Histology, microscopy, anatomy and disease
Histology, microscopy, anatomy and disease

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Histology, microscopy, anatomy and disease

1.1 A histology department in action

The following video shows the work of a busy histology department at Milton Keynes Hospital, in central England.

In the video, Peter Mooney, the Laboratory Manager for the department, takes you through the different steps involved in the production of sections, staining and initial analysis of slides for diagnostic histopathology. These steps will be described in more detail later in the course.

Please note that this video is around 17 minutes long, so you may prefer to watch it in stages.

Download this video clip.Video player: 39893_ou_futurelearn_mc1008_vid_002-540.mp4
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Transcript

DAVID MALE
Today we're at the histopathology department at Milton Keynes hospital. And I want to introduce you to Peter Mooney, who's the laboratory manager here. And this is a busy histopathology department. And Peter's going to introduce us to the kind of work that goes on here.
PETER MOONEY
Yes. Welcome, David. Welcome to a relatively busy histology department. We do about 400 to 500 specimens a week. We get specimens from GP surgeries. We got them from outpatient clinics, and of course, from inpatients and theatres.
That's the main source. We do get the occasional autopsy specimen. But that's few and far between. The majority are from clinics within the hospital. It could be dermatology, endoscopy.
DAVID MALE
OK. And from the time that you receive a specimen, how long does it take til it actually hits the desk of a pathologist?
PETER MOONEY
Right. Yeah. This is a problem we have with clinicians. They take a blood sample, press a button, out comes a result. With histology, it's quite different.
It depends very much on the size of the specimen. If it's a very small biopsy, it arrives midday, we could have a result maybe tomorrow. If it's a much larger specimen, like a breast or colon specimen, it can take one or two days to get a result. So it's very much dependent on the type of specimen and the size of the specimen.
DAVID MALE
So can you take us through the process as it goes here?
PETER MOONEY
Let's go. Let's follow the process--
DAVID MALE
So this is where we start?
PETER MOONEY
--starting where they book in.
DAVID MALE
OK. Good. So this is where you receive your specimens here?
PETER MOONEY
It is. Yep.
DAVID MALE
And then what you do with them?
PETER MOONEY
Well, this is our specimen reception area. So we get all the specimens from theatres and clinics, as I talked earlier. The first thing we have to do is check that the request card has got exactly the same details as the specimen cup. It's vital. If there's any discrepancy, it goes back to where it came from so it's done properly.
The specimens all arrive in various sized pots. You see here very small ones. There's a tiny little biopsy in the bottom of that one.
DAVID MALE
Oh, yes.
PETER MOONEY
And we have much larger pots, going up to five litres depending on what type of specimen it is. They are obviously from theatres, specimens, larger specimens. These tend to be from GPs or clinics.
DAVID MALE
OK. So what kind of specimen was that one?
PETER MOONEY
Well, that one there is a nesophogial biopsy.
DAVID MALE
OK.
PETER MOONEY
You can see how small it is.
DAVID MALE
Yes, it's tiny.
PETER MOONEY
Yeah.
DAVID MALE
And the large ones, what kind of specimens are they?
PETER MOONEY
In this one, there's a uterus and fallopian tubes. They're both uterus and fallopian tubes. But we see breast tissue, colon tissue, kidney. You name it all, we'll get it and we'll have a look at it. The thing to say of course is, these pots contain formaldehyde. Formaldehyde is a bit toxic. It's not very nice. So whenever we open the pots, we always do it on downdraft tables, which I'll show you later. And we monitor the levels in the lab on a regular basis.
So what happens then if we're quite happy with the specimen? It's booked in on the computer system. This is a pan pathology computer system, which the chemists have, the haematologists have, transfusion. And Rosia is booking a specimen now. And it will be given a unique lab number. The name then stops.
We don't relate to the name. We relate to the lab number. So it will be 16h, whatever the number is.
DAVID MALE
And does this go towards a central database then?
PETER MOONEY
It does. All the other patient's histories, only if the patient's been here before. It's all held centrally. Yeah. Yeah. When it's booked in, we get some labels come here. We get one for the pot, one for the card. And at the same time there's a cassette produced, which are these funny-colored things on the top of here.
