Tissue Processing: A Cornerstone to Quality Downstream Testing

Thank you for joining this Leica Symposium on tissue processing, embedding and microtomy. The cornerstone to quality downstream testing. In this presentation, we will cover the following objectives: 

• Pre analytics: steps to ensure molecular integrity
• Fixation: Steps that are required to ensure morphological and biomarkers integrity.
• Prosection: which involves prepping and sampling for good processing.
• And tissue processing: reconsidering loading and leaving.
• We’ll cover embedding: tips to make sectioning easier.
• And microtomy: techniques to ensure that you have beautiful sections.
• At the end we will have a short period of time for questions and answers. 

Workflow Overview 

First, let's look at the workflow overview that's involved with tissue processing. First step is arterial or vascular ligation. Collection, then transport, reception, prepping, processing, sectioning, formalin-fixed paraffin embedded sections, and molecular methods. I have taken a few of these items and color-coded them to break them into sections that we can talk about more in detail.

Pre-Analytics

On this slide, we will discuss in detail pre analytics involving warm and cold ischemia. Warm and cold ischemia are more accurately applied to transplant specimens, but for this presentation relates to the time of ligation to collection as warm ischemia and cold ischemia defined in this presentation as the time from collection to fixative.

Pre-Analytics: Warm Ischemia

When we talk about warm ischemia, the factor that impacts the tissue quality are several items. The most important in the warm ischemia field is the time to fixation. You'll see below that it is requested that tissue be placed in fixation within a 20 minute window. The quicker the better because our stress responses are initiated almost immediately after the tissue is deprived of its blood supply. If you need to, keeping tissues near 4°C prior to fixation will slow down some of these processes induced by oxygen and nutrient deprivation. Prior to fixation, tissue should be maintained in a neutral isotonic solution with physiologic calcium and magnesium concentrations to avoid unnecessary disruption of adhesion systems and activation of signaling cascades. So an example of that would be PBS plus the calcium and magnesium.

Pre-Analytics: Cold Ischemia

The other factors that impact tissue quality include temperature of the fixative, type of fixative, volume, total time in the fixative, and the size of the tissue sample. As I mentioned before, we've already touched on the time to fixation and the temperature.

Fixation - Temperature and Type

Let's move on to the next slide and talk about the type of fixative. So, we've already talked about the time we need to get the specimen to fixation and maybe even the temperature we need to keep it at. The type of fixation that is most common in the laboratory is neutral buffered formalin and/or paraformaldehyde. There is a lot of discussion around these two items. Is one better over the other? A lot of the literature indicates, and websites indicate that they're basically the same. The only caveat is I did see that some commercial options is that they might contain a small amount of methanol, which might impact some of the testing.

Fixation - Volume

When it comes to the volume of fixation needed, usually the general rule is 10 to 20 times the size of your tissue. That can be quite a challenge for large organs, but for most of what's seen in life science arena, a small container 4 oz, 8 oz, 16 oz is usually doable. It is important that if you are removing whole organs that you do want to prep those organs so that the fixative and subsequent reagents you're going to see in tissue processing have access to not only the exterior, but the interior, especially items such as kidneys, lungs and heart.

For those tissues that are whole, or especially if we're going to be processing rat brain, perfusion is ideal also for cardiac samples too.

Fixation - Time

After you've prepped your specimen, whether it's bisecting the kidney or even piercing the cirrhosa of some organs such as kidney uteri, maybe even liver, even those organs that maybe have a pseudo-cirrhosa as such as prostate, we definitely want to make some type of incision so the fixative can get access if it's a whole organ. After that, for small specimens that have been sectioned, you want 12 to 18 hours for small biopsies. 24 to 72 hours for standard tissue. It is critical because formalin and although it penetrates rapidly, it fixes very slowly and the whole goal, especially when we talk about molecular methods, is that RNA is degrades very quickly. As soon as the formalin can get to that tissue and make those methylene bridges the better preserved you will see for molecular testing and especially for IHC.

