Embedding of Tissue for Frozen Section

Abstract

Embedding of tissue for frozen sectioning is a vital step in the preparation of tissue for intra-operative consultation. In many cases, this task needs to be performed with great precision or risk suboptimal interpretation and loss of precious tissue. Traditional forms of face up and face down embedding techniques are described along with potential sources of difficulty. The Precision Cryoembedding System, a series of highly precise face down embedding techniques developed by the author is discussed in detail. The system capitalizes on the physical property of tissue to adhere to freezing temperature metal. On face, on edge and on point embedding can be accomplished with ease using a number of approaches. Advantages include high precision, speed, minimal tissue wastage and reduction of freeze artifact. Techniques include face down embedding using freezing temperature steel well bars, paper embedding, cut off technique, plastering techniques and frozen block cryoembedding.

3.1 Traditional Embedding Techniques

Now that we have taken our gross sections our next step is to embed the tissue. As in paraffin embedding, our goal is to embed the tissue in a medium which will fix the tissue to a chuck (or specimen holder) in the optimal position for microscopic examination. Unlike paraffin embedding which requires a lengthy processing step prior to embedding, in preparing slides by frozen section our processing is accomplished simply by freezing the fresh tissue. Traditional processing in paraffin embedding requires sequential infiltrations by formaldehyde, alcohols, xylene and paraffin so that our resulting tissue cuts with the consistency of solidified paraffin. Hardening the tissue to this paraffin-like consistency allows the tissue to cleanly shave into slices in the range of microns. In frozen section, the simple process of freezing the tissue will allow it to shave into slices as thin as a few microns. Fortunately, this processing works well with most tissues but as we will see later in Chap. 4, tissues containing mostly or all fat will remain soft at ideal cutting temperatures creating a host of difficulties.

Embedding tissue for frozen sectioning consists of freezing the tissue in precise position within a block of embedding medium which attaches the tissue to a chuck or specimen stage. Commercially available embedding media are viscous aqueous solutions of polyvinyl alcohol and polyethylene glycol designed to freeze and easily section at optimal cutting temperature. These media adhere well to most tissues and provide a frame that adds stability to the section. The specific position in which the tissue is to be embedded will be dictated by the microanatomy and the optimal plane to examine that tissue under the microscope.

First, let’s consider typical rectangular section of tissue. Such a piece will have two broad faces and four narrow edges. In most cases, embedding is carried out on face or on edge. Figure 3.1 illustrates on face and on edge embedding using face down embedding technique in well bars.

Fig. 3.1

(a) On Face embedding. In this example of face down embedding, the tissue is embedded with its broad face down on the well floor of a well bar. Using face up embedding the broad face is would be up; In both the cases, the broad surface will be visible under the microscope. (b) On-edge embedding. The illustration show the yellow tissue embedded by standing it on its narrower surface. The cross section of the edge will be visible under the microscope

On face refers to embedding the tissue so that one of the two broad sides of the section is the face of the prepared block. On edge refers to embedding the tissue so that one of the four narrow sides or edges of the tissue is the face to be embedded and sectioned. The resulting slide will show a cross section of the tissue block on microscopy. On edge embedding often requires standing the tissue on its edge and can present challenges with small specimens. The orientation will be dictated by the specific tissue, and questions we are hoping to answer.

3.1.1 Face Up Embedding

All cryostats offer a simple system for face up embedding. Typically, tissue is placed face up on the chuck and covered with embedding medium. The chuck is placed on a designated freezing surface or bar in the cryostat. After a period of partial freezing, a weighted heat extractor can be placed on the top of the chuck to flatten the tissue surface into a plane and complete the freezing process Fig. 3.2.

Fig. 3.2

Face up embedding. (a) Tissue is placed on top of a layer of embedding medium a top a chuck. (b) Embedding medium is applied liberally over the chuck surface. (c) The cryostat heat extractor is placed on top of the tissue flattening the surface into a plane. (d) The completed block

When embedding face up, there are several potential sources of difficulty. If the tissue is not uniform in thickness, the embedded surface will not be in a uniform plane parallel to the chuck face. On trimming, one will have to waste considerable tissue before reaching the desired tissue face in its entirety see Fig. 4.7. If multiple fragments of tissue of different thicknesses are placed on the chuck, we will have to trim across the thicker pieces before reaching the thinner pieces again wasting considerable tissue. The weighted heat extractor can serve to flatten the tissue closer to a desired plane. Unfortunately, the plane achieved is often not parallel to the chuck face and may require x–y adjustment to prevent unnecessary loss of tissue.

Some cryotomists will first prepare the chucks by applying a layer of embedding medium and placing on the heat extractor to create a flat surface. This serves to create a more uniformly flat layer than the initial chuck face and allows for a more uniform working surface. This is particularly helpful when handling small biopsies such as core biopsies or trying to stand tissue on edge. Unfortunately, in a very busy practice, it can be hard to keep up with the prepared chucks. It is helpful to have a good supply of chucks if you use this technique in a busy setting Fig. 3.3.

Fig. 3.3

Face up embedding on a chuck with a prepared flattened base of frozen embedding medium. (a) A layer of embedding medium is placed on the chuck. (b) A heat extractor is placed over the embedding medium. (c) The chuck with a flattened layer of embedding medium. (d) Tissue is placed on top of the prepared block face. (e) Embedding medium is applied to the tissue. (f) The completed block

3.1.2 Pre-freezing Tissue

Before arriving at the system of face down embedding to be discussed later in the chapter, I found I was able to improve my results with some tissues by first freezing the specimens as a whole. This was particularly useful in skin specimens. By first lightly freezing the tissues, I was able to cut more precisely flat pieces that were easier to position or stand on edge. In fact, it was this practice that lead to the idea of frozen block cryoembedding.

3.1.3 Face Down Embedding

Face down embedding refers to freezing the tissue face down similar to the way tissues are embedded in paraffin. The desired tissue face is frozen in position face down in a mold or on any freezing surface. The great advantage of face down embedding is that the tissue face which we will be cutting, freezes in a single flat plane. The surface will require less trimming and wastage of tissue before the entire tissue face is available for sectioning.

One traditional method of face down embedding of frozen section tissues has been performed using disposable plastic molds, sometimes referred to as boats . Tissue is placed on the floor of a mold which is then filled with medium. The chuck is then placed over the mold and freezing is accomplished by placing the filled well on the cryobar of the cryostat or by snap freezing in super-cooled liquids such as isopentane immersed in liquid nitrogen. One manufacturer offers cutouts for these plastic molds on their cryostat freezing bar to accommodate these molds. The prepared block will be embedded in a flat plane approximating the floor of the mold Fig. 3.4.

