A method for preparation of frozen sections
Start with a sharp blade
I find that I always get my best quality section with a new
sharp blade. I think some of the places we try to save money in
medicine are a bit "pound foolish". Your patients surgery is
costing thousands of dollars. Hundreds of dollars are spent on disposables
including lap pads, gloves, sponges, drapes, cautery, needles, needle magnets, BP
cuff, IV tubing, ……..
We are conserving pennies on what may be the most important decision
impacting on the procedure. Yet some will risk quality by trying to get
"20 shaves" out of a disposable blade.
In my practice I treat every patient to a new
section of blade. I will change it as soon as my section quality begins to
fall. Some tissues such as tough collagenous tissues or calcified tissues
will quickly dull the blade. If I'm having trouble getting a good section
with a new blade on occasion I have changed to a second new blade and
easily prepared a quality section.
If you cut yourself on a blade used on only one
patient, you will have minimized your risk of transmittable disease. If
you cut yourself on a blade that has been used for days, it is like
sleeping with numerous partners......without the fun! In this day and age
of doing FNA's with ridiculously flexible long safety needles and using
annoying safety scalpels, we can justify using a sharp blade for safety
reasons......and get the benefit of
awesome frozens every time!
Sitting or standing
always sit on a stool when I cut. I am hoping you learn to use the brush
as an articulate fine instrument, capable of the delicately maneuvering of
a microscopically thin snowflake of tissue while in flight!
would you want to do this hunched over with you neck hyper extended? This
position is fine if you are bending over to look in a hole in fear of an
animal jumping out at you! But for cutting a frozen section you want to be
relaxed and comfortable so that you will have maximum control in your left
I’m a brush user. I believe everyone must first learn to be good
with a brush. I consider starting a student on the anti-roll devise
like putting a child on crutches before they learn to walk. The
purpose of the brush is to grab and maneuver the section across the
stage. The unless you have perfect temperature, a cold section will by nature trying to curl up and pull away from
the brush. For this reason I use a brush with stiff bristles and a
fairly wide gripping surface. I have found Chinese boar
bristles to be the stiffest and work the best for me. You can buy and 1/4
inch #2 flat or bright brushes from an art supply store for about $3
and cut them at an angle. With this angled tip, the brush
meets the tissue flat like a broom because the brush is held at an
angle. I never understood why anyone would want to use the flimsy
camel hair brushes. The section can easily pull away from these
I am now making these brushes
availablefor anyone who would like to try them. See
Holding the brush
Hold the brush like a pen in
the left hand and stabilize the hand by gently resting the side of the
fifth finger on the stage (or where ever you can find a place
depending on your hand size and cryostat). This gives the operator great dexterity and
allows for conservation of movement. Focus on developing your
dexterity so you can control the brush like a fine instrument. Could
you catch a snowflake as it is falling? I cut the brush at an angle
which approximates the angle I hold the brush in my hand. This results
in the brush meeting the tissue flat over its 1/4 '' length.
Turning the wheel
the wheel in a continuous uniform motion without hesitation. I have
seen many frozen sectionists using a brush stop at the beginning of the
section, slowly grab the tissue and then start to turn the wheel. In my
experience this practice adds to potential artifacts at the beginning of
the section, potential variations in thickness, and leads to difficulties
when approaching tissues containing fat. With practice, by holding the
brush as I described, the operator is capable of grabbing the tissue in a
continuous motion, which began before the tissue meets the knife and
continues through the complete section. .
Movement of the brush
As the block begins to move toward the knife the brush moves downward in pace with the block. The brush can gently
rest on the bottom 2mm of the block and "ride the block" pulling away just as the block meets
the knife. It is the downward movement of the brush that allows you to
keep a continuous motion as you grab the section.
