Method for detecting blood coagulation reactions

Abstract

The present invention is a method for detecting a blood coagulation reaction, wherein the method comprises: 1) measuring a blood coagulation reaction in a blood sample to be tested, and obtaining a first derivative V(i) of the coagulation reaction up to the latest measurement point; 2) obtaining the pre-peak and post-peak area-under-the-curve (AUC) of V(i), where the peak top of V(i) is the maximum value cVmax(k) of V(i); and 3) detecting cVmax(k) as the true maximum value Vmax of V(i) when the duration L, during which both the pre-peak AUC and the post-peak AUC are equal to or greater than a first threshold AUCth1 and individually remain at fixed values, reaches a prescribed length.

Description

[Technical Field]

[0001] The present invention relates to a method for detecting a blood coagulation reaction. [Background technology]

[0002] Blood coagulation tests are tests to diagnose a patient's blood clotting ability by adding a specific reagent to a patient's blood sample and measuring the blood clotting time. Typical examples of blood clotting times include prothrombin time (PT), activated partial thromboplastin time (APTT), and thrombin time. Abnormal blood clotting ability causes prolongation of clotting time. Causes of prolonged clotting time include the effects of coagulation inhibitors, a decrease in coagulation-related components, a congenital deficiency of blood clotting factors, and the acquired appearance of autoantibodies that inhibit the clotting reaction.

[0003] In recent years, automated analyzers that automatically measure blood coagulation reactions have become widely used, making it possible to easily perform blood coagulation tests. For example, some automated analyzers irradiate a mixture obtained by adding a reagent to a blood sample with light and measure the coagulation reaction of the blood sample based on the resulting change in the amount of scattered light. In a typical blood coagulation reaction, the amount of scattered light increases rapidly as the coagulation reaction progresses after a certain amount of time has passed since the addition of the reagent. As the coagulation reaction approaches completion, the amount of scattered light then saturates and reaches a plateau, and the point at which the amount of scattered light reaches its maximum is the end of the coagulation reaction. Blood coagulation time can be calculated based on this temporal change in the amount of scattered light. Meanwhile, photometric data from an analyzer contains various noises due to the condition of the analyzer, reagent, and sample. For example, after adding a reagent to a blood sample, a slight increase in the amount of scattered light may be measured at the beginning of the reaction, before the amount of scattered light increases due to the actual coagulation reaction. This phenomenon is referred to as an initial reaction or pre-peak (Patent Documents 1 and 2). Noise in photometric data, such as an initial reaction, can lead to incorrect calculation of the coagulation time.

[0004] In automated measurement of blood coagulation reactions, methods have been proposed for eliminating the effects of early reactions or pre-peaks. For example, one method involves setting a threshold for the measurement time or measurement value in advance, and then calculating the coagulation time by considering the reaction after the measured time or data reaches the threshold as the true coagulation reaction. Furthermore, the aforementioned Patent Document 1 discloses a blood coagulation reaction analysis method characterized by detecting an early reaction by monitoring the amount and speed of optical changes due to the coagulation reaction at at least one checkpoint or check area from the start of measurement to the end of the coagulation reaction. Patent Document 2 discloses a blood coagulation analysis method characterized by repeating the steps of measuring the amount of scattered light and calculating the coagulation time as the point at which the measured amount of scattered light reaches 1 / N of the end point of the coagulation reaction until the calculated coagulation time is determined to be normal.

[0005] Several methods, such as the percentage detection method and differential method, are used to calculate clotting time using automated analyzers. For example, the percentage detection method measures the scattered light intensity until the end of the clotting reaction, and then detects the clotting time as the point at which the light intensity reaches X% (e.g., 50%) of the maximum scattered light intensity at the end of the clotting reaction. The percentage detection method enables accurate calculation of clotting time even for abnormal samples, such as low fibrinogen samples, chyle samples, and hemolyzed samples. Furthermore, by measuring the scattered light intensity until the end of the clotting reaction, the percentage detection method can detect the true clotting reaction rather than noise, thereby preventing erroneous calculation of clotting time due to the aforementioned initial reaction. On the other hand, the percentage detection method calculates the clotting time after measuring one sample for several minutes, enabling it to detect the end of the clotting reaction in various blood samples, including abnormal samples with prolonged clotting times. However, such a long measurement time is not necessary for normal samples, which account for the majority of blood samples.

[0006] Patent Document 3 describes a blood coagulation time measurement method in which scattered light intensity data obtained in real time from an analyzer is smoothed and an origin is adjusted to obtain reference data X, and reference integral data Y obtained by further integrating the reference data is calculated from the reference data to obtain reference ratio data Z, which is the ratio of the integrated values ​​of the reference data at adjacent short time intervals.Of the times when the reference ratio data Z reaches a predetermined reference ratio data value Zs, a reference data value Xd is selected at a time after the peak of the reference ratio data Z and at a time when the reference integral data Y is equal to or greater than a predetermined threshold value Ys, and the coagulation time is determined to be the time from the time of mixing to a time corresponding to 1 / N (N is a constant integer equal to or greater than 1) of Xd. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Japanese Patent Application Laid-Open No. 2003-169700 [Patent Document 2] Japanese Patent Application Laid-Open No. 2010-217059 [Patent Document 3] Japanese Patent Application Publication No. 6-249855 Summary of the Invention [Problem to be solved by the invention]

[0008] The present invention provides a method for detecting a blood coagulation reaction, which is capable of detecting a true coagulation reaction in real time during blood coagulation reaction measurement. [Means for solving the problem]

[0009] That is, the present invention provides the following. [1] A method for detecting a blood coagulation reaction, comprising: 1) Measuring the blood coagulation reaction of the test blood sample and obtaining a first derivative V(i) of the coagulation reaction up to the latest measurement point, where i represents a measurement point or time, i = k0 to k, k represents the latest measurement point or time for the latest V(i), and k0 represents any measurement point or time where k0 ≦ k; 2) The maximum value cVmax(k) of V(i) up to k is taken as the peak top of V(i), and the area under the curve (AUC) before and after the peak of V(i) is calculated; 3) The pre-peak AUC and post-peak AUC are both equal to or less than the first threshold AUC th1 When the period L during which the values ​​are equal to or greater than the predetermined value and remain constant reaches a predetermined length, cVmax(k) is detected as the true maximum value Vmax of V(i). A method comprising: [2] The method according to [1], The above 2) is cVmax(k), cVmaxT(k), AUC pre (k), and AUC post (k), where cVmax(k) represents the maximum value of V(i) (i=k0~k), cVmaxT(k) represents the measurement point or time when V(i) = cVmax(k), AUC pre (k) is the pre-peak AUC at k, which represents the AUC of V(i) from k1 to cVmaxT(k); AUC post (k) is the post-peak AUC at k, which represents the AUC of V(i) from cVmaxT(k) to k2; k1 is the latest measurement point or time among the measurement points or times at which V(i)≦cVmax(k)×Hr% occurs before cVmaxT(k), k2 is the earliest measurement point or time after cVmaxT(k) that satisfies V(i)≦cVmax(k)×Hr%, 0 <Hr<100であり; And the method comprises: If the true maximum value Vmax is not detected in the step 3), the steps 1) to 3) are repeated with k=k+x (x>0). A method comprising: [3] The above 3) is AUC pre (k) and AUC post (k) is AUC th1 or more, and AUC pre (k)=AUC pre (kx), and AUC post (k)=AUC post If (kx), then let L = L + 1, otherwise let L = 0; When L reaches a predetermined value, cVmax(k) is detected as the true maximum value Vmax of V(i); The method described in [2], comprising: [4] The method according to [2] or [3], wherein 10≦Hr≦70. [5] The method according to any one of [1] to [4], wherein in 3), Vmax is not detected if the measurement point or time at which cVmax(k) occurs is included in the detection exclusion zone. [6] When k exceeds the measurement end point or time without detecting the true maximum value Vmax, the pre-peak AUC at k is equal to the first threshold AUC th1 The method according to any one of [1] to [5], further comprising detecting cVmax(k) as the true maximum value Vmax of V(i) if cVmax(k) is equal to or greater than cVmax(k). [7] When k exceeds the measurement end point or time without detecting the true maximum value Vmax, the pre-peak AUC and post-peak AUC at k both exceed the second threshold AUC th2 The method according to any one of [1] to [6], further comprising detecting cVmax(k) as the true maximum value Vmax of V(i), when the measurement point or time at which the pre-peak AUC is maximum is the same as the measurement point or time at which cVmax(k) is reached and the measurement point or time at which the post-peak AUC is maximum is later than the measurement point or time at which cVmax(k) is reached. [8] The method according to any one of [1] to [7], further comprising determining the coagulation reaction up to the latest measurement point as reaction P(i). [9] The method described in [8], further comprising calculating the blood coagulation time of the test blood sample based on P(i) or V(i).