Fig. 3.4

Face down embedding in plastic molds. (a) Tissue placed on the floor of the plastic mold and covered with the embedding medium. (b) A chuck placed on top of the filled mold. (c) The completed block

Face down embedding can also be performed on any flat freezing temperature surface simply by freezing the tissue into position on the flat cold surface covering the tissue with embedding medium and fixing the tissue to a chuck. There are many individuals using variations of this technique. Some will freeze the tissue flat by pressing it against a freezing surface such as a heat extractor and then placing the pre-frozen flattened tissue face up on a chuck. If you are using freezing cold chucks with a particularly good gripping surface, you can simply freeze the tissue on a freezing cold surface, cover it with embedding medium and press on a cold chuck. A sharp tap with a hard object will free the chuck. See Chap. 9 for more on snap freezing.

3.2 Face Down Cryoembedding in Well Bars: The Precision Cryoembedding System

Over the course of a career all cryotomists are faced with embedding tasks that require great precision to even approximate the desired information. Many such cases require accuracy which is simply not possible with conventional methods. Our results are suboptimal more often than we would like to admit. Repeatedly frustrated by seemingly impossible tasks, I was inspired to begin experimenting with techniques to better handle these difficult cases. The result was development of a system of apparatus and techniques to embed tissue face down in wells precision machined into a bar of stainless steel (Peters 2003a). The well bars are kept at freezing temperature in the cryostat or freezer. There is a simple physical principal which gives the well bars nearly unlimited precision. The tissue adheres to the freezing temperature metal. It is common knowledge that if you touch your tongue to a metal flag pole at freezing temperature, it will stick to the pole in a most embarrassing way. It is this same physical property that allows the tissue to easily adhere to the well floor in any position either flat or on edge. Using these methods, tissues freeze significantly faster than conventional methods and require no monitoring during the freezing process. Multiple blocks can be prepared rapidly. The blocks are prepared with the entire tissue face in a precisely flat plane; requiring minimal trimming and resulting in little tissue wastage. It is my hope that this book will help disseminated these techniques which have served so well our practice. Apparatus for the Precision Cryoembedding System can be purchased through Pathology Innovations LLC, Wyckoff NJ www.pathologyinnovations.com.

3.3 Apparatus

3.3.1 Embedding Well Bars

These 1 inch thick bars of stainless steel have wells machined precisely flat into one surface. All machining is performed to a precision of 1/1000th of an inch. These bars are kept at freezing temperature in the cryostat. This substantial piece of steel acts as a heat sink to rapidly draw heat from the tissue. The colder the bars are kept, the faster they will freeze. Between temperatures of approximately –5°C and –40°C the tissue will adhere into place when touched to the well floor. Well bars are available in a variety in sizes and depths to accommodate a wide variety of sample sizes Fig. 3.5a

Fig. 3.5

Apparatus of the Precision Cryoembedding System. (a) Well bars (from top to bottom): 30 mm well bar; 24 mm well bar; 18 mm well bar. (b) Chucks or specimen stages. (c) Over-chuck freezing block. (d) Dispensing slides. (e) The interior of a Leica 1,850 cryostat accessorized for the use with the Precision Cryoembedding System. The embedding shelf (foreground), well bar storage platform (left) and chuck bin (right) are labeled

3.3.2 Chucks

These chucks are made of stainless steel and are designed to be used cold and act as a heat extractor for rapid freezing. The faces are designed with a deep waffle pattern to maximize the gripping power required to hold the embedded tissue block while allowing for extrusion of excess medium. The stem of the chuck provides the focal point for a sharp tap resulting in an easy release of the block from the well. These chucks fit many of the major brand cryostat and can be used in most cryostats with the use of an adaptor. Chucks can also be used warm with the use of the over-chuck freezing block (see below) Fig. 3.5b.

3.3.3 Over-chuck Freezing Blocks

These steel rectangular blocks are tools useful for a number of tasks. They serve as heat extractors, designed to fit over the stem of the chuck. The freezing block also serves as a dislodging tool. A sharp tap of the chuck stem cleaves the plane of adhesion holding the formed block to the well floor. These blocks also function as convenient hand held flat freezing surface useful in gently cooling the tissue without the use of sprays or to rapidly freeze embedding medium during plastering (see Chap. 4). The flat surfaces can also be used as extra heat extractors when using conventional cryostat embedding technique or as a stand for stemmed chucks Fig. 3.5c.

3.3.4 Dispensing Slides

These thin transparent vinyl slides serve as a surface to precisely orient the tissue into the desired position and accurately transfer the tissue to the embedding well floor. Tissue is applied face down to the end of the transparent slide, where it can be visualized from below and manipulated into position. The face that is visible will be laid onto the well floor and will ultimately be the embedded surface to be sectioned Fig. 3.5d.

3.3.5 Embedding Shelf

The removable shelf seen in Fig. 3.5e, can be installed below the opening of the cryostat in the most convenient and ergonomically comfortable location available. If the user chooses not to install a shelf, or if the cryostat does not accommodate a shelf, the embedding well bars can be placed on the brush holder, stage or other convenient place in the cryostat for the embedding process. Bars can be stacked to increase their elevation. Well bars can be removed to the work bench for the short time it takes to embed, but should not be left out for prolonged periods to avoid warming. If the setting calls for repeated or prolonged removal of well bars from the cryostat, it would be best to set the cryostat a bit colder in the range of −27°.

Figure 3.5e shows the inside of one of our Leica 1,850 cryostats with additional accessories which make the cryostat more accommodating. The picture shows the well bar platform which transforms the left side of the cryostat into an open garage to accommodate multiple well bars. A chuck bin is designed to fit into the space on the right side to store and segregate our cold clean chucks.

3.4 Face Down Cryoembedding in Well Bars: Technique

To finish the block, the shallow defects are repaired by a technique I call plastering, shown in figures n, o, and p and discussed in detail in Chap. 4.

Freezing time. It is recommended that well bars be kept at cryostat temperatures of −24° or colder. The bars should be kept at a convenient low point in the cryostat where the temperature will reflect the cryostat setting. As one gets more experienced at estimating freezing times, the freezing time can be shortened by lowering the temperature. I usually keep our cryostats at −27°C, however on very busy days, I have functioned nicely at cryostat temperature as low as −31°. The down side of keeping the cryostat very cold is that we are at the risk of starting with a very cold block far out of range if we do not get the block out on time. As we will learn in Chap. 5, adjusting the block to ideal cutting temperature plays an important role in achieving our best sections. If faced with a block of −31°, one will have to be very diligent in re-warming the block with the thumb or suffer a shattering ride through the tissue.

