Objective: To identify an accurate, reliable, and readily accessible histologic method to verify cell death in thermally ablated margins.

Methods: Thermal ablation of tissue leads to the formation of three distinct zones: a central region of heat fixation, a region of coagulative necrosis, and a transitional region. Cells in heat fixed regions are functionally dead, yet retain histologic characteristics of live cells. When ablated margins are evaluated histologically, heat-fixed cells may be confused with viable tumor cells, complicating the determination of negative margins. TriphenylTetrazolium Chloride (TTC) staining has been used to determine tissue viability. In viable tissue, the TTC is reduced to red formazan. Dead tissue remains unstained. TTC is not useful for histologic sections, however, as the dye fades during processing. In addition to examination of H&E sections, cell viability has been determined in histologic sections via immunohistochemistry against proliferating cell nuclear antigen (PCNA). However, microwave fixation does not reduce antigenicity of PCNA, and heat fixation may behave similarly. Although less frequently employed, fluorescence microscopy may be used to detect cell death, because necrotic cells exhibit autofluorescence in contrast to most viable cell populations. After ablation using conductive interstitial thermal therapy (CITT), swine mammary tissue and rabbit VX-2 carcinomas were resected, embedded in Histomer^®, sectioned at 4mm thickness, and incubated in TTC solution at 37ºC for 1 hour to identify non-viable and viable regions. Sections were then rinsed and placed into 10% neutral buffered formalin, and photographed. Non-viable and viable areas were trimmed, processed and embedded into paraffin, and sectioned at 5µm. PCNA staining was performed using immunoperoxidase labeling and evaluated semiquantitatively . Additional sections were stained with H&E, and autofluorescence was quantified using NIH Image-J analysis of digital images captured under fluorescence microscopy using a 488nm filter.

Results: Heat fixed cells in non-viable regions retain reactivity to PCNA, but exhibit increased autofluorescence compared to viable tissue.

Conclusions: PCNA staining is unreliable as an indicator of cell death in heat-fixed, ablated tissues. Examination of H&E stained sections using fluorescence microscopy may be a reliable and readily available method to accurately identify heat-fixed cells in ablated surgical margins.