CN116659997A

CN116659997A - Method and apparatus for preparing measurement sample

Info

Publication number: CN116659997A
Application number: CN202310170485.8A
Authority: CN
Inventors: 新和之; 玛丽莎·马顿; 佐佐木悠人; 正见圭一郎; 山口美悠
Original assignee: Sysmex Corp
Current assignee: Sysmex Corp
Priority date: 2022-02-28
Filing date: 2023-02-27
Publication date: 2023-08-29
Also published as: CN116660003A; JP2023125975A

Abstract

The present invention provides a method for producing a measurement sample, which is easy to automate and has little influence on the signal of a cell to be measured, and which produces a sample from a blood sample. The method for preparing the measurement sample comprises the following steps: removing red blood cells from a blood sample using a solid phase capable of binding to the red blood cells; the red blood cell-depleted blood sample is reacted with a reagent that immunostains the cells.

Description

Method and apparatus for preparing measurement sample

Technical Field

The present invention relates to a method and apparatus for preparing a measurement sample for preparing a sample from a blood sample.

Background

In order to analyze white blood cells contained in blood, flow cytometry is widely used for analysis. For example, the cell surface antigen and the intracellular antigen of the measurement target cell contained in blood are immunostained with a labeled antibody, and the immunostained cell is detected by optical measurement with a flow cytometer. In order to prepare a sample for analysis by a flow cytometer from a blood sample, it is necessary to remove red blood cells from the blood. Patent document 1 discloses that PBMCs (peripheral blood mononuclear cells) are recovered from blood by density gradient centrifugation, cell surface antigens and intracellular antigens of the recovered PBMCs are stained, and a stained sample is measured by a flow cytometer to identify regulatory T cells. Patent document 2 discloses a method for preparing a sample for flow cytometer analysis. Patent document 2 discloses a step of staining blood cells, a step of lysing erythrocytes with a lysing agent, and a step of immobilizing stained blood cells.

Prior art literature

Patent literature

Patent document 1: japanese patent application laid-open No. 2014-528697;

patent document 2: patent No. 6170511.

Disclosure of Invention

Technical problem to be solved by the invention

As disclosed in patent document 1, a method of collecting PBMCs by centrifugation is widely used as a method of removing erythrocytes, but an operation of removing only a PBMC layer formed in the middle of a centrifuge tube after centrifugation requires insertion of a pipette tip into the PBMC layer, and a manual operation of pipetting only PBMCs without pipetting layers other than PBMCs is difficult to automate. In addition, although the method using a lytic reagent of patent document 2 is easy to automate, the lytic reagent may directly or indirectly affect the protein expression amount, and the signal obtained from the cells may be changed. Therefore, in rare cells such as regulatory T cells disclosed in patent document 1, for example, there is a possibility that the detection performance may be lowered. Therefore, a method and an apparatus for preparing a measurement sample, which are easy to automate and have little influence on signals (fluctuation of measurement results), are desired.

The present invention is directed to a method and apparatus for preparing a measurement sample, which can be easily automated and has little influence on the signal of a cell to be measured, and which can prepare a sample from a blood sample.

Technical means for solving the technical problems

In order to achieve the above object, the method for producing a measurement sample according to claim 1 comprises: removing red blood cells from a blood sample using a solid phase capable of binding to the red blood cells; the red blood cell-depleted blood sample is reacted with a reagent that immunostains the cells.

As described above, the method for producing a measurement sample according to invention 1 uses a solid phase capable of binding to erythrocytes to remove erythrocytes from a blood sample. This allows red blood cells to be removed together with the solid phase to which the red blood cells are bound, and thus facilitates automation of the red blood cell removal process. In addition, unlike the case where red blood cells are lysed and removed by a lysing agent, the influence on the signal of the cell to be measured (fluctuation in measurement results) can be reduced.

As shown in fig. 2, the sample preparation device according to claim 2 is a sample preparation device (100) for preparing a measurement sample by reacting a blood sample with a reagent, comprising: the blood sample processing device includes a 1 st processing unit (20) for removing red blood cells from the blood sample using a solid phase capable of binding to the red blood cells, and a 2 nd processing unit (30) for reacting the red blood cell-removed blood sample with a reagent for immunostaining the cells.

As described above, the sample preparation device according to claim 2 includes the 1 st processing unit (20) for removing red blood cells from a blood sample using a solid phase capable of binding to red blood cells. This allows red blood cells to be removed together with the solid phase to which the red blood cells are bound, and thus facilitates automation of the red blood cell removal process. In addition, unlike the case where red blood cells are lysed and removed by a lysing agent, the influence on the signal of the cell to be measured (fluctuation in measurement results) can be reduced.

Effects of the invention

According to the present invention, a method and an apparatus for preparing a measurement sample, which are easy to automate and have little influence on the signal of a cell to be measured, can be realized.

Drawings

FIG. 1 is a schematic view of examples 1 to 3 of a sample preparation apparatus for removing red blood cells from a blood sample;

FIG. 2 is a schematic diagram schematically showing a configuration example of the sample preparation apparatus;

FIG. 3 is a schematic view of a process flow of the sample preparation apparatus;

FIG. 4 is a view for explaining a specific example of the sample preparation process;

FIG. 5 is a diagram showing the movement of a rack for transferring a reaction vessel in a sample preparation apparatus;

FIG. 6 is a diagram showing movement of a rack of sample dispensing of the sample preparation device;

FIG. 7 is a diagram showing a state in which a rack moves when a sample preparation apparatus is processed by the 1 st processing section;

FIG. 8 is a diagram showing the movement of the rack after processing by the 1 st processing section of the sample preparation apparatus;

FIG. 9 is a graph showing the detection results of Teff cells of examples and comparative examples;

fig. 10 is a graph showing the detection results of Treg cells of examples and comparative examples;

FIG. 11 is a scatter diagram showing the division of T cells having a large CD4 expression level among cells divided into lymphocytes according to the example;

FIG. 12 is a scatter diagram showing the division of T cells having a large CD4 expression level among cells divided into lymphocytes according to a comparative example;

FIG. 13 is a comparative example to CD4+Cells with low expression level of CD62L in T cells (CD4+/CD 62L) ^Low T cells) are partitioned histogram;

FIG. 14 shows the comparison of cells (CD4+/CD62L) with low expression level of CD62L in CD4+ T cells ^Low T cells) are partitioned histogram;

FIG. 15 is a scattergram of the example for dividing T cells (CD4+/CD25+/FOXP3+ T cells) with high expression of CD25 and FOXP3 in CD4+ T cells;

FIG. 16 is a scattergram of comparative example in which T cells (CD4+/CD25+/FOXP3+ T cells) having high expression levels of CD25 and FOXP3 were divided.

Detailed Description

The method for producing a measurement sample according to the present embodiment (hereinafter also referred to as "the method for producing the present embodiment") includes removing red blood cells from a blood sample using a solid phase capable of binding to the red blood cells, reacting the blood sample from which the red blood cells have been removed with a reagent for immunostaining the cells. According to the method of the present embodiment, a measurement sample from which red blood cells are removed and specific cells are immunostained is prepared.

