JP4843794B2 - Measuring system for mechanical properties of blood cells - Google Patents

Description

  The present invention relates to a measurement system and method capable of quantitatively measuring the mechanical properties of blood cells, particularly erythrocytes, with good reproducibility.

  Red blood cells, which play a major role in blood cells, have the function of passing through capillaries smaller than their own diameter. It is said that the deformability of erythrocytes is generally affected by the aging and osmotic pressure of erythrocytes themselves, and the influence of lipids dissolved in blood. Furthermore, in life-prolonging and treatment using circulatory system artificial organs such as artificial hearts, artificial valves, and dialysis, non-physiological shear stress that does not occur in vivo is applied to red blood cells, and Although it does not lead to destruction, membrane damage may occur, resulting in irreversible morphological changes and reduced deformability. This may reduce the life of red blood cells and induce anemia. On the other hand, since drugs for improving the deformability of erythrocytes by pharmaceuticals have been developed, development of a detailed erythrocyte deformability measuring apparatus is required in order to measure the effect.

  Conventionally, as a technique for evaluating the deformability of red blood cells, a membrane passage method (Patent Document 1) for measuring the time required for red blood cells to pass through fine pores, observation of the state of red blood cells passing through a flow path corresponding to capillaries with a CCD, Alternatively, a micro flow cell method (Patent Document 2, Patent Document 3, Patent Document 4) for measuring scattered light (diffracted light distribution), an ectosite method based on a rotational viscometer, or a micropipette method for measuring an erythrocyte membrane by a suction method and so on. There is also a method of capturing a change in the shape of red blood cells in a periodic flow field with a high-speed camera (Non-Patent Document 1).

  On the other hand, as a method of measuring the dynamic load of erythrocytes, that is, the durability, there is a method of estimating from the scattered light intensity of erythrocytes passing through the flow cell having two different inner diameters (Patent Document 5).

  The measurement of deformability of red blood cells includes a method of measuring the shape of red blood cells by direct and indirect methods and estimating the deformability of red blood cells from the measurement results. As a direct method, assuming the speed and shape change of red blood cells passing through capillaries, etc., in order to capture the state of flowing in a flow cell with an optional flow path shape under an arbitrary condition with a CCD camera, It is likely to be a subjective evaluation such as a shape change of the red blood cell or a state of blocking the flow path, and the quantitative evaluation is limited to information such as the moving speed of the red blood cell. There is also a method for measuring deformability from the shape change of red blood cells in a periodic flow field between parallel plates. However, in this method, in order to measure the shape change of erythrocytes under high shear stress, that is, the ellipsoidal shape, it is necessary to suspend erythrocytes in a high viscosity solvent several tens to several hundred times that of plasma. There is. For this reason, it is difficult to measure the deformability in consideration of the viscous effect inside the red blood cells.

On the other hand, as an indirect method, there is a method of irradiating red blood cells flowing in a flow cell or a double cylindrical tube with light, measuring its diffraction image or scattered light, and estimating the deformability from its light intensity characteristics. In this method, since the measurement is performed under a high shear stress, the red blood cells are suspended in the high-viscosity solvent.
In the measurement of the load accumulation of erythrocytes, there is a system that estimates the degree of damage by comparing scattered light indicating the shape change of erythrocytes in the flow field with a database of scattered light indicating the degree of damage. The conventional system is a system that separately examines the damage of a blood sample that has received a load, and is not suitable for a method of repeatedly applying a load to a specific sample such as fatigue fracture.

The fatigue fracture test for measuring the durability of erythrocytes includes a tester based on a rotational viscometer and a tester using a parallel plate. Many of these test machines are not suitable for a method for measuring the state of red blood cells in real time because the state of red blood cells after applying a load is observed or free hemoglobin is measured.
Although there is a method of floating red blood cell samples between parallel plates and applying shear stress to visualize the shape of red blood cells, as described above, this method uses image processing using a high-speed camera, so the apparatus is large. Become expensive.