Prosection

When we're preparing those samples to be processed in our tissue processor, it is important that we realize that we have to cut sections that will fit into the cassettes for adequate processing. We usually suggest sections less than 5mm thick or usually in the laboratories I'm used to working in, we asked residents or pH to cut tissues thinner than a nickel and the size of a postage stamp.

That is because tissue cassettes are limited in their size by three centimeters by 2 ½ usually, unless you're using mega cassettes. You want to allow for 5mm of space around each side of that tissue sample. It's also very critical that you select the right cassette. Usually for routine processing, you'll see the cassette at the top is most commonly used, but for smaller samples, especially for vessels; small biopsies, small skin sections that cassettes down below with smaller pores are usually ideal so that the sample is not lost.

Why does it matter when we're cutting our samples to be processed in those tissue cassettes? As you can see here from this section in the cassette, automatically you can see that it fits very tightly at the top and the bottom not allowing maybe for the fluids to be able to agitate through. If this tissue is too thick, you're going to see that the interior of the specimen as you cut into it on paraffin is not going to section as well, or maybe under processed. Also too, if the section is too large, it may not fit on the glass slide when you cut your sections. Those other items here too that you can run into when the section is not thinner than a nickel or the size of a postage stamp, or not even placed in the correct cassette. At this point, we are ready to begin our tissue processing.

Processing

Processing of tissue consists of three basic steps: 

1. Moving through alcohol to remove your free water.
2. Moving through xylene to remove the alcohols and get ready for paraffin.
3. Then the paraffin step, which certainly removes the remainder of any xylene that's residual. And then you're ready for embedding.

After the specimen is loaded into the processor, or sometimes it will go through another step of formalin fixation, but the first major step is to remove the free water, not bound water and usually you will see two to three graded alcohols and then usually three absolutes. This is a gradual removal of the free water, where it's a delicate balance of removing the free water, but not all for dehydrating the tissue.

The next step is xylene that removes the alcohol. Traditionally, we see in laboratories three xylene stations. It is advised that you have a small amount of heat on the last station before paraffin so that there's a nice transmission of one region to the next being that paraffin is a solid at room temperature.

The last step in processing is displacing that xylene with paraffin. I've seen a lot of literature and I'm seeing in life science laboratories where temperature is being taken more into account based on the testing that's done in the laboratory. Traditionally, we had temperature on every step of tissue processing it was a can I say necessary evil to make sure that tissue was processed because in the day we would rather have tissue a little over processed and under processed for sectioning. H&E and IHC was more forgiving for most of that, but as we move more to a molecular testing in the histology laboratory, it is critical to pay attention to the temperatures.

I see more low temp paraffins also being used. Please remember that it is very common to see pressure and vacuum used on every step in the processor, which is fine. But on the paraffin step it is strongly recommended that you only use vacuum because the PV which is a push and pull in the retort might be pushing that little bit of xylene that's residual in the tissue back and forth, instead of pulling it out. If there is a small amount left in the tissue, we'll certainly see that at H&E, which will give you a hazy, poorly defined morphology.

I have a little snapshot of the type of protocols that you might normally see in a laboratory. This is very common, you'll see that there's three graded alcohols, just two absolutes, three xylenes, and three parabens, which is very common in the laboratory. This slide doesn't include those temperatures or PV, but I did want to point out that it is becoming more and more common because of the molecular testing to customize protocols for tissue size, sometimes even for tissue type, especially for fatty specimens if anybody is using those in their testing.

One thing that we have been traditionally practicing in the histology laboratory that is, that we would take total time and just divide everything up among the steps as you can see. But I see that more labs are taking more thought into the times they want to set, where they're heavy on the front end of using 70% and lighter around the absolute because they're trying not to remove that bound water. The tissue coming out of the processors is actually easier to cut. The IHC and some of the molecular methods are more accurate because they are taking time to really delve into and customize the protocols with the tissues they're using and the testing that they want to do.

