Human DNA contamination of postmortem examination facilities: Impact of COVID‐19 cleaning procedure

Abstract The DNA contamination of evidentiary trace samples, included those collected in the autopsy room, has significant detrimental consequences for forensic genetics investigation. After the COVID‐19 pandemic, methods to prevent environmental contamination in the autopsy room have been developed and intensified. This study aimed to evaluate the level of human DNA contamination of a postmortem examination facility before and after the introduction of COVID‐19‐related disinfection and cleaning procedures. Ninety‐one swabs were collected from the surfaces and the dissecting instruments, analyzed by real‐time quantitative PCR (q‐PCR) and typed for 21 autosomal STRs. Sixty‐seven out of 91 samples resulted in quantifiable human DNA, ranging from 1 pg/μl to 12.4 ng/μl, including all the samples collected before the implementation of COVID‐19 cleaning procedures (n = 38) and 29 out of 53 (54.7%) samples taken afterward. All samples containing human DNA were amplified, resulting in mixed (83.6%), single (13.4%), and incomplete (3%) profiles. A statistically significant decrease in DNA contamination was found for dissecting instruments after treatment with chlorhexidine and autoclave (p < 0.05). Environmental decontamination strategies adopted during COVID‐19 pandemic only partially solved the long‐standing issue of DNA contamination of postmortem examination facilities. The pandemic represents an opportunity to further stress the need for standardized evidence‐based protocols targeted to overcome the problem of DNA contamination in the autopsy room.


| INTRODUC TI ON
It is well known that forensic science DNA profiling has a central role in the criminal justice community helping conviction of the guilty and exoneration of the innocent even in capital offenses.
However, the DNA profiling from biological material detected at a crime scene should always be interpreted with caution, considering that small amounts of "innocent DNA" could be found. The increased sensitivity of PCR-based methodologies has enabled genetic profiles to be obtained from degraded samples or from trace samples left by talking, sneezing, skin cells shedding, and DNA left on the surfaces by touch-so called "touch DNA" [1,2]. However, this has also exacerbated the issues of DNA persistence, background level and contamination [3,4]. Given that DNA transfer might occur as a consequence of criminal or noncrime-related activities, such as in contamination events, the main issue in forensic is represented by the mechanisms or actions that led to the deposition of the biological material concerned [5,6]. Recently, the DNA commission of the International Society for Forensic Genetics (ISFG) published recommendations for forensic geneticists to evaluate DNA and biological results, whose value is impacted by phenomena such as secondary (or tertiary) transfer, contamination or "fortuitous" presence of DNA in the environment [7,8].
The risk of contamination by exogenous DNA has been highlighted for samples collected from clothes or body surfaces of the victim in the mortuary, even if autopsy surfaces and instruments might appear falsely "clean" [9][10][11]. The DNA transfer at postmortem facilities does not represent a new matter, but the "mobility" of DNA is still an issue in court and a subject of research, as underlined by recent publications showing that DNA might be distributed even in the context of cleaning scenarios [12].
The COVID-19 pandemic outbreak has led to significant changes in the autopsy practices: personal protective equipment, hygiene precautions, hospital disinfection, and sterilization methods, including those related to autopsy room, have been recently updated and intensified, all of them devoted to the prevention of the infectious risk [13][14][15][16][17].
Given the long-standing issue of exogenous background DNA contamination and the recent attention devoted to the environmental cleaning from SARS-CoV-2, this study aimed to evaluate the level of human DNA contamination of a postmortem examination facility before and after the introduction of COVID-19-related disinfection and cleaning procedures, in order to assess their impact on the genetic typing of forensic evidentiary traces.

| Study design
The study was performed at the local postmortem examination facility of the University of Bologna, which is in use by several forensic pathologists, technicians, and occasionally by clinical pathologists.
Samples were collected from surfaces and dissecting instruments across 9 unannounced visits (V), which took place without any warning to pathologists and/or to cleaning services during two different time periods. Visits took place before the outbreak of COVID-19 pandemic (V1-V3) and after the implementation of a COVID-19 cleaning and decontamination plan (V4-V9) and were scheduled as follows: • V1, V4, and V7 on a random day, assumed to be representative of the daily forensic routine; • V2, V5, and V8 were done immediately before a scheduled postmortem examination; • V3, V6, and V9 took place after the scheduled postmortem examination, as soon as a complete cleaning and drying of room and instruments was achieved.

| Surfaces
Before the pandemic, all surfaces of the facilities were routinely cleaned with the following products: Cleaning products were provided diluted to a final sodium hypochlorite concentration of 0.1% and wiped up with absorbent material.

| Sampling
Ten (n = 10) surfaces and four (n = 4) dissection instruments, which were assigned a letter from A to N, were swabbed for sampling.
Details of the items, as well as the number of sampling per item, are shown in Figure 1 and Table 1. During V1, only surfaces samples A-J were available, since dissection instruments were not usually left in the local facility and were usually brought to the mortuary immediately before the autopsy.
On the basis of preliminary results, only dissection instruments were swabbed during V7-V9. PCR products were separated and detected by capillary electrophoresis on the SeqStudio ™ Genetic Analyzer (Applied Biosystems) following the manufacturer instructions. GlobalFiler ™ Allelic Ladder was included in each capillary electrophoresis run.

