JP2011164058A - Method and apparatus for evaluating degree of accumulated damage of red blood cell - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a measurement apparatus for quantitatively measuring the degree of accumulated damages in a red blood cell. <P>SOLUTION: The rotating shear load apparatus comprises: a means for continuously or intermittently applying a load as a high nonphysiological shear stress to the red blood cell in the sample to break a film of the red blood cell by using a rotating shear load apparatus, which puts a red blood cell-containing liquid sample between a plate A and a plate B oppositely disposed and induces a shear flow in the sample by rotating the plate A by a drive means through a fastening means; a light transmitting means for causing a light to enter into the sample; a light receiving means A for measuring an intensity of the light entering into the sample by the light transmitting means; a light receiving means B for receiving the light entering into the sample by the light transmitting means and passing through the sample; and a signal analysis means for analyzing a signal obtained by the light receiving means A, B. The apparatus then finds the degree of the accumulated damages of the red blood cell in the sample from a signal analysis result. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

Using a rotary type shear load device, a constant or temporal change in shear stress is applied to red blood cells continuously or intermittently, and the red blood cells are detected from the change in transmitted light intensity of the solution containing the red blood cells. The present invention relates to a method and an apparatus for evaluating the degree of damage accumulation.

Human erythrocytes occupy nearly half of the blood volume and play a role in oxygen transport. Red blood cells have deformability because they pass through blood vessels with a diameter smaller than the cell size.

When a blood contact type device such as an artificial heart blood pump or an artificial heart lung is used to circulate blood, a large non-physiological shear stress is applied to the erythrocytes locally in the device, and the erythrocyte membrane breaks and Red blood cell decay or hemolysis occurs. Even in a state where the device is not used, for example, when an exercise in which an excessive impact is applied to the sole is continued for a long time, similar hemolysis occurs.

In a general hemolysis test performed as a blood pump performance evaluation test, a fixed amount of blood is filled in a circulation circuit connected to a test blood pump, and the blood is circulated by driving the pump under predetermined conditions. Blood is collected from the circuit at each time interval and the plasma free hemoglobin concentration after centrifugation is measured. The blood compatibility of the blood pump is evaluated from the time change of plasma free hemoglobin concentration.

However, in such a test method, since only plasma free hemoglobin is used as an evaluation index, it is an evaluation that ignores the presence of blood cells that have not yet broken, although the erythrocyte membrane is damaged. Can not.

Conventionally, as a method for examining the degree of damage of red blood cells in a state where membrane rupture has not been reached, there is a method of paying attention to the deformability of red blood cells and measuring this directly or indirectly.

For example, the method using an ectacytometer, which measures the deformability of red blood cells from the diffraction pattern of red blood cells stretched by shear stress, is obtained for quantitative evaluation by placing the red blood cell-containing solution in a concentric cylindrical viscometer. Image processing means is required based on the image (Non-Patent Document 1).

The micropipette method, which uses a micropipette to suck a part of the erythrocyte membrane and measures the membrane elasticity from the suction pressure and the suction distance, has the advantage of being able to measure the deformability of individual erythrocytes. Tricky technique is required (Non-Patent Document 2).

In addition, as a prior art, there is a method of evaluating deformability from an image obtained by photographing and recording the shape of red blood cells that pass through a narrow channel or the time during which red blood cells pass through a narrow channel (Patent Document 1). In order to obtain highly reproducible measurement results by this method, a means for supplying the sample solution while keeping it constant at an extremely low flow rate is required.

