Method for detecting coagulation response

Abstract

A method for detecting a coagulation reaction, the method comprising: 1) measuring a coagulation reaction of a blood sample to be tested, obtaining a first-order differential V(i) of the coagulation reaction up to a latest measurement point; 2) setting a maximum value cVmax(k) of V(i) as a peak top of V(i), obtaining a pre-peak and post-peak area under curve (AUC) of V(i); 3) when both the pre-peak AUC and the post-peak AUC are a first threshold value AUC th1 or the value or more and a period L during which each of the values continues to be a certain value reaches a prescribed length, detecting cVmax(k) as a true maximum value Vmax of V(i).

Description

Technical Field

[0001] This invention relates to a method for detecting coagulation reactions. Background Technology

[0002] Coagulation tests are used to diagnose a patient's coagulation function by adding prescribed reagents to a blood sample and measuring the clotting time. Typical examples of clotting time include prothrombin time (PT), activated partial thromboplastin time (APTT), and thrombin time. Abnormal coagulation function leads to prolonged clotting time. Causes of prolonged clotting time include the effects of coagulation-inhibiting agents, a reduction in the components involved in coagulation, congenital deficiencies in coagulation factors, and the acquired presence of autoantibodies that inhibit the coagulation response.

[0003] In recent years, automated analytical devices for automatically measuring coagulation reactions have become widely used, enabling convenient coagulation testing. For example, in one such automated analytical device, light is irradiated onto a mixture obtained by adding reagents to a blood sample, and the coagulation reaction of the blood sample is measured based on the change in the amount of scattered light. In a typical coagulation reaction, after a certain period of time since the addition of reagents, the amount of scattered light increases sharply as the coagulation reaction progresses. Subsequently, as the coagulation reaction nears its end, the amount of scattered light saturates and reaches a plateau. The moment of maximum scattered light is the end of the coagulation reaction. The coagulation time can be calculated based on this change in the amount of scattered light over time. On the other hand, the photometric data from the analytical device contains various noises caused by the device, reagents, and sample conditions. For example, after adding reagents to a blood sample, a subtle increase in the amount of scattered light is sometimes measured in the early stages of the reaction before the initial increase in scattered light due to the original coagulation reaction. This phenomenon is called the initial reaction or leading edge (Patent Documents 1 and 2). Noise in the photometric data of the initial reaction, etc., can lead to incorrect calculations of the coagulation time.

[0004] A method for removing the influence of initial reactions or leading edges in the automated measurement of coagulation reactions is proposed. For example, one method involves pre-setting thresholds for measurement time and values, and treating the reaction after the measurement time and data reach the thresholds as a true coagulation reaction, thus calculating the coagulation time. Furthermore, Patent Document 1 discloses a method for analyzing coagulation reactions, characterized by detecting initial reactions by monitoring the amount and rate of optical changes caused by the coagulation reaction at at least one checkpoint or check area from the start of the coagulation reaction measurement to the end of the reaction. Patent Document 2 discloses a coagulation analysis method characterized by repeatedly measuring the amount of scattered light and calculating the time when the measured scattered light amount reaches 1 / N of the end point of the coagulation reaction as the coagulation time until the calculated coagulation time is determined to be normal.

[0005] Several methods, such as percentage detection and differential methods, have been used to calculate coagulation time based on automated analysis devices. For example, in the percentage detection method, the coagulation time is defined as the point where X% (e.g., 50%) of the scattered light intensity reaches the end of the coagulation reaction (maximum scattered light intensity). The percentage detection method can accurately calculate coagulation time even with abnormal samples such as those with low fibrinogen, chylous, or hemolyzed samples. Furthermore, by measuring the scattered light intensity until the coagulation reaction ends, the percentage detection method can detect a true coagulation reaction that is not noise, thus preventing erroneous calculations of coagulation time caused by the aforementioned initial reaction. On the other hand, in the percentage detection method, in order to detect the end of the coagulation reaction in various blood samples, including abnormal samples with prolonged coagulation times, the coagulation time is calculated after measuring a single sample for several minutes. However, such a long measurement time is unnecessary for the majority of normal blood samples.

[0006] Patent document 3 describes a method for measuring coagulation time, which smooths and adjusts the origin of scattered light data obtained in real time by an analysis device as reference data X. Based on the reference data, a reference integral data Y obtained by integrating the reference data is further calculated, and a reference ratio data Z is calculated as the ratio of the cumulative values ​​of each adjacent minute time of the above reference data. At the moment when the reference ratio data Z reaches a predetermined reference ratio data value Zs, a reference data value Xd is selected at the moment when the reference ratio data Z is below the peak value and the reference integral data Y reaches a predetermined threshold Ys or above. The time from the mixing moment up to the moment corresponding to the value of 1 / N (N is a certain integer greater than or equal to 1) of Xd is taken as the coagulation time.

[0007] Existing technical documents

[0008] Patent documents

[0009] Patent Document 1: Japanese Patent Application Publication No. 2003-169700

[0010] Patent Document 2: Japanese Patent Application Publication No. 2010-217059

[0011] Patent Document 3: Japanese Patent Application Publication No. 6-249855 Summary of the Invention

[0012] This invention provides a method for detecting coagulation reaction in real time during coagulation reaction measurement.

[0013] That is, the present invention provides the following methods.

[0014] [1] A method for detecting coagulation reaction, comprising the following steps:

[0015] 1) Measure the coagulation reaction of the tested blood sample and obtain the first differential V(i) of the coagulation reaction up to the latest measurement point. Here, i represents the measurement point or time, i = k0 ~ k, k represents the latest measurement point or time of the latest V(i), and k0 represents any measurement point or time where k0 ≤ k.

[0016] 2) Set the maximum value cVmax(k) of V(i) up to i = k as the peak of V(i), and calculate the area under the curve (AUC) before and after the peak of V(i).

[0017] 3) Both the pre-peak AUC and post-peak AUC are the first threshold AUC. th1 When the period L, which is above or equal to a certain value, reaches a specified length, cVmax(k) is detected as the true maximum value Vmax of V(i).

[0018] [2] According to the method described in [1], wherein 2) above includes calculating cVmax(k), cVmaxT(k), and AUC. pre (k) and AUC post (k), here

[0019] cVmax(k) represents the maximum value of V(i) (i = k0 ~ k).

[0020] cVmaxT(k) represents the measurement point or time at which V(i) = cVmax(k) is reached.

[0021] AUC pre (k) represents the pre-peak AUC at i = k, and is the AUC of V(i) from i = k1 to i = cVmaxT(k).

[0022] AUC post (k) represents the post-peak AUC at i = k, and is the AUC of V(i) from i = cVmaxT(k) to i = k2.

[0023] k1 is the latest measurement point or time among the measurement points or times that reach V(i)≤cVmax(k)×Hr% before cVmaxT(k).

[0024] k2 is the earliest measurement point or time among the measurement points or times that satisfy V(i) ≤ cVmax(k) × Hr% after cVmaxT(k).

[0025] 0 < Hr < 100;

[0026] The method includes the following steps: if Vmax is not detected in step 3) above, calculate k = k + x (x > 0), and repeat steps 1) to 3) above.

[0027] [3] According to the method described in [2], wherein 3) above includes the following steps:

[0028] AUC pre (k) and AUC post (k) are all AUC th1 Or above that value and AUC pre (k)=AUC pre (k-x) and AUC post (k)=AUC post (k-x) and when x>0, it is counted as L=L+1, and in the case that this is not the case, L=0;

[0029] When L reaches the specified value, cVmax(k) is detected as Vmax of V(i).

[0030] [4] The method according to [2] or [3], wherein 10 ≤ Hr ≤ 70.

[0031] [5] The method according to any one of [1] to [4], wherein, in the case where the measurement point or time that reaches cVmax(k) is included in the detection exclusion area in 3) above, Vmax is not detected.

