A Study of Policies and Guidelines for Collecting,Processing,and Storing Coronavirus Disease 2019 Patient Biospecimens for Biobanking and Research

Abstract

Biobanking has been playing a crucial role in the development of new vaccines, drugs, biotechnology, and therapeutics for the prevention and treatment of a wide range of human diseases. This puts biobanks at the forefront of responding to the ongoing worldwide outbreak of the severe pandemic, coronavirus disease 2019 (COVID-19). The leading public health institutions around the world have developed and established interim policies and guidelines for researchers and biobank staff to handle the infectious biospecimens safely and adequately from COVID-19 patients. A study of these important and complementary policies and guidelines is conducted in this study. It should be emphasized that the COVID-19 biospecimens must be collected, processed, and preserved by trained personnel equipped with right personal protective equipment to prevent the transmission of the coronavirus and ensure the specimen quality for testing and research. Six of the leading global public health organizations or institutions included in this study are the World Health Organization, the Pan American Health Organization, the U.S. Centers for Disease Control and Prevention, the Public Health England, the U.S. Food and Drug Administration, and the Office of Research at the University of California, San Francisco. In conclusion, following the recommended guidance and policies with extreme precautions is essential to ensure the quality of the collected COVID-19 biospecimens and accuracy of the conducted research or treatment, and prevent any possible transmission. Efforts from cryobiologist and biobanking engineers to optimize the protocol of COVID-19 biospecimen cryopreservation and develop the user-friendly and cost-effective devices are urgently required to meet the urgent and increased needs in the specimen biobanking and transportation.

Introduction

The coronavirus is a diverse group of RNA viruses that cause mild to severe autoimmune reactions in mammals and birds.¹ Currently, a novel strain of zoonotic coronavirus, named “severe acute respiratory syndrome coronavirus 2” (SARS-CoV-2) by the International Committee of Taxonomy of Viruses, has caused a pandemic respiratory disease coined and hereby referred to as “Coronavirus Disease 2019” (COVID-19). With its capability to spread quickly, and its high mortality risks to human beings, the COVID-19 has caused tremendous disruptions to the global health care systems and society.

As the pandemic rages on all over the world, collecting and biobanking biospecimens from patients with COVID-19 are critical for diagnostic testing as well as the research and development of new vaccines and drugs for the prevention and treatment of the disease. Although biobank staff are always required to take special precautions during the collecting, handling, and processing of infectious biospecimens to prevent transmission and secondary infections, the severity of the current pandemic has prompted the leading global public health institutions to create COVID-19-specific interim policies and guidelines for collecting, handling, preserving, and shipping the COVID-19 specimens. Here we conduct a study and review of these important and complementary guidelines and policies serving biomedical researchers and biobanking professionals. Six of the leading global public health institutions included in this study are the World Health Organization (WHO), the Pan American Health Organization, the U.S. Centers for Disease Control and Prevention (CDC), the Public Health England (PHE), the U.S. Food and Drug Administration, and the University of California, San Francisco (UCSF).

Specimen Collection

Since COVID-19 virus most often invades the human body through the respiratory system causing acute respiratory infections, most guidelines and policies recommend upper or lower respiratory specimen collection for the initial COVID-19 diagnostic testing. While upper respiratory specimens, such as nasopharyngeal and oropharyngeal swabs or wash, are recommended for ambulatory patients and asymptomatic carriers, patients under clinical circumstances (e.g., those who are hospitalized, receiving invasive mechanical ventilation) are recommended for lower respiratory collections from sputum, endotracheal aspirate, and bronchoalveolar lavage.^1–3 CDC also lists nasal mid-turbinate swab, anterior nares (nasal swab) samples, and nasal wash/aspirate samples as acceptable biospecimens.^1–3

Additional clinical COVID-19 specimens can also be collected in saliva, tissues,⁴ blood, feces, urine,^1,4,5 and cerebrospinal fluid.⁵ This can be useful for surviving patients, whose paired serum (acute and convalescent), in addition to respiratory materials, can be collected for retrospectively testing and analyses when serological assays become available in the future.¹ Finally, for deceased patients, autopsy tissue specimens, such as lung tissues, could be collected and examined.^1,3 This is especially important to study pathology and pathogenesis of the COVID-19 infection.

