Blood coagulation time measurement method

Abstract

Provided is a method for precisely measuring the coagulation time of a blood sample exhibiting various coagulation reactions. This method for measuring blood coagulation time comprises: acquiring the coagulation reaction P(i) and the reaction velocity V(i) of a test sample; calculating a start point of calculation Te using the ratio of V(i) to the maximum reaction velocity as an index; and calculating the time point Tc at which P(Tc) is equal to P(Te) x N% (0<N<100) and then determining the Tc as the blood coagulation time.

Description

Technical Field

[0001] The present invention relates to a method for measuring blood coagulation time.

Background Art

[0002] A blood coagulation test is a test for diagnosing a patient's blood coagulation ability by adding a predetermined reagent to a patient's blood sample and measuring the blood coagulation time and the like. Typical examples of blood coagulation time include prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time, and the like. The hemostatic ability and fibrinolytic ability of a patient can be examined by a blood coagulation test. An abnormality in blood coagulation ability mainly causes an extension of the coagulation time. For example, the extension of the coagulation time is caused by the influence of a coagulation inhibitor, a decrease in coagulation-related components, a deficiency of an innate blood coagulation factor, an autoantibody that inhibits an acquired coagulation reaction, and the like.

[0003] In recent years, an automatic analyzer that automatically measures a blood coagulation test has been widely used, and it has become possible to easily perform a blood coagulation test. For example, in a certain type of automatic analyzer, light is applied to a mixed solution obtained by adding a reagent to a blood sample, and the coagulation reaction of the blood sample is measured based on the change in the amount of light obtained. For example, when measuring the amount of scattered light, when a certain amount of time has elapsed since the addition of the reagent to the blood sample, the amount of scattered light rapidly increases due to the progress of coagulation, and then, as the coagulation reaction approaches completion, the amount of scattered light saturates and reaches a plateau. The blood coagulation time can be measured based on such a temporal change in the amount of scattered light.

[0004] As methods for calculating the coagulation time by an automatic analyzer, several methods such as the percentage method and the differential method are used (see Patent Document 1). In the case of calculating the coagulation time based on the scattered light amount, in the percentage method, typically, the time until the measured scattered light amount reaches a certain ratio of its maximum value is calculated as the coagulation time. The percentage method enables fairly accurate calculation of the coagulation time not only for normal specimens but also for abnormal specimens such as low fibrinogen specimens, milky specimens, and hemolyzed specimens. On the other hand, in automatic analysis based on the percentage method, it is necessary to set a long measurement time for the specimen so that the maximum scattered light amount can be detected even for abnormal specimens with low coagulation ability such as low fibrinogen specimens, so the analysis takes time.

[0005] In the differential method, typically, the time until the differential value of the scattered light amount reaches a peak or a certain ratio thereof is calculated as the coagulation time. However, in abnormal specimens with low coagulation ability such as low fibrinogen specimens, a clear peak may not be seen in the differential value of the scattered light amount. Also, in abnormal specimens, two or more peaks of the differential value may occur. A method of calculating the coagulation time based on a unimodal peak curve created by fitting the differential value curve may be used, but the fitting may lose accurate information regarding the coagulation ability of the specimen.

[0006] Furthermore, the photometric data in the analyzer includes various noises caused by factors such as the state of the apparatus, reagents, and specimens, and they can lead to misdetection of the coagulation time. In the automatic analysis of blood specimens, it is required to remove the adverse effects of the noises and calculate a reliable coagulation time.

Prior Art Documents

Patent Documents

[0007]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0008] The present invention relates to a blood coagulation time measurement method capable of accurately measuring the coagulation time of a blood specimen showing various blood coagulation reaction curves.