Processing - Maintenance

One item that's sometimes overlooked due to the busyness of the lab is maintenance. I will tell you there are many artifacts that you will see on your H&E, on your IHC, that will go back to the root cause of maintenance on the processor. I've put that exhausted reagents made for exhausting reworks, because it will require either reprocessing of the tissue or taking another sample because the tissues can't recover what damage has been done. It is very difficult to remove residual xylene once it's in the tissue, to get it out to go back. Usually another sample has to be taken. This ends this section when we talk about collection of samples, placing them in fixative long enough, making sure the sections are good and then processing them.

Embedding

We're going to move on. To some information about embedding. Embedding is pretty straightforward. There are some tips and tricks that are helpful. One thing to be mindful of, and I mentioned this previous, is that I see more life science people in the laboratory taking temperature into account, especially with molecular testing. Low temp paraffin is usually the option of choice. There are tools that we're going to talk about and one little note I made at the bottom and I'll explain this a little bit later is that this is not a slow cooker for your tissue.

Embedding - Temperature

Let's talk about embedding medium. Please be sure that we talked about the type of paraffin that's needed for your laboratory. Follow the temperature recommendations on whatever paraffin you may purchase. Please remember that this is not a slow cooker. I have been in laboratories where tissue cassettes were taken off the processor, placed on the embedder and either had intentions of embedding within an hour or so, and had an account where the cassettes had been in the embedder for a couple of weeks. This is a critical note as not only really your IHC be compromised, especially nuclear markers, P53, Ki67, HER2. It's really critical to make sure that they're embedded within a reasonable amount of time and not kept on the embedder. Also, the heat will degrade your RNA.

Embedding - Tools

Embedding tools were important. As we know there's a plethora of molds available. Whether you choose metal or plastic, create the consistency you need. I personally prefer metal. I've worked in clinical laboratories; it makes it a little quicker to pop those molds out. Also, too my tool of choice are forceps that are curved so that the ends of the forceps do not create any punctures in the tissue. As it transfers to the slide, I'm wondering why there are holes in my tissue, but allows you to press firmly on the tissue without creating any artificial appearance.

Also too is very popular, are tampers, they're just square pieces of metal, usually aluminum, and with a handle attached. You'll see the slide at the bottom right that allows you to keep the tissue on an even plane. This is critical, especially if you have more than one piece of tissue in the mold. We want to make sure that everything's on the same plane when you're getting ready to cut so that you don't have to sacrifice one portion of your sample to get to another part of that block.

Embedding – Techniques

In embedding the tissue, there are so many ways to embed tissue and everyone has their favorite methods that work for them because there's so many different tissue types, so many ways to embed, but a few tips I list here is that, making sure that tubular tissue such as vessels are always embedded on end so that you can view the lumen for samples that we mentioned earlier before cure readings that have multiple pieces, we want to make sure those are kind of toward the middle and all kept on the same plane by using that tamper.

With long tissue, diagonal always been great. Whether you put your block in horizontally or vertical, diagonally embedded tissue seems to take the impact off the blade when you're going to section and keeps the compression down. For membranes, it's always good if you can use an applicator stick and wrap that membrane around there and then place it in the middle and keep it in this central location.

With skin, one thing I found to be helpful is that a lot of times skin depending on where it's coming from; thick dermis or subcutaneous tissue attached, and so we would always embed those diagonally and I liked putting the skin down facing the blade so that I wouldn't be coming up from the sub Q which sometimes would cause the skin to separate at the epithelium. So that's one trick that might help with sectioning skin, that can be a little difficult at times.

Embedding - Tips

Embedding tips. These blocks are great examples of how you want to keep a 3mm border of wax around the perimeter. The only one I can see is at the nine o’clock position where it looks like some of that subcutaneous tissue touches the edge of that mold. This gives you the stability and the support you need with the tissue when you're taking those sections. I always want to make sure when you're finishing up your embedding and putting those cassettes on top, try not to capture any air bubbles. Avoid underfilling. If you can get to the top of that cassette, it gives you more support. If it is under filled, you risk the chance, especially with tissue that can be dense like bone, uteri, of popping off because it doesn't have that support on the backside.

