A novel human coronavirus, that is now named SARS-COV-2, was identified as the
cause of an epidemic of pneumonia cases in Wuhan, city in the Hubei Province of China. It rapidly spread throughout the world and is now causing a pandemic
1). A huge number of studies about the related clinical and pathological manifestations are in progress. Therefore, information and recommendations provided in this guideline are inherently provisional and future updates will be mandatory. Countries such as Germany and the United Kingdom have provisionally classified SARS-CoV-2 as a hazard group 3 biological agent. In general, four hazard groups of infectious biological agents are distinguished and categorised along three considerations: likelihood of causing disease by infection in humans, how likely the infection would spread to the community and the availability of any prophylaxis or treatment
2). This categorisation is primarily aimed at workers in diagnostic microbiology laboratories and infection research laboratories but could be adapted to autopsy practice to reflect the seriousness of many infections that could be transmitted from handling and dissecting infected deceased bodies
Person-to-person spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is thought to occur mainly via respiratory droplets. Respiratory droplets carrying pathogens are generated during coughing, sneezing, talking or during procedures such as suctioning and endotracheal intubation. The infection is transmitted when they travel directly from the respiratory tract of the infectious individual to susceptible mucosal surfaces of the recipient, generally over short distances. The maximum distance of droplet transmission is a currently unresolved matter, but historically, the area of risk has been defined as distance of less than 1 meter around the patient, based on epidemiologic and simulated studies of selected infections
6). A 2020 study showed that SARS-CoV-2 remained viable in experimentally generated aerosols for at least three hours
7). Given that, airborne precautions, which require the use of special air handling and
ventilation systems, are recommended
8). The infection transmission can also occur by the transfer of the infectious agent through a contaminated intermediate, such as the hands, touching eyes, nose, and mouth, after touching a contaminated surface
9). SARS-CoV-2 RNA samples were detected in blood and faeces of infected individuals
11). According to a joint WHO-China report, faecal-oral transmission is not yet well understood, but seems not to be a significant transmission factor
Clinical presentation of symptomatic infection ranges from mild to critical, although most infections are not severe. Most of the fatal cases tend to occur in patients with advanced age or underlying medical comorbidities
13). Pneumonia appears to be the most frequent serious manifestation of infection, characterized primarily by fever, dry cough, and dyspnoea. There are no specific clinical features that allow to reliably distinguish SARS-COV-2 from other viral respiratory infections, although development of dyspnoea several days after the onset of initial symptoms is strongly suggestive
14). Compared with non-severe patients, severe patients more commonly present neurologic symptoms, including headache, confusion, seizures, consciousness impairment. A few patients with SARS-COV-2 showed acute cerebrovascular disease
16). Smell and taste disorders, such as anosmia and dysgeusia have also been reported as common symptoms in patients with SARS-COV-2 infection
18). Whether this finding is a distinguishing feature of SARS-COV-2 is uncertain. Gastrointestinal symptoms (such as diarrhoea, nausea, vomiting and abdominal pain) and dermatological findings (urticarial eruptions) have also been reported
21). Studies have identified greater frequency and magnitude of
troponin elevations in hospitalized patients with severe SARS-COV-2 infection. It is not currently understood whether myocardial injury represents an independent risk marker in SARS-COV-2 or whether the risk is related to pre-existing cardiovascular susceptibility or disease
23). Some patients suddenly experience clinical deterioration that can result in sudden death. The level of D-dimer observed in critically ill patients is higher than in mild cases and suggests a higher thromboembolic risk. Rapid deterioration to death is usually associated to multi-organ failure related to: acute respiratory distress syndrome (ARDS); acute renal failure; septic shock; diffuse thrombosis and microvascular embolism configuring a framework of disseminated intravascular coagulation
Macroscopic findings: at dissection lungs display oedema and are increased in weight and consistency, in the absence of focal lesions
25). Sometimes pleural adhesions and signs of secondary bacterial pneumonia may be observed
27). CT findings of thromboembolism in major pulmonary arterial vessels have been reported in SARS-CoV-2 patients. Thus, during autopsy a comprehensive investigation and sampling of the lungs and pulmonary vessels is suggested
29). Right ventricle dysfunction, ARDS, and pulmonary thromboembolism may lead to develop cor pulmonale, which in the autopsy context can manifest with cardiac hypertrophy in some SARS-CoV-2 cases
30). Microscopic findings: microscopic examination of the lungs may reveal diffuse alveolar damage with congestion of alveolar septal capillaries, pneumocytes desquamation, fibromixoid exudate and hyaline membrane formation within the airspaces, indicating acute respiratory distress syndrome (ARDS). Interstitial inflammation at various stages may be observed, with patchy distribution, and composed mainly of lymphocytes
32). Atypical enlarged pneumocytes characterised by large nuclei, granular cytoplasm and prominent nucleoli have been identified in the intra-alveolar spaces, which may represent an expression of viral
33). Signs of inflammation within the bronchioles and the bronchial mucosa and, as it is common in diffuse alveolar damage, thrombi within small pulmonary artery branches have been reported
34). Substantial damage specifically related to SARS-COV-2 has not yet been identified in heart and liver tissues
In general, human coronaviruses represent a group of respiratory viruses that can reach the human central nervous system, by hematogenous or neuronal retrograde route, potentially causing neurological symptoms. Human coronaviruses reach the bloodstream through the disruption of the nasal epithelium or could reach directly the central nervous system through the olfactory route, causing encephalitis or acute flaccid paralysis. Additional possible complications of human coronaviruses include Guillain–Barré syndrome or acute disseminated encephalomyelitis (ADEM)
37). Since possible neurologic involvement, i.e. anosmia and dysgeusia, have been reported in SARS-COV-2 infection, the study of central nervous system may also be recommended.
