METHODS

Study Site and Ethical Approval
Autopsies were performed at the National Institute for Infectious Diseases Lazzaro Spallanzani-IRCCS Hospital, Rome, Italy. The study was approved by the local ethics committee (approval number 9/2020).

Study Cohort and Clinical Information
Patient demographic and clinical information was extracted from case records of 22 consecutive patients with COVID-19 who died and were autopsied.

Autopsy Procedures
Autopsies were performed using the specific guidance for post mortem, and collection according to submission of specimens and biosafety practices [11] to reduce the risk of transmission of infectious pathogens during and after postmortem examination. Because SARS-CoV-2 is classified as a biosafety level 3 (BSL3) organism, specific operating procedures for BSL3 pathogens were also followed. Autopsies were performed in a specific COVID-19 designated autopsy room with airflow control and airborne infection control procedures, including use of appropriated personal protective equipment (ie, National Institute for Occupational Safety and Health-certified disposable N-95 respirator).

SARS-CoV-2 RT-PCR
SARS-CoV-2 qualitative reverse transcriptase polymerase chain reaction (RT-PCR) testing for SARS-CoV-2 infection was performed on the following samples: ocular, nasopharyngeal, oropharyngeal, lung, and rectal swabs.

Tissues Sampled and Histological Stains
Tissues from the lung, heart, liver, kidney, spleen, and bone marrow were placed in formalin at room temperature for 72 hours before macroscopic analysis and processing for histological examination. The brain was not examined. Tissue samples were processed using hematoxylin and eosin staining (H&E), Masson trichrome stain, Perls stain, reticulin stain, periodic acid-Schiff reaction (PAS) and diastase-PAS. Giemsa and/or Grocott methenamine silver stains were performed where necessary.

Immunohistochemistry
Deparaffinized and rehydrated sections were used for immunohistochemistry. Organ sections were immersed in 10 mM sodium citrate, pH 6.0, and microwaved for antigen retrieval and stained on a BenchMark ULTRA system fully automated instrument (Roche) with an antibody directed against CD3 (2GV6; Ventana), CD4 (SP35; Ventana), CD8 (SP57; Ventana), CD15 (MMA; Ventana) CD26 (ab28340; Abcam), CD183 (1C6/CXCR3; BD Pharmigen), CD61 (ZfZ; Leica), CD20 (LZ6; Ventana), and CD68 (KP-1; Ventana).

Transmission Electron Microscopy
We only looked for evidence of SARS-CoV-2 in lung samples. Nonlung samples were not subject to transmission electron microscopy. Lung tissues were fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer for 4 hours at 4°C. Fixation was performed with 1% OsO4. Samples were then dehydrated in graded ethanol and embedded in Epon resin, as previously described [14, 15]. Ultrathin sections were stained with 2% uranyl acetate and examined under a transmission electron microscope (JEOL JEM 2100 Plus; Japan Electron Optics Laboratory). Images were captured with a digital camera (Tietz Video and Image Processing Systems).