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WO2018047311A1 - Agent de prétraitement pour la détection de cellules tumorales circulantes - Google Patents

Agent de prétraitement pour la détection de cellules tumorales circulantes Download PDF

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Publication number
WO2018047311A1
WO2018047311A1 PCT/JP2016/076699 JP2016076699W WO2018047311A1 WO 2018047311 A1 WO2018047311 A1 WO 2018047311A1 JP 2016076699 W JP2016076699 W JP 2016076699W WO 2018047311 A1 WO2018047311 A1 WO 2018047311A1
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WIPO (PCT)
Prior art keywords
cells
filter
pretreatment agent
antibody
fluorescent dye
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Ceased
Application number
PCT/JP2016/076699
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English (en)
Japanese (ja)
Inventor
勝也 遠藤
清太 中村
理美 八木
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Resonac Corp
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Hitachi Chemical Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Hitachi Chemical Co Ltd filed Critical Hitachi Chemical Co Ltd
Priority to PCT/JP2016/076699 priority Critical patent/WO2018047311A1/fr
Priority to US16/324,373 priority patent/US20190170755A1/en
Priority to EP17839611.5A priority patent/EP3499228A4/fr
Priority to PCT/JP2017/029329 priority patent/WO2018030548A1/fr
Priority to PCT/JP2017/029328 priority patent/WO2018030547A1/fr
Priority to PCT/JP2017/029326 priority patent/WO2018030546A1/fr
Priority to JP2018533584A priority patent/JPWO2018030547A1/ja
Publication of WO2018047311A1 publication Critical patent/WO2018047311A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer

Definitions

  • the present invention relates to a pretreatment agent for detecting circulating cancer cells in the blood.
  • CTC circulating tumor cells
  • the cells captured on the filter can be detected by fluorescently staining the cell nucleus and detecting this fluorescence.
  • the filter has a cell diameter similar to that of the CTC.
  • the leukocytes they have are also captured. Therefore, the captured cells are reacted with a fluorescently labeled antibody specific for each of leukocytes and CTCs, and the fluorescence emitted by each cell is detected to determine whether the captured cells are leukocytes or CTCs. Can be identified.
  • the captured leukocytes exhibit fluorescence indicating the cell nucleus and fluorescence indicating CTC (false positive), or in the captured cell, the cell nucleus is detected. In some cases, fluorescence showing fluorescence, fluorescence showing white blood cells, and fluorescence showing CTC were observed (triple positive).
  • the present invention provides a pretreatment agent for detecting circulating cancer cells in blood on a filter on which cells are captured, including animal serum and a surfactant.
  • the surfactant may have a nonionic surfactant and may have poly (oxyethylene) octylphenyl ether.
  • the concentration of the surfactant may be 0.05% by mass to 0.2% by mass.
  • the concentration of animal serum may be 2-10% by mass.
  • the filter for capturing cells may be treated with a secondary antibody that recognizes a marker protein of leukocytes and a secondary antibody that recognizes the primary antibody and is labeled with a first fluorescent dye.
  • the filter for capturing cells may be treated with an antibody that recognizes a marker protein of leukocytes and labeled with a first fluorescent dye.
  • the pretreatment agent may further include an antibody that recognizes a marker protein of epithelial cells and labeled with a second fluorescent dye, and a third fluorescent dye that stains nucleic acid.
  • the animal derived from animal serum, the primary antibody that recognizes the leukocyte marker protein or the animal derived from the antibody, and the animal derived from the antibody that recognizes the marker protein of epithelial cells may be the same animal, for example May be a mouse.
  • the leukocyte marker protein may be CD45.
  • the marker protein for epithelial cells may be cytokeratin.
  • FIG. 2 is a sectional view taken along line II-II in FIG.
  • the pretreatment agent according to one embodiment of the present invention includes animal serum and a surfactant. Such a pretreatment agent is used to detect circulating cancer cells (CTC) in the blood on the filter where the cells are captured.
