key: cord-0905914-qfqyk47d
authors: Khismatullin, Rafael R.; Ponomareva, Anastasia A.; Nagaswami, Chandrasekaran; Ivaeva, Rozalina A.; Montone, Kathleen T.; Weisel, John W.; Litvinov, Rustem I.
title: Pathology of lung‐specific thrombosis and inflammation in COVID‐19
date: 2021-09-28
journal: J Thromb Haemost
DOI: 10.1111/jth.15532
sha: fd761f6c85cebd80c168895c4cc6cf52e4cad6a3
doc_id: 905914
cord_uid: qfqyk47d

BACKGROUND: Infection by SARS‐CoV‐2 produces significant pulmonary pathology including endothelial damage with resultant thrombotic events. While pathologic features were described, there are limited data on the relationship of these changes to the inflammatory response and the production of thromboses. OBJECTIVE: To investigate pathology of COVID‐19‐related immunothrombosis. PATIENTS/METHODS: Tissue samples from lung, kidney, brain and heart that were collected from 45 patients who died of COVID‐19. Histopathological examination was performed after H&E and Picro‐Mallory staining in combination with (immuno)fluorescence to visualize neutrophil extracellular traps. Ultrastructural alterations in lungs were studied with scanning and transmission electron microscopy. RESULTS: Inflammatory changes and thrombosis were substantially more pronounced in the lung than in the kidney, heart, and brain. The most common pathologic finding was diffuse alveolar damage. In addition, most lung samples showed thrombi in vessels. The cause of death in single cases was massive pulmonary embolism. Ultrastructural examination revealed neutrophils attached to endothelium, perhaps as a step towards transendothelial migration. In addition, platelets were identified in the midst of fibrin as individual procoagulant balloon‐like cells. Ultrastructural examination demonstrated numerous virion‐like particles. CONCLUSIONS: Studying (ultra)structural features of the autopsy lung samples from patients with COVID‐19 has provided evidence for a pathogenic link between inflammation and thrombosis. The major features in the lungs of COVID‐19 patients comprised primary inflammatory thrombosis associated with diffuse alveolar damage. The lungs had pronounced circulatory changes with inflammation‐dependent intravascular blood clotting, whereas heart, brain, and kidneys had predominantly degenerative changes that were distinct from the lung pathology.

The disease caused by the SARS-CoV-2 virus and referred to as COVID-19 has been spread worldwide with an overall mortality rate between 1-2.3%. [1] [2] [3] [4] In the majority of cases, COVID-19 has prevailing respiratory clinical manifestations, ranging from mild upper airway symptoms to severe lower airway disorders, including the development of acute respiratory distress syndrome that frequently leads to respiratory failure. 5 At the same time, COVID-19 has extrapulmonary manifestations, such as cardiac insufficiency and renal failure. [6] [7] [8] In addition to the relatively well-studied regional and systemic inflammation, important pathogenic mechanisms in COVID-19 are related to hemostatic disorders, underlying a prothrombotic state and life-threatening thrombotic complications. 9, 10 It has been commonly accepted that inflammation and hypercoagulability associated with endothelial dysfunction can lead to pulmonary microthrombosis. 11 Accumulation of neutrophils and the presence of megakaryocytes that may be native to the pulmonary tissue and produce platelets, create proinflammatory and prothrombotic conditions necessary for formation of COVID-19-associated regional intra-and extravascular clots made of platelets and fibrin. 12, 13 Although the disease severity and unfavorable outcomes are determined mainly by the acute respiratory distress syndrome, extrapulmonary organ injury and multi-organ failure has also been marked as an important predictor of mortality. 14 In particular, patients with severe COVID-19 have a higher risk of kidney failure and cardiac insufficiency, both of which correlate with poor outcomes. [6] [7] [8] A combination of systemic inflammation and immunothrombosis has been considered one of the leading causes of death in However, the interplay between the inflammatory response to the SARS-CoV-2 infection and coagulopathies remain largely unclear.