The measurement sample prepared by the method of the present embodiment is a measurement sample in which cells contained in a blood sample are immunostained, and is particularly suitable for flow cytometry measurement. The blood sample is, for example, a sample derived from a living body, for example, whole blood collected from a subject. The subject is mainly a human, but may be an animal other than a human.

The blood sample includes at least cells to be measured and erythrocytes. The cells to be measured are, for example, white blood cells, more specifically lymphocytes. Further specifically T cells. More specifically, the target cell is a regulatory T cell (hereinafter also referred to as Treg cell) and an effector T cell (hereinafter also referred to as Teff cell) among T cells. As described later, the Treg cells and Teff cells are counted separately from each other by immunostaining cell surface antigens and intracellular antigens specifically expressed by the Treg cells and Teff cells, and obtaining fluorescent signals obtained by flow cytometry.

Signals from cells other than Treg cells and Teff cells become noise that prevents accurate differential counting. Especially, the blood sample contains a large amount of erythrocytes, which are noisy and therefore require removal before measurement by flow cytometry.

Conventionally, a lysing agent has been widely used for removing red blood cells contained in a blood sample. For example, ammonium chloride (ammonium chloride) and a surfactant are generally used as the dissolving agent. These lysing agents are inexpensive and can remove red blood cells only by mixing with a blood sample, and are therefore easy to prepare, and are therefore widely used for preparing measurement samples for measuring general antigen markers such as CD4 and CD 25. However, the inventors studied that when a conventional method of removing erythrocytes using a lytic agent is used in the measurement of specific cell surface antigens and intracellular antigens, the fluorescent signal is affected in the measurement of cells to be measured, particularly Treg cells and Teff cells described in examples described later. And the inventors have further studied that the influence on the fluorescence signal can be reduced by removing erythrocytes by using a solid phase capable of binding to erythrocytes, thereby removing erythrocytes instead of using a lytic agent. The method for producing the measurement sample according to the present embodiment will be described in detail below.

< erythrocyte removal >)

The solid phase capable of binding to erythrocytes is, for example, a solid phase capable of capturing and adsorbing erythrocytes suspended in a blood sample by contact with the blood sample. The solid phase capable of binding to erythrocytes specifically binds to erythrocytes and does not bind to cells to be measured, such as leukocytes. For specific binding to erythrocytes, an antibody capable of binding to erythrocytes is preferably immobilized on the surface of the solid phase. The antibody capable of binding to erythrocytes is preferably an antibody capable of binding to a surface antigen of erythrocytes (hereinafter also referred to as erythrocyte-capturing antibody) by antigen-antibody reaction, and examples thereof include CD235a antibody and CD55 antibody. Antibodies capable of binding specifically to erythrocytes are particularly preferred CD235a antibodies. In addition, to remove erythrocytes contained in blood derived from mice, a TER-119 antibody can be used.

The method of immobilizing the erythrocyte-capturing antibody on the solid phase surface is, for example, preferably using interaction of avidin or streptavidin with biotin. For example, the erythrocyte-capturing antibody can be immobilized on the solid phase surface by allowing a biotinylated erythrocyte-capturing antibody to react with a streptavidin-conjugated solid phase. The red blood cell-capturing antibody may be mixed with the blood sample in a state of being immobilized in advance on a solid phase, or the red blood cell-capturing antibody and the solid phase may be mixed separately with the blood sample, and the red blood cell-capturing antibody may be immobilized on the solid phase in the blood sample.

The solid phase is preferably a particle. By using the particles as a solid phase, erythrocytes can be adsorbed on the particle surface by adding the particles to a blood sample. The particles are dispersed in the blood sample, and thus can efficiently adsorb red blood cells, which is advantageous in this regard. Hereinafter, the particles as a solid phase are referred to as solid phase particles.

An example of red blood cell removal when solid particles are used will be described with reference to fig. 1. Examples of the method for removing erythrocytes when solid-phase particles are (A) BF (Bound. Free) separation method, (B) filtration method and (C) aggregation method.

The BF separation method will be described. In BF separation, magnetic particles are used as solid phase particles. As shown in fig. 1, in step 1, magnetic particles having a red blood cell capturing antibody immobilized on the surface are added to a blood sample. The mixture of the blood sample and the magnetic particles is preferably stirred, the magnetic particles being dispersed in the blood sample. In step 2, erythrocytes in the blood sample are adsorbed on the surface of the magnetic particles. In step 3, the magnetic particles are collected by the magnetic force by the magnet. For example, as shown in fig. 1, a magnet M is disposed near a container (side surface in the example of fig. 1) containing a mixture of a blood sample and magnetic particles, and the magnetic particles dispersed in the container are positioned on the inner side surface of the container. Thereby separating the red blood cells within the vessel. In step 4, a red blood cell-free portion, for example, a supernatant, is collected from the liquid in the container in which the magnetic particles are collected by the magnetic force. Thereby, red blood cells are separated from the blood sample.

The time for disposing the magnet M on the side of the container to gather the magnetic particles by the magnetic force is preferably 1 minute or more, more preferably 5 minutes or more. The position where the magnet M is disposed may be, for example, a side surface or a bottom surface of the container, as long as the magnet M is disposed near the container, can provide a magnetic force sufficient to position the magnetic particles in the container, and can avoid the complex including the positioned magnetic particles from sucking the supernatant liquid by suction. The 3 rd step of disposing the magnet M near the container can be achieved by relatively moving the magnet M and the container. For example, the container including the magnetic particles may be placed close to the magnet M, or the magnet M may be placed close to the container.

In the BF separation method, magnetic particles are allowed to accumulate on the side of the container due to magnetic force, whereby erythrocytes can be separated in a liquid phase, and thus there is an advantage of easy operation.

Next, a filtration method will be described. In the filtration method, as in the steps 1 and 2 of the BF separation method, solid phase particles are added to the blood sample, and erythrocytes are adsorbed on the surfaces of the solid phase particles. In step 3, a blood sample including solid-phase particles having red blood cells adsorbed thereon is dispensed to a column provided with a filter F. The filter F includes a porous member having a pore size that allows passage of target cells, such as leukocytes, but does not allow passage of solid phase particles. After the blood sample is dispensed to the upper surface of the filter, the blood sample is filtered by the filter F, the target cells pass through the filter F, and the solid-phase particles having adsorbed the red blood cells are captured by the filter F. Thereby, red blood cells are separated from a blood sample including the cells of the subject.

While the filtration method uses the filter F, a magnetic bead column may be used instead of the filter. When using a magnetic bead column, the column is filled with solid phase particles having a surface on which a red blood cell capturing antibody is immobilized, and a blood sample is injected into the column. When the blood sample passes through the gaps between the solid phase particles, the red blood cells contained in the blood sample are adsorbed on the surfaces of the solid phase particles.

