Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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Efficient Detection of BRAF Mutation in Plasma of Patients after Long-term Storage of Blood in Cell-Free DNA Blood Collection Tubes.

Author(s): Denis MG, Knol AC, Théoleyre S, Vallée A, Dréno B

Publication: Clin Chem, 2015, Vol. 61, Page 886-8

PubMed ID: 25896990 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study investigated the effects of delayed centrifugation of blood, tube type, and shipping on the detection of BRAF mutations in plasma. To investigate the effects of tube type, blood was drawn from ten melanoma patients with a known V600E or V600K BRAF mutation into K3EDTA and BCT tubes. Blood was stored at room temperature and aliquots were removed after 2 h, 4 days, 7 days, and 10 days. Plasma was obtained by centrifugation at 2000 x g for 10 min. To investigate the effect of shipping, blood was drawn from six patients into BCT tubes, an aliquot was removed and processed immediately, and the remainder of the specimen was sent by mail at ambient temperatures. Specimens were then processed 4-6 days after collection. DNA was extracted using the PureLink Virus Kit and mutations were detected using the Therascreen BRAF RGQ kit.

   Summary of Findings:

   Plasma obtained from blood stored uncentrifuged in EDTA tubes at room temperature had a decrease in the control quantification cycle value and comparable mutation specific quantification cycle values. Consequently, the amount of normal DNA in plasma was 6-, 44-, and 110-fold higher when uncentrifuged blood was stored at room temperature in EDTA tubes for 4, 7, and 10 days, respectively than when blood was centrifuged after 2 h. In contrast, the quantification cycle value was not affected in 8 of 10 specimens when uncentrifuged blood was stored in BCT tubes at room temperature, but smaller increases in normal DNA were observed in the remaining two specimens when compared to those stored in EDTA tubes. The quantification cycle values were comparable between blood that was processed immediately and blood shipped in BCT tubes and processed 4-6 days after collection.

   Biospecimens

   • Bodily Fluid - Plasma
   • Bodily Fluid - Blood

   Preservative Types

   • None (Fresh)
   • Streck/BCT

   Diagnoses:

   • Neoplastic - Melanoma

   Platform:

   Analyte	Technology Platform
   DNA	Real-time qPCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Acquisition	Type of collection container/solution	EDTA tube BCT tube
   Biospecimen Preservation	Type of fixation/preservation	Blood collection tube additive EDTA
   Real-time qPCR Specific	Targeted nucleic acid	Wildtype BRAF Mutated BRAF
   Storage	Between site transportation method	Not transported Mailed
   Storage	Time at room temperature	2 h 4 days 7 days 10 days

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