JP2010172251A - Cell dispersion apparatus and sample pretreatment apparatus - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a cell dispersion apparatus by which the efficiency of dispersing aggregated cells can be heightened while suppressing damage to the cells. <P>SOLUTION: Disclosed is an apparatus for dispersing aggregated cells, which are an aggregate of multiple cells, in a cell sample solution containing the aggregated cells into single cells. The apparatus comprises: a filter member which is so adapted that the single cells can pass therethrough; a dispersing member which is arranged apart from the filter member by a predetermined distance and can disperse aggregated cell located in a space formed between the filter member and the dispersing member; and a driving source 103 which can drive the dispersing member or the filter member. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a cell dispersion apparatus and a sample pretreatment apparatus. More specifically, the present invention relates to a cell dispersion apparatus that disperses aggregated cells obtained by aggregating a plurality of cells into a single cell, and a sample pretreatment apparatus including the cell dispersion apparatus.

  In cervical cancer screening using a flow cytometer, a cell sample obtained by rubbing the cervix with a dedicated brush and stored in a preservative solution mainly composed of alcohol is used as a measurement sample. Cells collected by rubbing the cervix consist of two types of cells, squamous epithelial cells and glandular cells, most of which are squamous epithelial cells, but the uterine body located in the back of the cervix is Since it is mainly composed of gland cells that secrete mucus, the collected cervical cell group is often in an aggregated state covered with mucus produced in the uterine body.

  When analyzing cells with a flow cytometer, the cells in the measurement sample aligned in a row are irradiated with laser light, and the size and shape of each cell are measured using scattered light and fluorescence from the cells. It is difficult to measure a specimen of cell aggregation, and it is necessary to be a single cell. Also, in order not to overlook the atypical cells and cancer cells that are present while the cells are aggregated, it is important to disperse the aggregated cells into a single cell. Furthermore, the aggregated cells may reach a size exceeding 500 μm, and in this case, the aggregated cells may be clogged in the apparatus and measurement may not be possible.

  Therefore, it has been proposed to stir a solution containing a cell specimen in order to disperse aggregated cells (for example, see Patent Document 1). In the method described in Patent Literature 1, a solution containing cells is immersed in a stirring device having a hollow body provided with a filter at the tip, and the hollow body is rotated to stir the solution containing cells. Thus, shearing force is generated in the cell group to disperse the cells. Next, the air inside the hollow body is sucked in a state where the hollow body is immersed in the solution. The filter is formed with a plurality of pores (openings) having a size that allows a single cell to be measured to pass, but allows passage of red blood cells or cell debris having a size smaller than that. By suction, a solution containing red blood cells and cell debris passes through the filter and is sucked into the hollow body, and cells that cannot pass through the filter are adsorbed on the filter surface. Then, after discharging the solution sucked into the hollow body, the filter surface on which the cells are adsorbed is brought into contact with the slide glass, whereby the cells adsorbed on the filter surface are distributed on the slide glass.

Japanese Patent Laid-Open No. 4-232834

  However, in the method described in Patent Document 1, a shearing force is applied by stirring the hollow body in a state where aggregated cells and cells dispersed from the aggregated cells are mixed. That is, a shearing force acts on both the aggregated cells and the dispersed cells. For this reason, there was a risk of damaging the dispersed cells. In addition, when a shearing force that does not damage the dispersed cells is applied, there is a problem that the cell dispersion efficiency of the aggregated cells decreases.

  The present invention has been made in view of such circumstances, and provides a cell dispersion apparatus capable of increasing the dispersion efficiency of aggregated cells while suppressing damage to cells, and a sample pretreatment apparatus including the same. It is an object.

The cell dispersion device of the present invention is a device for dispersing the aggregated cells in a cell sample solution containing aggregated cells in which a plurality of cells are aggregated into a single cell,
A filter member configured to allow passage of the single cell;
A dispersion member that is disposed at a predetermined interval from the filter member, and disperses aggregated cells in the interval;
And a drive source for driving the dispersion member or the filter member.

Moreover, the sample pretreatment apparatus of the present invention includes the cell dispersion apparatus,
An aspiration dispensing unit for aspirating and dispensing a cell sample solution containing cells separated by the cell dispersion device;
And a filtration unit for extracting predetermined cells from the cell sample solution dispensed by the suction dispensing unit.

  According to the cell dispersion device and the sample pretreatment device of the present invention, it is possible to increase the dispersion efficiency of the aggregated cells while suppressing damage to the cells.

It is a perspective view of the cell analyzer provided with the cell dispersion device concerning one embodiment of the present invention. It is a block diagram which shows the internal structure of a measuring device. It is a block diagram which shows the internal structure of a sample preparation apparatus. It is a block diagram which shows the internal structure of a data processor. It is a functional block diagram of the flow cytometer which comprises a main detection part. It is a side view which shows the optical system of a flow cytometer. It is a fluid circuit diagram of a preparation device part. It is a perspective explanatory view of one embodiment of a cell dispersion device of the present invention. It is a cross-sectional explanatory drawing of the front-end | tip part of the cell dispersion apparatus shown by FIG. It is a top view of a filter member. It is the perspective view which looked at the rotor which is a dispersion member from the upper direction. It is the perspective view which looked at the rotor which is a dispersion member from the downward direction. It is an expanded sectional view of a discrimination and substitution part. It is explanatory drawing which shows the filtration effect | action of a discrimination and substitution part. It is a flowchart which shows the process which each control part of a cell analyzer performs. It is a flowchart which shows the process which each control part of a cell analyzer performs. It is a flowchart which shows a discrimination and replacement process. (A) is a plane explanatory view of another example of a rotor, (b) is the perspective view. It is a perspective view of other examples of a distribution member.

Hereinafter, embodiments of a cell dispersion device and a sample pretreatment device of the present invention will be described in detail with reference to the accompanying drawings.
[Overall configuration of cell analyzer]
First, the overall configuration of a cell analyzer equipped with a cell dispersion device according to an embodiment of the present invention will be described, and then each device or each part constituting this cell analyzer will be described.

FIG. 1 is a perspective view of a cell analyzer 1 including a cell dispersion device according to an embodiment of the present invention. The cell analyzer 1 flows a measurement sample containing cells collected from a patient through a flow cell, irradiates the measurement sample flowing through the flow cell with laser light, and emits light (forward scattered light, side fluorescence, etc.) from the measurement sample. By detecting and analyzing the optical signal, it is used to determine whether a cancer cell is contained in the cell. More specifically, the cell analyzer 1 uses cervical epithelial cells as analysis targets and is used for screening cervical cancer.
As shown in FIG. 1, a cell analyzer 1 includes a measuring device 2 that performs optical measurement with a laser beam on a measurement sample, and a pretreatment such as washing and staining of a biological sample collected from a subject. And a sample preparation device 3 for producing a measurement sample to be supplied to the measurement device 2, and a data processing device 4 for analyzing a measurement result in the measurement device 2.

[Internal configuration of measuring device]
FIG. 2 is a block diagram showing an internal configuration of the measuring apparatus 2.
As shown in FIG. 2, the measurement apparatus 2 includes a main detection unit 6, a signal processing unit 7, a measurement control unit 8, and an I / O interface 9.
Among these, the main detection unit 6 detects the measurement target cell and the number and size of the nuclei thereof from the measurement sample. In this embodiment, the flow cytometer 10 shown in FIGS. 5 and 6 to be described later. Is adopted.

The signal processing unit 7 includes a signal processing circuit that performs signal processing necessary for the output signal from the main detection unit 6. The measurement control unit 8 includes a microprocessor 11 and a storage unit 12, and the storage unit 12 includes a ROM, a RAM, and the like.
The ROM of the storage unit 12 stores a control program for controlling the operation of the main detection unit 6 and the signal processing unit 7 and data necessary for executing the control program. The microprocessor 11 stores the control program. Can be loaded into RAM or executed directly from ROM.

