WO2012067259A1 - 高濃度夾雑細胞群から低濃度の特定細胞を検出する方法と検出した細胞を回収し解析する方法 - Google Patents
高濃度夾雑細胞群から低濃度の特定細胞を検出する方法と検出した細胞を回収し解析する方法 Download PDFInfo
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Definitions
- the present invention relates to a method for detecting specific cells from a group of high-concentration contaminated cells, such as a method for detecting peripheral circulating tumor cells (CTC) from the peripheral blood of cancer patients, and a therapeutic method such as cancer treatment using the same.
- CTC peripheral circulating tumor cells
- CTC peripheral circulating tumor cells
- CEC peripheral circulating endothelial cells
- vascular endothelial progenitor cells derived from bone marrow It has been reported that (CEP: circulating endothelial progenitor) is useful (Non-patent Document 1).
- Peripheral circulating tumor cell (CTC) detection has the following significance. That is, detection of cancer cell metastasis in blood is considered to be effective in the discovery of metastatic cancer and cancer treatment for the following reasons. (1) It is thought that cancer metastasis is that cancer cells drop off from cancer tissue, enter peripheral blood, and are carried to others to proliferate. (2) It has been reported that the number of CTCs correlates with cancer metastasis and prognosis. (3) Diagnosing the degree of progression, malignancy, and prognosis of breast cancer by the number of CTCs is clinically performed.
- CTC-chip there is a technology called CTC-chip.
- this technology is based on the total number of 80,000 microposts coated with an antibody against CD326 by passing blood through a chip formed of 80,000 microposts on a business card size silicon wafer.
- This is a method of discriminating and counting CTC adsorbed by image recognition.
- Patent Document 3 discloses a method of recovering CTC detected by the method of Patent Document 1 and performing genetic analysis of CTC by fluorescence in situ hybridization (FISH) method.
- FISH fluorescence in situ hybridization
- Non-Patent Document 2 discloses a method in which a virus that infects only cancer cells is used, GFP, which is a fluorescent protein, is expressed in the cancer cells by the virus, and detected with a fluorescence microscope.
- thermoresponsive magnetic particles are used to bind different types of antibodies to each other, and after antigen-antibody reactions with multiple types of cells, separation by gradient magnetic fields is performed.
- separation by gradient magnetic fields is performed.
- CD326 EpCAM
- Tumor Cell, Enrichment, and Detection, Kit
- This kit consists of CD326 (EpCAM) antibody magnetic beads and fluorescent antibody for cytokeratin, etc., and is based on the premise that detection is based on fluorescence of cytokeratin staining. All cells are dead during the staining process.
- a reagent kit (HUMAN EpCAM POSITIVE SELECTION KIT (Stem Cell Technologies, Catalog # 18356)) is also known for magnetically concentrating epithelial cells from Stem Cell Technologies, but its catalog is for concentrating CTC in blood. The protocol is not described.
- a reagent kit (Tumor Cell Enrichment Cocktail (Stem Cell Technologies, Catalog) that cross-links red blood cells and leukocytes, which are non-CTC contaminating cells, using antibodies and negatively selects them by density gradient centrifugation. # 15167)) is also known. However, there is no confirmation that the cells enriched with this kit are CTC only.
- a reagent kit (Tumor Cell Enrichment Cocktail (Stem Cell Technologies, Catalog # 14152)) that concentrates CTC by negative selection with antibody magnetic beads and magnets. Similarly, there is no assurance that the cells enriched with this kit are CTC only.
- an antibody magnetic bead (APC Selection Kit (Stem Cell Technologies, Catalog # 18451)) against allophycocyanin (hereinafter referred to as APC), which is a fluorescent molecule
- APC allophycocyanin
- Patent Document 5 describes the technical content of a conventional flow cytometer in which a liquid feeding system including a flow cell is fixed.
- How to detect the CTC counting is a method of detecting counting the number few specific cells of about 1 / mL from 1mL per 10 9 high concentration contaminating cell populations. About this method, CTC is concentrated in a live state under the condition of cross-contamination free between specimens, a live CTC and a dead CTC are distinguished and counted, and the CTC can be recovered alive There is no known example.
- a method for detecting CTC there is a method disclosed in Patent Document 1, and an apparatus based on the method is commercialized as a Cell search system.
- the method is a method in which a determiner determines whether a target cell is a target cell by using human image recognition from a plurality of candidate cell image groups acquired by the apparatus.
- the disadvantage of this method is that it is not an automatic determination, and in order to recognize CTC, it is necessary to make a hole in the cell membrane for fluorescent antibody staining of intracellular cytokeratin. It is a point. Therefore, the cell cannot be cultured.
- Patent Document 2 is a method of measuring an image of specific cells trapped using an antibody on about 80,000 cylinders formed in a chip. The problem is that it takes more time.
- Non-Patent Document 2 is a method of infecting CTC of peripheral blood collected by a patient with a virus that specifically infects cancer cells, and detecting CTC expressed by GFP with a fluorescence microscope. .
- the time required until sufficient GFP is expressed for detection is 1 day or more. Therefore, detection will be two days after blood collection at the earliest. Furthermore, since the virus is infected, the cells cannot be cultured.
- the flow cytometer in which the liquid feeding system including the conventional flow cell is fixed cannot make cross-contamination between specimens zero, and the sample in the sample tube Since the sample liquid is sucked up with a capillary and the sample liquid is allowed to flow into the flow cell, the sample liquid remains at the bottom of the sample tube, so that it is impossible in principle to measure the total amount.
- a conventional flow cytometer that cannot measure the total amount of a sample cannot evaluate the total number of cells contained in the sample.
- the cells settle by gravity, the cells concentrate on the bottom of the sample solution with time. Regardless of the phenomenon, it is essential to measure the total amount in order to enable absolute evaluation of the cell concentration.
- Patent Document 4 describes a technique of a flow cytometer that realizes contamination-free, but does not describe a total amount measurement method using the technique. Furthermore, Non-Patent Document 3 describes a method of concentrating CTC with magnetic beads and discriminating between life and death and measuring it. However, the recovery rate of CTC when CTC was added experimentally was 70%. Here, the method for measuring the total amount is not described.
- the first problem There is no technology that automatically determines and counts CTCs in a short time while they are still alive.
- the second problem A process that makes it possible to detect and count live CTCs and dead CTCs and then collect and culture live CTCs has not been realized. This process requires aseptic processing of cells to be cultured with cross-contamination free between specimens for culturing.
- the third problem In the flow cytometer technology that is considered to be able to automatically determine CTC in a short time, there is no known example that is free of contamination and can measure the total amount of the sample. Other known measurement methods do not satisfy the first problem.
- the present invention provides a method for simultaneously solving the above three types of problems.
- the inventors of the present invention have obtained a sample reservoir and a collection reservoir, and a bottom portion of the sample reservoir.
- the above problem can be solved by measuring the total amount of the sample liquid using a flow cytometer that uses a replaceable microchannel chip as a flow cell, which includes a microchannel from the substrate to the bottom of the recovery reservoir on the substrate. It was.
- the microchannel chip can flow the entire amount of the sample liquid directly from the bottom of the sample reservoir to the microchannel, and the end point of the total amount measurement by bubbles generated after the entire amount of the sample liquid flows. It was found that it is possible to detect.
- the present invention is based on such knowledge.
- the present invention [1] A method for evaluating the number of specific cells from a high concentration of cells, wherein the specific cells are magnetically concentrated alive, and a fluorescent staining process is performed, and the specific cells are subjected to fluorescence signal intensity. And counting all the specific cells in the sample, the process of counting the cells from the sample reservoir and the collection reservoir and from the bottom of the sample reservoir to the bottom of the collection reservoir.
- a flow cytometer that uses a replaceable microchannel chip as a flow cell, including a microchannel on a substrate, measures the total amount of sample liquid, and automatically counts specific cells. The end point of the measurement is characterized by detecting, as an end point, bubbles generated in the microchannel after the sample liquid in the sample reservoir has completely flowed.
