Skip to main content
SpringerLink
Log in

Cell-Free DNA: An Overview of Sample Types and Isolation Procedures

  • Protocol
  • First Online:
Cell-free DNA as Diagnostic Markers

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1909))

Abstract

The study of cell-free DNA (cfDNA) is often challenging due to genomic DNA contamination, low concentration, and high fragmentation. Therefore, it is important to optimize pre-analytical and analytical procedures in order to maximize the performance of cfDNA-based analyses.

In this chapter, we report the most common methods for the correct collection, centrifugation, storage, and DNA isolation from cell-free biological sources such as plasma, urines, cerebrospinal fluid, and pleural effusion fluid.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution

Subscribe and save

Springer+ Basic
$34.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or eBook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 139.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Hardcover Book
USD 179.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

Similar content being viewed by others

References

  1. Volckmar A-L, Sültmann H, Riediger A et al (2018) A field guide for cancer diagnostics using cell-free DNA: from principles to practice and clinical applications. Genes Chromosomes Cancer 57:123–139. https://doi.org/10.1002/gcc.22517

    Article  CAS  PubMed  Google Scholar 

  2. Xia Y, Huang C-C, Dittmar R et al (2016) Copy number variations in urine cell free DNA as biomarkers in advanced prostate cancer. Oncotarget 7:35818–35831. https://doi.org/10.18632/oncotarget.9027

    Article  PubMed  PubMed Central  Google Scholar 

  3. Salvi S, Gurioli G, Martignano F et al (2015) Urine cell-free DNA integrity analysis for early detection of prostate cancer patients. Dis Markers 2015:1–6. https://doi.org/10.1155/2015/574120

    Article  CAS  Google Scholar 

  4. Casadio V, Calistri D, Tebaldi M et al (2013) Urine cell-free DNA integrity as a marker for early bladder cancer diagnosis: preliminary data. Urol Oncol Semin Orig Invest 31:1744–1750. https://doi.org/10.1016/j.urolonc.2012.07.013

    Article  CAS  Google Scholar 

  5. Swinkels DW, de Kok JB, Hanselaar A et al (2000) Early detection of leptomeningeal metastasis by PCR examination of tumor-derived K-ras DNA in cerebrospinal fluid. Clin Chem 46:132–133

    CAS  PubMed  Google Scholar 

  6. Li Y, Pan W, Connolly ID et al (2016) Tumor DNA in cerebral spinal fluid reflects clinical course in a patient with melanoma leptomeningeal brain metastases. J Neurooncol 128:93–100. https://doi.org/10.1007/s11060-016-2081-5

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Pan W, Gu W, Nagpal S et al (2015) Brain tumor mutations detected in cerebral spinal fluid. Clin Chem 61:514–522. https://doi.org/10.1373/clinchem.2014.235457

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Kimura H, Fujiwara Y, Sone T et al (2006) EGFR mutation status in tumour-derived DNA from pleural effusion fluid is a practical basis for predicting the response to gefitinib. Br J Cancer 95:1390–1395. https://doi.org/10.1038/sj.bjc.6603428

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Jian G, Songwen Z, Ling Z et al (2010) Prediction of epidermal growth factor receptor mutations in the plasma/pleural effusion to efficacy of gefitinib treatment in advanced non-small cell lung cancer. J Cancer Res Clin Oncol 136:1341–1347. https://doi.org/10.1007/s00432-010-0785-z

    Article  CAS  PubMed  Google Scholar 

  10. Takahama T, Sakai K, Takeda M et al (2016) Detection of the T790M mutation of EGFR in plasma of advanced non-small cell lung cancer patients with acquired resistance to tyrosine kinase inhibitors (West Japan Oncology Group 8014LTR Study). Oncotarget 7:58492–58499. https://doi.org/10.18632/oncotarget.11303

