A novel technique to assess chromatin texture using pixel optical densitometry in oral squamous cell carcinoma

Background: The early detection of neoplasia is the prime aim of a diagnostician. Altered chromatin distribution is the earliest microscopic change observed in malignant transformation making it a valuable morphometric parameter. This study was aimed to assess and correlate the progressive changes in chromatin texture from normal to varying grades of premalignancies to malignancy of the oral cavity. Methods: Thirty-four archival tissue specimens categorized as normal buccal mucosa (5), low-grade epithelial dysplasia (11), high-grade epithelial dysplasia (7), and squamous cell carcinoma (11) were stained with Feulgen reaction. Pixel optical densitometry histograms were obtained from analysis of an average of 300 cells/case using ImageJ software. Nine histogram curve characteristics (including area under the curve, area integer percentage, center, centroid) were then analyzed statistically for differences between the four groups. Results: Area integer percentage (P = 0.002), center (P = 0.038), and centroid (P = 0.021) were statistically significant within the four groups The parameters showed a dip in their value from normal to low-grade dysplasia but showed a steady increase in high-grade dysplasia and carcinoma. Conclusion: The optical density characteristics had the ability to differentiate the progression of neoplastic changes by extracting information of the chromatin distribution not quantifiable by routine microscopy. The early clumping of chromatin in the periphery in low-grade dysplasia followed by progressively increasing chromatin aggregates in high-grade dysplasia and carcinoma correlated well with the shift observed in the histogram center and centroid. The novel pixel optical densitometry technique efficiently predicted malignant transformation.