It is like handing off a baton in a relay race. The second runner must
run along side the first runner for the handoff. The baton never slows
down. If the second runner was stopped the first runner would have to slow
to a stop and the second runner would have to accelerate with the baton
As the first few
millimeters of the section passes the knife there will be some degree of
curling of the section. As the curl begins, the brush in motion will catch
the edge of the tissue and change to a horizontal motion toward you
. The path of the brush is an "elbow" shape down and
then toward you in a continuous motion. It is important to pull the
tissue toward you rather than to press it to the cryostat stage. Pressing
tissue to the cryostat stage sometimes result in adhesion of the tissue to
the stage, especially if the tissue is fatty. This will result in a
smeared section and a need to clean the stage. This motion of
grabbing and guiding the tissue is like pulling a blanket over you in bed. As the
brush returns to grab the next section it completes a continuous
elliptical motion. The continuous repeated sectioning of a block
becomes like turning the pedals of a bicycle. Both hands are circling in
blocks prepared with a "handle" of embedding medium makes this job easier
without having to engage the tissue with the brush.
1) As the block descends toward the brush
the brush keeps pace with the block by gently resting on the bottom
2-3 mm of the block and
“Riding the block”
2) As the block meets the blade and the sections
begins it’s curl the brush leaves the block while catching the curling
edge of the section.
"Catching the curl"
3) The brush jumps off the block with the curl.
"The brush jumps over
3) The brush holding the curl pulls the section horizontally over
the stage like a pulling the covers over you in bed without pressing
the tissue to the stage.
"Pull over the blanket"
Tissue can be picked up from the cryostat stage or from the block. I
routinely pick sections up from the stage. When the section is complete
the tissue can be picked up by holding the slide just above the section
and angle the slide down to touch a portion of the tissue. Static
attraction will draw the section to adhere to and quickly melt on to the
warm slide Having a few millimeters "handle" of embedding medium
surrounding the tissue is an advantage. This extra medium allows a margin
of error for curling or flipping at either end before it involves the
tissue. If I am having particular difficulties with the section I can stop
the section before the last 2 mm. of embedding medium leaving
the section attached to the block This allows a fixed edge of the section
against which to stretch the section with the brush. Occasionally when
faced with a difficult situation I may have more luck retrieving the
section from the block. This can sometimes offer a solution to problems
arising from curling or fat sticking to the stage. Some operators prefer
this technique for the majority of their sections. To retrieve from the
block the tissue is cut through and stopped when the handle of medium on
the far side of the tissue is reached. At this point the crank is moved
backward and the block is reversed away from the knife. The section is
uncurled downward with the brush over the face of the block and the
section is picked up off the block face rather than the stage .
Retrieving from stage
levers down to gently touch the section which will float onto
the slide with static or cohesive attraction. Try avoid stretching or
folding the section during this process by keeping the a steady hand
and the transverse axis of the slide parallel to the section.
Retrieving from the block
section is cut leaving an attachment of medium at the top
2) The wheel is turned in
opposite direction bring the section back to the face of the block.
3) Section is retrieved by
placing the slide over the tissue on the face of the block.
As I mentioned earlier I hold the slide in my right hand as I turn the
wheel. The moment the section is complete, I immediately pick up the
tissue on the slide and in a moment it is placed into fixative.
Have your fixative opened in an immediately reachable location. Start with
the slide in your hand. If am using a staining rack I keep it
outside the fixative jar so it does not impede my swift motion. I first
fix the slide in 95% ETOH then put it in the rack
If there is delay in fixing the tissue there will be
significant drying artifact. In my experience when the frozen section is
sitting cold on the stage the effect of drying is minimal. From the time
the tissue touches a warm slide it starts to under go significant drying
artifact with loss of nuclear detail and leakage of fluids from the
cytoplasm. The examples below show the same tissue after 15 seconds
delay of fixation on a warm slide and sections fixed immediately . The
differences are striking. It also demonstrates the quality of cytology
possible by frozen section using this system.
