Commutability of ctDNA reference materials: native-plasma vs synthetic-matrix head-to-head and independent studies

1. Summary

Native plasma cfDNA has a modal fragment length of about 160–167 bp [1][4]. Two independent groups compared commercial reference materials with native patient or maternal plasma cfDNA and found measurable differences in fragment-size profile [1][2]. No study located this pass ran a formal IFCC/CLSI EP30-style commutability assessment of a commercial ctDNA reference material against patient plasma, and no study compared a native-plasma-matrix format with a synthetic-matrix format of the same material on the same assay. Both absences are recorded in section 5.

2. What Changed

_Baseline (v1). No prior version; this establishes the starting point for future diffs._

3. Details

Independent multi-material comparison against patient cfDNA (closest to target (b))

• Hallermayr et al. 2023. Three laboratories used a duplex-sequencing NGS (next-generation sequencing) assay to test six commercial cfDNA reference materials against 42 patient plasma samples collected in Streck or PAXgene tubes. Materials: Seraseq ctDNA Complete / mutation mix v1–v2, Seraseq Myeloid ctDNA Mix, SensID, Horizon OncoSpan cfDNA, Twist cfDNA Pan-cancer, and Covaris-sheared Coriell NA24385 gDNA. The laboratories analysed cfDNA isolated from the materials, so the two matrix formats were not compared [1] [single-source].
• Fragment size: "the Twist reference material was the only reference material tested with an insert size distribution similar to native cfDNA. The SeraCare reference material as well has a slightly similar insert size distribution … The remaining cfDNA reference materials (Horizon, SeraCare_Myo, SensID, Coriell) show a broad distribution of DNA fragment size comparable to mechanical DNA fragmentation" [1] [single-source].
• Background noise: Twist and SeraCare "showed significantly higher numbers of variants compared to the patient samples in all five VAF (variant allele frequency) intervals" (0.01%–5%). The authors attribute this to variants "present in the reference materials as a result of the manufacturing process" [1] [single-source].
• Conclusion (verbatim): "No reference material resembled native cfDNA in all performance metrics investigated." The word "commutability" does not appear in the full text [1] [single-source].
• Conflicts of interest: two authors were employed by Limbus Medical Technologies GmbH. No author declared an affiliation with a reference-material vendor [1].

Independent Seraseq vs native plasma comparison, NIPT context (adjacent to target (c))

• Giroux et al. (CHU de Québec, no competing interests declared) sequenced six replicates each of eight Seraseq NIPT genotypes using their natural-maternal-plasma workflow. "Fragment size profiles exhibited slight but consistent deviations from natural plasma, notably after size selection." The authors concluded these differences "did not affect analytical performance" [2] [single-source]. This is a prenatal material, not a ctDNA/MRD material. Abstract only.

Consortium ring trials using commercial contrived material (no patient-plasma comparator)

• BLOODPAC JFDI: nine laboratories tested "commercially available contrived materials" at 0.1%–5.0% VAF. Concordance and sensitivity were high above 0.1% VAF and lower at 0.1% [3] [single-source]. Read at abstract depth: no commutability comparison with patient plasma is described there. The author list includes a Horizon Discovery affiliation.
• IMI CANCER-ID: Seraseq ctDNA material was used "as purified ctDNA (study setup A) or encapsulated in a plasma-like matrix format (study setup B)". The two setups used different assays, so they are not a matrix head-to-head. cfDNA from diagnostic-leukapheresis and Streck patient plasma showed "the majority of fragment ranging around 160 bp and multiples thereof" [4] [single-source].

Native-plasma-background and nucleosomal designs (development papers, no commutability test)

• CANCER-ID multicentre extraction study: mononucleosomal DNA from lung-cancer cell lines was spiked into pooled healthy-donor plasma and extracted at 15 sites. "Some kits preferentially recovered spiked mnDNA over endogenous ccfDNA" [5] [single-source].
• China NIFDC panel (Xu et al.): sheared cell-line gDNA was spiked into "basal plasma cfDNA" and diluted to 0.1%–3% with basal plasma. At 0.1%, "15 samples had inconsistent results" across five manufacturers [11] [single-source].
• Blood-TMB reference materials: fragmented, size-selected cell-line DNA gave "DNA fragment sizes … similar to those found within patient plasma-derived cell-free DNA" [6] [single-source · manufacturer]. Three authors are LGC Clinical Diagnostics employees, and LGC "manufactured the reference materials used in this study."
• Micrococcal-nuclease digestion of cell-line nucleosomes gave dominant peaks at 128 bp and 143 bp [7] [single-source]. A Korean nucleosomal-DNA EGFR material showed "approximately 150–170 bp", with no VAF difference against paired gDNA by dPCR (digital polymerase chain reaction) (Mann-Whitney p > 0.05) [8] [single-source]. Neither paper tested its material against patient plasma.

