Fundamentals of Hematoxylin and Eosin Staining

I am Cindy Sampias, applications technical lead for Leica, and today we're going to talk about the fundamentals of hematoxylin and eosin staining. One of the things that's been really fun with my job is being able to interact with customers as part of our cancer diagnostics process. One of the biggest questions I get is about H&E.

Overview

So why do we do it? In this particular lecture, we're going to be looking at not only why we do it, we're going to talk about the components. Is it better to stain by hand or on a platform? We're going to suggest protocols and then try to find the right balance between your colors, what our artifacts look like, and how can we troubleshoot some of those? 

How do I fix these things? And of course at the very end, we'll talk about whatever questions you guys might have.

What Is It?

What is the H&E? Of course for our routine diagnosis, we're using this as our baseline. This is what’s getting us started. It really gives us a great opportunity to look at the cellular and tissue structure in detail. So whenever your slide reviewer is looking at a particular tumor type, whether it's benign or malignant, the stain intensity is one of the things that they're going to look at to determine just how bad things are.

In some cases, when you're talking about multiple chromosome anomalies in a nucleus, the more hematoxylin it picks up, is more DNA that's present in that cell. At the end of the day, most of the folks reviewing slides have their preferred coloration palette. Some people like it very, very pink. Others like it very, very blue. It just really depends on the end user and what they're looking for and of course, how they were trained initially.

We're going to talk about quite a bit of this today and ways to balance that stain out to get that side reviewer the exact coloration that they're needing for their diagnostics.

The Basic Components

We have 4 basic components. We have our hematoxylin, our differentiator, which of course is going to adjust the coloration of your hematoxylin, bluing, which is going to change the hematin from that reddish color to the blue color we're used to, and of course eosin which is our cytoplasmic stain.

Hematoxylin – An Overview

Hematoxylin is of course an acidophilic dye, which means it's acid loving. It's going to stain anything in your nucleus, which of course includes your DNA and your RNA.

There's a lot of different types of hematoxylin out there, and each one has a slightly different coloration appearance. Historically, we used to make all these things in our laboratories. You would have your hematoxylin powder and you'd mix it up. Now the challenge with that was making sure that if I made hematoxylin this week and you made hematoxylin next week, that we would get the nice balance there and our preparation techniques could influence exactly how those colors look.

Depending on who's making it, you're going to have a stronger intensity or a weaker intensity. The manufacturer commercially of the hematoxylin stain, whichever one you choose to use, has really made that quality nice and standardized. You don't see those variations based on preparation.

When you think about hematoxylin, oxidation is going to produce hematein, which is the actual dye in the stain. You can see this by looking at the top of your hematoxylin if you have it in a dish. You'll start to see it have almost a holographic appearance on top.

It reminds me of the sheen you see with E coli. That's the sign that your hematoxylin is being oxidized. One of the ways we stabilize our hematoxylin is by adding a mordant, and that gives the ability of the hematein to attach to an anion, something like iron, aluminum is another popular one that we use, and it helps stabilize it.

Hematoxylin – A Word About Mordants

Hematoxylins are usually classed by whichever mordant they're using. Like we said previously, the mordant is going to strengthen the positive ionic charge of the hematein. It's going to aid in the bonding of the hematein to the anionic tissue component, which of course is going to be your chromatin, your DNA, RNA.

Now because we're using different mordants that is going to influence the final color of the stain components. those changes are very, very subtle across the board. If you have a particular preference, you will notice the difference between Mayer’s hematoxylin, for example, and Harris hematoxylin.

The most common one that we use in routine histology is aluminum ammonium sulfate. This is going to cause a nuclei to stain red in color until we rinse it with a weakly-basic solution, i.e. our blueing solution. It's going to change it to the more familiar blue color that you're used to seeing. 

Hematoxylin-Harris

Harris hematoxylin is the most commonly used alum hematoxylin, and it can be used for progressive staining and regressive staining of cytology specimens as well. It tends to provide a little bit clearer nuclear detail and one of the things you should know about it is that it can be best differentiated with the mild acid as opposed to using hydrochloric acid, which is a very strong acid.

Historically, we would use that. You would do one dip of the slide into your hydrochloric acid differentiator and you'd have to immediately get it into water. If you had it in there for two seconds, you could potentially completely decolorize the slide. Of course, we want to avoid that. The mild acids with your Harris hematoxylin is going to make that change a lot more subtle and give you as much control as you could possibly want in order to make sure you get just the right color that you're looking for. Harris hematoxylin is an alcohol-based stain.