[0032] [6] The method according to any one of [1] to [5], further comprising the step of: if no Vmax is detected and k exceeds the measurement end point or time, if the pre-peak AUC at i = k is the first threshold AUC th1 If the value is greater than or equal to this value, then cVmax(k) is detected as the Vmax of V(i).

[0033] [7] The method according to any one of [1] to [6], further comprising the step of: when no Vmax is detected and k exceeds the measurement end point or time, the pre-peak AUC and post-peak AUC at i=k are both the second threshold AUC. th2 If the value is greater than or equal to the value above, and the measurement point or time of the maximum AUC before the peak is the same as the measurement point or time of reaching cVmax(k), and the measurement point or time of the maximum AUC after the peak is later than the measurement point or time of reaching cVmax(k), then cVmax(k) is detected as the Vmax of V(i).

[0034] [8] The method according to any one of [1] to [7], wherein the method further comprises the step of determining the solidification reaction up to the latest measurement point as reaction P(i).

[0035] [9] The method according to [8] further includes the following step: calculating the clotting time of the tested blood sample based on the above P(i) or V(i).

[0036]

[10] According to the method described in [9], where i ≥ k1.

[0037] According to the method of the present invention, false detections due to noise such as initial reactions in coagulation reaction measurements can be prevented, and the true coagulation reaction can be accurately detected in real time during the measurement. According to the method of the present invention, the coagulation time based on the true coagulation reaction can be accurately calculated. Furthermore, the real-time detection of the true coagulation reaction based on the method of the present invention allows for stopping the measurement of a blood sample after obtaining the data required to calculate the coagulation time of that sample, and moving on to the 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 coagulation testing. Attached Figure Description

[0038] Figure 1 This is an example of solidification reaction measurement data.

[0039] Figure 2 This refers to cVmax(k), cVmaxT(k), and AUC. pre (k) and AUC post (k) A concept map for explanation.

[0040] Figure 3 The AUC that accompanies the solidification reaction pre (k) and AUC post The change in (k). A represents the extent of the solidification reaction. B represents the AUC. pre (k) and AUC post The time variation of (k).

[0041] Figure 4 It is a solidification reaction with an initial reaction. A is the pre-peak AUC and post-peak AUC of V(i) for the initial reaction and the actual reaction, and B is an enlarged view of the initial reaction.

[0042] Figure 5 It refers to solidification reactions that have or do not have an initial reaction.

[0043] Figure 6 This is a flowchart illustrating one embodiment of the steps of the method of the present invention.

[0044] Figure 7 These are the coagulation reactions of three tested samples with different coagulation abilities.

[0045] Figure 8 This is a conceptual diagram illustrating the configuration of an automated analysis apparatus for performing a coagulation reaction detection method based on the present invention.

[0046] Figure 9It is the relationship between parameters calculated from the solidification reaction.

[0047] Figure 10 It is a curve of the time (Fix) at which Vmax was detected in Example 3 relative to the VmaxT measured in Example 1.

[0048] Figure 11 This is a flowchart of the Vmax detection steps used in Example 4.

[0049] Figure 12 This refers to the change in time of the parameters calculated from sample 1 in Example 4. Detailed Implementation

[0050] In coagulation tests, prescribed reagents are added to a blood sample, and the subsequent coagulation reaction is measured. The coagulation time is then calculated from this reaction. In this instruction manual, the blood sample is sometimes simply referred to as the sample. Coagulation reactions are measured using common methods, such as optical methods that measure scattered light, transmittance, absorbance, etc., or mechanical methods that measure plasma viscosity. Coagulation reactions are generally represented by a coagulation reaction curve, which shows the change in the amount of coagulation reaction over time. The coagulation reaction curve of a normal sample lacking significant factors for coagulation abnormalities, while depending on the measurement method, is generally S-shaped. For example, as... Figure 1 As shown, the coagulation reaction curve of normal samples based on the amount of scattered light typically rises sharply as coagulation occurs after a certain period of time since the reagent was added, and then reaches a plateau as the coagulation reaction nears its end. On the other hand, the coagulation reaction curve of abnormal samples, which have important factors for coagulation abnormalities, exhibits various shapes due to abnormal reasons such as delayed rise time and slow rise.

[0051] In determining the clotting time of a sample, data can be collected until the coagulation reaction ends, i.e., until the coagulation reaction curve reaches a plateau, and the clotting time can be calculated based on this data. For example, in the percentage detection method, the reaction amount from the start to the end of the reaction can be set to 100%, and the time until the reaction amount reaches a specified value (e.g., 50%) can be calculated as the clotting time. Alternatively, the rate of change of the coagulation reaction curve can be used to calculate the clotting time, for example, based on the peak of the coagulation reaction rate (so-called differential method) or the time-varying cumulative value of the coagulation reaction over a small time interval (see Patent Document 3). However, the percentage detection method is inefficient because it can measure the amount of scattered light until the end of the coagulation reaction from various blood samples, including abnormal samples with prolonged clotting time, requiring a sufficiently long measurement time for each sample. On the other hand, in the latter method, since the clotting time can be calculated before the end of the coagulation reaction, the clotting time can be calculated in a shorter time, but sometimes inaccurate clotting times are calculated due to noise.

[0052] In solidification reaction measurements, solidification reactions are sometimes detected prematurely due to initial instrument noise or initial reaction noise. Such false detections lead to inaccurate calculations of solidification time. To prevent these premature detections, one method is to pre-set thresholds for measurement time and values, treating reactions occurring after these thresholds are reached as genuine solidification reactions and calculating the solidification time accordingly. However, setting the threshold too high may prevent detection of genuine solidification reactions in anomalous samples with very small solidification rates. Conversely, lowering the threshold increases the likelihood of false detections due to noise.

[0053] In coagulation reaction measurements, it is desirable to accurately detect the true coagulation reaction without false detections of noise such as the initial reaction, and to accurately calculate the coagulation time. Furthermore, while existing coagulation reaction analysis methods that calculate the coagulation time after the initial coagulation reaction are completed can accurately calculate the coagulation time, their analytical efficiency is low due to the long measurement time per sample. A method that can prevent false detections of noise and shorten the measurement time per sample, thereby improving the analytical efficiency of blood samples, would be preferable.

[0054] [1. Methods for detecting coagulation reactions]

[0055] This invention provides a method for detecting coagulation reactions. The method for detecting coagulation reactions of this invention (hereinafter also referred to as the method of this invention) typically includes the following steps:

[0056] 1) Measure the coagulation reaction of the tested blood sample and obtain the first differential V(i) of the coagulation reaction at the latest measurement point. Here, i represents the measurement point or time, i = k0 ~ k, k represents the latest measurement point or time of the latest V(i), and k0 represents any measurement point or time where k0 ≤ k.

[0057] 2) Set the maximum value cVmax(k) of V(i) up to i = k as the peak of V(i), and calculate the area under the curve (AUC) before and after the peak of V(i).

[0058] 3) Both the pre-peak AUC and post-peak AUC are the first threshold AUC. th1 When the period L, which is above or equal to a certain value, reaches a specified length, cVmax(k) is detected as the true maximum value Vmax of V(i).

[0059] In the method of the present invention, the coagulation reaction of a blood sample under test (hereinafter also referred to as the test sample) is measured, and the true coagulation reaction (hereinafter also referred to as the true reaction) is detected in real time based on the time series data of the coagulation reaction obtained in the measurement. The method of the present invention can prevent false detections due to noise such as initial reactions in coagulation reaction measurements, and accurately detect the true reaction. The 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 initial reactions. Furthermore, according to the present invention, the measurement time can be optimized in a way that applies the respective coagulation times to various blood samples, including normal samples and abnormal samples, to calculate the minimum coagulation reaction measurement time required.

[0060] Examples of clotting times that can be calculated according to the present invention include prothrombin time (PT), activated partial thromboplastin time (APTT), and clotting time in fibrinogen (Fbg) concentration determination. In the following specification, the activated partial thromboplastin time (APTT) as a clotting time will be used as an example to describe the method of the present invention. Modifications to other clotting times (e.g., prothrombin time (PT) in the method of the present invention) are feasible for those skilled in the art.