[10] The method described in [9], wherein i≧k1. [Effects of the Invention]

[0010] According to the method of the present invention, false detection of noise such as an initial reaction in blood coagulation reaction measurement can be prevented, and true coagulation reactions can be correctly detected in real time during measurement. According to the method of the present invention, coagulation times based on true coagulation reactions can be correctly calculated. Furthermore, real-time detection of true coagulation reactions by the method of the present invention makes it possible to stop measurement of a blood sample once the data necessary to calculate the coagulation time of that sample has been acquired, and move on to measurement of the next blood sample. Therefore, according to the method of the present invention, the measurement time required for one blood sample can be shortened, improving the efficiency of blood coagulation tests. [Brief explanation of the drawings]

[0011] [Figure 1] An example of coagulation reaction measurement data. [Figure 2] 1 is a conceptual diagram illustrating cVmax(k), cVmaxT(k), AUCpre(k), and AUCpost(k). [Figure 3] Changes in AUCpre(k) and AUCpost(k) as the coagulation reaction progresses. A: Coagulation reaction progress. B: Changes in AUCpre(k) and AUCpost(k) over time. [Figure 4] Clotting reaction with an early reaction. A: Pre-peak and post-peak AUC of V(i) for the early reaction and the true reaction. B: Zoomed-in view of the early reaction. [Figure 5] Clotting reaction with and without an early reaction. [Figure 6] 1 is a flow chart illustrating one embodiment of the method steps of the present invention. [Figure 7] Clotting reactions of three test samples with different coagulation abilities. [Figure 8] 1 is a conceptual diagram showing the configuration of an automatic analyzer for carrying out the method for detecting a blood coagulation reaction according to the present invention. [Figure 9] Relationships between parameters calculated from the coagulation reaction. [Figure 10] A plot of the time (Fix) at which Vmax was detected in Example 3 against VmaxT measured in Example 1. [Figure 11]10 is a flowchart of a Vmax detection procedure used in Example 4. [Figure 12] Temporal changes in parameters calculated from sample 1 in Example 4. DETAILED DESCRIPTION OF THE INVENTION

[0012] In blood coagulation tests, a specific reagent is added to a blood sample, and the subsequent blood coagulation reaction is measured. The blood coagulation time is calculated from the coagulation reaction. In this specification, a blood sample may be simply referred to as a "sample." Blood coagulation reactions are measured using conventional methods, such as optical methods that measure scattered light intensity, transmittance, absorbance, etc., or mechanical methods that measure plasma viscosity. Blood coagulation reactions are generally represented by a coagulation reaction curve, which shows the change in the coagulation reaction amount over time. The coagulation reaction curve of a normal sample without coagulation abnormalities generally exhibits a sigmoid shape, although this depends on the measurement method. For example, as shown in Figure 1, the coagulation reaction curve based on the scattered light intensity of a normal sample typically rises sharply after a certain time has elapsed since the addition of the reagent, as coagulation progresses, and then plateaus as the coagulation reaction approaches completion. On the other hand, the coagulation reaction curve of an abnormal sample with coagulation abnormalities exhibits various shapes, such as a delayed rise time or a gradual rise, depending on the cause of the abnormality.

[0013] In measuring the blood clotting time of a sample, data is collected until the coagulation reaction is complete, i.e., until the coagulation reaction curve reaches a plateau, and the coagulation time can be calculated based on that data. For example, in the percentage detection method, the reaction volume from the start of the reaction to the end of the reaction is defined as 100%, and the time until the reaction volume reaches a predetermined value (e.g., 50%) can be calculated as the coagulation time. Alternatively, the coagulation time can be calculated based on the rate of change of the coagulation reaction curve, such as the peak of the coagulation reaction rate (the so-called differential method) or the time-dependent change in the integrated value of the coagulation reaction over a short time period (see Patent Document 3). However, the percentage detection method requires a sufficiently long measurement time per sample to enable measurement of the scattered light amount until the end of the coagulation reaction in various blood samples, including abnormal samples with prolonged coagulation times, and therefore has low analytical efficiency. On the other hand, the latter method allows for the calculation of the coagulation time before the end of the coagulation reaction, allowing for a shorter calculation time, but may result in inaccurate calculations due to noise.

[0014] In coagulation reaction measurement, coagulation reactions can be detected too early due to instrument noise at the beginning of the measurement or initial noise from the initial reaction. Such false detection of coagulation reactions leads to inaccurate calculation of coagulation time. To prevent false detection of coagulation reactions too early, one method is to set a threshold for the measurement time or measurement value in advance, and then consider the reaction after the measured time or data reaches the threshold as the true coagulation reaction in the coagulation time calculation process. However, if the threshold is set too high, true coagulation reactions may not be detected, especially in abnormal samples with small coagulation reactions. Conversely, lowering the threshold increases the possibility of false detection due to noise.

[0015] In measuring blood coagulation reactions, it is desirable to correctly detect the true coagulation reaction without falsely detecting noise such as an initial reaction, and to accurately calculate the coagulation time. Conventional blood coagulation reaction analysis methods, which calculate the coagulation time after detecting the end of the coagulation reaction, are capable of accurately calculating the coagulation time, but the long measurement time per sample results in low analytical efficiency. It would be more desirable to improve the analytical efficiency of blood samples by shortening the measurement time per sample while preventing false detection of noise.