- the pretreatment process includes a process of attaching magnetic beads to EpCAM expressed in epithelial cell-derived CTC and concentrating CTC using a magnet, and adding CTC to anti-EpCAM antibody or 5E11 antibody.
- the CTC identification and counting process includes a sample reservoir and a collection reservoir and a microfluidic flow on the substrate that includes a microchannel from the bottom of the sample reservoir to the bottom of the collection reservoir.
- the total amount of sample liquid is measured by a flow cytometer using a road chip as a flow cell, and CTC is counted.
- the end point of the total volume measurement of the sample liquid is the sample liquid in the sample reservoir.
- CTC concentration evaluation method characterized by detecting as an end point bubbles generated in the microchannel after the flow of [4]
- the pretreatment process further includes two kinds of nuclear staining treatments of cell membrane permeation and cell membrane non-permeability, and the discrimination counting process distinguishes and counts a live CTC and a dead CTC.
- the pretreatment process includes attaching a magnetic bead on which anti-EpCAM antibody is immobilized to CTC and concentrating CTC using a magnet, fluorescent labeling of CTC with APC-labeled anti-EpCAM antibody, and SYTO9
- the CTC identification and counting process includes CTC identification by identifying the APC fluorescence spectrum at a ratio of two types of fluorescence signal intensities generated by photoexcitation near the wavelength of 640 nm.
- a counting method comprising performing at least two antibodies in the group consisting of a CD326 antibody, an anti-CD146 antibody for CEC detection, and an anti-CD133 antibody for CEP detection, About.
- a method for evaluating a specific small number of cells from a high-concentration cell group comprising a pretreatment process in which specific cells from a high-concentration cell group are magnetically concentrated and subjected to fluorescence staining, A process of automatically identifying and counting cells based on their fluorescence signal intensity. The process of counting the cells is such that the entire liquid delivery system including the flow cell can be replaced for each specimen and the total amount in the total amount of the sample liquid.
- a method for evaluating the number of specific cells which automatically recognizes specific cells based on the fluorescence signal intensity and counts the cells according to life / death judgment by a flow cytometer capable of measuring the number of cells, [2]
- the flow cytometer used in the process of detecting the cells uses a disposable and replaceable micro-channel chip as a flow cell, and the sample reservoir formed on the micro-channel chip substrate is used for measuring the total amount of the sample liquid.
- the method for evaluating the number of specific cells according to [1], wherein a detection signal of bubbles generated in the microchannel immediately after flowing from the bottom of the microchannel is used as an end point detection signal, [3]
- the pretreatment process includes a process of attaching magnetic beads to EpCAM expressed in CTC derived from epithelial cells and condensing CTC using a magnet, and a surface marker of CTC epithelial cells.
- CTC includes a fluorescence labeling process using EpCAM antibody or 5E11 antibody, and a process of performing two types of nuclear staining, cell membrane permeation and non-permeability.
- CTC is automatically recognized based on the ratio of multiple fluorescent signal intensities, and live CTC and dead CTC are distinguished and counted.
- the flow cytometer used in the process of discriminating and counting the CTC uses a disposable and replaceable microchannel chip as a flow cell, and the total amount of sample liquid is measured on a sample formed on a microchannel chip substrate.
- the total amount of the sample is measured by using the detection signal of the bubbles generated in the flow channel as the end point detection signal of the sample liquid immediately after the total amount of the sample liquid has flowed through the microchannel connected to the bottom of the reservoir [3]
- CTC concentration evaluation method described in [5] The pretreatment process includes a reaction of attaching magnetic beads having a human EpCAM antibody immobilized to CTC, a fluorescent staining treatment of CTC with an APC-labeled human EPCAM antibody, and a nuclear staining treatment with SYTO9 and PI.
- the process of discriminating and detecting CTC occurs by identifying the CTC by identifying the APC fluorescence spectrum by the ratio of the two types of fluorescence signal intensities generated by photoexcitation near the wavelength of 640 nm and by photoexcitation near the wavelength of 480 nm.
- the method for evaluating the number of specific cells of the present invention is a method for evaluating a specific small number of cells from a high concentration group of cells, wherein a specific cell from a high concentration group of cells is attached with antibody magnetic beads to generate a magnetic field.
- a concentration process used Including a concentration process used, a pretreatment process for performing a fluorescent staining process for detection, and a process of counting specific cells based on a fluorescence signal, and the process of counting the cells includes a liquid feeding system including a flow cell.
- the cell concentration evaluation method is characterized in that it is a measurement process using a flow cytometer that can be exchanged for each specimen and that can measure the total amount of sample liquid.
- the cells in the process of counting the cells, may be counted according to whether the cells are alive or dead, or may be counted without determining whether the cells are alive or dead.
- the specific cell is not particularly limited as long as it is a small number of cells contained in a large amount of cells.
- CTC peripheral circulating tumor cells
- CEC peripheral circulating vascular endothelial cells
- CEP bone marrow-derived vascular endothelial precursors
- the “small number of cells” is not particularly limited, for example, as long as the concentration is 1/10 5 or less in cells in blood in a living body, but preferably 1/10 6.
- the concentration of CTC detected in early cancer patients is usually about 1 / mL.
- the total number of CTC contained in 5 mL of blood is estimated to be about 5;
- measuring the total amount of the sample solution is effective. This is because the cell concentration in the sample solution is uniform in the method of calculating the number of cells in the entire sample solution from the result of a small number of cells by partial measurement of the sample solution that is generally performed with a flow cytometer. This assumption is necessary, but since cells are affected by gravitational sedimentation, this assumption usually does not hold. Therefore, the effect of the present invention is remarkably obtained in the absolute measurement of the specific cell concentration contained in the sample solution.
- the flow cytometer used in the above-described cell detection process uses the disposable and replaceable micro-channel chip shown in FIG. 1 as a flow cell.
- a laser light source and forward scattered light are used. It has a detector 62, a side scattered light detector 67, and a plurality of fluorescence detectors 63, 64, 65, 66.
- the method for measuring the total amount of sample liquid using this device is to allow all sample liquid to flow through the microchannel connected to the bottom of the reservoir by applying air pressure to the sample reservoir 1 formed on the microchannel chip substrate. Therefore, it is sufficient to measure until all the sample liquid flows. As shown in a) of Fig.
- the bubbles are generated in the flow channel from the moment when the entire sample flow is completed, and the measurement is automatically terminated using the detection signal for the bubbles as the end point detection signal for the sample liquid.
- the number of cells is evaluated using only detection signal data that does not contain bubbles, and the absolute concentration of the cells is evaluated based on the number of cells included in the total volume of the measured sample.
- microchannel chip for example, described in Japanese Patent No. 4358888, “First channel formed on the substrate into which the sample liquid containing fine particles is introduced, and second and third channels that are arranged on both sides of the first channel and into which the sheath liquid is introduced. And a fourth flow path where the first to third flow paths merge, the flow cell having first to third flow paths upstream of the fourth flow path and downstream.
- the second and third flow paths for introducing the sheath liquid are provided with first to second water tanks and second water tanks, which are respectively formed on the upstream side and the downstream side thereof. Is connected to the first water tank, and the first flow path for introducing the sample liquid is connected to a third water tank provided inside the first water tank, and the second and third flows are connected to the first water tank.
- the channel has a common liquid level in the first water tank, the third water tank is independent of the first water tank, and the sheath liquid and the sample liquid are not mixed in the first water tank.
- the fifth flow path at the center of the flow is connected to the fourth water storage tank provided inside the second water storage tank, and the sixth and seventh separation flow paths on both sides are connected to the second water storage tank.
- the first to third channels on the upstream side of the first channel and the fifth to seventh channels on the downstream side have a symmetrical pattern, and the sample liquid is collected in the fourth water storage tank on the downstream side, and the second water storage A flow cell having a structure in which the sheath liquid is collected in the tank can be used, and these configurations are shown in FIG.