There has also been proposed a device that uses a parallel plate type shear load device to generate a periodic shear flow and measures the deformability of red blood cells therein using a photoelectric detector (Patent Document 2).
The parallel plate type shear load device is suitable for loading a red blood cell with repeated shear stress obtained by periodically changing the shear stress, but cannot apply a constant shear stress continuously for a long time. It is unsuitable for applying sufficient shear stress to cause the erythrocyte membrane to break. Usually, a mechanism for converting the rotational motion of the motor into a linear reciprocating motion is required, and the apparatus becomes complicated. In order to measure erythrocyte deformability, it is necessary to apply a shear stress that does not cause erythrocyte collapse. In particular, in order to evaluate the viscoelastic characteristics, it is necessary to change the shear stress with time. In these cases, a parallel plate type shear load device is suitable. However, this device is a device that can easily measure the deformability of erythrocytes, but in order to evaluate the degree of erythrocyte damage, it is sometimes insufficient to measure erythrocyte deformability. Furthermore, in the signal processing step, since at least one of frequency information and phase information obtained by frequency analysis is used for the signal from the light receiving means, a detection device capable of high-speed response is required as the light receiving means.

Since the non-physiological large shear stress is applied to the erythrocytes passing through the blood pump, a part of the erythrocyte membrane is broken, and hemolysis occurs in which the erythrocytes collapse and internal hemoglobin leaks. The development of blood pumps is designed to minimize the amount of hemolysis. When evaluating the blood compatibility performance of a blood pump, a circulation test is generally performed in accordance with US standard test method ASTM F1841-97. Using a simple circulation circuit consisting of a reservoir, tube, pump, and a device that adjusts the flow resistance, a certain amount of blood is continuously circulated under a predetermined pump drive condition, and the blood is circulated at regular intervals. A certain amount of blood is collected from the blood, the plasma component is separated from the blood by centrifugation, the hemoglobin concentration in the plasma is measured by an optical method, and the hemolysis index (NIH) calculated from the change over time of the concentration is obtained. Based on this, the blood compatibility performance of the test blood pump is evaluated. Since this evaluation method is based on the plasma free hemoglobin concentration, it is impossible to evaluate the damage accumulation state of erythrocytes before the erythrocyte membrane is broken.

In the present invention, the degree of damage accumulation state of erythrocytes before the rupture of the erythrocyte membrane is defined as the degree of erythrocyte damage accumulation. As the erythrocyte membrane is damaged, the fragility of erythrocytes increases, and in the blood circulation system of the living body, destruction of erythrocytes may occur locally, including peripheral blood vessels. In recent years, blood pumps with good hemocompatibility and low hemolysis volume have been developed, and in order to correctly compare the blood compatibility performance of each model, in addition to the hemolysis amount revealed in the hemolysis test, Using the accumulation degree is extremely effective.

The results of the hemolysis test vary depending on the initial blood cell damage caused by the type of blood used in the test, the blood collection method, and the storage method. It is common to evaluate the pump performance by applying statistical processing based on the test results. In order to accurately compare the results of each test, it is necessary to grasp the state of red blood cells before the test, and it is useful to measure the degree of red blood cell damage accumulation as one of the means.

Information about red blood cells obtained in a typical blood test includes the number of red blood cells per whole blood unit volume, the amount of hemoglobin per blood unit volume, the average volume of red blood cells, the average hemoglobin content of red blood cells, and the average hemoglobin concentration of red blood cells Is included. Easiness of erythrocyte deformation, that is, erythrocyte deformability, depends on the mechanical properties of the erythrocyte membrane, and can therefore be an indicator of the soundness of the erythrocyte membrane. As a method for measuring the deformability, the red blood cells passing through a flow channel having a width equal to or smaller than the diameter of the red blood cells are photographed, and the shape of the red blood cells is measured, or the light incident on the flow channel is measured by the scattered light by the red blood cells. There is a micro flow cell method based, but it requires a liquid feeder.

As means for investigating changes in the structure and mechanical properties of erythrocyte membranes due to red blood cell damage accumulation, observation and measurement of the membrane surface structure using an atomic force microscope and measurement of the local elastic modulus of the membrane using the microscope However, the need for an expensive device is a problem for practical use as a general blood test means. In addition, there is a method for estimating the membrane elastic modulus by sucking the erythrocyte membrane with a macropipette, but it requires a high level of expert skill.

