JPH08122328A - Method and apparatus for measurement of function of red blood corpuscle - Google Patents

Abstract

PURPOSE: To measure the deformation capability of a red blood corpuscle due to a change in an osmotic pressure and due to a shearing stress simply, quickly and by one apparatus by a method wherein a diluent whose osmotic pressure and viscosity have been subjected to a gradient change is mixed with a blood sample, a stress is applied to the red blood corpuscle in a sample suspension and a diffraction image is obtained. CONSTITUTION: A blood pipette 41 is moved into a blood-corpuscle sample container 4 by means of a pipette movement mechanism 43, and a blood sample in a prescribed quantity is sucked by a syringe 49 so as to be injected into a dispensing hole 31 in a mixing part 14. At the same time, a hypotonic high-viscosity liquid and an isotonic high-viscosity liquid are sucked from containers 21, 22 by means of syringes 21b , 22b , they are sent out to cavities 21c , 21c in a mixing part 12 so as to be mixed, a diluent in which an osmotic-pressure characteristic is changed continuously is generated, the diluent is sent to the dispensing hole 31 via a monitor part 13 so as to be mixed 14 with the blood sample, a sample suspension which has been obtained is made to stay 32 for a prescribed time, the sample suspension is introduced into a pipe passage for a flow cell 16, and the shape of a red blood corpuscle is measured on the basis of the beam of diffracted light of the red blood corpuscle which has been caught by a CCD camera 63. Also the change capability due to the shearing stress of the red blood corpuscle can be measured by nearly the same operation inside the same apparatus.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a red blood cell function measuring method and measuring apparatus, and more particularly to a red blood cell function measuring method and measuring apparatus capable of performing a red blood cell deformability test.

[0002]

2. Description of the Related Art Conventionally, a red blood cell test has been performed as one of the methods for diagnosing various diseases. As a red blood cell test,
An erythrocyte osmotic pressure resistance test (hemolysis test) and a shear stress deformability test are known. The osmotic resistance test is a test in which erythrocytes are destroyed by the difference between the osmotic pressure inside the erythrocytes and the osmotic pressure surrounding the erythrocytes. The piezoresistance, ie the fragility of red blood cells, can be measured. This can lead to some inferences about health or physiological status. Further, this test depends on the erythrocyte morphology and erythrocyte viscoelasticity, and is an extremely important test for the differential diagnosis of spherocytosis.

On the other hand, the shear stress deformability test is a test for observing the morphology of red blood cells which changes due to an external force. Erythrocytes generally have a shape of a hollow disc having a diameter of about 8 μm when the external force is removed, but the shape can be easily changed by a small external force. It is known that the deformability of erythrocytes decreases depending on the presence or absence of disease, and depends on the solubility and concentration of hemoglobin inside erythrocytes and the viscoelasticity of erythrocyte membranes (ATP amount, free cholesterol, spectrin properties of membrane protein). ing.

[0004]

The erythrocyte osmotic pressure resistance test is conducted in the range of 0.1% to 0.1% with a pH 7.4 buffer solution added.
Put erythrocytes in a 9% saline series test tube and keep at 30 ℃ at 30 ℃.
A method of measuring the hemoglobin concentration in the supernatant by centrifuging after standing still for a minute is known. In addition, a thin tube wound in a coil was filled with saline with a concentration gradient, a blood sample was added, and the mixture was allowed to stand at 37 ° C for 10 minutes, and then red blood cells were moved into the thin tube by a rotation centrifuge. Then, by measuring the red blood cell hemolysis site,
Red blood cell osmotic pressure resistance (CPC method, Coli Planet Centrifu
A method of measuring ge) is known.

However, in the former test, it is necessary to prepare several kinds of saline solutions having different concentrations, and
It is necessary to perform centrifugation or to measure the supernatant after centrifugation with a spectrophotometer, and the operation is complicated, and when treating a large number of samples, it is necessary to continuously perform particularly complicated operation. In the latter CPC method, the steps of filling a thin tube with a saline solution having a concentration gradient, operating by a revolving centrifuge, and operating a photometer for measuring the red color of hemoglobin hemolyzed in the tube are performed. Yes, and involves a very complicated operation. Both the former and the latter require a long time for the test.

On the other hand, as a method for measuring the deformability of red blood cells due to shear stress, conventionally, a red blood cell suspension is placed in the cylindrical gap of a double cylinder rotational viscometer and the outer cylinder is rotated to generate shear stress. An apparatus (trade name: ectocytometer) that measures the deformability of red blood cells by deforming the red blood cells, irradiating laser light and analyzing the diffraction image thereof is used. However, it takes a lot of time to clean the inside of the double-cylinder rotational viscometer for each measurement, and since the double-rotary cylinder is rotating, the position irradiated with the laser light continues to move relatively, and the rotation There is a defect that the diffraction image changes due to distortion and dirt of the cylinder.

Further, in the above-mentioned erythrocyte test method, since the device for measuring the deformability of red blood cells due to osmotic pressure change and the device for measuring deformability of red blood cells due to shear stress are separate, both measurements are combined. If a multi-faceted diagnosis is to be performed by doing so, the measuring device becomes complicated and the operation becomes complicated. Also, the flow rate of the solution used is 25
Since the amount is as small as 0 μl / sec, it is difficult to prepare a liquid having an osmotic pressure and a viscosity gradient by simply mixing. An object of the present invention is to provide a erythrocyte function measuring method and a device thereof capable of measuring the deformability by osmotic pressure change of red blood cells and the deformability by shear stress in a short time by a simple operation using one device. is there.