Means for Solving the Problems

[0009] That is, the present invention provides the following. 〔1〕A blood coagulation time measurement method, comprising: acquiring a coagulation reaction P(i) for a test specimen and a reaction rate V(i) which is a differential value of the reaction P(i), where i represents a measurement point; calculating a calculation start point Te using the ratio of V(i) to the maximum reaction rate as an index; and calculating a time Tc such that P(Tc) = P(Te) × N% (0 < N < 100), and determining the Tc as the blood coagulation time. A method including the above. 〔2〕The method according to 〔1〕, wherein the maximum reaction rate is the maximum value of V(i) obtained so far. 〔3〕The method according to 〔1〕 or 〔2〕, wherein the maximum reaction rate is the maximum value of V(i) obtained so far between after exceeding a threshold value Vs and the latest measurement point. 〔4〕The method according to any one of 〔1〕 to 〔3〕, wherein after V(i) reaches the maximum reaction rate, the calculation start point Te is calculated as the time when V(i) becomes S% (S is a predetermined value in the range of 5 to 95) of the maximum reaction rate. 〔5〕When after V(i) reaches the maximum reaction rate and the maximum value continuous width K(i) exceeds a threshold value Ks, the time when V(i) becomes S% (S is a predetermined value in the range of 5 to 95) of the maximum reaction rate is calculated as the calculation start point Te. The maximum value continuous width K(i) is the time or the number of measurement points during which V(i) continues to be a constant value after reaching the maximum reaction rate at i. The method according to 〔4〕. 〔6〕When the peak width W(i) exceeds a threshold value Ws, the time when V(i) becomes S% (S is a predetermined value in the range of 5 to 95) of the maximum reaction rate is calculated as the calculation start point Te. The peak width W(i) is the time width or the number of measurement points between the latest i and the point before V(i) reaches the maximum reaction rate and where V(i) is X% of the maximum reaction rate, where X is 1 or more and less than 100, The method according to [4] or [5]. 〔7〕A method for measuring the concentration of a coagulation factor, comprising measuring the concentration of the coagulation factor in a test subject based on the blood coagulation time of the test subject measured by the method according to any one of 〔1〕to 〔6〕 above. 〔8〕The method according to 〔7〕, wherein the coagulation factor is fibrinogen.

Advantages of the Invention

[0010] According to the method of the present invention, it is possible to accurately measure the coagulation time of a blood specimen showing various blood coagulation reaction curves including normal specimens and abnormal specimens. Further, when analyzing a large number of specimens in real time with an automatic analyzer, the method of the present invention can shorten the analysis time per specimen compared to the conventional percentage method, and can improve the analysis efficiency.

Brief Description of the Drawings

[0011]

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Mode for Carrying Out the Invention

[0012] In a blood coagulation test, a predetermined reagent is added to a blood specimen, and then the subsequent blood coagulation reaction is measured to measure the blood coagulation time from the coagulation reaction. In the following description of this specification, the blood specimen may sometimes be referred to as a specimen. For measuring the blood coagulation reaction, general means are used, for example, optical means for measuring the amount of scattered light, transmittance, absorbance, etc., or mechanical means for measuring the viscosity of plasma. The coagulation reaction curve of a normal specimen depends on the measuring means, but basically shows a sigmoid shape. For example, the coagulation reaction curve based on the amount of scattered light of a normal specimen usually rises sharply due to the progress of coagulation at a certain time after the addition of the reagent, and then reaches a plateau as the coagulation reaction approaches completion. On the other hand, the coagulation reaction curve of an abnormal specimen having a coagulation abnormality shows various shapes depending on the cause of the abnormality, such as a delay in the rise time of the curve and a gentle rise. The variety of the coagulation reaction curves of abnormal specimens makes it difficult to accurately measure the coagulation time with an automatic analyzer.

[0013] In conventional general blood coagulation time measurement, data is acquired at least until the end of the coagulation reaction, and the coagulation time is calculated based on the acquired data. For example, in the case of calculating the coagulation time based on the amount of scattered light, after determining that the time point when the amount of scattered light is saturated is the end of the coagulation reaction, the time point when the coagulation reaction curve reaches the maximum speed or 1 / N thereof between the reagent addition time point and the end time point of the coagulation reaction is determined as the coagulation time (differential method), the time point when the amount of scattered light at the end time point of the coagulation reaction reaches 1 / N is determined as the coagulation time (percent method, see Patent Document 1), and so on. However, due to the abnormal shape and noise of the coagulation reaction curve of an abnormal specimen as described above, false detection of the peak of the coagulation reaction rate and the end of the coagulation reaction occurs, and for example, it may be detected at a time point when the peak of the reaction rate and the end of the reaction are too early. Such false detection leads to an inaccurate calculation of the coagulation time.

