Rapid electrokinetic isolation of cancer-related circulating cell-free DNA directly from blood

Avery Sonnenberg, Jennifer Y. Marciniak, Laura Rassenti, Emanuela M. Ghia, Elaine A. Skowronski, Sareh Manouchehri, James McCanna, George F. Widhopf, Thomas J. Kipps, Michael (Mike) Heller

Research output: Contribution to journalArticle

28 Citations (Scopus)

Abstract

BACKGROUND: Circulating cell-free DNA (ccf-DNA) is becoming an important biomarker for cancer diagnostics and therapy monitoring. The isolation of ccf-DNA from plasma as a "liquid biopsy" may begin to replace more invasive tissue biopsies for the detection and analysis of cancer-related mutations. Conventional methods for the isolation of ccf-DNA from plasma are costly, time-consuming, and complex, preventing the use of ccf-DNA biomarkers for point-of-care diagnostics and limiting other biomedical research applications. METHODS: We used anACelectrokinetic device to rapidly isolate ccf-DNA from 25 μL unprocessed blood. ccf-DNA from 15 chronic lymphocytic leukemia (CLL) patients and 3 healthy individuals was separated into dielectrophoretic (DEP) high-field regions, after which other blood components were removed by a fluidic wash. Concentrated ccf-DNA was detected by fluorescence and eluted for quantification, PCR, and DNA sequencing. The complete process, blood to PCR, required <10 min. ccf-DNA was amplified by PCR with immunoglobulin heavy chain variable region (IGHV)-specific primers to identify the unique IGHV gene expressed by the leukemic B-cell clone, and then sequenced. RESULTS: PCR and DNA sequencing results obtained by DEP from 25 μL CLL blood matched results obtained by use of conventional methods for ccf-DNA isolation from 1 mL plasma and for genomic DNA isolation from CLL patient leukemic B cells isolated from 15-20 mL blood. CONCLUSIONS: Rapid isolation of ccf-DNA directly from a drop of blood will advance disease-related biomarker research, accelerate the transition from tissue to liquid biopsies, and enable point-of-care diagnostic systems for patient monitoring.

Original languageEnglish (US)
Pages (from-to)500-509
Number of pages10
JournalClinical chemistry
Volume60
Issue number3
DOIs
StatePublished - Jan 1 2014
Externally publishedYes

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Blood
DNA
Neoplasms
Cell Separation
B-Cell Chronic Lymphocytic Leukemia
Point-of-Care Systems
Biopsy
Polymerase Chain Reaction
Immunoglobulin Heavy Chains
DNA Sequence Analysis
Biomarkers
Plasmas
B-Lymphocytes
Clone cells
Tissue
Patient monitoring
Physiologic Monitoring
Tumor Biomarkers
Liquids
Fluidics

ASJC Scopus subject areas

  • Clinical Biochemistry
  • Biochemistry, medical

Cite this

Sonnenberg, A., Marciniak, J. Y., Rassenti, L., Ghia, E. M., Skowronski, E. A., Manouchehri, S., ... Heller, M. M. (2014). Rapid electrokinetic isolation of cancer-related circulating cell-free DNA directly from blood. Clinical chemistry, 60(3), 500-509. https://doi.org/10.1373/clinchem.2013.214874

Rapid electrokinetic isolation of cancer-related circulating cell-free DNA directly from blood. / Sonnenberg, Avery; Marciniak, Jennifer Y.; Rassenti, Laura; Ghia, Emanuela M.; Skowronski, Elaine A.; Manouchehri, Sareh; McCanna, James; Widhopf, George F.; Kipps, Thomas J.; Heller, Michael (Mike).

In: Clinical chemistry, Vol. 60, No. 3, 01.01.2014, p. 500-509.

