Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N1/00—Sampling; Preparing specimens for investigation
  • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
  • G01N1/40—Concentrating samples
  • G01N1/4077—Concentrating samples by other techniques involving separation of suspended solids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
  • A61B10/00—Instruments for taking body samples for diagnostic purposes; Other methods or instruments for diagnosis, e.g. for vaccination diagnosis, sex determination or ovulation-period determination; Throat striking implements
  • A61B10/0045—Devices for taking samples of body liquids
  • A61B10/007—Devices for taking samples of body liquids for taking urine samples
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
  • A61B10/00—Instruments for taking body samples for diagnostic purposes; Other methods or instruments for diagnosis, e.g. for vaccination diagnosis, sex determination or ovulation-period determination; Throat striking implements
  • A61B10/0096—Casings for storing test samples
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01D—SEPARATION
  • B01D27/00—Cartridge filters of the throw-away type
  • B01D27/08—Construction of the casing
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
  • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
  • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
  • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
  • C12N15/1017—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by filtration, e.g. using filters, frits, membranes
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
  • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
  • B01L2200/04—Exchange or ejection of cartridges, containers or reservoirs
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
  • B01L2200/06—Fluid handling related problems
  • B01L2200/0631—Purification arrangements, e.g. solid phase extraction [SPE]
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2300/00—Additional constructional details
  • B01L2300/04—Closures and closing means
  • B01L2300/041—Connecting closures to device or container
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2300/00—Additional constructional details
  • B01L2300/06—Auxiliary integrated devices, integrated components
  • B01L2300/0681—Filter
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2300/00—Additional constructional details
  • B01L2300/08—Geometry, shape and general structure
  • B01L2300/0832—Geometry, shape and general structure cylindrical, tube shaped
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2300/00—Additional constructional details
  • B01L2300/08—Geometry, shape and general structure
  • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
  • B01L2300/087—Multiple sequential chambers
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2300/00—Additional constructional details
  • B01L2300/12—Specific details about materials
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2400/00—Moving or stopping fluids
  • B01L2400/04—Moving fluids with specific forces or mechanical means
  • B01L2400/0475—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure
  • B01L2400/0478—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure pistons
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2400/00—Moving or stopping fluids
  • B01L2400/04—Moving fluids with specific forces or mechanical means
  • B01L2400/0475—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure
  • B01L2400/0487—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure fluid pressure, pneumatics
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/154—Methylation markers
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/158—Expression markers
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N1/00—Sampling; Preparing specimens for investigation
  • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
  • G01N1/40—Concentrating samples
  • G01N1/4077—Concentrating samples by other techniques involving separation of suspended solids
  • G01N2001/4088—Concentrating samples by other techniques involving separation of suspended solids filtration