    Article  PubMed  PubMed Central  Google Scholar 

  11. Asano H, Toyooka S, Tokumo M et al (2006) Detection of EGFR gene mutation in lung cancer by mutant-enriched polymerase chain reaction assay. Clin Cancer Res 12:43–48. https://doi.org/10.1158/1078-0432.CCR-05-0934

    Article  CAS  PubMed  Google Scholar 

  12. Sun K, Jiang P, Chan KCA et al (2015) Plasma DNA tissue mapping by genome-wide methylation sequencing for noninvasive prenatal, cancer, and transplantation assessments. Proc Natl Acad Sci 112:E5503–E5512. https://doi.org/10.1073/pnas.1508736112

    Article  CAS  PubMed  Google Scholar 

  13. Snyder MW, Kircher M, Hill AJ et al (2016) Cell-free DNA comprises an in vivo nucleosome footprint that informs its tissues-of-origin. Cell 164:57–68. https://doi.org/10.1016/j.cell.2015.11.050

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Leon SA, Shapiro B, Sklaroff DM, Yaros MJ (1977) Free DNA in the serum of cancer patients and the effect of therapy. Cancer Res 37:646–650

    CAS  PubMed  Google Scholar 

  15. Bettegowda C, Sausen M, Leary RJ et al (2014) Detection of circulating tumor DNA in early- and late-stage human malignancies. Sci Transl Med 6:224ra24. https://doi.org/10.1126/scitranslmed.3007094

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Diehl F, Schmidt K, Choti MA et al (2008) Circulating mutant DNA to assess tumor dynamics. Nat Med 14:985–990. https://doi.org/10.1038/nm.1789

    Article  CAS  PubMed  Google Scholar 

  17. El Messaoudi S, Mouliere F, Du Manoir S et al (2016) Circulating DNA as a strong multimarker prognostic tool for metastatic colorectal Cancer patient management care. Clin Cancer Res 22:3067–3077. https://doi.org/10.1158/1078-0432.CCR-15-0297

    Article  CAS  PubMed  Google Scholar 

  18. Thierry AR, Mouliere F, El Messaoudi S et al (2014) Clinical validation of the detection of KRAS and BRAF mutations from circulating tumor DNA. Nat Med 20:430–435. https://doi.org/10.1038/nm.3511

    Article  CAS  PubMed  Google Scholar 

  19. Abbosh C, Birkbak NJ, Wilson GA et al (2017) Phylogenetic ctDNA analysis depicts early-stage lung cancer evolution. Nature 545:446–451. https://doi.org/10.1038/nature22364

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Stroun M, Anker P, Maurice P et al (1989) Neoplastic characteristics of the DNA found in the plasma of cancer patients. Oncology 46:318–322. https://doi.org/10.1159/000226740

    Article  CAS  PubMed  Google Scholar 

  21. De Mattos-Arruda L, Weigelt B, Cortes J et al (2014) Capturing intra-tumor genetic heterogeneity by de novo mutation profiling of circulating cell-free tumor DNA: a proof-of-principle. Ann Oncol 25:1729–1735. https://doi.org/10.1093/annonc/mdu239

    Article  PubMed  PubMed Central  Google Scholar 

  22. Jamal-Hanjani M, Wilson GA, Horswell S et al (2016) Detection of ubiquitous and heterogeneous mutations in cell-free DNA from patients with early-stage non-small-cell lung cancer. Ann Oncol 27:862–867. https://doi.org/10.1093/annonc/mdw037

    Article  CAS  PubMed  Google Scholar 

  23. Murtaza M, Dawson S-J, Pogrebniak K et al (2015) Multifocal clonal evolution characterized using circulating tumour DNA in a case of metastatic breast cancer. Nat Commun 6:8760. https://doi.org/10.1038/ncomms9760

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Heitzer E, Ulz P, Geigl JB (2015) Circulating tumor DNA as a liquid biopsy for cancer. Clin Chem 61:112–123. https://doi.org/10.1373/clinchem.2014.222679

