Cardio-Pulmonary Histopathology with Clinical Correlations of Deceased Patients with COVID-19: A Case Series in Tehran, Iran

Background: SARS-CoV-2 may affect vital organs. The present study investigated the histopathology of pulmonary and cardiac tissues with clinical correlation in deceased patients with COVID-19. Methods: We obtained pulmonary and cardiac tissues from 30 deceased patients with COVID-19 in Tehran, Iran, from January to May 2021. Sampling was performed through a percutaneous needle biopsy. After slide preparation, two expert pathologists studied them. We assessed the correlation between clinical and pathological data by Fisher’s exact test. Results: The mean age of the patients was 73.8±13.4 years, and the male-to-female ratio was 23/7. The most common underlying disease was hypertension (HTN) in 25 patients (83%). Fifty-five tissue samples were achieved, including 28 pulmonary and 27 cardiac samples. Our results showed that all patients (100%) developed diffuse alveolar damage (DAD), and 26 (93%) developed hyaline membrane formation. The most common phase of DAD was the exudative-proliferative phase in 16 (57.1%). Three cardiac samples (11%) revealed myocarditis, and seven (26%) showed cardiomyocyte hypertrophy. In univariate analysis using Fischer’s exact test, myocarditis had significant relationships with C-reactive protein (CRP) levels higher than 80 mg/dL (P=0.008) and elevated cardiac troponin levels higher than two-fold (P=0.01). Conclusion: COVID-19 can affect the major vital organs. However, only myocarditis had a significant relationship with the circulating levels of inflammatory factors.


Introduction
SARS-CoV-2 enters the respiratory epithelial cells via angiotensin-converting enzyme II (ACE-II) receptors. 1everal chronic diseases such as diabetes mellitus (DM), hypertension (HTN), chronic lung disease (CLD), chronic renal failure (CRF), and ischemic heart disease (IHD) lead to increased expression of ACE-II receptors, higher viral binding, and a more severe immune response.This phenomenon explains the severe COVID-19 in the elderly with underlying diseases. 2,3The primary organ target of this virus is the lungs.In severe disease, respiratory distress and diffuse alveolar damage (DAD) constitute the leading cause of death. 4COVID-19 can affect other vital organs and cause fatal inflammation.6][7] Nevertheless, only a few studies performed multi-organ biopsies on these patients.We aimed to assess the clinical and histopathological features of pulmonary and cardiac tissues in deceased patients with COVID-19.

Study Design
We performed this case series on deceased patients with a polymerase chain reaction-confirmed COVID-19 in Khanevadeh Academic Hospital in Tehran, Iran, from January to May 2021.We selected our subjects by conventional sampling.

Participants and Sampling Methods
We only selected deceased patients whose first-degree relatives gave written informed consent for sampling in that period.The bodies underwent minimal invasive percutaneous needle biopsy 6-24 hours following death.An expert with a universal gun, two coats of gloves, a five-layered mask, and a face shield took the samples in a dissection room with negative pressure ventilation. 8A semi-automatic biopsy needle was used for cardiac and pulmonary sampling (Supplementary file 1, Figure S1).The pulmonary samples were taken from the areas with the most severe involvement on the computed tomography scan, often through the seventh or eighth intercostal space.The cardiac samples were taken through the fourth or fifth left anterior intercostal space.

Tissue Preparation
The tissues were prepared by the formalin-fixed paraffinembedded (FFPE) method and were cut into 10-μM sections using a microtome device.They were fixed on a slide and stained using hematoxylin and eosin dyes.Two expert pathologists studied each slide.The physicians and pathologists reviewed the histopathological findings and clinical data in three joint sessions.

Histopathological Findings
We obtained pulmonary and cardiac tissue from each patient.Three cardiac and two pulmonary tissues failed during the sampling procedure and tissue preparations.We studied 55 tissue samples, including 28 pulmonary and 27 cardiac specimens.
In univariate analysis by Fisher's exact test, we did not find any association between the clinical cause of death and troponin level (P = 0.470), C-reactive protein (CRP) level (P = 0.750), GGO extension in lung CT scan (P = 0.392), and BMI (P = 0.357).Only death due to pulmonary embolism was significantly associated with a 4-fold increase in D-dimer level (P = 0.011).Also, we did not find any association between the DAD staging and GGO extension (P = 0.702), D-dimer level (P = 0.268), CRP level (P = 0.211), and BMI (P = 0.558).Regarding cardiac pathology, we found an association between myocarditis and a two-fold rising of troponin (P = 0.010) and CRP over 80 mg/dL (P = 0.008).However, we did not find any association between myocarditis and pulmonary   GGO extension (P = 0.774) or BMI (P = 1.000).

