FLOW CYTOMETRIC ANALYSES OF CREVICULAR NEUTROPHILS

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Studies of circulating and crevicular neutrophil (PMN's) functions suggest that capabilities of these cells may be depressed by systemic as well as local factors with a resultant increase in periodontal disease. Specifically, patients with prepubertal periodontitis, juvenile periodontitis or periodontitis in diabetics have associated deficits in PMN function. Past studies of crevicular PMN function have focused on in-vitro microscopic and cell culture analyses. The flow cytometer rapidly measures the structure and function of suspended cells based upon cellular light scatter and fluorescence characteristics. While circulating PMN's have had their viability and functional status studied with flow cytometry, there have been no studies of crevicular PMN's using flow cytometry. Analysis of crevicular PMN cell populations using flow cytometry would permit more rapid sample analyses and more detailed characterizations. The specific aim of this study is to test the application of flow cytometric methods to characterize the functional capabilities of crevicular PMN's. Initially we will establish the flow cytometer's ability to quantitate viability, phagocytosis and respiratory stimulation in PMN's isolated from peripheral blood. These results will be compared to accepted light microscopic techniques. Flow cytometric assays of samples containing 10-3 to 10-6 cells also will be analyzed for effects due to reduced scale. Finally, saline washes will be used to collect crevicular PMN's from the diseased and healthy sites to subjects with periodontitis. Flow cytometric techniques will be applied to characterize the viability and functional status of these crevicular PMN's. Statistical analyses will test differences between light microscopic and flow cytometric techniques in circulating as well as differences in crevicular PMN functional data from healthy versus diseased sites. Future application of flow cytometric tests of crevicular PMN functional status could provide a new investigative tool for use during studies of periodontal diseases.