[0014] In an automatic analyzer, in order to efficiently analyze a large number of specimens, it is desirable to promptly terminate the measurement once the necessary data for one specimen has been acquired and then start the measurement of the next specimen. However, such a method has a risk that the false detection of the end of the coagulation reaction at too early a point as described above may lead to too early termination of the measurement, resulting in the omission of necessary data. On the other hand, if the coagulation reaction measurement time per specimen is fixed at a sufficiently long time, the omission of data due to false detection of the end of the coagulation reaction can be prevented. However, such a method increases the measurement time more than necessary for many specimens, thus reducing the overall analysis efficiency.

[0015] The present invention prevents false detection of the coagulation time caused by an abnormal shape of the coagulation reaction curve as described above and enables accurate measurement of the coagulation time. Further, according to the present invention, since the minimum necessary coagulation reaction measurement time for measuring the coagulation time can be applied to various blood specimens including normal specimens and abnormal specimens, the analysis time per specimen can be shortened.

[0016] [Method for Measuring Blood Coagulation Time] 1. Measurement of Coagulation Reaction The present invention relates to a method for measuring the blood coagulation time of a blood specimen. In the method for measuring the blood coagulation time of the present invention (hereinafter also referred to as the method of the present invention), the coagulation reaction of a test blood specimen mixed with a reagent is measured. Based on the time-series data of the coagulation reaction obtained by this measurement, the blood coagulation time is measured. Examples of the blood coagulation time measured by the method of the present invention include prothrombin time (PT), activated partial thromboplastin time (APTT), and coagulation time in fibrinogen (Fbg) concentration measurement. In the following specification, the method of the present invention will be mainly described by taking the activated partial thromboplastin time (APTT) as an example of the coagulation time. Modification of the method of the present invention to other coagulation times (for example, prothrombin time (PT)) is feasible for those skilled in the art.

[0017] In the method of the present invention, as the test blood sample, the plasma of the subject is preferably used. An anticoagulant usually used in coagulation tests may be added to the sample. For example, after blood is collected using a blood collection tube containing sodium citrate and then centrifuged, plasma can be obtained.

[0018] A coagulation time measurement reagent is added to the test sample to initiate a blood coagulation reaction. The coagulation reaction of the mixture after reagent addition can be measured. The coagulation time measurement reagent to be used can be arbitrarily selected according to the measurement purpose. Reagents for various coagulation time measurements are commercially available (for example, APTT reagent Coagu-PIA APTT-N; manufactured by Sekisui Medical Co., Ltd.). For measuring the coagulation reaction, general means such as optical means for measuring the amount of scattered light, transmittance, absorbance, etc., or mechanical means for measuring the viscosity of plasma can be used. The reaction start point of the coagulation reaction can typically be defined as the time when the reagent is mixed with the sample to initiate the coagulation reaction, but other timings may be defined as the reaction start point. The time for continuing the measurement of the coagulation reaction can be, for example, several tens of seconds to about 7 minutes from the time of mixing the sample and the reagent. This measurement time may be an arbitrarily determined fixed value, or may be until the time when the completion of the coagulation reaction of each sample is detected. Further, in the method of the present invention, before the completion of the coagulation reaction, at the time when the coagulation time of the sample is determined, the measurement of the sample may be terminated and the measurement of the next sample may be started. During this measurement time, the progress of the coagulation reaction (photometry when optically detecting) can be repeatedly measured at predetermined intervals. For example, measurement may be performed at 0.1-second intervals. The temperature of the mixture during the measurement is under normal conditions, for example, 30°C or higher and 40°C or lower, preferably 35°C or higher and 39°C or lower. Also, various conditions for the measurement can be appropriately set according to the test sample, reagent, measurement means, etc.