  • CTC cancer cells
  • Examples of CTC detection methods that can use the pretreatment agent according to the present embodiment include: (a) a step of filtering a blood sample through a filter and capturing cells on the filter; and (b) a filter in which cells are captured. A step of contacting a primary antibody that recognizes a leukocyte marker protein, and then a secondary antibody that recognizes the primary antibody and is labeled with a first fluorescent dye, and (c) a cell.
  • step (D) contacting the filter that captures the marker protein of the epithelial cell and labeled with a second fluorescent dye, and a third fluorescent dye that stains nucleic acid; ) First, second and third fluorescence emitted from each cell captured on the filter by irradiating the filter in which the cells are captured with excitation light of the first, second and third fluorescent dyes, respectively. And detecting the fluorescence of the unit, respectively, and a method comprising the.
  • the pretreatment agent can be used at any time after step (b) and before step (d).
  • a blood sample is filtered through a filter, and cells in the blood sample are captured on the filter.
  • “cell” means leukocyte or CTC unless otherwise specified.
  • CTC is not contained in the blood of a healthy person, but CTC is contained in the blood of a subject to whom cancer has metastasized. Therefore, when the blood of a subject to whom cancer has metastasized is filtered through a filter, CTC is captured on the filter. Further, since the diameter of white blood cells is approximately the same as the diameter of CTC, white blood cells are captured together with CTC on the filter.
  • blood collected from a subject can be used as it is, or blood diluted with a buffer solution such as phosphate buffered saline (PBS) or other suitable medium can be used.
  • PBS phosphate buffered saline
  • the blood sample may be added with additives that are usually added to blood samples, such as anticoagulants and fixatives.
  • the filter is not particularly limited as long as it can selectively capture white blood cells and CTCs present in the blood sample, and a conventionally known filter can be used.
  • the filter may be, for example, a metal filter, and preferably has a through hole having a pore diameter of 5 ⁇ m to 15 ⁇ m, more preferably 6 ⁇ m to 12 ⁇ m, and even more preferably 7 ⁇ m to 10 ⁇ m.
  • the hole diameter of the through hole is the maximum value of the diameter of a sphere that can pass through the through hole.
  • the filter in which the cells are captured may be washed (step (x)).
  • Step (x) is performed, for example, by bringing a cleaning solution containing a known buffer solution such as PBS into contact with the filter.
  • the washing solution may contain additives such as bovine serum albumin (BSA) or ethylenediaminetetraacetic acid (EDTA).
  • BSA bovine serum albumin
  • EDTA ethylenediaminetetraacetic acid
  • the step (x) is not limited to the step (a), and may be appropriately performed after each step.
  • contacting means passing the substance or a solution of the substance through the filter in which cells are trapped, or a filter in which cells are trapped.
  • the method is not limited to these methods.
  • step (b) the primary antibody that recognizes the leukocyte marker protein is brought into contact with the filter in which the cells have been captured, and then the secondary antibody that recognizes the primary antibody and is labeled with the first fluorescent dye. Contact with the secondary antibody.
  • the leukocytes captured on the filter are fluorescently labeled.
  • the filter in which the cells have been captured may be washed with a washing solution (step (x)) before being brought into contact with the secondary antibody.
  • Fluorescent labeling of leukocytes captured on the filter can be performed in two steps as described above, but may be performed in one step. That is, in step (b), an antibody that recognizes leukocyte marker protein and labeled with a first fluorescent dye is brought into contact with the filter in which the cells are captured, thereby capturing the filter on the filter.
  • White blood cells may be fluorescently labeled in one step.
  • leukocyte marker protein examples include CD45 expressed in all hematopoietic stem cells.
  • a primary antibody that recognizes a leukocyte marker protein, a secondary antibody that is labeled with a first fluorescent dye, and an antibody that recognizes a leukocyte marker protein and is labeled with a first fluorescent dye in particular, It is not limited, A polyclonal antibody or a monoclonal antibody may be sufficient.
