JP2005331506A - Microchip and fluorescent particle counting apparatus equipped with the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To accurately measure the number of leucocytes, while being simple operations. <P>SOLUTION: A blood-collecting device etc. not shown in Fig. are connected to an adaptor 4 to make blood L1 flow in a first inflow path B1 and a liquid L2 containing a surfactant and fluorescent pigments flow in a second inflow path B2. The blood L1 and the liquid L2 are mixed at a mixing part C, and blood platelets, erythrocytes, and leucocyte cell membranes in the blood are dissolved by the surfactant to fluorescent-stain leucocyte cell nuclei with the fluorescent pigments. The fluorescent-stained leucocyte cell nuclei are counted by a fluorescent particle counting part E, when they move through an outflow path D. Using the present invention, it is possible to accurately count the number of leucocytes by simple operations, such as connection of the blood collecting device etc. to the adaptor 4. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

    The present invention relates to a microchip for mixing two or more kinds of liquids, and a fluorescent particle counter equipped with the microchip.

    Conventionally, counting after fluorescent staining of particles is performed in various technical fields. For example, in the medical field, platelet preparations and erythrocyte preparations obtained by extracting platelets and erythrocytes from blood are used. However, it is preferable that these platelet preparations and erythrocyte preparations have no remaining white blood cells. Leukocyte removal is performed. And the white blood cell count for confirming whether the white blood cell was removed from the platelet preparation and the red blood cell preparation is performed (for example, refer patent document 1).

    FIG. 4 is a block diagram showing an example of a conventional structure of the white blood cell counter. In FIG. 4, reference numeral 100 denotes an imaging container for containing a fluorescently-stained platelet preparation, and 101 denotes the imaging container. Reference numeral 100 denotes a laser light source that emits a laser beam, reference numeral 102 denotes a mirror, and reference numeral 103 denotes a CCD camera. In such an apparatus, when a laser beam (excitation light) is irradiated to the imaging container 100 by the laser light source 101, an image of white blood cells is taken by the CCD camera 103. The image is captured by a computer (not shown), subjected to image processing, and the number of white blood cells is counted.

    By the way, in order to fluorescently stain the particles to be counted, it is necessary to mix a fluorescent dye into the solution containing the particles. For example, in order to fluorescently stain white blood cells in platelet preparations, platelets and red blood cells are removed with fluorescent dyes such as propidium iodide and platelet preparations (more precisely, surfactants such as Triton X, etc.) It is necessary to mix with the one in a state of being converted into a solid state).

    Although not intended for counting fluorescent particles, as a device (microchip) for mixing two or more kinds of liquids, a device having a structure shown in FIG. (See Patent Document 3). Reference numeral 200 in the figure indicates a sample supply port for supplying a liquid sample, reference numeral 201 indicates the flow path, reference numeral 202 indicates a reagent supply port for supplying a liquid reagent, and reference numeral 203 indicates the flow path. Indicates. The specimens and reagents supplied from these supply ports 200 and 202 are merged and mixed in the merge unit 204, and are discharged from the discharge port 206 via the outflow path 205.

JP 2001-83092 A JP 2002-221485 A JP 2003-181255 A

    However, in order to fluorescently stain the particles, it is necessary to sufficiently mix the fluorescent dye in the solution containing the particles. For example, when white blood cells are fluorescently stained with a fluorescent dye such as propidium iodide or ethidium bromide, the fluorescent dye needs to be slid between the bases of the DNA of the white blood cell nucleus and must be mixed sufficiently. However, even if a fluorescent dye is mixed with the structure shown in FIG. 5 and counting is performed with the counting device shown in FIG. 4, there is a problem that the fluorescent staining is insufficient and the accuracy of particle counting is deteriorated. It was. Moreover, when removing platelets and red blood cells and making white blood cells naked nuclei as described above, it is necessary to sufficiently mix and react the surfactant, but in the structure shown in FIG. Insufficient platelets, leukocyte cell membranes and the like remain, and there is a problem in that the accuracy of leukocyte count deteriorates.

When using the counting device shown in FIG.
・ Work to put fluorescently stained platelet preparations etc. into imaging container 100 ・ Process to rotate imaging container 100 to settle fluorescent particles on the bottom of the container (centrifugal processing)
There is a problem that the operation of tracing (specifying) the sample contained in the imaging container 100 is required, and the counting operation becomes complicated.

    Furthermore, in the case of the counting device shown in FIG. 4, it is necessary to switch the optical path in accordance with the concentration of the measurement sample, and accordingly, the counting operation becomes complicated and the structure of the device becomes complicated.

    An object of the present invention is to provide a microchip in which a plurality of types of solutions can be sufficiently mixed.