[0061] The steps of the method of the present invention will be described in detail below.

[0062] In the method of the present invention, plasma of the subject is preferably used as the test sample. An anticoagulant commonly used in coagulation tests may be added to this sample. For example, plasma is obtained by centrifugation after blood is collected using a blood collection tube containing sodium citrate.

[0063] In the measurement of coagulation reaction, a coagulation time assay reagent is added to the test sample to initiate the coagulation reaction. The coagulation reaction of the mixture containing the reagent and the test sample can be measured. The coagulation time assay reagent used can be arbitrarily selected depending on the purpose of the measurement. Commercially available products can be used as reagents for measuring various coagulation times (e.g., APTT reagent Coagpia APTT-N; manufactured by Sekisui Medical Co., Ltd.). The measurement of coagulation reaction can be performed using general methods, such as optical methods that measure the amount of scattered light, transmittance, absorbance, etc., or mechanical methods that measure the viscosity of plasma. In the following specification, the method of the present invention will be described using the measurement of coagulation reaction based on the amount of scattered light as an example.

[0064] The onset time of the coagulation reaction can typically be defined as the moment when the reagent is mixed in the sample and the coagulation reaction begins, but other moments can also be defined as the onset time. The measurement time for continuing the coagulation reaction can be, for example, from tens of seconds to about 8 minutes from the moment the sample and reagent are mixed. This measurement time can continue until the actual coagulation reaction of each sample is detected, until any other arbitrary condition is met, or it can be any arbitrarily determined fixed value. The measurement of the progress of the coagulation reaction can be repeated at predetermined intervals during this measurement time (photometric measurement in the case of optical detection). For example, measurements can be performed at 0.1-second intervals. The temperature of the mixture in this measurement is typically 30°C to 40°C, preferably 35°C to 39°C. Furthermore, various measurement conditions can be appropriately set according to the sample being tested, the reagent, the measurement method, etc.

[0065] The series of operations in the coagulation reaction measurement described above can be performed using an automated analytical device. An example of such an automated analytical device is the CP3000 coagulation automated analyzer (manufactured by Sekisui Medical Co., Ltd.). Alternatively, some operations can be performed manually. For example, the preparation of the test sample can be considered, with subsequent operations performed using an automated analytical device.

[0066] In step 1) of the method of the present invention, the coagulation reaction of the tested sample is measured to obtain the first differential V(i) of the coagulation reaction up to the latest measurement point.

[0067] First, measurement data D(i) (photometric values ​​of scattered light) are obtained sequentially through the above-described solidification reaction measurement. Here, "i" represents the measurement point, that is, the point from which the measurement is performed. Alternatively, "i" represents the time from the start of the solidification reaction (also simply referred to as time). For example, if the measurement (photometric) interval is 0.1 seconds, it is represented by time = 0.1 × i.

[0068] Next, the reaction P(i) is obtained from the measurement data D(i). Since the measurement data D(i) contains noise during photometry and variations unrelated to the reaction that occur after the initial 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 zeroing by subtracting the amount of scattered light from the sample mixture before the reaction. The smoothing of the measurement data can be performed using any of the various known methods related to noise removal. For example, as a smoothing process, filtering or integrating the differential value obtained by calculating the difference or the average slope within the interval (described later) can be used. For zeroing, for example, the smoothed measurement data can be adjusted to a value of 0 at the start of the measurement. It is preferable to smooth or zero the measurement data D(i) to obtain the reaction P(i). More preferably, it is preferable to smooth and zero the measurement data D(i) to obtain the reaction P(i). The reaction P(i) constitutes the coagulation reaction curve.

[0069] Based on the calculated reaction P(i), its first differential V(i) is obtained. The differentiation of V(i) from P(i) can be performed using any method; for example, it can be done by calculating the average slope value over the interval. In calculating the average slope value over the interval, constant measurement points before and after each measurement point i can be used, for example, 2K+1 measurement points from i-K to i+K. Here, K is any integer. For example, when K is 2, the (i-2), (i-1), i, (i+1), and (i+2)th measurement points can be used. The average slope value represents the slope value when linearly approximating these multiple measurement points. The linear approximation can be performed using conventional methods such as the least squares method. The average slope value of these measurement points can be considered as the first differential at measurement point i. The first differential V(i) constitutes a curve representing the rate of the solidification reaction.

[0070] The V(i) used in the method of the present invention is simply the first derivative value of the solidification reaction P calculated from any measurement point or time up to the latest measurement point or time. Therefore, the V(i) used in the method of the present invention can form a curve that elongates as the solidification reaction measurement proceeds. Specifically, in the method of the present invention, V(i) at i = k0 to k can be obtained. k represents the latest measurement point or measurement time of the latest V(i). Therefore, k increases as the solidification reaction measurement proceeds. It should be noted that "latest V(i)" refers to the V(i) most recently calculated using the solidification reaction P at the latest measurement point or time. The latest measurement point or time for that V(i) is the "latest measurement point or time of V(i)". For example, if V(i) is solidification reaction rate data up to 1 second to 100 seconds after the measurement, then 100 seconds is the "latest measurement time" for that V(i). k0 represents any measurement point or time before k (i.e., k0 ≤ k). k0 represents the measurement point or time at which the detection process of the actual reaction shown in step 2) begins, also referred to in this specification as the detection start point (or detection start time). k0 is the measurement point or the time corresponding to the point after which P(i) can be obtained through the smoothing process of D(i) or V(i) can be obtained through the differentiation of P(i). Therefore, the measurement point k0 is generally 2 or more. For example, when performing the interval average slope method using the above 5 measurement points, the measurement point k0 is 3 or more. In addition, for example, when using 20 measurement points in the smoothing or differentiation process, the measurement point k0 is 21 or more.

[0071] In one implementation, k0 represents the measurement point or time following the initial detection exclusion zone. The detection exclusion zone corresponds to the measurement area or time period in the initial stage of measurement where a true reaction cannot occur. Preferably, under standard APTT measurement conditions, the detection exclusion zone is set to a time period of approximately 10 seconds from the start of the measurement or a measurement area corresponding to that time period, where time k0 is greater than 10 seconds. The same applies to PT measurements. Under measurement conditions used to determine Fbg concentration, the detection exclusion zone is set to a time period of approximately 3-4 seconds from the start of the measurement or a measurement area corresponding to that time period, where time k0 is greater than 3 seconds. By setting the detection exclusion zone, noise generated in the initial stage of measurement can be removed from the detection process of a true reaction.

[0072] In another implementation, k0 is the threshold V that V(i) initially reaches. th The measurement point or time. This can be achieved by setting a threshold V. th This removes the minute noises mixed in during the solidification reaction from the actual reaction detection process.

[0073] In step 2) of the method of the present invention, the area under the curve (AUC) before and after the peak of V(i) obtained in step 1) is calculated.

[0074] The calculation of the pre-peak AUC and post-peak AUC uses the maximum value of V(i) below the measurement point or time k obtained in step 1) as the peak of V(i). In the following specification, the maximum value of V(i) at k is referred to as cVmax(k). cVmax(k) can vary depending on k. Therefore, the pre-peak AUC and post-peak AUC can also vary depending on k, and are therefore represented as variables AUC k in this specification. pre (k) and AUC post (k).

[0075] Preferably, in step 2), cVmax(k), cVmaxT(k), and AUC are calculated. pre (k) and AUC post (k).

[0076] cVmax(k) is the maximum value of V(i) obtained in step 1) at the measurement point or time k below, that is, the maximum value of V(i) (i=k0~k).

[0077] cVmaxT(k) represents the measurement point or time at which V(i) = cVmax(k) is reached.

