key: cord-1048761-akdpfih0
authors: Zhang, Heng; Zhou, Jun; Chen, Rong; Ren, Yong; Cai, Jun; Zhao, Lei; Fei, Xiaochun; Liu, Zhenhua; Zhang, Yu; Yuan, Ling; Wang, Chaofu
title: Autopsy and Histologic Findings of Patients with New Coronavirus Pneumonia: The Pathologic Associations with Hypoxemia
date: 2021-02-13
journal: Med Sci Monit
DOI: 10.12659/msm.928837
sha: 35af09b49027423162abda8ac0d372f6ee452b78
doc_id: 1048761
cord_uid: akdpfih0

BACKGROUND: Coronavirus 2 (SARS-CoV-2) was declared a pandemic by the World Health Organization (WHO) in March 2020. To further reveal the pathologic associations between coronavirus and hypoxemia, we report the findings of 4 complete systematic autopsies of severe acute respiratory syndrome coronavirus 2-positive individuals who died of multiple organ failure caused by severe hypoxemia. MATERIAL/METHODS: We examined the donated corpses of 4 deceased patients who had been diagnosed with severe acute respiratory syndrome coronavirus 2. A complete post-mortem examination was carried out on each corpse, and multiple organs were macroscopically examined. RESULTS: The 4 corpses were 2 males and 2 females, with an average age of 69 years. Bilateral lungs showed various degrees of atrophy and consolidation, with diffusely tough and solid texture in the sections. A thromboembolism was found in the main pulmonary artery extending into the atrium in 1 corpse, and significant atherosclerotic plaques tagged in the inner wall of the aortic arch were found in 2 corpses. Two corpses were found to have slightly atrophied bilateral renal parenchyma. Atrophic changes in the spleen were found in 2 corpses. Notably, there were significantly expanded alveolar septa and prominent fibroblastic proliferation. CONCLUSIONS: The laboratory data of these corpses showed a progressive decrease in blood oxygen saturation, followed by refractory and irreversible hypoxemia. Clinical and laboratory information and autopsy and histologic presentations of multiple organs showed insufficient air exchange due to abnormalities in the respiratory system, and reduced erythropoiesis in bone marrow may play a role.

Coronavirus is a large family of viruses known to cause Middle East Respiratory Syndrome (MERS) and Severe Acute Respiratory Syndrome (SARS). The wide spread of coronavirus 2 (SARS-CoV-2), a novel coronavirus named by the Coronavirus Study Group of the International Committee, has emerged as a global health emergency since December 2019 [1] . The coronavirus pneumonia induced by this virus has been officially named COVID-19 by the WHO. As of March 2020, it had caused more than 5 800 000 cases and 360 000 deaths globally. Most infections were non-severe, with common symptoms of upper-respiratory tract disease and pneumonia, including fever, cough, dyspnea, fatigue, and myalgia [2, 3] . However, some patients have severe symptoms clinically characterized by hypoxemia, acute lung injury (ALI), acute respiratory distress syndrome (ARDS), septic shock, or acute renal injury [2] .

Hypoxemia is defined as arterial oxygen tension (PaO2) below normal (normal PaO2=80-100 mmHg) [4] . Severe hypoxemia is also a characteristic of ARDS [5] . The hypoxia caused by hypoxemia and ensuing multiple organ failure is the main cause e928837-2 of disease progression and death for patients with coronavirus pneumonia [2] . Although there have been some publications describing the clinical, imaging, and treatment findings of patients with COVID-19, few histologic studies have been conducted and published, and most of them focused on the lungs [6] [7] [8] . Our team performed a systematic anatomy and detailed pathological analysis on the corpses of 4 COVID-19 patients who died of multiple organ failure caused by severe hypoxemia, aiming to assess the pathologic associations between COVID-19 and hypoxemia.

This study was approved by the Ethics Committee of Jin Yintan Hospital and Ruijin Hospital, Shanghai Jiaotong University (KY-2020-15.01). All the patients' families consented to using the patients' personal information. The corpses of 4 patients with COVID-19 with a complete clinical information were assessed. Clinical and laboratory data were collected and recorded.