- the CTC concentration evaluation method is a method for detecting low concentration of peripheral circulating tumor cells (CTC) from peripheral blood containing a high concentration of blood cells, pretreatment process including CTC concentration and fluorescence staining treatment and CTC.
- pretreatment process for example, magnetic beads fixed with antibodies against surface markers expressed in CTC derived from human epithelial cells are attached to CTC, and CTC is converted using a magnet.
- CTC is included in the treatment of specific enrichment, treatment of fluorescent labeling using antibodies that are surface markers of human epithelial cells, and treatment of two types of nuclear staining, cell membrane permeability and non-permeability.
- CTC alive and dead That CTC By counting to distinguish a CTC density evaluation method and evaluating the absolute concentration for the blood volume of life and death by CTC.
- the treatment of performing two types of nuclear staining of cell membrane permeation and non-permeability, and the distinction and counting of live CTCs and dead CTCs are optional steps, and these steps
- a CTC concentration evaluation method that does not perform these steps is also included in the present invention.
- CTCs are not limited to those derived from humans, but include those derived from mammals such as dogs, cats, cows, horses, rabbits, mice, or rats.
- the method of concentrating and staining CTC alive is magnetic by using an antibody against the surface marker of CTC derived from epithelial cells without performing cytokeratin staining, which requires a hole in the cell membrane.
- concentration and staining CTC is specifically concentrated and fluorescent staining is performed.
- the flow cytometer used in the process of discriminating and detecting CTC uses a disposable and replaceable microchannel chip as a flow cell, and the total amount of sample liquid is measured at the bottom of the sample reservoir formed on the microchannel chip substrate.
- the bubble detection signal generated in the flow path is used as the sample liquid end point detection signal to measure the total volume of the sample, thereby reducing the CTC count as much as possible. This can be done by a reduced CTC concentration evaluation method.
- CTC specific fluorescent labeling antibodies correspond to Type A and Type B antibody reaction steps in the process flow chart of FIG.
- Type A competition inhibition may occur because it is an antibody against the same surface marker as the magnetic bead antibody, but there is no practical problem according to actual data.
- TypeB there is no problem of competition inhibition.
- two types of lasers (a blue laser with an oscillation wavelength ranging from 470 to 490 nm and a red laser with an oscillation wavelength ranging from 630 nm to 650 nm) are simultaneously irradiated.
- an analysis for identifying CTC is performed as follows. That is, the fluorescence generated by red laser irradiation is detected as two types of fluorescence signals with different wavelengths as shown in Fig. 6a), and the APC fluorescence spectrum is identified by the ratio of the two signal intensities as shown in Fig. 6b). To do. Detection cells having a common ratio are distributed on a straight line as shown in FIG. 6b).
- the ratio is different, so that the spectra can be distinguished because they are distributed in different linear shapes. In this way, it is automatically recognized as CTC by identifying a cell having an APC fluorescence spectrum.
- non-specific fluorescent labeling reaction to leukocytes can be considered as APC fluorescence-labeled cells. Therefore, leukocytes are labeled using an APC-Cy7-labeled CD45 antibody and the spectrum is shifted from the APC fluorescence spectrum. To distinguish.
- CTC stained with SYTO9 alone is a live cell (LIVE-CTC) and CTC stained with both SYTO9 and PI is a dead cell (by a SYTO9 and PI fluorescence signal generated by blue laser irradiation). DEAD-CTC) and CTC life / death judgment is performed.
- fluorescence spectrum identification of APC and fluorescence spectrum identification of SYTO9 and PI, there are problems as can be seen from the fluorescence spectrum shown in a) of FIG. The problem is that the fluorescence spectrum of PI excited by blue laser and the fluorescence spectrum of APC excited by red laser overlap. That is, the APC fluorescence detection signal and the PI fluorescence detection signal interfere with each other.
- the output of the blue laser is reduced to one-tenth or less compared to the output of the red laser, so that the PI fluorescence signals in FL3 and FL4, which are APC detection signals, are made small enough to be ignored. That is, as shown in FIG. 7b), the APC fluorescence detection signal and the PI fluorescence detection signal can be analyzed independently by setting the 473 nm laser output to 1 mW or less and the 640 nm laser to 30 mW or more. As a result, both CTC identification by APC spectrum identification and CTC LIVE / DEAD determination were realized, as shown in Fig. 7a) b).
- One of the merits of this analysis method is that the troublesome fluorescence correction required in normal flow cytometry measurement is unnecessary.
- the process of concentrating CTC may include a human EpCAM antibody APC label and an antibody reaction treatment for adsorbing antibody magnetic beads against the APC. This corresponds to Type C of antibody reaction in the process flowchart of FIG. In this case, depending on the specific selectivity of one CD326 antibody, there is no problem of competitive inhibition.
- biomarkers such as CEC and CEP in addition to CTC
- at least two biomarkers selected from the group consisting of CTC, CEC, and CEP may be detected simultaneously, or three biomarkers may be detected simultaneously.
- the method of concentrating CEC and CEP to be detected at the same time uses negative selection for contaminating cells without performing positive selection in magnetic concentration with antibody magnetic beads for each surface marker. That is, after removing red blood cells by hemolysis, all white blood cells are trapped with antibody magnetic beads.
- the type of antibody magnetic beads used for the negative selection is selected from antibodies against surface markers that are expressed in all leukocytes and not expressed in CTC, CEC, and / or CEP.
- CD2, CD3, CD4, CD5, CD8, CD10, CD11b, CD14, CD15, CD16, CD19, CD20, CD24, CD25, CD27, CD29, CD33, CD36, CD38, CD41, CD45RA, CD45RO, CD56 Choose from CD66b, CD66e, CD69, CD124.
- Specific fluorescence labeling with the following fluorescence-labeled antibody is performed on the passed cells. That is, CTC is an APC fluorescently labeled CD326 antibody and CEC is an AlexaFluor660 fluorescently labeled CD146 antibody. CEP uses AlexaFluor680 fluorescently labeled CD34 antibody.
- Figure 11a shows the fluorescence spectra of the fluorescence-labeled molecules APC, AlexaFlou660 and AlexaFlou680, all of which can be excited with a 640 nm red laser, and the correlation between the fluorescence signal intensities of FL3 and FL4 is as shown in Figure 11b). , And can be counted separately by being distributed on separate lines. In this case, since leukocytes are removed by negative selection, the APC-Cy7 fluorescently labeled CD45 antibody is unnecessary. In this method, the LIVE / DEAD determination described in b) of FIG. 7 can be performed for CTC, CEC, and CEP by nuclear staining with SYTO9 and PI.
- the present invention has realized this.
- This function enables assessment of the concentration of live CTC that can metastasize and the concentration of CTC that cannot metastasize, so it is possible to determine the therapeutic effect of anticancer drug administration and radiotherapy, and to determine the progression of cancer metastasis.
- Information is obtained.
- CTCs detected from individual patients can be collected and cultured in a live state, and the CTCs can be used for genetic analysis and surface marker analysis to determine the treatment strategy after the cells have reached a sufficient number. Furthermore, it can contribute to molecular target antibody drug development in a pharmaceutical manufacturer.
- the evaluation method for the number of specific cells or the CTC concentration evaluation method of the present invention as described in the examples below, a small amount of cells are added to a large amount of cells, and the recovery rate is obtained by the method of the present invention. As a result, it was possible to obtain a recovery rate of almost 100%. This is because the entire amount of sample liquid can flow directly from the bottom of the sample reservoir to the microchannel, and the end point of the total amount measurement can be detected by bubbles generated after the entire amount of sample liquid flows. It is thought that.
- FIG. 1 It is a figure which shows the disposable chip
- a schematic diagram of the generation of bubbles used as an end point detection signal of the sample liquid (a), a scatter diagram (b) by data containing bubbles, and A scatter diagram (c) with bubbles removed is shown.
