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.

Keywords: autolysis; brain; canine; forensic; immunohistochemistry; neuropathology.

Figure 1.

Effect of experimentally induced autolysis via delayed formalin fixation on histomorphology in H&E-stained sections of canine brain. A. Control tissue. B. 2-h RT. C. 4-h RT. D. 8-h RT. E. 12-h RT. F. 24-h RT. G. 48-h RT. H. 72-h RT. I. 72-h 37°C. RT = room temperature. All images taken at identical magnification.

Figure 2.

Effect of experimentally induced autolysis via delayed formalin fixation or prolonged formalin fixation on IHC specificity and intensity in canine brain. A. OLIG2, control: labeling is nuclear. B. OLIG2, 72-h RT: labeling “bleeds” into the surrounding cytoplasm. C. OLIG2, 72-h 37°C: labeling is decreased in intensity, and there is similar bleeding of labeling into the cytoplasm of some cells. D. CNPase, control: labeling is cytoplasmic in both soma and processes of neurons. E. CNPase, 72-h RT: there is a loss of specificity but not of intensity. F. CNPase, 72-h 37°C: there is a loss of both specificity and intensity. G. NeuN, control: labeling is cytoplasmic in both soma and processes of neurons, with less intense nuclear labeling. H. NeuN, 72-h 37°C: there is a diffuse lack of labeling. I. NeuN, 2-mo formalin fixation: there is loss of labeling intensity, with some neurons failing to label at all. RT = room temperature. All images taken at identical magnification.

Figure 3.

Effect of delayed fixation on IHC specificity and intensity in canine brain. A. MAP2, control: labeling is cytoplasmic in both soma and, to a lesser extent, processes of neurons. B. MAP2, 72-h 37°C: there is marked loss of specificity and intensity. C. GFAP, control: labeling is cytoplasmic and highlights delicate astrocytic processes in the gray matter. D. GFAP, 72-h 37°C: labeling is similar in intensity to positive controls but has lost specificity, causing widespread off-target labeling of neuroparenchyma. E. PGP9.5, control: labeling is cytoplasmic in both soma and, to a lesser extent, processes of neurons. F. PGP9.5, 72-h 37°C: there is a loss of labeling specificity with no loss of intensity. RT = room temperature. All images taken at identical magnification.