In other words, how a small biopsy disappears piece by piece before we manage to do everything we expect from it

Tissue stewardship is the deliberate management of tissue material so that there is enough for histopathological diagnosis and immunohistochemistry, as well as for predictive testing and NGS.

There is a block. Small. Maybe even very small.

A few fragments of tissue taken from the lung. Nothing spectacular. The pathologist looks at the H&E and knows that a seemingly simple question needs to be answered:

What is it?

Except that in 2026, the answer “adenocarcinoma” is often only the beginning, because more questions immediately follow.

• Is PD-L1 expressed?
• Are there therapeutically relevant molecular alterations?
• Is the material suitable for NGS?
• Do we need FISH or ISH?
• Do we need additional immunohistochemistry?

And suddenly this unassuming block becomes one of the most valuable resources in the entire diagnostic process. The problem is that the block has one very unpleasant property.

It does not regenerate.

The first cut

First, trimming. Then H&E. Then another level because the first one is not ideal. Next, immunohistochemistry.

• TTF-1.
• p40.
• Maybe Napsin A.
• Maybe a neuroendocrine marker.

One more slide. And another one… let’s make a “series”.

Another cut bites the dust.

Every turn of the microtome takes us deeper into the block. Every slide uses a fragment of tissue that we will never recover in its original form. Usually, that is not a problem. Unless, of course, it suddenly is.

Small biopsy. Big expectations.

Modern lung cancer diagnostics is probably one of the best examples of this paradox. We obtain material using increasingly less invasive procedures, which is excellent. Smaller biopsies mean less burden for the patient. At the same time, however, we expect more and more from that small amount of tissue.

The College of American Pathologists, together with a number of organisations involved in pulmonology, cytopathology and molecular diagnostics, developed dedicated recommendations specifically for the collection, processing and triage of thoracic small biopsies and cytology specimens. The reason is simple: the material must be sufficient not only for morphological diagnosis, but also for ancillary testing — immunohistochemistry, FISH, molecular studies and other tests required for further treatment. Interestingly, these guidelines were reaffirmed by CAP in February 2026.

And this is where a concept appears that should probably hang above every microtome next to the safety instructions:

Tissue stewardship — sensible management of tissue material.

Not “save tissue at all costs”, not “let’s avoid testing”, but:

let’s perform exactly the tests that are needed — and leave enough material for the next stage of diagnostics.

The most expensive test may cost… one unnecessary IHC stain

Of course, this is not about the price of the reagent, but about the biological cost. If we have a large resection specimen, a few extra slides usually make little difference. But if we have three tiny biopsy fragments, the situation is completely different. That is why, in the diagnosis of small lung biopsies, the importance of a limited, carefully selected IHC panel has been emphasised for years.

In the right setting, morphology should do as much of the work as it can on its own. When immunohistochemistry is needed, however, a carefully selected combination of markers — classically including TTF-1 for glandular differentiation and p40 for squamous differentiation — may allow a large proportion of non-small cell lung carcinomas to be classified with minimal tissue consumption. In one analysis of limited biopsy samples, the use of TTF-1 and p40 correctly classified the great majority of cases that required immunohistochemical differentiation.

And this is exactly where a certain temptation appears. If we have an IHC stainer and the antibody is available… if it “might be useful”… it feels almost wasteful not to run it. But the block does not know that we are doing the test “just in case”. For the block, every slide costs exactly the same amount of tissue.

Death by a thousand sections

It is rare for tissue to be exhausted in one spectacular move. Rare does not mean never, but much more often it disappears slowly.

• H&E.
• An additional level.
• TTF-1.
• p40.
• Napsin A.
• PanCK.
• Synaptophysin.
• One more marker.
• A repeat stain.
• A few unstained slides.
• PD-L1.
• And suddenly someone asks:

“And the block for molecular testing?”…

Silence.

This is the real death by a thousand sections. Not one catastrophic decision, but a hundred small, completely reasonable-looking decisions that together lead to a situation in which there is simply too little material left. That is why test planning increasingly begins to resemble budget management.

