NEW YORK – As soon as a patient's nose is swabbed or spit is sampled for SARS-CoV-2 testing, the clock starts running. For accurate PCR test results, the sample typically has to be kept cold and must reach a lab to be processed within two to three days.
Recent extreme weather in US made shipping patient samples for PCR-based COVID-19 testing a major challenge. Beginning around Valentine's Day, a collapsing polar vortex struck much of the US leading to serious disruptions, including shutdowns of certain FedEx shipping superhubs, and as of Feb. 26 the delivery service company was still warning of substantial disruptions at the FedEx Express hub in Memphis.
Testing labs and firms like ARUP Laboratories and Biodesix, as well as large national reference labs like Quest Diagnostics and Laboratory Corporation of America, rely on commercial shipping, but have deployed a mix of strategies — such as maintaining alternate couriers and using specialized transport media — so for the most part they avoided having to dispose of any patient samples stuck too long transit during the recent storms.
Degradation of nucleic acids is correlated with time and temperature as well as with qualities of the transport media. The SARS-CoV-2 virus has an RNA-based genome, and RNA is particularly prone to degradation, but the problem is potentially mitigated by using a high-sensitivity test.
In order to be authorized to test shipped patient samples, labs and assay developers are typically required by the US Food and Drug Administration to conduct shipping studies to determine the limits of temperature and delay time beyond which test results are impacted.
The FDA said early on in the pandemic that for Emergency Use Authorization during the COVID-19 pandemic, shipping and stability studies could be abbreviated, and consist of labs shipping themselves contrived samples and also holding them at certain temperatures for varying amounts of time, then running their PCR method of choice. The agency guided, for example, that 20 contrived samples put through these rigors should remain positive and also not deviate by more than 3 cycle thresholds (Ct) from the Ct value of a control sample, according to its template for home specimen collection for molecular diagnostic testing.
David Grenache, president of the American Association for Clinical Chemistry and chief science officer at TriCore Reference Laboratories, said in an interview that shipping stability requirements are essential for reliable testing.
"Delays in shipping are certainly a problem for sample stability — not just for COVID-19 testing, but also for lots of different samples for lab tests that are sent to laboratories across the country," he said.
The maximum delay time before samples have to be rejected will vary with each test, Grenache said, and labs typically follow the stability guidelines in the IFU of whichever tests they use.
As an example, the US Centers for Disease Control and Prevention's test IFU states that labs shipping the agency samples send them on ice packs, or if the specimens are frozen, that they be shipped overnight on dry ice, with delivery within 72 hours. The CDC's also advises that if a delay in shipping is expected, specimens should be stored at minus 70 degrees Celsius or below, and specifies that refrigerated specimens received outside of the 72-hour window "will be rejected."
Some tests seem to be particularly resistant to shipping delays. A bioRxiv preprint posted in June from a team in Turkey, for example, claimed five days at room temperature did not impact PCR results using an assay from Coyote Bioscience. In August, researchers in South Korea claimed in American Journal of Clinical Pathology that seven days of storage at temperatures of up to 8 degrees didn't affect results using a PCR kit from SD Biosensor.
However, in the latter study, just one day of sample storage at temperatures above 20 degrees had detrimental impacts. "The higher the storage temperature, the faster the nucleic acid was degraded, and viral RNA was not amplified by RT-qPCR," the authors wrote.
Shipping delays in the cold snap led a research team in Grenache's lab to have to dispose of a batch of samples that were sent to a COVID-19 research project. And, shipping delays can also impact reagents and test kits, which also need to be shipped in a temperature-controlled way, Grenache noted, adding that this is happening on top of already problematic supply chain shortages.
Diverse mitigation strategies
Fortunately, reference labs large and small are capable of flexing their shipping strategies as needed.
David Rogers, the senior operations director of support services at Salt Lake City, Utah-based ARUP, said if specimens are significantly delayed in transit – "which rarely occurs" – the lab determines the status of the specimen upon arrival in conjunction with its own published stability limits.
And, if a specimen is deemed compromised and unacceptable for testing, ARUP uses a process it calls Except in conjunction with College of American Pathologists and regulatory guidelines to document the issue and notify clients.
"In general, we do not discard specimens earlier due to factors that may compromise specimens for testing," Rogers said, adding that this is consistent for all specimens submitted to ARUP for the wide menu of testing it provides.
Instead, ARUP uses an automated system for the majority of its specimen storage to ensure accuracy in its storage and discard timeframes and processes, and the lab stores all specimens for a predetermined length of time before discarding them so that clients and ARUP can have access to the specimen should any other questions or issues arise.