I've seen scientists sometimes take blocks and you know, always in a hurry, but just happen to take a rack or a tray of blocks and throw them in the freezer, thinking that that would help move things along a lot quicker. When they were pulled out, it was cooled too quickly. It had a lot of cracks that ended up in the tissue and gives you that artifact on your slide of cracked tissue. On the opposite side of that, it's not good to let those blocks just solidify room temperature. You need your chill plate on or have an ice tray out that you can use. I've done this in a hurry, we try to get our molds out quickly so that we can go ahead and section and get done. But premature mold popping certainly sometimes will leave some of the tissue behind in the mold, if it's not ready.

Microtomy - Clean & Tight

Moving on to microtomy. The first thing I'd like to talk about with microtomy is the chuck holder. Make sure it's clean and tight. Usually they're very simple, as you can see in the image at the top or a little bit more complex on the bottom right. You want to be sure that once you have it set to a zero plane, you want to just set it and forget it. There's nothing worse than sitting down trying to cut tissue that is inevitably, it's always the case that it is a small tissue, and having the plane change where you might have the risk of losing that tissue when you begin to section. Once it's set at zero on either plane, you just want to set it and forget it. With either chuck holder, you will see at the bottom when you pull the lever forward to put your block in, there are two springs at the bottom. We've already talked about how we keep the blocks cold and some people use ice trays. Well, that brings in an element of moisture and so that moisture can collect at the bottom of that little groove where the block sits. It's always important to dry that off at the end of your cutting session and lubricate it with a couple of drops of oil so that you won't have problems down the road.

Also important is that you'll know that when you put that block in its chuck holder in the corners where the block is kept firm, paraffin can build up in those corners and change your plane too.

Microtomy - Blades & Holders

There are a ton of different blades out in histology, and there's a lot of conversation and controversy about which blades are better. You can use high profile or low profile and it just depends what works for you. Most holders of blade assemblies are equipped now that they can hold either one. As we discussed with the chuck holder, some of the items we will repeat because it's important the blade assembly. Everything you see on this blade assembly to the right, you'll see there are three handles, one for the blade, one for the assembly that will move the blade holder back and forth, and then one for the angle.

You want to make sure that all of these are kept free of paraffin and lubricated according to manufacturer recommendations. You will see on the side of blade assembly, there is always a setting for the angle. Based on the design of the blade assembly, you will see an angle that needs to be set anywhere from three to four degrees. I have seen them go beyond that or under that based on the design of the blade assembly.

Why is that important? It's because you must have the right clearance angle to properly section your blocks. You can see here on this diagram that if it's not set correctly, you can get a variety of artefactual cutting, whether it's vibration, Venetian blinds, or actually the blade biting into the tissue and you're losing part of your sample. Some of the tips I offer when people are cutting on a microtome, is to make sure that the sides of your blocks are clean. That extra piece of paraffin like we discussed before, can get trapped in the chuck holder, or it can eventually when you're cutting and not keeping the back of that blade clean, you'll see a paraffin build up. That will keep you from getting ideal sections because it won't ribbon properly.

Microtomy - Problems

Let's talk about some of the things you might see in the laboratory that would be helpful. Keeping the blocks cold is important. You can see here from the block and the ribbon that came off of it, if it's not chilled properly, you'll get compression, and even more so beyond that if you keep cutting, you'll see where the section is not cutting evenly, and you'll have holes in the section, as you can see in the bottom right-hand image.

More problems, especially if the blades aren't changed very often, you will see compression of the tissue. As you can see on the water bth to the right, top right and the section on the left that came off of that. If the blade or the angle is not correct, here's an example of Venetian blinds.

Some of the tips too I've suggested is using a dull straight edge to take off the sides of your block because sometimes a fresh sharp edge of the block will help with the ribboning. If you have blocks that consist of tissue that tends to be on the dryer side, such as pig skin, especially blood. Sometimes a trick is just to dip it in a little bit of chilled water for a few seconds, put it back on the microtome, and then take sections right off the top.

