J. Greenlee

Grossing an Appropriate Tissue Thickness

Today's topic is all about cutting tissue to an appropriate thickness during gross dissection. It may not be the most glamorous aspect of histology, but it's crucial for proper tissue fixation and processing.  What happens at the grossing bench has an impact on all the downstream histology processes.  The old adage often heard in histology laboratories is "crap in... crap out."

Let's talk about why tissue thickness matters. It all has to do with the tissue's surface area to volume ratio, which I will explain.  The surface area of a tissue is, of course, the amount of space that makes up the surface.  This is what we see when we look at the tissue, the surface.  Think of this space and its area as the size of the doorway to get inside the tissue.  Fixatives like formalin and the processing reagents use this space to get into, or penetrate, the tissue.  The reagents are outside so need this doorway space to get into the tissue.  The volume of the tissue is all the space that makes up the inside of the tissue starting at the surface.  The volume is the room inside the tissue that needs to fill, whether penetrating with formalin or the processing reagents.  The surface area to volume ratio, then, is the ratio of the doorway space compared to the room space.

How does this ratio change with different thicknesses of tissue?  Let's look at some examples.  Imagine a 1 x 1 x 1 mm cube of tissue.  Of course, tissues are not usually perfect cubes, but for this example, just imagine we all live in the world of Minecraft, and even our GI biopsies are blocks.  The surface area of the tissue would be 1 x 1 mm on each side of the cube, which has 6 sides.  The combined surface area would be 6 mm2.  The volume would be 1 x 1 x 1 mm or 1 mm3.  The ratio of doorway to room is 6:1.  There is plenty of doorway space to get into the room.  Now, let's imagine a tissue cube that is 2 x 2 x 2 mm.  The surface area of a side of this cube is 2 x 2 mm, so the total surface area would be 4 mm2 x 6 sides or 24 mm2.  The volume would be 2 x 2 x 2 mm or 8 mm3.  Now the ratio is just 3:1.  The doorway space to get into the room compared to the smaller tissue is cut in half.  Now, for a final example, consider a 3 x 3 x 3 mm cube.  For this we have a surface area of 54 mm2 and a volume of 27 mm3 for a ratio of just 2:1.  Once again our doorway to room ratio is reduced.  Fewer doorways to get into the room means it will take longer for the formalin or processing reagents to get from the outside to the center of the tissue. 

As you can see from the examples, the thicker the tissue, the more difficult it is for reagents to penetrate.  If the tissue is too thick, then the fixative and reagents may not be able to fully penetrate the center of the tissue in the time provided, leading to incomplete fixation and processing. This can result in poor quality histological sections. So, in order to ensure proper fixation and processing, it's important to cut the tissue to an appropriate thickness.

But what exactly is an appropriate thickness? Well, it depends some on the tissue type and the amount of time the laboratory can allot for fixation and tissue processing.  If the laboratory can allow tissues to sit in formalin for days and are fine with a very long tissue processing protocol, then those fatty tissue sections that can barely cram into the cassettes may be OK.  However, most laboratories do not have all of the time in the world, and quality and efficiency are important.  In this case, generally, tissues should be cut to a thickness between 2-3 mm.  A really basic rule of thumb for formalin fixation is that formalin penetrates approximately 1 mm per hour.  In reality, that number changes based on tissue structures and slows as it penetrates deeper into tissues, but it is a good general rule of thumb.  For a tissue cut to "nickel" thick or 2-3 mm, that means that formalin penetrating from all sides should reach the center of the tissue within about 1 - 1.5 hours.  Don't forget, though, that penetration does not equal fixation.  It takes considerably longer for fixation and the cross-linking to occur, even for small tissue biopsies.

For some perspective, the standard depth of the common tissue cassette that a vast majority of laboratories use is 5 mm deep. Therefore, if the tissue is touching both the bottom and lid of the cassette, it is definitely too thick.  Closing the lid of a cassette should never be like trying to get our suitcase closed when getting ready for a long vacation.  If a grosser doesn't want to take out the ruler for every tissue, it is easy to remember that an appropriate thickness tissue should be about half the depth of the cassette. 

Now, let's talk about what can happen if tissues are cut to an inappropriate thickness at gross dissection. If tissues are cut too thick, they may not be fully fixed or processed, as we mentioned earlier. This can result in sections blowing up on the water bath at microtomy or that are uneven or poorly stained, making it difficult or impossible to accurately identify cellular structures or abnormalities.
On the other hand, if tissues are cut too thin, they may be difficult to handle and process as well. Thin tissues are more fragile and prone to tearing during processing, which can result in loss of tissue and poor quality sections. In addition, thin tissues may dry out more quickly, which can also impact processing and staining.  It is important when sectioning at grossing to use an appropriate thickness. 

So, what have we learned today? Cutting tissue to an appropriate thickness during gross dissection is crucial for proper tissue fixation and processing. The surface area to volume ratio of the tissue impacts the ability of fixatives and processing reagents to penetrate to the center of the tissue, so it's important to cut tissues to a thickness that allows for full penetration. If tissues are cut too thick or too thin, it can result in poor quality sections and unreliable results. Remember, tissue thickness may not be the most exciting aspect of histology, but it's essential for accurate results. So, don't be afraid to take your time and cut those tissues just right!
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