Diffusion Tensor Imaging of Healthy and Infarcted Porcine Hearts: Study on the Impact of Formalin Fixation

Abstract

Background Due to complexities of in-vivo cardiac diffusion tensor imaging (DTI), ex-vivo formalin-fixed specimens are used to investigate cardiac remodeling in diseases, and reported results have shown conflicting trends. This study investigates the impact of formalin-fixation on diffusion properties and optimizes tracking parameters based on controls to understand remodeling in myocardial-infarction (MI). Methods DTI was performed on 4 healthy (controls) and 4 MI induced formalin-fixed (PoMI) ex-vivo porcine hearts. Controls were scanned pre-fixation (PrCtrl) and re-scanned (PoCtrl) after formalin-fixation. Fractional anisotropy (FA) and apparent diffusion coefficient (ADC) were estimated in all hearts. Tracking parameters (FA, tract termination angle (TTA), fiber-length) were optimized in controls and then used to investigate structural remodeling in PoMI hearts. Results Fixation increased ADC and decreased FA. PoMI showed increased ADC but decreased FA in infarcted zone compared to remote zone. TTA showed sharp increase in slope from 5°–10°, which flattened after 25° in all groups. Mean fiber-length for different tracking length range showed that PoCtrl had shorter fibers compared to PrCtrl. Fibers around infarction were shorter in length and disarrayed compared to PoCtrl group. Conclusion Formalin-fixation affects diffusion properties and hence DTI parametric trends observed in pathology may be influenced by the fixation process which can cause contradictory findings.

Submillimeter diffusion tensor imaging and late gadolinium enhancement cardiovascular magnetic resonance of chronic myocardial infarction

“…Third, despite submillimeter resolution of the imaging with average of ~8 voxels across the wall in the infarct, we excluded the regions with wall thickness less than 2 mm to ensure reliable measurement of transmural angle profile with at least ~4 voxels across the wall; this excluded around 10% of the infarcted wall. Finally, while the fixation process could have affected the baseline values of diffusivity such as FA and MD in our study [45], it has been shown that it does not change the eigenvector orientation significantly after infarction [45]. So we do not believe it could have affected the main findings of the study.…”

Myoarchitectural disarray of hypertrophic cardiomyopathy begins pre‐birth

“…We might expect differences between in vivo and ex vivo data, and also between fresh and fixed ex vivo data. Although there are studies that have described that formalin-fixation affects diffusion properties and affect the microstructural organisation of cardiac tissue, there are no studies investigating the effects of fixation or dehydration on the structure tensor (Giannakidis et al 2016;Mazumder et al 2016). As stated by Giannakidis et al (2016) it is crucial to minimise the time of the start of tissue fixation after death to ensure that ex vivo measurements represent the structural characteristics of the living myocardium as accurately as possible.…”

Longitudinal assessment of tissue properties and cardiac diffusion metrics of the ex vivo porcine heart at 7 T: Impact of continuous tissue fixation using formalin

“…With 0.62 ± 0.1 (10 −3 mm 2 /s), our ADC values in this study are in good agreement with our previous work at 7 T 32 (0.51‐0.66 [10 −3 mm 2 /s]) using fresh hearts from animals of the same breed and comparable weight. ADC values of fresh hearts reported by Mazumder et al 20 (0.52 ± 0.026 [10 −3 mm 2 /s], Yorkshire, 41‐50 kg) and Agger et al 18 (0.54 ± 0.026 [10 −3 mm 2 /s], Danish Landrace, 50‐60 kg) are slightly lower and ADC values of fixed hearts reported by Wu et al 41 (0.671 ± 0.106 [10 −3 mm 2 /s], minipigs, 45‐50 kg) and Pashakhanloo et al 42 (0.633 ± 0.04 [10 −3 mm 2 /s], Yorkshire, 50 ± 18 kg) are slightly higher. Literature results of comparisons between fixed and unfixed heart tissue are sparse.…”

“…This tissue fixation, often performed using formalin, enables long scan times and, thus, ex vivo measurements remain an important scientific tool which provides high resolution and high fidelity, ground truth data. Historically, such data allowed validation of DTI against histology, 9,15–17 proof of concept studies, 18–20 as well as validation of in vivo results 3,21 . Furthermore, cardiac tissue samples can often be gained as a byproduct of other scientific or biomedical studies.…”

“…In order to allow an accurate comparison between in vivo and ex vivo measurements it is paramount to assess fixation‐induced changes of tissue properties. While there are studies analyzing the impact of tissue fixation on diffusion metrics of the heart, 18,20,22 methodology in studies throughout the years remained highly heterogeneous with regard to the procedure of fixation, storage times and temperature, the fixative itself, and the overall fixation duration.…”