Method of detecting administered drugs, and associated program and device

Abstract

To enable determination of whether a patient has been administered a direct oral anticoagulant (DOAC) or not.SOLUTION: A method, a program and a device for detecting administered drugs are provided, the method comprising adding a specimen to a dry reagent containing magnetic particles and causing the magnetic particles in the reagent to move, monitoring a magnetic particle motion signal, analyzing the monitored magnetic particle motion signal, and comparing a result of the analysis with a threshold.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for detecting an administered drug, and a program and device therefor. [Background technology]

[0002] Anticoagulants are used to prevent blood clotting. For example, anticoagulants may be used to prevent thromboembolism. A representative anticoagulant is warfarin. Warfarin prevents blood clotting by inhibiting the biosynthesis of multiple blood clotting factors in the liver. Other anticoagulants include direct acting oral anticoagulants (DOACs). DOACs act directly on certain coagulation cascade components such as thrombin and factor Xa. Examples of DOACs include dabigatran, rivaroxaban, apixaban, edoxaban, and betrixaban.

[0003] In patients receiving direct oral anticoagulants (DOACs), bleeding events can make hemostasis difficult due to the effects of the DOACs. If the administration of DOACs is known, it is possible to deal with the issue by using an antidote (or reversal agent) appropriate for each DOAC. However, in emergency care settings, the patient's medical history is often unknown, and there are times when it is difficult to identify the cause of hemostasis. In addition, the reversal agents used for warfarin and DOACs are different.

[0004] To date, efforts have been made to detect DOAC administration using the clot waveform of a blood sample (Non-Patent Document 1). However, this has not yet been put to practical use.

[0005] Patent Document 1 proposes the analysis of DOACs and the like by clotting waveform analysis. However, since the disclosed automatic analyzer is a large device used in a central laboratory, even if it is put into practical use, it takes about 30 minutes from sample collection to obtaining results. It is considered difficult for this method to meet the needs of the field of emergency medical care, where rapid judgment is required. In addition, although the first step of the algorithm of the disclosed determination method includes an extension of the clotting time, many types of liquid reagents require a high concentration of DOAC that exceeds the therapeutic range in order to extend the clotting time, and it is considered that in reality, DOACs cannot be detected except in special cases such as overdose.

[0006] Methods for analyzing blood coagulation include the prothrombin time (PT). Prothrombin time (PT) is a test that reflects the extrinsic coagulation mechanism, and is prolonged by decreased activity of coagulation factors VII, X, V, II, and I. PT can be analyzed by adding a thromboplastin reagent containing tissue factor and calcium to plasma (e.g., citrated plasma) or whole blood, and measuring the clotting time. Prothrombin time (PT) can vary depending on the reagent and device, but the variation between reagents and devices can be normalized by using the prothrombin time-international normalized ratio (PT-INR).

[0007] Another method for analyzing blood clotting is the activated partial thromboplastin time (APTT). The activated partial thromboplastin time (APTT) is a test that reflects the intrinsic clotting mechanism and is prolonged by decreased activity of coagulation factors XII, XI, IX, VIII, X, V, II, and I. APTT can be analyzed by adding phospholipids, activation promoters (e.g., silica, kaolin, ellagic acid, etc.), and calcium to plasma (e.g., citrated plasma) or whole blood and measuring the clotting time.

[0008] There is a need for practical analytical methods capable of detecting anticoagulants. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] Patent Publication No. 2022-62969 [Patent Document 2] Patent Publication No. 07-135999 [Non-patent literature]

[0010] [Non-Patent Document 1] Masatoshi Wakui Journal of Thrombosis and Hemostasis 2022;33(1):69-75 Summary of the Invention [Problem to be solved by the invention]

[0011] The present invention aims to provide a method and apparatus that at least partially solves the above problems, and more specifically, to provide a method and apparatus for determining what type of anticoagulant has been administered from a patient's blood sample. [Means for solving the problem]

[0012] The present inventors have conducted intensive research to solve the above technical problems. Then, when a sample administered with DOAC was measured using an apparatus and dry reagent for measuring blood coagulation by analyzing the kinetic signal of magnetic particles, it was found that the coagulation time was extended even in a relatively low concentration of DOAC. In addition, when the clot waveform of the sample administered with DOAC was analyzed, it was found that the attenuation was slower than that of other anticoagulants such as warfarin. From these findings, it was found that by analyzing the clot waveform of a sample administered with DOAC using an apparatus and dry reagent for measuring blood coagulation by analyzing the kinetic signal of magnetic particles, it is possible to detect DOAC from low concentrations that cannot be detected by conventional reagents, particularly liquid reagents. In addition, it was found that the presence or absence of DOAC administration can be quickly determined at a site with high urgency by using a blood coagulation measuring device that has a short measurement time and can be used as a point-of-care (POC). Based on these findings, the present invention, which includes these as one embodiment, was completed.

[0013] The present disclosure encompasses the following embodiments. [1] A testing method for determining whether or not a direct oral anticoagulant (DOAC) has been administered, comprising: (i) adding a sample to a dry reagent containing magnetic particles for measuring prothrombin time or a dry reagent containing magnetic particles for measuring activated partial thromboplastin time; (ii) after the addition of the sample, moving the magnetic particles in the reagent and monitoring the magnetic particle movement signal; (iii) analyzing the magnetic particle movement signal monitored in step (ii); and (iv) comparing the results of the analysis in step (iii) with a threshold value that can clearly distinguish between a non-DOAC-administered control 1 and a DOAC-administered control 2; The method comprising: [2] The method according to embodiment 1, wherein the threshold value is the arithmetic mean of the slope of the change in the magnetic particle movement signal over time of a DOAC-nonadministered control 1 and the slope of the change in the magnetic particle movement signal over time of a DOAC-administered control 2, and the slope of the change in the magnetic particle movement signal over time is the slope of the clot waveform obtained by subtracting from the maximum magnetic particle movement amount the magnetic particle movement amount when it is weakened by a certain percentage selected from the group consisting of 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, and 15% compared to the maximum magnetic particle movement amount. [3] The method according to embodiment 1, wherein the threshold value is the arithmetic mean of the minimum value of the differential value of the slope of the time-dependent change in the magnetic particle movement signal of DOAC-nonadministered control 1 and the minimum value of the differential value of the time-dependent change in the magnetic particle movement signal of DOAC-administered control 2, and the slope of the time-dependent change in the magnetic particle movement signal is the slope of the clot waveform obtained by subtracting from the maximum magnetic particle movement amount the magnetic particle movement amount when it is weakened by a certain percentage selected from the group consisting of 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, and 15% compared to the maximum magnetic particle movement amount. [4] The method according to embodiment 1, wherein the threshold value is the arithmetic mean of the minimum value of the second derivative of the slope of the change in the magnetic particle movement signal over time of DOAC-nonadministered control 1 and the minimum value of the second derivative of the slope of the change in the magnetic particle movement signal over time of DOAC-administered control 2, and the slope of the change in the magnetic particle movement signal over time is the slope of the clot waveform obtained by subtracting from the maximum magnetic particle movement amount the magnetic particle movement amount when it is weakened by a certain percentage selected from the group consisting of 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, and 15% compared to the maximum magnetic particle movement amount. [5] Step (iv) (iv-1) comparing the results of the analysis in step (iii) with a first threshold value that can clearly distinguish between a non-DOAC-administered control 1 and a DOAC-administered control 2; and (iv-2) The result of the analysis in step (iii) is compared with a second threshold value that can clearly distinguish between a control 3 administered an anticoagulant other than a DOAC and a control 2 administered a DOAC. 2. The method of embodiment 1, comprising: [6] The method according to embodiment 5, wherein the threshold value is the arithmetic mean of the slope of the change over time of the magnetic particle movement signal of control 3 administered an anticoagulant other than DOAC and the slope of the change over time of the magnetic particle movement signal of control 2 administered DOAC, and the slope of the change over time of the magnetic particle movement signal is the slope of the clot waveform obtained by subtracting from the maximum magnetic particle movement amount the magnetic particle movement amount when it is weakened by a certain percentage selected from the group consisting of 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, and 15% compared to the maximum magnetic particle movement amount. [7] The method according to embodiment 5, wherein the threshold value is the arithmetic mean of the minimum value of the differential value of the slope of the time-dependent change in the magnetic particle movement signal of control 3 administered an anticoagulant other than DOAC and the minimum value of the differential value of the time-dependent change in the magnetic particle movement signal of control 2 administered DOAC, and the slope of the time-dependent change in the magnetic particle movement signal is the slope of the clot waveform obtained by subtracting from the maximum value of the magnetic particle movement the magnetic particle momentum when it is weakened by a certain percentage selected from the group consisting of 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, and 15% compared to the maximum value of the magnetic particle momentum. [8] The method according to embodiment 5, wherein the threshold value is the arithmetic mean of the minimum value of the second derivative of the slope of the change in the magnetic particle movement signal over time of control 3 administered an anticoagulant other than DOAC and the minimum value of the second derivative of the slope of the change in the magnetic particle movement signal over time of control 2 administered DOAC, and the slope of the change in the magnetic particle movement signal over time is the slope of the clot waveform obtained by subtracting from the maximum magnetic particle movement the magnetic particle momentum when it is weakened by a certain percentage selected from the group consisting of 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, and 15% compared to the maximum magnetic particle momentum. [9] Step (iv) (iv-1) comparing the results of the analysis in step (iii) with a first threshold value that can clearly distinguish between a non-DOAC-administered control 1 and a DOAC-administered control 2; and (iv-2) comparing the results of the analysis in step (iii) with a third threshold value that can clearly distinguish between a control 4 not administered an anticoagulant and a control 2 administered a DOAC; 2. The method of embodiment 1, comprising:

[10] The method according to embodiment 9, wherein the threshold value is the arithmetic mean of the slope of the time-dependent change in the magnetic particle movement signal of control 4 not administered an anticoagulant and the slope of the time-dependent change in the magnetic particle movement signal of control 2 administered a DOAC, and the slope of the time-dependent change in the magnetic particle movement signal is the slope of the clot waveform obtained by subtracting from the maximum magnetic particle movement the magnetic particle momentum when it is weakened by a certain percentage selected from the group consisting of 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, and 15% compared to the maximum magnetic particle momentum.

[11] The method according to embodiment 9, wherein the threshold value is the arithmetic mean of the minimum value of the differential value of the slope of the time-dependent change in the magnetic particle movement signal of control 4 not administered an anticoagulant and the minimum value of the differential value of the slope of the time-dependent change in the magnetic particle movement signal of control 2 administered a DOAC, and the slope of the time-dependent change in the magnetic particle movement signal is the slope of the clot waveform obtained by subtracting from the maximum magnetic particle movement amount the magnetic particle movement amount when it is weakened by a certain percentage selected from the group consisting of 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, and 15% compared to the maximum magnetic particle movement amount.

[12] The method according to embodiment 9, wherein the threshold value is the arithmetic mean of the minimum value of the second derivative of the slope of the change in the magnetic particle movement signal over time of control 4 not administered an anticoagulant and the minimum value of the second derivative of the slope of the change in the magnetic particle movement signal over time of control 2 administered a DOAC, and the slope of the change in the magnetic particle movement signal over time is the slope of the clot waveform obtained by subtracting from the maximum magnetic particle movement the magnetic particle momentum when it is weakened by a certain percentage selected from the group consisting of 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, and 15% compared to the maximum magnetic particle momentum.

[13] The method according to embodiment 1, wherein the threshold is a threshold at which the slope of the clot waveform, calculated by subtracting the magnetic particle momentum when it is 5% weakened compared to the maximum magnetic particle momentum from the maximum magnetic particle momentum, is -1000 when a CG02N device is used, a dry hemato PT reagent is used as a dry reagent containing magnetic particles for measuring prothrombin time, and 25 μL of a citrated whole blood sample is added to the dry hemato PT reagent, or a corresponding threshold when another device and dry reagent are used.

[14] The method according to embodiment 13, comprising a step of determining that the sample is derived from a patient not administered a DOAC if the slope of the change in the magnetic particle movement signal over time is steeper (<-1000) than the threshold value of -1000, and determining that the sample is derived from a patient administered a DOAC if the slope of the change in the magnetic particle movement signal over time is the threshold value of -1000 or has a gentler slope (>-1000) than -1000, wherein the slope of the change in the magnetic particle movement signal over time is the slope of a clot waveform obtained by subtracting the magnetic particle momentum when it is 5% weakened compared to the maximum magnetic particle momentum from the maximum magnetic particle momentum.

[15] As a result of comparison with the threshold, if the sample is determined to be from a patient administered a DOAC, administering or suggesting administration of a reversal agent for the DOAC to the patient; 14. The method of embodiment 13, further comprising:

[16] The method of any one of embodiments 1 to 15, wherein the DOAC is selected from the group consisting of dabigatran, rivaroxaban, apixaban, edoxaban, and betrixaban.

[17] A program for executing the method according to any one of embodiments 1 to 16.

[18] An information recording medium having the program described in embodiment 17 recorded thereon.

[19] An anticoagulant determination device incorporating the program described in embodiment 17 or storing the information recording medium described in embodiment 18. Effect of the Invention

[0014] According to the present invention, it is possible to quickly determine whether or not a patient is receiving anticoagulant therapy. [Brief description of the drawings]

[0015] [Figure 1] 1 is an example of a typical reaction slide used for a prothrombin time measurement dry reagent. [Diagram 2] FIG. 2 is a partially exploded view of the reaction slide of FIG. 1. [Figure 3-1] 4 shows the clot waveform of a sample not administered a direct oral anticoagulant. [Figure 3-2] The clot waveforms of each edoxaban-administered sample are shown. [Figure 4] The clot waveforms of each rivaroxaban-administered sample are shown.

[0016] In one embodiment, the present disclosure provides a testing method for determining the presence or absence of administration of a direct oral anticoagulant (DOAC). In one embodiment, the method may be an automated testing method implemented by a device. In one embodiment, the method does not involve a physician's judgment. In one embodiment, the testing method or automated testing method includes: (i) adding a sample to a dry reagent containing magnetic particles for measuring prothrombin time; (ii) after the addition of the sample, moving the magnetic particles in the reagent and monitoring the magnetic particle movement signal; (iii) analyzing the magnetic particle movement signal monitored in step (ii); and (iv) comparing the results of the analysis in step (iii) with a threshold value that can clearly distinguish between a non-DOAC-administered control 1 and a DOAC-administered control 2; In one embodiment, the specimen may be citrated plasma. In another embodiment, the specimen may be citrated whole blood.

[0017] The dry reagent containing magnetic particles is for adding a sample, then moving the magnetic particles in the reagent, and monitoring the magnetic particle movement signal. Unless otherwise specified, the dry reagent containing magnetic particles in this specification is for measuring prothrombin time (PT). In one embodiment, the prothrombin time (PT) is the prothrombin time-international normalized ratio (PT-INR). The INR is calculated by the following formula. [Number 1] INR=(Patient PT / Normal PT) ISI (wherein, ISI is the International Sensitivity Index set for each PT reagent)

[0018] Examples of the dry reagent containing magnetic particles include Dry Hemato PT Reagent (A&T Corporation) and a reagent for measuring prothrombin time that exhibits a similar function. In one embodiment, the dry reagent containing magnetic particles is (i) tissue thromboplastin; (ii) magnetic particles; (iii) calcium salts, (iv) a dry reagent layer solubility enhancer; (v) a dry reagent layer reinforcement material; and (vi) pH adjusters (pH buffers) In another embodiment, the dry reagent for measuring prothrombin time may further comprise a heparin neutralizer and / or an antifoaming agent.