    Another object of the present invention is to provide a fluorescent particle counting apparatus that can easily perform counting.

The invention according to claim 1 is illustrated in FIG. 1 and includes a first inflow path (B1) into which the first liquid (L1) is introduced,
A second inflow channel (B2) into which the second liquid (L2) is introduced;
A mixing section (C) which is configured to bend and communicate with these inflow paths (B1, B2) and into which the first liquid (L1) and the second liquid (L2) are introduced and mixed; ,
An outflow passage (D) arranged to communicate with the mixing section (C) and through which the mixed liquid flows out;
This is for a microchip (A) constructed by providing

    The invention according to claim 2 is characterized in that, in the invention according to claim 1, the mixing portion (C) is curved in a substantially zigzag shape.

The invention according to claim 3 is illustrated in FIG. 1, wherein the first liquid (L1) containing particles to be counted and the second liquid (L2) containing a fluorescent dye are mixed in the mixing unit ( The microchip (A) according to claim 1 or 2, wherein the fluorescent particles (see reference numeral 3 in Fig. 2 and Fig. 3) are mixed in C) and sent out to the outflow passage (D),
A fluorescent particle counter (see symbol E in FIG. 1) for counting the fluorescent particles (3) sent out,
It is about the fluorescent particle counting device comprised by providing.

    The invention according to claim 4 is the invention according to claim 3, wherein a diluting liquid (L4) for diluting the first liquid (L1) and the second liquid (L2) is provided upstream of the mixing section (C). The third inflow path (B3) for supplying to the side is provided and configured.

As illustrated in FIGS. 1 and 2, the invention according to claim 5 is the invention according to claim 3 or 4, which is downstream of the mixing portion (C) and upstream of the outflow passage (D). A fourth inflow passage (B4) for supplying the fourth liquid (L4) to
The fluorescent particles (3) are aligned by supplying the fourth liquid (L4).

The invention according to claim 6 is the invention according to any one of claims 3 to 5, as illustrated in FIG. 1, wherein the fluorescent particle counting unit (E) is configured to emit fluorescence in the outflow channel (D). A light source (10) for irradiating the particles (3) with excitation light, and a fluorescence measuring means (11) for capturing the fluorescence generated by irradiating the fluorescent particles (3) with the excitation light. It is characterized by.

The invention according to claim 7 is the invention according to any one of claims 3 to 6, wherein the first inflow channel (B1) is an inflow channel into which the first liquid (L1) containing leukocytes is introduced. Yes,
The second inflow path (B2) is an inflow path through which a second liquid (L2) containing a surfactant for lysing the leukocyte cell membrane and a fluorescent dye for fluorescently staining the leukocytes is introduced. It is characterized by that.

    The invention according to claim 8 is characterized in that, in the invention according to claim 7, the first inflow passage (B1) has a connection portion (4) to which a blood collection device and a blood bag can be attached.

The invention according to claim 9 is the invention according to any one of claims 6 to 8, wherein sampling means (14) for acquiring the output of the fluorescence measuring means (11) at regular intervals;
Counting means (15) for counting fluorescent particles (3) based on the output of the sampling means (14) is provided.

    The invention according to claim 10 is the camera according to any one of claims 6 to 9, wherein the fluorescence measuring means (11) is a camera that sequentially acquires still images every predetermined time. And

According to an eleventh aspect of the present invention, in the invention according to the tenth aspect, the counting of the fluorescent particles by the counting means (15) is performed in a predetermined fluorescence distribution measuring region in each still image acquired by the fluorescent measuring means (11). (Refer to symbols K ₁ , K ₂ , K _{3 in} FIGS. 6 and 7)
The width w of the predetermined fluorescence distribution measurement region in the moving direction of the fluorescent particles satisfies the following formula.

In the invention according to claim 12, in the invention according to claim 11, as illustrated in FIGS. 7 (a) and 7 (b), the flow velocity of the liquid flowing through the outflow path (D) is When changing from the vicinity of the wall surface to the central portion, the width w _x of the fluorescence distribution measurement region (K ₂ , K ₃ ) at a distance _x from the wall surface satisfies the following expression.

The invention according to claim 13 is the invention according to claim 12, wherein the upstream edge (23, 25) in the fluorescence distribution measurement region (K ₂ , K ₃ ) is a straight line crossing the outflow path (D). The downstream edge (24, 26) in the region is a curved line protruding to the downstream side, and the region (K ₂ , K ₃ ) is shaped like a bullet.