Scope of the recommendations.
Since the handling of people who have died with or as a result of infectious disease may represent a source of nosocomial transmission, attention should be paid to safe post-mortem procedures. During autopsies transmission may occur by contact with body fluids, percutaneous inoculation, splashes to unprotected mucosa, and inhalation of infectious aerosols. The purpose of these recommendations is to advise on the rational approach and on the right personal protective equipment (PPE), those who conduct autopsies on possible SARS-COV-2 deceased cases and to indicate the optimal evaluation paths for diagnosing and studying the SARS-COV-2 infection.
A comprehensive knowledge of these safety and diagnostic criteria could be even more important for the forensic pathologist operating during the spread of SARS-COV-2 infection. Indeed, the forensic pathologist is often claimed not only to reconstruct the cause and the physiopathology of death but also to solve a number of further issues.
․ Formerly, during crime scene investigation the forensic pathologist may be the first physician facing to the death of a SARS-COV-2 infected subject. A comprehensive knowledge of clinical and pathological findings related to SARS-COV-2 infection may be of the utmost importance not only to pose the suspect of infection in the post-mortem setting but also to timely provide safety measures and adequate post-mortem sampling strategies, which may be time dependent due to decline of viral burden related to autolysis.
․ Several studies highlight the importance of a proper monitoring of SARS-COV-2 infections through wide testing also on asymptomatic subjects. Indeed, this strategy is the basis for a valid contact tracing of SARS-COV-2 positive subjects and for the related preventing measures aimed at containing the number of infected patients through quarantine. As a matter of fact, post-mortem investigations may be of the utmost importance for a comprehensive contact tracing of SARS-COV-2 infection. Indeed, also in the case of fatalities related to other causes (such as traumatic deaths) a concurrent SARS-COV-2 asymptomatic infection should not be overlooked. Therefore, post-mortem testing for SARS-COV-2 infection may be considered, especially if close contact with other subjects are supposed during life and SARS-COV-2 infection was not tested in-vitam.
․ In the elderly, SARS-COV-2 infection usually lead to a rapid and inevitable death because of frailty related to a number of pre-existing diseases. A comprehensive monitoring of the infection, also through post-mortem testing, may be of the utmost importance, especially for fatalities occurred in close contact to other fragile patients, such as in nursing homes for elderly or disabled people, to provide timely strategies aimed at preventing the diffusion of SARS-COV-2 infection.
․ Malpractice and medical liability claims are emerging problems related to SARS-
38). The forensic pathologist is often asked also to evaluate on the procedures and the timing of diagnosis, hospitalization, and therapy in SARS-COV-2 fatal cases. Such evaluations may be cumbersome, especially if we consider the evolving opinions about these issues.
․ Some suicidal cases have been reported during the SARS-COV-2 pandemia. Such suicidal behaviours have been related to the diagnosis of SARS-COV-2 infection, the contamination of a beloved or even the suspect of contamination of a beloved. Moreover, the suicide of some healthcare professional operating in intensive care department and facing with many SARS-COV-2 fatalities and resulting depressive state was related to their occupation. On these grounds sometimes it may be important for the forensic pathologist to take into account the SARS-COV-2 infection also to reconstruct the mechanism of death.
Lastly, although the Italian Ministry of Health (2 May 2020) stated that autopsies should be avoided in SARS-CoV-2 fatal cases, according to the ancient verse “mors ero tua mors” it appears of the utmost importance to complete clinical investigations with a comprehensive autopsy which may represent a unique opportunity to increase scientific knowledge on SARS-CoV-2 infection and of course to achieve important advances also in the clinical setting.
Health and safety aspects.
It is not possible to set to zero the risk of acquiring an infection in the autopsy room; therefore, the goal is to minimize this risk while maintaining adequate professional service standards. As for autopsies performed on corpses infected with SARS-CoV-2, if these are performed in adequately equipped rooms where good practices are respected, the risk of infection remains extremely low
39). Not only workers operating inside the autopsy room are exposed to the risk of SARS-CoV-2 infection. Indeed, malpractice may be related to the spread of infection to people living and or working in nearby facilities. The SARS-CoV-2 is considered a dangerous biological agent and should be managed in a biosafety level (BSL) 3 setting. A biosafety level (BSL) is a set of biocontainment precautions needed to isolate hazardous biological agents in a closed facility. Biosafety levels proceed from lowest to highest in a range of 1 (BSL1) to 4 (BSL4)
41). At the
lowest biosafety level, precautions consist of regular hand washing and minimal protective equipment. At higher levels, precautions include air flow systems, multiple containment rooms, sealed containers, positive pressure personnel suits, and high level of access control to the facility.
The autopsy suite.