[0016] 1. Method for detecting blood coagulation reactions The present invention provides a method for detecting a blood coagulation reaction. The method for detecting a blood coagulation reaction according to the present invention (hereinafter also referred to as the method of the present invention) typically includes the following steps: 1) Measuring the blood coagulation reaction of the test blood sample and obtaining a first derivative V(i) of the coagulation reaction up to the latest measurement point, where i represents a measurement point or time, i = k0 to k, k represents the latest measurement point or time for the latest V(i), and k0 represents any measurement point or time where k0 ≦ k; 2) The maximum value cVmax(k) of V(i) up to k is taken as the peak top of V(i), and the area under the curve (AUC) before and after the peak of V(i) is calculated; 3) The pre-peak AUC and post-peak AUC are both equal to or less than the first threshold AUC th1 When the period L during which the values ​​are equal to or greater than the predetermined value and remain constant reaches a predetermined length, cVmax(k) is detected as the true maximum value Vmax of V(i).

[0017] In the method of the present invention, while measuring the blood coagulation reaction of a test blood sample (hereinafter also referred to as the test sample), the true coagulation reaction (hereinafter also referred to as the true reaction) is detected in real time from the time-series data of the coagulation reaction obtained by the measurement. The method of the present invention prevents erroneous detection of noise such as an early reaction during blood coagulation reaction measurement, making it possible to correctly detect the true reaction. The blood coagulation time of the test sample can be calculated based on the obtained true reaction. According to the method of the present invention, the coagulation time can be accurately calculated without being affected by noise such as an early reaction. Furthermore, according to the present invention, the measurement time can be optimized for various blood samples, including normal and abnormal samples, so that the minimum coagulation reaction measurement time required to calculate each coagulation time can be applied.

[0018] Examples of blood clotting times that can be calculated according to the present invention include prothrombin time (PT), activated partial thromboplastin time (APTT), and clotting time determined by measuring fibrinogen (Fbg) concentration. The following description of the present invention will primarily use activated partial thromboplastin time (APTT) as an example of clotting time. Those skilled in the art can easily modify the method of the present invention to accommodate other clotting times (e.g., prothrombin time (PT)).

[0019] The procedure of the method of the present invention will be described in detail below.

[0020] In the method of the present invention, the test sample is preferably plasma from a subject. An anticoagulant commonly used in coagulation tests may be added to the sample. For example, plasma can be obtained by collecting blood using a blood collection tube containing sodium citrate and then centrifuging the blood.

[0021] In measuring a blood coagulation reaction, a coagulation time measuring reagent is added to a test sample to initiate the blood coagulation reaction. The coagulation reaction of a mixture containing the reagent and the test sample can be measured. The coagulation time measuring reagent used can be selected arbitrarily depending on the purpose of the measurement. Various reagents for measuring coagulation time are commercially available (e.g., APTT reagent Coagpia APTT-N; manufactured by Sekisui Medical Co., Ltd.). Coagulation reaction can be measured using common means, such as optical means for measuring the amount of scattered light, transmittance, absorbance, etc., or mechanical means for measuring plasma viscosity. In the following specification, the method of the present invention will be described using the example of coagulation reaction measurement based on the amount of scattered light.

[0022] The start of the clotting reaction is typically defined as the time when the sample is mixed with a reagent to initiate the clotting reaction, but other timings may also be defined as the start of the reaction. The time for continuing measurement of the clotting reaction may be, for example, several tens of seconds to approximately 8 minutes from the time when the sample and reagent are mixed. This measurement time may be until the true clotting reaction of each sample is detected, or may be until any other arbitrary condition is met, or may be an arbitrarily determined fixed value. During the measurement time, measurement of the progress of the clotting reaction (photometry in the case of optical detection) may be repeated at predetermined intervals. For example, measurements may be performed at 0.1-second intervals. The temperature of the mixed solution during the measurement is kept under normal conditions, for example, from 30°C to 40°C, preferably from 35°C to 39°C. Furthermore, various measurement conditions may be appropriately set depending on the test sample, reagent, measurement means, etc.

[0023] The above-described series of operations in measuring the coagulation reaction can be performed using an automatic analyzer. An example of an automatic analyzer is the CP3000 automatic blood coagulation analyzer (manufactured by Sekisui Medical Co., Ltd.). Alternatively, some operations may be performed manually. For example, the preparation of the test sample can be performed manually, and the subsequent operations can be performed by the automatic analyzer.

[0024] In step 1) of the method of the present invention, the blood coagulation reaction of the test sample is measured, and the first derivative V(i) of the coagulation reaction up to the most recent measurement point is obtained. First, measurement data D(i) (photometric values ​​of the amount of scattered light) is sequentially acquired by the above-described coagulation reaction measurement. Here, "i" represents the measurement point, i.e., the number of measurement points from the start of measurement. Alternatively, "i" represents the time (also simply referred to as "time") from the start of the coagulation reaction. For example, if the measurement (photometric) interval is 0.1 seconds, then time = 0.1 × i.

[0025] Next, a reaction P(i) is obtained from the measurement data D(i). Because the measurement data D(i) contains noise during photometry and fluctuations unrelated to the reaction that appear immediately after the start of photometry, it is preferable to smooth the measurement value using a known method. Furthermore, when measuring the coagulation reaction using the amount of scattered light, it is preferable to perform a zero-point adjustment process in which the amount of scattered light from the sample mixture before the reaction is subtracted. The smoothing process for the measurement data can be performed using any of a variety of known noise removal methods. Examples of smoothing processes include filtering, or a process of calculating a differential value by calculating a difference value or an average slope within a section (described below), and then integrating the differential value. Zero-point adjustment can be performed, for example, by adjusting the smoothed measurement data so that the value at the start of measurement is 0. Preferably, the measurement data D(i) is smoothed or zero-point adjusted to obtain the reaction P(i). More preferably, the measurement data D(i) is smoothed and zero-point adjusted to obtain the reaction P(i). The reaction P(i) constitutes a coagulation reaction curve.

[0026] The first derivative V(i) of the calculated reaction P(i) is obtained. The differentiation process for calculating V(i) from P(i) can be performed by any method, for example, by calculating the average slope within the interval. The calculation of the average slope within the interval can use a certain number of measurement points before and after each measurement point i, for example, 2K+1 measurement points from iK to i+K. Here, K is an integer. For example, when K is 2, five measurement points i-2, i-1, i, i+1, and i+2 can be used. The average slope refers to the slope value obtained by linearly approximating these multiple measurement points. A standard method, such as the least squares method, can be used to calculate the linear approximation. The average slope value of these measurement points can be considered the first derivative at measurement point i. The first derivative V(i) forms a curve representing the rate of the coagulation reaction.

[0027] V(i) used in the method of the present invention may be any first derivative calculated from the coagulation reaction P from any measurement point or time to the most recent measurement point or time. Therefore, V(i) used in the method of the present invention can form a curve that elongates as the coagulation reaction measurement progresses. Specifically, in the method of the present invention, V(i) can be obtained for i = k0 to k. k represents the latest measurement point or measurement time for the most recent V(i). Thus, k increases as the coagulation reaction measurement progresses. Note that the "latest V(i)" refers to the V(i) most recently calculated using the coagulation reaction P at the most recent measurement point or time. The latest measurement point or time for this V(i) is the "latest measurement point or time for V(i)." For example, if V(i) is coagulation reaction rate data from 1 second to 100 seconds after measurement, 100 seconds is the "latest measurement time" for V(i). k0 represents any measurement point or time before k (i.e., k0≦k). k0 represents the measurement point or time point at which the true reaction detection process shown in step 2) begins, and is also referred to herein as the detection start point (or detection start time). k0 is a measurement point or a time corresponding to a point after which it is possible to obtain P(i) by smoothing D(i) or obtain V(i) by differentiating P(i). Therefore, measurement point k0 is generally 2 or greater. For example, when the interval average slope method using the above-mentioned five measurement points is performed, measurement point k0 is 3 or greater. Also, for example, when 20 measurement points are used for smoothing or differentiation, measurement point k0 is 21 or greater.