Fragmentation and commutability — viral-load analogue

• Hayden et al. tested 42 CMV-positive and 41 EBV-positive plasma samples on 10 qPCR assays in 5 laboratories. "Commutability showed marked improvement following fragmentation of both CMV standards but not after fragmentation of the EBV standards" [9] [single-source].
• Across 11 extraction methods, 50 bp and 100 bp fragments showed "poor yields of less than 20%" [13] [single-source].

Expert and review statements

• The 2023 ELBS workshop (44 experts) emphasised materials that "closely mimic patient-derived ctDNA", including "plasma-like reference materials" [10] [single-source]. A 2026 review defines commutability as reference materials that "behave like patient specimens across diverse workflows" and proposes it as a harmonisation pillar [12] [single-source].

4. Sources

[1] Hallermayr A et al. Impact of cfDNA Reference Materials on Clinical Performance of Liquid Biopsy NGS Assays — Cancers (Basel) 15(20):5024, MDPI (published 2023-10-17; accessed 2026-09-25). PMID (PubMed identifier) 37894392; DOI 10.3390/cancers15205024; PMC10605119 — https://pmc.ncbi.nlm.nih.gov/articles/PMC10605119/ [peer-reviewed] [2] Giroux S et al. SeraSeq reference materials support non-invasive cfDNA screening methods — Clinical Biochemistry (published online 2025-11-11, issue 2026 Jan; accessed 2026-09-25). PMID 41232587; DOI 10.1016/j.clinbiochem.2025.111046 — https://pubmed.ncbi.nlm.nih.gov/41232587/ [peer-reviewed] [3] Hernandez KM et al. Contrived Materials and a Data Set for the Evaluation of Liquid Biopsy Tests: A BLOODPAC Community Study — J Mol Diagn (published 2023 Mar, day not given in the PubMed record; accessed 2026-09-25). PMID 36828596; DOI 10.1016/j.jmoldx.2022.12.003 — https://pubmed.ncbi.nlm.nih.gov/36828596/ [peer-reviewed] [4] Weber S et al. Technical Evaluation of Commercial Mutation Analysis Platforms and Reference Materials for Liquid Biopsy Profiling — Cancers (Basel) 12(6):1588 (published 2020-06-16; accessed 2026-09-25). PMID 32560092; DOI 10.3390/cancers12061588; PMC7352370 — https://pmc.ncbi.nlm.nih.gov/articles/PMC7352370/ [peer-reviewed] [5] Lampignano R et al. Multicenter Evaluation of Circulating Cell-Free DNA Extraction and Downstream Analyses for the Development of Standardized (Pre)analytical Work Flows — Clinical Chemistry (published 2020-01-01; accessed 2026-09-25). PMID 31628139; DOI 10.1373/clinchem.2019.306837 — https://pubmed.ncbi.nlm.nih.gov/31628139/ [peer-reviewed] [6] Raiber-Moreau EA et al. Development and validation of blood tumor mutational burden reference standards — Genes Chromosomes Cancer (published online 2022-12-07; accessed 2026-09-25). PMID 36326821; DOI 10.1002/gcc.23100; PMC10107199 — https://pubmed.ncbi.nlm.nih.gov/36326821/ [peer-reviewed] [7] Tang Y et al. Establishment of Reference Measurement Procedure for TP53 R175H/R248W Detection and a Novel Preparation Method for ctDNA Reference Material — Genes (Basel) (published 2025-05-14; accessed 2026-09-25). PMID 40428398; DOI 10.3390/genes16050576; PMC12110974 — https://pubmed.ncbi.nlm.nih.gov/40428398/ [peer-reviewed] [8] Hong SR et al. Development and characterization of nucleosomal DNA-based reference materials for the epidermal growth factor receptor gene liquid biopsy — Analytical and Bioanalytical Chemistry (published online 2026-04-06; accessed 2026-09-25). PMID 41936644; DOI 10.1007/s00216-026-06475-5; PMC13221357 — https://pmc.ncbi.nlm.nih.gov/articles/PMC13221357/ [peer-reviewed] [9] Hayden RT et al. Impact of Fragmentation on Commutability of Epstein-Barr Virus and Cytomegalovirus Quantitative Standards — J Clin Microbiol (published 2019-12-23; accessed 2026-09-25). PMID 31619529; DOI 10.1128/JCM.00888-19; PMC6935903 — https://pubmed.ncbi.nlm.nih.gov/31619529/ [peer-reviewed] [10] Deans ZC et al. Reference materials and external quality assessment for liquid biopsy assays: Expert opinions from the European Liquid Biopsy Society ctDNA workshop — Cancer Treatment and Research Communications (published online 2026-02-19; accessed 2026-09-25). PMID 41734680; DOI 10.1016/j.ctarc.2026.101151 — https://pubmed.ncbi.nlm.nih.gov/41734680/ [peer-reviewed] [11] Xu J et al. Construction of a reference material panel for detecting KRAS/NRAS/EGFR/BRAF/MET mutations in plasma ctDNA — J Clin Pathol (published online 2020-08-17, issue 2021 May; accessed 2026-09-25). PMID 32817175; DOI 10.1136/jclinpath-2020-206745; PMC8070650 — https://pubmed.ncbi.nlm.nih.gov/32817175/ [peer-reviewed] [12] Shibeeb S et al. Harmonising ctDNA Measurement in Haematological Malignancies: Traceability, Commutability and Reporting — Diagnostics (Basel) (published 2026-04-01; accessed 2026-09-25). PMID 41975767; DOI 10.3390/diagnostics16071056; PMC13072925 — https://pubmed.ncbi.nlm.nih.gov/41975767/ [peer-reviewed] [13] Cook L et al. Does Size Matter? Comparison of Extraction Yields for Different-Sized DNA Fragments by Seven Different Routine and Four New Circulating Cell-Free Extraction Methods — J Clin Microbiol (published 2018 Dec, day not given in the PubMed record; accessed 2026-09-25). PMID 30282788; DOI 10.1128/JCM.01061-18; PMC6258844 — https://pubmed.ncbi.nlm.nih.gov/30282788/ [peer-reviewed]

