LDTs

FDA Provides New Guidance on Validation of Sample Pooling for SARS-CoV-2 Test Development   

On June 16, the FDA issued new guidance to help labs and commercial manufacturers use test sample pooling to develop, validate and acquire Emergency Use Authorization (EUA) of qPCR-based SARS-CoV-2 detection tests.

Pooling & SARS-CoV-2 Test Development

Pooling is a technique that involves mixing aliquots, i.e., sub-samples extracted from individual samples into a pool or “batch” that can be tested with a single test. If the entire pool returns a positive result, the individual samples are retested to locate the source of the positive; if the batch tests negative, all of the constituent samples are also deemed to be negative.

Historically, developers of EUA tests have used pooling to modify how those tests are used. In the context of the current pandemic, pooling offers the advantage of conserving testing resources that have been in short supply. During her June 23 keynote address to the American Society for Microbiology Microbe conference, White House coronavirus task force response coordinator Deborah Birx called on labs to make greater use of pooling, suggesting that the technique “would give us the capacity to go from a half a million tests per day to potentially 5 million individuals tested per day.”

However, there’s also a downside of pooling to the extent that it dilutes the nucleic acids produced by the SARS-CoV-2 virus, creating the risk of false negatives. As a result, test producers who use pooling must account for the false negative effect in validating the modified use.

The New Guidance

The new FDA guidance, which takes the form of additions and revisions to the pre-existing EUA templates for laboratory developed tests (LDTs) and molecular diagnostic tests, outlines the agency’s “validation expectations” for the use of specimen pooling either to develop a new test for EUA or modify the use of one that has already received EUA clearance. Specifically, it recommends use of a Dorfman or simple pooling approach combining samples into non-overlapping pools and testing each sample pool.

The utility of pooling to scale up testing depends on prevalence, test sensitivity and the number of low-positive samples. The dilution of samples used in pooling may reduce sensitivity. Result: Positive cases may go undetected, particularly weakly-positives in which viral concentration may already be near the limit of detection.

Validation of Pooling

According to the template, a pool of five samples is a reasonable starting point for validation of pooling for a high-sensitivity test in populations with a positivity rate of approximately 5 percent to 6 percent; however, the agency suggests that smaller pools may be necessary in populations with higher prevalence rates.

The FDA suggests that labs and developers test a random sampling of patient samples without pooling to evaluate the positivity rate and percent of weak positive samples in the testing population and to identify differences in positivity rate between those tested individually and those tested through pooling.

To validate pooled testing, labs and developers should characterize the reduction in assay analytical sensitivity indicated by a shift in Ct score for RT-PCR assays with respect to the number of samples to be pooled to ensure the selected sample pooling strategy maintains appropriate sensitivity. They should also determine the maximum number of samples acceptable to pool for each specimen type.

The FDA recommends conducting a clinical validation study in the intended use population that includes testing each sample individually and using the proposed pooling strategy.

For validation, a clinical study of a five-sample pool strategy should include at least 20 individual positive samples and 180 negative samples, either archived samples or freshly collected. The study should compare the performance of the EUA-authorized assay when testing single specimens to the performance of the assay when testing sample pools.

Adding pooling to a test that already has EUA requires a clinical study large enough to include 20 positive samples. Labs and developers should submit an EUA amendment request with the appropriate validation data, but don’t have to establish assay performance with a separate comparator test.

The Template for Developers

The FDA strongly encourages developers to “work with their customers to gather existing data.” Example: 100 Ct scores from individually tested positive patient samples can be used to evaluate the percentage of samples with Ct scores close to the assay Limit of Detection (LoD), or weak positives.

A Ct shift of Log2(n), where “n” is the number of samples in a pool, can be estimated, e.g., so that a Ct shift of 2.3 is expected such that for a pool of five samples. According to the template. “Therefore, if a large percentage of positive patient samples are close to your assay LoD, you may want to consider a smaller n, which will reduce the observed Ct shift and maintain higher sensitivity.” The template recommends that at least 25 percent of the validation samples be within 2-3 Ct of the cut off, and no more than within 2-4 Ct.

For an LDT or commercial test that’s not previously authorized, pooling can be included in the EUA filing, but must also include performance characterization with a high-sensitivity comparator assay and a clinical study of pooling involving at least 30 individual positive samples and enough negative samples to generate 30 five-sample pools with one positive sample plus 30 five-sample pools with only negative samples.

Takeaway

Use of specimen pooling for purposes of developing new lab tests and expanding the use of previously approved ones is nothing new, of course. Historically, while the FDA “has encouraged all test developers to reach out to the agency to discuss appropriate validation approaches,” it hasn’t made any specific recommendations on the subject. So, the decision to add guidance on specimen pooling to its template is a significant policy change, which an agency statement describes as a “step forward. . . to help facilitate the preparation, submission, and authorization under an” EUA. In the larger context, the move represents a continuation of the FDA’s strategy of backing away from its initial laissez faire approach to COVID-19 testing quality in favor of a more active regulatory strategy. 

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