Tips & Tricks to Better Histology (Part 1): A Review of Key Factors Influencing the Quality of Tissue Preparation and Processing

Hello, everyone, and welcome to part one of our two-part series, Tips and Tricks to Better Histology in Tissue-Based Research, a review of key factors influencing tissue preparation and processing. I'm Suzy Valdez of LabRoots, and I will be your moderator for today's event. Today's educational web seminar is brought to you by LabRoots and sponsored by Leica Biosystems. For more information about our sponsor, please visit their site at leikabiosystems.com. Let's get started. I want to remind everyone that this event is interactive, and we encourage you to participate by submitting as many questions as you want at any time you want during the presentation. To do so, simply type them into the Ask a Question box and click Send. If you have any trouble seeing or hearing this presentation, click on the Support tab found at the top right of your presentation window or report your problem by clicking on that Ask a Question box located on the far left of your screen. I now want to introduce our presenter, Fiona Tarbet, a product manager at Leica Biosystems in Melbourne, Australia. For a complete biography of our presenters, please visit the biography tab at the top of your screen. Fiona, you may now begin your presentation. Welcome. 

Thank you for the introduction. Today I'd like to talk to you about just some different tips and tricks to better histology and better quality throughout the laboratory. These are just a compilation of things that we have learned over the long journey. And hopefully you'll find this a very informative couple of talks. First with our disclaimer. This presentation does provide educational material. This is not intended to be medical or regulatory or legal advice. 

First of all, I'd like to set the scene. It's often difficult, as anybody who's working in a laboratory, whether it be clinical laboratory or in a research setting, there's a lot of pressure today. There's often less time to think problems through when problems do occur. Often problems are not necessarily brought to your attention in a timely manner. You may find that there is a problem long after the blocks are cut and the tissue has been stained. There's more pressure to react quickly and therefore quick fixes. And the staff might be inexperienced or not fully trained. Those interpreting the sites may not be the people that would understand some of the problems caused by incomplete fixation, excessive sized specimens or protocols that are too short. You're often expected to do more with less resources. There's an accelerating pace of technological change and monitoring, maintaining and improving quality under these circumstances is a major challenge. 

You do need to be thinking both globally at a management level and locally at a bench level to identify the key factors affecting quality. So in simple terms, what we are trying to do every single day is produce high quality sections and stains in minimum time from blocks that cut easily and are stable and put on storage with accurate identification and labelling in minimum time and consistently every day. Simple, isn't it? As a challenge, just think of six actions you would take to improve the quality in your laboratory. You can probably think of more than six. What you can think about in terms of the actions are management actions and bench-level actions. The majority of this talk will be on a more technical level. 

We'll be talking about bench level actions, but just to go through some of the management level actions to think about. There's staff recruitment, staff induction and training. There's staff accountability and traceability, who did what and when. Staff shift rostering to make it more interesting for people and also to rotate staff through. Staff task rostering, ongoing professional development, staff morale, interaction of all staff with pathologists or senior scientists, selection of instruments and equipment, equipment maintenance, reagent quality and supply, lab layout, conditions and workflow. All critical. At a bench level, there are just so many different factors that do affect technical quality. As you can see from the graphic on the right-hand side here, we've put together just so many different factors from all the different stages of your workflow. And these are probably just a few. There's probably a lot more that people can think of. 

Today's webinar will be concentrating on the pre-analytics and up to the processing, and the next webinar will be concentrating on the following process from embedding through to coverslipping. First, here's a quote from Bracegirdle, and this was sometime in the last century, but nothing has changed in this aspect. If tissue is not in a healthy living state when it is fixed, it can only reveal abnormal details, whatever its subsequent treatment. Think about some of the prefixation damage where this could occur. Heat damage, where forceps or forceps or cautery happens and the tissue is left burnt, if you like. The morphology is lost, tissue is shrunken. Desiccation, often tissue does not get fixed in a timely manner. You know, sometimes you might find that the tissue is stuck to the lid of your container. Crushed artifact, tissue is treated a bit rough, and it is partially crushed. Autolysis, this can occur if fixation hasn't happened in a timely manner. 

