
DISADVANTAGES:
 • Temperature is difficult to control.
Because it is in an open environment, the
temperature of the tissue block, the
microtome knife, and even the
surrounding room is subject to
temperature variation – possible affecting
sectioning
• Sections do not form ribbons but rather
stick to the knife blade and should,
therefore, be removed with a camel hair
brush or finger moistened with water.
CRYOSTAT
 A cryostat functions to obtain thin (1-10
mm in thickness) sections from a piece of
tissue, but while a standard microtome
carries the operation at room
temperature, the cryostat enables the
operator to section the tissue at low
temperature (–20 to –30° C) within a
refrigerated chamber.
 An Optimal cutting temperature
compound (OCT compound) is used to
embed tissue samples prior to frozen
sectioning on a microtome-cryostat.
 Optimum cutting temperature (OCT)
compound is used as embedding
medium.
The OCT is made up of water-soluble glycols
and resin.
A. CRYOSTAT PROCESSING
Preparing the cryostat
 The cryostat is usually set at -18 to -
20°C. The cutting knife or blade should
be loaded along with all other required
materials (i.e. brush).
 The cryostat should be left on at all times
even when not in use, since it will require
several hours to reach operating
temperature from a room temperature
start.
A. Specimen Preparation
1. Grossing and cutting the specimen
• Identify the tissue sample of the patient
and the requisition form
• Salient clinical information are helpful as
it guides the pathologist to reach the
possible differential diagnosis.
• Tissue appearance: The gross
appearance of tissue (color, texture,
consistency, and any suture to mark the
anatomical position).
• Resection margin: It is very important to
identify the resection margins of the
tumor. The resection planes and margins
should be inked thoroughly. Different
colors of ink can be used for medial and
lateral margin identification.
2. Cutting the specimen
• The tissue should be fresh, preferably
without any fixative. The tissue should be
preferably dry, and it should not be
wrapped in gauze piece.
• Any suture, staple or sharp hard structure
should be removed from the tissue
sample.
• The tissue is cut into small pieces as it
facilitates freezing. Take multiple
sections of the tissue to understand the
main pathology and to minimize the error.
3. Cytology of the tissue
• At times, the imprint of the tissue on the
slide provides good morphological
details (lymphoma).
• Similarly, crushing of tissue also provides
excellent morphological details (tissue of
brain tumor).
B. CRYOSTAT PROPER
1. Tissue embedding in the mold
 The small piece of the tissue is kept in the
center of the mold, and then the OCT
(Optimal cutting temperature compound)
is poured over it in excess.
 Then the tissue holder or chuck is firmly
placed over the tissue with overflown
OCT.
2. Tissue loading in the frozen section
chamber
 The tissue is now placed in the frozen
section chamber, and cold spray can be
used to make the process faster.
Both the microtome knife and the tissue
block are left in the cryostat for 15
minutes at -20°C, to ensure that they are
cooled to the correct temperature.
3. The specimen holder is clamped
firmly onto the object head mounted
on the spindle of the microtome
4. Trimming the tissue
 The block is trimmed to remove the
excess OCT and to get the smooth tissue
surface for sectioning.
 Before facing, you may remove excess
plastic (OCT) by cutting it using blade.
 Adjust the micrometer setting of the
microtome to “trimming” thickness of
15um and begin to turn the microtome
hand wheel; the specimen will advance
by this set value and will make contact
with the knife and the surface of the block
will be sectioned.
 This process is known as “trimming” or
“facing” the block, and the purpose is to
achieve a full face section of the
specimen. This is similar to what you do
with paraffin blocks during sectioning.
5. Antiroll plate
 Carefully lift the antiroll plate and with a
cold brush, arrange or move the sections
on the pressure plate or knife surface
6. Sectioning
 Sections between 5-10 μm are then cut
slowly and steadily, removed from the
knife with a camel hair brush spread
gently over the antiroll plate.
 The tissue is now cut gently and is
spread over the antiroll plate with the
help of a brush. The brush should also be
cooled.
 The tip of the tissue is guided by the
brush.
7. Section lifting
 The glass slide of normal room
temperature is pressed firmly
over the tissue section, and
normally the tissue sticks
immediately.
 One edge of the glass slide is
lowered gently until it is about ½
to 1mm from the knife face.
 The section will automatically
transfer from the cold knife to the