[0019] Another example of a dry reagent containing magnetic particles is Dry Hemato APTT Reagent (A&T Corporation) or a reagent for measuring activated partial thromboplastin time that exhibits a similar function. In one embodiment, the dry reagent containing magnetic particles is (i) phospholipids, (ii) Activation promoter (iii) magnetic particles; (iv) calcium salts, (v) a dry reagent layer solubility enhancer; (vi) a dry reagent layer reinforcement material; and (vii) pH adjusters (pH buffers) In another embodiment, the dry reagent for measuring prothrombin time may further comprise a heparin neutralizer and / or an antifoaming agent.

[0020] In the method of the present disclosure, the result of the analysis in step (iii) can be compared with a threshold value that can clearly distinguish between the non-DOAC control 1 and the DOAC control 2, thereby determining whether the sample is from a patient administered a direct oral anticoagulant or from a patient not administered a direct oral anticoagulant. In one embodiment, this does not involve the judgment of a physician. In another embodiment, this may be determined by a physician.

[0021] In one embodiment, the threshold value may be the arithmetic mean of the slope of the time-dependent change in the magnetic particle motility signal of the DOAC-non-administered control 1 and the slope of the time-dependent change in the magnetic particle motility signal of the DOAC-administered control 2. In another embodiment, the threshold value may be the arithmetic mean of the minimum value of the differential value of the slope of the time-dependent change in the magnetic particle motility signal of the DOAC-non-administered control 1 and the minimum value of the differential value of the slope of the time-dependent change in the magnetic particle motility signal of the DOAC-administered control 2. In another embodiment, the threshold value may be the arithmetic mean of the minimum value of the second differential value of the slope of the time-dependent change in the magnetic particle motility signal of the DOAC-non-administered control 1 and the minimum value of the second differential value of the slope of the time-dependent change in the magnetic particle motility signal of the DOAC-administered control 2. In one embodiment, the slope of the time-dependent change in the magnetic particle motility signal may be the slope of the coagulation waveform obtained by subtracting the magnetic particle motility at a certain rate attenuated compared to the maximum value of the magnetic particle motility from the maximum value of the magnetic particle motility. In some embodiments, the attenuated percentage may be 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, for example 50%.

[0022] In one embodiment, step (iv) comprises: (iv-1) comparing the results of the analysis in step (iii) with a first threshold value that can clearly distinguish between a non-DOAC-administered control 1 and a DOAC-administered control 2; and (iv-2) The result of the analysis in step (iii) is compared with a second threshold value that can clearly distinguish between a control 3 administered an anticoagulant other than a DOAC and a control 2 administered a DOAC. This can include determining whether the patient is a DOAC-administered patient, as well as whether the patient is a non-DOAC-administered anticoagulant, including, but not limited to, warfarin, heparin (sometimes referred to as unfractionated heparin), low molecular weight heparin, danaparoid, fondaparinux, and argatroban.

[0023] In one embodiment, the second threshold value may be the arithmetic mean of the slope of the time course of the magnetic particle motion signal of the control 3 administered with an anticoagulant other than DOAC and the slope of the time course of the magnetic particle motion signal of the DOAC-administered control 2. In another embodiment, the second threshold value may be the arithmetic mean of the minimum value of the differential value of the slope of the time course of the magnetic particle motion signal of the control 3 administered with an anticoagulant other than DOAC and the minimum value of the differential value of the slope of the time course of the magnetic particle motion signal of the DOAC-administered control 2. In another embodiment, the second threshold value may be the arithmetic mean of the minimum value of the second differential value of the slope of the time course of the magnetic particle motion signal of the control 3 administered with an anticoagulant other than DOAC and the minimum value of the second differential value of the slope of the time course of the magnetic particle motion signal of the DOAC-administered control 2. The slope of the change over time of the magnetic particle movement signal can be the slope of the coagulation waveform obtained by subtracting the magnetic particle movement amount when it is weakened by a certain percentage compared to the maximum value of the magnetic particle movement amount from the maximum value of the magnetic particle movement amount.

[0024] In one embodiment, step (iv) (iv-1) comparing the results of the analysis in step (iii) with a first threshold value that can clearly distinguish between a non-DOAC-administered control 1 and a DOAC-administered control 2; and (iv-2) comparing the results of the analysis in step (iii) with a third threshold value that can clearly distinguish between a control 4 not administered an anticoagulant and a control 2 administered a DOAC; This can determine not only whether the patient is a DOAC-administered patient, but also whether the patient is not receiving an anticoagulant.

[0025] In one embodiment, the third threshold value may be the arithmetic mean of the slope of the time-dependent change in the magnetic particle motility signal of the control 4 not administered an anticoagulant and the slope of the time-dependent change in the magnetic particle motility signal of the control 2 administered DOAC. In another embodiment, the third threshold value may be the arithmetic mean of the minimum value of the differential value of the slope of the time-dependent change in the magnetic particle motility signal of the control 4 not administered an anticoagulant and the minimum value of the differential value of the slope of the time-dependent change in the magnetic particle motility signal of the control 2 administered DOAC. In another embodiment, the third threshold value may be the arithmetic mean of the minimum value of the second differential value of the slope of the time-dependent change in the magnetic particle motility signal of the control 4 not administered an anticoagulant and the minimum value of the second differential value of the slope of the time-dependent change in the magnetic particle motility signal of the control 2 administered DOAC. The slope of the time-dependent change in the magnetic particle motility signal may be the slope of the coagulation waveform obtained by subtracting the magnetic particle motility at a certain rate attenuated compared to the maximum value of the magnetic particle motility from the maximum value of the magnetic particle motility.

[0026] The above-mentioned certain percentage for determining the slope (or its derivative or second derivative) of the time-dependent change in the magnetic particle motion signal is not limited to 5%, but may be a certain percentage selected from the group consisting of 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, for example 50%.

[0027] In some embodiments, the non-DOAC control 1 can be calculated from a non-DOAC control population with n samples of 5 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 100 or more, e.g., 1000 or more. In some embodiments, the DOAC-treated control 2 can be calculated from a DOAC-treated control population with n samples of 5 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 100 or more, e.g., 1000 or more. In some embodiments, the non-DOAC anticoagulant-treated control 3 can be calculated from a non-DOAC anticoagulant-treated control population with n samples of 5 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 100 or more, e.g., 1000 or more. In one embodiment, the non-anticoagulant controlled population 4 may be calculated from a non-anticoagulant controlled population having a sample size n of 5 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 100 or more, e.g., 1000 or more.

[0028] In one embodiment, the threshold value may be a threshold value at which the slope of the clot waveform obtained by subtracting the magnetic particle momentum at a 5% attenuation compared to the maximum value of the magnetic particle momentum from the maximum value of the magnetic particle momentum is −1000 when a CG02N device (A&T Corporation) is used, a dry hemato PT reagent (A&T Corporation) is used, and 25 μL of a citrated whole blood sample is added to the dry hemato PT reagent. However, the method of the present disclosure is not limited to the device and the reagent. The method of the present disclosure may be similarly carried out when other devices and dry reagents for measuring prothrombin time are used. In this case, the threshold value used may be a threshold value for the device and the reagent corresponding to the threshold value at which the slope of the clot waveform obtained by subtracting the magnetic particle momentum at a 5% attenuation compared to the maximum value of the magnetic particle momentum from the maximum value of the magnetic particle momentum is −1000 when a CG02N device (A&T Corporation) is used, a dry hemato PT reagent (A&T Corporation) is used, and 25 μL of a citrated whole blood sample is added to the dry hemato PT reagent. Those skilled in the art, based on the teachings herein, can determine threshold values ​​corresponding to the above threshold of −1000 by routine confirmatory testing.