[0078] AUC pre (k) and AUC post (k) represents the pre-peak AUC and post-peak AUC at point k, respectively. pre (k) represents the AUC of V(i) for k1~cVmaxT(k), 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 that reach 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. In addition, the AUC is the rise from the peak of V(i) to the peak. post (k) = 0. k1 and k2 are respectively the AUC pre The starting point for calculating (k) and AUC post The endpoint of the calculation of (k). Hr is the height ratio, which determines the AUC. pre (k) and AUC post The height of the start or end point of V(i) used in the calculation of (k). Hr is set to any value in the range of greater than 0 and less than 100 (0 < Hr < 100), preferably 10 ≤ Hr ≤ 70.

[0079] Reference Figure 2 For cVmax(k), cVmaxT(k), AUC pre (k) and AUC post (k) will be explained. Figure 2 The first derivative V(i) of the solidification reaction curve is plotted against time. Figure 2 In the case of V(i), at measurement point k, the peak of V(i) is the maximum value cVmax(k), and the time at this point is cVmaxT(k). Draw a straight line (baseline) representing cVmax(k) × Hr% under the curve of V(i). There are two moments when V(i) ≥ cVmax(k) × Hr% (the intersection of V(i) and the baseline), one being k1 before cVmaxT(k) and the other being k2 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 AUC. pre The AUC after the peak of V(k) and V(i), i.e., from cVmaxT(k) to k2, is AUC. post (k).

[0080] Reference Figure 3 AUC of the reaction accompanied by solidification pre (k) and AUC post Explain the changes in (k). For example... Figure 3 As shown in Figure A, V(i) is divided into four intervals, T1 to T4, along the horizontal axis (reaction proceeds). A straight line representing the Hr% of the peak value Vmax of V(i) is defined as the baseline. The maximum point is the time when V(i) = Vmax. i and k represent time.

[0081] T1: Below the reaction's rise point

[0082] T2: Rising point ~ Maximum point

[0083] T3: Maximum point ~ baseline endpoint

[0084] T4: Below the baseline endpoint

[0085] Table 1 shows the cVmax(k) and AUC values ​​from T1 to T4. pre (k) and AUC post The behavior of (k). The AUC is shown at T1 before the solidification reaction begins. pre (k) and AUC post (k) are all 0. In the rising part of V(i) shown at T2, cVmax(k) increases along with k, i.e., cVmax(k) = V(k), therefore AUC pre(k) also increases along with k; on the other hand, since there is no post-peak region, AUC... post (k) is 0. T3 is the period after V(i) reaches its true maximum value Vmax and then decreases. cVmax(k) remains constant at Vmax, therefore AUC pre (k) is also constant; on the other hand, the AUC in the post-peak region... post (k) increases together with k. T4 is the period after V(i) exceeds the baseline endpoint (k2), and AUC pre (k), AUC post (k) are all constant. Figure 3 B, together with V(i), illustrates the AUC. pre (k) and AUC post The time variation of (k).

[0086] [Table 1]

[0087] cVmax(k) - V(k) Vmax Vmax <![CDATA[AUC pre (k)]]> zero rise A certain value A certain value <![CDATA[AUC post (k)]]> zero zero rise A certain value

[0088] Figure 4 The example shows the pre-peak AUC and post-peak AUC of V(i) for the initial reaction and the true reaction in a solidification reaction with an initial reaction. Figure 4 A represents the overall V(i) of the solidification reaction, including the initial reaction and the actual reaction. Figure 4 B is a magnified view of V(i) in the initial reaction. V(i) increases sharply after the reaction, forming a small peak in the initial reaction, and then decreases slowly to approach 0. The AUC before the peak in the initial reaction (AUC) is also shown. pre ) and post-peak AUC (AUC post The peak AUC of the subsequent true reaction is smaller than that of the peak-precession AUC. pre ) and post-peak AUC (AUC post ).

[0089] Thus, the AUC is determined based on the progress of the solidification reaction. pre (k) and AUC post (k) changes. Therefore, it can be based on AUC. pre (k) and AUC post (k) is used to monitor the progress of the solidification reaction.

[0090] Therefore, it can be based on AUC pre (k) and AUC post (k) is used to distinguish between initial noise and the actual reaction in the initial stage of the solidification reaction. Figure 5 Examples of solidification reactions with and without initial reactions are shown. The graphs in each row, from left to right, show the reaction P, the baseline Bh representing cVmax(k) × Hr%, and AUC. pre and AUCpost The time-based variation. The first differential V is shown together in each figure. In the case of no initial reaction during solidification (upper layer), Bh and AUC... pre As V increases, AUC rises, reaching a peak (Vmax) and then becoming constant. On the other hand, AUC... post After V reaches Vmax, it increases; once V is below Bh, it becomes constant. In the case of an initial reaction (middle and lower layers), Bh increases in two phases: during the initial reaction and during the actual reaction. AUC pre and AUC post It rises temporarily during the initial reaction, rises again corresponding to the shift in Bh caused by the occurrence of the actual reaction, and then becomes constant.

[0091] Thus, whether it's initial noise such as the initial reaction or the actual reaction, the AUC when V(i) reaches its peak pre (k) are all constant, and therefore AUC post (k) is also constant. However, the height and width of the peak of V(i) appearing in the initial noise are relatively small, therefore the AUC is... pre (k) and AUC post (k) are all limited to relatively small values. Additionally, the AUC for the initial noise peak... pre (k) and AUC post (k) are reset due to the occurrence of the actual reaction, so they do not remain at a certain value for long.

[0092] Therefore, the true reaction can be detected by 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, the peak used in the calculation of the pre-peak AUC and post-peak AUC is regarded as the peak of the true reaction.

[0093] More specifically, in step 3) of the method of the present invention, the pre-peak AUC and post-peak AUC of V(i) calculated in step 2) are both the first threshold AUC. th1 When the period L, which is above or equal to a certain value, reaches a specified length, cVmax(k) is detected as the true maximum value Vmax of V(i). Vmax is the true maximum value of the reaction in V(i).

[0094] First threshold AUC th1 Different values ​​can be set for the pre-peak AUC and post-peak AUC, as long as a common threshold is set for both. th1 The AUC can be appropriately set by considering the magnitude of the coagulation reaction and the level of noise in the sample being tested. Therefore, the AUC... th1The settings can be appropriately adjusted based on the reagents and equipment used in the coagulation reaction measurement. Under standard APTT measurement conditions, the AUC... th1 Preferably, it is about 1.5 to 2.5 times the maximum value of the AUC before the peak of the initial reaction, for example, in the range of 250 to 420. If the AUC... th1 Setting the value to a smaller value allows for the detection of Vmax even in samples with low coagulation reaction rates, but increases the risk of false positives from the initial reaction. In this case, setting the aforementioned exclusion region can prevent false positives from the initial reaction. On the other hand, if the AUC is set to a smaller value... th1 Setting a larger value can prevent false detections of the initial reaction, but it makes it difficult to detect Vmax with samples showing a small coagulation reaction. In this case, it can be addressed by performing the procedure described later. Figure 11 The steps shown are used to detect Vmax of samples with small coagulation reactions.

[0095] L represents the period during which the pre-peak AUC and post-peak AUC remain at a certain value. For example, if the initial value of L is 0, and the pre-peak AUC and post-peak AUC at the measurement point or time k are the same as the previously calculated values, then it is increased to L = L + 1. On the other hand, if one or both of the pre-peak AUC and post-peak AUC are different from the previously calculated values ​​(increase or decrease), it is reset to L = 0.

[0096] Preferably, AUC in step 3) pre (k) and AUC post (k) are all AUC th1 Or above that value and AUC pre (k)=AUC pre (k-x) and AUC post (k)=AUC post When (k-x) is true, it is counted as L=L+1; otherwise, it is counted as L=0. L reaches the specified value L0. th When cVmax(k) is used, it is considered the true maximum value Vmax of V(i). Here, the latest measurement point or time of V(i) is k, and the previous measurement point or time is k-x, where x>0. x can be appropriately set according to the time frame used for comparison with the previous calculated value. For example, when k is the number of measurement points, it is counted as x=1, and the latest AUC can be... pre (k) and the AUC calculated at the previous measurement point pre The comparison can be performed using (k-1), or x can be set to 2, 3, or higher to expedite the calculation process. The same applies when k is time. L th The value can be set appropriately, but under standard APTT measurement conditions, L is preferred. th The time range is 0.5 to 2 seconds.