[0019] The series of operations in the above-described coagulation reaction measurement can be performed using an automatic analyzer. As an example of the automatic analyzer, a blood coagulation automatic analyzer CP3000 (manufactured by Sekisui Medical Co., Ltd.) can be mentioned. Alternatively, some operations may be performed manually. For example, a human can prepare the test sample, and subsequent operations can be performed using an automatic analyzer.

[0020] An example of the measurement data is shown in FIG. 1. The horizontal axis in FIG. 1 represents the elapsed time (coagulation reaction time) after the addition of the calcium chloride solution (at the start of the reaction), and the vertical axis represents the amount of scattered light. As the coagulation reaction of the mixed solution progresses with the passage of time, the amount of scattered light increases. In this specification, a curve showing the change in the coagulation reaction amount with respect to such a coagulation reaction time is referred to as a coagulation reaction curve.

[0021] The coagulation reaction curve based on the amount of scattered light as shown in FIG. 1 is usually sigmoid-shaped. On the other hand, the coagulation reaction curve based on the amount of transmitted light is usually inverse sigmoid-shaped. In the following description of this specification, data analysis using the coagulation reaction curve based on the amount of scattered light will be explained.

[0022] 2. Data Analysis Hereinafter, the present invention will be described with reference to the basic flow of an embodiment of the blood coagulation time measurement method according to the present invention shown in FIG. 2.

[0023] In the method of the present invention, the analyzer sequentially acquires measurement data R(i) (the photometric value of the amount of scattered light) for the coagulation reaction from the start of the reaction. Here, "i" represents the measurement point number. For example, if the measurement (photometry) interval is 0.1 second, R(i) represents the measurement value 0.1×i seconds after the start of the measurement.

[0024] Since the measurement data R(i) includes noise during photometry and fluctuations unrelated to the reaction that appear immediately after the start of photometry, smoothing processing is performed on the measurement values by a known method. Also, when measuring the coagulation reaction by scattered light amount, zero point adjustment processing is performed to subtract the scattered light amount derived from the reaction solution before the reaction. Therefore, the acquired measurement data R(i) is sequentially smoothed and zero point adjusted to obtain the reaction P(i) (step 1). For the smoothing processing of the measurement data, any of various known methods related to noise removal can be used. For example, as the smoothing processing, filtering processing, or processing of obtaining a differential value by calculating a difference value or an average slope within an interval described later and then integrating it, etc. can be mentioned. In zero point adjustment, for example, the smoothed measurement value may be adjusted so that the value at the start of measurement becomes 0. Further, initial fluctuation removal processing may be performed on the measurement data R(i). The initial fluctuation removal processing may be performed so that all values from the start of photometry to a predetermined initial fluctuation removal time become 0. Basically, as shown in FIG. 3, the reaction P(i) constitutes a coagulation reaction curve that is smoothed and zero point adjusted.

[0025] From the obtained reaction P(i), the reaction rate V(i) is acquired (step 2). V(i) is obtained by differentiating P(i) once. The differentiation processing can be performed by an arbitrary method, but for example, it can be performed by calculating the average slope value within an interval. In the calculation of the average slope value within an interval, a fixed number of measurement points before and after each measurement point i, for example, 2K + 1 measurement points from i - K to i + K can be used. For example, the 5 measurement points of the i - 2, i - 1, i, i + 1, i + 2th can be used. The average slope value means the slope value when these multiple measurement points are linearly approximated. As the calculation method of the linear approximation, a fixed method such as the least squares method can be used. The average slope value of these measurement points can be regarded as the first derivative value at the measurement point i. In the following specification, P(i) and V(i) may be simply abbreviated as P and V, respectively.

