Temperature of the block
There is an ideal temperature for cutting the frozen section block. After years of research I find this to be exactly in between too cold and too warm. This is the magical temperature when the section cuts such that it flows over the knife in a smooth uniform sheet with minimal curling. At this perfect combination of temperature , tissue type and a sharp blade sections can float off without even using a brush. A block that is too cold will quickly curl in an unmanageable way or shatter creating a Venetian blind like artifact. If the tissue you're cutting is very tough when it is very cold it will be tougher and further stress the system, resulting in thick and thin sections or chatter. When the block is too warm it will bunch in a crumpled pile.
To warm a cold block many operators including myself will place the thumb on the face of the block. Just bare in mind that many an operator has also cut his finger on the knife blade this way. When using any part of the hand to touch the block, always turn the crank to bring the block as far from the blade as possible and be very careful. You should even have the wheel locked or be well aware that if you apply downward motion to the block it may take your thumb on a ride to meet the blade!!.
I find I must watch new residents like a hawk at the beginning of all stages the frozen section process. They will do some pretty scary things around the knife blade and with a scalpel for that matter.
To cool a warm block using the apparatus in the Precision Cryoembedding System, simply press the over-chuck freezing block to the block face for a few seconds. In most cryostats there is some kind of heat extractor that can be used in a similar way. There are freezing sprays available to rapidly cool blocks. These can be useful for super-cooling fat. Be careful to avoid breathing these sprays or any shavings that may be aerosolized in the cryostat. If you have a snowstorm in your cryostat these sprays will turn it into a blizzard!
My usual routine is to trim the block. I then try a section. If it needs warming I place the thumb on the tissue for about two seconds. I then take two a quick turns of the crank, brush those sections away and pick up the next section on the slide. I find the first section after warming is will usually be too thick often followed by one that is too thin.. If you follow this routine please know your cryostat and learn how to do this and avoid the blade.
Looking at the section
This refers to looking at the actual section as it flows over the knife. If you can tell a good section from a bad section at this stage you will know to go back for another cut. This is better than finding out you have a miserable section when you're reading the slide.
1) Too thick , too thin or just right
If your lucky enough to have a cryostat that always cuts the exact thickness no matter what the state or type of tissue or what the condition of the cryostat then skip this section. For the rest of us who find a variety of tissue thickness magically appearing despite the cryostat thickness setting it's important to recognize the thickness of the section. A section that is too thin will have a more translucent lens paper like quality. A section that is too thick will look almost opaque and seem a bit less flexible. To my eye a 5 or 6 micron section looks about like a thin printer paper.
Three microns thick
Six microns thick
Ten microns thick
This artifact is often a result of the block being to cold. It can be dealt with by warming the block. Tissues with high water content have greater tendency to shatter and must be the 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.
This kidney tumor is a good example of water content causing shattering. The tumor tissue on the left is shattering while the benign kidney on the right is cutting without shattering. I believe the tumor has a higher water content and becomes harder and icier when frozen at the same temperature.
Notice the regular periodicity of the shattering. I believe the shattering process can be likened to a piece of wet cloth which has frozen. If one pushed a wedge ( blade) under the cloth it would bend. The icy sheet which forms would break periodically as the wedge pushed under it. Each successive break would represent the ice passing its limit of flexibility. You can only bend a sheet of glass so far without breaking it.
The shattering is obvious in the three pictures in the top row.
Shattering is very subtle in the pictures in the bottom row. You have to look very closely to see this shattering. You may be able to feel it or hear it as its cutting if you pay close attention.
3)Stripes on the section
Nicks on the blade.
Thin stripes perpendicular to the blade can be a sign of nicks on the blade. This can result from cutting tissues with calcification, suture or staples.
Tissue adhering to the underside of the blade
Wider stripes, tearing or mysterious sudden difficulty cutting the tissue can be a result of tissue adhering to the underside of the blade. This is common in fatty tissues. The blade will have to be removed and wiped when this happens. Be extremely careful when wiping the blade. Always try to wipe perpendicular to and away from the edge of the blade to avoid being cut.