- 2 shows a flowchart of a sample processing process of the present invention. It is the schematic diagram which showed the adhesion state of the antibody magnetic bead adhering to CTC, and the fluorescence labeled antibody. (a) corresponds to TypeA, (b) corresponds to TypeB, and (c) corresponds to TypeC. It is the figure which described the APC spectrum using two types of fluorescence signals from which a wavelength differs, and the method of identifying them.
- (a) shows the principle of discriminating the APC fluorescence spectrum based on the ratio from the correlation between the fluorescence spectrum of APC and two types of fluorescence detection wavelength regions, and (b) the correlation between the two types of fluorescence detection signals.
- LIVE / DEAD determination was made after identification of the APC-labeled fluorescence spectrum.
- (a) A procedure for automatically identifying a LIVE-CTC, and (b) a procedure for automatically identifying a DEAD-CTC.
- the method of detecting and counting CTC is a method of detecting and counting a small number of specific cancer cells of about 1 cell / mL from a group of 10 9 red blood cells and white blood cells per mL.
- the following examples will be described using a method and data for detecting several CTCs from 4 mL of peripheral blood.
- the process of processing peripheral blood collected from patients includes a process of hemolysis to remove red blood cells.
- the amount of blood collected should be constant within the range from 4 mL to 7 mL.
- This section describes a method for concentrating epithelial cell-derived CTCs alive and a method for specific fluorescent labeling.
- the following three types of magnetic beads of type A, B, and C and fluorescently labeled antibodies are used.
- Type A CD326 antibody magnetic beads contained in Catalog # 130-090-500 of Miltenyi Biotec can be used. However, when using these magnetic beads, it is desirable to use a magnet for magnetic concentration from Miltenyi Biotec.
- APC labeled CD326 antibody is used.
- the APC-Cy7 fluorescently labeled CD45 antibody distinguishes APC fluorescently labeled cells adsorbed non-specifically to leukocytes by specifically labeling leukocytes.
- the necessity of labeling leukocytes with this APC-Cy7 fluorescently labeled CD45 antibody can be understood from the graph of c) of FIG. That is, the number of leukocytes is large even after magnetic concentration, and EpCAM should be expressed only in epithelial CTC in blood, but the APC-labeled CD326 antibody is non-specifically adsorbed on leukocytes.
- the area indicated by the broken line in Fig. 8c) shows that a line shifted from the APC spectrum appears when APC-Cy7 fluorescence is labeled with a CD45 antibody that specifically adsorbs to leukocytes. Indicated by By eliminating this, it is possible to perform correct CTC counting.
- Type B magnetic beads are available from Stem Cell Technologies Catalog # 18356. In this case, it is desirable to use a Stem Cell Technologies magnet.
- As the fluorescently labeled antibody an APC-labeled 5E11 antibody is used. This fluorescently labeled antibody is specially prepared.
- an APC fluorescently labeled CD326 antibody is used as a specific fluorescently labeled antibody for CTC, and a magnetic bead adsorbed on the APC is used.
- APC antibody magnetic beads Catalog # 18451 of Stem Cell Technologies
- Magnetic concentration is a step in which contaminant cells are washed away with a buffer in a state where CTC adsorbed by magnetic beads is trapped by magnetic force.
- the optimum magnet is selected because the necessary magnetic force differs depending on the magnetic beads to be used as described above. Since antibody magnetic beads may adsorb to contaminating cells non-specifically, the cell purity after the magnetic concentration step is not perfect. For this reason, it is important to take measures against fluorescent labeling with the APC-Cy7 fluorescently labeled CD45 antibody.
- Nuclear staining for LIVE / DEAD determination Use BacLight kit (Cat. No L34856) manufactured by Invitrogen.
- This reagent kit contains two types of nuclear staining reagents, SYTO9 that permeates the cell membrane and non-permeable PI, and is a live / dead determination reagent for bacteria. It can be applied to cells by diluting the concentration to about 100 times.
- the scatter diagram on the right side of FIG. 7 b) is a graph in which LIVE / DEAD of a cell called PC-9 was determined using this reagent kit. Cells judged to be LIVE are stained with SYTO9 only, and those judged to be DEAD are cells stained with both SYTO9 and PI. It is determined whether the fluorescence spectrum is LIVE or DEAD.
- FIG. 1 shows the structure of a disposable replaceable chip, where a) is a cross-sectional view, b) is a photograph, and c) a photograph of a microchannel pattern.
- the sample liquid 2 containing CTC is put into the sample reservoir 1 after removing the cover at the top of the sheath liquid reservoir 3 on the right side of the chip.
- This maximum capacity is 200 ⁇ L.
- the maximum capacity of the sheath fluid reservoir is 3 mL, and the sheath fluid 7 is placed in the sheath fluid reservoir 3.
- air pressure is applied to the upstream reservoir 3 to flow the sample liquid downstream without contact.
- the volume of the sheath liquid is such that the sheath liquid remains.
- FIG. 2 shows the entire measurement system. Two types of lasers are used: a blue laser 65-1 having an oscillation wavelength ranging from 470 nm to 490 nm and a red laser 65-2 having an oscillation wavelength ranging from 630 to 640 nm.
- the scattered light detection system includes a forward scattered light (FS) detector 62 and a side scatter (SS) detector 67, and the fluorescence detection optical system uses an apparatus configuration having a detector that detects four or more colors of fluorescence.
- the four types of fluorescence detection regions FL1, FL2, FL3, and FL4 shown in FIG. 7 correspond to detectors 63, 64, 65, and 66, respectively.
- the detection wavelength range is determined by the reflection characteristics of the dichroic mirrors 55, 56, and 57 and the transmission characteristics of the bandpass filters 58, 59, 60, and 61.
- the wavelengths of FL1, 2, 3, and 4 are shown in Fig. 7 (A). Regions are illustrated.
- the side scattered light detector 67 detects side scattered light obtained by totally reflecting the side scattered light generated in the micro flow path at the inclined surface of the chip end face.
- the detection signal of one cell is digitized by the AD converter 68 and the calculation processing is performed by the control computer 69.
- the flow rate is controlled by pressurizing the pressure specified by the air pump 80 to the chip reservoir.
- Signal diagram a) schematically shows a situation in which bubbles are generated at the merging portion of the flow path when the sample liquid 2 completely flows out of the sample reservoir 1.
- FIG. FIG. 3 b) shows the distribution including the detection data of bubbles generated after the entire sample liquid has flowed.
- the distribution of APC-labeled cells is selected by the fluorescence intensity correlation graph of FL3 and FL4. This corresponds to CTC identification.
- the fluorescence intensity correlation graph of FL1 and FL2 distinguishes SYTO9-only nuclear stained cells and cells stained with both SYTO9 and PI. This identifies LIVE and DEAD.
- LIVE-CTC counting the number is obtained by selecting the LIVE distribution.
- FIG. 8 (b) shows the DEAD-CTC count, and the number is determined by selecting the DEAD distribution in the correlation graph of FL1 and FL2.
- the data in FIG. 9 is a graph in which the detection rate was determined by mixing PC-9, which is a cell line derived from lung cancer, into peripheral blood with a blood collection volume of 4 mL and counting using the Type A antibody reagent kit according to the above steps. is there.
- the horizontal axis is the number of PC-9 contamination evaluated with a hemocytometer, and the vertical axis is FISHMAN-R, which is the total number of LIVE-CTC and DEAD-CTC.
- the microchannel pattern of the chip to be used is that the three branch channels on the downstream side and the three combined channels on the upstream side are almost the target pattern, so the sample liquid and the sheath liquid flowing between both sides are downstream. Separate again in the side branch channel. Therefore, the sample solution can be collected in the central reservoir.
- the photograph of the cells is a culture of MCF-7 collected by detection by mixing with peripheral blood.
- the method of concentrating CEC and CEP that are detected simultaneously uses negative selection.