[0097] To eliminate false detections due to initial noise such as early reactions, Vmax detected too early in the measurement or time range where a true reaction can occur (e.g., the aforementioned detection exclusion region) can be discarded as an error. In this case, Vmax will not be detected as long as the time for the detected Vmax (i.e., cVmaxT(k)) is included in the detection exclusion region. When setting the detection exclusion region, AUC can be used... th1 The value is set relatively small, so even samples with small solidification reactions can detect the true maximum value Vmax.

[0098] The detection of the true maximum value Vmax refers to the measurement at the measurement point or time k, where the measured reaction is not an initial noise such as an initial reaction, but a true solidification reaction. Therefore, the peak of V(i) at which the detected Vmax is set is considered the true V(i) of the reaction. The time when Vmax is detected is AUC. post (k) is a point with a certain value, i.e., after V(i) decreases below the baseline, the solidification reaction reaches its end at that moment. Therefore, in the method of the present invention, the solidification reaction measurement of the test sample can be ended after obtaining the measurement data required for calculating the solidification time after Vmax is detected. For example, the solidification reaction measurement of the test sample can be ended at the moment Vmax is detected. Alternatively, for example, the solidification reaction measurement of the test sample can be ended when V(i) has sufficiently decreased after Vmax is detected. If the solidification reaction measured until the end of the measurement, such as P(i) or V(i), is used, the solidification time of the test sample can be accurately calculated.

[0099] On the other hand, in the method of the present invention, when the true maximum value Vmax is not detected at the measurement point or time k in step 3), that is, the pre-peak AUC or post-peak AUC at k does not reach the AUC. th1 If L does not reach the specified length, continue the solidification reaction measurement and repeat steps 1) to 3). Preferably, k = k + x (x as described above) and repeat steps 1) to 3). This step is repeated as long as Vmax or k does not exceed the measurement end point or time (the maximum measurement point or time for the pre-specified solidification reaction measurement of the tested sample). When Vmax or k exceeds the measurement end point or time, the repetition of steps 1) to 3) ends. The measurement end point or time can be set appropriately, preferably in the range of 4 to 8 minutes under standard APTT measurement conditions.

[0100] As one embodiment of the present invention, a flowchart illustrating the steps for detecting a true solidification reaction according to the method of the present invention is shown below. Figure 6 .

[0101] S01: Setting of settings (Settings: Hr, AUC)th1 L th wait)

[0102] S02: Set the number of measurement points to "i = 1" (initial value).

[0103] S03: Acquisition of measurement data D(i)

[0104] S04: When the operation of reaction P(i) can be performed, proceed to S05 (because the smoothing process requires a specified number of D(i)).

[0105] S05: Operation of P(i)

[0106] S06: When performing the first-order differential V(i), proceed to S07 (because the differential process requires specifying the number of P(i)).

[0107] S07: Operations of V(i)

[0108] S08: Setting cVmax(i) (Setting i that reaches cVmax(i) to cVmaxT(i))

[0109] S09: Calculation of the baseline Bh(i) for cVmax(i) (=cVmax(i)×Hr%)

[0110] S10: AUC pre (i) and AUC post (i) calculation

[0111] S11: Determine AUC pre (i) and AUC post (i) Whether these two are suitable as threshold AUC th1

[0112] S12: Determine AUC pre (i) and AUC post (i) Both of these are suitable as threshold AUC th1 And whether it maintains a certain value (whether L≥L) th )

[0113] S13: This step is not required: Determine whether cVmaxT(i) is in the detection exclusion region.

[0114] S14: When cVmaxT(i) is not included in the detection exclusion region, cVmax(i) is determined as the true maximum value of V(i), Vmax (the true maximum value of the reaction).

[0115] S21: Counting of measurement points

[0116] S22: Determine if the number of measurement points exceeds the set measurement time; if not, return to S03.

[0117] S23: If the number of measurement points exceeds the measurement time, end the measurement to determine whether Vmax has been detected.

[0118] S24: Based on the obtained calculated values, evaluate the measurement data offline.

[0119] When the coagulation ability of the tested 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, because there is no true peak of V(i) for the actual reaction, or V(i) does not decrease sufficiently after the peak, the pre-peak AUC does not reach a certain value and the post-peak AUC is 0, or even if the pre-peak AUC reaches a certain value, the post-peak AUC does not reach a certain value. As a result, in the method of the present invention, Vmax may not be detected until the measurement end point or time. Therefore, in one embodiment of the method of the present invention, if the true maximum value Vmax is not detected and k exceeds the measurement end point or time, if the AUC at the measurement end point... pre (k) is the AUC th1 If the value is higher than or equal to this value, then cVmax(k) is detected as the true maximum value Vmax of V(i). This step allows for the detection of the true reaction of the test sample where the reaction has not ended at the measurement end point or within the time frame. If necessary, additional information related to the coagulation ability of the test sample can be provided by outputting information indicating that Vmax was detected in this step, along with Vmax.

[0120] When the coagulation reaction of the tested sample is relatively small, the pre-peak AUC or post-peak AUC may not reach the required AUC. th1 Therefore, sometimes Vmax cannot be detected until the end of the measurement or the time. Thus, in one embodiment of the method of the present invention, when the true maximum value Vmax is not detected and k exceeds the end of the measurement or the time, the maximum value of the pre-peak AUC and post-peak AUC is the second threshold AUC. th2 If the measured point or time of the maximum AUC before the peak is the same as cVmaxT(k), and the measured point or time of the maximum AUC after the peak is later than cVmaxT(k), then cVmax(k) is detected as the true maximum value Vmax of V(i) (e.g., using the method described later). Figure 11 (Steps shown). AUC th2 Preferred value is less than AUC th1 And preferably in AUC th1The range is 10% to 30%. This procedure allows for the detection of the true response in samples with relatively low clotting function. If necessary, additional information related to the clotting ability of the sample can be provided by outputting information along with Vmax indicating that this step detected Vmax.

[0121] Sometimes the coagulation reaction of the tested sample is absent or very weak, or for other reasons the true reaction of the tested sample is unclear. In such cases, it is difficult to distinguish the true reaction from noise. Samples with unclear true reactions can typically be identified by the fact that the pre-peak AUC has not yet reached the true AUC. th1 (and thus AUC) th2 Alternatively, it can be determined by ensuring that V(i) does not exceed the aforementioned threshold V. th To distinguish. For example, by only achieving V in V(i). th In the case of starting step 2), false detections due to noise can be prevented. In samples where the true response is unclear, the true response may be removed along with the noise, making Vmax undetectable. In this case, outputting Vmax as an undetected result indicates that this is because the threshold AUC cannot be detected. th1 The peak AUC, or the inability to detect V. th The information in V(i) above can provide additional information related to the coagulation ability of the tested sample.

[0122] As an example Figure 7 A through C in the figure represent the response P and first derivative V of three tested samples with different coagulation abilities. Figure 7 In the diagram, A represents a sample where the actual response is unclear. Figure 7 B in the figure indicates the measurement end point or a sample where the actual response did not end within the time frame. Figure 7 The C value in the figure represents samples where no real reaction was detected. These samples can be analyzed based on AUC, as described below. pre (k) and AUC post (k) is used for classification.

[0123] Figure 7 A: AUC pre (k)(and AUC) post (k) did not reach AUC th1 and AUC th2 →No response

[0124] Figure 7 B: AUC pre (k)(or AUC) post (k) reaches AUC th1 However, if a certain value is not reached → reaction occurs midway.

[0125] Figure 7C: AUC pre (k) and AUC post (k) reaches AUC th1 And it remains at a certain value → a reaction was detected.

