JP2020030217A - Device for analysis of blood coagulation system, and method and program for analysis of blood coagulation system - Google Patents

Abstract

To propose a device for analysis of a blood coagulation system and a method and a program for the analysis of the blood coagulation system, capable of analyzing the blood coagulation system with high precision.SOLUTION: A blood coagulation system is observed prior to timing of viscoelasticity manifestation (timing at which blood starts to clot from a viewpoint of viscoelasticity (more specifically, timing at which active polymerization of fibrin monomers starts)) as temporal change in permittivity. The permittivity of the blood placed across a pair of electrodes is measured at prescribed time intervals after anticoagulant effect acting on the blood is ended, and degree of activity of the blood coagulation system is analyzed from the measured result.SELECTED DRAWING: Figure 10

Description

  The present invention relates to a field of technology for acquiring information on blood coagulation from the dielectric constant of blood.

  Conventionally, prothrombin time and activated partial thromboplastin are widely known as blood coagulation tests. In addition, these tests are important for examination of blood coagulation factor deficiency and the like and monitoring of blood to which an anticoagulant has been administered, and a standardization method for the tests has been proposed (for example, see Patent Document 1).

JP 2006-349684 A

  However, the above test does not (quantitatively) measure the degree of easiness of blood clotting, but under the condition that the coagulation reaction is accelerated by adding an excessive coagulation initiator, It is a piece-wise (qualitative) observation of whether or not coagulation is completed. In other words, the above test is a technique for evaluating the risk (bleeding tendency) due to the difficulty of blood clotting, and cannot evaluate the risk (thrombosis tendency) due to the tendency of blood to clot. It is important to see the degree of easiness of blood clots (quantitatively) because it has an advantage such as a large amount of information compared to fragmentation, and in recent years, it is higher than fragmentation. What can analyze the blood coagulation system with high accuracy is required.

  The present invention has been made in consideration of the above points, and has as its object to propose a blood coagulation system analysis apparatus, a blood coagulation system analysis method, and a program capable of analyzing the blood coagulation system with high accuracy.

  In order to solve such a problem, the present invention relates to a blood coagulation system analyzing apparatus, comprising: a pair of electrodes; an applying unit that applies an alternating voltage to the pair of electrodes at predetermined time intervals; The measurement means for measuring the permittivity of the blood to be measured and the permittivity of the blood measured at time intervals after the anticoagulant action acting on the blood is released, the degree of the function of the blood coagulation system is determined. Analysis means for performing analysis;

  The present invention also relates to a blood coagulation system analysis method, which comprises applying an alternating voltage to a pair of electrodes at predetermined time intervals, and measuring a dielectric constant of blood disposed between the pair of electrodes. After the step and the anticoagulant effect acting on the blood are released, an analyzing step is performed to analyze the degree of the function of the blood coagulation system using the dielectric constant of the blood measured at time intervals.

  Further, the present invention is a program, by applying an alternating voltage to a pair of electrodes at a predetermined time interval to an application unit for applying an alternating voltage, and a pair of electrodes for a measurement unit for measuring a dielectric constant. Let the dielectric constant of the blood placed between the electrodes be measured, and to the analysis unit that analyzes the blood coagulation system, the blood coagulation measured at time intervals after the anticoagulant action acting on the blood is released. Using the dielectric constant, the degree of the function of the blood coagulation system is analyzed.

  The blood coagulation system appears as a time change in the dielectric constant before the time when blood starts to solidify from the mechanical viewpoint of viscoelasticity (the time when active polymerization of fibrin monomer starts).

  In this regard, the present invention uses a dielectric constant measured after the anticoagulant action acting on the blood is released, and therefore, before the blood starts to solidify from the mechanical viewpoint of viscoelasticity. You can clearly observe the process.

  Therefore, the present invention can analyze the function of the early blood coagulation system by the time change of the dielectric constant before the time when the blood starts to solidify from the mechanical viewpoint of viscoelasticity. The analysis accuracy can be improved as compared with the conventional system, as much as the trend of the system is grasped by the time change of the dielectric constant. In addition, the degree of action of the blood coagulation system can be analyzed earlier than in the past.

It is a figure which shows roughly the relationship between the ion of a cell and an electric field. It is a graph which shows a simulated complex permittivity spectrum. It is a figure which shows the structure of a sample introduction part schematically. It is a graph which shows the dielectric spectrum (when thrombin is given) of the blood coagulation system obtained by the experiment. It is a graph which shows the dielectric spectrum (when thrombin is not given) of the blood coagulation system obtained by the experiment. 6 is a graph showing a temporal change of a dielectric constant at a specific frequency (in the case of model blood). It is a graph which shows the measurement result of mechanical viscoelasticity using the damping vibration type rheometer. 5 is a graph showing a temporal change of a dielectric constant at a specific frequency (in the case of human blood). It is a figure showing roughly composition of a dielectric blood coagulation measuring device. It is a flowchart which shows a blood coagulation system analysis processing procedure. It is a photograph which shows the red blood cell which formed the coin. 5 is a graph showing a time change of a dielectric constant after stopping stirring. It is a graph which shows a time change when a dielectric constant corresponding to two specific frequencies is expressed as a ratio with a dielectric constant at a reference time. 6 is a graph showing a time change when a difference in a change rate of a dielectric constant corresponding to each frequency is taken. It is a graph which shows the solidification start time obtained by the rheology measurement, and the inclination in the time change of the rate of change of the dielectric constant corresponding to two frequencies.

