Cargando…
Preanalytical blood sample workup for cell‐free DNA analysis using Droplet Digital PCR for future molecular cancer diagnostics
In current molecular cancer diagnostics, using blood samples of cancer patients for the detection of genetic alterations in plasma (cell‐free) circulating tumor DNA (ctDNA) is an emerging practice. Since ctDNA levels in blood are low, highly sensitive Droplet Digital PCR (ddPCR) can be used for dete...
Autores principales: | , , , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
John Wiley and Sons Inc.
2017
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5633557/ https://www.ncbi.nlm.nih.gov/pubmed/28940814 http://dx.doi.org/10.1002/cam4.1184 |
_version_ | 1783269912156831744 |
---|---|
author | van Ginkel, Joost H. van den Broek, Daan A. van Kuik, Joyce Linders, Dorothé de Weger, Roel Willems, Stefan M. Huibers, Manon M. H. |
author_facet | van Ginkel, Joost H. van den Broek, Daan A. van Kuik, Joyce Linders, Dorothé de Weger, Roel Willems, Stefan M. Huibers, Manon M. H. |
author_sort | van Ginkel, Joost H. |
collection | PubMed |
description | In current molecular cancer diagnostics, using blood samples of cancer patients for the detection of genetic alterations in plasma (cell‐free) circulating tumor DNA (ctDNA) is an emerging practice. Since ctDNA levels in blood are low, highly sensitive Droplet Digital PCR (ddPCR) can be used for detecting rare mutational targets. In order to perform ddPCR on blood samples, a standardized procedure for processing and analyzing blood samples is necessary to facilitate implementation into clinical practice. Therefore, we assessed the technical sample workup procedure for ddPCR on blood plasma samples. Blood samples from healthy individuals, as well as lung cancer patients were analyzed. We compared different methods and protocols for sample collection, storage, centrifugation, isolation, and quantification. Cell‐free DNA (cfDNA) concentrations of several wild‐type targets and BRAF and EGFR‐mutant ctDNA concentrations quantified by ddPCR were primary outcome measurements. Highest cfDNA concentrations were measured in blood collected in serum tubes. No significant differences in cfDNA concentrations were detected between various time points of up to 24 h until centrifugation. Highest cfDNA concentrations were detected after DNA isolation with the Quick cfDNA Serum & Plasma Kit, while plasma isolation using the QIAamp Circulating Nucleic Acid Kit yielded the most consistent results. DdPCR results on cfDNA are highly dependent on multiple factors during preanalytical sample workup, which need to be addressed during the development of this diagnostic tool for cancer diagnostics in the future. |
format | Online Article Text |
id | pubmed-5633557 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2017 |
publisher | John Wiley and Sons Inc. |
record_format | MEDLINE/PubMed |
spelling | pubmed-56335572017-10-17 Preanalytical blood sample workup for cell‐free DNA analysis using Droplet Digital PCR for future molecular cancer diagnostics van Ginkel, Joost H. van den Broek, Daan A. van Kuik, Joyce Linders, Dorothé de Weger, Roel Willems, Stefan M. Huibers, Manon M. H. Cancer Med Clinical Cancer Research In current molecular cancer diagnostics, using blood samples of cancer patients for the detection of genetic alterations in plasma (cell‐free) circulating tumor DNA (ctDNA) is an emerging practice. Since ctDNA levels in blood are low, highly sensitive Droplet Digital PCR (ddPCR) can be used for detecting rare mutational targets. In order to perform ddPCR on blood samples, a standardized procedure for processing and analyzing blood samples is necessary to facilitate implementation into clinical practice. Therefore, we assessed the technical sample workup procedure for ddPCR on blood plasma samples. Blood samples from healthy individuals, as well as lung cancer patients were analyzed. We compared different methods and protocols for sample collection, storage, centrifugation, isolation, and quantification. Cell‐free DNA (cfDNA) concentrations of several wild‐type targets and BRAF and EGFR‐mutant ctDNA concentrations quantified by ddPCR were primary outcome measurements. Highest cfDNA concentrations were measured in blood collected in serum tubes. No significant differences in cfDNA concentrations were detected between various time points of up to 24 h until centrifugation. Highest cfDNA concentrations were detected after DNA isolation with the Quick cfDNA Serum & Plasma Kit, while plasma isolation using the QIAamp Circulating Nucleic Acid Kit yielded the most consistent results. DdPCR results on cfDNA are highly dependent on multiple factors during preanalytical sample workup, which need to be addressed during the development of this diagnostic tool for cancer diagnostics in the future. John Wiley and Sons Inc. 2017-09-21 /pmc/articles/PMC5633557/ /pubmed/28940814 http://dx.doi.org/10.1002/cam4.1184 Text en © 2017 The Authors. Cancer Medicine published by John Wiley & Sons Ltd. This is an open access article under the terms of the Creative Commons Attribution (http://creativecommons.org/licenses/by/4.0/) License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Clinical Cancer Research van Ginkel, Joost H. van den Broek, Daan A. van Kuik, Joyce Linders, Dorothé de Weger, Roel Willems, Stefan M. Huibers, Manon M. H. Preanalytical blood sample workup for cell‐free DNA analysis using Droplet Digital PCR for future molecular cancer diagnostics |
title | Preanalytical blood sample workup for cell‐free DNA analysis using Droplet Digital PCR for future molecular cancer diagnostics |
title_full | Preanalytical blood sample workup for cell‐free DNA analysis using Droplet Digital PCR for future molecular cancer diagnostics |
title_fullStr | Preanalytical blood sample workup for cell‐free DNA analysis using Droplet Digital PCR for future molecular cancer diagnostics |
title_full_unstemmed | Preanalytical blood sample workup for cell‐free DNA analysis using Droplet Digital PCR for future molecular cancer diagnostics |
title_short | Preanalytical blood sample workup for cell‐free DNA analysis using Droplet Digital PCR for future molecular cancer diagnostics |
title_sort | preanalytical blood sample workup for cell‐free dna analysis using droplet digital pcr for future molecular cancer diagnostics |
topic | Clinical Cancer Research |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5633557/ https://www.ncbi.nlm.nih.gov/pubmed/28940814 http://dx.doi.org/10.1002/cam4.1184 |
work_keys_str_mv | AT vanginkeljoosth preanalyticalbloodsampleworkupforcellfreednaanalysisusingdropletdigitalpcrforfuturemolecularcancerdiagnostics AT vandenbroekdaana preanalyticalbloodsampleworkupforcellfreednaanalysisusingdropletdigitalpcrforfuturemolecularcancerdiagnostics AT vankuikjoyce preanalyticalbloodsampleworkupforcellfreednaanalysisusingdropletdigitalpcrforfuturemolecularcancerdiagnostics AT lindersdorothe preanalyticalbloodsampleworkupforcellfreednaanalysisusingdropletdigitalpcrforfuturemolecularcancerdiagnostics AT dewegerroel preanalyticalbloodsampleworkupforcellfreednaanalysisusingdropletdigitalpcrforfuturemolecularcancerdiagnostics AT willemsstefanm preanalyticalbloodsampleworkupforcellfreednaanalysisusingdropletdigitalpcrforfuturemolecularcancerdiagnostics AT huibersmanonmh preanalyticalbloodsampleworkupforcellfreednaanalysisusingdropletdigitalpcrforfuturemolecularcancerdiagnostics |