  • the animal from which the antibody is derived is not particularly limited as long as the animal from which the primary antibody is derived is different from the animal from which the secondary antibody is derived.
  • the first fluorescent dye is not particularly limited as long as it is a fluorescent dye usually used for fluorescent labeling of antibodies.
  • the first fluorescent dye is a fluorescent dye different from the second and third fluorescent dyes. Each fluorescent dye is distinguishable because it has a different fluorescence wavelength.
  • Alexa Fluor registered trademark
  • the first fluorescent dye can be used as the first fluorescent dye.
  • the cells captured on the filter may be immobilized (step (y1)).
  • the cells can be fixed by bringing a known fixing agent such as formaldehyde into contact with the filter in which the cells are captured. By fixing the cells, cell spoilage or aggregation can be further reduced.
  • the cells captured on the filter may be further permeabilized (step (y2)).
  • the cells can be permeabilized by bringing a known permeabilizing agent into contact with the filter in which the cells are captured.
  • a known permeabilizing agent for example, poly (oxyethylene) octylphenyl ether can be used.
  • Steps (y1) and (y2) are preferably performed between step (b) and step (c).
  • an antibody that recognizes the marker protein of the epithelial cell and is labeled with a second fluorescent dye, and a third fluorescence that stains the nucleic acid on the filter in which the cells are captured Contact the dye.
  • the antibody labeled with the second fluorescent dye and the third fluorescent dye that stains the nucleic acid may be contacted with the filter simultaneously or in any order.
  • Examples of an epithelial cell marker protein that is an antibody that recognizes an epithelial cell marker protein and is labeled with a second fluorescent dye include cytokeratin, epithelial cell adhesion molecule (EpCAM), CD146, Examples include CD176 and tumor markers such as EGFR or HER2. Cytokeratin or tumor markers are preferred. Since CTC is derived from epithelial cells, it has a marker protein for these epithelial cells.
  • the second fluorescent dye is not particularly limited as long as it is a fluorescent dye usually used for fluorescent labeling of antibodies. As the second fluorescent dye, for example, fluorescein such as fluorescein isothiocyanate (FITC) can be used.
  • the antibody is not particularly limited, and may be a polyclonal antibody or a monoclonal antibody. The animal from which the antibody is derived is not limited.
  • the third fluorescent dye for staining nucleic acid is not particularly limited as long as it is a fluorescent dye capable of binding to nucleic acid, and a fluorescent dye usually used for fluorescent staining of nucleic acid can be used.
  • the third fluorescent dye include 4 ′, 6-diamidino-2-phenylindole (DAPI) and 2 ′-(4-ethoxyphenyl) -5- (4-methyl-1-piperazinyl) -2,5. And '-bi-1H-benzimidazole trihydrochloride (Hoechst 33342).
  • the filter in which the cells are captured is irradiated with the excitation light of the first, second and third fluorescent dyes, respectively, and the first emitted from each cell captured on the filter.
  • the fluorescence of the second and third fluorescent dyes is detected.
  • White blood cells are labeled with the first and third fluorescent dyes. Therefore, when detecting the fluorescence of the first, second and third fluorescent dyes, the fluorescence by the second fluorescent dye is not detected (negative), and the fluorescence by the first and third fluorescent dyes is detected. (Positive) cells are identified as white blood cells.
  • CTC is labeled with second and third fluorescent dyes. Therefore, a cell in which fluorescence from the first fluorescent dye is not detected (negative) and fluorescence from the second and third fluorescent dyes is detected (positive) is identified as CTC.
  • the first, second and third fluorescent dyes are detected (positive), that is, triple positive, it is not possible to identify whether the cell is a leukocyte or a CTC.
  • the cells are leukocytes, the fluorescence by the first fluorescent dye is not detected (negative), and the fluorescence by the second and third fluorescent dyes is detected (positive) is false positive. .
