Abstract
DNA released from cells into the peripheral blood is known as cell-free DNA (cfDNA), representing a promising noninvasive source of biomarkers that could be utilized to manage Diffuse Large B-Cell Lymphoma (DLBCL), among other diseases. The procedure for purification and handling of cfDNA is not yet standardized, and various preanalytical variables may affect the yield and analysis of cfDNA, including the purification kits, blood collection tubes, and centrifugation regime. Therefore, we aimed to investigate the impact of these preanalytical variables on the yield of cfDNA by comparing three different purification kits DNeasy Blood & Tissue Kit (Qiagen), QIAamp Circulating Nucleic Acid Kit (Qiagen), and Quick-cfDNA Serum & Plasma Kit (Zymo Research). Two blood collection tubes (BCTs), EDTA-K2 and Cell-Free DNA (Streck), stored at four different time points before plasma was separated and cfDNA purified, were compared, and for EDTA tubes, two centrifugation regimes at 2000 × g and 3000 × g were tested. Additionally, we have tested the utility of long-term archival blood samples from DLBCL patients to detect circulating tumor DNA (ctDNA). We observed a higher cfDNA yield using the QIAamp Circulating Nucleic Acid Kit (Qiagen) purification kit, as well as a higher cfDNA yield when blood samples were collected in EDTA BCTs, with a centrifuge regime at 2000 × g. Moreover, ctDNA detection was feasible from archival plasma samples with a median storage time of nine years.
Highlights
Diffuse Large B-Cell Lymphoma (DLBCL) accounts for 30– 40% of all newly diagnosed non-Hodgkin lymphoma cases [1]
To investigate the effects of preanalytical steps on the quantity of cell-free DNA (cfDNA) in plasma samples, purification kits, blood collection tubes, storage time before processing whole blood, and centrifugation regimen were tested as variables for cfDNA yield and quality
The yield of cfDNA from blood plasma was assessed using three different DNA purification kits: DNeasy Blood & Tissue Kit (Qiagen), QIAamp Circulating Nucleic Acid Kit (Qiagen), and Quick-cfDNA Serum & Plasma Kit (Zymo Research). cfDNA was purified from blood samples drawn from three healthy volunteers who simultaneously had 3 × 10 ml peripheral blood drawn for each purification kit, and cfDNA was purified from plasma in duplicates
Summary
Diffuse Large B-Cell Lymphoma (DLBCL) accounts for 30– 40% of all newly diagnosed non-Hodgkin lymphoma cases [1]. Tissue biopsies are currently used to diagnose DLBCL patients with the genetic limitation that the biopsied tissue might not represent the whole somatic mutational profile of individual patients due to the existence of subclonal mutations and metastasis [6]. Obtaining several biopsies during follow-up is usually unfeasible in clinical practice once the response is achieved due to its invasive nature and diminished or undetectable tumor size [6]. To overcome these obstacles, cell-free DNA (cfDNA) can potentially be used as a malignant DNA source, genuinely representing the whole genetic profile of individual DLBCL patients
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