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
• • 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
• • 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
• • 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
  • B01L2300/0861—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
  • 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
  • B01L2400/06—Valves, specific forms thereof
  • B01L2400/0677—Valves, specific forms thereof phase change valves; Meltable, freezing, dissolvable plugs; Destructible barriers
  • B01L2400/0683—Valves, specific forms thereof phase change valves; Meltable, freezing, dissolvable plugs; Destructible barriers mechanically breaking a wall or membrane within a channel or chamber
• • 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
  • B01L3/02—Burettes; Pipettes
  • B01L3/021—Pipettes, i.e. with only one conduit for withdrawing and redistributing liquids
  • B01L3/0217—Pipettes, i.e. with only one conduit for withdrawing and redistributing liquids of the plunger pump type
  • B01L3/0231—Pipettes, i.e. with only one conduit for withdrawing and redistributing liquids of the plunger pump type having several coaxial pistons
• • 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
  • B01L3/5025—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures for parallel transport of multiple samples
  • B01L3/50255—Multi-well filtration
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L9/00—Supporting devices; Holding devices
  • B01L9/54—Supports specially adapted for pipettes and burettes
• • 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
  • G01N1/4044—Concentrating samples by chemical techniques; Digestion; Chemical decomposition
• • 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 a method, a device and a kit for molecular biology for extracting amplified genetic material from isolated cells on a filter and detecting mutations and levels of gene expression for sensitivity and resistance to targeted therapies. .
• Some particular blood cells for example tumor cells or trophoblastic cells, are in very low concentration and must be concentrated for cytopathological analysis. However, compared to blood cells, they are larger.
• the present invention aims to remedy these drawbacks and to respond to this need by making it possible to collect, in conditions compatible with routine laboratory tests, a large proportion of the cellular material, in particular, RNA and DNA, cells considered, in particular good condition.
• the present invention provides a method for collecting cellular material from particular cells present in a liquid, characterized in that it comprises:
• the method that is the subject of the present invention makes it possible to collect cellular material quickly and efficiently.
• the cellular material recovered through the practice of the present invention is the genetic material of the cells.
• the method which is the subject of the present invention thus makes it possible to recover, directly on the filter and with virtually no loss, the genetic material of rare cells, up to a single isolated cell. A large proportion of the genetic material of the cells in question is thus collected, in good condition, under conditions compatible with routine laboratory tests.
• the process which is the subject of the present invention comprises, following the lysis step, a step of amplification of the DNA and / or RNA and, during the recovery step, amplified genetic material is recovered from the lysed cells.
• a uniform amplification is carried out, preserving the quantitative aspect of the DNA and the RNA.
• the method which is the subject of the present invention, as briefly described above, comprises, in in addition, a detection step, during which the amplified DNA is used as a template to detect at least one gene mutation of sensitivity or resistance to at least one targeted therapy.
• the method which is the subject of the present invention furthermore comprises a detection step, during which the amplified DNA is used as a matrix to detect a variation in the level of d expression of genes of sensitivity or resistance to targeted therapies.
• the method which is the subject of the present invention further comprises a detection step during which RNA is converted into cDNA and said cDNA is used to detect the cDNA.
• a quantitative and real-time PCR (acronym for polymerase chain reaction) is used.
• At least one pair of sense and antisense primer pairs are used to amplify a predetermined sequence of interest.
• At least one pair of probes is implemented.
• At least two probes of a pair of probes are coupled to two different fluorochromes and are defined so that one recognizes the mutated sequence and the other recognizes the normal sequence.
