EXERCISE 3: Basic Laboratory TISSUE ORIENTATION

Techniques in 1. The tubular tissue (fallopian

Histopathology tube, vas deferens, artery, etc.)
should be placed in such a
manner so that we get a
EXERCISE 4b: Trimming And transverse section with all the
Cutting Of Paraffin layers
Sections
TISSUE ORIENTATION
ROUTINE TISSUE PROCESSING 2. Tissue with epithelial surface
should be placed vertically and
Exercise 4a right angle to the surface so that
we can get all the layers
ROUTINE TISSUE PROCESSING TISSUE ORIENTATION
Specimens have been processed 3. Multiple section of tissue such as
properly and are now prepared for endometrial curetting should be
trimming & sectioning. placed all in central position.
Multiple sections of tissue can also be
PROPERLY EMBEDDED arranged in a line. The 4 samples on the
SPECIMENS left contain lymph nodes, and the 4
TISSUE SIZE samples on the right are the lymph nodes
- An appropriately sized specimen is only.
crucial in producing ribbons. A Both come from the same GIT sites.
larger tissue can allow more
microscopic features to be viewed TISSUE ORIENTATION
under the microscope, but may be 4. Linear long tissue should be
difficult to section placed diagonally.
- Avoid placing the specimen near
the edge of the block in order for TISSUE ORIENTATION
the paraffin wax between sections 5. Muscle biopsy should be placed
to connect together into ribbons. both in longitudinal and
- A recommended size of at least transverse plane
2cm 2 is preferred.
6. Long membranous tissue such as
TISSUE ORIENTATION amniotic membrane should make
as Swiss roll.
- Specimen orientation is the most
critical step in embedding, ORIENTATION: CONSIDERATIONS
because microtomy may ruin Microtomy of bone is much easier if the
incorrectly oriented tissue with the section is embedded diagonally in the
first section cut. block.
- Many sections are large and flat, Bone tissue is hard (especially if not
and so are easily oriented. decalcified). Orienting the tissue
diagonally can spread the contact surface
area of the knife against the tissue.
If orientated in a straight line, the knife
will come into contact with the hard
tissue entirely, increasing the chance of
breaking/tearing the specimen, and
make cutting ribbons harder.
ORIENTATION:
CONSIDERATIONS
The tissue in this block has not been
properly centered.
When the paraffin margin is inadequate
(top of block), ribbons are more difficult
to obtain.
ORIENTATION: CONSIDERATIONS
This block shows incorrect embedding of
multiple pieces.
This type of embedding makes sectioning
more difficult and increases the chances
of the pathologist overlooking 1 of the
pieces.
ORIENTATION: CONSIDERATIONS
When embedding into the paraffin
medium, all pieces of tissue must be
pressed down gently to ensure they are
of the same level.
The 2 pieces of tissue were not
embedded flat and at the same level in
this block.
Mild pressure should be placed on tissue
so that it will be embedded flat.
TRIMMING
Trimming is also known as “facing”, or
“roughing”.
1. The solidified paraffin tissue block is
removed from the mold and the
accession number is noted.
TRIMMING
2. The sides of the paraffin block is
trimmed down with a scalpel blade
so that the tissue block can fit the
block holder of the microtome.
Please note that all sides must be
parallel to each other
TRIMMING
3. Excess paraffin wax is removed from
the block to partly expose the issue
surface.
The sides, top and bottom of the tissue
block are trimmed until perfectly level
and all sides are parallel, almost to the
edge of the tissue.
The goal of properly trimming a block is
to conservatively expose the tissue down
to a level where a representative section
can be obtained.
TRIMMING
Trimming away excess paraffin wax can
produce shorter ribbons, allowing you to
place multiple sections in a single slide.
CUTTING OF SECTIONS IN A ROTARY
MICROTOME
1. Arrange the blocks in a numerical
order on a cooling device to give
both the tissue and the paraffin
similar consistency.
Sectioning is generally improved when
the specimen and the wax are well
matched in hardness.