Next, the flocculation method will be described. In the flocculation method, solid-phase particles are added to a blood sample in the same manner as in steps 1 and 2 of the BF separation method. The red blood cells are adsorbed to the surface of the solid phase particles, whereby the red blood cells and the solid phase particles form a clot (Clump). In step 3, the blood sample including the solid phase particles having erythrocytes adsorbed thereon is left to stand for a predetermined period of time. The clot formed by the red blood cells and the solid phase particles settled to the bottom of the vessel due to its own weight. In this state, the supernatant including the subject cells is separated, whereby the erythrocytes are separated from the blood sample.

In the agglutination method, solid phase particles that are liable to form a clot with erythrocytes are preferably selected, and latex particles are preferably used, for example.

< immunostaining >)

Cells contained in the blood sample from which red blood cells are removed as described above are immunostained. In immunostaining, the target antigen is labeled, for example, by mixing an antibody reagent including a labeled antibody capable of binding to the target antigen of the subject cell with a blood sample.

The labeled antibody is, for example, an antibody modified with a labeling molecule that generates a fluorescent signal of a specific wavelength in response to irradiation with excitation light having a specific center wavelength.

The number of antigens to be labeled by immunostaining is not particularly limited, and may be 1 or plural. When a plurality of antigens are simultaneously stained, a plurality of target antigens can be stained by mixing a blood sample with a mixed reagent including a plurality of labeled antibodies labeled to emit fluorescent signals different from each other.

The antigen may be expressed on the surface of a cell, expressed in a cell, or a combination of these. The type of the combined antigen can be changed according to the antigen of interest. For example, in the examples described below, treg cells and Teff cells are used as target cells. Teff cells can be identified as cells that are low in expression of CD62L antigen among T cells positive for CD4 antigen (cd4+ T cells). In addition, treg cells can be identified as cells positive for the CD25 antigen and positive for the FOXP3 antigen in cd4+ T cells. In this example, CD4, CD62L, CD25 can be immunostained as a cell surface antigen and FOXP3 can be immunostained as an intracellular antigen.

The inventors studied and found that, among the above exemplified antigens, CD62L, CD and FOXP3 reduced fluorescence signals when treated with a lytic agent to remove red blood cells contained in a blood sample. This is because lytic agents have an effect on cells, and thus antigens on the cell surface and in the cells are stimulated or damaged. In contrast, it was confirmed that the influence on the signal of the antigen was reduced by removing erythrocytes using a solid phase capable of binding to erythrocytes instead of the lysing agent.

When staining cell surface antigens (e.g., CD4, CD62L, CD 25) and intracellular antigens (e.g., FOXP 3) by immunostaining, the following procedure is preferably included: an antibody 1 reagent comprising a labeled antibody that specifically binds to a cell surface antigen, and an antibody 2 reagent comprising a labeled antibody that specifically binds to an intracellular antigen are mixed with a blood sample. It is also preferable that the order of mixing with the blood sample is the 1 st antibody reagent and the 2 nd antibody reagent. The blood sample is preferably mixed with an immobilizing agent and a membrane penetrating agent after staining the cell surface antigen with the 1 st antibody reagent and before adding the 2 nd antibody reagent. Macromolecules such as labeled antibodies cannot penetrate the cell membrane into the cell interior, and thus membrane penetrating agents allow the labeled antibodies to penetrate the cell membrane into the cell. The immobilizing agent passes through the cell membrane treated with the membrane penetrating agent to immobilize so that the intracellular antigen, i.e., the protein, does not diffuse.

< measurement by flow cytometer >)

The immunostained assay samples were supplied for flow cytometer measurements. Flow cytometry obtains an optical signal, such as a fluorescent signal, from immunostained cells by flow cytometry and analyzes the cells based on the fluorescent signal.

The method for classifying and counting Treg cells and Teff cells when a measurement sample from which erythrocytes have been removed by the above method and cells have been immunostained is measured by a flow cytometer will be described below.

First, lymphocytes are classified from white blood cells contained in a blood sample. Lymphocytes are classified, for example, according to parameters based on forward scattered light intensity and side scattered light intensity.

Then, cells having a CD4+ signal equal to or higher than a predetermined value among lymphocytes are classified. Specifically, a cell in which a fluorescent signal emitted from a labeled antibody that binds to the CD4 antigen is equal to or greater than a predetermined value is specified. In order to improve the classification performance, for example, the scattered light intensity, more preferably the side scattered light intensity, is combined as a parameter in addition to the fluorescence signal corresponding to CD 4.

Classification of CD4+ cells into cells with low expression of CD62L (CD 62L) ^low ). Specifically, a cell in which a fluorescent signal emitted from a labeled antibody that binds to the CD62L antigen is equal to or less than a predetermined value is specified. When a histogram is formed with the intensity of a fluorescent signal emitted from a labeled antibody that binds to CD62L antigen as the horizontal axis and the number of cells corresponding to the intensity as the vertical axis, a so-called bimodal histogram is drawn in which a peak of a cell highly expressing CD62L appears on the high-value side of the fluorescent signal and a peak of a cell lowly expressing CD62L appears on the low-value side. Cells plotted on the low-value side of the fluorescence signal compared with the portion corresponding to the valley between the 2 peaks were designated as CD62L ^low 。

Cells of CD4+ cells, FOXP3+ and CD25+ were classified. Specifically, a cell in which a fluorescent signal (FOXP 3 signal) from a labeled antibody that binds to FOXP3 antigen is equal to or higher than a predetermined value and a fluorescent signal (CD 25 signal) from a labeled antibody that binds to CD25 antigen is equal to or higher than a predetermined value is specified. When a two-dimensional distribution map is formed with FOXP3 signal intensity as the 1 st axis and CD25 signal intensity as the 2 nd axis, 4 quadrants are obtained, each of which is divided into a region having a predetermined value or more and a region having a smaller value than the predetermined value with respect to 2 signals. Cells plotted in quadrants where FOXP3 signal is equal to or greater than a predetermined value and CD25 signal is equal to or greater than a predetermined value are specified as foxp3+ and cd25+. The number and ratio of Treg cells and Teff cells in blood were thus determined. The proportion of Treg cells and the proportion of Teff cells in blood are known to be effective for predicting the efficacy of immune checkpoint inhibitors (Nivolumab).

Specifically, CD4+ and CD62L ^low The ratio of cells to lymphocytes was determined as% Teff, and the ratio of cells of cd4+ and foxp3+ and cd25+ to lymphocytes was determined as% TregThen, the index of the drug effect prediction can be obtained by substituting the following formula (1).

Index = [% Teff ] ² ／［％Treg］・・・（1）

For example, by comparing the index to an empirically set threshold, patients can be stratified into a responsive group and a non-responsive group of nivolumab. According to the sample preparation method of the present invention, fluorescent signals from Treg cells and Teff cells can be detected with high accuracy, and accurate count results can be obtained.