DAVID MALE
Is there any difference between the colours on these?
PETER MOONEY
Yes, there is. The cassette, which is going to have the tissue in it eventually, has the lab number on, which is there, and what's in the tissue, what's in the pot. The colours denote urgency. If you come in, if your GP has referred you to the hospital because they're worried about a condition you have, and that it could be serious, we treat those as urgent. So a prostate biopsy, a breast biopsy, anything like that that somebody thinks that will treat very urgent--
DAVID MALE
So you would be looking for a diagnosis of cancer in this case?
PETER MOONEY
Yes. Yes. So the red cassettes denote urgency. There is a reason why these large specimens are urgent. Clinicians suspect something.
DAVID MALE
OK.
PETER MOONEY
OK. These ones are-- this is a skin lesion. The patient may well have had it for 20 years and decided to go to a GP.
DAVID MALE
Less urgent.
PETER MOONEY
Less urgent. Yes. Yes.
DAVID MALE
OK. So you've booked your specimens in. And now what are you going to do with them.
PETER MOONEY
Well, this is the first real step in the process, David. What we do is we examine the specimens macroscopically. The specimens are arriving throughout the day. And we have biomedical scientists look at specimens, as well as pathologists.
And we make a microscopic description, and we dictate it. The person will be wearing headphones. And we have secretarial staff upstairs typing as they speak, more or less. The tissue is put in the cassettes. Now, it depends, again, on the size of the tissue. The one we showed earlier, which was a tiny, little about a millimetre, two millimeter-sized esophageal biopsy, that would be put in the cassette with a lid put on it.
DAVID MALE
As is?
PETER MOONEY
As is.
DAVID MALE
Yes.
PETER MOONEY
The much larger specimens, of course, we couldn't possibly sample it all. So if it's got a tumour, we'd sample the tumour, the resection margins. And that's very much an experiential thing.
DAVID MALE
So it's really important to get exactly the right bit of the tissue to look at?
PETER MOONEY
Absolutely. Yes. Sampling tumours limits every section, which is very much an experienced pathologist would do that.
DAVID MALE
So it's the pathologist that will be looking at the initial cutting of--
PETER MOONEY
Of the largest specimens. Absolutely. More and more labs are trying to train bio-medical scientists to do this. But it's a lot of training involved.
DAVID MALE
Very skilled.
PETER MOONEY
Very skilled. Yes. Yes. So it's very important that the block of sample tissue that goes in the cassette is what's going to be seen down the microscope later on.
DAVID MALE
OK.
PETER MOONEY
You can always go back to the specimen, of course, and take more. But, of course, that will slow the process up.
DAVID MALE
Of course.
PETER MOONEY
It's better to get it right the first time.
DAVID MALE
OK. So we can we go and have a look at the next stage now?
PETER MOONEY
Yes, of course.
DAVID MALE
Right. Let's go. So you've got your samples now on the cassettes. What happens to them here?
PETER MOONEY
Well, this is the tissue processing stage. What we're trying to do is to try to get a very thin section of tissue onto a microscope slide so we can look down a microscope. To do that, we need a nice, firm piece of tissue.
We're replacing all the water in the tissue-- the tissue is about 80 per cent water-- with wax, a bit like candle wax. It's called fibre wax, slightly specialised wax. Quite a slow process, usually overnight. Most of the specimens go overnight.
So as I alluded to earlier with haematology chemistry, we get results very quickly. Histology is much slower because the tissue cassettes are going in here. And overnight, these reagents, IMS, and distilled methylated spirit, and xylene, and finally, wax, will visit the reaction chamber. And over that length of time, by 8 o'clock tomorrow morning, all the water will have been removed from the tissue, and it will be replaced by wax.
DAVID MALE
So obviously, the whole of that process is effectively automated by this machine.
PETER MOONEY
It is. Yes.
DAVID MALE
You just have to put the material in.
PETER MOONEY
Put the material in. Press the button.
DAVID MALE
And then it comes out first thing the next morning.
PETER MOONEY
The following morning at 8 o'clock, or whenever, you look in there. You think nothing's different. Only the tissue, instead of sitting in formalin, is sitting in wax.
DAVID MALE
OK. And then where do you take the tissue?
PETER MOONEY
From there we take it for embedding.
DAVID MALE
OK. And that's back in the main lab.
PETER MOONEY
That's back in the main lab.
DAVID MALE
OK. Let's go and have a look at that then. So this next process is embedding here. What happens at the station?
PETER MOONEY