These complex and interrelated pathogenic reactions must include cells and mediators of innate and adaptive immunity, vessel walls and other epithelial barriers, activated platelets and clotting factors -all aimed at fighting against the viral pathogen and preventing it from invading into the blood. 15 Despite the vital importance, the pathogenic mechanisms of tissue damage and structural alterations in COVID-19 are not completely understood. This study is aimed at elucidating light and ultrastructural morphological features at autopsy in patients who died of COVID-19. In this work, we studied the lung and other organs from 45 autopsies and put a special emphasis on the microthrombosis and inflammation in the lungs as the major determinant of the clinical course and outcomes of COVID-19. As a methodological advancement, we described and analyzed samples of lung tissue using multiple complementary morphological techniques, which clearly demonstrated a combination of leukocytes, multiple microthrombi and diffuse alveolar damage. In particular, we were able to show neutrophil extracellular traps using (immuno)fluorescent light microscopy and ultrastructural alterations in lungs using scanning and transmission electron microscopy. Microscopic images obtained at various resolution scales enabled us to glean new and important information about the lung-specific inflammatory thrombosis and circulatory disorders that provides the structural basis for pathologic tissue alterations in COVID-19. kidney, brain and heart were procured and processed for morphological examination.

For histopathological examination, the tissue samples were fixed in 10% neutral buffered formalin, washed in water, cut into smaller pieces, then treated with in ascending concentrations of isopropanol and xylene using a tissue processor (STP420ES, Thermo Scientific), and embedded in paraffin. Four-micrometer-thick sections were stained with histological stains (H&E and Picro-Mallory histochemical kits). Twelve lung samples were used to visualize neutrophil extracellular traps (NETs), where NET-specific citrullinated histones H3 (Cit-H3) were stained using primary rabbit anti-human histone H3 antibodies (Abcam; cat. ab5103; 1:200). The samples were washed ESSENTIALS • The disease caused by the SARS-CoV-2 virus has been spread worldwide with a high mortality rate.

• The structural features of inflammation and thrombosis in various organs were analyzed with the emphasis on the lung-specific immunothrombosis.

• The pathological basis of the acute respiratory distress syndrome and a major cause of death in COVID-19 is the inflammatory thrombosis (immunothrombosis) in lungs.

• Heart, brain, and kidneys had predominantly degenerative changes and less pronounced thrombotic and inflammatory alterations that were distinct from the lung pathology. 

Twelve freshly incised samples of the lung tissue or the freshly extracted pulmonary thromboembolus were rinsed with saline and fixed in an excess volume of 2% glutaraldehyde in isotonic 50 mM cacodylate buffer containing 150 mM NaCl, pH 7.4. Following fixation, the samples were cut into smaller pieces; then each of them was cut open so that the parenchymal part and the vessels could be visualized. The fixed tissues were washed in the same cacodylate buffer, then dehydrated in ascending concentrations of ethanol (30- 100 v/v%), dried using hexamethyldisilazane, and sputter-coated with gold-palladium (Polaron e5100). High-resolution micrographs were obtained from randomly chosen areas of each sample to eliminate selection bias and imaged using an FEI Quanta 250FEG scanning electron microscope (FEI, Hillsboro, OR). increasing temperatures from 37°C to 60°C, ultrathin sections were cut using an Ultramicrotome-III (LKB, Sweden) and stained using the standard procedure with saturated aqueous uranyl acetate and lead citrate. The specimens were examined using a Hitachi HT7700 electron microscope (Hitachi HTC, Japan).

The overall demographic and clinical characteristics of the patients involved in this study are presented in Table 1 

In all patients, postmortem examination revealed that the lungs were heavy (>700 g) compared to normal lung weights (350-450 g) and

congested. The lung surface often had a distinct patchy structure with dense dark reddish hypervascular areas alternating with pale softer zones. The lungs were characterized by the outflow of a large amount of pink foaming fluid upon incision, which corresponded to abundant intra-alveolar microscopic tissue edema.

