FIXATION OF WHOLE EYES - THE ROLE OF FIXATIVE OSMOLARITY IN THE PRODUCTION OF TISSUE ARTIFACT

Background: Whole eyes fixed in 4% buffered formaldehyde (10% neutral buffered formalin) demonstrate a variety of artifacts, including separ ation of the neurosensory retina from the retinal pigment epithelium. We postulate that the osmolarity of 4% buffered formaldehyde causes co ntraction of the internal compartments of the eye leading to several a rtifactual changes commonly observed in routine histologic sections. M ethods: In part I of the study, enucleated animal eyes were examined h istologically after immersion in different concentrations of formaldeh yde. The variables of fixation and processing were kept constant excep t for the concentration and osmolarity of formaldehyde. In part II, en ucleated animal eyes were used to empirically determine the optimal mi xture of formaldehyde and glutaraldehyde for fixation based on subject ive assessment of histologic sections. Results: In the first part of t he study, the post-fixed volume of the anterior chamber and vitreous i ncreased as the concentration (and osmolarity) of formaldehyde decreas ed. In part II of the study, fixation of whole eyes was optimal with a mixture of 1% buffered formaldehyde and 1.25% glutaraldehyde. The neu rosensory retina was less likely to detach from the retinal pigment ep ithelium, and the anterior chamber retained a more normal shape with t his 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) i n 1% buffered formaldehyde/1.25% glutaraldehyde reduces tissue distort ion without compromising cellular preservation.