  The microprocessor 11 of the measurement control unit 8 is connected to the data processing device 4 and the microprocessor 19 of the adjustment control unit 16 to be described later via the I / O interface 9. Data necessary for processing can be transmitted and received between the data processing device 4 and the microprocessor 19 of the adjustment processing unit 16.

[Internal configuration of sample preparation device]
FIG. 3 is a block diagram showing the internal configuration of the sample preparation device 3.
As shown in FIG. 3, the sample preparation device 3 automatically performs sub-detection unit 14, signal processing unit 15, preparation control unit 16, I / O interface 17, and component adjustment for a biological sample. And a preparation device unit 18.

Among these, the sub-detection unit 14 detects the number of cells to be measured included in the biological sample. In the present embodiment, the sub-detection unit 14 is also shown in FIGS. 5 and 6. The same flow cytometer 10 is employed.
The signal processing unit 15 includes a signal processing circuit that performs signal processing necessary for the output signal from the sub-detection unit 14. The preparation control unit 16 includes a microprocessor 19, a storage unit 20, a sensor driver 21, and a drive unit driver 22. The storage unit 20 includes a ROM, a RAM, and the like.

  The preparation device unit (sample pretreatment device) 18 in the present embodiment includes a sample setting unit 24, a cell dispersion unit 25 as a cell dispersion device, a sample pipette unit 26 as an aspiration dispensing unit, and a sample quantification unit 27. And a reagent determination unit 28 and a discrimination / substitution unit 29 as a filtration unit.

  Among these, the specimen setting unit 24 is for setting a plurality of biological containers 53 and product containers 54 (see FIG. 7) that contain biological samples. The cell dispersion unit 25 forcibly disperses the aggregated cells contained in the biological sample in the biological container 53 into single cells, and details thereof will be described later.

  In addition, the specimen pipette unit 26 takes out a biological sample in which cells are dispersed from the biological container 53 and introduces the biological sample into the fluid circuit of the preparation device unit 18, or returns the prepared product to the product container 54. The specimen quantification unit 27 quantifies the biological sample supplied to the fluid circuit.

  Furthermore, the reagent quantification unit 28 quantifies a reagent such as a staining solution added to the biological sample, and the discrimination / substitution unit 29 mixes and replaces the biological sample and the diluent, and also measures the measurement target cell and the other cells. It is for discriminating cells (red blood cells, white blood cells, etc.) and bacteria. In addition, the structure (FIG. 7) of the fluid circuit of the preparation device part 18 which has the said each parts 24-29 is mentioned later.

  The ROM of the storage unit 20 stores a control program for controlling the operation of the sub-detection unit 14, the signal processing unit 15, the sensor driver 21, and the drive unit driver 22, and data necessary for executing the control program. The microprocessor 19 can load the control program into the RAM or execute it directly from the ROM.

  The microprocessor 19 of the preparation control unit 16 is connected to the microprocessor 11 of the measurement control unit 8 via the I / O interface 17, thereby allowing the data processed by itself and the data necessary for the processing to be processed. Data can be transmitted to and received from the microprocessor 11 of the measurement processing unit 8.

  Further, the microprocessor 19 of the preparation control unit 16 is connected to the sensors of the units 24 to 29 of the preparation device unit 18 and the drive motor constituting the drive unit via the sensor driver 21 and the drive unit driver 22. The control program is executed based on the detection signal from the control unit to control the operation of the drive unit.

[Internal configuration of data processor]
FIG. 4 is a block diagram showing an internal configuration of the data processing device 4.
As shown in FIG. 4, the data processing device 4 according to the present embodiment includes a personal computer such as a notebook PC (may be a desktop type), and includes a processing main body 31, a display unit 32, and an input unit 33. It is mainly composed.

The processing body 31 includes a CPU 34, a ROM 35, a RAM 36, a hard disk 37, a reading device 38, an input / output interface 39, and an image output interface 40. These units are communicably connected via an internal bus. Has been.
The CPU 34 can execute a computer program stored in the ROM 35 or a computer program loaded in the RAM 36.

The ROM 35 is configured by a mask ROM, PROM, EPROM, EEPROM, or the like, and stores a computer program executed by the CPU 34 and data used for the computer program.
The RAM 36 is composed of SRAM, DRAM, or the like, and is used as a work area for the CPU 34 when reading various computer programs recorded in the ROM 35 and the hard disk 37 and executing these computer programs.

The hard disk 37 is installed with various computer programs to be executed by the CPU 34 such as an operating system and application programs, and data used for executing the programs.
The hard disk 37 is installed with an operating system that provides a graphical user interface environment such as Windows (registered trademark) manufactured and sold by Microsoft Corporation.

  Further, the hard disk 37 has an operation program for transmitting operation commands to the measurement control unit 8 and the preparation control unit 16, receiving and analyzing the measurement results performed by the measurement apparatus 2, and displaying the processed analysis results. 41 is installed. The operation program 41 operates on the operating system.

The reading device 38 is configured by a flexible disk drive, a CD-ROM drive, a DVD-ROM drive, or the like, and can read a computer program or data recorded on a portable recording medium.
The input / output interface 39 includes, for example, a serial interface such as USB, IEEE 1394, and RS-232C, a parallel interface such as SCSI, IDE, and IEEE1284, and an analog interface including a D / A converter and an A / D converter. Has been.

An input unit 33 including a keyboard and a mouse is connected to the input / output interface 39, and data can be input to the computer by a user operating it.
Further, the input / output interface 39 is also connected to the I / O interface 9 of the measuring device 2, whereby data can be transmitted and received between the measuring device 2 and the data processing device 4.
The image output interface 40 is connected to the display unit 32 such as an LCD or a CRT, and causes the display unit 32 to output a video signal corresponding to the image data from the CPU 34.

[Configuration of main detector (flow cytometer)]
FIG. 5 is a functional block diagram of the flow cytometer 10 constituting the main detection unit 6, and FIG. 6 is a side view showing an optical system of the flow cytometer 10.
As shown in FIG. 5, the lens system 43 of the flow cytometer 10 condenses the laser light from the semiconductor laser 44 that is a light source onto the measurement sample that flows through the flow cell 45. The forward scattered light of the cells in the measurement sample is collected on a scattered light detector composed of a photodiode 47.

In FIG. 5, for ease of understanding, the lens system 43 is illustrated as a single lens, but the lens system 43 is specifically configured as shown in FIG. 6, for example.
In other words, the lens system 43 in the present embodiment includes, in order from the semiconductor laser 44 side (left side in FIG. 6), a collimator lens 43a, a cylinder lens system (plano-convex cylinder lens 43b + biconcave cylinder lens 43c), and a condenser lens system (condenser). Lens 43d + condenser lens 43e).

  Returning to FIG. 5, the side condensing lens 48 condenses the side scattered light and the side fluorescence of the measurement target cell or the nucleus in the cell on the dichroic mirror 49. The mirror 49 reflects side scattered light to the photomultiplier 50 that is a scattered light detector, and transmits side fluorescent light toward the photomultiplier 51 that is a fluorescence detector. These lights reflect the characteristics of cells and nuclei in the measurement sample.

The photodiode 47 and each of the photomultipliers 50 and 51 convert the received optical signal into an electrical signal, and forward scattered light signal (FSC), side scattered light signal (SSC), and side fluorescent signal, respectively. (SFL) is output. These output signals are amplified by a preamplifier (not shown) and sent to the signal processing unit 7 (see FIG. 2) of the measuring apparatus 2.
Each signal FSC, SSC, SFL processed by the signal processing unit 7 of the measuring device 2 is transmitted from the I / O interface 9 to the data processing device 4 by the microprocessor 11.

