JPH0230457B2 - KETSUKYUNOTOKUSEIOSOKUTEISURUSOCHI - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a device for measuring the characteristics of blood cells, and particularly to a device for measuring red blood cell membrane function.

Conventionally, as a method for measuring red blood cell membrane function, there is a red blood cell osmotic fragility test, which is generally performed as one of the tests for determining the presence or absence of increased hemolysis of red blood cells. In this method, when red blood cells are suspended in a hypotonic sodium chloride solution, the inside of the red blood cells is more hypertonic than the outside, so water is taken inside and the red blood cells expand and become spherical.When more water is taken in, the membrane ruptures and hemolysis occurs. It is something to be used. Erythrocyte osmotic fragility is said to be increased if normal red blood cells undergo hemolysis in hypertonic hemolysis rather than in normal red blood cells, and normal red blood cells undergo hemolysis in hypotonic solutions rather than hemolysis. You say that red blood cell osmotic fragility is enhanced. In either case, if it deviates from the normal value, it is determined to be abnormal.

There are several types of testing methods, but the most commonly used method is PAR PART.
There is a law. This method involves creating a series of buffered NaCl solutions with a pH of 7.4 (for example, preparing 20 test tubes,
NaCl concentration 0.50%, 0.48%, 0.46%…0.30%,
In this method, a fixed amount of test blood is added to the sample (prepared as 0.28%) and the degree of hemolysis produced is measured colorimetrically using a spectrophotometer. However, this Perpart method requires quantitative measurement of many types of NaCl.
It is necessary to prepare a concentrated solution, requires a large amount of test tubes and reagents, cannot measure a large number of specimens, is very time-consuming, and has the disadvantage of large errors.

Recently, automated methods have been proposed, and a representative method is a method using a coil planet centrifuge (CPC). A coil planet type centrifuge uses a coil (equivalent to a centrifuge tube).
While revolving around the center axis of the centrifuge at high speed,
It is a double rotating device that rotates at low speed around the axis of the coil itself, and is an instrument that can produce a centrifugation effect comparable to that of using a centrifuge tube several meters long. To measure red blood cell osmotic fragility, a long and thin polyethylene tube wrapped around a glass rod into a coil is used as a centrifuge tube, and an osmotic concentration gradient of 300 mOsM to 0 mOsM is applied from the top to the bottom of the coil. Fill with NaCl solution, inject a small amount of blood into the upper tube, and centrifuge using CPC. The red blood cells then settle toward the lower end, the low osmotic pressure side, and move through the medium, whose osmotic pressure is gradually lower than that of the red blood cells. When it reaches a point where it can no longer withstand the drop in osmotic pressure, it collapses and lyses, creating a hemoglobin solution. Since hemoglobin is a much smaller particle than red blood cells, it does not sediment due to the centrifugal force of the CPC, but remains in the coil area where it occurs, creating a hemolytic zone. By observing the location and formation of this hemolytic zone, the magnitude of red blood cell osmotic fragility can be determined. in this way
The CPC method does not require the preparation of many test tubes and performs measurements automatically, but it is time-consuming to inject blood into the coil and requires a long time to set up. The disadvantage was that the accuracy decreased. In addition, there is no easy way to measure the start of hemolysis, maximum hemolysis, end point of hemolysis, etc., and it is necessary to use a scale or the like to measure it. Furthermore, the coil must be injected with NaCl solution having an osmotic concentration gradient in advance.
When conducting research on hemolysis for NaCl solutions and drugs with different concentration gradients, separate coils must be created, which is impractical.

Additionally, the applicant has already filed a patent application for an apparatus that adds water or a reagent at a constant rate to a liquid in which blood cells are suspended, causing a change in osmotic pressure, and determining the change in mean volume (MCV) of blood cells at this time. (Japanese Patent Application Laid-Open No. 143692/1983). However, this method calculates MCV = total blood cell volume/number of red blood cells, and after a predetermined time, divides the red blood cell pulse height information by the number of red blood cells.As the number of red blood cells decreases, the denominator becomes smaller, resulting in an error in the detected red blood cells. The present inventors have found that the magnitude of the MCV gradually increases, resulting in variations in MCV values and making it impossible to perform accurate measurements. Furthermore, the present inventors have found that the maximum hemolysis point in the conventional CPC method does not necessarily match the results obtained by the continuous measurement of MCV.

The present invention has been made in view of the above points, and it is an object of the present invention to provide an apparatus that can easily measure the characteristics of blood cells without requiring a physiological saline pipe with a concentration gradient.

Hereinafter, the configuration of the present invention will be explained based on the drawings. Figure 1 shows an example of the surroundings of a particle detection device.
FIG. 2 shows one embodiment of the device of the invention. The apparatus of the present invention has a sample container 1 whose temperature can be controlled.
, a dropping mixing means 3 for dropping and mixing a predetermined amount of water or a medical solution into the blood diluent (blood cell suspension) 2 in the sample container 1; A particle detection device 6 that detects blood cells based on the electrical difference between the blood cells and the diluent and generates an electric signal proportional to the size of the blood cells; a detection circuit 7 that detects an electric signal proportional to the size of the particle and converts it into a pulse signal;
a first gate 8 and a second gate 10 connected in parallel to each other; a first counter 11 connected to the first gate 8; a second counter 12 connected to the second gate 10; A reference pulse generation circuit 13 connected to the second gate 10, the first counter 11 and the second counter 12, and the first counter 1
It consists of a switching circuit 14 connected to the first and second counters 12, a data processing circuit 15 connected to this switching circuit 14, and a display/recording device 16 connected to this data processing circuit 15. It is structured so that you can ask for it.