Histologically, in the vast majority of the samples (87%), multiple microthrombi in capillaries ( Figure 1A ) and larger primary thrombi in arterioles ( Figure 1B) were present. These thrombi were considered to be primary in nature as there was no evidence for venous occlusion of the lower limbs and pelvic floor at autopsy and no premortem clinical records indicating deep vein thrombosis. In only 9% of cases, the cause of death was a massive pulmonary embolism that included a large embolus in the trunk as well as emboli in small branches of the pulmonary artery (Table 1 ). In these patients, formation of these multiple large and small pulmonary emboli was associated with the clinically diagnosed deep vein thrombosis and/or thrombosis of the right chamber of the heart.

In these thromboemboli that were 10-13 mm in diameter, fibrin was accumulated at the periphery and the adjacent lung tissue was infiltrated with leukocytes, mainly mononuclear cells. The internal structure of pulmonary emboli showed alternation of fibrin and erythrocyte layers, with a significant predominance of erythrocytes. It is noteworthy that fibrin in pulmonary emboli was a few days or more old, as indicated by the blue color with the Picro-Mallory stain ( Figure 1C ), suggesting that clots formed at least 24 h before they were removed and fixed. 17 In addition to microthrombosis, the most common histological findings in the lungs were inflammatory cells (in 80% of samples), distinct hyaline membranes (73%), capillary congestion and blood stasis detected as vasodilation combined with increased content of blood elements in the microvasculature (60%), hemorrhage (82%) and interstitial edema (100%). Numerous samples of lungs showed diffuse alveolar damage ( Figure 1D ). Some lungs had no significant diffuse alveolar damage, but rather extensive neutrophilic infiltration resembling bacterial bronchopneumonia (27%) ( Figure 1E ). As a valid sign of inflammation, accumulation of neutrophils and neutrophil extracellular traps (NETs) associated with thrombosis were clearly detected in the vascular lumens ( Figure 1F ), using (immuno) fluorescence of NET-specific citrullinated histones H3 and DNA.

Notably, the NETs were associated with intravital clinical manifestations of severe inflammation, including tachycardia, tachypnea, and high fever.

In addition to the alterations related to the acute COVID-19, long-term or chronic pulmonary pathological changes were frequently observed, such as destruction of alveolar septae (100%) and

focal lymphocytic infiltration of the bronchi (11%), likely associated with pre-existing conditions and comorbidities.

Scanning and transmission electron microscopy revealed detailed structural alterations in the COVID-19 lungs at a much higher level of spatial resolution. Presumable fibrin masses recognized as amorphous proteinaceous depositions on septal membranes were the most abundant structure on the surface of the alveolar septa, which may correspond to the histologically revealed hyaline membranes, the main microscopic feature of diffuse alveolar damage (Figure 2A ).

These extremely dense agglomerates of fibrin, dead cells, and surfactant 18 were characterized by close adherence to the septa and lack of porosity. There were also other morphologic types of fibrin, yet present in a much smaller amount. Structurally, they were less dense and sparser than those described above and had a fibrillar (fibers and bundles) or spongy structure; they were freely located in the lumens of the alveoli, not attached or tightly adherent to the alveolar septa ( Figure 2B ).

The blood vessels were dilated and contained blood cells, mainly erythrocytes ( Figure 2C ). The endothelial cells of the inner vascular wall were often flattened. In 83% of samples, blood cells were found in a similar quantity both inside the vessels and in the lumen of alveoli; however, within the vessels, erythrocytes were tightly packed, which is a sign of microthrombosis. It is noteworthy that compressed and deformed erythrocytes often adhered tightly to the inner wall Figure 3A ), but they were often found in the extracellular space, either as sparse structures or large clusters ( Figure 3B ).

Most of the virion-like particles had distinct boundaries and a dense internal matrix. Figure 3C shows a single virion-like particle with clearly seen protrusions on the surface; the size of the particle body was 60 nm, while with the spike-like frame it reached 100 nm ( Figure 3C ).

