Assessment of Epithelial Cell Trauma Following Mechanical Separation by Means of Trypan Blue Staining

Abstract

Abstract: : Purpose: In epi–LASIK, the epithelial layer is mechanically separated by means of a specially designed surgical instrument, is temporarily reflected and finally repositioned on the surface of the cornea following laser ablation for the correction of ametropias. It is the purpose of the present study to employ trypan blue staining and an image processing technique in order to quantify the degree and pattern of trypan blue staining of epithelial sheets following mechanical separation. Moreover, to correlate the macroscopic staining parameters to the histological appearance of the separated epithelium and to study the effect of surgically induced trauma during separation to the postoperative clinical course. Methods: In a group of three patients the epithelial sheets were excised, prepared by means of trypan blue staining and photographed under controlled conditions. Additionally, the sheets were examined microscopically both by light and electron microscopy. In ten patients, epithelium was excised from both eyes and sheets were examined in order to evaluate staining symmetry between fellow eyes. Following the initial observations on staining symmetry, thirty patients underwent bilateral treatment whereas the epithelium was excised and stained from one eye (randomly selected) while in the fellow eye the sheet was repositioned after laser ablation. A purposely–developed MATLAB script was used to quantify the degree and pattern of staining for the excised sheets. The clinical course of the fellow eye was documented for a postoperative period of three months. Study parameters were re–epithelialization time, pain score, and stability of refraction. Results: In stained areas electron microscopy revealed discontinuities of the basal membrane and partial separations between adjacent basal epithelial cells, while in the non–stained areas the basal membrane and basal epithelial layer appeared intact. Degree and pattern of staining are subject to high inter–subject variability. This is supported by the fact that there seems to be a strong bilateral symmetry in the staining patterns. None of the study parameters of the postoperative course in epi–LASIK eyes was correlated to the degree of staining. Conclusions: Trypan blue staining can be used to quantify cellular level trauma in mechanical separation of the epithelial layer. Amount of staining in intact sheets, is not correlated to clinical parameters.

Keywords: refractive surgery: other technologies • cornea: epithelium • wound healing