Whole blood of 4 healthy donors was collected into either EDTA or PAXgene Blood ccfDNA Tubes. Blood was left at room temperature for up to 7 days. Each day, ccfDNA was isolated from samples, bisulphite converted (Epitect FAST kit, QIAGEN) and analyzed with the GEMINI assay (Clinical Genomics) based on 2 methylated (BCAT1 and IKZF1) and 1 control (ACTB) target. Three PCR replicates per target sequence were analyzed. ccfDNA from blood stored in EDTA tubes generated more false positives than from blood stored in PAXgene Blood ccfDNA Tubes (data courtesy of Clinical Genomics).
With the standard protocols (STA), predominantly small ccfDNA fragments are isolated. The large fragment protocols (LAF) efficiently co-purify large and small ccfDNA fragments. Both protocol lines consist of one protocol for 2.4 ml and one protocol for 4.8 plasma input.
The change in delta-CT values of target versus control signal for ccfDNA isolated from whole blood stored in EDTA and PAXgene Blood ccfDNA Tubes was measured at different storage intervals. Shown are the relative signals of the 3 mutations covered by the therascreen EGFR assay: [A] point mutation T790M, [B] deletion in exon 19 and [C] L858R. Each PCR was done in duplicate. To calculate the delta-CT value, the average CT of each mutant was subtracted from the average CT of the wildtype sub-assay. The average of all donors is shown in the figure for all tested time points. The therascreen EGFR assay defines a threshold for each sub-assays. Delta-CT values above this threshold result in false-negative calls. Results from the ccfDNA isolated from EDTA tubes exceeded this threshold over time whereas results from the PAXgene Blood ccfDNA Tubes remained stable.
A magnetic rod protected by a rod cover enters a well containing the sample and attracts the magnetic particles. The magnetic rod cover is positioned above another well and the magnetic particles are released. The QIAsymphony SP uses a magnetic head with an array of 24 magnetic rods, and can therefore process up to 24 samples simultaneously. Collection, transfer and release of magnetic particles is repeated several times during sample processing.
Results from two studies assessing the performance of the QIAsymphonyPAXgene Blood ccfDNA Kit. Study A compares ccfDNA yield and portion of double-stranded DNA of eluates generated with the QIAsymphonyPAXgene Blood ccfDNA Kit to eluates from the manual QIAamp Circulating Nucleic Acid Kit. Study B summarizes performance parameters for the PAXgene ccfDNA system. The QIAsymphonyPAXgene Blood ccfDNA Kit provides consistently accurate results.