We're following the processing, which has happened overnight. The following morning all the cassettes are taken out of the machine and embedded in moulds, so various sized moulds. And what Liz is doing at the moment is she's taking a mould, put the tissue in the mould. In here, there's molten wax. And this is a dispenser, which dispenses wax.
This stage is very important. However the tissue's being cut, whether it's a skin biopsy, there's a transverse section, it must be oriented correctly. Because the way the tissue's orientated in that mould will dictate what it looks like on a microscope slide.
DAVID MALE
And that determines whether the pathologist can see what they're really interested in seeing at this stage.
PETER MOONEY
Exactly. Yes. And you have to get it right first time. If it's a piece of skin, and it's been poorly orientated, and it's been cut, you could try to reorient it in the wax. But chances are that's been cut away.
DAVID MALE
So it's almost back to square one at that stage if it's not right.
PETER MOONEY
Yes. But you're always aware that this is one only sample. It's not like a blood sample. You can't go back to that patient for a blood sample. This is a once only lesion, possibly. So this stage is very important.
So this is a case in point. Liz is being very careful to orientate the sample the correct way around. It's a piece of skin with the epidermis and the dermis. So when you look at the microscope slide, it's the right way around.
So this is topped up with wax. And this is a cold plate to solidify the wax.
DAVID MALE
OK. So that was a liquid one, and this is now a solid one?
PETER MOONEY
So this one was done a little while ago. And it may come out the mould looking like that.
DAVID MALE
OK.
PETER MOONEY
OK? So we got the details along there. We've got the tissue embedded in there.
DAVID MALE
And that will go on now for sectioning.
PETER MOONEY
It will do. Yes, which is the next stage.
DAVID MALE
OK. Let's go and take a look at that then. So you've got the specimens now in their wax blocks. And they're going to be sectioned on the microtome.
PETER MOONEY
That's right. A microtome. Is where the sections are cut from. Every time Vickie turns the handle, it advances the cassette by three thousandth of a millimetre. That's about a cell thick.
So it's very important the sections are thin. You don't want to be looking down a microscope and seeing a thick section with cells piled on top of each other. It's got to be nice and thin.
So again, quite labour intensive, like the embedding stage, and quite skilled. Often people refer to histology more of an art than a science because it takes practise and experience. What Vickie's done is she's taken a ribbon of tissue, floated it on some warm, distilled water, picked it up with a microscope slide.
DAVID MALE
How many sections would be taken from a single specimen, normally?
PETER MOONEY
If it's a large block, we'll just take the one section. If it's a smaller piece of tissue, like the esophageal biopsy we looked at earlier, you'd take three sections at different levels through the tissue. Because it's so small, you can't judge by eye that you've actually caught everything. There maybe something lurking further down, so we do three levels.
DAVID MALE
Again, it's just to hit exactly the right place in the tissue so that you don't miss a lesion or something really important.
PETER MOONEY
Yeah. Yeah.
DAVID MALE
OK. So we've now got these sections on the slides. What happens to them now?
PETER MOONEY
When the water's drained from them, it will be put in an oven and the wax will be melted onto the slide to make sure the tissue sticks to the slide. And that's the end of this process. And then it will be staining after that.
DAVID MALE
OK. Can we go and look at some staining machines?
PETER MOONEY
Yeah.
DAVID MALE
OK. Let's go.
PETER MOONEY
So the next stage, we've got the sections on the glass slide. But, of course, we can't see anything down the microscope. So now we have to stain the tissue. Now, this is a multi-stainer. It will do various stains on here. But the one we're interested in is called a hemotoxylin and eosin, or H&E, we will refer to it.
It's a purple stain and a pink stain. And the purple stain stains the cell nucleus purple. And the cytoplasm stains pink. The machine's preprogramed, so it knows what to do.
We have various coloured clips. So if we put a red clip on it, the machine knows to stain it with H&E stains. We have other staining techniques.
DAVID MALE
Oh, it's a coding system or it.
PETER MOONEY
It's a coding system so it recognises it. So basically, you pop it in the machine, close the drawer, an arm will come across, and what this machine do is doing the reverse of the processing machine. It's taking all the wax off the tissue, and it's replacing it with water. And it's put these stains in the relevant places. So it goes in to xylene, then alcohol, then water. The tissue's stained, and then it's cover slipped.