Hematoxylin-Mayers Another one of our hemotoxins that we're very familiar with, of course, is Mayer’s hematoxylin. It’s another alum hematoxylin and it can be used both progressively and regressively, just like our Harris. It's also used as a nuclear counterstain for many special stains and IHC. It works really well in that scenario. It doesn't give you too much blue and it doesn't tend to stain other components of the cells.

When it's used as a counter stain, we want to see the nuclei and of course, then we're going to blue the slide without using any differentiator because we’re not concerned about removing any excess hematoxylin at this point. It is a water-based stain as well.

Hematoxylin-Gill’s

Gill’s hematoxylin is another one of what I consider to be the big three. It's an alum hematoxylin often used progressive or regressive and you can get it in lots of different concentrations of predominantly one, two, and three. It is typically used for cytology stains, but sometimes folks will use that in histology as well. It's made with water and ethylene glycol.

The oxidation of the stain is prevented over months because of this, and it just makes it more stable than Harris hematoxylin. Now the nature of Gill’s is that there is some extra nuclear staining that may occur. So this is where your differentiator can be really important for getting rid of that extra staining. And Mucin and even other adhesives on the slide can actually stain blue in the background. So you will see some more background stain than you would be experiencing with some of the other hemotoxins out there.

Hematoxylin - Others

There are quite a few other ones out there that use iron salts as a mordant, but they're usually in special stains. This is because they generally will demonstrate more tissue structures than the alum hematoxylin, so you can see things like myelin or elastin fibers. 

One of the best known out there is Weigert’s, which we use in the Verhoff-Van Gieson stain or elastic stain.

Differentiator

Now the differentiator, we've talked about that just a little bit. What this is basically doing is allowing you to selectively remove stain from tissues to get the coloration that you're comfortable with.

When you're talking about hematoxylin, hydrochloric acid was used historically. Of course, it's a very rapid differentiator. Acetic acid or citric acid are the ones that are mild acids. Those are going to be for a much more controlled differentiation and those are the ones you're going to commonly see in laboratories.

While hydrochloric acid was the standard, it's really started to become more fashionable, I guess, to use milder acid because we are starting to use these things on platforms, automated staining instruments, that sort of thing. You're going to get a much gentler dye removal with that.

You know, automated staining has really brought some amazing opportunities into the laboratory for it increases in quality and production because we are able to do the same thing over and over again because you have those robotic arms on there. It makes it very easy for your techs to load rack; the instrument takes off, does its thing and now you've got your techs free to do other things in the laboratory.

This slide is an interesting one. This one has not had any differentiation at all. And if you notice there is a purple haze in the background and the tissue itself, this is tonsil, looks almost a little blurred. Now in this case I am using charge slides. So you can see in the lower right- and left-hand corner of the slide itself, there are little plus signs indicating that they are charged.

All that means is that the slides are going to have the tissue adhere much more strongly. Your differentiator would be used, and preferably a mild acid in this case, 30 seconds will get rid of this blue background for you. If you go up to a minute and now you're going to actually start being able to pull just a little bit of color out of the nuclei. If you have overstained it, you can see where you have a little bit more room to play if you want to pull out more color or not. Thirty seconds is going to be the minimum in order to get rid of all this blue background that we're seeing here on this charge slide.

Things like colon for example, which you can see in this image, depending on what you're looking at and who's looking at it, of course. You can notice that the goblet cells here are just… …do a progressive stain and that way you'll be able to capture the blue of this mucin in the goblet cells.

Bluing

Now let's take a look at bluing reagents. One of the most common ones out there is Scott's Tap Water. Certainly there are a wide variety of commercially available bluing agents out there that you can get to use in your laboratory, but they all function in the same way and that they are a mildly basic solution that's going to change that hematoxylin from the red to the traditional blue color that we're used to seeing microscopically.

Here's a great example of how bluing works, so if you take a look at this piece of tonsil here, you're seeing nice, delineated line. The top half of the slide hasn't been exposed to the bluing agent, but the bottom half has, and this is one of those things in your protocols you're going to see that the bluing is typically about a minute. Mostly that's to accommodate movement of the robotic arms on any of the stainers that are out there on the market.

