Hello. Welcome back in the new year to our master class series on the challenges of IHC staining. I’m Bettina Winkler from Visiopharm. And today, Colin Tristram will talk about the advantages of using cell lines as control tissue.

Colin has spent the last twenty years developing, marketing, and commercializing a range of products for the IVD market, specifically tissue diagnostics.

Colin is a founder and current CEO of HistoCyte Laboratories.

He studied medical microbiology at Newcastle University, specializing in immunology.

Heading over to Colin to start the webinar.

So I’d like to start by thanking Visiopharm for inviting me to talk in this webinar.

I’ve been asked to talk about the cell lines. So HistoCyte is a business built around, quality control.

When we employ the use of cell lines as a means of creating a standardized material for use in immunohistochemistry and indeed in situ hybridization in laboratories around the world.

The question of how this all came to be is really having an understanding of, where variation occurs in the laboratories and how it affects not only the quality of the tissue, but the output of, these assays that are employed in the laboratories.

So to really understand this, one has to look at what happens to samples in the lab and where variation occurs.

And I believe two biggest critical areas are around the sample collection and tissue processing. And sample collection is affected by a number of factors.

The fixatives, certainly fixatives can vary around the the world, the quality of the fixatives and the time and fixation as well.

But probably more critical to that is the is the cold scheme time. So the time the sample takes to to be fixed.

One has to understand or appreciate at least that once a sample is taken from a body, a whole series of cascading events occur that, begin the degradation of that that sample. So protein starts to denature.

That’s the whole point of fixatives. We prevent that, and we we try and preserve the the tissue in in as closer state as possible to to when it was in the body in order to make the diagnosis.

The second critical area is around tissue processing. So it’s true that laboratories try and standardize these processes, and they have protocols that are validated. And these protocols that are are kind of a one size fits all. So you will have a whole number of of samples put in at the same time, to be processed.

And those samples can vary significantly in terms of content.

Breast samples, some of them could be very fatty, for example. All those components are innate variation. And, and as a consequence, they can all have subtle differences between them. But nevertheless, the this is the the best practice. We use protocols that, are kind of applied to to everything.

We also have to understand that these, instruments use reagents, various alcohols, xylenes, and they get diluted over time. They get contaminated. And, of course, depending on when those reagents have been changed, they, of course, can have an effect. So, you know, samples processed with fresh reagents can sometimes show differences to those processed with, old reagents.

When you look at the other areas, embedding, so stuff can be, material can be, wrongly embedded. You know, just melt out and and reembed it.

The other areas around sectional, so sometimes sections, thickness can have quite an effect on, the immuno just by virtue of there being more, protein there. So you will get stronger results with thicker sections, for example. But those tend to be standardized and and validated in the laboratory around whether they use routinely three, four, or five microns.

Step five with assay is conducted is really the only point you can use controls to establish that the assay has been run effectively. So you want to demonstrate that the assay has worked the right way, the same way as it worked the last time you used it, the, you know, yesterday, the week before, or a month before. This is the importance of controls. It’s very difficult to control for those pre analytical variations. But if you can demonstrate that your assay has been, effectively run properly, it gives you a greater degree of diagnostic confidence.

That’s not to say one shouldn’t look at internal controls in the tissue. That’s vital that that analysis is done as well. Lastly, with, interpretation, having the confidence to know that the assays run properly, one can can assess it more comfortably.

But the cell lines are lending themselves to not only that, but the analysis of support performed fire image analysis.

So these cell lines are addressing some of the challenges that affect the laboratories.

And there are three big drivers really around the use of cell lines. One is, as I mentioned before, all those variations create an issue with tissue.

The second big driver is the laboratories struggle for much of this material, particularly for some of the rare mutations.

And the last part that isn’t always appreciated is the the cost to laboratories to actually manage their control materials, particularly for some of these high volume assays. So we’re gonna look at those in a bit more detail.

Variation that you can see in the laboratories and in the the samples, heterogeneity is probably the greatest with a lot of the tumor markers. Now a lot of controls that will suggest you use normal tissue because the expression tends to be more stable.

But the problem with normal tissue is these are cancer diagnostic labs, and they tend to be diagnosed in cancer. So you don’t necessarily always have an abundance of of normal tissue.

And, certainly, even when you have kind of, samples, receptions that have normal elements in, they are often in close proximity to the cancer and, as a consequence, can have some, localized change. But, nevertheless, those samples are not necessarily in abundance. And if they are, they’re they’re used, for wide variety of different markers.

But the heterogeneity you see in cancer can be quite stark. So, for example, a composite block that has, you know, HER2 samples in a three two one plus.

They can start off that. That’s how they’ve been selected. But, you know, within microns, your samples can change considerably, and it raises the question for the operator of, has my assay varied, or are all my tissues just heterogeneous?

And, more often than not, it will be heterogeneity, and they need to discard it and find more control material.

The image there from a publication by Nita et al shows you in very close proximity three two one, expression for her too.

You also get other things in materials like retraction artifacts and crush artifacts, which can give you false staining.

