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

TL;DR

A new three-part cytological method improves the accuracy of diagnosing canine oral lesions by combining shape analysis, cell proliferation markers, and DNA damage indicators.

Contribution

A trimodal cytological framework integrating cytomorphometry, AgNOR staining, and micronuclei assay is developed and validated for canine gingival mass diagnosis.

Findings

Malignant neoplasms showed significantly higher AgNOR counts and micronuclei frequency compared to benign and hyperplastic lesions.

The nuclear-to-cytoplasmic ratio was highest in benign neoplasms and correlated with AgNOR and micronuclei counts.

The trimodal approach provides a quantitative framework that reduces subjective interpretation in diagnosing canine oral lesions.

Abstract

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.

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.

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).

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.

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