The thing to know with that is I literally dipped this slide in and pulled it right back out. You can just see after one-to-two second exposure to the Scott’s Tap Water, it made a nice blue color here on the slide.

Eosin

Eosin is the most used counterstain. This is what's going to distinguish your connective tissue, your cytoplasm from your nuclei. It's generally going to be pink and there are different shades of pink depending on what type of tissue fiber it's staining.

So Eosin Y is the most common form and you can use it in both water as a base or alcohol as a base. Just a small amount of acetic acid added to it will also sharpen that detail in your eosin. It's kind of nice, especially if your eosin is weak. Just add a drop of acetic acid to it, and that should perk it right back up.

Eosin with phloxine is another common mixture that we'll see when it comes to eosin. There are a lot of folks that really enjoy how it enhances the reds on their H&Es. So when you're talking about cytology, there are other mixtures. In the case of eosin, we talk about EA50, EA65. These are going to add a little bit of light green, yellowish, and bismarck brown to the eosin. When you look at a cytology slide for example, a pap smear, you'll see the teals, blues, pinks, all of those shades of color and all of that's coming from your eosin.

Just adding those two extra guys is really what's going to make the difference. And of course the concentration of the eosin is determined by the designation of whether it's EA50 or EA65, which is going to have the most eosin Y added to it.

Eosin Differentiation

Now does eosin really need to be differentiated? Well, of course the right answer is yes, a graded alcohol, most commonly 95. Some folks will go as low as 70% alcohol and that's ideal to remove any of the excess eosin and that may be in the tissue. So what you'll be able to see is three different shades of pink. When you have too much erosion, just like with too much hematoxylin, if you don't get some of that excess out, then the whole thing is just going to be very bland, one color. Of course, that's not what we're looking for, right?

Higher grades of alcohol can also be used following eosin but remember, it's the water that's doing the differentiation for you. That's what's going to pull out that excess pink. If you go straight from eosin to 100, that's going to impact your coloration. 

Staining by Hand or on a Platform?

What's better? Should we stain the stuff by hand, or should we use the platform? I can tell you as somebody who has done a lot of staining by hand over the years, the platform has just improved the quality and the quantity of work that we're able to get through in the laboratory.

For one thing you're staining is going to be very consistent. It's going to be reproducible. Of course, your technologists hands on time. They can go off and do the other things that may need to be done in the laboratory.

Now there are some concerns about potentially longer protocol times. Back in the day we used to say, I must have a 25-minute H&E stain. We were putting a focus, in my opinion, on the wrong thing. When you're looking at a platform, you can have 10-12 racks in process at the same time. Your protocol length maybe 50 minutes, but you're going to constantly have material coming off of your stainer. Whereas when we were doing things by hand back in the day, it was oftentimes a lot easier to have just one or two racks staining at the same time, so you didn't have 5 or 6 timers going off all over the place. This made it a lot easier. The other thing that's nice is you're much, much more able to follow your reagent management and make sure that your reagents are refreshed after a specified amount of time and a specified number of slides or runs going through them.

Protocols – Putting Them All Together

When we look at protocols, this is usually where folks struggle. Everybody has their own preference of what they want to see under the scope. What's the shortest possible time I can get? Turnaround time is huge and that's usually one of the things that kind of trips people up a little bit, because again, when you're talking about platforms, you're talking about having many things happening at the same time as opposed to just one or two racks at the same time.

We want to reduce those touch points, but then depending on what you're doing, how many touch points are there actually? Is the cost of doing it on a platform worth it? Depending on what you're doing, it may not be worth it. In most cases it is, but it depends on your laboratory and what your end game is.

Remember at the end of the day we have we want to have good quality and reproducible results. We want it to be cost effective and that's going to be the biggest concerns from most laboratories, the quality and overall cost.

Protocols – What Type?

What kind of protocols do we want to run? There are generally three different types. Progressive, which means there's no differentiation step. It goes hematoxylin, water, bluing, water, alcohol, eosin.

Now modified regressive or progressive depending on, you know if your glass is half empty, glass is half full person. There is a differentiation step utilized. However, the only purpose of that differentiation step is to get rid of the blue background. It's not going to impact your nuclear detail at all.

Regressive staining means we're going to overstain with our hematoxylin. Then we're going to use that acid to gently bring back and remove that background but also remove any excess dye from the nuclei.