- This negative selection includes a method not using magnetic beads (for example, a method using Tumor Cell Enrichment Cocktail (Stem Cell Technologies, Catalog # 15167)) and a method using magnetic beads.
- a method using Tumor Cell Enrichment Cocktail (Stem Cell Technologies, Catalog # 15167)
- a method using magnetic beads When using magnetic beads, after removing red blood cells by hemolysis, all white blood cells are trapped with antibody magnetic beads.
- the antibody magnetic beads used for the negative selection are selected from antibodies against surface markers that are expressed in all leukocytes and not expressed in CTC, CEC, or CEP.
- CTC is an APC fluorescently labeled CD326 antibody
- CEC is an AlexaFluor660 fluorescently labeled CD146 antibody.
- CEP uses AlexaFluor680 fluorescently labeled CD34 antibody.
- Figure 11a shows the fluorescence spectra of the fluorescence-labeled molecules APC, AlexaFlou660 and AlexaFlou680, all of which can be excited with a 640 nm red laser, and the correlation between the fluorescence signal intensities of FL3 and FL4 is as shown in Figure 11b). , And can be counted separately by being distributed on separate lines. In this case, since leukocytes have been removed by negative selection, APC-Cy7 fluorescently labeled CD45 antibody is unnecessary. In this method, CTC, CEC, and CEP are subjected to LIVE / DEAD determination described in b) of FIG. 7 by nuclear staining with SYTO9 and PI.
- the present invention has the following advantages. 1) Benefits of detecting cancer by detecting CTC only by blood sampling of patients, 2) Benefits of patients who have surgically removed cancer tissue to be able to detect recurrence early by blood sampling, 3) CTC from individual patients It is possible to investigate which molecular target therapies are effective by detecting and recovering them and analyzing their gene analysis, expression analysis of surface markers, and drug screening. Contributes to the development of targeted therapeutics. As mentioned above, although this invention was demonstrated along the specific aspect, the deformation
- Spatial filter for blocking transmitted laser light 62 ... Photodiode 63, 64, 65, 66... Photomultiplier tube 65-1 ... Laser light source driver circuit with wavelength 473nm 65-2 ... Laser light source driver circuit with wavelength of 640nm 68 ... AD converter 69... AD converter 70 ... Keyboard 71... Display 72 ... Mouse 80 ... Air pump 81 ... Air pump driver circuit
Abstract
Description
(1) 癌組織から癌細胞が脱落し末梢血中に入り、他に運ばれて増殖することががん転移であると考えられる。
(2) CTC数と、癌の転移及び予後とは相関することが報告されている。
(3) 乳癌の進行度や悪性度、予後をCTC数で診断することが臨床的に行われている。
更に、非特許文献3には、CTCを磁気ビーズによって濃縮し、そして生死を判別して測定する方法が記載されている。しかしながら、実験的にCTCを添加した場合の、CTCの回収率は70%であった。ここでは、全量測定の方法を記載していない。
CTCを生きたままの状態で短時間に自動判定し計数する技術がない。
生きた状態のCTCと死んだ状態のCTCを区別して検出計数し、その後生きたCTCを回収し培養する可能とするプロセスが実現していない。このプロセスは、培養するために検体間クロスコンタミネーションフリーと培養する細胞の無菌処理が必須条件である。
CTCを短時間に自動判定可能と考えられるフローサイトメーター技術において、コンタミネーションフリーかつサンプル全量を測定可能な公知例が存在しない。その他の公知の測定方法は第一課題を満足しない。
本発明は、このような知見に基づくものである。
[1]高濃度の細胞から特定の細胞の数を評価する方法であって、特定の細胞を生きたまま磁気濃縮し、蛍光染色処理を行う前処理過程と、特定の細胞をその蛍光信号強度に基づいて自動的に識別し、試料中の全ての特定細胞を計数する過程とを含み、前記細胞を計数する過程は、サンプルリザーバー及び回収リザーバー、並びにサンプルリザーバーの底部から回収リザーバーの底部へのマイクロ流路を基板上に含む、交換可能なマイクロ流路チップをフローセルとして用いるフローサイトメーターにより、サンプル液を全量測定し、自動的に特定細胞を計数するものであって、前記サンプル液の全量測定の終点は、サンプルリザーバーのサンプル液が流れきった後にマイクロ流路内で発生する気泡を終点として検出することを特徴とする、特定細胞の数の評価方法、
[2]前記前処理過程において、特定の細胞を生きたまま濃縮し、前記細胞を計数する過程において、細胞を生死判定別に計数する、[1]に記載の特定細胞の数の評価方法、
[3]高濃度の血球を含む末梢血から末梢循環腫瘍細胞(CTC)を検出するCTC濃度評価方法であって、CTCの濃縮と蛍光染色処理とを含む前処理過程と、CTCを識別計数する過程とを含み、前処理過程は、上皮性細胞由来のCTCに発現しているEpCAMに磁気ビーズを付着させて磁石を利用してCTCを濃縮する処理と、CTCを抗EpCAM抗体又は5E11抗体を利用して蛍光標識する処理とを含み、CTCの識別計数過程は、サンプルリザーバー及び回収リザーバー、並びにサンプルリザーバーの底部から回収リザーバーの底部へのマイクロ流路を基板上に含む、交換可能なマイクロ流路チップをフローセルとして用いるフローサイトメーターにより、サンプル液を全量測定し、CTCを計数するものであって、前記サンプル液の全量測定の終点は、サンプルリザーバーのサンプル液が流れきった後にマイクロ流路内で発生する気泡を終点として検出することを特徴とする、CTC濃度評価方法、
