Molecular Detection of SARS-CoV-2 in Formalin Fixed Paraffin Embedded Specimens

Abstract

<jats:title>Abstract</jats:title><jats:p>Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of human coronavirus disease 2019 (COVID-19), emerged in Wuhan, China in December 2019. The virus rapidly spread globally, resulting in a public-health crisis including more than one million cases and tens of thousands of deaths. Here, we describe the identification and evaluation of commercially available reagents and assays for the molecular detection of SARS-CoV-2 in infected formalin fixed paraffin embedded (FFPE) cell pellets. We identified a suitable rabbit polyclonal anti-SARS-CoV spike protein antibody and a mouse monoclonal anti-SARS-CoV nucleocapsid protein (NP) antibody for cross detection of the respective SARS-CoV-2 proteins by immunohistochemistry (IHC) and immunofluorescence assay (IFA). Next, we established RNAscope <jats:italic>in situ</jats:italic> hybridization (ISH) to detect SARS-CoV-2 RNA. Furthermore, we established a multiplex fluorescence ISH (mFISH) to detect positive-sense SARS-CoV-2 RNA and negative-sense SARS-CoV-2 RNA (a replicative intermediate indicating viral replication). Finally, we developed a dual staining assay using IHC and ISH to detect SARS-CoV-2 antigen and RNA in the same FFPE section. These reagents and assays will accelerate COVID-19 pathogenesis studies in humans and in COVID-19 animal models.</jats:p>

Discordant anti‐SARS‐CoV‐2 spike protein and RNA staining in cutaneous perniotic lesions suggests endothelial deposition of cleaved spike protein

“…Furthermore, for reasons that are incompletely understood, IHC is known to sometimes produce non‐specific staining, 34 and an alternative explanation for the discordant SP and RNA staining via IHC and RNA ISH is a false positive IHC signal. Notably, IHC and ISH results do not always correlate with the presence of virions as IHC or ISH positivity alone can be because of free viral antigens or degenerating RNA fragments 35 . As both PCR testing 36 and RNA ISH are able to detect a very low number of virions, negative PCR testing of 29 lesions of PDC 9,21 and negative RNA ISH in 3 cases in this series would suggest that SARS‐CoV‐2 virions are not directly present within the tested samples.…”

SARS-CoV-2 leads to a small vessel endotheliitis in the heart

“…Immunofluorescence (IF) labeling of the spike (S) glycoprotein and nucleocapsid protein (NP) of SARS-CoV-2 was performed on formalin-fixed paraffin embedded (FFPE) myocardial tissue (which was positive with qRT-PCR) using following clones and antibodies (all purchased from www.sinobiological.com ). We adapted a previously described procedure for FFPE tissue [30] and used the following antibodies: Polyclonal rabbit anti-S antibody, Cat#40150-T62-COV2, Dilution at 1/2000, Pretreatment Ultra CC1 er, visualized with anti-FITC fluorescent labeling, Monoclonal rabbit anti-NP antibody (labelled with orange tape), Cat# 40143-R001, Dilution at 1/5000, Pretreatment Ultra CC1 er, visualized with anti-FITC fluorescent labeling, Monoclonal mouse anti-NP antibody (labelled with purple tape) Cat#40143-MM05. Dilution at 1/8000, Pretreatment Ultra CC1 mild, visualized with goat anti-mouse Alexa Fluor 488 fluorescent labeling.…”

Les lésions histologiques pulmonaires associées à l’infection par le Sars-CoV-2

“…Différentes protéines virales peuvent être identifiées dans d’autres types cellulaires, comme les cellules alvéolaires pulmonaires, les cellules glandulaires et, dans des études récentes, au niveau des cellules endothéliales [ 24 , 26 ]. Les techniques d’HIS ont permis de détecter des fragments d’ARN du Sars-CoV-2 dans le cytoplasme des cellules épithéliales ciliées de l’arbre respiratoire [ 23 ]. En combinant l’IHC et l’HIS, il est possible d’identifier simultanément les protéines et l’ARN virale [ 23 ].…”

“…Les techniques d’HIS ont permis de détecter des fragments d’ARN du Sars-CoV-2 dans le cytoplasme des cellules épithéliales ciliées de l’arbre respiratoire [ 23 ]. En combinant l’IHC et l’HIS, il est possible d’identifier simultanément les protéines et l’ARN virale [ 23 ].…”