4) Wavy lines "Chatter"
This is a sign of movement in the system and possibly a cry for servicing. I see these fine regularly spaced lines in my cryostat when there is movement in the blade because there is movement in the stage which holds the blade. To achieve a good clean section there must be absolutely no extraneous movement in any part of the mechanism that holds the chuck or the blade. All knobs, screws and levers securing of the knife, knife holder, chuck, chuck holder and microtome must always be tight and free of any debris that could cause movement. A simple bit of embedding medium on the back of the chuck, or beneath the blade can cause drastic changes in the cutting of the block.
There must be no movement in the system
The blade and every thing holding it must be tight.
-The blade must be completely inserted to proper depth.
-Blade holder must clamp tight.
-Blade angle must be correct.
All clamping knobs holding knife holder and stage must be tight.
The specimen and everything holding it must be tight.
-Specimen must be secured to the chuck
-Chuck must be clamped tight.
-X-Y adjustment knob clamped tight
-Blade and chuck should be free of medium which could cause rocking or movement
Wavy lines "Chatter"
Wrestling the fat one!
Fat is without question the arch nemesis of the frozen sectionist. It is a simple matter of nature that fat will not freeze. It can be made hard enough to cut thin sections at very low temperatures, but these temperatures are too cold for obtaining good sections of the non fatty tissues that are often present in the same sample. The problem will become obvious when the fat begins to smear and prevents cutting of any tissue in its path. I have had moderate success answering most of my questions in fat containing tissues using the following techniques.
1) Whenever possible dissect off any unnecessary fat from the tissues.
When I examine lymph nodes I am very meticulous about removing all of the fat from the surface and medulla if present. By pulling and scraping the fat off of the node using a scalpel and then slicing the fat away at the capsule line I am able to remove the fat without incising the lymph node capsule. There are those that will say I have removed tissue that may make me miss a positive node. It is my rational that I have a much better chance of finding a positive node with a good clean section than of a smeared miserable section containing a lot of fat.
The Fat Gouge Trick You can even remove fat after you have started to trim the block. A frequent problem in sentinel lymph node frozens. If an area of fat appears in the section as you are trimming down and is preventing you from a clean section see you can remove the fat using this technique. Described under "Plastering" below.
2) Orient the tissue so that fat hits the blade last or by itself as I described above.
Avoid having the fat hit the blade first if possible. The fat will pull away from the embedding medium and start a hole, If a little non fatty tissue is next to the embedding medium it gives it a little better bind to the medium and a better start to the section. As I said earlier, if fat must hit the blade first it should have a handle of embedding medium
to get the section started. Otherwise the fat will immediately smear on the stage and ruin the section.
3) Start with a clean stage and a very cold block.
The stage and blade must be clean. If you smear a section it must be cleaned again with a dry gauze.. Be very careful of the blade. If you see streaks in the tissue or it begins to bunch there may be tissue stuck to the underside of the blade. Remove it and clean it very carefully.
The colder the block the easier to cut fat. Unfortunately the non fatty tissues will become harder and more difficult to cut. Watery tissues will shatter. It may be worth combining sections taken colder to optimize the fatty portion and somewhat warmer to optimize the non fatty portion. By following the technique I have outlined I am usually able to get a satisfactory exam by cooling it to the -24C temp of the well bars. Freezing sprays can be useful but beware of a snowstorm in a filthy cryostat!
4) Here is where the clean swift turn of the wheel without hesitation and a sharp blade is most important.