  The CPU 34 of the data processing device 4 creates a scattergram for analyzing cells and nuclei from the signals FSC, SSC, SFL by executing the operation program 41, and in the measurement sample based on the scattergram. It is determined whether or not these cells are abnormal, specifically, whether or not they are cancerous cells.

  As the light source of the flow cytometer 10, a gas laser can be used instead of the semiconductor laser 44. However, it is preferable to employ the semiconductor laser 44 because of its low cost, small size, and low power consumption. The adoption of the laser 44 can reduce the product cost and reduce the size and power consumption of the apparatus.

  In this embodiment, a blue semiconductor laser having a short wavelength that is advantageous for narrowing the beam is used. Blue semiconductor lasers are also effective for fluorescence excitation wavelengths such as PI. Of the semiconductor lasers, a red semiconductor laser that has a low cost and a long life and is stably supplied from the manufacturer may be used.

By the way, the average size of epithelial cells in the cervix is about 60 μm, and the size of the nucleus is 5 to 7 μm. When this cell becomes cancerous, the frequency of cell division increases abnormally, and the nucleus becomes 10 to 15 μm in size. This increases the N / C ratio (nucleus size / cell size) compared to normal cells.
Therefore, by detecting the size of the cells and nuclei, it can be used as an index for determining whether or not the cells have become cancerous.

Therefore, in the present embodiment, scattered light from the measurement sample flowing through the flow cell 45 is detected by the photodiode 47, and fluorescence from the measurement sample flowing through the flow cell 45 is detected by the photomultiplier 51.
The signal processing unit 7 of the measurement device 2 acquires the pulse width of the scattered light signal, which is a value reflecting the size of the measurement target cell, from the scattered light signal output from the photodiode 47, and from the photomultiplier 51. From the output fluorescence signal, the pulse width of the fluorescence signal, which is a value reflecting the size of the nucleus of the measurement target cell, is acquired.

Then, based on the value reflecting the size of the measurement target cell obtained by the signal processing unit 7 and the value reflecting the size of the nucleus of the measurement target cell, whether or not the measurement target cell is abnormal is determined. Is determined by the CPU 34 of the data processing device 4 constituting the analysis unit.
Specifically, the CPU 34 of the data processing device 4 determines that the measurement target cell is abnormal when the value obtained by dividing the pulse width of the fluorescence signal by the pulse width of the scattered light signal is larger than a predetermined threshold.

[Configuration of sub-detection unit]
The sub-detection unit 14 of the sample preparation device 3 detects the number of cells to be measured from the biological sample in the pre-processing step of preparing the sample. In this embodiment, the sub-detection unit 14 is illustrated in FIGS. 5 and 6. The flow cytometer 10 having the same configuration as that of the first embodiment is employed.
However, since the sample preparation device 3 in the present embodiment preliminarily measures the concentration of the measurement target cell before the main measurement by the measurement device 2, the sub-detection unit 14 counts the number of cells. It is enough to output a signal for this purpose.

For this reason, in the case of the flow cytometer 10 constituting the sub-detecting unit 14, it is sufficient if the forward scattered light signal (FSC) can be acquired. Therefore, the side scattered light signal (SSC) and the side fluorescent signal (SFL) are acquired. There is no photomultiplier 50, 51 for the purpose, and only a photodiode 47 for obtaining the FSC is provided.
The optical signal received by the photodiode 47 of the sub-detection unit 14 is converted into an electric signal, amplified, and sent to the signal processing unit 15 (see FIG. 3) of the sample preparation device 3.

The signal FSC processed by the signal processing unit 15 of the sample preparation device 3 is sent to the preparation control unit 16. The microprocessor 19 of the preparation control unit 16 counts the number of cells to be measured based on the signal FSC.
Further, the microprocessor 19 obtains the volume of the biological sample preliminarily collected by the sample pipette unit 26 for concentration measurement from a flow sensor (not shown) provided in the pipette unit 26. The concentration of the biological sample is calculated by dividing the number of cells obtained from the FSC by its volume.

However, it is not always necessary to calculate the concentration of the biological sample itself. When the collected amount of the biological sample is always kept constant, the number of cells itself becomes information reflecting the concentration. That is, the concentration information generated by the microprocessor 19 of the preparation control unit 16 may include other concentrations substantially equivalent to this concentration as well as the concentration of the biological sample.
Then, the microprocessor 19 of the preparation control unit 16 controls a preparation operation (an operation such as quantification of the biological sample or reagent) performed by the preparation device unit 18 on the biological sample based on the concentration information generated by itself. A control command is issued to The specific contents of this control will be described later.

[Fluid circuit of the preparation device section]
FIG. 7 is a fluid circuit diagram of the preparation device unit 18.
As shown in FIG. 7, the specimen setting unit 24 includes a circular rotary table 24A and a drive unit 24B that rotationally drives the rotary table 24A, and a biological container that contains a biological sample at the outer peripheral edge of the rotary table 24A. 53 and a holding part capable of setting a product container (microtube) 54 for storing a product after preparing a biological sample is provided.

  The cell dispersion unit 25 includes a filter 101 that is a filter member, a rotor 102 that is a dispersion member disposed at a predetermined interval from the filter 101, and a motor 103 that is a drive source that rotationally drives the rotor 102. The rotor 102 is rotationally driven via a rotating shaft 104 connected to an output shaft (not shown) of the motor 103. A pipe 105, which is a guide cylinder, is provided outside the filter 101 and the rotor 102. By inserting these pipes 105 into the biological container 53 and rotating the rotor 102, the biological sample in the biological container 53 is obtained. By stirring, the aggregated cells contained in the biological sample are dispersed into single cells. Details including the specific configuration of the cell dispersion unit 25 will be described later.

  The sample pipette unit 26 sucks the biological sample in the biological container 53 and the measurement sample in the product container 54 and supplies the sample to the sample quantitative unit 27 and the preparation device unit 18. And a second pipette 26 </ b> B for returning the product after the predetermined treatment to the product container 54.

The second pipette 26B is connected to a container 57 of the discrimination / replacement unit 29, which will be described later, through a conduit, and the liquid in which the measurement target cells are discriminated by the discrimination / replacement unit 29 is produced by the second pipette unit 26B. It can be returned to the container 54.
The sample quantification unit 27 includes a quantification cylinder 27A and a drive unit 27B that moves the quantification piston inserted through the cylinder 27A up and down. The metering cylinder 27A is connected to the first pipette 26A through a pipe via a direction switching valve V1.

The biological sample sucked from the biological container 53 by the first pipette 26A is introduced into the quantitative cylinder 27A through the direction switching valve V1. The introduced biological sample is sent to the next discrimination / substitution unit 29 through the direction switching valve V1 by the movement of the fixed amount piston by the drive unit 27B.
In the present embodiment, the quantification cylinder 27A of the sample quantification unit 27 is also connected to a dilution liquid unit 55 that prepares a dilution liquid for the biological sample by a pipe line.

The reagent quantification unit 28 includes a pair of quantification cylinders 28A and 28B, and a drive unit 28C that vertically moves the quantification pistons inserted through the cylinders 28A and 28B. Each fixed quantity cylinder 28A, 28B is connected with the 2nd pipette 26B by the pipe line via supply switching valve V2, V3, respectively.
The reagent in the reagent container is supplied to each quantitative cylinder 28A, 28B. The supplied reagent is quantified by a predetermined amount by the movement of the quantification piston by the drive unit 28C, and the quantified reagent is sent to the second pipette 26B through the supply switching valves V2 and V3.

  Therefore, a plurality of types of predetermined amounts of reagent determined by the reagent quantitative unit 28 can be mixed with the discriminated sample returned to the product container 54 of the sample setting unit 24.