The consent for body donation and autopsy for scientific research was obtained from the bereaved families. Four autopsy pathologists participated from Department of Pathology, Ruijin Hospital. The research team assured that the corpses were treated with respect. Complete post-mortem external and internal examinations were performed on the 4 corpses at the Military Hospital in Wuhan within 12 h after death. The pathologists involved in the anatomy used strict positive-pressure protection, and the dissection was performed in a negative-pressure square cabin dedicated to autopsy. 

lung, trachea and primary bronchus, thoracic cavity, heart, aorta, liver and gall bladder, spleen, pancreas, intestine, genital organs, kidney, adrenal gland, and thyroid gland.

Representative tissues from organs were sampled, followed by fixed in 10% formalin and embedding in paraffin blocks. The slides were stained by hematoxylin and eosin (HE) staining and evaluated independently by 2 senior pathologists. Any morphologic changes were recorded. Immunohistochemical staining was also carried out using commercially available antibodies (all were prediluted, from Dako, Carpinteria, US, if not 

The detailed clinical features are summarized in 

Autopsy Results Table 2 . The thoracic cavity of the 4 corpses had low to medium volume of pale-yellow effusion, and varying degree of adhesion was found between the lungs and thoracic wall. Bilateral lungs showed various degrees of atrophy and consolidation, with diffusely tough and solid texture in the sections. Mucus exudate was seen in the pulmonary section in Case 2. In all 4 corpses, a small amount of mucus in trachea and enlarged hilar lymph nodes were also found.

The sizes of the hearts of 4 corpses were normal, but 1 showed myocardial infarction (Case 2). A thromboembolism was found in the main pulmonary artery extending into the atrium in Case 3, and significant atherosclerotic plaques tagged in the inner wall of the aortic arch were found in 2 cases (Case 3, Case 4). None of the 4 had any obvious liver abnormalities. The gallbladder of Case 1 had a spherical enlargement with dark-brown bile pigment deposits and stones in its cyst. Two corpses (Case 1 and Case 4) had slightly atrophied bilateral renal parenchyma, and Case 4 had multiple serous cysts in both kidneys. Atrophic changes in the spleen were found in 2 coprses (Case 1 and Case 2). There were no obvious abnormalities in the pancreas, small intestine, large intestine, uterus, ovaries, fallopian tubes, or testes ( Table 3) .

Microscopic examination of the lungs revealed that the significantly expanded alveolar septa had prominent fibroblastic proliferation. The hyperplastic fibrous tissue filled the alveolar cavity, forming solid interalveolar fibroblastic plugs, resembling organizing pneumonia (Figure 1A, 1B) . Some areas showed the alveolar epithelial shedding and extensive alveolar injury; most showed the widening alveolar septa were irregular with e928837-6 rupture, leading to the formation of bullae ( Figure 1C) . The lining of the alveolar walls was extensively replaced by a thick, homogeneous, eosinophilic hyaline membrane (Figure 1D ). Serous and fibrous exudates and hemorrhage could be seen in the alveolar spaces (Figure 1E, 1F) . The mucous plugs were formed due to the exfoliated ciliated columnar epithelium intermingled with secreted mucous in the small bronchi, bronchioles, terminal bronchioles, and respiratory bronchioles, which may have blocked the airway. Residual mucus plugs were also seen in the focal alveolar ducts (Figure 2A, 2B) . The interstitial capillaries showed loss of endothelial cells, formation of thrombosis, and recanalization (Figure 2C, 2D) . Type I alveolar epithelial cells (AECs) were decreased or vanished, and instead there was a florid proliferation of type II AECs, usually with tufted or papillary growth with or without exfoliation (Figure 3A, 3D) . Obvious aggregation of macrophages deposited in the alveolar spaces. Viral inclusion could be seen in the cytoplasm of both II alveolar epithelial cells and macroiphages (Figure 4A, 4B) . Immunohistochemically, the proliferated or exfoliated Type II AECs were further labeled by AE1/AE3, TTF1, and CK7 (Figure 3B, 3C, 3E) ; the 2019-nCoV-specific marker RN3-NP was also expressed in these cells (Figure 3F) . The aggregated macrophages were stained by CD68 ( Figure 4D ) and RN3-NP ( Figure 4E ). RN3-NP was found to localize in type II alveolar epithelial cells and macrophages by immunofluiorescence assay (Figure 4C, 4F) .

