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         Frozen section technique             

 

A method for preparation of frozen sections

The basics                                    

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. 

Safety Tip   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

I 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! Why 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 hand.

   The Brush 

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 hold on to 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 flexible hairs.

I am now making these brushes available for anyone who would like to try them. See apparatus.

 

   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.
 

 

 

 

 

 

 

Body Position

I recommend sitting as comfortable as possible when cutting frozen sections. I am hoping you learn to use the brush as an articulate fine instrument. Why would we want to do this hunched over with our neck hyper extended?  When cutting a frozen section we need to be relaxed and comfortable in order to have maximum control of the left hand. I suggest sitting on an adjustable stool, at a height that allows your arms to most comfortably drape to the stage, best allowing your left hand to assume the hand position discussed below. 

 

Turning the wheel

Turn 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 again.

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 gently lift up over the blade and  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. I like to compare the motion to a child coming down a slide. 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 synchrony.

Having blocks prepared with a "handle" of embedding medium makes this job easier without having to engage the tissue with the brush.  The handle provides margin of error when both cutting and retrieving the tissue. When cutting the block we can grasp this handle with the brush without having to engage the tissue. In sections having a tendency to form holes such as fatty and necrotic tissues, the handle will hold the section together and resist crumpling and tearing. Blocks made with a handle of embedding medium will have fewer tendencies to smear if this handle is pressed to the stage.

 

 

1) Ride the block - 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

2)  Gently lift up while crossing the blade - As the brush and block meet the blade, the section will begin to form under the brush. The brush gently lifts up as it reaches the blade while holding onto the edge of the newly forming section.

3) Hold onto the curl - 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 moving brush is in position on top of this curl of the tissue and is holding onto it. With the curl in hand, the brush changes to a horizontal motion toward you.   The path of the brush is an "elbow" shape down and then toward you in a continuous motion. The motion is much like a child coming down a slide.

4) Glide the section across the stage - The motion continues horizontally as the forming section is drawn across the stage. Figure 4c illustrates that by being extremely delicate with the brush; one can hold onto the edge of the curl and glide the section across the stage. Avoid pressing tissue to the cryostat stage which can result in adhesion of the tissue to the stage, especially in fatty tissues. This will result in a smeared section and a need to clean the stage.

Retrieving the section                      

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

 Slide 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

 1) A 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.

 

 Rapid fixation

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.

 1) Bronchiolo-alveolar Carcinoma - 15 seconds drying  

 2) Same tissue immediately fixed 95% ETOH

1) Kidney tubules -15 seconds drying

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 crystal artifact.
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.
 

Staining the sections

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 desiccation. "Less glue"

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.

4) 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 medium 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.

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