  In the present embodiment, there are two types of reagents to be quantified by the quantification cylinders 28A and 28B of the reagent quantification unit 28. Among these, the reagent weighed with one quantitative cylinder 28A and added to the biological sample is a dye solution for performing PI staining, and the reagent weighed with the other quantitative cylinder 28B and added to the biological sample is treated with RNA. RNase for performing PI staining is performed with propidium iodide (PI), which is a fluorescent staining solution containing a dye. In PI staining, the nucleus is selectively stained, so that fluorescence from the nucleus can be detected. The RNA treatment is a treatment for dissolving RNA in cells. Since the dye solution stains both RNA and DNA of the epithelial cells, the RNA treatment dissolves the RNA and does not stain with the dye solution, so that the DNA of the cell nucleus can be accurately measured.

  The discriminating / replacement unit 29 includes an upper-opening storage container 57, a filtration cylinder 58 inserted through the storage container 57 so as to be movable up and down, and a drive unit 59 that moves the filtration cylinder 58 up and down in the storage container 57. It has.

The container 57 is connected to the quantitative cylinder 27A of the sample quantitative unit 27 through a direction switching valve V1, V7, V4. For this reason, the biological sample quantified by the specimen quantification unit 27 is supplied to the storage container 57 through the direction switching valves V1, V7, and V4, and can be temporarily held in the container 57.
The container 57 is connected to the second pipette 26B via a direction switching valve V4 through a conduit, and the biological sample in the container 57 that has been discriminated is sent from the direction switching valve V4 to the second pipette 26B. It is done.

Furthermore, the flow cell 45 of the sub-detecting unit 14 is interposed in the pipe line portion between the direction switching valve V4 and the second pipette 26B. For this reason, the sub-detection unit 14 can count the number of cells with respect to the distinguished biological sample discharged from the storage container 57.
The filtration cylinder 58 is formed of a hollow cylinder having a filter 60 at a lower portion thereof that does not allow measurement target cells (epithelial cells) to pass therethrough and allows cells having smaller diameters (red blood cells, white blood cells, etc.) to pass therethrough. Is connected to the diluent unit 55 via a switching valve V5.

For this reason, the diluent of the diluent unit 55 can be supplied to the inside of the filtration cylinder 58 by opening the switching valve V5.
The drive unit 59 of the discrimination / substitution unit 29 moves the filtration cylinder 58 downward with respect to the storage container 57 containing the mixture of the biological sample and the diluent, and the filter 60 of the filtration cylinder 58 is placed in the storage container 57. Move downward in the mixed liquid. As a result, the liquid containing only the measurement target cells remains as a residual liquid below the filter 60, and the liquid containing other cells and impurities remains as a filtrate above the filter 60 (inside the filtration cylinder 58).

Further, the filtration cylinder 58 is connected to the filtrate disposal unit 61 via a switching valve V6 through a pipe line. For this reason, the filtrate filtered by the lowering of the filtration cylinder 58 is discarded outside through the switching valve V6.
On the other hand, the residual liquid in the filtration cylinder 58 is sent to the flow cell 45 of the sub-detecting section 14 through the direction switching valve V4 after the filtrate is discarded, and then sent to the second pipette 26B of the sample pipette section 26.

  In addition, when the residual liquid after filtration is for measuring the concentration of a biological sample, it is discharged from the second pipette 26B, and when the residual liquid is for preparing a measurement sample, the second pipette 26B is returned to the product container 54.

As shown in FIG. 7, the quantification cylinder 27A of the sample quantification unit 27 is also connected to the main detection unit 6 of the measurement apparatus 2 via a direction switching valve V1, V7.
The measurement sample in the product container 54 is quantified by the sample quantification unit 27 via the first pipette 26A and supplied to the main detection unit 6 of the measurement device 2 through the valves V1 and V7. Yes.
In addition, the operation control with respect to the drive parts and switching valves (electromagnetic valves) V1 to V7 shown in FIG. 7 is performed based on a control command from the preparation control part 16 (microprocessor 19). The pretreatment process performed by the preparation control unit 16 will be described later.

[Specific configuration of cell dispersion unit]
FIG. 8 is a perspective explanatory view showing a more specific configuration of the cell dispersion portion 25, and FIG. 9 is a cross-sectional explanatory view of the tip portion of the cell dispersion portion 25.
As shown in FIGS. 8 to 9, the cell dispersion unit 25 in the present embodiment includes a filter 101, a rotor 102 disposed at a predetermined interval from the filter 101, and the rotor 102 being driven to rotate. A motor 103 is provided, and a pipe 105 having a function of guiding the flow of the biological sample is disposed outside the filter 101 and the rotor 102.

  The filter 101 is made of stainless steel in consideration of chemical resistance, strength, etc., and is made of a circular film-like body having a thickness of about 100 μm as shown in FIG. The filter 101 is provided with a large number of holes 101A having a size through which a single cell can pass, except for the peripheral edge 101B. Since the measurement target cells in the present embodiment are cervical epithelial cells (usually about 20 to 80 μm, most of which are about 60 μm), the pore diameter of the hole 101A of the filter 101 is small. Is 100 μm, and the aperture ratio is 40%. The filter 101 is fixed to a predetermined location on the inner peripheral surface of the pipe 105 by laser spot welding.

If the hole diameter of the hole 101A of the filter 101 is set to 80 to 120 μm, if it is less than 80 μm, the single cell to be measured cannot pass through the filter 101, and the single cell may be damaged by shearing force. When the thickness exceeds 120 μm, the aggregated cells may pass through the filter 101, and the cell dispersion efficiency of the aggregated cells may be lowered. The hole diameter of the hole 101A is more preferably around 100 μm.

  Similarly to the filter 101, the rotor 102 is made of stainless steel in consideration of chemical resistance and strength. As shown in FIGS. 11 to 12, a short cylinder having a diameter of about 7 mm and a thickness (height) of about 5 mm. It consists of a body. A hole 102 </ b> A is formed at the center of the rotor 102, and the other end of the rotating shaft 104 whose one end is connected to the output shaft 103 </ b> A of the motor 103 is press-fitted into the hole 102 </ b> A. Further, around the hole portion 102A, four flow paths 102D penetrating from the upper surface 102B (the upper surface in FIGS. 8 to 9) of the rotor 102 to the lower surface 102C are formed at equal intervals in the circumferential direction. The hole diameter of the flow path composed of the through holes is about 2 mm.

  Fins 106 are provided on the upper surface 102B of the rotor 102 so as to correspond to the respective flow paths 102D. Each of the fins 106 is an approximately “he” -shaped member obtained by bending a tongue-shaped plate in the middle, and its short side portion 106A is press-fitted into a groove 107 formed on the upper surface 102B of the rotor 102. ing. The groove 107 is formed radially outward with the hole 102A as the center. The long side portion 106B of the fin 106 forms a surface inclined with respect to the upper surface 102B of the rotor 102, and a part of the long side portion 106B desires an opening portion of the flow channel 102D, or an opening portion of the flow channel 102D. The position of the groove is set so as to cover. Thereby, when the rotor 102 rotates, the biological sample that hits the lower surface 106B1 of the long side portion 106B of the fin 106 is introduced into the flow path 102D, and the flow of the biological sample is formed. . The corner portion 106B2 of the long side portion 106B is subjected to curved surface processing or chamfering processing in order to reduce damage to cells.

  On the lower surface 102 </ b> C of the rotor 102, a convex portion 108 formed of a cross-shaped protruding portion 108 </ b> A is formed. Each flow path 102D opens between adjacent protrusions 108A. The protruding height of the protruding portion 108A varies depending on the size of the aggregated cells to be dispersed, but is usually in the range of 100 to 300 μm. By forming the convex portion 108, the aggregated cells can be reliably dispersed. Further, although not shown, the corner portion of the protruding portion 108A is subjected to curved surface processing or chamfering processing in order to reduce damage to the cells, similarly to the corner portion 106B2 of the long side portion 106B.