The overall structure and composition of a large pulmonary blood clot were analyzed using 48 scanning electron micro- 

Histologically, microthrombosis of cerebral vessels was observed in about 47% of the samples analyzed. In all the samples, without exception, the brain tissue had signs of significant pericellular and Figure 5C ). There were no other significant and consistent changes revealed in the brain tissue.

In 

To reveal COVID-19-related pathological changes, we examined autopsy samples of lung, heart, kidney and brain tissues from patients All this allowed us, in contrast to many previous investigations, to obtain an extremely detailed and comprehensive morphological description of ultrastructural tissue lesions, as well as to visualize NETs and virion-like particles.

Inflammation and thrombosis were substantially more pronounced in the lungs than in kidneys, heart, and brain, in which we observed only degenerative and minor circulatory changes (Table 2) , which is consistent with other studies. 27 The most common morphological signs of tissue damage in the lungs comprised alveolar destruction (often manifested as diffuse alveolar damage) with pulmonary edema, which corresponds to clinically observed respiratory failure ( Figures 1D and 2A) . Strictly speaking, these pathological alterations could be not fully related to the inflammatory lung injury.

Given the older age of the deceased patients and the presence of comorbidities (hypertension, obesity, type II diabetes) (Table 1) , the edema itself could be caused and aggravated by a combination of pulmonary and extrapulmonary causes. The acute pneumonitis, pulmonary thrombotic embolism, and regional thrombosis of the lung microvasulature were often combined with ischemic heart disease, cardiomegaly, and sepsis that could exaggerate the respiratory problems. Irrespective of the underlying pathogenic mechanisms, respiratory failure was the ultimate cause of death in all of the patients analyzed.

There is a controversy in the literature as to whether the COVID-19-related micro-and macrothrombi in lungs are formed primarily in situ or they comprise secondary thrombotic emboli originating from an extrapulmonary thrombus. 11 In the vast majority of the samples analyzed here, the thrombotic obstruction of lung vessels associated with acute inflammation was observed in the absence of detected deep vein thrombosis, which is an argument for primary microthrombosis in the lungs, rather than pulmonary embolism. 28 Other authors have also found that pulmonary microthrombi in COVID-19

are due to the local hypercoagulability and are not originating from thrombi of the lower extremities. 29, 30 However, in a few cases we observed a typical secondary pulmonary embolus in a COVID-19 patient (Tables 1 and 2, Figure 1C ). Given a high risk of deep venous thrombosis in COVID-19, 31-33 pulmonary thrombotic embolism is highly likely and can further exaggerate the respiratory dysfunction.

As for the large pulmonary clot presented in Figure 4 , based on the absence of clinical and autopsy data for deep vein thrombosis and strong contraction, which is not typical for thrombotic emboli, 34 this is a primary thrombus rather than an embolus. Therefore, in COVID-19 the primary pulmonary thrombosis and thrombotic embolism in the lung artery and/or its branches seem probable, but with a prevalence of the primary immunothrombosis. The morphology of the virion-like particles revealed with scanning and transmission electron microscopy ( Figure 3 ) is generally consistent with the ultrastructure of SARS-CoV-2 virions observed in tissues of COVID-19 patients, [45] [46] [47] including the cells of the respiratory tract. 48 However, without specific identification, we prefer calling them "coronavirus-like" structures, as suggested by others. 47 heart, brain, and kidneys.

We thank Dr. Ivan S. Raginov for the administrative support and Dr. Vladimir A. Anokhin for helpful discussions. Transmission electron microscopy was carried out in the Interdisciplinary Center for Analytical Microscopy of Kazan Federal University.

Authors declare that they have no competing financial interests in relation to the work. 