    Article  CAS  PubMed  Google Scholar 

  25. Fan HC, Blumenfeld YJ, Chitkara U et al (2008) Noninvasive diagnosis of fetal aneuploidy by shotgun sequencing DNA from maternal blood. Proc Natl Acad Sci U S A 105:16266–16271. https://doi.org/10.1073/pnas.0808319105

    Article  PubMed  PubMed Central  Google Scholar 

  26. Page K, Guttery DS, Zahra N et al (2013) Influence of plasma processing on recovery and analysis of circulating nucleic acids. PLoS One 8:e77963. https://doi.org/10.1371/journal.pone.0077963

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. El Messaoudi S, Rolet F, Mouliere F, Thierry AR (2013) Circulating cell free DNA: preanalytical considerations. Clin Chim Acta 424:222–230. https://doi.org/10.1016/j.cca.2013.05.022

    Article  CAS  PubMed  Google Scholar 

  28. Volik S, Alcaide M, Morin RD, Collins C (2016) Cell-free DNA (cfDNA): clinical significance and utility in cancer shaped by emerging technologies. Mol Cancer Res 14:898–908. https://doi.org/10.1158/1541-7786.MCR-16-0044

    Article  CAS  PubMed  Google Scholar 

  29. Underhill HR, Kitzman JO, Hellwig S et al (2016) Fragment length of circulating tumor DNA. PLoS Genet 12:e1006162. https://doi.org/10.1371/journal.pgen.1006162

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Board RE, Williams VS, Knight L et al (2008) Isolation and extraction of circulating tumor DNA from patients with small cell lung cancer. Ann N Y Acad Sci 1137:98–107. https://doi.org/10.1196/annals.1448.020

    Article  CAS  PubMed  Google Scholar 

  31. Jung M, Klotzek S, Lewandowski M et al (2003) Changes in concentration of DNA in serum and plasma during storage of blood samples. Clin Chem 49:1028–1029

    Article  CAS  Google Scholar 

  32. Lee TH, Montalvo L, Chrebtow V, Busch MP (2001) Quantitation of genomic DNA in plasma and serum samples: higher concentrations of genomic DNA found in serum than in plasma. Transfusion 41:276–282

    Article  CAS  Google Scholar 

  33. Tuck MK, Chan DW, Chia D et al (2009) Standard operating procedures for serum and plasma collection: early detection research network consensus statement standard operating procedure integration working group. J Proteome Res 8:113–117. https://doi.org/10.1021/pr800545q

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Chang Y, Tolani B, Nie X et al (2017) Review of the clinical applications and technological advances of circulating tumor DNA in cancer monitoring. Ther Clin Risk Manag 13:1363–1374. https://doi.org/10.2147/TCRM.S141991

    Article  PubMed  PubMed Central  Google Scholar 

  35. Kang Q, Henry NL, Paoletti C et al (2016) Comparative analysis of circulating tumor DNA stability in K3EDTA, Streck, and CellSave blood collection tubes. Clin Biochem 49:1354–1360. https://doi.org/10.1016/j.clinbiochem.2016.03.012

    Article  CAS  PubMed  Google Scholar 

  36. Barra GB, Santa Rita TH, Vasques J de A et al (2015) EDTA-mediated inhibition of DNases protects circulating cell-free DNA from ex vivo degradation in blood samples. Clin Biochem 48:976–981. https://doi.org/10.1016/j.clinbiochem.2015.02.014

    Article  CAS  PubMed  Google Scholar 

  37. Norton SE, Lechner JM, Williams T, Fernando MR (2013) A stabilizing reagent prevents cell-free DNA contamination by cellular DNA in plasma during blood sample storage and shipping as determined by digital PCR. Clin Biochem 46:1561–1565. https://doi.org/10.1016/j.clinbiochem.2013.06.002

    Article  CAS  PubMed  Google Scholar 

  38. van Dessel LF, Beije N, Helmijr JCA et al (2017) Application of circulating tumor DNA in prospective clinical oncology trials - standardization of preanalytical conditions. Mol Oncol 11:295–304. https://doi.org/10.1002/1878-0261.12037