[0008]

According to the present invention, a sample solution containing red blood cells, a diluting solution whose osmotic pressure is gradually changed at a constant viscosity or a diluting solution whose viscosity is gradually changed at a constant osmotic pressure is provided. Are mixed with each other, stress is applied to the red blood cells in the obtained sample suspension, and then the stressed red blood cells are irradiated with light to obtain a diffraction image, and the deformability of the red blood cells is measured from this diffraction image. A method for measuring red blood cell function is provided.

According to the present invention, a plurality of liquid delivery means for supplying diluting liquids having different viscosities and / or osmotic pressures, a selection drive means for selectively driving the plurality of liquid delivery means, and a liquid delivery means. Of the sample suspension obtained in the second mixing section, a first mixing section for mixing the different diluents sent from the second mixing section, a second mixing section for mixing the mixed diluent and the sample solution containing red blood cells, Stress applying means for applying stress to red blood cells, diffractive image forming means for irradiating the stressed red blood cells with light to form a diffraction image, imaging means for imaging the diffraction image, and the diffracted imaged There is provided an erythrocyte function measuring device provided with an analyzing means for analyzing the deformability of erythrocytes from an image. A method for testing deformability of red blood cells by changing osmotic pressure or shear stress.

The measurement of deformability of red blood cells in the present invention refers to both the measurement of deformability by osmotic pressure change of red blood cells and the measurement of deformability by shear stress, and it is preferable that these measurements are performed in one device. The diluent used in the present invention has a constant viscosity in the range of about 5 to 25 cP in the case of measuring deformability by osmotic pressure change of red blood cells.
It is preferable that the osmotic pressure in the range of about 300 to 30 mOsm changes the slope. Where the isotonic osmotic pressure is 300 ±
It is defined as 50 mOsm.

The diluting solution used in the present invention has a constant osmotic pressure in the range of about 250 to 350 mOsm and about 1 when it is used for measuring the deformability of red blood cells due to shear stress change.
It is preferable that the viscosity changes in the range of 25 to 25 cP. The gradient-diluted diluent in the present invention means, for example, when a deformability test of erythrocytes by a change in osmotic pressure is performed, for example, a liquid having the above-mentioned high viscosity and hypotonic osmotic pressure is continuously added at a constant ratio. And isotonic with high viscosity, that is, the liquid is adjusted so as to be continuously supplied at a constant rate while continuously decreasing the liquid on the high osmotic pressure side at a constant rate. The respective inclination directions are not limited to the above, and the opposite cases are possible. Further, in the case of carrying out the deformability test of red blood cells by the change of shear stress, for example, the above-mentioned osmotic pressure isotonic and highly viscous liquid is continuously increased at a constant rate, and osmotic pressure is isotonic and low. It is a liquid adjusted so as to be supplied while keeping the total flow rate constant while continuously decreasing the viscosity liquid at a constant rate. The respective inclination directions are not limited to the above, and the opposite cases are possible.

The application of stress in the present invention refers to applying an external force to red blood cells in order to observe the morphology of red blood cells that changes due to an external force. For example, a flow cell having a passage with an inner diameter of about 0.2 mm contains red blood cells. A method of flowing a sample suspension and applying shear stress to red blood cells is preferable. The flow cell preferably has a rectangular cross section, and the aspect ratio of the flow path cross section is preferably 5 or more, and particularly preferably 10 from the viewpoint of imparting stress to blood cells. In the present invention, laser light is preferably used as the light with which red blood cells are irradiated to obtain a diffraction image. The types of laser light are
The light flux is not particularly limited as long as it does not spread.

In the present invention, it is preferable that the second mixing section has a retention section for retaining the sample suspension until the osmotic pressure reaches equilibrium. Second in this invention
It is preferable that the mixing section is provided with a turbulent flow generating means for generating a turbulent flow in the sample suspension and uniformly mixing the sample suspension. Here, the turbulent flow generating means includes, for example, a device in which a flow path filled with fine particles is provided in the mixing portion and a turbulent flow is generated by increasing the flow velocity when passing through a slight gap between the particles. In the present invention, the diluting liquid sent out by any one of the plurality of liquid sending means contains a dye, and the mixing ratio detecting means for detecting the mixing ratio of the dye-containing diluting liquid and the other diluting liquid by measuring the dye concentration. Is preferably provided. The mixture ratio detecting means may be, for example, an optical detecting means including an LED and a photodiode.

In the present invention, the image pickup means preferably comprises a CCD camera for picking up a diffraction image. In the present invention, it is preferable that the analyzing means calculates the red blood cell deformation index by analyzing the major axis and the minor axis of the imaged diffraction image. The red blood cell deformation index is defined as DI = (A−B) / (A + B), where the major axis is A and the minor axis is B when the imaged diffraction image is elliptical. I mean something. In the present invention, the stress applying means is preferably a flow cell that allows red blood cells in the sample suspension to pass through. The flow cell preferably has the above-mentioned predetermined aspect ratio.

It is preferable that the red blood cell function measuring device is provided with a liquid delivery means for delivering a liquid having a low viscosity and a low osmotic pressure with a variable flow rate. As a result, the conventional erythrocyte deformability test with a low-viscosity isotonic isotonic diluent and a low-viscosity hypotonic diluent is performed under the above-mentioned high-viscosity osmotic pressure or shear stress. It can be done in one device together with the variability test of red blood cells by changes.

