From Flab to Fab: How to Properly Handle Fatty Tissue for Optimal Pathology Results

Ladies and gentlemen, today we're going to talk about fatty tissue, otherwise known as the bane of the histologist’s existence.  Adipose (the fancy term for fat) is one of the more difficult types of tissue to handle in histology and is often the most commonly re-processed tissue in many histology laboratories.   Throughout much of the histology process, fatty tissue can pose challenges, but properly processing fatty tissues like breast, colon, brain, and others are an essential part of the pathology process. According to the American Cancer Society, after skin cancer, breast cancer is the most common cancer impacting women in America.¹   After skin, breast cancer was also the leading cause of cancer globally making up 12.5% of new cases of cancer in 2020 regardless of gender and 25.8% of new cases for women according to World Cancer Research Fund International.²   There is no way we can escape it, so how as histologists do we properly handle fatty tissue to give us the best opportunity for optimal pathology results?

First things first, let's talk about tissue thickness.  When it comes to fatty tissue, it's important to get that tissue thickness just right. Too thick, and we are just setting ourselves up for failure.  When tissues are cut too thick, we reduce the surface to volume area ratio of the tissue.  That is just a glorified way of saying that with thick tissue we have a whole lot of space inside the tissue that needs to be filled or penetrated with either our fixative or the processing reagents, yet we do not have a whole lot of surface space to let those fixatives or processing reagents get access into the tissue.   Especially for fatty tissue, that means that we are likely to have difficulties properly fixing and processing the tissue.  We also do not want to cut too thin either because we want to submit adequate tissue, and we also do not want to make it unnecessarily difficult for the grosser. So, what's the sweet spot? Typically, you want to aim for slices between 2-3mm in thickness.  I can already hear the collective groan from all the grossers out there, and as someone who has spent many hours grossing myself, I feel your pain.  However, doing things the right way is not always the easiest way, and for the sake of patients who may be receiving a life-altering diagnosis at the end of this process, we owe it to them to do the right thing even if inconveniences us a bit.  There are also some tips to help us out, too.  For example, bread-loafing large fatty tissues to increase the surface area and giving them a chance to fix some before grossing will help firm up the tissue to make it easier to cut those standard nickel-thick slices.  There are also grossing tools that are available from various vendors that can help make sectioning to the appropriate thickness much easier. 

Next up on the road to better fatty tissue results, fixation. If you don't fix your tissue properly, especially fatty tissue, you're going to have a bad time.  Fixation is what helps preserve the tissue and prevent any further degradation, so it is important that the tissue be placed in fixative, commonly formalin, as soon as possible.  The cold ischemia time, or the time between removing the tissue from the body and placing it into fixative, should ideally be kept to less than 1 hour.  With fatty tissue, it's especially important to use a fixative that can penetrate through the adipose tissue.  Formalin is a common choice, but make sure you're using the right concentration and giving the tissue enough time to fully fix.  Using 10% neutral-buffered formalin is the common choice.  Remember, however, that formalin is an additive fixative, so as it molecularly binds to the tissue, the solution gets “used up” over time.  The formalin at the grossing bench or even on the tissue processor may start out at 10%, but the longer it has been used, the less effective it will be.  Keep that formalin fresh, especially when dealing with fatty tissue, for the best results.  Otherwise, you'll end up with some mushy, unrecognizable tissue after processing that no one wants to deal with.  Using alcoholic formalin for the fixation of fatty tissue prior to tissue processing can also help with penetration and fixation of fatty tissue.  Alcoholic formalin can be made in the laboratory, but for consistency and to avoid the risk of human error, ready-to-use alcoholic formalin solutions are readily available from several vendors.

Using a proper volume of fixative is also crucial.  It is common for large fatty tissues like breast and others to arrive in the laboratory in formalin containers that simply are not large enough to accommodate a volume of fixative appropriate for the tissue size.  You have likely heard of either the 10x or 80/20 rules when it comes to fixative volume for formalin.  The objective is to have a significantly larger volume of fixative compared to the volume of the tissue.  For very large tissues, it may not be practical for the tissue to be transported in a large enough container for proper fixation, so in some cases it may not be until the tissue is grossed and cut down into cassettes that it is finally placed in a container with an appropriate fixative volume for adequate fixation.  Remember this when you are considering the next important aspect, which is time.