It is for this reason that most paraffin
blocks must be cold when sections are
cut. The actual method used to chill the
block is important.
CUTTING OF SECTIONS IN A ROTARY
MICROTOME
- Cold wax provides better support
for the harder elements in a
specimen allowing thinner
sections to be obtained.
- The blocks are placed on a cold
plate or a cold wet surface for a
few minutes (surface of melting
ice).
- Water penetrates a small distance
into the block face, swelling the
tissues and making them more
amenable to cutting. This is
particularly important to over-
dehydrated, dry or crumbly
tissues.
CUTTING OF SECTIONS IN A ROTARY
MICROTOME
Prepare the water bath
- 40° - 45° (5°-10° lower than the
melting point of paraffin)
CUTTING OF SECTIONS IN A ROTARY
MICROTOME
2. Clamping the specimen
Orient the cassette/block in the block
holder. The orientation of the specimen
to the blade during the cutting stroke can
affect the ease with which a ribbon can
be obtained and directly influence section
quality.
 Intestine: blade passes through
the mucosa last.
 Cervix: it is better to present a
point of dense tissue to the blade
rather than a straight edge.
 Skin: blade passes through the
epidermis last.
ROTARY MICROTOME
 Currently the most common type
used for both routine and research
laboratories, especially for
sectioning paraffin-embedded
tissues.
 Although the flywheel in many
microtomes can be operated
manually, they are generally
automated or semi-automated.
 Typically, sections are cut between
3-5 μm
ROTARY MICROTOME
MECHANISM:
As the handwheel (1) is rotated, the
tissue block attached to the specimen
holder (2) will rock up-and-down against
the blade held by the knife holder (3).
The specimen holder also moves forward
as more and more of the tissue block is
cut as set by the section thickness via the
digital console (4), or analog mechanism
of the specific microtome.
ROTARY MICROTOME
MECHANISM:
The sections/ribbons formed through
cutting will rest on the knife guard/holder
(3).
Rejected sections can be rolled off into
the waste tray (5). This can be done
using a brush/forceps.
CUTTING OF SECTIONS IN A ROTARY
MICROTOME
3. Clamping the knife/disposable
blade
Insert the knife blade into the holder and
tighten to lock in place.
Be sure that the knife blade is secure to
prevent uneven sectioning.
CUTTING OF SECTIONS IN A ROTARY
MICROTOME
4. Advance the block close to the knife*
Generally, you bring the knife guard
closer to the block, not the other way
around (dependent on the model of
microtome being used. Some microtomes
have knife guards that can be manually
adjustable.
CUTTING OF SECTIONS IN A ROTARY
MICROTOME
* Extending the sample holder/block
holder can cause vibrations during
sectioning, and produce chattering
artifacts in the slide.
CHATTERING ARTIFACTS
Chatter, or microvibration, most often
results from overdehydration during
processing, but may also result from a
dull blade, too much blade tilt, or
cutting too rapidly.
Soaking the faced block with moistened
cotton will help correct this problem if it
is caused by excessive dehydration of
the tissue.
CUTTING OF SECTIONS IN A ROTARY
MICROTOME
5. Select the appropriate tissue section
thickness by adjusting the thickness
scale/ micrometer gauge.
Other factors such as speed of rotation,
clearance angle setting and the condition
of the cutting edge can influence the
actual thickness achieved.
CUTTING OF SECTIONS IN A ROTARY
MICROTOME
6. Adjusting the clearance angle
While the thickness gauge sets thickness
of the section cut, the knife holder sets
the clearance angle.
Clearance angle must be set for optimum
performance
Set the correct clearance angle required
(0 - 15°)
CUTTING OF SECTIONS IN A ROTARY
MICROTOME
 The clearance angle, or blade tilt,
is too slight. This most frequently
results in sections that are missed
or skipped, alternately thick and
thin, wrinkled and jammed, or
lifted from the blade.
 The clearance angle, or blade tilt,
is too great. This frequently
causes chatter, microvibration,
washboarding, or undulations in
sections and may make it
impossible to obtain a ribbon.
CUTTING OF SECTIONS IN A ROTARY of ten to allow easy location of serial
MICROTOME sections.
10. Set aside ribbons of paraffin sections
7. Bring the specimen to the cutting in a wooden tray with their
position by turning the hand wheel. accession numbers.
You may also float them immediately into
 Hard tissues cut best with firm, the water bath to continue
relatively rapid stroke. processing.