Research output: Contribution to journalArticle

Sonnenberg, A, Marciniak, JY, Rassenti, L, Ghia, EM, Skowronski, EA, Manouchehri, S, McCanna, J, Widhopf, GF, Kipps, TJ & Heller, MM 2014, 'Rapid electrokinetic isolation of cancer-related circulating cell-free DNA directly from blood', Clinical chemistry, vol. 60, no. 3, pp. 500-509. https://doi.org/10.1373/clinchem.2013.214874
Sonnenberg A, Marciniak JY, Rassenti L, Ghia EM, Skowronski EA, Manouchehri S et al. Rapid electrokinetic isolation of cancer-related circulating cell-free DNA directly from blood. Clinical chemistry. 2014 Jan 1;60(3):500-509. https://doi.org/10.1373/clinchem.2013.214874
Sonnenberg, Avery ; Marciniak, Jennifer Y. ; Rassenti, Laura ; Ghia, Emanuela M. ; Skowronski, Elaine A. ; Manouchehri, Sareh ; McCanna, James ; Widhopf, George F. ; Kipps, Thomas J. ; Heller, Michael (Mike). / Rapid electrokinetic isolation of cancer-related circulating cell-free DNA directly from blood. In: Clinical chemistry. 2014 ; Vol. 60, No. 3. pp. 500-509.
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AU - Skowronski, Elaine A.

AU - Manouchehri, Sareh

AU - McCanna, James

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N2 - BACKGROUND: Circulating cell-free DNA (ccf-DNA) is becoming an important biomarker for cancer diagnostics and therapy monitoring. The isolation of ccf-DNA from plasma as a "liquid biopsy" may begin to replace more invasive tissue biopsies for the detection and analysis of cancer-related mutations. Conventional methods for the isolation of ccf-DNA from plasma are costly, time-consuming, and complex, preventing the use of ccf-DNA biomarkers for point-of-care diagnostics and limiting other biomedical research applications. METHODS: We used anACelectrokinetic device to rapidly isolate ccf-DNA from 25 μL unprocessed blood. ccf-DNA from 15 chronic lymphocytic leukemia (CLL) patients and 3 healthy individuals was separated into dielectrophoretic (DEP) high-field regions, after which other blood components were removed by a fluidic wash. Concentrated ccf-DNA was detected by fluorescence and eluted for quantification, PCR, and DNA sequencing. The complete process, blood to PCR, required <10 min. ccf-DNA was amplified by PCR with immunoglobulin heavy chain variable region (IGHV)-specific primers to identify the unique IGHV gene expressed by the leukemic B-cell clone, and then sequenced. RESULTS: PCR and DNA sequencing results obtained by DEP from 25 μL CLL blood matched results obtained by use of conventional methods for ccf-DNA isolation from 1 mL plasma and for genomic DNA isolation from CLL patient leukemic B cells isolated from 15-20 mL blood. CONCLUSIONS: Rapid isolation of ccf-DNA directly from a drop of blood will advance disease-related biomarker research, accelerate the transition from tissue to liquid biopsies, and enable point-of-care diagnostic systems for patient monitoring.

AB - BACKGROUND: Circulating cell-free DNA (ccf-DNA) is becoming an important biomarker for cancer diagnostics and therapy monitoring. The isolation of ccf-DNA from plasma as a "liquid biopsy" may begin to replace more invasive tissue biopsies for the detection and analysis of cancer-related mutations. Conventional methods for the isolation of ccf-DNA from plasma are costly, time-consuming, and complex, preventing the use of ccf-DNA biomarkers for point-of-care diagnostics and limiting other biomedical research applications. METHODS: We used anACelectrokinetic device to rapidly isolate ccf-DNA from 25 μL unprocessed blood. ccf-DNA from 15 chronic lymphocytic leukemia (CLL) patients and 3 healthy individuals was separated into dielectrophoretic (DEP) high-field regions, after which other blood components were removed by a fluidic wash. Concentrated ccf-DNA was detected by fluorescence and eluted for quantification, PCR, and DNA sequencing. The complete process, blood to PCR, required <10 min. ccf-DNA was amplified by PCR with immunoglobulin heavy chain variable region (IGHV)-specific primers to identify the unique IGHV gene expressed by the leukemic B-cell clone, and then sequenced. RESULTS: PCR and DNA sequencing results obtained by DEP from 25 μL CLL blood matched results obtained by use of conventional methods for ccf-DNA isolation from 1 mL plasma and for genomic DNA isolation from CLL patient leukemic B cells isolated from 15-20 mL blood. CONCLUSIONS: Rapid isolation of ccf-DNA directly from a drop of blood will advance disease-related biomarker research, accelerate the transition from tissue to liquid biopsies, and enable point-of-care diagnostic systems for patient monitoring.

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