Definitions

• the present invention relates to biological fluid filtration assemblies and to methods of using such assemblies.
• Bladder cancer is the sixth most common cancer in the world.
• the symptoms include microscopic or macroscopic hematuria, painful urination and polyuria; however, none of these symptoms is specific for the disease.
• the gold standard for diagnosing bladder cancer is cystoscopy and subsequent transurethral resection of the bladder tumour (TURBT) .
• the sensitivity of cystoscopy for non-muscle invasive bladder cancer (NMIBC; stage Ta, Tl and Tis) is around 80% with white-light cystoscopy and >95% with fluorescence (hexaminolevulinate) -guided cystoscopy.
• bladder tumour patients 70-80% are diagnosed with NMIBC, which has a relatively good prognosis.
• the recurrence rate for these tumours is very high, with around 70% of the patients experiencing relapses, and up to 25% of these recurrences will progress to muscle invasive cancers (MIBC; stage T2-4) with a poor prognosis.
• MIBC muscle invasive cancers
• the high recurrence rate and the risk of progression require life-long surveillance with periodic cystoscopy, making bladder cancer the most expensive cancer to treat (Avritscher et al . , 2006) .
• cystoscopy is an invasive method that causes considerable discomfort to the patients, there is an unmet need for
• Voided urine from bladder tumour patients may contain exfoliated tumour cells that can be identified by cytology.
• Urine cytology has been used for decades and is still the most common
• Bladder tumour cells contain a large number of genome
• driver and passenger events may have a potential as biomarkers for bladder cancer, provided that they are cancer specific (i.e., not found in normal tissues or present at a different level of expression) and recurrent (i.e., occur in independently arising tumours at appreciable frequencies) .
• the most frequently mutated genes in bladder cancer include the proto-oncogenes FGFR3, RAS, and PIK3CA, and the tumour suppressor gene TP53. Mutations in FGFR3 are common in NMIBC, with reported frequencies of >60%, whereas TP53 mutations are predominantly found in MIBC. In addition, hundreds of genes have been shown to be differentially methylated between bladder tumours and normal bladder epithelium.
• the present invention is based on the inventors' insight that a convenient and efficient assembly for capturing and storing biological material obtained from biological fluids may offer significant advantages for patients and medical practitioners in the diagnosis and long-term monitoring of conditions and
• the present invention relates to filtration assemblies for easy and low-cost collection of biological material from biological fluids and to methods using these filtration
• the present invention further relates to assemblies for the storage of biological material collected from such fluids, and methods of using the same.
• Assemblies of the present invention also offer advantages in the provision of medical care in a patient's home by visiting medical practitioners and carers. Captured material may be stored immediately, either for mailing to an appropriate medical centre or testing facility or transport there by the medical
• assemblies and methods described herein may be of relevance to the collection and filtering of urine for the capture and detection of cells associated with genitourinary disorders.
• These disorders may include genitourinary cancers such as for example, and not by way of limitation, bladder, prostate and renal cancer.
• genitourinary cancers such as for example, and not by way of limitation, bladder, prostate and renal cancer.
• gynecological cancers such as endometrial cancer or cancers that have metastasized to the genitourinary site from other sites.
• Uses of the assemblies described herein directed to urine filtration were prompted by the inventors' insights into the limitations of current procedures for bladder tumour diagnosis and the disadvantages of cystoscopy, which is commonly used for the diagnosis and long-term monitoring of patients, both in terms of discomfort to the patient and the burdens placed by this approach on health care systems.
• cells and other biological material associated with urological disorders other than cancer may also be captured and stored using assemblies of the present invention.
• assemblies of the invention may also be used for the collection of cells (such as for example, and not by way of limitation, normal epithelial, cancer, bacterial or yeast cells) and other biological material (such as for example, and not by way of limitation, proteins or nucleic acids) from other biological samples, such as for example, and not by way of limitation, saliva, sera, blood, and washes, for example, bladder washes .
• cells such as for example, and not by way of limitation, normal epithelial, cancer, bacterial or yeast cells
• other biological material such as for example, and not by way of limitation, proteins or nucleic acids
• the assembly may comprise a filtration device and a storage unit.
• the method may comprise an initial step of capturing biological material by forcing fluid through a filter that is housed in a support, for example, a removable filter cartridge. After filtration, the support with filter content can be removed from the filtration device and placed into the storage unit, which may contain an appropriate solution for facilitating storage and/or analysis of the captured biological material.
• the present invention may provide a biological fluid filtration assembly comprising a filtration device for filtering a biological fluid sample, and a storage unit, the filtration device having a collection chamber, a waste reservoir, and a filter support platform, the filter support platform housing a removable filter cartridge having a filter suitable for capturing biological material present in the biological fluid sample; wherein the collection chamber, waste reservoir and filter support platform are connectable to permit passage of a biological fluid from the collection chamber into the waste reservoir through the filter of the filter cartridge; and the storage unit having a body configured to engage with the removable filter cartridge such that, when engaged, the filter of the filter cartridge is sealed within the body of the storage unit .
• the filter cartridge may be slidably retained in the filter support platform. That is, the filter support platform may have a recess of a size and shape suitable for receiving the filter cartridge such that, when the filter cartridge is inserted, the filter is positioned as described so that, in use, fluid passes from the collection chamber into the waste reservoir through the filter.
• This slidable engagement may be provided with
• the storage unit body may comprise a recess for slidably
• the recess of the storage unit body is configured to engage with the filter cartridge such that the filter cartridge may not be removed accidentally.
• This may be through use of a sufficiently close fit, or by the provision of complementary protrusions and recesses on the filter cartridge and in the recess to improve the fit and hold and/or to provide a snap fit-type interaction to retain the filter cartridge in place.
• the storage unit body may have an opening to permit access to the filter and/or filter content of the filter and/or a liquid surrounding the filter when the filter cartridge is in place.
• the storage unit may further comprise a removable lid covering the opening. It will be appreciated that depending on the intended use and on the nature of the lid, in some
• the lid may be arranged to provide access only to the filter content, that is, the biological material trapped on the filter following use, or to the filter content and/or any surrounding liquid following use.
• the captured biological material it may be preferable for the captured biological material to be exposed to a solution prior to
• a suitable solution may, for example, be a. buffer suitable for inducing cell lysis, a fixative/preservative, a culture medium, an isotonic buffer, or an appropriate buffer for elution, each as described herein. It will be appreciated that the provision of a solution chamber, and the inclusion of a solution, is an optional feature .
