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Review
. 2023 Oct;115(5):263-274.
doi: 10.32074/1591-951X-933.

COVID-19: detection methods in post-mortem samples

Ilaria Tedesco  1 , Federica Zito Marino  1 , Andrea Ronchi  1 , Amaro Nunes Duarte Neto  2 , Marisa Dolhnikoff  2 , Maurizio Municinò  3 , Carlo Pietro Campobasso  4 , Giuseppe Pannone  5 , Renato Franco  1
Affiliations
Review

COVID-19: detection methods in post-mortem samples

Ilaria Tedesco et al. Pathologica. 2023 Oct.

Abstract

COVID-19 identification is routinely performed on fresh samples, such as nasopharyngeal and oropharyngeal swabs, even if, the detection of the virus in formalin-fixed paraffin-embedded (FFPE) autopsy tissues could help to underlie mechanisms of the pathogenesis that are not well understood.

The gold standard for COVID-19 detection in FFPE samples remains the qRT-PCR as in swab samples, contextually other methods have been developed, including immunohistochemistry (IHC), and in situ hybridization (ISH). In this manuscript, we summarize the main data regarding the methods of COVID-19 detection in pulmonary and extra-pulmonary post-mortem samples, and especially the sensitivity and specificity of these assays will be discussed.

Keywords: COVID-19; autopsy samples; immunohistochemistry; in situ hybridization.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1.
Figure 1.
Workflow qRT-PCR COVID-19 detection in post-mortem samples. qRT-PCR of SARS-CoV-2 RNA from FFPE post-mortem biomaterial and preparation of the mix for analysis. Detection of N, S, ORF1ab and RNase P genes. qRT-PCR, Real-Time Quantitative Reverse Transcription-PCR; ORF1ab, Open reading frame 1ab; N, Nucleocapsid; E, Envelope; S, Spike; M, Membrane; RNase P, Ribonuclease P; dsDNA, double strand DNA.
Figure 2.
Figure 2.
Workflow ISH assays to detect COVID-19 in post-mortem samples. Detection of SARS-CoV-2 N, S and ORF1ab genes using Z probes for in situ hybridization. Tissue pretreatment, hybridization of Z probe to RNA, binding of pre-amplifiers to probe and binding of amplicons to each pre-amplifier, followed by binding of chromogenic enzyme to amplicons and optical microscope visualization. FFPE, formalin-fixed paraffin-embedded; N, nucleocapsid; S, spike; ORF1ab, Open Reading Frame 1ab; ISH, In situ hybridization; DAB, 3,3′-Diaminobenzidine.
Figure 3.
Figure 3.
Representative results of immunohistochemistry (IHC) and in situ hybridization (ISH) for SARS-CoV-2 detection in pulmonary and extra-pulmonary post-mortem samples. Nucleocapsid IHC in lung tissue (A), Nucleocapsid IHC in brain tissue (B), Nucleocapsid IHC in heart tissue (C), Spike ISH in lung tissue (D), Spike ISH in liver tissue (E), Spike ISH in heart tissue (F).
Figure 4.
Figure 4.
Workflow IHC assays to detect SARS-CoV-2 in post-mortem samples. Detection of SARS-CoV-2 using antibody anti-N and anti-S for immunohistochemistry. Tissue pretreatment, binding of primary antibody to the S and N antigens, binding of a secondary antibody conjugated to horseradish peroxidase polymers (HRP) which provides an enzyme to convert DAB into a precipitate visible under optical microscope. FFPE, formalin-fixed paraffin-embedded; N, nucleocapsid; S, spike; IHC, immunohistochemistry; DAB, 3,3′-Diaminobenzidine.
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