Autopsies on corpses infected with SARS-CoV-2 should be performed in BSL3 rooms
42). The facility should have, preferably hands-free hand-washing sink and eyewash station, sharps containers, biohazard signs and self-closing, lockable doors separating the workspace from the rest of the structure
43). Clean sinks should be positioned so that they do not require unnecessary travel during routine work or in the event of an emergency
44). Work surfaces should have integrated waste-containment and drainage features that minimize the leakage of body fluids and wastewater
45). The floor and the surface of the dissection table must be water repellent and easy to clean
46). It is good practice to predispose several areas in the autopsy facility, basing on their degree of contamination. It is recommended to designate separated environments to a contaminated area (referred also as red zone), a potentially contaminated area (referred also as yellow zone) and a clean area (referred also as green zone). The contaminated area corresponds to the dissection room, with the dissection table at the centre. Potentially contaminated areas represent the personnel cleaning and disinfection sites. Contaminated, potentially contaminated and clean areas should be isolated as much as possible, and access must be restricted to authorized personnel
47). Autopsy rooms and in particular contaminated and potentially contaminated areas should have a separate air supply and should be physically separated from the administrative areas of the facility
48). Autopsy rooms should have a minimum of 6 up to 12 air changes per hour and should be under negative pressure compared to the surrounding spaces
51). The air in the autopsy room should flow unidirectionally from clean to less clean areas and should then be exhausted directly outside from the facility, away from other air intake systems. Alternatively, exhausted air
may be filtered through HEPA filters in case of recirculation
53). Downdraft dissection tables are recommended because SARS-CoV-2 corpses may harbor the risk of infection through aerosol
54). When combined with appropriate techniques, Class I and Class II biologic safety cabinets are suitable to contain moderate and high-risk microorganisms (Biosafety Level 2 and 3 agents), such as SARS-CoV-2, protecting workers from infectious aerosols generated within the cabinet
Personal protective equipment (PPE).
Since the survival time of the virus in the deceased is not known, workers who perform autopsies on corpses infected by SARS-CoV-2 need protection from both contact and aerosol transmission of the pathogen. A good practice is to consider that all body fluids (blood, secretions, excretions), non-intact skin and mucous membranes can contain the infectious agent. Also, equipment or items likely to have been contaminated with body fluids, must be handled in order to prevent transmission of SARS-COV-2. All workers, for protection purposes, should wear a surgical scrub suit, a surgical cap, a waterproof gown or apron with full sleeve coverage and shoe covers or rubber boots
57). Three pairs of gloves must be worn: one external and one internal latex pair and in the middle a pair of cut-resistant gloves
58). Metal and synthetic mesh gloves worn underneath surgical gloves may mitigate the risk of injuries from sharp objects, but do not prevent needle punctures
60). Gloves must not be washed for subsequent reuse because the continued glove integrity cannot be guaranteed
61). The use of gloves that fit perfectly around the wrist is preferable because in covering the gown cuff they provide a more reliable continuous barrier for arms, wrists, and hands, so it might be useful to stock gloves in several sizes
62). It is essential for the worker to wear devices that protect the eyes and the face. Personal eyeglasses do not represent an adequate eye protection. Although the goggles may represent an effective eyes protection, they do not prevent contact with splashes in other parts of the face. The combined use of goggles and masks to protect healthcare professionals from exposure to infectious agents transmitted through respiratory droplets has been studied for respiratory syncytial virus. Reports published in the mid-1980s showed that this combined protection reduced professional
transmission of the disease
64). Face shield extending from chin to crown can provide protection to other facial areas in addition to the eyes. Surgical masks mitigate the risk of splashing body fluids and droplets and help in preventing contact between the worker’s hands and nose or mouth
65). However, because of substantial marginal air leakage, standard surgical masks do not protect autopsy participants from inhaling airborne contaminants
67). When at risk for an aerosol pathogen (as is probably SARS-CoV-2) respiratory protection is required, and respirators with N95 or higher filtration should be worn (FFP3 when performing aerosol-generating procedures, according to European guidelines)
71). The fabric of these mask respirators is designed to filter at least 95% of particles of 1 µm in diameter
72). It is important to ensure that facial hair does not cross the respirator sealing surface and if the respirator has an exhalation valve, hair within the sealed mask area should not contact the valve
73). Those who are unable to properly wear N-95 respirators because of the beard, or other fit limitations, should utilize purified air respirators (PAPR) equipped with appropriate N-95 or high-efficiency particulate air (HEPA) cartridge filters
76). Most valved respirators are not fluid resistant, therefore they should be worn with a full-face shield if body fluid splashing is anticipated
77). To provide the right protection, respiratory protective masks must adhere well to the wearer’s face. All personnel wearing a respirator must undergo suitability tests for the model in question to ensure adequate tightness
effective protection against aerosols cannot be granted
79). An orientative fit test
80)should be done every time a respirator is put on, to ensure that the device is fitted properly
81). A controversial debate is underway on the value of taping of PPE components, such as gloves, respirators, boots, and goggles, because additional taping brings both benefits and risks
82). An improvement which additional taping might grant is the facilitation of the doffing process: if gloves or boots are connected to the coveralls by adhesive tape, they can be taken off in ‘one stroke’, limiting the possibilities for secondary contamination. Another improvement is represented by the closure of gaps between adjacent PPE components. On the other hand, taping of PPE components has some drawbacks. First, if carried out improperly the connection can break during doffing, increasing secondary contamination risk. Moreover, inexperienced users may be prone to tape over essential functional parts of PPE (covering the respirator with tape easily results in breathing difficulties)
83). Generally, proper additional taping of PPE requires adequate training and experience, moreover, most PPE manufacturers explicitly state in the product manuals, that taping compromises the integrity and functionality of PPE components
Donning of PPE.
It is critical to never don a PPE without proper active assistance.
Suggested steps for donning:
․ Put on scrubs and surgical cap.
․ Perform hand hygiene.
․ Put on the waterproof gown.
․ Put on foot protection.
․ Put on inner pair of gloves.