• the storage unit is arranged such that the lid has a solution chamber containing a solution selected to facilitate storage and/or analysis of the biological material, wherein engagement of the lid with the storage unit body causes the solution to be released such that it contacts the filter.
• the filter cartridge may be inserted into the storage unit without the lid in place. The lid may then be fitted, thereby releasing the solution.
• the storage unit may alternatively be configured to have a solution chamber arranged such that engagement of the filter cartridge with the storage unit causes the release of the solution into contact with the filter.
• the storage unit has a piston retained within the recess, the piston and recess defining a solution chamber distal from the recess opening, the solution chamber containing a solution selected to facilitate storage, processing and/or analysis of the biological material, the piston being configured such that insertion of the filter cartridge into the recess causes the piston to move further in to the recess, such that the solution contained within the chamber is forced around the piston into contact with the filter, are therefore with any filter content present.
• the storage unit may be provided with a solution in place in the chamber, or may be provided separately for inclusion in the storage unit by a user.
• assemblies described herein may be used to filter biological fluids using only gravity, that is, through gravitational percolation, it is preferable to provide a means of, or for, facilitating passage of the biological liquid through the filter. This may be achieved by creating a pressure differential, for example, by providing means for applying pressure to the liquid in the collection chamber to push the biological fluid through the filter, or by providing means for creating a vacuum in the waste reservoir to pull the biological fluid through the filter.
• the filtration device has means to enable application of pressure to a fluid contained within the collection chamber when the device is assembled to force the fluid through the filter into the waste reservoir.
• the collection chamber may itself be compressible such that when the filtration device is assembled and the collection chamber contains a fluid sample, compression of the collection chamber applies pressure to the fluid, thereby forcing the fluid through the filter into the waste reservoir.
• the collection chamber may be a cylindrical bag with a spring surrounding the cylindrical bag along its cylindrical axis, thereby permitting compression of the cylindrical bag in the direction of its cylindrical axis.
• the collection chamber may be provided with a piston configured to force biological fluid through the filter from the collection chamber to the waste reservoir when the filtration device is assembled following sample provision.
• a pump system may also be used to apply pressure.
• means may be provided for
• generating a vacuum to pull/suck the fluid through the filter This may be through use of a pump arranged to draw air out of the waste reservoir, thereby creating a vacuum, or the waste
• reservoir may itself be provided with chamber under vacuum. This chamber may then be opened to the remainder of the waste
• a pressure differential is to be used to force/draw the biological fluid through the filter, it may be desirable to include one or valves configured to allow pressure within the device to equilibrate during and after application of
• the biological fluid is urine or a bladder wash, most preferably urine.
• the fluid may be blood or serum.
• the waste reservoir may contain an absorbent and/or deodorising material, which may be especially advantageous for the filtration of urine samples.
• the filter may be selected to capture biological material as desired and as described herein.
• the filter is selected to capture biological material associated with the diagnosis and/or prognosis of a disease, condition or disorder, for example, with cancer.
• the biological material is cells suspended in the biological fluid, more preferably, cells suspended in urine.
• the biological material may be tested for the presence of, for example, markers associated with the diagnosis and/or prognosis of a disease, condition or disorder.
• the biological material may be cells suitable for testing for the presence of a marker that is indicative of a particular disease, condition or disorder, for example, markers associated with the diagnosis and/or prognosis of urological cancers.
• the present invention may provide method of capturing biological material from a biological sample using an assembly as described herein, the method comprising:
• the method may further comprise the step of applying pressure to the biological fluid sample in the collection chamber to force flow of the biological fluid sample from the collection chamber into the waste reservoir through the filter, for example, by compressing the collection chamber, if the assembly is suitably arranged.
• the method may further comprise the step of generating a vacuum within the waste reservoir to suck the biological fluid sample through the filter.
• the filter cartridge and storage unit combination may provide a convenient sealed unit for storage and/or transportation of the captured biological material.
• the filter cartridge and storage unit combination may then be stored prior to testing, given to an appropriate care giver, for example, a medical practitioner, or transported using, for example, a national mail carrier or internal mail system, in each case conveniently and hygienically .
• the captured biological material may be retrieved from the filter and/or any surrounding liquid and tested as described herein. This testing may assist in the diagnosis and/or prognosis of conditions as described herein.
• the present invention provides a method wherein, having filtered a biological fluid sample using an assembly and/or method as described herein, a method
• assemblies as described herein will typically be provided to a user, who may be the patient
• the present invention provides a kit comprising a collection chamber, a filter support platform, a waste reservoir, and a storage unit, as any one embodiment described herein, and, optionally, instructions for a method as described herein. It will be appreciated that in some circumstances, the individual elements of the assembly may be provided separately, and that the invention also provides a filter cartridge as described herein and a storage unit as described herein which may be supplied separately to the remainder of the assembly.
• the present invention includes any combination of the aspects and preferred features described herein except where such a
• Figure 1 shows a technical drawing showing an exploded view of a filtration assembly according to the present invention.
• Figure 2 shows a perspective view of a collection chamber (left) and filtration unit (right) .
• Figure 3 shows a side view of an assembled device of the present invention.
• Figure 4 shows an alternative storage unit according to the present invention, and the assembly thereof.
• FIG. 5 shows denaturing gradient gel electrophoresis (DGGE) analysis of HRAS exon 2.
• the human cell line T24 is homozygous for the G12V mutation.
• Figure 6 shows pyrosequencing analysis of 6 CpG sites in the BCL2 promoter.
• Figure 7 shows MethyLight analysis of the BCL2 promoter in urine samples from pt .
• X diagnosed with a high-grade Ta tumour
• a positive signal was obtained only for the filter sample;
• A labels the "Filter”,
• B labels the "Sediment”.
• Figure 8 shows pyrosequencing analysis of the BCL2 promoter.
• Figure 9 shows capture of tumor cells from fluid by filtration using a device mounted with an 8- ⁇ pore size polycarbonate membrane filter. Data of triplicate measurements (each on 4% of total DNA) from one experiment are represented as means ⁇ SD. Percentages above bars represent the number of recovered cells relative to the number of input cells.
