JPS634146B2 - - Google Patents

Description

[Detailed description of the invention] The present invention relates to a device for measuring red blood cell deformability.

In the microcirculation region constituted by peripheral blood vessels, red blood cells flow while being deformed into various shapes under the influence of external forces in the blood. In particular, deformation of red blood cells is essential when blood flows through peripheral blood vessels such as capillaries, which have a diameter (3 to 5 μ) less than half the diameter of red blood cells (7 to 8 μ). High flexibility (called deformability) is very important for maintaining health. When the deformability of red blood cells decreases due to various diseases or aging, red blood cells become difficult to pass through capillaries and aggregate or aggregate, causing various obstacles to the supply of oxygen and nutrients to tissues and further worsening the disease condition. It is known.

Recently, with the development of drugs that improve the deformability of red blood cells, there is an increasing need to quickly and accurately measure the deformability of red blood cells in the prevention and diagnosis of circulatory system diseases, testing of drug efficacy, etc. ing. The main conventional methods for measuring the deformability of red blood cells include:

A. Microfilter method Negative pressure or positive pressure is applied to a microfilter with a pore size of 3 to 8μ, red blood cells are passed through the filter, and the amount of red blood cells passing per unit time or the time it takes for a unit volume to pass is measured.

B. Micropipette method A constant negative pressure is applied to a micropipette with an inner diameter of approximately 3 μm, and the amount of suction of one red blood cell into the pipette, or conversely, the negative pressure required for one red blood cell to be sucked into the pipette is measured. do.

C. Laser diffraction method A red blood cell is placed in a rotational viscometer and rotated, and a laser beam is applied to the red blood cell, and the deformability is measured by the degree of change in the shape of the obtained diffraction image.

All of these measurement methods have advantages and disadvantages, but the microfilter method can be said to be the most advantageous method when considering simplicity, short measurement time, cost, etc.

The conventionally used microfilter method will be explained below with reference to FIGS. 1 and 2.

A holder 2 containing a filter is connected to a 1 ml syringe 1, a blood sample is placed therein, and the suction pressure generated by operating the manual aspirator 7 is applied to the holder 2 through the vacuum holding container (5) and the tap 4. Blood is passed through the filter, and the resulting liquid is received in a liquid receiver 3. The degree of suction pressure is determined by the height of the water column (for example, 20
cm) measured and adjusted. The interior of the holder 2 consists of a clamp ring 8, a cap 9 with a dead space 10, a filter 11, a support grid 12 and a base 13 with a discharge nozzle, as shown in FIG. 2, and can be freely disassembled into various parts. It's becoming like that.

Using such a device, a fixed amount (usually 1
The deformability of red blood cells is determined by measuring the time it takes for a blood sample (ml) to pass through a filter under constant suction pressure (e.g. -20 cm water column). ) The flow of blood through the filter is not uniform, and each operation is performed manually, resulting in large human errors. (B) It takes a considerable amount of time to become proficient in the operation. (C) The operator (measuring) (d) The measurement time required for each sample is long, making it impossible to efficiently process a large number of samples in a short period of time. I couldn't reach it.

The biggest problem with the microfilter method is that the injected blood does not completely fill the dead space 10 in the holder, and the viscosity of the blood causes air bubbles to form on the filter surface and inside the holder, which causes the blood to evaporate. This obstructed the smooth flow, resulting in a negative impact on the reproducibility of measured values.

The inventor of the present invention avoided the above-mentioned drawbacks of the microfilter method, and as a result of making extensive efforts to develop an accurate and efficient method for measuring red blood cell deformability using simple operations, the inventors have discovered that when blood is passed through the holder, the filter is replaced with physiological saline. A method that prevents the formation of air bubbles by washing the blood with a liquid before passing through it, combines this operation with a step of measuring deformability by flowing blood, and also automates the switching of each step. We have now completed a device to carry out the project.

Next, FIG. 3 showing an example of the method and apparatus of the present invention will be explained.

The device of the present invention is broadly divided into a measuring section, a physiological saline supply section, a suction pressure applying mechanism, and a solenoid valve section for switching the direction of liquid flowing between these sections according to each process, and its control section. has been done.

