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Clinical Chemistry and Laboratory Medicine (CCLM)

Published in Association with the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM)

Editor-in-Chief: Plebani, Mario

Ed. by Gillery, Philippe / Greaves, Ronda / Lackner, Karl J. / Lippi, Giuseppe / Melichar, Bohuslav / Payne, Deborah A. / Schlattmann, Peter


IMPACT FACTOR 2018: 3.638

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1437-4331
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Volume 56, Issue 2

Issues

Results of the first external quality assessment scheme (EQA) for isolation and analysis of circulating tumour DNA (ctDNA)

Verena Haselmann
  • Corresponding author
  • Institute for Clinical Chemistry, Medical Faculty Mannheim of the University of Heidelberg, University Hospital Mannheim, Mannheim, Germany
  • Email
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/ Parviz Ahmad-Nejad
  • Institute for Medical Laboratory Diagnostics, Centre for Clinical and Translational Research (CCTR), HELIOS Hospital, Witten/Herdecke University, Wuppertal, Germany
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/ Wolf J. Geilenkeuser
  • Reference-Institute for Bioanalytics, German Society for Clinical Chemistry and Laboratory Medicine (DGKL), Bonn, Germany
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/ Angelika Duda
  • Institute for Clinical Chemistry, Medical Faculty Mannheim of the University of Heidelberg, University Hospital Mannheim, Mannheim, Germany
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/ Merle Gabor
  • Institute for Clinical Chemistry, Medical Faculty Mannheim of the University of Heidelberg, University Hospital Mannheim, Mannheim, Germany
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/ Romy Eichner
  • Institute for Clinical Chemistry, Medical Faculty Mannheim of the University of Heidelberg, University Hospital Mannheim, Mannheim, Germany
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/ Simon Patton
  • European Molecular Genetic Quality Network (EMQN), Manchester Centre for Genomic Medicine, St Mary’s Hospital, Manchester, UK
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/ Michael Neumaier
  • Corresponding author
  • Institute for Clinical Chemistry, Medical Faculty Mannheim of the University of Heidelberg, University Hospital Mannheim, Mannheim, Germany
  • Email
  • Other articles by this author:
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Published Online: 2017-08-25 | DOI: https://doi.org/10.1515/cclm-2017-0283

Abstract

Background:

Circulating tumour DNA (ctDNA) is considered to have a high potential for future management of malignancies. This pilot external quality assessment (EQA) scheme aimed to address issues of analytical quality in this new area of laboratory diagnostics.

Methods:

The EQA scheme consisted of three 2-mL EDTA-plasma samples spiked with fragmented genomic DNA with a mutant allele frequency ranging from 0% to 10% dedicated to the analysis of nine known sequence variations in KRAS codon 12/13 and of BRAF V600E. Laboratories reported: (1) time elapsed for processing, (2) storage temperatures, (3) methods for extraction and quantification, (4) genotyping methodologies and (5) results.

Results:

Specimens were sent to 42 laboratories from 10 European countries; 72.3% reported to isolate cell-free DNA (cfDNA) manually, 62.5% used the entire plasma volume for cfDNA isolation and 38.5% used >10% of cfDNA extracted for downstream genotyping. Of the methods used for quantification, PicoGreen demonstrated the lowest coefficient of variation (33.7%). For genotyping, 11 different methods were reported with the highest error rate observed for Sanger sequencing and the lowest for highly sensitive approaches like digital PCR. In total, 197 genotypes were determined with an overall error rate of 6.09%.

Conclusions:

This pilot EQA scheme illustrates the current variability in multiple phases of cfDNA processing and analysis of ctDNA resulting in an overall error rate of 6.09%. The areas with the greatest variance and clinical impact included specimen volume, cfDNA quantification method, and preference of genotyping platform. Regarding quality assurance, there is an urgent need for harmonisation of procedures and workflows.

This article offers supplementary material which is provided at the end of the article.

Keywords: circulating DNA; external quality assessment scheme; liquid biopsy; liquid profiling; plasma

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About the article

Corresponding authors: Dr. med. Verena Haselmann and Professor Dr. med. Michael Neumaier, Institute for Clinical Chemistry, Medical Faculty Mannheim of the University of Heidelberg, University Hospital Mannheim, Theodor-Kutzer-Ufer 1-3, 68167 Mannheim, Germany, Phone: 0049-621-383-3561 (V. Haselmann); 0049-621-383-2222 (M. Neumaier), Fax: 0049-621-383-3819


Received: 2017-04-01

Accepted: 2017-07-25

Published Online: 2017-08-25

Published in Print: 2018-01-26


Author contributions: Scheme organisers: V.H., P.A.N., S.P., M.N.; scheme administrator: W.J.G.; technical performance: M.G., A.D., R.E., V.H.; data analyses: V.H., W.J.G.; writing of paper: V.H., P.A.N., S.P., M.N. All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.

Research funding: None declared.

Employment or leadership: None declared.

Honorarium: None declared.

Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.


Citation Information: Clinical Chemistry and Laboratory Medicine (CCLM), Volume 56, Issue 2, Pages 220–228, ISSN (Online) 1437-4331, ISSN (Print) 1434-6621, DOI: https://doi.org/10.1515/cclm-2017-0283.

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