• Figure 10 shows filtration-based enrichment of bladder cancer cells in a background of normal lymphocytes.
• Percentages above the bars represent the number of recovered cells relative to the number of input cells.
• Figure 11 shows detection of tumor-derived DNA in paired urine samples prepared by device filtration and sedimentation.
• FIG. 1 An exploded view of an assembled biological fluid filtration assembly according to the present invention is shown in Figure 1.
• the assembled device and use thereof is shown in Figure 3, while Figure 2 shows the collection chamber (left) and a filtration unit assembled from the filter support platform and waste reservoir (right) prior to their coupling to afford the assembled device .
• the collection chamber 1 is open-topped for convenience of sample provision.
• the collection chamber is formed of a cylindrical bag 3 of water-impermeable material, which is approximately 100 mm in length and 95 mm in diameter and is suitable for housing a volume of approximately 500 mL for convenience of sample provision and maximal DNA when analysing urine samples.
• the collection chamber is suitable for housing up to 400 mL, 300 mL, 250 mL, 100 mL, 50 mL, or up to 20 mL . While larger volumes may be appropriate for urine collection, smaller volumes may be preferable for the filtration of, for example, saliva.
• the cylindrical bag is contained within a spring 5 which imparts some rigidity to the cylindrical bag of the collection chamber.
• a lid 7 At the sealed end of the cylindrical bag is a lid 7 and at the open end of the cylindrical bag is an annular spring attachment portion 9 which encircles the open end of the cylindrical bag without substantially occluding the open portion.
• the spring 5 is connected to or abuts the lid 7 at one end and the annular attachment portion 9 at the other end.
• the lid 7 and the annular spring attachment portion 9 are rigid and made of plastics material, although other suitable rigid materials, for example, a metal such as stainless steel, may be used.
• the lid 7 is circular and imperforate, and of a diameter slightly larger than the diameter of the cylindrical bag.
• collection chamber has a locking ring attachment 11 to which annular spring attachment portion 9 can be fixed by means of a snap fit interaction.
• Other fixing means may be used, including complementary screw threads and rotatably engaging lugs.
• the collection chamber is connectable via the locking ring attachment 11 to the filtration unit 13 to assemble the complete filtration device.
• This connection is necessarily substantially watertight to permit use of the device as described herein without loss of fluid before filtration, with O-ring 14 which is retained in an annular groove around the top of the filtration unit improving the seal.
• the filtration unit 13 has a filter support platform 15. Small protrusions on the filter support platform 15 are located to engage with complementary indents in the annular attachment portion 9. It will be appreciated that other attachment means may alternatively be provided.
• the filter support platform 15 has a removable filter cartridge 17 with a membrane filter 19, and is connectable to a waste reservoir 21. It will be appreciated that other filter materials as described herein may also be used.
• the waste reservoir 21 is a rigid cylindrical container made of plastics material able to accommodate a volume of at least 500 mL (that is, the entire volume of liquid contained in the collection chamber prior to filtration) . Other suitable rigid materials suitable for receiving fluids may be used in place of plastics material.
• the waste reservoir 21 and the filter support platform 15 are connectable to form, in combination with the filter cartridge 17, the filtration unit 13. This connection is necessarily
• the waste reservoir 21 and the filter support platform 15 are connectable by a snap fit connection between a protrusion on the outside of waste reservoir 21 and an annular groove on the inside of the filter support platform 15.
• the waste reservoir 21 contains a moisture absorbing material and/or a deodorant.
• Suitable moisture absorbing materials may include absorbent material such as paper, cotton wool or sponge, or silica gel and/or other water-absorbent polymers known in the art. The inclusion of a moisture absorbent material improves ease of disposal of the waste reservoir after use.
• Suitable deodorants may include carbonates such as
• FIG. 1 shows the component parts of the filter support platform 15.
• the filter support platform is connectable to both the connection chamber at its open end and to the waste reservoir, and when the device is fully-assembled separates the two.
• the filter support platform has an opening 23 to allow fluid communication between the collection chamber and the reservoir and the filter 19 of the filter support portion, in this case, filter cartridge 17,
• the filter support platform has a slotted recess 25 suitable for receiving a filter cartridge 17 such that the filter of the filter cartridge occludes the opening as described.
• the filter cartridge may be inserted and removed from the slotted recess in a sliding movement.
• the filter support platform is assembled from a top portion 27 and a bottom portion 29, which clip together by means of a snap-fit connection between protrusions on the top portion and complementary recesses on the bottom portion.
• Other connecting means may be envisaged including other snap-fit interactions and complementary screw threads.
• the embodiment shown in Figure 1 has two handles, 31 and 32 to facilitate ease of use. It will be understood that handles are not necessary, and that other handle arrangements, for example, a single handle, a continuous annular handle, or one or more D-shaped handles may be used.
• O-ring 33 is provided to prevent leakage during use.
• the filter support platform further comprises a back flow membrane 35 and a pressure relief valve 37.
• the pressure relief valve is configured to activate at a certain pressure to allow liquid to pass into the waste chamber should the filter becomes saturated.
• the backflow membrane 35 is adapted to allow air to pass from the reservoir 21 into the collection chamber 1 during intermittent application of pressure to prevent the filter content becoming disturbed due to turbulence.
• the relief valve is an umbrella-type valve that opens at 10-12 kg pressure, but other suitable valves may be used.
• the filter cartridge 17 has a body of a width complementary to the width of the slot, and is sufficiently longer in length to cause a portion of the body to protrude from the slotted recess during use (as shown in Figure 2) to facilitate ease of removal of the cartridge from the device.
• the filter cartridge housing may have one or more indentations or perforations 41 to improve grip and aid removal.
• the filter 19 is housed on a ledge within an opening in the filter cartridge housing and maintained in place by a perforated over support 43 which is connected to the housing by means of a snap-fit connection between protrusions on the perforated over support and complementary recesses in the housing. Other connecting means may be envisaged including other snap-fit interactions and complementary screw threads.
• O-rings 45, 47, and 48 improve the seal.
• O-ring 47 improves the seal of the assembled filter cartridge 17 around the filter 19, while 0- rings 45 and 48 are present on the external surface of the filter cartridge 17 and serve both to improve the seal when the filter cartridge 17 is housed within the filter support platform for filtration of the biological fluid and to improve the seal when the filter cartridge 17 is inserted into a storage unit 49
• Figure 1 further shows such a storage unit 49 according to the present invention.
• the filter cartridge 17 may be inserted into the storage unit 49 after use to facilitate ease of storage and transportation and may preserve the sample during storage.
• the storage unit 49 further provides a means for ease of access to the filter content (and any surrounding liquid) for analysis without the need to remove the cartridge from the storage unit.