What can we do? We can document problem sources. Particularly around cold ischemic time, this is critical and is often missed. Get the word out, education, training, information sheets where people have to list how long a tissue is fixed for at the time, when it was put into fixation, the role of the pathologist, internal and external communication. Improve specimen transport if possible. Think about what the role of the lab is. How are tissues treated when they come into the lab? Are large tissues automatically opened and allowed to fix? What does the lab do? 

Fixation, some common problems. Inappropriate containers, inadequate volume of formalin, poor quality fixative, or tissue fragments that are roughly handled. Think about the size of the container. You should be having a container that fits the specimen. You should-- there is generally a rule of thumb of 1 in 20, certainly no less than 1 in 10 volume tissue to fixative. The fixative pH is critically important to make sure that fixation happens correctly. Some of the consequences of inadequate fixation. There's nuclear evacuation. You might have a shrinkage and separation, particularly if the tissue has been fixed in alcohol. There's formalin pigment if the pH is incorrect or zonal fixation where the tissue has not been penetrated accurately and therefore the external part of the tissue might be fixed in a different way than the internal areas of the tissue. Again, some of the consequences that you might see on downstream processes. 

On the left, you can see a breast tissue where there is uneven staining due to zonal fixation. You can see a tonsil that has been stained for a kappa light chain where well-fixed tissue is showing very strong, well-demarcated staining and poorly fixed and weak reaction on the right-hand side. Same tissue, just poorly fixed. Here we can see some of the differences that you would see if a tissue is correctly fixed in formalin or has had inadequate fixation in formalin and it's fixed in ethanol. As you can see, there's quite a large amount of shrinkage, but it's actually, they look like different tissues. We have the ability and many of these slides we have created internally. We’ve used the same tissue, just cut in parallel, one fixed in formalin and the other fixed in ethanol. And as you can see, there is a very big difference between that fixed in formalin and that fixed in ethanol. 

What would make an ideal fixative? An ideal fixative should react rapidly and completely with tissue, fixing all the constituents without removing any of them. It would penetrate rapidly and deeply. Tissue would be stabilized to withstand all the chemicals that are used in later processing. They should be minimally distorted by swelling or shrinkage. It should be hardened sufficiently to make handling easy. And you should be able to perform a full range of staining methods. And that would include IHC or any molecular assays that are needed. Tissue should be left in fixative indefinitely without harm, and should be cheap, non-toxic, and non-inflammable. Unfortunately, there is no such thing as an ideal fixative. 

A lot of what we need to do is a compromise, and formalin is the world's most common fixative. There are pros and cons to formalin. It is relatively stable, it is relatively cheap, it does favor basophilic staining, most stains are possible, though obviously with IHC, anyone familiar with that will know that you do need to do antigen retrieval. The tissue consistency is okay, the tissue shrinkage is acceptable, morphology is acceptable, and prolonged fixation is generally okay. But the cons are that it does penetrate slowly, it fixes slowly, it is unpleasant, and it's toxic. Not as good results with acid dyes, requires retrieval for IHC and ISH. If it is left in an unbuffered state, you can get formalin pigment, and it does require buffering. 

There are many commercial fixatives and commercial non-formalin fixatives on the market. It's always good to be aware of what the non-formalin fixatives are. Most people looking at tissue nowadays are very familiar with the artefacts that come with the formalin fixation. There may be differences with the different fixatives. So always understand what the fixatives are. Always understand too. There are some, for instance, that require only up to seven days, that only last up to seven days. It might require refrigeration. Yes, just be very aware of the choice of fixative. 