[0026] Referring to Fig. 3, the relative differences in the shapes of reaction P and reaction rate V for a normal sample with normal APTT and a sample lacking factor VIII (FVIII) (hereinafter referred to as the prolonged sample) with prolonged APTT are explained. In Fig. 3, the normal sample is shown on the left and the prolonged sample is shown on the right. The horizontal axis is converted to the time from the start of the reaction. In the normal sample (left), for reaction P, the rising point is early, the rising slope is large, and for reaction rate V, the position of the peak top is high and the shape is almost symmetric about the left and right. However, in the prolonged sample (right), for reaction P, the rising point is late, the rising slope is small, and for the peak top position of reaction rate V, it is low (less than one-sixth of that of the normal sample in this example), the shape is asymmetric about the left and right, and the peak is not single but bimodal.

[0027] In the method of the present invention, the coagulation time Tc is calculated using the values of the above-mentioned reaction P(i) and reaction rate V(i). In the method of the present invention, the acquisition of P(i) and V(i) can be performed in parallel with the coagulation reaction measurement procedure.

[0028] First, based on the V(i) obtained so far, the maximum reaction rate Vmax is determined (step 3). Vmax most simply represents the maximum value of the V(i) obtained so far. On the other hand, when determining Vmax, it is necessary to eliminate the influence of noise from the measuring instrument and initial reaction abnormalities (so-called early reactions) seen in the initial stage of the coagulation reaction. Therefore, preferably, Vmax is the value of the peak top of the maximum peak after V(i) exceeds a predetermined threshold for noise removal.

[0029] Preferably, in the method of the present invention, when the peak of sequentially obtained V(i) has a predetermined width, the value of the peak top of the peak is determined as Vmax. Fig. 4 shows a conceptual diagram of an embodiment of the method for determining Vmax. In Fig. 4, S1 to S6 show V(i) obtained from the initial reaction time point (T1) to the Vmax determination time point (Tt) in time series. In each figure, the obtained data of V(i) is represented by a solid line and the unobtained data is represented by a broken line. Finally, V(i) forms a curve having a peak top (Vmax). ·S1 represents V(i) up to time point T1 immediately after the start of the solidification reaction. V(i) at T1 (=V(T1)) is below the velocity threshold Vs. Vs is the velocity threshold for excluding initial noise. When V(i) exceeds Vs, the search for Vmax is started. ·S2 represents V(i) up to time point T2 when the solidification rate is increasing. Vp is a provisional peak and represents the maximum value of V(i) from after V(i) exceeds Vs to the latest measurement point. Vp is represented by the function Vp(t) of time t or the function Vp(i) of the measurement point. V(i) at T2 (=V(T2)) exceeds Vs and is the maximum value of V(i) at time point T2 (i.e., Vp(T2)=V(T2)), but has not reached the peak top. ·S3 represents V(i) up to time point T3 when V(i) reaches the peak top. Vp(T3)=V(T3). ·S4 represents V(i) up to time point T4 when V(i) starts to decrease after passing the peak top. The maximum value Vp(T4) of V(i) at T4 remains at the peak top (=V(T3)). K is the maximum value continuation width and represents the width (or number of measurement points) of the time during which Vp remains at a constant value, i.e., the peak top (=V(T3)). K is represented by the function K(t) of time t or the function K(i) of the measurement point. For example, K(T4) in the figure represents K at time point T4 and corresponds to the time width from time point T3 at the peak top to T4. K(i) at time point T4 is the value obtained by multiplying K(T4) by the measurement frequency (number of measurement points per second) of the solidification reaction measurement. ·S5 represents V(i) up to time point T5 when V(i) further decreases. Until time point T5, Vp continues to be the peak top V(T3) and K(T5) exceeds the maximum value continuation width threshold Ks (i.e., K(T5)>Ks). At this time, Vp(T5)(=V(T3)) is determined as the provisional maximum value Vmax0. ·S6 represents V(i) at the detection end point Tt of Vmax. Tt is the point (time point or measurement point) at which, after the detection of Vmax0, V(i) decreases to a predetermined value while K(i) > Ks. Although Tt is defined in S6 as the time point when V(i) decreases to 50% of Vmax0, it can be set arbitrarily. Preferably, Tt is the point where V(i) is between 40% and 95% of Vmax0. At the point where Tt is reached, Vmax0 is determined as the true maximum value Vmax, and the time satisfying V(i) = Vmax is determined as the maximum value time VmaxT. On the other hand, after the detection of Vmax0, if V(i) rises again without decreasing to the predetermined value and exceeds Vmax0, K(i) is reset to 0, and S2 - S5 are executed again to detect a new Vmax0. Table 1 shows the states of V(i) in S1 - S5.