 Soft tissues cut best with slow SECTIONING: TROUBLESHOOTING

gentle motion. Compressed, Wrinkled, or Jammed
* Make sure that the blade is parallel to Sections
the tissue block.
TROUBLESHOOTING
CUTTING OF SECTIONS IN A ROTARY CAUSE
TECHNIQUES
MICROTOME
Resharpen the
The entire face of the block was not Use of dull knife/blade
knife or move the
edge
parallel to the blade, so that ribboning existing blade to
was very uneven, and a major part of one an unused surface
side of the block has been cut away. or replace the
blade with a new
one.
The block is not parallel to the blade
Keep the edge free
edge. The top and bottom edges of the Paraffin sticking in
of paraffin by
block should be parallel, and the bottom the back side of the
wiping (up, never
holder
of the block should be parallel to the down) with gauze
blade edge, or crooked ribbons and poor slightly dampened
adherence of one section to the next may in xylene. Remove
excess xylene with
result. another gauze
soaked in absolute
CUTTING OF SECTIONS IN A ROTARY alcohol.
MICROTOME
Too little knife/blade Increase the tilt of
8. Sectioning tilt the blade (greater
As sections are cut, a ribbon is created clearance angle).
because successive sections stick edge- Decrease
Too rapid cutting
to-edge due to local pressure with each cutting
stroke. To ensure proper sectioning and speed (1
revolution
a good ribbon, the wheel should be per
turned clockwise, at a moderate and second) for
even phase. very thin
sections.
CUTTING OF SECTIONS IN A ROTARY Too warm room Cool/change the room
MICROTOME temperature or cool
9. A length of about 6 inches is sufficient the block in ice water
to detach the tissue ribbon from the Unparalleled block Trim the block edges
microtome. This is around ribbons edges until they are parallel.
SECTIONING: TROUBLESHOOTING SECTIONING: TROUBLESHOOTING
Skipping of Sections Scratches, Lines, and Splits

TROUBLESHOOTING CAUSE TROUBLESHOOTING

CAUSE
TECHNIQUES TECHNIQUES
Specimen needs to be Defect or nicks in the Move the blade to an unused
Very large blocks or treated by softening the knife/ blade edge area or change the
blocks with hard block: blade/knife, then cut the
regions that may - Chill the block block.
damage the spring further Change the blade or re-
knife/blade edge while - Soak the block sharpen the knife.
sectioning or materials in a bowl of
not properly hardened soapy water Dirt/calcium, bone or Check the tissue for
other hard particle/ foreign bodies, staples,
material present in the calcium, etc. If present,
Too much tilting of
Adjust the block remove carefully to avoid
the knife/ blade
knife/blade so damaging the tissue.
which scrapes off
that the (Note: If the defect
the section instead
clearance angle remains in exactly the
of cutting it
between the same area of the new
cutting facet of section, then the
the knife/blade problem is the
and specimen is specimen).
correct.
Refilter dirt in paraffin.

Decalcify the tissue.