[0029] In one embodiment, if the gradient of the time-dependent change in the magnetic particle motion signal is steeper (<-1000) than the threshold value of -1000, the sample is determined to be derived from a patient not administered DOAC, and if the gradient of the time-dependent change in the magnetic particle motion signal is the threshold value of -1000 or is gentler (>-1000) than -1000, the sample is determined to be derived from a patient administered DOAC. The same applies to the corresponding threshold values.

[0030] In some embodiments, if the comparison with the threshold indicates that the sample is from a patient administered a DOAC, the patient may be administered or recommended to be administered a reversal agent for the DOAC. In some embodiments, the recommendation to administer is performed by an automated device or system. In some embodiments, the recommendation to administer does not include a physician's action.

[0031] In certain embodiments, the DOAC can be selected from the group consisting of dabigatran, rivaroxaban, apixaban, edoxaban, and betrixaban.

[0032] In one embodiment, a program, software, or algorithm for executing the method of the present disclosure is provided. In another embodiment, an information recording medium on which the program, software, or algorithm is recorded is provided. In another embodiment, an anticoagulant determination device is provided in which the program is incorporated or an information recording medium is stored. In one embodiment, the anticoagulant determination device may be CG02N (A&T Corporation) or a device having a similar function. The CG02N device is a device that applies a combination of an oscillating magnetic field and a stationary permanent magnetic field at 0.5 second intervals to a reagent to which a sample has been added, and monitors a magnetic particle movement signal at the same intervals. For example, see the product catalog or manual of CG02N (A&T Corporation).

[0033] For convenience, CG02N (A&T Corporation) will be described in this specification. After the start of measurement, the CG02N device switches the electromagnet at the bottom of the reaction cell ON and OFF to move the magnetic particles in the reaction cell. The movement of the magnetic particles is monitored as a change in scattered light, and the change over time is analyzed to determine the coagulation time. When using the CG02N device, the change over time in the magnetic particle movement signal is inversely correlated with the change in viscosity in the dry reagent. As the coagulation reaction progresses, the viscosity in the reaction cell increases, and the movement of the magnetic particles also slows down accordingly. When analyzing the coagulation waveform, the difference between the waveform value (scattered light) at a point a certain time after the electromagnet is turned ON and the waveform value (scattered light) at a point a certain time after the electromagnet is turned OFF is obtained. The difference in waveform value is plotted over time, and the point where it drops by a certain rate from the maximum value (also called the peak TOP) is set as the end point. The time from the peak TOP to the end point is measured, and a measured value (seconds) is obtained. The coagulation time referred to in this specification refers to the time from the peak TOP to the end point. However, this is merely for convenience and does not prevent the occurrence of a coagulation reaction at a time point before the maximum value (peak TOP) of the magnetic particle momentum. From the acquired clot waveform data, (1) the maximum value of the magnetic particle momentum, (2) the time when the maximum value of the magnetic particle momentum was obtained, (3) the magnetic particle momentum when the magnetic particle momentum attenuated by 5% from the maximum value, and (4) the time when the magnetic particle momentum attenuated by 5% from the maximum value can be obtained as feature quantities of the clot waveform. From the feature quantities (1) to (4), it is possible to obtain the slope of the clot waveform between the maximum value of the magnetic particle momentum and the magnetic particle momentum attenuated by a certain percentage, for example, 5%, selected from the group consisting of 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, and 15% compared to the maximum value of the magnetic particle momentum.

[0034] An example of a method for preparing a dry reagent for measuring prothrombin time is to first prepare a buffer solution containing a calcium salt, an amino acid or its salt, and a sugar, then dissolve highly active tissue thromboplastin in the buffer solution, and then add magnetic particles to the solution to obtain a final solution, which is then dispensed in a certain amount onto any reaction slide, frozen, and lyophilized. The buffer solution may further contain a heparin neutralizer and / or an antifoaming agent. The tissue thromboplastin solution may be prepared, for example, by dissolving a lyophilized product containing rabbit brain-derived tissue thromboplastin and rabbit brain-derived phospholipids in purified water.

[0035] The reaction slide used in the above preparation method is not particularly limited as long as it is a reaction slide that can optically monitor the increase in viscosity in the dry reagent for measuring prothrombin time as the attenuation of the motion signal of the magnetic particles during the measurement of prothrombin time. Examples include reaction slides as shown in Figures 1 and 2. Figure 1 is a top view of the reaction slide. The area surrounded by the dotted line in Figure 1 is a reaction cell part consisting of a dispensing port for the final solution for preparing the dry reagent for measuring prothrombin time and a sample addition port. Figure 2 shows the details of the structure of the reaction cell part. First, a transparent polyester plate B is attached to a white polyester plate C, and then a transparent polyester plate A is attached on the attached transparent polyester plate B to form a reaction cell part. First, a surfactant aqueous solution is filled from the dispensing port shown in Figure 1 and removed by suction to make the part D hydrophilic. Then, the final solution for the dry reagent for measuring prothrombin time is injected from the dispensing port, and the final solution is filled in the part D. When using this type of reaction slide, the above-mentioned final solution for the dry reagent for measuring prothrombin time can usually be dispensed in an amount of 20 to 30 μL. For a method for measuring prothrombin time using such magnetic particles, see, for example, Japanese Patent Application Laid-Open No. 07-135999. The entire contents of the document are incorporated herein by reference.

[0036] In the present specification, "undiluted whole blood" refers to whole blood that has not been diluted by adding a dilution buffer to the whole blood sample after collection. Therefore, even if blood is diluted by citric acid or the like contained in a blood collection tube at the time of collection (such blood is generally called citrated whole blood), if the whole blood after collection has not been diluted in any particular way, it corresponds to the undiluted whole blood referred to in the present specification. Therefore, undiluted whole blood includes citrated whole blood and heparinized whole blood that have not been diluted. In addition, in the present specification, "undiluted plasma" refers to a supernatant obtained by centrifuging undiluted whole blood, and has not been diluted by adding a dilution buffer. Therefore, undiluted plasma includes citrated plasma and heparinized plasma that have not been diluted. In the present specification, undiluted and undiluted are synonymous.

[0037] In one embodiment, the dry reagent for measuring prothrombin time contains tissue thromboplastin. The amount of tissue thromboplastin contained in the dry reagent for measuring prothrombin time is not particularly limited, but may be, for example, 1 ng to 1 mg of rabbit brain-derived tissue thromboplastin per measurement and 1 ng to 1 mg of rabbit brain-derived phospholipid per measurement.

[0038] In an embodiment, the dry reagent for measuring prothrombin time includes magnetic particles. Known magnetic particles can be used without any limitation as the magnetic particles used in the dry reagent for measuring prothrombin time. Examples of magnetic particles include, but are not limited to, iron tetraoxide particles, iron sesquioxide particles, iron particles, cobalt particles, nickel particles, and chromium oxide particles. In an embodiment, the magnetic particles can be fine particles of iron tetraoxide. That is, in a specific embodiment, fine particles of iron tetraoxide are preferably used in terms of the strength of the kinetic signal of the magnetic particles obtained. The particle size of the magnetic particles is not particularly limited, but can be an average particle size of 0.05 to 5 μm, 0.1 to 3.0 μm, for example, 0.25 to 0.5 μm, but is not limited thereto. In an embodiment, the magnetic particles can have an average particle size of 0.1 to 3.0 μm. In this specification, the average particle size refers to the particle size (D50) at 50% of the integrated value in the particle size distribution determined by the laser diffraction / scattering method, unless otherwise specified. The amount of magnetic particles contained in the dry reagent for measuring prothrombin time is not particularly limited, and is preferably in the range of, for example, 4 to 40 mg / 1 mL of the final solution.