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Norton SE, Luna KK, Lechner JM et al (2013) A new blood collection device minimizes cellular DNA release during sample storage and shipping when compared to a standard device. J Clin Lab Anal 27:305–311. https://doi.org/10.1002/jcla.21603

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Fernando MR, Chen K, Norton S et al (2010) A new methodology to preserve the original proportion and integrity of cell-free fetal DNA in maternal plasma during sample processing and storage. Prenat Diagn 30:418–424. https://doi.org/10.1002/pd.2484

    Article  CAS  PubMed  Google Scholar 

  41. Medina Diaz I, Nocon A, Mehnert DH et al (2016) Performance of Streck cfDNA blood collection tubes for liquid biopsy testing. PLoS One 11:e0166354. https://doi.org/10.1371/journal.pone.0166354

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Page K, Powles T, Slade MJ et al (2006) The importance of careful blood processing in isolation of cell-free DNA. Ann N Y Acad Sci 1075:313–317. https://doi.org/10.1196/annals.1368.042

    Article  CAS  PubMed  Google Scholar 

  43. Wong IH, Lo YM, Yeo W et al (2000) Frequent p15 promoter methylation in tumor and peripheral blood from hepatocellular carcinoma patients. Clin Cancer Res 6:3516–3521

    CAS  PubMed  Google Scholar 

  44. Liggett T, Melnikov A, Yi Q et al (2010) Differential methylation of cell-free circulating DNA among patients with pancreatic cancer versus chronic pancreatitis. Cancer 116:1674–1680. https://doi.org/10.1002/cncr.24893

    Article  CAS  PubMed  Google Scholar 

  45. Lecomte T, Berger A, Zinzindohoué F et al (2002) Detection of free-circulating tumor-associated DNA in plasma of colorectal cancer patients and its association with prognosis. Int J Cancer 100:542–548. https://doi.org/10.1002/ijc.10526

    Article  CAS  PubMed  Google Scholar 

  46. Wang S, An T, Wang J et al (2010) Potential clinical significance of a plasma-based KRAS mutation analysis in patients with advanced non-small cell lung cancer. Clin Cancer Res 16:1324–1330. https://doi.org/10.1158/1078-0432.CCR-09-2672

    Article  CAS  PubMed  Google Scholar 

  47. Christensen E, Nordentoft I, Vang S et al (2018) Optimized targeted sequencing of cell-free plasma DNA from bladder cancer patients. Sci Rep 8:1917. https://doi.org/10.1038/s41598-018-20282-8

    Article  PubMed  PubMed Central  Google Scholar 

  48. Wang BG, Huang H-Y, Chen Y-C et al (2003) Increased plasma DNA integrity in cancer patients. Cancer Res 63:3966–3968

    CAS  PubMed  Google Scholar 

  49. Jiang W-W, Zahurak M, Goldenberg D et al (2006) Increased plasma DNA integrity index in head and neck cancer patients. Int J Cancer 119:2673–2676. https://doi.org/10.1002/ijc.22250

    Article  CAS  PubMed  Google Scholar 

  50. Pinzani P, Salvianti F, Cascella R et al (2010) Allele specific Taqman-based real-time PCR assay to quantify circulating BRAFV600E mutated DNA in plasma of melanoma patients. Clin Chim Acta 411:1319–1324. https://doi.org/10.1016/j.cca.2010.05.024

    Article  CAS  PubMed  Google Scholar 

  51. Pinzani P, Salvianti F, Zaccara S et al (2011) Circulating cell-free DNA in plasma of melanoma patients: qualitative and quantitative considerations. Clin Chim Acta 412:2141–2145. https://doi.org/10.1016/j.cca.2011.07.027

    Article  CAS  PubMed  Google Scholar 

  52. Salvianti F, Pinzani P, Verderio P et al (2012) Multiparametric analysis of cell-free DNA in melanoma patients. PLoS One 7:e49843. https://doi.org/10.1371/journal.pone.0049843