Too little tilting of the
knife/bladeresulting in Increase the tilt of the
compression of the blade (greater clearance SECTIONING: TROUBLESHOOTING
block on the return angle)
TROUBLESHOOTING
stroke. CAUSE
TECHNIQUES
Too much knife/ Decrease tilt of the
SECTIONING: TROUBLESHOOTING blade tilt knife/blade so it will
Grooved, Scored, Smeared, and cut rather than
Deformed Sections scrape.
Dirty knife/blade Clean the knife/blade
edge edge
TROUBLESHOOTING Tissue too hard Use celloidin-
CAUSE
TECHNIQUES for paraffin embedding materials
or soak in water to
Dull knife/bladeedge Re-sharpen the knife soften
or move existing blade Dirty stock solution Filter the stock
to an unused surfaceor solution
replace blade with a
new one.
SECTIONING: TROUBLESHOOTING
Brittle and Shattered Sections
Room too warm Cool/change the
temperature ofthe
TROUBLESHOOTING
room or cool the CAUSE
TECHNIQUES
block in ice water
Incomplete Rehydrate the block
dehydration or ribbons until the
clearing holes disappear
Incomplete Re-infiltrate tissues Excessive Expose the tissue,
infiltration of soft with paraffin and dehydration or then soak the block
and mushy tissue reembed. improperly in ice water or with a
with embedding processed tissue wet piece of cotton
material before sectioning
Too long in Try chloroform or
paraffin bath or toluene instead of SECTIONING: TROUBLESHOOTING
too hot bath xylene, or a mixture
of toluene and cedar
Washboarding and Undulations in
oil as clearing agents. the Sections
TROUBLESHOOTING
CAUSE
SECTIONING: TROUBLESHOOTING TECHNIQUES
A pleated (a) and folded (b) tissue Worn microtome pants This commonly occurs in
that allow too much very hard tissues such as
section. tolerance in some of the the uterus, or in
TROUBLESHOOTING moving parts overfixed tissues. This is
CAUSE
TECHNIQUES easily seen when sections
Imperfections in the - Trim the are floating on top of
blade edge, i.e., the block parallel the water bath.
horizontal edges (top to the other
and bottom) are not edge or re-
parallel or the lower embed them Loose clamping Make certain that the
block edge is not in molds so of the blade or microtome is in good
parallel to the knife that the edge block working condition and is
edge when sectioning of block is routinely scheduled for
parallel to the maintenance.
knife edge.
Loose screws Tighten all screws.
- Try another Ensure that the block
part of the
holder shaft is not
knife edge.
overextended.
The block is not evenly Ensure that the block Loose knife holder Clamp tightly. The block
chilled or the hardness is evenly chilled and and blade should be
of the paraffin varies hardened. tightly clamped in the
from one side of the
microtome.
block to the other

SECTIONING: TROUBLESHOOTING SECTIONING: TROUBLESHOOTING

Holes in the Sections Poorly processed liver that will not
CAUSE
TROUBLESHOOTING produce a cohesive section.
TECHNIQUES
Block positioned Position the block Indicates under-processing. The tissue is
too aggressively less aggressively with
smaller micrometer
not completely dehydrated and/or
advances of the block cleared and is therefore not properly
for each removed infiltrated (the wax could not penetrate
section due to the presence of water).
Small flecks of If there is sufficient Reprocessing should help.
Tissue removed from tissue in the block,
the block cut and discard
SECTIONING: TROUBLESHOOTING
- Wrong micrometer setting
- Sectioning at too great a speed
- Microtome needs recalibration

SECTIONING: TROUBLESHOOTING
- Temperature of bath too high
- Section left for too long on water
- Poor fixation and/or processing
(residual solvent)

SECTIONING: TROUBLESHOOTING

Compressed sections, as seen in this

figure, may result from a dull blade, too
little blade tilt, paraffin accumulation on
the blade, and cutting too rapidly.

The results of a major nick, or defect, in

the blade edge can be seen in this
paraffin ribbon. This defect possibly
resulted from improper handling of the
forceps used in sectioning.

Any metallic object that is allowed to

come into contact with the blade edge
can damage the edge and cause this type
of artifact.