․ Wear respiratory protection and perform orientation fit test.
․ Put on eye protection.
․ Perform inner gloves disinfection and put on outer gloves (i.e. second and third layer).
․ Put on apron (optional).
․ Test the fit of the PPE components together.
․ Ready to pass through the yellow zone and to enter the red zone.
Doffing of PPE.
Active assistance in doffing of the worker leaving the red zone is essential for preventing him or her from manipulating contaminated PPE without having direct sight
85). Active assistance implies a close collaboration between two operators; therefore, the instructions below refer to the conduct that both will have to assume, respectively.
․ Remove the optional apron (in the red zone)
․ Step out of the red zone
․ PPE inspection of the worker ready for doffing to identify cuts or contamination
․ Disinfect the PPE (wiping with disinfectant)
․ Remove the outer gloves
․ Put on a new pair of outer gloves
․ Remove tape from face area (if present)
․ Remove the goggles.
․ Use new pair of outer gloves.
․ Roll down the waterproof gown.
․ Roll down the sleeves (with eventually the integrated taped gloves)
․ Remove the foot protection.
․ Use new pair of outer gloves.
․ Remove the PPE user’s respirator.
․ Hand hygiene and step into the green zone
The last PPE user to remove the PPE ensemble will have to perform all tasks without external assistance. Receiving guidance without being touched is essential. The ‘last one doffing’ can be the last active assistant withdrawing from the red zone but also a person who had performed the last tasks. However, doffing alone after obvious exposure to bodily fluids or waste should be avoided at all costs, even when a qualified supervisor is
available. A mirror, placed close to the doffing zone enables self-monitoring during the process, providing additional control
Comprehensive illustrated material outlining both donning and doffing procedures is available at the dedicated section of the European Centre for Disease Prevention and Control website.
Clinical information relevant to the autopsy.
The choice to prudentially consider each corpse a possible source of SARS-COV-2 infection regardless medical history is still a matter of debate. In particular, this plan of action might be taken into account almost during the pandemic phase. On the other hand, the criteria to assess whether a deceased may have been infected by SARS-COV-2 are mainly the same as those used to assess the infection risk in the living
89). Moreover, a comprehensive knowledge of these informations should guide the following dissection and sampling activities.
․ Evaluation of clinical history:
○ signs and symptoms which suggest SARS-COV-2 infection during life (fever, dry cough, dyspnoea, anosmia and dysgeusia);
○ hospitalization for pneumonia;
○ laboratory data and microbiology data;
○ radiologic findings during life
․ Post-mortem imaging:
○ chest radiography or chest computerized tomography (CT)
○ bilateral and peripheral ground-glass opacification, with or without consolidative abnormalities, consistent with viral pneumonia may be observed
○ the corpse should be sealed in triple sealed bags, to prevent leaking and contamination
The autopsy procedure.
This document does not provide a detailed description of the autoptic techniques and procedures but will give a summary of good practices and recommendations for a forensic pathologist dealing with a SARS-COV-2 corpse.
Behaviour during autopsy.
The movement of the corpse should be as gentle as possible, to avoid blood and stool leakage. A standard systematic external and internal organ examination is recommended. The dissection method must be adapted for each single case aiming to highlight the specific relevant pathological conditions. All major organs (heart, lungs, liver, and kidneys) must be isolated and weighed
97). Cuts and pricks while using scalpels, knives or needles are the most obvious source of mechanical hazards
98). Universal precautions implemented to reduce such mechanical risk are even more recommended, and include:
․ placing used disposable syringes, needles, scalpel blades, and other sharp items in puncture-resistant containers for disposal, located as close to the use area as is practical
․ eliminating the unnecessary use of needles and implementing the use of devices with safety features
․ not hand re-sheathing of needles after fluid sampling
․ not removing used needles from disposable syringes by hand
․ using a tray or an instrument to pass items
․ picking up needles or sharp objects from the floor using a rolling magnet or magnetic needle pad and gloves
․ using of round-ended scissors and blades with blunted points
․ operating within the body cavity one practitioner at a time
․ slicing of unfixed organs holding them firm on the table with a sponge
Moreover, precautions against air and droplet transmission should be implemented in
case of SARS-COV-2 deceased body dissection. Aerosols consist of suspensions of solid or liquid particles in a gas. Infectious aerosols are composed of airborne particles with a size of approximately 1 to 5 µm in diameter, which can reach the pulmonary alveoli via inhalation
109). The main source of aerosols in autopsy facilities are bone-cutting procedures
110). Sawing the skull with an oscillator saw with suction extraction of the bone aerosol into a removable chamber, or alternatively with a hand saw, is mandatory in SARS-COV-2 infected corpses. Indeed, it can reduce significantly the exposure to infected aerosolized bone dust
111). Moreover, protection against aerosols requires also specialized and well-fitted PPE
Biological samples collection.