The measuring part consists of a holder 23 containing a syringe 21 and a filter 22, which are commonly used in this type of device, and the upper end of the syringe 21 is preferably connected to a suction nozzle tube 24 with a lid that can fit with the syringe. and can be freely removed. Further, the holder 23 has an opening at its lower end for inflowing and outflowing the liquid, and the measuring section is further covered in whole or in part with a constant temperature jacket 25 so as to maintain a constant temperature during measurement. Measurements are made by visually or photoelectrically observing changes in blood volume, ie, decreases in liquid level.

The physiological saline supply section consists of a physiological saline tank 26 and a constant temperature chamber 27 for maintaining it at or near body temperature, and this constant temperature chamber is operated by a common heat medium circulation system with the constant temperature jacket 25 of the holder section. However, in some cases, each may be independently controlled by a separate circulatory system.

The suction mechanism is connected to an aspirator 28 with a constant suction pressure via a solenoid valve, and has a discharge pipe 29 at the bottom.
a liquid distillate 30 having a variable suction pressure, an aspirator 31 with variable suction pressure, and a suction buffer 3 connected thereto.
2. It consists of a pressure gauge 33 and a liquid distillate 34 with a discharge pipe at the bottom.

These parts are connected by three-way solenoid valves A, B, C, and D, two-way solenoid valves E and F, and a pipe communicating between them.

In the embodiment in which the speed of blood passing through the filter is photoelectrically measured, in addition to these, a light source unit 3 is installed around the holder injector in order to automatically detect the liquid level of blood flowing down from the injector into the holder.
5. A photoelectric detection device consisting of a combination of a light receiving element 36 and a timing device (not shown) may be provided. It can also be connected to a device for displaying or recording it as a print or image, if necessary.

Next, the method of the present invention will be explained in order of operation steps using the apparatus illustrated in FIG.

1. First cleaning step: Set the filter 22 on the filter holder 23. A syringe 21 is connected to this. By connecting the upper end of the syringe 21 with the suction nozzle 24 and opening the electromagnetic valves A, B, and F and closing E and C, the maximum suction pressure of the constant pressure aspirator 28 is applied to the syringe 21 and the suction nozzle 24 through the liquid reservoir 30 and the suction nozzle 24. It hangs on the holder 23, and this action causes the tank 2 to
6, physiological saline maintained at a predetermined temperature (usually body temperature) by a thermostat 27 is drawn up into the syringe 21 through the filter 22. This removes air bubbles in the filter and blood remaining from previous measurement steps.

2. Second cleaning step By switching the valve operation circuit, solenoid valves A, C, D, and E are opened, F is closed, and B is released to the atmosphere. All the suction pressure of the constant pressure aspirator 28 is applied to the liquid reservoir 34. The saline present in the syringe 21 descends and flows into the reservoir 34 . At this time, it is preferable to leave the holder filled with physiological saline to prevent air from being trapped in the holder in the next measurement step. Note that the first cleaning step and the second cleaning step are repeated 2 to 3 times as necessary.

3 Measurement process Close solenoid valves C and F, and with the syringe 21 filled with sample blood in contact with the holder, close solenoid valves D and E.
When opened, the suction pressure of the variable suction pressure aspirator 31 is applied to the liquid reservoir 34, and blood flows down inside the syringe 21 and flows into the liquid reservoir 34 through the filter. Adding a constant suction pressure measured by the pressure gauge 33,
The deformability of red blood cells is measured by measuring the time it takes for a given volume of blood to pass through a filter, or by measuring the amount of blood that flows in a given amount of time. At this time, it is preferable to keep the measuring parts such as the syringe 21 and the holder 23 at a constant temperature (for example, body temperature) with a constant temperature jacket in order to reduce measurement errors.

Note that time and volume measurements may be performed by reading the scale with the naked eye or by manual operation such as using a stopwatch, but in order to reduce errors and improve measurement efficiency, quantitative changes in the blood in the syringe can be measured. Preferably, this is carried out automatically by providing a photoelectric detection device, such as a combination of a light source 35 and a light receiving element 36, located near the syringe and connecting this to an appropriate time or capacitance measuring and display device. Furthermore, by connecting these automatic measuring mechanisms to a display/recording device using an image or a printer, etc., they can be automatically recorded, and for example, changes over time in the amount of blood passing through a filter, which could not be measured with conventional devices, can be learned. It also becomes possible to perform more precise measurements.

