A novel method for detecting single glandular intestinal metaplasia in the mucosal surface of the fixed stomach using methylene blue.

A close association between intestinal metaplasia of the stomach and the well-differentiated type of gastric cancer is well recognized. The etiological relationship and how intestinal metaplasia contributes to gastric carcinogenesis are, however, still unclear. In order to answer this question, precise mapping and identification of the smallest lesion of intestinal metaplasia are desired. Establishment of an accurate and easy method for detecting intestinal metaplasia was the goal of this study. Surgical specimens of stomachs resected for gastric cancer were used. The specimens were stained with methylene blue, an oxidation-reduction marker, in whole mount, after fixation with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4), and observed under a stereo-microscope. Normal gastric mucosa was stained blue, whereas intestinal metaplasia mucosa was not stained and had white or sky-blue island-like features. Intestinal metaplasia of complete type was unstained and showed white island-like features, while intestinal metaplasia of incomplete type showed sky-blue staining. With this method, we were able to detect even intestinal metaplasia composed of a single gland, when the intestinal metaplasia was of complete type. When stomach samples were stained in the presence of diphenyleneiodonium chloride (DPI), an inhibitor of nicotineamide adenine dinucleotide phosphate reduced form (NADPH) reductase, all the samples were homogeneously stained blue. Loss of the color of methylene blue was caused by the reductase activity of NADPH reductase, which is strongly and specifically expressed in intestinal metaplasia. A novel method for detecting intestinal metaplasia, even a single gland, was established.