Introduction

The risk for transmission of SARS-CoV-2 through transplantation of allogeneic human tissues (tissues) is unknown. The identification of the angiotensin 2 converting enzyme (ACE-2) receptor as the primary receptor for virus to enter cells provides an imperfect guide to potential target tissues. There have been many reports describing the detection of SARS-CoV-2 RNA in tissues beyond the respiratory tract (Bradley 2020; Best 2021; Gaussen 2021; Penfield 2020; Trypsteen 2020). This includes reports that viral RNA (vRNA) is detectable in the blood of some patients, suggesting systemic viral dissemination (Yang 2020; Beyerstedt 2021) and broad organ involvement. While, historically, respiratory viruses were not thought to pose a significant risk for transmission via allograft tissue implantation, SARS-CoV-2 may behave differently due to this systemic dissemination. Considering the severity of illness, rapid community spread, and uncertainty surrounding tropism in human tissue, the American Association of Tissue Banks (AATB) issued guidance for screening and exclusion of donors who may be infected with SARS-CoV-2 (American Association of Tissue Banks 2020).

In the United States, human tissues for transplantation are regulated by the U.S. Food and Drug Administration (FDA) as human cells, tissues, and cellular and tissue-based products (HCT/Ps) under 21 CFR Part 1271. This study evaluated multiple different human tissues intended for transplantation, including bone, tendon, skin, fascia lata, vascular tissues, and heart valves. FDA requires all HCT/P donors to be screened and tested for relevant communicable disease agents or diseases for use in making a donor eligibility determination to exclude donors with the potential to transmit communicable diseases. Typically, when potential tissue donors are identified, recovery establishments perform a preliminary review for donor suitability, i.e., obtain donor information to identify any data that would obviously render the donor ineligible for donation according to standards established by FDA, AATB, and each tissue establishment. If a potential donor appears to be suitable for donation, tissues are then recovered, sent to the tissue processing facility where they are held or processed while completing the collection and review of donor medical and social history data.

Determining whether viral RNA (vRNA), and infectious virus, is regularly present in tissues typically used for transplantation collected from donors with a history of prior SARS-CoV-2 infection would better inform decisions regarding whether to exclude potential donors. AATB sponsored two studies to help evaluate the potential role of SARS-CoV-2 transmission in tissue transplantation. The first (Greenwald 2022) was to examine the risk of SARS-CoV-2 viremia in blood of deceased tissue donors, showing an incidence of RNAemia of approximately 1 in 1000. However, in that study, the results of SARS-CoV-2 RNA testing of donor nasopharyngeal (NP) swabs, if performed, was unknown, and tissues from these donors were not tested. To further characterize the risk of viral transmission and inform tissue safety policy, this retrospective study was performed to examine various tissues obtained from deceased donors whose NP swabs collected within 24 h of death tested positive for SARS-CoV-2 RNA for evidence of the SARS-CoV-2 via reverse transcriptase polymerase chain reaction testing (RT-PCR).

Materials/methods

Between April 2020 and April 2021, tissue establishments identified stored, frozen human tissues collected from research-authorized deceased donors who had NP samples positive for SARS-CoV-2 RNA using nucleic acid amplification testing (NAT) for the presence of viral RNA (Table 1) and meeting all other study criteria (Table 2) for study inclusion. Research authorization was provided by the individual authorizing donation after death, or at the time of donation registration (first-person authorization), and all donor samples were anonymized by the establishment providing the research tissue and its associated recovery data. At the time of tissue collection, the donors were not suspected of being at risk of SARS-CoV-2 infection and there was no information available at the time tissues were recovered to indicate the donor would be ineligible for donation (Table 3). Tissue donor NP swabs were collected at the time of tissue recovery. Tissues were handled, stored, and processed according to the information provided in Table 4. None of the tissues underwent processing that included viral inactivation steps, and none of the antibiotics or antifungals used are known to have virucidal properties.

Table 1 Donor testing for SARS-CoV-2
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Table 2 Study criteria
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Table 3 Donor information
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Table 4 Tissue handling and processing
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Because there were finite lab resources available, amongst the available stored donor tissue identified, a convenience sample representing a variety of the tissue types available for transplantation was selected for testing. Human tissue and blood specimens were shipped frozen on dry ice to the Miller Laboratory at University of California Davis (ML/UCD) where tissues were kept in dry ice storage from receipt until they were prepared and tested between January 2021 and February 2022. All tissues and blood had been frozen once, and not thawed, at the time that they were shipped.