[0028] In one embodiment, k0 is a measurement point or time after the detection exclusion range set at the beginning of measurement. The detection exclusion range corresponds to a measurement range or time range in which a true reaction cannot occur at the beginning of measurement. Preferably, under standard APTT measurement conditions, the detection exclusion range is set as a time range of about 10 seconds from the start of measurement or a measurement range corresponding thereto, and in this case, the time k0 is greater than 10 seconds. The same applies to PT measurement. Under measurement conditions for Fbg concentration measurement, the detection exclusion range is set as a time range of about 3 to 4 seconds from the start of measurement or a measurement range corresponding thereto, and in this case, the time k0 is greater than 3 seconds. By setting the detection exclusion range, noise occurring at the beginning of measurement can be excluded from the detection process of a true reaction. In another embodiment, k0 is set to the value when V(i) is first set to the threshold V th The measurement point or time when the threshold V th By setting the above, it is possible to exclude minute noises mixed in the clotting reaction from the detection process of the true reaction.

[0029] In step 2) of the method of the present invention, the areas under the curve (AUC) before and after the peak of V(i) obtained in step 1) are calculated. The calculation of the pre-peak AUC and post-peak AUC uses the peak of V(i) whose peak top is the maximum value of V(i) up to the measurement point or time k obtained in step 1). The maximum value of V(i) at k is referred to as cVmax(k) in the present specification. cVmax(k) may vary depending on k. Therefore, the pre-peak AUC and post-peak AUC may also vary depending on k, and therefore, in the present specification, the variable AUC of k is referred to as AUC. pre (k), and AUC post It is expressed as (k).

[0030] Preferably, in step 2), cVmax(k), cVmaxT(k), AUC pre (k), and AUC post Calculate (k). cVmax(k) is the maximum value of V(i) up to the measurement point or time k acquired in step 1), that is, the maximum value of V(i) (i=k0 to k). cVmaxT(k) represents the measurement point or time at which V(i) = cVmax(k). AUC pre (k) and AUC post (k) are, respectively, the pre-peak AUC and post-peak AUC at k as described above. AUC pre (k) represents the AUC of V(i) from k1 to cVmaxT(k), and AUC post (k) represents the AUC of V(i) from cVmaxT(k) to k2, where k1 is the latest measurement point or time among the measurement points or times where V(i) ≤ cVmax(k) × Hr% before cVmaxT(k), and k2 is the earliest measurement point or time among the measurement points or times that satisfy V(i) ≤ cVmax(k) × Hr% after cVmaxT(k). Therefore, basically k1 ≤ k2, and from the rise of the peak of V(i) to the peak top, AUC post (k) = 0. k1 and k2 are, respectively, the starting point for calculating AUC pre (k), and the ending point for calculating AUC post (k). Hr is the height ratio and determines the height of the starting point or ending point of V(i) used in the calculation of AUC pre (k) and AUC post (k). Hr is set to any value in the range greater than 0 and less than 100 (0 < Hr < 100), preferably 10 ≤ Hr ≤ 70.

[0031] Referring to Figure 2, cVmax(k), cVmaxT(k), AUC pre (k), and AUC postNext, we will explain (k). In Figure 2, the first derivative V(i) of the coagulation reaction curve is plotted against time. In Figure 2, at measurement point k, the peak top of V(i) is the maximum value cVmax(k), and the time at that point is cVmaxT(k). A line (baseline) indicating cVmax(k) x Hr% is drawn under the curve of V(i). There are two points (intersections of V(i) and the baseline) where V(i) = cVmax(k) x Hr%, one of which is k1, which exists before cVmaxT(k), and the other is k2, which exists after cVmaxT(k). k1 and k2 correspond to the start and end points of the baseline, respectively. The AUC before the peak of V(i), i.e., from k1 to cVmaxT(k), is the AUC pre (k), and the AUC after the peak of V(i), i.e., from cVmaxT(k) to k2, is AUC post (k).

[0032] AUC with progression of coagulation reaction pre (k) and AUC post The change in (k) will be explained with reference to Figure 3. As shown in Figure 3A, V(i) was divided into four sections, T1 to T4, along the horizontal axis (reaction progress). A line representing the Hr% of the peak top Vmax of V(i) was set as the baseline. The maximum point is the time when V(i) = Vmax. i and k represent time. T1: Up to the start of the reaction T2: From the rising point to the maximum point T3: From the maximum point to the end point of the baseline T4: After the end of the baseline Table 1 shows the cVmax(k) and AUC pre (k) and AUC post Before the start of the coagulation reaction, indicated by T1, the AUC pre (k) and AUC post (k) are both 0. In the ascending part of V(i), denoted by T2, cVmax(k) increases with k, i.e., cVmax(k) = V(k), and therefore AUC pre (k) also increases with k, while the AUC post(k) is 0. T3 is the period when V(i) decreases after reaching the true maximum value Vmax, and cVmax(k) remains constant at Vmax, so AUC pre (k) is also constant, while the post-peak area AUC post (k) increases with k. T4 is the time after V(i) passes the baseline endpoint (k2), and AUC pre (k), AUC post (k) are both constant. Figure 3B shows the AUC pre (k) and AUC post The temporal change of (k) is shown together with V(i).

[0033] [Table 1]

[0034] Figure 4 illustrates the pre-peak AUC and post-peak AUC of V(i) for the initial and true reactions in a coagulation reaction with an initial reaction. Figure 4A shows V(i) for the entire coagulation reaction, including the initial and true reactions, and Figure 4B is an enlarged view of V(i) for the initial reaction. V(i) increases rapidly immediately after the reaction, forming a small peak for the initial reaction, and then gradually decreases and approaches 0. The pre-peak AUC (AUC pre ) and post-peak AUC(AUC post ) is small compared to that of the true response that follows.

[0035] Thus, the AUC pre (k) and AUC post (k) varies. Therefore, AUC pre (k) and AUC post Based on (k), the progress of the coagulation reaction can be grasped.

[0036] Furthermore, AUC pre (k) and AUC postBased on (k), it is possible to distinguish between initial noise, such as an initial reaction in a clotting reaction, and a true reaction. Figure 5 shows examples of clotting reactions with and without an initial reaction. Each figure in each row shows, from the left, the reaction P, the baseline Bh showing cVmax(k) × Hr%, and the AUC pre and AUC post The graphs show the time-dependent changes in Bh and AUC. Each graph also shows the first derivative V. When the coagulation reaction does not have an initial reaction (upper row), pre increases with increasing V and remains constant after V reaches its peak (Vmax). On the other hand, AUC post AUC increases after V reaches Vmax and remains constant after V falls below Bh. When the coagulation reaction has an initial reaction (middle and bottom rows), Bh increases in two stages: at the time of the initial reaction and at the time of the true reaction. pre and AUC post rises once during the initial reaction, then rises again in response to the shift in Bh due to the emergence of the true reaction, and then becomes constant. In this way, whether it is the initial noise such as the initial response or the true response, when V(i) reaches the peak top, the AUC pre (k) becomes constant, followed by AUC post (k) may also be constant. However, the peaks in V(i) that appear due to initial noise are relatively small in both height and width, so AUC pre (k) is also AUC post (k) also remains relatively small. Also, the AUC pre (k) and AUC post (k) are reset with the appearance of a true response, so they do not remain constant for long.