  An interval (clearance) CL1 of 30 to 80 μm is provided between the tip end surface 108A1 (the lower surface in FIGS. 8 to 9) of the protrusion 108A formed on the lower surface 102C of the rotor 102 and the upper surface of the filter 101. . By providing this distance CL1, shearing force or dispersion force can be effectively applied to the aggregated cells. That is, the size of the cervical epithelial cells to be measured in this embodiment is 60 μm on average, and when this aggregates, the size exceeds 120 μm. Moreover, the hole diameter of the filter 101 in this Embodiment is 100 micrometers. In the case of a single epithelial cell, it can pass through the hole 101A of the filter 101, but the aggregated cell cannot pass through and is captured by the hole 101A of the filter 101. At this time, if the interval CL1 is smaller than the size of the aggregated cells, the aggregated cells captured in the holes 101A are surely given a shearing force or a dispersion force to reliably disperse the aggregated cells. Can be made.

If the distance CL1 is set to 30 to 80 μm, the cell may be crushed if it is less than 30 μm, and if it exceeds 80 μm, the dispersion force applied to the aggregated cells may be reduced. The interval CL1 is more preferably around 50 μm.

  The motor 103 is fixed to the upper member 109A of the U-shaped support bracket 109, and its output shaft 103A projects downward from a hole (not shown) formed in the upper member 109A. One end of the rotating shaft 104 is connected to the output shaft 103 </ b> A, and the other end of the rotating shaft 104 is connected to the rotor 102. Thereby, the rotor 102 can be rotated by rotating the motor 103.

  The pipe 105 is a stainless steel circular pipe having an outer diameter of about 10 mm and a wall thickness of about 1.5 mm, and one end thereof is connected to a hollow body 110 provided on the lower surface of the lower member 109B of the support bracket 109. Has been. At the other end (end side end portion) of the pipe 105, a tapered portion 105A having a sectional area gradually decreasing toward the tip is formed. The hole diameter of the hole 105B at the tip of the tapered portion 105A is about 2.5 mm. The rotor 102 is disposed such that the outer peripheral surface 102E thereof is close to the inner peripheral surface 105D of the pipe 105, and the interval CL2 between the outer peripheral surface 102E and the inner peripheral surface 105D is set to about 100 μm.

  Two elongated openings 105C are formed in the pipe peripheral wall slightly above the position where the rotor 102 is disposed. The two openings 105C are formed at positions facing each other with the core of the pipe 105 as the center. As will be described later, the biological sample outside the pipe 105 is introduced into the pipe 105 through the opening 105C.

Next, the operation of the cell dispersion unit 25 having the above configuration will be described.
When the motor 103 is rotated with the pipe 105 immersed in the biological sample in the biological container 53, the rotor 102 connected to the output shaft 103A of the motor 103 via the rotation shaft 104 rotates. The rotational speed of the motor 103 is not particularly limited in the present invention, but is generally 2000 to 20000 rpm. The preferred number of rotations varies depending on the biological sample and the shape of the rotor 102, but if the number of rotations is too small, the aggregated cells cannot be sufficiently dispersed. On the other hand, if the number of rotations is too large, the cells may be crushed. is there. The rotation time of the motor 103 required for one processing operation (dispersion operation) varies depending on the type and amount of the biological sample, the number of rotations, and the like, but is usually 20 to 300 seconds. Further, it is preferable to periodically rotate the motor 103 in order to stir the aggregated cells stagnating in the corners or to release the aggregated cells entangled with the fins 106 and the like. Specifically, in this embodiment in which cervical epithelial cells are measured, for example, by repeating a cycle of 18 seconds for normal rotation and 2 seconds for inversion, the aggregated cells can be sufficiently dispersed. .

  When the rotor 102 rotates, the fin 106 provided integrally with the rotor 102 also rotates. As shown in FIG. 11, since the long side portion 106B of the fin 106 is inclined in the normal rotation direction of the rotor 102, the biological sample is introduced into the flow path 102D of the rotor 102 by the rotation of the fin 106. .

  The biological sample introduced into the channel 102D is ejected toward the filter 101 from the lower opening of the channel 102D. The ejected biological sample includes a single epithelial cell and an aggregated cell obtained by aggregating a plurality of single epithelial cells. Of these, the single cell passes through the hole 101 of the filter 101 and is piped. It is ejected to the outside of the pipe 105 from the hole 105B at the tip of 105. At this time, in this embodiment, since the tip portion of the pipe 105 is a tapered portion 105A whose diameter is reduced toward the tip, the biological sample that has passed through the filter 101 is accelerated by the taper portion 105A to be the tip of the pipe 105. Is ejected from the outside.

  On the other hand, the aggregated cells are trapped in the hole 101A of the filter 101 and cannot pass through the filter 101. Between the filter 101 and the rotor 102, the flow of the biological sample caused by the rotation of the rotor 102, and the rotor 102 By contact with the convex portion 108 formed on the lower surface of the substrate, a dispersion force is applied to disperse into single cells. Then, the dispersed single cells pass through the hole 101 of the filter 101 and are ejected to the outside of the pipe 105 from the hole 105B at the tip of the pipe 105.

  In the present embodiment, the flow from the rotor 102 toward the filter 101 is formed by the fins 106 provided integrally with the rotor 102. Thereby, single cells dispersed from the aggregated cells between the rotor 102 and the filter 101 can be discharged more efficiently below the filter 101.

  Further, in the present embodiment, a pipe 105 having an opening in the peripheral wall is disposed outside the rotor 102 and the filter 101 to form a closed space between the rotor 102 and the filter 101. . For this reason, the flow of the biological sample formed by the fin 106 includes the flow path 102D of the rotor 102, the filter 101, the hole 105B at the tip of the pipe 105, the outside of the pipe 105, the opening 105C of the pipe 105, and the flow of the rotor 102. By forming a circulation flow returning to the path 102D and forming such a flow of the biological sample, aggregated cells in the biological sample in the biological container 53 can be efficiently dispersed.

The cells collected from the cervix of the subject were dispersed using the cell dispersion part 25 described above, and the dispersion effect was confirmed.
As a dye for staining cells, 0.5% trypan blue staining solution (product code: 29853-34) manufactured by Nacalai Tesque, Inc. was used, and this staining solution was diluted with PBS solution (PBS solution: 0.5%). -Trypan blue stain = 1: 4).

  A mixture of 0.3 ml of a sample (PreservCyt (CYTYC) stock specimen) and 0.3 ml of the diluted trypan blue staining solution was subjected to a dispersion treatment for 60 seconds using the cell dispersion unit 25 described above. The number of revolutions of the motor was 10,000 rpm, and the cycle was repeated 3 cycles, with 18 seconds for forward rotation and 2 seconds for reverse rotation as one cycle.

After the dispersion treatment, 300 μl of a sample was added to MPC-200 (trade name: plankton calculator manufactured by Matsunami Glass Industrial Co., Ltd.) and left at room temperature for 10 minutes, and then 200 masses (corresponding to 0.05 ml) under a microscope. The number of cells in the cell (those classified as single cells) were counted.
The above operation was repeated three times (Samples 1 to 3). Moreover, the number of single cells was similarly counted about the sample which did not perform a dispersion | distribution process. The results are shown in Table 1.

  As can be seen from Table 1, the number of single cells is increased by performing the dispersion treatment.

[Specific configuration of discrimination / replacement unit]
FIG. 13 is an enlarged cross-sectional view showing a more specific configuration of the discrimination / substitution unit 29, and FIG. 14 is an explanatory diagram showing its filtering action.
As shown in FIG. 13, the discrimination / substitution unit 29 in the present embodiment does not allow the container 57 that can contain the liquid L containing the biological sample and the first cell C <b> 1 in the biological sample to pass through the first container C. And a filter 60 that allows the second cells C2 having a smaller diameter than the cells C1 to pass therethrough.