The invention according to a fourteenth aspect is the invention according to any one of the eleventh to thirteenth aspects, wherein the counting means (15) includes the fluorescence distribution measurement region (K ₁ , K ₂ , K) in the still image. ₃ ) passing through the upstream fluorescent particles (3 ₁ ) passing through the upstream edges (21, 23, 25) and the downstream edges (22, 24, 26) in the fluorescence distribution measurement region Only one of the fluorescent particles (3 ₂ ) at a certain moment is counted, and the other is not counted.

    Note that the numbers in parentheses are for the sake of convenience indicating the corresponding elements in the drawings, and therefore the present description is not limited to the descriptions on the drawings.

    According to the first aspect of the present invention, since the mixing portion is configured to bend, the first liquid and the second liquid can be sufficiently mixed.

    According to the second aspect of the present invention, since the mixing portion is formed to be curved in a substantially zigzag shape, the first liquid and the second liquid can be sufficiently mixed. Moreover, since it takes time to pass through the mixing portion, the first liquid and the second liquid can be sufficiently reacted during that time.

    According to the inventions according to claims 3, 6 and 9, the first liquid and the second liquid can be sufficiently mixed so that the particles can be fluorescently stained, and the fluorescent particle counting unit accurately counts the fluorescent particles. be able to. Moreover, the inflow path and the outflow path of the microchip can be narrowed to reduce the amounts of the first liquid and the second liquid. For example, even if the first liquid is a platelet preparation or blood, loss of a useless platelet preparation or the like can be suppressed.

    According to the invention of claim 4, since the first liquid and the second liquid can be diluted with the diluent, the fluorescent particles can be thinly aligned even if the first liquid and the second liquid are high in viscosity. And counting of fluorescent particles can be performed accurately.

    According to the fifth aspect of the present invention, since the fluorescent particles can be thinly aligned by the supply of the fourth liquid, the fluorescent particles are accurately detected by the fluorescent particle counter regardless of the concentrations of the first liquid and the second liquid. It can be measured. Therefore, unlike Patent Document 1, there is no need to switch the optical path according to the concentration of the liquid to be measured, and the counting operation and the structure of the apparatus can be simplified. Further, unlike the above-described conventional apparatus, it is not necessary to perform the centrifugal process to align the fluorescent particles, so that the counting operation can be simplified and the counting accuracy can be improved. Information error recognition due to human error can be eliminated, and the mental and physical distress of the inspector can be reduced.

    According to the seventh aspect of the invention, the platelets and red blood cells in the first liquid can be removed, and the white blood cell nuclei can be dissolved and the white blood cell nuclei can be fluorescently stained, so that the white blood cells can be accurately counted.

    According to the eighth aspect of the present invention, white blood cells can be counted simply by connecting a blood collection device or a blood bag to the connection portion, and a separate container (for example, the imaging container 100 shown in FIG. 4) as in the conventional device. Since there is no need to transfer the measurement sample to, the counting operation can be simplified. Moreover, since it is possible to count with a blood collection device or a device directly connected to a blood bag, it is not necessary to trace information, so that erroneous information recognition due to human error can be eliminated, and the mental and physical pain of the inspector can be reduced.

    According to the invention which concerns on Claim 10, a commercially available camera can be used and cost can be reduced by the part which does not need to produce a fluorescence measurement means for exclusive use.

    According to the invention of claim 11, since the fluorescent particles are counted using a certain virtual region, the counting accuracy can be increased as compared with the case where the region is not used.

    According to the invention which concerns on Claim 12 and 13, even when the flow rate changes from the wall surface vicinity part of an outflow channel to the center part, the counting precision of fluorescent particles can be improved.

    According to the invention which concerns on Claim 14, duplication count can be reduced and counting precision can be improved.

    Hereinafter, the best mode for carrying out the present invention will be described with reference to FIGS. 1 to 3 and FIGS. 6 and 7.

    FIG. 1 is a schematic diagram showing an example of the structure of a microchip and a fluorescent particle counter according to the present invention, and FIG. 2 is a schematic diagram showing how fluorescent particles are aligned by the inflow of a fourth liquid. 3 (a) is a schematic diagram for explaining a method of counting fluorescent particles, and FIG. 3 (b) is a waveform diagram showing a fluorescence intensity distribution along the gate 20 in FIG. 3 (a). FIG. 6 is a schematic diagram for explaining another method for counting fluorescent particles, and FIG. 7 is a schematic diagram for explaining still another method for counting fluorescent particles.

    The microchip according to the present invention includes a first inflow path B1 into which the first liquid L1 flows (liquid feeding) and a second inflow path B2 into which the second liquid L2 flows in, as indicated by reference numeral A in FIG. A mixing portion C that is configured to bend and communicate with the inflow paths B1 and B2, and into which the first liquid L1 and the second liquid L2 are introduced and mixed, and the mixing portion C. And an outflow path D through which the mixed liquid flows out. According to the present invention, since the mixing part C is configured to bend, the first liquid L1 and the second liquid L2 can be sufficiently mixed.