[0030]

Table 1

[0031] If V(i) does not exceed the speed threshold Vs within the measurement time, it is determined that there is no coagulation reaction. Also, if V(i) exceeds Vs within the measurement time but the maximum value continuous width K does not exceed the threshold Ks, it is determined that the coagulation reaction has not ended. Further, even when the detection end point Tt cannot be detected and Vmax cannot be determined after Vmax0 is detected, it is determined that the coagulation reaction has not ended.

[0032] In another embodiment, Vmax can be determined based on the peak width W of V(i). The peak width W is the width (time or number of measurement points) between the latest point and the point at which V(i) was at X% of the height of the temporary peak Vp before that and before Vp. X may be any value greater than or equal to 1 and less than 100, but is preferably between 40 and 95. X is preferably greater than the parameter S used for calculating the calculation start point Te described later, but may be the same as or smaller than S. More specifically, when the coagulation time to be measured is APTT, X = 41 to 80 is preferred, and when the coagulation time is PT, X = 40 to 95 is preferred. In this specification, the peak width W may be referred to as the "X% peak width" according to the reference X. In this method, as in the above procedure, after V(i) exceeds Vs, Vp is sequentially detected, and the peak width W(t) at time t or the peak width W(i) at measurement point i is obtained. When the peak width W exceeds the peak width threshold value Ws, Vp is determined as the temporary maximum value Vmax0, and at the point where Tt (the definition is the same as above) is reached thereafter, Vmax0 is the true maximum value Vmax, and the time satisfying V(i) = Vp is the maximum value time VmaxT. Alternatively, more simply, Vp at Tt can be taken as the true maximum value Vmax. However, if the peak width W does not exceed the threshold value Ws within the measurement time, it is determined that the coagulation reaction has not ended.

[0033] For the speed threshold Vs, the maximum value continuous width threshold Ks, and the peak width threshold Ws, predetermined fixed values can be used respectively. For example, coagulation time measurement is performed on a reference specimen population including normal specimens and various abnormal specimens, and appropriate Vs, Ks, and Ws can be set using the data of V(i) for the obtained population. For example, Vs is set to a value smaller than the minimum Vmax among the Vmax values of each specimen obtained from the measurement data of the reference specimen population. For Ks and Ws, the maximum value continuous width K and the peak width W for each specimen in the reference specimen population are obtained by the above procedure, and appropriate values can be set as Ks or Ws based on the obtained K and W. Preferably, Ks is set to a value smaller than the minimum value among the maximum values (hereinafter referred to as Kmax) of K(i) obtained for each specimen, and Ws is set to a value smaller than the minimum value among the maximum values (hereinafter referred to as Wmax) of W(i) obtained for each specimen.

[0034] Alternatively, the speed threshold Vs, the maximum value continuous width threshold Ks, and the peak width threshold Ws may be functions of time t or the measurement point i. Even when Vs, Ks, and Ws are functions of t or i, the functions can be set based on the values of Vmax, Ks, and Ws obtained from each specimen in the reference specimen population in the same manner as above. For example, Vs can be set as a function represented by a curve that always shows a value smaller than Vmax on a graph where Vmax obtained from each specimen is plotted against VmaxT. Similarly, Ks and Ws can be set as functions represented by curves that always show values smaller than Kmax and Wmax respectively on a graph where Kmax and Wmax obtained from each specimen are plotted against VmaxT. In a preferred embodiment, the functions Vs, Ks, and Ws are calculated, for example, by the procedure described in Reference Example 1 below and can be obtained by the following equations. Vs = b + (a ÷ t 2 ) Ks = b’ + (a’ × t 2 ) Ws = b” + (a” × t 2 ) [a, a’, a”, b, b’, b” are coefficients, t is time (seconds)] Alternatively, by converting t in the above equation to i, Vs, Ks, and Ws can be obtained as functions of measurement point i. By using Vs, Ks, and Ws as variable values, Vmax can be appropriately detected even when the reaction rate V(i) is small or bimodal, as often seen in abnormal specimens.