[0037] Therefore, a true response can be detected from V(i) based on the values ​​of the pre-peak AUC and post-peak AUC. In the method of the present invention, if the pre-peak AUC and post-peak AUC exceed a predetermined threshold and this state continues for a certain period of time or longer, the peaks used in calculating the pre-peak AUC and post-peak AUC are considered to be the peaks of the true response.

[0038] More specifically, in step 3) of the method of the present invention, both the pre-peak AUC and the post-peak AUC of V(i) calculated in step 2) are set to a first threshold AUC th1 When the period L during which the value is equal to or greater than the predetermined value and remains constant reaches a predetermined length, cVmax(k) is detected as the true maximum value Vmax of V(i). Vmax is the maximum true response in V(i).

[0039] First threshold AUC th1 Although different values ​​may be set for the pre-peak AUC and the post-peak AUC, it is sufficient to set a common threshold for both. th1 can be appropriately set taking into consideration the magnitude of the coagulation reaction of the test sample and the magnitude of noise. th1 can be set appropriately depending on the type of reagent and equipment used for measuring the coagulation reaction. Under standard APTT measurement conditions, AUC th1 is preferably about 1.5 to 2.5 times the maximum AUC before the peak of the initial response, for example, in the range of 250 to 420. th1 If AUC is set smaller, it becomes possible to detect Vmax even from specimens with a small coagulation reaction, but the risk of false positives of early reactions increases. In that case, setting the detection exclusion zone mentioned above can prevent false positives of early reactions. On the other hand, AUC th1 Setting Vmax to a larger value can prevent false detection of the initial reaction, but it becomes difficult to detect Vmax in specimens with a low clotting reaction. In such cases, Vmax for specimens with a low clotting reaction can be detected by following the procedure shown in Figure 11, which will be described later. L represents the period during which the pre-peak AUC and post-peak AUC remain constant. For example, the initial value of L is 0, and if both the pre-peak AUC and post-peak AUC at measurement point or time k are the same as the previously calculated values, L is increased to L+1. On the other hand, if one or both of the pre-peak AUC and post-peak AUC are different (increased or decreased) from the previously calculated values, L is reset to 0.

[0040] Preferably, in step 3), AUC pre (k) and AUCpost (k) is AUC th1 or higher and AUC pre (k)=AUC pre (kx), and AUC post (k)=AUC post If (kx), L = L + 1, otherwise L = 0. th When the AUC reaches the maximum value of V(i), cVmax(k) is detected as the true maximum value Vmax of V(i). Here, the latest measurement point or time for V(i) is k, and the previous measurement point or time is kx, where x>0. x can be set appropriately depending on the time frame for comparison with the previous calculated value. For example, if k is the number of measurement points, then x=1, and the latest AUC pre (k) and the AUC calculated at the previous measurement point pre (k-1) may be compared, or x may be set to 2, 3 or more to speed up the calculation process. Similarly, when k is time. L th The value of can be set appropriately, but under the conditions of standard APTT measurement, L th It is preferable that the time is in the range of 0.5 to 2 seconds.

[0041] In order to eliminate false detections of initial noise such as initial reactions, Vmax detected in a measurement range or time range (for example, the detection exclusion range described above) that is too early for a true reaction to occur can be excluded as an error. In this case, if the time for the detected Vmax (i.e., cVmaxT(k)) is included in the detection exclusion range, Vmax will not be detected. When the detection exclusion range is set, AUC th1 can be set to a relatively small value, making it possible to detect the true maximum value Vmax even from a specimen with a small coagulation reaction.

[0042] The detection of the true maximum value Vmax means that the reaction being measured at the measurement point or time k is a true coagulation reaction, not an initial noise such as an initial reaction. Therefore, the peak of V(i) with the detected Vmax as its peak top is considered to be the V(i) of the true reaction. The time point at which Vmax is detected is determined by the AUC postThis is the point at which (k) becomes a constant value, that is, after V(i) has fallen below the baseline, and therefore the coagulation reaction has reached the end stage at this point. Therefore, in the method of the present invention, the coagulation reaction measurement of the test sample can be terminated after Vmax is detected and after the measurement data required for calculating the coagulation time has been obtained. For example, the coagulation reaction measurement of the test sample can be terminated when Vmax is detected. Also, for example, the coagulation reaction measurement of the test sample can be terminated when V(i) has sufficiently fallen after Vmax is detected. By using the coagulation reaction measured up to the end of the measurement, for example, P(i) or V(i), the coagulation time of the test sample can be accurately calculated.

[0043] On the other hand, in the method of the present invention, if a true maximum value Vmax is not detected at the measurement point or time k in step 3), that is, if the pre-peak AUC or post-peak AUC at k is AUC th1 If L does not reach the predetermined length, or if L does not reach the predetermined length, the coagulation reaction measurement continues, and steps 1) to 3) are repeated again. Preferably, steps 1) to 3) are repeated with k = k + x (x is as described above). This procedure is repeated unless Vmax is detected or k exceeds the measurement end point or time (the maximum measurement point or time at which the coagulation reaction measurement of the test sample is performed, as specified in advance). When Vmax is detected or k exceeds the measurement end point or time, the repeating process of steps 1) to 3) ends. The measurement end point or time can be set appropriately, and under standard APTT measurement conditions, it is preferably in the range of 4 to 8 minutes.

[0044] As one embodiment of the present invention, a flow chart illustrating the procedure for detecting a true clotting reaction according to the method of the present invention is shown in FIG. S01: Setting the setting value (Setting value: Hr, AUC th1 ,L th etc.) S02: Set the measurement counter to "i=1" (initial value) S03: Acquisition of measurement data D(i) S04: When the reaction P(i) can be calculated, proceed to S05 (because a predetermined number of D(i) are required for the smoothing process) S05: Calculation of P(i) S06: If the first differential V(i) can be calculated, proceed to S07 (because a predetermined number of P(i) are required for the differential processing) S07: Calculation of V(i) S08: Set cVmax(i) (i which becomes cVmax(i) is set as cVmaxT(i)) S09: Calculate the baseline Bh(i) (= cVmax(i) × Hr%) for cVmax(i). S10:AUC pre (i) and AUC post Calculate (i) S11:AUC pre (i) and AUC post (i) Both threshold AUC th1 Determine whether it matches S12:AUC pre (i) and AUC post (i) Both threshold AUC th1 and maintain a constant value (L ≧ L th (Whether S13: This step is optional: Determine whether cVmaxT(i) is in the detection exclusion zone. S14: If cVmaxT(i) is not included in the detection exclusion zone, cVmax(i) is determined to be the true maximum value Vmax of V(i) (the true maximum response value). S21: Count up the measurement counter S22: Determine whether the measurement counter exceeds the set measurement time, and if not, return to S03 S23: If the measurement counter exceeds the measurement time, the measurement is terminated and it is determined whether Vmax is detected or not. S24: Evaluate the measurement data offline based on the calculated values.

