Grocott Methenamine Silver Positivity in Neutrophils.

Grocott methenamine silver (GMS) stain is commonly used for the identification of fungi on cytosmears and tissue sections. It imparts a black color to the fungal profiles and a pale green color to the background. It stains all pathogenic and nonpathogenic fungi and melanin. Mucins and glycogen may also be stained as dark brown colored structures. We routinely perform GMS stain in our lab using the standard procedure and reagents.[1] We have observed that the cytoplasm of neutrophils in the smear take up the black color. [Figure 1a and b] The black stained neutrophils may be mistaken for yeast forms of fungus. On a casual look the black stained neutrophils may be confused with pneumocystis during evaluation of bronchoalveolar lavage fluid. Careful evaluation of the cell will reveal the unstained lobated nuclei of the neutrophils, whereas the organism will show typical “cup and saucer” appearance. [Figure 1c and d] On the other hand, neutrophils may also serve as a positive internal control to ensure the quality of the stain. Other pitfalls of Grocott stain include its aberrant staining in nonfungal organisms such as the internal organs of Strongiloides stercoralis larvae, in the intranuclear inclusions of Cytomegalovirus infected cells, endospores of Bacillius cereus, and surface of Nocardia[2]. Awareness of these facts is essential to avoid misdiagnosis.

Figure 1

a) Pleural fluid smears showing numerous neutrophils, (Periodic acid Schiff stain ×100). b) Smears showing positivity in neutrophils (Grocott methanamine silver stain x200) c) Smear showing positivity in neutrophil cytoplasm with lobulated unstained nuclei (Grocott methanamine silver stain x400) d) Pneumocystis jiroveci seen on Grocott stain. Careful observation will reveal lobated nuclei of neutrophils, whereas the organism will show typical “cup and saucer” appearance. (Grocott methanamine silver stain x 400)

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