The diagnostic tissue budget

You can think of the block as an account. At the beginning, we have a certain amount of capital. We do not know exactly how much will be needed later, but we know that the subsequent stages may include:

morphology → diagnostic IHC → predictive biomarkers → molecular diagnostics.

If we spend everything at the first stage, nobody is going to increase our limit.

CAP/IASLC/AMP recommendations for molecular testing in lung cancer have long highlighted the need to preserve material for tests predicting response to treatment. They also pointed to the benefit of panel-based testing of multiple molecular alterations rather than repeatedly consuming material in a sequence of single-gene assays where appropriate. And that changes the way we think. The question is no longer:

“Can we do another test?”

but rather:

“Will the result of this test change the diagnosis or further management enough to justify using another part of the tissue?”

It may seem like a small difference. In practice, it is enormous.

Why not simply take more tissue?

Of course, that would be ideal. Except that the patient is not a warehouse of paraffin blocks. A repeat biopsy may mean another procedure, more waiting, additional risk and a delay in treatment decisions. This is associated not only with patient discomfort but, above all, with additional costs.

That is why CAP guidelines on thoracic small biopsies address not only what happens once the specimen reaches the laboratory, but also how the material is collected, the number of passes, adequacy assessment and the use of ROSE where available and justified. One of the aims is to increase the chance of obtaining material sufficient both for diagnosis and for ancillary testing.

Tissue stewardship therefore begins before the block ever reaches the microtome.

In fact — before the block even exists.

When the dust comes back to life

And now we come to perhaps the most interesting part of this story. What if the tissue really has been exhausted? Is a previously used slide now only documentation?

Not necessarily.

In April 2026, Pathology published a paper with a wonderfully fitting title:

“Upcycling pathology”

The authors set out to determine whether tissue from previously prepared H&E slides and immunohistochemically stained slides could be reused for molecular diagnostics.

The study included 40 lung cancer biopsies. The slides were first digitised to preserve their diagnostic image. Tissue from the previously used slides was then subjected to DNA extraction and targeted NGS. The result?

Sequencing was successful in 33 of 40 cases — 82.5%. In 30 cases, the detected variants were fully concordant with the reference material, while in the remaining three the concordance was partial. The authors also noted that the quality of the recovered DNA was poorer and that the method requires further validation. This obviously does not mean:

“No problem, use up the block — we can always scrape the H&E later.”

Quite the opposite. The best solution is still to manage the material properly from the very beginning, but the study demonstrates something extremely interesting. A slide that until recently would have been considered the final product of the diagnostic process may potentially become source material again for another test. A kind of pathological recycling.

Or — as the authors called it — upcycling.

It is not about doing fewer tests

This is probably the most important point. Tissue stewardship is not about doing less diagnostics. It is about doing diagnostics more intelligently.

• Using morphology when morphology can genuinely provide the answer.
• Using targeted panels instead of an immunohistochemical “shotgun”.
• Being aware of which tests may be needed in an hour, tomorrow or next week.
• Communication between the pathologist, histotechnologist, cytologist, molecular biologist and the clinician obtaining the specimen.
• And finally — remembering that a thin, several-micrometre section of tissue may carry far more value than its size would suggest.

Because sometimes that one fragment contains the information that will determine the patient’s next treatment. So the next time the microtome makes another turn…

it is worth thinking for a second:

Another cut bites the dust.

Hopefully not the one we will need later.

PS: what if the problem starts before the first cut?

There is one more part of this story worth remembering.

The material used in the previously described “upcycling” experiment involving H&E and IHC slides was formalin-fixed, and formalin is not neutral to nucleic acids.

Formalin fixation leads, among other things, to biomolecular cross-linking, nucleic acid fragmentation and artefact formation, all of which can complicate subsequent molecular analyses. And although modern NGS assays can work very well with FFPE material, to a large extent we have simply learned how to cope with the limitations of a fixative that has been used for more than a century.

And this is where things become really interesting.

