Pre-analytical variables of circulating cell-free nucleosomes containing 5-methylcytosine DNA or histone modification H3K9Me3.
Author(s): Rasmussen L, Herzog M, Rømer E, Micallef J, Bulut O, Wilhelmsen M, Christensen IJ, Nielsen HJ
Publication: Scand J Clin Lab Invest, 2016, Vol. , Page 1-10
PubMed ID: 27291394 PubMed Review Paper? No
Purpose of Paper
This paper investigated the effects of tourniquet usage, time of day of collection, timing of collection relative to colonoscopy/polypectomy or surgery, delayed centrifugation, platelet contamination, inter- and intra-individual variability, and the presence of colorectal cancer on circulating levels of 5-methylcytosine DNA (5mC) and the histone modification H3K9Me3.
Conclusion of Paper
Although serum from blood stored at room temperature had 20% higher levels of 5mC and 22% higher levels of H3K9Me3 than serum from blood stored on ice, there were no storage duration dependent effects observed. There was no effect of tourniquet usage, time of collection, or platelet contamination on levels of 5mC and H3K9Me3 in serum or plasma. Day-to-day inter-individual variation in the levels of 5mC and H3K9Me3 in serum was greater than intra-individual variation. Patients with colorectal cancer had higher levels of 5mC and H3K9Me3 than healthy patients, but there were no differences in levels of 5mC and H3K9Me3 between serum collected prior to or after colonoscopy or colorectal cancer resection.
Studies
-
Study Purpose
This study investigated if levels of 5mC and H3K9Me3 in plasma and serum were affected by tourniquet usage or platelet contamination. Blood was collected from 20 healthy fasting patients using a tourniquet for collection from the left arm and no tourniquet for collection from the right. Blood was collected into EDTA and coagulation enhancer tubes. Plasma and serum were obtained by centrifugation at 3000 x g for 10 min. To determine the effects of platelet contamination, the top, middle, and bottom thirds from each tube were carefully transferred to new tubes while leaving 0.5 cm of plasma or serum above the buffy coat. All specimens were frozen at -80˚C. Levels of circulating 5mC and H3K9Me3 were quantified by ELISA.
Summary of Findings:
The authors report that levels of 5mC and H3K9Me3 in plasma and serum were not affected by the use of a tourniquet during blood collection. Further, the authors report that levels of 5mC and H3K9Me3 were comparable in the three layers of plasma or serum, indicating no effect of platelet contamination.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Normal
Platform:
Analyte Technology Platform Protein ELISA DNA ELISA Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Aliquots and Components Biospecimen components Top third
Middle third
Bottom third
Biospecimen Acquisition Method of fluid acquisition Tourniquet times compared
-
Study Purpose
This study investigated the effects of the time of day of blood collection on levels of 5mC and H3K9Me3 in plasma and serum and determined the intra- and inter-individual variation in 5mC and H3K9Me3 levels over 29 days. To investigate within-day variations, blood was collected from 12 healthy non-fasting patients at 8:00 AM, noon, and 3:00 PM on the same day. For the study of day-to-day variations, blood was obtained from 20 healthy non-fasting patients at 8:00 AM on day 1, 8, 15, 22, and 29. Blood was collected into EDTA and coagulation enhancer tubes. Plasma and Serum were obtained by centrifugation at 3000 x g for 10 min and transferred to tubes leaving 0.5 cm above the buffy coat. All specimens were frozen at -80˚C. Levels of circulating 5mC and H3K9Me3 were quantified by ELISA.
Summary of Findings:
Day-to-day inter-individual variation was greater than intra-individual variation of serum levels of 5mC and H3K9Me3 (98.1% versus 12.7% and 60.8% versus 11.5%, respectively). The authors report no effect of time of day of collection on serum or plasma levels of 5mC and H3K9Me3.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Normal
Platform:
Analyte Technology Platform Protein ELISA DNA ELISA Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Time of biospecimen collection 8:00 AM
Noon
3:00 PM
Day 1
Day 8
Day 15
Day 22
Day 29
-
Study Purpose
This study investigated the effects of pre-centrifugation storage of blood at room temperature or on ice on serum levels of 5mC and H3K9Me3 and compared serum levels of 5mC and H3K9Me3 in healthy individuals and those with colorectal cancer. Blood from each of 10 healthy non-fasting patients and 10 patients with colorectal cancer was collected into 14 coagulation enhancer tubes. Specimens were stored on ice and at room temperature for 30 min, 1 h, 2 h, 6 h, 24 h, 48 h, and 72 h prior to centrifugation at 3000 x g for 10 min. Specimens stored on ice were centrifuged at 4˚C and specimens stored at room temperature were centrifuged at 21˚C. Serum was frozen at -80˚C. Levels of circulating 5mC and H3K9Me3 were quantified by ELISA.
Summary of Findings:
Compared with serum from blood stored at 4˚C, serum from blood stored at room temperature had 20% higher levels of 5mC (P=0.0005) and 22% higher levels of H3K9Me3 (P <0.0001). Additionally, thawed serum from blood stored on ice had large fibrin clots that were not observed when the blood had been stored at room temperature. However, only a non-significant 8-10% increase in levels was observed with 24 h or more of storage at either temperature. Further, there was no significant interaction between storage temperature and duration. Levels of 5mC were 36% higher and levels of H3K9Me3 were 50% higher in individuals with colorectal cancer than in healthy individuals (P<0.0001, both).
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Normal
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform Protein ELISA DNA ELISA Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Aliquots and Components Centrifugation Centrifugation delays investigated
Preaquisition Diagnosis/ patient condition Healthy
Colorectal cancer
Storage Storage temperature Room temperature
On ice
Storage Storage duration 30 min
1 h
2 h
6 h
24 h
48 h
72 h
-
Study Purpose
This study investigated if serum levels of 5mC and H3K9Me3 were affected by the timing of blood draw relative to colonoscopy or surgery for colorectal cancer. For the study on the effects of colonoscopy, blood was collected in coagulation enhancer tubes from 43 patients (20 with adenomas, 23 without) just before colonoscopy and 1 h after. For the study of the effects of surgical trauma, blood was collected in coagulation enhancer tubes prior to surgical incision and 6, 24, 48, 72, and 96 h post-incision. Serum was obtained by centrifugation at 3000 x g for 10 min, transferred to new tubes, and frozen at -80˚C. Levels of circulating 5mC and H3K9Me3 were quantified by ELISA.
Summary of Findings:
Levels of 5mC and H3K9Me3 in serum were comparable before and 1 h after colonoscopy and between polypectomy and no polypectomy specimens. Similarly, the surgical trauma of colon cancer resection had no effect on levels of serum 5mC and H3K9Me3 as they were comparable before and 6, 24, 48, 72, and 96 h post-incision.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Normal
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform Protein ELISA DNA ELISA Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Time of biospecimen collection Pre-colonoscopy
1 h post-colonoscopy
Pre-surgical incision
6 h post-surgical incision
24 h post-surgical incision
48 h post-surgical incision
72 h post-surgical incision
96 h post-surgical incision
Preaquisition Surgical procedure type Colonoscopy
Polypectomy
Surgical resection