  An apparatus based on a rotational viscometer can measure fatigue fracture in real time, but an apparatus based on a rotational viscometer is very excellent in a method for applying a constant stress. However, in the method of applying a cyclic repetitive load, it is difficult to adjust the amplitude and the cycle because the direct rotation shaft is used as a power source.

JP 2001-242166 A JP 2006-145345 A JP-A-8-122328 Special table 2001-507122 gazette JP 2004-138561 A Biophysical Journal, September 2006, Vol.91, No.5, p.1984-1998

As described above, conventionally, measurements such as deformability and durability have been separately measured as mechanical characteristics of red blood cells. However, considering the direction of reducing the amount of collected blood as much as possible, the same sample and the same measuring device can be used. It is desirable to be able to measure the mechanical properties of red blood cells.
In addition, it is expected to develop a device that can measure the mechanical properties of red blood cells that is inexpensive and does not depend on the user. Furthermore, in order to reduce the influence of the viscosity inside erythrocytes, it is also expected to measure erythrocyte deformability in a solvent having the same viscosity as blood.

  Conventionally, in order to measure mechanical properties such as deformability and durability of red blood cells, a technique is known in which red blood cells are subjected to periodic shear stress load and red blood cells are photographed with a camera. However, in order to give a sufficient shear stress load to erythrocytes, it is necessary to apply a high shear rate. To cope with this, it is necessary to use an expensive high-speed camera or a large light source. Even in this case, the shape change rate of the red blood cells exceeds the performance of the high-speed camera, so that it is necessary to increase the viscosity of the solvent in order to give a sufficient shear stress load to the red blood cells within the performance of the high-speed camera. However, when a solvent having a viscosity higher than that of blood is used, there has been a problem that red blood cell movement in actual blood cannot be reproduced. Furthermore, the analysis method using a camera has a problem that a system for image processing of shape changes relating to individual red blood cells is required separately. A system based on a rotational viscometer is also known, but in such a system, the mechanical load applied to the red blood cells becomes constant, and it is difficult to adjust the amplitude and period of the mechanical load. There was also a problem.

  In order to solve the above problems, the present invention provides a measurement system capable of quantitatively measuring the mechanical characteristics of blood cells, particularly erythrocytes, without using a high-speed camera or a high-viscosity solvent. And to provide a method.

In order to achieve the above object, the present invention has the following configuration.
A blood cell mechanical property measurement system for measuring mechanical properties of a blood cell by applying a periodic shear stress load to the blood cell, wherein the first flat plate is at least partially transparent, and the first flat plate. A second flat plate arranged in parallel with a gap therebetween, a sample containing blood cells arranged in a gap between the first flat plate and the second flat plate, and blood cells in the sample In order to apply a periodic shear stress load to the plate, plate vibration means for parallelly vibrating at least one of the first plate and the second plate at a predetermined vibration frequency, light irradiation means for irradiating the sample with light, and A light receiving means for receiving transmitted light, reflected light or scattered light from a sample; and a signal analyzing means for analyzing a signal from the light receiving means to obtain a mechanical characteristic of the blood cell, the signal analyzing means is received from said light receiving means Signal has a means for frequency analysis, determine the frequency and phase information of the light receiving signal by said frequency analysis, and control information including a vibration frequency of the flat plate vibrating means, frequency and phase information of the light receiving signal A blood cell mechanical property measurement system for obtaining the mechanical property of the blood cell from the relationship between the blood cell and the blood cell.