[0126] [2. Calculation of clotting time and other analyses]

[0127] In one embodiment, in the method of the present invention, after detecting the true reaction of the tested sample, the detected true reaction can be further used to calculate the coagulation time of the tested sample. The true coagulation reaction obtained in the method of the present invention, such as P(i) or V(i), can be used for coagulation time calculation or various other blood analyses.

[0128] In one example, in the method of the present invention, V(i) obtained from the data measured by the solidification reaction can be used to detect the true maximum value Vmax, and then V(i) can be used to calculate the solidification time. In another example, in the method of the present invention, P(i) and V(i) can be obtained from the data measured by the solidification reaction, V(i) can be used to detect the true maximum value Vmax, and then P(i) and / or V(i) can be used to calculate the solidification time.

[0129] In one embodiment, the method of the present invention may terminate the coagulation reaction measurement of the tested sample at the moment Vmax is detected, and the coagulation time may be calculated using the P(i) and / or V(i) of the true reaction obtained so far. In another embodiment, the coagulation time may be calculated using the P(i) and / or V(i) after the rise of the peak of the detected Vmax, for example, the P(i) and / or V(i) at i≥k1.

[0130] In another embodiment of the method of the present invention, the coagulation reaction measurement of the tested sample can continue even after Vmax is detected, so as to obtain data related to the coagulation reaction required for the calculation of coagulation time or other analyses.

[0131] The method for calculating the solidification time from the solidification reaction obtained by the method of the present invention is not particularly limited. Examples of methods for calculating solidification time include: calculating the time when P(i) reaches N% of its maximum value Pmax as the solidification time (so-called percentage detection method); calculating the time when V(i) reaches its maximum value Vmax or N% of its maximum value as the solidification time (so-called differential method); calculating the solidification time based on the time-varying cumulative value of P(i) over a small time period (see Patent Document 3 and Japanese Patent Application 2019-237427); calculating the solidification time based on the weighted average time of V(i) (see Japanese Patent Application 2020-039344); and calculating the time when P(i) reaches N% of P(Te) as the solidification time, taking the time when V(i) reaches its maximum value Vmax and then reaches a predetermined value as the calculation starting point Te (see Japanese Patent Application 2020-068877), etc.

[0132] [3. Application to other solidification reaction measurement methods]

[0133] The above description uses the case of coagulation reaction measurement based on the amount of scattered light as an example to illustrate the detection method of the coagulation reaction of the present invention. However, anyone skilled in the art can apply the method of the present invention to the detection method of the coagulation reaction using other coagulation reaction measurement methods (e.g., coagulation reaction measurement methods based on transmittance, absorbance, viscosity, etc.), and therefore such application is included within the scope of the present invention. For example, the reaction P(i) obtained from an inverse S-shaped coagulation reaction curve such as based on the amount of transmitted light is opposite in sign to the reaction P(i) based on the amount of scattered light described above. In such a case, it is obvious to those skilled in the art that in steps 1) to 3) above, the signs of P(i) and V(i) are reversed, the minimum value of V(i) below k is calculated cVmin(k) instead of cVmax(k), and the area under the curve (AOC) is calculated instead of the area under the curve (AUC).

[0134] [4. Procedures and apparatus]

[0135] The coagulation reaction detection method of the present invention described above can be performed automatically using a computer program. Therefore, one aspect of the present invention is a program for performing the coagulation reaction detection method of the present invention described above. Furthermore, the series of steps in the method of the present invention described above can be performed automatically using an automated analysis device. Therefore, one aspect of the present invention is an apparatus for performing the coagulation reaction detection method of the present invention described above.

[0136] Hereinafter, one embodiment of the device of the present invention will be described. One embodiment of the device of the present invention is as follows: Figure 8 The automatic analysis device 1 shown is equipped with a control unit 10, an operation unit 20, a measurement unit 30, and an output unit 40.

[0137] The control unit 10 controls the overall operation of the automatic analysis device 1. The control unit 10 can be, for example, a personal computer (PC). The control unit 10 includes a CPU, memory, RAM, and a communication interface (I / F), and performs tasks such as processing instructions from the operation unit 20, controlling the operation of the measurement unit 30, storing measurement data received by the measurement unit 30, performing data analysis, storing analysis results, and controlling the output of measurement data and analysis results based on the output unit 40. Furthermore, the control unit 10 can be connected to external media, a host computer, or other devices. It should be noted that in the control unit 10, the PC controlling the operation of the measurement unit 30 and the PC performing measurement data analysis can be the same or different.

[0138] The operation unit 20 receives input from the operator and transmits the input information to the control unit 10. For example, the operation unit 20 may have a user interface (UI) such as a keyboard or touch panel. Under the control of the control unit 10, the output unit 40 outputs the measurement data from the measurement unit 30, its analysis results, such as P(i), V(i), the detection results of the actual coagulation reaction (Vmax, etc.), and the coagulation time of the blood sample. For example, the output unit 40 may have a display device such as a monitor.

[0139] The measuring unit 30 performs a series of operations for coagulation testing to obtain measurement data on the coagulation reaction of a sample containing a blood sample. The measuring unit 30 is equipped with various instruments and analytical modules required for coagulation testing, such as a sample container for holding the blood sample, a reagent container for holding the test reagents, a reaction container for reacting the sample with the reagents, a probe for dispensing the blood sample and reagents into the reaction container, a light source, a detector for detecting scattered or transmitted light from the sample in the reaction container, a data processing circuit for transmitting data from the detector to the control unit 10, and a control circuit for receiving instructions from the control unit 10 to control the operation of the measuring unit 30.

[0140] The control unit 10 analyzes the coagulation reaction of the sample based on the data measured by the measuring unit 30. This analysis may include obtaining P(i) and V(i) as described above, detecting the actual coagulation reaction (Vmax, etc.), and calculating the coagulation time using the detected actual coagulation reaction. Alternatively, the coagulation reaction curve P(i) or the first derivative V may be generated by the control unit 10 based on measurement data from the measuring unit 30, or generated by other devices such as the measuring unit 30 and transmitted to the control unit 10. The control unit 10 may store parameters used in the detection of the actual coagulation reaction, such as AUC. th1 L Th The set value, etc., or the control unit 10 can also obtain the set value stored on an external device or network during analysis.

[0141] The above analysis can be implemented using a program for performing the method of the present invention. Therefore, the control unit 10 can be equipped with a program for performing the coagulation reaction detection method of the present invention.

[0142] The analysis results in control unit 10 are transmitted to output unit 40 and output. The output can take any form, such as displaying on a screen, sending to a host computer, or printing. The output information from the output unit can include P(i), V(i), the actual detection results of the solidification reaction, solidification time, etc. The type of output information from the output unit can be controlled by the program of this invention.

[0143] In one embodiment of the apparatus of the present invention, the measuring unit 30 continues to measure the sample under test until the coagulation reaction ends, and the data is sequentially transmitted 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), and AUC. pre (k), AUC post The calculation of (k) is then used to determine whether L≥L. Th The Vmax is then detected. Upon detection of Vmax, the control unit 10 further calculates the solidification time of the sample. The obtained analysis results are transmitted 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), and AUC are also output sequentially as needed. pre (k), AUC post (k). After Vmax is detected, output Vmax, its time, and solidification time in a timely manner.

[0144] Example

[0145] The present invention will be described in more detail below with reference to specific embodiments, but the present invention is not limited to these embodiments.

[0146] Example 1: Parameters reflecting the solidification reaction

[0147] 1) Samples being tested

[0148] The following 187 samples were tested: normal plasma (10 cases), heparinized plasma (47 cases), LA-positive plasma (11 cases), coagulation factor deficient plasma (14 cases), inhibitory plasma (41 cases), low-concentration fibrinogen plasma (5 cases), and other plasmas with prolonged clotting time (59 cases). Initial reactions were observed in 6 cases.

