Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

Search BRD Papers

Optimal preservation condition for the extraction of cell-free DNA from bile of patients with biliary tract cancer.

Author(s): Yang J, Zhao T, Nian B, Yang C, Zhu Y, Xu Y, Xu X, Chen S, Li Z, Zhang W, Zhang D, Lu K, Zhang F

Publication: Transl Cancer Res, 2024, Vol. 13, Page 5328-5338

PubMed ID: 39525024 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared the yield, purity and fragment size distribution of cell-free DNA (cfDNA) isolated from bile specimens collected with or without protectant and stored for up to 10 days at room temperature or for 2 months at 4, −20, or −80°C. Bile was collected from 8 patients with biliary tract cancer by percutaneous transhepatic cholangial drainage (PTCD) either before or during surgery. Bile from seven patients was collected into BEAVER cfDNA tubes with protectant and bile from 1 patient into plain polypropylene tubes. Bile specimens were shipped (<24 h) and then centrifuged at 1,600 g for 10 min at 4 ℃ followed by at 16,000 g for 15 min at 4 ℃. The supernatant was mixed and then aliquoted for experiments. Aliquots of bile from 2 patients (one collected with and one without protectant) were stored at room temperature for 1, 4, 7 and 10 days before analysis. Bile aliquots from 5 patients (four collected with and one without protectant) were stored at 4, −20, and −80°C for 2 months before analysis.  cfDNA was extracted using Qiagen reagents and QIAquick columns. cfDNA was quantified by Qubit 3.0 fluorometer and purity was assessed by NanoDrop Spectrometry. The fragment size profile was determined using an Agilent 2100 bioanalyzer.

   Summary of Findings:

   Room temperature storage of aliquots of bile collected without protectant led to progressive degradation of the DNA (shift in electropherogram and of the electronic DNA bands) and decreased recovery of DNA. After 4 days of storage at room temperature, bile collected without protectant had a higher percentage of fragments <376 bp (P≤0.01) and ≥376 bp to <3000 bp (P<0.05) and a lower percentage of fragments  ≥3000 bp (P≤0.01) and less cfDNA yield (P≤0.05) than in matched bile specimens from the same patient that were collected without protectant and stored for 1 day. While the fragment size and yield were not affected by storage of bile collected without protectant at -20 or -80°C for 2 months, bile collected without protectant that was stored at 4°C for 2 months had a higher percentage of fragments ≥376 bp to <3000 bp (P<0.05) and a nonsignificant trend toward a reduced yield and a lower percentage of fragments ≥3000 bp.  In contrast, the fragment size profile and yield were not affected by storage for up to 10 days at room temperature or 2 months storage at 4, −20, or −80°C when bile was collected in tubes containing protectant. DNA purity was unaffected by storage regardless of collection tube. The authors conclude that bile for cfDNA studies should be stored frozen or collected with protectant.

   Biospecimens

   • Bodily Fluid - Bile

   Preservative Types

   • None (Fresh)
   • Frozen
   • Other Preservative

   Diagnoses:

   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   DNA	Automated electrophoresis/Bioanalyzer
   DNA	Spectrophotometry
   DNA	Fluorometry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Storage	Time at room temperature	1 day 4 days 7 days 10 days
   Biospecimen Preservation	Type of fixation/preservation	Blood collection tube additive Frozen None (fresh) Refrigeration
   Biospecimen Acquisition	Type of collection container/solution	BEAVER cfDNA tubes with protectant Plain polypropylene tube
   Storage	Storage temperature	4°C −20°C −80°C

   View more