  • the animal serum contained in the pretreatment agent acts in the direction of reducing false positives and triple positives in the detection of CTC.
  • the mechanism by which false positives and triple positives are reduced is not clear, but it is presumed that nonspecific binding of antibodies can be reduced by contacting animal serum with cells.
  • the animal serum is not particularly limited as long as it is a commonly used animal serum, but may be serum derived from the same animal as the animal from which the antibody recognizing the epithelial cell marker protein is derived. It may be a serum derived from the same animal as the animal from which the antibody to be recognized is derived, and the primary antibody to recognize the leukocyte marker protein or the animal from which the antibody is derived. For example, when the primary antibody or antibody that recognizes the leukocyte marker protein and the antibody that recognizes the epithelial cell marker protein are mouse-derived antibodies, the animal serum is preferably mouse serum.
  • the surfactant contained in the pretreatment agent acts in a direction to reduce false positives and triple positives in CTC detection.
  • the mechanism by which false positives and triple positives are reduced is not clear, but it is presumed that nonspecific binding of antibodies can be reduced by bringing a surfactant into contact with cells.
  • the surfactant preferably has a nonionic surfactant.
  • Nonionic surfactants include, for example, poly (oxyethylene) octylphenyl ether, polyethylene glycol sorbitan monolaurate (polysorbate), and n-octyl ⁇ -D-glucopyranoside, and poly (oxyethylene) octylphenyl Ether is preferred.
  • the pretreatment agent includes a buffer solution such as PBS or other appropriate medium.
  • the concentration of animal serum in the pretreatment agent is preferably 2% by mass to 10% by mass, more preferably 4% by mass to 6% by mass, and even more preferably 5% by mass. When the concentration of animal serum is 2% by mass or more, false positives and triple positives are further reduced. When the concentration of animal serum is 10% by mass or less, contamination of the filter with animal serum is reduced.
  • the concentration of the surfactant in the pretreatment agent is preferably 0.05% by mass to 0.2% by mass, more preferably 0.05% by mass to 0.1% by mass, and further preferably 0.05% by mass.
  • concentration of the surfactant is 0.05% by mass or more, false positives and triple positives are further reduced.
  • concentration of the surfactant is 0.2% by mass or less, the physical form of the cell is well maintained.
  • the combination of the concentrations of animal serum and surfactant in the pretreatment agent is preferably 2% by mass to 10% by mass of animal serum and 0.05% by mass to 0.2% by mass of surfactant. More preferably, the animal serum is 4% to 6% by mass and the surfactant is 0.05% to 0.1% by mass, the animal serum is 5% by mass and the surfactant is 0.05% by mass. % To 0.1% by mass is more preferable, animal serum is 5% by mass and surfactant is particularly preferably 0.05% by mass.
  • the false positive and tolyl positive are sufficiently reduced by bringing the combination of animal serum and surfactant into contact with the filter in which the cells are captured, and the filter is contaminated.
  • the background staining due to is reduced and the physical morphology of the cells is maintained.
  • the third fluorescent dye that stains the nucleic acid is brought into contact with the filter in which the cells are captured. It is not essential to carry out in the step (c).
  • the third fluorescent dye can be contacted with the filter in which the cells are captured at any stage after step (a) and before step (d). Even in such a case, the effects of the present invention can be achieved.
  • the pretreatment agent according to another embodiment of the present invention comprises, in addition to animal serum and a surfactant, an antibody that recognizes an epithelial cell marker protein and labeled with a second fluorescent dye. Contains a third fluorescent dye for staining.
  • the pretreatment agent according to this embodiment is used in the CTC detection method, cells are captured because the pretreatment agent itself contains an antibody labeled with the second fluorescent dye and the third fluorescent dye.
  • the step (c) of separately contacting the antibody labeled with the second fluorescent dye and the third fluorescent dye with the filter becomes unnecessary.