• the storage unit comprises a base 51 having a recess 53 suitable for receiving the filter
• This base has an opening 55 located to permit access to the filter content for analysis and processing when the filter cartridge is inserted.
• the opening is covered by a lid 57 to preserve the sample and to permit storage and transportation.
• the lid connects to the base by means of complementary screw threads, although other connection means may be envisaged including a suitable snap-fit interaction or hinged lid.
• the lid 57 comprises a chamber containing an appropriate liquid that is released during engagement of the lid with the base 51.
• the lid may be an OG-250 lid from Oragene®, developed by DNA Genotek® and containing a DNA lysis buffer.
• Base 51 has sharp protrusions which break a seal of the chamber in the lid when the lid is screwed onto the base, thereby releasing the solution.
• FIG. 4 shows an alternative storage unit according to the present invention, and the assembly thereof.
• the storage unit 490 comprises a housing 510 having a recess 530 suitable for receiving the filter cartridge and a first opening 550 to permit access to the filter content when a filter cartridge is inserted.
• the lid 570 engages with the housing by a sliding cooperation between protrusions on the housing and complementary recesses on the lid, and is retained in place by abutment against a stop plate and by a retaining clip.
• the storage unit also has a bottom 571 that engages with the housing in a manner analogous to that of the lid.
• a piston 600 is retained within the recess at a point beyond the first opening and defines a chamber 602 at the end of the recess distal from the recess opening.
• the housing has a second opening 603 into this chamber.
• the chamber 602 is suitable for receiving a fluid, for example, and not by way of limitation, a buffer for lysis of cells and preservation of nucleic acids and/or proteins, a fixative/preservative to prepare cells with the retention of the characteristic morphology (for cytological examination) , a culture medium to sustain cell growth or an isotonic buffer suitable for the storage of biological material, or an appropriate buffer for the elution of the biological material from the filter.
• the storage unit is provided with a suitable fluid of this type contained within the chamber. It will be
• the fluid may be selected in accordance with the nature of the sample to be stored and the subsequent analysis required .
• the piston 600 is retained within the recess but application of pressure, for example, by insertion of a filter cartridge, is able to push the piston further into the recess, reducing the size of the chamber and forcing the fluid therein into the remainder of the recess, and into contact with the filter and filter content.
• the filter cartridge 17 and storage unit 490 form a water tight seal around the filter, the filter content, and any surrounding liquid that may be present. It will be appreciated that varying the filter cartridge and storage unit dimensions and the provision and location of suitable O-rings in order to achieve said water-tight seal will be apparent to the skilled person.
• Assemblies of the invention may be provided as a kit directly to the user, who can then:
• assemblies according to the present invention may be used in the home, with samples stored,
• a storage unit permits samples captured on filters according to methods of the present invention to be sent hygienically and efficiently using, for example, regular national mail services.
• a kit comprising a collection chamber as described herein and a filtration unit as described herein, and, optionally, instructions for using the assembly in a method as described herein.
• the filtration unit is provided fully assembled.
• a sample is then provided, for example, through normal urination, into the collection chamber.
• the filtration unit is then fastened to the collection chamber.
• the user then flips the assembled device so that the collection chamber is now upside down at the top of the device, as shown in Figure 3, and then provides manual pressure to force the liquid through the filter into the waste reservoir.
• the provision of one or more valves and/or backflow membranes allows pressure to equalise within the device.
• kits of the invention may further comprise a storage unit as described herein. The user then inserts the filter cartridge into the storage unit as shown in, for example, Figure 4 for convenient storage and transport.
• the storage unit is provided as a lid and a base (denoted 57 and 51, respectively, in Figure 1) .
• the filter cartridge is first inserted into the base 51.
• Lid 57 is then added, with the engagement of the lid with the base causing the seal to a chamber containing solution within the lid to break, thereby releasing the solution into contact with the filter content .
• the storage unit is provided as a single unit (denoted 490 in Figure 4) .
• This storage unit comprises a piston retained within the recess at a point beyond the first opening and defining a chamber at the end of the recess distal from the recess opening.
• the base of the storage unit has a second opening into this chamber.
• the chamber contains a solution, for example, and not by way of limitation, a buffer for lysis of cells and preservation of nucleic acids, a
• the combined filter cartridge and storage unit may then be conveniently and hygienically transported to a testing/ analysing facility or appropriate medical centre. Access to the filter content is facilitated by removal of the lid (denoted 57 or 570 in Figure 1 or Figure 4, respectively) to reveal the relevant opening in the storage unit housing.
• Filter content for example, DNA, may be analysed using methods known in the art and methods described herein, with the presence or absence of certain known markers used to provide a diagnosis.
• the assembly may be provided as a kit comprising a waste reservoir and filter support base that have not yet been fastened together.
• the user must first assemble the filtration unit.
• filter cartridges and storage units optionally comprising a solution housed within a chamber as described herein, may be provided separately to the remainder of the assembly as these may be selected specifically with regard to the intended application. Suitable Filters
• the present invention is based on the inventors' insight that devices comprising certain suitable filters may be utilised for capturing material from biological fluids for efficient analysis, for use in the diagnosis and monitoring of relevant conditions and diseases.
• the assemblies and methods of the present invention may be used to capture cells from biological fluids. Previous studies have shown that it is possible to capture and separate cells from fluids using mechanical filtering (Wilding et al., 1998; Mohamed et al . , 2004; Zheng et al., 2007; Lin et al., 2010) .
• assemblies and methods of the present invention provides the convenience and efficacy associated with the assemblies and methods of the present invention, that is, the provision of an assembly for the inexpensive and easy collection and processing of a sample which may be used by the patient or another caregiver to provide a sample of captured cells suitable for storing and sending through the post to a testing facility or appropriate medical centre.
• Any filter material having the necessary character to capture material of interest may be used in assemblies and methods of the present invention. It will be appreciated that assemblies and methods of the present invention may be used for the capturing of different types of biological material from various biological fluids, for the detection, diagnosis and monitoring of a variety of diseases and conditions.
• the filter may be selected from filter media known in the art to have certain desirable characteristics, and in some cases it may be desirable to provide multiple filters in series. Where multiple filters are used, each filter may be identical to, or have different characteristics to, any other filter in the assembly .
