Aim: Donor-derived cell-free DNA has emerged as a valuable biomarker in transplant medicine, providing non-invasive insights into graft health. Efficient extraction of cell-free DNA is a critical step for accurate downstream monitoring assays and the potential early detection of allograft rejection. However, most manual cell-free DNA extraction methods are labor-intensive and require significant hands-on time. Moreover, the manual approach poses technical challenges that can result in potential contamination or poor recovery rates. In this study, we evaluated an automated system for the extraction and purification of cfDNA.
Method: Thirty-three samples were processed in total, and the cfDNA yield and purity was measured using the Qubit Fluorometer and the Tapestation 4150 System, respectively. Additionally, the total time to completion was calculated and the hands-on time required. Blood samples from healthy donors were collected into collection tubes (Paxgene Blood ccfDNA Tubes or Streck Cell-Free DNA BCT RUO Tubes). Up to 8 mL of plasma was then centrifuged and processed according to the manufacturer's instructions. Samples were prepared following the Maxwell ccfDNA Plasma Kit protocol and loaded onto the HSM 2.0 Instrument. After completion, samples were transferred to prepared Maxwell cartridges, and the LV ccfDNA method was performed on the Maxwell CSC instrument in research mode. Varying elution volumes were also considered, to maximize concentration without compromising total recovery. All samples were subsequently sequenced utilizing AlloSeq cfDNA chemistry and sequenced on a Miseq with v3 150-cycle flow cells.
Results: Total yield per ml of plasma ranged from 16 ng to 245 ng. Concentrations ranged from 0.133ng/ul to 4.91 ng/ul. Decreasing the elution volume from the standard 75ul to 50 ul resulted in a 39% increase in concentration on average. The total yield lost was acceptable at a 7% average.
Conclusion: In conclusion, the utilization of an automated system for the extraction and purification of cell-free DNA presents a promising advancement in transplant medicine. This study demonstrates the efficiency of this automated approach in achieving high yields of cell-free DNA. Additionally, the automated system offers improved consistency and reduces the risk of contamination, addressing key challenges associated with manual extraction techniques.
