15.4 Special Procedures: Frozen Sections, Special Embedding, Immunohistochemistry, Electron Microscopy, Autopsy, Museum Techniques, and Laboratory Records

Special Procedures: Frozen Sections, Special Embedding, Immunohistochemistry, Electron Microscopy, Autopsy, Museum Techniques, and Laboratory Records

The histopathology blueprint gives 10% to special tissue processing and 5% each to autopsy, special procedures, and clerical work and logging. These are practical topics. The board asks what the histotechnologist does when a surgeon needs an answer in 20 minutes, when a specimen will not section in paraffin, when an antibody stain needs antigen retrieval, when a body is autopsied, when a specimen goes on display, and when a label is missing. This section covers each in turn.

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1. Frozen Sections and Intraoperative Consultation

• Purpose: A rapid diagnosis while the patient is still on the operating table, such as whether a lesion is malignant or whether a surgical margin is clear. Frozen sections are also used for fat stains (Oil Red O, Sudan black B), enzyme histochemistry, and direct immunofluorescence, all of which paraffin processing would destroy.
• Freezing: Fresh, unfixed tissue is embedded in OCT (optimal cutting temperature) compound and frozen rapidly. Slow freezing allows large ice crystals to form and leaves holes ("Swiss cheese" artifact). Snap-freezing in isopentane cooled by liquid nitrogen avoids both ice crystals and the insulating vapor layer that forms when tissue is dropped directly into liquid nitrogen. Aerosol coolants, dry ice, and the cryostat's cold block are also used.
• Cryostat: A microtome housed in a refrigerated cabinet, commonly operated around -18 °C to -25 °C. Fatty tissue such as breast needs colder settings, while brain and liver cut better slightly warmer. Sections are usually 5 to 10 µm thick, picked up on a room-temperature slide, fixed at once (formalin or alcohol for a few seconds), and stained with a rapid H&E or toluidine blue.
• Turnaround: Laboratories commonly aim to report most single-specimen frozen sections within about 20 minutes of receipt.
• Safety: Tissue in a cryostat is unfixed and potentially infectious. Tuberculosis is a particular concern in the Philippines, so suspected TB specimens should not be frozen when it can be avoided. The cryostat is decontaminated regularly.

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2. Special Embedding Media

Double embedding infiltrates tissue with celloidin and then paraffin to support hard, brittle material.

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3. Immunohistochemistry (IHC) and Immunofluorescence

• Principle: An antibody binds a specific antigen in the tissue section, and an enzyme label converts a chromogen into a colored precipitate at that site.
• Antigen retrieval: Formalin cross-links mask many epitopes. Heat-induced epitope retrieval (HIER) heats sections in a buffer such as citrate (pH 6) or Tris-EDTA (pH 9) in a pressure cooker, steamer, or water bath. Enzyme-induced retrieval uses proteinase K, trypsin, or pepsin.
• Blocking: Endogenous peroxidase (abundant in red cells and granulocytes) is quenched with dilute hydrogen peroxide, commonly 3%. Endogenous biotin is blocked when a biotin-based detection system is used, and protein blocking reduces nonspecific background.
• Chromogens: DAB (diaminobenzidine) gives a permanent brown product that tolerates alcohol and xylene. AEC gives a red product that is alcohol-soluble, so it requires an aqueous mounting medium.
• Controls: A positive tissue control known to contain the antigen, a negative reagent control in which the primary antibody is omitted or replaced, and internal controls (normal structures in the patient's own section that should or should not stain).
• Common markers: Cytokeratins (epithelium and carcinoma), vimentin (mesenchymal cells), CD45 or leukocyte common antigen (lymphoma), CD20 (B cells), CD3 (T cells), S-100 and SOX10 (melanoma and nerve sheath tumors), desmin (muscle), chromogranin and synaptophysin (neuroendocrine tumors), and estrogen receptor, progesterone receptor, and HER2 (breast carcinoma).
• Direct immunofluorescence on unfixed frozen sections detects immunoglobulin and complement deposits in kidney and skin biopsies. Specimens for immunofluorescence are sent fresh or in a transport medium such as Michel's medium, never in formalin.