Automation of pretreatment device

The method for producing a measurement sample according to the present embodiment has an advantage that the influence on the fluorescent signal of a target cell can be reduced, and further has an advantage that automation is easy. An example of automation of measurement sample preparation when magnetic particles are used as a solid phase capable of binding to erythrocytes will be described below.

[ outline of sample preparation apparatus ]

Referring to fig. 2, an outline of the sample preparation apparatus 100 according to an embodiment will be described.

As shown in fig. 2, the sample preparation apparatus 100 includes a 1 st processing unit 20 and a 2 nd processing unit 30. The 1 st processing unit 20 performs a process of removing red blood cells from a blood sample using a solid phase capable of binding to red blood cells. The 2 nd processing unit 30 performs a process of reacting the red blood cell-removed blood sample with a reagent for immunostaining cells.

The 1 st processing unit 20 includes a rack 10a for housing the processing container 11, a rack 10b for housing the reaction container 12, and a rack 10c for housing the sample container 13 containing the blood sample. The 1 st processing unit 20 includes a 1 st moving unit 15, a 2 nd moving unit 16, and a 3 rd moving unit 17 for moving the racks 10a, 10b, and 10c in the lateral directions (X1 and X2 directions), respectively.

The sample preparation apparatus 100 includes a 1 st dispensing section 41 and a container transfer section 42. The 1 st dispensing section 41 and the container transfer section 42 are supported by a common transfer shaft 43 and movable in the Y direction. The 1 st dispensing section 41 includes a pipette 41a. The 1 st dispensing unit 41 dispenses the blood sample stored in the sample container 13 to the processing container 11 using the pipette 41a. The container transfer portion 42 includes a pair of hands 42a that can be moved toward and away from each other. The container transfer section 42 holds the reaction container 12 by the hand 42a, and transfers the reaction container 12 between the rack 10b and the 2 nd processing section 30.

The sample preparation device 100 includes a stirring section 50 for stirring a blood sample. The stirring section 50 takes out the sample container 13 from the rack 10c and performs the tip-over stirring, thereby stirring the blood sample in the sample container 13.

The sample preparation apparatus 100 includes a 2 nd dispensing section 60. The 2 nd dispensing section 60 is supported by a transfer shaft 61 so as to be movable in the lateral direction (X1 and X2 directions). The transfer shaft 61 is supported by a transfer shaft 62 so as to be movable in the Y direction. Thus, the 2 nd dispensing unit 60 can move in the horizontal direction in the apparatus. The 2 nd dispensing unit 60 includes a pipette 60a. The 2 nd dispensing unit 60 sucks the supernatant from the processing container 11 using a pipette 60a, and dispenses the supernatant into the reaction container 12. The 2 nd dispensing unit 60 dispenses the reagents set in the reagent setting units 70a and 70b described later to the process container 11 of the 1 st processing unit 20 or the reaction container 12 in the 2 nd processing unit 30.

The sample preparation device 100 includes a reagent setting section 70a and a reagent setting section 70b. The reagent setting section 70a includes a cold storage chamber for cooling and placing the reagent. The reagent setting part 70b sets the reagent at room temperature.

The sample preparation apparatus 100 includes a nozzle cleaning section 80. The nozzle cleaning unit 80 cleans the nozzles of the 2 nd dispensing unit 60.

The sample preparation apparatus 100 includes a control unit 90 for controlling each unit of the apparatus. The control unit 90 includes a processor and a storage unit. The processor is constituted by, for example, a CPU. The storage section may include a memory and a storage. The processor functions as the control unit 90 of the sample preparation device 100 by executing the program stored in the storage unit.

The 1 st processing unit 20 includes a magnet 21 at a position adjacent to the 1 st moving unit 15. More specifically, the magnet 21 is provided at a position adjacent to the side surface of the processing container 11 where the rack 10a is placed when the 1 st moving unit 15 moves the rack 10a where the processing container 11 is placed to the origin (rightmost side (X2 side)). In the example of fig. 2, the magnet 21 is a permanent magnet or may be an electromagnet.

The 2 nd processing unit 30 is a centrifugal separation unit capable of accommodating a plurality of reaction vessels 12 as centrifugal tubes in the circumferential direction and capable of centrifugally separating a sample in the accommodated reaction vessels 12 by rotating at a high speed about an axis. The 2 nd processing unit 30 includes a rotor 31 rotating at a high speed and a plurality of holder units 32 provided on the outer periphery of the rotor 31. The holder 32 has a cylindrical shape, for example, and can house the reaction vessel 12 therein. When the rotor 31 is stopped, the holder 32 holds the reaction vessel 12 in a state in which the opening thereof is upward.

[ outline of method for preparing measurement sample ]

Referring to fig. 3, a schematic method of preparing a measurement sample in the sample preparation apparatus 100 will be described.

The 1 st dispensing unit 41 dispenses a part of the blood sample stored in the sample container 13 mounted on the rack 10c to the processing container 11 mounted on the rack 10 a. The 2 nd dispensing unit 60 dispenses a reagent including an anti-erythrocyte antibody which has been biotinylated to the processing container 11 in which the blood sample has been dispensed. The rack 10b transfers the reaction vessel 12 to the 2 nd processing unit 30 by the vessel transfer unit 42.

The 2 nd dispensing unit 60 dispenses a reagent including streptavidin-conjugated magnetic particles as a solid phase to the processing container 11 in which the blood sample is dispensed. Streptavidin binds to the magnetic particles and reacts with the biotinylated anti-erythrocyte antibody and binds, thereby forming a complex comprising erythrocytes and a solid phase within the processing vessel 11. The 1 st moving part 15 positions the processing container 11 laterally to the magnet 21, thereby concentrating the compound laterally to the processing container 11.

The 2 nd dispensing unit 60 sucks the supernatant from the processing container 11 in which the complex is collected on the side, and dispenses the supernatant into the reaction container 12 of the 2 nd processing unit 30. Thereby separating the supernatant including the cells to be measured from the erythrocytes contained in the complex.

The 2 nd dispensing unit 60 dispenses the antibody reagent to the reaction container 12 in which the blood sample is dispensed. The antibody reagent is an antibody mix reagent comprising a labeled antibody that binds to a cell surface antigen of a leukocyte (e.g., a labeled antibody that binds to CD4, CD25, CD 62L). After the reaction between the white blood cells in the sample and the antibody reagent, the 2 nd processing unit 30 centrifugally separates the sample in the reaction vessel 12. By centrifugation, the white blood cells settle to the bottom of the reaction vessel 12. The 2 nd dispensing section 60 aspirates and removes the supernatant of the centrifuged sample. Thus, the white blood cells reacted with the labeled antibody remain in the reaction vessel 12, and the supernatant including the unreacted antibody component is removed.

