Optimizing histopathological processing of human eyes - fixative conditions

Abstract

Purpose : Human globes provide an important resource for the vision research community; however, standard histopathological methods commonly used for other tissues create artifacts when applied to eyes. Due to their unique anatomy and delicate tissue relationships, eyes present unique challenges. Animal research has found alcohol-acid formalin preparations useful as ocular preservatives. There is limited information available regarding the utility of such fixatives for human globes.

Methods : Sixteen pairs of human cadaveric globes from regional eye banks were obtained with short post-mortem collection intervals. The right and left eyes were submerged in buffered 10% formalin with and without various concentrations of acetic acid and ethanol. Macroscopic internal structures and fundus were documented photographically. The tissues were dehydrated through graded ethanols and xylenes and embedded in paraffin. Serial 5 um sections were prepared along a plane through the optic nerve, foveola, and pupil and mounted on electrostatic slides. Stains: H&E, PAS, Immunohistochemistry using Avidin Biotiin Complex, and Impress polymer IHC with diaminobenzadine and Vector red chromogens. Counter stain: Azure blue metachromasia to distinguish DAB from melanin.

Results : Globes fixed in 10% formalin showed characteristic deformations including shrinkage and indentation of the cornea and sclera often at its thinnest locations. 10% formalin typically caused distortion of the angle architecture and choroidal and extensive retinal detachment. Conversely, acid-ethanol formalin-fixed globes remained spherical without shrinkage or indentations. Furthermore, the neural retina remained attached and angle structures better maintained their normal anatomic relationships. The subretinal zone often appeared compressed compared to those fixed in formalin alone. Immunohistochemical studies confirmed preservation of the RPE-IPM-retinal interface.

Conclusions : In the general surgical pathology and animal ocular research literature, various formulations of acid-alcohol formalin formulations have been described under numerous names (Davidson's, modified Davidson's, TB-fix, etc.). The advantage of these fixatives over 10% formalin is maintaining the overall eye shape and minimizing retinal detachment. The mechanism(s) responsible for these features and formula optimization to preserve the subretinal zone and immunoreactivity are under study in our laboratory.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.