[4]前記前処理過程が、更に細胞膜透過性及び細胞膜非透過性の2種類の核染色処理を含み、前記識別計数過程が、生きているCTCと死んでいるCTCとを区別して計数する、[3]に記載のCTC濃度評価方法、
[5]前記前処理過程が、CTCに対して抗EpCAM抗体を固定した磁気ビーズを付着させて磁石を利用してCTCを濃縮する処理、APC標識抗EpCAM抗体によるCTCの蛍光標識処理、及びSYTO9及びPIによる核染色処理とを含み、CTCの識別計数過程は、波長640nm近傍の光励起で発生する波長が異なる2種類の蛍光信号強度の比率でAPC蛍光スペクトルを識別することでCTCの同定を行い、波長480nm近傍の光励起で発生するSYTO9とPIの蛍光信号によりCTCの生死判定を行う、[4]に記載のCTC濃度評価方法、
[6]前記前処理過程において、APC標識抗EpCAM抗体に代えて、APC標識5E11抗体を用いる、[5]に記載のCTC濃度評価方法、
[7]前記CTCを濃縮する処理が、APC標識抗EpCAM抗体によるCTCの蛍光標識、及び抗APC抗体磁気ビーズによるCTCへの磁気ビーズの付着を含む、[3]に記載のCTC濃度評価方法、
[8]APCの蛍光信号によるCTC識別と、PIによる死菌識別とを同時に解析可能とするために、PI蛍光励起用青色レーザー出力をAPC蛍光励起用赤色レーザー出力より、1/10以下に設定することを含む、[5]に記載のCTC濃度評価方法、又は
[9]末梢血から、CTC、CEC、及びCEPからなる群の少なくとも2つを同時に計数する方法であって、白血球に発現している表面マーカーのうちCEC及び/又はCEPに発現していない抗原に対する抗体磁気ビーズを利用したネガティブセレクションにより白血球を除き、前記抗体磁気ビーズと反応しなかった細胞に対して、CTC検出用として抗CD326抗体、CEC検出用として抗CD146抗体、及びCEP検出用として抗CD133抗体からなる群の少なくとも2つの抗体を用いて、異なる蛍光標識を行うことを含む、計数方法、
に関する。
[1]高濃度の細胞群から特定の少数の細胞数を評価する方法であって、高濃度細胞群から特定の細胞を生きたまま磁気濃縮し蛍光染色処理を行う前処理過程と、特定の細胞をその蛍光信号強度に基づいて自動的に識別し、計数する過程とを含み、細胞を計数する過程は、フローセルを含む送液系全体が検体毎に交換可能でかつサンプル液全量中の総細胞数が測定可能であるフローサイトメーターにより、蛍光信号強度により自動的に特定細胞を認識し、かつその細胞を生死判定別に計数する、特定細胞数の評価方法、
[2]前記細胞を検出する過程で用いるフローサイトメーターは、使い捨て交換可能なマイクロ流路チップをフローセルとして用いるものであって、サンプル液の全量測定はマイクロ流路チップ基板上に形成したサンプルリザーバーの底から流れきった直後にマイクロ流路内で発生する気泡の検出信号を終点検出信号として利用する、[1]に記載の特定細胞数の評価方法、
[3]高濃度の血球群を含む末梢血から低濃度の末梢循環腫瘍細胞(CTC)を検出する方法であって、CTCを濃縮と蛍光染色処理を含む前処理過程と、CTCを識別計数する過程とを含み、前処理過程は、上皮性細胞由来のCTCに発現しているEpCAMに磁気ビーズを付着させて磁石を利用してCTCを濃縮する処理と、CTCの上皮性細胞の表面マーカーをEpCAM抗体又は5E11抗体を利用して蛍光標識する処理と、細胞膜透過性と非透過性の2種類の核染色を行う処理とを含み、CTCを識別計数過程は、フローセルを含む送液系全体が検体毎に交換可能でかつサンプル液全量計測が可能であるフローサイトメーターを用い、複数の蛍光信号強度の比率によりCTCを自動認識し、生きているCTCと死んでいるCTCを区別して計数することで、生死別CTCの血液量に対する絶対濃度を評価することを含む、CTC濃度評価方法、
[4]前記CTCを識別計数する過程で用いるフローサイトメーターは、使い捨て交換可能なマイクロ流路チップをフローセルとして用いるものであって、サンプル液の全量測定はマイクロ流路チップ基板上に形成したサンプルリザーバーの底に接続するマイクロ流路にサンプル液が全量流れ終えた直後に流路内に発生する気泡の検出信号をサンプル液の終点検出信号として利用することでサンプル全量測定を行う、[3]に記載のCTC濃度評価方法、
[5]前記前処理過程が、CTCに対してヒトEpCAM抗体を固定した磁気ビーズを付着させる反応と、APC標識ヒトEPCAM抗体によるCTCの蛍光染色処理、SYTO9とPIによる核染色処理とを含み、CTCを識別検出する過程は、波長640nm近傍の光励起で発生する波長が異なる2種類の蛍光信号強度の比率でAPC蛍光スペクトルを識別することでCTCの同定を行い、波長480nm近傍の光励起で発生するSYTO9とPIの蛍光信号によりCTCの生死判定を行う、[3]に記載のCTC濃度評価方法、
[6]前記CTCを濃縮する過程が、ヒトEpCAM抗体磁気ビーズと蛍光標識ヒト5E11抗体の反応を含む、[3]に記載のCTC濃度評価方法、
[7]前記CTCを濃縮する過程が、ヒトEpCAM抗体APCラベルとそのAPCに対する抗体磁気ビーズを吸着させる抗体反応処理を含む、[3]に記載のCTC濃度評価方法、
[8]前記CTCの識別計数する過程が、CTC識別とCTCの生死判定とを行うこと含む、[3]に記載のCTC計数方法、
[9]前記CTCを蛍光染色する過程が、PIによる死細胞の核染色と、EpCAM抗体APCラベル染色とを含み、APCの蛍光信号によるCTC識別とPIによる死菌識別とが同時に解析可能とするために、PI蛍光励起用青色レーザー出力をAPC蛍光励起用赤色レーザー出力より、1/10以下に設定していることを含む、[3]に記載のCTC数の評価方法、
[10]末梢血からCTCの他に別のバイオマーカーであるCECやCEPを同時に検出計数する方法であって、又は
赤血球を溶血した後に、白血球に発現している複数の表面マーカーのうちCECとCEPに発現していない抗原に対する抗体を利用したネガティブセレクションにより白血球を除き、前記抗体磁気ビーズと反応しなかった細胞に対して、CTC検出用としてCD326、CEC検出用としてCD146、CEP検出用としてCD133を用いて、それぞれ異なる蛍光標識を行うことを含む、複数バイオマーカー濃度の同時計数方法
を開示する。
本発明の特定細胞の数の評価方法は、細胞を計数する過程において、細胞を生死判定別に計数してもよく、細胞の生死を判別せずに計数してもよい。特定細胞は、大量の細胞に含まれる少数の細胞であれば、特に限定されるものでないが、例えば末梢循環腫瘍細胞(CTC)、末梢循環血管内皮細胞(CEC)、又は骨髄由来の血管内皮前駆細胞(CEP)を挙げることができる。また、本明細書において、「少数の細胞」とは、例えば、生体内の血液中の細胞において1/105以下の濃度であれば特に限定されるものではないが、好ましくは1/106以下であり、より好ましくは1/107以下であり、更に好ましくは1/108であり、最も好ましくは1/109である。例えば、初期のがん患者において検出されるCTC濃度は通常1個/mL程度であることが知られており、例えば血液5mL中に含まれるCTCの総数は約5個程度と見積もられるが、このような低濃度の細胞濃度を正確に測定するためには、サンプル液の全量測定が効果的である。何故ならば、一般的にフローサイトメーターで行われているサンプル液の部分測定による少ない細胞数の結果からサンプル液全体の細胞数を計算で求める方法では、サンプル液中の細胞濃度が均一であるという前提が必要であるが、細胞は重力沈降の影響があるのでその前提は成り立たないのが普通である。従って、サンプル液中に含まれる特定の細胞濃度の絶対測定において、本願発明の効果が顕著に得られる。
「基板上に形成された、微粒子を含む試料液が導入される第1の流路と、第1の流路の両側に配置されてシース液が導入される第2及び第3の流路と、第1~第3の流路が合流する第4の流路とを備えるフローセルであって、前記フローセルは、第4の流路の上流側に第1~第3の流路を有し下流側に第5~7流路を有し、更にその上流側と下流側にそれぞれ形成された第1貯水槽と第2貯水槽とを備え、シース液を導入する第2及び第3の流路は第1貯水槽に接続されており、試料液を導入する第1の流路は第1貯水槽の内側に設けられた第3貯水槽に接続しており、前記第2及び第3の流路は第1貯水槽内で共通の液面を有し、第3貯水槽は第1貯水槽とは独立しており第1貯水槽内でシース液と試料液が混合しない構造となっており、下流の中央の第5の流路は第2貯水槽の内側に設けられた第4貯水槽に接続し、両側の第6及び第7の分離流路が第2貯水槽に接続し、大気圧より高い一定の圧力の気体を第1貯水槽内に取り外し可能なキャップ構造を利用して導入し、当該気体圧力の制御によって試料液の流速を一定に制御する液体中微粒子計測装置において、第4の流路の上流側の第1~第3の流路と下流側の第5~7流路が対称パターンであって、下流側の第4貯水槽内に試料液が回収され、第2貯水槽にシース液が回収される構造となっているフローセル」を用いることができ、これらの構成は図1に示されているものである。