Hereinafter, embodiments for carrying out the invention will be described. The description will be made in the following order.
<1. Electrical properties of cells>
<2. Discussion of Dielectric Spectrum of Blood Coagulation System>
[2-1. experimental method]
[2-2. Experimental result]
<3. Embodiment>
[3-1. Configuration of Dielectric Blood Coagulation Measurement Device]
[3-2. Blood coagulation system analysis processing procedure]
[3-3. Effects]
<4. Other Embodiments>

<1. Electrical properties of cells>
Cells contain positive ions and negative ions (FIG. 1 (A)). When there are cells in the alternating electric field, these ions move (follow) according to the positive / negative direction change in the alternating electric field. In this case, since the cell membrane has high insulating properties, cations and anions are unevenly distributed at the interface between the cell membrane and the cytoplasm, and ionic interfacial polarization occurs (FIG. 1B).

  Here, the dielectric spectrum of a simulated cell suspension cited in the literature and the like is shown in FIG. As can be seen from FIG. 2, when the frequency of the alternating electric field is sufficiently low, since the cell undergoes interfacial polarization at the interface between the cell membrane and the cytoplasm, the real part of the complex permittivity is obtained as a large value. Hereinafter, the real part of the complex permittivity is referred to as permittivity.

  On the other hand, when the frequency of the alternating electric field is about several tens [MHz], positive and negative switching occurs in the alternating electric field before cations and anions move to the interface between the cell membrane and the cytoplasm. That is, the interface polarization cannot follow the change in the alternating electric field.

  Therefore, the higher the frequency of the alternating electric field, the smaller the dielectric constant. The phenomenon in which the dielectric constant decreases is called “dielectric relaxation”, and the imaginary part of the complex dielectric constant increases in a portion where the dielectric relaxation occurs. Incidentally, the imaginary part of the complex permittivity is generally called dielectric loss.

  This dielectric relaxation occurs at a specific frequency band depending on the size and structure of the cell, and the number of dielectric relaxations depends on the number of main interfaces included in the cell. For example, a cell such as a red blood cell having no cell nucleus has one dielectric relaxation, and a nucleated cell having one or more nuclei has two or more dielectric relaxations.

  Thus, the complex permittivity of a cell has a relationship that depends on the frequency of the electric field for both the real part and the imaginary part. This is called "dielectric dispersion".

<2. Discussion of Dielectric Spectrum of Blood Coagulation System>
[2-1. experimental method]
The reaction was started by adding thrombin to the model blood, and the model blood immediately after the addition of the thrombin was introduced into the sample introduction section to measure the dielectric constant (real part of complex permittivity) in the blood coagulation system.

  The model blood was prepared by mixing an erythrocyte suspension obtained by washing rabbit-preserved blood from Kojin Bio Co. with PBS with bovine fibrinogen from Sigma to adjust to a hematocrit of 25% and a fibrinogen concentration of 0.22%. What was done was used.

  Thrombin was prepared by adjusting bovine thrombin from Sigma to 0.01% (10 [units / ml]) and 10 [μl] per 1 [ml] of model blood (that is, 100 milliunits / ml). Alternatively, a volume of 5 μl (ie, 50 milliunits / ml) was added.

  The dielectric constant was measured using an impedance analyzer (4294A) manufactured by Agilent. Further, a frequency band to be measured (hereinafter, also referred to as a measurement frequency range) is 40 [Hz] to 110 [MHz], and a time interval to be measured (hereinafter, also referred to as a measurement interval) is 1 [minute]. The temperature of the object to be measured was 37 ° C.

  The sample introduction part had the structure shown in FIG. That is, a pair of gold-plated cylindrical electrodes E1 and E2 are inserted into both ends of a polypropylene cylindrical body CL to be sealed, and an injection needle for supplying blood between the electrodes E1 and E2. HN was penetrated through the outer wall of the cylindrical body CL. In addition, by closing the penetrating portion of the injection needle HN with grease, a sealed state of a space surrounded by the cylindrical body CL and the electrodes E1 and E2 is maintained.

[2-2. Experimental result]
FIG. 4 shows a dielectric spectrum obtained under the above-described experimental method, and FIG. 5 shows a dielectric spectrum observed under the same conditions as the above-described experimental method in a state where thrombin is not added to the model blood. . FIG. 4 is a dielectric spectrum when 10 [μl] is added per 1 [ml] of model blood.

