Evaluating Ex Vivo Fluorescence Confocal Microscopy for Intraoperative Diagnosis in Pediatric Surgery: A Feasibility Study

Abstract

Background

Ex vivo fluorescence confocal microscopy (FCM) is an emerging technology that enables real-time, high-resolution digital imaging of freshly excised tissues without requiring standard histological preparation. This study aims to evaluate the diagnostic performance of FCM compared with conventional histology in a pediatric population with suspected oncological pathology.

Materials and Methods

A total of 18 tissue samples from pediatric patients with suspected oncological lesions were analyzed using FCM. The results were compared with the definitive diagnoses obtained via conventional histology, serving as the gold standard. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated to assess the diagnostic accuracy. Cohen's kappa coefficient (Κ), 95% confidence interval (95% CI), and p-value were computed to evaluate the interobserver agreement and the concordance between FCM and histology.

Results

FCM correctly identified 10 positive cases (true positives) and 7 negative cases (true negatives), with 1 false positive and no false negatives. Interobserver agreement was perfect, with a Cohen's kappa coefficient of 1.00 (95% CI: 0.81–1.00, p < 0.001). Sensitivity was 100% (95% CI: 72.2–100%), specificity 87.5% (95% CI: 52.9–97.8%), PPV 90.9% (95% CI: 62.3–98.4%), and NPV 100% (95% CI: 64.6–100%). The Cohen's kappa coefficient was 0.89, indicating excellent agreement between FCM and histology (95% CI: 0.67–1.10, p < 0.001). FCM provided rapid diagnostic results, significantly reducing turnaround time compared with conventional methods.

Conclusion

FCM demonstrates exceptional diagnostic accuracy, with excellent sensitivity and specificity in evaluating pediatric specimens. Its ability to deliver reliable intraoperative results highlights its potential as a valuable adjunct to conventional histopathology in pediatric surgery. Larger studies are warranted to confirm these findings and establish their role in clinical practice.

Keywords

ex vivo fluorescence confocal microscopy - pediatric - children - conventional histology - real-time