If the tissue is a mixture of fat and connective tissues it will cut better than pure fat. Sometimes surprisingly well if you follow all of the techniques I have mentioned. Catch the edge of the tissue in motion and quickly and pull the section across the stage with the brush. Do not press the brush and tissue against the stage. It will stick and you won't be able to cut anything until you clean it. A good sharp blade is imperative. If you can make this swift cut you will be able to get reasonable sections of some fairly fatty tissues. The fat may appear as large empty spaces but the fibrous strands between them will be cut well. In that piece of breast tumor and margin where there is a few mm of fat which is covered by ink if you orient the fatty margin perpendicular to the blade, the tumor will cut fine, the fat will cut to varying degree and leave some empty space but there will often be a line of ink to indicate the position of the margin. A good swift clean section will show the strands of fibrous tissue that extend to the margin. I have found carcinoma in a fatty margin this way on many occasions.
1) Breast tissue with inked fatty margin.
2) Orient tissue so margin hits perpendicular and the fat hits the blade alone.
3) A smooth even turn of the wheel results in a clean section.
4) Here there are holes left where pure fat was present, yet the section is intact and the margin is interpretable.
5) Try a thick section in very fatty tissues
Taking thicker sections can be a good adjunct to reading your best attempt at a thin section. Thicker sections can be made by in a variety of ways. On my automated cryostat I first try with a single press of the fine advance button. If conservation of tissue is not an issue I may try a press of the course advance this will produce a very thick section. You can also adjust the section thickness dial or take a double click of the wheel by cranking forward a quarter turn, then backward , and again forward. This variety of maneuvers will produce a range of section thickness. If you then turn the wheel in a continuous very deliberate fashion while using your best brush technique, you can end up with a slice of butter to pick up on the slide. Follow all the steps in fixing and staining but double or triple the times depending on the thickness. Be very gentle with the tissue in the solutions. No vigorous movements. For all this effort you will be rewarded with an amazing fairly well stained three dimensional section which you can actually see through because the fat is so transparent (Figure 16).You will see tiny capillaries coursing in all directions. I have also had luck recognizing structures in the non fatty portions. This type of preparation of a margin will contain the entire tissue face and can help interpret the thin section taken from a fatty margin where some of the tissue has been lost. Highly malignant nuclei and necrosis of a comedo carinoma are still recognizable in these preparations. But realize if this is precious tissue you will be using it up quickly if you take more than two or three tries. Practice with a piece of fat before trying that breast margin.
1) Thick section of fatty tissue. Relationships and architecture preserved.20 x
2) Thick section of fatty tissue 200x. Can read through clear fatty cells.
3) Duct carcinoma in situ. Thick but still interpretable in conjunction with thin section above.
Plastering Technique Basic block repair
Plastering is a very simple and rapid way to address problems caused by defects in the block. In the section on details in face down embedding I discussed the first example below. When embedding tissues "super flat" with a very thin film of embedding medium on the dispensing slide, there will be a slight retraction of medium away from the tissue. If one needs to take a section with minimal trimming ( in the first few hundred microns) the retraction space must be "plastered" with medium. If not, when the section is cut the tissue will separate from the medium making it difficult to get an intact section with out having the tissue "curl away" from the medium. This simple maneuver takes only a second and results in a neatly filled block face. This is illustrated in the first example.
Plastering "super flat" embedded specimen
1) Close up of a flat embedded block showing slight retraction of medium around the tissue.
2) Place a drop of embedding medium on the chuck face.
3) Press the block face to a flat freezing surface such as cryostat stage or any of the freezing apparatus.
4) Remove the chuck with a tap of the over chuck freezing block.
5) Trimmed block with defects filled.
Plastering can be used to repair defects in blocks in a variety of common predicaments.
Removing a staple
In our practice we frequently get a variety of specimens riddled with staples. Every now and then one gets a way in the gross and ends up in a block. It will make a real mess of the knife blade and your section will be split in two. Here is an easy way to deal with them.
1) Staple in the tissue. This is a mock up using a much larger paper staple. The technique is the same. Remove the blade while performing any of these maneuvers.
2) Grab hold of the staple with a forceps or hemostat.