[0016]

When a deformability test is performed by changing the osmotic pressure of red blood cells, two types of liquids having the same viscosity but different osmotic pressures, for example, the first and second liquids are delivered while continuously changing the flow rate ratio. Send out from the means. For example, the liquid delivery means is driven so that the total flow rate is constant and the first liquid is continuously increased at a constant ratio, and the second liquid is continuously decreased at a constant ratio. Next, these are delivered by the liquid delivery means and mixed in the first mixing section. Next, a blood sample is added to the mixed solution at a constant ratio in the second mixing section to form a blood cell suspension. In the retention section of the second mixing section, the blood cell suspension is retained until the osmotic pressure reaches equilibrium. At low osmotic pressure, red blood cells become spherical and increase in volume. The blood cell suspension is guided to a flow cell having a large aspect ratio and irradiated with laser light. This produces a scattered diffraction image.

The scattering diffraction image changes depending on the degree of deformation of red blood cells. The deformability (area) of red blood cells based on the change in osmotic pressure is measured by capturing the scattered diffraction image with a CCD camera and processing the captured diffraction image. By performing the above-described measurement while continuously changing the flow rate ratio of the two kinds of liquids in this manner, the deformability test of red blood cells with osmotic pressure as a parameter can be performed. When the deformability is measured by changing the osmotic pressure in a highly viscous state, a large shear stress can be applied, so an accelerated test can be performed and the measurement can be speeded up.

In the erythrocyte function measuring method according to the present invention, when the deformability test is performed by changing the shear stress of erythrocytes, first, two kinds of liquids having the same osmotic pressure and different viscosities, for example, the second and the third liquids are flow rate ratios. Is continuously changed while the liquid is delivered from the liquid delivery means. The liquid delivery means is driven so that the second liquid is continuously increased at a constant rate and the third liquid is continuously decreased at a constant rate while keeping the total flow rate constant. Next, these are delivered by the liquid delivery means and mixed in the first mixing section. Next, a blood cell sample is added to the mixed solution at a constant ratio in the second mixing section to obtain a blood cell suspension. Next, the blood cell suspension is introduced into a flow cell having a large aspect ratio. In the flow cell, erythrocytes undergo a morphological change by being subjected to a stress (shear stress) determined by the flow velocity and viscosity of the mixed solution, and deform. The flow cell is irradiated with laser light, and the scattered diffraction image thereof changes depending on the degree of deformation of red blood cells. The deformability (deformation index) of red blood cells is measured by capturing the scattered diffraction image with a CCD camera and processing the captured diffraction image. As described above, by performing the above-described measurement while continuously changing the flow rate ratio of the two types of suspensions, the deformability of erythrocytes with the viscosity as a parameter can be measured.

It is considered that the stress applied to red blood cells can be changed by changing the flow velocity without changing the viscosity, but a large amount of liquid must be introduced to the flow cell in order to apply a large stress. Further, it is difficult to maintain the blood cell concentration in the blood cell suspension constant, and the flow rate of blood cells in the laser light irradiation region is not constant, which causes variations in measurement conditions. Therefore, the above problem does not occur in the method of the present invention for changing the viscosity.

In the erythrocyte function measuring apparatus according to the present invention, the mixed liquid is delivered to the second mixing section in any of the deformability tests described above. At this time, if the dye premixed in the liquid is monitored by the mixing detection means, the osmotic pressure or viscosity of the liquid can be confirmed by the state of the liquid at the moment of measurement. That is, if the relationship between the osmotic pressure or viscosity and the dye concentration, and the relationship between the dye concentration and the absorbance are obtained by calculation and experiment, the osmotic pressure or viscosity can be obtained in real time from the absorbance. This makes it possible to determine whether or not the mixture has been made according to the setting.

The two liquids delivered are mixed with the sample liquid containing blood cells delivered by the sample liquid delivery means in the second mixing section to form a sample suspension. If the second mixing section has a retention section,
The sample suspension reaches equilibrium before being delivered to the flow cell. Therefore, the measurement can be performed under the optimum conditions. Furthermore, if the second mixing section has the turbulent flow generating means as described above, highly efficient mixing is possible even if the volume of the mixing section is reduced. It is more preferable that the turbulent flow generating means is one filled with fine particles. Further, in the device of the present invention, for example, a fourth liquid having a low viscosity and a low osmotic pressure, and a third liquid having a low viscosity and a higher osmotic pressure than the first liquid are selected and delivered in the same manner as above. For example, a deformability test can be performed by changing the osmotic pressure of red blood cells under low viscosity. As a result, the deformability test based on the osmotic pressure change of red blood cells under low viscosity, which is conventionally performed, can be performed by one device together with the above-described deformability test under high viscosity.

[0022]

1 shows an apparatus for measuring red blood cell function according to an embodiment of the present invention. The red blood cell function measuring device 10 includes a liquid supply means 11 for supplying a diluting liquid, a first mixing part 12, a monitor part 13, a second mixing part 14, a liquid sending part 15, and a flow cell 16.
And a measuring unit 17. The liquid delivery means 11 is a container 21.
-24, solenoid valves 21a-24a and syringes 21b-2
4b is connected by piping. Syringe 21b
24b have drive sources not shown. Thereby, up to four types of suspensions can be delivered to the downstream side of the syringes 21b to 24b. Syringe 21b-24b
The first mixing unit 12 is connected to the downstream side of the.

The first mixing section 12 has cavities 21c-2c.
4c and solenoid valves 21d to 24d.
The cavities 21c to 24c are formed in a constant temperature block 26 having a heater 25, as shown in FIG. A monitor unit 13 is connected to the downstream side of the first mixing unit 12. The monitor unit 13, as shown in FIG.
It comprises an ED 27, a photodiode 28 and a holder 29. The holder 29 is made of a light shielding material and coaxially fixes the LED 27 and the photodiode 28. LE
The first mixing unit 1 is provided between the D27 and the photodiode 28.
The piping 30 derived from 2 is arranged. Piping 30
Is a transparent tube made of Teflon (PTFE), and is arranged so as to penetrate the holder 29. With such a configuration, when the liquid passing through the monitor unit 13 contains a dye described later, this liquid can be monitored. The second mixing unit 14 is connected to the downstream side of the monitor unit 13.