A blood cell mechanical property measurement method for measuring mechanical properties of a blood cell by applying a periodic shear stress load to the blood cell, wherein the first flat plate is at least partially transparent, and the first flat plate A sample preparation step of arranging a sample containing blood cells between a second flat plate arranged in parallel with a gap therebetween, and for applying a periodic shear stress load to the blood cells in the sample , A flat plate vibration step for causing at least one of the first flat plate and the second flat plate to vibrate in parallel at a predetermined vibration frequency, a light irradiation step for irradiating the sample with light, and transmitted light, reflected light or scattering from the sample. A light receiving step of receiving light by the light receiving means, and a signal analyzing step of analyzing a signal from the light receiving means to obtain a mechanical characteristic of the blood cell, the signal analyzing step from the light receiving means frequency of the received light signal Has a step of analyzing determines the frequency and phase information of the received light signal to said frequency analysis, and control information including a vibration frequency of the flat plate vibrating means, the relationship between the frequency and phase information of the light receiving signal A method for measuring the mechanical properties of blood cells, wherein the mechanical properties of the blood cells are determined from

Moreover, it has the following embodiments.
Similar to the first flat plate, the second flat plate is at least partially transparent. When obtaining the reflected light from the sample, it is sufficient if only one of the two flat plates is transparent. You can definitely get.
The frequency of vibration by the flat plate vibration means is several Hz to several kHz. The vibration frequency of the parallel vibration of the flat plate is not particularly limited, but is preferably about several Hz to several kHz.
Vibration by the flat plate vibration means is performed by an actuator, and the actuator further has a function of adjusting a distance between the first flat plate and the second flat plate. By adjusting the distance between the first flat plate and the second flat plate, the shear stress on the sample containing blood cells can be adjusted. In addition, the introduction and exchange of the sample can be facilitated by increasing the interval during the introduction and exchange of the sample.

The blood cells are red blood cells. Although the present invention can be used for blood cells other than red blood cells (for example, white blood cells), it can be suitably used particularly for measuring the mechanical properties of red blood cells.
The sample containing blood cells is blood. The mechanical properties can be measured even if blood cells are contained in a solvent other than blood, but in the present invention, the mechanical properties of blood cells can be measured even if blood is used as it is as a solvent.
The light irradiation unit can irradiate the sample with light having a plurality of different wavelengths. Although the present invention can be measured even with light of a single wavelength, more precise measurement is possible by performing measurement using light of a plurality of different wavelengths.
The sample containing blood cells further contains a fluorescent agent. By introducing a fluorescent agent into a sample containing blood cells, changes in blood cell membrane cell characteristics can be evaluated at the membrane structure level.

  By adopting the above configuration, the present invention can measure the mechanical properties of blood cells, particularly erythrocytes, with good reproducibility and quantitatively. In the present invention, transmitted light, reflected light or scattered light from a sample containing blood cells is received by a light receiving means, and the received light signal is subjected to frequency analysis to measure the mechanical characteristics of the blood cells from frequency information and phase information. Therefore, blood cells can be moved at a higher speed than a conventional measurement system using a camera. Therefore, a sufficient shear stress load can be applied to blood cells even in a solvent having a low viscosity, and blood itself can be used as a sample. In addition, the configuration of the apparatus is simpler than that of the conventional technique for observing with a camera or measuring a diffracted light pattern. Furthermore, in the present invention, the mechanical load is applied to the red blood cells by the parallel vibration of the flat plate, and therefore it is easy to adjust the period and amplitude of the mechanical load applied to the red blood cells.

Hereinafter, an example of an embodiment of the present invention will be described with reference to the drawings.
FIG. 1 shows a mechanism for applying a shear stress load to blood cells. The mechanism for applying a shear stress to erythrocytes is designed so that two flat plates move as a pair in parallel. When the moving speed of the flat plate is u and the gap between the working flat plate and the fixed flat plate is h, the shear rate generated between the parallel flat plates is du / dh. When a solution in which red blood cells are dispersed is placed in the gap, the shear stress generated between the parallel plates is defined by the following equation.