なお、本発明において、細胞膜透過性と非透過性の2種類の核染色を行う処理、及び生きているCTCと死んでいるCTCを区別して計数することは、任意の工程であり、これらの工程を行うことが好ましいが、これらの工程を行わないCTC濃度評価方法も本願発明に含まれる。また、CTCはヒト由来のものに限定されるものではなく、イヌ、ネコ、ウシ、ウマ、ウサギ、マウス、又はラットなどの哺乳類由来のものを含む。
本発明の特定細胞の数の評価方法又はCTC濃度評価方法によれば、後述の実施例に記載のように、大量の細胞中に、少量の細胞を添加して、本発明の方法により回収率を測定したところ、ほぼ100%の回収率を得ることができた。これは、サンプルリザーバーの底部から直接マイクロ流路へサンプル液を全量流すことが可能であり、そして全量測定をサンプル液が全量流れた後に発生する気泡により全量測定の終点を検出することができたからであると考えられる。
1-1 採血工程
CTCを生きた状態で計数するためには、血液保存管が細胞にダメージを与えるものであってはならない。CTC検出用の血液保存管としては、CellSearchシステムで使用されているCellSave保存管があるが、この保存管は、細胞にダメージを与えるので、生きたまま計測し、計測後に回収して培養する目的に使用することは不可能である。そのため、CellSaveシステムによるCTC計測では、生きたままCTCを計測することは不可能である。これを解決するために、保存管としては生きた状態でCTCを保存できるEDTA保存管などを用いるのが望ましい。
患者からの採血した末梢血を処理する過程に、赤血球を除去するために溶血する工程を含める。密度勾配遠心によって赤血球層と白血球層を分離する血球分離方法があるが、CTCの密度が様々であり赤血球より密度が低いという保証もない為、その心配が不要な溶血方法により夾雑細胞群からまず赤血球を除去することが望ましい。採血量は4mLから7mLまでの範囲で一定量とする。
上皮性細胞由来のCTCを生きたまま濃縮する方法と特異的に蛍光標識する方法を述べる。このためには、以下のTypeA、B、Cの3通りの抗体磁気ビーズと蛍光標識抗体を用いる。TypeAは、Miltenyi Biotec社のCatalog # 130-090-500に含まれるCD326抗体磁気ビーズが利用可能である。但し、この磁気ビーズを利用する場合は磁気濃縮のための磁石は、Miltenyi Biotec社のものを利用するのが望ましい。APC標識CD326抗体を用いる。APC-Cy7蛍光標識CD45抗体は、白血球に特異的に標識することで、白血球に非特異的に吸着したAPC蛍光標識細胞を区別する。このAPC-Cy7蛍光標識CD45抗体による白血球の標識の必要性は、図8のc)のグラフで理解することができる。つまり、磁気濃縮後も白血球の数が多く、血液中では上皮性由来のCTCにしかEpCAMが発現していない筈であるが、白血球にAPC標識CD326抗体が非特異的に吸着する。この非特異的な吸着を区別するために、白血球に特異的に吸着するCD45抗体でAPC-Cy7蛍光標識すると、APCスペクトルからシフトしたラインが現れていることが図8c)の破線で示した領域で示される。これを排除することで正しいCTCの計数を行うことが可能となる。
TypeCの場合は、CTCの特異的な蛍光標識抗体としてAPC蛍光標識CD326抗体を利用し、磁気ビーズはそのAPCに吸着するものを利用する方法である。その磁気ビーズとしては、APC抗体磁気ビーズ(Stem Cell Technologies社のCatalog # 18451)を利用することができる。この場合、磁石はStem Cell Technologies社のものを利用するのが望ましい。やはり、APC-Cy7蛍光標識CD45抗体を用いて白血球に標識することで、白血球に非特異的に吸着したAPC蛍光標識細胞を区別する。
磁気濃縮は、磁気ビーズが吸着したCTCを磁力でトラップした状態で、夾雑物細胞をバッファーで洗い流す工程である。磁石については、前述したように使用する磁気ビーズに応じて必要な磁力が異なるために最適なものを選択する。抗体磁気ビーズは非特異的に夾雑細胞に吸着する場合があるので、磁気濃縮工程後の細胞純度は完全ではない。このために、 APC-Cy7蛍光標識CD45抗体による蛍光標識による対策が重要である。
Invitrogen社製BacLightキット(Cat.No L34856)を用いる。この試薬キットは、細胞膜を透過するSYTO9と非透過性のPIの2種類の核染試薬を含んでおり、細菌用のLIVE/DEAD判定用試薬である。濃度を100倍程度に希釈することで細胞に適用することが可能である。図7のb)の右側の散布図は、この試薬キットを利用し、PC-9という細胞のLIVE/DEADを判定したグラフである。LIVEと判定されるのはSYTO9のみで染色された細胞であり、DEADと判定されるのはSYTO9とPIの両方で染色した細胞となる。この蛍光スペクトルの相違でLIVEかDEADかを判定する。
2-1 CTCのサンプル全量測定工程
医療検体に含まれる極微量のCTCを短時間で計数し、その後それを回収し培養する目的のためには、検体間クロスコンタミネーションフリーのフローサイトメーターを利用するのが最適である。このフローサイトメーターとしては、唯一オンチップバイオテクノロジーズ社の製品であるFISHMAN-Rが対応する。このフローサイトメーターは特許文献4に関連しており、図1に示した様にフローセルが使い捨て交換型チップを利用することで、チップ交換により送液系の全交換が可能で、完全な検体間クロスコンタミネーションフリーを実現している。本発明では、この装置で実現したサンプル全量測定方法を、図1と図2と図3を用いて説明する。図1は、使い捨て交換可能なチップの構造を示しており、a)は断面図であり、b)は写真であり、c)マイクロ流路パターンの写真を示している。CTCを含むサンプル液2は、チップの右側のシース液リザーバー3の上部のカバーを取り外して、サンプルリザーバー1に入れる。この最大容量は200μLである。シース液リザーバーの最大容量が3mLであり、シース液7はシース液リザーバー3に入れる。送液は上流のリザーバー3内に空気圧を加えることで非接触的にサンプル液を下流側に流す。サンプル液を全量流した場合はシース液が残る程度の余分なシース液の容量とする。下流側には図1c)に示したマイクロ流路で接続しており、シース液7によってサンプル液2が絞られて流れる。レーザー光9はそのサンプルが流れる位置に照射する。下流ではシース液とサンプル液が再度分離し、CTCを含む回収液5は回収リザーバー4から回収する。図2は測定系全体を示している。照射するレーザーは、2種類のレーザー(発振波長が470nmから490nmまでの範囲の青色レーザー65-1、及び発振波長が630から640nmまで範囲の赤色レーザー65-2)を利用する。散乱光検出系は前方散乱光(FS)検出器62と側方散乱(SS)検出器67を含み、蛍光検出光学系は、蛍光4色以上検出する検出器を有する装置構成を利用する。図7に示した4種類の蛍光検出領域FL1、FL2、FL3、FL4は、それぞれ検出器63、64、65、66の検出器が対応する。それぞれの検出波長範囲は各ダイクロイックミラー55、56、57の反射特性と、バンドパスフィルター58, 59, 60, 61の透過特性によって定まり、図7(A)にFL1, 2, 3, 4の波長領域を図示してある。側方散乱光用の検出器67はマイクロ流路内で発生した側方散乱光をチップ端面の斜面で全反射した側方散乱光を検出するものである。一個の細胞の検出信号はAD変換器68によってデジタル化して、制御コンピュータ69で演算処理が行われる。流速は空気ポンプ80で指定された圧力をチップリザーバーに加圧することで制御する。信号図3のa)は、サンプル液2がサンプルリザーバー1から全て流れ切った場合に、流路の合流部で気泡が発生する状況を模式的に示したものである。図3のb)はサンプル液を全量流しきった後で発生する気泡の検出データも含めた分布を示したものである。気泡は、サンプル液がなった直後から発生するので、検出時刻が最後のほうからデータを除去することで気泡を含まない細胞のデータのみのグラフを得ることができる(図3(c))。そのため、気泡が分布する領域において一定数以上カウントした場合に自動終了するように設定し、更に検出データから最後から気泡のデータを除去する。
