Handheld confocal laser endomicroscopy (CLE) enables an instantaneous assessment of the cellular morphology of different tumor types and margin assessment ex vivo. Here, we describe the correlation between CLE images and histology and the feasibility of incorporating this technology in assessing surgical biopsy specimens. A CLE microscope (Optiscan, ViewnVivo) acquired high-definition confocal images of surgical biopsy cases and a single autopsy case (15 canine, 1 feline, and 1 equine cases). Following minimal training on the imaging modality, 14 participants (6 anatomic pathologists and 8 anatomic pathology residents) were asked to assess 17 cases with and without signalment and clinical history. The CLE captured detailed cellular morphology and tissue microarchitecture in various surgical biopsy and select autopsy specimens. Averaging the diagnostic accuracy with and without clinical history, study participants were able to distinguish abnormal tissue in 15/17 cases, neoplasia from inflammation in 12/17 cases, and the cellular morphology of neoplasia in 8/13 cases. Across all questionnaires, there was a statistically significant improvement in the diagnostic accuracy when clinical history was provided (P-value: 3.07E−07 to .005, paired t-test). CLE enables the capture of the high-resolution microarchitecture of tissue and can be readily correlated to histology images. We demonstrated that with little training, anatomic pathologists could evaluate surgical biopsy specimens that can provide a rapid preliminary diagnosis and margin assessment.
AcerbiFPolloBDe LaurentisC, et al. Ex vivo fluorescein-assisted confocal laser endomicroscopy (CONVIVO® System) in patients with glioblastoma: results from a prospective study. Front Oncol. 2020;10:606574.
2.
AlcazarJLDominguez-PirizJJuezL, et al. Intraoperative gross examination and intraoperative frozen section in patients with endometrial cancer for detecting deep myometrial invasion: a systematic review and meta-analysis. Int J Gynecol Cancer. 2016;26(2):407–415.
3.
AlgabaFArceYLópez-BeltránA, et al. Intraoperative frozen section diagnosis in urological oncology. Eur Urol. 2005;47(2):129–136.
4.
AliJPMallickBARashidK, et al. Diagnostic accuracy of intraoperative frozen section for margin evaluation of oral cavity squamous cell carcinoma. BMC Res Notes. 2024;17(1):43.
5.
BlackCMarottiJZarovnayaE, et al. Critical evaluation of frozen section margins in head and neck cancer resections. Cancer. 2006;107(12):2792–2800.
6.
ChapelEEChapelDBKunjuLP, et al. The anatomic pathology hospitalist model. Arch Pathol Lab Med. 2025;149:469–475.
7.
ChengEJenningsRNChenCL, et al. Optical coherence tomography for surgical margin evaluation of excised canine cutaneous and subcutaneous tumours. Vet Comp Oncol. 2022;20(4):836–845.
8.
ColemanMJSelmicLESamuelsonJP, et al. Diagnostic accuracy of optical coherence tomography for surgical margin assessment of feline injection-site sarcoma. Vet Comp Oncol. 2021;19(4):632–640.
9.
DornbuschJACoccaCJenningsR, et al. The feasibility and utility of optical coherence tomography directed histopathology for surgical margin assessment of canine mast cell tumours. Vet Comp Oncol. 2021;19(4):616–623.
10.
ChengJSohnEHJiaoC, et al. Correlation of optical coherence tomography and retinal histology in normal and Pro23His retinal degeneration pig. Transl Vis Sci Technol. 2018;7(6):18.
11.
DunbarKBOkoloPMontgomeryE, et al. Confocal laser endomicroscopy in Barrett’s esophagus and endoscopically inapparent Barrett’s neoplasia: a prospective, randomized, double-blind, controlled, crossover trial. Gastrointest Endosc. 2009;70(4):645–654.
12.
FabeloCSelmicLEHuangP-C, et al. Evaluating optical coherence tomography for surgical margin assessment of canine mammary tumours. Vet Comp Oncol. 2021;19(4):697–706.
13.
FaragallaHDavoudiBNofech-MosesN, et al. The use of optical coherence tomography for gross examination and sampling of fixed breast specimens: a pilot study. Diagnostics. 2022;12(9):2191.
14.
FoerschSHeimannAAyyadA, et al. Confocal laser endomicroscopy for diagnosis and histomorphologic imaging of brain tumors in vivo. PLoS ONE. 2012;7(7):e41760.
15.
GageISchnittSJNixonAJ, et al. Pathologic margin involvement and the risk of recurrence in patients treated with breast-conserving therapy. Cancer. 1996;78(9):1921–1928.
16.