The post-mortem collection of different samples is recommended for diagnosing the presence of SARS-CoV-2 in a deceased body. Samples should include:
․ cerebrospinal fluid
․ nasal-oropharyngeal (NP) swabs
․ lung tissue swabs
․ anal swabs
Although the collecting procedure of the NP swab is well-known in the clinical setting it deserves to be reminded to the forensic pathologist. Insert a flexible wire shaft minitip swab through the nostrils parallel to the palate (not upwards) until resistance is encountered, indicating contact with the nasopharynx. The swab should reach a depth equal to the distance from the nostrils to the external opening of the ear. Gently rub and roll the swab. Leave the swab in place for a few seconds to absorb the secretions. Slowly remove the swab by twisting it
132). Since the collection of NP swabs from deceased bodies will not induce coughing or sneezing, BSL 3 facility and N-95 respirator are not mandatory
133). When collection of a post-mortem NP swab is not possible, acceptable alternatives are oropharyngeal, nasal mid-turbinate, anterior nares swabs and nasopharyngeal wash/aspirate or nasal aspirate specimen
134). For nasal swabs, a single polyester swab with a plastic stem should be used to sample both nostrils
135). Only synthetic fibre swabs with plastic shafts should be used. Do not use calcium alginate swabs or swabs with wooden shafts, as they may contain substances that inactivate some viruses and inhibit PCR testing. Swabs must be placed immediately into sterile tubes containing 2-3 ml of viral transport media
136). Nasal swabs or nasal mid-turbinate swabs should be placed in a transport tube containing either viral transport medium, Amies transport medium, or sterile saline. If both NP and oropharyngeal swabs are collected, they should be combined in a single tube to maximize test sensitivity
137). Moreover, lung swab collection should be performed inserting one swab into the tracheobronchial tree on either side, after the removal of the heart-lung block, or, alternatively, inserting the swab to collect sample on either side in a slit of the lung, made using a sterile scalpel after wiping the surface of each lung with an iodine-containing disinfectant
In the authors’ experience SARS-CoV-2 virological tests performed on oropharyngeal, NP and broncho-pulmonary swabs, usually produce consistent results. Nevertheless, multi-sampling from different tissues and districts is recommended because improves diagnostic accuracy, reducing the false negative rate
140). On blood, similarly to in-vivo tests, SARS-Cov-2, infection might be diagnosed both through nucleic acid research test and serological tests
143). Post-mortem serological tests on blood are
suggested both for diagnostic purposes and for scientific purposes. Indeed, the behaviour of SARS-Cov-2 antibodies after death has not been established yet. However, according to literature, although a positive result at post-mortem analyses may be regarded as valuable, a negative result must be interpreted with extreme caution because autolysis may determine the degeneration of both viral nucleic acids and proteins, leading to a wrong diagnosis
144). SARS-CoV-2 RNA might still be detected up to 3 days after death and possibly longer based on data collected from human coronaviruses; however sensitivity is probably reduced with a longer post-mortem interval, but no official data is currently available
145). There are no data available on the infectivity of SARS-CoV-2 RNA in cadavers, and therefore cell cultures are needed. Indeed, molecular biology techniques allow to identify the presence of the virus, but do not provide information on its infectivity. Aiming at investigating on the presence of the viral RNA for scientific research purposes, the collection of faecal microbiota, intraocular fluid and bone marrow is also recommended
146). In addition to post-mortem samples, any specimen (e.g. swab, sputum, serum, stool) that has been collected prior to death should be retained
147). Considering that different techniques are available for the diagnosis of SARS-Cov-2 infection on post-mortem specimens, sampling and sample storage should be planned with the local virology laboratory. In general, samples should be sent immediately to the laboratory, or can be stored at 2-8°C for up to 72 hours after collection. If a delay in testing or shipping is expected, specimens should be stored preferably at -80 ° C
149). Transported specimens should be positioned into a two-layers container to minimize the risk of breakage or spillage. Fresh specimens should be transported on an ice pack
150). Post-mortem samples for histological evaluation should be collected from heart, lungs (parenchyma and bronchi), kidneys, liver, lymph nodes, intestinal mucosa (small and large intestine), spleen, pancreas, adrenal glands, and striated muscle. Head dissection is often mandatory during a forensic autopsy, on the other hand during an autopsy performed for clinical purposes the dissection of the head can be avoided, and of course should be avoided in cases of SARS-CoV-2 infection considering the related safety issues. On these grounds forensic autopsies can be considered a unique opportunity to study the brain also for scientific purposes and thus sampling of the brain in-toto should be rec
ommended. For histological and immunohistochemical analysis, tissue pieces must be fixed in 10% formalin long enough to allow the pathogen inactivation (at least 48-72 hours, time is proportional to size of the sample and amount of formalin)
154). Formalin-fixed, and formalin-fixed-paraffin-embedded tissue specimens, obtained at autopsy, can be used to establish a post-mortem diagnosis of SARS-CoV-2 infection by using immunohistochemical and molecular techniques
155). According to the update CDC guidance for collection of post-mortem specimens (April 2020) a minimum of three representative sections of lung parenchyma from different locations and a minimum of two sections of airway, to include trachea, bronchi, or both airways are recommended. To minimize potential viral contamination of non-involved tissues, lung and airway specimens should be collected immediately following removal of the chest plate. It is appropriate to consider that prolonged immersion in formalin can diminish the sensitivity of virus detection assays
156). Human coronaviruses have been shown to induce T cell apoptosis and immunosuppression, which can facilitate opportunistic infections
157). When infections related to pathogens other than SARS-CoV-2 are suspected, the collection of additional swabs and tissue specimens is also recommendable.