Publication History

Received: 13 April 2025

Accepted: 03 August 2025

Accepted Manuscript online:
05 August 2025

Article published online:
20 August 2025

• References

• 1 Di Fabrizio D, Bindi E, Ilari M, Filosa A, Goteri G, Cobellis G. Ex vivo fluorescence confocal microscopy meets innovation and revolutionary technology, for “real-time” histological evaluation, in pediatric surgical oncology. Children (Basel) 2024; 11 (12) 1417
• 2 Au M, Almeida-Magana R, Al-Hammouri T, Haider A, Shaw G. Accuracy of ex-vivo fluorescence confocal microscopy in margin assessment of solid tumors: A systematic review. J Histochem Cytochem 2023; 71 (12) 661-674
• 3 Ragazzi M, Longo C, Piana S. Ex vivo (fluorescence) confocal microscopy in surgical pathology: State of the art. Adv Anat Pathol 2016; 23 (03) 159-169
• 4 Razi S, Ouellette S, Khan S, Oh KS, Truong TM, Rao BK. Role of VivaScope 2500 ex vivo confocal microscopy in skin pathology: Advantages, limitations, and future prospects. Skin Res Technol 2023; 29 (06) e13388
• 5 Titze U, Sievert KD, Titze B. et al. Ex vivo fluorescence confocal microscopy in specimens of the liver: A proof-of-concept study. Cancers (Basel) 2022; 14 (03) 590
• 6 Malvehy J, Pérez-Anker J, Toll A. et al. Ex vivo confocal microscopy: revolution in fast pathology in dermatology. Br J Dermatol 2020; 183 (06) 1011-1025
• 7 Krishnamurthy S, Brown JQ, Iftimia N, Levenson RM, Rajadhyaksha M. Ex vivo microscopy: A promising next-generation digital microscopy tool for surgical pathology practice. Arch Pathol Lab Med 2019; 143 (09) 1058-1068
• 8 Rocco B, Sighinolfi MC, Bertoni L. et al. Real-time assessment of surgical margins during radical prostatectomy: a novel approach that uses fluorescence confocal microscopy for the evaluation of peri-prostatic soft tissue. BJU Int 2020; 125 (04) 487-489
• 9 Kamm M, Hildebrandt F, Titze B. et al. Ex vivo fluorescence confocal microscopy for intraoperative examinations of lung tumors as alternative to frozen sections-a proof-of-concept study. Cancers (Basel) 2024; 16 (12) 2221
• 10 De Benedetto L, Moffa A, Baptista P. et al. Potential use of vivascope for real-time histological evaluation in endoscopic laryngeal surgery. J Pers Med 2023; 13 (08) 1252
• 11 Eissa A, Puliatti S, Rodriguez Peñaranda N. et al. Current applications of ex-vivo fluorescent confocal microscope in urological practice: a systematic review of literature. Chin Clin Oncol 2024; 13 (04) 52
• 12 Villarreal JZ, Pérez-Anker J, Puig S. et al. Ex vivo confocal microscopy performs real-time assessment of renal biopsy in non-neoplastic diseases. J Nephrol 2021; 34 (03) 689-697
• 13 Puliatti S, Bertoni L, Pirola GM. et al. Ex vivo fluorescence confocal microscopy: the first application for real-time pathological examination of prostatic tissue. BJU Int 2019; 124 (03) 469-476
• 14 Bennàssar A, Vilata A, Puig S, Malvehy J. Ex vivo fluorescence confocal microscopy for fast evaluation of tumour margins during Mohs surgery. Br J Dermatol 2014; 170 (02) 360-365
• 15 Espinasse M, Cinotti E, Grivet D. et al. ‘En face’ ex vivo reflectance confocal microscopy to help the surgery of basal cell carcinoma of the eyelid. Clin Exp Ophthalmol 2017; 45 (05) 442-447
• 16 Krishnamurthy S, Ban K, Shaw K. et al. Confocal fluorescence microscopy platform suitable for rapid evaluation of small fragments of tissue in surgical pathology practice. Arch Pathol Lab Med 2019; 143 (03) 305-313
• 17 Rocco B, Sighinolfi MC, Sandri M. et al. Digital biopsy with fluorescence confocal microscope for effective real-time diagnosis of prostate cancer: A prospective, comparative study. Eur Urol Oncol 2021; 4 (05) 784-791
• 18 Bertoni L, Puliatti S, Reggiani Bonetti L. et al. Ex vivo fluorescence confocal microscopy: prostatic and periprostatic tissues atlas and evaluation of the learning curve. Virchows Arch 2020; 476 (04) 511-520
• 19 Panarello D, Compérat E, Seyde O, Colau A, Terrone C, Guillonneau B. Atlas of ex vivo prostate tissue and cancer images using confocal laser endomicroscopy: A project for intraoperative positive surgical margin detection during radical prostatectomy. Eur Urol Focus 2020; 6 (05) 941-958
• 20 Mathieu MC, Ragazzi M, Ferchiou M. et al. Breast tissue imaging atlas using ultra-fast confocal microscopy to identify cancer lesions. Virchows Arch 2025; 486 (02) 299-311
• 21 Nackenhorst MC, Kasiri M, Gollackner B, Regele H. Ex vivo fluorescence confocal microscopy: chances and changes in the analysis of breast tissue. Diagn Pathol 2022; 17 (01) 55
• 22 Messner L, Deußing M, Maurer M. et al. Ex vivo confocal laser scanning microscopy in rare skin diseases. Cancers (Basel) 2024; 16 (09) 28
• 23 Kose K, Fox CA, Rossi A. et al. An international 3-center training and reading study to assess basal cell carcinoma surgical margins with ex vivo fluorescence confocal microscopy. J Cutan Pathol 2021; 48 (08) 1010-1019