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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 EMAIL logo , Sara Pizzamiglio , Paolo Verderio , Mario Pazzagli , Claudio Orlando , Chiara Maura Ciniselli , Kalle Günther and Stefania Gelmini
Published/Copyright: April 17, 2015
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Clinical Chemistry and Laboratory Medicine (CCLM)
From the journal Clinical Chemistry and Laboratory Medicine (CCLM) Volume 53 Issue 12

Abstract

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).

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

Acknowledgments

We gratefully acknowledge the contribution of all partners and participating laboratories.

Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.

Financial support: The SPIDIA-DNAplas study has been supported by European Union (FP7, “SPIDIA: Standardisation and improvement of generic pre-analytical tools and procedures for in vitro diagnostic,” grant agreement no. 222916).

Employment or leadership: None declared.

Honorarium: None declared.

Competing interests: The funding organisation(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.

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Supplemental Material

The online version of this article (DOI: 10.1515/cclm-2014-1161) offers supplementary material, available to authorized users.

Received: 2014-11-26
Accepted: 2015-3-19
Published Online: 2015-4-17
Published in Print: 2015-11-1

©2015 by De Gruyter

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https://doi.org/10.1515/cclm-2014-1161
Keywords for this article
circulating cell-free DNA; External Quality Assessment; pre-analytical phase

Articles in the same Issue

  1. Frontmatter
  2. Editorial
  3. Biomarkers of inflammatory bowel disease: ready for prime time?
  4. Review
  5. Discriminant indices for distinguishing thalassemia and iron deficiency in patients with microcytic anemia: a meta-analysis
  6. Mini Reviews
  7. Cell-free DNA for diagnosing myocardial infarction: not ready for prime time
  8. The Laboratory Medicine and the care of patients infected by the Ebola virus. Experience in a reference hospital of Madrid, Spain
  9. Opinion Papers
  10. Theranos phenomenon − part 2
  11. Considerations in parathyroid hormone testing
  12. Quantity quotient reporting. Comparison of various models
  13. Genetics and Molecular Diagnostics
  14. Assessing quality and functionality of DNA isolated from FFPE tissues through external quality assessment in tissue banks
  15. 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
  16. Non-invasive fetal ABO genotyping in maternal plasma using real-time PCR
  17. Screening non-deletion α-thalassaemia mutations in the HBA1 and HBA2 genes by high-resolution melting analysis
  18. General Clinical Chemistry and Laboratory Medicine
  19. European views on patients directly obtaining their laboratory test results
  20. Diagnostic performances of clinical laboratory tests using Triton X-100 to reduce the biohazard associated with routine testing of Ebola virus-infected patients
  21. Thrombin generation, D-dimer and protein S in uncomplicated pregnancy
  22. Multi-analyte analysis of non-vitamin K antagonist oral anticoagulants in human plasma using tandem mass spectrometry
  23. Second generation analysis of antinuclear antibody (ANA) by combination of screening and confirmatory testing
  24. Faecal leukocyte esterase activity is an alternative biomarker in inflammatory bowel disease
  25. Reference Values and Biological Variations
  26. Total folate and 5-methyltetrahydrofolate in the cerebrospinal fluid of children: correlation and reference values
  27. Effect of age and gender on reference intervals of red blood cell distribution width (RDW) and mean red cell volume (MCV)
  28. Infectious Diseases
  29. Inosine triphosphate pyrophosphohydrolase activity: more accurate predictor for ribavirin-induced anemia in hepatitis C infected patients than ITPA genotype
  30. The ratio of calprotectin to total protein as a diagnostic and prognostic marker for spontaneous bacterial peritonitis in patients with liver cirrhosis and ascites
  31. Letter to the Editors
  32. Misleading high-sensitivity troponin algorithm for NSTEMI in the ESC guidelines
  33. Adjustment of serum potassium for age and platelet count. A simple step forward towards personalized medicine
  34. The impact of Tween 20 on repeatability of amyloid β and tau measurements in cerebrospinal fluid
  35. Effect of storage time and temperature on the generation of reactive oxygen species in peripheral blood leukocytes
  36. Iohexol interference in the α2-globulin fraction of the serum protein capillary electrophoresis
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