  • animal serum, surfactant, and marker protein for epithelial cells are added to the filter in which the cells are captured.
  • An antibody that recognizes the second fluorescent dye that is labeled with the second fluorescent dye and a third fluorescent dye that stains the nucleic acid can be contacted simultaneously.
  • a filter a primary antibody recognizing a leukocyte marker protein, a secondary antibody labeled with a first fluorescent dye, and an antibody recognizing a leukocyte marker protein labeled with a first fluorescent dye
  • the details of the prepared antibody, the pretreatment agent, the antibody labeled with the second fluorescent dye, the third fluorescent dye for staining the nucleic acid, and other reaction solutions are as described in the above embodiment. .
  • a CTC capturing cartridge (cartridge) 100 shown in FIGS. 1 and 2 has a housing having an inlet 130 to which an inflow pipe 125 into which liquid flows is connected and an outlet 140 to which an outflow pipe 135 from which liquid flows out is connected.
  • a body 120 and a filter 105 are provided.
  • the filter 105 is fixed by a casing 120 including an upper member 110 and a lower member 115.
  • the blood sample, the cleaning liquid, the pretreatment agent, and other reaction liquids are introduced into the housing 120 through the inflow pipe 125, and are discharged to the outside through the filter 105 through the outflow pipe 135.
  • Such a liquid flow can be created, for example, by connecting a pump upstream of the inflow pipe 125 or downstream of the outflow pipe 135.
  • a cock may be provided upstream of the inflow pipe 125 and / or downstream of the outflow pipe 135 to control the flow of the liquid.
  • a blood sample is introduced into the cartridge 100 from the inflow tube 125, and the blood sample is filtered by the filter 105 (step (a)).
  • White blood cells and CTC in the blood sample cannot pass through the through hole 106 of the filter 105 and remain on the surface of the filter 105.
  • Other components in the blood sample pass through the through hole 106 and are discharged out of the cartridge 100.
  • the cleaning liquid may be passed through the filter 105 to clean the filter 105 (step (x)).
  • cleaning process (x) can be suitably performed after each following process.
  • a solution containing a primary antibody recognizing a leukocyte marker protein is introduced into the cartridge 100 and held in the cartridge 100 for a predetermined time, thereby causing the cells captured on the filter 105 to react with the primary antibody.
  • a solution containing the secondary antibody labeled with the first fluorescent dye is introduced into the cartridge 100 and held in the cartridge 100 for a predetermined time, whereby the primary antibody and the secondary antibody are reacted ( Step (b)).
  • the filter 105 may be washed by passing a washing solution through the filter 105 (step (x)).
  • the step (b) may be performed in two steps using the primary antibody that recognizes the leukocyte marker protein and the secondary antibody labeled with the first fluorescent dye, as described above.
  • An antibody that recognizes a protein and labeled with a first fluorescent dye may be used in one step.
  • a solution containing an antibody that recognizes a leukocyte marker protein and labeled with a first fluorescent dye is introduced into the cartridge 100, and the cartridge 100 is filled with a predetermined amount. By maintaining the time, the cells captured on the filter 105 are reacted with the antibody.
  • the cells captured on the filter 105 may be fixed by introducing a solution containing a fixing agent into the cartridge 100 and holding the solution in the cartridge 100 for a predetermined time (step (y1)).
  • a solution containing a permeabilizing agent may be introduced into the cartridge 100 and held in the cartridge 100 for a predetermined time, so that the cells captured on the filter 105 may be permeabilized (step (y2)).
  • a pretreatment agent containing animal serum and a surfactant, a solution containing an antibody labeled with a second fluorescent dye, and a solution containing a third fluorescent dye for staining nucleic acid are introduced into the cartridge 100. Then, it is held in the cartridge 100 for a predetermined time to react with the cells captured on the filter 105 (step (c)).