Approximate freezing times with bars and chucks at −24°C:

• 18-mm well – 20 s

• 24-mm well – 35 s

• 30-mm well – 60 s

When the extruded beads of medium are frozen, the freezing is nearing completion. If the block is removed a bit early, the over-chuck freezing block can be pressed to the tissue face for intervals of a few seconds until the block cuts optimally just beyond the crumple stage . I consider this as the ideal temperature to cut most tissues. Tissue sections will lie flat, without curling or shattering just beyond the temperature of this crumple stage. The cutting behavior of tissues at different temperatures is discussed in Chap. 5. Freezing will take longer at warmer cryostat temperatures; if the bar is allowed to warm; if the chuck is warm; or if the tissue is very thick.

Do not try to trim any block embedded by any technique unless the embedding medium has turned completely white. If there is a grayish cast to block, the medium and tissue are not completely frozen. The medium will not firmly hold the tissue, and when not completely frozen the tissue will not shave. Rather any fibrous elements in the tissue will resist cutting and result in the unfrozen tissue being pulled from the block. This dilemma is known as having the tissue chunked out . Unless the cryostat has been cleaned of all shavings prior to starting, there will be little hope of retrieving the tissue without contamination if at all. This is one situation where meticulous cryostat hygiene can be a savior.

3.5 Face Down Cryoembedding in Well Bars – The Elements in Detail

3.5.1 Use of the Dispensing Slide

These simple transparent vinyl slides provide an easy way to precisely place tissues in the embedding wells. The slides allow precise placement in any orientation to the desired site on the well floor. They also act as a conveyance to carry tissue to the cryostat. These are best kept conveniently in a container or jar next to the cutting board. It is handy to keep an absorbent pad or open paper towel in reach of the cryostat to place the slides on when loaded with a specimen. I call this a launch pad . If the cryostat is a distance from the cutting board it would be useful to have a small tray to place the loaded slides on.

There are two different sized slides. Each with two different sized tips. Use the tip which best accommodates the tissue and well size.

3.5.2 Application of Embedding Medium and Tissue

A thin layer of embedding medium coats the end of the dispensing slide. This coating of medium is an essential step and must not be overlooked. The film of medium will create a clean plane of separation between the tissue and the well floor and will prevent sticking of any tissue residue. We start with a drop of embedding medium at the end of the slide and then gently glide the drop over a paper towel. This produces a thin uniform layer of medium on the dispensing slide which will give us the flattest surface when embedded Fig. 3.5a–c.

Place the tissue at the end of the slide. Orient the tissue so that the most important aspect can be addressed optimally. You can first address a critical margin or particular edge of the tissue by having it touch the well floor first. It may be better to have the critical area such as the epidermis of a thin strip of skin leave the slide longitudinally and slowly pressed into position as in the above pictorial Fig. 3.5d.

The dispensing slides also provide a means of transporting the tissue to the cryostat. The embedding medium provides a degree of adhesion of tissue to the slide and so it will not easily fall off if handled gently. Support the tissue when inverting heavier pieces more than 3 mm. thick.

3.5.3 Super Flat Embedding

Tissues that are very thin need to be embedded in the flattest possible plane and at optimal x–y orientation in order to achieve the entire tissue face in a section without first exhausting any part of the tissue. Examples include thin core biopsy specimens, very thin sheets of tissue, and multiple tiny specimens. To embed tissues in the flattest possible plane, use a very thin and uniform layer of embedding medium on the dispensing slide. A thick and irregular coating of medium will end up as an irregular layer of frozen medium on the face of the tissue. On sectioning the tissue face will be further from our ideal flat plane. For a random chunk of a tumor this is of no consequence, but if you want to section four core biopsies and have all four left for permanent sections, we need to embed the tissues as close to a uniform flat plane as possible. In order to cut very thin tissues in single plane, the block holder must be in the optimal x–y orientation. Adjustment of x–y orientation is discussed in Chaps. 1 and 4.

3.5.4 Looking Through the Dispensing Slide

While looking through the back of the slide, adjust the tissue into the desired position. Make sure the desired surface is visible. Particularly, check that an inked margin surface has not folded under the tissue. If you are doing Mohs surgery, make sure the epidermis is visible and not folded under Fig. 3.6e.

Fig. 3.6

Face down Cryoembedding in Well Bars Preparing the dispensing slide (a)Place a drop of medium at the end of the dispensing slide. (b)Gently glide the end of the slide over a paper towel. (c)A thin uniform layer of medium on the end of the slide is our goal. (d)Place tissue FACE DOWN on the dispensing slide (e)Look through the underside of the slide while adjusting the tissue so that the desired surface is visible in position. (f)Touch the leading edge of the tissue to the well floor. It will adhere. First, consider the best ositioning of tissue in the well. How will it fit best? How do I want the tissue to meet the blade? (g)Pull the dispensing slide out from under the tissue. (h) Tissue requiring precise positioning can be flattened or manipulated and guided into the desired position by carrying out this step slowly and carefully. (i)Fill the well so that a meniscus of medium is bulging above the brim. (j)Press a COLD chuck over the well as quickly as possible. (k)An over chuck freezing block can be placed over the chuck to speed freezing. (l)After a period of freezing, remove the block with a sharp tap to the chuck stem using the over-chuck block. (m)The face of the prepared block. The arrow shows retraction of embedding medium away from the tissue at the block surface which will require plastering in our next step. (n)Apply a drop of embedding medium to the block face. (o)Press the block to the stage (or any freezing bar) of the cryostat for a second. The block can be released with a tap. (p)The finished block is skim coated with medium, filling any defects. (q)The trimmed block with all pieces easily reached in the same plane. (r)Micrograph (20× magnification)

3.5.5 Flimsy Tissues and Friction

When very flimsy loosely adherent membranous tissues are moved along the slide, friction will create a tendency for the flimsy parts to be dragged out of position or run beneath the specimen. If the flimsy aspect is a critical part of the tissue such as a margin of a breast biopsy, position the tissue such that this flimsy aspect is right at the edge of the slide, or have it come off the slide longitudinally and manipulate this flimsy aspect into position. With flimsy tissues, precision can be maximized using paper embedding technique (see below).

3.5.6 Dealing with Multiple Samples

As in all of the complicated multi-step tasks we perform, it is best to create a specific routine to handle multiple specimens and adhere to it. This will help to minimize errors. When using multiple slides for multiple specimens, place them in order on your launch pad so that you will not confuse the specimens. See Fig. 3.7 . The dispensing slides can be labeled with a marker if confusion is a consideration. If you’re working in a small sub-optimal space (like so many of us) it is a good idea to have pre-labeled areas on the launch pad or a labeled tray to place the slides. Always use a separate slide for different specimens to avoid cross contamination. Multiple samples from a large tumor can be placed together on a single slide.