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Utting M, Werner W, Dahse R et al (2002) Microsatellite analysis of free tumor DNA in urine, serum, and plasma of patients: a minimally invasive method for the detection of bladder cancer. Clin Cancer Res 8:35–40

    CAS  PubMed  Google Scholar 

  54. Kamel AM, Teama S, Fawzy A, El Deftar M (2016) Plasma DNA integrity index as a potential molecular diagnostic marker for breast cancer. Tumor Biol 37:7565–7572. https://doi.org/10.1007/s13277-015-4624-3

    Article  CAS  Google Scholar 

  55. Leng S, Zheng J, Jin Y et al (2018) Plasma cell-free DNA level and its integrity as biomarkers to distinguish non-small cell lung cancer from tuberculosis. Clin Chim Acta 477:160–165. https://doi.org/10.1016/j.cca.2017.11.003

    Article  CAS  PubMed  Google Scholar 

  56. deVos T, Tetzner R, Model F et al (2009) Circulating methylated SEPT9 DNA in plasma is a biomarker for colorectal cancer. Clin Chem 55:1337–1346. https://doi.org/10.1373/clinchem.2008.115808

    Article  CAS  PubMed  Google Scholar 

  57. Ren CC, Miao XH, Yang B et al (2006) Methylation status of the fragile histidine triad and E-cadherin genes in plasma of cervical cancer patients. Int J Gynecol Cancer 16:1862–1867. https://doi.org/10.1111/j.1525-1438.2006.00669.x

    Article  CAS  PubMed  Google Scholar 

  58. Feng J, Gang F, Li X et al (2013) Plasma cell-free DNA and its DNA integrity as biomarker to distinguish prostate cancer from benign prostatic hyperplasia in patients with increased serum prostate-specific antigen. Int Urol Nephrol 45:1023–1028. https://doi.org/10.1007/s11255-013-0491-2

    Article  CAS  PubMed  Google Scholar 

  59. Chan KCA, Yeung S-W, Lui W-B et al (2005) Effects of preanalytical factors on the molecular size of cell-free DNA in blood. Clin Chem 51:781–784. https://doi.org/10.1373/clinchem.2004.046219

    Article  CAS  PubMed  Google Scholar 

  60. Bronkhorst AJ, Aucamp J, Pretorius PJ (2015) Cell-free DNA: preanalytical variables. Clin Chim Acta 450:243–253. https://doi.org/10.1016/j.cca.2015.08.028

    Article  CAS  PubMed  Google Scholar 

  61. McGann LE, Yang H, Walterson M (1988) Manifestations of cell damage after freezing and thawing. Cryobiology 25:178–185. https://doi.org/10.1016/0011-2240(88)90024-7

    Article  CAS  PubMed  Google Scholar 

  62. Sozzi G, Roz L, Conte D et al (2005) Effects of prolonged storage of whole plasma or isolated plasma DNA on the results of circulating DNA quantification assays. J Natl Cancer Inst 97:1848–1850. https://doi.org/10.1093/jnci/dji432

    Article  CAS  PubMed  Google Scholar 

  63. Sherwood JL, Corcoran C, Brown H et al (2016) Optimised pre-analytical methods improve KRAS mutation detection in circulating tumour DNA (ctDNA) from patients with non-small cell lung cancer (NSCLC). PLoS One 11:e0150197. https://doi.org/10.1371/journal.pone.0150197

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Tänzer M, Balluff B, Distler J et al (2010) Performance of epigenetic markers SEPT9 and ALX4 in plasma for detection of colorectal precancerous lesions. PLoS One 5:e9061. https://doi.org/10.1371/journal.pone.0009061

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Holdenrieder S, Burges A, Reich O et al (2008) DNA integrity in plasma and serum of patients with malignant and benign diseases. Ann N Y Acad Sci 1137:162–170. https://doi.org/10.1196/annals.1448.013