As a means of investigating changes in the mechanical properties of erythrocyte membranes due to accumulation of erythrocyte damage, the deformation behavior of erythrocytes can be enhanced when shear stress is applied to erythrocytes in a time-varying manner using a rotary or parallel plate type shear load device. There is a viscoelastic property evaluation method based on analysis of time-lapse images. The above method requires an expensive device, which is a problem when put into practical use as a general blood test means.

As a means of damaging the erythrocyte membrane, there is a means of chemically lysing the erythrocyte membrane using a drug in addition to a means by mechanical load. In order to quantitatively evaluate the progress of erythrocyte membrane destruction, It is necessary to take a picture of hemoglobin flowing out through the membrane and estimate the outflow amount by image analysis.

JP 2006-145345 A JP 2008-170390 A

Blood Cells, 1975, Volume 1, pp. 315-321 Red Cell Shape: Physiology, Pathology, Ultrastructure, Springer, New York, 1973

As shown in the previous section, in the prior art that can be applied to examine directly or indirectly changes in the structure and mechanical properties of erythrocyte membranes associated with erythrocyte damage accumulation, in order to perform highly accurate measurements on individual erythrocytes, The problem is that it requires the use of expensive equipment such as atomic force microscopes and high-speed cameras, and the need for highly trained procedures. In addition, even in the method used when measuring a large amount of red blood cells contained in a sample, high-speed image acquisition means and image processing means are required, and there are many factors that hinder simple characteristic evaluation. . There is also a problem in terms of speed of testing.
The present invention is intended to solve these problems, and provides a means for easily measuring the degree of red blood cell damage accumulation.

When a shear flow is induced in an erythrocyte-containing liquid sample and the erythrocytes in the sample are continuously subjected to shear stress, the difference in the average damage accumulation degree of the erythrocytes leads to complete membrane breakage, that is, hemolysis Based on this finding, the present invention has been completed as a means for solving the above-mentioned problems.

The present invention has the following configuration. This will be described with reference to FIGS.
Rotation that induces a shear flow in the sample 5 by sandwiching the red blood cell-containing liquid sample 5 between the plates B2 disposed so as to face the plate A1 and rotating the plate A1 by the driving means 4 through the fastening means 3. Means for continuously or intermittently applying a non-physiological high shear stress to the red blood cells contained in the sample 5 by using a mold shear load device, and causing the membrane of the red blood cells to break. , A light transmitting means 6 for making light incident on the sample 5, a light receiving means A 7 for measuring the intensity of light incident on the sample 5 by the light transmitting means 6, and an incident on the sample 5 by the light transmitting means 6. A light receiving means B8 for receiving the light transmitted through the sample 5, a signal analyzing means 11 for analyzing the signals obtained by the light receiving means A7 and the light receiving means B8, a control means A12 for controlling the driving means 4, Control means B13 for controlling the means 3 comprises a measuring device.

The magnitude of the shear stress applied to the erythrocytes by the shear flow induced in the sample is adjusted by at least one of the fastening means 3 and the drive means 4, the distance between the plate A1 and the plate B2, the drive It can be changed by the viscosity of the liquid sample 5 adjusted by the rotational speed of the plate B2 adjusted by the means 4 and the viscosity of the solvent for dispersing red blood cells.

Since the red blood cell damage accumulation degree measuring device according to claim 1 of the present invention is a combination of a rotary shear load device and an optical measuring device, it is possible to continuously apply a desired shear stress to a red blood cell-containing sample. The signal of the sample transmitted light can be measured.

In addition, a part or all of the sample contact surface of the rotating plate that is a component of the shear load means according to claim 2 reflects light incident on the sample from the light transmitting means through the stationary plate. It can arrange | position to a stationary plate side with a light transmission means.