[0045] When the coagulation ability of the test sample is low, the reaction may not end even at the measurement end point or time, and P(i) may continue to rise. In such cases, the peak top of V(i) of the true reaction does not appear, or after the peak top, V(i) does not drop sufficiently, so that the AUC before the peak does not reach a constant value and the AUC after the peak is 0, or even if the AUC before the peak reaches a constant value, the AUC after the peak does not reach a constant value. As a result, Vmax may not be detected by the measurement end point or time by the method of the present invention. Therefore, in one embodiment of the method of the present invention, when k exceeds the measurement end point or time without detecting the true maximum value Vmax, the AUC at the measurement end point is calculated. pre (k) is AUC th1 If it is equal to or greater than this, cVmax(k) is detected as the true maximum value Vmax of V(i). This procedure makes it possible to detect the true reaction of a test sample whose reaction does not end within the measurement endpoint or time. If necessary, additional information regarding the coagulation ability of the test sample can be provided by outputting information indicating that Vmax was detected by this procedure along with Vmax.

[0046] When the coagulation reaction of the test sample is relatively small, the AUC before the peak or the AUC after the peak is th1 Therefore, in one embodiment of the method of the present invention, if k exceeds the measurement end point or time without detecting the true maximum value Vmax, the maximum value of the pre-peak AUC and post-peak AUC is set to the second threshold AUC th2 If the measurement point or time at which the pre-peak AUC is maximum is the same as cVmaxT(k) and the measurement point or time at which the post-peak AUC is maximum is later than cVmaxT(k), then cVmax(k) is detected as the true maximum value Vmax of V(i) (for example, using the procedure shown in FIG. 11, which will be described later). AUC th2 is preferably AUC th1 and preferably AUC th1This procedure allows detection of the true reaction of test specimens with relatively low blood coagulation ability. If necessary, additional information regarding the coagulation ability of the test specimen can be provided by outputting Vmax together with information indicating that this Vmax was detected by this procedure.

[0047] When the clotting response of the test sample is absent or very small, or due to other reasons, the true response of the test sample may be unclear. In such cases, it is difficult to distinguish between the true response and noise. In samples where the true response is unclear, the AUC before the peak is typically AUC th1 (and AUC th2 Alternatively, V(i) can be distinguished by the fact that it does not reach the threshold V th For example, V(i) can be distinguished by the fact that it does not exceed V th By starting step 2) only when Vmax is reached, false detection of noise can be prevented. In a sample where the true reaction is unclear, the true reaction may be excluded along with the noise, and Vmax may not be detected. In this case, the result that Vmax is not detected and the threshold AUC th1 If the AUC before the peak is not detected, or if the AUC before the peak is greater than V th By outputting information indicating that this is because V(i) cannot be detected, additional information regarding the coagulation ability of the test sample can be provided.

[0048] As an example, Figures 7A to 7C show the response P and first derivative V of three test samples with different coagulation abilities. Figure 7A shows a sample in which the true response is unclear, Figure 7B shows a sample in which the true response did not end at the measurement end point or within the time, and Figure 7C shows a sample in which a true response was detected. These samples were analyzed using the AUC pre (k) and AUC post They can be classified based on (k). Figure 7A: AUC pre (k) (and AUC post (k)) is AUC th1 AUC th2 Not even reached → No response Figure 7B: AUC pre (k) (or AUC post (k)) is AUC th1 reaches a certain value but does not reach a certain value → reaction is in progress Figure 7C: AUC pre (k) and AUC post (k) is AUC th1 and remains constant → Reaction detected

[0049] 2. Calculation of blood clotting time and other analyses In one embodiment, the method of the present invention detects the true reaction of a test sample, and then the detected true reaction can be used to calculate the clotting time of the test sample. The true clotting reaction, for example, P(i) or V(i), obtained by the method of the present invention can be used to calculate the blood clotting time or for various other blood analyses. In one example, the method of the present invention can detect the true maximum value Vmax using V(i) obtained from the coagulation reaction measurement data, and then calculate the coagulation time using V(i). In another example, the method of the present invention can obtain P(i) and V(i) from the coagulation reaction measurement data, detect the true maximum value Vmax using V(i), and then calculate the coagulation time using P(i) and / or V(i). In one embodiment, in the method of the present invention, the coagulation reaction measurement of the test sample is terminated when Vmax is detected, and the coagulation time can be calculated using the P(i) and / or V(i) of the true reaction obtained up to that point. In one embodiment, the coagulation time can be calculated using P(i) and / or V(i) after the rise of the peak containing the detected Vmax, for example, P(i) and / or V(i) where i≧k1. In another embodiment, in the method of the present invention, the coagulation reaction of the test sample can be continued to be measured even after Vmax is detected, and data regarding the coagulation reaction necessary for calculating the coagulation time or other analyses can be obtained.

[0050] The method for calculating the clotting time from the clotting reaction obtained by the method of the present invention is not particularly limited. Examples of methods for calculating the clotting time include a method in which the clotting time is calculated when P(i) reaches N% of the maximum value Pmax (the so-called percentage detection method); a method in which the clotting time is calculated when V(i) reaches the maximum value Vmax or N% thereof (the so-called differential method); a method in which the clotting time is calculated based on the time-dependent change in the integrated value of P(i) over a short time period (see Patent Document 3 and Patent Application No. 2019-237427); a method in which the clotting time is calculated based on the weighted average time of V(i) (see Patent Application No. 2020-039344); and a method in which the clotting time is calculated when P(i) reaches N% of P(Te), with the calculation starting point Te being the time when V(i) reaches a predetermined value after reaching the maximum value Vmax (see Patent Application No. 2020-068877).

[0051] [3. Application to other coagulation reaction measurement methods] The blood coagulation reaction detection method of the present invention has been described above using the example of coagulation reaction measurement based on the amount of scattered light. However, those skilled in the art will understand that the method of the present invention can be applied to blood coagulation reaction detection methods using other coagulation reaction measurement methods (e.g., blood coagulation reaction measurement methods based on transmittance, absorbance, viscosity, etc.), and such applications are within the scope of the present invention. For example, the response P(i) obtained from an inverted sigmoidal coagulation reaction curve based on the amount of transmitted light will have the opposite sign to that based on the amount of scattered light described above. In such cases, it will be clear to those skilled in the art that the signs of P(i) and V(i) will be reversed in the above steps 1) to 3), that the minimum value cVmin(k) of V(i) up to k will be calculated instead of cVmax(k), and that the area over the curve (AOC) will be calculated instead of the area under the curve (AUC).

[0052] 4. Programs and Devices The above-mentioned method for detecting a blood coagulation reaction of the present invention can be performed automatically using a computer program. Therefore, one aspect of the present invention is a program for performing the above-mentioned method for detecting a blood coagulation reaction of the present invention. Furthermore, a series of steps of the above-mentioned method of the present invention can be performed automatically by an automatic analyzer. Therefore, one aspect of the present invention is an apparatus for performing the above-mentioned method for detecting a blood coagulation reaction of the present invention.

[0053] One embodiment of the device of the present invention will be described below. One embodiment of the device of the present invention is an automatic analyzer 1 as shown in Figure 8. The automatic analyzer 1 includes a control unit 10, an operation unit 20, a measurement unit 30, and an output unit 40.