    The mixing portion C described above is preferably curved in a substantially zigzag shape so that the first liquid L1 and the second liquid L2 are sufficiently mixed. In such a case, the first liquid L1 and the second liquid L2 can be sufficiently mixed. Moreover, since it takes time to pass through the mixing part C, the first liquid L1 and the second liquid L2 can be sufficiently reacted during that time. For example, leukocyte cell membranes, erythrocytes and platelets can be sufficiently dissolved by the surfactant described later.

    The inflow paths B1 and B2, the mixing section C, and the outflow path D described above are preferably formed by forming a groove shape on the surface of the substrate 1 and attaching the cover member 2 to the surface.

    On the other hand, the fluorescent particle counting apparatus according to the present invention includes the microchip A configured as described above. Into the first inflow channel B1 of the microchip A, a measurement sample (for example, blood, platelet preparation, erythrocyte preparation, etc.) containing particles (for example, white blood cells) to be counted is flowed as the first liquid L1. A solution containing a fluorescent dye may be allowed to flow into the second inflow path B2 as the second liquid L2. In such a case, the first liquid L1 and the second liquid L2 are mixed in the mixing section C to generate fluorescent particles (see reference numeral 3 in FIG. 2), and the fluorescent particles 3 pass through the outflow path D. Will be sent out. And it is good to count this sent-out fluorescent particle 3 by the fluorescent particle counter (refer the code | symbol E of FIG. 1). According to this fluorescent particle counting device, the first liquid L1 and the second liquid L2 can be sufficiently mixed to fluorescently stain the particles, and the fluorescent particle counting unit E can accurately count the fluorescent particles 3 It can be carried out. In addition, the inflow paths B1 and B2 and the outflow path D of the microchip A can be narrowed to reduce the amounts of the first liquid L1 and the second liquid L2. For example, even if the first liquid L1 is a platelet preparation or blood, loss of a useless platelet preparation or the like can be suppressed.

    In addition, when counting white blood cells in a platelet preparation or a red blood cell preparation, it is necessary to remove the platelets and red blood cells and to make the white blood cells naked (to remove the white blood cell membrane). For this purpose, a surfactant for dissolving leukocyte cell membranes, platelets and red blood cells may be mixed in the first liquid L1 (preparation described above) or the second liquid L2 (solution containing a fluorescent dye). . For example, the first inflow channel B1 is an inflow channel into which the first liquid L1 containing leukocytes is introduced, and the second inflow channel B2 is fluorescent with a surfactant for dissolving the leukocyte cell membrane and the leukocytes. An inflow path into which the second liquid L2 containing a fluorescent dye for staining is introduced is preferable. In such a case, platelets and red blood cells can be removed, and white blood cell nuclei can be lysed and white blood cell nuclei can be fluorescently stained, so that white blood cells can be accurately counted. Here, examples of the surfactant include Triton X-100, and examples of the fluorescent dye include propidium iodide and ethidium bromide.

    In FIG. 1, there is one second inflow path B2, but two or more second inflow paths B2 may be provided so that the surfactant and the fluorescent dye are supplied from different inflow paths B2.

    By the way, the 3rd inflow path B3 is provided in the upstream from the said mixing part C, The dilution liquid (3rd liquid) L3 for diluting the said 1st liquid L1 and the said 2nd liquid L2 is provided in the said mixing part. It is good to be able to supply to the upstream side of C. In such a case, since the first liquid L1 and the second liquid L2 can be diluted, the first liquid L1 and the second liquid L2 have high viscosity (for example, hyperlipidemic tendency) Even with highly viscous blood or chyle blood), the fluorescent particles can be thinly aligned, and the fluorescent particles can be accurately counted. As the diluent, it is preferable to use a colorless and transparent buffer in which cells can stably exist, and specifically, a PBS buffer is preferably used. The first liquid L1 or the second liquid L2 is diluted before the first liquid L1 flows into the first inflow path B1 or before the second liquid L2 flows into the second inflow path B2. The liquid is diluted in advance so as to have an appropriate viscosity (at least the viscosity of the liquids L1 and L2 flows smoothly through the inflow channels B1 and B2), and then diluted from the third inflow channel B3. It is preferable that the liquid is allowed to flow so as to have an optimum viscosity (for example, an optimum viscosity that allows the fluorescent particles 3 to flow in a state of being separated one by one). For example, a diluted liquid is mixed with high-viscosity blood to adjust the first liquid L1 having an appropriate viscosity, the adjusted first liquid L1 is allowed to flow into the first inflow path B1, and then the second inflow path B2 Alternatively, fluorescent staining of white blood cells may be performed by mixing a fluorescent dye, and then a final adjustment of the viscosity may be performed by mixing a diluent from the third inflow path B3. By the way, in FIG. 1, the third inflow path B3 is joined on the downstream side (see reference numeral H2) from the joining section (see reference sign H1) of the first inflow path B1 and the second inflow path B2. (That is, the diluting liquid L3 is mixed after the first liquid L1 and the second liquid L2 are mixed). However, the present invention is not limited to this. . For example, after the first liquid L1 and the diluting liquid L3 are mixed with the merging part of the first inflow path B1 and the third inflow path B3 being upstream of the merging part of the second inflow path B2, Even when the second liquid L2 is mixed, the second liquid L2 is formed such that the joining portion of the second inflow passage B2 and the third inflow passage B3 is located upstream of the joining portion of the first inflow passage B1. In addition, the first liquid L1 may be mixed after the diluent L3 is mixed.

