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Filtration Device for On-Site Collection, Storage and Shipment of Cells from Urine and Its Application to DNA-Based Detection of Bladder Cancer.

Andersson E, Dahmcke CM, Steven K, Larsen LK, Guldberg P - PLoS ONE (2015)

Bottom Line: In the group of patients with benign histopathology, urine DNA was positive for methylation markers in 13 out of 26 cases (50%).Only one patient in this group was positive for a FGFR3 mutation.This patient had a stage Ta tumor resected 6 months later.

View Article: PubMed Central - PubMed

Affiliation: Danish Cancer Society Research Center, Copenhagen, Denmark.

ABSTRACT
Molecular analysis of cells from urine provides a convenient approach to non-invasive detection of bladder cancer. The practical use of urinary cell-based tests is often hampered by difficulties in handling and analyzing large sample volumes, the need for rapid sample processing to avoid degradation of cellular content, and low sensitivity due to a high background of normal cells. We present a filtration device, designed for home or point-of-care use, which enables collection, storage and shipment of urinary cells. A special feature of this device is a removable cartridge housing a membrane filter, which after filtration of urine can be transferred to a storage unit containing an appropriate preserving solution. In spiking experiments, the use of this device provided efficient recovery of bladder cancer cells with elimination of >99% of excess smaller-sized cells. The performance of the device was further evaluated by DNA-based analysis of urinary cells collected from 57 patients subjected to transurethral resection following flexible cystoscopy indicating the presence of a tumor. All samples were tested for FGFR3 mutations and seven DNA methylation markers (BCL2, CCNA1, EOMES, HOXA9, POU4F2, SALL3 and VIM). In the group of patients where a transitional cell tumor was confirmed at histopathological evaluation, urine DNA was positive for one or more markers in 29 out of 31 cases (94%), including 19 with FGFR3 mutation (61%). In the group of patients with benign histopathology, urine DNA was positive for methylation markers in 13 out of 26 cases (50%). Only one patient in this group was positive for a FGFR3 mutation. This patient had a stage Ta tumor resected 6 months later. The ability to easily collect, store and ship diagnostic cells from urine using the presented device may facilitate non-invasive testing for bladder cancer.

No MeSH data available.


Related in: MedlinePlus

Outline of the filtration device and its components.The device is composed of a filtration unit (A, individual components; B, assembled device; C, after filtration) and a water-tight storage cassette (D, individual components; E, assembled cassette).
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pone.0131889.g001: Outline of the filtration device and its components.The device is composed of a filtration unit (A, individual components; B, assembled device; C, after filtration) and a water-tight storage cassette (D, individual components; E, assembled cassette).

Mentions: Voided urine samples (100–400 ml) or suspensions of cultured cells were processed using a filtration device (Fig 1) mounted with a Nuclepore track-etched polycarbonate hydrophilic membrane filter (diameter 25 mm, pore size 8 μm; Whatman, Maidstone, UK). After filtration, the filter cartridge was transferred to the storage cassette, which was then mounted with the lid from an Oragene DNA Self-Collection Kit containing 1.7 ml of lysis/stabilization buffer (disc format OG-250; DNA Genotek, Ottowa, Ontario, Canada). According to the manufacturer, DNA is stable in this solution for >5 years at room temperature. DNA was purified from 0.5 ml of sample using the Oragene DNA purifying solution (DNA Genotek), according to the ethanol precipitation protocol provided by the manufacturer. For collection of the total cellular component, 25 ml of urine were centrifuged at 2,000 g for 10 min. The pellet was resuspended in 200 μl of phosphate-buffered saline (PBS) and stored at -80°C. DNA was extracted using the Qiagen Mini Prep kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer’s instructions (protocol for DNA purification from tissues). DNA yield was estimated by quantitative real-time PCR analysis, using a LightCycler 2.0 system (Roche, Mannheim, Germany), the FastStart DNA MasterPLUS SYBR Green I Kit (Roche), Human Genomic DNA (Roche) as reference and the following primers for GAPDH: 5’-TAGTGTCCTGCTGCCCACAGTCCAG-3’ and 5’-GGCGACGCAAAAGAAGATGC-3’.