These collected COVID-19 specimens should be immediately placed into a sterile transport tube containing viral transport medium,^1–3,6 Amies transport medium, or sterile saline,^1,3 unless the original specimen is to be used or analyzed directly.³

The high quality of biospecimens is crucial for accurate testing and further research, meaning that samples should be collected by trained personnel. Proper infection prevention and control practice emphasize the use of recommended personal protective equipment (PPE), such as an N95 or higher-level respirator (or N95 facemask if a respirator is not available), eye protection, gloves, and a gown,^3,7 to minimize the risk of human-to-human transmission.^1–4

Specimen Storage, Package, and Shipment

Following their collection, all biospecimens should be labeled accordingly and then shipped to the laboratory within 72 hours, while being maintained at 2°C–8°C. If a delay in testing or shipping is expected, it is recommended to freeze specimens to at least −20°C,¹ ideally −70°C or below,^1–3 for storage before shipping them out using dry ice (−78.5°C). WHO recommends against repeated freezing and thawing of specimens, which may cause potential cross-contamination issues^1,8 or the potential denaturation of the virus.

Because COVID-19 specimens are high risk and time sensitive for use, it is strongly advised to alert the laboratory or receivers before sending the specimens, accompanied by a request form to facilitate timely and proper handling and processing of the specimens.^1,5,7

WHO also mandates that the national package and transport of specimens should comply with applicable national regulations, while international ones should comply with UN Model Regulations.¹ In general, all COVID-19 testing samples should be packaged with UN3373 packaging,^3,5–7 transported in accordance with Department of Transportation's Category B transportation regulations,^2,3,5–7 and labeled “Priority 10.”⁶ The cultured samples must be transported in accordance with the Department of Transportation's Category A transportation regulations⁵ as shown in Table 1. Finally, shipment by air must follow compliance with all international standards established by International Air Transport Association.^2,3

Table 1.

World Health Organization Regulations for the Transport of Infectious Substances 2019 to 2020, Used by the Public Health England

Sample type	Dangerous goods classification	Additional external labeling required for samples sent to PHE	Recommended method of transportation
Suspect patient sample	Category B	White label with Priority 10 printed in red	Same-day delivery courier
Presumptive positive patient sample	Category B	White label with Priority 20 printed in red	Same-day delivery courier
Confirmed positive samples for follow-up	Category B	White label with Priority 20 printed in red	Same-day delivery courier
Contacts of known positive patients	Category B	White label with Priority 10 printed in red	Same-day delivery courier
Cultured samples for research or calibration	Category A	None	Mandatory ADR-approved Category A courier (ADR stands for “Agreement concerning the International Carriage of Dangerous Goods by Road”)

World Health Organization.¹

ADR, Agreement concerning the International Carriage of Dangerous Goods by Road; PHE, Public Health England.

Laboratory Facilities and Safety

COVID-19 biospecimen handling for molecular testing requires biosafety level (BSL)-2 or higher laboratories, while attempts to culture the virus require a minimum of BSL-3 facilities.^1,4,7 In England, PHE requires that samples of known or suspected positive sources must be processed in at least containment level (CL) 2, and any propagation, culturing, or deliberate work be conducted in full CL3.⁵ In addition, all laboratories are independently responsible for performing site-specific and activity-specific risk assessments to identify and decrease risks.^5,7

The biospecimens collected and stored for laboratory examinations or further research should be assumed as potentially infectious.¹ Therefore, all specimen containers that may contain COVID-19 virus should be opened and handled within a certified Class II Biological Safety Cabinet⁷ (i.e., tissue culture biosafety cabinet with high-efficiency particulate air [HEPA] filters)⁴ or a microbiological safety cabinet.⁵ As for tissue culture growth of any biospecimen from patients under investigation, UCSF recommends using dedicated, HEPA-filtered incubators under BSL-3.⁴

Regular training of even experienced personnel plays a significant role in maintaining laboratory safety. Good laboratory practice includes the use of standard precautions, additional droplet and contact precautions,⁹ and well-documented standard operating procedures. Standard precautions emphasize hand and respiratory hygiene and the use of PPE,^1,5,7,9 such as laboratory coats or gowns, disposable gloves, and eye protection.⁷ CDC and WHO suggest additional airborne-related precautions for aerosol-generating procedures, such as wearing additional PPE, for example, a surgical mask, face shield, or other physical barriers like a splash shield, and using centrifuge safety cups and sealed centrifuge rotors.⁷

