HISTOPATHOLOGY
CUTTING PROCESS
CUTTING SECTIONS
 We use microtome for thin slices
Sectioning - process whereby tissues are cut into
uniformly thin slices or sections.
THREE GENERAL TYPES
1. Paraffin Sections- embedded tissue
blocks which may be cut by rocking and
rotary microtome.
 Only thin slices are taken out at a time
to prevent the block from cracking.
 Samples of small biopsy tissue may be
trimmed only to the depth of the first
representation of several levels that will
be collected.
 Tissue that was embedded improperly
may not reveal the entire tissue surface
and will have to be re-embedded.
UNDER PARAFFIN SECTION:
 coarse trimming, a heated spatula is
held between the tissue block and the
block holder until the wax begins to
melt. The block is then placed in the
microtome for fine trimming and
cutting.
o Re-chilling of the block may
be required if the block face
becomes warm or if deeper
levels are required.
 Fine trimming – uses knives like
biconcave knives.
o The knife is usually tilted at 0-1
5° angulation on a microtome to
allow a clearance angle between
the cutting facet and the tissue
block.
o Most of the paraffin wax should
be cold when sections are cut.
Chilling of the block is
important.
o The surface block is then
trimmed away until the entire
tissue surface has been partly
exposed.
 Cold Wax - the blocks on a cold plate
or a cold wet surface for a few minutes.
o The sections are then floated out
on a water bath set at 45-50°C,
approximately 6-10°C lower
than the melting point of the
wax used for embedding the
tissue.
o Sections are very easily
damaged when dislodging
wrinkles or bubbles with brush
or forceps.
o Leave the section on the water
surface just long enough for it to
flatten.
o NOTE: Overexpansion can
spoil the morphology in
susceptible sections.
 Flotation - use lint-free Kleenex or
Kim-wipe to thoroughly wipe clean the
surface of the water and the edges of the
flotation bath to prevent floaters or
cross-contamination.
o It usually pose problems in
tissue processing.
2. Celloidin Sections- embedded tissues
which are usually cut by sliding
microtome.
 Celloidin can be purchased in
solution or dump in the liquid to
reduce flammability.
 Celloidin is used in form of
solution, usually in a 1:1
mixture of ethanol-ether at
concentrations of 2%, 4% and
8%. Our ether is lipid solvent,
can remove from fat tissue.
 Celloidin sections do not come
off in ribbons and tend to roll up
during cutting and moistening
the block and sectioning in
alcohol by means of camel hair
brush. It will serve as flatten
section at a time.
3. Frozen Sections- cut tissues that have
been fixed and frozen with CO2 or
frozen cryostat
 HISTOPATHOLOGY
CUTTING PROCESS
PARAFFIN SECTIONS
 Trimming –is a procedure of the
excess wax is cut off from the block
to expose the tissue surface in
preparation for actual cutting.
Note: Tissue blocks are trimmed until
perfectly level and all sides are parallel,
almost to the edge of tissue.
4-60 micrometers block surface taking
appropriate cuts.
Coarse facing should be done on the
microtome at approximately 30 microns until
the entire tissue surface is exposed.
COARSE TRIMMING
• -heated spatula is held between the
tissue block and the block holder until
the wax begins to melt.
-placing blocks in a freezer can cause surface
cracking, where the friable tissue separates
from the surrounding wax cohesive sections
become difficult to obtain.
Coarse facing should be done on the
microtome at approximately 30 microns until
the entire tissue surface is exposed.
FINE TRIMMING
-may be done by either setting the thickness
adjuster at 15mm .The block is advance into
the knife and cutting is continued until
complete sections come out of the block and a
regular rhythm is maintained.
-sections are cut between 4-6 micrometer in
thickness for routine histologic procedures
after the block has been fixed and secured to
the block holder.
-provides better support for the harder
elements in a specimen allowing thinner
sections to be obtained. Important to over-
dehydrated ,dry or crumbly tissues.
-
COLD WAX
 may be done by either setting the
thickness adjuster at 15mm .The
block is advance into the knife and
cutting is continued until complete
sections come out of the block and a
regular rhythm is maintained.
 sections are cut between 4-6
micrometer in thickness for routine
histologic procedures after the block
has been fixed and secured to the
block holder.
FLOATATION
 the temperature will need to be 5-9
degrees below the melting point of
the wax.
 make sure the water is clean and free
of bubbles.
 slides must be grease-and dust free
and stored and handled correctly.
 Molecular grade water must be for
floating sections for RNA extraction.
FAULTS/PROBLEMS OBSERVED
DURING SECTION-CUTTING
• Mostly due to some faults in the
technique or cutting itself.
CELLOIDIN EMBEDDING
• -is a slow process, usually taking
weeks and does not produce sections
as thin as those produced by paraffin
embedding.
 HISTOPATHOLOGY
CUTTING PROCESS

• -used to form a solution, usually in a

1:1 mixture of ethanol-ether at
concentrations of 2%, 4% and 8%.
Advantages: It completely avoids the use of
heat at any stage.
Disadvantages: longer time to cut , the
thickness of the sections, the necessity for
staining to be done on free floating section.
After cutting the sections, they are
immediately collected into 70 % alcohol
instead of being mounted on to glass slides.
They are then stored in the same solution in
jars with tightly fitting lids, and finally
mounted on to slides after they have been
stained.
HISTOPATHOLOGY
CUTTING PROCESS
HISTOPATHOLOGY
CUTTING PROCESS