Some of the fixation factors. There's the rate of penetration and diffusion and the rate of reaction with formalin. It's often described as the penetration reaction paradox. While it does, formalin does penetrate relatively rapidly, the actual fixation is quite slow. First forming a methylene bridge reversible with the primary amines, and then more complex structures as fixation continues. And this is why this is sort of the basis of antigen retrieval, where the initial reversible part of fixation allows us to retrieve tissue so that we can then perform IHC. More in a clinical world. There are more and more rules to try and standardize fixation. Obviously in research, this is an absolute opportunity to try and standardize because it is just so important. Fixation is continuous. Fixation is necessary to happen immediately. If not immediately, need to understand just how long that time was between when a tissue was removed from a living body and put into fixative.

In the clinical world, there are rules about between 6 and 72 hours, so limiting it on both the upper and lower ends. Just remember the basics. With fresh tissue, fix it as soon as possible. If fixation isn't possible, refrigerate, but don't freeze. Freezing can cause an artefact or ice crystal artefact. Fresh tissue may be infectious. Do not distort and label very carefully.

Remember proper penetration. Fixation, the fixative should penetrate from all sides. Make sure that your container allows for that. Make sure that your cassettes allow for that. The cavity should be opened. If you have a large organ, make sure that you do open before fixation or if it's come in. Make sure that you inspect and open them just to allow the fixative to get in. Perfusion of some specimens could be advantageous. The thickness of the specimen is very important. Some agitation is useful just to move the fluid around and allow for diffusion. An adequate volume of fixative is vital. As I said earlier, one in 20 is a rule of thumb, certainly never less than one in 10. Sufficient time is required for penetration and fixation. Up to 24 hours, as you can see, the 6 to 72 hours is given for breast tissues, particularly if someone wants to do an ER or PR, which is an IHC. But 24 hours, if you have 24 hours, is considered ideal. Room temperature is satisfactory, but heat will increase penetration. But remember, excessive heat during fixation might also cause other metabolic reactions to occur. It's a balance. 37 degrees would be perhaps sufficient for fixation heat. 

Again, the right choice of fixatives. Carefully made-up from reagents and a suitable quality and at the correct pH. If you are making up your own fixative, it is important to understand exactly what the proportions of the salts, if you're using buffered, or to realize what is going to happen if you're using an unbuffered formalin. Formalin is particularly susceptible to pH. And as well as fixation, you do need that combination of the correct pH, seven, or slightly alkaline. And obviously, fixatives aren't going to be so affected by the pH that if it is more acid, you will not only start becoming formic acid and therefore having those artifacts, but you would also-- the fixation reaction, the chemical reaction just won't occur. Many commercial formalin fixatives do contain up to 10% methanol. This is to stabilize them. But again, remember that if the commercial fixative does contain a large amount of methanol, then they will be the alcoholic fixative first and then the formalin chemical reactions. Check specimens received in fixative and replaced if necessary. If a specimen has been in a fixative or formal and fixative for too long, again, it starts to become ineffective as a fixative. You do need to replace it. Use only one for primary fixation. And if you do need to do a post-fixation step, then make sure that, again, it is appropriate for what you want to demonstrate later on. Fixed sticks are also toxic and irritant, so be careful. 

Moving on to grossing. Getting the message through, thick specimens will not be properly fixed. Fixed specimens process poorly when normal routine schedules are used. So how do you enforce this? On the right, you can see some images. The most routine cassettes are only about five millimeters in depth, so you do need to cut the tissue quite thinly. And I believe in the US, it's often considered the size of a dime is appropriate. Also with larger tissues, I talked earlier about opening cavities. This is another, a large liver, I think, that we have what we would call breadboarding. And that just means slicing at appropriate intervals just so that the fixative can get in. You can also soak paper towels and put them within the sections that you've cut and then sort of reform and reshape so that you don't distort the tissue, but you also ensure that you get adequate fixation. 

