Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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Performance of ImproGene cfDNA blood collection tubes for mutation analysis in cancer patients.

Author(s): Zhang S, Zhou D, Li S, Bai Y, Huang B, Han J, Xu M, Wang S, Deng G

Publication: Scand J Clin Lab Invest, 2022, Vol. , Page 1-7

PubMed ID: 35861435 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This paper compared RNA and cell-free DNA (cfDNA) yields, cfDNA fragment size, and cfDNA mutational profiles between blood specimens collected in Streck and ImproGene Cell-Free DNA tubes. The authors also investigated if room temperature storage of blood in ImproGene tubes affects cfDNA yield.  Blood was collected from 39 patients with malignant gastric (5 patients), intestinal (8 patients), breast (7 patients), lung (2 patients), liver (9 patients), ovarian/uterine (4 patients), bladder (2 patients) or head and neck (2 patients) tumors into case-matched Streck Cell-Free DNA and ImproGene Cell Free DNA tubes. Tubes were transported (<2 h) and stored at room temperature for less than 24 h.  An aliquot of blood was removed, and genomic RNA was isolated using the RNeasy Plus mini Kit and quantified by nanoDrop. Plasma was separated from the remaining blood by centrifugation at 1,600 g at 4°C for 10 min followed by 16,000 g at 4°C for 10 min.  DNA was extracted from plasma using the Magnetic Serum/Plasma DNA Maxi Kit and quantified using Qubit and/or real-time PCR amplification of LINE-1, β-actin and an exogenous control. The cfDNA fragment size profile was evaluated by Bioanalyzer.  The effect of tube type on mutational profile was analyzed by next generation sequencing of 13 genes in case-matched specimens from 24 patients. The effect of delayed processing on cfDNA concentration was investigated by storing blood from 25 patients in ImproGene Cell Free DNA tubes for 0, 3 and 7 days at room temperature before plasma isolation and DNA extraction.

   Summary of Findings:

   No visible hemolysis was observed in plasma from any of the specimens regardless of storage duration and tube type. Total cfDNA concentration (Qubit), and plasma levels of β-Actin, and LINE-1 were unaffected by when blood was stored in ImproGene Cell Free DNA tubes at room temperature for ≤7 days. Yields of cfDNA and RNA were comparable between blood collected in Streck and ImproGene Cell Free DNA tubes. The cfDNA fragment size distribution was consistent between plasma from Streck and ImproGene tubes. Mutational analysis identified a total of 12 mutations, 5 were detected in both specimen types, and 6 were only detected in the specimen collected in the ImproGene tube, and the KRASG12C mutation was only identified in a specimen collected in a Streck tube. The detected variant allele frequency (VAF) was also higher for two of the mutations identified in specimens collected in ImproGene tubes compared to Streck tubes.

   Biospecimens

   • Bodily Fluid - Plasma
   • Bodily Fluid - Blood

   Preservative Types

   • Other Preservative
   • Streck/BCT

   Diagnoses:

   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   DNA	Fluorometry
   DNA	Real-time qPCR
   RNA	Spectrophotometry
   DNA	Next generation sequencing
   DNA	Automated electrophoresis/Bioanalyzer

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Acquisition	Type of collection container/solution	Streck Cell-Free DNA BCT ImproGene Cell Free DNA tube
   Biospecimen Aliquots and Components	Centrifugation	Centrifugation delays investigated
   Storage	Storage duration	0 days 3 days 7 days

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