μ is the viscosity coefficient of the solvent containing red blood cells, u is the moving speed of the parallel plate, h is the size of the gap, ω is the angular frequency of the plate, and ν is the kinematic viscosity coefficient. The magnitude of the shear stress applied to the red blood cells in the present invention can be adjusted by the parameters of the above three terms (μ, u, h) as shown in the equation (1).
Especially to make the flow field a Kuwait flow

The above items may be set to satisfy

  FIG. 2 is a schematic diagram illustrating an example of an embodiment of the present invention. First flat plate 1 and second flat plate 2 made of transparent glass, sample 3 containing blood cells, light irradiation means 4 for irradiating the sample with light, light receiving means 5 for receiving transmitted light, reflected light or scattered light from the sample. , A fixed part 6 and an operating part 7. The light irradiation means 4 includes a light source, an optical filter, a chopper, and the like. Similarly, the light receiving means 5 includes a light receiving element, an optical filter, and the like. The first flat plate 1 and the operating portion 7 can be controlled in parallel vibration and height with respect to the second flat plate 2 fixed to the fixing portion 6 by an actuator (not shown). A sample 3 containing blood cells is disposed between the first flat plate 1 and the second flat plate 2. The light irradiating means 4 and the light receiving means 5 are appropriately arranged depending on whether transmitted light, reflected light or scattered light from the sample is received.

  The operating portion 7 is a hollow cylindrical shape, and a transparent material (first flat plate 1) such as a glass surface and a transparent material (second flat plate 2) serving as a fixed portion are provided on the contact surface with the blood sample 3. Use. By using a transparent material, it is possible to directly irradiate the red blood cell sample 3 with light, and the measurement of transmitted light can be facilitated. In addition, by making the operating portion 7 into a hollow cylindrical shape, it becomes possible to easily install a sensor (optical fiber bundle) in which an optical element and an optical fiber connected to a light receiving element are bundled as shown in FIG. In addition, it is possible to easily measure the shape change state of the red blood cell sample with the operating unit 7 as absolute coordinates. Further, the reflected light can be measured by connecting the optical fiber bundle to the fixed portion.

  Based on FIG. 3, a method of measuring the shape of red blood cells from light passing through the measurement object will be described. As the light source of the light irradiation means 4, a light source having a wavelength effective for a measurement target, such as a laser or an LED, is used. For example, when a laser is used as a light source, the incident laser light is attenuated, polarized, divided (for reference and measurement) using optical components. In the case of an LED, light is transmitted using an optical fiber, and division is performed. Next, the light that has been divided, attenuated, chopped, etc. passes through the sample 3 as it is, passes through an optical filter such as dimming and polarization, and then reaches the light receiving portion of the light receiving means 5. The received light is converted into a voltage signal by an optical sensor, and only the signal intensity having the same period as the reference light is detected. The signal detection with the same period is performed using a lock-in amplifier or the like.

  The optical signal that has passed through the lock-in amplifier passes through an AD conversion circuit or the like and is taken into a personal computer. On the other hand, the shear stress load on the red blood cells during measurement is controlled by an actuator or the like connected to the operating unit 7 of the first flat plate 1. The actuator is controlled by a personal computer or the like in order to synchronize with signal measurement. Using a personal computer or the like, the received light signal is subjected to fast Fourier transform to obtain frequency information and phase information, and together with actuator control information, the mechanical characteristics of red blood cells are obtained.

  By providing an optical axis that measures transmitted or reflected light, it is possible to measure the state of red blood cells when passing through a specific position with transmitted light. It is possible to measure characteristics synchronized with movement. In particular, the reflected light can be measured by installing an optical probe equipped with light emission and light reception in the cylindrical portion of the operating portion 7. By incorporating the blood cells to be measured in the flow field to which a mechanical load is applied, measurement from deformability to durability evaluation can be performed without separately replacing the blood sample. By separately analyzing the captured data, it becomes possible to measure the viscoelastic properties of red blood cells under vibration load and the time to reach fatigue destruction. Moreover, by changing the wavelength of the light source and introducing a fluorescent agent into the blood sample, changes in blood cell membrane cell characteristics can be evaluated from the membrane structure level.