  Comparing FIG. 4 with FIG. 5, in the model blood coagulation system, dielectric relaxation can be observed in the same manner as a general cell suspension, and the dielectric response increases with the progress of coagulation (with time). Was observed.

  Here, FIG. 6 shows a temporal change of the dielectric constant corresponding to a specific frequency (here, 758 [kHz]). A curve W1 in FIG. 6 is a case where thrombin is not added to the model blood (a two-dimensional graph obtained by cutting out a portion of 758 [kHz] in FIG. 5).

  Curve W2 is a case where 10 [μl] of thrombin is added per 1 [ml] of model blood (two-dimensional graph cut out from 758 [kHz] in FIG. 4). Curve W3 is a case where 5 [μl] of thrombin is added per 1 [ml] of model blood (not shown, but a two-dimensional graph cut out of 758 [kHz] as in FIG. 4).

  As is clear from the curve W2, a peak of the dielectric constant was observed about 8 [minutes] from the time when thrombin was added. Further, as is apparent from the curve W3, when the amount of thrombin was halved, the time at which the peak of the dielectric constant was shifted, and this peak was observed approximately 18 minutes after the addition of thrombin.

  On the other hand, in the case where 10 [μl] thrombin was added per 1 [ml] of model blood and the case where 5 [μl] thrombin was added per 1 [ml] of the model blood, a free damping oscillation type rheometer was used. A mechanical viscoelasticity measurement (rheological measurement) was also performed. FIG. 7 shows the measurement results.

  7A shows a case where 10 [μl] of thrombin is added per 1 [ml] of model blood, and FIG. 7 (B) shows a case where 5 [μl] of thrombin is added per 1 [ml] of model blood. I have. The peaks in FIGS. 7A and 7B are the times when a certain viscoelastic characteristic appears. That is, it is a time when the blood starts to solidify from the viewpoint of viscoelasticity (mechanics) (hereinafter, this is also referred to as a viscoelastic flowering time).

By the way, the actual blood coagulation system undergoes a complex biological reaction in which a large number of coagulation factors are involved, and finally fibrinogen in blood is converted to fibrin by the involvement of thrombin. The part where fibrinogen turns into fibrin can be thought of as a process that plays a substantial role in coagulating blood.

  Specifically, in this process, fibrinogen is converted into fibrin monomer by the involvement of thrombin, which polymerizes with each other to form a fibrin polymer. The fibrin polymer is then cross-linked by the involvement of factor XIII into stabilized fibrin, causing substantial blood clotting.

  From this, the viscoelastic flowering time obtained as a peak in the measurement of the free damping vibration type rheometer (the peak may not be obtained, in which case the log reduction rate starts to decrease), the fibrin monomers are mutually separated. It is considered to correspond to the process of polymerizing to a fibrin polymer, or the process of cross-linking of this fibrin polymer to stabilized fibrin. At least at a time later than the time when thrombin starts to be involved in fibrinogen.

  As is apparent from FIG. 7, similarly to the case of the dielectric spectrum shown in FIG. 6, approximately 8 [minutes] from the time when thrombin was added (FIG. 7 (A)), thrombin was added in an amount half that of the thrombin. About 18 [minutes] from the time point (FIG. 7 (B)), a sharp increase in the viscoelastic modulus (viscoelastic flowering time) was observed.

  From this, it is understood that the time change of the dielectric constant reflects the blood coagulation system. It should be noted that when measuring the dielectric constant, it is clear from the comparison between FIG. 6 and FIG. 7 that the process before the viscoelastic flowering time, which cannot be observed with the free damping oscillation type rheometer, is performed. It can be clearly observed. As shown in FIG. 8, this process could be similarly observed for blood collected from humans.

  Incidentally, FIG. 8 shows that the blood at the end of a human was collected in a test tube containing an anticoagulant (citrate), and immediately after adding a coagulation initiator (calcium chloride) to the blood, a specific frequency (here, 758 [kHz]) shows the change over time of the dielectric constant corresponding to 758 [kHz].

  As is clear from these experiments, the fact that the process before the viscoelastic flowering time is reflected as an increase in the dielectric constant means that the change in the degree of the coagulation system before the viscoelastic flowering time (in other words, the coagulation The degree of enhancement or coagulation ability) can be referred to as an index that quantitatively indicates the degree of enhancement or coagulation ability.

  Specifically, the gradient of the dielectric constant in the initial stage from the start of the measurement to the peak is large when the viscoelastic flowering time is early (FIG. 6: curve W2), and when the viscoelastic flowering time is late. It becomes smaller (FIG. 6: curve W3). Therefore, the working degree of the blood coagulation system (the degree of hypercoagulation or the degree of coagulation ability) can be analyzed in a short time and finely from the transition of the dielectric constant in the initial stage up to the peak.