  • Each solution may be independently introduced into the cartridge 100, or a mixed solution obtained by mixing each solution in any combination may be introduced into the cartridge 100 in any order. Further, in the step (c), it is not essential to introduce the third fluorescent dye into the cartridge 100, and the third fluorescent dye may be any one after the step (a) and before the step (d).
  • the cartridge 100 can be introduced in stages.
  • step (c) when using a pretreatment agent further comprising an antibody labeled with a second fluorescent dye and a third fluorescent dye, in addition to the pretreatment liquid, it is separately labeled with a second fluorescent dye. It is not necessary to introduce the solution containing the antibody and the solution containing the third fluorescent dye into the cartridge 100 with the third fluorescent dye.
  • the fluorescence emitted from each cell captured on the filter 105 is detected by irradiating the cartridge 100 with excitation light of each fluorescent dye using a fluorescence microscope (step (d)).
  • the fluorescence is detected by, for example, observing the cartridge 100 from the upper surface in the vertical direction of the cartridge 100 and processing the fluorescence observation image. Depending on the detected fluorescence combination, it is identified whether the cell is CTC or leukocyte.
  • Example 1 Using a CTC capture cartridge (cartridge) in which a thin-film metal filter (membrane area 6 mm ⁇ 6 mm, film thickness 18 ⁇ m) having many through-holes having a major axis of 100 ⁇ m and a minor axis of 8 ⁇ m is incorporated into the cartridge, As detected.
  • the CTC capture cartridge corresponds to the cartridge 100 described in the above embodiment.
  • the process from the process (a) to the process (c) was performed using the CTC capture device.
  • the CTC capture device includes a reservoir for introducing a blood sample and other reaction solutions.
  • the cartridge was filled with a PBS solution containing 0.5% BSA and 2 mM EDTA (hereinafter referred to as “cleaning solution”). 7 mL of the cleaning solution was placed in the reservoir, and 3 mL of healthy human blood collected with a Streek Cell Free DNA Blood Collection Tube was added under the cleaning solution so that the blood and the cleaning solution were layered.
  • the CTC capturing device was activated, blood in the reservoir and washing solution were introduced into the cartridge at a flow rate of 200 ⁇ L / min, and leukocytes in the blood were captured on the filter. A washing solution was introduced into the cartridge to wash away blood components remaining on the filter.
  • 1.25 mL of anti-human CD45 mouse monoclonal antibody clone: 2D1 was introduced into the cartridge at a flow rate of 200 ⁇ L / min and reacted at room temperature for 30 minutes. 1.40 mL of the washing solution was introduced into the cartridge at a flow rate of 400 ⁇ L / min, and the reaction solution in the cartridge was discharged. 1.25 mL of Alexa Fluor (registered trademark) 594-labeled anti-mouse IgG goat polyclonal antibody was introduced into the cartridge at a flow rate of 400 ⁇ L / min and reacted at room temperature for 30 minutes. 1.40 mL of the washing solution was introduced into the cartridge at a flow rate of 400 ⁇ L / min, and the reaction solution in the cartridge was discharged.
  • FITC-labeled anti-human cytokeratin mouse monoclonal antibody clone CK3 / 6H5 / AE1 / AE3 mixture, DAPI, 5% by weight mouse serum, 0.05% by weight Triton X-100, and pretreatment agent containing washing solution 25 mL was introduced into the cartridge at 400 ⁇ L / min and reacted at room temperature for 30 minutes. 3.00 mL of the cleaning solution was introduced into the cartridge at a flow rate of 400 ⁇ L / min, and the reaction solution in the cartridge was discharged. The cartridge was then removed from the CTC capture device.
  • the cartridge was set on a fluorescence microscope. Using fluorescent mirror units, fluorescent dyes on cells (FITC, Alexa Fluor 594, and DAPI) were each excited. The fluorescence emitted from each fluorescent dye was photographed, and the resulting images were synthesized. From the synthesized image, cells showing triple positive and cells showing false positive were extracted visually or using image analysis software, and the number of each cell was determined. The results are shown in Table 1. Here, the cells showing triple positive are FITC positive, Alexa Fluor594 positive and DAPI positive cells. Moreover, the cell which shows a false positive is a cell of FITC positive, Alexa Fluor594 negative, and DAPI positive.
