Recommendations for Cell-Free DNA Assay Validations
Open accessAbstract
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.
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Pre-analytical variables including collection tube type, sample volume, time to processing, centrifugation protocol, storage conditions, freeze-thaw exposure, DNA extraction method, cfDNA input, sequencing depth, and error-suppression strategy can affect assay performance.
“Collection tube type, sample volume, time to processing, centrifugation protocol, storage conditions, freeze–thaw exposure, DNA extraction method, cfDNA input, sequencing depth, and error-suppression strategy can all affect assay performance [ 11 , 12 ].”
From Signals Alongside Scans: A Genomics-Guided Framework for Liquid Biopsy in Bone and Soft-Tissue Sarcomas (2026) · cites this paper as [12]
Tumor-informed assays can improve sensitivity by tracking patient-specific alterations identified from tissue sequencing.
“Tumor-informed assays can improve sensitivity by tracking patient-specific alterations identified from tissue sequencing”
From Signals Alongside Scans: A Genomics-Guided Framework for Liquid Biopsy in Bone and Soft-Tissue Sarcomas (2026) · cites this paper as [12]
MRD detection requires substantially greater analytical sensitivity than baseline genotyping because tumor-derived DNA may be present at very low levels after surgery or definitive therapy.
“MRD detection is the most clinically ambitious use of ctDNA in sarcoma. It requires substantially greater analytical sensitivity than baseline genotyping because tumor-derived DNA may be present at very low levels after surgery or definitive therapy [ 5 , 9 , 10 , 11 , 12 ].”
From Signals Alongside Scans: A Genomics-Guided Framework for Liquid Biopsy in Bone and Soft-Tissue Sarcomas (2026) · cites this paper as [12]
Blood collection tube type, sample volume, time from venipuncture to plasma separation, centrifugation protocol, storage temperature, freeze-thaw exposure, and cfDNA extraction method can influence cfDNA yield, leukocyte DNA contamination, fragment size, tumor fraction, and downstream assay performance.
“Blood collection tube type, sample volume, time from venipuncture to plasma separation, centrifugation protocol, storage temperature, freeze–thaw exposure, and cfDNA extraction method can all influence cfDNA yield, leukocyte DNA contamination, fragment size, tumor fraction, and downstream assay performance [ 5 , 11 , 12 , 40 ].”
From Signals Alongside Scans: A Genomics-Guided Framework for Liquid Biopsy in Bone and Soft-Tissue Sarcomas (2026) · cites this paper as [12]
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Vitale, S. R., Sieuwerts, A. M., Beije, N., Kraan, J., Angus, L., Mostert, B., Reijm, E. A., Van, N. M., van Marion, R., Dirix, L. Y., et al. (2018) · Journal of Molecular Diagnostics · doi:10.1016/j.jmoldx.2018.08.010 · Cited by 21 · see citing works
Zheng, F.-F., Shen, W.-H., Gong, F., Hu, Z.-D., Lippi, G., Šimundić, A.-M., Bossuyt, P. M., Plebani, M., & Zhang, K. (2021) · Annals of Translational Medicine · doi:10.21037/atm-21-1665 · Cited by 18 · see citing works
Pan-cancer methylome analysis for cancer diagnosis and classification of cancer cell of origin
Shimizu, D., Taniue, K., Matsui, Y., Haeno, H., Araki, H., Miura, F., Fukunaga, M., Shiraishi, K., Miyamoto, Y., Tsukamoto, S., et al. (2021) · Cancer Gene Therapy · doi:10.1038/s41417-021-00401-w · Cited by 17 · see citing works
Swystun, V. A., Green, F. H. Y., Dennis, J., et al. (2018) · Trials · doi:10.1186/s13063-018-2720-6 · Cited by 12 · see citing works
Taylor, F., Bradford, J. R., Woll, P. J., et al. (2016) · Advances in experimental medicine and biology · doi:10.1007/978-3-319-42044-8_6 · Cited by 8 · see citing works
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Alabid, I., Yousef, A. J., Patni, M. M., Aloti, R. A., Qassim, Z. A.-A. M., Alothman-Agha, A. A., Hamdy, H. A., Noury, F. M., & Abdelfattah, M. T. (2026) · Cells · doi:10.3390/cells15141271
- Authors
- Lockwood, C. M., Borsu, L., Cankovic, M., Earle, J. S. L., Gocke, C. D., Hameed, M., Jordan, D., Lopategui, J. R., Pullambhatla, M., Reuther, J., Rumilla, K. M., Tafe, L. J., Temple-Smolkin, R. L., Terraf, P., & Tsimberidou, A. M.
- Affiliations
- University of Washington (US)
- Memorial Sloan Kettering Cancer Center (US)
- Henry Ford Hospital (US)
- Hartford Hospital (US)
- Johns Hopkins University (US)
- Association for Molecular Pathology (US)
- Cedars-Sinai Medical Center (US)
- Invitae (United States) (US)
- Mayo Clinic (US)
- Dartmouth–Hitchcock Medical Center (US)
- The University of Texas MD Anderson Cancer Center (US)
- Corresponding author
- Lockwood, Christina M.
- ORCID
- Venue
- The Journal of Molecular Diagnostics
- Publisher
- Elsevier BV
- Published
- 2023-12
- Volume
- 25
- Issue
- 12
- Pages
- 876-897
- ISSN
- 1525-1578 (ISSN-L)
- OpenAlex
- W4387398478
- Cited by
- 96
- References
- 82
- Citations by year
- 2026: 45 · 2025: 27 · 2024: 23 · 2023: 1
- FWCI
- 12.23
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Licence
- Locations
- Journal of Molecular Diagnostics (publishedVersion)
- PubMed (publishedVersion)
- Funders
- Association for Molecular Pathology
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › Cancer Genomics and Diagnostics
- Health Sciences › Medicine › Physiology › Clinical Laboratory Practices and Quality Control
- Health Sciences › Medicine › Pediatrics, Perinatology and Child Health › Prenatal Screening and Diagnostics
- Keywords
- circulating tumor DNA, liquid biopsy, assay standardization, pre-analytical variables, lymphoma, solid tumors, precision oncology
- MeSH terms
- Pathologists, Cell-Free Nucleic Acids, Humans, Neoplasms, United States, Consensus, Pathology, Molecular
About these checks
Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.
Statistics and arithmetic are recomputed, not judged.
Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.
What counts as a claim ›How we measure confidence ›How we check this ›
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