• At least one pair of primers and a pair of probes are adapted to detect the G12D mutation of the K-ras gene of Erlotinib and Gefitinib resistance.
• At least one primer comprises at least 80% of the sequence
• At least one primer comprises at least 80% of the sequence
• At least one probe comprises at least 80% of the TTGGAGCTGGTGGCGT sequence.
• At least one probe comprises at least 80% of the TGGAGCTGATGGCGT sequence.
• At least one pair of primers and a pair of probes are adapted to detect the G12V mutation of the K-ras Erlotinib and Gefitinib resistance gene.
• At least one primer comprises at least 80% of the sequence
• At least one primer comprises at least 80% of the sequence
• At least one probe comprises at least 80% of the TTGGAGCTGGTGGCGT sequence.
• At least one probe comprises at least 80% of the sequence TTGGAGCTGTTGGCGT.
• At least one pair of primers and a pair of probes are adapted to detect the G13C mutation of the K-ras Erlotinib and Gefitinib resistance gene.
• At least one primer comprises at least 80% of the sequence
• At least one primer comprises at least 80% of the sequence
• At least one probe comprises at least 80% of the TTGGAGCTGGTGGCGT sequence. According to particular characteristics, at least one probe comprises at least 80% of the sequence TTGGAGCTGGTTGCGT.
• At least one pair of primers and a pair of probes are adapted to detect the L858R mutation of the EGFR gene of increased sensitivity to Erlotinib and Gefitinib.
• At least one primer comprises at least 80% of the sequence GCAGCATGTCAAGATCACAGATTT.
• At least one primer comprises at least 80% of the sequence
• At least one probe comprises at least 80% of the sequence CAGTTTGGCCAGCCCA.
• At least one probe comprises at least 80% of the sequence CAGTTTGGCCCGCCCA.
• the process as briefly described above comprises, in advance of the filtration step, a step of applying a formaldehyde-free fixing buffer to the liquid containing the particular cells.
• the particular cells sought are specifically hardened without altering the genetic material.
• the compartment has the general shape of a syringe in which the filter is positioned between the two openings.
• vacuum suction is applied below the filter. Thanks to these provisions, the filtration is more efficient and faster than in the absence of suction.
• the compartment is placed on a tube of the Eppendorf type. There is thus a direct passage from the filter to an Eppendorf-type tube.
• a piston is placed in the upper opening of the compartment, comprising a central punch movable inside the piston.
• the mobile central punch has a pointed lower end.
• the pointed lower end has a star shape.
• pressure is applied to the upper part of the punch to perforate the filter with the tip of the punch.
• the piston having a means for locking the punch longitudinally, during the recovery step, the punch is pivoted inside the piston to release it from said locking means, before applying a pressure on its upper part.
• a downward vertical pressure is applied to the piston to pass the contents of the compartment into a tube placed below the compartment.
• the cellular material of the particular cells can be extracted without deterioration through the perforation of the filter.
• the contents of the compartment are isolated by plugging the lower opening with a membrane.
• said membrane is positioned under a bar surrounding the lower opening of each compartment. This ensures the sealing and the maintenance of the lysis liquid above the filter.
• said membrane is an adhesive membrane.
• the filter is made of polycarbonate with a hydrophilic surface treatment.
• the use of such a filter improves the retention rate of the particular cells and reduces the adhesion of the cellular material to be recovered.
• the filter has a pore diameter centered on 7.5 ⁇ m.
• the diameters due to the dispersion of the diameters, virtually no pore has a diameter greater than 8 microns.
• this pore diameter which is less than the pore diameter traditionally used for the cytological analysis filters, makes it possible to remove them, which reduces the number of contiguous pores and avoids the loss of particular cells.
• the present invention relates to a device for collecting genetic material from particular cells present in a liquid, characterized in that it comprises:
• a compartment comprising an upper opening and a lower opening, between the two openings being positioned a filter whose micropores have a diameter intermediate between that of said particular cells and that of other cells,
• a filtration means passing the principal of the liquid and said other cells through the filter
• the present invention provides a molecular biology kit comprising at least two primers and / or two probes among the following sequences:
• the kit object of the present invention further comprises a device object of the present invention, as briefly described above.
• FIG. 1 schematically represents, in perspective, parts of two particular embodiments of the device that is the subject of the the present invention, implemented in a first phase of the process which is the subject of the present invention
• FIG. 2 schematically represents, in section, parts of the two particular embodiments of the device that is the subject of the present invention, implemented in a first phase of the method that is the subject of the present invention
• FIG. 3 schematically represents, in perspective, parts of a first particular embodiment of the device that is the subject of the present invention
• FIG. 4 represents, schematically, in elevation, parts of the first particular embodiment of the device that is the subject of the present invention
• FIG. 5 represents, schematically, in section, parts of the first particular embodiment of the device that is the subject of the present invention
• FIG. 6 schematically represents, in perspective, parts of a second particular embodiment of the device that is the subject of the present invention.
• FIG. 7 schematically represents, in section, parts of the second particular embodiment of the device that is the subject of the present invention.
• FIG. 8 schematically represents, in perspective, parts of the two particular embodiments of the device that is the subject of the present invention, used in a second phase of the method that is the subject of the present invention, and
• FIG. 9 represents, in the form of a logic diagram, the steps implemented in a particular embodiment of the method that is the subject of the present invention.
• the device for collecting cellular material here genetic, comprises (here four) syringe-shaped compartments 105 presenting, each , an upper opening 106 and a lower opening 107.
• a washer or ring 118 placed in the lower opening 107 retains a filter 115.
• the filters 115 are micro-perforated and glued on the washers or rings 118 and then inserted at the distal end, that is to say say, at the bottom, of the four syringe-shaped compartments 105.
• the filter 115 is made of polycarbonate with a hydrophilic surface treatment.
• the use of such a filter improves the retention rate of the particular cells and reduces the adhesion of the cellular material to be recovered.
• the filter 115 has, for example, a pore diameter centered on 7.5 ⁇ m. Due to the dispersion of the diameters, practically no pore then has a diameter greater than 8 ⁇ m.
• each compartment 105 At the small opening, or bottom opening, of each compartment 105 is placed, tightly, a tip (English "tip") 117 to avoid potential contamination of the end of the compartment 105 by splashing from the tank 112.
• These compartments 105 are assembled by a bar 116, made of plastic, to constitute a single piece.
• the assembly, consisting of the four compartments 105, the upper bar 116, the four pistons 140 and the bar-cap 120 (described below) is for single use.
• the compartments 105 are supported by a plate 110 inserted into a drawer holder 111 having a compartment connected to a tank 112 under the lower surface of the plate 110 through which an aspiration can be carried out. performed.
• An O-ring 113 seals the connection between a nozzle 117 and the plate 110.
• An O-ring 114 seals the connection between the plate 110 and the slide gate 111. The seal provided by the O-rings 113 and 114 allows aspiration of the contents of the compartments.
• the compartments 105 are removed from the portion of the device illustrated in FIGS. 1 and 2 and the tips 117 are removed from the lower openings of the compartments 105.
• a cap strip 120 is inserted at the tip of the compartments 105 in the form of syringes. It is a bar with four perforations that receive the lower end of the compartments.
• a plastic film is glued to the underside of this plug-bar 120, and constitutes a sealed membrane, preferably adhesive.
• the contents of the compartment 105 are thus kept away from the environment until the recovery of the cellular material of the particular cells.
• the bar 116 carrying the compartments 105, associated with the bar of plugs 120, is placed in abutment on a rack 133 made of plastic material. Then, in an oven, the lysis of the cells present in the compartment 105 is carried out in an oven. For this purpose, after the addition of the reagents for the lysis of the cell membranes of the desired cells and the uniform amplification of the DNA or the RNA, the rack 133, now the vertical compartments 105, is transported in an oven. At the outlet of the oven, after cooling, the Eppendorf-type tubes are positioned on the lower support 131 of the gantry 130. The bar 116 carrying the compartments 105, associated with the bar of plugs 120, is positioned on the gantry 130 and place the gantry 130 on the lower support 131.