In the cardiac tissues, vacuolar degeneration of myocardial cells was prominent, showing a focal infarct and hyaline degeneration with scattered or small aggregates of chronic inflammatory cell infiltration (Figure 5A-5C) . of granulocytes (Figure 5D, 5E) . In the spleen, hemophagocytosis could be seen (Figure 5F ).

The laboratory data of these 4 corpses showed a progressive decline in their oxygen saturation of blood, followed by refractory and irreversible hypoxemia. Combining the clinical and laboratory information with autopsy and histologic presentations of multiple organs, the potential causes of hypoxemia, appears to lie in several causes described below.

Mechanical obstruction in the outflow tract leads to difficulty exchanging air with the outside. The pulmonary conducting portion is the from trachea down to the level of the terminal bronchioles [9] . Under normal circumstances, the luminal surfaces of the bronchial tubes at all levels of the airway are lined with intact ciliated columnar epithelium, and as the cilia consistently swing toward the pharynx, the foreign bodies, including mucus, are pushed in the same direction to the pharynx and then coughed out; instead, the respiratory portion generally does not produce mucus due to absence of ciliated cells [10] . In our cases, some ciliary columnar epithelial cells in the small bronchi, bronchioles, terminal bronchioles, and respiratory bronchioles of the lungs fell off, and then mixed with the mucus to form a mucus plug, which severely hindered downstream air from entering the lung tissues, thus affecting the efficiency of ventilation. Moreover, the mucus plugs were found to exist in a varying extent in terminal respiratory sites such as alveolar ducts, alveolar sacs, and alveoli, interfering with gas exchange. These mucus plugs in the terminal location may have originated from the following: 1) mucus backflow and accumulation caused by prolonged bed rest; 2) the e928837-8 ventilator blows the mucus in the upstream bronchus into the downstream bronchus; 3) extensive shedding of the ciliated columnar epithelium from all levels of the conducting tract and the squamous metaplasia may result in a huge loss of ciliated cells, with ensuing significant decrease in the ability of the respiratory tract to clear mucus and diminished ventilation and gas exchange. Only 1 corpse showed a normal PaO2, partly because of consistent oxygen inhalation assisted by the mechanical ventilation. The other 3 patients underwent a decrease of PaO2, and alkalemia as well as high BE and BB, indicating the development of hypoxemia.

Histologically, the respiratory portion, containing respiratory bronchioles, alveolar ducts, alveolar sacs, and alveoli, is located at the end of the air-conducting portion, where gas-blood exchange is completed, allowing full oxygenation of the blood, and oxygen-laden erythrocytes are transported to all parts of the body to participate in the biochemical reaction, while carbon dioxide is removed [11] . The focal hemorrhage and mucus plugs and aggregated macrophages deposited in the alveolar cavities and occupying the spaces functioning in air exchanges decreased the amount of oxygen that can participate in airblood exchange. The air-blood barrier consists primarily of a liquid layer on the alveolar surface, type I AECs, basement membrane, thin connective tissue, capillary basement membrane, and continuous capillary endothelial cells, which is an important structure involved in the air-blood exchange process [12] . Several changes during the process of COVID-19 may affect the air-blood barrier, causing impaired air exchange: the hyaline membrane fully covering the inner surface of involved alveoli prevent rapid diffusion of oxygen and carbon dioxide, and the deposited mucus retention further aggravate this pathologic e928837-9 process; type I AECs, a component of the air-blood barrier, are substantially reduced due to damage and exfoliation, but instead, type II AECs compensatory hyperplasia featuring tufted or papillary growth may greatly reduce the effective areas of air exchange [6] ; the distended alveolar septa due to organizing connective tissues may result in an increased air diffusion distance; the endothelial cells, as the innermost layer of airblood barrier, are damaged and the secondary thrombogenesis may be responsible for the impairment of the complete histologic air-exchanged structure and the focally reduced blood flow, and hence could affect the air diffusion.

The lack of sufficient air exchange because of these abnormalities in the respiratory portion discussed above can directly lead to hypoxemia. In addition, reduced erythropoiesis in bone marrow may lead to a decrease in the production of red blood cells and therefore a diminished ability to transport enough oxygen far away.

None.

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