Think about some of the common problems. If a specimen is too thick or too large for a cassette, because of the thickness, the specimens will not process using your standard schedule. In extreme cases, cassette bars will distort the specimen. And you can see here some extreme examples where a tissue on the left was too large when it was put into the cassette and has now retained the outside or the bars of the cassette as it has shrunk as it has fixed. The middle shows that tissue often is jammed into cassettes. If it doesn't fit in a cassette and then it's not going to process correctly. Specimens shrink during processing. The cassette choice must always allow for this. We saw in the previous slide where the specimen was too large for the cassette. Equally, you must think about, is the tissue too small? Is it friable? Will it fall through the cassette? Make sure that the cassette choice is the correct choice. There's always a risk of specimen-to-specimen contamination if tissue comes through the holes in the cassettes. 

We've also seen that for many different techniques, it is appropriate to use what we would call a mega cassette. So mega cassettes are, as you can see, there's an example where at least twice the size of a routine cassette. This does create challenges for correct fixation, making sure that we do not treat a mega cassette or a tissue that fits in a mega cassette in the same way or fixed for the same time. It will need longer fixation and grossing. We need to make sure that we correctly gross so that it still fits within the large cassette. And again, particularly as there is so much material that it isn't crammed in. And for processing and cutting, we may need new microtomes, we may need new equipment. 

Moving on to the next stage, our tissue processing. What to look for when assessing and monitoring processing quality. There's a theoretical approach to optimizing processing. Advantages of batch processing are based on specimen type and dimensions, the causes of poor processing quality, and getting the best results from your tissue processor. What are the effects of poor processing? First, there are the macro effects, where the tissue has an unusual physical property at embedding. Section preparation may be difficult, and blocks might deteriorate rapidly. Then there's the micro effects, which is the physical quality of section is poor, the tissue morphology is poor, or the staining quality is poor. So just going through some of the effects of poor processing. 

You might see this demonstrated by the time you cut your tissue. And as you can see there, the tissue has not been processed correctly, leaving a large hole in the center. It might still look in its native state, but obviously, once you start to section it, there is nothing on-- you are unable to section and you lose the tissue. You might see some damage, large cracks, which again are shown on that slide on the right as cracked and lost tissue. Here we can see some examples of poor processing, not always what you might expect. This is a tissue that has been inadequately the tissue has been too large for the processing protocol that we saw. On the outside, we can see that the tissue processing has been reasonable. But as we move further into the tissue, we can see the gross cracking because the tissue has not been supported. And then in the very center of this tissue, we can see that there still remains some water in the tissue, so that we have a number of different effects in this tissue where the processing protocol was not long enough to penetrate, to allow the reagents to penetrate in to remove the water, but then also left sort of alcohol within the tissue and cause drying and cracking. 

What you might see is when you're preparing your section that the block has poor texture, that it's not uniform, it's not cohesive, the sections might compress, or the ribboning might be poor. Again, this is not the microtome. This is the processing. You might see when you are floating out your tissue that the sections sweat on the water bath, the components separate on the water bath, or that the sections are impossible to flatten. You may also see that the block stability reduces on storage so that the specimens start to shrink in the block or the block contains opaque patches, which means that there's still some residue solvent left in the block. 

We talked about the micro effects. So physical quality of the sections. You may see that the sections are disrupted, that they adhere poorly to the slides, that they're cracked, or that they're uneven in thickness. The quality of tissue preservation. Sections can show poor nuclear detail. They might show poor cytoplasmic detail. Some special tissue features can be disrupted, fibrous elements poorly preserved, or preservation is not uniform throughout the specimen or the quality of staining. 

The staining may not be uniform. Nuclear staining can be poor. We can see in this example, there is still water left in this section, and the nuclear staining has almost completely disappeared. You're just seeing what we would describe as a blue hue. The cytoplasmic staining could be poor, as it may have an effect with alcohol, or again, in this instance here, we can see that there is still a bit of water left in that tissue affecting the cytoplasmic stain as well, or that the extracellular components are poorly demonstrated. All of these could be effects in different ways. And it is a way of assessing your tissue to have a look at the different aspects from physical quality, from the quality of the morphology, but also the staining. You may have poor staining because you have poor staining, but often staining is an effect of the poor pressing as well. Consider what is causing any sort of artefacts you see. 

