Tissue fixation (formalin)

Principle

• Fixation preserves tissue close to its living state, stops autolysis and putrefaction, and hardens tissue for processing.
• Formaldehyde forms methylene cross-links between proteins (mainly via amino groups).
• 10% neutral buffered formalin (NBF) is 1 part stock formalin (37–40% formaldehyde) to 9 parts water, i.e. about 4% formaldehyde, phosphate-buffered to about pH 7.

Specimen

• Fresh surgical and biopsy tissue placed into fixative as soon as possible after removal.

Reagents and equipment

• 10% NBF, leak-proof containers of suitable size, labels; fume hood or ventilated grossing station.

Procedure

• Keep cold ischemia time (removal to fixation) as short as possible, ideally under 1 hour.
• Use a fixative-to-tissue volume ratio of at least 10:1 (15–20:1 preferred).
• Slice large specimens so tissue is thin (about 3–5 mm); open hollow organs.
• Formalin penetrates about 1 mm per hour, but cross-linking takes much longer; most tissues need about 24 hours at room temperature.
• For breast tissue tested for ER, PR and HER2, fixation in 10% NBF for 6–72 hours is required (ASCO/CAP).

Quality control and pitfalls

• Under-fixation: poor morphology, autolysis, alcohol fixation of the center during processing, and uneven IHC staining.
• Over-fixation masks antigens; antigen retrieval is then needed.
• Formalin pigment (acid formaldehyde hematin): brown-black, birefringent deposits in blood-rich tissue fixed in acidic, unbuffered formalin; removed with alcoholic picric acid; prevented by buffering.
• Stock formalin can form white paraformaldehyde precipitate; methanol is added as a stabilizer.
• Formaldehyde is a carcinogen and sensitizer; monitor exposure, use ventilation, gloves and eye protection, and have spill kits available.

Clinical use

• Standard fixative for routine histopathology, H&E, most special stains, IHC and many molecular tests.
• Alternatives for specific purposes include Bouin's (some biopsies), B5 or zinc formalin (lymphoid/bone marrow) and alcohol (cytology, some glycogen and nucleic acid work).