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4. Electron Microscopy Processing

1. Primary fixation: Tissue cubes no larger than about 1 mm³ in 2.5% glutaraldehyde (cross-links proteins).
2. Post-fixation: 1% osmium tetroxide, which fixes lipids and adds electron density to membranes.
3. Dehydration in graded ethanol or acetone, then propylene oxide as the transitional solvent.
4. Embedding in epoxy resin and polymerization by heat.
5. Semithin sections (0.5 to 1 µm) stained with toluidine blue to choose the area of interest, then ultrathin sections (60 to 90 nm) cut with a glass or diamond knife on an ultramicrotome and collected on copper grids.
6. Contrast staining with uranyl acetate and lead citrate.

Electron microscopy remains important for renal biopsies (location of immune deposits and basement membrane changes), some tumors, and identifying viral particles.

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5. Autopsy

Philippine Legal Basis

Section 95 of the Code on Sanitation of the Philippines (PD 856) governs autopsies:

• Who may perform autopsies: Health officers, medical officers of law enforcement agencies, and members of the medical staff of accredited hospitals.
• When autopsies are performed: When required by special laws; on orders of a competent court, a mayor, or a provincial or city fiscal; on the written request of police authorities; when the Solicitor General or a fiscal deems it necessary to disinter remains to determine the cause of death; and when the nearest kin requests it in writing.
• Hospital deaths: The hospital director notifies the next of kin and requests permission. The autopsy may proceed when permission is granted or when no objection is raised within 48 hours after death. If there is no next of kin, permission comes from the local health authority.

Types and Evisceration Techniques

• Medicolegal (forensic) autopsies establish the cause and manner of death for legal purposes; hospital (clinical) autopsies clarify disease and treatment effects with family consent. An autopsy may be complete or limited to selected organs.
• Virchow technique: Organs are removed one by one and examined individually.
• Rokitansky technique: Organs are dissected largely in situ.
• Ghon technique: Organs are removed in blocks (cervical and thoracic organs, abdominal organs, and urogenital organs).
• Letulle technique: All organs are removed en masse and then dissected; it best preserves relationships between organs.

The Histotechnologist's Role

Autopsy assistants prepare the instruments, label containers, and help collect specimens: tissue blocks in formalin for histology, sterile tissue and heart blood for microbiology, and toxicology specimens such as femoral blood, vitreous humor, urine, and gastric contents. Full personal protective equipment and good ventilation protect against infectious agents, formalin vapor, and sharps injuries.

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6. Museum Techniques

Gross specimens for teaching are fixed, color-restored, and mounted so they keep their natural appearance:

• Kaiserling method:
  1. Kaiserling I (fixing solution): Formalin with potassium nitrate and potassium acetate. Specimens are fixed until firm.
  2. Kaiserling II (color restoration): About 80% to 95% ethyl alcohol, which restores the red color of hemoglobin pigments. Exposure is watched closely because too long bleaches the color.
  3. Kaiserling III (mounting and preserving solution): Glycerin with potassium acetate in water, with a preservative.
• Pulvertaft's modification adds sodium hydrosulfite (sodium dithionite) to the mounting fluid to keep restoring color.
• Specimens are displayed in sealed acrylic (Perspex) or glass jars, labeled with the diagnosis, and topped up as the fluid evaporates.

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7. Clerical Work: Accessioning, Logging, and Records

• Requisition and labeling: Every container and requisition carries at least two patient identifiers (such as full name and date of birth or hospital number), the specimen site, the procedure, the clinical history, and the collection time. For breast specimens tested for hormone receptors and HER2, the time of removal and the time placed in formalin are recorded because cold ischemia time should not exceed about 1 hour and fixation should last 6 to 72 hours.
• Accessioning: Each specimen receives a unique accession number (often year-prefixed, such as S26-01234) entered in the logbook or laboratory information system. The same number follows the specimen through grossing, cassettes (with block letters or numbers written in solvent-resistant ink or printed), slides, and the final report.
• Mismatches and unlabeled specimens: Biopsies cannot be recollected, so they are never discarded. The laboratory holds the specimen, contacts the submitting clinician to correct the identification under a documented procedure, and records the discrepancy.
• Reconciliation: Blocks and slides are matched against the log at embedding, sectioning, and sign-out so that no block is lost and no slide is mislabeled.
• Retention: Laboratories keep wet tissue until well after the report is final and keep blocks, slides, and reports for years. Commonly cited College of American Pathologists minimums are wet tissue 2 weeks after the final report, and paraffin blocks, surgical pathology slides, and reports 10 years. Philippine laboratories follow DOH licensing requirements and institutional policy.
• Report corrections: Changes after sign-out are issued as clearly labeled amended or supplementary reports. The original report is never overwritten without a trace.