[0035] Figure 5 shows the relationships between V(i), Vp(i), W(i), and K(i). Figures 5A - D show the relationships between V(i) and Vp(i), W(i), and K(i) in normal specimens with normal APTT. Figures 5E - H show the relationships between V(i) and Vp(i), W(i), and K(i) in abnormal (FVIII - deficient) specimens. In each figure, V(i) is shown by a dashed - dotted line up to the point when it reaches 70% of Vmax (detection end point Tt) after Vmax, and by a dotted line thereafter. Vp(i), W(i), and K(i) are shown by solid lines. W(i) uses the 70% peak width. As shown in Figures 5A - D, the V(i) of normal specimens shows a unimodal peak. Vp(i) increases until V(i) reaches the peak (Vmax), and then remains constant. W(i) increases until the detection end point Tt and then remains constant. K(i) increases until the detection end point Tt. On the other hand, the V(i) of the abnormal specimens shown in Figures 5E - H shows an approximately bimodal peak containing multiple small peaks, and the second peak becomes the maximum value Vmax. Vp(i) increases step - by - step as V(i) reaches the higher peak and remains constant after V(i) reaches Vmax. W(i) increases until the detection end point Tt and then remains constant. K(i) is reset to 0 each time a higher V(i) peak appears, resulting in the appearance of peaks of K(i) corresponding to the peaks of V(i).

[0036] Once Vmax is determined, a point Te at which V(i) is S% of Vmax (100%) at a time point after VmaxT is determined (step 4). Te is a time point (or measurement point) at which it is considered that the coagulation reaction has proceeded to a considerable extent, which is calculated based on the ratio of V(i) to the maximum reaction rate Vmax (hereinafter also referred to as the "maximum speed ratio"). Here, S% corresponds to the maximum speed ratio, and VmaxT < Te. S can be determined in the range greater than 0 and less than 100, preferably S = 1 to 95, more preferably 5 to 95. The smaller S is, the more the measurement accuracy of the coagulation time tends to improve, but the required measurement time becomes longer. When the coagulation time to be measured is APTT, more preferably S = 5 to 40, even more preferably S = 5 to 20. When the coagulation time is PT, more preferably S = 5 to 40, even more preferably S = 5 to 15. In the method of the present invention, Te is used as the calculation starting point of the coagulation time.

[0037] Next, using P(i) obtained up to Te, a time point Tc at which P(i) is N% of P(Te) (P(Tc) = P(Te) × N%) is determined (step 5). The obtained Tc is determined as the coagulation time. N may be any value as long as 0 < N < 100, preferably 10 to 70, more preferably 20 to 60.

[0038] As shown in the examples described later, the coagulation time Tc based on the maximum speed ratio calculated by the method of the present invention has a high correlation with the coagulation time by the conventional general method, the percent method (for example, the method of calculating the time until P(i) reaches a certain ratio of its maximum value Pmax as the coagulation time). Therefore, by the method of the present invention, the coagulation time of the test specimen can be accurately determined. Furthermore, in the method of the present invention, the step of detecting the true peak of the reaction rate V(i) (step 3 above) enables a more reliable coagulation time measurement that is less affected by measurement noise and early reactions.

[0039] Furthermore, in the method of the present invention, after the reaction rate V(i) reaches the maximum reaction rate Vmax, it becomes possible to determine the coagulation time when a predetermined threshold value (Vmax×S%) is reached. Therefore, in the method of the present invention, it is not necessary to continue the measurement until the coagulation reaction reaches a plateau as in the conventional percentage method. Further, even when analyzing multiple specimens with an automatic analyzer, according to the method of the present invention, it is not necessary to set a long measurement time in preparation for abnormal specimens with low coagulation ability as in the conventional percentage method. Therefore, according to the present invention, the analysis time of the specimen can be shortened or optimized to improve the efficiency of the analysis.