Further, the discrimination / substitution unit 29 separates the liquid L into the first liquid L1 containing the first cells C1 and the second liquid L2 containing the second cells C2 by allowing the liquid L to pass through the filter 60. A filtration cylinder 58 that functions as a liquid separator is provided.
The container 57 integrally includes a hollow body portion 57A having an axial center that is directed in the vertical direction, and a bottom portion 57B that is connected to a lower portion of the body portion 57A and has an inner surface with a central portion recessed downward. ing.

On the other hand, the filtration cylinder 58 is a hollow cylindrical cylinder 63 that is inserted into the storage container 57 via a seal member 62 so as to be movable up and down, and the filter 60 provided so as to close the lower end opening of the cylinder 63. And.
In the present embodiment, cervical epithelial cells are assumed as the first cells C1, and the size of the epithelial cells is approximately 20 to 80 μm (the average is about 60 μm). The size of red blood cells, which are the second cells C2, smaller than the first cells C1, is approximately 7 to 10 μm, and the size of white blood cells, which are also the second cells C2, is approximately 8 to 15 μm. Furthermore, the size of contaminants such as bacteria that are the second cells C2 is approximately 1 to several μm.

  Therefore, in the present embodiment, a metal CVD (Chemical Vapor Deposition) filter having a through hole with a diameter of 10 to 20 μm is employed as the filter 60. Such a metal CVD filter has the advantage that even if it is made of a resin or a metal filter, the deformation of the through-hole is less than that of a mesh filter and the aperture ratio can be increased.

  In addition, the pore size of the filter 60 is set to 10 to 20 μm, and if it is less than 10 μm, there are many phenomena that cells and contaminants are clogged early in the through-hole, and if it exceeds 20 μm, epithelial cells pass through. This is because there are many things. The pore diameter of the filter 60 is more preferably around 15 μm.

  Connected to the bottom 57B of the container 57 is a residual liquid pipe 64 for discharging the quantified biological sample and discharging and collecting the residual liquid L1 after filtration to the outside. The direction switching valve V4 is provided. Therefore, the remaining liquid pipe 64 and the direction switching valve V4 constitute a liquid acquisition unit for acquiring the first liquid L1 separated by the filtration cylinder 58 which is a liquid separation unit.

A dilution liquid pipe 65 communicating with the dilution liquid unit 55 is connected to the upper wall of the cylinder 63, and the switching valve V <b> 5 is provided in the middle of the pipe 65.
Further, a filtrate pipe line 66 leading to the waste part 61 for discarding the filtrate L2 after filtration to the outside is connected to the upper wall part of the cylinder 63, and the switching valve V6 is provided in the middle part of the pipe line 66. Is provided. Therefore, the filtrate pipe line 66 and the switching valve V6 constitute a liquid discharge part for discharging the second liquid C2 separated by the filtration cylinder 58, which is a liquid separation part, to the outside.

Connected to the upper end portion of the cylinder 63 is a moving mechanism portion 67 that converts the rotational movement of the driving portion 59 made of a motor or the like into vertical movement of the cylinder 63.
The drive unit 59 of the moving mechanism unit 67 drives the filtration cylinder 58 in accordance with a control command from the preparation control unit 16 (microprocessor 19).
For example, as shown in FIG. 9A, the preparation control unit 16 moves the filter 60 from above the liquid level of the liquid L (mixed liquid of biological sample and diluent) in the container 57 toward the liquid. The filtration cylinder 58 is lowered so as to move downward.

Then, as shown in FIG. 9B, a liquid in which most of the cells contained therein are the first cells (epithelial cells) C1 remains as a residual liquid L1 below the filter 60 in the container 57, The liquid containing other second cells C2 (contaminants such as red blood cells, white blood cells, and bacteria) remains above the filter 60 (inside the filtration cylinder 58) as the filtrate L2.
Therefore, the preparation control unit 16 controls the drive unit 59 of the moving mechanism unit 65 so that the filter 60 that has moved into the liquid L returns upward by a predetermined distance.

Specifically, the preparation control unit 16 lowers the filter cylinder 58 until the filter 60 reaches the lower limit set at or near the bottom 57B, and then moves the filter 60 so that the filter 60 returns upward by a predetermined distance. By controlling the portion 67 and returning upward, the first cells C1 attached to the lower surface of the filter 60 can be detached from the filter 60.
Then, the preparation control unit 16 first opens the switching valve V6 to discharge the filtrate L2 to the outside prior to the residual liquid L1, as shown in FIG. 9C, and thereafter, the residual liquid L1 In order to acquire the direction switch valve V4.

[Contents of analysis processing]
15 and 16 are flowcharts showing processing performed by the control units 8, 16, and 31 of the cell analyzer 1.
In FIG. 15, the processing flow performed by the control unit (processing body) 31 of the data processing device 4 is shown in the right column, and the processing flow performed by the control unit 8 of the measuring device 2 is shown in the left column. Further, in FIG. 16, the processing flow performed by the control unit 16 of the sample preparation device 3 is shown in a row, but this processing flow is connected to the processing flow of FIG. 15 at points A, B, and C shown in the figure. Hereinafter, the processing content performed by the cell analyzer 1 will be described with reference to FIGS. 15 and 16.

First, the control unit 31 of the data processing device 4 displays a menu screen on the display unit 32 (step S1). Thereafter, when a measurement start instruction according to the menu screen is received from the input unit 33 (step S2), the control unit 31 of the data processing device 4 transmits a measurement start signal to the measurement device 2 (step S3).
When receiving the measurement start signal (step S4), the control unit 8 of the measurement device 2 transmits a preparation start signal to the sample preparation device 3 (step S5 and point A).

When receiving the preparation start signal (step S6), the control unit 16 of the sample preparation device 3 sucks a reagent (staining solution, RNase) used for preparation of the measurement sample into the flow path in the device, Then, the biological sample in the biological container 53 containing the alcohol main component storage solution is dispersed by the cell dispersion unit 25 (steps S7 and S8). The biological sample is dispersed in the cell dispersion unit 25 according to the embodiment described with reference to FIGS. That is, when the motor 103 is driven to rotate by the control unit 16, the rotor 102 and the fin 106 provided integrally with the rotor 102 are rotated by the rotation of the motor 103. The biological sample is introduced into the channel 102D of the rotor 102 by the rotation of the fin 106, and is ejected toward the filter 101 from the lower opening of the channel 102D. The single cells in the ejected biological sample pass through the hole 101 of the filter 101 and are ejected from the hole 105B at the tip of the pipe 105 to the outside of the pipe 105. On the other hand, the aggregated cells are trapped in the hole 101A of the filter 101 and cannot pass through the filter 101. Between the filter 101 and the rotor 102, the flow of the biological sample caused by the rotation of the rotor 102, and the rotor 102 By contact with the convex portion 108 formed on the lower surface of the substrate, a dispersion force is applied to disperse into single cells. Then, the dispersed single cells pass through the hole 101 of the filter 101 and are ejected to the outside of the pipe 105 from the hole 105B at the tip of the pipe 105. By performing the above operation for a predetermined time, the cell dispersion unit 25 disperses the aggregated cells in the biological sample into single cells.
Thereafter, the control unit 16 of the sample preparation device 3 sucks the dispersed biological sample from the biological container 53 into the flow path in the device by a predetermined amount (step S9) and sends it to the storage container 57 of the discrimination / substitution unit 29. Then, the discrimination / substitution unit 29 is caused to perform discrimination / substitution processing on the biological sample (step S10).

[Details of discrimination / replacement processing]
FIG. 17 is a flowchart showing the discrimination / substitution process.
As shown in FIG. 17, first, the control unit 16 of the sample preparation device 3 mixes the diluent in the container 57 containing the biological sample (step T1), and lowers the cylinder 63 to its lower limit position ( Step T2).

