Identification of asbestos fibers within single cells

Identification of asbestos fibers and other mineral particles in tissues is important for the diagnosis of interstitial lung disease. Conventional procedures to identify mineral particles are applicable to tissue digests, homogenates, or thin sections prepared for transmission electron microscopy. Positive identification of mineral particles in these samples is achieved by energy dispersive x-ray analysis or crystalline diffraction patterns. These analytical techniques are difficult to use for identification of long, thin asbestos fibers within cells collected from effusions or by saline lavage. A new preparative procedure is presented which allows intracellular visualization of fibers in these samples. Mice were injected intraperitoneally with 100 micrograms of crocidolite asbestos. After 1 to 30 days, the free peritoneal cell population was collected by saline lavage and allowed to attach to Formvar/carbon coated grids in vitro. Cell spreading was induced by exposure to phorbol-12-myristate-13-acetate for an additional 4 hours. The flattened cells were fixed, dehydrated and air-dried before examination by transmission electron microscopy. This procedure allows direct visualization of intracellular fibers. The characteristic Fe and Si peaks of crocidolite asbestos were confirmed by energy dispersive x-ray analysis. This technique was used to study the kinetics of clearance of asbestos fibers from the free peritoneal macrophage population of mice.