Tissue preparation for sampling

The ML/UCD received 47 tissues from 17 donors, and 6 donor blood tubes (serum or plasma) from 4 of the 17 donors. Tissues were kept in dry ice storage until time of preparation for testing. Fascia lata, dermis, tendon, femur, and tibia were thawed overnight at 4 degrees Celsius. Cryopreserved cardiovascular tissues were thawed and rinsed per clinical instructions for use (cryopreservative solutions were properly removed), while the remaining cardiovascular tissues that were not control-rate frozen and without cryopreservation solution were thawed in the same manner as other tissues.

Sample collection

Tissues were thawed to collect samples for testing. Three distinct sites/subsets (designated A, B, and C) were selected as samples for testing from all tissue sets (when possible). For bone, the subset sites included endosteum (A), periosteum (B) and cancellous bone (C), and for the other tissue types, the three subset sites were taken different areas of the selected tissue. For each distinct site, samples were collected in duplicate to allow ribonucleic acid (RNA) extraction using two different methodologies (i.e., Trizol or RNAeasy), and all RNA extracted from the samples was then suspended in RNAlater (Thermo Fisher Scientific/ Invitrogen) and snap-frozen on dry ice.

RNA/cDNA processing

Tissue samples were thawed and immediately transferred from RNAlater preservation solution to a sterile bead beater tube containing 1 ml Trizol (Thermo Fisher Scientific/ Invitrogen) and a 7 mm stainless steel bead. Tissues were homogenized for 3–5 min. Homogenate was transferred to a new tube. RNA was extracted using either Trizol or the RNAeasy Mini Kit (Qiagen). Frozen plasma and serum samples were thawed, and RNA was extracted using the Qiagen Ultrasens Virus Kit. One mg of complementary deoxyribonucleic acid (cDNA) was synthesized from the RNA extracted from each sample using Superscript IV (Thermo Fisher Scientific/ Invitrogen).

Real-time PCR

Plasma and serum cDNA samples were tested immediately after cDNA synthesis using the Qiagen Ultrasens Virus Kit using primers and probes from Integrated DNA Technologies as described in Carroll (2022), Shaan Lakshmanappa (2021), and Deere (2021).

Tissue cDNA samples, which underwent two freeze–thaw cycles (one of which was part of the RNA assessment protocol), were run on the Quant Studio 6 Flex real-time PCR system (Qiagen) as described in Carroll (2022), Shaan Lakshmanappa (2021), and Deere (2021). All samples were tested in quadruplicate for both subgenomic RNA (sgRNA) and genomic nucleocapsid RNA (gRNA) of SARS-CoV-2, and in duplicate for the housekeeping gene Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which served as the positive control. Positive controls were included for all three targets on every plate. Human control RNA was used for GAPDH positive control. The human control RNA was also screened for sgRNA and gRNA (negative for both).

Results

Results are reported across the 17 NP swab SARS-CoV-2-positive donors with donor identification designations as D1 through D17. Individual tissue sample identifiers are represented as the secondary number to the donor identification (i.e., D1.01, D1.02., etc.). Sample testing replicates for each tissue sample are identified with an alpha character (i.e., D1.01.A, D1.01.B, etc.).

Table 3 outlines the donor demographic and clinical data that were provided for the study. Twelve (12) male and five (5) female donors were evaluated; donors ranged in age from 5 to 69 years old with a median age of 47. Donor tissues were recovered between 4/17/2020 and 4/29/2021 with a median recovery date of 5/27/2020. Donations were recovered within the continental United States.

A range of circumstances and causes surrounding donor death were observed, including, but not limited to, sudden death, cardiac events, asphyxiation, and drug overdose. Cardiac histology data were limited; however, for the donors where histology data were provided, there were no indications of abnormal tissue pathology with the one exception being a donor showing contraction band necrosis/myocytolysis consistent with ischemic injury.

The results of all testing are provided in detail in Tables 5, 6, 7, 8, 9. No viral RNA was detected in any tissue or blood specimens provided.

Table 5 Blood testing results
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Table 6 Cardiac tissue results
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Table 7 Vascular tissue results
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Table 8 Musculoskeletal tissue results
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Table 9 Dermis tissue results
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Testing results for cadaveric (post cessation of heartbeat) blood available for D11 (plasma only) and D15–D17 (plasma and serum) are outlined in Table 5 and indicate negative real-time PCR cycle threshold (Ct) results for nucleocapsid sgRNA and gRNA. The remaining donors did not have cadaveric blood samples available for testing in this study.