3) Rotate the hemostat and pull the staple out by rolling the staple on the tip of the hemostat.
4) A drop of embedding medium is placed on the defect. Embedding medium can be applied directly from the bottle or with a slide.
5) Press the over chuck freezing block against the block face.
6) The repaired block before and after trimming.
7) The repaired block before and after trimming.
Removing a suture
Similar to removing a staple. The black silk suture shown here will not do too much damage to the section and may not need this procedure. The chromic and proline sutures can cause the section to split in which case it is worth pulling them.
Same basic technique as above. First remove the blade. The hemostat is best for grabbing the suture.
This technique can be used to remove tissues which are not needed and are preventing us from obtaining a high quality section. The best example is fat interfering with the cutting of the section. This is a common problem in lymph nodes removed to evaluate for metastatic tumor. If fat hits the blade in front of the more manageable tissues you can first try rotating the block. If this is not a solution we can "gouge out" the fat or unwanted tissue and plaster over it. Remove the blade first.
The sample is a piece of skin and subcutis rolled in a circle. Fat is in the center. When cutting the tissue (picture 2) there is a central hole. Picture 3 shows "gouging out" of the fat in the center. Here I am using a home made spatula. Any DULL implement with a narrow tip can be used. A narrow tipped spatula or butter knife or would work fine.
For very precise gouging it may be easier to scrape the unwanted tissue away with the chuck in your hand and using a finer scraping tool. Just be careful not to "gouge" your hand!
Plaster the defect as above (picture 4). Picture 5 show the repaired block. Notice I am using two hands to best steady the implement while gouging.
Thoughts on the conservation of tissue.
When asked to perform a frozen section on a small sample the pathologist is often faced with the decision of how much tissue to use for the frozen section and how much to save for permanent sections. I take a very practical approach to this situation.
The decision is based on the reason for the frozen section.
Decision dictates the course of surgery
I will make every attempt to provide the necessary information while preserving some unused tissue. If it is a diagnosis that can be made on an H & E preparation I will use every possible measure to prepare a section of the quality of a permanent section. I am never hesitant to ask the surgeon for more tissue. If it means doing something risky they will usually not go further unless the circumstances are extreme. They may seem a bit cranky when asking for more tissue but the are a lot crankier the next day when you tell them that you do not have diagnostic tissue. We are doing them a service by assuring that they have an adequate sample to justify their surgery.
Surgery was performed is to provide diagnostic tissue
I will examine the least amount of tissue necessary to assure that the surgeon has provided diagnostic tissue. If my initial frozen section does not yield this information I will examine more and ask for more if necessary. If at anytime the surgeon tells me he cannot get more tissue I will examine only what I need to decide further disposition of the tissue such as flow cytometry, snap freezing a sample, culturing, or genetic studies.
Experience has taught me another cold fact. If I have sampled the tissues carefully and the specimen was insufficient for diagnosis on frozen section, it very often will be insufficient on permanent sections. I try not to leave the frozen section suite without a good sample of diagnostic tissue.
Touch preps, crush preps and smears.
These simple techniques provide extremely useful information to contrast with the findings in the frozen section particularly when examining neoplastic tissues. Smears provide a dimension of information that cannot be seen in sectioned tissue either on permanent or frozen section. Smears tell us something about the cohesive nature of the tissue. A lymphoma will dissociate into single cells where epithelial tissues will demonstrate cohesion. Some tumor cells such as small cell carcinomas demonstrate great nuclear fragility. Some tumors have delicate cytoplasm and yield naked nuclei on smearing. Connective tissue tumors and glial tumors will demonstrate important clues in the fibrillarity of the cytoplasm. By air drying slides and performing the diff quick stain a entire new compendium of cytoplasmic details appear including vacuoles, cytoplasmic and extracellular mucins and colloid become apparent. These preparations also provide the nuclear detail to fill any void that may be obscured in the frozen section.