Processing Fatty Specimens
One of the most critical steps in histology is fixation, especially when it comes to fatty tissue.
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- Review the process of specimen handling before the processor
- Analyze the reasons why fatty specimens are so difficult to process
- Discuss the different technologies for processing and solutions used
- Identify good quality techniques for processing fatty specimens
Processing Fatty Specimens. Let’s talk about our objectives.
Throughout this presentation, we’re going to review the process of handling specimens, especially fatty specimens, before the processor. Next, we will analyze the reasons why fatty specimens are so difficult to process, and we also would like to discuss the different technologies. Finally, we will also identify good quality techniques for processing fatty specimens.
Today, we’re going to walk through the stages of tissue processing, from grossing all the way through infiltration.
Let’s start with grossing, one of the most important things in histology. If your specimen is not grossed properly, you’re not going to get the proper results that you’re looking for. The next step after grossing is going to be fixation.
Now, in the case of us presenting fatty tissue specimens today, I’m thinking more of breast specimens, so we do have to follow the CAP regulations about using neutral buffered formalin or NBF. Once we’re done with the fixation, we can speak to dehydration on the tissue processor and then talk about clearing reagents.
Next, we will speak to paraffin infiltration. This is so important because, there are numerous opinions about paraffin in the histology world. Last, but not least, we will focus on best practices for processing fatty specimens.
Fatty Specimens; why are they so difficult to process?
Fatty Specimens; why are they so difficult to process? It all comes down to fat being fundamentally hard to penetrate. This can lead to underfixation or underprocessing. Ultimately, often causing tissue that is difficult or even impossible to section. So, the question of today, what can we do to aid this process?
Well, first, it all starts in grossing. The specimens arrive into histology, have the proper identification and information about where the specimen taken from and a bit of patient history. Once checked, the grossing process can be initiated. With fatty tissue, it is imperative to select the most representative piece or pieces.
As we discussed earlier, fatty tissue is fundamentally hard to penetrate, so when selecting your representative piece of tissue, you will need to keep in mind that some trimming will be required. You want to get the specimen down to an ideal size. Optimally we have 1 to 2-millimeter biopsies for rapid processing and we have 3 to 4 millimeters for routine overnight processing. Once selected and trimmed, special inking of margins may be needed, to maintain a visual cue of the potentially abnormal tissue. Also, important to remember, no oversized tissue should ever be forced into a cassette.
Grossing Fatty Tissue
Cassette selection is also important. You can see that I have a picture here highlighting multiple types of cassettes. Importantly, when selecting a cassette, ensure there’s enough space in that cassette for fluid penetration throughout the tissue. If that cassette was totally filled there would be no room for a proper fluid penetration. The tissue is now ready for fixation.
The goal of fixation is to create the matrix of cross-links to immobilize proteins that make up the tissue. As our body is made up of all kinds of proteins and lipids, carbohydrates, etc., creating a matrix of cross-links is incredibly important. Once we do that, we stop autolysis which is the self-destruction of the tissue. Stopping autolysis prevents the tissue from acting upon itself as well as breaks down those intracellular proteins that will also prevent decomposition.
Driving the fixation process, is a fixative agent. There are several criteria that can and should be used when selecting the proper fixative. First, a general or routine fixative could be used when demonstrating general morphology. Should specific cell types of cell constituents need to be demonstrated, special fixatives can and should be used to preserve these structures. For example, there are fixatives specifically recommended for dense tissue, called fix-all. Lastly, the choice of fixative must be compatible with later staining steps. For example, Immunohistochemistry vs. routine staining. In IHC, you will want to avoid or limit the time in formalin, which could inadvertently mask the antigen, generating a potentially false negative outcome.
What are some of the properties of a good fixative? It should penetrate cells rapidly, as fixation works from the outside in. When specifically speaking about fatty specimens, it often helps to cut them open and make sure that it has multiple points of entry into that specimen, so the middle and outer edges get fixed equally.
When dealing with fatty or elongated tissue, thin pieces of cork, stapled to fatty tissue can be used to immobile the tissue, ensuring that the fixative can penetrate the tissue evenly. The cork prevents curling and uneven fixation throughout the tissue.
For reference, here is an image that highlights what can possibly happen when a large specimen isn’t fixed properly as a result of poor grossing practice. Please take a look at the picture on the right-hand side and there’s a little blue box.
What’s important is the pinkish area at the top of the uterus. The area is pink because it was not properly fixed. This could lead to an all too familiar conversation that goes something like this…
You are in the laboratory and you are approached by a pathologist. The pathologist brings you a slide, and states that everything is beautiful on this slide, except for one thing in one particular area. Then, you get the dreaded question, “why doesn’t this area look right? There’s a lot of distortion. The tissue doesn’t look fixed. I’m not sure how or why, because the rest of the slide looks so good.”
The reason it happened was because the specimen was likely improperly grossed and did not achieve fixation across all tissue.
The other thing to remember are the variables that can impact fixation. First is the choice of fixative. For example, once you open up a bottle of NBF, it starts crosslinking to itself. This can impact downstream results. Next, the volume ratios are important. You should have a minimum of 1:10 tissue to fixative ratio, ideally 1:20. This should help ensure optimal slides and images. Next, Tissue thickness is also an important variable, as it can impact the migration to the center of the tissue. While 4 mm would be ideal, when dealing with fatty specimens, the thinner the tissue, the better. Target 2 mm if possible. Many of the chemicals that we are speaking about have trouble penetrating the fatty layer, so the thinner the better.
When looking at desirable properties of fixatives, we are focused on the following. First, the fixative should provide reproducible and consistent results. When fixing tissue, we know that the tissue will shrink inside formalin, up to 10%, but what is more important is that cross-linking of the tissue results in sufficient hardening to retain cellular components. So, when you have successful crosslinking, the tissue hardens. To reinforce, formalin crosslinks proteins. Once the fixation is set, decomposition is going to stop.
Lastly, all fixatives should inactivate enzymes, kill bacteria and viruses, and enhance downstream staining properties.
Fixation of Fatty Tissue
One of the most common fixatives is formalin. Formalin is aqueous solution of formaldehyde. 10% formalin contains approximately 4% formaldehyde. The formalin needs to be buffered to a pH of 6.8 – 7.2 and to do this, buffered salts are added. The reason this is so important, is if formalin is not buffered, it will leave a pigment behind. Additionally, methanol is also added to minimize polymerization of formaldehyde molecules.
As we are speaking about fatty specimens, it is interesting to note that formalin is a required fixative for