The 2 nd dispensing section 60 dispenses the fixing and penetrating agent into the reaction vessel 12. By the immobilizing/penetrating agent, proteins in the cell membrane are immobilized, and the membrane is penetrated so that the labeled antibody is allowed to enter the cell membrane. After the reaction with the fixing/penetrating agent, the 2 nd processing unit 30 performs centrifugal separation of the sample in the reaction vessel 12. By centrifugation, the white blood cells settle to the bottom of the reaction vessel 12. The 2 nd dispensing section 60 aspirates and removes the supernatant of the centrifuged sample. Thus, the white blood cells treated with the fixative/penetrant remain in the reaction vessel 12, and the supernatant including the fixative/penetrant is removed.

The 2 nd dispensing unit 60 dispenses the antibody reagent into the reaction vessel 12 through the 2 nd dispensing unit 60. The antibody reagent is a reagent comprising a labeled antibody that binds to an intracellular antigen (e.g., a labeled antibody that binds to FOXP 3). After the reaction between the white blood cells in the sample and the antibody reagent, the 2 nd processing unit 30 centrifugally separates the sample in the reaction vessel 12. By centrifugation, the white blood cells settle to the bottom of the reaction vessel 12. The 2 nd dispensing section 60 aspirates and removes the supernatant of the centrifuged sample. Thus, the white blood cells reacted with the labeled antibody remain in the reaction vessel 12, and the supernatant including the unreacted antibody component is removed.

The 2 nd dispensing section 60 dispenses a buffer (e.g., phosphate buffer) into the reaction vessel 12. Thus, a measurement sample was prepared. The container transfer section 42 transfers the reaction container 12 containing the measurement sample to the rack 10 b.

[ operation of sample preparation device ]

The operation of measuring a sample by the sample preparation apparatus 100 will be described with reference to fig. 4. Hereinafter, the 1 st moving part 15, the 2 nd moving part 16, and the 3 rd moving part 17 move the frames 10a to 10c, and further refer to fig. 5 to 8.

As shown in fig. 5, the racks 10a, 10b, 10c are each capable of receiving a plurality (e.g., 6) of containers. The racks 10a, 10b, and 10c are transported in the X1 direction and the X2 direction by the 1 st moving unit 15, the 2 nd moving unit 16, and the 3 rd moving unit 17. In the present embodiment, the description has been given of a mode in which the 1 st moving portion 15, the 2 nd moving portion 16, and the 3 rd moving portion 17 integrally move the rack in the X1 direction and the X2 direction, but the 1 st moving portion 15, the 2 nd moving portion 16, and the 3 rd moving portion 17 may independently move the rack. The 1 st moving unit 15, the 2 nd moving unit 16, and the 3 rd moving unit 17 can convey the racks 10a, 10b, and 10c in the X1 direction and the X2 direction at a distance corresponding to the interval of the containers placed on the racks. In fig. 5 to 8, in order to make the illustration easier to understand the positions of the respective containers, lattices of a size corresponding to the container spacing are illustrated.

Sample split charging treatment

Prior to the sample dispensing process, as shown in fig. 5 (a), an empty processing container 11 is placed on the rack 10a by an operator. And the empty reaction vessel 12 is placed on the rack 10b by the operator. The sample container 13 containing the blood sample is also placed on the rack 10c by the operator.

In step S201 in fig. 4, the centrifuge tube as the reaction vessel 12 is transported from the rack 10b to the 2 nd processing unit 30. As shown in fig. 5 (B), the rack is moved in the X1 direction so that the leftmost reaction vessel 12 is located at the position P1. The position P1 is the position of the 12 th lattice from the rightmost position. At the position P1, the reaction vessel 12 is taken out by the vessel transfer section 42 and transferred to the 2 nd processing section 30. As shown in fig. 5 (C), the rack is moved in the X1 direction so that the next reaction vessel 12 is located at the position P1. Then, the reaction vessels 12 are taken out by the vessel transfer section 42 and transferred to the 2 nd processing section 30 in sequence. This process is repeated until the final reaction vessel 12 is moved to the position P1, and the vessel transfer section 42 transfers the reaction vessel to the 2 nd processing section 30.

In step S202, the blood sample in the sample container 13 is stirred. As shown in fig. 6 (a), the 3 rd moving unit 17 moves the rack in the X2 direction so that the leftmost sample container 13 is positioned at the position P2. The position P2 is the position of the 8 th lattice from the rightmost position. In the position P2, the sample container 13 of the rack 10c is taken out by the stirring section 50 and is turned upside down to be stirred.

In step S203, the blood sample is sucked from the sample container 13, discharged to the processing container 11 of the rack 10a, and packaged. As shown in fig. 6 (B), the rack is moved in the X1 direction so that the sample container 13 stirred in step S202 is located at the position P1. At the position P1, the 1 st dispensing unit 41 aspirates a part of the blood sample in the sample container 13, and dispenses the aspirated blood sample to the processing container 11 mounted on the rack 10 a. As shown in fig. 6 (C), the rack is moved in the X2 direction so that the next sample container 13 is located at the position P2. In the position P2, the sample container 13 is stirred by the stirring section 50. The stirring operation of the sample container 13 at the position P2 and the sub-packaging operation of the blood sample at the position P1 immediately thereafter are repeated until all the sample containers 13 have been subjected. Fig. 6 (D) is a diagram showing a state in which the stirring and dispensing operations are completed for all the sample containers 13.

BF separation treatment

In step S204, the antibody is dispensed to the processing container 11 from which the blood sample is discharged. As shown in fig. 7 (a), the rack is moved in the X2 direction so that the leftmost process container 11 is located at the position P3. At position P3, the 2 nd dispensing unit 60 dispenses the biotinylated anti-erythrocyte antibody to the processing vessel 11. As shown in fig. 7 (B), the rack is moved in the X1 direction so that the next process container 11 is located at the position P3. Then, at the position P3, the anti-erythrocyte antibody is dispensed to the processing container 11 by the 2 nd dispensing unit 60.

After the completion of the dispensing into all the processing containers 11, the agitation of the blood sample in the processing container 11 is performed in step S205. The 1 st moving unit 15 repeatedly reciprocates the rack in the X1 direction and the X2 direction, and agitates the blood sample in the processing container 11. After that, the rack 10a housing the processing container 11 is left to stand for a predetermined time (for example, 20 minutes).

In step S206, the buffer is dispensed to the processing container 11. The 1 st moving unit 15 moves the rack 10a, and the buffer is dispensed into each processing container 11 at the position P3 by the 2 nd dispensing unit 60. The 1 st moving unit 15 moves the rack 10a at a high speed to agitate the blood sample in the processing container 11. And then standing still. For example, BSA solution and Phosphate Buffered Saline (PBS) were dispensed as buffers. And agitating the blood sample separately filled with the buffer.

In step S207, the magnetic particles are dispensed into the processing container 11. For example, streptavidin is dispensed as magnetic particles, combined with magnetic particles. As shown in fig. 7 (C), the rack is moved in the X1 direction so that the leftmost process container 11 is located at the position P3. At position P3, streptavidin-coupled magnetic particles as a solid phase are dispensed to the processing vessel 11 by the 2 nd dispensing unit 60. As shown in fig. 7 (D), the rack is moved in the X1 direction so that the next process container 11 is located at the position P3. Then, at the position P3, the solid phase containing the magnetic particles is dispensed to the processing container 11 by the 2 nd dispensing unit 60.