SARS-CoV-2 may remain viable for hours to days on different materials
158). The sanitization is part of the correct maintenance of the area and the instrumentation dedicated to autoptic dissection and must be carried out with disinfectants effective against SARS-CoV-2. Using data obtained from similar human coronaviruses, SARS-COV-2 is supposed to be efficiently inactivated within one minute by 0.1% sodium hypochlorite, 62 to 71% of ethanol and 0.5% hydrogen peroxide
160). SARS-CoV-2 is susceptible also to quaternary ammonium compounds and phenolic compounds, if used according to the manufacturer’s recommendations
161). Other biocidal agents such as 0.05% to 0.2% benzalkonium chloride or 0.02% chlorhexidine digluconate could be less
163). Irradiation with ultraviolet light for 60 minutes on several coronaviruses in culture medium results in undetectable viral infectivity levels
1) Van Doremalen N et al.. Aerosol and Surface Stability of SARS-CoV-2 as Compared with SARS-CoV-1. N Engl J Med. 2020 Mar 17.
2) Osborn M et al.Briefing on COVID-19 Autopsy practice relating to possible cases of COVID-19 (2019-nCov, novel coronavirus from China 2019/2020). The Royal College of Pathologists. 2020.
3) Federal Institute for occupational healt and safety (BAUA). Novel virus SARS-CoV-2 (previously 2019-nCoV) classified by the ABAS in risk group 3 and recommendations for laboratory diagnostics available on www.baua.de/DE/Angebote/Aktuelles/Meldungen/2020/2020-02-19-Coronavirus.html Accessed 11 May 2020.
4) Advisory committee on dangerous pathogens (ACDP) COVID-19 safe handling and processing for samples in laboratories available on www.gov.uk/government/publications/wuhan-novel-coronavirus-guidance-for-clinical-diagnostic-laboratories/wuhan-novel-coronavirus-handling-and-processing-of-laboratory-specimens #fnref:3 Accessed 11 May 2020.
5) Feigin RD et al.. Epidemic meningococcal disease in an elementary-school classroom. N Engl J Med. 1982.
6) Siegel JD et al.2007 Guideline for Isolation Precautions: Preventing Transmission of Infectious Agents in Healthcare Settings. Last update July 2019. American journal of infection control. 2007.
7) Van Doremalen N et al.., op. cit. sub.15.
8) Siegel JD et al., op. cit. sub. 20.
10) Tang A et al. Detection of Novel Coronavirus by RT-PCR in Stool Specimen from Asymptomatic Child, China. Emerg Infect Dis. 2020 Jun 17.
11) Chen W et al. Detectable 2019-nCoV viral RNA in blood is a strong indicator for the further clinical severity. Emerg Microbes Infect. 2020 Feb 26.
12) Report of the WHO-China Joint Mission. 16-24 February 2020. available on https://www.who.int/docs/default-source/coronaviruse/who-china-joint-mission-on-covid-19-final-report.pdf. Accessed 11 May 2020.
13) Zhou F.Clinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan, China: a retrospective cohort study. The Lancet. 2020.
14) Wang D et al. Clinical Characteristics of 138 Hospitalized Patients With 2019 Novel Coronavirus-Infected Pneumonia in Wuhan, China. JAMA. 2020.
15) Mao L et al.Neurological Manifestations of Hospitalized Patients with COVID-19. JAMA. 2020.
16) Asadi-Pooy AA et al. Central nervous system manifestations of COVID-19: A systematic review. Journal of the Neurological Sciences. 2020.
17) Giacomelli A et al. Self-reported olfactory and taste disorders in SARS-CoV-2 patients: a cross-sectional study., Clin Infect Dis. 2020.
18) Desforges M et al. Human Coronaviruses and Other Respiratory Viruses. Viruses. 2019.
19) Wang D et al.. Clinical Characteristics of 138 Hospitalized Patients With 2019 Novel Coronavirus-Infected Pneumonia in Wuhan, China. JAMA. 2020.
20) Huang C et al. Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. Lancet. 2020.
21) Recalcati S. Cutaneous manifestations in COVID-19: a first perspective, J Eur Acad Dermatol Venereol. 2020.
22) Shi S et al. Association of Cardiac Injury With Mortality in Hospitalized Patients With COVID-19 in Wuhan, China. JAMA. 2020.
23) Guo T et al. Cardiovascular Implications of Fatal Outcomes of Patients With Coronavirus Disease 2019 (COVID-19). JAMA Cardiol. 2020.
24) Mei H et al.Characteristics, causes, diagnosis and treatment of coagulation dysfunction in patients with COVID-19. Chinese Journal of Hematology. 2020.
25) Barton LM et al. COVID-19 Autopsies, Oklahoma, USA. Am J Clin Pathol. 2020.
26) Osborn M et al. op. cit. sub. 16.
27) Barton LM et al. op. cit. sub. 39.
28) Jafari R et al. Large saddle pulmonary embolism in a woman infected by COVID-19 pneumonia. Eur Heart J. 2020.
29) Danzi GB et al.Acute pulmonary embolism and COVID-19 pneumonia: a random association? Eur Heart J. 2020.
30) Ioan AM et al. Pulmonary embolism in COVID-19. When nothing is what it seems. Rev Esp Cardiol. 2020.
31) Barton LM et al. op. cit. sub. 39.
32) Zhe XU et al. Pathological findings of COVID-19 associated with acute respiratory distress syndrome. Lancet Respir Med. 2020.
34) Barton LM et al. op. cit. sub. 39.
36) Zhe XU et al. op. cit. sub. 46.
37) Desforges M et al. op. cit. sub. 32.