Clinical predictors of mortality due to COVID-19 based on an analysis of data of 150 patients from Wuhan, China

A novel coronavirus from patients with pneumonia in China

Characteristics of and important lessons from the coronavirus disease 2019 (COVID-19) outbreak in China: summary of a report of 72 314 cases from the Chinese center for disease control and prevention

A pneumonia outbreak associated with a new coronavirus of probable bat origin

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and coronavirus disease-2019 (COVID-19): the epidemic and the challenges

Cardiovascular implications of fatal outcomes of patients with coronavirus disease

Association of cardiac injury with mortality in hospitalized patients with COVID-19 in Wuhan, China

Kidney disease is associated with inhospital death of patients with COVID-19

Anticoagulant treatment is associated with decreased mortality in severe coronavirus disease 2019 patients with coagulopathy

ISTH interim guidance on recognition and management of coagulopathy in COVID-19

SARS-2 coronavirus-associated hemostatic lung abnormality in COVID-19: is it pulmonary thrombosis or pulmonary embolism?

Pulmonary and cardiac pathology in African American patients with COVID-19: an autopsy series from New Orleans

COVID-19 update: Covid-19-associated coagulopathy

Clinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan, China: a retrospective cohort study

Thrombosis as an intravascular effector of innate immunity

Prevention, diagnosis and treatment of the new coronavirus infection (COVID-19)". Ministry of Health of the Russian Federation

Agedependent differential staining of fibrin in blood clots and thrombi

Acute respiratory distress syndrome and diffuse alveolar damage. New insights on a complex relationship

Ectosomes and exosomes: shedding the confusion between extracellular vesicles

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Multiorgan and renal tropism of SARS-CoV-2

Cardiac involvement in a patient with coronavirus disease 2019 (COVID-19)

Inhibition of SARS-CoV-2 infections in engineered human tissues using clinical-grade soluble human ACE2

Endothelial dysfunction: a marker of atherosclerotic risk

Dying with SARS-CoV-2 infection-an autopsy study of the first consecutive

Autopsy findings and venous thromboembolism in patients with COVID-19: a prospective cohort study

In situ thrombosis of the pulmonary arteries: an emerging new perspective on pulmonary embolism

Pulmonary embolism, pulmonary microvascular thrombosis, or both in COVID-19?

Pathological findings of COVID-19 associated with acute respiratory distress syndrome

Incidence of thrombotic complications in critically ill ICU patients with COVID-19

High incidence of venous thromboembolic events in anticoagulated severe COVID-19 patients

Acute pulmonary embolism in patients with COVID-19 at CT angiography and relationship to d-dimer levels

The distinctive structure and composition of arterial and venous thrombi and pulmonary emboli

Rouse RV Acute interstitial pneumonia -diffuse alveolar damage

Fibrin mimics neutrophil extracellular traps in SEM

Neutrophil activation and NETosis are the major drivers of thrombosis in heparin-induced thrombocytopenia

Neutrophil extracellular traps contribute to immunothrombosis in COVID-19 acute respiratory distress syndrome

Reciprocal coupling of coagulation and innate immunity via neutrophil serine proteases

Platelet TLR4 activates neutrophil extracellular traps to ensnare bacteria in septic blood

Platelets in host defense: experimental and clinical insights

Covid-19: the rollercoaster of fibrin(ogen), d-dimer, von Willebrand factor, p-selectin and their interactions with endothelial cells, platelets and erythrocytes

SARS-CoV-2-triggered neutrophil extracellular traps mediate COVID-19 pathology

Fc-modified HIT-like monoclonal antibody as a novel treatment for sepsis

Why misinterpretation of electron micrographs in SARS-CoV-2-infected tissue goes viral

Histopathology and ultrastructural findings of fatal COVID-19 infections in Washington State: a case series

Renal histopathological analysis of 26 postmortem findings of patients with COVID-19 in China

Pathology and pathogenesis of SARS-CoV-2 associated with fatal coronavirus disease, United States

Best practices for correctly identifying coronavirus by transmission electron microscopy

Pathological findings in the postmortem liver of patients with coronavirus disease 2019 (COVID-19)

Immunothrombotic dysregulation in COVID-19 pneumonia is associated with respiratory failure and coagulopathy

Pathology of lung-specific thrombosis and inflammation in COVID-19