A Friday-Afternoon Fragment: Frozen Section Versus PD-L1 and NGS

First: stop and inspect. Measure and describe the fragment, photograph it, and record the time out of the patient. A 5 mm fragment can be consumed by one frozen block, and once frozen it is generally unusable for validated PD-L1 and most NGS assays.

Do first:

• Make touch imprints (4-6 slides) from the fresh cut surface for immediate cytologic adequacy and to reserve cellular material; air-dry and stain some, fix the rest.
• Label and segregate: one portion for frozen, the remainder for formalin fixation.

Put in the cryostat: at most a 2-3 mm slice, and only if the answer changes intraoperative management. Here it may - a malignant diagnosis lets the surgeon place the port in the same anesthetic.

Do not freeze: the entire fragment, and do not freeze the portion you intend for PD-L1 or sequencing. Most validated PD-L1 assays and targeted NGS pipelines are validated on formalin-fixed, paraffin-embedded tissue; frozen tissue changes the antigen and nucleic acid substrate and falls outside the validated claim.

Fix the remainder in 10% NBF for 6-24 hours, then process - and start it now, not Monday, because fixation time is a variable you control.

Do not chase the diagnosis in the cryostat. A nondiagnostic frozen section is a result; the answer is 'defer to permanent sections,' communicated to the surgeon within minutes so he does not wait or act on nothing.

Next moves, in order:

1. Protect the FFPE portion. Confirm it is in formalin with an adequate volume-to-tissue ratio and an accurate in/out time. This tissue is now the sole source of PD-L1 and NGS material.
2. Harvest the saline. The 20 mL of saline the fragment traveled in contains shed cells. Centrifuge it and make a cell block (or a cytospin preparation); if tumor cells are present, this is a legitimate, sometimes the only, molecular substrate, and it may complement the FFPE block.
3. Use the touch imprints. They can confirm malignancy cytologically and give the pathologist a rapid read even when the frozen block fails; they are also a backup for molecular testing.
4. Estimate tumor cellularity on the H&E before block selection - necrotic debris and crush reduce the effective cellularity, and the 20% requirement applies to viable tumor cells, not to the area that looked abnormal on frozen.

Communicate to the surgeon that the diagnosis will be on permanent sections (typically the next business day) and that a port decision should not rest on a nondiagnostic frozen.

Document, on the requisition and in the report:

• Time of collection, time received, time in saline (approximately 90 minutes), transport temperature.
• Exactly what was taken for frozen, what was fixed, and the fixation start and stop times.
• The frozen diagnosis and the fact that permanent sections and ancillary studies were deferred.
• Estimated tumor cellularity and the block(s) selected for molecular testing.

Quality issues this case exposes:

1. No collection medium. Normal saline is isotonic but not a fixative; nucleic acids and antigens degrade, and cells lyse. Stock a transport medium, or instruct radiology to send fresh tissue dry on saline-moistened gauze with a hard time limit.
2. Friday-afternoon triage. Tissue arriving after the sequencing cutoff needs a written pathway: who decides block selection, who authorizes fixation, and how the case is handed to Monday's run without sitting in formalin over the weekend (over-fixation is its own failure mode).
3. Communication loop. A nondiagnostic frozen section must be called to the surgeon, not merely written in the log.

Change for next time: a preprinted fresh-specimen triage form with fields for intended tests, and a standing instruction that any specimen with a molecular or PD-L1 request is divided before freezing, with the FFPE portion placed in formalin immediately on receipt.