As shown in FIG. 4, the second mixing section 14 is mainly composed of a dispensing hole 31 and a retention section 32 for retaining the passing liquid for an arbitrary time. Dispensing hole 3
1 has a diameter through which the tip of a pipette 41 for blood dispensing described later can be inserted. A retention portion 32 is connected to the lower end of the dispensing hole 31. The retention section 32 is mainly composed of a coil-shaped thin tube 33 and beads 34 before and after the thin tube 33 as turbulent flow generating means. Beads 3
4 is fixed by stoppers 35 in the front and rear in the liquid feeding direction. The beads 34 are preferably made of a material that does not deteriorate or adversely affect blood, such as glass coated with silicon, alumina, or zirconia. A liquid feeding unit 15 is arranged above the second mixing unit 14 in the drawing (FIG. 1).

In this embodiment, the beads 34 are filled as the turbulent flow generating means, but other means such as, for example,
It is also possible to use a method of repeatedly shrinking and expanding the flow path, or a method of filling a filler having an irregular shape. The liquid feeding unit 15 includes a pipette 41 for dispensing, a holder 42 that holds the pipette 41, and a pipette moving mechanism 43 that moves the holder 42 in two axial directions. The pipette moving mechanism 43 includes air cylinders 44 and 45 that move the holder 42 horizontally and vertically, and switching valves 46 and 47 that control the air cylinders 44 and 45.
It is mainly composed of The pipette moving mechanism 43
The tip of the pipette 41 can be moved between the dispensing hole 31 and the blood cell sample container 48 arranged near the dispensing hole 31. The switching valve 46 is for horizontal movement,
It is an air cylinder switching valve for moving the switching valve 47 in the vertical direction.

The upper end of the pipette 41 is connected to the discharge side of the first syringe 49 for dispensing the sample solution containing blood cells. The first syringe 49 has a drive source (not shown). The suction side of the first syringe 49 has a solenoid valve 51 and a pipe 52.
It is connected to the discharge side of the cleaning liquid hermetic container 53 via. One end of a pipe 54 is opened inside the closed container 53. The other end of the pipe 54 is connected to a pressure source 56 via a valve 55. A branch pipe 57 is connected to the pipe 52. The pipe 57 is connected to the first mixing unit 12 via the solenoid valve 58.
It is connected to the. With such a configuration, the pipette 4
1 can dispense the blood sample in the blood cell sample container 48 into the dispensing hole 31, and can inject the cleaning liquid stored in the closed container 53 into the dispensing hole 31. A flow cell 16 is arranged below the second mixing section 14.

As shown in FIG. 5, the flow cell 16 is composed of a transparent rectangular parallelepiped block, and has a conduit 59 having a rectangular cross section about the longitudinal direction of the block. The conduit 59 has a sectional shape of 2 mm × 0.2 mm, that is, an aspect ratio value of 10 (FIG. 1).
A measuring unit 17 is arranged before and after the flow cell 16. The measuring unit 17 includes a helium-neon laser 61 as a light emitting means, a screen 62, a CCD camera 63,
The image processing unit 64 is mainly included. Laser 61
Are arranged so that the optical path axis is orthogonal to the longitudinal direction of the cross section of the conduit 59. The screen 62 is arranged to face the irradiation portion of the laser 61 with the flow cell 16 interposed therebetween. As shown in FIG. 6, the screen 62 is provided with a direct light blocking portion 62a capable of blocking direct light having high intensity. The position of the direct light shielding portion 62a is adjusted so as to directly shield the light. As a result, the laser light emitted from the laser 61 is scattered and diffracted according to the shape of the red blood cells flowing in the cell 16, and even weak scattered light is scattered on the screen 6.
2 is projected.

The projected scattered diffracted light image is displayed on the screen 6.
The image is captured by the CCD camera 63 installed at the rear of the
Images are obtained every 1/30 second. The picked-up image is binarized at an appropriate level, and is taken into the image processing unit 64 as input data. The image processing unit 64 performs image processing based on the input data. The result is displayed on the CRT 70 connected to the image processing unit 64. On the other hand, below the flow cell 16, a drainage chamber 66 is connected via an electromagnetic valve 65. Further, a fluid drive unit including an electromagnetic valve 67 and a pump 68 is connected to the drainage chamber 66. With such a configuration, a predetermined amount of the sample liquid and the cleaning liquid can be circulated from the retention section 32 to the flow cell 16.

FIG. 7 is a block diagram of the red blood cell function measuring device 10. The red blood cell function measuring device 10 includes a CPU,
It has a control unit 71 including a microcomputer having a ROM, a RAM, a timer, a counter and the like. The control unit 71 includes a key input unit 72 and a photodiode 28.
Is connected. In addition, the control unit 71 includes the solenoid valves 2
1a, 22a, 23a, 24a, 51, 55, 58, 6
7, 21d, 22d, 23d, 24d, syringe 21
c, 22c, 23c, 24c, pumps 46, 47, LE
D27, laser 61, CCD camera 63, heater 25,
The CRT 70 and the other input / output unit 73 are connected.

Next, the red blood cell function measuring device 1 of this embodiment
The operation of 0 will be described based on the flowchart of FIG. In step S1, initial setting is performed. Step S2
Then, the 1st mixing process as a liquid supply process is performed. In the first mixing step, at least two types of diluents stored in the containers 21 to 24 are mixed. In step S3, a second mixing process as a sample suspension mixing process is executed. In the second mixing step, the diluent prepared in the first mixing step and the sample solution containing blood cells are mixed.