In step S208, the blood sample in the processing container 11 is stirred to react. The time required is, for example, 5 minutes. The rack is moved at a high speed by the moving unit 15, and the blood sample in the processing container 11 is stirred. And then standing still. Thereby, a complex including red blood cells and a solid phase is formed in the treatment vessel 11.

In step S209, magnetism is collected. As shown in fig. 7 (E), the rack is moved in the X2 direction so that each processing container 11 is located at a position P4. The position P4 corresponds to 6 squares from the rightmost square. The magnet 21 is provided at a position adjacent to the position P4. By the magnet 21, the complex including the red blood cells and the solid phase is accumulated on the inner side surface of the processing container 11 due to the magnetic force. The time required is, for example, 10 minutes.

In step S210, the supernatant (for example, 700 μl) of the blood sample in the magnetically concentrated processing container 11 is aspirated, and the aspirated supernatant is discharged into the reaction container 12 of the 2 nd processing unit 30 and dispensed. As shown in fig. 8 (a), the supernatant is sucked from each processing container 11 at the position P4 by the 2 nd dispensing unit 60, and dispensed to each reaction container 12 moved to the 2 nd processing unit 30.

In step S212, the sample in the reaction vessel 12 is centrifuged. The 2 nd treatment unit 30 allows white blood cells to settle to the bottom of the reaction vessel 12 by rotating the rotor 31 at a high speed.

In step S213, the supernatant liquid in the reaction vessel 12 is removed. The 2 nd dispensing section 60 aspirates and removes the supernatant (e.g., 600. Mu.L) of the reaction vessel 12 in which the white blood cells were settled by centrifugal separation.

In step S214, the sample in the reaction vessel 12 is stirred. Through steps S212 and S213, white blood cells settle at the bottom of the reaction vessel 12. To disperse the settled white blood cells, the reaction vessel 12 is stirred. The 2 nd processing unit 30 agitates the sample in the reaction vessel 12 by repeating acceleration and deceleration of the rotor 31 in one direction and rotating the same.

Dyeing treatment 1

In step S215, the antibody reagent is dispensed to the reaction container 12. The 2 nd dispensing unit 60 dispenses a mixed reagent including a CD 25-labeled antibody, a CD 4-labeled antibody, and a CD 62L-labeled antibody as an antibody reagent into the reaction vessel 12 mounted in the 2 nd processing unit 30.

In step S216, the 2 nd processing unit 30 rotates the rotor 31 while repeatedly accelerating and decelerating in one direction, thereby stirring the sample in the reaction vessel 12. The predetermined time is, for example, 30 minutes.

In step S217, a cleaning solution for the sample before fixation is dispensed to the reaction container 12. The 2 nd dispensing section 60 dispenses PBS as a washing liquid into the reaction vessel 12.

In step S218, the 2 nd processing unit 30 rotates the rotor 31 while repeatedly accelerating and decelerating in one direction, thereby stirring the sample in the reaction vessel 12.

In step S219, the 2 nd processing unit 30 rotates the rotor 31 at a high speed in one direction, thereby centrifugally separating the sample in the reaction vessel 12. Thus, the white blood cells reacted with the antibody reagent settle.

In step S220, the 2 nd dispensing unit 60 aspirates and removes the supernatant from the reaction vessel 12. Through the above steps, the surface antigens CD25, CD4, CD62L are respectively stained with the corresponding labeling substances.

Cell fixation and penetration treatment

In step S221, the 2 nd dispensing unit 60 dispenses the fixing and penetrating agent into the reaction vessel 12.

In step S222, the 2 nd processing unit 30 repeats acceleration and deceleration of the rotor 31 in one direction and rotates the rotor, thereby stirring the sample in the reaction vessel 12 to perform a reaction. The predetermined time is, for example, 30 minutes.

In step S223, the 2 nd dispensing unit 60 dispenses the cleaning liquid for the fixed sample to the reaction container 12.

In step S224, the 2 nd processing unit 30 rotates the rotor 31 while repeatedly accelerating and decelerating in one direction, thereby stirring the sample in the reaction vessel 12.

In step S225, the 2 nd processing unit 30 rotates the rotor 31 at a high speed, thereby centrifugally separating the sample in the reaction vessel 12.

In step S226, the 2 nd dispensing unit 60 aspirates and removes the supernatant from the reaction vessel 12.

In steps S227 to S230, the operations of dispensing the cleaning liquid, stirring, centrifuging, and removing the supernatant are performed. That is, the cleaning process of the sample is repeated. The sample may be washed 1, 2, or 3 times or more. Through the above steps, the cells in the reaction vessel 12 are subjected to the immobilization treatment and the penetration treatment.

Dyeing process 2

In step S231, the 2 nd dispensing unit 60 dispenses the antibody reagent into the reaction container 12. For example, as the antibody reagent, a reagent including an anti-FOXP 3-labeled antibody is dispensed.

In step S232, the 2 nd processing unit 30 rotates the rotor 31 while repeatedly accelerating and decelerating in one direction, thereby stirring the sample in the reaction vessel 12 to perform a reaction. The predetermined time is, for example, 30 minutes.

In step S233, the 2 nd dispensing unit 60 dispenses the cleaning liquid for the fixed sample to the reaction container 12. In steps S234 to 236, the stirring, centrifugal separation, and supernatant removal operations are performed in the same manner as described above, and in steps S237 to S240, the washing process of the sample including the washing liquid dispensing, stirring, centrifugal separation, and supernatant removal operations is repeated. The sample may be washed 1, 2, or 3 times or more. Through the above steps, FOXP3 in the reaction vessel 12 is stained with the corresponding labeling substance.

Container return

In step S241, the 2 nd dispensing unit 60 dispenses the buffer solution into the reaction vessel 12. The sample in the reaction vessel 12 is adjusted to a predetermined liquid amount and a predetermined pH suitable for supply to the measuring apparatus by dispensing. For example, BSA solution and PBS were dispensed as buffers.

In step S242, the 2 nd processing unit 30 rotates the rotor and agitates the same as described above.

In step S243, the container transfer section 42 takes out the reaction container 12 mounted in the 2 nd processing section 30 from the rotor 31, and places it in the rack 10b mounted in the 2 nd moving section 16. After all the reaction containers 12 are transferred to the rack 10b, the rack is moved in the X2 direction by the moving parts 15 to 17 to return to the initial position as shown in fig. 8 (D). This allows the operator to take out the reaction vessel 12.

Through the above steps, the sample preparation by the sample preparation device 100 is completed.

Comparative data of separation and hemolysis

The influence on the detection performance of Treg cells and Teff cells was compared by the isolation method according to the present application and the hemolysis method according to the comparative example by the following experiments.

