ABSTRACT
For the first time, we have used phase-contrast x-ray tomography to characterize the three-dimensional (3d) structure of cardiac tissue from patients who succumbed to Covid-19. By extending conventional histopatholocigal examination by a third dimension, the delicate pathological changes of the vascular system of severe Covid-19 progressions can be analyzed, fully quantified and compared to other types of viral myocarditis and controls. To this end, cardiac samples with a cross section of 3.5mm were scanned at the synchrotron in a parallel beam configuration. The vascular network was segmented by a deep learning architecture suitable for 3d datasets (V-net), trained by sparse manual annotations. Pathological alterations of vessels, concerning the variation of diameters and the amount of small holes, were observed, indicative of elevated occurrence of intussusceptive angiogenesis, also confirmed by scanning electron microscopy. Further, we implemented a fully automated analysis of the tissue structure in form of shape measures based on the structure tensor. The corresponding distributions show that the histopathology of Covid-19 differs from both influenza and typical coxsackie virus myocarditis.
Subject(s)
COVID-19 , MyocarditisABSTRACT
Background Multiorgan tropism of SARS-CoV-2 has previously been shown for several major organs. Methods We have comprehensively analyzed 25 different formalin-fixed paraffin-embedded (FFPE) tissues/organs from autopsies of fatal COVID-19 cases (n=8), using detailed histopathological assessment, detection of SARS-CoV-2 RNA using polymerase chain reaction and RNA in situ hybridization, viral protein using immunohistochemistry, and virus particles using transmission electron microscopy. Finally, we confirmed these findings in an independent external autopsy cohort (n=9). Findings SARS-CoV-2 RNA was mainly localized in epithelial cells, endothelial and mesenchymal cells across all organs. Next to lung, trachea, kidney, heart, or liver, viral RNA was also found in tonsils, salivary glands, oropharynx, thyroid, adrenal gland, testicles, prostate, ovaries, small bowel, lymph nodes, skin and skeletal muscle. Viral RNA was predominantly found in cells expressing ACE2, TMPRSS2, or both. The SARS-CoV-2 replicating RNA was also detected in these organs. Immunohistochemistry and electron microscopy were not suitable for reliable and specific SARS-CoV-2 detection in autopsies. The findings were validated using in situ hybridization on external COVID-19 autopsy samples. Finally, apart from the lung, correlation of virus detection and histopathological assessment did not reveal any specific alterations that could be attributed to SARS-CoV-2. Interpretation SARS-CoV-2 could be observed in virtually all organs, colocalizing with ACE2 and TMPRSS2 mainly in epithelial but also in mesenchymal and endothelial cells, and viral replication was found across all organ systems. Apart from the respiratory tract, no specific (histo-)morphologic alterations could be assigned to the SARS-CoV-2 infection. Research in context Evidence before this study SARS-CoV-2 has been shown to infect the respiratory tract and affect several other major organs. However, on a cellular level, the localization of SARS-CoV-2 and its targets ACE2 and TMPRSS2 have not been described comprehensively. Added value of this study We have analyzed tissue SARS-CoV-2 RNA using RT-PCR and visualized its localization together with ACE2 and TMPRSS2 using in situ hybridization (ISH) in 25 different autopsy tissues. SARS-CoV-2 sense and antisense RNA were detected in 16 tissues/organs, mainly in epithelial cells and, to a lesser extent, in endothelial or stromal cells. Detection of viral protein using immunohistochemistry or viral particles using transmission electron microscopy did not yield specific results. Interestingly, apart from the respiratory tract and specifically the lungs, we have not found a specific pathology that would be associated with extrapulmonary viral spread. Implications of all the available evidence We provide a recommendation on using these methods in autopsy diagnostics for SARS-CoV-2. Our data extend the current hypothesis of severe COVID-19 being multisystemic diseases. Our data also provide clear evidence of infection and replication of SARS-CoV-2 in the endothelial cell across all organs, extending the hypothesis on the (micro)vascular involvement in COVID-19.
Subject(s)
COVID-19 , DiseaseABSTRACT
We present a new approach of three-dimensional (3d) virtual histology and histopathology based on multi-scale phase contrast x-ray tomography, and use this to investigate the parenchymal architecture of unstained lung tissue from patients who succumbed to Covid-19. Based on this first proof-of-concept study, we propose multi-scale phase contrast x-ray tomography as a novel tool to unravel the pathophysiology of Covid-19, extending conventional histology by a third dimension and allowing for full quantification of tissue remodeling. By combining parallel and cone beam geometry, autopsy samples with a maximum cross section of 4 mm are scanned and reconstructed at a resolution and image quality which allows for the segmentation of individual cells. Using the zoom capability of the cone beam geometry, regions-of-interest are reconstructed with a minimum voxel size of 167 nm. We exemplify the capability of this approach by 3d visualisation of the diffuse alveolar damage with its prominent hyaline membrane formation, by mapping the 3d distribution and density of lymphocytes infiltrating the tissue, and by providing histograms of characteristic distances from tissue interior to the closest air compartment.