One potential alternative is GAF — Glyoxal Acid-Free, an acid-free glyoxal-based fixative. In a multicentre comparative study, GAF demonstrated diagnostic quality and preservation of morphology that were non-inferior to buffered formalin in the tissues evaluated. Earlier studies also showed comparable immunohistochemical and molecular results, together with better preservation of longer DNA fragments in GAF-fixed material. More recent publications have also highlighted a potential advantage of GAF in preserving DNA and RNA relevant to modern molecular methods, including NGS. This is especially interesting in the context of tissue stewardship — because if we begin to think of every tissue fragment as potential material not only for H&E and IHC, but also for later molecular diagnostics, the way the specimen is fixed may matter long after the first slide has been prepared.

And here we can ask a rather provocative question.

If DNA suitable for NGS could be recovered from previously stained H&E and IHC slides that had been formalin-fixed…

what might be possible if the material had been fixed from the outset using a method that preserves nucleic acids more effectively?

We do not yet have enough data to answer: “problem solved”. Further studies and validation of specific procedures are needed, but that is exactly why this is such an interesting direction. Perhaps one day the H&E slide will no longer be seen exclusively as the final product of morphological diagnostics. In situations of critical tissue shortage, it may become a backup source of material for further molecular testing.

And one more thing: safety

Formaldehyde is toxic and carcinogenic, which is why systems designed to reduce staff exposure have been developed for years. Containers in which formalin is released only after closure, as well as closed systems, are undoubtedly a step forward. Studies show that they can significantly reduce contact with formaldehyde during specimen collection and transport. But not necessarily for pathology laboratory staff.

And, above all, they do not change one basic fact:

there is still formalin inside.

Such a system may protect the person collecting the biopsy very effectively. It does not, however, make formaldehyde magically disappear from the rest of the process.

In conversations with clinicians performing procedures, we once asked what they thought about containers with formalin stored in the cap. The answer was: “I don’t care.” Why? Because they were not the ones placing the biopsy specimens into the containers — “the assistants do that”. Brutal, but true. Later, however, the material still has to be received, opened, grossed and processed. It is precisely during work in pathology laboratories — particularly during gross examination — that occupational exposure may still occur, which is why adequate ventilation, grossing stations and formaldehyde concentration monitoring remain necessary. So what does “modern protection from exposure” actually give us? Not much, if the same hazard remains embedded in the process.

That is why it is worth asking one more question:

do we want to spend the next few decades building increasingly complex systems to protect people from formalin — or should we, in parallel, investigate much more seriously whether formalin itself can be replaced?

GAF should not be treated as a magical solution that can simply be introduced into every laboratory tomorrow without validation. Changing the fixative requires validation of morphology, IHC, ISH and the molecular methods used in a given laboratory, but the available results make it increasingly difficult to treat formalin alternatives as merely a laboratory curiosity. We have tested a substitute ourselves in multiple laboratories — with remarkable results.

If the future of pathology means extracting more and more information from smaller and smaller tissue fragments, then tissue stewardship may come to mean not only:

“how many more times can we cut this block?”, but also

“What was this block fixed in?”

Publications and guidelines

Roy-Chowdhuri S. et al. — Collection and Handling of Thoracic Small Biopsy and Cytology Specimens for Ancillary Studies. CAP guideline on the collection, preparation and management of thoracic small biopsies and cytology specimens; originally published in 2020 and reaffirmed by CAP in February 2026. CAP — guideline and full information

“Upcycling pathology”: targeted next-generation sequencing is feasible in re-purposed H&E and immunostained FFPE sections in lung cancer biopsies. Pathology, 2026. DOI: 10.1016/j.pathol.2025.12.009. Abstract in Pathology

CAP/IASLC/AMP — Molecular Testing Guideline for Lung Cancer. CAP — Molecular Testing Guideline

Best Practices Recommendations for Diagnostic Immunohistochemistry in Lung Cancer

Application of the 5th WHO Guidelines for the Diagnosis of Lung Carcinoma in Small Lung Biopsies.

Characterization of NSCLC in Limited Biopsy Samples and Identifying Optimal Immunohistochemical Marker Combinations.