A specific example of steps S2 and S3 will be described with reference to the flowchart of FIG. First, as preparation, four kinds of diluting liquids as the first to fourth liquids, that is, containers 21 to 24 respectively containing a hypotonic high viscosity liquid, an isotonic high viscosity liquid, an isotonic low viscosity liquid, and a hypotonic low viscosity liquid Set. In this device 10, a hypotonic high-viscosity liquid and an isotonic high-viscosity liquid are selectively used when the deformability test by changing the osmotic pressure of red blood cells is performed under high viscosity. Further, when performing a deformability test by changing the shear stress of red blood cells, an isotonic high viscosity liquid and an isotonic low viscosity liquid are selectively used. Furthermore, when the deformability test by changing the osmotic pressure of red blood cells is performed under low viscosity, an isotonic low viscosity liquid or a hypotonic low viscosity liquid is selectively used.

In this example, isotonic means an osmotic pressure of 3
00 ± 50 mOsm and hypotonicity are defined as 30 ± 25 mOsm.
High viscosity is defined as 5 to 25 c.P. (centipoise), and low viscosity is defined as 1.0 to 2.0 c.P. These solutions are phosphate buffer or Hepes buffer (5-40 mM, p
When the viscosity is increased based on H7.40), polyvinylpyrrolidone (average molecular weight about 360,000) or dextran (average molecular weight about 40,000) added to have an appropriate viscosity is used. The osmotic pressure is adjusted by the concentration of sodium chloride. The viscosity of the liquid is 5-25c.
When it exceeds P., it becomes difficult to transfer the liquid.

It is preferable that one of the four types of the above liquids contains a dye. The dyes to be added must meet the conditions that the erythrocytes do not change their morphology, the erythrocyte mean volume (MCV) does not change, they are water-soluble, and their absorbance changes sufficiently at low concentrations. In this example, sulfoledamine-B, astrazone red 6B, diallyl brilliant pink RN and brilliant pink 6B are preferred. In FIG. 9, first,
In step S11, the measurement items are initialized. Here, the input is made from the key input unit 72. First, the case where the deformability test by changing the osmotic pressure of red blood cells is performed under high viscosity will be described. In this case, the diluting liquid used is a hypotonic high-viscosity liquid and an isotonic high-viscosity liquid among four types of diluting liquids. In one of the above liquids, for example, the dye is 0.
About 01 w / v% is added. When the initial setting is completed, the process proceeds to step S12.

In step S12, the pipette moving mechanism 4
3 is driven to move the tip of the pipette 41 into the blood cell sample container 48. In step S13, the first syringe 49
Is absorbed. A predetermined amount of blood sample solution is stored in the first syringe 49.
When it is sucked in, the driving of the first syringe 49 is stopped in step S14. Next, in step S15, the pipette moving mechanism 43 is driven to move the tip of the pipette 41 to the dispensing hole 31 of the second mixing section 14.

Next, in step S16, the solenoid valve 21a
open. Next, in step S17, the second syringe 21
Drive b by suction. Next, in step S18, the solenoid valve 21a is closed and the solenoid valve 21d is opened. Step S above
At about the same time as 16, the solenoid valve 22a is opened in step S19. Next, in step S20, the third syringe 22b
Drive suction. As a result, a predetermined amount of liquid is injected into each syringe 21b, 22b. Next, step S21
Then, the solenoid valve 22a is closed and the solenoid valve 22d is opened.

Next, in step S22, the second syringe 21b and the third syringe 22b are driven to discharge. At this time, the two syringes discharge each liquid so that the total flow rate is constant and the flow rate ratio continuously changes with time. That is, the flow rate of one liquid gradually increases and the flow rate of the other liquid gradually decreases. Each discharged liquid is stored in the cavity 21.
It is sent to c and 22c, and two kinds of liquids are mixed. In this way, a diluting liquid whose osmotic pressure characteristics change continuously can be prepared. Next, in step S23, the monitor unit 13 monitors the liquid mixed in the first mixing unit 12. Here, the liquid prepared in the first mixing unit 12 by the dye contained in the liquid in advance becomes a liquid in which the dye concentration continuously changes. This suspension is the monitor unit 13.
When it flows through the pipe 30, the change in the concentration of the dye is monitored as the change in the absorbance. If the relationship between the dye concentration and the flow rate ratio is investigated beforehand, the properties of the flowing liquid can be confirmed in real time.

Next, in step S24, the first syringe 49 is driven to discharge. As a result, a predetermined amount of blood sample solution is injected into the dispensing hole 31 of the second mixing section 14. Then, the blood sample solution and the diluting solution are mixed and guided to the retention section 32. Next, in step S25, it is determined whether or not a predetermined retention time has elapsed in the retention section 32. Retention part 3
At 2, the erythrocytes change due to osmotic pressure and the sample suspension stays until this change reaches equilibrium. When the predetermined residence time has elapsed, the process proceeds to step S4 in the flowchart of FIG. In step S4, the suspension that has passed through the retention section 32 is introduced into the conduit 59 of the flow cell 16, and the morphology of red blood cells is measured from the diffraction image of red blood cells.

