Dispensing Slides

Slides narrow to various tip widths. The width of the tip is chosen based on the tissue size and the well size in which the tissue is being placed. Looking through the slide offers a view of the tissue face that will be placed down on the well floor. While looking through the back of the slide, tissue can be adjusted into the precise orientation desired.

This may be such that the epidermis or margin is visible, or to place a tissue on edge. Then as the tissue leaves the slide, it is guided to adhere in the desired position.

A thin coat of embedding medium assures a clean easy release of the block and assures adhesion of tissue to the well floor. This layer of medium also provides a support for the tissue, aiding in manipulation on the dispensing slide and providing adhesion to the slide during transport. If excess embedding medium is used there will be a thicker layer of frozen medium than desired over the embedded tissue. This is an issue when extreme flatness is required such as when embedding multiple minute samples or needle biopsies flat. Figure 3 demonstrates an artistic example requiring extremely flat embedding. The samples in this case are colored sesame seeds. This could not have been accomplished without use of a very thin layer of embedding medium to adhere the sesame seeds to the well floor.

If there is no risk of cross contamination, several samples of tissue can be placed on a single slide and pulled to the end for placement in one or more wells. For example in sampling a large tumor, four 1 cm squares can be placed in a 30 mm well. This allows a rather large amount of tissue to be sampled from a single sectioned block. If one were looking at separate specimens such as lymph nodes from four different sites however, separate dispensing slides must be used to avoid cross contamination.

Placing Tissue in the Well

Orientation and location of the tissue in the well should be considered before placing the tissue. If a large portion of tissue is being placed on the floor of the well, 鈥渢ouch-down" must take place at a point in the well that will allow room for the whole piece. For example, if a 2 cm length of tissue is placed in a 30 mm well, it should be touched down beginning near the edge rather than in the middle. If four 1 cm squares were placed in the 30 mm well, they should be placed squarely near the walls in the comers allowing for room (Figure 4). As in all frozen section preparation, the orientation of the tissue to the blade should be considered. For example, many practitioners agree that the epidermis should be oriented perpendicular to the blade when cutting skin.

The process of pulling the slide out from under the tissue can require a great deal of precision toward achieving the final result. In samples requiring no orientation, the slide can be removed in a swift motion. In samples requiring precise orientation this process can be carried out more slowly and meticulously. If the shape of the sample allows, the most critical edge of the sample should be first to touch down. The slide can then be slowly pulled out from under the sample with care being taken using forceps to guide the critical area to adhere to the steel in the desired orientation. If an inked margin is being examined, extra care can be taken to assure that the margin is first adhered, after which the remaining portion can be dealt with. In a thin strip of skin from a Mohs surgery case, great care can be taken to assure the epidermis is completely visualized and that the specimen is embedded flat. Specimens requiring extreme flattening can be pressed flat with the bottom of an angled forceps. Tubules, such as ureter margins, can be easily touched to adhere to the well floor in vertical orientation. Thin strips can be guided, so the edge adheres down for on edge orientation. Membranous fragments can be rolled or folded to stand on edge on the dispensing slide and pulled off and embedded in the same orientation. With a little foresight and imagination there are endless opportunities to achieve the desired embedding goals.

Handling Liquids

Liquids and very soft specimens, such as endometrial curettings and products of conception, can be scraped up with a plastic spoon or spatula and placed neatly in wells without crush artifact (Figures 5A and 5B). In very bloody liquid specimens, stirring in a few drops of embedding medium will allow the bloody portion to cut with less shattering. Figure 6 shows an example of the precision possible handling liquids using this system.

Filling the Well with Embedding Medium and Placing the Chuck

There must be a bulging meniscus of embedding medium to penetrate the channels in the chuck face for firm bonding to the formed block. The chuck should be placed over the well as quickly as possible after filling. The chuck is placed by firmly pressing the chuck face to the well bar surface. The excess medium is extruded through the grooves in the chucks.

Freezing Times

The recommended freezing times are approximate times experienced by the author when well bars and chucks are fully cooled. Freezing time will increase if well bars are allowed to warm, if chucks are used warm or if samples are very thick. The user quickly develops a sense for when the block is ready. Well bars should be returned to the lower level of the cryostat as soon as chucks are removed to maintain optimal temperature. Chucks should be cleaned, dried, and replaced in the cryostat as soon as possible for maximum performance. There is considerable variation of temperature at different levels of the cryostat. The embedding shelf is installed at a high level to optimize the operator鈥檚 ability to perform these critical fine manual tasks. Well bars, chucks or blocks will warm above optimum working temperatures if left on the shelf for prolonged periods.

Handling Numerous Specimens

When confronted with numerous specimens, this system lends itself to processing multiple blocks very quickly. Dispensing slides should be prepared in advance in an effort to keep the slides and specimens in order prior to filling the wells. Chucks are easily labeled after they are placed over the well. If the chucks are fully cooled, the blocks can be removed from the well in around 15 seconds before freezing is complete. The freezing process is completed on any freezing surface while additional blocks are prepared. In the time it takes to fill the four wells, the first ones filled can be removed, and additional specimens placed in the empty wells. By the time the practitioner is ready to cut the blocks, they will be fully frozen. Eight blocks can be frozen in under two minutes.

Removing the Block

A sharp tap to the stem of the chuck using the over-chuck freezing block easily frees the blocks. The sharp tap causes a cleavage in the plane of cohesion that forms between the cold steel and the frozen medium. If one tries to pull the block out by the stem, they will find it firmly attached in the larger wells, yet a sharp tap will release the block with ease.

Blocks will free intact even if incompletely frozen, provided the surface beneath the chuck has had time to freeze to the chuck.

If a chuck pulls off, it is either the result of underfilling of the well with medium or using a warm chuck without the over-chuck freezing block. An easy quick repair is possible by adding an additional layer of embedding medium and a new chuck.

Plastering

The plastering technique is used to repair any holes, defects, or crevices encountered in the face of a block prepared using this system or conventional methods (Figure 7). At times a small contraction space may form at the surface of the block between the medium that coated the tissue and medium that filled the well. This space is very superficial and will be quickly trimmed away in the early surfacing of the block. In minute samples where there is only minimal allowable trimming, the plastering process will fill any defect in a few seconds. Filling holes arising from bubbles in embedding medium or the defect left behind after removing an uninvited staple from the block will improve ability to get a complete section. Figure 7A shows an example of surface irregularities which can be seen in frozen block cryoembedding quickly filled by plastering. The plastering technique is performed as follows:

Medium can be applied as a drop from the bottle, a thin smear with the finger, a straight edge of cardboard, or any device which acts as a putty knife. If trimming has begun, this process can be performed while the block is in the holder by applying medium to the block face and then pressing the over-chuck freezing block to the face of the tissue block. This technique can be used to apply a "handle of embedding medium" to the edge of the block if a critical portion of the tissue is too close to the edge to get a quality section.

Orienting the Tissue to the Knife

This is an extremely important aspect of frozen section preparation. Using the embedding system described in this article, it is quite easy to prepare and orient tissues in the wells so that the knife meets the tissue in a specific orientation. Based on extensive experience, the following rules of thumb apply:

I am sure there are many other ideas and suggestions on this subject. We all learn so much of this in our own experience. The point is to consider the outcome as a result of how the tissue meets the blade. This system offers the ability to arrange the tissue however you would like to.