Fig. 3.7

Launch Pad. A paper towel with multiple loaded dispensing slides lined up in order prior to embedding

3.5.7 Use Your Imagination

The dispensing slides allow for a lot of creativity. The thick embedding medium offers support to fold tissues, stand them on edge, or make membrane rolls that stand nicely on edge when pulled off the slide. Try experimenting with different tips on the slides. They can be cut easily with a scissor. You may find it useful to have one with a finer point for tiny biopsies. Using the narrow tipped slides allows a great deal of articulation within the larger wells. Tissues can be neatly arranged or maneuvered in a variety of situations. Figure 3.8 shows how a long strip of tissue can be rolled on the dispensing slide to make a jelly roll.

Fig. 3.8

Jelly Roll on the dispensing slide. (a) A strip of skin about 6 cm long. (b) The skin wound into a jelly roll to be transferred to the well floor. (c) The completed block

3.5.8 Placement of Tissue in the Well

Choose a well that will fit the tissue while leaving space for a rim of embedding medium surrounding the tissue. I will refer to this rim of embedding medium as the handle. The handle gives brush users a leading edge to grab with the brush instead of the tissue. This handle provides a margin of error before the blade meets the tissue so if there is curling and flipping at the beginning of the section it may not involve the tissue. A good handle will act as a frame of frozen medium to support tissues with less structural integrity such as necrotic and fatty tissues helping to prevent the section from smearing or crumbling. Consider how the tissue will fall when it is placed in the well so that you avoid overlapping the tissue on the edge of the well. The dispensing slides make this quite easy. Just look through the slide as it’s placed over the well and you will see if and how it fits Fig. 3.6e.

To begin, touch the leading edge of the tissue to the well floor. It will adhere in place to the floor. See Fig. 3.6f. Next, we slowly pull the dispensing slide out from under the adherent tissue much like pulling a spatula out from under a pizza placed in an oven. We can do this slowly or quickly depending on the precision required. As the tissue leaves the slide, using a forceps, guide the tissue to adhere in the desired position. When performing a highly difficult task it may make it easier to use a larger size well. The extra room in the well allows the dispensing slide to be held at a lower angle making it easier to manipulate the tissues in precise position.

3.5.9 Using Forceps to Embed

Certain tissues can be embedded using forceps alone. Tiny biopsies can be easily placed in position on the well floor. A stiff piece of tissue can be held with a forceps and stood on edge by adhering it to the well floor. If you try to embed flimsy tissues with a forceps, they will be less manageable than with the dispensing slide. When placing large pieces of tissue approaching the size of the well with forceps, you will find the tissue adheres where ever you touch the tissue including places you don’t want it to. Adherent tissue can be pushed off with the forceps when this happens. The dispensing slides make these larger pieces much more manageable.

3.5.10 Standing Tissue on Edge and on Point

Due to the cohesive nature of the freezing temperature steel, it is possible to stand thin tissues on edge or vertically on point. The tissue must be firm enough to support its own weight when standing. Tissue can be held with a forceps, touched to a drop of embedding medium and touched to adhere to the well floor. Similarly, a cylindrical or pyramidal ­portion of tissue can be dipped in embedding medium and stood vertically by touching the wetted tip to the well floor and holding it for a second to freeze in place Fig. 3.9.

Fig. 3.9

Embedding on edge and on point (a) Holding the tissue with forceps, the edge of the tissue which has been wetted with embedding medium is touched and adhered to the well floor. (b) Tissue frozen into position on edge on the well floor. (c) Trimmed frozen section block of on edge section showing both epidermis and margin (inked black). (d) Forceps standing a triangular portion of skin on-point on the well floor. (e) Skin frozen into position. (f) Trimmed block showing a cross section of the skin point

This technique can be used to stand cores up to make microarrays and stand up the pointed “tip margins” in skin biopsies. These tissues must be firm enough to support its weight. Tissues can be made firmer to stand on point by pre-freezing the tissue or taking the sections from frozen tissue. This can be accomplished on any freezing temperature surface.

The still life picture in Fig. 3.10 was created by standing small pieces of flower petals on edge.

Fig. 3.10

Petals. This still life picture is created by standing flower petals and leaves on edge as in Fig. 3.8a

3.5.11 Use of the Flattening Forceps

Our system offers a useful pair of 7 inch angled forceps called flattening forceps. These forceps have a blunted end bent at a 60° angle that functions as a flattening iron. In Fig. 3.11 , this large flat portion of skin can be ironed flat over its broad surface with use of this simple tool.

Fig. 3.11

Ironing a large portion of skin with the flattening forceps. Figure (a) shows a 2 × 2 cm portion of skin at the end of a wide dispensing slide. (b) The leading edge of the tissue is pressed to the well floor with the flattening forceps. (c) As the dispensing slide is pulled out from under the tissue, the bottom of the forceps is used like an iron to press the tissue flat to the well floor as it comes off the dispensing slide. (d) The completed block showing uniformly flat surface

3.5.12 Filling the Well with Medium

The well must be filled to the brim so that the medium bulges above the level of the surface of the bar. The medium must engages the channels in the chuck face in order to solidly bind the chuck. If the well is under filled the chuck will not adhere. Overfilling the well is not a problem as the excess medium will be extruded out the grooves in the chuck.

3.5.13 Parallel Faces

The well bar is machined in a such a manner that the base of the well is parallel to the surface of the bar. When the chuck is placed flush over the well, its surface is parallel to the well base. Therefore, the surface of a perfectly prepared block will have its tissue face parallel to the chuck face. I will use the term parallel faces throughout this text to describe this relationship of tissue face and chuck face. Figure 8.6 illustrates the problems arising if chucks are prepared deviating from having parallel faces in a Mohs surgery specimen. In tissues that are very thin or contain thin structures that can be easily trimmed across, we will be most successful if we maximize this parallel relationship of chuck and chuck face. This is accomplished by applying the chuck flush to the well bar surface as quickly as possible when the well is filled so that the excess medium is extruded before freezing. We also need to fill the well as precisely as possible. When copious amounts of embedding medium overflow the well, there is more likely to be a slight loss of the parallel relationship of the chuck and block face. This parallel relationship can be maximized by using just enough embedding medium to fill the well and form a meniscus that bulges above the surface of the bar. In this situation, there will be minimal extrusion of medium and the chuck can be quickly placed very flush to the surface of the well bar. Using a large chuck on a smaller well will help accomplish this task. This parallel relationship of the block face and chuck face is most important if the chuck holder is stationary. As you develop a sense for filling the wells just right, and have a precisely adjusted cryostat, all of your chucks will approach the blade at the same angle. Trimming will be minimal and quite fast.

3.5.14 Use of the Over-chuck Freezing Block

If the chucks are used cold, placing an over-chuck freezing block is optional. Well cooled chucks have the freezing power to freeze the blocks without the over-chucks blocks. When using the extra large and deep wells the freezing process will be aided by placement of the over-chuck blocks. If the chucks are used warm, the over-chuck block is mandatory to cool the chuck and freeze the block.

