Fig 1.
Flow chart of the experimental setup.
(A) Blood collection: Blood from at least 20 subjects per experiment (S1-S20+) was drawn in random order with 10 replicate tubes per subject divided in 5 PAXgene Blood ccfDNA Tubes and 5 Streck CellFree DNA BCTs. Only completely drawn tubes (10ml blood) were included. (B) Spike-in & storage: 4 concentrations (c1-c4) of spike-in material was pipetted in the blood directly after draw, The fifth tube was left un-spiked as negative control (c0). All tubes were stored for 3 days at 25°C. (C) Plasma-generation & -pooling: Plasma was separated with a centrifugation protocol recommended by the tube suppliers. Complete plasma was removed from each tube. All plasma aliquots of one spike-level within one tube type were pooled. Plasma volume was measured of each pool and calculation of tube equivalent aliquot volume was done. (D) ccfDNA isolation, PCR assays and LOD calculation: Plasma pools were split in 20+ aliquots with a tube equivalent volume each. CcfDNA was isolated from each aliquot using the QIAamp Circulating Nucleic Acid Kit and the 5ml plasma input protocol. For eluate QC all eluates were analyzed by Qubit and Bioanalyzer measurements. QPCR assays targeting the spiked EGFR or BRAF gene mutations were run with all eluates. Probit based calculation of the LOD95 values were carried out for all qPCR (sub-)assays.
Fig 2.
Quality control: CcfDNA yield and fragment distribution.
(A) Qubit 1x dsDNA HS Assay-Kit. (B) Exemplary Bioanalyzer electropherograms. A 2100 Bioanalyzer instrument and the associated High Sensitivity DNA Kit were used. FU stands for fluorescence units and s for seconds run time.
Fig 3.
Detection rates and confidence intervals.
Calculations and graphs were executed by probit-model-based Arcus Quickstat Statistical Software Version 1.1.0.137. Green lines represent the calculated LOD and purple lines represent the corresponding confidence intervals. CI stands for confidence interval.
Fig 4.
Formaldehyde content in plasma was analyzed and quantified using Quantofix Formaldehyde stripes.