Patients’ clinical data and pathological finding
A total of 55 lung samples obtained from deceased COVID-19 patients were included. Patients included 38 men and 17 women, with a median age of 49 years (SD 17.27; range 13–82) at death. All patients tested positive for SARS-CoV-2 using nasopharyngeal swab PCR. Shortness of breath (49%), fever (40%), and cough (21.8%) were the most common symptoms at onset. Status regarding time spent in the hospital was available for 36 patients, and the mean length of stay of these patients in the critical care unit or intermediate medical ward was 12.6 days. Respiratory failure or multiorgan failure involving the respiratory system was the leading causes of death. In terms of previous comorbidities, 11 (20%) had diabetes, 15 (27.27%) had cardiovascular complications, 9 (16.3%) had previous respiratory illnesses, 18 (32.7%) had liver or renal diseases, and five (9%) had cancer. Additionally, 22 patients had other complications such as hypothyroidism, anaemia, steroid-induced hypoglycaemia, or dyslipidaemia (Table 1 and supplemental figure S1).
Table 1
Patient clinicopathological information
Characteristics
|
N or mean
|
% or range
|
COVID-19 cohort (55 patients)
|
|
|
Age
|
46y
|
13-82y
|
Sex
|
M 38, F17 (2.2:1)
|
|
Hospitalisation time
|
12.6d
|
1-88d
|
Major symptoms on admission
|
|
|
Fever
|
22
|
40.00%
|
Cough
|
12
|
21.82%
|
Shortness of breath
|
27
|
49.09%
|
Major comorbidities
|
|
|
Diabetes
|
11
|
20.00%
|
Cardiovascular complications
|
15
|
27.27%
|
Respiratory complications
|
9
|
16.36%
|
Cancer
|
5
|
9.09%
|
Liver or kidney complications
|
18
|
32.73%
|
Thyroid complications
|
5
|
9.09%
|
Aplastic anaemia
|
2
|
3.64%
|
Patients with no prior comorbidities
|
4
|
7.27%
|
Most common lung pathological features
|
|
|
Exudative phase of diffuse alveolar damage
|
25
|
45.45%
|
Organizing phase of diffuse alveolar damage
|
13
|
23.64%
|
Acute bronchopneumonia
|
5
|
9.09%
|
Acute lung injury
|
9
|
16.36%
|
No changes
|
3
|
5.45%
|
Coexisting acute bronchopneumonia with DAD
|
12
|
|
Control cohort (15 subjects)
|
|
|
Age
|
43.1y
|
18-70y
|
Sex
|
M 8, F 7 (1:1)
|
|
Lung pathology
|
|
|
Normal lung
|
13
|
86.66%
|
Diffuse alveolar haemorrhage
|
1
|
6.66
|
Diffuse alveolar damage
|
1
|
7%
|
On histopathology, majority of cases demonstrated histological evidence of exudative and organising phases of diffuse alveolar damage (69%), acute bronchopneumonia (9%), and acute lung injury (16.3%) (Figure 1 and Table 1, and supplementary figure S1). Additionally, two individuals had bacterial microabscesses assumed to have developed during hospitalisation. Minority cases (5.4%) did not show any significant histomorphological changes (Table 1).
SARS-CoV-2 expression
A subset of lung sections (n=10) from COVID-19 patients showed presence of SARS-CoV2 viral antigen using IHC (Figure 1B). Among the ten subjects, eight have diffuse alveolar damage on histopathology (Supplementary Table S1).
Control patients’ clinical data and histopathological findings
A total of 15 lung sections from control patients were included for comparison. The median age of this cohort was 48 years with male: female ratio of 1:1 (Table 1). All control patients had tested negative for SARS-CoV-2 using nasopharyngeal swab PCR. The control cohort's
characteristics are summarised in Table 1 and Supplementary Table S1. Histopathology was normal in all control patients, except for two patients whose lung biopsy showed evidence of diffuse alveolar haemorrhage and diffuse alveolar damage (Table 1 and Supplementary Table S2). Two sections, each, of normal kidneys, testes, and adrenal glands were also stained for ACE2 (Figure 2A-C).
ACE2 protein expression in cases and controls
ACE2 immunohistochemistry was performed in 44 cases and 15 controls (Supplementary Figure S1 and Table S1). ACE2 protein was largely undetectable (n=6) or was detected exclusively in the cytoplasm of rare type-II pneumocytes (n=7) in the histologically normal control lungs (Supplementary Table S1). Expression was observed at multiple foci in the two control biopsies that showed diffuse alveolar haemorrhage (DAH) and diffuse alveolar damage (DAD) (Supplementary Table S1). The mean intensity for ACE2 protein was 2.8±0.63 while was 14.5 and 4.9 in the two cases with DAD and DAH respectively.
ACE2 protein expression was higher in sections from the normal kidney, testes and adrenal glands (figure 2A-C). Additionally, quantitative imaging analysis revealed that the mean intensity of ACE2 protein is substantially lower in control lung (2.8±0.63) sections than in control kidney (19.5±2.57), testis (66.3±7.38), and adrenal (12.1±1.52) tissues (Figure 2F).
In lung tissues from COVID-19 patients, the mean ACE2 expression was 7±0.56, with 86% cases showing higher mean intensity than the control lung samples (2.8±0.63) (Figure 2D, 2E and 2G), including one sample with relatively normal histomorphology (3.12 mean intensity). This difference reached strong statistical significance (P = < 0.0001) (Figure 2G). Random sampling analysis further confirmed the significant increase in ACE2 protein in the lungs of COVID-19 patients (Figure 2H), negating any bias in the number of samples and images in the control group.
CD163 and CD61 expression levels
CD163 and CD61 staining were used to examine the inflammatory and coagulopathy components of COVID-19. CD163 and CD61 immunohistochemistry was performed in 36 and 26 cases (Figure S1) and 6 control samples each of normal lungs, respectively. Among control tissue and cases, macrophage-specific inflammation and platelet plugs and/or platelet-rich microthrombi were found exclusively in COVID-19 lung sections (Figure 3A-F). Quantitative IHC analysis demonstrated that patients with COVID-19 had significantly higher mean intensities of CD163 (8.5±2.20 vs. 26.3±1.15, P = <0.0001) and CD61 (0.28±0.04 vs. 10±0.78, P = <0.0001) staining than those without (Figure 3C and 3F). Even after random sampling analysis, CD163 and CD61 protein expression remained significantly higher in COVID-19 patients (Figure 3G and 3H). Linear regression analysis revealed a significant association between ACE2 expression and coagulation and inflammatory marker levels in paired samples from cases and controls (n= 26 for CD163 and n= 16 for CD61) (Figure 3I and 3J).