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Pooled Testing: Guidance from the CAP’s Microbiology Committee

Given the worldwide shortage of SARS-CoV-2 NAAT testing reagents, multi-stage, group testing of pooled specimens (sometimes called pooled testing or batch testing) has been proposed as a means of conserving limited test reagents. With this method, aliquots from multiple patient specimens (eg, 4-16) are pooled together and tested as a single specimen.

If the result from the pooled test is negative, then no additional testing is performed and the test results for each of the specimens that had been pooled together are reported as negative.

However, if the result of the pooled specimen is positive, then each specimen will be tested again individually to identify one or more specimens that have a positive result.

In infectious disease screening, pooled testing is most effective in situations in which the prevalence of a particular pathogen is low, so that repeat testing of individual, non-pooled specimens, is an uncommon occurrence.

Pooled testing is commonly employed for screening blood donors for a variety of transfusion-transmitted pathogens using nucleic acid amplification tests that are approved/cleared by the U.S. Food and Drug Administration (FDA) specifically for pooled testing. Laboratories can employ a pooled testing strategy for SARS-CoV-2 when using a test that has received emergency use authorization for this purpose by the FDA. Further guidance is available on the CDC's website. 

At this time, there are several pros and cons of pooled testing to consider:


  • Pooling can help to conserve testing reagents and facilitate surveillance testing of large populations with low prevalence of infection. Pooling can also aid in increasing the amount of testing that can be performed utilizing test instrumentation with limited throughput.


  • There is no well-established infrastructure currently available to support sample pooling for diagnostic testing in clinical laboratories, such as automated specimen aliquoting and software for managing pooling and de-construction of positive pools for individual sample testing. These systems are essential for minimizing human errors and require both expertise, laboratory information system support, and thorough evaluation prior to implementation. Without them, laboratories must perform manual aliquoting for specimen pooling which increases the risk for specimen mix-up and cross-contamination.
  • Pooling will also lead to a reduction in the sensitivity for detecting SARS-CoV-2 RNA in a given sample, because specimens are combined, leading to overall specimen dilution. In general, the larger the specimen pool, the greater the risk of generating a false-negative result from testing pooled specimens. The clinical implications of this potential loss in sensitivity needs to be considered for the patient populations being tested. The FDA requires performing laboratories to conduct their own population-specific (symptomatic vs asymptomatic) assay validation studies for relevant patient populations at the testing institutions, to demonstrate minimum loss of analytical sensitivity going from individual testing to pool testing. For institutions wishing to use multiple assays with pooled samples, each assay would have to be individually validated. This requirement places a tremendous time and financial burden on clinical laboratories that are already strained. See the CDC's website for more information.
  • Although testing reagents are conserved during pool testing, sample collection vials, swabs, and other plastic consumables used in testing that are also facing supply constraints are not necessarily conserved. Additionally, pooling does not provide significant savings in time or efficiency for laboratory personnel, because every specimen tube still need to be handled by laboratory personnel during manual pooling. The hands-on time required per specimen may actually increase in many laboratories because of the increased manual pipetting work required during the testing process.
  • Pooling of samples within the laboratory will not address the logistical challenges associated with sample collection including prolonged wait times at sample collection sites.
  • Finally, while pooling can reduce turn-around time for negative results, it actually will increase turn-around-time for positive results, because it is a multistage testing process for positive pools. Because the prevalence of infection may change over time, the utility and cost-effectiveness of a pooling strategy may also change. If the prevalence of infection increases, more positive pools will be detected, resulting in more repeat testing and longer turn-around-times for reporting.