Formalin Tissue Fixation Biases Myelin-Sensitive MRI

Abstract

PurposeChemical fixatives, such as formalin, form cross-links between proteins and affect the relaxation times and diffusion properties of tissue. These fixation-induced changes likely also affect myelin density measurements produced by quantitative magnetization transfer (qMT) and myelin water imaging (MWI). In this work, we evaluate these myelin-sensitive MRI methods for fixation-induced biases. MethodsWe perform qMT, MWI, and D2O-exchanged zero echo-time (ZTE) imaging on unfixed human spinal cord tissue, and repeat these measurements after 1 day and 31 days of formalin fixation. ResultsThe qMT bound pool fraction increased by 30.7{+/-}21.1% after 1 day of fixation and by 42.6{+/-}33.9% after 31 days of fixation. Myelin water fraction increased by 39.7{+/-}15.5% and 37.0{+/-}15.9% at these same time points, and mean T2 of the myelin water pool nearly doubled. Reference-normalized D2O-exchanged ZTE signal intensity increased by 8.17{+/-}6.03% after 31 days of fixation, but did not change significantly after 1 day of fixation. After fixation, specimen cross-sectional area decreased by approximately 5%; after correction for shrinkage, changes in D2O-exchanged ZTE intensity were nearly eliminated. ConclusionF and MWF are significantly increased by formalin fixation, while D2O-exchanged ZTE intensity is minimally affected. Changes in qMT and MWI may be due, in part, to delamination and formation of vacuoles in the myelin sheath. D2O-exchanged signal intensity may be altered by fixation-induced changes in myelin lipid solid-state 1H T1. We urge caution in the comparison of these measurements across subjects or specimens in different states, especially unfixed vs. fixed tissue.