  • the number of cells showing triple positive was expressed as a relative value when the number of cells showing triple positive in Comparative Example 1 was taken as 100.
  • the number of cells showing false positives was expressed as a relative value when the number of cells showing false positives in Comparative Example 1 was taken as 100. The results are shown in Table 1.
  • the filter staining was evaluated as follows. The fluorescence intensity was measured using spots on the upper right, lower right, upper left, lower left, and center of the filter without spots. The obtained fluorescence intensity was expressed as a relative value when the fluorescence intensity of background staining in Comparative Example 1 was defined as 100. The results are shown in Table 1.
  • the physical morphology of the cells was evaluated as follows. A plurality of cells trapped on the filter were randomly extracted and the morphology was visually observed. The results are shown in Table 1. In Table 1, A shows a case where no deformation was observed in the cells, and B shows a case where slight deformation of the cells was observed but there was no problem in observing the cells.
  • Example 1 The number of cells showing triple positive, the number of cells showing false positive, and the fluorescence intensity of background staining were the same as in Example 1 except that the pretreatment agent not containing mouse serum and Triton X-100 was used. And the physical morphology of the cells was evaluated. The results are shown in Table 1.
  • Example 2 The cell permeation treatment was performed in the same manner as in Example 1. Thereafter, 1.25 mL of a pretreatment agent containing 5% by mass mouse serum, 0.05% by mass Triton X-100, and a washing solution was introduced into the cartridge at 400 ⁇ L / min and reacted at room temperature for 30 minutes. 1.50 mL of the washing solution was introduced into the cartridge at a flow rate of 400 ⁇ L / min, and the reaction solution in the cartridge was discharged. 1.25 mL of PBS solution containing Anti-Cytokeratin-FITC and DAPI was introduced into the cartridge at 400 ⁇ L / min and reacted at room temperature for 30 minutes.
  • Examples 1 and 2 When the pretreatment agent according to one embodiment of the present invention was used (Examples 1 and 2), triple positive and false positive were reduced as compared with Comparative Example 1. In addition, background staining was reduced and no cell deformation was observed. On the other hand, when a pretreatment agent containing only one of animal serum or surfactant is used (Comparative Examples 2 and 3), false positives are not sufficiently reduced (Comparative Example 2), or the staining intensity of background staining is large. Increased (Comparative Example 3).
  • Example 3 Comparative Examples 4 to 6
  • Example 3 and Comparative Examples 4 to 6 were performed in the same manner as Example 1 and Comparative Examples 1 to 3, respectively, except that blood collected from a healthy person different from Test Example 1 was used.
  • the results are shown in Table 2.
  • the relative value of the number of cells showing triple positive, the relative value of the number of cells showing false positive, and the relative value of the fluorescence intensity of background staining are the relative values when the corresponding value in Comparative Example 1 is 100. did.
  • Example 4 The experiment was performed in the same manner as in Example 3 except that the concentration of Triton X-100 in the pretreatment agent was 0.1% by mass. The results are shown in Table 2.
  • Examples 3 and 4 When the pretreatment agent according to one embodiment of the present invention was used (Examples 3 and 4), triple positive and false positive were reduced as compared with Comparative Example 4. Moreover, background staining was reduced and cell deformation was not observed (Example 3), or even if deformation was observed, it was a slight deformation within a range that did not hinder cell observation (Example 4). . On the other hand, when a pretreatment agent containing only one of animal serum or surfactant is used (Comparative Examples 5 and 6), triple positive is not sufficiently reduced (Comparative Example 5), or the staining intensity of background staining is large. Increased (Comparative Example 6).