<3. Embodiment>
Next, a dielectric blood coagulation measuring apparatus will be described as an embodiment.

[3-1. Configuration of Dielectric Blood Coagulation Measurement Device]
FIG. 9 shows a schematic configuration of the dielectric blood coagulation measuring device 1. This dielectric blood coagulation measuring apparatus 1 has a sample introduction unit 2 and a signal processing unit 3. In the dielectric blood coagulation measuring apparatus 1, a temperature sensor (not shown) and a thermoelectric element (not shown) are provided in the sample introduction section 2 or the signal processing section 3. The dielectric blood coagulation measuring apparatus 1 measures the temperature of the blood to be measured using a temperature sensor, and gives a signal amount corresponding to the measurement result to the thermoelectric element, so that the temperature of the blood can be adjusted. ing.

  The sample introduction part 2 has a pair of electrodes, and human blood is introduced between the electrodes. The human blood has an anticoagulant effect, and the anticoagulant effect is released immediately before or after the human blood is introduced between the electrodes.

  As the sample introduction unit 2, for example, the one shown in FIG. 3 can be adopted. However, the structure of the sample introduction part 2 or the shape and material of each material are not limited to those shown in FIG. For example, a sample introduction part having a structure in which both ends of a cylinder having a polygonal cross section (triangle, square or more) are sealed, and a pair of electrodes and a wiring connected thereto are printed on one inner surface of the cylinder. can do. In short, it is sufficient that the anticoagulant effect acting on the blood can be released immediately before or after the blood is introduced between the electrodes, and the blood can be retained between the electrodes for a predetermined period.

  The signal processing unit 3 is configured to include a voltage application unit 11, a dielectric constant measurement unit 12, and a blood coagulation system analysis unit 13.

  The voltage applying unit 11 applies the alternating voltage at a time when a command to start measurement or when power is turned on is set as a start time.

  Specifically, the voltage applying unit 11 sets a frequency set as a target frequency for the pair of electrodes arranged in the sample introduction unit 2 at each set measurement interval (hereinafter, also referred to as a target frequency). Is applied.

  As the measurement interval and the frequency of interest, specifically, 1 [minute] and 758 [kHz] used in the above experiment can be adopted. However, these numerical values are merely examples, and the present invention is not limited to these numerical values. Further, the measurement interval and the frequency of interest can be set to various values via input means such as a mouse and a keyboard.

  The permittivity measuring unit 12 measures the permittivity at the time when a command to start the measurement or when the power is turned on is started.

  Specifically, the permittivity measuring unit 12 measures a current or impedance between a pair of electrodes arranged in the sample introduction unit 2 at a predetermined cycle, and derives a permittivity from the measured value. To derive the permittivity, a known function or relational expression indicating the relationship between the current or impedance and the permittivity is used.

  The blood coagulation system analysis unit 13 is provided with data indicating a dielectric constant corresponding to the frequency of interest (hereinafter, also referred to as dielectric constant data) from the dielectric constant measurement unit 12 at each measurement interval.

  The blood coagulation system analysis unit 13 performs a blood coagulation system analysis process when receiving, from the dielectric constant data given from the dielectric constant measurement unit 12, the dielectric constant data after the anticoagulant action of human blood is solved. Start.

  The determination at the time when the anticoagulant action of human blood is released is determined, for example, by using a threshold value that is larger than the permittivity presented before the anticoagulant action is released by a predetermined amount as a threshold value and a permittivity indicating a dielectric constant not less than the threshold value. A method is adopted in which the time when the data is received is the time when the anticoagulant effect of human blood is released.

When this method is adopted, roughly, in the case where the anticoagulant effect of human blood is released after being introduced between the electrodes in the sample introduction section 2, the first dielectric constant data from the time of release is obtained. The point in time when the data is received is set as the start point of the analysis processing. On the other hand, in the case where the anticoagulant effect of human blood is released immediately before being introduced between the electrodes in the sample introduction part 2, the point of time when the first dielectric constant data given from the dielectric constant measurement part 12 is received is the time point of the analysis processing. It is the starting point.

  In the blood coagulation system analysis unit 13, the period from the start of the blood coagulation system analysis process to the elapse of the set period is set as the analysis period. The blood coagulation system analysis unit 13 detects a straight line that is closest to the dielectric constant indicated by each of the plurality of (measured) dielectric constant data received during the analysis period. Then, the blood coagulation system analysis unit 13 obtains the gradient of the detected straight line as a parameter indicating the amount of increase in the dielectric constant before the viscoelastic flowering time, and predicts the viscoelastic flowering time from the gradient.

  This prediction is based on the assumption that the larger the slope of the straight line, the faster the viscoelastic flowering time is. For example, the relationship between the database of the straight line slope and the straight line slope or the slope of the straight line and the viscoelastic flowering time This is performed on the basis of a function indicating gender (regularity).