Microtomy - waterbath: boring, but critical to quality

Once you get your ribbon, then we're going to place it on the water bath to be able to pick it up. Let's talk a little bit about that. I know the water bath is boring, but it's critical to the quality we're looking for. Temperature is especially critical because if it's not hot enough, the section won't be able to flatten out and lose all those wrinkles. Then if it's too high then it can cause the section to break apart and we don't want that either. You can see at the bottom here the water bath is a little too warm and the section is starting to separate. Under H&E you can actually see that separation.

With water baths too, I wanted to make sure I mentioned that if you are doing molecular testing, it might be important to use ultra-pure water. I think it's called DEPC water, which some of you are probably already familiar with too. If you are doing molecular testing such with RNA, it's probably wise to use RNAse to clean everything up around your instrument and on the water bath so not to contaminate it.

I promised you a histology hack, and there's a ton of them out in the field, but I'll bring you this one when it comes to sectioning. If your sections, and sometimes this happens with tissue that's just dense, you just can't seem to get a good ribbon and every section you take, you've chilled it, you've moistened it, you've got your temperature at the right temp in the water, and you still just can't get that ribbon. As you can see this one on the upper left is full of wrinkles. Take a beaker or a glass dish and make up a really weak solution of absolute, like maybe around 40 to 50%. We can't use charged slides on this, but you can pick up the final product with a charged slide. Make your ribbon and then lay those few sections on that alcohol bath. Then take a non-charged slide, which are usually just the basic cheap slides, right? Pick up that ribbon and then gently place it on your water bath. You will be amazed at how that section will gently spread out and usually get rid of all the wrinkles so that it's ready to be picked up with your charge slide or slides that are designed for IHC.

Microtomy - Slides: they all look alike

One thing about slides after they are picked up out of the water baths, that I think some of us don't think about is; whether we put these right away in the oven or if we're going to leave them out at room temp. The goal, whether you do either one, depending on if you're doing molecular, is that we want to remove all the water that's behind that section that's on the slide. With charge slides, it's a little bit more of a challenge because it's charged to hold the section, but it also tends to hold water. Every time you pick up that section, you want to make sure that you set it upright on a paper towel or something that will cause it to drain well. Be careful with charge slides. If you pick up a section and it didn't position right, or if you got a little crease in it, you want to lay it back out with charge slides, you can't double dip in the water bath. You might just need to cut another section. Also, with these slides that are designed for IHC, they are not only charged, but they're also designed that sometimes they're hydrophilic. It's tough to try to reposition or pull that section off. You may just have to cut another section. With the goal being in mind of just trying to get the water out from behind the section.

In more conventional settings, if you're not doing molecular, sometimes you could leave slides out overnight and that would work just as well. In a molecular setting, we have to be more cognizant of RNA degradation and for antigenicity when it comes to IHC also. So I've been told that recommendations are usually a low temp oven around 60°C for about anywhere from. 40 to 60 minutes.

You can see there's a couple of examples of things you'll see if water is trapped. The upper right-hand side, there's a water droplet that got trapped and you can see it's quite evident once you stain it with H&E. On the left, the same issue happened with residual water. The morphology in this area too will be hazy and poorly defined sometimes, especially if they put the slides in a high-heating oven.

Microtomy - Oven: not too hot, not too cold, just right

This is just a repeat of the oven tips that we don't want it too cold or too hot, just right. Although H&E is for the most part forgiving, you still will see artifacts if it is too hot or too cold. If the water has not been removed or you boiled the little bit of water was behind that section, we certainly see it with IHC. You also see, as I mentioned before, the loss of cellular detail. No doubt, you'll lose the quality that you want to see with some of your molecular methods.

There has been literature out there that talks about sectioning tissue for IHC, not too far in advance. If the sections are taken and not stored properly, even in clinical settings, they used to do it where it would sit at room temperature for a few days until they did it and we're seeing that we even at that short amount of time you see loss of energy. I think especially in a lab that is doing a lot of molecular testing that would want sections that would cut right away. There's a recent article out that talks about if the sections do need to be cut, then coating them with paraffin will preserve that RNA and IHC targets. I hope you've enjoyed this presentation. We'll now open up for questions and answers.