EXAMPLE (preparation of samples by separation)

And subpackaging the whole blood sample collected from the heparin blood collection tube into test tubes. 50. Mu.L of biotinylated CD235a antibody was added to the tube and stirred and allowed to stand at room temperature for 20 minutes. To the tube was added 0.6mL of Phosphate Buffered Saline (PBS). 200. Mu.L of a streptavidin-conjugated magnetic particle-containing solution (Mojoport (registered trademark)) was added to the test tube and stirred, and allowed to stand at room temperature for 5 minutes. Permanent magnets were placed so as to be adjacent to the side of the test tube, and allowed to stand at room temperature for 10 minutes, whereby the complex of erythrocyte-biotinylated CD235a antibody-streptavidin-conjugated magnetic particles was concentrated on the inner wall of the test tube. The supernatant of the in-tube solution was collected by pipette at 0.7mL and dispensed into centrifuge tubes. The centrifuge tube was centrifuged at 300G for 5 minutes at room temperature, and 600. Mu.L of the supernatant was removed, whereby a sample of the example was obtained.

(staining of cell surface antigen)

50. Mu.L of an antibody mixture reagent of CD4/CD25/CD62L was added to the sample, followed by stirring and standing at room temperature for 30 minutes. Antibody mixing reagents include FITC-labeled CD4 antibody, PE-Cy 7-labeled CD25 antibody, and APC-labeled CD62L antibody. 1mL of PBS was added to the sample after completion of the reaction with the antibody-mixed reagent, and the mixture was stirred. The sample was centrifuged at 300G for 5 minutes at room temperature, and the supernatant was removed and stirred.

(fixation, membrane penetration)

To the centrifuged sample, 0.5mL of a fixing/membrane penetrating agent (eBioscience Foxp3/Transcription Factor Fixation/Permeabilization Concentrate and Diluent, manufactured by Invitrogen) was added and stirred, and the mixture was allowed to stand at room temperature for 30 minutes. 1mL of a washing solution (eBioscience Permeabilization Buffer (10X), manufactured by Invitrogen) was added to the sample and stirred. The sample was centrifuged at 400G for 5 minutes at room temperature and the supernatant removed. 1mL of the washing solution (as above) was added to the centrifuged sample and stirred, and the mixture was centrifuged at 400G for 5 minutes at room temperature to remove the supernatant and stirred.

(staining of intracellular antigen)

To the obtained sample, 50. Mu.L of an antibody reagent including a PE-labeled FOXP3 antibody was added, and the mixture was allowed to stand at room temperature for 30 minutes. 1mL of a washing solution (eBioscience Permeabilization Buffer (10X), manufactured by Invitrogen) was added to the sample and stirred. The sample was centrifuged at 400G for 5 minutes at room temperature and the supernatant removed. 1mL of the washing solution (as above) was added to the centrifuged sample and stirred, and the mixture was centrifuged at 400G for 5 minutes at room temperature to remove the supernatant and stirred.

(measurement by flow cytometer)

To the sample, 0.3mL of PBS including 0.5% bsa was added and stirred. The obtained sample was measured by a commercially available flow cytometer FACS Canto II (manufactured by Becton Dickinson Co.) and was used for the production of Teff cells (CD 62L ^low And cd4+ T cells) and Treg cells (cd25+, foxp3+, and cd4+ T cells) were counted separately.

Comparative example (preparation of sample by hemolysis)

And subpackaging the whole blood sample collected from the heparin blood collection tube into a centrifuge tube. To the centrifuge tube, 2mL of a hemolyzing agent (CyLyse; manufactured by Hizichikun Co., ltd.) was added and allowed to stand at room temperature for 10 minutes, thereby hemolyzing erythrocytes in blood. After stirring the centrifuge tube with a vortex mixer, 2mL of PBS was added to the centrifuge tube. Centrifuge the tube at room temperature for 5 minutes at 300G and remove the supernatant. To the centrifuge tube, 2mL of PBS was added, and the centrifuge tube was centrifuged at 300G for 5 minutes at room temperature, and the supernatant was removed to obtain a sample of comparative example. The samples of the comparative examples were subjected to the same procedure as in the examples, and the measurement was performed by a flow cytometer.

(control experiment)

A5 mL whole blood sample taken into heparin tubes was diluted with 5mL of liquid medium (10 mM HEPES/PRMI-1640). 10mL of a medium for lymphocyte separation (Ficoll-Paque Plus, manufactured by Cytiva Co.) was added to the 50mL tube. Diluted whole blood samples were added to the tube and stratified. PBMC were isolated in tubes according to the instructions described in Ficoll Paque Plus, suspended to 1X10≡6 cells/mL using CELLBANCER 2 (manufactured by Nippon pharmaceutical Industry Co., ltd.), and frozen with an ultra-low temperature freezer. After the freezing treatment, PBMCs were transferred to liquid nitrogen (in liquid phase) 1 week after 24 hours. After washing the thawed cells with 10% FBS/RPMI1640, 10% FBS/RPMI1640 is added to make 1×10≡6 cells/mL. 2mL of the cell suspension was added to a 24-well plate (manufactured by Corning Co.) and cultured for 48 hours. After the culture, the cell culture solution was transferred to a 15mL tube and centrifuged at 400G for 5 minutes to obtain a sample of a control example. The same procedure as in example was carried out for the samples of the comparative example, and measurement was carried out by a flow cytometer.

(measurement results)

The measurement results are shown in fig. 9 and 10.

As shown in fig. 9, in the detection performance of Teff cells, the measurement sample prepared by the separation method of the example showed good correlation with the sample using frozen PBMCs of the control experiment (correlation coefficient= 0.9419). And the examples showed significantly higher values in the correlation coefficient compared with the comparative example (correlation coefficient= 0.7064).

As shown in fig. 10, the results of the measurement samples prepared by the separation method of the examples and the use of frozen PBMCs in the control experiment showed good correlation (correlation coefficient= 0.8679) in the detection performance of Treg cells. And the examples showed significantly higher values in the correlation coefficient compared with the comparative example (correlation coefficient=0.0065).

From the above results, it is clear that the separation method according to the present invention shows high measurement accuracy for both Teff cells and Treg cells, compared to the hemolysis method. The isolation method was found to have the same measurement accuracy as when the sample was prepared using frozen PBMC.

(Signal correlation investigation from markers)

Fig. 11 and 12 are scatter diagrams of T cells (cd4+ T cells) having a large expression level of CD4 among lymphocytes. FIG. 11 is a scattergram of the example (isolation method), and FIG. 12 is a scattergram of the comparative example (hemolysis method). The count of CD4+ T cells was 44.4% in the example (isolation method) and 43.0% in the comparative example (hemolysis method), with no significant difference.