38) Parisi S G, Viel G, Cecchi R, Montisci M. COVID-19: the wrong target for healthcare liability claims. Legal Medicine https://doi.org/10.1016/j.legalmed.2020.101718
39) Petrosillo N et al. Procedura per l’esecuzione di riscontri diagnostici in pazienti deceduti con infezione da SARS-CoV-2 - Gruppo di Lavoro ISS Cause di morte COVID-19” 23 March 2020. Available on: https://www.iss.it/documents/20126/0/Rapporto+ISS+COVID-19+n.+6_2020+autopsie.pdf/004df480-4222-6f44-bfee-0ef8b91c108a?t=1587106915706 accessed on 12 May 2020.
40) Chosewood LC et al.Biosafety in Microbiological and Biomedical Laboratories (5th ed.). Centers for Disease Control and Prevention. 1 April 2020.
41) Directive 2000/54/EC of the European Parliament and of the Council of 18 September 2000 on the protection of workers from risks related to exposure to biological agents at work (seventh individual directive within the meaning of Article 16(1) of Directive. 2000.
42) Petrosillo N et al. op. cit. sub. 52.
43) Le AB et al. U.S. Medical Examiner/Coroner capability to handle highly infectious decedents. Forensic Sci Med Pathol. 2018.
44) Nolte KB et al. Medical Examiners, Coroners, and Biologic Terrorism. CDC. 2004. Available on: https://www.cdc.gov/mmwr/preview/mmwrhtml/rr5308a1.htm accessed on 12 May 2020.
46) Directive 2000/54/EC op. cit. sub. 54.
48) Nolte K B et al.Biosafety Considerations for Autopsy. The American Journal of Forensic Medicine and Pathology. 2002.
49) Petrosillo N et al. op. cit. sub. 52.
50) Nolte KB et al. op. cit. sub. 61.
51) Nardell EA et al.Airborne infection: theoretical limits of protection achievable by building ventilation. Am Rev Respir Dis. 1991.
52) Petrosillo N et al. op. cit. sub. 52.
53) Nolte KB et al. op. cit. sub. 61.
55) Nolte KB et al. op. cit. sub. 57.
56) Nolte KB et al. op. cit. sub. 61.
57) Petrosillo N et al. op. cit. sub. 52.
59) Nolte KB et al. op. cit. sub. 61.
60) Zugibe FT et al.Protective gloves for high-risk autopsies. Am J Forensic Med Pathol. 1995.
61) Siegel J.D. et al. op. cit. sub.20.
64) Gala C.L. et al.The use of eye-nose goggles to control nosocomial respiratory syncytial virus infection. JAMA. 1986.
65) Nolte KB et al. op. cit. sub. 61.
67) Pippen DJ et al. Efficacy of face masks in preventing inhalation of airborne contaminants. J Oral Maxillofac Surg. 1987.
68) Siegel J.D. et al., op. cit. sub.20.
69) Nolte KB et al. op. cit. sub. 61.
70) Jensen PA et al.Guidelines for preventing the transmission of Mycobacterium tuberculosis in health-care facilities - CDC”. Morb Mortal Wkly Rep. 1994.
71) Guidance for wearing and removing personal protective equipment in healthcare settings for the care of patients with suspected or confirmed COVID-19 - ECDC. 2020. Available on: https://www.ecdc.europa.eu/sites/default/files/documents/COVID-19-guidance-wearing-and-removing-personal-protective-equipment-healthcare-settings-updated.pdf accessed 12 May 2020.
72) Nolte KB et al. op. cit. sub. 61.
73) Available on https://www.gov.uk/coronavirus. accessed on April 24, 2020.
74) Petrosillo N et al. op. cit. sub. 52.
75) Nolte KB et al. op. cit. sub. 57.
76) Nolte KB et al. op. cit. sub. 61.
77) Available on https://www.gov.uk/coronavirus. accessed on April 24, 2020.
78) Fit testing: procedure aimed at verifying that the contaminated air does not enter the respiratory device, which is mandatory by law in USA and in UK. There are two types of fit tests: qualitative and quantitative. Qualitative fit testing is a pass/fail test method that uses the sense of taste or smell, or the reaction to an irritant in order to detect leakage into the respirator facepiece. Quantitative fit testing uses a machine to measure the actual amount of leakage into the facepiece.
79) Safe use of personal protective equipment in the treatment of infectious diseases of high consequence - ECDC. 2014. Available on: https://www.ecdc.europa.eu/sites/default/files/media/en/publications/Publications/safe-use-of-ppe.pdf accessed on 12 May 2020.
80) The fit check of a respirator is performed through an inspiration and an expiration manoeuvre. For the inhalation test an assistant covers the respirator surface with both hands and asks the user to inhale deeply. If airflow is entering around the nose, the nose metal clip must be adapted. If air is entering from the respirator edges, the straps need to be re-adjusted. In the exhalation test an assistant covers the respirator surface (unvalved respirator) or the exhalation valve (valved respirator). When sharply exhaling in the respirator, the PPE user should feel no air blowing into the eyes or cheeks. If air is escaping, the respirator needs to be adapted.
81) Safe use of personal protective equipment in the treatment of infectious diseases of high consequence - ECDC. 2014. op. cit. sub. 92.
89) Osborn M et al. op. cit. sub. 16.
91) Barton LM et al. op. cit. sub. 39.
92) Wong HYF et al. Frequency and Distribution of Chest Radiographic Findings in COVID-19 Positive Patients. Radiology. 2019.
93) Barton LM et al. op. cit. sub. 39.
94) Shi H et al.Radiological findings from 81 patients with COVID-19 pneumonia in Wuhan, China: a descriptive study. Lancet Infect Dis. 2020.