Cardiac and vascular tissue type RNA target results are included in Tables 6 and 7. Cardiac tissues, including pulmonic and aortic valves, ascending aorta, and pulmonary arteries were tested from ten (10) donors. Vascular tissues, femoral veins and arteries, saphenous veins, and aortoiliac arteries, were tested from six (6) donors. Cardiac tissue was negative for sgRNA and gRNA using real-time PCR, while the mean GAPDH Ct values ranged from 17.41 to 34.954. Vascular tissue sgRNA and gRNA targets were negative using real-time PCR with mean Ct-GAPDH values ranging from 17.4 to 27.736.

Table 8 outlines musculoskeletal (MS) tissue RNA target results. Seven (7) donors tested both bone and soft tissues, femur, tibia, humerus, gracilis tendon, semitendinosus tendon, and fascia lata. Viral RNA targets (sgRNA and gRNA) were negative for all MS tissue types. Mean Ct-GAPDH values ranged from 21.908 to 30.722.

Table 9 outlines dermis tissue RNA target results from one (1) donor where this tissue type was procured. Dermis tissue sgRNA and gRNA targets were negative using real-time PCR. Mean Ct-GADPH values ranged between 24.045 and 24.972.

Discussion

This study tested human tissues, intended for transplantation, for evidence of SARS-CoV-2. There was a focus on oversampling for tissue types that tend to be minimally processed and tropic for the virus, as those have been the tissue types most likely to transmit infection in past outbreaks involving a variety of pathogens (Tugwell 2005; CDC 2011; Schwartz 2022; Lu 2018; Greenwald 2012). We did not detect SARS-CoV-2 sgRNA or gRNA in any of the samples tested. However, we did detect GAPDH mRNA in all samples, except for one replicate of each of two samples of cardiovascular cissue, indicating that the RNA extraction and PCR methods used were valid. Further, the level of GAPDH in a sample was consistent with the cellularity of that sample. Thus, the samples that contained very few cells, tendon, heart valves, had lower levels of GAPDH (higher CT values) than more cellular samples from the dermis and vasculature. We quantified sgRNA because SARS-CoV-2 sgRNA is a good surrogate marker of infectivity (Santos Bravo 2022).

Many early exploratory studies detected SARS-CoV-2 by various testing methods in multiple organs and biospecimens, only few of which were designed to determine whether live virus was present (Bradley 2020; Best 2021; Gaussen 2021; Penfield 2020; Trypsteen 2020). While the ACE-2 receptor is required for cellular infection, the extent to which cells with ACE-2 receptors become infected with SARS-CoV-2 remains unclear. Furthermore, it is not clear whether end-organ damage observed is a result of direct viral activity or if it was immune-mediated, while emerging evidence indicates much of the observed damage outside of the respiratory system is likely immune-mediated (Merad 2022).

In October 2020, Trypsteen and colleagues reviewed available data regarding SARS-CoV-2 tropism. At that time, only respiratory and GI tract samples (mostly stool) demonstrated evidence that viral particles from a biopsy were capable of reinfecting target cells in-vitro. For cardiac tissue, there was discussion of the high level of acute cardiac injury that occurred in individuals hospitalized in the ICU with COVID-19, and the need to further investigate whether this was due to direct viral effects versus immune-mediated damage. Furthermore, some studies detected viral particles in or around cardiac tissue, but not within the myocytes. They concluded that the evidence at that time suggested immune mediated injury is more likely the culprit for the observed cardiac damage, but additional studies would be required.

In July 2021, Gaussen and colleagues reviewed literature for evidence of SARS-CoV-2 transmissibility via cell, tissue, and organ transplantation. Most studies included in this review article looked for evidence of the virus (e.g., NAT testing, direct visualization) while only few performed viral infectivity assays. As presented in the Gaussen review article, the evidence was strong for SARS-CoV-2 presence in lungs and the respiratory tract, both by NAT testing and some viral infectivity assays. Kidneys had some intermittent positive NAT results amongst tissues obtained from individuals who died of COVID-19, while only one study had found evidence of a small subset of kidneys having evidence of viral infectivity (Braun 2020). Ocular tissue testing largely did not have positive NAT in SARS-CoV-2-infected individuals, but one cited study showed NAT positivity and positive viral infectivity in an individual with COVID and bilateral conjunctivitis at the time of death (Colavita 2020). Studies including heart tissue demonstrated mostly histopathological evidence of damage with only rare instances of myocarditis. There was one cited study where testing of heart tissue from 39 individuals deceased from COVID demonstrated PCR positive results in 24/39 individuals and among those, 5 individuals with the highest viral load also demonstrated viral replication determined by cDNA synthesis (Lindner 2020).