DAVID MALE
So there are a lot of different things in there. Does this machine do things other than hemotoxylin and eosin?
PETER MOONEY
There's a stain called periodic acid Schiff, and we use that. We use it for staining immuno sections, immunohistochemistry sections, so we can set it up to how we want when we want easily.
DAVID MALE
Is all your staining done automatically now or do you do any hand staining?
PETER MOONEY
No. We do hand staining as well. This is the automated section. But we do do, on this bench, we do do special stains. So you can see all these bottles of tinctorial stains. We can stain for bacteria, for acid-fast bacilli, amyloid fat, lots of different elements whether it is--
DAVID MALE
Those would be much less commonly done. It's normally H&E isn't it?
PETER MOONEY
H&E, yes, much, most of it's common. If a pathologist looks down the microscope, and they see something that might be tuberculous, they would ask for a Ziehl-Neelsen stain.
DAVID MALE
So how long does this machine take to do the H&E staining?
PETER MOONEY
It takes about 30 minutes or so to do a H&E as it goes to the ends up in xylene, and then it has a cover slip put on. Actually, there's one happening at the moment where the slide is being raised up. It's had some mountant and a thin cover slip put on it. And this is done automatically.
It is very important at this stage not to get any air bubbles under the cover slip because down a microscope, the air bubbles look huge. So this machine achieves that without air bubbles.
DAVID MALE
So, and it's assembling them on a rack here ready to go to microscopy?
PETER MOONEY
That's right.
DAVID MALE
OK, Peter, so you've got your stained sections now. And you need to marry them up to the original blocks from which they came.
PETER MOONEY
That's right. This is a very important step. We have to ensure that the tissue on the glass slide marries up exactly with the tissue in the block. So we check the numbers. We do a visual check at this stage that it's correct.
Throughout the process in the lab, of course, the card has been annotated with initials of all the members of staff to ensure that the tissue is correct, the names are correct, the numbers are correct.
DAVID MALE
So every single stage you've got checks going through to make sure that you've got absolutely the right tissue and the right sections.
PETER MOONEY
Absolutely. Absolutely vital, because it we make an error, the worst error would be to send a report out on a John Smith when it really belongs to a Jane Smith.
DAVID MALE
A wrong diagnosis, in other words.
PETER MOONEY
Absolutely. So we have as many checks as possible built into the system to ensure that doesn't happen.
DAVID MALE
OK. So it's a very rigorous quality control right the way through.
PETER MOONEY
Absolutely. Yes. Yes. both microscopically, like we're doing here, and later microscopically.
DAVID MALE
So presumably, the first thing is that you check that you've got the right tissue and everything else. And you do that on a microscope in the lab here?
PETER MOONEY
That's right. We'll do a visual check here to make sure that the shape of the tissue, if you like, resembles the shape of the block.
DAVID MALE
OK.
PETER MOONEY
But then we'll do a microscopic quality control as our next stage.
DAVID MALE
So this is the final stage of your quality control. You're starting to look at the sections under the microscope now.
PETER MOONEY
Yes, another important step. What Kimmy's doing at the moment, she's checking the quality of the section. We check beforehand that the numbers all match and the names all match with the card. She's done that again. It's always a different person.
She's checking down the microscope for the quality, that the staining is correct, that the quality of the section that was cut earlier is correct. There are no scores in it because a score down a microscope looks very big, that it's representative of all the section, and if it's a piece of skin that it isn't being superficial and there's bits missing. So we're looking at the qualities.
Kim has checked that, decided it can go out to the pathologist. And initialled the card. So that's not ready for the patho--
DAVID MALE
OK. So that's the end of your process here and you are satisfied that this is ready for diagnostic pathology?
PETER MOONEY
Absolutely. Yes.
DAVID MALE
OK. It's an absolutely fascinating process. I've been really interested seeing this. So thank you very much, Peter, for showing this, and all your staff too.
PETER MOONEY
Yeah, of course, great pleasure. Thank you.
End transcript
 
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