  When predicting the viscoelastic flowering time, the blood coagulation system analyzer 13 notifies one or both of the prediction result and the dielectric constant used for the prediction.

  This notification is performed, for example, by graphing and displaying on a monitor or printing on a predetermined medium.

[3-2. Blood coagulation system analysis processing procedure]
Next, the blood coagulation system analysis processing procedure will be described with reference to the flowchart shown in FIG.

  That is, the dielectric blood coagulation measuring device 1 develops the program stored in the ROM into the RAM, for example, when receiving a command to start the measurement or when the power is turned on, and proceeds to step SP1 to execute the blood coagulation system. Start the analysis process.

  In step SP1, the dielectric blood coagulation measuring apparatus 1 starts applying the alternating voltage of the frequency of interest to the voltage application unit 11 at each set measurement interval to the pair of electrodes arranged in the sample introduction unit 2. Then, the process proceeds to the next step SP2.

  In step SP2, the dielectric blood coagulation measuring apparatus 1 causes the dielectric constant measuring unit 12 to start measuring the dielectric constant, and proceeds to a subsequent subroutine (hereinafter, also referred to as a blood coagulation system analysis routine) SRT to analyze the blood coagulation system. The unit 13 is started to analyze the blood coagulation system.

  That is, in step SP11 of the blood coagulation system analysis routine, the blood coagulation system analysis unit 13 waits for permittivity data indicating a permittivity that is equal to or higher than a predetermined threshold. If the dielectric constant data equal to or larger than the predetermined threshold value is received in step SP11, the blood coagulation system analyzing unit 13 determines that the anticoagulant action of human blood has been released, and proceeds to the next step SP12.

  In step SP12, the blood coagulation system analysis unit 13 starts measuring the time of the set analysis period, and accumulates the dielectric constant data given from the dielectric constant measurement unit 12 until the analysis period elapses.

  Next, in step SP13, the blood coagulation system analysis unit 13 detects a straight line that is the closest to the permittivity indicated by the permittivity data accumulated during the analysis period, and calculates the gradient of the straight line as the permittivity of the permittivity before the viscoelastic flowering time. It is obtained as a parameter indicating the amount of increase.

Then, in step SP14, the blood coagulation system analysis unit 13 predicts the viscoelastic flowering time from the parameter (gradient of the initial waveform of the dielectric spectrum) obtained in step SP13, and ends the blood coagulation system analysis process.

  In this way, the dielectric blood coagulation measuring device 1 executes the blood coagulation system analysis processing according to the program developed in the RAM.

[3-3. Effects]
In the above configuration, the blood coagulation system analyzing apparatus 1 applies an alternating voltage of a frequency of interest to a pair of electrodes facing each other so as to sandwich a position where blood is to be arranged at predetermined time intervals ( (See FIG. 9).

  The blood coagulation system analyzer 1 also measures the permittivity of blood disposed between the pair of electrodes at predetermined time intervals after the action of the anticoagulant acting on the blood is released, and the measurement result is obtained. Analyzes the degree of the blood coagulation system from.

  The blood coagulation system is observed as a time change in the dielectric constant before the viscoelastic flowering time (the time when blood starts to solidify in terms of viscoelasticity (more specifically, at least the time when active polymerization of fibrin monomer starts)) (See FIGS. 6 and 7).

  In this regard, the blood coagulation system analyzer 1 uses a dielectric constant measured after the anticoagulant action acting on the blood is released, and therefore, the viscosity which cannot be observed with a free damping vibration type rheometer. The process before the elastic flowering time can be clearly observed.

  That is, the blood coagulation system analyzing apparatus 1 can quantitatively capture a blood coagulation factor that acts early before the viscoelastic flowering time by a temporal change of the dielectric constant before the viscoelastic flowering time.

  By the way, it is known that a series of coagulation reactions that cause venous thrombosis may be initiated by the slow activation of blood coagulation factor IX on the erythrocyte membrane (Masaru Kaihara, Experimental Medicine Vol. 22, No. 13, 2004, pp. 1869-1874).

  Activated Partial Thromboplastin Time (APTT) is widely known as a conventional measurement method for the blood coagulation system as described above.

  However, this APTT does not look at the degree of easiness of blood clotting, but determines whether coagulation is completed within a normal time under the conditions of adding an excessive coagulation initiator to accelerate the coagulation reaction. By observing the results in a fragmentary (qualitative) manner, the difficulty of blood clotting (bleeding tendency) is evaluated. Therefore, the APTT is useless when easiness of blood clots (thrombotic tendency) is a problem, such as venous thrombosis, which progresses relatively slowly due to stagnation of blood flow in the veins, and moreover, the risk of the disease is evaluated. It is not possible.