As a theoretical approach to optimize processing, you need to think about, do you need to separate out your tissues? You may have small tissues that don't require long processing protocols. or that your processing protocol that you have, or the instrumentation that you have has a slightly higher ambient temperature. And therefore, your protocol might be optimally shorter for smaller tissues or little friable tissues. You may also have medium-sized tissues, which is sort of optimally processed, say at around four to six hours. But then you may have large tissues. So how do they process? I mean, the larger the tissue, the more time you need to penetrate into those tissues. You might have more dense tissues, more fatty tissues. You need to think about what your tissue type is. And if you can separate out or optimize the processing for these tissues, it is always a good idea. 

Here's just some examples where we might consider one to two hours for a two-millimeter endoscopy biopsy, a slightly longer time for myocardium. We might see a wedge of cervix, which is quite dense, hard tissue, and often a larger section might be taken, and that would require a longer time. There's sort of a minimal acceptable quality If you do need to put all of your tissues in together, just be aware of where that line might be. To optimize processing quality, you could process tissues in batch based on specimen types and dimensions. The challenge is always to do that with the number of instruments you have to process tissues as well as maintaining an effective workflow. Think about what instrument choice you're having to satisfy demands. Think about the workflow of your laboratory and whether you can optimize the workflow to fit in with the needs of your specimens to optimize the tissue processing quality. Another challenge, so think of listing 12 different causes of poor-quality processing. Probably you could think again of a lot more. 

Here are just some that we've prepared earlier. And as you can see, it is a can of worms. There's incorrect solvent, there's a specimen type, there might be incorrect fixation, there might be faulty dehydration, poor clearing, poor grossing, under filtration, under infiltration, process of malfunction, process is not maintained properly, poor quality reagent, inappropriate sets, excessive specimen load, scheduled too long, scheduled too short. There might be calcium in the tissue, or the temperature isn't correct. If the tissue isn't fixed before processing, as we went through some of the reasons for the fixation, you can see the effect of short fixation, then the processing will not be will not be good. The specimens are too thick, poor grossing techniques or tissue that no one can decide on which part not to put in, so they put in the whole lot into a cassette, the tissue is too dense for that particular protocol, it's too fatty and it, again, needed a special protocol to deal with the fat. Tissue contains calcium deposits or foreign bodies such as sutures or staples or synthetic grafts, always best to remove it grossing before we get to the microtome site. The schedule was too short, so specimens were not properly fixed or not properly dehydrated, not properly cleared, not fully infiltrated with wax, or a combination of all of the above. The schedule was too long. Tiny or delicate specimens were exposed to dehydrants for too long or exposed to a higher concentration of dehydrants too soon. And this is particularly important when you have poorly fixed tissue. As you saw earlier in the presentation, if the tissue is fixed in ethanol rather than formalin, it causes a great deal of shrinkage and a completely different picture. It could be exposed to clearing agents for too long, exposed to hot wax for too long, high temperatures for too long, or a combination of all of the above. Think about the proportions of your protocol, not just haven't using any one-size-fits-all protocol. It's important to think about every aspect. 

As I said, with the incorrectly proportioned for the specimen type, you may have insufficient dehydration, excessive dehydration before clearing, insufficient clearing before wax, insufficient time in wax, excessive time in wax. And remember that the time in wax is also not just when the protocol finishes, but when you remove the tissue from your tissue processor. You may process overnight, having an end time at six or seven in the morning, and then the tissue is not retrieved from the tissue processor till nine. That is all extra time in wax. That by definition is still heated. Remember to think about that when you're thinking about your tissue processing protocols. 