By the lowering of the cylinder 63, as described above, the liquid L in the container 57 is discriminated into a predetermined amount of residual liquid L1 containing the first cell C1 to be measured and filtrate L2 containing other cells C2. Is done.
Thereafter, when the lower limit position of the cylinder 63 is reached, the control unit 16 of the sample preparation device 3 raises the cylinder 63 by a predetermined distance (step T3), thereby the first cells attached to the lower surface of the filter 60. (Epithelial cells) C1 is suspended in the concentrate L1 below the filter 60.

Therefore, the ascending movement of the cylinder 63 in this process (step T3) may be a speed and stroke that can physically remove the first cell C1 from the filter 60, and the number of movements may be multiple. .
After the processing, the control unit 16 of the sample preparation device 3 discharges the filtrate L2 above the filter 60 to the outside (step T4), and then raises the cylinder 63 to the initial position (step T5). It is determined whether or not the descent is the second time (step T6).

If the lowering of the cylinder 63 is not the second time, the control unit 16 of the sample preparation device 3 repeats the filtration again from the mixing of the diluent, and if this is the second time, the processing routine of the discrimination / substitution process is ended. To do. This discrimination / replacement operation is not limited to two times, and may be performed more than twice.
Returning to FIG. 16, when the discrimination / replacement process (step S <b> 10) is completed, the control unit 16 of the sample preparation device 3 adds the concentrated liquid (residual liquid L <b> 1 that is mostly epithelial cells) to the flow cell 45 of the sub-detection unit 14. (Step S11), and using this sub-detecting unit 14, pre-measurement of the concentrated liquid L1 is performed by the flow cytometry method (Step S12).

  This pre-measurement is for obtaining concentration information reflecting the concentration of the measurement target cell (epithelial cell) C1 contained in the biological sample before the main measurement performed by the measurement device 2 for cancer determination. This is performed by detecting the number of cells C1 contained in the concentrate L1.

[Use of concentration information]
Next, the control unit 16 of the sample preparation device 3 discharges the concentrate L1 out of the fluid circuit (step S13), calculates the concentration of the biological sample (step S14), and based on this concentration information, A sample suction amount of the biological sample for preparing a measurement sample for measurement is determined (step S15).

For example, when the amount of suction of the biological sample used for the pre-measurement is 200 μl and the number of cells C1 found from the result of the pre-measurement is 10,000, the concentration of the biological sample is 10,000 / 200 μl = 50 ( Pieces / μl) = 50000 (pieces / ml).
On the other hand, if the number of significant cells necessary for detection of cancer cells in this measurement is, for example, 100,000, collection of a biological sample necessary for performing this measurement to such an extent that this number of significant cells is ensured. The fee can be calculated as 100,000 / 50000 (pieces / ml) = 2 ml.

Therefore, the control unit 16 of the sample preparation device 3 sucks the biological sample from the biological container 53 by the necessary amount (step S16), and performs the discrimination / substitution process described above again on the biological sample (step S16). S17).
As described above, the control unit 16 of the sample preparation device 3 determines and determines the amount of the biological sample for preparation of the measurement sample used for the main measurement based on the concentration information of the cell C1 generated by itself. The preparation device unit 18 of the sample preparation apparatus 3 is controlled so as to acquire an amount of biological sample.

In this case, the control unit 16 of the sample preparation device 3 determines a larger amount of the biological sample used for preparing the measurement sample as the concentration of the cell C1 in the biological sample is lower, and the concentration of the cell C1 in the biological sample. The higher the value, the smaller the amount of biological sample used to prepare the measurement sample.
In the control unit 16 of the sample preparation device 3, the number of cells C1 detected by the main detection unit 34 is not limited to the 100,000 cells that are the number of significant cells for cancer detection. The amount of the biological sample used for the preparation of the measurement sample may be determined so that it falls within a predetermined range such as 100,000 to 150,000.

Further, the control unit 16 of the sample preparation device 3 determines not only the amount of the biological sample but also the amount of the reagent for preparing the measurement sample based on the concentration information of the cell C1 generated by itself. It is also possible to control the preparation device unit 18 of the sample preparation device 3 so as to obtain a sufficient amount of reagent.
Further, in the control unit 16 of the sample preparation device 3, the ratio between the number of cells C1 in the biological sample and the amount of reagent (for example, staining solution) is within a predetermined range based on the concentration information of the cells C1 generated by itself. It is also possible to determine the amount of at least one of the biological sample and the reagent so that the preparation device unit 18 of the sample preparation apparatus 3 is controlled so as to acquire the determined amount of the biological sample or reagent. .

[Preparation of measurement sample]
Next, the control unit 16 of the sample preparation device 3 supplies the concentrate L1 obtained by the second discrimination / substitution process to the product container (microtube) 54 (step S18) and stores it in the device. The staining solution and RNase thus prepared are supplied from the reagent quantification unit 28 to the product container 54 (step S19), and DNA staining and RNA treatment are performed in the product container 54 to prepare a measurement sample (step S20).

When the processing is completed, the control unit 16 of the sample preparation device 3 sends the obtained measurement sample to the main detection unit 6 of the measurement device 2 (step S21 and point B).
Note that the control unit 16 of the sample preparation device 3 always determines whether or not the shutdown signal from the measurement device 2 has been received (step S22 and point C). Returning to step S6 for determining whether or not a preparation start signal has been received, if the signal is received, shutdown is performed and the sample preparation process is terminated (step S23).

[Main measurement by measuring equipment and data analysis]
Returning to FIG. 15, after transmitting the preparation start signal, the control unit 8 of the measurement device 2 always determines whether or not the measurement sample is supplied from the sample preparation device 3 (step S <b> 24).
Therefore, when the measurement sample is fed from the sample preparation device 3 (point B), the control unit 8 of the measurement device 2 sends the measurement sample to the flow cell 45 of the main measurement unit 14, and the measurement sample with respect to the cell C1 is measured. The actual measurement is performed (step S25), and the measurement data is transmitted to the data processing device 4 (step S26).

On the other hand, after transmitting the measurement start signal, the control unit 31 of the data processing device 4 always determines whether or not measurement data has been received from the measurement device 2 (step S27).
When the measurement data is received from the measurement device 2, the control unit 31 of the data processing device 4 analyzes the cells and nuclei using the measurement data and determines whether or not the cells in the measurement sample are cancerous. (Step S28).

The control unit 31 of the data processing device 4 displays the analysis result on the display unit 32 (step S29), and determines whether there is a shutdown instruction by a user input (step S30).
When there is the shutdown instruction, the control unit 31 of the data processing device 4 transmits a shutdown signal to the measuring device 2 (step S31).

  The control unit 8 of the measuring device 2 always determines whether or not the shutdown signal from the data processing device 4 has been received (step S32). If the signal has not been received, the measurement start signal Returning to step S4 for determining whether or not to receive the signal, if the signal is received, the shutdown signal is transferred to the sample preparation device 3 (step S33), and the shutdown is executed to complete the measurement process (step S34). .

  As described above, according to the cell analyzer 1 of the present embodiment, the sample preparation device 3 performs the pre-measurement on the biological sample, and includes the concentration adjustment of the measurement target cell C1 based on the measurement result. Since the processing step is performed, a measurement sample suitable for analysis of the measurement target cell C1 can be well prepared even if the concentration of the measurement target cell C1 varies greatly between biological samples. In addition, since the main measurement of the measurement target cell C1 is performed using the measurement sample having the concentration adjusted for the measurement target cell C1 obtained by the concentration adjustment, the concentration of the measurement target cell C1 is large for each biological sample. Even if there is variation, the measurement target cell C1 in the biological sample can be analyzed with high accuracy.

[Other variations]
The above-described embodiments are examples of the present invention and are not restrictive. The scope of the present invention is shown not by the above-described embodiment but by the scope of claims for patent, and further includes all modifications equivalent to the configurations of the scope of claims for patent.

