Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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Preanalytical Variables in the Analysis of Mitochondrial DNA in Whole Blood and Plasma from Pancreatic Cancer Patients.

Author(s): Randeu H, Bronkhorst AJ, Mayer Z, Oberhofer A, Polatoglou E, Heinemann V, Haas M, Boeck S, Holdenrieder S

Publication: Diagnostics (Basel), 2022, Vol. 12, Page

PubMed ID: 36010255 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to compare levels of mtDNA and nDNA in specimens collected in PAXgene versus K₃EDTA tubes, in blood versus plasma, in blood stored for six days at room temperature versus -20°C, after isolation using two different kits, in specimens from cancer patients versus healthy volunteers, and in archival specimens stored at -80°C and those prospectively collected in the present study. Blood was collected from ten healthy volunteers and ten pancreatic cancer patients into K₃EDTA and PAXgene Blood DNA tubes. Unless otherwise specified, blood was stored for 2 h at room temperature after which one tube of each type was centrifuged at 1600 g for 10 min at room temperature followed by 2800 g for 15 min to obtain plasma.  Whole blood and plasma were aliquoted into LoBind tubes and stored at -20°C. Unless otherwise specified, DNA was isolated from blood and plasma using the Wizard Plus SV Miniprep DNA purification Kit and the Promega Maxwell instrument. mtDNA and nDNA were quantified by real-time amplification of TL1 and β-globin, respectively. DNA fragment size was analyzed using the Bioanalyzer DNA 1000 Kit and DNA was quantified using the Qubit dsDNA HS Assay. To investigate possible storage effects, case-matched whole blood was stored at room temperature and -20°C for 6 days in each tube type. To investigate the effect of extraction method, DNA was also extracted from plasma and blood using the QIAprep Kit.  To investigate potential effects of long-term specimen storage, levels of mtDNA and nDNA were compared between prospectively collected specimens from pancreatic cancer patients and 10 archived blood specimens collected from pancreatic cancer patients (from two prior studies) that were stored at -80°C for approximately 8 years.

   Summary of Findings:

   mtDNA levels were significantly higher in blood than plasma regardless of whether specimens were collected from healthy volunteers or pancreatic cancer patients, or whether K₃EDTA or PAXgene tubes were used (P<0.001, all). Case- matched blood from healthy volunteers stored at room temperature for six days had higher levels of mtDNA than when specimens were stored at -20°C, although the observed differences between storage temperatures was larger when specimens were stored in K₃EDTA tubes compared to PAXgene tubes. Interestingly, no effect of blood storage temperature was observed on mtDNA levels in blood from pancreatic cancer patients mtDNA levels did not differ between K₃EDTA and PAXgene specimens when stored under the same conditions, with the exception of higher levels in PAXgene blood from healthy patients stored at room temperature. Overall, the yield of mtDNA from blood was higher when the MAXwell Kit was used for extraction, but differences were not significant when blood was stored at room temperature in K₃EDTA tubes. In contrast, mtfDNA yield did not differ between extraction methods in plasma from healthy patients, while the QIAprep kit yielded more mtDNA  for plasma collected from cancer patients. Generally, nDNA  yields were comparable between extraction kits; however, the yield of nDNA from blood stored at -20°C was higher when the MAXwell Kit was used for extraction. For specimens stored at room temperature, extraction with the MAXwell Kit rather than QIAprep Kit resulted in a higher ratio of mtDNA to nDNA but this was not observed for specimens collected from cancer patients stored in K₃EDTA tubes. While the mtDNA to nDNA ratio was higher when DNA was extracted from blood specimens collected from cancer patients and stored at-20°C using the QIAprep Kit rather than the MAXwell Kit, no difference was observed among blood specimens collected from healthy volunteers. mtDNA yields were higher in whole blood specimens from cancer patients than healthy volunteers when specimens were stored at room temperature, but not when stored at -20°C.  Although nDNA yield did not differ between blood collected from pancreatic cancer patients in 2021 and stored at -20°C versus those collected in 2014-2015 and stored at -80°C, mtDNA yields were higher in the 2021 cohort.

   Biospecimens

   • Bodily Fluid - Plasma
   • Bodily Fluid - Blood

   Preservative Types

   • None (Fresh)
   • PAXgene
   • Frozen

   Diagnoses:

   • Normal
   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   DNA	Real-time qPCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Acquisition	Type of collection container/solution	K3EDTA tube PAXgene tube
   Biospecimen Aliquots and Components	Blood and blood products	Whole blood Plasma
   Biospecimen Preservation	Type of fixation/preservation	PAXgene EDTA Frozen
   Preaquisition	Diagnosis/ patient condition	Pancreatic cancer Healthy
   Storage	Storage duration	< 1 year 7-8 years
   Analyte Extraction and Purification	Analyte isolation method	MAXwell kit QIAprep kit
   Storage	Storage temperature	Room temperature -20°C -80°C

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