3.5.15 Releasing the Block

Holding the top of the over-chuck freezing block, give the chuck stem a sharp quick tap. This will break the bond holding the block to the well bar Fig. 3.6l. If you try to pull the blocks up by hand, you will be surprised at how firmly the forces hold the block. Try to avoid slow week taps as this can hammer off the chuck off of the block while the block remains fixed to the well floor. If the chuck comes away from the block it is because the medium has not engaged, adhered to or has been broken away from the grooves in the chucks. Causes include: The well was under filled and the medium never made it to the channels; the chuck was used warm without a heat extractor; the chuck was removed too early; the chuck was covered in oil or alcohol; or the chuck was hammered off the block with weak taps.

To fix the separated block, add a generous application of embedding medium over the frozen filled well and place a new cold chuck over the well. Remove the block with a sharp tap after a period of freezing.

3.6 Reducing Freezing Artifact

Freeze artifact refers to morphologic variations in the microanatomy that result from ice crystal formation as water freezes in the tissue. Artifacts will be much more prominent in tissues having high water content. Rapidly freezing the tissue will reduce freeze artifact by formation of vitreous ice crystal which result in less artifacts microscopically. Freeze artifact will be discussed in greater detail in Chaps. 7 and 9. For the purpose of this discussion, there are several ways we can reduce freeze artifact by speeding the freezing process using well bars to freeze the tissue.

3.6.1 Colder Well Bar Temperature

I suggest new users start with well bars at –24°C. At this temperature, there is less tendency to over cool the block while the user is getting used to estimating the freezing times. As one gains experience, the bars can be used at colder temperatures. At –27 to −30°C, the freezing is more rapid and artifact is considerably reduced.

3.6.2 Pre-chilling Tissue

The tissues we receive in clinical practice are usually somewhere between body temperature and room temperature and often quite warm to the touch. We can considerably reduce the time it takes to freeze the tissue if we start the freezing process with the tissue chilled to 3°C rather than 37°C. Pre-chilling can be easily accomplished on any small piece of metal or ceramic kept in the refrigerator. Place the tissue on the cold surface for a few seconds on each side to bring the tissue temperature closer to freezing temperature before placing the tissue in the well.

3.6.3 Cold Embedding Medium

Using well bars, the tissue face rapidly begins freezing on touching the well floor. Small pieces will essentially completely freeze in the first few seconds. However, the tissue is re-warmed when we fill the well with warm embedding medium. By using refrigerated embedding medium we will prevent re-warming, complete the freeze considerably faster and reduce the freeze artifact. If it is possible to locate a refrigerator next to the cryostat, using cold embedding medium is no inconvenience at all. When embedding tissue that has been snap frozen always prepare the blocks using cold embedding medium to prevent melting of the block and introduction of freeze artifact.

3.6.4 Freezing Semi-liquid Samples

Soft, partially liquid specimens such as uterine curettings can be prepared into three dimensional blocks using well bars to freeze the tissue. Keep a household teaspoon to scrape up these specimens off of the Telfa pad or whatever surface they are received on. Very bloody or water density specimens often shatter when cut because they get icy hard on freezing. We can decrease the shattering considerably by stirring in a small quantity of embedding medium in the spoon before putting tissue into the well. Tissue can be scraped off of the spoon into the well using the narrow tip of a dispensing slide Fig. 3.12.

Fig. 3.12

Liquids (a) A few drops of embedding medium being stirred into curettings in a spoon. The tissue is then scraped from the spoon into an embedding well. (b) Completed block containing a three dimensional layer of the curettings

3.6.5 Embedding Snap Frozen Tissue Samples

Embedding of snap frozen samples can be accomplished easily and precisely using deep well bars designed for research applications. Well bars designed with wells of both 6 mm and 9 mm depth will accommodate most snap frozen samples. The pre-frozen tissue can be adhered into desired position by applying a drop of embedding medium to the surface to be embedded down and touching it to the well floor. Fill the well with cold embedding medium to avoid warming of the tissue and apply a cold chuck.

3.7 The Cut Off Technique

At times, the task of grossly cutting a 3 mm. section for embedding can be quite ­difficult due to the softness of the tissue or the irregularities of the anatomy. If possible we do not want to freeze tissues much thicker than 3 mm. if it is not necessary because the thicker tissue will slow the freezing time and increase freezing artifact. The cut off technique begins by placing a thick portion of tissue on the well floor which will be held fixed by the bond of the freezing temperature steel. By fixing the tissue to the well bar floor we can easily cut across the tissue in a uniform plane using the well bar surface as a guide for the scalpel. Figure 3.13 shows the example of taking a section from the margin of a 1 cm segment of ureter. Cutting the margin off freehand with a scalpel often results in displacement and telescoping of the mucosa. Using the cut off technique, the margin is placed face down on the well floor as the segment is stood vertically. The remaining ureter can be cut away at the surface of the well bar leaving a clean 3.5 mm section; the depth of the well bar. Another useful application is taking a section of bronchial margin. Cutting a three of four millimeter shave margin off the bronchial margin is not always easy because the various structures at the hilum are in a multitude of planes. By taking all of the hilar structures in a thicker piece, then pressing it margin down on the well floor the hilar structures will adhere together in a single plane. The excess tissue can be cut away with a sharp scalpel at the face of the well bar using the surface of the well bar as a guide.

Fig. 3.13

Cut off technique using a mock up of a ureter margin. (a) Wet the surface of the tissue with medium. (b) Holding the tissue with a forceps adhere face of the tissue to the well floor. (c) While holding the tissue upward with a forceps cut the tissue off with a scalpel blade using the well bar surface as a guide. (d) Fill the well and apply the chuck

The tissue being cut away must be held upward with the forceps to avoid tissue sticking to the well bar surface. Make sure your blade is sharp and use a gentle slicing motion with the scalpel. This technique will allow easy trimming of a 3 mm section with very soft tissues. If you find yourself cutting into heavily calcified tissue you can finish the cut with a sturdy scissor Figs. 3.13 and 3.14.