In FIG. 1, a sample container 1 containing a blood diluent 2 is placed in a thermobath 18 whose temperature is controlled by a constant temperature device 17, and a detection hole 5 is provided below the surface of the blood diluent 2. A drop mixing means 3 is provided for dropping and mixing a predetermined amount of water or a chemical solution from the outside, and continuously sucking a suspension of blood cells from the pores 5 to detect blood cells. . In FIG. 1, as an example of the drip-mixing means 3, a predetermined amount is dropped into a sample container from a tank 20 containing water or a chemical solution by a pump 21.
A case where stirring is performed using the stirrer 22 is shown. When water is dropped, the impedance of the liquid changes, such as the salt concentration, so an auxiliary electrode 23 may be provided as necessary.
The correction circuit 24 corrects the sensitivity. Since the rate of change in salt concentration is usually known from a predetermined program, it is sufficient to provide only the change in sensitivity with respect to time. When the salt concentration decreases, the impedance of the fluid that suspends blood cells increases,
Changes in sensitivity occur due to the apparent increase in output pulses from blood cells. Therefore, the apparent sensitivity change is corrected by the correction circuit 24 so that a predetermined particle sensitivity is always maintained. Note that since the impedance of the liquid changes depending on the temperature of the liquid, a thermobath 18 is used to always maintain a constant temperature.

The apparatus of the present invention is constructed by adding a circuit configuration as shown in FIG. 2 to the particle detection apparatus 6 described above. The output signal of the detection circuit 7 is amplified by the amplifier circuit 25, corrected by the correction circuit 24, and sent to the two gates 8, 1.
Two up-down counters 11,1 through 0
Sent to 2. On the other hand, the output of the reference pulse generation circuit 13 is sent to gates 8 and 10 and up-down counters 11 and 12, respectively. Note that the NOT circuit 26 sends alternate gate signals to the upper half and the lower half. As a result, as shown in Figure 3,
For example, at time _t3 , the first counter 11
counts up, and the second counter 12 counts down. At the end of t ₃ the output of the switching circuit 14 provides the output B of the second counter 12. Note that at _t1 , the output of the first counter 11 becomes the output of the switching circuit 14 as it is because it also serves as a count for setting an initial value, that is, setting a denominator for displaying the blood cell concentration as a percentage. At the end of t ₂ , the output of the difference between t ₁ and t ₂ of the first counter 11 is obtained, and t ₂
At , the second counter 12 is counted up. Furthermore, a timer 27 measures the time by counting the reference pulses and sends a signal to the data processing circuit 15 together with the output of the switching circuit 14 . The data processing circuit 15 stores the signal sent from the switching circuit 14 and the signal from the timer 27, and a hemolysis curve is created using the display/recording device 16 (printer). The hemolytic curve thus obtained is as shown by the solid line in FIG. 4, and it agrees well with the hemolytic curve determined by the conventional coil planet type centrifugation method. The broken line in FIG. 4 indicates the number of blood cells, and as the osmotic concentration changes over time, blood cells are hemolyzed one after another starting from those with lower osmotic resistance, and the number of detected particles decreases. In Figure 4, point a is the hemolysis starting point, b
The point is the maximum hemolysis point, and the c point is the end point of hemolysis. Note that time corresponds to osmotic pressure. An alarm 28 is used to generate an alarm and prompt re-measurement when the values of the counters 11 and 12 are extremely large and exceed a set value due to a blockage in the detector or erroneous counting due to external noise.

As explained above, according to the device of the present invention,
Blood cell characteristics can be easily measured without the need for saline pipes with concentration gradients, and there is no need to optically detect hemoglobin elution. Another advantage is that it can be applied not only to red blood cells but also to other particles such as platelets.

[Brief explanation of drawings]

Fig. 1 is an explanatory diagram showing an example of the particle detection device and its surroundings in the device of the present invention, Fig. 2 is an explanatory diagram showing an embodiment of the device of the present invention, and Fig. 3 is an explanatory diagram showing the operating state of the counter etc. , FIG. 4 is a curve diagram showing an example of a hemolysis curve obtained using the apparatus of the present invention. DESCRIPTION OF SYMBOLS 1... Sample container, 2... Blood dilution liquid, 3... Dripping mixing means, 4... Detector, 5... Pore, 6...
Particle detection device, 7...Detection circuit, 8...First gate, 10...Second gate, 11...First counter, 12...Second counter, 13...Reference pulse generation circuit, 14...Switching Circuit, 15... Data processing circuit, 16... Display recording device, 17... Constant temperature device, 18... Thermobath, 20... Tank, 21
... pump, 22 ... stirrer, 23 ... auxiliary electrode, 24 ... correction circuit, 25 ... amplifier circuit, 26
...NOT circuit, 27...timer, 28...alarm.

Claims (1)

[Claims] 1. A sample container whose temperature can be controlled, a drip mixing means for dropping and mixing a predetermined amount of water or drug solution into the blood diluent in the sample container, and a droplet mixing means for dropping and mixing the blood diluent in the sample container into a thin tube provided at the lower end of the detector. A particle detection device that detects blood cells based on the electrical difference between the blood cells passed through the hole and a diluent and generates an electrical signal proportional to the size of the blood cells, and A detection circuit that detects an electric signal and converts it into a pulse signal, a first gate and a second gate connected in parallel to the detection circuit, a first counter connected to the first gate, and a first counter connected to the second gate. a second counter connected to the first gate, the second gate, the first counter and the second counter; a switching circuit connected to the first counter and the second counter; 1. A device for measuring characteristics of blood cells, comprising a data processing circuit connected to the device and a display/recording device connected to the data processing circuit, and configured to directly obtain a hemolysis curve.