    By the way, when a plurality of fluorescent particles 3 are attached (in a lump) and pass through the place where the fluorescent particle counting unit E is arranged, a plurality of fluorescent particles 3 are counted as one. As a result, the counting accuracy is deteriorated, so that the fluorescent particles 3 must pass through the place where the fluorescent particle counting section E is disposed in a state where they do not stick (separate each other). Therefore, the fluorescent particle counting unit E or other means detects whether or not the fluorescent particles 3 are stuck, and the information detected there is a means for putting a diluent into the third inflow path B3 ( It is made to feed back to the means (4th liquid inflow means) which puts in the 4th liquid L4 (detailed later) and the inflow of the dilution liquid and the 4th liquid L4 according to the state of the fluorescent particles 3 It is preferable to adjust the amount so that the fluorescent particles 3 are fed one by one in a separated state.

    Further, a fourth inflow path B4 for supplying the fourth liquid L4 is provided downstream of the mixing section C and upstream of the outflow path D, and a flow (laminar flow) from the mixing section C is provided. The fluorescent particles 3 may be aligned by supplying the fourth liquid L4 without disturbing the state flow) (see FIG. 2). As the fourth liquid L4, the above-described buffer may be used. In this case, the laminar flow from the mixing part C is made thin by adjusting the inflow direction and the inflow speed of the fourth liquid L4 (see reference signs d1 and d2), and the fluorescent particles 3 are formed on the wall surface of the outflow path D. It is preferable to flow in a state where only one layer is arranged along the line. In such a case, the fluorescent particles 3 can be accurately measured by the fluorescent particle counter E regardless of the concentrations of the first liquid L1 and the second liquid L2. Therefore, unlike Patent Document 1, there is no need to switch the optical path according to the light absorption characteristics and optical characteristics of the liquid to be measured, and the counting operation and the structure of the apparatus can be simplified. That is, since the thickness of the outflow channel D in the light irradiation direction of the light source 10 (layer thickness of the fluorescent particles 3) is formed so thin as to be photographed with a camera, the platelet preparation and the whole blood / erythrocyte preparation In order to prevent the influence of background light, the difference in transmittance is prevented as much as possible from affecting the amount of fluorescence as background light excited from the fluorescent dye remaining in the liquid. It is not necessary to switch the optical path from the light source to the outflow path D. In addition, unlike the conventional apparatus, it is not necessary to align the fluorescent particles by performing a centrifugal process, so that the counting operation can be simplified (the mental and physical pain of the inspector can be reduced) and the counting accuracy is also improved. It can be improved. Information error recognition due to human error can be eliminated, and the mental and physical distress of the inspector can be reduced. Of course, when an optical system with a deep focal depth is used, it is not necessary to perform this white blood cell nucleus alignment process, so a simpler flow path pattern than that shown in FIG. 1 can be employed.

    On the other hand, the first inlet B1 may be provided with a connection part (adapter) 4 to which a blood collection device or a blood bag can be attached. In that case, white blood cells can be counted simply by connecting a blood collection device or a blood bag to the connecting portion 4, and the measurement sample is transferred to another container (for example, the imaging container 100 shown in FIG. 4) as in the conventional device. Since there is no need to change, counting work can be simplified (the mental and physical pain of the inspector can be reduced). For those whose leukocyte count exceeds the standard, leukocyte removal may be performed using a leukocyte removal filter or the like. Since the counting work is simplified as described above, it is also easy to perform a total inspection of the collected blood or increase the number of sampling tests, and when the total number of sampling tests are performed or the number of sampling tests is increased. Can more reliably prevent the occurrence of defective products as compared to the case where a small number of sampling inspections are performed. Furthermore, since it is possible to count with a blood collection device or a device directly connected to a blood bag, there is no need to trace information (that is, when the imaging container 100 shown in FIG. 4 is used, which sample is stored in each container 100). It is necessary to specify whether or not it is contained, but it is not necessary to perform such identification when a blood collection device or the like is directly connected to the counting device). Can reduce mental and physical distress.