• a piston 140 provided with a central axial punch 141 ending in a tip 142.
• this tip 142 has a star shape, for example to four branches, the point being cruciform.
• each compartment 105 is positioned an Eppendorf tube 125 held in position by a shelf of the gantry 130.
• Figures 3 and 4 illustrate the respective successive positions of the piston 140 and the punch 141 during the second phase of the process.
• the piston 140 and the punch 141 are substantially entirely outside the compartment 106 and the upper part of the punch 141 protrudes vertically above the piston 140.
• the punch is passed from a safety position in which the punch 141 can not slide longitudinally inside a piston 140 because of a mechanical stop, at a position activation device in which the punch can slide longitudinally inside the piston 140.
• a vertical force is applied downward on the top of the punch 141, the tip 142 of the punch 141 is lowered into the body of the compartment 106, to the filter 115.
• the tip 142 first pierces the filter 115 and the membrane 120 to penetrate slightly into the tube 125.
• the punch 141 is then held in position by contact on the opening bottom of the compartment 105.
• the Eppendorf type tubes 125 are then removed from the lower support 131, by withdrawing, upwardly from the gantry 130, the compartments 105 and the membrane 120, for analysis of the amplified genetic material, in particular the DNA or the RNA of the cells. of interest, collected in these tubes 125.
• the plate 110, the rack 133, the gantry 130 and the support 131 are reusable.
• a support 131 of the gantry 130 is replaced by a support 132 having eight openings instead of four.
• the four tubes of Eppendorf type 125 are replaced by a bar of eight tubes of Eppendorf type 126 of lower capacity, typically 0.25 ml. Instead of 1, 5 ml.
• These Eppendorf-type tubes are adapted to another mode of recovery of cellular material making it possible to extract the amplified genetic material directly on the filter but in a smaller volume. Such a volume can then be contained in Eppendorf type tubes directly adapted to a RT-PCR apparatus (acronym for "reverse transcription polymerase chain reaction" for reverse transcription and polymerase chain reaction) in real time. It is noted that when using eight Eppendorf-type tubes, four tubes are used to collect amplified genetic material and four tubes are used to make controls, positive or negative.
• the method which is the subject of the present invention first comprises, in a known manner, a step 200 of sampling a sample of liquid to be analyzed, for example from blood to which, optionally, a dilution or filtration.
• a formaldehyde-free fixing buffer is applied in order to fix, that is to say harden, specifically the particular cells sought, without altering their genetic material.
• this binding buffer is composed of "PBS", a phosphate buffer, saponin for lysing red blood cells of BSA, bovine serum albumin to preserve the morphology of ETDA cells calcium chelating, sodium hydroxide (NaOH ) to adjust the pH to 7.2 and RCL2, a cell fixer that does not alter their genetic material. It is noted that formaldehyde is not used because it induces breaks in the genetic material.
• step 210 the liquid resulting from step 205 is placed in a compartment 105 which ends with a filter whose micropores have an intermediate diameter between that of the desired cells and that of the other cells of the sample. liquid.
• step 205 is fixed inside compartment 105 after step 210.
• step 215 vacuum suction is applied below the filter.
• the principal of the liquid as well as the cells of diameter smaller than that of the pores of the filter 115 then pass through the filter 115.
• the desired cells of diameter smaller than that of the pores of the filter 115 are retained above the filter 115, in the compartment 105.
• the compartments are removed from the plate 110, the tips 117 are removed from the compartments 105 and this remaining content is isolated in the compartment 105 by plugging the lower opening with a cap bar covered, in its lower part with an adhesive membrane 120.
• the compartments 105 associated on the one hand with the bar of plugs 120 and the bar 116, are inserted into the rack 133, the bar 116 being held by this rack 133.
• the cells retained on the filter are lysed according to known techniques.
• the rack 133 maintaining the vertical compartments 105, is kept in an oven for a period of time. known duration.
