Circulating Cell-Free DNA (ccfDNA) Isolation from Patients with Diffuse Large B-Cell Lymphoma (DLBCL): Comparative Analysis of Commercial Kits
Authors: de Oliveira Karolliny Silva , Culler Hebert Fabricio , Levy Débora , Filho José Roberto Assis , Nogueira Daniel Silva , de Almeida Lis Vilela , da Silva Luiz Henrique , Fonseca Fernando Luiz Affonso , Alves Sarah Isabel Pinto Monteiro do Nascimento , Lage Luis Alberto de Padua Covas , Pereira Juliana 
Published: November 2021

Abstract
Introduction: Isolation of circulating cell-free DNA (ccfDNA) and circulating tumor DNA (ctDNA) are useful methodologies for studies of neoplastic genetic material and can provide information about cancer heterogeneity, prognosis and minimal residual disease (MRD) [1,2]. Therefore, the recovery of ccfDNA in sufficient quantity and adequate purity is crucial to provide accurate and reproducible results. In this study, we investigate the efficiency of four different commercial kits to recover short fragments of DNA compatible with the ccfDNA.
Methods: Ten mL of total peripheral blood from seven patients with Diffuse Large B-Cell Lymphoma (DLBCL) were collected in EDTA and were immediately processed. Plasma was obtained by centrifugation at 1.900 x g for 10 min at 4 ºC, followed by other centrifugation at 20.000 x g for 10 min at 4 ºC to remove cellular debris. Aliquots of 1.0 mL of plasma were stored at -80 ºC until ccfDNA extraction. The ccfDNA extraction was performed using the same samples by the following kits: QIAamp MinElute ccfDNA kit (CFDNA - Qiagen, Hilden, Germany), QIAamp DNA Mini Kit (QBLOOD - Qiagen, Hilden, Germany), Illustra Blood GenomicPrep Mini Spin Kit (GE - GE Healthcare Bio-Sciences Corp, UK) and ccfDNA isolation kit Quick-ccfDNA Serum & Plasma Kit (ZYMO - Zymo Research, Irvine, USA) [Table 1] according to the manufacturer's recommendations. The ccfDNA obtained was eluted in 30 µL of elution buffer for the subsequent experiments. The measurement of the ccfDNA obtained for each kit was performed in a Qubit 2.0 fluorometer using Qubit dsDNA HS assay kit (Thermo Fischer Scientific, Waltham, USA). The analysis of integrity, purity and the size of the fragments of DNA were performed in the Agilent 2100 Bioanalyzer (Agilent Technologies, Germany) equipment using the High Sensitivity DNA kit (Agilent Technologies, Germany).
Results: The results obtained for each commercial kit in the Qubit-analysis were: CFDNA (0.27 ng/uL), QBLOOD (0.26 ng/uL), ZYMO (0.03 ng/uL) and GE (0.02 ng/uL) [Figure 1]. The Bionalyzer eletrophoresis showed that CFDNA kit was able to recover two different fragments sizes of ccfDNA one of 360-380 bp (0.114 ng/uL) and other of 160-180 bp (1.808 ng/uL). The DNA recovered with QBLOOD revealed sizes of 360-380 bp (0.031 ng/uL) and 160-180 bp (0.079 ng/uL). Both kits, GE and ZYMO were not able to isolate fragments of DNA smaller than 10.380 pb .
Conclusion: We demonstrated that QIAamp MinElute ccfDNA kit (CFDNA) was capable to recovery high quantity of small fragments of DNA compatible with ccfDNA. This result supports the use of protocols based in magnetic beads in experiments working with ccfDNA. Additionally, we highlight the importance to verify the quality and not only the quantity of the DNA extracted to confirm the presence of ccfDNA.
Figure 1 Figure 1.

Disclosures
No relevant conflicts of interest to declare.