Because of the coronavirus' unknown survival time length on surfaces, it is required to decontaminate work surfaces and equipment with appropriate disinfectants following the local policies and manufacturer's instructions.^5,7 For example, UCSF recommends using 10% bleach solutions (0.5% sodium hypochlorite) to disinfect both inside and outside of a biosafety cabinet.⁴

All laboratory waste from suspected or confirmed COVID-19 patients must be handled and disposed using the same standard protocol used for all biohazardous wastes,^4,7 without additional packaging or disinfection procedures.⁷ The protocol states that all solid waste should be packed into autoclavable waste bags with a biohazard symbol. Both the solid waste bags and liquid biohazardous waste should be put into a rigid, leak-proof container with a biohazard symbol. Moreover, the sharp waste should be collected into red plastic sharp containers with a biohazard symbol and tight-fitting lid.¹⁰

The key points of the guidelines/policies regarding the COVID-19 specimen handling and processing, which are established by the six leading global public health institutions, are highlighted, respectively, in Table 2.

Table 2.

The Highlighted Points of the Guidelines and Policies Regarding the Coronavirus Disease 2019 Specimen Collecting, Handling, Processing, Storing, and Shipping

Source	Highlights of the policies and guidelines on the collecting, processing, and storing of biospecimens from COVID-19 patients
General requirements and information	• All biospecimen types procured from COVID-19 patients/PUIs are to be managed in only BSL-2 or higher laboratories. This includes sampling of nasopharyngeal swabs, saliva, urine, blood, feces, and tissues. Early studies out of China did indicate that COVID-19's viral RNA has been found in saliva, feces, and blood.^13,14 • All laboratories should perform a site-specific and activity-specific risk assessment to identify and mitigate risks. Risk assessments and mitigation measures depend on the following: ○ The procedures performed. ○ Identification of the hazards involved in the process and/or procedure. ○ The competency level of the personnel who perform the procedures. ○ The laboratory equipment and facility. ○ The resources available.
UCSF⁴	• When collecting biospecimens from COVID-19 patients/PUIs, all personnel should follow current infection control precautions related to PPE to minimize risk of transmission to self and others. • All specimen containers (vacutainers, cups, test tubes, conical tubes, vials, etc.) that may contain SARS-CoV-2 should be opened and processed within a certified Class II BSC (i.e., tissue culture biosafety cabinet with HEPA filter). • Following collections, biospecimens from COVID-19 patients/PUIs should be taken directly to a BSL-2 laboratory and not stored and/or opened at workstations (such as cubicles and desks). If the research project does not have access to a BSL-2 space, it is suggested to consider utilizing services provided by BIOS', Acquisition and Processing Core, CTSI's, CRS, or the ASB. • All faculty and laboratory managers must ensure their personnel are using aseptic techniques when processing specimens. This includes disinfection of all surfaces and equipment using 10% bleach solutions (0.5% sodium hypochlorite), both inside and outside of the biosafety cabinet. • All waste must be disposed as biohazardous waste. • Use of dedicated, HEPA-filtered incubators for tissue culture growth of any biospecimen from PUIs is recommended.
PHE^5,6	• Laboratory staff must wear PPE when conducting work in the laboratory. PPE must be removed on leaving the laboratory and hygiene practices, including hand washing, must be rigorously maintained. PPE must include disposable gloves and a laboratory coat or gown as a minimum, and may also include eye protection and other equipment, as identified by risk assessment. The following work may also be conducted at CL2 following standard laboratory precautions, which is consistent with the terms of the local risk assessment for those activities: • Diagnostic assays using whole blood, serum, and plasma, including routine biochemistry and hematology, unless there is a risk of generating aerosols. • Assays using virus-inactivated specimens, including molecular testing of inactivated specimens. • Examination of bacterial or fungal cultures. • Staining and microscopy of heat-fixed or chemically fixed smears. • Centrifugation of routine blood samples. However, if there is infectious potential, samples must be centrifuged using sealed centrifuge rotors or sample cups, which are loaded and unloaded in an MSC. The following work must be conducted at CL3: • Any propagation, culturing, or deliberate work on SARS-CoV-2 for diagnostic or research purposes.
WHO¹	WHO mandates that the national package and transport of specimens should comply with applicable national regulations, while international ones should comply with UN Model Regulations.¹ In general, all COVID-19 testing samples should be packaged with UN3373 packaging,^3,5–7 transported in accordance with Department of Transportation's Category B transportation regulations,^2,3,5–7 and labeled “Priority 10.”⁶
CDC^3,7	• Specimens should be labeled accordingly, and the laboratory should be alerted to ensure proper specimen handling. • For providers who are handling specimens, but are not directly involved in collection (e.g., self-collection) and not working within 6 feet of the patient, follow standard isolation precautions.¹¹ Health care personnel are always recommended to wear facemask or cloth face covering while in the health care facility. • Storage: Store specimens at 2°C–8°C for up to 72 hours after collection. If a delay in testing or shipping is expected, store specimens at −70°C or below. • Virus isolation in cell culture and initial characterization of viral agents recovered in cultures of SARS-CoV-2 specimens should only be conducted in a BSL-3 laboratory using BSL-3 practices. Site- and activity-specific biosafety risk assessments should be performed to determine if additional biosafety precautions are warranted based on situational needs.
FDA¹²	FDA is issuing the guidance to provide a policy to help accelerate the availability of COVID-19 tests developed by laboratories and commercial manufacturers for the duration of the public health emergency. Rapid detection of COVID-19 cases in the United States requires wide availability of testing to control the emergence of this rapidly spreading, severe illness. This guidance describes a policy for laboratories and commercial manufacturers to help accelerate the use of tests they develop to achieve more rapid and widespread testing capacity in the United States.
PAHO²	• Samples should be kept refrigerated (4°C–8°C) and sent to the laboratory where they will be processed within 24–72 hours of the collection. If samples cannot be sent within this period, freezing at −70°C or below is recommended until samples are shipped (ensuring the cold chain is maintained). • Cultured samples for research or calibration must be transported in accordance with Category A transportation regulations.

