JPH0247692B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to the properties of red blood cells, in particular the ability of red blood cells to transform into various states as they pass through capillaries;
That is, the present invention relates to a method and apparatus for measuring deformability.

[Conventional technology]

Among the various characteristics of red blood cells, red blood cells constantly circulate within blood vessels, and those with a diameter of approximately 8 microns can freely pass through capillaries that are less than a few microns in diameter. This is because it has this deformability, and examining this deformability can be useful in diagnosing microcirculatory disorders. On the other hand, one method for quantifying the deformability of red blood cells is to measure the negative pressure (suction pressure) required to be sucked into a glass capillary of about 3μ using a constant pH and osmotic pressure, or other methods. , a method was used in which red blood cells were passed through a porous filter of about 3 to 5 microns and the flow rate was measured.

[Problem to be solved by the invention]

However, the former method involves measurement of a single red blood cell under a microscope, and the measurement result does not necessarily represent the average value of all red blood cells, requiring skill and is not practical. Although the latter allows obtaining an average value for a large number of red blood cells, it also has errors due to the destruction of red blood cells.
There was a drawback that the flow rate could not necessarily be determined to be a parameter related to the deformability because it was affected by the viscosity, temperature, etc. of the liquid in which the red blood cells were suspended. Also,
Both methods require time and effort for measurement, and the time required for measurement per sample is quite long. In the conventional measurement method described above, when focusing on a single blood cell, an average value cannot be obtained.On the other hand, in the method of measuring deformability from flow rate, it is difficult to obtain an average value for each red blood cell. The problem was that it was unclear.

The present invention was made in view of the above points, and is a detection method in which a blood diluent is passed through a pore and red blood cells are detected based on the electrical difference between the red blood cells and the diluent. The object of the present invention is to provide a method and an apparatus for accurately measuring the deformability of red blood cells by passing the red blood cells through a filter having a large number of pores, and then passing the red blood cells through the pores.

[Means and actions for solving the problem]

In order to achieve the above object, the method of measuring the characteristics of red blood cells according to the present invention involves passing a blood diluent 2 through the pores 4 and mixing the red blood cells with the diluent, as shown in FIGS. 1 and 2. In the detection method of detecting red blood cells based on electrical differences, the blood diluent 2 is
The red blood cell is characterized in that the red blood cell is passed through a filter 5 having a large number of pores of about 3 μm, and then passed through the pores 4 to measure the deformation of the red blood cell.

The total area of the pores of the filter 5 is 100,000 to 100,000 times the area of the pores 4, and the number of red blood cells passing through the pores 4 in a predetermined time is detected. The total area of the pores 4 is 1 to 0.1 times the area of the pores 4, and the passage time of a predetermined volume of blood diluent is measured.

In addition, as shown in FIGS. 1 and 2, the device for measuring the characteristics of red blood cells of the present invention allows the blood dilution solution to pass through a pore 4 provided at the lower end of the detector 3, thereby allowing the red blood cells and the dilution solution to interact. In a particle detection device that detects red blood cells based on electrical differences, an adapter 6 is fitted to the lower end of a detector 3 having pores 4, which is provided with a filter 5 having a large number of pores with a diameter of about 3 μm at the bottom. It is characterized by

The total area of the pores in the filter 5 is 100,000 to 100,000 times the area of the pores 4, or the total area of the pores in the filter 5 is 1 to 0.1 times the area of the pores 4. Configure it as such.

In the method of claim 2, the flow rate of the liquid is kept constant and the deformability is measured by the number of red blood cells detected within a predetermined time. Therefore, compared to the conventional method of measuring deformability from the flow rate per unit time, the influence of the viscosity and temperature of the liquid on the measurement results can be reduced because the flow rate of the liquid, that is, the flow velocity, is kept constant. As an effective means of keeping the flow rate of liquid constant, pores 4 with a diameter of around 100μ are provided in a disk of ruby, sapphire, etc.
Apply suction pressure at a predetermined pressure from the back side of the pore.
By setting the pressure at a constant value of, for example, 200 mmHg, the flow rate is kept approximately constant unless the pores 4 become clogged. In order to prevent the viscosity of the liquid itself from being affected by changes in the temperature of the liquid, it is desirable to maintain the temperature at a constant temperature using a constant temperature bath or the like. The pore 4 is not only an orifice for adjusting the flow rate to keep the flow rate constant, but also detects blood cells when they pass through the orifice based on the difference in impedance between the blood cells and the liquid that suspends the blood cells. It also serves as a detection hole. Furthermore, the front part (upstream side) of the pore 4
A filter 5 having at least 10,000 or more holes with a gap diameter of about 3 μm is provided. In other words, the diameter of the detection hole (pore) is around 100μ and the diameter of the gap is around 3μ, so one detection hole and the gap
With around 1000 holes, the area of both holes is equal,
Unless the total area of the pores in the filter 5 is at least 10 times the area of the detection holes, the flow rate will be regulated by the filter. Therefore, if the hole area of the filter is about 10 times or more the area of the detection hole, the flow rate is regulated only by the detection hole.