Allowing enough time for fixation is extremely important, especially when using formalin for fatty tissue.  The general rule of thumb is that formalin penetrates tissue at approximately 1mm per hour.  Of course, that rate depends on the tissue thickness, which we’ve already discussed, as well as the structure of the tissue.  Fat and water don’t get along, so with formalin solutions consisting of primarily water, the lipids within the tissue do not cooperate well when it comes to letting the solution in.  Once the formalin does finally penetrate, though, it takes significantly longer for the cross-linking to occur to complete fixation.  For some advanced staining methods and molecular testing, we must take care with formalin fixation to not over-fix the tissues since it can have an impact on those results, but the problem most often seen with fatty tissue is under-fixation.  Unfortunately, there seems to have been a bit of a misunderstanding that has occurred in histology since the release of the fixation guidelines for HER2/neu, ER, and PgR, which states that breast tissues should be fixed between 6 to 72 hours.  Some have incorrectly assumed that the minimum threshold provided of 6 hours in formalin means that the fatty tissue is completely fixed by that time, but that is not the case.  That minimum time threshold is not a measure of the completeness of fixation but rather simply the minimum time in formalin that seems to not negatively impact the results of the testing.  In other words, the tissue may not be fully fixed at 6 hours, but the results are likely not to be negatively impacted.  For large resection fatty tissues, allowing 24 hours or more for the tissue to fix can help yield better outcomes for tissue processing and microtomy while still being well under the 72-hour maximum threshold for the guidelines. 

Finally, let’s move onto tissue processing.  As many of us have experienced, tissue processing can get a bit tricky with fatty tissue.  If we're not careful, we can end up with some pretty disappointing results, but if we’ve covered the first two points and submitted an appropriate tissue thickness at grossing and allowed it to properly fix, we will have set ourselves up for better success at processing.  When it comes to the most used conventional processing reagents, defatting of the tissue is primarily accomplished with xylene.  Unfortunately, the clearing steps in conventional processing protocols happen after all the fixation and dehydration steps have typically concluded.  Prior to the defatting occurring, the lipids within the fat cells in the tissue act to impede fixation and dehydration, generally slowing the processes down and often requiring the processing protocol to run for an extended period.  Inadequate time in xylene can also leave lipids in the tissue that can impede paraffin infiltration, resulting in an oily, under-processed tissue that ends up blowing up on the water bath at microtomy. 

All is not lost, however.  Here are a few tips to help with processing fatty tissue.  As I already mentioned, using an alcoholic formalin prior to processing can get us going on the right track.  This combination reagent helps with penetrating the tissue as well as starting a bit of the dehydration process while the tissue is still fixing.  By using a combination reagent, you get a bit of action from both without having to extend the protocol time like you would if the reagents were separate.

Another processing tip is to add a third change of xylene into the solution configuration for the protocols on the tissue processor.  Many conventional processing protocols use just two changes of xylene.  Two changes are fine, but as the primary defatting reagent, when processing a lot of fatty tissue, it can be helpful having three xylene changes on the processor instead to help ensure complete defatting prior to the paraffin infiltration. 

An alternative to using a third xylene station in the solution configuration is to use a mixed reagent station of both alcohol and xylene instead.  A combination reagent of both alcohol and xylene between the end of dehydration and the beginning of clearing allows some defatting to begin to get those pesky lipids out of the tissue while there is still some alcohol present to help complete dehydration.  Like the alcoholic formalin, the benefit of using a combination reagent is that you can extend the dehydration and clearing without extending the overall protocol time since they are occurring simultaneously during that step.  This is not a new trick and was used more often earlier in histology when turnaround time and staffing were not quite the issues they are today.  Using mixed reagent stations on the processor has also become a bit of a forgotten tactic because of the inconsistency and risk of human errors associated with having to create those mixed reagents manually and offline the processor.  As technology has changed, though, processors now exist that can automate the creation of mixed reagent stations onboard the processor to take the risk and time issues out of the equation.  By incorporating some of these tips along with a protocol of adequate length and reagent ratios, we can achieve efficient, fat-busting tissue processing.

And there you have it, folks! A quick guide with some tips and tricks to dissecting, fixing, and processing fatty tissue for optimal pathology results. Just remember, the key to success is in the details - proper tissue thickness, adequate and proper fixation, and careful tissue processing can make all the difference.