First, the second or third syringe 21b, 2
By driving the suspension 2b to drive the suspension into the flow cell 16, a predetermined shear stress is applied to the suspension in the conduit 59, that is, a surface / volume and an internal viscosity which are erythrocyte deformability regulating factors due to osmotic pressure change. The external force acts to deform the red blood cells that have changed. As a result, red blood cells are deformed by the shear stress. The light emitted from the laser 61 causes a scattered diffracted light image of the deformed red blood cells to be projected on the screen 62. The scattered diffracted light image captured by the CCD camera 63 is image-processed in real time by the image processing unit 64, and the degree of deformation of red blood cells is continuously obtained in time. The degree of deformation of red blood cells can be obtained, for example, from the deformation index. As shown in FIG. 10, when the captured diffraction image has an elliptical shape as shown in the figure, assuming that the major axis is A and the minor axis is B, the deformation index DI is defined by the following equation.

DI = (A−B) / (A + B) [Equation 1] In this high viscosity osmotic pressure deformability test, as described above, among the four types of diluents, the hypotonic high viscosity liquid and Uses an isotonic high viscosity liquid. Since these liquids have high viscosities, large shear stress acts on the red blood cells flowing through the cells 16. When the osmotic pressure is low, erythrocytes change from their original shape of a hollow disc into a spherical shape, and their volume increases, making them difficult to deform. Therefore, the deformation index DI becomes small. Monitor unit 1
A high-viscosity hemolysis curve can be obtained by graphing this deformation index using the osmotic pressure obtained by the absorbance method in 3 as a parameter (FIG. 12). A measurement example is shown in FIG. further,
Differentiating FIG. 12 results in FIG. In the figure, they are named A: maximum deformability point, B: maximum change point of deformability, and C: minimum point of deformability.

When the measurement in step S4 is completed, the process proceeds to step S5. In step S5, the pipette 4
1, 1st and 2nd mixing parts 12, 14 and flow cell 16
Wash. First, the piston of the first syringe 49 is returned to the suction position. Next, the solenoid valves 51, 55, 58 are opened and the pump 56 is driven. As a result, the cleaning liquid 53
Flows through the pipe 52, the first syringe 49 and the pipette 41 into the second mixing section 14. In addition, the cleaning liquid 53
Flows to the second mixing section 14 through the pipes 52 and 30. The cleaning liquid 53 that has passed through the second mixing section 14 passes through the flow cell 16 and is stored in the drainage chamber 66. When the cleaning liquid 53 is stored in the drainage chamber 66, the solenoid valve 67
Is opened and the pump 68 is driven to discharge the cleaning liquid 53 out of the system. When the cleaning is completed, the process proceeds to step S6 to wait for the next measurement and perform other processes.

[When Performing Deformability Test by Shear Stress Change of Red Blood Cells] In this test, an isotonic high-viscosity liquid of the container 22 and an isotonic low-viscosity liquid of the container 23 are used among the four types of suspensions. To do. A dye is added to the container 24. In this case, the viscosity is a parameter in the isotonic state. When the viscosity is high, shear stress acts on the red blood cells, resulting in a stretched shape. The scattered diffraction image in this case also has a similar elliptical shape. When the same amount of shear stress is applied, how much is elongated depends on the deformability of red blood cells themselves. In other words, the deformability of red blood cells can be examined by examining the degree of deformation. The shear stress curve can be obtained by continuously calculating the deformation index using the viscosity obtained by the absorbance method by the monitor unit 13 as a parameter and plotting it into a graph (FIG. 17). 14 to 16 show measurement examples. FIG. 14 is a deformation index, FIG. 15 is an area of a diffraction image, and FIG.
Is the measurement of the major axis and the minor axis. The shear stress is used as a parameter. Differentiating FIG. 17 results in FIG. In the figure, A: Deformation end shear stress, B: Change maximum shear stress, C: Change start shear stress are named.

[When Deformability Test by Changing Osmotic Pressure of Red Blood Cells is Performed at Low Viscosity (Conventional Test)] In this test, 4
Of the types of suspensions, the isotonic low viscosity liquid in container 23 and the hypotonic low viscosity liquid in container 24 are used. In this case, the osmotic pressure is a parameter in the isotonic state. When the osmotic pressure is low, the red blood cells become spherical from the original hollow disk shape, which is a hollow disc, to increase their volume. When the osmotic pressure is isotonic, red blood cells have a hollow disc shape and flow in various directions in the flow, so the diffraction image on the screen is circular. When the osmotic pressure decreases, red blood cells become spherical and increase in volume. The spherical scattering makes the random scattering uniform and reduces the size of the diffraction image. The hemolysis curve in FIG. 22 can be obtained by graphing the size area of this diffraction image using the osmotic pressure (which can be obtained from the monitor by the absorbance method) as a parameter. The horizontal axis shows the osmotic pressure decreasing from left to right. Further, in this case, the perimeter and the diameter are also important information (which can be area information). FIG. 19 shows the area and FIG.
FIG. 21 shows a graph of measurement examples of the perimeter and FIG.

The area graph of FIG. 22 will be described as an example. Here, A: hemolysis start point, B: hemolysis peak point, C: 50% hemolysis point, and D: hemolysis end point. It can be seen whether the resistance of the membrane is increased or decreased depending on whether these points shift to the low osmotic pressure side or the high osmotic pressure side. As described above, in the erythrocyte function measuring method according to the above-described embodiment, four kinds of liquids having different osmotic pressures or viscosities can be arbitrarily selected, and the mixing ratio thereof can be arbitrarily continuously changed. The sex test and various red blood cell deformability tests can be performed continuously. Furthermore, in the deformability test by osmotic pressure change, the osmotic pressure is changed under high viscosity. Therefore, the influence of the change in the surface area / volume ratio (ie, spheroidization), which is the erythrocyte deformability regulating factor due to the osmotic pressure change, and the internal viscosity There is an advantage that the effect of changes (that is, changes in hemoglobin concentration in red blood cells) can be understood. Also, osmotic pressure resistance test (hemolysis test) that changes osmotic pressure under low viscosity as in the past
Can be done together.