By the above method, blood diluted approximately 50,000 times is set so that the filter 5 and the detection hole are below the liquid level, and when suction pressure is applied from the back side of the detection hole, the red blood cells are absorbed into the filter of approximately 3 μm. After passing through 5, one piece is detected by the detection hole. By counting the number of blood cells detected for a predetermined period of time, the number of blood cells that have passed through the filter 5 is measured. That is, the diameter of red blood cells is usually about 8μ, but the pore size of the filter 5 is around 3μ, and the red blood cells must be deformed in order to pass through this filter. Therefore, only red blood cells that have deformability can pass through. For example, red blood cells that pass in a hemolyzed state cannot be counted as one red blood cell, so the deformability can be expressed numerically. The measurement time is set to about 10 seconds to 1 minute, but if it is set too long,
Red blood cells or other particles that do not have deformability cause clogging, causing errors in measurement values.

In claims 2 and 5,
The total area of pores in filter 5 is 10 of the area of pores 4.
~100,000 times more. In principle, there is no upper limit, but when considering the practical size of the detector 3, the size of the filter 5 provided in the detector 3 is also limited, and therefore there is a limit to the total area of the pores of the filter 5. . If it exceeds about 100,000 times, the size of the filter 5 becomes too large and it becomes difficult to configure it as a device.

The method according to claim 3 is a method in which the number of pores in the filter 5 is 1000 or less, that is, the total area of the pores in the filter is equal to or less than the area of the detection holes. In this method, since almost the same pressure as the suction pressure applied to the detection hole is applied to the filter itself, it is necessary to prevent the influence of changes in liquid viscosity due to temperature. Therefore, it is necessary to perform measurements under more advanced temperature control. This method measures how many seconds it is possible to aspirate a predetermined amount of sample (blood diluent), and the detection hole is used to monitor whether the red blood cells are in a normal state. Alternatively, by measuring the number of particles per unit diluted liquid before passing through the filter, it is possible to obtain the number of particles that can pass through the filter as well as the important parameter of the passage rate. Together with the parameters, the deformability characteristics of red blood cells can be expressed in a two-dimensional histogram.

In claims 3 and 6,
The total area of the pores in the filter 5 is 1 of the area of the pores 4.
~0.1 times. It measures how many seconds a predetermined amount of sample can be aspirated, and how easily blood cells pass through the filter 5. Therefore, it is meaningless unless the sample flow rate is determined (limited) by the total area of the filter.
The total area of the filter is larger than the area of the pores (1
(more than double), the flow rate will be restricted by the pores, making it impossible to perform the desired measurement. Conversely, if the total area of the filter is too small, it will take too much time for a predetermined amount of sample to pass through, making it impractical. Therefore, the limit is about 0.1 times.

〔Example〕

Next, an embodiment of the apparatus of the present invention for carrying out the above method will be described based on the drawings. FIG. 1 shows the entire device of the present invention, and FIG. 2 shows the detection hole and the area around the filter. 1 is a particle detection device in which a blood diluent 2 is passed through a pore 4 (detection hole) provided at the lower end of a detector 3 and having a pore diameter of approximately 100μ;
The apparatus is configured to detect red blood cells based on electrical differences between the red blood cells and the diluent. At the lower end of the detector 3 having the pores 4, an adapter 6 is fitted, which is provided with a filter 5 having a large number of pores with a diameter of about 3 μm at its lower part. Reference numeral 7 denotes a disk made of a hard material such as ruby or sapphire, which has pores 4. This disk 7 is fixed to the tip (lower end) of the detector 3 made of glass or synthetic resin, and the pores 4 are used as electric circuits. , detection electrodes 8 and 9 are provided inside and outside the detector. 10 is a sample container, 11 is a detection circuit, 12 is a fluid control device, and 13 is a counting circuit.