relatively warm slide
8. Slide fixation
 The tissue should be
immediately fixed in methanol for
1 min or 95% ethanol for few
seconds. Rapid fixation within
few seconds is mandatory.
 In case of delayed fixation, the
cells are swollen, and the
cytoplasmic margin may be
ruptured giving hazy appearance
of the margin of the cells.
TROUBLESHOOTING
FREEZING ARTIFACT
 When tissue is frozen, ice crystals are
formed within the tissue.
 Even if the tissue is placed in formalin,
ice crystal gaps or clefts will remain.
SHATTERING
 This artifact is often a result of the block
being too cold. It can be dealt with by
warming the block.
 Tissues with high water content have
greater tendency to shatter and must be
 warmed to a temperature that will cut with
the least shattering. Edematous or
bloody tissues will show this problem.
 Brain biopsies are notorious for this
problem. Imagine trying to cut an ice
cube. It will shatter as it is cut.
Nuclear ice crystals
 Nuclei will show varying tendency to form
ice crystals. The thinner tissue is cut, the
more these crystals appears as holes.
FREEZING PREVIOUSLY FIXED TISSUE
SPECIAL PROCESSING TECHNIQUES
Freeze-Drying
 Freeze-drying is a special way of
preserving tissues by rapid freezing
(quenching) of fresh tissue at -160°C and
subsequently removing ice water
molecules (desiccation) by transferring
the still frozen tissue block into a vacuum
chamber at a higher temperature, e.g. -
40°C (sublimation) without the use of any
chemical fixative.
 This technique is generally not used in
routine surgical laboratories, and is
restricted to specialized or research
laboratories.
 A tissue around 2mm thick is plunged
into isopentane or propane-isopentane
mixture which has been chilled to -160°
to -180°C with liquid nitrogen.
 This will effectively solidify the tissue in 2-
3 seconds, thus preventing the formation
of large ice crystals, autolysis and
putrefaction.
 The frozen tissue is then transferred into
a high vacuum drying chamber
maintained at a temperature of -30° to -
40°C depending upon the size of the
tissue. Water is sublimated and
dehydrated from the tissue, thereby
completing the desiccation process
within 24-48 hours.
 Once drying is completed, the tissue is
removed, fixed and embedded, either in
molten paraffin wax, water soluble waxes
or celloidin.
 Infiltration and impregnation are usually
performed in a vacuum embedding oven
or VEU.
 The tissue is then sectioned in the usual
routine manner and specific staining is
applied, depending upon individual
necessity. This technique is generally
time-consuming and expensive.
 Drying is by far the most time consuming
part of the process, as certain tissues
contain 70- 80% water by weight that has
to be removed without damage to the
tissue.
FREEZE DRYING ADVANTAGES
• Produces minimum tissue shrinkage
• Allows tissues to be processed in a fresh
state.
• Causes minimal chemical change on the
cells, most especially on the protein
components, and less displacement of
tissue and cellular constituents.
• Avoids the chemical alteration of cellular
components, the denaturation of
proteins, destruction of enzymes, and
loss of tissue constituents that usually
occur in the usual histological
processing.
FREEZE-SUBSTITUTION
• Freeze-substitution is a process of
dehydration, performed at temperatures
low enough to avoid the formation of ice
crystals and to circumvent the damaging
effects observed after ambient-
temperature dehydration.
• It is similar to freeze-drying in preparing
and preserving tissue blocks for
subsequent sectioning because both
involve the rapid freezing of tissues and
the subsequent infiltration and
embedding of the frozen tissue block in
paraffin or celloidin. 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 section

• Freeze-substitution is based on rapid

freezing of tissues followed by solution
("substitution") of ice at temperatures
well below 0°C.

• A 1 mm to 3 mm specimen is thrown into

3:1 propane-isopentane that is super
cooled by liquid nitrogen to -175°C (with
precautions).

• Cryostat sections are cut 8-10 μm, and

transferred to water-free acetone
(substituting fluid), and cooled to -70°C
for 12 hours to 1 week in order to dissolve
ice slowly without distorting tissue
structure.

• The sections are floated onto coverslips

or slides and allowed to dry for
subsequent histochemical staining.

• This technique is relatively more

economical and less time-consuming
than freeze-drying.

• For best morphological and

histochemical preservation, substituting
fluids should in general contain both
chemical fixing agent and solvent for ice,
e.g., 1% solutions of osmium tetroxide in
acetone, mercuric chloride in ethanol, or
picric acid in ethanol.