WO2018047311A1 - Pretreatment agent for detecting circulating tumor cells - Google Patents

Abstract

The present invention provides a pretreatment agent for detecting circulating tumor cells on a filter for capturing cells, the pretreatment agent including animal serum and a surfactant.

Description

Pretreatment agent for detecting circulating cancer cells in the blood

The present invention relates to a pretreatment agent for detecting circulating cancer cells in the blood.

As a method of predicting the presence or absence of metastasis of cancer, there is a method of detecting circulating tumor cells (circulating tumor cells, hereinafter also referred to as “CTC”) circulating in the body through blood vessels and lymphatic vessels. The detection of CTC is performed by first capturing CTC on the filter using a filter (for example, Patent Document 1) due to the difference in cell size and deformability, and then staining the captured CTC. it can.

JP 2013-42689 A

The cells captured on the filter can be detected by fluorescently staining the cell nucleus and detecting this fluorescence. In addition to the CTC, 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. However, in the conventional CTC detection method, when 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).

In view of such a situation, as a result of intensive studies, the present inventors have found that treating cells captured on a filter with a surfactant and animal serum may result in false positives and triple positives in CTC detection. As a result, the present invention was completed.

That is, 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. In addition, 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.

According to the present invention, false positives and triple positives in CTC detection can be reduced.

It is a perspective view which shows one Embodiment of a cell capture | acquisition cartridge. 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.

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. (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).

First, the steps of the detection method will be described.

First, in step (a), a blood sample is filtered through a filter, and cells in the blood sample are captured on the filter. In the present specification, “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.

As a blood sample, 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. 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 μm to 15 μm, more preferably 6 μm to 12 μm, and even more preferably 7 μm to 10 μm. The hole diameter of the through hole is the maximum value of the diameter of a sphere that can pass through the through hole.

After the step (a), 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). The step (x) is not limited to the step (a), and may be appropriately performed after each step.

In this specification, “contacting” a substance with a filter in which cells are trapped 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. However, the method is not limited to these methods.

Next, in 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. By this step, the leukocytes captured on the filter are fluorescently labeled. After the primary antibody is brought into contact with the filter in which the cells have been captured, 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.

Examples of leukocyte marker protein 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. For example, Alexa Fluor (registered trademark) can be used as the first fluorescent dye.

After step (b), 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.

Further, after the step (y1), 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. As the permeation treatment agent, for example, poly (oxyethylene) octylphenyl ether can be used.

Steps (y1) and (y2) are preferably performed between step (b) and step (c).

Next, in 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. Examples of 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).

Finally, in the step (d), 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. On the other hand, 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. Here, when 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. In addition, although 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. .

In the above CTC detection method, when the pretreatment agent is brought into contact with the filter in which the cells are captured, 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.

In the above CTC detection method, when a pretreatment agent is brought into contact with a filter in which cells are trapped, 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.

In addition, even when animal serum adheres to the filter and the filter is contaminated, the animal serum adhering to the filter is removed by the surfactant by bringing the surfactant into contact with the filter.

The surfactant preferably has a nonionic surfactant. Nonionic surfactants include, for example, poly (oxyethylene) octylphenyl ether, polyethylene glycol sorbitan monolaurate (polysorbate), and n-octyl β-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. When the concentration of the surfactant is 0.05% by mass or more, false positives and triple positives are further reduced. When the 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. When the combination of the concentrations of animal serum and surfactant is in the above range, the effects of the present invention can be exhibited more remarkably. In addition, background staining due to filter contamination can be reduced, and the physical morphology of the cells can be maintained.

When only animal serum and surfactant are contacted with a filter in which cells are trapped, false positives and triple positives cannot be reduced sufficiently, or animal serum adheres on the filter. (The filter is contaminated). When animal serum adheres to the filter, the secondary antibody or antibody labeled with the first fluorescent dye present on the filter binds to the adhering animal serum (background staining), whereby step (d) In this case, the fluorescence from the first fluorescent dye is detected over a wide range of the filter, making it difficult to detect CTC. If only the surfactant of animal serum and surfactant is brought into contact with cells, false positives and triple positives cannot be reduced sufficiently, or the physical morphology of the captured cells is destroyed. , CTC detection becomes difficult. According to the pretreatment agent according to this embodiment, 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.

In the example of the detection method, in the step (c), 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. When 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. By using the pretreatment agent according to the present embodiment after step (b) and before step (d), 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.

Next, another example of a CTC detection method that can use a pretreatment agent will be described with reference to FIGS. In this method, 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. Further, 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.

First, 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. Next, the cleaning liquid may be passed through the filter 105 to clean the filter 105 (step (x)). In addition, the washing | cleaning process (x) can be suitably performed after each following process.

