Generic Protocols for the Analytical Validation of Next-Generation Sequencing-Based ctDNA Assays: A Joint Consensus Recommendation of the BloodPAC’s Analytical Variables Working Group
Open accessAbstract
Liquid biopsy, particularly the analysis of circulating tumor DNA (ctDNA), has demonstrated considerable promise for numerous clinical intended uses. Successful validation and commercialization of novel ctDNA tests have the potential to improve the outcomes of patients with cancer. The goal of the Blood Profiling Atlas Consortium (BloodPAC) is to accelerate the development and validation of liquid biopsy assays that will be introduced into the clinic. To accomplish this goal, the BloodPAC conducts research in the following areas: Data Collection and Analysis within the BloodPAC Data Commons; Preanalytical Variables; Analytical Variables; Patient Context Variables; and Reimbursement. In this document, the BloodPAC's Analytical Variables Working Group (AV WG) attempts to define a set of generic analytical validation protocols tailored for ctDNA-based Next-Generation Sequencing (NGS) assays. Analytical validation of ctDNA assays poses several unique challenges that primarily arise from the fact that very few tumor-derived DNA molecules may be present in circulation relative to the amount of nontumor-derived cell-free DNA (cfDNA). These challenges include the exquisite level of sensitivity and specificity needed to detect ctDNA, the potential for false negatives in detecting these rare molecules, and the increased reliance on contrived samples to attain sufficient ctDNA for analytical validation. By addressing these unique challenges, the BloodPAC hopes to expedite sponsors' presubmission discussions with the Food and Drug Administration (FDA) with the protocols presented herein. By sharing best practices with the broader community, this work may also save the time and capacity of FDA reviewers through increased efficiency.
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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 [11]
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 [11]
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 [11]
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 [11]
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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
- Godsey, J. H., Silvestro, A., Barrett, J. C., Bramlett, K., Chudova, D., Deras, I., Dickey, J., Hicks, J., Johann, D. J., Leary, R., Lee, J. S. H., McMullen, J., McShane, L., Nakamura, K., Richardson, A. O., Ryder, M., Simmons, J., Tanzella, K., Yee, L., & Leiman, L. C.
- Affiliations
- Illumina (United States) (US)
- Novartis Pharmaceuticals, Cambridge, MA
- AstraZeneca (United States) (US)
- Thermo Fisher Scientific (United States) (US)
- Guardant (United States) (US)
- Personal Genome Diagnostics, Baltimore, MD
- University of Southern California (US)
- University of Arkansas at Little Rock (US)
- National Institutes of Health (US)
- Sysmex Inostics, Baltimore, MD
- BloodPAC, Chicago, IL
- Corresponding author
- Leiman, Lauren C
- ORCID
- Venue
- Clinical Chemistry
- Publisher
- Oxford University Press (OUP)
- Published
- 2020-09-01
- Volume
- 66
- Issue
- 9
- Pages
- 1156-1166
- ISSN
- 0009-9147 (ISSN-L)
- 1530-8561
- PMC
- PMC7462123
- OpenAlex
- W3082663460
- Cited by
- 104
- References
- 25
- Citations by year
- 2026: 15 · 2025: 23 · 2024: 9 · 2023: 23 · 2022: 20 · 2021: 12 · 2020: 2
- FWCI
- 4.88
- Type
- journal article
- Language
- en
- Open access
- hybrid
- doi.org
- Licence
- Locations
- Clinical Chemistry (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- Funders
- National Cancer Institute
- National Institutes of Health
- NIH
- NCI
- BloodPAC Consortium
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › Cancer Genomics and Diagnostics
- Health Sciences › Medicine › Physiology › Clinical Laboratory Practices and Quality Control
- Life Sciences › Immunology and Microbiology › Immunology › Biosimilars and Bioanalytical Methods
- Keywords
- circulating tumor DNA, liquid biopsy, next-generation sequencing assay, analytical method validation, cell-free DNA, false negative detection, FDA regulatory review
- MeSH terms
- Liquid Biopsy, Circulating Tumor DNA, Humans, Neoplasms, Reference Standards, Biomarkers, Tumor, Guidelines as Topic, Validation Studies as Topic, High-Throughput Nucleotide Sequencing
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