5. Sourcing & Gaps

Well established: Native plasma cfDNA has a modal fragment length of about 160–167 bp. This rests on two independent patient cohorts [1][4].

Thin: Every study-specific finding is [single-source]. [6] is also [manufacturer] because LGC co-authored it and made the material. [2], [3], [5], [6], [7], [9]–[13] were read at abstract depth only. [1], [4] and [8] were read in full text through PMC. The full text of [3] (ScienceDirect) and of [1] on the MDPI site both returned HTTP 403; [1] was read through PMC instead. [3]'s author list includes Horizon Discovery, Illumina and Bio-Rad affiliations.

Rescoped from class 3: None in this report.

Out of scope: CLSI EP30 (2nd edition) and IFCC commutability recommendations (PMID 29348163) are behind a purchase wall or were not opened. They were not used as sources. Vendor product-page commutability statements are covered in the reference-products overview. JCO 2024 commentary PMID 38754043 was found, but PubMed has no abstract for it and it was not read. bioRxiv preprint 10.1101/2023.09.06.554514 ("Standardisation of cell-free DNA measurements", an international comparability study) was found by search. The fetch returned HTTP 429 and the bioRxiv API returned nothing, so it was not retrieved and is not cited.

Not searched vs. not found:

• (a) Head-to-head of the same material in native-plasma vs synthetic-matrix format on one assay: not found. Searched with literature ("ctDNA reference standard synthetic plasma matrix", "ctDNA reference material human plasma matrix spiked", "cell-free DNA reference material commutable plasma") and WebSearch. [4] uses both formats, but in different study setups with different assays.
• (b) An independent EP30/IFCC-design commutability study (regression or difference-in-bias against native clinical samples, run across two or more measurement procedures) of a commercial ctDNA reference material: not found. Searched with literature ("ctDNA reference material commutability", "commutability cfDNA", "commutability circulating DNA", "commutability digital PCR reference material mutation") and WebSearch. The only ctDNA-titled commutability hit was a review [12]. The closest study is [1]: it compares materials with native cfDNA on one assay chemistry, but it is not a commutability design and does not use the term. No independent study of Seraseq ctDNA v4 or of the HDx synthetic-matrix formats specifically was found.
• (c) Fragment-size comparisons: found only in [1] (six materials vs patients) and [2] (NIPT). The other papers [6]–[8] report their own material's profile without a patient comparator, or with a comparator stated only in words.
• MRD (molecular residual disease)-range VAF (below 0.01%): no study above tested commutability at that level. Not found.

[inference] In [1], one material resembled native cfDNA in fragment size (Twist) but was among those with excess low-VAF variants. The materials suited to sensitivity work were those with mechanical-shear-like size profiles. This suggests that "native-like fragmentomics" and "fit for LoD" can pull apart. That bears on idea-003 claims 31 and 63, but it is one duplex-sequencing chemistry and not a commutability result.

[inference] [9] is the only located study that directly tested fragmentation against commutability. Its result was analyte-dependent (CMV improved, EBV did not). It is a virology analogue, and any carry-over to tumour-informed MRD assays is untested.

6. Claim Candidates