Quantitative Ex Vivo MRI Changes due to Progressive Formalin Fixation in Whole Human Brain Specimens: Longitudinal Characterization of Diffusion, Relaxometry, and Myelin Water Fraction Measurements at 3T

Purpose Mesorectal lymph node staging plays an important role in treatment decision making. Here, we explore the benefit of higher‐order diffusion MRI models accounting for non‐Gaussian diffusion effects to classify mesorectal lymph nodes both 1) ex vivo at ultrahigh field correlated with histology and 2) in vivo in a clinical scanner upon patient staging. Methods The preclinical investigation included 54 mesorectal lymph nodes, which were scanned at 16.4 T with an extensive diffusion MRI acquisition. Eight diffusion models were compared in terms of goodness of fit, lymph node classification ability, and histology correlation. In the clinical part of this study, 10 rectal cancer patients were scanned with diffusion MRI at 1.5 T, and 72 lymph nodes were analyzed with Apparent Diffusion Coefficient (ADC), Intravoxel Incoherent Motion (IVIM), Kurtosis, and IVIM‐Kurtosis. Results Compartment models including restricted and anisotropic diffusion improved the preclinical data fit, as well as the lymph node classification, compared to standard ADC. The comparison with histology revealed only moderate correlations, and the highest values were observed between diffusion anisotropy metrics and cell area fraction. In the clinical study, the diffusivity from IVIM‐Kurtosis was the only metric showing significant differences between benign (0.80 ± 0.30 μm2/ms) and malignant (1.02 ± 0.41 μm2/ms, P = .03) nodes. IVIM‐Kurtosis also yielded the largest area under the receiver operating characteristic curve (0.73) and significantly improved the node differentiation when added to the standard visual analysis by experts based on T2‐weighted imaging. Conclusion Higher‐order diffusion MRI models perform better than standard ADC and may be of added value for mesorectal lymph node classification in rectal cancer patients.

“…9 also exhibited a large spread in the same benign nodes; however, no significant correlations have been found between ADC and

T_{2}^{*}

Section: Discussioncontrasting

confidence: 65%

Higher‐order diffusion MRI characterization of mesorectal lymph nodes in rectal cancer

Ianuş

Santiago

Galzerano

et al. 2019

“…However, many factors including natural tissue decomposition and chemical fixation may alter the MRI properties of post‐mortem tissue causing undesirable biases in the measurement. It is known that aldehyde fixation leads to gradual shortening of T 1 /T 2 /T 2 * relaxation times, increased myelin water fraction, and apparent diffusivity reductions . The most rapid change in proton MR signal relaxation occurs during the first 3 weeks after fixation, which gradually plateaus before complete tissue penetration by the fixative agent, ~3 months after fixation .…”

Section: Introductionmentioning

confidence: 99%

The choice of embedding media affects image quality, tissue R₂^*, and susceptibility behaviors in post‐mortem brain MR microscopy at 7.0T

Dušek

Madai

Huelnhagen

et al. 2018

Purpose The quality and precision of post‐mortem MRI microscopy may vary depending on the embedding medium used. To investigate this, our study evaluated the impact of 5 widely used media on: (1) image quality, (2) contrast of high spatial resolution gradient‐echo (T1 and T2*‐weighted) MR images, (3) effective transverse relaxation rate (R2*), and (4) quantitative susceptibility measurements (QSM) of post‐mortem brain specimens. Methods Five formaldehyde‐fixed brain slices were scanned using 7.0T MRI in: (1) formaldehyde solution (formalin), (2) phosphate‐buffered saline (PBS), (3) deuterium oxide (D2O), (4) perfluoropolyether (Galden), and (5) agarose gel. SNR and contrast‐to‐noise ratii (SNR/CNR) were calculated for cortex/white matter (WM) and basal ganglia/WM regions. In addition, median R2* and QSM values were extracted from caudate nucleus, putamen, globus pallidus, WM, and cortical regions. Results PBS, Galden, and agarose returned higher SNR/CNR compared to formalin and D2O. Formalin fixation, and its use as embedding medium for scanning, increased tissue R2*. Imaging with agarose, D2O, and Galden returned lower R2* values than PBS (and formalin). No major QSM offsets were observed, although spatial variance was increased (with respect to R2* behaviors) for formalin and agarose. Conclusions Embedding media affect gradient‐echo image quality, R2*, and QSM in differing ways. In this study, PBS embedding was identified as the most stable experimental setup, although by a small margin. Agarose and Galden were preferred to formalin or D2O embedding. Formalin significantly increased R2* causing noisier data and increased QSM variance.

“…Cohen-Adad et al observed this same delamination in electron microscopic images. 49 Shatil et al observed a nearly 150% increase in MWF in whole human brains fixed by immersion in formalin for approximately 40 days, 25 but MWI was performed without first rinsing the fixative solution out of the tissue. The presence of formaldehyde decreases the T 1 and T 2 of tissue independently of its fixative effects, which may explain the much greater increase in MWF observed by Shatil et al Formaldehyde also diffuses slowly, requiring approximately 3 to 5 weeks to penetrate and fix a whole brain specimen.…”

Section: Discussionmentioning

confidence: 99%

“…Chemical fixatives such as formalin form cross‐links between proteins, which decrease the T 1 and T 2 of tissue water and enhance the magnetization transfer effect . These fixation‐induced changes likely bias the myelin density measurements produced by qMT and MWI …”

Section: Introductionmentioning

confidence: 99%

“…[22][23][24] These fixation-induced changes likely bias the myelin density measurements produced by qMT and MWI. 25 Solid-state MRI methods, including zero TE (ZTE) 26,27 and UTE, [28][29][30] are emerging techniques that have the potential to directly image and quantify the short-lived 1 H signal arising from myelin lipids. [31][32][33][34] The very presence of tissue water, however, is a significant impediment to quantification.…”

Section: Introductionmentioning

confidence: 99%

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Formalin tissue fixation biases myelin‐sensitive MRI

Seifert

Umphlett

Hefti

et al. 2019

Purpose Chemical 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 and myelin water imaging. In this work, we evaluate these myelin‐sensitive MRI methods for fixation‐induced biases. Methods We perform quantitative magnetization transfer, myelin water imaging, and deuterium oxide‐exchanged zero TE imaging on unfixed human spinal cord tissue at 9.4 Tesla and repeat these measurements after 1 day and 31 days of formalin fixation. Results The quantitative magnetization‐transfer 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 deuterium oxide‐exchanged zero TE 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 deuterium oxide‐exchanged zero TE intensity were nearly eliminated. Conclusion Bound pool fraction and myelin water fraction are significantly increased by formalin fixation, whereas deuterium oxide‐exchanged zero TE intensity is minimally affected. Changes in quantitative magnetization transfer and myelin water imaging may be due in part to delamination and formation of vacuoles in the myelin sheath. Deuterium oxide‐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 versus fixed tissue.