Why did your lab start pooling SARS-CoV-2 specimens?

A: Like all other laboratories, when SARS-CoV-2 first reached the U.S. we found ourselves needing to ramp up testing faster than the supply chain for reagent and ancillary supplies could support. Every week was a struggle to ensure that we did not run out of reagent, and we were forced to make difficult decisions about which patient samples we would run and which ones we would save until our next reagent shipment. This was what led us to consider pooled testing.

When we initially presented the pooling concept to our medical directors, they rejected the idea. As supply shortages continued, though, everyone realized that we would either have to restrict SARS-CoV-2 testing or move forward with pooling.

How does your pooling process work?

First, we create a mock accession number and label the pooled test tube, then we manually pipette four 400 μL patient samples into the accession tube and vortex to ensure adequate mixing. We use a 10 x 5 rack so that we can rack the individual specimens directly behind the mock/pooled accession tube, and specimens remain in the rack until we have verified all results. After we pool the specimens, we then load the pooled accession specimens onto the polymerase chain reaction (PCR) platform, where they are treated as individual specimens. 

What challenges have you experienced with pooled testing?

Implementing and sustaining pooled testing has presented our staff with numerous challenges. For starters, we are a mid-volume lab with automation and very few processes offline from our laboratory information system (LIS) or middleware. To implement pooling, we therefore had to create a process that gives each pool specimen a unique barcode identifier so that the PCR analyzer can associate results with individual patients within the pool. This enables us to break apart the pool and rerun individual samples in the event of a positive pool, or to result negative pools as individual patients.

Pooling has increased our handling of SARS-CoV-2 specimens, which in turn has increased the staff’s exposure risk. Instead of processing specimens once and loading them onto the analyzer, we are now processing and handling specimens multiple times. Storage has also become an issue as all pools are stored along with individual specimens until the test results are cleared.

Resulting pools is an offline process and requires the laboratory to manually enter all results. This has added to our turnaround time as it requires intervention instead of our LIS releasing results once each run is validated. 

Unanticipated delays occur because we have to hold off on testing until we have the 94 specimens needed to run a full plate. This is particularly problematic on weekends, as our collection station is open on Saturdays but closed on Sundays.

Additionally, as the positivity rate continues to increase in our community, pooling is becoming less efficient, and it’s becoming more difficult for laboratory staff to ensure that known positive samples aren’t included in pools.

What is the clinical impact of pooling?

Our validation of pooled testing did not show a significant loss of sensitivity or specificity. Pooled cycle counts were also within acceptable limits compared to individual cycle counts, which indicates that it is unlikely that pooling is generating false negatives.

Most of the clinical impact we’ve seen is due to the extended turnaround times caused by having to retest individual samples from positive pools. With that said, we’ve saved more than 40% of our reagents by pooling, and it continues to be worth the extra effort to test as many patients as we can.

Nanette West, MBA, MT (ASCP), is the system director of laboratory operations at TMC in Kansas City, Missouri. +Email: [email protected]

The author wishes to acknowledge and thank her Truman Medical Center (TMC) colleagues Kamani Lankachandra, MD, medical director of laboratory operations; Jeffrey Kelleher, MBA, MLT (ASCP), core laboratory supervisor; and Debra Engblom, MT (ASCP), quality assurance manager, for their support with developing responses to these questions.