There could also be a problem with loading a processor. The wrong schedule is chosen. How do people know which is the correct protocol? Make sure your protocols are named very clearly. The baskets may be overloaded with cassettes. the cassettes overloaded with tissue or inappropriate cassettes used. You saw earlier that there are different cassettes. Some will be mesh, some will have larger holes. Think about your choice of cassettes that must support the tissue, but you may use other things like papers or biopsy pads and keep the same cassettes just so that you get consistent movement of fluid. There may be maintenance issues with your instrument. Instruments, every instrument will need to be maintained well. Make sure that the maintenance is kept up. 

Also make sure that we always use a correct reagent, that when you do replace the solvents, if you're taking them out manually, that they go back in in the right place. Be careful of heavily contaminated reagents, using recycled reagents of unsatisfactory quality, Recyclers will give recommendations on how pure the final product will be. Remember, most recycling of alcohols, the recyclers themselves would say that their product at the end is perhaps 98% pure. Remember that if you are using as 100%, that you take that into consideration, that you'll now have 2% and then maybe more. of water in your final product. Some people do make a mistake and purge to start the cleaning cycle before they've removed the specimens. While never ideal, they can be recovered, but just really make sure that there are adequate processes within your laboratory so that this doesn't happen. 

Inadequate training of staff. Often the people doing the changing the tissue processes are not well trained on just how critical the tissue processing is to the overall workflow. Make sure that they know how important their job is. There's also a lot of different factors that affect carryover during processing. And again, this is the calculation whether your instrument does calculate correctly what the concentration of reagents are, or whether you are changing reagents based on a time of the week or the time somebody can come in to do the change or the number of cassettes. But just remember, there are a number of different factors that will affect the reagent carryover. 

There is the number, the type, size of the specimens themselves, the number and type of cassettes. Cassettes that have got more infrastructure, if you like, might carry over more reagent to the next-- to the following reagent than a cassette, a more standard cassette. The drain time between the steps can be important. The actual retort design. Reagent viscosity, if you are using a xylene substitute, some of these may be more viscous than xylene. Remember that in considering the carryover into the wax. The reagent volume, the properties of the previous reagent. If you have a lot of graded alcohols or if you are having a format with fewer graded alcohols, remember that you will be carrying over from the previous reagent. Reagent temperature is important. 

Basket design. Does your basket allow you to stack your cassettes in too tightly? Or have you got spoons, which allows for more fluid movement? The number of biopsy pads and the type of biopsy pads. Biopsy pads, while very efficient at holding small tissues in place, do carry over at least 10 times the amount of reagent as a standard cassette. Remember if there's a lot of biopsy pads that your reagents will becoming more contaminated than you perhaps are expecting. 

Miscellaneous. A failure to fill, maybe specimens are left in the air for a prolonged period, allowing drying to occur. Maybe the specimen's being immersed in particular reagent for an excessive time, such as wax, when they're not removed, at the end of the run. Specimens being subjected to excessive heat, could that be at the embedding center? Specimens being placed in contaminated reagents, such as wax contaminated with formalin. Think about where you place your basket that contains formalin. Does that sit on top of the tissue processor, drinking formalin into the wax? It's important to think about these things. Specimens can sometimes be lost during processing if the cassettes were not correct. So that's a total of 36 causes. I'm sure there's more. 

