Biofluid Biopsy-based Diagnostics and Sample Prep: Rapid Isolation and Detection of DNA/RNA Biomarkers Directly from Blood and Plasma

Held in conjunction with Biofluid Biopsies and Companion Diagnostics 2014

27 Oct 2014, at 18:15-20:15 in San Diego, California, USA

The analysis of circulating cell free (cf) DNA and RNA continues to become more widely used for cancer diagnostics and management. The parallel development of new methods that allow rapid isolation directly from small amounts of blood or plasma will also be necessary to advance biological fluid biopsies-based point of care (POC) diagnostic applications.

We have now demonstrated a dielectrophoretic (DEP) based approach that allows cf-DNA and RNA biomarkers from chronic lymphocytic leukemia (CLL) patients to be isolated in about 10 minutes directly from a small volume (25ul) of patient whole blood or plasma. 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 cf-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 Vp-p 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. cf-DNA was then removed from device and PCR amplified using VHL patient specific primers. Elution of the cf-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, cf-DNA for “biofluid 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 cf-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.

Dr. Heller will Frame the Sample Prep Challenges and Approaches in the Point-of-Care Biofluid-based Diagnostics Development Space.

Key Topics Addressed in this Short Course:

• Circulating Free DNA (cfDNA) Sample Prep Approaches for Developing Biofluid Biopsies and Point-of-Care (POC) Diagnostics
• Isolation of Various Circulating Biomarkers (cfDNA, cfDNA, and Exosomes) from Biological Fluids--Review of Current Methodologies & Approaches
  
• New Devices using DEP for Isolating cfDNA from Various Cancer Classes
• Emerging Trends in Circulating Biomarker-based Product Development

All Short Course Attendees Receive Electronic Copies on CD of Publications, Scientific Reviews, and PowerPoint Presentations on the Market and Technology Landscape of Biofluid Biopsies and Sample Preparation for use in R&D Planning, Business Planning--An Excellent Reference Resource.

Who Should Attend:

• Academic Researchers working in the cfDNA, CTCs, extracellular vesicles (EVs) fields as well as those seeking to study various categories of circulating biomarkers
• Industry Researchers working in companies involved in biomarker analysis and diagnostics product development