• the capturing of cells of different sizes and different types may be achieved by use of a filter, or use of multiple filters, configured to exclude certain sizes or forms of cells, most likely by selection of filter pore size and or/ pore arrangement.
• Size exclusion may be achieved by use of particular pore or other aperture size, or by use of a particular pore form.
• Filters may also be used that are made from materials, or have coatings, designed specifically to capture certain materials, for example, macromolecules such as proteins, DNA, RNA and
• filter characteristics that may be suitable for use in some embodiments of the present invention are provided by way of illustration and are not intended to limit the invention to any particular filter type. These and other suitable filters are known in the art, and may be commercially available.
• a pore size of about 0.5 ⁇ to 4 ⁇ may be preferred.
• a pore size of about 20 nm to 300 nm, more preferably of about 20 nm to about 50 nm may be used.
• a pore size of about 4 m to 10 ⁇ may be preferred.
• a pore size of about 7 ⁇ to 12 ⁇ may be preferred.
• a pore size of about 8 ⁇ to 20 ⁇ may be preferred, with about 8 ⁇ to 12 ⁇ being especially preferred, about 8 ⁇ most preferred.
• ultrafiltration membrane filters with a specific molecular weight cut off limit (for example, but not by way of limitation, 50 kDa) selected to capture the macromolecules of interest may be used.
• the filter may be made of a suitable polymer material such as polycarbonate, nylon, or parylene, or a suitable non-polymer material such as silicone, as appropriate.
• membrane filters may be preferred, for example, in the capturing of cells from, for example, urine.
• the membrane filter may be a polycarbonate membrane, preferably a polycarbonate hydrophilic membrane, for example, a track-etched polycarbonate hydrophilic membrane.
• the filter may have a pore size of about 5-10 ⁇ , preferably about 8 um.
• Preferred membrane filters may include micromembrane filters such as commercially available polycarbonate filters, for example, Whatman Nuclepore track-etched polycarbonate hydrophilic filters, (diameter 25 mm, pore size 8 ⁇ ) .
• the storage unit contains a solution selected to facilitate storage and/or analysis of the biological material.
• the solution may be, for example,
• a buffer suitable for inducing cell lysis to permit analysis of nucleic acids or proteins released from the cell a buffer suitable for inducing cell lysis to permit analysis of nucleic acids or proteins released from the cell
• fixative/preservative to prepare cells with the retention of the characteristic morphology a culture medium to sustain cell growth, an isotonic buffer suitable for storage of biological material, for example, phosphate buffered saline solution, or an appropriate buffer for the elution of the biological material from the filter.
• an isotonic buffer suitable for storage of biological material for example, phosphate buffered saline solution, or an appropriate buffer for the elution of the biological material from the filter.
• the solution will preferably be selected to correspond to the biological material to be captured and the analysis to be performed.
• assemblies of the invention could be used for the collection of exfoliated tumour cells from urine with the aim of analysing alterations in their DNA.
• this may be using a polycarbonate membrane filter with a pore size of 8 ⁇ to capture the tumour cells, then inserting the filter cartridge into the storage unit, and, optionally, releasing a cell-lysis and nucleic acid-preserving solution such as those commercially available from Qiagen or DNA Genotek [for example, as described in WO2003104251 A9] onto the filter content.
• the solution released onto the filter content may, for example, be a cell-lysis and protein-preserving solution such as RIPA buffer (commercially available from
• Millipore or cell extraction buffer (commercially available from Invitrogen) .
• the solution released onto the filter content may, for example, be a preservative buffer, for example one commercially available from Hologic (PreservCyt Solution, containing methanol) or a cellular growth medium, for example DMEM supplemented with 10% FBS, 1% L- glutamine, 100 U/ml penicillin and 100 ⁇ g/ml streptomycin.
• a preservative buffer for example one commercially available from Hologic (PreservCyt Solution, containing methanol) or a cellular growth medium, for example DMEM supplemented with 10% FBS, 1% L- glutamine, 100 U/ml penicillin and 100 ⁇ g/ml streptomycin.
• assembles of the invention may be used for the collection of a particular cell-free protein from urine, for example, by using filter composed of Protein A/G coated sepharose beads to which an antibody which binds to the protein of interest has been attached, the filter cartridge then being placed into the storage unit and, optionally, an isotonic buffer such as phosphate buffered saline being released onto the filter content.
• RNA isolated from urine sediments has been analysed for diagnosis of acute
• Assemblies and methods of the invention may be used to capture free macromolecules (e.g. proteins, DNA, RNA or metabolites) in urine or other fluids.
• free macromolecules e.g. proteins, DNA, RNA or metabolites
• ovarian cancer patients have been shown to have altered levels of Glycosylated eosinophil- derived neurotoxin, COOH-terminal osteopontin fragments and the ⁇ -subunit core fragment of human chorionic gonadotrophin, SMRP and Bcl-2 in their urine (Das and Bast Biomark Med. 2008; 2(3) : 291-303) .
• Detection of the S100A6 and S100A9 proteins in urine may have utility in the detection of upper GI tract cancers (Husi et al Proteomics Clin Appl . 2011; 5 ( 5-6) : 289-99 ) , whilst
• the assemblies and methods described herein may also be used for the collection and filtration of other biological fluids, such as saliva, sputum and blood, and bodily fluids obtained using more invasive methods such as, for example, pleural effusions, lavage fluid (for example ductal,, bronchoalveolar ) and sera for the analysis of captured material including via detection of genomic alterations associated with certain diseases and disorders including cancers such as lung and breast cancer (Belinsky et al Proc. Natl. Acad. Sci. USA, 95: 11891-11896, 1998; Ahrendt et al J . Natl. Cancer Inst., 91: 332-339, 1999; Evron et al Lancet, 357 : 1335-1336, 2001) .
• other biological fluids such as saliva, sputum and blood, and bodily fluids obtained using more invasive methods such as, for example, pleural effusions, lavage fluid (for example ductal,, bronchoalveolar )
• Filtration and concentration of blood may also be used in the isolation and analysis of circulating tumour cells (CTCs) .
• CTCs circulating tumour cells
• CTCs have been identified in cancers such as in breast, prostate, lung, ovarian and colon cancer patients, where they have been shown to provide predictive and prognostic information. CTCs have also been identified in pancreatic patients, although no pivotal study using CTCs to guide clinical treatment has been undertaken (Cen et al
• markers known to be associated with a particular disease or condition may be genetic markers, genomic alterations, the presence of or elevated/decreased levels of proteins (for example, antibodies), the presence of or elevated/decreased levels of bacteria or yeast, both as described herein and as documented in the art.
• the marker may be a marker known to be associated with cancer.
• the cancer may be urinary, or gynecological cancer, for example, bladder cancer, prostate cancer, renal cancer, urethral cancer, ureteral cancer,
• the cancer may be a cancer associated with other organs, for example, liver cancer, melanoma, colorectal cancer, head and neck cancer, lung cancer, breast cancer, pancreatic cancer, or a cancer of the upper GI tract.
• the cancer may be a metastatic cancer. Markers associated with these and other cancers are known in the art.
• the marker is associated with a genitourinary cancer, preferably, bladder, prostate, or renal cancer.
• the marker is associated with bladder cancer, more preferably non- muscle invasive bladder cancer.
