Attachment D Laboratory Validation Application.docx

ATTACHMENT D

LABORATORY VALIDATION APPLICATION

INSTRUCTIONS:

Submission of Laboratory Validation Application

1. The Bidder must submit the all information set forth in this Attachment D for each laboratory that it proposes to use for Pooled Testing under the K-12 COVID-19 Testing program for which an EUA is not available through the FDA. For example, if the Bidder proposes to use two separate laboratories, it must submit the information set forth in this attachment separately for each such laboratory.

1. The Bidder must submit the information set forth in this Attachment in the order requested and under section headings that correspond to the section headings set forth herein.

1. If an EUA is available through the FDA for a laboratory’s pooled test or has an application pending, the Bidder must complete Attachment C – Laboratory Validation Information.

1. Bidders should refer to the published literature and FDA’s pooling recommendations for guidance on best practices in designing the Bidder’s validation depending on the type of pooling performed: https://www.fda.gov/medical-devices/coronavirus-disease-2019-covid-19-emergency-use-authorizations-medical-devices/vitro-diagnostics-euas.

1. The attachment to the Bidder’s response containing the information in this Attachment D must follow the naming convention, “Attachment D – [bidder]_[lab name].docx”.

1.

Pooling Validation Application

1. Summary of Pooling Strategy:

a. The Bidder shall provide a full description of the test method (extraction, amplification, detection) and sample type (e.g., dry anterior nasal swab or other sample type), type of pooling scheme (sample/media pooling or swab pooling), and availability of pooled result only or pooled result with reflex testing.

b. The Bidder shall provide background information on its laboratory’s experience and credentials.

2. Test Method Overview:

a. The Bidder shall provide an overview of the test; instruments used; qualitative/quantitative data output (cycle threshold or other value); and assay cut off value.

b. If applicable, the Bidder shall document how its lab has addressed any manufacturer’s notification letters associated with the test.

c. The Bidder shall provide a test result table including all results (positive/negative/inconclusive/invalid, etc.) by assay target(s); and the expected turn around times (TAT) for its proposed validated pooling scheme.

3. Individual Test Performance Characteristics:

a. Limit of Detection (LoD)- Analytical Sensitivity: The Bidder shall provide data demonstrating your assay’s limit of detection (LOD) from a 10-fold serial dilution of quantitated whole viral genomic RNA or virus spiked in your negative clinical matrix. A ≥95% detection (detection of 19/20 positive replicates) must be demonstrated for the LoD.

b. Clinical Evaluation: The Bidder shall provide data from 30 positive /30 negative of previously characterized samples. These samples may be banked clinical samples or contrived samples spiked with a range of positive material to reflect high, medium, and low cycle threshold values. Note: a minimum 95% positive and negative agreement is required.

4. Cycle Threshold Shift (Ct shift)- (for all types of pooling schemes)

a. To evaluate the impact of pooling on test performance, the Bidder will need to evaluate the cycle threshold shift (Ct shift) for the individual versus pooled test result for your assay. To account for variables unique to each pooling scheme, the study design should demonstrate a range of Ct threshold values (from 20-35) detected for a specified pool size versus the individual result for 20 positive pools and 20 individual samples tested in replicates. Data should be presented in tables with the Ct value for each individual versus pooled samples and its difference. Provide the average Ct change observed for the entire study and the standard deviation. Additionally, Ct data for the internal control targets should be presented. Laboratories should design their validation based on the intended maximum pool size and include any considerations for modifying the pool size based on the possibility of an increase in prevalence.

b. A set of 5-10 negative pools should also be tested and the average Ct data and standard deviation for the internal control targets should be presented in a table.

5. Swab Pooling Validation: to validate a swab pooling method, data from the following two studies should be presented:

a. For the Bidder’s intended swab pooling method, test 10 sets of the Bidder’s maximum swab pool size at 3-4X the LOD of the Bidder’s assay to ensure that test interference does not occur with multiple swab specimens in the single tube. Data should be presented in tables; provide the average Ct observed and the standard deviation.

b. For the Bidder’s intended swab pooling method, 5-10 sets of the expected final swab pool size should be spiked with 1 or more positive swabs representing a high viral concentration (e.g., 100,000-300,000X the LOD) to demonstrate that excess viral loads do not inhibit the performance of the assay. While this is less urgent in a low prevalence setting, it is imperative in a high prevalence setting and may precipitate the need to drop to smaller swab pool sizes.

6. General Comments: Laboratories must confirm that they have implemented processes to reduce the potential for inadvertent laboratory contamination (careful sample handling, stringent surface/equipment decontamination, uni-directional work flows, and monitoring of extraction controls) in additional to monitoring their environment for laboratory contamination. Identification of laboratory quality issues must be immediately reported to the submitter if they impact test results.

Attachment D: Laboratory Validation Application 1 K-12 COVID-19 Testing Services Program RFR