Furthermore, the red blood cell damage accumulation degree measuring apparatus according to claim 3 can measure the degree of red blood cell damage accumulation on the basis of the time change of the signal detected by the light receiving device, without requiring an advanced image processing means. It is possible to evaluate quantitatively by the test.

In addition, the red blood cell damage accumulation measuring apparatus according to claim 4 can apply a shear stress that changes with time to the red blood cells by changing the rotational speed of the rotating plate that is a component of the shear load means. It is possible to apply to the red blood cells a shear stress equivalent to the shear stress received by the red blood cells flowing in the blood circulation system of the pump or the living body.

In short, the damage accumulation degree of erythrocytes can be evaluated by a measuring device constituted by a shear load device and an optical measuring device with a simple configuration and simple signal processing.

It is the schematic of embodiment. It is a figure showing the flow of each signal in the implementation of this invention, and the process of a process. It is explanatory drawing of a shear load means. It is a figure which shows an experimental result.

The plate B2 provided in the measuring device is at least partially transparent in order to cause the light transmitted from the light transmitting means to enter the sample. In order to efficiently receive the sample transmitted light by the light receiving means B8, it is desirable to provide a light reflecting means on at least a part of the surface of the plate A1 that contacts the sample.

The measuring device can use an image sensor for the light receiving means B8, and can measure the red blood cell damage accumulation degree as well as the red blood cell shape. When an image sensor is used for the light receiving means B8, image processing is performed in the signal processing means, and a red blood cell damage accumulation degree is obtained based on luminance information.

Even if a light source that is inferior in output stability is used as the light transmitting means 6 by providing the light receiving means A7 equipped in the measuring device with an optical element that divides a part of the light transmitted from the light transmitting means. The light absorption characteristics of the sample can be obtained from the light receiving signals of the light receiving means A7 and the light receiving means B8.

Hereinafter, the present invention will be described based on specific examples.

As the plate A1 shown in FIG. 2, a conical plate having an inclined surface in contact with the red blood cell-containing liquid sample is used. The plate A1 is made of metal, and the sample contact surface can be mirror-finished to reflect light. The plate B2 is a transparent flat plate and is disposed horizontally. The plate A1 is connected to the driving means via the fastening means, and the rotation axis thereof is arranged to be perpendicular to the plate B2.

Polymer dextran is dissolved in a solution in which the osmotic pressure is adjusted to the same level as that in blood, and a viscosity adjusting solution in which the viscosity is adjusted to a predetermined value is prepared. A red blood cell-containing liquid sample prepared by adding a small amount of fresh blood to this solution is placed between plate A1 and plate B2. At this time, contact friction between the plate A1 and the plate B2 can be prevented when the plate A1 rotates by providing a minute gap between the plate A1 conical apex and the flat plate.

As shown in FIG. 3, a constant shear stress is applied to the red blood cells in the sample solution between the plates A1 and B2 by rotating the plate A1 at a constant rotational speed. The magnitude τ of the shear stress can be adjusted by the inclination angle θ of the plate A1 cone, the viscosity μ of the sample solution, the rotational angular velocity ω of the plate A1, and the distance g between the plate A1 cone tip and the plate B2 flat plate, The radial distance r from the rotation axis of the plate A1 is expressed by the following equation.

The inclination angle of the plate A1 cone is 3 °, the distance between the plate A1 cone tip and the plate B2 flat plate is 20 μm, the amount of the sample solution sandwiched between the plate A1 and the plate B2 is 1 ml, and the viscosity of the sample solution is 32.3 Pa · When the rotational angular velocity of the plate A1 is 62.8 rad / s, the average shear stress applied to the red blood cells contained in the sample solution 5 is 37.2 Pa.

Using a laser light source as the light transmitting means and an optical power meter as the light receiving means, the sample solution 5 is irradiated with light from the laser light source into the sample solution 5 in which a certain shear stress is applied to the internal red blood cells under the above conditions. The power of the light that has passed through is measured with the optical power meter of the light receiving means B8. Further, a part of the incident light is divided by the beam splitter 9, and the optical power is measured by the optical power meter of the light receiving means A7.