ASB, AIDS Specimen Bank; BIOS, Biospecimen Resource Program; BSC, Biological Safety Cabinet; BSL, biosafety level; CDC, Centers for Disease Control and Prevention; CL, containment level; CRS, Clinical Research Services; COVID-19, coronavirus disease 2019; CTSI's, Clinical and Translational Science Institute; FDA, Food and Drug Administration; HEPA, high-efficiency particulate air; MSC, microbiological safety cabinet; PAHO, Pan American Health Organization; PPE, personal protective equipment; PUIs, patients under investigation; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; UCSF, University of California, San Francisco; WHO, World Health Organization.

An Urgent Need to Develop Optimal Cryopreservation Protocols and Technology for Biobanking of COVID-19 Specimens

Aerosolized coronavirus can remain in the air for only a few hours.¹⁵ The cell-associated coronavirus (i.e., the virus in the host cells) will die in hours or days after the host cells or tissues are isolated as biospecimens from human body. There is an urgent and increased need for cryopreservation and biobanking of the coronavirus for medical research and development of novel vaccines and drugs to prevent and treat COVID-19. Unfortunately, currently, there are no policies, guidelines, or references providing optimal cryopreservation protocols to ensure the long-term viability and functionality of the frozen coronavirus (in either solutions or inside host cells and tissues), although some investigations were previously conducted for the preservation of other types of viruses from human, animals, and plants by cryopreservation or freeze-drying.^16–19 A few empirical and simple methods were developed and used in the past for the virus cryopreservation, including the snap freezing in a −80°C freezer or in liquid nitrogen (−196°C) with or without using cryoprotective agents. Some general recommendations for the virus preservation are as follows^16–19: (1) The storage temperature should be below −70 °C. The lower the storage temperature, the longer time the virus infectivity can be retained. (2) The use of proteins (e.g., using serum) and cryoprotectants (such as DMSO, glycerol, and sucrose) can be helpful for virus cryopreservation to increase virus cryo-survival rate. (3) If samples are stored in the liquid nitrogen tank, storage in the vapor phase of nitrogen is recommended to prevent or reduce cross contamination of specimens inside the tank. (4) Extreme precautions should be taken in each of cryopreservation steps to avoid the exposure of operators to the viruses and the risk of the cross contamination.