In the present embodiment, the experiment was performed with the gap between the flat plates being 200 μm, the driving frequency of the operating unit 7 being 3 Hz, and the amplitude being 4 mm. The measurement objects used were water and blood samples in which erythrocytes were suspended at an isotonic pressure and high osmotic pressure of Ht 8.5% (Ht at isotonic pressure). A PBS solution was used to adjust Ht. The light source chopper was 150 Hz. The measurement result was obtained by performing frequency analysis on the actually measured voltage signal. In solutions containing red blood cells, characteristic intensities were measured at specific frequencies. As for the influence of osmotic pressure, the strength in the frequency region of the measurement target was generally stronger in the erythrocyte sample (isotonic pressure) excellent in deformability than in the erythrocyte under the condition of high osmotic pressure.
From the above results, the behavior of red blood cells affects the attenuation of light relative to the light passing through the flow field, and the light passing through the sample becomes a periodic optical signal indicating the movement of red blood cells, that is, deformability. It was confirmed that From this, it can be seen that red blood cell deformability diagnosis is possible by detecting a signal indicating the behavior of red blood cells in a periodically changing flow field.

Although an example of the embodiment of the present invention has been described above, the present invention is not limited to this, and it goes without saying that various modifications can be made within the scope of the technical idea described in the claims. Yes.

Illustration of shear rate generation by parallel plates Schematic diagram of this embodiment Block diagram of this embodiment Example of light irradiation means and light receiving means

Explanation of symbols

1 First flat plate (transparent glass, etc.)
2 Second flat plate (transparent glass, etc.)
3 Sample containing blood cells 4 Light irradiation means 5 Light receiving means 6 Fixed part 7 Working part

Claims (2)

A blood cell mechanical property measurement system that measures mechanical properties of blood cells by applying periodic shear stress load to the blood cells,
A first flat plate at least partially transparent;
A second flat plate disposed parallel to the first flat plate with a gap therebetween;
A sample containing blood cells disposed in a gap between the first flat plate and the second flat plate;
In order to apply a periodic shear stress load to the blood cells in the sample, flat plate vibration means for parallelly vibrating at least one of the first flat plate and the second flat plate at a predetermined vibration frequency ;
Light irradiation means for irradiating the sample with light;
A light receiving means for receiving transmitted light, reflected light or scattered light from the sample;
Signal analyzing means for analyzing the signal from the light receiving means to determine the mechanical properties of the blood cells,
The signal analyzing means has means for analyzing the frequency of the received light signal from the light receiving means, and obtains frequency information and phase information of the received light signal by the frequency analysis, and includes a vibration frequency of the flat plate vibrating means. A blood cell mechanical property measurement system that obtains a mechanical property of the blood cell from information and the relationship between frequency information and phase information of the received light signal . A method for measuring the mechanical properties of blood cells by measuring the mechanical properties of blood cells by applying periodic shear stress load to the blood cells,
A sample preparation step of arranging a sample containing blood cells between a first flat plate at least partially transparent and a second flat plate arranged parallel to the first flat plate with a gap;
A plate vibration step of causing at least one of the first plate and the second plate to vibrate at a predetermined vibration frequency in order to apply a periodic shear stress load to the blood cells in the sample;
A light irradiation step of irradiating the sample with light;
A light receiving step of receiving light transmitted from the sample, reflected light or scattered light by a light receiving means;
Analyzing the signal from the light receiving means to determine the mechanical properties of the blood cells, and
The signal analyzing step includes a step of analyzing a frequency of a light reception signal from the light receiving means, and obtaining frequency information and phase information of the light reception signal in the frequency analysis, and including a vibration frequency of the flat plate vibration means. A blood cell mechanical property measuring method for obtaining a mechanical property of the blood cell from a relationship between information and frequency information and phase information of the received light signal .