The optical power measured by the light receiving means A7 and the light receiving means B8 is recorded in the computer 11 and the ratio of input / output optical power is calculated, and the rate of change over time is used as an evaluation index for the degree of red blood cell damage accumulation.

The present invention has been implemented in the form described above. Human red blood cells contained in blood immediately after blood collection are separated into high-aged old red blood cells and low-density young red blood cells by centrifugation, and 5 μl of each is added to another 1 ml of the viscosity adjusting solution to prepare a red blood cell-containing liquid sample. The shear stress was applied to each erythrocyte by the shear load means of FIG. As a result, as shown in FIG. 4, since the temporal change in the ratio of the optical power before and after passing through the sample containing each erythrocyte of the aging red blood cell and the young red blood cell is different, the transmitted light power is measured, It can be seen that the age of erythrocytes, ie the membrane damage, can be evaluated.

The rotation speed of the plate A1 of the shear load means of this apparatus is controlled by the drive control means A12, and the distance between the plates A1 and B2 is controlled by the control means B13. The rotational speed of the plate A1 is changed constantly, time-varying or intermittently, and a shearing load which is complicated in time and quantity is given to the red blood cells.

The results of hemolysis tests performed to assess blood compatibility of blood contact medical devices depend on the degree of red blood cell damage in the blood used in the test. The erythrocyte damage accumulation degree evaluation apparatus of the present invention can be used for simply evaluating the damage accumulation degree of erythrocytes in blood used in the test.

1 Board A
2 Board B
3 Fastening means 4 Driving means 5 Red blood cell-containing liquid sample 6 Light transmitting means 7 Light receiving means A
8 Light receiving means B
9 Beam splitter 10 Mirror 11 Computer 12 Control means A
13 Control means B

Claims (4)

In a red blood cell damage accumulation measuring device that measures the degree of red blood cell damage accumulation in a red blood cell-containing liquid sample,
A rotary type in which a red blood cell-containing liquid sample 5 is sandwiched between plates B2 arranged so as to face the plate A1, and the plate A1 is rotated by the driving means 4 through the fastening means 3 to induce a shear flow in the sample. Using a shear load device, applying a non-physiological high shear stress to the erythrocytes in the sample continuously or intermittently to cause the erythrocyte membrane to break;
A light sending means 6 for making light incident on the sample 5; a light receiving means A7 for measuring the intensity of light incident on the sample 5 by the light sending means 6;
A structure in which the light receiving means is disposed on the stationary plate side together with the light transmitting means in order to reflect the light incident on the sample from the light transmitting means 6 through the plate B2.
A light receiving means B8 for receiving the light incident on the sample 5 by the light transmitting means 6 and transmitted through the sample 5,
Signal analyzing means 11 for analyzing the signals acquired by the light receiving means A7 and the light receiving means B8;
A measuring device comprising a control means A12 for controlling the driving means 4 and a control means B13 for controlling the fastening means 3. As a means for applying a shear stress to the erythrocytes and damaging the erythrocyte membrane, a shear load means based on a rotary viscometer is used, and the shear load means is a stationary plate in which all or part of the sample contact surface is transparent The apparatus according to claim 1, further comprising a rotating plate devised so that part or all of the sample contact surface reflects light. Intensity of light transmitted through the sample while continuously or intermittently applying a shear stress to the red blood cells by generating a shear flow in the red blood cell-containing liquid sample between the stationary plate and the rotating plate of the shear load means Alternatively, the apparatus measures the luminance using a light receiving device. By changing the rotation speed of the drive means with time by the control means A12, the rotation speed of the plate B2 is changed with time, and the red blood cell containing liquid sample between the stationary plate and the rotary plate of the shear load means The apparatus according to claim 1, which has a function of continuously and variably controlling the magnitude of the shear stress applied to the red blood cells.