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Abstract

L'invention concerne un agent de prétraitement qui permet de détecter des cellules tumorales circulantes sur un filtre servant à capturer des cellules, ledit agent de prétraitement comprenant du sérum animal et un tensioactif.
PCT/JP2016/076699 2016-08-12 2016-09-09 Agent de prétraitement pour la détection de cellules tumorales circulantes Ceased WO2018047311A1 (fr)

Priority Applications (7)

Application Number Priority Date Filing Date Title
PCT/JP2016/076699 WO2018047311A1 (fr) 2016-09-09 2016-09-09 Agent de prétraitement pour la détection de cellules tumorales circulantes
US16/324,373 US20190170755A1 (en) 2016-08-12 2017-08-14 Detection Method of Circulating Tumor Cells and Pretreatment Method for Detecting Circulating Tumor Cells
EP17839611.5A EP3499228A4 (fr) 2016-08-12 2017-08-14 Procédé de détection de cellules cancéreuses circulantes dans le sang, et procédé de prétraitement destiné à détecter des cellules cancéreuses circulantes dans le sang
PCT/JP2017/029329 WO2018030548A1 (fr) 2016-08-12 2017-08-14 Kit destiné à détecter des cellules cancéreuses circulantes dans le sang
PCT/JP2017/029328 WO2018030547A1 (fr) 2016-08-12 2017-08-14 Procédé de détection de cellules cancéreuses circulantes dans le sang, et procédé de prétraitement destiné à détecter des cellules cancéreuses circulantes dans le sang
PCT/JP2017/029326 WO2018030546A1 (fr) 2016-08-12 2017-08-14 Agent de prétraitement destiné à détecter des cellules cancéreuses circulantes dans le sang
JP2018533584A JPWO2018030547A1 (ja) 2016-08-12 2017-08-14 血中循環癌細胞の検出方法及び血中循環癌細胞を検出するための前処理方法

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US20120021435A1 (en) * 2010-07-23 2012-01-26 Siemens Aktiengesellschaft Detection of living, circulating, or disseminated cells or cell constituents in blood or bone marrow following filtration of blood
JP2014025918A (ja) * 2012-06-20 2014-02-06 Arkray Inc 血液成分を含む試料の処理方法
WO2014198242A1 (fr) * 2013-06-14 2014-12-18 Metacell, S.R.O. Procede pour la separation de cellules sporadiques a partir de liquides organiques et appareil pour la mise en oeuvre dudit procede
JP2015087382A (ja) * 2013-09-25 2015-05-07 アークレイ株式会社 血液検体の処理方法
JP2016512197A (ja) * 2013-03-05 2016-04-25 ボード・オブ・リージエンツ，ザ・ユニバーシテイ・オブ・テキサス・システム 間葉及び上皮間葉形質転換循環腫瘍細胞のための特異的検出ツール

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090317836A1 (en) * 2006-01-30 2009-12-24 The Scripps Research Institute Methods for Detection of Circulating Tumor Cells and Methods of Diagnosis of Cancer in Mammalian Subject
US20120021435A1 (en) * 2010-07-23 2012-01-26 Siemens Aktiengesellschaft Detection of living, circulating, or disseminated cells or cell constituents in blood or bone marrow following filtration of blood
JP2014025918A (ja) * 2012-06-20 2014-02-06 Arkray Inc 血液成分を含む試料の処理方法
JP2016512197A (ja) * 2013-03-05 2016-04-25 ボード・オブ・リージエンツ，ザ・ユニバーシテイ・オブ・テキサス・システム 間葉及び上皮間葉形質転換循環腫瘍細胞のための特異的検出ツール
WO2014198242A1 (fr) * 2013-06-14 2014-12-18 Metacell, S.R.O. Procede pour la separation de cellules sporadiques a partir de liquides organiques et appareil pour la mise en oeuvre dudit procede
JP2015087382A (ja) * 2013-09-25 2015-05-07 アークレイ株式会社 血液検体の処理方法

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