On the other hand, since the erythrocyte deformability test can be performed with isotonicity and high viscosity, there is an advantage that the deformability under physiological osmotic pressure, that is, the deformability of red blood cells in the living body can be estimated.
In the erythrocyte function measuring apparatus 10, since the containers 21 to 24 containing four kinds of diluting liquids having different osmotic pressures or viscosities are connected to the first mixing section 12 so as to be selectable, any dilution can be performed by switching the piping. The liquid and the blood cell sample liquid can be mixed. Therefore, it becomes easy to grasp the functions of red blood cells from various aspects with a simple structure. Further, since the washing liquid can be delivered to the flow path using the pipette 41 into which the blood cell sample has been dispensed after the completion of the predetermined test, the complexity of the washing operation is eliminated, and the test is continuously performed using different parameters. can do.

In this device 10, since the second mixing section 14 has the retention section 32, the sample suspension can be delivered to the flow cell 16 after the osmotic pressure of the blood suspension reaches equilibrium. it can. Therefore, the measurement accuracy can be improved. Since the monitor unit 13 can monitor the osmotic pressure and the viscosity of the mixed suspension based on the concentration of the dye added to the suspension, the accurately mixed suspension can be delivered to the flow cell 16. Therefore, the measurement accuracy can be improved.

Since the measuring unit 17 has a CCD camera 63 for picking up a scattering diffraction image from the laser 61, the size and area of the scattering diffraction image can be output as data and easily digitized. You can Since the flow cell 16 has a large aspect ratio, shear stress can be effectively applied to blood cells. Further, unlike the conventional ectocytometer using a double cylinder rotational viscometer, cleaning the inside does not take time. Further, since the screen 62 is fixed, the diffraction image does not change due to rotational distortion, stains, etc., unlike the case where the above-described rotation meter is used. Furthermore, unlike the conventional hemolysis test, it is not necessary to prepare several kinds of saline solutions having different concentrations, and it is complicated to perform centrifugation or to measure the supernatant after centrifugation with a spectrophotometer. No operation required. Also, like the CPC method,
The problem that only erythrocyte osmotic pressure resistance can be measured is solved, and a large number of specimens can be rapidly processed.

[0047]

In the red blood cell function measuring method according to the present invention, the deformability test is carried out by changing the osmotic pressure of red blood cells by mixing the sample solution containing blood cells and the diluting solution whose osmotic pressure is gradually changed at a constant viscosity. It is possible to perform a deformability test by changing the shear stress of erythrocytes by mixing a sample solution containing blood cells with a diluting solution whose viscosity is gradually changed at a constant osmotic pressure. Since these tests are performed by one device, the test device can be simplified and the operation can be facilitated. Furthermore, if the erythrocyte function measuring device is provided with a liquid delivery means for delivering a diluting liquid having low viscosity and low osmotic pressure, the conventional osmotic pressure deformability test (hemolysis test) that changes the osmotic pressure under low viscosity The test) can also be performed with one device. If the above deformability test is performed by mixing a sample solution containing blood cells with a diluting solution having a high viscosity and an osmotic pressure gradient changed, an accelerated test becomes possible and the measurement time can be shortened.

In the deformability test based on change in osmotic pressure, the osmotic pressure is changed under high viscosity. Therefore, the change in the surface area / volume ratio, which is the erythrocyte deformability regulating factor due to the change in osmotic pressure (ie,
There is an advantage that the effects of spheronization) and changes in internal viscosity (that is, changes in hemoglobin concentration in red blood cells) can be understood. On the other hand, since the erythrocyte deformability test can be performed with high viscosity under isoosmotic pressure, there is an advantage that the deformability under physiological osmotic pressure, that is, the deformability of red blood cells in the living body can be estimated. Second
If the mixing section is provided with a retention section that retains the prepared sample suspension until the osmotic pressure reaches equilibrium, the sample suspension with well-conditioned conditions can be delivered to the flow cell. Furthermore, if the second mixing section is provided with the turbulent flow generating means, it becomes possible to mix the diluent having a small flow rate and high viscosity so as to form a predetermined gradient change. In particular, if the turbulent flow generating means is provided with a mixing section filled with minute particles, efficient mixing can be achieved by the turbulent flow passing through the gaps between the particles. Thus, not only the erythrocyte deformability test, but also the deformability test by osmotic pressure change and the erythrocyte osmotic pressure resistance test can be performed with one device. In addition, the measurement accuracy of the erythrocyte osmotic pressure resistance test can be improved.

The dye is dissolved in at least one of the two liquids to be mixed among the plurality of diluting liquids, and the mixing ratio for detecting the mixing ratio of the liquids mixed in the first mixing portion due to the change in the concentration of the dye is mixed. If the ratio detecting means is provided, the characteristics of the liquid, that is, the viscosity and the osmotic pressure can be monitored from the color of the liquid mixed in the first mixing section, so that more accurate measurement can be performed. Since the image pickup means is provided with a CCD camera for picking up a diffraction image, numerical processing of the diffraction image can be easily performed.
Since the analysis means calculates the red blood cell deformation index by analyzing the major axis, the minor axis, the perimeter, and the area of the captured scattering diffraction image, the electrical detection method as in the conventional method or the visual observation is performed. Even without it, accurate and quick measurement is possible.

[Brief description of drawings]

FIG. 1 is a piping diagram of a red blood cell function measuring device according to an embodiment of the present invention.

FIG. 2 is a schematic view of a first mixing unit that constitutes the red blood cell function measuring device of FIG.

FIG. 3 is a schematic view of a monitor unit that constitutes the red blood cell function measuring apparatus of FIG.