    Further, an opening (output port) 5 is provided in the outflow passage D, and the first liquid L1 and the second liquid L2 flow in a laminar flow state in each inflow passage by being sucked from the opening 5. It is good to make it.

    By the way, the fluorescent particle counting unit E described above captures (measures) the light source 10 that irradiates the fluorescent particles 3 in the outflow path D with the excitation light and the fluorescence generated by irradiating the fluorescent particles 3 with the excitation light. It is good to comprise by the fluorescence measurement means 11.

    The light source 10 needs to be selected in accordance with the light absorption characteristics of a fluorescent dye (a dye for fluorescently staining particles such as leukocytes). For example, when propidium iodide (PI) is used as a fluorescent dye, a light source that generates green light (excitation light in a wavelength range of about 500 to 550 nm) is good, and when ethidium bromide (EtBr) is used as a fluorescent dye. A light source that generates ultraviolet light is preferable.

    Further, as the fluorescence measuring means 11, a camera that sequentially acquires still images every predetermined time can be used, and specifically, a camera such as a video camera, a CCD camera, or a USB camera can be used. In such a case, a commercially available camera can be used, and the cost can be reduced to the extent that it is not necessary to create a dedicated fluorescence measuring means.

    In addition, between the fluorescence measurement means 11 and the said outflow path D, the optical filter 12 for allowing only the light produced by the fluorescence to pass (not allowing the light in other wavelength ranges to pass), or for condensing the light It is preferable to arrange the lens 13 in advance.

    On the other hand, it is preferable that a sampling means 14 is connected to the fluorescence measuring means 11 so that the output of the fluorescence measuring means 11 is acquired at regular intervals. Further, it is preferable that a counting unit 15 is connected to the sampling unit 14 so that the fluorescent particles 3 can be counted based on the output of the sampling unit 14. That is, as shown by an arrow L5 in FIG. 3A, the fluorescent particles 3 move along the outflow path D, but a gate 20 is provided virtually across the outflow path D. A one-dimensional fluorescence intensity distribution along 20 is acquired by the fluorescence measuring means 11 and the sampling means 14. As a result, a one-dimensional fluorescence intensity distribution G1, G2, G3,... Is obtained at regular intervals as shown in FIG. The fluorescent particles 3 that pass through the gate 20 may be counted. For example, when a camera equipped with the fluorescence measuring unit 11 and the sampling unit 14 is used to capture a moving image at a video rate of 30 fps, a still image can be obtained every 1/30 seconds. As a result, a fluorescence intensity distribution as shown in FIG. 3B can be obtained. The number of fluorescent particles 3 may be obtained by analyzing the obtained fluorescence intensity distribution. Alternatively, the motion detection technology used in the moving image compression / analysis technology may be used to capture and count the number of objects that appear in the moving image.

Note that the counting of the fluorescent particles 3 based on the one-dimensional fluorescence intensity distribution measurement as described above is such that all the fluorescent particles 3 passing through the gate 20 are photographed only once by the camera at the moment of passing through the gate 20. It is necessary to The reason is that if there are fluorescent particles flowing without being photographed at the moment of passing through the gate 20, the fluorescent particles will not be counted, and the counting accuracy will deteriorate accordingly. On the contrary, the shooting interval of the camera (the time from when the camera acquires one still image until the next still image is acquired as described later) is too short and passes through the gate 20. This is because if there are fluorescent particles that have been photographed a plurality of times, the fluorescent particles will be counted repeatedly, and the counting accuracy will deteriorate. For all fluorescent particles, the moment of passing through the gate 20 To shoot only once, video rate and velocity of the video camera (there is shown by reference numeral L ₅ in FIG. 3 (a), outlet channel It is necessary to optimally adjust the relationship of the flow velocity of the liquid flowing through D.

However, in the case where it is difficult to avoid miscounting of the fluorescent particles in the one-dimensional fluorescence intensity distribution measurement as described above, the fluorescence measuring unit 11 acquires the count of the fluorescent particles by the counting unit 15. It may be performed for a predetermined fluorescence distribution measurement region in each still image. The width (the length in the direction in which the fluorescent particles move) w of the predetermined fluorescence distribution measurement region should satisfy the following formula. In such a case, since a certain virtual region is used to count fluorescent particles, counting errors can be reduced and counting accuracy can be increased as compared to a case where no region is used.