As there's so many different possible causes, so what do you do to analyze your problem? When faced with blocks that are difficult dissection, the first question to ask is, what was different about that run that produced problem specimens to the previous successful runs? If you've been using the same protocol for the same types of tissues for a long time, then it's probably not the protocol. Was the intended schedule or protocol used? Does my problem affect all the specimens in a batch or just a small number? Are they all of a similar type? Are they all from the same source? Were my specimens at the very top of the retort? Do I know with certainty what has gone wrong? Often assumptions are made about what might have gone wrong or where the problem may be. Ask, do I know? Were my specimens processed using the usual schedule that generally produces good results for this type of specimen? Is it likely that the schedule was too long or too short for my problem specimens? Do I definitely know that the problem was caused by the process of malfunction? Did the software indicate any error? Did a visual check of reagent bottles help the levels? Is there a possibility that an error has been made when replacing solvents on the processor or any reagents over the recommended purity thresholds? Was normal fixation applied to my problem specimens? All of these questions acting as a checklist can help you determine what was wrong, what has gone wrong. And if you remain uncertain as to the cause of the problems, just gather as much information as you can by examining your specimens, thinking about different possible causes, and thinking about the situation as well. Close examination of the blocks can confirm what you suspect may have happened, and your nose can help if there's still solvent left in the tissue. It can often help to describe your blocks too. We often use words which like crisp or crunchy or brittle. It's often better to be much more descriptive. Think about your macro effects, think about your micro effects. Think about the terms you're using to describe the tissue. Is it, is there a tolysis or, you know, have you lost morphology? What does the nuclei look like? 

We do use terms like crisp, crunchy, brittle, shrunken, shriveled, cooked. Some of the reasons for this might be, it could be over-processed, it could be under-processed, sludgy, could be under-processed with fat, over-processed, dry, powdery. If there's hard fragments, is it calcium or other material, smells of clearing reagent, probably under-processed, soft and compressible, under-processed. Reprocessing can be done, but we need to be careful about how you reprocess. Because remember, you're coming from a situation where your tissue is not in perfect condition anymore. If it's over-processed, reprocessing will probably not help. If it's under processed you can carefully consider what you need to do in order to proceed and there are different methods to reprocess, some of which might be more suitable for others depending on what's happened to your tissue. Is the outer room satisfactory that the sensory is poor? Is there an ovular example? It's probably under processed. Is there poor consistency Poor consistency throughout. Maybe there's a problem with your processing reagents or the fixation. Is the outer rim brittle, but the center is satisfactory?

Over processing. There may be discrete layers or areas that are poor. There could be under processing of susceptible tissues. You may have a tissue that has got soft elements and then quite dense collagen, perhaps There is one where you needed to consider the different type of tissue and, you know, perhaps use a medium length tissue processing protocol. Specimen separated from surrounding wax. Could it be an embedding fault or is it under processing? 

As a final question, optimizing processing. We can look at these two examples; both are processed on an eight-hour schedule. One is kidney block A, and one is kidney block B. But I will tell you that it is exactly the same tissue. It is cut in parallel. It's the same source, it's the same kidney, it's cut in parallel. They're both put on an eight-hour schedule. Think of the different steps that we just went through in what do I know? What do I know and how would I understand what has happened to this tissue? Why is there such a difference between a well-processed kidney in block A and a pretty poor tissue in block B? Think about what your answer will be. What is the difference between these two tissues? I'll go to the next slide and let you know. The difference was the block on the left is 3 millimeters in thickness, and the block on the right was 4.5 millimeters in thickness. This is enough to change the appearance of this tissue and to change the processing. Again, if everybody got that completely correct, then well done. It is worth thinking and challenging your suppositions of what may go wrong during processing. 

That's the end of this session. The next session will be continuing on from embedding to handing out the slides. We will be taking questions at the end of the next session. Thank you very much for your attention today. I hope that it was informative and any questions, let me know. Thank you. 

Thank you, Fiona, for that informative presentation. Questions that come in during today's broadcast will be answered during part two of our webinar series airing on Thursday at 9 a.m. Pacific and 12 p.m. Eastern time. We want to thank you again, Fiona, for your time today and for your important research. We also want to thank LabRoots and our sponsor, Leica Biosystems, for underwriting today's educational webcast. You can view the webinar on demand and LabRoots will alert you via e-mail when it's available for replay. We hope you enjoyed today's presentation, and we look forward to seeing you on Thursday for part two, tips and tricks to better histology in tissue-based research, a review of key factors influencing the quality of sections and stains. Take care everyone. Be safe, stay healthy.