The present invention has the following technical advantages due to the above-described configuration.

1. By completely eliminating air bubbles in the holder and filter, blood flow can be made smoother, measurement precision can be increased, and good reproducibility can be obtained.

2. Since a large part of the operations such as cleaning and measurement, which were previously performed completely manually, is automated, it is possible to prevent measurement errors due to unfamiliar operation.
Work is done faster.

3. Because measurement errors are reduced and there is less loss of sample blood, the amount of blood sample required for one time is about half (0.5ml) compared to conventional methods.

4. Since the operation is simplified and the measurement time is shortened, a large number of samples can be processed in a fixed period of time. This is particularly useful for the prevention and early diagnosis of ischemic diseases, since red blood cell deformability is closely related to adult diseases.

Next, the present invention will be explained by examples.

A holder with a 0.5ml glass syringe installed according to the arrangement shown in Figure 3 (Nuclepore No.
Attach the filter (Nuclepore No.N500CPR01300, GE product) inside the filter (FH013PL0010, GE product). Physiological saline is introduced into the holder from a plastic tank 1, maintained at 37°C by a heat exchanger type incubator. In liquid distillates 30 and 34
Using a 500ml glass bottle, three-way solenoid valves A, B, C,
For D, use US-M5-37 type (manufactured by Chukyo Electric Co., Ltd.), and for two-way Teflon solenoid valves E and F, use MTV-2-M6 (manufactured by Takasago Electric Works, Ltd.). As an aspirator, an electric compressor (water circulation drive WP-45 type,
100W, 6-7/min), the constant suction pressure side is approximately -50mmHg, and the variable suction pressure side is approximately -7.4mmHg.
It was activated with. Surround the outside of the injector with a hot water jacket with two windows, and insert a filament lamp (2.4W, ROYAL H0853 type, Hamai Bulb Co., Ltd.) through the windows.
product) and photo sensor (18 array, P884
A photoelectric measurement unit is equipped with a light-receiving element for liquid level detection consisting of a type (type, product of Hamamatsu TV Co., Ltd.), and when the blood reaches the specified scale of the syringe, the photo sensor detects the liquid level, and the output is measured. Measure the time by activating the digital timer. The electromagnetic valves and photoelectric measurements are operated by a control box that includes a suitable combination of relays, switches, microcomputers, power supplies, etc.

When the red blood cell deformability of healthy adult blood was measured using the above-mentioned device at a suction pressure of about -7.4 mmHg, the transit time for 0.5 ml was about 10 to 14 seconds. It was possible to perform about 30 measurements per hour by simply replacing the filter each time and injecting a new blood sample into the syringe.

[Brief explanation of the drawing]

Figures 1 and 2 are schematic diagrams of a conventional microfilter-based red blood cell deformability measurement device.
FIG. 3 is an example of the method and apparatus of the present invention.

Claims (1)

[Scope of Claims] 1. In measuring the deformability of red blood cells by the rate of passage of blood through a filter under suction pressure, the filter is filled with a physiological saline solution before the blood to be measured passes through the filter. A method for measuring red blood cell deformability, the method comprising washing a filter by discharging the red blood cell deformability. 2. The method according to claim 1, wherein the filter is washed with physiological saline in a direction opposite to the flow of blood. 3. The method according to claim 1 or 2, characterized in that changes in the amount of blood passing through the filter are photoelectrically detected and measured. 4. A measuring section consisting of a holder housing a filter and a blood injector connected to the holder, a physiological saline supply section for cleaning the measuring section, a suction pressure applying mechanism, the aforementioned measuring section, a physiological saline supply section, and a suction a valve inserted between each of the pressure applying mechanisms;
A cleaning process in which the measurement unit is cleaned with physiological saline, a measurement process in which blood is passed from the syringe through a filter, and a washing and discharge process in which residual blood is drained out with physiological saline are connected to a suction pressure applying mechanism by operating a valve. A red blood cell deformability measuring device comprising a control unit for sequential switching. 5. The apparatus according to claim 4, further comprising a thermostat for keeping the physiological saline solution and the blood in the measuring section warm. 6. The device according to claim 4 or 5, characterized in that the measurement section is provided with a photoelectric detection device for photoelectrically measuring changes in blood volume caused by passing through the filter.