[0054] The control unit 10 controls the overall operation of the automatic analyzer 1. The control unit 10 may be configured, for example, by a personal computer (PC). The control unit 10 includes a CPU, memory, storage, a communication interface (I / F), and performs tasks such as processing commands from the operation unit 20, controlling the operation of the measurement unit 30, saving and analyzing measurement data received from the measurement unit 30, saving analysis results, and controlling the output of measurement data and analysis results by the output unit 40. The control unit 10 may also be connected to other devices such as external media or a host computer. In the control unit 10, the PC that controls the operation of the measurement unit 30 and the PC that analyzes the measurement data may be the same or different.

[0055] The operation unit 20 acquires input from an operator and transmits the acquired input information to the control unit 10. For example, the operation unit 20 includes a user interface (UI) such as a keyboard or a touch panel. Under the control of the control unit 10, the output unit 40 outputs the measurement data of the measurement unit 30 and its analysis results, such as P(i), V(i), the detection results of the true coagulation reaction (Vmax, etc.), and the coagulation time of the blood sample. For example, the output unit 40 includes a display device such as a display.

[0056] The measuring unit 30 performs a series of operations for a blood coagulation test and acquires measurement data of the coagulation reaction of a sample including a blood sample. The measuring unit 30 includes various equipment and analysis modules required for a blood coagulation test, such as a sample container for storing a blood sample, a reagent container for storing a test reagent, a reaction container for reacting the sample with the reagent, a probe for dispensing the blood sample and the reagent into the reaction container, a light source, a detector for detecting scattered light or transmitted light from the sample in the reaction container, a data processing circuit for sending data from the detector to the control unit 10, and a control circuit for controlling the operation of the measuring unit 30 in response to commands from the control unit 10.

[0057] The control unit 10 analyzes the coagulation reaction of the sample based on the data measured by the measurement unit 30. This analysis may include obtaining the above-mentioned P(i) and V(i), detecting the true coagulation reaction (Vmax, etc.), and calculating the coagulation time using the detected true coagulation reaction. Alternatively, the coagulation reaction curve P(i) or first derivative V may be created by the control unit 10 based on the measurement data from the measurement unit 30, or may be created by another device, for example, the measurement unit 30, and sent to the control unit 10. The control unit 10 may store parameters used to detect the true coagulation reaction, such as AUC th1 , L Th Alternatively, the control unit 10 may retrieve the setting values ​​stored in an external device or on a network during analysis.

[0058] The above-described analysis can be carried out by a program for carrying out the method of the present invention. Therefore, the control unit 10 can be provided with a program for carrying out the method of detecting a blood coagulation reaction of the present invention.

[0059] The analysis results from the control unit 10 are sent to the output unit 40 and output. The output may take any form, such as display on a screen, transmission to a host computer, or printing. The output information from the output unit may include P(i), V(i), the true coagulation reaction detection result, coagulation time, etc. The type of output information from the output unit may be controlled by the program of the present invention.

[0060] In one embodiment of the device of the present invention, the measurement unit 30 continues to measure the test sample until the coagulation reaction is completed, and the data is sequentially sent to the control unit 10. The control unit 10 sequentially performs calculations to obtain P(i) and V(i), as well as cVmax(k), cVmaxT(k), AUC pre (k), AUC post (k) etc., and then L ≧ L Th When Vmax is detected, the control unit 10 further calculates the clotting time of the test sample. The obtained analysis results are sent to the output unit and output. For example, P(i) and V(i) are output sequentially in parallel with the measurement, and cVmax(k), cVmaxT(k), AUC pre (k), AUC post (k) is also output sequentially. After Vmax is detected, Vmax, its time, and coagulation time are output in a timely manner. [Example]

[0061] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0062] Example 1 Parameters reflecting the coagulation reaction 1) Test specimen The test samples were 187 samples: normal plasma (10 samples), heparinized plasma (47 samples), LA-positive plasma (11 samples), coagulation factor-deficient plasma (14 samples), inhibitor plasma (41 samples), low fibrinogen plasma (5 samples), and other plasma samples with prolonged clotting time (59 samples). An early reaction was observed in 6 samples.

[0063] 2) Clotting reaction measurement The measurement reagent used was Coagpia APTT-N (manufactured by Sekisui Medical Co., Ltd.), an APTT measurement reagent, and the calcium chloride solution was Coagpia APTT-N Calcium Chloride Solution (manufactured by Sekisui Medical Co., Ltd.). The coagulation reaction of samples containing specimens was measured using a CP3000 automated blood coagulation analyzer (manufactured by Sekisui Medical Co., Ltd.). After 45 seconds of heating at 37°C in a cuvette, 50 μL of the test reagent at approximately 37°C was added. After 171 seconds, 50 μL of 25 mM calcium chloride solution was added to initiate the coagulation reaction. The reaction was carried out at 37°C. For the coagulation reaction measurement, the cuvette was irradiated with 660 nm light from an LED light source, and the amount of scattered light at a 90° side angle was measured at 0.1-second intervals. The maximum measurement time was 400 seconds (measurement points i = 1 to 4000).

[0064] 3) Obtaining the reaction P(i) and reaction rate V(i) The photometric data from each sample was subjected to smoothing, including noise removal, and then zero-point adjustment was performed so that the amount of scattered light at the start of photometry was 0 to create the response P(i). The first derivative V(i) was calculated from P(i).

[0065] 4) APTT calculation The clotting time (APTT) of the test sample was calculated by the percentage method. The time when P(i) reached 50% of its maximum value (Pmax) was defined as the clotting time.

[0066] Example 1 Parameters reflecting the coagulation reaction 1) Calculation of parameters Using the obtained P(i) and V(i) (i = 21 to 3980), the following parameters were calculated with the height ratio (Hr) set to 20%. Vmax: Maximum value of V(i) VmaxT: Time of Vmax Pmax: Maximum value of P(i) AUC pre : AUC before the peak of V(i) AUC post : AUC after the peak of V(i)

[0067] Examples of calculated parameters are shown in Table 2. The nine samples shown in Table 2 were as follows: Sample 1: Sample with the smallest Pmax Sample 2: The sample with the smallest VmaxT Sample 3: Sample with bimodal V(i) Samples 4-9: Samples in which an initial reaction appeared

[0068] [Table 2]

[0069] The relationships between parameters are shown in Figures 9A–F. The APTT values ​​of the 187 test samples ranged from a minimum of 24.9 seconds to a maximum of 283.7 seconds, and VmaxT showed an almost linear relationship with APTT (Figure 9A). The two samples in Figure 9A that deviated from the linear relationship were coagulation factor VIII (FVIII)-deficient samples with a bimodal V(i) profile with a larger first peak. There was no clear trend between the maximum P(i) value, Pmax, and VmaxT (Figure 9B). The relationship between Vmax and VmaxT tended to be inversely proportional (Figure 9C). This trend indicates that the V(i) peak increases with increasing coagulation reaction rate and decreases with decreasing coagulation reaction rate. There was no clear relationship between the pre-peak AUC, post-peak AUC, and VmaxT (Figures 9D and E). The post-peak AUC tended to be larger than the pre-peak AUC (Figure 9F).

[0070] 2) Parameters of test specimens with initial reactions For test samples with an initial reaction (samples 4 to 9 in Table 2), the initial reaction part and the true reaction part were determined from the obtained coagulation reaction curve, and the parameters Vmax, VmaxT, Pmax, and pre-peak AUC (AUC pre ), and post-peak AUC (AUC post ) was calculated. The results are shown in Table 3. The VmaxT of the true reaction ranged from approximately 70 seconds to 275 seconds, but the VmaxT of the initial reaction was approximately 4 seconds in all cases, confirming that it appeared early in the measurement.

