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Dead or alive? Autofluorescence distinguishes heat-fixed from viable cells

Purpose: A proof-of-concept study to evaluate a new autofluorescence method to differentiate necrotic thermally fixed cells from viable tissue following thermal ablation. Methods: A conductive interstitial thermal therapy (CITT) device was used to ablate swine mammary tissue and rabbit VX-2 carcinomas in vivo. The ablated regions and 10-mm margins were resected 24 h following treatment, embedded in HistOmer® and sectioned at 3 mm. The fresh sections were evaluated for gross viability with triphenyl tetrazolium chloride, 1 h post-resection. Representative non-viable and viable areas were then processed and embedded into paraffin, and sectioned at 5 µm. Standard H&E staining and proliferating cell nuclear antigen (PCNA) immunohistochemistry were compared against autofluorescence intensity, at 488-nm wavelength, for cellular viability. Results: Heat-fixed cells in non-viable regions exhibit increased autofluorescence intensity compared to viable tissue (area under receiver operating characteristics (ROC) curve = 0.96; Mann-Whitney P < 0.0001). An autofluorescence intensity-based classification rule achieved 92% sensitivity with 100% specificity for distinguishing non-viable from viable samples. In contrast, PCNA staining did not reliably distinguish heat-fixed, dead cells from viable cells. Conclusions: Examination of H&E-stained sections using autofluorescence intensity-based classification is a reliable and readily available method to accurately identify heat-fixed cells in ablated surgical margins.