Dielectrophoretic isolation and detection of cancer‐related circulating cell‐free DNA biomarkers from blood and plasma

Abstract

Conventional methods for the isolation of cancer-related circulating cell-free (ccf) DNA from patient blood (plasma) are time consuming and laborious. A DEP approach utilizing a microarray device now allows rapid isolation of ccf-DNA directly from a small volume of unprocessed blood. In this study, the DEP device is used to compare the ccf-DNA isolated directly from whole blood and plasma from 11 chronic lymphocytic leukemia (CLL) patients and one normal individual. Ccf-DNA from both blood and plasma samples was separated into DEP high-field regions, after which cells (blood), proteins, and other biomolecules were removed by a fluidic wash. The concentrated ccf-DNA was detected on-chip by fluorescence, and then eluted for PCR and DNA sequencing. The complete process from blood to PCR required less than 10 min; an additional 15 min was required to obtain plasma from whole blood. Ccf-DNA from the equivalent of 5 µL of CLL blood and 5 µL of plasma was amplified by PCR using Ig heavy-chain variable (IGHV) specific primers to identify the unique IGHV gene expressed by the leukemic B-cell clone. The PCR and DNA sequencing results obtained by DEP from all 11 CLL blood samples and from 8 of the 11 CLL plasma samples were exactly comparable to the DNA sequencing results obtained from genomic DNA isolated from CLL patient leukemic B cells (gold standard).

Detecting cancer biomarkers in blood: challenges for new molecular diagnostic and point-of-care tests using cell-free nucleic acids

“…To overcome these limitations, techniques have been developed that employ both immunologic and DEP (size-based) strategies to separate CTC more efficiently from other blood cells [99]. Other recent innovations include continuous flow DEP systems and newer DEP microarray devices capable of selecting by size [98,100,101]. Clearly, techniques to capture and analyze CTC for use in cancer diagnostics are rapidly evolving.…”

DNA methylation as a promising landscape: A simple blood test for breast cancer prediction

“…Presence of circulatory tumor DNA in low amounts and possibility of their contamination with normal cell DNA are serious limitations to consider them as reliable source. However, undergoing techniques may overcome these limitations to catch circulatory tumor cell DNA in good quantity and purity, giving them a momentum to be apply for diagnostic and prognostic purposes [9]. On the contrary, high amount and good quality of WBC DNA and more importantly its predictive potentials and implications concluded by several case-control studies make it a practical source to examine the epigenetic changes of WBCs before and during breast cancer development.…”

Liquid Biopsies in Oncology and the Current Regulatory Landscape

“…Others have reported data to suggest that the QIAamp Ò is superior in terms of cfNA yield and reproducibility [52]. Finally, several groups have been utilizing dielectrophoretic devices to rapidly isolate and detect DNA fragment sizes directly from whole blood [53] with the goal of eliminating an extraction step.…”