[0149] 2) Measurement of solidification reaction

[0150] As reagents for the assay, Coagpia APTT-N (manufactured by Sekisui Medical Co., Ltd.) was used as the reagent for APTT assay, and Coagpia APTT-N calcium chloride solution (manufactured by Sekisui Medical Co., Ltd.) was used as the calcium chloride solution. The coagulation reaction measurement of the sample containing the test material was performed using a CP3000 automated coagulation analyzer (manufactured by Sekisui Medical Co., Ltd.). 50 μL of the sample was heated in a test tube at 37°C for 45 seconds, followed by the addition of 50 μL of the assay reagent at approximately 37°C. 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. During the coagulation reaction measurement, the test tube was illuminated with light of 660 nm wavelength using an LED as the light source, and the amount of scattered light from a 90-degree side scattering was measured at 0.1-second intervals. The maximum measurement time was 400 seconds (measurement points i = 1–4000).

[0151] 3) Obtaining reaction P(i) and reaction rate V(i)

[0152] After smoothing the photometric data from each sample to remove noise, the reaction P(i) is generated by zeroing the scattered light at the start of the photometric measurement. The first derivative V(i) is then calculated from P(i).

[0153] 4) APTT calculation

[0154] The settling time (APTT) of the tested sample was calculated using the percentage method. The settling time was defined as the time during which P(i) reached its maximum value (Pmax).

[0155] Example 1: Parameters reflecting the solidification reaction

[0156] 1) Parameter calculation

[0157] Using the obtained P(i) and V(i) (i = 21 to 3980), the following parameters are calculated by setting the height ratio (Hr) to 20%.

[0158] Vmax: The maximum value of V(i)

[0159] VmaxT: The time of Vmax

[0160] Pmax: The maximum value of P(i)

[0161] AUC pre : Pre-peak AUC of V(i)

[0162] AUC post Post-peak AUC of V(i)

[0163] Examples of the calculated parameters are shown in Table 2. The nine samples shown in Table 2 are described below.

[0164] Sample 1: The sample with the smallest Pmax

[0165] Sample 2: The sample with the smallest VmaxT

[0166] Sample 3: Samples where V(i) is bimodal

[0167] Samples 4-9: Samples showing initial reactions

[0168] [Table 2]

[0169] Sample 1 139.0 569 1.7 207 285 Sample 2 21.0 4,133 35.3 799 2,669 Sample 3 60.9 7,046 6.5 377 6,076 Sample 4 165.3 13,435 10.2 4,044 8,163 Sample 5 70.3 12,885 25.7 1,265 8,322 Sample 6 90.3 11,071 27.4 3,748 5,232 Sample 7 259.9 12,210 11.9 4,518 6,097 Sample 8 274.6 12,280 12.0 5,144 5,644 Sample 9 166.2 9837 19.8 3,700 4,717

[0170] The relationship between the parameters will be shown in Figure 9 The APTT values ​​of the 187 tested 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 9 A in the middle. Figure 9 In A, the two samples that deviate from the linear relationship are the coagulation factor VIII (FVIII) lacking samples with a larger first peak and bimodal V(i). There is no clear trend between the maximum value Pmax of P(i) and VmaxT. Figure 9 (B in the original text). The relationship between Vmax and VmaxT shows an inverse proportional trend. Figure 9 (C in the text). This trend indicates that the peak of V(i) increases when the solidification reaction proceeds rapidly and decreases when the solidification reaction proceeds slowly. There is no clear relationship between the pre-peak AUC and post-peak AUC and VmaxT. Figure 9 (D and E in the text). The post-peak AUC tends to be larger than the pre-peak AUC. Figure 9 (F in the middle).

[0171] 2) Parameters of the tested sample exhibiting an initial reaction

[0172] For the tested samples exhibiting an initial reaction (samples 4-9 in Table 2), after determining the initial reaction region and the true reaction region based on the obtained solidification reaction curves, the parameters Vmax, VmaxT, Pmax, and pre-peak AUC (AUC) for each region were calculated. pre ) and post-peak AUC (AUC post The results are shown in Table 3. The true reaction time VmaxT was confirmed to be in the range of approximately 70 seconds to 275 seconds, while the initial reaction time VmaxT was approximately 4 seconds and appeared at the beginning of the measurement.

[0173] [Table 3]

[0174]

[0175] Example 2: Real-time detection of the actual solidification reaction - 1

[0176] Using the coagulation reaction data of 187 samples measured in Example 1, according to Figure 6 The flowchart illustrates the steps for implementing real-time detection of Vmax.

[0177] 1) Threshold AUC th1 decision

[0178] The results of Example 1 show that the AUC in the tested sample pre and AUC post The minimum values ​​are 207 and 285 in sample 1, where Pmax is the smallest. On the other hand, the AUC of the initial reaction portions of samples 4–9, which have initial reactions, is... pre The maximum value is 168 in sample 5. AUC th1 If the AUC is too low, it may falsely detect sample 5 and other initial reaction parts. On the other hand, the AUC... th1 When the value is too large, smaller reactions in samples 1, 3, etc., cannot be detected. Based on the above results, the AUC... th1 The value was determined to be 255, which is approximately 1.5 times the maximum value of the initial reaction section (168 in sample 5).

[0179] 2) Testing conditions

[0180] Implement real-time detection of Vmax under the following conditions.

[0181] This continues until i = 21 to Vmax (maximum 3980) is detected.

[0182] Hr (%): 20%

[0183] AUC th1 255

[0184] L th 10 (1 second)

[0185] Exclusion area detected (step S13): None

[0186] 3) Results

[0187] Vmax was detectable by the true reaction in 186 samples, excluding sample 1.

[0188] Example 3: Real-time detection of the actual solidification reaction - 2 (with low threshold and detection exclusion region)

[0189] Based on the results of Example 1, it is known that the peak of the initial reaction appears at the beginning of the measurement. Therefore, real-time detection of Vmax is performed under the condition that a smaller reaction than that of Sample 1 in Example 1 can be detected, but the detection of the reaction in the range where the initial reaction is assumed to occur (low threshold, present in step S13) is not performed. Specifically, the following changes are made, except that real-time detection of Vmax is performed under the same conditions as in Example 2.

[0190] AUC th1 100

[0191] Exclusion zone (step S13): Yes. Vmax is detected only when cVmaxT(i) is below 50 (5 seconds).

[0192] Vmax was detectable by the true reaction in all 187 samples. Figure 10 This is a curve showing the time (Fix) at which Vmax was detected in this embodiment relative to VmaxT measured in Example 1. Since all Fixes are later than VmaxT, it is confirmed that the steps according to this embodiment can accurately detect the true reaction. Furthermore, the results of this embodiment show that by setting a detection exclusion region (step S13), the true reaction can also be accurately detected in samples with small reactions.

[0193] Example 4: Real-time detection of the actual solidification reaction – 3 (with high threshold, offline detection)

[0194] Assuming a larger initial reaction than samples 4-9 of Example 1 occurs, a higher AUC than that of Examples 2 and 1) is set. th1 Prediction is achieved by making AUC th1 Increased so that a portion of the samples (e.g., sample 1) follow Figure 6 The steps described above cannot detect the actual reaction. Therefore, in Figure 6 The period from S24 to the end is introduced Figure 11 The steps are as follows. Specifically, the following changes are made, except that real-time detection of Vmax is performed under the same conditions as in Example 2.

[0195] AUC th1 510

[0196] AUC th2 100

[0197] Follow the steps outlined in S24:

[0198] S31: Determine AUC pre (i) and AUC post The maximum value of (i) is AUC pre Max and AUC postMax values ​​all exceeded the threshold AUC. th2

[0199] S32: When S31 is true, the AUC is determined to be reached. pre Max's time (AUC) pre MaxT) takes the same amount of time as cVmaxT(i) and achieves AUC. post Max's time (AUC) post MaxT) is below cVmaxT(i)

[0200] S33: When S32 is true, detect cVmax(i) as Vmax.

