Storing high-quality samples

Most of the current knowledge on diseases and the consequent diagnostic and therapeutic possibilities is based on the study of biological samples and the data associated with them. Following the exceptional health emergency caused by the outbreak of the COVID-19 epidemic, unprecedented action is underway to collect biological samples and related personal and health data from the population. These samples and data, collected for diagnostic procedures and the consequent care of the affected people are also pivotal for effective research to defeat COVID-19.

In this context, structuring a coordinated and extensive collection, handling and storage of samples (nasopharyngeal swabs, sputum, blood and its derivatives, broncho-alveolar lavage, post-mortem tissues, etc.) from all the infected and recovered populations takes priority. It is crucial to collect and store the biological materials of the population concerned according to the established international criteria, and in compliance both with ELSI (ethical-legal social) requirements and the rights of the patients involved, in order to make them available over time for collaborative research projects.

From this perspective, it is recommended to store COVID-19 samples according to prevailing country requirements, and quality standards in a stable and continuous manner for future research and/or diagnosis. Inadequate or inappropriate patient specimen collection, storage, and transport are likely to impact specimen quality and yield false test results. Therefore, training in specimen handling is highly recommended, and there is a critical need for tight environmental uniformity to ensure specimen viability. This article gives a brief overview of collecting, transporting, and storing COVID-19 specimens.

Specimen storage

• In order to ensure accurate results, it is essential to understand and implement the storage and transport conditions mandated by the regulatory bodies.
• According to the interim guidance provided by WHO (1), specimens for virus detection should reach the laboratory as soon as possible after collection.
• In addition, as per official guidelines, specimens that are delivered promptly to the laboratory can be stored and shipped at 2-8°C.
• However, when there is a higher likelihood of delay in the specimens reaching the laboratory, the use of viral transport medium is strongly recommended.
• Specimens may be frozen to -20°C or ideally to -70°C and shipped on dry ice if further delays are expected.

Table 1 – Specimen Collection and Storage

*For transport of samples for viral detection, use a viral transport medium (VTM) containing antifungal and antibiotic supplements. Avoid repeated freezing and thawing of specimens. If VTM is not available, then sterile saline may be used instead (in which case, duration of sample storage at 2-8 °C may be different from what is indicated above).

Aside from specific collection materials indicated in the table, also assure other materials and equipment are available: transport containers, specimen collection bags and packaging, coolers, cold packs or dry ice, sterile blood-drawing equipment (e.g. needles, syringes and tubes), labels and permanent markers, PPE, materials for decontamination of surfaces, etc.

Before storing samples, you should consider:

• How long should the samples be stored?
• Should there be a differentiation between positive and negative samples (and non-conform or inconclusive results)?
• Are there any other guidelines for storing the samples?

Reasons for storage of samples

• Clinical specimens or a subset of the clinical specimens may need to be retained for various purposes such as performing additional tests, for quality control purposes, or for use as control materials to assess newer diagnostic tests.
• In addition, a laboratory may need to store specimens for projects aimed at studying genomic epidemiology of the SARS-CoV-2 virus across regions and over time

Existing guidelines: storage duration

• The WHO or ECDC do not have guidelines about the duration of storage of COVID-19 PCR samples.
• In August 2020, the Indian Council of Medical Research (ICMR), Government of India, advised government laboratories to retain all positive COVID-19 samples at ultra-low temperatures for 30 days, then disinfect the samples before disposal. Some respiratory samples will need to be kept even longer — even a year or more — in ultra-low temperature freezers, for future testing and quality control purposes.
• According to the new ICMR guidelines, all samples in long-term cold storage must be appropriately labelled with laboratory identifiers and collection dates. Positive COVID-19 test samples must be kept in -80 C deep freezers.
• Beyond that, ICMR says many positive COVID-19 samples must be kept in government laboratories for one year. The number of retained samples will vary among laboratories, but ICMR is looking for 50 positive samples at each laboratory, or 1–2% of total monthly samples, or whichever is smaller.

Existing guidelines: storage conditions

• WHO recommends the storage of dacron or polyester flocked swabs with a viral transport medium (VTM) containing antifungal and antibiotic supplements for up to 12 days until analysis at 2-8°C. (1) If phosphate buffered saline is used instead of VTM, it recommends storage for up to 7 days at 4°C. ].
• In case other viruses such as influenza should also be tested, the WHO recommends storage of samples for no longer than 5 days at 4-8 °C, counting from the date of sampling until analyses.
• ECDC recommends that all specimens should be stored at 2-8°C for up to 48 hours after collection. For handling or shipping after 48 hours, storage at -70°C (dry ice) is recommended.

Storage of Specimens

• Specimens must be stored in containers with adequate strength, integrity and volume to contain the specimen, leak-proof when the cap or stopper is correctly applied, made of plastic whenever possible, free of any biological material on the outside of the packaging, correctly labelled, marked and recorded to facilitate identification, and made of an appropriate material for the type of storage required.
• Set the temperature for shipment in line with the expected shipment time.
• Avoid repeated freezing and thawing.
• At least two aliquots of VTM should be made before the specimens are stored or shipped. One of two aliquots should be stored at −70°C or −80°C as soon as possible.

Storage of Nucleic acid extract:

The RNA from the viral specimen is extracted and stored at -80°C or lower freezer.

Storage of cDNA product:

The amplified cDNA used for SARS-CoV-2 genome amplification is stored at -80°C in a freezer.

Specimen handling of SARS-CoV-2 samples

• Personnel collecting the specimens should wear appropriate PPE (N95, KF94, or equivalent respirators, protective clothing, disposable gloves, etc.) and handle the specimens in a Class II or higher biosafety cabinet (BSC) at a biosafety level 2 (BL2) laboratory.
• Aerosol-producing procedures should always be performed within the BSC.
• If the container has to be opened outside of the BSC, PPE, such as an N95, KF94, or a higher-grade respirator (powered air-purifying respirator recommended), should be worn by the personnel and the bench should be disinfected after the procedure.
• Personal hand hygiene: Wash hands thoroughly, preferably with warm running water and soap, after handling biological material and/or animals, before leaving the laboratory and when hands are known or believed to be contaminated.
• All work surfaces and equipment to be disinfected with appropriate disinfectants.
• Controlled ventilation system – A controlled ventilation system maintains inward directional airflow into the laboratory room.

References

China CDC COVID-19: Laboratory Testing Guideline

US CDC Interim Guidelines for Collecting and Handling of Clinical Specimens for COVID-19 Testing

ICMR India Specimen Collection, Packaging and Transport Guidelines for 2019 novel Coronavirus (2019-nCoV)

PHE UK Packaging requirements for COVID-19 samples

WHO Guidance on regulations for the transport of infectious substances 2019 – 2020

WHO Laboratory testing for coronavirus disease (‎COVID-19)‎ in suspected human cases: interim guidance, 19 March 2020

Advice on storage duration and conditions of human samples collected for COVID-19 PCR analysis – in the context of the national platform BIS

Guidelines for storage of respiratory specimens collected for COVID-19 diagnosis by RT PCR platforms in Government laboratories

WHO Diagnostic testing for SARS-CoV-2: Interim guidance September 2020

ECDC Laboratory support for COVID-19 in the EU/EEA

Coronavirus Disease 2019 (COVID-19) Re-infection by a Phylogenetically Distinct Severe Acute Respiratory Syndrome Coronavirus 2 Strain Confirmed by Whole Genome Sequencing