The filter 5 is a porous membrane filter or the like treated with paraffin to remove the necessary number of pores so that it has a predetermined number of filters, and is fixed to an adapter 6 for fixing the filter. The adapter 6 has a cylindrical container shape with the filter 5 fixed to the bottom thereof, and its inner diameter is adapted to fit with the outer diameter of the tip (lower end) of the detector 3. The filter 5 is provided so that the total area of the pores is 100,000 to 100,000 times the area of the pores 4, or the total area of the pores is 1 to 0.1 times the area of the pores 4. Note that when the total area of the pores in the filter 5 is sufficiently larger than the area of the pores 4, a soft synthetic resin molded product may be sufficient as the adapter 6;
, the adapter 6 must be made of conductive metal. This is filter 5
When the total area of the pores in the filter 5 is sufficiently large compared to the pores 4, an electric path is secured through the pores in the filter 5, but when the total area of the pores in the filter 5 is small, particles are detected in the pores themselves in the filter 5. This is because noise is generated. Further, the fluid control device 12 has a built-in manometer, a pump, etc.
The manometer contacts turn the pump on and off to generate a predetermined suction force. Furthermore, the detection circuit 11 has a built-in timer, and in the method of claim 2, it issues measurement start and measurement end signals, and in the method of claim 3, the fluid is quantified by the fluid control device. Measure the time of the liquid. Although it is desirable that the filter and the adapter be disposable, it is also possible to reuse it by backflowing distilled water to lyse and wash the filter. For each measurement, it is necessary to sufficiently wipe off the remaining red blood cells from the previous measurement to prevent contamination.

(Effect of the invention) In any method of the present invention, red blood cells are
In order to detect each individual particle, the height of the detection pulse is proportional to the size of the particle, so by looking at the size of the detection pulse, the state of the red blood cells can be monitored. is obtained as a direct measurement result, the measurement is easy, and the average measurement result of red blood cells in the blood can be obtained.
Furthermore, conventionally, there is no parameter that directly indicates the deformability of red blood cells, and depending on the measurement method, various parameters such as flow rate (ml/min), filter-passed red blood cells (%), or flow rate x number of red blood cells (ml/RBC/min) are used.
Although it was generally a laboratory measurement method, by adding a filter function to a general-purpose particle counting device and providing a timer, it became possible to easily measure the deformability. The measurement parameters are also accurate in terms of numerical display, and have the effect of being sufficiently valuable for general routine use.

[Brief explanation of drawings]

FIG. 1 is an explanatory side view showing one embodiment of the apparatus of the present invention, and FIG. 2 is an enlarged sectional view of the pores and filter portion of the detector. 1...Particle detection device, 2...Blood diluent, 3...
...Detector, 4...Pore, 5...Filter, 6...
Adapter, 7... Disk, 8, 9... Electrode, 10...
... Sample container, 11 ... Detection circuit, 12 ... Fluid control device, 13 ... Counting circuit.

Claims (1)

[Scope of Claims] 1. A detection method in which blood diluent 2 is passed through pores 4 and red blood cells are detected based on the electrical difference between the red blood cells and the diluent. After passing through a filter 5 having
A method for measuring the characteristics of red blood cells, which comprises passing the red blood cells through the pore 4 and measuring the deformation of the red blood cells. 2 The total area of the pores in the filter 5 is the area of the pores 4.
10. The method for measuring the characteristics of red blood cells according to claim 1, wherein the number of red blood cells passing through the pores 4 in a predetermined time is detected. 3. The method for measuring the characteristics of red blood cells according to claim 1, wherein the total area of the pores of the filter 5 is 1 to 0.1 times the area of the pores 4, and the passage time of a predetermined volume of blood diluent is measured. . 4. In a particle detection device that detects red blood cells based on the electrical difference between the red blood cells and the diluent by passing the blood diluent through the pore 4 provided at the lower end of the detector 3,
Detector 3 with pore 4 at the lower end, diameter 3μ at the bottom
A device for measuring the characteristics of red blood cells, characterized in that an adapter 6 is fitted with a filter 5 having a large number of front and rear pores. 5 The total area of the pores of the filter 5 is the area of the pores 4.
The apparatus for measuring the characteristics of red blood cells according to claim 4, which is 100,000 to 100,000 times larger. 6. The apparatus for measuring the characteristics of red blood cells according to claim 4, wherein the total area of the pores of the filter 5 is 1 to 0.1 times the area of the pores 4.