[0201] Twelve samples, including sample 1 which has the smallest Pmax, were detected as Vmax after passing through S24. All other samples were detected as Vmax without passing through S24. Figure 12 The diagram shows the time variation of the parameters calculated when performing the steps of this embodiment on sample 1. According to... Figure 12 From B to D, we know that cVmaxT(i) becomes maximum at the time VmaxT when it reaches its true maximum value, and AUC pre (i) It becomes maximum at VmaxT, AUC post (i) Vmax becomes the maximum after VmaxT. It was confirmed that Vmax accurately detected the maximum value of the true response in the 12 samples including sample 1 detected by S24.

Claims

1. A method for detecting coagulation reaction, the method comprising: 1) Measure the coagulation reaction of the tested blood sample and obtain the first derivative V(i) of the coagulation reaction P(i) up to the latest measurement point. Here, i represents the measurement point or time, i = k0 ~ 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) Set the maximum value cVmax(k) of V(i) up to i=k as the peak of V(i), and calculate the area under the curve before and after the peak of V(i), i.e., AUC. 3) Both the pre-peak AUC and post-peak AUC are the first threshold AUC. th1 When the period L, which is above or equal to a certain value, reaches a specified length, cVmax(k) is detected as the true maximum value Vmax of V(i). 4) Under the condition of detecting the true maximum value Vmax, calculate the clotting time of the tested blood sample based on k corresponding to Vmax.

2. The method according to claim 1, wherein, The second step includes calculating cVmax(k), cVmaxT(k), and AUC. pre (k) and AUC post (k), here, cVmax(k) represents the maximum value of V(i), where i = k0 ~ k. cVmaxT(k) represents the measurement point or time at which V(i) = cVmax(k) is reached. AUC pre (k) represents the pre-peak AUC at i=k and is the AUC of V(i) from i=k1 to i=cVmaxT(k). AUC post (k) represents the post-peak AUC at i=k, and is the AUC of V(i) from i=cVmaxT(k) to i=k2. k1 is the latest measurement point or time among the measurement points or times that reach V(i) ≤ cVmax(k) × Hr% before cVmaxT(k). k2 is the earliest measurement point or time among the measurement points or times that satisfy V(i) ≤ cVmax(k) × Hr% after cVmaxT(k). 0＜Hr＜100； Furthermore, the method includes the following steps: if Vmax is not detected in step 3), calculate k = k + x, x > 0, and repeat steps 1) to 3).

3. The method according to claim 2, wherein, The third part includes: At AUC pre (k) and AUC post (k) are all AUC th1 Or above that value and equal to AUC pre (k) = AUC pre (k-x) and AUC post (k) = AUC post (k-x) where x > 0 is counted as L = L+1, and L = 0 is counted as L otherwise. When L reaches the specified value, cVmax(k) is detected as Vmax of V(i).

4. The method according to claim 2 or 3, wherein, 10≤Hr≤70。 5. The method according to any one of claims 1 to 3, wherein, In the case described in 3), Vmax is not detected if the measurement point or time that reaches cVmax(k) is contained in the detection exclusion area.

6. The method according to claim 4, wherein, In the case described in 3), Vmax is not detected if the measurement point or time that reaches cVmax(k) is contained in the detection exclusion area.

7. The method according to any one of claims 1 to 3, wherein, Further includes: if Vmax is not detected and k exceeds the measurement end point or time, and the pre-peak AUC at i=k is the first threshold AUC. th1 If the value is above this, then cVmax(k) is detected as the Vmax of V(i).

8. The method according to claim 4, wherein, Further includes: if Vmax is not detected and k exceeds the measurement end point or time, and the pre-peak AUC at i=k is the first threshold AUC. th1 If the value is above this, then cVmax(k) is detected as the Vmax of V(i).

9. The method according to claim 5, wherein, Further includes: if Vmax is not detected and k exceeds the measurement end point or time, and the pre-peak AUC at i=k is the first threshold AUC. th1 If the value is above this, then cVmax(k) is detected as the Vmax of V(i).

10. The method according to claim 6, wherein, Further includes: if Vmax is not detected and k exceeds the measurement end point or time, and the pre-peak AUC at i=k is the first threshold AUC. th1 If the value is above this, then cVmax(k) is detected as the Vmax of V(i).

11. The method according to any one of claims 1 to 3, wherein, Furthermore, in the case where Vmax is not detected and k exceeds the measurement end point or time, both the pre-peak AUC and post-peak AUC at i=k are the second threshold AUC. th2 If the measurement point or time of the maximum AUC before the peak is the same as the measurement point or time of reaching cVmax(k), and the measurement point or time of the maximum AUC after the peak is later than the measurement point or time of reaching cVmax(k), then cVmax(k) is detected as Vmax of V(i).

12. The method according to claim 4, wherein, Furthermore, in the case where Vmax is not detected and k exceeds the measurement end point or time, both the pre-peak AUC and post-peak AUC at i=k are the second threshold AUC. th2 If the value is greater than or equal to the value above, and the measurement point or time of the maximum AUC before the peak is the same as the measurement point or time of reaching cVmax(k), and the measurement point or time of the maximum AUC after the peak is later than the measurement point or time of reaching cVmax(k), then cVmax(k) is detected as Vmax of V(i).

13. The method according to claim 5, wherein, Furthermore, in the case where Vmax is not detected and k exceeds the measurement end point or time, both the pre-peak AUC and post-peak AUC at i=k are the second threshold AUC. th2 If the value is greater than or equal to the value above, and the measurement point or time of the maximum AUC before the peak is the same as the measurement point or time of reaching cVmax(k), and the measurement point or time of the maximum AUC after the peak is later than the measurement point or time of reaching cVmax(k), then cVmax(k) is detected as Vmax of V(i).

14. The method according to claim 6, wherein, Furthermore, in the case where Vmax is not detected and k exceeds the measurement end point or time, both the pre-peak AUC and post-peak AUC at i=k are the second threshold AUC. th2 If the value is greater than or equal to the value above, and the measurement point or time of the maximum AUC before the peak is the same as the measurement point or time of reaching cVmax(k), and the measurement point or time of the maximum AUC after the peak is later than the measurement point or time of reaching cVmax(k), then cVmax(k) is detected as Vmax of V(i).

15. The method according to claim 7, wherein, Furthermore, in the case where Vmax is not detected and k exceeds the measurement end point or time, both the pre-peak AUC and post-peak AUC at i=k are the second threshold AUC. th2 If the value is greater than or equal to the value above, and the measurement point or time of the maximum AUC before the peak is the same as the measurement point or time of reaching cVmax(k), and the measurement point or time of the maximum AUC after the peak is later than the measurement point or time of reaching cVmax(k), then cVmax(k) is detected as Vmax of V(i).

16. The method according to claim 8, wherein, Furthermore, in the case where Vmax is not detected and k exceeds the measurement end point or time, both the pre-peak AUC and post-peak AUC at i=k are the second threshold AUC. th2 If the value is greater than or equal to the value above, and the measurement point or time of the maximum AUC before the peak is the same as the measurement point or time of reaching cVmax(k), and the measurement point or time of the maximum AUC after the peak is later than the measurement point or time of reaching cVmax(k), then cVmax(k) is detected as Vmax of V(i).

17. The method according to claim 9, wherein, Furthermore, in the case where Vmax is not detected and k exceeds the measurement end point or time, both the pre-peak AUC and post-peak AUC at i=k are the second threshold AUC. th2 If the measurement point or time of the maximum AUC before the peak is the same as the measurement point or time of reaching cVmax(k), and the measurement point or time of the maximum AUC after the peak is later than the measurement point or time of reaching cVmax(k), then cVmax(k) is detected as Vmax of V(i).

18. The method according to claim 10, wherein, Furthermore, in the case where Vmax is not detected and k exceeds the measurement end point or time, both the pre-peak AUC and post-peak AUC at i=k are the second threshold AUC. th2 If the measurement point or time of the maximum AUC before the peak is the same as the measurement point or time of reaching cVmax(k), and the measurement point or time of the maximum AUC after the peak is later than the measurement point or time of reaching cVmax(k), then cVmax(k) is detected as Vmax of V(i).

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