[0039] In an embodiment, the dry reagent for measuring prothrombin time may contain a heparin neutralizer as an optional component. As the heparin neutralizer, any known one may be used without any limitation, and examples thereof include, but are not limited to, polybrene, protamine sulfate, and heparinase. In an embodiment, as the heparin neutralizer, polybrene may be preferably used in terms of good storage stability and cost. The amount of the heparin neutralizer contained in the dry reagent for measuring prothrombin time may be appropriately set and is not particularly limited. In an embodiment, when polybrene is used as the heparin neutralizer, the amount of polybrene contained in the dry reagent for measuring prothrombin time is preferably in the range of, for example, 50 to 300 μg / 1 mL of the final solution.

[0040] In one embodiment, the dry reagent for measuring prothrombin time contains a calcium salt. The calcium salt used in the dry reagent may be any known one without any limitation. For example, salts of calcium with inorganic acids include calcium chloride, calcium nitrite, calcium sulfate, and calcium carbonate. Salts of calcium with organic acids include calcium lactate and calcium tartrate. In one embodiment, calcium chloride is preferred as the calcium salt. The amount of calcium salt contained in the dry reagent for measuring prothrombin time may be appropriately set and is not particularly limited. When calcium chloride dihydrate is used as the calcium salt, the amount of calcium chloride dihydrate contained in the dry reagent for measuring prothrombin time is preferably in the range of 0.2 to 2 mg / 1 mL of final solution.

[0041] In an embodiment, the dry reagent for measuring prothrombin time includes a dry reagent layer solubility enhancer. Examples of the dry reagent layer solubility enhancer include amino acids or salts thereof or sugars. As the amino acids or salts thereof or sugars used, any of neutral amino acids or salts thereof, acidic amino acids or salts thereof, basic amino acids or salts thereof, monosaccharides and polysaccharides may be used. Representative examples of acidic amino acids or salts thereof include glutamic acid, sodium glutamate, aspartic acid, sodium aspartate, etc. Representative examples of neutral amino acids or salts thereof include glycine, glycine hydrochloride, alanine, etc. Representative examples of basic amino acids or salts thereof include lysine, lysine hydrochloride, arginine, etc. Furthermore, examples of monosaccharides include glucose, fructose, etc. Furthermore, examples of polysaccharides include sucrose, lactose, dextrin, etc. Among them, glycine is most preferred in terms of good solubility of the reagent when a sample is added to the dry reagent for measuring prothrombin time, good reproducibility of the obtained motion signal of the magnetic particles, and good impact resistance. Thus, in certain embodiments, the dry reagent layer solubility enhancer can be glycine.

[0042] The amount of the dry reagent layer solubility enhancer, for example, amino acid or its salt or sugar, contained in the dry reagent for measuring prothrombin time may be appropriately set and is not particularly limited. In an embodiment, when glycine is used as the dry reagent layer solubility enhancer, the amount of glycine contained in the dry reagent for measuring prothrombin time is 1.5 wt% or more, 1.6 wt% or more, 1.7 wt% or more, 1.8 wt% or more, 1.9 wt% or more, 2.0 wt% or more, 2.1 wt% or more, 2.2 wt% or more, 2.3 wt% or more, 2.4 wt% or more, 2.5 wt% or more, 2.6 wt% or more, 2.7 wt% or more, 2.8 wt% or more, 2.9 wt% or more, It can be 3.0% by weight or more, 3.1% by weight or more, 3.2% by weight or more, 3.3% by weight or more, 3.4% by weight or more, 3.5% by weight or more, 3.6% by weight or more, 3.7% by weight or more, 3.8% by weight or more, 3.9% by weight or more, 4.0% by weight or more, 4.1% by weight or more, 4.2% by weight or more, 4.3% by weight or more, 4.4% by weight or more, 4.5% by weight or more, 4.6% by weight or more, 4.7% by weight or more, 4.8% by weight or more, 4.9% by weight or more, for example, 5.0% by weight. In one embodiment, when glycine is used as the dry reagent layer solubility enhancer, the amount of glycine contained in the dry reagent for measuring prothrombin time is 5.0% by weight or less, 4.9% by weight or less, 4.8% by weight or less, 4.7% by weight or less, 4.6% by weight or less, 4.5% by weight or less, 4.4% by weight or less, 4.3% by weight or less, 4.2% by weight or less, 4.1% by weight or less, 4.0% by weight or less, 3.9% by weight or less, 3.8% by weight or less, 3.7% by weight or less, 3.6% by weight or less, The amount of glycine contained in the dry reagent for measuring prothrombin time may be 3.5% by weight or less, 3.4% by weight or less, 3.3% by weight or less, 3.2% by weight or less, 3.1% by weight or less, 3.0% by weight or less, 2.9% by weight or less, 2.8% by weight or less, 2.7% by weight or less, 2.6% by weight or less, 2.5% by weight or less, 2.4% by weight or less, 2.3% by weight or less, 2.2% by weight or less, 2.1% by weight or less, 2.0% by weight or less, 1.9% by weight or less, 1.8% by weight or less, 1.7% by weight or less, 1.6% by weight or less, for example, 1.5% by weight. In the present specification, the amount of glycine contained in the dry reagent for measuring prothrombin time includes any combination in which the lower limit and the upper limit are set to any of the above values.For example, in certain embodiments, the amount of glycine contained in the dry reagent for measuring prothrombin time is 1.5 to 5.0% by weight, 2.0 to 5.0% by weight, 2.5 to 5.0% by weight, 3.0 to 5.0% by weight, 3.5 to 5.0% by weight, 4.0 to 5.0% by weight, 4.5 to 5.0% by weight, 1.5 to 4.5% by weight, 2.0 to 4.5% by weight, 2.5 to 4.5% by weight, 3.0 to 4.5% by weight, 3.5 to 4.5% by weight, 4.0 to It may be 4.5% by weight, 1.5 to 4.0% by weight, 2.0 to 4.0% by weight, 2.5 to 4.0% by weight, 3.0 to 4.0% by weight, 3.5 to 4.0% by weight, 1.5 to 3.5% by weight, 2.0 to 3.5% by weight, 2.5 to 3.5% by weight, 3.0 to 3.5% by weight, 1.5 to 3.0% by weight, 2.0 to 3.0% by weight, 2.5 to 3.0% by weight, 1.5 to 2.5% by weight, 2.0 to 2.5% by weight, or 1.5 to 2.0% by weight. In one embodiment, when glycine is used as the dry reagent layer solubility enhancer, the amount of glycine contained in the dry reagent for measuring prothrombin time is preferably in the range of 1.5 to 4.0% by weight. In another embodiment, when glycine is used as the dry reagent layer solubility enhancer, the amount of glycine contained in the dry reagent for measuring prothrombin time is preferably in the range of 2.0 to 3.0% by weight. When undiluted plasma is measured, when glycine is used as a dry reagent layer solubility enhancer, the amount of glycine contained in the dry reagent for measuring prothrombin time can be in the above range, for example, 1.5 to 4.0% by weight. When undiluted whole blood is measured, when glycine is used as a dry reagent layer solubility enhancer, the amount of glycine contained in the dry reagent for measuring prothrombin time can be in the above range, for example, 1.5% by weight or more. For example, when undiluted whole blood is measured, when glycine is used as a dry reagent layer solubility enhancer, the amount of glycine contained in the dry reagent for measuring prothrombin time can be 1.5 to 5.0% by weight, 1.5 to 4.5% by weight, for example, 1.5 to 4.0% by weight. When both undiluted plasma and undiluted whole blood can be measured, when glycine is used as a dry reagent layer solubility enhancer, the amount of glycine contained in the dry reagent for measuring prothrombin time may be various combinations of these ranges. In this specification, unless otherwise specified, weight % refers to the concentration in the final solution, that is, the final concentration.