| Genetic analyses
Data collection software was used to collect raw data and genemapper idx v 1.6 (Thermo Fisher) for allele calls and profile analysis, using an analytical threshold value of 100 RFU.

| Global filer profile interpretation
The interpretation of electrophoretic data was carried out according to the national Ge.F.I. recommendations [18]. DNA profiles were classified in incomplete profiles: <10 STR loci detected; single source profiles: ≥10 STR loci successfully amplified and characterized by no more than two alleles at each locus; mixed profiles: ≥10 STR loci successfully amplified with more than two alleles detected in at least two different loci.

| Statistical analyses
Descriptive statistics were provided, including median and in-   This rate of positive sample, slightly higher than previously reported [19], might be also due to the higher sensitivity of DNA analysis techniques achieved in the last years [11]. The median human DNA content of the whole sample was 0.007 ng/μl (IQ = 0.136-0.000).

| RE SULTS AND D ISCUSS I ON
Detailed results for surfaces and instruments are shown in Table 2 and Figure 2.
All the 67 amplified samples showed 56 (83.6%) mixed profiles of which 44 (78.6%) contained DNA from three or more contributors (data not shown); nine profiles (13.4%) were single source and 2 (3.0%) were incomplete. Among mixed profiles, 48 were generated from samples collected from the surfaces and eight from dissecting instruments. Detailed results are shown in Table 3.

| Surfaces
Contaminating DNA was found on all the work surfaces of the On the other hand, the presence of single profiles on surfaces also appears worrisome, since this might also appear as the main   have been shown to act as contamination vectors in the autopsy room, as already reported [11,19].

| Dissection instruments
In our study, before the COVID-19 pandemic, each instrument showed a high DNA amount and was thus considered a potential source of contamination (

| Pre-and post-COVID outbreak and cleaning procedures
After No inhibition or degradation was observed for all analyzed swabs.
By comparing samples collected pre and post-COVID-19 decontamination plan, no statistically significant difference in DNA content was found for the whole sample (p = 0.052) as well as for the facility surfaces (p > 0.05); for dissecting instruments, a statistically significant decrease in DNA content was found (p < 0.05).
The cleaning procedures of work surfaces adopted during the pandemic are known to be effective for bacteria, fungi, and even for high hazard viruses [16,17]. In our study, human quantifiable DNA Autoclave has been shown to not be completely effective in DNA removal [10], so that chlorhexidine gluconate likely played a major role in the undetectability of DNA on dissection instruments.
According to our results, it might also be useful for the cleaning of those work surfaces that showed unaltered contamination level

| Limitations
One limitation is that samples were not taken directly on the body areas usually swabbed for trace DNA evidence collection, but only on surfaces and dissection instruments in the autopsy room. We acknowledge, that, in order to be forensically significant, human contaminant DNA present on these surfaces must be transferred to the body in a quantity sufficient to then be transferred to a swab.
However, even small amounts of DNA from transfer events could interfere with data interpretation. In selected cases, it could be useful to preliminarily swab the surfaces on which the body will be placed, as background control.
In addition, as previously discussed, even if the dissection instruments are not used for collection of DNA evidence, which typically is performed before any dissection, they could represent a vector for secondary transfer events and were for this reason included in this study.
The high number of observed mixed profiles indicates that more than one individual contributed to the background DNA of the surfaces and instruments, possibly by primary or higher-order DNA transfer. By using software for statistical interpretation [24], profiles could be deconvoluted and compared to a database containing reference profiles, as would be done when performing a staff elimination search in forensic genetics laboratory. An elimination database for exclusionary purposes, which has been proposed to identify the source of contamination, was not available for our postmortem facility and this is acknowledged as a major limitation.
Nevertheless, an elimination database would not be sufficiently informative to interpret the complex DNA mixtures which originated from more than three contributors, which, as reported in our study, represented a frequent occurrence, given the cumulative effect of the DNA contamination over time.
The challenges represented by DNA decontamination, the large number of dead bodies and living persons who pass through the autopsy room, and the nature of the autopsy process, all make it unreasonable to expect that the autopsy room could ever be a completely DNA-free environment, as opposed to clinical or forensic PCR laboratories. The awareness that the autopsy room should be considered a potentially contaminated environment, similar to a crime scene or a transport vehicle, suggests the need to adopt dedicated strategies for trace DNA collection in the autopsy setting to serve as guidelines for best practice policy.
In conclusion, environmental decontamination strategies adopted during the COVID-19 pandemic only partially solved the long-standing issue of DNA contamination of postmortem examination facilities. However, the pandemic represents an opportunity to further emphasize the need for standardized evidence-based protocols, to mitigate the risk of potential DNA contamination in the autopsy room. Recommendations include collecting samples for trace DNA prior to any significant manipulation of the body and while it is still in the body bag, using disposable, sterile instruments, using gloves and changing gloves in-between one sampling and another.
Considering the high sensitivity of forensic genetic analyses and the awareness of the occurrence and consequences of DNA transfer events, the development of an effective protocol requires the involvement of a cross-disciplinary forensic science team.

ACK N OWLED G M ENT
The authors acknowledge Alessandra Iuvaro for the technical support in DNA extraction from samples and in the interpretation of electrophoretic data. Open Access Funding provided by Universita degli Studi di Bologna within the CRUI-CARE Agreement.