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A 4 mm Lung Core and Six Requests

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A 4 mm Lung Core and Six Requests

A 4 mm lung core biopsy from a never-smoker with a 3.1 cm mass arrives for reflex testing. The pathologist requests: H&E, TTF-1, p40, ALK (D5F3), PD-L1 (22C3), and EGFR/ALK molecular testing. The block yields an estimated 12–14 usable sections. Each IHC needs one 4 µm section on a charged slide; the molecular lab requires four 5 µm unstained sections on uncharged slides and will not accept tissue that has been melted at high temperature, stained, or floated on a shared water bath. The tumor is estimated at 15% of the core cross-sectional area. The block is the only one available and there is no cell block.

1. Plan the sectioning order and slide allocation. What do you cut first, and what rules govern how you cut it?

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Budget first. Roughly: 1 H&E + 4 molecular + 4 IHC = 9 sections, leaving 3–5 for repeats and deeper levels. That is workable but leaves almost no slack - so nothing gets cut 'just in case.'

Order:

  1. One H&E at 4 µm to confirm tumor is present and to estimate tumor cellularity on the actual sections. If the tumor is not in the first levels, everything downstream changes.
  2. The four molecular sections next, before the IHC block face gets worn. Cut at 5 µm on uncharged slides, from a fresh blade, floated on a dedicated clean water bath with fresh water (nuclease-free if available), and handled with clean forceps. Do not bake or melt these slides. Cutting them early also minimizes the number of times the block face is cut and re-cut, which is the main source of DNA/RNA degradation and tissue loss at the face.
  3. Then the IHC sections, one per slide: TTF-1, p40, ALK, PD-L1.

Rules throughout:

  • Cut on demand where possible, but keep the block face protected (paraffin film or a spare block) so it does not oxidize between cutting sessions.
  • Never float two blocks in the same bath; never let a section from another case enter the bath.
  • Label every slide with the block and section number so a repeat can be traced.
  • Record how many sections were taken off the block - this is a finite resource and the record is what lets the next person plan.

2. Given 15% tumor in a 4 mm core, which requests would you flag, defer, or drop, and why?

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The binding constraint is not the number of slides - it is the amount of tumor. At 15% of a 4 mm core, several of these assays may be uninterpretable no matter how carefully the sections are cut.

Prioritize:

  • Lineage markers (TTF-1, p40). These are the cheapest in tissue terms and determine whether this is adenocarcinoma or squamous, which in turn determines whether molecular testing is even indicated. Run them first.
  • Molecular (EGFR/ALK). In a never-smoker with a 3.1 cm lung mass, the pretest probability of a driver alteration is meaningful and the result is actionable for first-line therapy. Protect these sections above all: molecular testing cannot be salvaged if the tissue is exhausted or the nucleic acid is degraded.

Flag for the pathologist before cutting:

  • PD-L1 (22C3). Scoring requires a minimum number of viable tumor cells, and at 15% of a 4 mm core the tumor cell count may fall short. There is a real chance the result will be reported as insufficient for interpretation - which means the section was spent for nothing.
  • ALK IHC. Same concern: a negative result on a core with scant tumor is unreliable, and the molecular ALK result (if ordered as a fusion assay) may be the more robust answer.

The judgment call: don't silently cut everything. Tell the pathologist the arithmetic - estimated tumor cellularity, sections remaining, and which assay is most likely to be non-contributory - and ask which they would sacrifice if only one can be dropped. The default in most algorithms is to drop PD-L1 first, because lineage and driver testing drive immediate management and PD-L1 can be repeated later on a new sample.

3. TTF-1 comes back with no staining in the tumor and also no staining in the adjacent benign bronchial epithelium. What is your interpretation and next step?

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The run is invalid - this is not a negative result.

The benign bronchial epithelium and normal pneumocytes in the same core are the internal control for TTF-1, and they should show nuclear positivity. When the internal control fails, a negative tumor result carries no information. Reporting it as 'TTF-1 negative' could push the case toward a squamous or other lineage designation on the basis of a failed stain.

Work-up, in order of likelihood:

  1. Check the external control on the same slide - if it is also negative, the problem is reagent, detection, or retrieval. If the external control is positive while the internal control is negative, suspect incomplete antigen retrieval or inadequate fixation of this particular block (over-fixation with prolonged NBF can mask the epitope in a small core).
  2. Verify the basics: antibody lot and expiry, detection kit, retrieval solution pH and time, and whether the slide was appropriately charged and not dried during staining. A section that dries during the protocol gives diffuse or absent staining.
  3. Repeat on the next section with fresh reagents and a proper control. Only if the repeat shows a valid internal control and a genuinely negative tumor do you report a negative result.
  4. Consider a different clone or an alternate lineage marker if the repeat is equivocal, but only after checking with the pathologist about tissue budget - this is exactly the spare section you deliberately left.

The general rule: an IHC result is only as good as its control. A negative stain with a failed control is an unanswered question, not a negative answer, and it should never be signed out as data.