  On the other hand, the blood coagulation system analyzing apparatus 1 can quantitatively grasp a blood coagulation factor that acts early due to a temporal change in the dielectric constant before the viscoelastic flowering time. For this reason, the blood coagulation system analyzer 1 has a higher degree of accuracy in blood coagulation than a conventional method in which whether or not coagulation is completed within a normal time is fragmentarily (qualitatively) determined. Monitoring or risk assessment of diseases involved in Being able to assess the risk of disease is also very useful from a preventive medicine perspective.

For example, since surgery is a risk factor for the onset of venous thromboembolism, it is possible to easily determine the risk after surgery and determine the medication based on the risk. Elderly people, obesity, smoking, pregnancy, etc. are also risk factors for the development of venous thromboembolism, so that it is possible to easily determine these risks and determine medication based on these risks. In addition, since diabetes patients often have increased blood coagulation, judging the degree of the coagulation facilitates the judgment of medication.

  In the analysis of the blood coagulation system analyzing apparatus 1 in this embodiment, the viscoelastic flowering time is predicted from a parameter indicating the amount of increase in the dielectric constant before the viscoelastic flowering time. Therefore, the blood coagulation system analyzer 1 can not only present the trend of the blood coagulation system before the viscoelastic flowering time on the display unit as a change in the dielectric constant, but also is predicted from the change in the dielectric constant. The viscoelastic flowering time can be promptly presented.

  Generally, the viscoelastic flowering time requires several tens [minutes] from the start of the coagulation system, but the fact that the viscoelastic flowering time can be presented in a few [minutes] is particularly useful from the viewpoint of disease monitoring and risk evaluation. Will be useful.

  According to the above configuration, the blood coagulation system analyzer 1 is used to analyze the function of the blood coagulation system at an early stage based on the time change of the dielectric constant after the anticoagulant action acting on the blood is released. Was. This makes it possible not only to monitor a disease that was impossible with the conventional measurement method for the blood coagulation system, but also to evaluate the risk of the disease, and as a result, blood coagulation that can improve the analysis accuracy compared to the conventional method The system analysis device 1 can be realized.

<4. Other Embodiments>
In the above embodiment, human peripheral blood (venous blood) was used as a sample to be measured. However, the sample is not limited to human blood, but may be an animal other than human. It may also be arterial blood.

  In the above-described embodiment, the alternating voltage of the frequency of interest is applied to the pair of electrodes arranged in the sample introduction unit 2. However, the frequency of the alternating voltage to be applied may be the entire frequency in a certain band including the frequency of interest or a frequency for each predetermined width.

  In this case, the blood coagulation system analysis unit 13 is provided with data indicating the dielectric constant corresponding to a plurality of frequencies from the dielectric constant measurement unit 12 at each measurement interval. Therefore, the blood coagulation system analysis unit 13 can correct the dielectric constant using the dielectric constant corresponding to a plurality of frequencies.

  By the way, as shown in FIG. 11, erythrocytes often agglutinate (form clumps) in a coin-like or irregular manner. It is known that when red blood cells aggregate, the dielectric constant of the red blood cells increases, as shown in FIG.

  FIG. 12 shows the results of measurement of the change in the dielectric constant after the stirring of whole blood, blood with a plasma component of 50%, and blood with a blood component of 0% was stopped, according to A. lrimajiri et al, elsevier science, Biochim. Biophys, vol. 1290, pp. 207-209.

  When the rate of change of the permittivity is viewed as a ratio to the permittivity at the reference time, the time change of the permittivity caused by the agglutination of red blood cells is more frequency dependent than the time change of the permittivity caused by blood coagulation. (Difference in time change of permittivity due to difference in frequency) was found to be small.

  Therefore, by taking the difference between the rate of change of the permittivity corresponding to a specific frequency and the rate of change of the permittivity corresponding to a frequency different from the frequency, while maintaining the permittivity change due to blood coagulation, the red blood cells The degree of influence of the dielectric constant caused by the aggregation can be reduced.

  Here, the experimental results are shown in FIGS. FIG. 13 shows a case where human venous blood is collected in a test tube containing an anticoagulant (citric acid), and immediately after adding a coagulation initiator (calcium chloride) to the blood, the sample corresponds to two specific frequencies. This is a temporal change when the dielectric constant is expressed as a ratio with the dielectric constant at the reference time.

  The specific frequency is 150 [kHz] (waveform W10 shown by a solid line in FIG. 13) and 260 [kHz] (waveform W11 shown by a broken line in FIG. 13), and the permittivity at the reference time is measured first. (Ε′t = 0).

  On the other hand, FIG. 14 shows a time change (that is, a difference between the waveform W10 and the waveform W11 shown in FIG. 13) when a difference in a change rate of the dielectric constant corresponding to each frequency shown in FIG. 13 is obtained.

  Incidentally, this experiment was performed under the same conditions as those in the experiment results (FIGS. 4, 5, and 8). That is, the measurement frequency range is 40 [Hz] to 110 [MHz], the measurement interval is 1 [minute], the temperature of the object to be measured is 37 [° C], and the dielectric constant is an impedance analyzer (4294A) manufactured by Agilent. It measured using.