And then, of course, there are issues affecting sometimes larger samples, around fixation gradient. But as I mentioned in the beginning, the the critical element is is getting them fixed. And under fixation is is probably to the greatest detriment of the sample rather than over fixation.

Our cells are fixed and processed in the standard method that’s equivalent to tissue, and thus we aren’t affected by these same problems that affect tissue.

The other driver is the struggle for for samples in the laboratories, particularly those, low or mid expressing, samples, particularly things like HER2, ER, PR. But But as I mentioned before, a lot of labs struggle, certainly that smaller labs struggle for, some of the rarer biomarkers such as ALK or ROS1 in lung. Sometimes labs will will have a a large resection, and they will have plenty of control material. But for most small, to medium sized laboratories, they really struggle to find these.

And it’s not surprising. I mean, these mutations occur in maybe less than three or two percent of non small cell lung cancers. Whilst the assays often recommend the use of things like appendix, you know, looking for, wild type stain and in ganglia, the appendix is already kind of ubiquitously used for for controls for many other assays. So, again, to my point before about, you know, the those normal tissues that are used, the demand on them is so great that they they run out very quickly.

The other thing we see in in labs is, and it’s a hang up from from back in the day, when we used to use things like pressure cookers or microwaves for epitope retrieval.

Batch controls are used, and they were they were appropriate at the time because all the slides were generally treated together in a batch. But now slides are independently treated on these machines. All of them are kind of, separate from one another. So you can’t really use a batch control in the sense that all these slides will be treated the same way. They’re just not on the machine anymore.

So the last thing I think that affects them is the greater volume that comes from this. So having to use same slide controls means that the the amounts of material they need is really great, and there’s a great demand on these laboratories.

So the they tend to run out quickly. So for the large hospitals, even though they they might get the resections and they might have a a greater abundance of material in their archive, they just simply run out due to their volume. And as mentioned already, the small hospitals tend to, struggle just because they don’t have such a depth of samples in in their own collections.

The last factor that is becoming a a greater driver is hospitals are laboratories are becoming more aware of the real cost to them in, managing their controls. So often when asked how much their controls cost, the immediate answer is, well, it doesn’t cost us anything because it comes from our archive.

But when laboratory start looking at just how many scientists are involved in going through the database, identifying potential samples, potentially retesting them, there’s a cost to retest them. There’s a cost to get the pathologist involved, their time taken away from actually doing the diagnosis and and looking at to archive samples so that all of this is a drain, in time, resource, and cost.

Some laboratories make their own TMAs, and, it’s not uncommon for course to fall out, making the block redundant.

As I mentioned before, you know, variation that can occur through heterogeneity, just the innate heterogeneity of these these proteins in the samples means that, you know, in some cases, within a few microns, a a block becomes, useless.

And and some cases, they they just don’t have as much sample in that block as they first thought, so they cut through very quickly.

When laboratories take into consideration all these costs, they frequently outweigh the cost of buying controls.

And what a lot of laboratories are starting to do now is they they will buy, you know, four to six blocks, and they get in up to three hundred tests from a block. They will buy what they need for the year, and they will validate in the first block. And then this all these blocks are from the same batch. They don’t need to do any more validation for the year, and they can not have to worry about the, you know, sourcing controls for, you know, be it HER2 or ER or PR as an example. They’re good for the for the following twelve months. We are often asked about how the cells are fixed and processed, because laboratories want to use controls that are fixed and processed. It’s the same as the own material, the material that they’re testing in the laboratories.

But they can’t really control quality control for that. I mean, we can. We know how they’re fixed in process. We know how they work, and we know from the QC that they work in appropriate manner with, the standardized assays that laboratories use around the world. And probably the biggest consideration for them is whilst it’s, it’s nice to have samples that fix the process the same, a lot of them are testing samples from other hospitals. So, you know, most of the time, the samples they’re testing aren’t necessarily fixed and processed exactly the same as the the tissue controls they might wish to employ.

And each tissue is different. As I mentioned at the very beginning, it it it’s very hard to control just by the very nature of tissue and how it’s made up. I think this is very well illustrated when, you know, labs can demonstrate good EQA results on their tissue.

It’s not surprising. It’s it’s what they use day in, day out, and their assays are generally kind of, adapted for the material that they see in their laboratory.

What they often complain about is the referred samples from the EQA bodies. So this is, you know, centrally sourced tissue disseminated to these laboratories and they test it. And most of the time, they will complain that, we you know, the material from the EQA is isn’t that good. We can’t get it to work as well as ours.

And that just shows you that there is variation from, laboratory to laboratory just based on the on their how they’re fixing and processing their their samples. It’s not what they do is is wrong. What they do is is fit for the purpose in their laboratory to get appropriate results. But to think that everybody is working in a perfect manner is is perhaps not so true.

So with ourselves, what we look to do is answer a couple of issues, which were that cells typically don’t look like tissue. So they’re quite abstract. So we wanted something that would be more familiar. After all, immunoscedochemistry is very aesthetic. We feel comfortable, looking at things that are familiar with us. So that was the first part of this, for HistoCyte was creating a solution that looks more like tissue.

