Effects of autolysis and prolonged formalin fixation on histomorphology and immunohistochemistry of normal canine brain tissue: an experimental study

Abstract

CNS tumor diagnosis in dogs often relies on immunohistochemistry (IHC) given similar histologic features among tumors. Most CNS tissue samples encountered by diagnostic pathologists are collected during autopsy, and postmortem specimens can be susceptible to autolysis and prolonged formalin fixation, both of which have the potential to influence IHC results and interpretation. Here we evaluated the effects of experimentally controlled autolysis induced by delayed tissue fixation (sections of brain held for 2, 4, 8, 12, 24, 48, and 72 h in 0.9% NaCl at either room temperature or 37°C prior to fixation) as well as the effects of prolonged formalin fixation times (1 wk, 1 mo, 2 mo) on a panel of 8 IHC markers (CNPase, GFAP, Iba1, OLIG2, PGP9.5, MAP2, NeuN, synaptophysin) relevant to brain tumor diagnosis. Prolonged fixation of up to 2 mo had no detrimental effect on any immunomarker except NeuN, which had reduced immunolabeling intensity. Delayed fixation led to autolytic changes as expected, on a gradient of severity corresponding to increased time in saline prior to fixation. Several immunomarkers should be used with caution (CNPase, OLIG2) or avoided entirely (MAP2, NeuN) in markedly autolyzed brain and brain tumor tissues. Our results suggest that autolysis has minimal effect on most immunomarkers, but that advanced autolysis may cause a loss of specificity for GFAP, MAP2, and PGP9.5, a loss of intensity of CNPase and OLIG2, and loss of labeling with MAP2 and NeuN. Prolonged fixation affected only NeuN, with mildly decreased intensity.

Onset and progression of postmortem histological changes in the central nervous system of RccHan™: WIST rats

“…In this regard, laboratory rodents represent an optimal model to evaluate these findings due to their genetic homogeneity and the tightly controlled environmental factors. Results obtained in the present work can likely be translated to other animal species or even humans (10,16,(28)(29)(30)(31). However, although the nature of postmortem changes may be shared among species, the onset and progression of these findings can differ due to Frontiers in Veterinary Science 14 frontiersin.org intrinsic differences in body weight, cooling time, or metabolic activity of cells.…”

“…Glial cells encompass a broad category of cells in the central nervous system with homeostatic functions and include astrocytes, oligodendrocytes, and microglia. In the present study, glial cells were evaluated all together due to their morphological similarities on routine H&E sections and the difficulties to accurately differentiate them at later time points after death ( 10 ). Glial cells showed pericellular haloes, chromatin condensation and nuclear shrinkage.…”

“…Forensic, diagnostic and toxicologic pathologists frequently face the challenge of differentiating autolytic changes from real findings. Moreover, autolytic changes can modify the performance of immunohistochemistry and histochemical stains (9)(10)(11)(12). Despite its importance, investigation of microscopic postmortem changes is limited, with most of the studies covering only limited neuroanatomical locations, focusing on late stages of autolysis, or employing techniques other than light microscopy on routine H&E stains, such as transmission electron microscopy, immunohistochemistry, or biochemical analyses (11,(13)(14)(15)(16)(17)(18).…”

“…The nomenclature proposed in the present work to characterize postmortem findings reflects a shared consensus among the 14 pathologists evaluating the slides. The terminology chosen aims to be simple and descriptive, applying whenever possible pre-existing terms already established in the pathology literature and avoiding the Frontiers in Veterinary Science 13 frontiersin.org use of specific diagnostic terms (7,8,10,16,18,19). Some terms employed in the present work (i.e., chromatin condensation, nuclear shrinkage, nuclear fading) reflect histological features that are also found in physiologic and pathologic processes of cell degeneration and/or cell death (e.g., pyknosis is evinced by chromatin condensation and shrinking; karyolysis is characterized by nuclear fading due to the action of endonucleases) (20)(21)(22).…”

“…All animals employed in the present study were surplus naïve animals from regulatory studies approved by the Ethical Committee of the Generalitat de Catalunya (Departament d' Acció Climática, Alimentació y Agenda Rural) and licensed under ref. 10,832. Requirements of the Spanish Policy for Animal Protection (RED118/2021 and RED1386/2018) and the European Union Directive 2010/63 on the protection of experimental animals were always fulfilled.…”