The coronavirus (SARS-CoV-2 or so-called COVID-19 virus), as an enveloped RNA virus, is more heat labile and therefore needs more cautions in the preservation. However, optimal cryopreservation protocols for the coronavirus and COVID-19 specimens have not been developed yet. This is largely because of a lack of research on the cryobiology of coronavirus (SARS-CoV-2). To solve this important problem, a collaboration of cryobiologists and biobanking engineers is required to develop and optimize the following key conditions for cryopreservation of both coronavirus and its associated host cells/tissues based on the cryobiology principles^20–23: (1) the formulation of cryoprotective media with cryoprotectants, (2) the controlled cooling and rewarming conditions to prevent lethal intracellular ice crystallization during cooling and the recrystallization during rewarming,²¹ (3) the storage temperatures below −70°C, and (4) the methods for addition and removal of cryoprotectants in cells/tissues to prevent the osmotic injury.^24–27 In addition, a series of user-friendly and cost-effective devices should be further developed to realize the developed optimal cryopreservation conditions. Besides, the optimal lyophilization methods^28,29 should also be explored and developed, especially for the biobanking at the regions where the liquid nitrogen is not easily available.

It should also be noted that since the COVID-19 viruses can survive being frozen, this means, in theory it is possible that virus infection could spread through the “cold chain” for the transportation and storage of frozen biomaterials or foods, which are contaminated with the virus. Indeed, it has been reported that the “cold-chain” transmission has currently become one possible virus transmission route through frozen food like fishes.³⁰ It has been discovered that some viruses are relatively robust to both low temperature and to the cooling/rewarming process. Repeated cycles of freeze-thawing have been used to lyse the virus-host cells to release the viruses into a culture medium for cryopreservation,¹⁶ which is a proof of the robustness of viruses to the freezing/thawing process and low temperature itself. Therefore, extreme precautions are recommended to the “cold-chain” workers and the general public when they handle, purchase, and use frozen food during the COVID-19 pandemic. The new methods, regulations, and policies on the “cold-chain” quality control to prevent the virus transmission through the frozen foods should be established as soon as possible.

Conclusion

Biobanking of COVID-19 biospecimens is indispensable for investigating and developing vaccines, drugs, and therapeutics in preventing and protecting the public from the worsening COVID-19 pandemic. To maintain laboratory safety and ensure a high quality of the specimens for diagnostic testing and biomedical research, biobank staff must follow the up-to-date guidelines and policies for handling and processing all COVID-19 specimens. It is urgently needed to develop and standardize optimal techniques and devices for long-term cryopreservation and biobanking of the COVIC-19 specimens.

Footnotes

Author Disclosure Statement

No conflicting financial interests exist.

Funding Information

No funding support to declare for this work.

References

Laboratory testing for 2019 novel coronavirus (2019-nCoV) in suspected human cases. World Health Organization. Available at https://www.who.int/publications/i/item/10665-331501 (accessed July 20, 2020).

2020 3 F. Laboratory Guidelines for Detection and Diagnosis of the Novel Coronavirus (2019-nCoV) Infection. PAHO/WHO | Pan American Health Organization. Available at https://www.paho.org/en/node/68888 (accessed July 20, 2020).

Interim Guidelines for Clinical Specimens for COVID-19. Centers for Disease Control and Prevention. 2020. Available at https://www.cdc.gov/coronavirus/2019-ncov/lab/guidelines-clinical-specimens.html (accessed July 20, 2020).

COVID-19 Biospecimen Guidelines. Office of Research. Available at https://research.ucsf.edu/covid-19-biospecimen-guidelines (accessed July 20, 2020).

COVID-19: Safe handling and processing for samples in laboratories. GOV.UK. Available at https://www.gov.uk/government/publications/wuhan-novel-coronavirus-guidance-for-clinical-diagnostic-laboratories/wuhan-novel-coronavirus-handling-and-processing-of-laboratory-specimens (accessed July 20, 2020).

COVID-19: Laboratory investigations and sample requirements for diagnosis. GOV.UK. Available at https://www.gov.uk/government/publications/wuhan-novel-coronavirus-guidance-for-clinical-diagnostic-laboratories/laboratory-investigations-and-sample-requirements-for-diagnosing-and-monitoring-wn-cov-infection (accessed July 20, 2020).