FIGS. 13 and 14 show the results of the expression of CD62L in CD4+ T cells (CD4+ and CD62L) ^Low T cells of (a) are divided, and obtained from the same sample. FIG. 13 is a bar chart of the example (separation method), and FIG. 14 is a bar chart of the comparative example (hemolysis method). In the histogram, the fluorescence signal of CD62L is CD4+ and CD62L within a predetermined critical value range ^Low T cells, i.e., teff cells, were counted. In the example (isolation method), the Teff cells were 8.48%, whereas in the comparative example (hemolysis method), the Teff cells were 15.4%. In the bar graph of the example (separation method), CD62L ^Low Peak of T cells and T cells with high CD62L expression (CD 62L ^High T cells) are significantly separated, with few cells plotted between the valleys between the peaks. On the other hand, the histogram of the comparative example (hemolysis) is compared with the histogram of the example (separation), CD62L ^High The peak of T cells decreased and the number of cells plotted between the valleys of 2 peaks was greater. This is because insufficient staining of CD62L in the hemolysis method, the fluorescence signal intensity decreases as a whole, resulting in cells with high expression level of CD62L (CD 62L ^High T cells) to lowThe value side slides. This data indicates that in the hemolysis method, the signal of CD62L is overall reduced.

FIGS. 15 and 16 are scatter plots obtained from the same sample, which are obtained by dividing T cells (CD4+/CD25+/FOXP3+ T cells) having high expression levels of CD25 and FOXP3 in CD4+ T cells. FIG. 15 is a scattergram of the example (isolation method), and FIG. 16 is a scattergram of the comparative example (hemolysis method). Cells that showed a value higher than the predetermined threshold value with respect to the fluorescence signal intensity (horizontal axis) corresponding to CD25 and the fluorescence signal intensity (vertical axis) corresponding to FOXP3, respectively, were identified as cd25+/foxp3+ T cells, that is, treg cells (cells in the Q2 region). Treg cells divided into Q2 were 3.88% in the example (isolation method) and 1.94% in the comparative example (hemolysis method). The scattergram of the comparative example (hemolysis) showed an increase in the number of cells contained in Q3 as compared with the scattergram of the example (isolation). That is, in the hemolysis method, the cell mapping was decreased in the vertical axis direction, and thus it was shown that the signal of FOXP3 was decreased as a whole.

(modification)

The embodiments disclosed herein are illustrative in all respects, and are in no way limiting. The scope of the present invention is shown not by the description of the above embodiments but by the claims, and includes all modifications equivalent in meaning and scope to the claims.

Numbering represents

10a: a processing container transfer unit (10 b): reaction vessel supply unit, 10c sample placement unit, 11: treatment vessel, 12: reaction vessel, 13: sample container, 20: 1 st processing unit, 30: treatment unit 2, 41: 1 st sub-packaging unit, 42: container transfer unit, 60: 2 nd sub-packaging part, 100: sample preparation device

Claims

1. A method for producing a measurement sample, comprising:

removing red blood cells from a blood sample using a solid phase capable of binding to the red blood cells;

reacting the blood sample from which the red blood cells have been removed with a reagent that immunostains the cells.

2. The method for preparing an assay according to claim 1, wherein:

removing red blood cells from the blood sample includes: forming a complex comprising red blood cells and said solid phase, and separating the formed complex and a supernatant of said blood sample.

3. The method for preparing an assay according to claim 2, wherein:

in the complex, the erythrocytes and the solid phase are bound via a binding substance having a binding force to the erythrocytes and the solid phase.

4. The method for preparing an assay according to claim 3, wherein:

forming the complex comprising red blood cells and the solid phase comprises: the complex is formed by contacting the blood sample, the binding substance, and the solid phase.

5. The method for preparing an assay according to claim 3, wherein:

the binding substance includes an antibody capable of binding to the red blood cells.

6. The method for preparing a measurement sample according to any one of claims 2 to 5, wherein:

the solid phase is provided with magnetic particles,

removing red blood cells from the blood sample includes: the complexes are caused to aggregate by magnetic force by a magnet and the supernatant of the blood sample is pipetted.

7. The method for preparing a measurement sample according to any one of claims 2 to 5, wherein:

removing red blood cells from the blood sample includes: the complex formed is filtered off through a filter, whereby the supernatant of the blood sample is separated.

8. The method for preparing a measurement sample according to any one of claims 2 to 5, wherein:

removing red blood cells from the blood sample includes separating a supernatant of the blood sample by settling the complex formed by centrifugation.

9. The method for preparing a measurement sample according to any one of claims 1 to 5, wherein:

reacting the blood sample with red blood cells removed with a reagent that immunostains cells includes: the blood sample is centrifuged.

10. A sample preparation device for preparing a measurement sample by reacting a blood sample with a reagent, comprising:

a 1 st processing unit for removing red blood cells from a blood sample using a solid phase capable of binding to the red blood cells;

and a 2 nd treatment unit for reacting the blood sample from which the red blood cells have been removed with a reagent for immunostaining the cells.

11. The sample preparation device of claim 10, wherein:

the 1 st processing section includes a separation section that forms a complex including red blood cells and the solid phase, and separates the formed complex and a supernatant of the blood sample.

12. The sample preparation device of claim 11, wherein:

13. The sample preparation device of claim 12, wherein:

the 1 st processing unit forms the complex by bringing the blood sample, the binding substance, and the solid phase into contact with each other.

14. The sample preparation device of claim 12, wherein:

15. The sample preparation device according to any one of claims 11 to 14, wherein:

the solid phase is provided with magnetic particles,

the 1 st processing section includes a magnet that causes the complex to aggregate due to magnetic force, and separates the complex from a supernatant of the blood sample by causing the complex to aggregate due to magnetic force and pipetting the supernatant of the blood sample through the magnet.

16. The sample preparation device according to any one of claims 11 to 14, wherein:

the treatment section 1 includes a filter that filters out the complex formed to separate a supernatant of the blood sample.

17. The sample preparation device according to any one of claims 11 to 14, wherein:

the 1 st processing section includes a 2 nd centrifugal separation section that separates a supernatant of the blood sample by settling the formed complex by centrifugal separation.

18. The sample preparation device according to any one of claims 10 to 14, wherein:

the 2 nd processing unit includes a 1 st centrifugal separation unit that performs centrifugal separation on the blood sample.

19. The sample preparation device according to any one of claims 10 to 14, further comprising:

A sample placement unit for placing a sample container in which the blood sample is stored;

a reaction container supply unit for placing a reaction container for receiving the blood sample reacted in the 2 nd processing unit;

a container transfer section for transferring the reaction container between the reaction container supply section and the 2 nd processing section;

a 1 st dispensing unit for dispensing the blood sample from the sample container to a processing container for receiving the blood sample processed by the 1 st processing unit;

and a 2 nd dispensing unit for dispensing the reagent set in the reagent setting unit into the processing container of the 1 st processing unit or the reaction container in the 2 nd processing unit.

20. The sample preparation device of claim 19, wherein:

the 2 nd dispensing unit sucks the supernatant from the processing container and dispenses it into the reaction container in the 2 nd processing unit.