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One of the most critical steps in histology is fixation, especially when it comes to fatty tissue. If a specimen is not well fixed, the lack of full fixation could affect staining steps and possible diagnosis. A laboratory thoughts of what alcohols to use in dehydration steps and the solutions used in clearing steps in optimum processing of fatty tissue is an important step to get these tissues to ultimate processing and fixation. This presentation will review the process of processing fatty tissue, processors types, and good quality measures for ultimate fixation of fatty tissues.
- Review the process of specimen handling before processor.
- Review Fatty Specimens and why they are so difficult to process.
- Overview of the different technologies for processing and solutions used.
- Expand on good quality techniques for processing fatty specimens.
MS. MARI ANN MAILHIOT: Good morning everybody and thank you for attending.
First off let’s talk about our objectives this morning. We’re going to review the process of handling specimens for the processor. That’s really important how we handle them, what we do with them when we get them into the laboratory, and things like that. If there’s no formalin we really should add formalin unless the surgeon specifically says don’t do it. We want to analyze the reasons why fat is so difficult to process, and we also would like to discuss the different technologies that are available to us now to process fat, including the different solutions that are on the market, and we want to also identify good quality control for fatty specimens.
Of course, we’re going to have to start off with grossing, that is 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. Are you going to use 10% buffered formalin?
Now, in the case of us presenting fat today, I’m thinking more of breast specimens so we do have to follow the CAP regulations about using neutral buffered formalin. Once we’re done with the fixation of course, we can go to dehydration on our tissue processor, it starts there and we have graded alcohols that will help us with that dehydration, and then we're going to talk about clearing reagents. We all know that xylene is one of the best reagents in histology, but we also know that there are some affects to our health from using xylene, so now the world of histology is thinking about maybe we should be xylene-free, and that’s not a bad idea from someone who suffers from adult-onset asthma as being exposed to xylene in the workforce long before we had proper air exchanges, and we didn’t even have air conditioning at University of Chicago, we had to open up the windows. You can understand how important this is to me to know that we’re going in that direction.
Last, but not least, we have infiltration with paraffin and the thing with paraffin we have to remember is you have to use the paraffin that’s the best one for you and your lab. There are so many opinions about paraffin in the histology world that I could not begin to say use this one, this one, this one. It’s entirely up to you and what works the best for you. There's no way you want to be frustrated sitting and cutting at a microtome because your paraffin is not the best. Last, but not least, is we have protocols for processing fatty specimens on some of the higher end tissue processors today.
We get our specimens into histology and to the lab and let’s hope there's proper identification on those specimens, let’s hope the surgeon has given us information about where that specimen was taken from, and possibly a little bit of history about that patient. Once that’s done and we’ve checked everything, we start our grossing process. Most cases if it is a breast we’re going to have to ink margins. Now we do inking in other types of histology to skin when we have an elliptical piece of skin, and I’m mentioning skin also because it does have a fat pad on it and sometimes it’s really hard to process skin specimens when you don’t get that fat fixed properly on that specimen.
The selection of the most represented pieces you’ve got this big breast specimen or this big skin specimen and you have to look at it and say which do I think is going to be the best piece for me to take and put into the cassette. Sometimes that’s a thinking process as you look at that specimen. Then 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 5 millimeters for routine overnight processing. We have to really think about this process ahead of time to make sure we get our specimen to the optimal size so that means no more stomping on the cassette to close it.
[Slide 6 00:06:31] You can see that I have a picture here of a cassette with a piece of tissue inside and there’s actually two pieces of tissue. There’s enough space in that cassette for fluid penetration throughout the tissue. That’s really, really important and throughout the cassette you want the fluid to get into the cassette and get into the tissue. If that cassette was totally filled there would be no room for a proper fluid penetration.
Our body is made up of all kinds of proteins and lipids, carbohydrates, and inorganic salts. What we need to do in fixation is to coagulate everything in our body including the proteins. That’s really, really important. Once we do that then we stop autolysis which is the self-destruction of the tissue. Stopping autolysis does prevent the tissue from acting upon itself and that’s really important. We break down those intracellular proteins and we also prevent decomposition.
It probably would be important for me to bring up something I think is important. I have pleasure last summer of working with someone in a histology lab that does regular histology but they also have been asked by the medical examiner in their area to take on their specimens and process them for them. They did not have a regular histology lab. She called me because she was really frustrated about the fact that her specimens were not processing properly. She could not get any proper sections and she was extremely upset about that.
Basically, her and I worked through all of the details of her processing and I have to say she really was on top of everything, she did all of the things that are required of an excellent tech, and we whittled it down to the fact that the medical examiner was offsite so they put their specimens in a bucket of formalin. One of the things that she told them is she wanted the formalin changed once before it comes to the lab. Probably that was not done at that lab.