Next, 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. . Next, 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)). After reacting the cells with the primary antibody and before reacting the primary antibody and the secondary antibody, 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. When the step (b) is performed in a single 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.

Here, 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)). Next, 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)).

Next, 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.

When using a pretreatment agent further comprising an antibody labeled with a second fluorescent dye and a third fluorescent dye, in step (c), 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.

Finally, 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.

<Test Example 1>
(Example 1)
Using a CTC capture cartridge (cartridge) in which a thin-film metal filter (membrane area 6 mm × 6 mm, film thickness 18 μm) having many through-holes having a major axis of 100 μm and a minor axis of 8 μm is incorporated into the cartridge, As detected. The CTC capture cartridge corresponds to the cartridge 100 described in the above embodiment. In addition, 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.

First, 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 μL / 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 μL / 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 μL / 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 μL / 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 μL / min, and the reaction solution in the cartridge was discharged.

1.25 mL of a PBS solution containing 0.5% by mass to 4% by mass of formaldehyde was introduced into the cartridge at a flow rate of 400 μL / min and reacted at room temperature for 10 minutes to immobilize the cells. 1.40 mL of the washing solution was introduced into the cartridge at a flow rate of 400 μL / min, and the reaction solution in the cartridge was discharged.

1.25 mL of PBS solution containing 0.05% to 0.1% by mass of Triton X-100 (manufactured by Sigma-Aldrich) was introduced into the cartridge at a flow rate of 400 μL / min and reacted at room temperature for 10 minutes. The cells were permeabilized. 1.40 mL of the washing solution was introduced into the cartridge at a flow rate of 400 μL / 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 μL / 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 μL / 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. Similarly, 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 (background 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.

Also, 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.

(Comparative 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 μL / 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 μL / 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 μL / 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 μL / min, and the reaction solution in the cartridge was discharged. The cartridge was then removed from the CTC capture device. Thereafter, in the same manner as in Example 1, 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 were obtained, and the physical form of the cells was determined. evaluated. The results are shown in Table 1.

(Comparative Example 2)
Except that a pretreatment agent containing no mouse serum was used, the relative value of the number of cells showing triple positive, the relative value of the number of cells showing false positive, and the fluorescence intensity of background staining were the same as in Example 1. Relative values were determined and the physical morphology of the cells was evaluated. The results are shown in Table 1.

(Comparative Example 3)
Except for using a pretreatment agent not containing Triton X-100, the same as in Example 1, the relative value of the number of cells showing triple positive, the relative value of the number of cells showing false positive, and the fluorescence of background staining The relative value of intensity was determined and the physical morphology of the cells was evaluated. The results are shown in Table 1.

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).

<Test Example 2>
(Example 3, Comparative Examples 4 to 6)
Experiments of 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.

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).

DESCRIPTION OF SYMBOLS 100 ... CTC capture cartridge, 105 ... Filter, 106 ... Through hole, 110 ... Upper member, 115 ... Lower member, 120 ... Housing, 125 ... Inflow pipe, 130 ... Inlet, 135 ... Outlet, 140 ... Outlet

Claims (12)

1. A pretreatment agent for detecting circulating cancer cells in blood on a filter where cells are trapped, including animal serum and a surfactant.
2. The pretreatment agent according to claim 1, wherein the surfactant has a nonionic surfactant.
3. The pretreatment agent according to claim 1, wherein the surfactant has poly (oxyethylene) octylphenyl ether.
4. The pretreatment agent according to any one of claims 1 to 3, wherein the concentration of the surfactant is 0.05 mass% to 0.2 mass%.
5. The pretreatment agent according to any one of claims 1 to 4, wherein the concentration of animal serum is 2% by mass to 10% by mass.
6. The filter for capturing cells is treated with a primary antibody that recognizes a marker protein of leukocytes and a secondary antibody that recognizes the primary antibody and labeled with a first fluorescent dye. The pretreatment agent according to any one of 1 to 5.
7. The filter according to any one of claims 1 to 5, wherein the filter for capturing cells is treated with an antibody that recognizes a marker protein of leukocytes and labeled with a first fluorescent dye. Processing agent.
8. The pretreatment agent according to claim 6 or 7, further comprising 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.
9. The animal from which the animal serum is derived, the primary antibody that recognizes the leukocyte marker protein or the animal from which the antibody is derived, and the animal from which the antibody that recognizes the epithelial cell marker protein is derived are the same animal. The pretreatment agent as described.
10. The pretreatment agent according to claim 9, wherein the derived animal is a mouse.
11. The pretreatment agent according to any one of claims 6 to 10, wherein the leukocyte marker protein is CD45.
12. The pretreatment agent according to any one of claims 8 to 10, wherein the marker protein of epithelial cells is cytokeratin or a tumor marker.

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