Dead or alive? Autofluorescence distinguishes heat-fixed from viable cells.
OBJECTIVE
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 microm. 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.
Hennings
Leah
L
Department of Pathology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205, USA. LHennings@uams.edu
Kaufmann
Yihong
Y
Griffin
Robert
R
Siegel
Eric
E
Novak
Petr
P
Corry
Peter
P
Moros
Eduardo G
EG
Shafirstein
Gal
G
D000818:Animals; D002470:Cell Survival; D005260:Female; D006358:Hot Temperature / Q000009:adverse effects*; D006979:Hyperthermia, Induced; D008321:Mammary Glands, Animal / Q000528:radiation effects; D008856:Microscopy, Fluorescence / Q000379:methods*; D009336:Necrosis / Q000473:pathology*; D009374:Neoplasms, Experimental / Q000473:pathology; D018809:Proliferating Cell Nuclear Antigen / Q000032:analysis; D011817:Rabbits; D012867:Skin / Q000166:cytology; D013552:Swine
D018809:Proliferating Cell Nuclear Antigen