[0043] In one embodiment, the dry reagent for measuring prothrombin time contains a pH buffer (also called a pH adjuster). Prior to freeze-drying, the buffer solution containing tissue thromboplastin, magnetic particles, heparin neutralizer, calcium salt, and dry reagent layer solubility enhancer is not particularly limited as long as it has a buffering effect between pH=6.0 and 8.0. In one embodiment, the pH adjuster (pH buffer) may adjust the pH of the reagent to pH6.0 to pH8.0, for example, about pH7.35 or about pH7.5. For example, 40 mM HEPES buffer (pH=7.35) or 40 mM Tris-HCl buffer (pH=7.5) is a suitable buffer.

[0044] In one embodiment, the dry reagent for measuring prothrombin time according to the present invention includes a dry reagent layer reinforcing material. Examples of the dry reagent layer reinforcing material include, but are not limited to, bovine serum albumin and human serum albumin. When bovine serum albumin is used as the dry reagent layer reinforcing material, the amount of the dry reagent layer reinforcing material contained in the quantitative dry reagent is preferably in the range of 0.6 to 2.0 mg / 1 mL of the final solution.

[0045] In an embodiment, the dry reagent for measuring prothrombin time according to the present invention may contain an antifoaming agent as an optional component. Examples of the antifoaming agent include, but are not limited to, sorbitan monolaurate, silicone-based antifoaming agents, and polypropylene glycol-based antifoaming agents. When sorbitan monolaurate is used as the antifoaming agent, the amount of the antifoaming agent contained in the quantitative dry reagent is preferably in the range of about 0.001 to about 0.010% by weight.

[0046] The method for drying the buffer solution containing the above components is preferably freeze-drying in terms of the solubility of the dry reagent for measuring prothrombin time, the strength of the obtained motion signal of the magnetic particles, and reproducibility. In one embodiment, the drying method does not include drying by air drying.

[0047] The freezing and freeze-drying methods are not particularly limited. For example, the final solution for the prothrombin time measurement dry reagent is dispensed onto a reaction slide from the dispensing port shown in FIG. 1, and then the reaction slide is stored overnight in a freezer kept at -40°C or lower to freeze, or the reaction slide is set in a freeze-dryer with a shelf temperature of -40°C or lower and stored overnight to freeze, or the reaction slide is flash-frozen with liquid nitrogen, or other general freezing methods can be used. In addition, the freeze-drying method of the frozen reaction slide is also not particularly limited. For example, the freeze-drying method includes a method in which the temperature of the frozen reaction slide is linearly increased from -30°C to -20°C in 24 hours in a vacuum state, then linearly increased from -20°C to 30°C in 20 hours, and finally kept at 30°C for 3 hours, and then the vacuum is released with dry air.

[0048] The dry reagent for measuring prothrombin time after freeze-drying is preferably immediately sealed with an aluminum film in a dehumidified environment. The dehumidified environment is not particularly limited, but is preferably an environment with a relative humidity of 35% or less at room temperature of 22 to 27°C. The specifications of the aluminum film are not particularly limited, but a five-layer aluminum film (86 μm thick) made of polyester film (12 μm thick), polyethylene resin (15 μm thick), aluminum foil (9 μm thick), polyethylene resin (20 μm thick), and polyethylene film (30 μm thick) bonded with an AC coating agent is preferable. The entire dry reagent for measuring prothrombin time is wrapped in the aluminum film and sealed by heat welding. The dry reagent for measuring prothrombin time is preferably stored in a sealed state in a refrigerator until it is used to measure prothrombin time.

[0049] The dry reagent for measuring activated partial thromboplastin time in the present invention can be prepared by adding phospholipids and an activation promoter instead of tissue thromboplastin among the components of the dry reagent for measuring prothrombin time described above. As the phospholipid, various phospholipids derived from natural products or artificially synthesized can be used. As the phospholipid derived from natural products, cephalin derived from rabbit brain, lipid derived from pig, lipid derived from chicken, lipid derived from soybean, etc. can be used. As the artificially synthesized phospholipid, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, phosphatidylinositol, etc. can be used. As the activation promoter, inorganic substances such as silica and kaolin, or organic substances such as ellagic acid and actin can be used. EXAMPLES

[0050] Having generally described the invention, the invention can be further understood by reference to the following specific examples, which are intended for purposes of illustration and illustration only and are not intended to be limiting of the invention in any way.

[0051] [Example 1] Discrimination of samples not administered with direct oral anticoagulant and samples administered with edoxaban using a dried PT reagent A lyophilized product containing rabbit brain-derived tissue thromboplastin and rabbit brain-derived phospholipids (manufactured by Thermo Fisher Scientific) was dissolved in 10 mL of purified water to obtain a tissue thromboplastin solution. The tissue thromboplastin solution was centrifuged at 4,000 rpm, 4°C, and 30 minutes, and the supernatant was removed to obtain a pellet of rabbit brain-derived tissue thromboplastin. A 40 mM HEPES buffer (pH 7.35) containing 10 mM CaCl2·2H2O, 2.5 (wt / v)% glycine, 3.0 mg / mL bovine serum albumin, 0.005 (wt / v)% sorbitan monolaurate, and 150 μg / mL GPRP-amide was added to the pellet of rabbit brain-derived tissue thromboplastin, and the pellet was stirred and dispersed to obtain a prothrombin time reagent solution. 0.47 g of iron oxide (product name AAT-03; average particle size 0.35 μm; manufactured by Toda Kogyo Co., Ltd.) was added to 35 mL of the reagent solution and suspended to obtain a final solution. 25 μL of the final solution was dispensed into the reaction slide shown in FIG. 1. The reaction slide was frozen by storing it overnight in a freezer kept at -40°C. The frozen reaction slide was then freeze-dried. The freeze-drying conditions were as follows: the temperature was linearly increased from -30°C to -20°C in 24 hours in a vacuum state, then linearly increased from -20°C to 30°C in 20 hours, and finally kept at 30°C for 3 hours, and then the vacuum was released with dry air. The freeze-dried reagent was immediately sealed in aluminum film in a dehumidified environment.

[0052] The method of distinguishing between samples not administered with direct oral anticoagulants and samples administered with edoxaban using the dried PT reagent was as follows. First, 26 samples not administered with direct oral anticoagulants (including 11 samples administered with warfarin, 2 samples administered with heparin, and 3 samples not administered with anticoagulants) and 37 samples administered with edoxaban were prepared. Next, the above-mentioned freeze-dried reagent was set in a blood coagulation analyzer CG02N (manufactured by A&T Corporation), 25 μL of the prepared samples were added, and the coagulation time and PT-INR of each sample were obtained. Samples with a PT-INR of less than 2.0 were excluded as not showing coagulation abnormalities, and for the 17 samples not administered with direct oral anticoagulants and the 34 samples administered with edoxaban that were not excluded, coagulation waveform data obtained when each sample was measured using the CG02N was obtained. Furthermore, from the acquired clot waveform data, (1) the maximum value of magnetic particle momentum, (2) the time when the maximum value of magnetic particle momentum was obtained, (3) the magnetic particle momentum when the magnetic particle momentum attenuated by 5% from the maximum value, and (4) the time when the magnetic particle momentum attenuated by 5% from the maximum value were obtained as feature quantities of the clot waveform. From the feature quantities (1) to (4), the slope of the clot waveform between "maximum value of magnetic particle momentum - magnetic particle momentum when the magnetic particle momentum attenuated by 5% from the maximum value" was obtained. Figures 3-1 and 3-2 show the clot waveforms of a specimen not administered a direct oral anticoagulant and a specimen administered edoxaban, respectively.