    Article  CAS  PubMed  Google Scholar 

  66. Sorber L, Zwaenepoel K, Deschoolmeester V et al (2017) A comparison of cell-free DNA isolation kits. J Mol Diagn 19:162–168. https://doi.org/10.1016/j.jmoldx.2016.09.009

    Article  CAS  PubMed  Google Scholar 

  67. Ali N, Rampazzo RCP, Costa ADT, Krieger MA (2017) Current nucleic acid extraction methods and their implications to point-of-care diagnostics. Biomed Res Int 2017:9306564. https://doi.org/10.1155/2017/9306564

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  68. Skvortsova TE, Rykova EY, Tamkovich SN et al (2006) Cell-free and cell-bound circulating DNA in breast tumours: DNA quantification and analysis of tumour-related gene methylation. Br J Cancer 94:1492–1495. https://doi.org/10.1038/sj.bjc.6603117

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  69. Melnikov A, Scholtens D, Godwin A, Levenson V (2009) Differential methylation profile of ovarian cancer in tissues and plasma. J Mol Diagn 11:60–65. https://doi.org/10.2353/jmoldx.2009.080072

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  70. Madhavan D, Wallwiener M, Bents K et al (2014) Plasma DNA integrity as a biomarker for primary and metastatic breast cancer and potential marker for early diagnosis. Breast Cancer Res Treat 146:163–174. https://doi.org/10.1007/s10549-014-2946-2

    Article  CAS  PubMed  Google Scholar 

  71. Stötzer OJ, Lehner J, Fersching-Gierlich D et al (2014) Diagnostic relevance of plasma DNA and DNA integrity for breast cancer. Tumor Biol 35:1183–1191. https://doi.org/10.1007/s13277-013-1158-4

    Article  CAS  Google Scholar 

  72. Gautschi O, Huegli B, Ziegler A et al (2007) Origin and prognostic value of circulating KRAS mutations in lung cancer patients. Cancer Lett 254:265–273. https://doi.org/10.1016/j.canlet.2007.03.008

    Article  CAS  PubMed  Google Scholar 

  73. Swisher EM, Wollan M, Mahtani SM et al (2005) Tumor-specific p53 sequences in blood and peritoneal fluid of women with epithelial ovarian cancer. Am J Obstet Gynecol 193:662–667. https://doi.org/10.1016/j.ajog.2005.01.054

    Article  CAS  PubMed  Google Scholar 

  74. He Q, Chen H-Y, Bai E-Q et al (2010) Development of a multiplex MethyLight assay for the detection of multigene methylation in human colorectal cancer. Cancer Genet Cytogenet 202:1–10. https://doi.org/10.1016/j.cancergencyto.2010.05.018

    Article  CAS  PubMed  Google Scholar 

  75. García Moreira V, Prieto García B, de la Cera Martínez T, Álvarez Menéndez FV (2009) Elevated transrenal DNA (cell-free urine DNA) in patients with urinary tract infection compared to healthy controls. Clin Biochem 42:729–731. https://doi.org/10.1016/J.CLINBIOCHEM.2008.12.021

    Article  PubMed  Google Scholar 

  76. Devonshire AS, Whale AS, Gutteridge A et al (2014) Towards standardisation of cell-free DNA measurement in plasma: controls for extraction efficiency, fragment size bias and quantification. Anal Bioanal Chem 406:6499–6512. https://doi.org/10.1007/s00216-014-7835-3

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  77. Pérez-Barrios C, Nieto-Alcolado I, Torrente M et al (2016) Comparison of methods for circulating cell-free DNA isolation using blood from cancer patients: impact on biomarker testing. Transl lung Cancer Res 5:665–672. https://doi.org/10.21037/tlcr.2016.12.03

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  78. Yuan H, Zhu Z-Z, Lu Y et al (2012) A modified extraction method of circulating free DNA for epidermal growth factor receptor mutation analysis. Yonsei Med J 53:132. https://doi.org/10.3349/ymj.2012.53.1.132

