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Different trephination methods may lead to differences in degree of tissue damage and endothelial cell loss, which both influence the outcome of penetrating keratoplasty. Light, transmission, and scanning electron microscopy were used to compare the ultrastructural appearance of the cut edges and the endothelial cell loss in 26 human corneal donor buttons obtained by trephination with the suction fixated guided trephine system (GTS) and with the free hand posterior punch technique (PPT).Human corneas were stored between 5 and 14 days in Optisol. One cornea from each pair was used for each technique. Trephinations (7.5 mm) were performed either from the anterior direction with the GTS (n=13) or from the posterior direction with the PPT (n=13) using Pharmacia Superblade trephines. Light microscopy, transmission electron, and scanning electron microscopy were performed according to standard procedures. Widening of the cut edges and the extent of endothelial cell loss were measured at three different areas per corneal button and analysed statistically.In contrast with the PPT, the GTS trephine produced considerable fibrillar disorder at the cut edges of the corneal buttons. The distance to which the endothelial cell loss extended from the edges of the cuts was significantly (p<0.001) lower for the GTS (42.2 (SD 50.8) μm from the edge) than for the PPT (109.3 (68.1) μm). Stromal widening at the edges (measured as percentage increase in stromal thickness, compared with the thickness of the central cornea) was observed with both techniques. However, the mean stromal widening produced by the GTS was significantly greater than that produced by PPT (106% (24%) v 69% (21%); p<0.002).Both trephination techniques produced only minor tissue damage. Nevertheless, there were distinct differences in the fine appearance of the cuts produced by the GTS and the PPT techniques. The extent of the fibrillar dislocation and stromal widening was greater at the edges of the GTS buttons. The GTS technique produced significantly less endothelial cell loss at the cut edges than did the free hand punching technique, PPT.