A place of pride for pathology labs

In the early days of the COVID-19 pandemic, labs scrambled to meet demand for tests. Testing sites were overwhelmed, and the facilities that processed the specimens sometimes took up to a week to return results. Laboratories around the country started stepping up; they shifted focus from their day-to-day operations in order to stem the tide of coronavirus tests. But increasing testing capacity isn’t just a matter of labs adding COVID-19 testing to their offerings; it is a major undertaking.

Scientists at Yale’s Molecular Diagnostics Lab, part of the Department of Pathology, rose to this challenge. In March 2020, the pathologists had none of the equipment or space necessary and far too few technicians qualified to process COVID-19 tests. By December 2020, they had the capacity to run an estimated 1,200 tests a day. Over the course of the pandemic, the Molecular Diagnostics Lab evolved from manually processing nasopharyngeal swabs to playing a crucial role in the validation and execution of Yale’s own FDA-approved SalivaDirect test.

Here’s how they did it.

On a typical day, the Molecular Diagnostics Lab performs tissue-based molecular testing related to cancer diagnoses. Extracting RNA from nasal swabs to detect the presence of an infectious pathogen was not a part of their routine. “Actually, we started from zero,” said Jianhui Wang, PhD, a research scientist. Wang and his colleagues did not even have an appropriate place to run the tests. Their current facility was too small for the added operation; moreover, safe handling of the coronavirus pathogen required a separate stand-alone space.

In just two weeks, the lab had borrowed the space, the equipment, and even the workforce that it needed to get started from other groups or labs. “Everything was backordered. Borrowing was the only way,” Wang said. “Every time I’d come into the lab,” Susan Bell, interim manager of the Molecular Diagnostics Lab, recalled, “different things were missing—pipettes, biosafety cabinets—everything was disappearing as they moved it over to the other space.”

As for the borrowed workforce, staff from other labs learned the safety protocols for handling the coronavirus pathogen—which included working in a hood, a mask, a face shield, and a hazmat suit. Then, in addition to continuing their own professional responsibilities, they volunteered for a few hours a week in the new COVID-19 lab.

At the same time, Pathology’s in-house software engineering team moved quickly to create a platform for ordering tests, uploading results, and reporting positive results to the patient’s home state according to that state’s reporting regulations. Among its many functions, the platform allows clinics to print barcodes on a generic label printer and send specimens to the lab already labeled and ready to scan.

“It’s pretty unique to have an engineering team as part of pathology,” said Peter Gershkovich, MD, MHA, director of pathology informatics. “But without this, it wouldn’t have been possible to scale up for COVID testing.”

Like the lab, the engineers had their system up and running in two weeks.

By the first week in April 2020, the lab was running COVID-19 nasopharyngeal tests. To perform this relatively complicated test, technicians must extract RNA from inside human cells and make copies of it to see whether it is viral. At first, the lab didn’t have equipment that would automate this process. “You put the reagent in the tubes yourself, put the tubes in the machine, and turned them with your fingers,” Wang said. “It was a very tedious job.” The technicians continued to work this way until they acquired polymerase chain reaction (PCR) machines that would do much of this work for them.

Meanwhile, Nathan Grubaugh, PhD, assistant professor of epidemiology, and Anne Wyllie, PhD, associate research scientist in epidemiology at Yale School of Public Health, had come up with a saliva-based COVID-19 test called SalivaDirect™ that would eliminate this tedious step. In June, the diagnostic test seemed ready for clinical use, but the researchers needed a lab to validate it. Just a few months earlier, the testing capacity required to validate this new diagnostic test might have seemed ambitious. By early summer, the Molecular Diagnostics Lab’s COVID-19 operation was ready to help SalivaDirect make the final push into the clinic.

Over the next few weeks, Wang and his team ran COVID-19 tests on the nasal swabs and the saliva of 3,779 people—pulled from NBA players, staff members, and contractors. Their job was to confirm that SalivaDirect got the same results as the gold standard nasal swab test. The team confirmed the test results against the nasal swabs twice with equipment and supplies from different vendors to safeguard against potential supply chain shortages in the future. This crucial assist helped SalivaDirect obtain FDA-accelerated emergency use authorization (EUA) as a COVID-19 test.

Running COVID-19 tests can be grueling work. Manual RNA extraction, the method that Wang and his team used initially to process nasal swabs, takes about two hours, and technicians cannot stop during the process—a challenge given the required safety gear. “You look like an astronaut,” Wang said. “But you can’t stop till the manual extraction is done because it’s very fragile and you have to move fast.”

Researchers use pipettes to add chemicals, and then spin the vials to separate the mucus and other nasal matter from the human cells they need to test. Another chemical coaxes the cells open so the RNA can be extracted. Then a chemical reaction replicates the RNA multiple times so that there is enough of it to test for the presence of coronavirus.

After ushering the samples through these steps, the researchers would be soaked in sweat. They’d break, change clothes, and then start the next batch. Once the researchers acquired PCR machines that automated this process, the work moved faster and their testing capacity increased.

SalivaDirect makes things easier still. It’s not necessary to extract viral RNA from the cells to perform the test. Technicians load vials containing saliva samples into a 96-well plate—a tray with a hole for each tube. They use heat to inactivate the virus and then place the plate in the PCR machine, which copies the DNA and searches within it for the presence of coronavirus. A single “run” takes about an hour and 20 minutes.

The lab had fielded numerous PCR machines by winter 2020, each of which could perform seven or eight 96-sample runs per day. The lab receives hundreds of saliva and nasal samples daily.

A lot has happened since the Molecular Diagnostics Lab launched its COVID-19 testing lab in April 2020. Several COVID-19 vaccines gained official approval around the world; national vaccination campaigns have been undertaken in many countries; and new genetic variants of the virus, some of which may be more infectious or escape protection from vaccines, have emerged.

The lab of Jeffrey Sklar, MD ‘77, PhD ‘77, professor of pathology and of laboratory medicine, has taken up the next step in testing for SARS-CoV-2—the identification of viral variants. Mutations arise spontaneously as the virus replicates during infections. “Identification of variants requires analysis of the entire viral genome, which resembles a string of 30,000 beads (chemically termed nucleotides),” said Sklar of challenges facing surveillance for known and previously unknown variants. “Even a single change in one nucleotide among the 30,000 can potentially define a new viral variant.”

Wang, too, knows there’s still plenty of work ahead on the testing front, especially as schools and businesses look toward a full reopening that will depend on quick, effective diagnostics.

The lab continues to develop innovative ways to increase access and speed. The researchers recently applied for FDA approval of a point-of-care saliva-based test that they developed in their lab. A technician would administer the test on-site at a clinic and deliver the results in about an hour. “We’ve gained a lot of experience from SalivaDirect,” Wang said. “If we ever need to scale up testing quickly for an epidemic or pandemic, we’ll be ready.”