Cell-Free DNA Assay Validations
Summary of Recommendations for Clinical ctDNA Testing, Reporting, and Publications
- Laboratories should clearly define and describe during validation the medical indication for an assay, including the clinical scenarios to which the performance characteristics apply and the methods used.
- Laboratories should clearly define and describe during validation clinical assay performance characteristics (sensitivity, specificity, positive predictive value, negative predictive value, accuracy, and concordance) appropriate for the medical indication for the test.
- Laboratories should evaluate and describe during validation key clinical assay performance characteristics on an individual variant basis, but these may be aggregated for each variant class. Note: variant classes could include SNVs, indel, copy number alteration, structural variant (eg, fusion), or signature (microsatellite instability, homologous recombination deficiency, or tumor mutational burden).
- Laboratories should define and describe during validation the analytical sensitivity (limit of detection) of the assay/s for each variant and/or variant class.
- Laboratories should define and describe during validation both in vivo and in vitro potential sources of assay interference.
- Laboratories should evaluate and address during validation both in vivo and in vitro potential sources of result interpretation error.
- Laboratories should define and describe during validation orthogonal method confirmations, to include the parameters above.
- Laboratory reporting for clinical ctDNA assays should describe pre-analytical variables pertinent to both pathologist and clinician understanding of reported results. Note: these may include but are not limited to volume and type of collected fluid, collection tube, and details of storage and processing (eg, refrigeration and centrifugation).
- Laboratory reporting for clinical ctDNA assays should describe analytical variables pertinent to both pathologist and provider understanding of reported results. Note: these may include but are not limited to nucleic acid extraction method, library preparation method, volume/concentration of nucleic acid extracted, volume/concentration of nucleic acid used in assay, minimum/mean sequencing depth (for NGS) and droplet scoring parameters (for ddPCR), and details of the bioinformatic pathway.
- Laboratory reporting for clinical ctDNA assays should state the analytical sensitivity (limit of detection) of the assay/s for each variant and/or variant class.
- Laboratory reporting for clinical ctDNA assays should describe interpretive variables pertinent to both pathologist and provider understanding of reported results. Note: these may include but are not limited to methods employed for variant/signature annotation/interpretation and cutoffs.
- Although universal orthogonal confirmation is not required, laboratory reporting for clinical ctDNA assays should have and provide information regarding the laboratory's policy for orthogonal method confirmations and specify any exceptions that may apply.
- Publications describing ctDNA assays intended for clinical applications, including description of clinical validation, should include key performance characteristics.
Cell-Free DNA Assay Validations
October 6, 2023
Last Updated Month/Year
November 6, 2023
Country of Publication
Diagnosing, selecting therapy for, and monitoring cancer in patients using a minimally invasive blood test represents a significant advance in precision medicine. Wide variability exists in how circulating tumor DNA (ctDNA) assays are developed, validated, and reported in the literature, which hinders clinical adoption and may negatively impact patient care. Standardization is needed for factors affecting ctDNA assay performance and reporting, including pre-analytical variables, analytical considerations, and elements of laboratory assay reporting. The Association for Molecular Pathology Clinical Practice Committee's Liquid Biopsy Working Group (LBxWG), including organizational representation from the American Society of Clinical Oncology and the College of American Pathologists, has undertaken a full-text data extraction of 1228 ctDNA publications that describe assays performed in patients with lymphoma and solid tumor malignancies. With an emphasis on clinical assay validation, the LBxWG has developed a set of 13 best practice consensus recommendations for validating, reporting, and publishing clinical ctDNA assays. Recommendations include reporting key pre-analytical considerations and assay performance metrics; this analysis demonstrates these elements are inconsistently included in publications. The LBxWG recommendations are intended to assist clinical laboratories with validating and reporting ctDNA assays and to ensure high-quality data are included in publications. It is expected that these recommendations will need to be updated as the body of literature continues to mature.
Male, Female, Adolescent, Adult, Child, Infant, Older adult
Health Care Settings
Ambulatory, Laboratory services
Laboratory technician, nurse, nurse practitioner, physician, physician assistant
Assessment and screening
D000074141 - Circulating Tumor DNA, D004247 - DNA
DNA, Cell-free DNA, dna assay, circulating tumor DNA
Lockwood CM, Borsu L, Cankovic M, Earle JSL, Gocke CD, Hameed M, Jordan D, Lopategui JR, Pullambhatla M, Reuther J, Rumilla KM, Tafe LJ, Temple-Smolkin RL, Terraf P, Tsimberidou AM. Recommendations for Cell-free DNA Assay Validations: A Joint Consensus Recommendation of the Association for Molecular Pathology and College of American Pathologists. J Mol Diagn. 2023 Oct 6:S1525-1578(23)00219-2. doi: 10.1016/j.jmoldx.2023.09.004. Epub ahead of print. PMID: 37806433.