Detection of Low-Copy Human Virus DNA upon Prolonged Formalin Fixation

Formalin fixation, albeit an outstanding method for morphological and molecular preservation, induces DNA damage and cross-linking, which can hinder nucleic acid screening. This is of particular concern in the detection of low-abundance targets, such as persistent DNA viruses. In the present study, we evaluated the analytical sensitivity of viral detection in lung, liver, and kidney specimens from four deceased individuals. The samples were either frozen or incubated in formalin (±paraffin embedding) for up to 10 days. We tested two DNA extraction protocols for the control of efficient yields and viral detections. We used short-amplicon qPCRs (63–159 nucleotides) to detect 11 DNA viruses, as well as hybridization capture of these plus 27 additional ones, followed by deep sequencing. We observed marginally higher ratios of amplifiable DNA and scantly higher viral genoprevalences in the samples extracted with the FFPE dedicated protocol. Based on the findings in the frozen samples, most viruses were detected regardless of the extended fixation times. False-negative calls, particularly by qPCR, correlated with low levels of viral DNA (<250 copies/million cells) and longer PCR amplicons (>150 base pairs). Our data suggest that low-copy viral DNAs can be satisfactorily investigated from FFPE specimens, and encourages further examination of historical materials.

Volumetric assessment and longitudinal changes of brain structures in formalinized Beagle brains

High field MRI represents an advanced technique both for diagnostic and research purposes on animal models such as the Beagle dog. The increasing interest in non-invasive neuroscience, aging, and neuropathological research led to a need of reference values (in terms of volumetric assessment) for the typical brain structures involved and, nowadays, several canine brain MRI atlases have been provided. Since no reports are available regarding the measurements reproducibility and few information are available about formalin fixation effect on brain structures when applied to MRI segmentation, we assessed the segmentation variability of selected structures as a function of the operator (two operators segmented the same data) and their intrinsic variability within a sample of 11 Beagle dogs (9 females and 2 males, 1.6 ± 0.2 years). Then, we analyzed for one further Beagle dog (2 years old) the longitudinal changes in the brain segmentations of these structures corresponding four conditions: in vivo, post mortem (after euthanasia), ex vivo (brain extracted and studied after 1 month in formalin and after 11 months); all the MRI images were collected with a 3 T MRI scanner. Our findings suggest that the segmentation procedure can be considered overall reproducible since only slight statistical differences were detected, apart from the ventricles.Furthermore, in the post mortem/ ex vivo comparison, the majority of the structures showed a higher contrast leading to more reproducible segmentations across operators and a net increase of volume of the studied structures; this could be justified by the intrinsic relaxation time changes observed as a consequence of formalin fixation, that led to an improvement of brain structures visualization and then segmentation.To conclude, MRI based segmentation seems to be a useful and accurate tool that allows longitudinal studies, especially when applied to formalin fixed brains.

Changes in magnetic resonance imaging relaxation time on postmortem magnetic resonance imaging of formalin-fixed human normal heart tissue

Background Postmortem magnetic resonance imaging (MRI) has been used to investigate the cause of death, but due to time constraints, it is not widely applied to the heart. Therefore, MRI analysis of the heart after formalin fixation was previously performed. However, the changes in MRI signal values based on the fixation time of formalin were not investigated. The objective was to investigate changes over time in the T1- and T2-values of MRI signals in normal areas of hearts removed during autopsy, hearts subsequently fixed in formalin, and heart specimens sliced for the preparation of pathological specimens. Methods The study subjects were 21 autopsy cases in our hospital between May 26, 2019 and February 16, 2020 whose hearts were removed and scanned by MRI. The male:female ratio was 14:7, and their ages at death ranged from 9 to 92 years (mean age 65.0 ± 19.7 years). Postmortem (PM)-MRI was conducted with a 0.3-Tesla (0.3-T) scanner containing a permanent magnet. A 4-channel QD head coil was used as the receiver coil. Scans were performed immediately after removal, post-formalin fixation, and after slicing; 7 cases were scanned at all three time points. Results The T1- and T2-values were calculated from the MRI signals of each sample organ at each scanning stage. Specimens were sliced from removed organs after formalin fixation, and the changes in T1- and T2-values over time were graphed to obtain an approximate curve. The median T1-values at each measurement time point tended to decrease from immediately after removal. The T2-values showed the same tendency to decrease, but this tendency was more pronounced for the T1-values. Conclusion MRI signal changes in images of heart specimens were investigated. Formalin fixation shortened both T1- and T2-values over time, and approximation formulae were derived to show these decreases over time. The shortening of T1- and T2-values can be understood as commensurate with the reduction in the water content (water molecules) of the formalin-fixed heart.