This was put to the test recently when ARUP experienced significant delays in receiving specimens and shipments routed through FedEx during the recent severe weather issues, Rogers said.
The delays correlated with the Feb. 13 weather-related shutdown of FedEx's Memphis superhub and subsequent shutdown of two other backup hubs.
However, ARUP quickly engaged "a comprehensive network of airline carriers" and immediately started routing specimens through other carriers for specimen transport.
"This minimized the impact of the FedEx shutdowns; however, the specimens that had been shipped just prior to the FedEx shut-downs were impacted by these issues," Rogers said.
The lab is now using its Except process to document and handle these specimens as they arrive, and is still determining the final impact of excessive FedEx delays for specimens shipped prior to FedEx notifications, he said.
Overall, the impact of the delays was minimized due to ARUP's ability to use other commercial carriers for specimen transport, Rogers said.
Boulder, Colorado-based Biodesix also confirmed that a proportion of shipments sent to the lab through FedEx recently were impacted by the extreme weather. Bobbi Coffin, Biodesix's chief growth officer, said that the lab mitigated the problem in part by using alternate couriers and same-day local car couriers for in-state deliveries.
For out-of-state samples, which are typically sent by FedEx, it also uses an alternate transport media — specifically PrimeStore MTM from Longhorn Vaccines and Diagnostics — that preserves samples for significantly longer.
"Because of our use of our recommended media, this was not as impactful as it could have otherwise been," Coffin said, noting that the lab experienced less than 1 percent loss due to recent weather-related delays.
For its SARS-CoV-2 testing, Biodesix uses Bio-Rad Laboratories' droplet digital PCR systems.
Similar to the CDC's shipping recommendations, the Bio-Rad IFU specifies that specimens can be stored at 2 to 8 degrees Celsius for up to 72 hours after collection, and if a delay in extraction is expected, they should be stored at minus 70 degrees or lower.
But, Biodesix has extended that time by using the PrimeStore MTM transport media. "In our out-of-state WorkSafe COVID-19 test program, we accept specimens if received within seven days from collection and transported in PrimeStore MTM and according to our shipping instructions," Coffin said.
The lab bases its polices on multiple industry requirements for establishing sample collection, shipping and handling, and acceptance activities to ensure sample stability, such as from the New York State Clinical Laboratory Evaluation Program, Clinical Laboratory Improvement Amendments and CAP, the FDA, and the Clinical Laboratory Standards Institute.
Coffin said that the demand remains high for SARS-CoV-2 testing but is also continually evolving as the market dynamics surrounding the pandemic evolve. The lab runs ddPCR molecular testing and antibody serology testing from its central labs, and supports point-of-care rapid antigen testing nationwide. In addition, it is assessing newer technologies and test modalities "that may play an increasing role through the coming year and beyond," Coffin also said.
Large national reference labs also appear to have weathered the recent bad weather well.
Quest Diagnostics' IFU specifies the use of FedEx to transport its self-collected patient samples tested using a SARS-CoV-2 molecular test on the Roche Cobas 6800/8800. Samples need to be received within two days of collection, and the lab's shipping and temperature excursion studies in which contrived samples were kept in cycles of high and low ambient temperatures showed stability out to 56 hours.
A Quest representative confirmed that the lab relies on FedEx for its self-collection kits but said it did not need to cancel any tests due to recent weather-related delays.
This was because the lab has established "a robust logistics network," which sometimes includes third parties, to help meet challenges such as extreme weather. "We operate under the idea that behind every specimen is a person, so we know how important these results are," the representative commented.
Labcorp also mitigates delays through alternate carriers. The lab's IFU for its EUA Pixel home-collection kit notes that its sample shipping stability study found no impact of 72-hour-shipping using FedEx specifically as a carrier. And a temperature excursion study also did not impact Ct values for Labcorp's assay, even with storage at 4 degrees for an additional 94 hours.
A Labcorp representative commented in an email that while overall testing volumes were impacted by the recent inclement weather, the firm's operations saw limited disruption.
"We prepared accordingly, and where necessary, diverted samples to alternate Labcorp facilities using our own ground and air transport resources," the representative said, adding that Labcorp also leveraged a common laboratory information system and standardized platforms to help expedite testing for areas impacted by the weather.
Even with the possibility that severe weather events may be part of normal life, AACC's Grenache said that send-out testing is likely here to stay.
"I don't think we'll ever get away from the need to ship specimens for tests, because not every laboratory in the country is capable of doing every laboratory test that's available," he said.