図8(a)は、サンプル全量測定後に気泡データを除去したデータに対して、最初にFS信号の閾値がある一定以上という条件を設定する。この条件は、細胞に吸着していないフリーな蛍光標識抗体を除去するために行う。
図10を用いて説明する。使用するチップのマイクロ流路パターンは、流路が下流側の3分岐流路と上流側の3合流路とがほぼ対象パターンであるため、試料液とその両側をはさんで流れるシース液は下流側の分岐流路において再度分離する。そのため、試料液は中央のリザーバーで回収することができる。
細胞の写真は、末梢血中に混入して検出して回収したMCF-7を培養したものである。
患者毎にCTCを計数したのちに回収し、必要に応じて培養を行い、FISH法やPCR法によって、CTCの遺伝子解析を行う。例えば、 HER2遺伝子増幅の検出やEGFR遺伝子変異の検出などにより、患者の治療に効果的な分子標的治療薬の選択を行う。
以上は、CTCを生きたままの状態で生死を区別して計数を行って、生きたまま回収して培養可能なCTCを得てCTCの細胞診断を行う技術である。
CTCの他に同時に検出するCECやCEPを濃縮する方法は、ネガティブセレクションを利用する。このネガティブセレクションには、磁気ビーズを利用しない方法(例えば、Tumor Cell Enrichment Cocktail (Stem Cell Technologies, Catalog # 15167)を用いる方法)と、磁気ビーズを用いる方法がある。磁気ビーズを用いる場合は、赤血球を溶血で除いた後は、全ての白血球を抗体磁気ビーズでトラップする。そのネガティブセレクションするための抗体磁気ビーズの抗体の種類は、全ての白血球で発現しており、CTC, CEC, CEPで発現していない表面マーカーに対する抗体から選択する。例えば、CD2, CD3, CD4, CD5, CD8, CD10, CD11b, CD14, CD15, CD16, CD19, CD20, CD24, CD25, CD27, CD29, CD33, CD36, CD38, CD41, CD45, CD45RA, CD45RO, CD56, CD66b, CD66e, CD69, CD124から選ぶのがよい。磁気ビーズを用いない場合でも、用いる場合でも、ネガティブセレクション後の細胞液に対して、以下の蛍光標識抗体による特異的蛍光標識を行う。すなわち、CTCはAPC蛍光標識CD326抗体であり、CECはAlexaFluor660蛍光標識CD146抗体である。CEPはAlexaFluor680蛍光標識CD34抗体を用いる。
以上、本発明を特定の態様に沿って説明したが、当業者に自明の変形や改良は本発明の範囲に含まれる。
2…サンプル
3…シース液リザーバー
4…回収リザーバー
5…回収サンプル
6…廃液シース液リザーバー
7…シース液
8…廃液シース液
9…照射レーザー光
10…空気
11…気泡
12…チップ基板
20…CD326抗体磁気ビーズ
21…APC蛍光標識CD326抗体
22…APC蛍光標識5E11抗体
23…APC抗体磁気ビーズ
50-1…波長473nmのレーザー光源
50-2…波長640nmのレーザー光源
51…対物レンズ
52, 53, 54, 55, 56…ダイクロイックミラー
57, 58, 59, 60, 61…バンドパスフィルター
60…透過レーザー光遮断用空間フィルター
62…フォトダイオード
63, 64, 65, 66…光電子増倍管
65-1…波長473nmのレーザー光源のドライバー回路
65-2…波長640nmのレーザー光源のドライバー回路
68…AD変換器
69…AD変換器
70…キーボード
71…ディスプレイ
72…マウス
80…空気ポンプ
81…空気ポンプのドライバー回路
Claims (9)
- 高濃度の細胞から特定の細胞の数を評価する方法であって、
特定の細胞を濃縮し、蛍光染色処理を行う前処理過程と、
特定の細胞をその蛍光信号強度に基づいて自動的に識別し、試料中の全ての特定細胞を計数する過程とを含み、
前記細胞を計数する過程は、サンプルリザーバー及び回収リザーバー、並びにサンプルリザーバーの底部から回収リザーバーの底部へのマイクロ流路を基板上に含む、交換可能なマイクロ流路チップをフローセルとして用いるフローサイトメーターにより、サンプル液を全量測定し、自動的に特定細胞を計数するものであって、前記サンプル液の全量測定の終点は、サンプルリザーバーのサンプル液が流れきった後にマイクロ流路内で発生する気泡を終点として検出することを特徴とする、特定細胞の数の評価方法。 - 前記前処理過程において、特定の細胞を生きたまま濃縮し、前記細胞を計数する過程において、細胞を生死判定別に計数する、請求項1に記載の特定細胞の数の評価方法。
- 高濃度の血球を含む末梢血から末梢循環腫瘍細胞(CTC)を検出するCTC濃度評価方法であって、
CTCの濃縮と蛍光染色処理とを含む前処理過程と、
CTCを識別計数する過程とを含み、
前処理過程は、
上皮性細胞由来のCTCに発現しているEpCAMに磁気ビーズを付着させて磁石を利用してCTCを濃縮する処理と、
CTCを抗EpCAM抗体又は5E11抗体を利用して蛍光標識する処理とを含み、
CTCの識別計数過程は、
サンプルリザーバー及び回収リザーバー、並びにサンプルリザーバーの底部から回収リザーバーの底部へのマイクロ流路を基板上に含む、交換可能なマイクロ流路チップをフローセルとして用いるフローサイトメーターにより、サンプル液を全量測定し、CTCを計数するものであって、前記サンプル液の全量測定の終点は、サンプルリザーバーのサンプル液が流れきった後にマイクロ流路内で発生する気泡を終点として検出することを特徴とする、CTC濃度評価方法。 - 前記前処理過程が、更に細胞膜透過性及び細胞膜非透過性の2種類の核染色処理を含み、
前記識別計数過程が、生きているCTCと死んでいるCTCとを区別して計数する
請求項3に記載のCTC濃度評価方法。 - 前記前処理過程が、CTCに対して抗EpCAM抗体を固定した磁気ビーズを付着させて磁石を利用してCTCを濃縮する処理、APC標識抗EpCAM抗体によるCTCの蛍光標識処理、及びSYTO9及びPIによる核染色処理とを含み、
CTCの識別計数過程は、波長640nm近傍の光励起で発生する波長が異なる2種類の蛍光信号強度の比率でAPC蛍光スペクトルを識別することでCTCの同定を行い、波長480nm近傍の光励起で発生するSYTO9とPIの蛍光信号によりCTCの生死判定を行う、請求項4に記載のCTC濃度評価方法。 - 前記前処理過程において、APC標識抗EpCAM抗体に代えて、APC標識5E11抗体を用いる、請求項5に記載のCTC濃度評価方法。
- 前記CTCを濃縮する処理が、APC標識抗EpCAM抗体によるCTCの蛍光標識、及び抗APC抗体磁気ビーズによるCTCへの磁気ビーズの付着を含む、請求項3に記載のCTC濃度評価方法。
- APCの蛍光信号によるCTC識別と、PIによる死菌識別とを同時に解析可能とするために、PI蛍光励起用青色レーザー出力をAPC蛍光励起用赤色レーザー出力より、1/10以下に設定することを含む、請求項5に記載のCTC濃度評価方法。
- 末梢血から、CTC、CEC、及びCEPからなる群の少なくとも2つを同時に計数する方法であって、
白血球に発現している表面マーカーのうちCEC及び/又はCEPに発現していない抗原に対する抗体磁気ビーズを利用したネガティブセレクションにより白血球を除き、前記抗体磁気ビーズと反応しなかった細胞に対して、CTC検出用として抗CD326抗体、CEC検出用として抗CD146抗体、及びCEP検出用として抗CD133抗体からなる群の少なくとも2つの抗体を用いて、異なる蛍光標識を行うことを含む、計数方法。
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US20160146825A1 (en) | 2016-05-26 |
US9341550B2 (en) | 2016-05-17 |
US9506927B2 (en) | 2016-11-29 |
JPWO2012067259A1 (ja) | 2014-05-19 |
JP5943521B2 (ja) | 2016-07-05 |
US20130302828A1 (en) | 2013-11-14 |
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