Interim Guidelines for Biosafety and CO

VID-19. Centers for Disease Control and Prevention. 2020. Available at https://www.cdc.gov/coronavirus/2019-ncov/lab/lab-biosafety-guidelines.html (accessed July 20, 2020).

Tedder

, Zuckerman

, Brink

, et al. Hepatitis B transmission from contaminated cryopreservation tank. Lancet, 1995; 346:137–140.

Disease commodity package—Novel Coronavirus (COVID-19). World Health Organization. Available at https://www.who.int/publications/i/item/disease-commodity-package—novel-coronavirus-(ncov) (accessed July 20, 2020).

10.

Biohazardous Waste. EHS. Available at https://www.ehs.washington.edu/biological/biohazardous-waste (accessed July 20, 2020).

11.

Isolation Precautions. Centers for Disease Control and Prevention. 2019. Available at https://www.cdc.gov/infectioncontrol/guidelines/isolation/index.html (accessed July 20, 2020).

12.

Center for Devices and Radiological Health. Policy for COVID-19 Tests During the Public Health Emergency (Revised). U.S. Food and Drug Administration. Available at https://www.fda.gov/regulatory-information/search-fda-guidance-documents/policy-coronavirus-disease-2019-tests-during-public-health-emergency-revised (accessed July 20, 2020).

13.

Wang

, Xu

, Gao

, et al. Detection of SARS-CoV-2 in different types of clinical specimens. JAMA, 2020; 323:1843–1844.

14.

Zhang

, Du

R-H

, Li

, et al. Molecular and serological investigation of 2019-nCoV infected patients: Implication of multiple shedding routes. Emerg Microbes Infect, 2020; 9:386–389.

15.

Doremalen

, Morris

, Wit

, et al. Aerosol and surface stability of SARS-CoV-2 as compared with SARS-CoV-1. N Engl J Med, 2020; 382:1564–1567.

16.

Gould

. Methods for long-term virus preservation. Mol Biotechnol, 1999; 13:57–66.

17.

Malenovska

. The influence of stabilizers and rates of freezing on preserving of structurally different animal viruses during lyophilization and subsequent storage. J Appl Microb, 2014; 117:1810—1819.

18.

Rightsel

, Greiff

. Freezing and freeze-drying of viruses. Cryobiology, 1967; 3:423–431.

19.

Costa

, Teixeira

, Aguiar

TDDF

, et al. Rabies virus viability after short-term cryopreservation using cryoprotectant agents. Rev Inst Adolfo Lutz, 2011; 70:106–112.

20.

Mazur

. Cryobiology: The freezing of biological systems. Science, 1970; 168:939–949.

21.

Gao

, Critser

. Mechanisms of cryoinjury in living cells. ILAR J, 2000; 41:187–196.

22.

Chaves

, Silva

, Freitas

, Neilson

, Catharina

, Gadelha

. Rabies virus viability after short-term cryopreservation using cryoprotectant agents. Rev Inst Adolfo Lutz (Impr.), 2011; 70:106–112.

23.

Pan

, Ren

, Sekar

, et al. Investigation of electromagnetic resonance rewarming enhanced by magnetic nanoparticles for cryopreservation. Langmuir, 2018; 35:7560–7570.

24.

Mazur

. The role of intracellular freezing in the death of cells cooled at supraoptimal rates. Cryobiology, 1977; 14:251–272.

25.

Fahy

. The relevance of cryoprotectant “toxicity” to cryobiology. Cryobiology, 1986; 23:1–13.

26.

Gao

, Liu

, et al. Prevention of osmotic injury to human spermatozoa during addition and removal of glycerol. Hum Reprod, 1995; 10:1109–1122.

27.

Meryman

. Cryoprotective agents. Cryobiology, 1971; 8:173–183.

28.

Rowe

. Optimization in freeze-drying. Dev Biol Stand, 1976; 36:79–97.

29.

Heckly

. Principles of preserving bacteria by freeze-drying. Dev Ind Microbiol, 1985; 26:379–395.

30.

Mystery grows over coronavirus spread via contaminated food packaging; The Japan Times. Available at https://www.japantimes.co.jp/news/2020/08/19/world/coronavirus-contaminated-food-packaging (accessed September 9, 2020).