Organ Retention Litigation

The common practice of retaining organs and tissues after the autopsy dissection is a potential legal liability for forensic pathologists and the offices they work for. In 1988, a coroner and the coroner’s office were successfully sued for corneal procurements without the consent of the next of kin. That led to later lawsuits of forensic pathologists and medical examiner offices over the retention of brains for formalin fixation and neuropathologic examination without consent, although the autopsies were not consent based. National Association of Medical Examiners became involved in this ligation and filed numerous amicus briefs. These later lawsuits were not successful. After caselaw on the subject, such lawsuits have largely abated. Nonetheless, the threat of such lawsuits remains, and statutory language can be promulgated to mitigate the risk.

Comparison of Heart Valve Circumference Examined Before and After 10% Formalin Fixation

Objective: To compare the heart valve circumference before and after 10% formalin fixation. Materials and Methods: The study analyzed 63 Thai human cadaveric hearts. Each heart valve circumference was separately measured in the fresh state by specifically designed equipment. After that, the hearts were fixed in 10% formalin for 3 days. Then each heart valve circumference was measured by the same equipment and by the thread and ruler technique. The results were analyzed using SPSS package to find the association between the heart valve circumference before and after formalin fixation. Results: This study showed that the average circumferences of the heart valve measured in the fresh state were 13.329 cm in the tricuspid valve, 10.617 cm in the mitral valve, 8.416 cm in the pulmonic valve, and 7.122 cm in the aortic valve. The average circumferences of the heart valve measured after 10% formalin fixation were 11.019 cm in the tricuspid valve, 8.714 cm in the mitral valve, 6.751 cm in the pulmonic valve, and 6.089 cm in the aortic valve. The average ratios of the heart valve circumference measured fresh and after 10% formalin fixation were 0.8267 in the tricuspid valve, 0.8235 in the mitral valve, 0.8050 in the pulmonic valve, and 0.8573 in the aortic valve. There were significant differences in the heart valve circumference between the fresh state and after formalin fixation (p < 0.001). Conclusion: This study revealed important information on the dimensional changes of all the formalin-fixed heart valves. We found that the heart valve shrank after formalin fixation, with the formalin-fixed hearts an estimated 0.8 times smaller than the fresh cadaveric hearts.

Comparison of Heart Valve Circumference Examined Before and After 10% Formalin Fixation

Objective: To compare the heart valve circumference before and after 10% formalin fixation.Materials and Methods: The study analyzed 63 Thai human cadaveric hearts. Each heart valve circumference was separately measured in the fresh state by specifically designed equipment. After that, the hearts were fixed in 10% formalin for 3 days. Then each heart valve circumference was measured by the same equipment and by the thread and ruler technique. The results were analyzed using SPSS package to find the association between the heart valve circumference before and after formalin fixation.Results: This study showed that the average circumferences of the heart valve measured in the fresh state were 13.329 cm in the tricuspid valve, 10.617 cm in the mitral valve, 8.416 cm in the pulmonic valve, and 7.122 cm in the aortic valve. The average circumferences of the heart valve measured after 10% formalin fixation were 11.019 cm in the tricuspid valve, 8.714 cm in the mitral valve, 6.751 cm in the pulmonic valve, and 6.089 cm in the aortic valve. The average ratios of the heart valve circumference measured fresh and after 10% formalin fixation were 0.8267 in the tricuspid valve, 0.8235 in the mitral valve, 0.8050 in the pulmonic valve, and 0.8573 in the aortic valve. There were significant differences in the heart valve circumference between the fresh state and after formalin fixation (p < 0.001).Conclusion: This study revealed important information on the dimensional changes of all the formalin-fixed heart valves. We found that the heart valve shrank after formalin fixation, with the formalin-fixed hearts an estimated 0.8 times smaller than the fresh cadaveric hearts.