Carcinoma - 15 seconds drying
2) Same tissue immediately fixed 95% ETOH
1) Kidney tubules -15
2) Same tissue immediately
fixed in 95% ETOH
Thickness of the section
For general surgical pathology I recommend
cutting at six microns. This thickness will give a
rich stain which is easier to interpret at scanning powers of 2x and 4x
where pathologists gather much of their information. Very thin sections
will often look pale at these powers and fine details are easy to miss. A
six micron section will afford a moment more time to avoid drying
artifact. There will also be less nuclear “holes” visible from ice
Specialized situations may call for thinner or thicker sections.
Thickness must be confirmed visually. In FS technique III I will show
sections of various thicknesses. In my experience cryostats will not
repeatedly cut perfect six micron sections unless all conditions are
correct and the sections are being cut repeatedly in uniform motion. The
change of surface temperature of the block resting between sections will
result in warming and expansion and a thicker section will be cut. This is
often followed by a very thin section.
When warmed with the hand the first section will often be thicker. When
cutting I always let the first two sections pass then continue on to take to
the next section if it appears to be the correct thickness. This is
another reason why we want to become skilled with the brush so that we can
continuously cut the sections until we are satisfied that we have a
section of the correct thickness without other artifacts.
Individual staining recipes are a matter of pathologist preference. My
only advice is not to rush any step of the staining process and to keep
all stains and solutions fresh and well maintained. Gentle agitation is
helpful in speeding the process and keeping the staining uniform, but the
type of tissue and its adhesive nature should be considered (see below).
When staining I find
that looking at the slide after it leaves the bluing agent is a good way
to access the adequacy of the stain.
Get to know the color and
shade of a well stained slide as compared to a lightly stained slide. In
our hematoxolin I look for a specific navy blue tone to tell me it is
stained well. Keep in mind the thickness of the tissue and the amount of
nuclear material will make the slide appear lighter or darker. However the
is a particular tone of blue that tells me the slide is well stained. Make
your own observations. The idea is to check the slide before continuing on
with the staining.
I like many pathologists
make the large part of my observations at scanning powers i.e. 2x or 4x
magnification. At these powers looking at an under stained slide is like
driving in a snowstorm, you really don't see much. When looking at lymph
nodes for metastatic disease I give particular attention to deep rich
staining, especially the eosin. If the eosin stain is rich, the the pale
pink cytoplasm of a sinus histiocyte can be more easily distinguished from
the tumor cell cytoplasm which may be more eosinophilic or clear more clear.
Why did my tissue fall off?
I'm not sure what the scientific
explanation for the adhesion of tissue to the glass slide but I would
guess it has something to do with weak bonding at a molecular level
conveyed by the fluids in the fresh tissue to the glass. Anyone who knows
the explanation please let me know. I arrived at this explanation because
in my experience the dryer the
tissue tissue the less tendency it has to adhere and if it has been in
formalin it seems to have had any tendency to adhere "washed away"; the
"juicier" tissues adhere better. I like to think of them as
having "more glue" In
my experience I can list several situations where I have experienced
sections coming off the slide in the staining process. Obviously over
agitating loosely held tissues will shake then off.
1) Very dry tissues either by nature or
2) Large ratio of perimeter to area.
Thin strips that have a large perimeter to catch the turbulence of the
motion in the stain jars can easily fall off the slide. This is especially
true if thin fibrous walled cysts which are not very "juicy" tissues to
begin with. Also includes in this are amorphous necrotic tissues which
have no integrity holding them together. This also applies to very thick sections which have a thicker wall to grab the turbulence.
3) Ammonia bluing reagent is too
concentrated.-If you use ammonia for
bluing my rule is if I can smell it without putting my nose up too
it its too strong.
100 % Etoh instead of 95%. I have on occasion
had someone place 100% Etoh in my fixing jar instead of 95%. In my
experience tissue will not stay on the slide.
5) A section is placed over embedding
which is already on the slide. When placing
multiple sections on a slide be careful to not overlap the tissue onto the
embedding medium of the neighboring section.