Fig. 3.14

(a) Cut off technique of a quarter of the cervix with attached vaginal margin (arrow). Check the section by looking through the dispensing slide. (b) The desired margin is placed face down on the well floor. (c) Cut across the tissue using the surface of the bar as a guide. (d) Trimmed section after the cut off. (e) The trimmed block showing complete cervical and vaginal margin (arrow)

3.8 Plastering Technique

Plastering is a very simple and rapid way to repair defects in the face of the prepared block. When embedding tissues very 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. Figure 3.6n–p. If we skip this step, when the section is cut the tissue will separate from the medium. This will make it difficult to get an intact section without having the tissue curl away from the medium (see Chap. 4). The actual depth of this retraction is quite shallow and in thicker specimens it is reached quickly during superficial trimming requiring no attention. Very thin specimens should be plastered before trimming to avoid curling away. Defects seen around tissue pieces prepared by frozen block cryoembedding will also require plastering so we can begin cutting on a completely flat surface. Plastering can also be used to repair defects caused by removing sutures or staples that find their way into the block. If the block is already in the chuck holder it may be best not to remove it for plastering in order to avoid unnecessary trimming and tissue wastage. We can plaster the block while it is still in the chuck holder by applying medium to the defect using a slide or other flat applicator as one would use a putty knife. Freeze the medium by pressing an over-chuck freezing block against the block face Fig. 3.15.

Fig. 3.15

Plastering technique and suture removal. (a) Extract the suture by grasping the suture with a hemostat or forceps and rolling tip of the instrument. (b) Apply embedding medium to the defect. (c) Press an over-chuck freezing block to the chuck face to freeze the medium. (d) Trimmed block showing the filled defect

3.9 Paper Embedding

Paper embedding is a face down embedding technique which employs a small piece of lens paper to transfer tissues to the well bars in precise position. It allows accurate orientation using the most flimsy tissues or complex arrangements of tissues (Peters, Fitzgerald et al 2003).

The lens paper is wetted with medium on both sides and then placed at the end of a dispensing slide with a 2 mm tab of paper overlapping the end of the slide. Excess medium is pressed out of the paper with the back of a forceps so that the paper is applied flat to the dispensing slide. Tissue is then placed in position on the paper. The paper and tissue are transferred in position to the well floor by touching the tab of paper to adhere to the well floor. The tissue is transferred to the well floor by pulling the slide out from under the paper Fig. 3.16.

Fig. 3.16

Paper embedding (a)A thin strip of skin (epidermis seen up) less than 1 mm thick. (b)A drop of embedding medium is placed on the dispensing slide. A square of lens paper is at the lower portion of the photograph. (c)The lens paper is wetted in the drop of embedding medium, then flipped over and placed on the drop of medium wetting both sides. Excess medium is pressed out from under the paper using the back of the forceps (not shown). Our goal is to have the paper applied flat to the slide like wallpaper, leaving just enough medium so that the paper will slide unhindered from the dispensing slide as in Fig. 3.16 d. (d)The lens paper is pulled to the end of the slide so that a tab of 2–3 mm of paper overhangs the dispensing slide. (e)Place the tissue in precise position on the paper. (f)Observe the tissue from below and from the side. Make any adjustments to the position. (g)Press the wetted tab of the lens paper to the well floor where it will adhere. (h)Pull out the dispensing slide from under the paper, letting the paper fall to the well floor. (i)Fill the well, freeze and remove the block in the usual fashion. (j)Untrimmed prepared block looking through the lens paper. (k)Block trimmed across lens paper to reach tissue. (l)Photograph of slide (above) and micrograph (below; 20× magnification)

3.9.1 Paper Embedding Multiple Tissue Sections on Edge: The Book

This technique is useful for on edge embedding of multiple sections from large flat surfaces such as cysts walls, synovium and broad margins that require embedding on edge. Cut a piece of lens paper to fit the tissue sample and the well size as in Fig. 3.14 leaving a 2–3 mm tab of paper to overlap the edge of the dispensing slide. The paper should be no larger than 3 mm narrower than the well floor diameter. The tab should not overlap the wall of the well Fig. 3.17.

Fig. 3.17

Paper embedding: The book (a) Wetted skin strips can be stood on the edge by leaning the tissues against each other to help them stand up on paper wetted with embedding medium. The overlapping tab of paper has been touched to the well floor and the dispensing slide is being pulled away. (b) The trimmed block. (c) The stained slide

3.9.2 Paper Embedding Membrane Roll

This technique allows us to embed a large area of paper-thin membranes on edge which are too flimsy to stand on edge in a book. Figure 3.16 demonstrated a membrane roll prepared from a 3 cm length of fetal membrane. Start by cutting the membrane into 3 mm. wide strips and wet them with embedding medium. Prepare the lens paper as shown above. Start by rolling a strip of membrane to form the central core on the paper. Add additional layers to the roll by wrapping strips of membrane around the central core Fig. 3.18a–e.

Fig. 3.18

Paper Embedding: Membrane rolls. (a) Start by rolling a strip on the paper to form the center. (b) Add additional strips by wrapping them around the central core. (c) Touch tab paper to the well floor and pull the dispensing slide out from under the paper and jelly roll which falls to the well floor. (d) Trimmed jelly roll block showing numerous layers of tissue. (e) Frozen section slide showing multiple layers of fetal membrane in this jelly roll preparation. Micrograph 20× magnification

3.9.3 Paper Embedding very Thin Tissues on Face

Embedding and sectioning paper thin highly delicate tissues such as brain slices and membranes can be accomplished by placing the tissue on face on a piece of lens paper and then transferring it to the well floor. Figure 3.19 illustrates this technique using fetal membrane measuring approximately 0.4 mm in thickness. The entire face is sectioned within the first 200 microns with ample tissue remaining Fig. 3.19.

Fig. 3.19

Paper Embedding membranes on face. (a) Fetal membrane measuring 10 mm across prepared on lens paper. (b) Trimmed block. (c) Micrograph (20× magnifications)

3.9.4 Further Details on Paper Embedding

• Touch the wetted tab to the well floor along its entire width so it will pull the paper evenly off the slide.

• For very thin tissues less than a mm. in thickness, the layer of medium under and on top of the paper should be as thin and uniform as possible to achieve the most uniformly flat and parallel specimen. It must be nearly wallpapered to the slide leaving just enough medium so the paper will not stick to the slide. It is not as critical in thicker specimens which can afford a bit more trimming. In thick pieces, you can leave a nicely wetted surface for easy dispensing.

• If the embedding process is complex and will take more than a minute or two, make sure the medium has not dried to the point that the paper no longer slides easily from the dispensing slide. If there is drying the paper can be kept moistened with a drop of water or saline so that it is adequately wetted at the time of dispensing.

• When cutting the section, the lens paper can be trimmed away from the face of the block without significantly dulling the blade. The lens paper can also be peeled off by first briefly rubbing the face on a warm surface. Next, peal the paper away and then quickly refreeze the block face by pressing the block face to a cold bar or over chuck block. A thin plastering will take care of any defects Fig. 3.20.