  As can be seen from FIG. 13, when the dielectric constant corresponding to a specific frequency and the dielectric constant corresponding to a frequency different from the frequency are viewed as the ratio of the dielectric constant at the reference time, the shorter the measurement time, the higher the dielectric constant. Wrap. This is because, on the short time side, the influence of the change in the dielectric constant due to the aggregation of red blood cells is relatively large, and the frequency dependence is relatively small. On the other hand, on the long-time side, the influence of the change in the dielectric constant caused by blood coagulation is relatively large, and the frequency dependence is relatively large.

  On the other hand, as can be seen from FIG. 14, the difference in the rate of change of the dielectric constant corresponding to each frequency is due to the effect of the dielectric constant caused by red blood cell aggregation, while the effect of the dielectric constant change caused by blood coagulation remains. Degrees tend to decrease and increase linearly.

  FIG. 15 shows another experimental result. FIG. 15 shows the relationship between the solidification start time obtained by the rheological measurement and the analysis using the difference in the rate of change in the dielectric constant corresponding to the two frequencies.

  The plot in FIG. 15 shows the difference in the change rate of the dielectric constant 10 minutes after the start of measurement corresponding to two frequencies (150 [kHz] and 260 [kHz]) for five healthy subjects. The waveform in FIG. 15 is an optimal approximate curve based on these plots.

  As can be seen from FIG. 15, the tendency was obtained that the shorter the solidification start time obtained by the rheological measurement, the smaller the slope of the difference between the rates of change in the dielectric constants corresponding to the two frequencies. However, this tendency may be different depending on the frequency to be measured, or the difference in the structure or material of the electrode used in the sample introduction unit 2.

  As described above, the blood coagulation system analyzing unit 13 expresses the measurement value (dielectric constant corresponding to two specific frequencies) given from the dielectric constant measuring unit 12 at predetermined intervals as a ratio with the dielectric constant at the reference time. By performing the division, it is possible to accurately pay attention to the time change of the dielectric constant reflecting the initial process of the blood coagulation reaction. As a result, the accuracy of the analysis of the dielectric constant caused by blood coagulation can be further improved.

As another example, the permittivity corresponding to the frequency of interest may be corrected using a permittivity other than the permittivity corresponding to the frequency of interest. For example, a correction method for correcting the gradient of the dielectric constant corresponding to the frequency of interest so that the greater the degree of deviation of the gradient of the dielectric constant corresponding to the frequency approximating the frequency of interest is weighted on the side where the gradient bias is greater, Applicable. The above-described correction method is an example, and various other correction methods can be adopted.

  As a method of applying the alternating voltage of a plurality of frequencies, any one of a so-called frequency domain method, a frequency superposition method, and a time domain method can be adopted, and a combination technique of these techniques can also be adopted.

  The frequency domain method is a method of applying an alternating voltage while rapidly switching all or every predetermined width in a frequency band to be measured. The frequency superposition method is a method of applying an alternating voltage in which all or some frequency components in a frequency band to be measured are mixed. The time domain method is a method of applying a step voltage. In the time domain method, a process of performing a Fourier transform on a current responding to an alternating voltage in which a plurality of frequency components are mixed as a function of time and detecting frequency dependency is required.

  Examples of a wave in which a plurality of frequency components are mixed include a differential Gaussian wave, a surface shear wave (STW), a Rayleigh wave (Surface Acoustic Wave), a BGS (Bleustein-Gulyaev-Shimizu) wave, a Lamb wave, a surface skimming bulk wave, or an SH. (Shear horizontal) waves or the like can be applied.

  In the above-described embodiment, the gradient of a straight line that is the closest to the dielectric constant measured during the analysis period is applied as the parameter indicating the amount of increase in the dielectric constant before the viscoelastic flowering time. However, this parameter is not limited to the slope of a straight line. For example, it can be an average of the rate of change of the dielectric constant measured within the analysis period.

  Specifically, every time the blood coagulation system analysis unit 13 receives the dielectric constant data from the dielectric constant measurement unit 12 during the analysis period, the blood coagulation system analysis unit 13 indicates the dielectric constant data and the dielectric constant data received immediately before the reception. Take the difference in dielectric constant. This difference is an increase in the dielectric constant per observation interval.

  The blood coagulation system analysis unit 13 acquires the increase amount of the dielectric constant per observation interval until the analysis period elapses, and when the analysis time elapses, calculates the average of the increase amount of the acquired dielectric constants as the viscoelastic flowering time. It is obtained as a parameter indicating the amount of increase in the previous dielectric constant.

  As described above, as the parameter indicating the amount of increase in the dielectric constant before the viscoelastic flowering time, the average of the rate of change in the dielectric constant measured during the analysis period can be applied. Of course, other than the average of the change rate of the dielectric constant and the gradient of the straight line, a parameter indicating the amount of increase in the dielectric constant before the viscoelastic flowering time can be used.

