Pre-analytical variables of circulating cell-free nucleosomes containing 5-methylcytosine DNA or histone modification H3K9Me3.
OBJECTIVE
To evaluate pre-analytical variables of circulating cell-free nucleosomes containing 5-methylcytosine DNA (5mC) or histone modification H3K9Me3 (H3K9Me3).


METHODS
Six studies were designed to assess the possible influence of pre-analytical variables. Study 1: influence of stasis and contamination with white-cells and platelets. Study 2: influence of within-day variations. Study 3: influence of day-to-day variation. Study 4: influence of temperature during handling and storage, and of neoplastic disease. Study 5: influence of colonoscopy. Study 6: influence of the surgical trauma. 5mC and H3K9Me3 measurements were performed using enzyme-linked immunosorbent assays.


RESULTS
Stasis, white-cell and platelet contamination, within-day variations, varying storage time before centrifugation, colonoscopy, and surgical trauma had no significant influence on levels of 5mC or H3K9Me3. Day-to-day variations of 12.7% and 11.5% (intra-individual) and 98.1% and 60.8% (inter-individual) were shown for 5mC and H3K9Me3, respectively. Levels of 5mC or H3K9Me3 were significantly higher in samples stored at room temperature until centrifugation compared to samples stored on ice. Patients with cancer had significantly lower levels of 5mC or H3K9Me3 compared to levels in healthy individuals.


CONCLUSIONS
Levels of 5mC or H3K9Me3 appear stable in most pre-analytical settings if blood samples are stored at room temperature until centrifugation.
a Department of Surgical Gastroenterology , Hvidovre Hospital, University of Copenhagen , Hvidovre , Denmark ;
Louise
L
Rasmussen
b Belgian Volition SA, Rue du Séminaire 20A, Centre Technologique , Namur , Belgium ;
Marielle
M
Herzog
a Department of Surgical Gastroenterology , Hvidovre Hospital, University of Copenhagen , Hvidovre , Denmark ;
Eva
E
Rømer
b Belgian Volition SA, Rue du Séminaire 20A, Centre Technologique , Namur , Belgium ;
Jake
J
Micallef
a Department of Surgical Gastroenterology , Hvidovre Hospital, University of Copenhagen , Hvidovre , Denmark ;|c Institute of Clinical Medicine, University of Copenhagen , Copenhagen , Denmark.
Orhan
O
Bulut
a Department of Surgical Gastroenterology , Hvidovre Hospital, University of Copenhagen , Hvidovre , Denmark ;
Michael
M
Wilhelmsen
a Department of Surgical Gastroenterology , Hvidovre Hospital, University of Copenhagen , Hvidovre , Denmark ;
Ib Jarle
IJ
Christensen
a Department of Surgical Gastroenterology , Hvidovre Hospital, University of Copenhagen , Hvidovre , Denmark ;|c Institute of Clinical Medicine, University of Copenhagen , Copenhagen , Denmark.
Hans J
HJ
Nielsen
D044503:5-Methylcytosine / Q000097:blood*; D014408:Biomarkers, Tumor / Q000097:blood*; D016022:Case-Control Studies; D015179:Colorectal Neoplasms / Q000097:blood* / Q000175:diagnosis; D019175:DNA Methylation; D004273:DNA, Neoplasm / Q000097:blood; D006657:Histones / Q000097:blood*; D006801:Humans; D009707:Nucleosomes / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D015203:Reproducibility of Results
D014408:Biomarkers, Tumor; D004273:DNA, Neoplasm; D006657:Histones; D009707:Nucleosomes; D044503:5-Methylcytosine
Biomarkers; DNA methylation; colorectal neoplasms; early detection of cancer; epigenomics; histone code; tumor