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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

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Volume 53, Issue 12


Influence of storage conditions and extraction methods on the quantity and quality of circulating cell-free DNA (ccfDNA): the SPIDIA-DNAplas External Quality Assessment experience

Francesca Malentacchi
  • Corresponding author
  • Department of Biomedical, Experimental and Clinical Sciences, University of Florence, Florence, Italy
  • Email
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
/ Sara Pizzamiglio / Paolo Verderio / Mario Pazzagli
  • Department of Biomedical, Experimental and Clinical Sciences, University of Florence, Florence, Italy
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
/ Claudio Orlando
  • Department of Biomedical, Experimental and Clinical Sciences, University of Florence, Florence, Italy
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
/ Chiara Maura Ciniselli / Kalle Günther / Stefania Gelmini
  • Department of Biomedical, Experimental and Clinical Sciences, University of Florence, Florence, Italy
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
Published Online: 2015-04-17 | DOI: https://doi.org/10.1515/cclm-2014-1161


Background: Circulating cell-free DNA (ccfDNA) has been confirmed as a useful biomarker in cancer and pre-natal clinical practice. One of the main critical points in using ccfDNA is a lack of standardisation for sample processing methods, storage conditions, procedures for extraction, and quantification that can affect ccfDNA quality and quantity. We report the results obtained from the SPIDIA-DNAplas, one of the EU SPIDIA (Standardisation and improvement of generic pre-analytical tools and procedures for in vitro diagnostics) subprojects based on the implementation of an External Quality Assessment scheme for the evaluation of the influence of the pre-analytical phase on ccfDNA. This is the first reported quality control scheme targeting ccfDNA for pre-analytical phase studies.

Methods: Fifty-six laboratories throughout Europe were recruited. The participating laboratories received the same plasma sample and extracted ccfDNA by using their own procedures, at defined plasma storage conditions, and sent the isolated ccfDNA to the SPIDIA facility for analyses. Laboratory performance was evaluated by using specific quality parameters such as ccfDNA integrity (by multiplex PCR) and yield (by qPCR).

Results: The analysis of the ccfDNA extracted by the laboratories showed that most of them (53 of 56) were able to recover ccfDNA but only 12.5% recovered non-fragmented ccfDNA. Extraction methods specifically designed for ccfDNA preserved the integrity profile.

Conclusions: The evidence-based results of the SPIDIA-DNAplas EQA have been proposed as a basis for the development of a Technical Specification by the European Committee for standardisation (CEN).

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

Keywords: circulating cell-free DNA; External Quality Assessment; pre-analytical phase


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About the article

Corresponding author: Francesca Malentacchi, Department of Biomedical, Experimental and Clinical Sciences, University of Florence, Viale G. Pieraccini, 6, 50139 Florence, Italy, Phone: 0039 055 2758251, Fax: 0039 055 4271371, E-mail:

Received: 2014-11-26

Accepted: 2015-03-19

Published Online: 2015-04-17

Published in Print: 2015-11-01

Citation Information: Clinical Chemistry and Laboratory Medicine (CCLM), Volume 53, Issue 12, Pages 1935–1942, ISSN (Online) 1437-4331, ISSN (Print) 1434-6621, DOI: https://doi.org/10.1515/cclm-2014-1161.

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