Filtration Device for On-Site Collection, Storage and Shipment of Cells from Urine and Its Application to DNA-Based Detection of Bladder Cancer.

Andersson E, Dahmcke CM, Steven K, Larsen LK, Guldberg P - PLoS ONE (2015)

Outline of the filtration device and its components.The device is composed of a filtration unit (A, individual components; B, assembled device; C, after filtration) and a water-tight storage cassette (D, individual components; E, assembled cassette).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4495058&req=5

pone.0131889.g001: Outline of the filtration device and its components.The device is composed of a filtration unit (A, individual components; B, assembled device; C, after filtration) and a water-tight storage cassette (D, individual components; E, assembled cassette).
Mentions: Voided urine samples (100–400 ml) or suspensions of cultured cells were processed using a filtration device (Fig 1) mounted with a Nuclepore track-etched polycarbonate hydrophilic membrane filter (diameter 25 mm, pore size 8 μm; Whatman, Maidstone, UK). After filtration, the filter cartridge was transferred to the storage cassette, which was then mounted with the lid from an Oragene DNA Self-Collection Kit containing 1.7 ml of lysis/stabilization buffer (disc format OG-250; DNA Genotek, Ottowa, Ontario, Canada). According to the manufacturer, DNA is stable in this solution for >5 years at room temperature. DNA was purified from 0.5 ml of sample using the Oragene DNA purifying solution (DNA Genotek), according to the ethanol precipitation protocol provided by the manufacturer. For collection of the total cellular component, 25 ml of urine were centrifuged at 2,000 g for 10 min. The pellet was resuspended in 200 μl of phosphate-buffered saline (PBS) and stored at -80°C. DNA was extracted using the Qiagen Mini Prep kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer’s instructions (protocol for DNA purification from tissues). DNA yield was estimated by quantitative real-time PCR analysis, using a LightCycler 2.0 system (Roche, Mannheim, Germany), the FastStart DNA MasterPLUS SYBR Green I Kit (Roche), Human Genomic DNA (Roche) as reference and the following primers for GAPDH: 5’-TAGTGTCCTGCTGCCCACAGTCCAG-3’ and 5’-GGCGACGCAAAAGAAGATGC-3’.

Bottom Line: In the group of patients with benign histopathology, urine DNA was positive for methylation markers in 13 out of 26 cases (50%).Only one patient in this group was positive for a FGFR3 mutation.This patient had a stage Ta tumor resected 6 months later.

View Article: PubMed Central - PubMed

Affiliation: Danish Cancer Society Research Center, Copenhagen, Denmark.

ABSTRACT
Molecular analysis of cells from urine provides a convenient approach to non-invasive detection of bladder cancer. The practical use of urinary cell-based tests is often hampered by difficulties in handling and analyzing large sample volumes, the need for rapid sample processing to avoid degradation of cellular content, and low sensitivity due to a high background of normal cells. We present a filtration device, designed for home or point-of-care use, which enables collection, storage and shipment of urinary cells. A special feature of this device is a removable cartridge housing a membrane filter, which after filtration of urine can be transferred to a storage unit containing an appropriate preserving solution. In spiking experiments, the use of this device provided efficient recovery of bladder cancer cells with elimination of >99% of excess smaller-sized cells. The performance of the device was further evaluated by DNA-based analysis of urinary cells collected from 57 patients subjected to transurethral resection following flexible cystoscopy indicating the presence of a tumor. All samples were tested for FGFR3 mutations and seven DNA methylation markers (BCL2, CCNA1, EOMES, HOXA9, POU4F2, SALL3 and VIM). In the group of patients where a transitional cell tumor was confirmed at histopathological evaluation, urine DNA was positive for one or more markers in 29 out of 31 cases (94%), including 19 with FGFR3 mutation (61%). In the group of patients with benign histopathology, urine DNA was positive for methylation markers in 13 out of 26 cases (50%). Only one patient in this group was positive for a FGFR3 mutation. This patient had a stage Ta tumor resected 6 months later. The ability to easily collect, store and ship diagnostic cells from urine using the presented device may facilitate non-invasive testing for bladder cancer.

No MeSH data available.


Related in: MedlinePlus