Such fluorescence distribution measurement region, there may be mentioned two-dimensional rectangular region K ₁ as indicated by hatching in FIG. 6, the width w ₁ of the region, it is preferable to the following expression.

If you set the width w ₁ of the rectangular region K ₁ as such, all fluorescent particles flowing in the outflow path, it has been taken once theoretically in the region K ₁ in one of the still images Thus, counting errors of the fluorescent particles (count omission and overlapping count as described above) can be reduced, and the counting accuracy of the fluorescent particles can be increased. However, as shown in FIG. 6, a moment that passes through the rectangular region K ₁ on the upstream side of the edge (edge) 21, fluorescent particles 3 ₁ only right half is photographed in a state that has entered the rectangular region K ₁ It is the next imaging timing takes the position indicated by reference numeral 3 ₂ moved by w _1, after all, there, it would be shot twice, counting accuracy when it counts both deteriorates. Therefore, in all photographed still image, a rectangular region (fluorescence distribution measurement region) moments of the fluorescent particles passing through the upstream side of the edge 21 of the K ₁ (for example, fluorescent particles indicated by reference numeral 3 _1), and the rectangular region K ₁ downstream side moment of the fluorescent particles passing through (rim) 22 (e.g., reference numeral 3 ₂ fluorescent particles shown) instead of the counting means 15 both counts, counts only one, the other It is better not to count. In such a case, duplication count can be reduced and counting accuracy can be increased.

By the way, the method shown in FIG. 6 is effective when the flow velocity in the central portion of the outflow passage D (see symbol L _{51 in} FIG. 6) and the flow velocity in the vicinity of the wall surface (see symbol L _{50 in} FIG. 6) are substantially equal. In reality, there are more cases where there is a difference between the flow rates. That is, when trying to flow a liquid in the outflow passage D at a slightly higher flow velocity, the flow velocity becomes slow due to the influence of the wall surface 27 in the portion close to the wall surface 27, and eventually the flow velocity L ₅₁ in the central portion of the outflow passage D is It becomes faster than the flow velocity L ₅₀ in the vicinity of the wall surface, and a large difference occurs between these flow velocity. In such a case (that is, when the flow velocity of the liquid flowing through the outflow path D changes from the vicinity of the wall surface to the center of the outflow path D), the fluorescence distribution measurement region is set to a constant width w as shown in FIG. ₁ , instead of making it a rectangular shape, the region width (see symbol w ₂₁ ) at the center of the outflow channel D is changed to the region width (see symbol w ₂₀ ) in the vicinity of the wall surface, as illustrated in FIG. It is better to set it wider. Then, the width w _x of the fluorescence distribution measurement region at a location x away from the wall surface 27 of the outflow channel D should satisfy the following equation. In such a case, the counting accuracy of the fluorescent particles can be increased even when the flow velocity changes from the vicinity of the wall surface of the outflow passage D to the central portion.

Specifically, the following equation should be used.

If the difference between the flow velocity L _{51 at} the central portion of the outflow passage D and the flow velocity L _{50 at the} vicinity of the wall surface is increased, the region width (reference symbol w) at the central portion of the outflow passage D is shown in FIG. ₃₁ reference), it may rather broadly set than the area width (reference numeral w ₃₀₎ in the near-wall portion.

Incidentally, even in a region K ₂ shown in FIG. 7 (a), a moment of passing through the upstream side (edge) 23 of the region K _2, is photographed with only the right half has entered the region K ₂ fluorescent particles 3 _1, the next photographing timing takes the position indicated by reference numeral 3 _2, after all, there, it would be shot twice, when counting both counting accuracy deteriorates. Therefore, in all of the still image shooting, the moment of fluorescent particles passing through the upstream side edge 23 in the region K ₂ (for example, fluorescent particles indicated by reference numeral 3 ₁₎ and downstream edge regions K ₂ (boundary moment of fluorescent particles passing through a line) 24 (e.g., reference numeral 3 ₂ instead of counting both fluorescent particles) showing, may be adapted to count only one. In such a case, duplication count can be reduced and counting accuracy can be increased.

By the way, in the areas K ₂ and K ₃ shown in FIGS. 7A and 7B, the upstream sides 23 and 25 are straight lines crossing the outflow path D, and the downstream edges 24 and 26 are on the downstream side. It is a protruding curve, and the regions K ₂ and K ₃ have a bullet-like shape (in other words, a shape like a baseball home base). When the upstream edges 23 and 25 are straight, it is easy to count the fluorescent particles at the moment of passing through the edges 23 and 25, which is preferable. However, if the above formulas for the region widths w ₂₀ and w ₂₁ are satisfied, the upstream edge may be a curved line instead of a straight line.