And in doing so, we wanted to retain the morphology, better morphology.

So we fix or harvest the cells in a proprietary manner.

That means we’ve retained some of that original morphology.

And, and that’s critical, we think, because it’s not just about the amount of brown that you get. The localization is is obviously very critical. You want to see membrane markers localize in the membrane and nuclear markers in the nuclei and so on.

The morphology can tell you how the the slide’s being treated generally. So those images in the bottom there are, showing you, the image one is a gold standard for in situ hybridization looking at HPV two and three. Clearly, the sample’s been overdigested.

So that morphology is is really important in situations like that. And show we’ll show you when, samples are are retrieved, for example.

So because we QC with standardized diagnostics from the main vendors, we’re able to show that they work in an appropriate manner. And therefore, when the laboratory use, samples, they’re not having to use tissue in their lab that can vary and can sometimes mean that, assays need to be adapted in order to cope with the variation that they see in the samples. It’s not it’s not uncommon.

The problem with that is you start getting drift drift in quality tissue drift in in the the the assays.

So our controls will prevent that. And this is where it’s important that laboratories are always looking at the quality of tissue in their laboratory and looking at internal controls. So the controls are one one thing, but looking at internal controls in the patient sample are critical. Like, with ER, for example, looking at normal ducts if they’re there to see that, you know, the the this they stain appropriately.

If they don’t, then there is potentially issues at a clinical level.

However, our controls are making sure that the standard and quality of the assays are maintained and they don’t drift.

For QC only, I think it’s really important, to stress the importance of of tissue. Really, what we’re doing by providing these cell lines is reducing the the drain on resource in the laboratory, reducing the the need to be creating, you know, countless, tissue blocks.

The QC will simply say the assay has worked. And if you’re using the, the semi positive controls that you’ve got an assay working at the right sensitivity.

And it’s important that laboratories validate what’s normal in their lab so they develop a a a baseline of performance in their lab with these these controls because each lab has its own, subtle variations mainly through the the protocols they employ and some of the local issues that will affect them.

It does not aid diagnosis. It can’t be used as a an equivalence. So level of staining in this in a core is equivalent to the level of staining in the tissue. That’s that’s not what they’re there for. They’re there to show you that the assays work the same as it did yesterday and the day before.

So with regards to tissue, they complimented by cells, but you still need tissue for validating assays.

I have had customers want to use a cell lines to validate assays. You can use it as a control in the validation of an assay, but you can’t use it to validate the assay. Tissue is vital. These are clinical assays that they need to be validated on clinical samples.

You can use a tissue for troubleshooting through your internal quality assurance.

I think most EQA groups that review samples want to see tissue or tissue as well as cells if you use those routinely.

But, also, it’s part of your incoming QC. Not all laboratories necessarily do it in a specific manner, but you should be looking at receive new reagents and how they perform. And, of course, the cell lines can be used, but you want to see it on your your own tissue. So I I think it’s important to stress that you still need tissue. What we’re doing is relieving burden on the laboratory for the volume that they require. As I said before, you know, each of these assays work appropriately with our controls, but they have their own subtle differences, be it created locally or the fact that, you know, they’re different chemistries. So they have different aesthetics, but they should produce the same result in terms of, you know, you have a a negative low, mid, high sample.

They should look appropriately negative, low, mid, high, on on each of the the platforms and the chemistries that they employ. They just might have different, shades of brown, for example.

But even with, the LDTs that some of the laboratories have, you know, they still need to create what’s normal for their laboratory. We call this this baseline.

Laboratories need to determine what that looks like across the platforms they employ. So if they use one for the breast work, for example, or two or three, they they need to show that they they work consistently across those. And through the week, the working week varies, the operators vary. And with the samples, they may cut and store them for when they need them. They need to determine that they are stable in that period. If they keep them on the bench, they keep them in the fridge, that needs to be validated in in their laboratory.

Without that, it’s hard to see how you confer, you know, greater confidence in your results. You need to essentially validate them for use in your lab. And with the advent of Qualitopix, we are seeing with our QC a a means of being able to create a greater metric around measuring our QC.

So we’re able to determine that how the assays work because immunosy chemistry is not kind of a a linear reaction. You know? It doesn’t work in a consistence. There is a there is a tolerance, and when it falls out of that, that’s where we want to to capture it. It’s not an exact science by virtue of the, multiple steps involved with the chemistry and the samples that we use.

So we rely on our eyeballs to to to to measure that. But with the advancing, image analysis and the the technologies that are available, we’re able to get a better handle on those. And with time, I would like to think that in combination with with enough data, with the the technology available, we’re not just gonna be able to determine whether the assays worked and how well it’s worked. But if it’s not worked, what’s gone wrong, and what, what was the cause of of that error so that we can fix these things in a in a a better real time fashion.

And the minute we often find that the the problems disappear as soon as they come and and none the wiser as to why they’ve happened. And I and I think in future, creating a greater degree of robustness around the element of the industry will only be a good thing for both industry and for the laboratories.

And thank you for your time.