FIG. 4 is a schematic view of a second mixing section which constitutes the red blood cell function measuring device of FIG.

5 is a schematic view showing a laser beam and a screen which pass through a flow cell which constitutes the red blood cell function measuring apparatus of FIG.

6 is a front view of the screen of FIG.

FIG. 7 is a block diagram of a control unit of the red blood cell function measuring device.

FIG. 8 is a flowchart of a control unit of the red blood cell function measuring device.

9 is a flowchart of the mixing process of FIG.

FIG. 10 is an explanatory diagram for explaining a red blood cell deformation index.

FIG. 11 is a graph showing a measurement example of a deformability test based on a change in osmotic pressure.

FIG. 12 is a graph of a deformability curve obtained by a deformability test based on changes in osmotic pressure.

FIG. 13 is a graph obtained by differentiating the deformability curve by osmotic pressure change in FIG.

FIG. 14 is a graph showing a measurement example of a deformability test due to a change in shear stress, showing a relationship between a red blood cell deformation index and viscosity.

FIG. 15 is a graph of a measurement example of a deformability test due to a change in shear stress, showing the relationship between the area of the scattering diffraction image of red blood cells and the viscosity.

FIG. 16 is a graph of a measurement example of a deformability test due to shear stress change, which shows the relationship between the major axis or the minor axis and the viscosity.

FIG. 17 is a graph of a deformability curve obtained by a shear stress test.

FIG. 18 is a graph obtained by differentiating the deformability curve of FIG.

FIG. 19 is a measurement example graph of a hemolysis curve showing the relationship between the area of a red blood cell scattering diffraction image and the osmotic pressure.

FIG. 20 is a measurement example graph of a hemolytic test showing the relationship between the perimeter of a red blood cell scattering diffraction image and the osmotic pressure.

FIG. 21 is a measurement example graph of a hemolysis test showing the relationship between the outer diameter of an erythrocyte scattering diffraction image and the osmotic pressure.

FIG. 22 is a graph of a hemolytic curve obtained by a hemolytic test.

23 is a graph obtained by differentiating the hemolysis curve of FIG. 22.

[Explanation of symbols]

 10 Red Blood Cell Function Measuring Device 11 Liquid Delivery Means 12 First Mixing Section 13 Monitor Section (Mixing Ratio Change Detection Means) 14 Second Mixing Section 15 Liquid Delivery Section (Sample Solution Delivery Means) 16 Flow Cell 17 Measuring Sections 21-24 Containers 21b- 24b Syringe (selecting means) 21c to 24c Cavity 32 Retaining part 41 Pipette 62 Screen (diffraction image generating means) 63 CCD camera (imaging means) 64 Image processing part (analyzing means)

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁶ Identification number Internal reference number for FI Technical indication G01N 21/47 A 21/85 A // G01N 33/50 L

Claims (11)

[Claims] 1. A sample suspension obtained by mixing a sample solution containing erythrocytes with a diluent whose osmotic pressure is gradually changed at a constant viscosity or a diluent whose viscosity is gradually changed at a constant osmotic pressure. A method for measuring the function of red blood cells, which comprises applying a stress to the red blood cells therein, then irradiating the red blood cells to which stress has been applied to obtain a diffraction image, and measuring the deformability of the red blood cells from the diffraction image. 2. The method for measuring red blood cell function according to claim 1, which is performed in one device. 3. The diluent used is for measuring the deformability of red blood cells by osmotic pressure change, and has a constant viscosity in the range of about 5 to 25 cP and a gradient change in the osmotic pressure in the range of about 300 to 30 mOsm. The method for measuring red blood cell function according to claim 1, wherein 4. The diluting solution used is for measuring the deformability of red blood cells due to shear stress change, and is about 250 to 350 m.
The method for measuring red blood cell function according to claim 1 or 2, wherein the erythrocyte function is graded with a constant osmotic pressure in the Osm range and a viscosity in the range of about 1 to 25 cP. 5. A plurality of liquid delivery means for supplying diluents having different viscosities and / or osmotic pressures, a selection drive means for selectively driving the plurality of liquid delivery means, and a plurality of liquid delivery means First mixing different diluents
A mixing section, a second mixing section for mixing the mixed diluent and a sample solution containing red blood cells, a stress applying means for applying stress to the red blood cells in the sample suspension obtained in the second mixing section, Diffraction image forming means for irradiating the attached red blood cells with light to form a diffraction image, imaging means for capturing the diffraction image, and analysis means for analyzing the deformability of the red blood cells from the captured diffraction image Erythrocyte function measuring device. 6. The red blood cell function measuring device according to claim 5, wherein the second mixing section comprises a retention section for retaining the sample suspension until the osmotic pressure reaches equilibrium. 7. The second mixing section is provided with a turbulent flow generating means for generating a turbulent flow in the sample suspension and mixing it uniformly.
Or the red blood cell function measuring device according to 6. 8. A mixing ratio in which the diluting liquid sent out by any one of the plurality of liquid sending means contains a dye, and the mixing ratio between the dye-containing diluting liquid and the other diluting liquid is detected by measuring the dye concentration. The erythrocyte function measuring device according to any one of claims 5 to 7, comprising a detecting means. 9. The red blood cell function measuring device according to claim 5, wherein the imaging means comprises a CCD camera for capturing a diffraction image. 10. The red blood cell function measuring device according to claim 5, wherein the analyzing means calculates the red blood cell deformation index by analyzing the major axis and the minor axis of the imaged diffraction image. 11. The red blood cell function measuring device according to claim 5, wherein the stress applying means is a flow cell for passing red blood cells in the sample suspension.