FIG. 1 is a schematic diagram showing an example of the structure of a microchip and a fluorescent particle counter according to the present invention. FIG. 2 is a schematic diagram showing how fluorescent particles are aligned by the inflow of the fourth liquid. FIG. 3 (a) is a schematic diagram for explaining a method of counting fluorescent particles, and FIG. 3 (b) is a waveform diagram showing a fluorescence intensity distribution along the gate 20 in FIG. 3 (a). . FIG. 4 is a block diagram showing an example of a conventional structure of the white blood cell counter. FIG. 5 is a schematic diagram showing an example of a conventional structure of an apparatus for mixing two or more kinds of liquids. FIG. 6 is a schematic diagram for explaining another method of counting fluorescent particles. FIG. 7 is a schematic diagram for explaining still another method of counting fluorescent particles.

Explanation of symbols

4 connections (adapter)
DESCRIPTION OF SYMBOLS 10 Light source 11 Fluorescence measuring means 14 Sampling means 15 Count means A Microchip B1 1st inflow path B2 2nd inflow path B3 3rd inflow path B4 4th inflow path C Mixing part D Outflow path E Fluorescent particle counting part L1 1st liquid L2 2nd liquid L4 4th liquid

Claims (14)

A first inflow path through which the first liquid flows,
A second inflow path through which the second liquid is introduced;
A mixing portion that is configured to bend and communicate with these inflow paths, and into which the first and second liquids are introduced and mixed;
An outflow passage arranged so as to communicate with the mixing section and through which the mixed liquid flows out;
A microchip constructed by providing The mixing portion is curved in a substantially zigzag shape,
The microchip according to claim 1. The microchip according to claim 1 or 2, wherein the first liquid containing particles to be counted and the second liquid containing a fluorescent dye are mixed in the mixing unit and the fluorescent particles are sent out to the outflow path,
A fluorescent particle counter for counting the fluorescent particles sent out;
A fluorescent particle counting apparatus configured by providing A third inflow path for supplying a dilution liquid for diluting the first liquid and the second liquid to the upstream side of the mixing unit;
The fluorescent particle counting device according to claim 3, comprising: A fourth inflow path for supplying a fourth liquid downstream of the mixing section and upstream of the outflow path;
Aligning the fluorescent particles by supplying the fourth liquid;
The fluorescent particle counting apparatus according to claim 3 or 4, wherein The fluorescent particle counting unit is composed of a light source for irradiating the fluorescent particles in the outflow path with excitation light, and a fluorescence measuring means for capturing the fluorescence generated by irradiating the fluorescent particles with the excitation light.
The fluorescent particle counter according to any one of claims 3 to 5, wherein The first inflow path is an inflow path through which a first liquid containing white blood cells is introduced,
The second inflow path is an inflow path into which a second liquid containing a surfactant for lysing the leukocyte cell membrane and a fluorescent dye for fluorescently staining the leukocytes is flowed.
The fluorescent particle counting device according to claim 3, wherein the fluorescent particle counting device is a fluorescent particle counter. The first inflow path has a connection part to which a blood collection device and a blood bag can be attached,
The fluorescent particle counter according to claim 7. Sampling means for obtaining the output of the fluorescence measuring means at regular intervals;
Counting means for counting fluorescent particles based on the output of the sampling means;
The fluorescent particle counting device according to claim 6, wherein the fluorescent particle counting device is provided. The fluorescence measuring means is a camera that sequentially acquires still images every predetermined time.
The fluorescent particle counter according to any one of claims 6 to 9, wherein The counting of the fluorescent particles by the counting unit is performed on a predetermined fluorescence distribution measurement region in each still image acquired by the fluorescence measuring unit,
The width w of the predetermined fluorescence distribution measurement region in the direction in which the fluorescent particles move satisfies the following formula:

The fluorescent particle counter according to claim 10. When the flow velocity of the liquid flowing through the outflow path varies from the vicinity of the wall surface to the center of the outflow path, the width w _x of the fluorescence distribution measurement region at a distance _x from the wall surface satisfies the following equation: ,

The fluorescent particle counter according to claim 11. The upstream edge in the fluorescence distribution measurement region is a straight line that crosses the outflow path, and the downstream edge in the region is a curve protruding to the downstream side, and the region is shaped like a bullet,
The fluorescent particle counting apparatus according to claim 12, wherein In the still image, the counting means is a fluorescent particle at an instant passing through an upstream edge in the fluorescence distribution measurement region, and a fluorescent particle at an instant passing through a downstream edge in the fluorescence distribution measurement region Configured to count only one of the other and not the other,
The fluorescent particle counting device according to claim 11, wherein the fluorescent particle counting device is a fluorescent particle counter.

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