    Article  CAS  PubMed  Google Scholar 

  79. Hufnagl C, Stöcher M, Moik M et al (2013) A modified phenol-chloroform extraction method for isolating circulating cell free DNA of tumor patients. J Nucleic Acids Invest 4:1. https://doi.org/10.4081/jnai.2013.4282

    Article  Google Scholar 

  80. Mauger F, Dulary C, Daviaud C et al (2015) Comprehensive evaluation of methods to isolate, quantify, and characterize circulating cell-free DNA from small volumes of plasma. Anal Bioanal Chem 407:6873–6878. https://doi.org/10.1007/s00216-015-8846-4

    Article  CAS  PubMed  Google Scholar 

  81. Hung M-S, Lin C-K, Leu S-W et al (2006) Epidermal growth factor receptor mutations in cells from non-small cell lung cancer malignant pleural effusions. Chang Gung Med J 29:373–379

    PubMed  Google Scholar 

  82. Izumchenko E, Chang X, Brait M et al (2015) Targeted sequencing reveals clonal genetic changes in the progression of early lung neoplasms and paired circulating DNA. Nat Commun 6:8258. https://doi.org/10.1038/ncomms9258

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  83. Wang Y, Springer S, Mulvey CL et al (2015) Detection of somatic mutations and HPV in the saliva and plasma of patients with head and neck squamous cell carcinomas. Sci Transl Med 7:293ra104. https://doi.org/10.1126/scitranslmed.aaa8507

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  84. Wu S-G, Gow C-H, Yu C-J et al (2008) Frequent epidermal growth factor receptor gene mutations in malignant pleural effusion of lung adenocarcinoma. Eur Respir J 32:924–930. https://doi.org/10.1183/09031936.00167407

    Article  CAS  PubMed  Google Scholar 

  85. Tsai T-H, Su K-Y, Wu S-G et al (2012) RNA is favourable for analysing EGFR mutations in malignant pleural effusion of lung cancer. Eur Respir J 39:677–684. https://doi.org/10.1183/09031936.00043511

    Article  CAS  PubMed  Google Scholar 

  86. Chang HW, Tsui KH, Shen LC et al (2007) Urinary cell-free DNA as a potential tumor marker for bladder cancer. Int J Biol Markers 22:287–294

    Article  CAS  Google Scholar 

  87. Su Y-H, Song J, Wang Z et al (2008) Removal of high-molecular-weight DNA by carboxylated magnetic beads enhances the detection of mutated K-ras DNA in urine. Ann N Y Acad Sci 1137:82–91. https://doi.org/10.1196/annals.1448.019

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  88. Gyurászová M, Kovalčíková A, Bábíčková J et al (2018) Cell-free nucleic acids in urine as potential biomarkers of kidney disease. J Appl Biomed 16:157. https://doi.org/10.1016/J.JAB.2018.01.007

    Article  Google Scholar 

  89. Botezatu I, Serdyuk O, Potapova G et al (2000) Genetic analysis of DNA excreted in urine: a new approach for detecting specific genomic DNA sequences from cells dying in an organism. Clin Chem 46:1078–1084

    CAS  PubMed  Google Scholar 

  90. Su Y-H, Wang M, Brenner DE et al (2004) Human urine contains small, 150 to 250 nucleotide-sized, soluble DNA derived from the circulation and may be useful in the detection of colorectal cancer. J Mol Diagn 6:101–107. https://doi.org/10.1016/S1525-1578(10)60497-7

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  91. Tencer J, Frick I-M, Öquist BW et al (1998) Size-selectivity of the glomerular barrier to high molecular weight proteins: upper size limitations of shunt pathways. Kidney Int 53:709–715. https://doi.org/10.1046/j.1523-1755.1998.00797.x

    Article  CAS  PubMed  Google Scholar 

  92. Melkonyan HS, Feaver WJ, Meyer E et al (2008) Transrenal nucleic acids: from proof of principle to clinical tests. Ann N Y Acad Sci 1137:73–81. https://doi.org/10.1196/annals.1448.015