Protocols – General Rule

General rules when you're looking at protocols, your H&E had that dewax set to get rid of the paraffin. Then you're going to hydrate it. That's usually done after going from xylene, to pure alcohol, to a graded alcohol, to water. Of course, in our hematoxylin differentiator bluing. Now we're going to start to introduce alcohol back in so we can do our eosin setup.

Then once it comes out of eosin, we're going to dehydrate it. Starting usually with a graded alcohol, going to pure alcohol. And then for our clearing step moving into xylene, toluene or some other aliphatic hydrocarbon solvent. And then we'll go to cover slipping.

The thing to remember with this suit is whatever you're using for your clearing, it’s often going to be the best reagent that you use in your cover slipper. Or if you're manually cover slipping, you want to make sure that it’s going to be compatible with whatever mounting media you choose to use.

The thing that's nice about this basic protocol is it's easy to reproduce and your reagents are going to be resilient enough to allow for a large number of slides to go through before your reagents need to be changed. That is so huge, especially if you're a laboratory where you're running 1000 slides every day. You want to know that your reagents and your setup is going to be able to tolerate that much workload.

Dewaxing and Hydrating

When we look at dewaxing and hydrating, xylene is going to be the most common solvent, at least in the United States we use it exclusively or some type of xylene substitute. There are lots of other things that you can use in its place. Some folks use toluene. Of course. We talked about xylene substitutes. There are things with citric oils in them like citrus, salt, limonene. Those are all different solvents. The thing is, you want to make sure that whatever you are using, you're going to be able to tolerate the slide volume that you're putting through the reagent. These things are all materials that are going to have to have special waste, just like xylene. Depending on where on you're at, those rules may vary slightly, but by the end of the day, they really are important to understand exactly what your local regulations are regarding waste of any of those solvents.

Once the paraffin has been removed during the dewaxing step, you can go into your alcohols and then your water and then move on for the rest of your staining. The water washes at the end of your graded alcohol is what's actually going to finish that rehydration, and it's getting it ready for the hematoxylin.

Hematoxylin – The Nuclear Stain

Depending on which hematoxylin you use, you want to make sure that there's plenty of time to get whatever coloration you're looking for. Once you've got to that sweet spot where you're like, this is what I like, now the slides are going to be rinsed. That's essentially going to stop the process and it's just going to remove any excess stain. Then if you want to differentiate your slide, you can certainly do that and then even control it even more by how long you leave it in that differentiator. Keep in mind, this is also going to include your background staining. You want to make sure that if you are going to use a differentiator, keep that in the back in your mind.

Any additives you may have added to your water bath, some folks use albumin. Some folks use sayon, or if you're using charged glass slides, all of those things are going to pick up your hematoxylin to some extent. So your differentiator here is going to be certainly something to consider while you're doing this. And of course. Once you're done with the differentiation, the water rinse is going to stop that now you're going to go into your bluing. You can use other buffers. Scott's Tap Water is one of the most common ones. It's very easy to make and it's not going to be something that's going to necessarily have as big of an impact on quality as, say, not being able to mix your own hematoxylin correctly. But it is going to change that reddish cast to the desired blue, as we saw in the previous slides. 

Eosin and Beyond!

Before we add eosin of course, we want to make sure we use that 95% alcohol to get that sample ready to accept this alcohol-based dye. What's really fun about eosin is that you can adjust set coloration just with minor time changes. Eosin is amazing in that it will turn anything pink very, very quickly. If you've ever made hematoxylin from powder, you know that that powder is finer than confectioner sugar. If you get it on anything, you may not see it right away, but as soon as water hits it, it turns pink.

When I optimize slides for customers, I will do anything as low as 15 minutes, generally up to one minute, and I'll make those changes in 15 second increments. Any shorter than that, you're probably not going to get enough of a difference to be able to go, “Ah.” If you do it too long, then you're going to go from light pink to extraordinarily bright pink. That can be painful on the eyes if you're not used to seeing that.

The eosin of course is differentiated typically with the 95% ethanol. It's going to stop the staining process; some folks will use a 50 or 70%. It just depends on what you want to do. The more water in that alcohol following, the eosin is going to pull out more of that pink color for you. If you're tending to over stain with your eosin, you can use a 50 percent, 70%. Those will pull out the eosin because they have such high water content.

Normally, we would do 95% of ethanol and then you're going to go from that to a pure alcohol and then your clearing so that you can get that slide coverslipped and ready to view.

A Word About Solvents