key: cord-0948636-493suex3
authors: Quondamatteo, Fabio; Corzo‐Leon, Dora E.; Brassett, Cecilia; Colquhoun, Ian; Davies, David C.; Dockery, Peter; Grenham, Sue; Guild, Simon; Hunter, Amanda; Jones, James; Lee, Thomas C.; Tracey, Chris; Wilkinson, Tracey; Munro, Carol A.; Gillingwater, Thomas H.; Parson, Simon H.
title: Neutralisation of SARS‐CoV‐2 by anatomical embalming solutions
date: 2021-10-11
journal: J Anat
DOI: 10.1111/joa.13549
sha: ac64ea9312846a1b12479819b697b682520a15e5
doc_id: 948636
cord_uid: 493suex3

Teaching and learning anatomy by using human cadaveric specimens has been a foundation of medical and biomedical teaching for hundreds of years. Therefore, the majority of institutions that teach topographical anatomy rely on body donation programmes to provide specimens for both undergraduate and postgraduate teaching of gross anatomy. The COVID‐19 pandemic has posed an unprecedented challenge to anatomy teaching because of the suspension of donor acceptance at most institutions. This was largely due to concerns about the potential transmissibility of the SARS‐CoV‐2 virus and the absence of data about the ability of embalming solutions to neutralise the virus. Twenty embalming solutions commonly used in institutions in the United Kingdom and Ireland were tested for their ability to neutralise SARS‐CoV‐2, using an established cytotoxicity assay. All embalming solutions tested neutralised SARS‐CoV‐2, with the majority of solutions being effective at high‐working dilutions. These results suggest that successful embalming with the tested solutions can neutralise the SARS‐CoV‐2 virus, thereby facilitating the safe resumption of body donation programmes and cadaveric anatomy teaching.

The use of human cadaveric specimens for anatomy teaching remains a cornerstone of medical and scientific education (Ghosh, 2017) . There are currently 47 medical schools in the United Kingdom and Ireland, and at least 2600 worldwide (Duvivier et al., 2014) , the vast majority of which rely upon body donation and embalming preservation techniques (Habicht & Kiessling, 2018) . The ongoing COVID-19 pandemic resulted in immediate, widespread cessation of the acceptance of donor bodies by medical schools and universities, largely because of the perceived risks to staff and students (Brassett et al., 2020) . Moreover, the challenge of community-based testing as well as the poor specificity and sensitivity of some tests meant that there could not be any guarantee that accepted donors were virus-free (Morgan et al., 2021) .

Critically, there have been no data published about the efficacy of anatomical embalming solutions to neutralise SARS-CoV-2. Such data are urgently required in order to inform decisions about the safe resumption of body donation programmes and cadaver-based anatomy education. Therefore, in the current study, the ability of 20 different, bespoke and commercial anatomical embalming solutions used in the United Kingdom and Ireland, to neutralise SARS-CoV-2, was tested. All 20 solutions tested were effective, several of them at high dilutions, resulting in effective neutralisation of anti-SARS-CoV-2 at concentrations well below those used to embalm a body. 

Vero E6 cells were cultured in 96-well plates as described for the cytotoxicity assay above. Each embalming solution was diluted twofold (1:2-1:128) in 2% FCS supplemented DMEM in a final volume of 50 µl. The viral inoculum was prepared in 2% FCS supplemented DMEM at a range of 4.5 × 10 8 -4.5 × 10 10 PFU/ml. A 50 µl aliquot of viral inoculum (~2.3 × 10 9 PFU) was added to 50 µl diluted embalming solution and incubated for 1 h at 37°C and 5% CO 2 . After this incubation period, each diluted embalming solution w/virus was further diluted 10-fold (10 1 -10 10 ) in 2% FCS supplemented DMEM.

Depending on the cytotoxicity, 50 µl samples of each dilution from 10 4 or 10 5 to 10 9 or 10 10 were transferred into wells of 96-well plates containing the previously prepared 3 × 10 4 Vero E6 cells. Then, Vero cells plus embalming solution/virus were incubated for 1 h at 37°C and 5% CO 2 (adsorption phase). After the adsorption phase, 100 µl of 1.2% Avicel ® PH-101 solution in 2% FCS DMEM were added to each well before the cells were incubated again for 72 h at 37°C and 5% CO 2 . After 72 h, the cells were fixed in 150 µl of 10% neutral buffered formalin for 3 h before being stained with 1% CV in 20% ethanol solution. After staining, plaques were quantified, and can neutralise the SARS-CoV-2 virus, thereby facilitating the safe resumption of body donation programmes and cadaveric anatomy teaching. viral titres were determined as previously published (Baer & Kehn-Hall, 2014) . Each embalming solution and its two-fold dilutions were tested in three independent experiments.

In order to rule out a potential direct cytotoxic effect of the embalming solutions tested on the Vero E6 cell system, dilutions of the embalming solutions that were non-toxic to Vero E6 cells were determined. The non-toxic dilutions for each embalming fluid tested (safe dilution) are listed in Table 1 where donor material is not embalmed, given that SARS-CoV-2 remains stable and viable for longer at low temperatures (Aboubakr et al., 2021; Chin et al., 2020) .

Interestingly, the soft embalming solutions tested, that is 4, 11, 15, 16, 17, 18, 20 , also showed efficacy in neutralising the virus and these may represent a suitable alternative during the COVID era for anatomical units otherwise routinely using fresh frozen bodies.

In particular, as noted above, some of them do not contain formal- Table 1 and Table S1 . In summary, our finding that all 20 anatomical embalming solutions tested effectively neutralise the SARS-CoV-2, and a number of them at concentrations much lower than they are normally used, provides a robust scientific basis upon which to make informed decisions about the safe resumption of body donor programmes and the subsequent deployment of embalmed donors for undergraduate and postgraduate teaching.

Stability of SARS-CoV-2 and other coronaviruses in the environment and on common touch surfaces and the influence of climatic conditions: a review

Viral concentration determination through plaque assays: using traditional and novel overlay systems

COVID-19 and anatomy: stimulus and initial response

Stability of SARS-CoV-2 in different environmental conditions

Overview of the world's medical schools: an update

Cadaveric dissection as an educational tool for anatomical sciences in the 21st century

Bodies for anatomy education in medical schools: an overview of the sources of cadavers worldwide

History and future of human cadaver preservation for surgical training: from formalin to saturated salt solution method

Disease surveillance for the COVID-19 era: time for bold changes

Neutralisation of SARS-CoV-2 by anatomical embalming solutions

The authors are grateful to Ms Catherine MacRobbie of the University of Glasgow for administrative help with this project. The authors wish also to thank Prof Gordon Findlater, her Majesty's Inspector for Anatomy in Scotland, for helpful advice.

The authors have no conflicts of interest.

All data will be made freely available, upon request, from the authors.