Fig. 3.20

Tulip. This picture of a tulip is a trimmed block made by embedding numerous flower petals and leaves on the edge using paper cryoembedding

3.10 Frozen Block Cryoembedding

Frozen Block Cryoembedding (Peters 2003) is a simple two stage embedding technique which will allow precise on-edge embedding of virtually anything. Using the simple apparatus described below, the tissue is first frozen in a block of embedding medium. The frozen block containing the tissue is then cut transversely in the appropriate plane. The resulting slices of the frozen block are firm rectangular and have flat surfaces and can be easily placed on their side on the floor of the well. The well is then filled with embedding medium and the chuck is applied to complete the block. The process transforms tiny specimens into large ones, flimsy soft tissues into firm easy to cut specimens, straightens out rubbery curled tissues and makes torn and perforated tissues whole again.

3.11 Apparatus

3.11.1 Cutting Board/Freezing Griddle

The steel Freezing Griddle serves as a flat freezing surface on which to prepare the frozen block and to keep cut sections cold. The attached cutting board serves as a cold surface to cut the frozen block while maintaining the freezing temperature. This simple piece of apparatus is kept at freezing temperature in the cryostat or freezer and can be briefly taken to the work bench during the process of cutting the frozen block Fig. 3.21 (left).

Fig. 3.21

Frozen block cryoembedding apparatus. Cutting board freezing griddle (left); elevated freezing block (right); epoxy coated forceps (bottom)

3.11.2 Elevated Freezing Block

This steel block is kept cold in the cryostat and is used in conjunction with the freezing griddle. The two broad sides of the block are equipped with feet measuring 3.0 mm on one side and 4.5 mm on the other. The feet create a space between the two steel bars in which the frozen blocks are prepared Fig. 3.21 (right).

3.12 Frozen Block Cryoembedding Technique

The technique is illustrated using a 3 mm skin ellipse. The ellipse is sectioned and embedded with margins taken both transversely across the ellipse and longitudinally to include the tips of the ellipse. The cuts correspond to the lines on the frozen block specimen in Fig. 3.22d.

Fig. 3.22

Frozen block cryoembedding a small skin ellipse (a)Place tissue on a thin film of embedding medium face down on the dispensing slide and place the tissue on the freezing griddle. (b)Cover the tissue with an ample quantity of embedding medium. (c)Place the elevated freezing block over the tissue. Allow a freezing time of 1 min. (d)Frozen block after removal of the elevated freezing block. The lines show the cuts to be made in numerical order. (e)Remove the cutting board to the workbench. Trim the edges of the frozen medium while testing the frozen block for hardness. The block should be with a very firm fudge like consistency. (f)If there is any tendency for the cut piece to fly apart it is too hard. Warm the frozen block for a few seconds between the gloved hands and retest. (g)Cut the frozen block into the appropriate sections to achieve the desired orientation. (h)When cutting these longitudinal margins push the blade toward the medium and make sure the tissue surface is well frozen to the medium to prevent dislodging. (i)Cut pieces shown face up. (j)Using cold epoxy coated forceps, the frozen slices are placed face down on the embedding well floor leaving a 2 mm of space between pieces for medium to penetrate (not shown). Pieces are shown in position on the well floor filled with medium. (k)Trimmed block face. (l)Photograph of frozen section slide. (m)Photomicrograph composite showing the tissue embedded on edge with all margins visible (50× magnification)

3.12.1 Bowel on the Griddle

Examining resection margins from bowel and esophagus resections are commonplace in the frozen section lab. It can be quite difficult to get a well embedded cross section of these flimsy tissues because of the slippery and loosely held submucosal layer and the retraction of the cut mucosal and muscularis propria tissues. By first freezing the tissue in a block of embedding medium, the tissue can be cut and rotated 90° creating a rectangular section that is positioned and embedded precisely in the cross section Fig. 3.23.

Fig. 3.23

Frozen block cryoembedding of bowel. (a)Portion of small Intestine covered with medium on the freezing griddle. (b)Frozen block being cut. (c)Embedded small intestine, four slices in a block. All four are layers visible. (d)Photograph of frozen section slide. (e)Micrograph showing transverse section with all layers of the wall visible and vertical villi in the mucosa 20× magnification)

3.13 Frozen Block Cryoembedding Details

3.13.1 Making the Frozen Block

Before making the frozen block, we must first ask ourselves; Do we need to visualize the tissue when we are sectioning the frozen block? In a small skin ellipse as shown in Fig. 3.20, we need to be able to visualize the inked side so that we can accurately cut the tissue with a scalpel. We must use very little embedding medium in the side that we have to see. Therefore, we want to use very little embedding medium on the dispensing slide beneath the tissue. When the embedding medium freezes it becomes opaque, and if there is a thick layer over the tissue we will not be able to see the tissue during cutting. If we are simply looking to cross section the tissue and three dimensional orientation is not critical, we can start by putting a thin layer of embedding medium on the griddle, placing the tissue on top and add more embedding medium before freezing. The resulting frozen block will have embedding medium on both sides and cut in larger flatter slices which are less delicate and a bit easier to handle.

Another consideration is flattening artifact. If flattening is not acceptable, then make sure there is enough room under the elevated block for the tissue and apply an ample layer of embedding medium over the tissue. If we would like to freeze small intestinal tissue so that the villi are vertical and not flattened, the tissue should be placed on the griddle mucosa side up, and covered with medium allowing the tissue time to freeze partially before placing on the elevated freezing block.

3.13.2 How Do I Know When the Block Is Frozen?

Give the tissue about a minute to freeze. Then, very gently try to rotate the elevated freezing block a degree or so. If there is movement, the block is not fully frozen yet. If it feels solid it is frozen. If you accidentally separate the blocks too early it can be easily repaired by removing the embedding medium from the side without the tissue, applying another squirt of embedding medium to the tissue and replacing the elevated freezing block.

3.13.3 Removing the Frozen Block

Use a plastic putty knife or similar tool to lift the frozen block from the griddle. Avoid using a scalpel of other sharp instrument for this purpose.

3.13.4 Cutting the Frozen Block Fig. 3.22g–i

The tissue will cut easily at the right temperature. If the block is too cold the tissue will fly apart on cutting. Always first test the block by trimming away the excess medium. This is a necessary task so that the pieces will fit more easily fit in the well. At the optimal temperature, the tissue cuts with a very firm fudge like consistency. It can be likened to cutting a hard ice cream cake. If the excess medium flies apart when cut, it is to cold and will require warming between the gloved hands. This maneuver should be done in short doses and not left between the gloves for more than a few seconds. Retest the tissue and warm additionally until the tissue reaches the proper consistency for cutting. The added few seconds it takes to warm the tissue is a lot less time than trying to find a piece of tissue that grew wings!

