Trimodal cytological integration of micronuclei assay, argyrophilic nucleolar organizer region staining, and cytomorphometry enhances diagnostic discrimination of canine gingival masses

Vet World Vol.19 January-2026 Article - 6

Research Article

Veterinary World, 19(1): 65-80

https://doi.org/10.14202/vetworld.2026.65-80

Poppapak Hoonpo¹

, Tawewan Issarankura Na Ayudhaya²

, Kridsada Chaichoun²

, Panpanga Sangsuriya²

, Thanongsak Mamom³

, and Parin Suwannaprapha²

1. Faculty of Veterinary Science, Mahidol University, Salaya Campus, 999 Phutthamonthon Sai 4 Road, Salaya, Phutthamonthon, Nakhonpathom, 73170, Thailand.

2. Department of Preclinic and Applied Animal Sciences, Faculty of Veterinary Science, Mahidol University , Salaya Campus, 999 Phutthamonthon Sai 4 Road , Salaya, Phutthamonthon , Nakhonpathom , 73170 , Thailand.

3. Faculty of Veterinary Medicine, Mahanakorn University of Technology 140 Cheum-Sampan Rd., Nong Chok, Bangkok , 10530, Thailand.

Background and Aim: Canine gingival masses are common oral lesions with variable biological behavior, ranging from reactive hyperplasia to malignant neoplasia. Although routine cytology is widely used for initial evaluation, diagnostic overlap between benign and malignant lesions may limit accuracy when relying solely on morphology. This study aimed to develop and validate a trimodal cytological framework that integrates cytomorphometric analysis, argyrophilic nucleolar organizer region (AgNOR) staining, and micronuclei assay to enhance cytological differentiation and objectively characterize proliferative and genotoxic alterations in canine gingival masses.

Materials and Methods: Cytological specimens were obtained through fine-needle aspiration from gingival masses of 46 dogs and classified as epithelial hyperplasia (n = 11), benign neoplasms (n = 14), and malignant neoplasms (n = 21), with histopathology serving as the reference standard. Cytomorphometric parameters (nuclear diameter, nuclear area, cytoplasmic area, cellular diameter (CD), and nuclear-to-cytoplasmic [N:C] ratio) were measured using digital image-analysis. Cellular proliferation was evaluated by AgNOR silver staining, while genomic instability was assessed with acridine orange-based micronuclei assay. Group comparisons were conducted using one-way analysis of variance, and relationships among parameters were examined using Pearson’s correlation coefficient.

Results: Significant differences were observed among lesion categories for AgNOR count, micronuclei frequency, and most cytomorphometric parameters (p < 0.01), except for CD. Malignant neoplasms showed the highest AgNOR count (4.04 ± 2.81) and micronuclei frequency (7.76 ± 2.10), indicating increased proliferative activity and genotoxic damage. Epithelial hyperplasia presented larger nuclear and cytoplasmic dimensions, while the N:C ratio was highest in benign neoplasms (0.44 ± 0.23). The N:C ratio showed significant correlations with AgNOR (r = 0.319, p = 0.030) and micronuclei counts (r = 0.317, p = 0.032). A strong positive correlation was found between AgNOR and micronuclei counts (r = 0.631, p < 0.01).

Conclusion: The integration of cytomorphometry, AgNOR staining, and the micronuclei assay creates a strong, quantitative cytological framework that improves diagnostic accuracy for canine gingival masses. This three-part approach decreases subjective interpretation, enhances detection of malignant changes, and can easily be adapted to digital and AI-supported cytopathology systems in veterinary clinical practice.

Keywords: AgNOR staining, canine gingival masses, cytomorphometry, diagnostic cytopathology, genotoxicity biomarkers, micronuclei assay, oral tumors in dogs, veterinary oncology.

How to cite this article: Hoonpo P, Issarankura Na Ayudhaya T, Chaichoun K, Sangsuriya P, Mamom T, Suwannaprapha P. Trimodal cytological integration of micronuclei assay, argyrophilic nucleolar organizer region staining, and cytomorphometry enhances diagnostic discrimination of canine gingival masses. Vet World. 2026;19(1):65–80.

Received: 08-08-2025 Accepted: 15-12-2025 Published online: 08-01-2026

Corresponding author: Parin Suwannaprapha E-mail: parin.suw@mahidol.edu

DOI: 10.14202/vetworld.2026.65-80

Copyright: Hoonpo, et al. This article is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.