Background: The products released by neutrophils are highly associated with the destruction seen in periodontitis. Previous works have demonstrated that elevated oral neutrophil levels correlate with the severity of oral inflammation and periodontitis. However, these studies relied on either lengthy serial rinses or cumbersome counting techniques for the collection and quantification of oral neutrophils. Objectives: To develop and validate a single, rapid, non-invasive oral rinse assay to enable quantification of oral neutrophils to facilitate the monitoring of a patient's periodontal disease status and effectiveness of periodontal therapy. Methods: 53 Patients with moderate and severe chronic periodontal disease and 16 periodontally healthy controls provided 30 second oral rinse samples prior to any periodontal treatment. Samples were stained with acridine orange, a fluorescence lysosomal marker that helps differentiate neutrophils from the other cellular components in the oral rinse. These stained samples were then assessed for neutrophil numbers using a fluorescence-based hemocytometer. 34 patients who completed phase I sanative therapy donated a second rinse sample at re-evaluation. Results: A statistically significant difference in oral neutrophil counts was seen between diseased and control groups. The lowest counts were observed in the control group (n=16) followed by the moderate (n=21), and the severe disease group (n=32) having more than five times the number of neutrophils compared to controls (p<0.001, Tukey). Patients who responded to phase 1 therapy demonstrated a 38% reduction in oral neutrophil counts when compared to their pre-treatment levels (n=19, p<0.05, Student's t-test). Furthermore, patients who did not respond to treatment showed no significant difference in oral neutrophil levels (n=15, p>0.05, Student's t-test). Conclusion: A single, rapid, non-invasive oral rinse assay is an effective means of collecting and quantifying oral neutrophil levels for the purpose of diagnosing and monitoring oral inflammation and periodontal disease in patients with moderate and severe periodontal disease.

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