• the marker is associated with a condition other than cancer.
• the marker may be associated with acute rejection in kidney transplants, which has the advantage of potentially obviating the need for invasive renal biopsies, or markers associated with bacterial and/or yeast infections, for example urinary tract infections such as cystitis and pyelonephritis. It will also be appreciated that in some methods of the
• the marker may not in itself be associated with a disease or condition but may instead be a genetic marker
• a key application of the invention is the diagnosis and
• the present invention was developed to provide a simple means for capturing bladder tumour cells from urine and storing/preserving DNA from these cells for later analysis. Important advantages include:
• the fraction of tumour cells may be increased by size-based filtration, increasing the sensitivity of detection;
• the filter content (e.g. captured cells) can be shipped by regular mail to an appropriate medical centre or testing
• Voided morning urine samples were collected from bladder cancer patients admitted for cystoscopy and transurethral resectioning (TURBT) at Herlev Hospital, Denmark and from healthy volunteers without known urological malignancies. Samples were sent to th Danish Cancer Research Center where they were processed within 6 hours after collection.
• Whatman Nuclepore track-etched polycarbonate hydrophilic filters were used, (diameter 25 mm, pore size 8 ⁇ ) and the corresponding filter holders. The sample was passed through the filter until saturation, with a maximum of 125 ml. The filter was rinsed with PBS before removal from the filter holder. Both urine sediment and the filter were stored at -80 °C until further processing.
• the filter cartridge was transferred to the storage cassette, which was mounted with the lid from an Oragene DNA Self-Collection Kit (disk format OG-250, DNA Genotek, Ottowa, Ontario, Canada) .
• Oragene DNA Self-Collection Kit disk format OG-250, DNA Genotek, Ottowa, Ontario, Canada
• DNA was isolated from urine sediment and filter by QiaAmp DNA Mini Kit (Qiagen GmbH, Hilden, Germany) . Filter samples and urine sediments were incubated with ATL buffer and proteinase K at 56 °C for at least 1 hour (filter) or overnight (sediments) . Subsequent processing was done according to manufacturer's instructions. DNA from filters and sediments were eluted in 50 ⁇ and 100 ⁇ of buffer AE, respectively, and stored at -80 °C. DNA concentration was measured using a NanoDrop 1000 spectrometer. The samples from 16 patients and 9 healthy controls did not contain sufficient DNA for analysis and were discarded.
• Bisulfite conversion was done using the EZ DNA Methylation-Gold Kit (Zymo Research) according to the manufacturer's protocol.
• the bisulfite-treated DNA was eluted in 20 ⁇ of M-Elution Buffer and stored at -80°C.
• paired samples sediment and filter sample
• the same amount of DNA was used, with a maximum of 500 ng.
• the maximum sample volume (20 ⁇ ) was used for bisulfite treatment.
• Methylation levels were calculated as percent methylated reference (PMR; Ref. [Wei senberger DJ, Campan M, Long TI, Kim M, Woods C et al .
• Methylation analysis was performed using MethyLight, a
• the human ureter transitional cell carcinoma cell line 639V was purchased from DSMZ (Braunschweig, Germany) . Cells were
• Lymphocytes from a healthy donor were prepared from peripheral blood
• Lymphocytes and 639V cells were mixed in different ratios in 100 ml of PBS and processed using the filtration device. Mutation analysis
• FGFR3 mutations R248C, S249C, G370C and Y373C
• ddPCR droplet digital PCR
• QX200 Bio-Rad Laboratories, Hercules, CA
• hydrolysis probe-based assays Purge-based assays; Bio-Rad
• the PCR mixture contained 11 ⁇ of ddPCR droplet supermix for probes (no dUTPs), 1.1 ⁇ of mutation primer/probe mix (FAM), 1.1 ⁇ of wildtype primer/probe mix (HEX) and 2 ⁇ of DNA in a final volume of 22 ⁇ .
• the sensitivity was 81% when urine sediments from the 204 bladder tumour patients were analyzed for the seven DNA methylation markers, while it was 87% for the corresponding filter samples (Table 1) .
• the sensitivity increased from 75% in sediments to 84% in filter samples.
• NMIBC carcinoma in situ
• CIS carcinoma in situ
• Ta tumours a carcinoma in situ
• Tl tumours two Tl tumours.
• three were positive (two in both filter and sediment; one in filter only) .
• One of these had been misclassified and had a bladder tumour.
• the second had prior problems with the bladder, and subsequent cystoscopy showed the presence of a hyperplastic lesion.
• the third was negative on cystoscopy.
• micromembrane filters for examples, commercial polycarbonate membrane filters
• the present invention relates to a method of passing a biological fluid sample, such as a urine sample, through a micromembrane filter.
• a biological fluid sample such as a urine sample
• the filter sample was positive for tumour-specific DNA methylation markers.
• the corresponding urine sediments were positive in 81% of the cases .
• Morning urine samples were collected from 30 patients admitted for bladder cystoscopy at Herlev Hospital. The samples were processed within 3-6 hours at the Danish Cancer Research Center. The sample volume varied between 150 and 400 ml, average 240 ml (Table 2) .
• the filtration devices were mounted with an 8 um pore size, track-etched polycarbonate filter (Whatman) . After filtration, the filters were removed from the filtration device and stored at -80 °C until further processing. DNA was isolated from the filters as described in above. DNA was eluted in 50 ⁇ of AE buffer and stored at -80°C. Bisulfite conversion of DNA was performed as described above. The DNA concentration was determined by quantitative PCR analysis of GAPDH. The methylation status of seven methylation markers ( CCNA1 , BCL2, EOMES, POU4F2, SALL3, HOXA9 and VIM2) was
• the average DNA yield for the 30 urine samples was 242 ng (range 6 to 1, 000 ng; Table 2) .
• Table 3 MethyLight analysis of seven DNA methylation markers in urine DNA from 18 bladder tumour patients. The pathology of these cases is indicated in Table 2.
• the inventors used 639V bladder cancer cells, which have a point mutation (p.R248C; c.742C>T) in the gene encoding fibroblast growth factor receptor 3 (FGFR3) with loss of the corresponding wildtype allele.
• FGFR3 fibroblast growth factor receptor 3
• the inventors spiked between 10 3 and 5 ⁇ 10 5 639V bladder cancer cells into 100 ml of PBS containing 10 7 normal purified cultured human lymphocytes (diameter 7-8 ⁇ ) and processed the suspension using the filtration device.
• Analysis of DNA extracted from filters by ddPCR showed signals for both mutant (R248C) and wildtype FGFR3 ( Figure 10A) .
• Vertical lines represent manually set cutoff settings.
• DNA was extracted from the filters and tested for mutant FGFR3 (R248C) molecules using ddPCR.
• DNA from normal peripheral blood lymphocytes (PBL) was used as a control for wildtype FGFR.
• the device is capable of isolating low abundant tumour cells, and therefore may therefore be useful for diagnosing smaller less aggressive tumours earlier.
• the size and stage of the tumour is normally reflected by the number of cells expected in a urine sample. The smaller less aggressive tumours would not shed as many cells into the urine as a more established tumour and therefore could potentially be missed on standard diagnostic techniques.
• DNA can be isolated from tumour cells spiked into PBS containing normal peripheral blood lymphocytes, showing that the device can isolate tumour cells from normal blood cells.
• lymphocytes The performance of the device was further evaluated by DNA-based analysis of cells collected from urine from patients with bladder cancer, including some with low-grade Ta tumors. The ratio of tumor-to-normal DNA was higher in filtered samples compared with the same samples processed by sedimentation and showed high sensitivity. The ability to easily collect, process and ship diagnostic cells from urine may broaden the use of noninvasive tests for detection and follow-up of bladder cancer.