I asked her when she received the specimens in her lab to change out the formalin. When we started doing that, her specimens increased so much, everything went so much clearer and much better on the slide so there’s a lot of things that we have to remember with fixation and one of them is to make sure we have clean reagents, we change our formalin on a more normal basis, so if you start grossing at 8:00 in the morning maybe at 12:00 or 1:00 you dump that formalin out and you either add more specimens to that tray or you start up another tray, but make sure you empty out your formalin. It is a big concern of mine.
The other thing that we have to remember, and I can tell you, you can see this in your H and E slides your hematoxylin will be a grey color and your eosin will be very pale. That’s because the fixation started in formalin but it finished in alcohol. You know that you did not have enough time in your fixative.
What are some of the properties of a good fixative? It should penetrate that cell very rapidly, it should also, starts from the outside, goes to the inside of that specimen and it takes a while to reach the area that it needs to fix. What am I saying about this? I’m talking about big specimens. You better cut them open and make sure that it has two or three or four points of entry into that specimen, so the middle can get fixed and so can the outer edges get fixed and hopefully the two will meet at some point.
When you bisect something you’ll always come back, especially if it’s a big specimen, and then take representative specimens from that. Columns need to be pinned on a board. They have fat also and if they’re not pinned and opened properly they will not fix property and we know that for a fact when we sit down to cut specimens.
Here is something that I thought would be a good reminder of what happens when we don’t cut a large specimen open. You take a look at the picture on the right-hand side and there’s a little blue box around it and a red box. There is a pink area at the top of the uterus that is not fixed. The next day you come into the lab and the pathologist brings you the slide, Mari Ann everything was so beautiful on these slides except this one particular area. I don’t understand. There’s a lot of distortion and it doesn’t look like it was fixed well but I don’t know how that happened because everything else is so well. It happened because we did not open up that specimen and submit that specimen to formalin and that’s very, very important.
If your fixative has a job to do that job is going to be stabilize the tissue. Why would you want that stabilized tissue? Because we’re going to subject that its use to really rough tissue processing. Going through alcohols and going into a clearing agent is tough on the tissue and then going into paraffin so we have to make sure that we have taken care of toughening up that specimen for the rigors of processing.
First thing we can talk about here is my choice of fixative. We know that formalin is our fixative. We’re going to use this for fixing all specimens and I did find some interesting information when I was preparing the presentation. Back in the later 1900s there was a gentleman by the name of Frederick Blum and he discovered formalin but used it as a disinfectant. I’m glad I wasn’t back there when he was disinfecting the laboratory because that stuff is really bad. Also, in 1987, the government said we’re going to standardize fixation and embalming and we’re going to use 10% buffered formalin. That’s just a little bit of information for you. Those of you that have been in histology for a long time, I’ve seen changes in histology that I just can’t imagine. I’ll be honest with you. It’s all for the positive, so we’ve really improved how we can process specimens and how we can get the best results to our patient. That’s really, really important.
The other thing I want to talk about is tissue thickness. I made the comment about no longer stepping on the cassette to close it. That is very true. Back in the day the specimen was falling out the sides of the cassette and that just is not acceptable. The other thing too is postmortem storage. I can address that a little bit because if come from a big teaching hospital and we stored everything from the 1920s on up and some of it was never thrown out. You know that some of those specimens that were stored in the early twenties the formalin was never changed out so now we have formalin acid and really and truly that specimen isn’t that useful if we wanted to any research on it.
The other thing to remember once you open up the bottle of formalin it starts crosslinking to itself. Second of all once you’re all done with a specimen and it’s properly fixed and you want to store it and you put it back in the bottle and store it, that formalin is going to continue to crosslink that specimen. I don’t think I really thought too [Slide 12 00:15:38] much about that crosslinking myself. I did later on when I was doing IHC and stuff like that, but I really did not give it that much of a thought. Good information for you to thinking about definitely when you’re storing your tissue.
What about fixation and desirable properties of a fixative the thing that I like the most is that it prevents shrinkage of tissue. We don’t want our tissue to shrink. The other thing is it provides the hardening of the tissue for intracellular components to be retained during processing. That is important. That’s what I was talking about the rigors of tissue processing. It activates the enzymes so they don’t destroy the protein. That’s really important. We’re going to always make a reference to staining properly. You want it to enhance staining. You do enhance staining if it’s properly fixed.
Formalin crosslinks proteins creating a gel and that stops that protein from acting on its job in our body. That’s really what that means. Once that action stops then decomposition is going to stop and some other things that go on if we don’t have that specimen in formalin.
The other thing that I think I want to stress here is there’s a relatively short time for fixation. Again, I have to bring up CAP because it’s a minimum of 6 hours to 48 hours for breast specimens if it’s a