Stability of Nucleosomal DNA Fragments in Serum

Holdenrieder, Stefan; Mueller, Susanne; Stieber, P.

doi:10.1373/clinchem.2005.048454

Cited by 22 publications

(26 citation statements)

References 14 publications

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“…Apart from the method quality itself, preanalytical handling of samples prior to analysis in the laboratory can influence the final results, as is known for several research and routine parameters [25][26][27] . Hence, we examined the stability of the tested markers.…”

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confidence: 99%

Methodical and pre-analytical characteristics of a multiplex cancer biomarker immunoassay

Hermann

Dressen

Schildberg

et al. 2014

WJM

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AIM:To test the methodical and pre-analytical performance of a new multiplex cancer biomarker panel using magnetic beads. METHODS: The MILLIPLEX® MAP Human Circulating Cancer Biomarker Magnetic Bead Panel 1 comprises the tumor markers carcinoembryonic antigen, alpha-fetoprotein, total prostate-specific antigen, cancer antigen 15-3, cancer antigen 19-9, cancer antigen 125, cytokeratine 19-fragment, β-human chorionic gonadotropin, human epididymis protein 4, osteopontin, prolactin, the cell death and angiogenesis markers soluble Fas, soluble Fas-ligand, tumor necrosis factor related apoptosisinducing ligand, vascular endothelial growth factor and the immunological markers interleukin-6 (IL-6), IL-8, tumor necrosis factor-α, transforming growth factor α, fibroblast growth factor-2, macrophage migration inhibitory factor, leptin, hepatocyte growth factor, and stem cell factor. We determined intra-and inter-assay imprecision as well as dilution linearity using quality controls and serum pools. Furthermore, the stability of the 24 biomarkers examined in this panel was ascertained by testing the influence of different storage temperatures and time span before centrifugation. RESULTS:For all markers measured in the synthetic internal quality controls, the intra-assay imprecision ranged between 2.26% and 9.41%, while for 20 of 24 measured markers in the physiological serum pools, it ranged between 1.68% and 12.87%. The inter-assay imprecision ranged between 1.48%-17.12% for 23 biomarkers in synthetic, and between 4.59%-23.88% for 18 biomarkers in physiological quality controls. Here, single markers with very low concentration levels had increased imprecision rates. Dilution linearity was acceptable (70%-130% recovery) for 20 biomarkers. Regarding pre-analytical influencing factors, most markers were stable if blood centrifugation was delayed or if serum was stored for up to 24 h at 4 ℃ and 25 ℃ after centrifugation. Comparable results were obtained in serum and plasma for most markers. However, great changes were observed for single markers. CONCLUSION: MILLIPLEX® MAP Human Circulating Cancer Biomarker Magnetic Bead Panel 1 assay is a stable and precise method for detection of most biomarkers included in the kit. However, single markers have to be interpreted with care.© 2014 Baishideng Publishing Group Inc. All rights reserved.Key words: Multiplex immunoassay; Tumor marker; Cytokines; Cell death markers; Methodical evaluation Core tip: In this study, the methodological quality of a new research-use-only multiplex magnetic bead assay,

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confidence: 99%

Methodical and pre-analytical characteristics of a multiplex cancer biomarker immunoassay

Hermann

Dressen

Schildberg

et al. 2014

WJM

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“…The fact that the ELISA analysis of circulating nucleosomes requires the presence of histone-DNA complexes makes cross-linking and DNA fragmentation unnecessary. Accordingly, circulating nucleosomes have been used in immunologic assays (20 ) without any indication of a requirement for cross-linking (27 ).…”

Section: Discussionmentioning

confidence: 99%

“…Finally, soluble chromatin is immunoprecipitated with an antibody that recognizes the protein of interest. Because positive ELISA signals indicate the presence of soluble and stable histone complexes and short stretches of associated DNA (monoand oligonucleosomes) in the circulation and because there is no indication in the literature of a cross-linking requirement in immunologic assays (27 ), we omitted these steps and started directly with the preanalytical steps of immunoprecipitation. We modified the protocol of the Protein Agarose A Immunoprecipita-tion Kit (Roche Diagnostics) to immunoprecipitate nucleosomes from plasma.…”

Section: Chromatin Immunoprecipitationmentioning

confidence: 99%

Sequence-Specific Histone Methylation Is Detectable on Circulating Nucleosomes in Plasma

et al. 2008

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BACKGROUND: Alterations in DNA methylation and histone modifications have been implicated in carcinogenesis. Although tumor-specific alterations in DNA methylation can be detected in the serum and plasma of cancer patients, no data are available on the presence of histone modifications in circulating blood. We investigated whether histone methylation, as a model of histone modifications, is detectable in plasma. Because methylation at histone 3 lysine 9 (H3K9) has been demonstrated to be enriched at sites of repetitive ALU elements, we addressed the specificity of histone-methylation detection and hypothesized that if monomethylated H3K9 (H3K9me1) is detectable in plasma, the concentrations in mononucleosomes and oligonucleosomes would be different. We also analyzed a single-copy gene, CDKN2A.

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“…cfDNA Concentration -State of the Art One of the most extensive studies of this phase was by Holdenrieder et al [41]. Even if it cannot be generalized to cfDNA analysis since the study was performed on serum and measures nucleosome levels only, this work indicates the sensitivity of nucleosomes to temperature variations: they seem to be more sensitive at +37 C than at either +4 C or RT.…”

Section: Influence Of Storage Conditionsmentioning

confidence: 99%

Pre-analytical Requirements for Analyzing Nucleic Acids from Blood

Messaoudi

Thierry

2014

Advances in Predictive, Preventive and Personalised Medicine

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Circulating nucleic acids have received an increasing scrutiny over the past decade with some applications, such as in prenatal diagnosis and oncology, being on the verge of use in clinical practice. It is crucial to implement optimal standardization of pre-analytical procedures. Currently, this domain has been poorly studied and there is no well-established procedure. This chapter examines the literature on the pre-analytical factors affecting nucleic acids from blood drawing to the storage of circulating cell-free DNA extracts ready for analysis and provides some elements as guidelines for a set procedure. In particular, this chapter reports on the choice between serum and plasma as the biological source but does not concern the actual nucleic acid extraction procedures (these will be dealt with in chapter "Circulating DNA and miRNA Isolation"). Currently, the lack of a standard operating procedure for the application of blood handling in a clinical setting is due to the lack of dispensing and sharing data among researchers as well as head-to-head comparative studies between techniques. This has led to in-house specific procedures that are, undoubtedly, prejudicial to the smooth translation of nucleic acid analysis into clinical practice. Hence, the proposed procedure should overcome this gap in technique.

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Stability of Nucleosomal DNA Fragments in Serum

Cited by 22 publications

References 14 publications

Methodical and pre-analytical characteristics of a multiplex cancer biomarker immunoassay

Methodical and pre-analytical characteristics of a multiplex cancer biomarker immunoassay

Sequence-Specific Histone Methylation Is Detectable on Circulating Nucleosomes in Plasma

Pre-analytical Requirements for Analyzing Nucleic Acids from Blood

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