95) Zhao W et al.Relation Between Chest CT Findings and Clinical Conditions of Coronavirus Disease (COVID-19) Pneumonia: A Multicenter Study. AJR Am J Roentgenol. 2020.
96) Le AB et al. op. cit. sub. 57.
97) Petrosillo N et al. op. cit. sub. 52.
98) Wenner L. Aerosol Generation During Bone-Sawing Procedures in Veterinary Autopsies. Veterinary Pathology. 2017.
99) Osborn M et al. op. cit. sub. 16.
100) Update: Universal Precautions for Prevention of Transmission of Human immunodeficiency Virus, Hepatitis B Virus, and Other Bloodborne Pathogens in Health-Care Setting - CDC. 1988.
101) Preventing Needlestick Injuries in Health Care Settings - CDC. 1999. Available on https://www.cdc.gov/niosh/docs/2000-108/pdfs/2000-108.pdf?id=10.26616/NIOSHPUB2000108 accessed on 12 May 2020.
104) FRY D. E. et al. Prevention of Blood Exposure. Surgical Clinics of North America. 1995.
106) Osborn M et al. op. cit. sub. 16.
109) Wenner L. op. cit. sub. 111.
111) Osborn M et al. op. cit. sub. 16.
113) Siegel J.D. et al., op. cit. sub.20.
114) Osborn M et al. op. cit. sub. 16.
116) Barton LM et al. op. cit. sub. 39.
117) Petrosillo N et al. op. cit. sub. 52.
118) Minghetti L et al. Raccomandazioni per raccolta, trasporto e conservazione di campioni biologici COVID-19 - Gruppo di lavoro ISS Ricerca traslazionale COVID-19. 2020. Available on: https://www.epicentro.iss.it/coronavirus/pdf/rapporto-covid-19-13-2020.pdf accessed on 13 May 2020.
119) Osborn M et al. op. cit. sub. 16.
120) Minghetti L et al. op. cit. sub. 131.
121) Jianguo W et al.Detection and analysis of nucleic acid in various biological samples of COVID-19 patients. Travel Medicine and Infectious Disease 2020.
122) Lee YL et al.Dynamics of anti-SARS-Cov-2 IgM and IgG antibodies among COVID-19 patients. Journal of Infection. 2020.
123) Osborn M et al. op. cit. sub. 16.
124) Barton LM et al. op. cit. sub. 39.
125) Petrosillo N et al. op. cit. sub. 52.
126) Minghetti L et al. op. cit. sub. 131.
127) Osborn M et al. op. cit. sub. 16.
128) Minghetti L et al. op. cit. sub. 131.
129) JIANGUO W. et al. op. cit. sub. 134.
130) Minghetti L et al. op. cit. sub. 131.
131) Jianguo W et al. op. cit. sub. 134.
132) Collection and Submission of Postmortem Specimens from Deceased Persons with Known or Suspected COVID-19 - CDC. 2020. Available on: https://www.cdc.gov/coronavirus/2019-ncov/hcp/guidance-postmortem-specimens.html accessed on 13 May 2020.
139) Barton LM et al. op. cit. sub. 39.
140) Jianguo W et al. op. cit. sub. 134.
142) Lee YL et al. op. cit. sub. 135.
143) Jiuxin Q et al.Profile of IgG and IgM antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Infectious Diseases Society of America.
144) Tryland M et al.Persistence of antibodies in blood and body fluids in decaying fox carcasses, as exemplified by antibodies against Microsporum canis. Acta Vet Scand.2006.
145) Collection and Submission of Postmortem Specimens from Deceased Persons with Known or Suspected COVID-19 - CDC. 2020. op. cit. sub. 145.
146) Petrosillo N et al. op. cit. sub. 52.
147) Collection and Submission of Postmortem Specimens from Deceased Persons with Known or Suspected COVID-19 - CDC. 2020. op. cit. sub. 145.
149) Stefanelli P et al. Raccomandazioni per il corretto prelievo, conservazione e analisi sul tampone oro/nasofaringeo per la diagnosi di COVID-19 - Gruppo di Lavoro ISS Diagnostica e sorveglianza microbiologica COVID-19: aspetti di analisi molecolare e sierologica. 2020. Available on: https://www.epicentro.iss.it/coronavirus/pdf/rapporto-covid-19-11-2020.pdf accessed on 13 May 2020.
150) Minghetti L et al. op. cit. sub. 131.
151) Petrosillo N et al. op. cit. sub. 52.
152) Collection and Submission of Postmortem Specimens from Deceased Persons with Known or Suspected COVID-19 - CDC. 2020. op. cit. sub. 145.
153) Iwen PC et al.Safety Considerations in the Laboratory Testing of Specimens Suspected or Known to Contain the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). American Society for Clinical Pathology. 2020.
154) Hemwood AF.Coronavirus disinfection in histopathology. Journal of Histotechnology. 2020.
155) Collection and Submission of Postmortem Specimens from Deceased Persons with Known or Suspected COVID-19 - CDC. 2020. op. cit. sub. 145.
157) Mei H et al. op. cit. sub. 38.
158) Van Doremalen N. et al.. op. cit. sub. 15.
159) Minghetti L et al. op. cit. sub. 131.
160) Hemwood AF. op. cit. sub. 167.
161) Minghetti L et al. op. cit. sub. 131.
163) Hemwood AF. op. cit. sub. 167.
164) Hemwood AF. op. cit. sub. 167.
165) Duan SM et al.Stability of SARS coronavirus in human specimens and environment and its sensitivity to heating and UV irradiation. Biomed Environ Sci. 2003.