Dr. Stephen Peters
Pathology Innovations, LLC
Orienting the tissue to the knife
This is an extremely important aspect of the frozen section preparation. Using the embedding system described in the previous articles 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 my experience I have come up with certain rules I try to follow:
1) Fat should be the last thing to hit the blade or should hit the blade by itself whenever possible. 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. Sectioning fat will be discussed in greater detail below. If I find myself having difficulty getting a good section because fat appears in the plane I suggest rotating the chuck to avoid the fat or try "The Gouge".2) 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. Let's consider a section as having a beginning, middle, and an end. At the beginning there is a risk of curling, or brush damage, possibly thickness issues because hesitation engaging the tissue. These are opportunities for artifacts. Similarly at the end curling becomes an issue and possibly stretching in picking up the tissue. The middle is the place where we are less likely to see artifacts and have the cleanest histology. This is where I want to see that critical portion of the slide such as an inked margin.
3) Epithelial and mucosal lined tissues such as skin and GI, bladder, uterus and cervix should be oriented with the plane of the epithelium perpendicular to the blade.
When embedding a skin ellipse the end which hits the blade first has a tendency to curl. This diagram below shows an approach to embedding a small skin ellipse, so that the longitudinal margins (2-5) will hit the blade last. This arrangement is quite simple to do using specimens prepared by frozen block cryoembedding.
Skin Embedding Tip: Karen Courtney of
Metropolitan Methodist Hospital, San Antonio Texas embeds
skins epidermis to epidermis.
that skin should be embedded vertically.
When cutting skin the medium does not adhere to the
epidermis. I believe the outer later of
squamous cells in the stratum corneum
which are designed to "flake away" from our skin, simply
come apart from the rest of the epidermis. The result is
that we see a separation of the epidermis from the medium.
This is one way to tell if you have reached the level of the
epidermis when you are trimming.
"epidermis to epidermis" these lines of separation will be
gathered together and result in the instability of the
section over a narrow range. The rest of the skin will have
its own channels of preservation.
Please, always start with a sharp blade!
I am sure others may not agree with these suggestions and may have other ideas on this subject. We all learn so much of this in our own experience. The point I am trying to make is to consider the outcome as a result of how the tissue meets the blade. This system will allow you to arrange the tissue however you would like to.
How much tissue can be put in a single block?
The amount of tissue that can be cut in a single block depends on two factors.
1) The ability of the cryostat to cut
through large tough portions of tissue.
In order to get a smooth evenly cut section the knife must pass freely through the tissue with no movement, stretching, loosening or bending of any part of the mechanism that holds the chuck and tissue and the blade. This implies that the chuck is tightly secured and that any shafts or supports that hold the chuck are sound. The blade, the blade holder and any structure that holds the blade holder in place must be secures and tight. This ability will vary with makes and models of cryostat and with the state of maintenance of a given cryostat. The type of blade and the flexibility of the blade are another factor.
2) The toughness or hardness of the tissue.
Tissues vary considerably in toughness or hardness. On my cryostat tissues such as liver or kidney can be easily cut even in very large pieces. But very tough collagenous tissues like scalp or cervix will cut thick and thin if too large a section is attempted. The colder these tough tissues are the more difficult they are to cut. So before taking a very large panoramic portion of tissue for a frozen section, consider the consistency of the tissue.
When the system is being stressed by too much tissue resistance I have observed the phenomenon of thick and thin sections. This happens when the stress moves the knife to take a thicker section. The following section will then be thinner.
When cutting very tough tissues keep the tissue block as warm as you can and still get a section.
Listen to the blade
Get to know the sound of a good frozen coming off the blade. There is almost no sound at all. When a block is too cold there is distinct sound as the blade scratches the icy block. The blade will make a variety of grating or vibrating sounds when it is at the wrong blade angle, showing movement or out of positioned because of a bit of medium in the blade holder.
Trimming the block
This refers to the shaving away the surface of the block to a depth at which the complete desired tissue face is available for the frozen section. This rather important step is often taken for granted. If trimmed too little, the section will not include some critical feature, if trimmed too much there will be unnecessary wastage of tissue and potential loss of some precious portion of the sample. Two key skills must be developed to carryout this task. The operator must learn to grossly recognize the anatomy and landmarks visible on the block face. I refer to this as "reading the block". Although this is often a small picture to read grossly, one must be well aware when such things as the inked margin, the mucosal surface or the epidermis becomes available for the section. There is a brief period before these structures are reached when they look as though they have been reached. At this slightly "premature stage" it is like looking at the structure through frosted glass. One will think the structure is present but it will not appear in the section. This "premature face" must be learned to be distinguished from the "mature" face. The keratin layer of the epidermis has a tendency of separating from the embedding medium on the cut section and can serve as an indicator that the epidermis has been reached.