HayashiMHariyaMKayanoM, et al. Distribution of follicles in canine ovary—a simple and rapid method for counting follicles. Cryobiology. 2015;71(3):514–517.
17.
HoltDParthasarathyABOkusanyaO, et al. Intraoperative near-infrared fluorescence imaging and spectroscopy identifies residual tumor cells in wounds. J Biomed Opt. 2015;20(7):76002.
18.
HoltDSinghalSSelmicLE.Near-infrared imaging and optical coherence tomography for intraoperative visualization of tumors. Vet Surg. 2020;49(1):33–43.
19.
KamstockDAEhrhartEJGetzyDM, et al. Recommended guidelines for submission, trimming, margin evaluation, and reporting of tumor biopsy specimens in veterinary surgical pathology. Vet Pathol. 2011;48(1):19–31.
20.
KaufmanZLewSGriffelB, et al. Frozen-section diagnosis in surgical pathology. A prospective analysis of 526 frozen sections. Cancer. 1986;57(2):377–379.
21.
KeereweerSVan DrielPBAASnoeksTJA, et al. Optical image-guided cancer surgery: challenges and limitations. Clin Cancer Res. 2013;19(14):3745–3754.
22.
LaakmanJMChenSJLakeKS, et al. Frozen section quality assurance. Am J Clin Pathol. 2021;156(3):461–470.
23.
LechagoJ.The frozen section: pathology in the trenches. Arch Pathol Lab Med. 2005;129(12):1529–1531.
24.
LégerHSantanaEBeltranWA, et al. Preparation of mouse retinal cryo-sections for immunohistochemistry. J Vis Exp. 2019;(149):59683.
25.
LevitinHAFossKDHagueDW, et al. The utility of intraoperative impression smear cytology of intracranial granular cell tumors: three cases. Vet Clin Pathol. 2019;48(2):282–286.
26.
LiptakJM.Histologic margins and the residual tumour classification scheme: is it time to use a validated scheme in human oncology to standardise margin assessment in veterinary oncology?Vet Comp Oncol. 2020;18(1):25–35.
27.
MartirosyanNLGeorgesJEschbacherJM, et al. Potential application of a handheld confocal endomicroscope imaging system using a variety of fluorophores in experimental gliomas and normal brain. Neurosurg Focus. 2014;36(2):E16.
28.
MeutenDJMooreFMDonovanTA, et al. International guidelines for veterinary tumor pathology: a call to action. Vet Pathol. 2021;58(5):766–794.
29.
MilovancevMTownsendKLGormanE, et al. Shaved margin histopathology and imprint cytology for assessment of excision in canine mast cell tumors and soft tissue sarcomas. Vet Surg. 2017;46(6):879–885.
30.
NewtonAPredinaJMisonM, et al. Intraoperative near-infrared imaging can identify canine mammary tumors, a spontaneously occurring, large animal model of human breast cancer. PLoS ONE. 2020;15(6):e0234791.
31.
PavlovVMeyronetDMeyer-BischV, et al. Intraoperative probe-based confocal laser endomicroscopy in surgery and stereotactic biopsy of low-grade and high-grade gliomas: a feasibility study in humans. Neurosurgery. 2016;79(4):604–612.
32.
PolglaseALMcLarenWJSkinnerSA, et al. A fluorescence confocal endomicroscope for in vivo microscopy of the upper- and the lower-GI tract. Gastrointest Endosc. 2005;62(5):686–695.
33.
PradiptaARTaneiTMorimotoK, et al. Emerging technologies for real-time intraoperative margin assessment in future breast-conserving surgery. Adv Sci. 2020;7(9):1901519.
34.
RatnaveluNDGBrownAPMallettS, et al. Intraoperative frozen section analysis for the diagnosis of early stage ovarian cancer in suspicious pelvic masses. Cochrane Database Syst Rev. 2016;3(3):CD010360.
35.
SankarTDelaneyPMRyanRW, et al. Miniaturized handheld confocal microscopy for neurosurgery: results in an experimental glioblastoma model. Neurosurgery. 2010;66(2):410–417.
36.
TanJQuinnMAPymanJM, et al. Detection of cervical intraepithelial neoplasia in vivo using confocal endomicroscopy. BJOG. 2009;116(12):1663–1670.
37.
WardJMUnoHFrithCH.Immunohistochemistry and morphology of reactive lesions in lymph nodes and spleen from rats and mice. Toxicol Pathol. 1993;21(2):199–205.
38.
WhitehairJGGriffeySMOlanderHJ, et al. The accuracy of intraoperative diagnoses based on examination of frozen sections. A prospective comparison with paraffin-embedded sections. Vet Surg. 1993;22(4):255–259.
