Microcalcifications as seen in epoxy-embedded carotids using a trichromic staining

Atherosclerotic plaques that undergo rapid progression to stenosis are responsible of stroke or acute coronary syndrome. The vulnerability of the atherosclerotic plaque may be enhanced due to the presence of microcalcifications. Our aim was to analyze microcalcifications on epoxy resin-embedded semithin sections from bioptic samples of carotid endarterectomies. An original trichromic method consisting of methylene blue, azur B, basic fuchsin and alizarin red was used. Particular attention was given to the discrimination of calcifications with a diameter < 10 mm which are difficult to be detected in routine paraffin embedded tissue. A comparison was performed among samples stained either with this original method, methylene blue-azur B alone or together with Von Kossa staining, methylene blue-azur B-basic fuchsin alone or together with von Kossa staining. Our simple and fast method (about 2 min) yields a general sharp contrast to all structures and allows to easily recognize collagen and elastin. In addition, foam cells are evident due to their gray-greenish color associated to intracellular lipid droplets. Metachromatic granules in mast cells are also well recognized. Calcifications > 0,5 mm are clearly recognizable. In conclusion, in spite of their reduced size, microcalcifications can be clearly distinguished from the extracellular matrix. This trichromic method is thus reproducible and allows an easy identification of microcalcifications during the morphological analysis of atherosclerotic plaques.