    Article  CAS  PubMed  Google Scholar 

  93. Tsui NBY, Jiang P, Chow KCK et al (2012) High resolution size analysis of fetal DNA in the urine of pregnant women by paired-end massively parallel sequencing. PLoS One 7:e48319. https://doi.org/10.1371/journal.pone.0048319

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  94. Yao W, Mei C, Nan X, Hui L (2016) Evaluation and comparison of in vitro degradation kinetics of DNA in serum, urine and saliva: a qualitative study. Gene 590:142–148. https://doi.org/10.1016/j.gene.2016.06.033

    Article  CAS  PubMed  Google Scholar 

  95. Szarvas T, Kovalszky I, Bedi K et al (2007) Deletion analysis of tumor and urinary DNA to detect bladder cancer: urine supernatant versus urine sediment. Oncol Rep 18:405–409

    CAS  PubMed  Google Scholar 

  96. Ghanjati F, Beermann A, Hermanns T et al (2014) Unreserved application of epigenetic methods to define differences of DNA methylation between urinary cellular and cell-free DNA. Cancer Biomark 14:295–302. https://doi.org/10.3233/CBM-140407

    Article  CAS  PubMed  Google Scholar 

  97. Vaara ST, Lakkisto P, Immonen K et al (2016) Urinary biomarkers indicative of apoptosis and acute kidney injury in the critically ill. PLoS One 11:e0149956. https://doi.org/10.1371/journal.pone.0149956

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  98. Zancan M, Galdi F, Di Tonno F et al (2009) Evaluation of cell-free DNA in urine as a marker for bladder cancer diagnosis. Int J Biol Markers 24:147–155

    Article  CAS  Google Scholar 

  99. Stewart CM, Kothari PD, Mouliere F et al (2018) The value of cell-free DNA for molecular pathology. J Pathol 244:616. https://doi.org/10.1002/path.5048

    Article  CAS  PubMed  Google Scholar 

  100. Wang Y, Springer S, Zhang M et al (2015) Detection of tumor-derived DNA in cerebrospinal fluid of patients with primary tumors of the brain and spinal cord. Proc Natl Acad Sci U S A 112:9704–9709. https://doi.org/10.1073/pnas.1511694112

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  101. Liu B-L, Cheng J-X, Zhang W et al (2010) Quantitative detection of multiple gene promoter hypermethylation in tumor tissue, serum, and cerebrospinal fluid predicts prognosis of malignant gliomas. Neuro-Oncology 12:540–548. https://doi.org/10.1093/neuonc/nop064

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  102. Soh J, Toyooka S, Aoe K et al (2006) Usefulness of EGFR mutation screening in pleural fluid to predict the clinical outcome of gefitinib treated patients with lung cancer. Int J Cancer 119:2353–2358. https://doi.org/10.1002/ijc.22190

    Article  CAS  PubMed  Google Scholar 

  103. Zhang X, Zhao Y, Wang M et al (2008) Detection and comparison of epidermal growth factor receptor mutations in cells and fluid of malignant pleural effusion in non-small cell lung cancer. Lung Cancer 60:175–182. https://doi.org/10.1016/j.lungcan.2007.10.011

    Article  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Medical Biotechnologies, University of Siena, Siena, Italy

    Filippo Martignano

Authors
  1. Filippo Martignano
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. Biosciences Laboratory, IRCCS I.R.S.T, Meldola, Italy

    Valentina Casadio

  2. Biosciences Laboratory, IRCCS I.R.S.T, Meldola, Italy

    Samanta Salvi

Rights and permissions

Reprints and permissions

Copyright information

© 2019 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Martignano, F. (2019). Cell-Free DNA: An Overview of Sample Types and Isolation Procedures. In: Casadio, V., Salvi, S. (eds) Cell-free DNA as Diagnostic Markers. Methods in Molecular Biology, vol 1909. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8973-7_2

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-8973-7_2

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-8972-0

  • Online ISBN: 978-1-4939-8973-7

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation