Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus
  • C12M3/06—Tissue, human, animal or plant cell, or virus culture apparatus with filtration, ultrafiltration, inverse osmosis or dialysis means
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
  • C12M47/02—Separating microorganisms from the culture medium; Concentration of biomass
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01D—SEPARATION
  • B01D35/00—Filtering devices having features not specifically covered by groups B01D24/00 - B01D33/00, or for applications not specifically covered by groups B01D24/00 - B01D33/00; Auxiliary devices for filtration; Filter housing constructions
  • B01D35/20—Vibrating the filters
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01D—SEPARATION
  • B01D37/00—Processes of filtration
  • B01D37/04—Controlling the filtration
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M1/00—Apparatus for enzymology or microbiology
  • C12M1/02—Apparatus for enzymology or microbiology with agitation means; with heat exchange means
• • 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
  • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/02—Separating microorganisms from their culture media
• • 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
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/0081—Purging biological preparations of unwanted cells
• • 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
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0602—Vertebrate cells
  • C12N5/0693—Tumour cells; Cancer cells
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M27/00—Means for mixing, agitating or circulating fluids in the vessel

Definitions

• the invention relates to the field of biological sample purification. More specifically, the invention relates to devices and methods which can separate trace target cells from a fluid sample having a plurality of other cells and/or contaminants.
• US 7,494,809 B2 discloses an automated process in which a fluid biological sample is passed through a series of filters and treated with reagents in order to provide a stained, enriched cell population for evaluation.
• One cell population which can be of interest is circulating tumour cells or CTC. This because the greatest risk for mortality among cancer patients is often not the original malignant tumour, but the formation of metastases in tissues or organs distant from the primary tumour. Small numbers of cells can break free from the primary tumour and enter the circulatory system, becoming CTC. While the majority do not survive, some not only survive, but at some point translocate across the endothelial wall, establishing a new tumour in the surrounding tissue.
• CTC detection has largely depended upon antibody-based positive selection.
• the present invention meets this need where the present technology fails by not only separating target cells from a plurality of other cells and/or contaminants in a liquid sample, but by providing the purified, enriched target cell population in a relatively native state. Yet another benefit of the present invention may be the provision of faster diagnostic information. Because the invention allows for a rapid separation of target cells, and offers those cells in a native state, the time from sample-taking to target cell analysis is minimised. The effectiveness of the invention can also reduce the need for expensive and time-consuming repeat sampling.
• the target cells are effectively and efficiently removed from the greater sample and provided in a native state.
• a further benefit of the present invention is that the non-target components of the sample are also preserved in their native state. In many cases these components are also of interest. A device and method which allows for observation and analysis these components as well is therefore an even greater advance in the art.
• a device for sample purification and enrichment comprises a filtration unit having a first side and a second side and comprising an agitator, a sample input in elective fluid communication with the first side of the filtration unit, a first liquid reservoir in elective fluid communication with the first side of the filtration unit, a first suction means having a first suction reservoir and in elective fluid communication with the first side of the filtration unit, a second liquid reservoir in elective fluid communication with the second side of the filtration unit, a pressure measurer in fluid communication with the second side of the filtration unit and having a flow stop ability, and a second suction means in elective fluid communication with the second side of the filtration unit.
• the second suction means can have a second suction reservoir. At least one of the first suction means and the second suction means can be a peristaltic pump.
• the pressure measurer flow stop ability can involve a clamp or a piezo-electric membrane.
• a cartridge for use in the inventive device which comprises a filtration unit having a first side and a second side and comprising a filter and being connectable to an agitator, a sample input tube in fluid communication with the first side of the filtration unit, a first liquid reservoir tube in fluid communication with the first side of the filtration unit, a first suction reservoir tube in fluid communication with the first side of the filtration unit, a second liquid reservoir tube in fluid communication with the second side of the filtration unit, and a second suction tube in fluid communication with the second side of the filtration unit, wherein the cartridge has at least one access point for a pressure measurer.
• a method of purifying a sample comprises the steps of providing a sample to a sample input in fluid communication with a first side of a filter, applying suction to a second side of a filter to draw fluid from the sample input through the filter until a predetermined pressure is measured, placing the second side of the filter in exclusive fluid communication with a reservoir containing a liquid, applying suction to the first side of the filter to draw fluid from the reservoir through the filter, and collecting the fluid drawn from the reservoir through the filter in a container.
• the filter is subject to agitation during at least a significant portion of the process and the purified sample is collected in the container.
• the pre-determined pressure can be in the range of
• a sample can be from any source, such as an organism.
• a sample can include an extract of a swab, a sputum, urine, or fecal sample, a wash of an internal bodily area such as bronchial washes, cord blood, bone marrow aspirates, ascites fluid, or internal or peripheral blood. It may comprise additives such as anticoagulants or stabilisers.
• a target cell is a specific type of cell of interest which may or may not be present in the sample. Non-target cells are those which are preferably removed, even though their analysis may be of interest.
• a "filter” is a structure that comprises one or more openings or pores of a particular dimension which allows the passage of particles smaller than that dimension to an opposite side of the filter while preventing larger particles from passing through the filter. It may be formed of any material, for example, plastics, ceramics, silicon, metal, or glass.
• the filter may be microfabricated or micromachined, alternatively, it may be a more conventional filter such as mono- or multi-fibrous filters made of nylon, polycarbonate, celluloses, etc.
• a filter may be treated to alter its surface properties, such as to increase hydrophilicity.
• Figure 1 is a schematic view of an embodiment of the invention
• Figure 2 is a schematic view of a further embodiment of the invention.
• Figure 3 shows an untreated bladder washing sample
• FIG 4 shows the sample of Figure 3 after treatment according to the invention
• Figure 5 shows an untreated ascites sample
• Figure 6 shows the sample of Figure 5 after treatment according to the invention.
• a device of the invention 10 comprises a filtration unit 11 having a first side 12 and a second side 13 and comprising an agitator (not shown).
• the first and second sides of the filter 12, 13 are generally opposing, but not necessarily strictly opposite each other. Depending on the configuration and placement of the actual filtration material, the filter, their relative positions may be closer together or further apart.
• Filtration unit 11 comprises a filter (not shown) which may be any suitable filter as known in the art.
• the pore size of the filter should be selected based on a consideration of both the target cells of interest and the predicted non-target cells and/or contaminants expected to be in the sample. For example, when using a device 10 of the invention to purify and enrich CTCs from samples including blood cells, a filter size in the range of 10pm is preferred.
• the size of the filter itself can vary depending on the device and the purpose. In an embodiment of the invention a standard commercial 22 mm diameter filter is used, and is capable of filtering about 15,000 - 20,000 cells.
• the agitator which is provided in conjunction with filtration unit 11 allows mechanical vibration to be used during filtration to maximise efficiency and precision of the device 10.
• Mechanical agitation in any direction may be effective, however, agitation in a horizontal direction has been found to work particularly well with biological samples in a device 10 of the invention.
• agitating a filtration unit which contains some headspace for gases in a back and forth horizontal motion can result in the fluid within the filtration unit swirling in a circumferential motion. This motion appears to be effective while at the same time gentle.
• Agitation in the range of 1000-2000 Hz may be preferred. This range generally allows for effective use of the entire surface area of the filter while not being so strong as to damage or cause rupture to components in the sample.
• tubes 14 of the various components are in fluid communication with one another via a series of tubes 14.
• the broad range of uses of the device is accompanied by a significant degree of choice in commercially-available tubing. It will be preferred to employ materials which are inert with respect to biological fluids and components thereof. Furthermore, the length and diameter, particularly the inner diameter, of the tubing should be taken into consideration when determining system parameters such as speed and pressure. In some cases it may be preferred to use silicone tubing with an internal diameter in the range of 2 mm. While described herein as generally flexible cylindrical forms, tubes 14 of the invention can comprise non-flexible and/or non-cylindrical materials. For example, glass or metal formed into cylinders or other shapes.
• the tubing serves the purpose of conveying liquids from and between various components of the device and as such a great deal of variety of forms may be used depending on the chosen dimensions and configuration of the overall unit. Skilled persons will appreciate that the inner dimensions of the tubes need be sufficiently large to accommodate the cells in the sample, knowing that diameters which approach the size of the cells may cause pulsing or other undesirable effects on fluid flow within the device.
• a plurality of selection means 15 are provided which allow selective fluid communication between various components of the device.
• Selection means can direct and stop fluid passage via manual or automated control.
• a two-way valve can be used which could be adjusted into a first position to put a first component into fluid communication with the filter, then rotated into a second position putting a second component into fluid communication with the filter.
• selection means 15 may differ, for example some may be manual and some may be automated.
• Sample input 16 can be a port or other means to allow a fluid sample access to the system.
• Sample input 16 may comprise a holder or container, such as a test tube.
• a tube 4 which can be placed in communication with a sample, which could be provided in, for example, a test tube. Due to concerns regarding, i.e., contamination and security, the contents of the sample may preferably be enclosed, for example by providing the sample in a stoppered test tube and configuring the sample input as a needle at the end of a tube, which needle is inserted into the stopper.
• First liquid reservoir 17 is provided to the device 10. In initial stages of use a liquid is present in first liquid reservoir 17. Its use will be further described in the examples, below, however it can be noted that the fluid in first liquid reservoir serves at least a general flushing or rinsing purpose and as such is preferable physiologically neutral. Suitable fluids include phosphate buffered saline (PBS), Hank's buffered saline solution, (hydroxymethyl)aminomethane (TRIS) buffer, and other buffers with a physiologically neutral pH and osmolarity of approximately 270-330 mOsm/kg.
• PBS phosphate buffered saline
• TMS hydroxymethyl)aminomethane
• First suction means 18 is capable of providing suction to a first side of the filter 12, effectively drawing fluid from the second side of the filter 13 to the first side of the filter 12.
• First suction means 18 is provided with a first suction reservoir (not shown) which is capable of collecting fluid and particles suspended therein.
• first suction means 18 may be a syringe the stopper of which can be drawn out to create suction and the inner cavity of which provides a reservoir.
• the first suction means 8 can be a peristaltic pump.
• Second liquid reservoir 19 is initially supplied to device 10 with fluid therein. Use will be further described below, however, the fluid is preferably physiologically neutral and as a consequence the material used to form second liquid reservoir 19 is preferably non-reactive. The size and/or capacity of second liquid reservoir 19 will depend on the intended use of device 10 and will also depend on whether second liquid reservoir 9 is adapted as a fixed portion or if it is interchanged after every use of device 10.
• Pressure measurer 20 allows a user (or automated monitoring device) to monitor the degree of vacuum present within the device. Through calibration, this measure will allow a determination of when the filter is sufficiently saturated with target cells. Pressure measurer 20 may include a visual display.
• Pressure measurer 20 further allows a user to normalize a sample. By relying on pressure of the internal system one can avoid the need for timely pre-screening steps which would otherwise be beneficial for determining the concentration of particulate matter in the sample. As the samples and organisms from which they originate will differ, so too will the samples evaluated. Furthermore, users may in some cases choose to dilute a sample so that it can be used for a number of purposes, or to make the purification and enrichment method function most efficiently.
• An example of a suitable pressure measurer 20 is a column of water. When such a pressure measurer 20 is employed, a reasonable operating range for the device 10 is 130-150 mm H 2 0. The range of 135-145 mm H 2 0 may be preferred.
• Approximately 140 mm H 2 0 may be particularly effective.
• pressure measurer 20 may be connected to a selection means 15 or other flow control means such that, upon reaching certain predetermined pressures, the aspects of the system are engaged or disengaged.
• pressure measurer 20 need not include a display means as is inherent in a water column. Instead, it may comprise an electronic sensor which detects pressure.
• the information obtained by pressure measurer 20 could be transmitted to a central control unit, which could send commands which, using the flow stop ability of the pressure measurer 20, stops or starts flow in chosen areas of the device.
• Flow stop ability could be achieved using, for example, a membrane.
• a membrane One example is a piezo-electric membrane.
• Simple flow stops are also contemplated, such as a clamp or crimp which can physically deform a flexible tube when engaged, thus stopping flow through the tube. By disengaging the clamp or crimp flow may begin again.
• Pressure measurer 20 can be a device which directly measures pressure
• pressure measurer 20 serves a purpose when conducting the initial pass of the sample through the filtration unit 11 , in subsequent steps using the device 10 it may not be required. Nonetheless, particularly in automated systems, one can continue to measure the internal system pressure for statistical purposes and/or as a safety check to possibly detect malfunctions in the system.
• Second suction means 21 is capable of providing suction to a second side of the filter 13, effectively drawing fluid from the first side of the filter 12 to the second side of the filter 13.
• An optional second suction reservoir may be provided (not shown) which could be capable of collecting fluid and particles suspended therein.
• a second suction means 21 could be a syringe the stopper of which can be drawn out to create suction.
• the inner cavity of the syringe could serve as a reservoir if desired.
• second suction means 21 could be a pump, such as a peristaltic pump.
• FIG. 2 A further embodiment of a device 10 of the invention is shown in Figure 2. In that figure, for ease of illustration, not every piece of tubing 14 is marked with a reference numeral.
• a plurality of selection means 15 are shown.
• the selection means 15 in fluid communication with the first side of the filter 12 is in this embodiment a three-way valve, whereas that in fluid communication with the second side of the filter 13 is a two-way valve.
• Figure 2 has an alternative configuration of elements, grouping all components connected to the first side of the filter 12 via a single selection means 15 and tube 14.
• the components connected to the second side of the filter 13 are also in a different configuration relative to the Figure 1
• pressure measurer 20 is not connected via a selection means but instead one of second liquid reservoir or second suction means 21 may be in fluid communication with the system at any time. It should be apparent to skilled persons that further configurations are possible and within the scope of the invention. Furthermore, a plurality of devices can be provided in sequence or in parallel.
• an entire device according to the invention can be provided in a refrigerated unit or cold room.
• the liquid reagents used within the system are provided at a particular temperature, for example, between about 0-4°C.
• most or all components of a device according to the invention may be disposable. In particular, it is
• disposable cassettes or cartridges may be supplied with components of a device according to the invention.
• a cassette can preferably include a barcode or space to adhere a barcode to help facilitate electronic processing of samples.
• electronic identifiers which can be incorporated into or affixed to the cassette.
• Cassettes or cartridges useful with the present invention comprise a filtration unit and may comprise an agitator. Alternatively, the filtration unit may simply be capable of being connected or engaged by an agitator.
• a plurality of tubes are also provided which connect respective first and second sides of the filtration unit to the reservoirs and inputs of the device.
• At least an access point is provided for a pressure measurer, the pressure measurer itself may be included in the cassette. The access point could simply be a place in the tubing where a sensor is inserted.
• a device of the invention may be operated manually; alternatively it may be partially or entirely automated.
• Figure 1 is referred to herein.
• the system and/or filter can be primed with a liquid.
• a sample is provided to a sample input 16 which is in fluid communication with the filtration unit 11.
• the sample may be pre-treated prior to performing the method of the invention. For example, to decrease the propensity of cells to flex and thus be able to pass through a pore smaller than their general diameter, they can be fixed.
• the plasma can be separated from the cells treated with a standard 0.37% formaldehyde solution. PBS or other neutral liquid can then be added up to a volume of the original sample. While not wanting to be bound by theory, it is believed that the cells may be fixed or more rigid after the formaldehyde addition, making them pass or fail to pass through the filter in a manner more closely related to their diameter, and less closely related to their ability to flex through a filter pore.
• the relevant selection means 15 is configured so that second suction means 21 is in fluid communication with the filtration unit 11. Second suction means 21 is engaged thus drawing fluid and suspended particles via sample input 6 into filtration unit 11.
• a range of suitable pressures can be used, depending on the type of sample and configuration of the device. Through the use of pressure measurer 20 it is possible to calculate approximately how saturated the filtration unit 11 has become with target cells. At the appropriate time, for example, when a column of water used to indicate pressure indicates approximately 140 mm H2O, second suction means 21 ceases to apply suction and sample input 16 is taken out of fluid communication with filtration unit 11.
• This step has the effect of drawing non-target cells and contaminants smaller than the pore size of the filter through the filter, leaving target cells on the first side of the filtration unit.
• Another optional pre-treatment would be to use known materials and methods to achieve selective cellular lysis. This may allow for a faster or more efficient filtration using the device of the invention.
• First liquid reservoir 17 is placed in fluid communication with filtration unit 11 and second suction means 21 is again engaged, drawing fluid from first liquid reservoir 17 through filtration unit 11.
• This step has the effect of rinsing or cleaning the system of remaining non-target cells.
• First liquid reservoir 17 is removed from fluid communication with the filtration unit and second liquid reservoir 19 is put into communication with the system.
• First suction means 18 is engaged, drawing fluid out of second liquid reservoir 19 and through filtration unit 11. The fluid thus drawn is collected in a first suction reservoir (not shown).
• target cells which were left on the first side of filter 12 are suspended in the passing fluid and carried into the first suction reservoir. From that point they may undergo direct observation or analysis, treatment or staining, or transfer to another device or storage.
• second suction means 21 is again used to draw fluid from first fluid reservoir 17 through filtration unit 11 , followed by first suction means 8 drawing additional fluid from second fluid reservoir 19.
• first suction means 8 drawing additional fluid from second fluid reservoir 19.
• the immediately aforementioned dual fluid transfer step is repeated twice or three or more times.
• a bladder washing is performed on an organism and the washing fluid, with probable suspended cells and contaminants is collected.
• Process time is about 10 minutes.
• the maximum pressure reached in the device during the first suction of the sample through the filter is 140 mm H 2 O.
• the material captured in the first suction reservoir is placed on a slide and stained. Upon microscopic inspection, the purified and enriched target cells are readily visible in their natural state, see Figure 4.
• a second suction reservoir is used. The material captured in the second suction reservoir is placed on a slide and stained. Upon microscopic inspection, it reveals a plurality of red blood cells and epithelial cells. Based on analysis of the target and non-target cells captured, the recovery yield of target cells is calculated to be >95%.
• Example Two Ascites Fluid Contaminated with Epithelial Cells A sample of ascites fluid is obtained. A stained droplet of the fluid is examined microscopically, showing significant amounts of epithelial cells and small numbers of potential target cells, see Figure 5. The 10 ml remaining sample was placed in a 200 ml sample input vessel and filtered through a device of the present invention. The process was concluded in approximately 10 minutes, during which the maximum pressure reached during the first suction of the sample through the filter was 140 mm H 2 O. The material captured in the first suction reservoir was placed on a slide and stained. Upon microscopic inspection, the purified and enriched target cells were readily visible in their natural state, see Figure 6.
• a sample containing cells including peripheral blood cells, is provided.
• the cells are chemically lysed according to known techniques.
• the cell contents are placed in a sample input vessel of a device according to the invention.
• a first process is carried out using a filter whose pores allow free passage to most subcellular components, but retain the largest: cell nuclei. After this run of the system the nuclei are set aside for later use and the remaining components collected in the second suction reservoir are removed. The system is primed again for use and the components previously collected in the second suction reservoir are loaded in the sample input.
• a second process is carried out with a filter provided with pores which allow free passage to most cellular components, but retain the large mitochondria from this fraction. Subsequent to this process the nuclei, mitochondria, and the remaining components collected in the second suction reservoir are separately analysed.

Landscapes

• Engineering & Computer Science (AREA)
• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Zoology (AREA)
• Organic Chemistry (AREA)
• Wood Science & Technology (AREA)
• Biotechnology (AREA)
• Biomedical Technology (AREA)
• Genetics & Genomics (AREA)
• General Engineering & Computer Science (AREA)
• Biochemistry (AREA)
• General Health & Medical Sciences (AREA)
• Microbiology (AREA)
• Sustainable Development (AREA)
• Cell Biology (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Virology (AREA)
• Medicinal Chemistry (AREA)
• Tropical Medicine & Parasitology (AREA)
• Oncology (AREA)
• Molecular Biology (AREA)
• Water Supply & Treatment (AREA)
• Sampling And Sample Adjustment (AREA)
• Apparatus Associated With Microorganisms And Enzymes (AREA)
• Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
• Separation Of Solids By Using Liquids Or Pneumatic Power (AREA)
• Investigating Or Analysing Biological Materials (AREA)

Priority Applications (8)

Applications Claiming Priority (2)

Publications (1)

Family

ID=44196041

Family Applications (1)

Country Status (10)

Cited By (2)

Families Citing this family (6)

Citations (10)

Family Cites Families (10)

• 2009
  • 2009-12-22 SE SE0951009A patent/SE0951009A1/sv not_active Application Discontinuation
• 2010
  • 2010-12-22 CA CA2783007A patent/CA2783007A1/en not_active Abandoned
  • 2010-12-22 BR BR112012015643A patent/BR112012015643A2/pt not_active IP Right Cessation
  • 2010-12-22 US US13/517,086 patent/US20120270312A1/en not_active Abandoned
  • 2010-12-22 CN CN201080058448.9A patent/CN102803467B/zh active Active
  • 2010-12-22 RU RU2012122009/10A patent/RU2012122009A/ru not_active Application Discontinuation
  • 2010-12-22 WO PCT/SE2010/051473 patent/WO2011078784A1/en not_active Ceased
  • 2010-12-22 EP EP10839905.6A patent/EP2516619B1/en active Active
  • 2010-12-22 KR KR1020127019068A patent/KR20120121883A/ko not_active Withdrawn
  • 2010-12-22 JP JP2012545908A patent/JP2013514806A/ja active Pending

Patent Citations (10)

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events