Assessment of inorganic fibres burden in biological samples by Scanning Electron Microscopy - Energy Dispersive Spectroscopy

A protocol to detect inorganic fibres in samples of biological tissues by SEM-EDS is proposed. The sample (500mg in the case of lung tissue) is digested by NaClO, filtered using a sample holder and fixed onto a SEM stub by clarification. A total of 800 microscopic fields (MF) at 2000_ are scanned along 5 parallel strips of the filter preparation at regular intervals for a total area of 1.85mm2, representing 0.7% of the total accessible area. In order to test the method and to show that the investigation of animals (sentinel animals) instead of human tissues can provide information on the lung burden of inorganic fibres, the data obtained from a control group I (animals which lived in an environment free of fibre bearing rocks) consisting of 12 cattle and a test group II (animals which lived in alpine valleys with serpentine outcrops) consisting of 6 cattle and 6 wild animals are compared. As expected, group I shows by far a lesser burden than group II. The proposed SEM-EDS method is a first attempt to standardize SEM-EDS investigations of inorganic particles in biological tissues and is shown to provide results able to significantly discriminate the lung burden between populations even when subjected to non natural environmental exposure alone.