This "premature" block shows the area at the left where there is a frosted slightly blurred look. This may look obvious here but it can be very subtle at a distance in the cryostat.
The tissue is fully exposed and seen as a crisp discrete line. This skin is dyed red on the epidermis and black in the deep aspect. It is much less apparent in less colorful samples. Look close!
|Agar Depth markers|
|Ervin Shaw, M.D., of Pathology Associates of Lexington, P.A. has some ingenious suggestions for using layered blocks of colored agar as markers of depth and orientation in paraffin blocks. Markers are made by layering different colors of agar in a Petri dish at a quantitative thicknesses. Placing a depth marker level with the tissue face, one can approximate how deep they have cut into the tissue by the color of the agar. This technique could be applied to frozen section by placing a marker on the well floor. Knowing the thickness of the marker and the thickness of the tissue one can estimate how much tissue has been cut through. By preparing the colored layers in intervals at a fraction of a mm. it could be a useful in estimation the depth of very thin specimens. See more at Dr Shaw's site: http://www.palpath.com/agards.htm|
The well adjusted cryostat
Pathologists, residents, PA's and histotechs should have a basic knowledge of maintenance of their cryostat. It is a good idea to read the manual. This should include:
Lubrication- where and when to apply oil periodically and especially after warming and decontamination. Oil must be formulated for cryostat temperatures.
Blade angle- This varies with make of cryostat and type of blade. Be aware of the proper setting for your cryostat and how to adjust it. It is not uncommon for a cryostat to be left out of adjustment by some covering tech, service person, new resident ect. You will be faced by a strange sound and poor sectioning.
To achieve the ideally trimmed block the path of the plane of the face of the block must be in the same plane as the blade. If these planes are parallel then trimming will start uniformly across the block face. Using face down embedding in wells, the ability to prepare blocks with faces parallel to the chuck face becomes a reality. In a properly adjusted cryostat these flat surfaces can be reached with minimal trimming and yield a complete section with minimal trimming and wastage. But how do we "properly adjust" our cryostat? Here is where our technology has left something to be desired. Many cryostats designed and priced for used in the clinical laboratory offer only rudimentary manual adjustability of the "X-Y" axis. Some are totally stationary. Therefore in order to adjust the block face to the plane of the knife blade it is a difficult coarse adjustment. In order to achieve the flat minimally trimmed block faces such as seen in the sesame seed art in the earlier articles, painstaking fine adjustments need to be accomplished to reach the entire plane of sesame seeds before cutting through any. If you need this degree of alignment be aware that manually moving these chuck holders even a degree will result in a drastic change in the way the knife meets the block. Ideally, using the embedding wells and preparing blocks to optimize "parallel faces" then an initial adjustment to the cryostat to meet the center of the block will yield the least possible trimming.
Keep It Clean
I believe we should leave the cryostat clean after each use. It takes seconds to empty the shavings tray and wipe out the loose shavings with a gauze. There are many who feel cleaning the shavings once a day by a "histo-slave" is adequate cryostat hygiene. Having personally inhaled shavings from a cryostat snowstorm I cannot agree. I think a filthy cryostat offers several risks:
1) Risk of infection by inhalation or other route of entry.
2) Risk of cross contamination of a slide with an aberrant section.
3) If tissue or a chuck is dropped into a snowstorm it comes up looking like its covered in coconut.
I think we should leave the cryostat the same way we leave the bathroom. We do not wait for our mother to come flush for us! .......or do we?
Clean your brushes When the frozen section brush gets dirty there is a greater tendency for the sections to cling to the brush. They can be cleaned in seconds by:
1) quick touch of soap and water and dry
2) quick dip and dry in 100% ETOH
3) quick dip and dry in xylene.
4) cool for a few seconds against a cold surface.
There must be no movement in the system ( see FS technique III)
We are very grateful for Dr. Stephen Peters generous contribution of the valuable technique. Please visit the original source at http://www.pathologyinnovations.com/ for more updated information.