Detection of SARS-CoV-2 in formalin-fixed paraffin-embedded tissue sections using commercially available reagents

Abstract

Coronavirus Disease-19 (COVID-19), caused by the coronavirus SARS-CoV-2, was initially recognized in Wuhan, China and subsequently spread to all continents. The disease primarily affects the lower respiratory system, but may involve other organs and systems. Histopathologic evaluation of tissue from affected patients is crucial for diagnostic purposes, but also for advancing our understanding of the disease. For that reason, we developed immunohistochemical (IHC) and in situ hybridization (ISH) assays for detection of the. virus. A total of eight autopsy lungs, one placenta, and ten kidney biopsies from COVID-19 patients were stained with a panel of commercially available antibodies for IHC and commercially available RNA probes for ISH. Similarly, autopsy lungs, placentas and renal biopsies from non-COVID-19 patients were stained with the same antibodies and probes. All eight lungs and the placenta from COVID-19 patients stained positive by IHC and ISH, while the kidney biopsies stained negative by both methodologies. As expected, all specimens from non-COVID-19 patients were IHC and ISH negative. These two assays represent a sensitive and specific method for detecting the virus in tissue samples. We provide the protocols and the list of commercially available antibodies and probes for these assays, so they can be readily implemented in pathology laboratories and medical examiner offices for diagnostic and research purposes.

Fig. 1

SARS-CoV-2 IHC and ISH in lung. SARS-CoV-2 ISH (a–c) and IHC (d–f) in lung parenchyma from the autopsy of a patient who died secondary to COVID-19. Positive reaction for the (a) control probes to the housekeeping gene peptidylprolyl isomerase B (PPIB) by in situ hybridization confirms the presence of intact nucleic acid. b There is a positive reaction for the probes directed against SARS-CoV-2 (arrow) and c a negative reaction for the negative control probes (bacterial gene dapB) in a serial section (Periodic acid-Schiff counterstain; original magnifications ×200; bar = 50 µm). d–f Immunohistochemical analysis for SARS-CoV-2 is positive (arrow) in the tissue (d with higher power of outlined region in e) with negative staining in the f negative control (hematoxylin counterstain; original magnifications ×100 in D and ×200 in e and f; bar = 100 µm in d and bar = 50 µm in e and f).

Fig. 2

SARS-CoV-2 IHC and ISH in kidney. ISH in a renal biopsy tissue from a patient with active COVID-19 at the time of biopsy shows (a) reactivity for the positive control probe to the housekeeping gene peptidylprolyl isomerase B (PPIB) (original magnification ×400; bar = 25 µm) and b, c negativity for the presence of SARS-CoV-2 RNA (Periodic acid-Schiff counterstain; original magnifications ×400 and ×200; bar = 25 µm for B and bar = 50 µm for c). d–f IHC staining of renal biopsy tissue from three different patients with active COVID-19 at the time of biopsy shows negative staining in the parenchyma (hematoxylin counterstain; original magnification ×200; bars = 50 µm).