  In the above-described embodiment, the viscoelastic flowering time is predicted from the parameter indicating the amount of increase in the dielectric constant before the viscoelastic flowering time. However, the analysis items are not limited to the prediction of the viscoelastic flowering time. For example, the risk of thrombus can be evaluated.

  In this evaluation, the larger the parameter indicating the amount of increase in the dielectric constant before the viscoelastic flowering time (the slope of the gradient of the straight line closest to the dielectric constant measured during the analysis period or the average of the rate of change in the dielectric constant), the larger the thrombus Risk is high. Specifically, for example, the determination is performed based on a database or a parameter in which the parameters are associated with the risk of thrombus, and a function indicating a relationship (regularity) with the risk of thrombus. It should be noted that the risk of thrombus can be graded.

INDUSTRIAL APPLICABILITY The present invention can be used in the bioindustry, such as biological experiments, monitoring or diagnosis, or drug creation.

  DESCRIPTION OF SYMBOLS 1 ... Dielectric blood coagulation measuring device, 2 ... Sample introduction part, 3 ... Signal processing part, 11 ... Voltage application part, 12 ... Dielectric constant measurement part, 13 ... Blood coagulation system analysis part, CL ... Cylindrical body, E1, E2 ... electrode, HN ... injection needle.

In order to solve such a problem, the present invention relates to a blood coagulation system analyzing apparatus, comprising: a pair of electrodes; an applying unit that applies an alternating voltage to the pair of electrodes at predetermined time intervals; Measuring means for measuring the dielectric constant of blood to be measured, and the function of the blood coagulation system based on the time change of the dielectric constant of blood measured at time intervals after the anticoagulant action acting on the blood is released. Analysis means for analyzing the degree is provided.

The present invention also relates to a blood coagulation system analysis method, which comprises applying an alternating voltage to a pair of electrodes at predetermined time intervals, and measuring a dielectric constant of blood disposed between the pair of electrodes. And an analyzing step of analyzing the degree of the function of the blood coagulation system based on the time change of the dielectric constant of the blood measured at time intervals after the anticoagulant action acting on the blood is released.

Further, the present invention is a program, by applying an alternating voltage to a pair of electrodes at a predetermined time interval to an application unit for applying an alternating voltage, and a pair of electrodes for a measurement unit for measuring a dielectric constant. Let the dielectric constant of the blood placed between the electrodes be measured, and to the analysis unit that analyzes the blood coagulation system, the blood coagulation measured at time intervals after the anticoagulant action acting on the blood is released . Analyzing the degree of the function of the blood coagulation system based on the time change of the dielectric constant is executed.

Claims (7)

A pair of electrodes;
Applying means for applying an alternating voltage to the pair of electrodes at predetermined time intervals,
Measuring means for measuring the dielectric constant of blood disposed between the pair of electrodes,
Analysis means for analyzing the degree of the function of the blood coagulation system using the dielectric constant of the blood measured at the above time intervals after the action of the anticoagulant working on the blood is released; and . The analysis means is
The time when a certain viscoelastic property appears is estimated from a parameter indicating an increase amount of a dielectric constant of a frequency of interest in a predetermined period after the anticoagulant action acting on the blood is released. Blood coagulation system analyzer. The analysis means is
The blood coagulation system analyzer according to claim 1, wherein a risk of thrombus is evaluated from a parameter indicating an increase amount of a dielectric constant of a frequency of interest in a predetermined period after the anticoagulant action acting on the blood is released. . The blood coagulation analyzer according to claim 2, wherein the pair of electrodes are opposed to each other so as to sandwich a position where blood is to be arranged. The analysis means is
The dielectric constant of a specific frequency in a predetermined period after the anticoagulant action acting on the blood is released, and the dielectric constant of a frequency different from the frequency, expressed as a ratio of the dielectric constant at a reference time. The blood coagulation system analyzer according to claim 1, wherein the division is performed, and the degree of operation of the blood coagulation system is analyzed using the result of the division. Applying an alternating voltage to the pair of electrodes at predetermined time intervals,
A measuring step of measuring the dielectric constant of blood disposed between the pair of electrodes,
Analyzing the degree of the function of the blood coagulation system using the dielectric constant of the blood measured at the time intervals after the action of the anticoagulant working on the blood is resolved. . Applying an alternating voltage to a pair of electrodes at a predetermined time interval with respect to an applying unit for applying the alternating voltage;
For the measurement unit that measures the dielectric constant, to measure the dielectric constant of the blood disposed between the pair of electrodes,
For the analysis unit that analyzes the blood coagulation system, the degree of blood coagulation system operation is determined using the dielectric constant of blood measured at the above time intervals after the anticoagulant action acting on the blood is resolved. The program that executes the analysis.