Landscapes

• Health & Medical Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Engineering & Computer Science (AREA)
• General Health & Medical Sciences (AREA)
• Analytical Chemistry (AREA)
• Organic Chemistry (AREA)
• Pathology (AREA)
• Biomedical Technology (AREA)
• Molecular Biology (AREA)
• Wood Science & Technology (AREA)
• Genetics & Genomics (AREA)
• Zoology (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Physics & Mathematics (AREA)
• Immunology (AREA)
• Biochemistry (AREA)
• Bioinformatics & Cheminformatics (AREA)
• General Engineering & Computer Science (AREA)
• Biotechnology (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Hematology (AREA)
• Public Health (AREA)
• Animal Behavior & Ethology (AREA)
• Heart & Thoracic Surgery (AREA)
• Veterinary Medicine (AREA)
• Surgery (AREA)
• Medical Informatics (AREA)
• Biophysics (AREA)
• Microbiology (AREA)
• General Physics & Mathematics (AREA)
• Clinical Laboratory Science (AREA)
• Plant Pathology (AREA)
• Hospice & Palliative Care (AREA)
• Oncology (AREA)
• Crystallography & Structural Chemistry (AREA)
• Apparatus Associated With Microorganisms And Enzymes (AREA)
• Sampling And Sample Adjustment (AREA)
• Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Priority Applications (11)

Applications Claiming Priority (2)

Related Child Applications (2)

Publications (1)

Family

ID=49552641

Family Applications (1)

Country Status (11)

Cited By (8)

Families Citing this family (21)

Citations (4)

Family Cites Families (9)

• 2013
  • 2013-09-13 GB GB201316347A patent/GB201316347D0/en not_active Ceased
• 2014
  • 2014-09-12 CN CN201480062490.6A patent/CN105722466B/zh active Active
  • 2014-09-12 WO PCT/GB2014/052776 patent/WO2015036781A1/en not_active Ceased
  • 2014-09-12 DK DK14784509.3T patent/DK3043718T3/da active
  • 2014-09-12 JP JP2016542375A patent/JP6552504B2/ja active Active
  • 2014-09-12 ES ES14784509T patent/ES2808664T3/es active Active
  • 2014-09-12 CA CA2924086A patent/CA2924086C/en active Active
  • 2014-09-12 US US15/021,470 patent/US20160223442A1/en not_active Abandoned
  • 2014-09-12 AU AU2014320077A patent/AU2014320077B2/en active Active
  • 2014-09-12 EP EP14784509.3A patent/EP3043718B1/en active Active
  • 2014-09-12 PL PL14784509T patent/PL3043718T3/pl unknown

Patent Citations (4)

Also Published As

Similar Documents

Legal Events