Our study did not include donations of birth tissue (e.g., placenta, amniotic membrane) or reproductive tissue (i.e., semen or oocytes). Best and colleagues evaluated the semen of men who were diagnosed with acute SARS-CoV- 2 by RT-PCR testing of NP swab specimens. Among the 30 semen samples provided by subjects during the 11–64 days after testing positive for SARS-CoV-2 infection, 16 semen samples were tested for SARS-CoV-2 by RT-PCR (in addition to the semen analysis performed on all samples) and found to be negative. Penfeld et al. (2020) reported a study of 32 pregnant patients who delivered at the time of being diagnosed with COVID-19 infection. They obtained placental swabs of 11 of those patients, and found 3 of those 11 placental swabs to be positive for SARS-CoV-2 by RT-PCR—the positive placental specimens were found in women with severe COVID disease at the time of delivery. Follow-up studies to verify whether positive results on placenta represent viable virus should be performed.

To date, there has been no known transmission via tissue or ocular transplantation (FDA 2021) or blood transfusion (FDA 2022), and the only verified organ transmission was via lung transplantation (Kaul et al. 2021). In a study testing blood specimens from deceased tissue donors without COVID-19 symptoms, the rate of SARS-CoV-2 NAT-positive results (about 1 in 1000) was found, while infectivity data are unavailable (Greenwald 2022). Considering that, through Spring 2022, most tissue establishments have excluded donors with positive NP swab results for SARS-CoV-2, the true risk of transmission is difficult to measure. Although no transmission events have been reported through human tissue, since there are recognized challenges of identifying donor-derived transmission amongst allograft recipients (Greenwald 2012), the actual risk of transmission via tissues cannot be reliably confirmed by absence of transmission alone.

In our study, amongst the 17 donors who died of causes not known to be related to COVID but were found to have positive NP swabs, 45 tissue samples and 6 blood samples were tested by two different methodologies and no SARS-CoV-2 RNA was detected. Follow-up viral culture to determine infectivity was therefore not possible. It is notable that in the few studies where replication-competent SARS-CoV-2 was detected in non-respiratory organs and tissues, the virus was found only intermittently and in tissues or organs from individuals who were known to have died of COVID-19 infection (Gaussen 2021). Available evidence therefore indicates that asymptomatic tissue donors, regardless of their SARS-CoV-2 NP swab test results, are unlikely to have SARS-CoV-2 present in their tissues, much less a replication-competent virus. While it is notable that among the 17 donors in our study, 10 died suddenly of unknown causes (often attributed to “cardiac death” or “sudden cardiac death”), and another donor died of ST-elevation myocardial infarction (STEMI), there is not enough information available to draw any conclusions about the potential role of undiagnosed COVID-19, including hypercoagulability, in the donor deaths.

Our study has limitations

First, our study was not a random sample of donors who tested positive by NP swabs for SARS-CoV-2, but rather a convenience sample of donors provided by participating US tissue processors in which tissues were recovered, available, and met acceptability criteria for recovery. As such, included donors were most likely not severely ill with COVID-19, and perhaps therefore less likely to have disseminated infection with the virus to involve recovered tissues. No viral RNA was detected in blood from donors tested in this study, but this subsample and the overall study sample size were small. Second, the donor nasopharyngeal swab testing was performed using assays under Emergency Use Authorization since there were no available FDA-cleared or approved test kits, see Table 10 for information on assay performance characteristics. Third, tissue storage (Auer et al. 2014; Bao 2013), freeze thaw cycles (Dzung 2021; Botling 2009), and any minimal processing performed (e.g., rinsing) could have impacted results (Schilling-Loeffler 2022; Nogueira 2022). The bone and tendon were not rinsed prior to testing and thus represent a worst case for viral load. The dermis, heart, and vascular tissue were rinsed either in transport solution (all) or during processing (heart and veins). As the virus is an intracellular pathogen, it will not be passively rinsed off the tissue; however, cells such as endothelium that may contain virus could be dislodged. These rinses are representative of processing for human tissues and thus representative of the potential viral load after rinsing. Finally, the effect of different SARS-CoV-2 variants circulating during the study, with possible differences in virulence and tropism, are unknown since genotyping was not available.

Table 10 Performance characteristics of EUA assays used in testing donor NP specimens
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In conclusion, this study did not detect SARS-CoV-2 in MS, vascular, and skin types of human tissue collected from donors with positive NP swabs. There were no data collected on birth or reproductive tissue. The preponderance of evidence, from this research data and others, is reassuring that the likelihood of transmission of SARS-CoV-2 through the human tissue studied is likely low.

Data availability statement

All data generated or analyzed during this study are included in this published article.