After warming, press the tissue face flat to the griddle again for a second or two to assure that the tissue is held firmly by the frozen medium when cutting begins. This is particularly important in very thin specimens such as a thin skin specimen. The tissue is being held by a thin layer of frozen medium on the edge of the skin. If slightly melted, the tissue can dislodge on when cutting. To prevent dislodging the tissue on cutting, always push the scalpel toward the medium. Don’t move the scalpel in a direction away from the medium to avoid of dislodging the tissue from the medium with the scalpel stroke. Use mild scalpel pressure in an even movement without trying to push it through tissue like your chopping it. If the frozen block is at the ideal temperature, it will cut without too much effort. This will also vary with the water content of the tissue. If you find yourself having to lever through the block like you are using a chef’s knife, the block should be warmed a bit more. The smoother you cut the tissue, the flatter the surface of each piece will be and the flatter the final preparation will be. The completed block will have fewer and shallower defects to plaster over, and the tissue will be closest to a single plane.

3.13.5 Putting the Tissue Pieces in the Wells Fig. 3.22j

Tissue is placed face down in the well. Use epoxy coated forceps to handle frozen tissue and keep them cold in the cryostat. If you try to use uncoated warm steel forceps to handle the pieces of the frozen block, the pieces will stick to the forceps and make it difficult and frustrating to arrange tissues in the well. If you don’t have epoxy coated forceps and must use steel forceps, keep a pair in the cryostat and coat the tips with a bit of paraffin to prevent sticking.

Leave 2 mm of space between each piece for medium to penetrate. This is very important. If the pieces are very close together there will be deep crevices where medium cannot penetrate between the pieces. These crevices can only be plastered superficially. These thin crevices will cause separations in the sections when cut beyond the depth of the plastering. If the tissue pieces are separated by about 2 mm, then there will be ample space to fill with medium.

3.13.6 Filling the Well

Gently apply the embedding medium to fill the well taking care not to push the pieces out of position with the flowing medium. Make sure the bottle of embedding medium contains enough medium and has been inverted long enough for medium to fill the nozzle. If the bottle first blows out a puff of air it can blow the pieces out of position. Apply the chuck quickly with firm pressure to extrude the excess medium.

3.14 Orienting the Anatomy of Tissue Relative to the Blade

When we cut our frozen section, the way the anatomy of the tissue is oriented relative to the blade will impact our ability to get our best sections. First, let us consider the blade as it passes through our tissue. The blade first meets our handle of embedding followed by the beginning, middle and end of the tissue and more handle of medium. If you master the continuous motion technique I offer in Chap. 4, this will happen in a smooth uniform motion. If you are cutting from a standstill, inching along or hesitating, the areas of difficulty will be magnified. Which part of the anatomy hits the blade first, and how the layers of the anatomy pass through the blade can often give varying results and difficulties. I will offer suggestions that I have found successful in my experience.

Always approach the block where there is a handle of embedding medium. Always prepare your blocks with a handle of medium surrounding the tissue. This will serve as the handle for the brush to grab on our new section and a frame to stabilize our less substantial tissues such as fat, necrotic tissues and highly delicate cystic processes. It also serves as our running start to a section destined to give us difficulty. The section will start with a solid easy section of only embedding medium before plunging our problem tissues. The handle is our margin of error when mischief arises during cutting and retrieving such as tissue sticking to the brush, and stretching the section on retrieving. A handle on the back side of the block will also allow for easy retrieving from the block. See Chap. 5 p. 26.

The most critical aspect of the tissue should be perpendicular or diagonal to the blade and not the first or last aspect of the tissue to hit the blade. The part of the section where the tissue first meets the blade has more propensities for artifacts including flipping of the section, thickness issues and wrinkling. Avoid having the critical aspect of the tissue such as mucosal surfaces or margins, hit first and parallel to the blade. The last part of the section is again at the risk of curling, or problems on retrieval. The middle of the section is where cutting tends to be cleanest is less at risk for artifacts. This is where I want to see that critical portion of the slide such as an inked margin. Mucosal lined tissues such as GI, bladder, uterus and cervix should be oriented with the plane of the epithelium perpendicular to the blade Fig. 3.24a.

Fig. 3.24

Illustration of a frozen section block in the cryostat and the suggested orientation of the tissue relative to the blade. (a) The block shows the bowel tissue oriented with the mucosal surface perpendicular to the blade. (b) The block shows a piece of skin with the epidermis embedded perpendicular to the blade

Skin should be oriented with the epidermis perpendicular or diagonal to the blade (Fig. 3.22b). The epidermis of human skin has a propensity to separate from the embedding medium. I believe the stratum corneum which desquamates naturally, creates a plane of separation from the medium. As a result, there is always a propensity for the epidermis to pull away from the embedding medium and curl over and flip. I refer to this as curling away . Skin cut in a horizontal position with the epidermis toward the blade will have a strong tendency to flip over the entire epidermis and is discouraged. When we embed skin perpendicular to the blade the separation of the epidermis from the medium creates a tendency for the tip closest to the blade to curl or flip. This can be minimized by warming the tissue to reduce curling. See Chap. 4 for more on curling away. Figure 3.25 shows an approach to embedding a small skin ellipse, sectioned as in Fig. 3.22d. The skin pieces that have a margin on only one side, should be oriented so that the margin end or tips are last to hit the blade to avoid curling (Fig. 3.25 arrow heads). In this case, only the complete transverse sections will be prone to a flipping tip. This arrangement is quite simple to do using samples prepared by frozen block cryoembedding.

Fig. 3.25

Orienting a skin biopsy to minimize curling of the tips. (a)The trimmed block with five pieces of skin from Fig. 3.20k.The arrow points to the cryostat blade. The arrowheads point out the tips of the four longitudinal margin sections. Orienting the block with the tips away from and last to reach the blade will reduce the problem of tips curling away and flipping. (b) The cut section forming over the blade without flipping of tips. The arrow points to an area of separation of epidermis from embedding medium visible as a fine cleft

Fat should be the last thing to hit the blade or should hit the blade by itself whenever possible (Fig. 5.3). Fat does not get hard enough to cut well at temperatures that are best for cutting most other tissues. When fat hits the blade before the more manageable tissues it may smear and ruin the rest of the section. I find by hitting the knife last or by itself fat won’t interfere with the other tissues as much. The worst possible situation is to have fat meet the blade first without a “handle” of embedding medium. The handle gives the section a start. Without this handle, the fat will smear and wipe out the whole section. Sectioning fat will be discussed in greater detail in Chap. 5. If we are having difficulty getting a good section because fat appears in the, plane we can rotate the chuck to get the tissues that will easily cut out of the path of the fat or using a technique I refer to as the gouge . See Chap. 5 Fig. 5.6.

Very tough tissues should be embedded vertically or on a diagonal to reduce the effective “width” of the tissue and reduce knife stress. Cut tough tissues as warm as possible. Tissue characteristics are covered in detail in Chap. 5.