Ancillary Techniques (Molecular, Cytology, Frozen Section, Education)
Frozen section
Performed on a cryostat at about -20 to -25 °C, tissue embedded in OCT and cut at 4-6 µm, stained with rapid H&E or toluidine blue within minutes. Indications include intraoperative diagnosis, margin assessment and confirming that lesional tissue is present for permanent processing. Pitfalls: freezing artifact, fatty tissue that will not cut, bone needing decalcification, small biopsies consumed by the frozen block, and a deferred diagnosis when the sample is not representative.
Cytology
Alcohol-based fixation (95% ethanol) or liquid-based collection preserves nuclear detail. The Pap stain uses hematoxylin, OG-6 (orange) and EA-50 (green-pink); Giemsa or Diff-Quik highlights cytoplasm and extracellular material. Cell blocks permit ancillary testing on cytology material.
Molecular
PCR, FISH and sequencing require intact nucleic acid. Acid decalcifiers (nitric, hydrochloric) fragment DNA and RNA, so EDTA is preferred whenever molecular testing is planned; prolonged formalin fixation also lowers yield. FISH depends on adequate fixation and balanced protease digestion: underdigestion gives weak signals, overdigestion loses nuclei.
Education
Competency assessment is required for new hires and at defined intervals thereafter, with documented continuing education and training records for every new method.
Traps
Calling freezing artifact a diagnostic finding; sending acid-decalcified bone for molecular testing; assuming a Pap-stained smear can be used for IHC without validation; miscounting FISH signals.
Rule of thumb: For any tissue that might go to molecular testing, fix in NBF and decalcify with EDTA, never with acid.