Pulmonary stromal expansion and intra-alveolar coagulation are primary causes of COVID-19 death

Abstract

Most COVID-19 victims are old and die from unrelated causes. Here we present twelve complete autopsies, including two rapid autopsies of young patients where the cause of death was COVID-19 ARDS. The main virus induced pathology was in the lung parenchyma and not in the airways. Most coagulation events occurred in the intra-alveolar and not in the intra-vascular space and the few thrombi were mainly composed of aggregated thrombocytes. The dominant inflammatory response was the massive accumulation of CD163 + macrophages and the disappearance of T killer, NK and B-cells. The virus was replicating in the pneumocytes and macrophages but not in bronchial epithelium, endothelium, pericytes or stromal cells. The lung consolidations were produced by a massive regenerative response, stromal and epithelial proliferation and neovascularization. We suggest that thrombocyte aggregation inhibition, angiogenesis inhibition and general proliferation inhibition may have a roll in the treatment of advanced COVID-19 ARDS.

Keywords: ARDS; Autopsy; COVID-19.

Figure 1

Characteristics of COVID-19 lungs. Consolidations in COVID-19 lungs show good correlation between the CT image (A), the macro image of the lung surface (B), the cut surface of the lower lobe (C) and the low magnification HE stained histology slide (D) showing fluid and cell filled areas that are sharply demarcated by the lobules border. (Patient C3). Sharply demarcated areas of mildly affected and massively consolidated lung areas are shown by HE staining (E). Surfactant positive type II pneumocytes that are the primary site of viral replication are primarily associated with the mildly affected areas (F). SARS-CoV-2 Spike RNA (brown signal) mainly associated with CK18 positive epithelial cells (red) as shown by combined RNAScope in situ hybridization and immunohistochemistry (G). The denuded alveolar walls are populated by CD68 positive macrophages in the consolidated area (H). Bystander damage of alveolar capillary wall (CD34 - red) shown in the vicinity of SARS-CoV-2 infected alveolar cell (RNAScope Spike in situ – brown) that led to intra-alveolar bleeding. No infection of endothelial cells or pericyte is observable (I).

Figure 2

Cut surfaces of the lower lung lobes showed complete consolidation in all cases with total lack of air in the alveolar structures.

Figure 3

The upper airways were mostly unaffected.

Figure 4

Identification of “COVID cells”. Desquamated atypical alveolar epithelial cells, “COVID cells” are one of the histological hallmarks of the virus induced lung damage.

Figure 5

Distribution of virus infected cells in the airways. Virus RNA positive material was restricted to the lumen of pharynx (A), epiglottis (B), trachea (C), main bronchus (D) and minor bronchi (E,F) whereas there were ample virus signal carrying cells in the alveoli (G,H,I). Productive virus infection of the alveolar macrophages is demonstrated by combination of RNAScope detection of viral Spike RNA and staining of CD68 macrophage marker (J). Deposition of virus carrying debris in the hyaline membranes (L). Hyaline membrane fragment on the surface of the trachea (M).

Figure 6

Thrombo-embolic complications are rarely observed. Vacuolising cytopathic effect on the capillary endothelial cells (A) that leads to fenestration (B), plasma leakage and bleeding (C) into the alveolar space (top row TEM images). The extent of plasma leakage is conveniently demonstrated by staining for von Willebrand factor in the affected lung (D). Coagulated plasma in the alveoli constitute the largest amount of filamentous fibrin in the body of the COVID-19 victims (E) HE staining. Intra-alveolar bleeding from alveolar wall capillaries (F) HE staining. Collapsed alveolar walls in progressing consolidation where the alveolar lumens are filled with coagulated blood (Masson trichrome). Thromboembolic complications were uncommon in our cohort. The multiple thromb-emboli that was observed in C8 (left panel upper row) was mainly composed of aggregated CD61 positive thrombocytes (K) rather than fibrin coagulum (left panel bottom). Disseminated intravascular coagulation was observed only in one case (C2) as shown in the glomerulus of the kidney (right panel).

Figure 7

Intra-alveolar hyaline membranes, the histological hallmarks of ARDS. Molecular composition of the hyaline membranes reveals that they are composed of debris of lung epithelial cells, macrophages, serum components and surfactant.

Figure 8

Cellular events of pulmonary consolidation reaction. Large number of CD163 positive myeloid cells accumulate in the lung parenchyma (A) and in the hilar lymph nodes (B), a sign of MAS (macrophage activation syndrome). Robust angiogenesis is observed with the production of large number of new capillaries with patent lumen (C). Proliferation of podifyllin (D2-40 positive stromal cells is demonstrated (D). Subdued lymphocytic reaction with decreased amount of B cells (CD29 – red) and modest amount of T cells (CD3-brown) (E). Rare event of an immune engagement of a virus infected cell (RNAScope brown) with a Granzyme B positive effector cells (F). Preservation of the collagen IV scaffold inside the heavily consolidated area might provide mechanical framework for later tissue regeneration after clearing off the solid elements (G and H).

Figure 9

Generalized effect of tissue hypoxia on the heart of a Covid-19 victim. Massive contract band necrosis in all tested myocardium samples despite that all coronaries are fully open.

Figure 10

Minimal histological change in the most affected brain restricted to focal perivascular bleedings and subtle cytopathic effect in a few cortical neurons.

Figure 11

In vitro isolation of the virus from infected lung. Isolation of the virus from the lung of the patient C9 using VeroE6 cells (A - uninfected). Cytopathic effects on the VeroE6 48 hours after infection (B). Phase contrast (A and B). Scanning electron microscopy (SEM) images of the surface of uninfected VeroE6 cells (C) and infected Vero E6 cells 48 hours post-infection (D). Intra-alveolar coagulum (E). Budding virus particles on the surface of a type II pneumocyte (F). Virus infected dead cell in the bronchiolar lumen, on the top of cilliated epithelium (G) Virus particles on the alveolus wall (G). E,F,G and H SEM images from the lung of patient C9. Size markers A and B 10 μm, C and D 300 nm, E 10 μm, F 100 nm, G 2 μm, H 100 nm.