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Research ArticleSpecial Report

Assessment of Digital PCR as a Primary Reference Measurement Procedure to Support Advances in Precision Medicine

Alexandra S. Whale, Gerwyn M. Jones, Jernej Pavšič, Tanja Dreo, Nicholas Redshaw, Sema Akyurek, Muslum Akgoz, Carla Divieto, Maria PaolaSassi, Hua-Jun He, Kenneth D. Cole, Young-Kyung Bae, Sang-Ryoul Park, Liesbet Deprez, Philippe Corbisier, Sonia Garrigou, Valérie Taly, Raquel Larios, Simon Cowen, Denise M. O'Sullivan, Claire A. Bushell, Heidi Goenaga-Infante, Carole A. Foy, Alison J. Woolford, Helen Parkes, Jim F. Huggett, Alison S. Devonshire
DOI: 10.1373/clinchem.2017.285478 Published June 2018
Alexandra S. Whale
Molecular and Cell Biology Team, LGC, Teddington, Middlesex, UK;
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Gerwyn M. Jones
Molecular and Cell Biology Team, LGC, Teddington, Middlesex, UK;
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Jernej Pavšič
National Institute of Biology, Department of Biotechnology and Systems Biology, Ljubljana, Slovenia;Jožef Stefan International Postgraduate School, Ljubljana, Slovenia;
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Tanja Dreo
National Institute of Biology, Department of Biotechnology and Systems Biology, Ljubljana, Slovenia;
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Nicholas Redshaw
Molecular and Cell Biology Team, LGC, Teddington, Middlesex, UK;
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Sema Akyurek
TUBITAK National Metrology Institute (TUBITAK UME), Bioanalysis Laboratory, Gebze, Kocaeli, Turkey;
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Muslum Akgoz
TUBITAK National Metrology Institute (TUBITAK UME), Bioanalysis Laboratory, Gebze, Kocaeli, Turkey;
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Carla Divieto
INRIM Istituto Nazionale di Ricerca Metrologica, Turin, Italy;
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Maria PaolaSassi
INRIM Istituto Nazionale di Ricerca Metrologica, Turin, Italy;
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Hua-Jun He
Material Measurement Laboratory, National Institute of Standards and Technology, Gaithersburg, MD;
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Kenneth D. Cole
Material Measurement Laboratory, National Institute of Standards and Technology, Gaithersburg, MD;
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Young-Kyung Bae
Center for Bioanalysis, KRISS, Yuseong-gu, Daejeon, Republic of Korea;
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Sang-Ryoul Park
Center for Bioanalysis, KRISS, Yuseong-gu, Daejeon, Republic of Korea;
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Liesbet Deprez
Directorate for Health, Consumers and Reference Materials, Joint Research Centre (JRC), European Commission, Geel, Belgium;
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Philippe Corbisier
Directorate for Health, Consumers and Reference Materials, Joint Research Centre (JRC), European Commission, Geel, Belgium;
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Sonia Garrigou
INSERM UMR-S1147, CNRS SNC5014, Equipe labellisée Ligue Nationale contre le cancer, Paris Descartes University, Paris, France;
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Valérie Taly
INSERM UMR-S1147, CNRS SNC5014, Equipe labellisée Ligue Nationale contre le cancer, Paris Descartes University, Paris, France;
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Raquel Larios
Inorganic Analysis Team, LGC, Teddington, Middlesex, UK;
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Simon Cowen
Statistics Team, LGC, Teddington, Middlesex, UK;
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Denise M. O'Sullivan
Molecular and Cell Biology Team, LGC, Teddington, Middlesex, UK;
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Claire A. Bushell
Molecular and Cell Biology Team, LGC, Teddington, Middlesex, UK;
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Heidi Goenaga-Infante
Inorganic Analysis Team, LGC, Teddington, Middlesex, UK;
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Carole A. Foy
Molecular and Cell Biology Team, LGC, Teddington, Middlesex, UK;
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Alison J. Woolford
Molecular and Cell Biology Team, LGC, Teddington, Middlesex, UK;
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Helen Parkes
Molecular and Cell Biology Team, LGC, Teddington, Middlesex, UK;
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Jim F. Huggett
Molecular and Cell Biology Team, LGC, Teddington, Middlesex, UK;School of Biosciences and Medicine, Faculty of Health and Medical Science, University of Surrey, Guildford, UK.
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  • For correspondence: alison.devonshire@lgcgroup.comj.huggett@surrey.ac.uk
Alison S. Devonshire
Molecular and Cell Biology Team, LGC, Teddington, Middlesex, UK;
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  • For correspondence: alison.devonshire@lgcgroup.comj.huggett@surrey.ac.uk
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Abstract