[0053] The table below shows the average and standard deviation of the slope of the clot waveform for samples not administered direct oral anticoagulants and samples administered edoxaban. The average slope of the clot waveform for samples not administered direct oral anticoagulants and samples administered edoxaban was -1219.8 and -664.0, respectively, indicating that the samples administered edoxaban showed a larger slope than the samples not administered direct oral anticoagulants.

[0054] [Table 1]

[0055] To distinguish between samples not administered direct oral anticoagulants and samples administered edoxaban, an algorithm was used to distinguish samples with a clot waveform slope of -1000 or more as samples administered edoxaban, and samples with a slope of less than -1000 as samples not administered direct oral anticoagulants. A cross-tabulation of the results is shown in the table below.

[0056] [Table 2]

[0057] The sensitivity of determining whether or not edoxaban was administered, calculated from the table above, was 0.85, and the specificity was 0.94. It was found that determining whether or not edoxaban was administered based on the slope of the clot waveform has high sensitivity and specificity.

[0058] [Example 2] Discrimination between samples not administered with direct oral anticoagulant and samples administered with rivaroxaban using dried PT reagent The dry PT reagent was the freeze-dried reagent of Example 1, and the method for discriminating between samples not administered with a direct oral anticoagulant and samples administered with rivaroxaban was as follows.

[0059] First, 26 specimens not administered with direct oral anticoagulants (including 11 specimens administered with warfarin, 2 specimens administered with heparin, and 3 specimens not administered with anticoagulants) and 24 specimens administered with rivaroxaban were prepared. Next, the above-mentioned freeze-dried reagent was set in a blood coagulation analyzer CG02N (manufactured by A&T Corporation), 25 μL of the prepared specimens were added, and the coagulation time and PT-INR of each specimen were obtained. Specimens with a PT-INR of less than 2.0 were excluded as not showing coagulation abnormalities, and 17 specimens not administered with direct oral anticoagulants and 22 specimens administered with rivaroxaban that were not excluded were measured using CG02N to obtain clot waveform data. Furthermore, from the obtained clot waveform data, (1) the maximum value of the magnetic particle momentum, (2) the time when the maximum value of the magnetic particle momentum was obtained, (3) the magnetic particle momentum when the magnetic particle momentum was attenuated by 5% from the maximum value, and (4) the time when the magnetic particle momentum was attenuated by 5% from the maximum value were obtained as feature quantities of the clot waveform. From the feature quantities (1) to (4), the slope of the clot waveform between the "maximum value of the magnetic particle momentum - the magnetic particle momentum when the magnetic particle momentum is attenuated by 5% from the maximum value" is obtained. Figure 4 shows the clot waveforms of each of the rivaroxaban-administered samples.

[0060] The table below shows the average and standard deviation of the slope of the clot waveform for samples not administered direct oral anticoagulants and samples administered rivaroxaban. The average slope of the clot waveform for samples not administered direct oral anticoagulants and samples administered rivaroxaban was -1219.8 and -805.9, respectively, indicating that the samples administered rivaroxaban showed a larger slope than the samples not administered direct oral anticoagulants.

[0061] [Table 3]

[0062] To distinguish between samples not administered with a direct oral anticoagulant and samples administered with rivaroxaban, an algorithm was used to distinguish samples with a clot waveform slope of -1000 or more as samples administered with rivaroxaban, and samples with a slope of less than -1000 as samples not administered with a direct oral anticoagulant. A cross-tabulation of the results is shown in the table below.

[0063] [Table 4]

[0064] The sensitivity of determining whether or not rivaroxaban has been administered, calculated from the table above, was 0.86, and the specificity was 0.94. It was found that determining whether or not rivaroxaban has been administered based on the slope of the clot waveform has high sensitivity and specificity.

[0065] [Example 3] Discrimination of dabigatran administration using a dry APTT reagent 20 mL of 60 mM HEPES buffer (pH 7.35) containing 4.0 (wt / v)% glycine, 20 mM CaCl2·2H2O, and 6.0 mg / mL bovine serum albumin was added to 20 mL of rabbit brain-derived cephalin, and the mixture was stirred and dispersed to obtain an activated partial thromboplastin reagent. 1.41 g of iron oxide (product name AAT-03; average particle size 0.35 μm; Toda Kogyo Co., Ltd.) was added to 35 mL of the reagent solution and suspended to obtain a final solution. 25 μL of the final solution was dispensed into the reaction slide shown in Figure 1. The reaction slide was frozen overnight in a freezer kept at -40°C. The frozen reaction slide was then freeze-dried. The freeze-drying conditions were as follows: in a vacuum, the temperature was raised linearly from -30°C to -20°C over 24 hours, then raised linearly from -20°C to 30°C over 20 hours, and finally kept at 30°C for 3 hours, after which the vacuum was released with dry air. The freeze-dried reagents were immediately sealed in aluminum film in a dehumidified environment.

[0066] The method of measuring dabigatran-administered samples using the dried APTT reagent was as follows. DMSO was added to 10 mg of dabigatran to a total volume of 10 mL to prepare a 1,000 μg / mL dabigatran solution. The dabigatran solution was diluted with physiological saline to prepare a dilution series of 0, 0.9, 1.8, 5, and 10 μg / mL. 100 μL of each of the dabigatran dilution series was added to 900 μL of normal plasma to prepare dabigatran-added plasma of 0, 90, 180, 500, and 1,000 ng / mL. The above freeze-dried reagent was set in a blood coagulation analyzer CG02N (manufactured by A&T Corporation), and 25 μL of the prepared sample was added to determine the coagulation time of each sample.

[0067] The clotting time of dabigatran-added plasma is shown in Table 5. It was observed that the clotting time of the lyophilized reagent was prolonged depending on the concentration of dabigatran.

[0068] [Table 5] [Industrial Applicability]

[0069] According to the present invention, it is possible to quickly determine whether or not a patient has been administered an anticoagulant, such as a DOAC. This makes it possible to quickly determine whether or not a patient with an unknown medication history has been administered an anticoagulant, such as a DOAC, in a highly urgent medical setting such as a critical care emergency. By determining whether or not a DOAC has been administered, it is possible to develop an appropriate hemostatic strategy. In addition, by determining whether or not a DOAC has been administered, the survival rate of patients administered a DOAC in a bleeding event can be improved. In addition, by determining whether or not a DOAC has been administered, the time until hemostasis can be shortened, thereby reducing the amount of blood products used.

[0070] All documents mentioned herein are hereby incorporated by reference in their entirety. [Explanation of symbols]

[0071] A Transparent resin plate B Transparent resin plate C White resin board D Reagent filling section

Claims

1. A test method for determining whether or not direct oral anticoagulants (DOACs) have been administered, (i) A step of adding a sample to a dry reagent containing magnetic particles for measuring prothrombin time, or to a dry reagent containing magnetic particles for measuring activated partial thromboplastin time. (ii) A step of moving magnetic particles in the reagent after adding the sample and monitoring the magnetic particle motion signal, (iii) a step of analyzing the magnetic particle motion signal monitored in step (ii), and (iv) a step of comparing the results of the analysis in step (iii) with a threshold that can distinguish between DOAC-free control 1 and DOAC-administered control 2. The method, including the method described above.

