Abstract

Rapid Isolation and Detection of DNA/RNA Biomarkers from Blood and Plasma “Key to Liquid Biopsy Diagnostics”

Michael Heller, Distinguished Scientist - Knight Cancer Institute, Center for Cancer Early Detection and Research (CEDAR), Oregon Health & Science University (OHSU)

Cfc-DNA isolated from fifteen CLL patient blood samples and eleven CLL patient plasma samples was PCR amplified to identify the VHL genotype and then sequenced. The DNA sequencing results for ccf-DNA isolated by DEP were compared to two gold standard methods for CLL analysis. New DEP microarray devices specifically designed for the isolation of ccf-DNA/RNA from high conductance buffer, blood, plasma and serum samples were obtained from Biological Dynamics (La Jolla, CA). DEP experiments were carried out by adding about 25ul of blood or plasma sample to the DEP microarray device. The DEP field was applied at 10 kHz and 20 V for 3 minutes. The microarray was then washed and examined by epifluorescent microscopy. Overall, the fluorescent ccf-DNA levels were higher in most of the CLL patient samples when compared to the fluorescent DNA levels obtained from the normal blood samples. Ccf-DNA was then removed from device and PCR amplified using VHL patient specific primers. Elution of the ccf-DNA from the DEP device for quantification and PCR analysis required less than ten minutes. The PCR and DNA sequencing results for ccf-DNA isolated from 15 CLL patient blood samples by the DEP process were exactly comparable to results obtained using conventional methods, which take several hours to complete, are more laborious (many steps) and require much larger blood/plasma samples. The results for eight of eleven CLL plasma samples compared well with the gold standards. Generally, ccf-DNA for “liquid biopsies” is isolated form plasma using relatively long and involved processes which are not suitable for point of care (POC) diagnostics. The new DEP devices are now being used to collect ccf-DNA/RNA from solid tumor cancer and TBI patient blood and plasma samples. The stage for new “seamless sample to answer” liquid biopsy POC diagnostics is being set.

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