Background: Genetic testing of tumor tissue and circulating cell-free DNA for somatic variants guides patient treatment of many cancers. Such measurements will be fundamental in the future support of precision medicine. However, there are currently no primary reference measurement procedures available for nucleic acid quantification that would support translation of tests for circulating tumor DNA into routine use.

Methods: We assessed the accuracy of digital PCR (dPCR) for copy number quantification of a frequently occurring single-nucleotide variant in colorectal cancer (KRAS c.35G>A, p.Gly12Asp, from hereon termed G12D) by evaluating potential sources of uncertainty that influence dPCR measurement.

Results: Concentration values for samples of KRAS G12D and wild-type plasmid templates varied by <1.2-fold when measured using 5 different assays with varying detection chemistry (hydrolysis, scorpion probes, and intercalating dyes) and <1.3-fold with 4 commercial dPCR platforms. Measurement trueness of a selected dPCR assay and platform was validated by comparison with an orthogonal method (inductively coupled plasma mass spectrometry). The candidate dPCR reference measurement procedure showed linear quantification over a wide range of copies per reaction and high repeatability and interlaboratory reproducibility (CV, 2%–8% and 5%–10%, respectively).

Conclusions: This work validates dPCR as an SI-traceable reference measurement procedure based on enumeration and demonstrates how it can be applied for assignment of copy number concentration and fractional abundance values to DNA reference materials in an aqueous solution. High-accuracy measurements using dPCR will support the implementation and traceable standardization of molecular diagnostic procedures needed for advancements in precision medicine.

  • Received for publication December 12, 2017.
  • Accepted for publication May 24, 2018.
  • © 2018 American Association for Clinical Chemistry

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Clinical Chemistry: 65 (2)
Vol. 65, Issue 2
February 2019
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Assessment of Digital PCR as a Primary Reference Measurement Procedure to Support Advances in Precision Medicine
Alexandra S. Whale, Gerwyn M. Jones, Jernej Pavšič, Tanja Dreo, Nicholas Redshaw, Sema Akyurek, Muslum Akgoz, Carla Divieto, Maria PaolaSassi, Hua-Jun He, Kenneth D. Cole, Young-Kyung Bae, Sang-Ryoul Park, Liesbet Deprez, Philippe Corbisier, Sonia Garrigou, Valérie Taly, Raquel Larios, Simon Cowen, Denise M. O'Sullivan, Claire A. Bushell, Heidi Goenaga-Infante, Carole A. Foy, Alison J. Woolford, Helen Parkes, Jim F. Huggett, Alison S. Devonshire
Clinical Chemistry Jan 2018, clinchem.2017.285478; DOI: 10.1373/clinchem.2017.285478
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Assessment of Digital PCR as a Primary Reference Measurement Procedure to Support Advances in Precision Medicine
Alexandra S. Whale, Gerwyn M. Jones, Jernej Pavšič, Tanja Dreo, Nicholas Redshaw, Sema Akyurek, Muslum Akgoz, Carla Divieto, Maria PaolaSassi, Hua-Jun He, Kenneth D. Cole, Young-Kyung Bae, Sang-Ryoul Park, Liesbet Deprez, Philippe Corbisier, Sonia Garrigou, Valérie Taly, Raquel Larios, Simon Cowen, Denise M. O'Sullivan, Claire A. Bushell, Heidi Goenaga-Infante, Carole A. Foy, Alison J. Woolford, Helen Parkes, Jim F. Huggett, Alison S. Devonshire
Clinical Chemistry Jan 2018, clinchem.2017.285478; DOI: 10.1373/clinchem.2017.285478

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