  For example, in the above-described embodiment, the flow path 102D parallel to the axial direction of the rotor 102 made of a short cylindrical body is formed. This flow path has one end surface (upper end surface) as shown in FIG. ) And the position of the opening 202D2 on the other end face (lower end face) may be inclined channels 202D that are shifted from each other in the circumferential direction. In the case of such an inclined channel 202D, the biological sample can be smoothly introduced into the inclined channel 202D when the rotor 202 is rotated in the direction of arrow N in FIG.

  In the above embodiment, the circulation flow of the biological sample is generated by the fin 106 provided integrally with the rotor 102. However, a pump mechanism is disposed in the biological container 53, and this pump mechanism is driven. Thus, convection can be generated in the biological sample. Further, such a pump mechanism can be disposed outside the biological container 53. In this case, the biological sample is discharged into the biological container 53 using tubes connected to the discharge port and the suction port of the pump mechanism. In addition, a flow of the biological sample can be created by inhaling from the biological container 53.

  In the above embodiment, the circulating flow of the biological sample is generated by the fins 106 provided integrally with the rotor 102. However, a spiral groove is formed on the side surface of the rotor 102 made of a short cylinder. Thus, the flow of the circulating flow can be made stronger.

  In the above embodiment, the filter 101 is disposed below the rotor 102, but the filter 101 may be disposed above the rotor 102. In this case, the filter 101 is provided with a hole through which the rotary shaft 104 for rotating the rotor 102 is driven, and the fin 106 is arranged so that the biological sample flows from the rotor 102 toward the filter 101, that is, from below to above. Provided on the lower end surface of the rotor 102.

  In the above embodiment, the rotor 102, which is a dispersion member, is rotated, but a dispersion member 302 made of a ring body having a cross-shaped central interference portion 302A as shown in FIG. When the dispersion member 302 is fixed near the end of the pipe, for example, at a position where the filter 101 is disposed in FIG. 9, the aggregated cells can be dispersed by rotating the filter. In this case, the filter is fixed so as to form a predetermined distance from the dispersion member 302 on the lower surface of the cylindrical body (the convex portion is not formed) in FIGS. The rotor functions not as a dispersion member but as a support for supporting the filter.

  Moreover, in the said embodiment, although the hole 105B is formed in the front-end | tip of the pipe 105, a side hole can also be formed in the taper part 105A. When the flow rate of the biological sample can be secured to some extent, the biological sample is discharged from the pipe 105 through the side hole, so that the stagnation of the biological sample in the biological container 53 can be reduced.

  In the above embodiment, the filter member is a filter made of a film-like body in which a large number of micropores are formed. For example, nylon can be used as long as it can pass a single cell to be measured. It is also possible to use a mesh body knitted with synthetic resin fibers. Furthermore, a disc body in which slits having a size (for example, 1 mm) considerably larger than the size of a single cell can be used as a filter member. The material of the filter 101 is not limited to stainless steel, and synthetic resin such as polycarbonate can be used, for example.

  In the above embodiment, the cervical epithelial cell C1 is used as a measurement target cell. However, it is also possible to determine canceration of oral cells, other epithelial cells such as bladder and pharynx, and organ epithelial cells. Can do.

  In the above embodiment, the prepared measurement sample is measured with a flow cytometer. When the prepared measurement sample is smeared on a slide glass to produce a smear, and this smear is observed, In addition, the cell dispersion device of the present invention can be preferably used. According to the present invention, a measurement sample having a uniform concentration of cells to be measured can be prepared, so that a smear suitable for analysis can be easily produced.

  Moreover, in the said embodiment, even if the density | concentration of a measuring object cell has a big dispersion | variation for every biological sample, in order to prepare the measuring sample suitable for the analysis of a measuring object cell well, the sub detection part is provided. However, such a sub-detection unit can be omitted when the density variation is relatively small and the measurement accuracy is not substantially affected.

  Note that the various dimensions (for example, the diameter of the pipe 105, the thickness and the aperture ratio of the filter 101), the number (for example, the number of the flow paths 102D), and the like in the above embodiment are merely examples, and the amount of the biological sample. It can be appropriately changed according to the cell to be measured and the like.

DESCRIPTION OF SYMBOLS 1 Cell analyzer 2 Measuring apparatus 3 Sample preparation apparatus 4 Data processing apparatus 6 Main detection part 8 Measurement control part 10 Flow cytometer 14 Sub detection part 16 Preparation control part 18 Preparation device part 24 Sample setting part 25 Cell dispersion part 26 Sample pipette Section 27 Specimen quantification section 28 Reagent quantification section 29 Discrimination / replacement section 31 Processing body 44 Semiconductor laser 45 Flow cell 47 Photo diode 101 Filter 102 Rotor 102D Flow path 103 Motor 105 Pipe 105A Taper section 105C Open section 106 Fin 108 Projection section CL1 Interval ( clearance)

Claims (13)

An apparatus for dispersing the aggregated cells in a cell sample solution containing aggregated cells in which a plurality of cells are aggregated into a single cell,
A filter member configured to allow passage of the single cell;
A dispersion member that is disposed at a predetermined interval from the filter member, and disperses aggregated cells in the interval;
A cell dispersing device comprising: a driving source for driving the dispersing member or the filter member.   The cell dispersion apparatus according to claim 1, further comprising convection forming means for forming a flow of the cell sample solution in a direction from the dispersion member to the filter member. Further comprising a guide cylinder disposed outside the filter member and the dispersion member and having an opening in the peripheral wall thereof;
When one of the front end opening and the opening of the guide cylinder is an inlet and the other is an outlet, the cell sample solution is introduced from the inlet to the inside of the guide cylinder, the outlet, and the guide cylinder. The cell disperser according to claim 2, wherein a circulating flow that circulates to the outside and the inlet is formed. A guide cylinder disposed outside the filter member and the dispersion member;
When one of the front end opening and the upper end opening of the guide cylinder is an inflow port and the other is an outflow port, the cell sample solution flows from the inflow port to the inside of the guide tube, the outflow port, and the outside of the guide tube. The cell disperser according to claim 2, wherein a circulating flow that circulates to the inlet is formed.   The said convection formation means is integrally provided in the said dispersion member, The flow of the said cell sample liquid is formed when the said dispersion member is driven by the said drive source. Cell dispersion device.   The cell dispersion device according to any one of claims 1 to 5, wherein the dispersion member is formed of a cylindrical body, and the cylindrical body has at least one flow path penetrating from one end surface to the other end surface.   The convection forming means is composed of a fin that is inclined on the end surface of the cylindrical body, and the fin is provided so that a cell sample solution can be introduced into the opening in the vicinity of the opening of the flow path. The cell dispersion apparatus according to claim 6.   The cell dispersion device according to claim 3, wherein a tapered portion whose cross-sectional area gradually decreases toward the tip is formed at the tip of the guide cylinder.   The cell dispersion device according to claim 1, wherein the dispersion member has a convex portion on a surface facing the filter member.   The cell dispersion device according to claim 1, wherein the filter member is formed of a film-like body or a mesh body having a plurality of openings having a size capable of capturing the aggregated cells.   The cell dispersion device according to any one of claims 2 to 10, wherein the convection forming means can form a flow of the cell sample solution in a direction from the filter member to the dispersion member.   The cell dispersion device according to any one of claims 1 to 11, wherein the cell sample solution is a sample solution containing cells collected from the cervix. A cell dispersion device according to claim 1;
An aspiration dispensing unit for aspirating and dispensing a cell sample solution containing cells separated by the cell dispersion device;
A sample pretreatment device for preparing a cell sample, comprising: a filtration unit for extracting predetermined cells from the cell sample solution dispensed by the suction dispensing unit.

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