[0071] [Table 3]

[0072] Example 2 Real-time detection of true coagulation reaction-1 Using the coagulation reaction data of 187 samples measured in Example 1, real-time detection of Vmax was carried out according to the procedure shown in the flowchart of FIG. 1) Threshold AUC th1 Decision As a result of Example 1, AUC pre and AUC post The minimum values ​​of AUC for the initial reaction part of samples 4 to 9, which had the smallest Pmax, were 207 and 285. pre The maximum value of AUC was 168 in sample 5. th1 If AUC is too low, sample 5 and other early reaction sites will be falsely detected. th1 If AUC is too large, it will not be possible to detect small reactions such as those in sample 1 and sample 3. th1 was determined to be 255, or about 1.5 times the maximum value of the initial reaction portion (168 in sample 5).

[0073] 2) Detection conditions Real-time detection of Vmax was performed under the following conditions. i=21 to Vmax detection (max 3980) Hr(%):20% AUC th1 :255 L th :10(1 second) Detection exclusion zone (step S13): None

[0074] 3) Results Vmax could be detected from the true reaction in 186 samples except for sample 1.

[0075] Example 3: Real-time detection of true coagulation reaction - 2 (low threshold, detection exclusion zone) The results of Example 1 confirmed that the peak of the initial reaction appeared early in the measurement. Therefore, real-time detection of Vmax was performed under conditions that could detect a smaller reaction than Sample 1 in Example 1, but did not detect the reaction in the section where the initial reaction is expected to appear (low threshold, with step S13). Specifically, real-time detection of Vmax was performed under the same conditions as in Example 2, except for the following changes. AUC th1 :100 Detection exclusion zone (step S13): Yes. Vmax is detected only when cVmaxT(i) is 50 (5 seconds) or later.

[0076] Vmax could be detected from the true reaction in all 187 samples. Figure 10 is a plot of the time (Fix) at which Vmax was detected in this example against VmaxT measured in Example 1. Since all Fix times were later than VmaxT, it was confirmed that the true reaction could be correctly detected using the procedure of this example. Furthermore, the results of this example showed that by setting a detection exclusion zone (step S13), true reactions can be correctly detected even in samples with small reactions.

[0077] Example 4: Real-time detection of true coagulation reaction - 3 (high threshold, offline detection available) Assuming that a larger initial reaction than that of samples 4 to 9 in Example 1 occurs, a higher AUC than that of Example 2, 1) th1 AUC was set. th1 It was predicted that increasing Vmax would prevent true reactions from being detected in some samples (e.g., sample 1) using the procedure in Figure 6. Therefore, the procedure in Figure 11 was incorporated between S24 and the end of Figure 6. Specifically, real-time detection of Vmax was performed under the same conditions as in Example 2, except for the following changes. AUC th1 :510 AUC th2 :100 On your S24: S31:AUC pre (i) and AUC post (i) Maximum AUC preMax and AUC post Max is the threshold AUC th2 Check whether it exceeds When S32:S31 holds, AUC pre Maximum time (AUC pre MaxT) is the same time as cVmaxT(i) and AUC post Maximum time (AUC post MaxT) is determined to be equal to or greater than cVmaxT(i). S33: When S32 is true, cVmax(i) is detected as Vmax.

[0078] Twelve specimens, including specimen 1 with the smallest Pmax, passed through S24 and Vmax was detected, while all other specimens had Vmax detected without passing through S24. Figure 12 shows the temporal changes in the parameters calculated when specimen 1 was subjected to the procedure of this example. As shown in Figures 12B to 12D, cVmaxT(i) reaches a maximum at the time VmaxT of the true maximum value, and AUC pre (i) is maximized at VmaxT, and AUC post (i) reached its maximum after VmaxT. It was confirmed that Vmax correctly detected the true maximum response in 12 samples, including sample 1, which was detected after S24.

Claims

1. A method for detecting a blood coagulation reaction, comprising: 1) Measuring the blood coagulation reaction of the test blood sample and obtaining the first derivative V(i) of the coagulation reaction P(i) up to the latest measurement point, where i represents the measurement point or time, and i=k 0 ∼k, where k represents the latest measurement point or time for the most recent V(i), and k 0 Ha K 0 represents any measurement point or time where k≦k, 2) Calculating the area under the curve (AUC) before and after the peak of V(i) using the maximum value cVmax(k) of V(i) up to k as the peak top of V(i); 3) The pre-peak AUC and the post-peak AUC are both a first threshold AUC th1 When the period L during which V(i) is equal to or greater than V(i) and remains constant reaches a predetermined length, cVmax(k) is detected as the true maximum value Vmax of V(i). 4) calculating the blood coagulation time of the test blood sample based on k corresponding to the true maximum value Vmax under the condition where the true maximum value Vmax is detected; A method comprising:

2. 2. The method of claim 1, The above 2) is cVmax(k), cVmaxT(k), AUC pre (k), and AUC post (k), where cVmax(k) is V(i) (i=k 0 represents the maximum value of cVmaxT(k) represents the measurement point or time at which V(i) = cVmax(k), AUC pre (k) is the pre-peak AUC at k, 1 represents the AUC of V(i) from cVmaxT(k) to cVmaxT(k), AUC post (k) is the post-peak AUC at k, calculated from cVmaxT(k) to k 2 represents the AUC of V(i) up to k 1 is the latest measurement point or time at which V(i)≦cVmax(k)×Hr % occurs before cVmaxT(k), k 2 is the earliest measurement point or time after cVmaxT(k) that satisfies V(i)≦cVmax(k)×Hr%, 0<Hr<100; And the method comprises: If the true maximum value Vmax is not detected in the step 3), the steps 1) to 3) are repeated with k set to k+x (x>0). A method comprising:

3. The above 3) is AUC pre (k) and AUC post (k) are all AUC th1 or more, and AUC pre (k) = AUC pre (k-x), and AUC post (k) = AUC post If (k-x), then let L = L + 1, otherwise let L = 0; When L reaches a predetermined value, cVmax(k) is detected as the true maximum value Vmax of V(i); 3. The method of claim 2, comprising:

4. 4. The method according to claim 2, wherein 10≦Hr≦70.

5. 5. The method according to claim 1, wherein in 3), Vmax is not detected if the measurement point or time at which cVmax(k) occurs is included in the detection exclusion zone.

6. When k exceeds the measurement end point or time without detecting the true maximum value Vmax, the pre-peak AUC at k exceeds the first threshold AUC th1 6. The method of claim 1, further comprising detecting cVmax(k) as the true maximum Vmax of V(i) if cVmax(k) is equal to or greater than cVmax(k).

7. When k exceeds the measurement end point or time without detecting the true maximum value Vmax, both the pre-peak AUC and post-peak AUC at k exceed the second threshold AUC. th2 The method according to any one of claims 1 to 6, further comprising detecting cVmax(k) as the true maximum value Vmax of V(i) when the measurement point or time at which the pre-peak AUC is maximum is the same as the measurement point or time at which cVmax(k) is reached and the measurement point or time at which the post-peak AUC is maximum is later than the measurement point or time at which cVmax(k) is reached.

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