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Interim Guidance for Use of Pooling Procedures in SARS-CoV-2 Diagnostic and Screening Testing

Interim Guidance for Use of Pooling Procedures in SARS-CoV-2 Diagnostic and Screening Testing
Updated June 30, 2021

Summary of Updates

  • Removed information about pooling specimens for public health surveillance testing. The majority of testing for SARS-CoV-2 using a pooling strategy is for diagnostic and screening testing purposes. Although pooling specimens for Public Health Surveillance purposes is still acceptable, this guidance focuses on the use of pooling for diagnostic and screening testing.

Key Points

  • Pooling specimens is a testing methodology that can be used when performing Nucleic Acid Amplification Tests (NAATs) provided the test has received an FDA authorization for pooling.
  • Pooling specimens allows laboratories to increase the volume of testing while using fewer testing materials. Pooling can also increase efficiency (time and labor) and reduce the overall cost of testing.
  • Pooling strategies depend on the incidence of infection in the community
  • Pooling reduces test sensitivity

Note: This document provides guidance on the appropriate use of pooling and does not dictate the determination of payment decisions or insurance coverage of testing involving the pooling of specimens, except as may be otherwise referenced (or prescribed) by another entity or federal or state agency.

Introduction

Pooling—sometimes referred to as pool testing or pooled testing—means combining the same type of specimen from several people and conducting one NAAT laboratory test on the combined pool of specimens to detect SARS-CoV-2, the virus that causes COVID-19.  Pooled tests that return positive results will require each specimen in the pool to be retested individually to determine which individual(s) are positive.

The advantages of pooling include preserving testing reagents and resources, reducing the amount of time required to test large numbers of specimens (increasing throughput), and lowering the overall cost of testing.

The optimal pooling strategy depends on the incidence of infection in the community, and pool size will need to be adjusted accordingly. CDC recommends that laboratories should determine incidence based on a rolling average of the positivity rate of their own SARS-CoV-2 testing over the previous 7–10 days. Laboratories should use a standardized methodology or calculations that factor in the sensitivity of the test they are using and the cost of testing to determine when the positivity rate is low enough to justify the implementation of a pooling strategy. Laboratories should also understand and, where appropriate, communicate the limitations associated with pooled testing.

Using a pooled testing procedure for SARS-CoV-2 has some limitations. In a pooling procedure, the laboratory cannot ensure the diagnostic adequacy of an individual specimen because it is combined with other specimens before testing. Specimen integrity can be affected by the quality of specimen collection. There is a risk that some specimens may have limited amounts of viral genetic material for detection. Inadequate individual specimens, including those with limited amounts of viral genetic material, may be included in the pool before testing. Even if each individual specimen in a pool is adequate, the specimens in a pooled procedure are diluted, which could result in a concentration of viral genetic material below the limit of detection of a specific test.

These limitations mean that monitoring the incidence of disease, collecting quality specimens, and properly validating the test and the instrumentation are important to limit the potential for false-negative results. In general, the larger the pool of specimens, the higher the likelihood of generating false-negative results.

The incidence of COVID-19 in a population also affects the efficiency of pooled testing strategies. In general, lower disease incidence may enable a laboratory to use a larger optimal pool size. A study by the Nebraska Public Health Laboratoryexternal icon found that nucleic acid tests for SARS-CoV-2 reliably returned a positive result when one positive specimen was mixed with four negatives and could reduce the number of tests needed by >50% in certain scenarios (such as a COVID-19 incidence of 5%). However, as the incidence of COVID-19 increases the cost savings of a pooling strategy decreases because more pooled tests will return positive results and those specimens will need to be retested individually to determine which individual(s) are positive.

Diagnostic or Screening Testing Using a Pooling Strategy

Regulatory Requirements

Laboratories certified under the Clinical Laboratory Improvement Amendments (CLIA) can use a specimen pooling strategy to expand SARS-CoV-2 nucleic acid diagnostic or screening testing capacity when using a test authorized for such use by FDA. Any SARS-CoV-2 in vitro diagnostic device authorized by FDA for use with specimen pooling will be included on FDA’s list of In Vitro Diagnostics EUAsexternal icon. FDA has also outlined various policies regarding the use of COVID-19 tests prior to authorization. A laboratory that wishes to use pooling with a SARS-CoV-2 nucleic acid amplification test would be expected to evaluate and validate the performance of a test for a pooling strategy. Recommendations for doing so are included in FDA’s COVID-19 molecular diagnostic templates for EUA submissionexternal icon. FDA has more specific guidance for laboratories and test manufacturers interested in using a combination of specimen pooling and serial testing. These recommendations and the process by which laboratories can request this claim may be found in the FDA’s umbrella Pooling and Serial Testing Amendment Letterexternal icon.

If the laboratory modifies an authorized test, whether for pooling or otherwise, by incorporating alternative components, including extraction methods, polymerase chain reaction (PCR) instruments, and software versions, the laboratory should evaluate and validate the performance of the component changes, and recommendations for doing so as outlined in FDA’s Policy for COVID-19 Testsexternal icon. Please note that the modified test is no longer covered by the manufacturer’s EUA for the test; it is considered a laboratory-developed test (LDT).

Laboratories must ensure their diagnostic test and pooling approach are in compliance with FDA requirements. For more information on FDA’s policies and recommendations, see FDA’s Policy for COVID-19 Testsexternal icon, FDA’s Molecular Diagnostic Template for Laboratoriesexternal icon, and FDA’s FAQs on Testing for SARS-CoV-2external icon. Questions about regulatory requirements for COVID-19 diagnostics may be submitted to FDA at CDRH-EUA-Templates@fda.hhs.gov.

Laboratories that conduct diagnostic or screening testing for COVID-19 must also comply with CLIA regulations. If at any time a facility intends to report patient-specific test results, it must first obtain a CLIA certificate and meet all requirements to perform testing. For more information, see the Centers for Medicare & Medicaid Services’ (CMS’) summary of CLIA regulationsexternal icon.

Interpreting Results

If a pooled test result is negative then all the specimens can be presumed negative with the single test. In other words, all of the people who provided specimens can be assumed to test negative for SARS-CoV-2 infection and no additional testing is necessary.

If the pooled test result is positive, each of the specimens in the pool will need to be tested individually to determine which specimen(s) is (are) positive. Sufficient volume of the specimen must be available for any subsequent individual retesting. If a matrix pooling strategy (specimens are tested more than once by inclusion in different pools) is used, there is no need to test individual specimens as long as the positive specimen(s) can be identified in the matrix pools.

Reporting Results to Public Health Departments

A CLIA-certified laboratory that allows for pooling of specimens must report diagnostic or screening negative test results to the participants in the pool according to the instructions for use of the FDA-authorized SARS-CoV-2 in vitro diagnostic device. The test report given to the individuals in the pool should indicate that the testing procedure involved specimen pooling and explain the limitations of that type of testing. See the CDC guidance on How to Report Laboratory Data for reporting results to local, state, tribal or territory health departments.

The CLIA-certified laboratory should not report positive or indeterminate results of a pooled test to either the participants in the pool, or the local, state, tribal, or territory health department. All participant specimens that were in a pooled test with a positive or indeterminate result should be retested separately, and the subsequent individual diagnostic or screening results must be reported to the person tested and the local, state, tribal, or territory health department.