Discussion
Our study is the first comprehensive multi-organ autopsy study with a generous sample size of patients with COVID-19 in Iran.Our findings revealed that all pulmonary tissues developed DAD, while 11% of the cardiac tissue samples showed inflammation.Other findings were cardiomyocyte hypertrophy with interstitial edema in the cardiac tissues.DAD occurred most often in the proliferative and organizing phases, suggesting that most patients expired during the late stage of the disease.Previous studies reported DAD in various stages, most often in the early phase of the disease.A study in Spain on 18 deceased patients with COVID-19, with a mean age of 61 years, reported that the proliferative phase of DAD occurred in 16 patients (89%), and micro-thrombosis occurred in 6 patients (33%). 9Another study in Austria on 14 deceased patients with a mean age of 81 years showed that the organizing phase of DAD occurred in 13 (93%), and the exudative phase occurred in 12 patients (86%).In that study, all patients developed pulmonary hemorrhage, and 11 (79%) developed bronchopneumonia and microthrombosis. 10One study in Germany on 10 deceased patients showed an acute phase of DAD in 9 (90%) and an organizing phase in one (10%). 11We also found anthracosis in 11 of 28 pulmonary tissues (39%).A study on eight deceased patients in the United States reported anthracosis in all samples. 12Other studies did not report anthracosis, and the relationship between this finding and the severity or prognosis of COVID-19 continues to be investigated.
Earlier studies showed that numerous patients with COVID-19 have a mild elevation of D-dimer levels, but a D-dimer level higher than 2590 ng/mL was reported to be a significant risk of thromboembolism. 13In our study, 3 out of 30 patients (10%) had D-dimer levels higher than 2590 ng/mL, and only one of the 28 pulmonary tissues (3.5%) revealed thrombosis.The frequency of thrombosis in our pulmonary tissue samples was much lower than other reports.One study on 76 deceased patients in Hamburg showed that 32 patients (40%) developed deep vein thrombosis, and 17 patients (21%) developed pulmonary artery embolism. 14Another multicenter study on 68 pulmonary tissues of deceased patients with COVID-19 in the United States and Italy showed that 59 patients (87%) developed DAD, 57 patients (84%) developed micro-thrombosis, and 29 patients (43%) developed large-vessel thrombosis. 15A study by Carsana et al on 38 deceased patients with COVID-19 in Italy reported that all patients developed a 10-fold rise in D-dimer levels, and 33 (87%) developed thrombosis. 168][19][20] This discrepancy may be due to differences in sampling techniques, study populations, and anticoagulant treatments.We used minimally invasive tissue sampling methods, which may underestimate the existing pathology.Cardiac complications are a leading cause of death in some patients with COVID-19.They may arise from exacerbation of a pre-existing cardiac disease or an acute condition during the illness, such as infarction, thrombosis or myocarditis.In our study, 7 out of 27 (25.9%)cardiac tissues showed cardiomyocyte hypertrophy, and 3 out of 27 (11.1%)samples showed myocarditis.The frequency of myocarditis in COVID-19 varies in different studies.A study on 22 cardiac samples of deceased patients with COVID-19 with a mean age of 68 years in the United States showed a mild increase of troponin in 20 patients (90.9%), but no case of myocarditis was reported. 21nother multicenter study in the United States and Italy reported myocarditis in 3 out of 21 patients (14.2%). 22A survey of nine deceased patients with a mean age of 72 years reported only one case of myocarditis (11.1%). 23One study on four deceased patients revealed the initial stages of myocarditis in all cardiac tissue samples. 24imited multi-organ studies were also performed on deceased patients with COVID-19.A study in South Africa on 75 deceased patients with a mean age of 60 years indicated the exudative phase of DAD in 47 (62.6%) and myocarditis in 7 (9.3%)patients. 25Another study on 21 deceased patients in Switzerland showed the exudative phase of DAD in 16 patients (76.2%), but no case of myocarditis was reported in cardiac tissues. 26A study on 11 patients in Austria showed that only one patient (9%) had myocarditis. 27Two additional multi-organ studies on deceased patients, including 21 deceased patients in the Netherlands and 10 deceased patients in the United Kingdom, showed myocarditis in 55% of the Dutch and 0% of the UK patients. 28,29Other studies on 17 and 12 deceased patients in the United States showed myocarditis in 0% and 7%, respectively. 30,31A multi-organ study on 22 deceased patients with a mean age of 68 years in Italy showed that 12 patients (54.5%) had myocarditis. 32Furthermore, a survey of 32 deceased patients with a mean age of 68 years demonstrated myocarditis in only one patient (3%). 33We did not find any similarity between our findings and other studies about myocarditis.In our research, myocarditis did not have a significant association with the severity of the disease.So, it may be attributed to other risk factors, such as viral tropism or race.We were unsure that all the histopathological findings were related to COVID-19, especially pulmonary anthracosis, increased type 2 pneumocytes, cardiac myocytes hypertrophy, and edema.Like the previous studies, our most common findings in the pulmonary tissues were DAD and hyaline membrane formation.The authors believe that these findings are related to COVID-19.However, other nonspecific findings such as hemorrhage, anthracosis, and the appearance of type II pneumocytes may be seen in other patients with underlying pulmonary diseases.In the cardiac tissues, the authors also believe that mononuclear infiltration in the patients with elevated cardiac troponin are related to COVID-19, as myocarditis.Nevertheless, other findings, such as myocyte hypertrophy and interstitial edema, are unspecific.So, we tried to avoid any complex analysis and only expressed our observations.Nevertheless, in contrast to previous studies, we found a lower frequency of microthrombosis in our pulmonary samples.

Conclusion
The most common pulmonary histological findings in our study was diffuse alveolar damage, similar to previous studies.Still, in cardiac tissues, we found a higher frequency of myocarditis and cardiac myocyte degeneration than in the earlier studies.

Limitations
The sampling technique was our major limitation Percutaneous needle biopsy provides less histopathological information compared to open biopsy and may underestimate pathological changes, such as thrombosis.Another limitation was the absence of a control group to compare our pathology findings between the COVID and non-COVID patients.Unavailability of immunohistochemical staining was also a limitation in differentiating inflammatory cells and immunological assays of the tissue samples.

Table 1 .
Main Clinical Data of the Study Population

Table 2 .
Pulmonary Lesions and Cardiac Lesions