Fixation of whole eyes: the role of fixative osmolarity in the production of tissue artifact

Abstract

• Background: Whole eyes fixed in 4% buffered formaldehyde (10% neutral buffered formalin) demonstrate a variety of artifacts, including separation of the neurosensory retina from the retinal pigment epithelium. We postulate that the osmolarity of 4% buffered formaldehyde causes contraction of the internal compartments of the eye leading to several artifactual changes commonly observed in routine histologic sections. • Methods: In part I of the study, enucleated animal eyes were examined histologically after immersion in different concentrations of formaldehyde. The variables of fixation and processing were kept constant except for the concentration and osmolarity of formaldehyde. In part II, enucleated animal eyes were used to empirically determine the optimal mixture of formaldehyde and glutaraldehyde for fixation based on subjective assessment of histologic sections. • Results: In the first part of the study, the post-fixed volume of the anterior chamber and vitreous increased as the concentration (and osmolarity) of formaldehyde decreased. In part II of the study, fixation of whole eyes was optimal with a mixture of 1% buffered formaldehyde and 1.25% glutaraldehyde. The neurosensory retina was less likely to detach from the retinal pigment epithelium, and the anterior chamber retained a more normal shape with this fixative. • Conclusions: Volume contraction of whole eyes fixed in 4% buffered formaldehyde is caused by the relatively high osmolarity of the fixative. Immersion fixation of whole eyes for 36 h (or longer) in 1% buffered formaldehyde/1.25% glutaraldehyde reduces tissue distortion without compromising cellular preservation.