2. The method according to claim 1, wherein the threshold is the arithmetic mean of the slope of the change over time of the magnetic particle motion signal of DOAC-free control 1 and the slope of the change over time of the magnetic particle motion signal of DOAC-administered control 2, and the slope of the change over time of the magnetic particle motion signal is the slope of the coagulation waveform obtained by subtracting the magnetic particle momentum when the magnetic particle momentum is attenuated from the maximum value of the magnetic particle momentum by a certain percentage selected from the group consisting of 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, and 15% compared to the maximum value of the magnetic particle momentum.

3. The method according to claim 1, wherein the threshold is the arithmetic mean of the minimum derivative of the slope of the change over time of the magnetic particle motion signal of DOAC-free control 1 and the minimum derivative of the slope of the change over time of the magnetic particle motion signal of DOAC-administered control 2, and the slope of the change over time of the magnetic particle motion signal is the slope of the coagulation waveform obtained by subtracting the magnetic particle momentum when the magnetic particle momentum is attenuated from the maximum value of the magnetic particle momentum by a certain percentage selected from the group consisting of 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, and 15% compared to the maximum value of the magnetic particle momentum.

4. The method according to claim 1, wherein the threshold is the arithmetic mean of the minimum second derivative of the slope of the change over time of the magnetic particle motion signal of DOAC-free control 1 and the minimum second derivative of the slope of the change over time of the magnetic particle motion signal of DOAC-administered control 2, and the slope of the change over time of the magnetic particle motion signal is the slope of the coagulation waveform obtained by subtracting the magnetic particle momentum when the magnetic particle momentum is attenuated from the maximum value of the magnetic particle momentum by a certain percentage selected from the group consisting of 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, and 15% compared to the maximum value of the magnetic particle momentum.

5. Step (iv) is, (iv-1) The results of the analysis in step (iii) above are compared with a first threshold that can distinguish between DOAC-free control 1 and DOAC-administered control 2, and further, (iv-2) The results of the analysis in step (iii) above are compared with a second threshold that can distinguish between control 3, who was administered an anticoagulant other than a DOAC, and control 2, who was administered a DOAC. The method according to claim 1, including the following:

6. The method according to claim 5, wherein the threshold is the arithmetic mean of the slope of the change over time of the magnetic particle motion signal of control 3 administered with an anticoagulant other than a DOAC and the slope of the change over time of the magnetic particle motion signal of control 2 administered with a DOAC, and the slope of the change over time of the magnetic particle motion signal is the slope of the coagulation waveform obtained by subtracting the magnetic particle momentum when the magnetic particle momentum is attenuated from the maximum value of the magnetic particle momentum by a certain percentage selected from the group consisting of 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, and 15% compared to the maximum value of the magnetic particle momentum.

7. The method according to claim 5, wherein the threshold is the arithmetic mean of the minimum derivative of the slope of the change over time of the magnetic particle motion signal of control 3 administered with an anticoagulant other than a DOAC, and the minimum derivative of the slope of the change over time of the magnetic particle motion signal of control 2 administered with a DOAC, and the slope of the change over time of the magnetic particle motion signal is the slope of the coagulation waveform obtained by subtracting the magnetic particle momentum when the magnetic particle momentum is attenuated from the maximum value of the magnetic particle momentum by a certain percentage selected from the group consisting of 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, and 15% compared to the maximum value of the magnetic particle momentum.

8. The method according to claim 5, wherein the threshold is the arithmetic mean of the minimum second derivative of the slope of the change over time of the magnetic particle motion signal of control 3 administered with an anticoagulant other than a DOAC, and the minimum second derivative of the slope of the change over time of the magnetic particle motion signal of control 2 administered with a DOAC, and the slope of the change over time of the magnetic particle motion signal is the slope of the coagulation waveform obtained by subtracting the magnetic particle momentum when the magnetic particle momentum is attenuated from the maximum value of the magnetic particle momentum by a certain percentage selected from the group consisting of 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, and 15% compared to the maximum value of the magnetic particle momentum.

9. Step (iv) is, (iv-1) The results of the analysis in step (iii) above are compared with a first threshold that can distinguish between DOAC-free control 1 and DOAC-administered control 2, and further, (iv-2) A step of comparing the results of the analysis in step (iii) with a third threshold that can distinguish between control 4, who has not been administered an anticoagulant, and control 2, who has been administered a DOAC. The method according to claim 1, including the method described in claim 1.

10. The method according to claim 9, wherein the threshold is the arithmetic mean of the slope of the change over time of the magnetic particle motion signal of control 4, which was not administered an anticoagulant, and the slope of the change over time of the magnetic particle motion signal of control 2, which was administered a DOAC, and the slope of the change over time of the magnetic particle motion signal is the slope of the coagulation waveform obtained by subtracting the magnetic particle momentum when the magnetic particle momentum is attenuated from the maximum value of the magnetic particle momentum by a certain percentage selected from the group consisting of 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, and 15% compared to the maximum value of the magnetic particle momentum.

11. The method according to claim 9, wherein the threshold is the arithmetic mean of the minimum derivative of the slope of the change over time of the magnetic particle motion signal of control 4, which was not administered an anticoagulant, and the minimum derivative of the slope of the change over time of the magnetic particle motion signal of DOAC-administered control 2, and the slope of the change over time of the magnetic particle motion signal is the slope of the coagulation waveform obtained by subtracting the magnetic particle momentum when the magnetic particle momentum is attenuated from the maximum value of the magnetic particle momentum by a certain percentage selected from the group consisting of 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, and 15% compared to the maximum value of the magnetic particle momentum.

12. The method according to claim 9, wherein the threshold is the arithmetic mean of the minimum second derivative of the slope of the change over time of the magnetic particle motion signal of control 4, which was not administered an anticoagulant, and the minimum second derivative of the slope of the change over time of the magnetic particle motion signal of DOAC-administered control 2, and the slope of the change over time of the magnetic particle motion signal is the slope of the coagulation waveform obtained by subtracting the magnetic particle momentum when the magnetic particle momentum is attenuated from the maximum value of the magnetic particle momentum by a certain percentage selected from the group consisting of 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, and 15% compared to the maximum value of the magnetic particle momentum.

13. The method according to claim 1, wherein the threshold is the threshold at which the slope of the coagulation waveform obtained by subtracting the magnetic particle momentum at which the magnetic particle momentum is attenuated by 5% compared to the maximum value from the maximum value of the magnetic particle momentum when using a CG02N instrument and dry hemato PT reagent as a dry reagent containing magnetic particles for measuring prothrombin time is -1000, or the corresponding threshold when using other instruments and dry reagents.

14. The method according to claim 13, comprising the steps of determining that if the slope of the change in the magnetic particle motion signal over time is steeper than the threshold of -1000 (<-1000), the sample is derived from a patient who has not been administered a DOAC, and determining that if the slope of the change in the magnetic particle motion signal over time is -1000 or a gentler slope than -1000 (>-1000), the sample is derived from a patient who has been administered a DOAC, wherein the slope of the change in the magnetic particle motion signal over time is the slope of the coagulation waveform obtained by subtracting the magnetic particle momentum at which the magnetic particle momentum is attenuated by 5% compared to the maximum value of the magnetic particle momentum from the maximum value of the magnetic particle momentum.

15. Based on the comparison with the threshold, If the sample is determined to originate from a patient who has been administered a DOAC, the process involves administering an antidote to the patient or suggesting that such an antidote be administered. The method according to claim 13, further comprising:

16. The method according to claim 1, wherein the DOAC is selected from the group consisting of dabigatran, rivaroxaban, apixaban, edoxaban, and betrixaban.

17. A program for performing the method described in any one of claims 1 to 16.

18. An information recording medium that stores the program described in claim 17.

19. An anticoagulant determination device incorporating the program described in claim 17.

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