• each compartment 105 is placed above an Eppendorf type tube 125, or 126 by positioning this tube on the lower support, 131 or 132 respectively, then the gantry 130 is positioned with the bar 116 compartments 105 and the cap strip 120 on lower portion 131, or 132, respectively.
• a piston 140 provided with a central axial punch 141 movable relative to the piston 140 and ending, inside the compartment 105, by a tip 142.
• this tip 142 has a star shape, for example with four branches, the tip then being cruciform.
• a pressure is applied on the upper part of the punch 141 to lower it along the longitudinal axis of the compartment 105, being guided by the piston 140.
• the tip 142 of the punch 141 perforates , successively, the filter 115 and the plug or the adhesive membrane 120.
• a pressure is applied on the rest of the piston 140 so that the remaining contents of the compartment 105 passes through the filter 115 and the plug or the membrane 120, and reaches the Eppendorf-type tube, by opening surrounding the tip 142 of the punch 141.
• the genetic material of the particular cells is extracted without deterioration through the perforation of the filter.
• the support, 131 or 132, and the Eppendorf type tubes, 125 or 126 are removed after removal, upwardly of the gantry 130, from the compartments 105 and the membrane 120.
• steps 265 and 270 an analysis of the genetic material, in particular the DNA or RNA of the desired cells, is carried out in these tubes 125 or 126.
• the amplified DNA is used as a template to detect the mutations of sensitivity or resistance to targeted therapies.
• cDNA derived from RNA by RT conversion and amplified is used as a template to detect the level of expression of susceptibility genes. or resistance to targeted therapies.
• a defined volume of the amplified genetic material, in particular DNA, is taken to detect mutations in sensitivity or resistance to targeted therapies using primer and antisense pairs of primers. pairs of probes and during a quantitative and real-time PCR (acronym for polymerase chain reaction).
• the principle of the search for mutations of sensitivity or resistance to targeted therapies implemented in the embodiment shown is as follows.
• the allele discrimination or "SNP genotyping assay” (SNP being the acronym for "single nucleotide polymorphism” for single nucleotide polymorphism) provides information on the presence or absence of a point mutation in a gene.
• the first step, 265, of allelic discrimination is a real-time quantitative PCR reaction carried out with two primers to amplify the sequence of interest and two probes, for example of the TaqMan (registered trademark) type. One of the probes recognizes the mutated sequence and the other recognizes the normal sequence.
• the two probes are associated with different fluorochromes, for example "VIC” for the probe hybridizing to the normal sequence and "FAM” for the probe hybridizing to the mutated sequence.
• the second step, 270 uses an allelic discrimination program measuring the initial fluorescence and the final fluorescence emitted by the FAM or / and VIC fluorochromes. This program makes it possible to distinguish between the different sequences present in each sample:
• sequences of the following primers and probes are used, for example:
• a defined volume of the genetic material including RNA converted to cDNA by RT (acronym for "reverse transcription") and amplified is taken to detect the level of gene expression of sensitivity or resistance to targeted therapies using sense and antisense primer pairs and a probe and during a quantitative and real-time polymerase chain reaction (PCR), for example with 50 cycles.
• PCR polymerase chain reaction
• the method, the device and the kit object of the present invention make it possible to collect and amplify under a condition compatible with routine laboratory tests, a large proportion of the genetic material of the present invention. cells considered, in good condition, even if the sample has only one desired cell.

Applications Claiming Priority (3)

Publications (1)

Family

ID=39217983

Family Applications (1)

Country Status (5)

Families Citing this family (9)

Family Cites Families (12)

• 2007
  • 2007-09-21 FR FR0757779A patent/FR2921490B1/fr active Active
• 2008
  • 2008-01-18 US US12/016,222 patent/US20090081772A1/en not_active Abandoned
  • 2008-09-01 FR FR0855849A patent/FR2921491B1/fr active Active
  • 2008-09-15 US US12/679,027 patent/US20110104670A1/en not_active Abandoned
  • 2008-09-15 WO PCT/FR2008/051650 patent/WO2009047436A1/fr active Application Filing
  • 2008-09-15 EP EP08838264A patent/EP2191250A1/de not_active Withdrawn
  • 2008-09-15 JP JP2010525397A patent/JP2010538672A/ja active Pending

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events