[0040] 3. Measurement of coagulation factor concentration Normal blood contains coagulation factors such as coagulation factors I to XIII, and abnormalities or deficiencies in these coagulation factors result in abnormalities in coagulation ability. Usually, the coagulation factor concentration of a test specimen can be measured based on the coagulation time of a measurement sample prepared from the test specimen using a dedicated reagent for each coagulation factor. Therefore, the coagulation factor concentration of the test specimen can be measured using the coagulation time of the measurement sample measured by the method of the present invention. Usually, the coagulation factor concentration is measured based on a calibration curve showing the relationship between the coagulation time and the coagulation factor concentration. Therefore, the coagulation factor concentration of the test specimen can be calculated by applying the coagulation time of the measurement sample measured by the method of the present invention to the previously prepared calibration curve. Preferred examples of the coagulation factors measured by the method of the present invention include factor I (fibrinogen), factor VIII, factor IX, and the like.

[0041] In calculating the clotting time used for measuring the coagulation factor concentration by the method of the present invention, the threshold value of V(i) (「Vmax×S%」) for determining the calculation starting point Te based on the maximum speed ratio is a predetermined value set preferably in the range of 1 to 95% of Vmax (i.e., S = 1 to 95), more preferably in the range of 5 to 95% of Vmax (i.e., S = 5 to 95), still more preferably in the range of 20 to 95% of Vmax (i.e., S = 20 to 95), and even more preferably in the range of 30 to 75% of Vmax (i.e., S = 30 to 75). Also, for the X% peak width W for determining Vmax, it is preferable to use a 75% peak width to 95% peak width, where X is preferably larger than S, but may be the same as or smaller than S.

[0042] 4. Application to Other Coagulation Reaction Measurement Methods As described above, the blood coagulation time measurement method of the present invention has been described by taking the case of coagulation reaction measurement based on the scattered light amount as an example. However, those skilled in the art can apply the method of the present invention to a blood coagulation time measurement method using other coagulation reaction measurement methods (for example, blood coagulation reaction measurement methods based on transmittance, absorbance, viscosity, etc.). For example, the reaction P(i) obtained from an inverse sigmoid-shaped coagulation reaction curve based on the transmitted light amount has the opposite sign to that based on the scattered light amount described above. In such a case, it is obvious to those skilled in the art that the signs of P(i) and V(i) are reversed in Steps 1 to 4 described above, for example, the minimum reaction speed Vmin is determined instead of the maximum reaction speed Vmax.

Examples

[0043] The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.

[0044] Example 1 Measurement of Clotting Time (APTT) 1. Method 1.1) Sample A total of 24 samples were used as test samples, including 9 normal plasma samples and 15 abnormal plasma samples with prolonged APTT. Normal plasma was Normal Donor Plasma manufactured by CliniSys Associates, Ltd. Abnormal plasma was used, including 2 samples each of FVIII-deficient plasma, FIX-deficient plasma, FXI-deficient plasma, and FXII-deficient plasma as coagulation factor deficient plasma (Factor Deficient Plasma), 2 samples of lupus anticoagulant plasma, and 5 samples of unfractionated heparin-containing plasma (Anticoagulant Plasma) (all manufactured by CliniSys Associates, Ltd.).

[0045] 1.2) Reagents Coagpia APTT-N (manufactured by Sekisui Medical Co., Ltd.) was used as the APTT reagent.

[0046] 1.3) Clotting reaction measurement The coagulation reaction was measured using an automatic blood coagulation analyzer CP3000 (manufactured by Sekisui Medical Co., Ltd.). 50 μL of the sample was dispensed into a cuvette (reaction vessel) and heated at 37°C for 45 seconds, then 50 μL of APTT reagent heated to about 37°C was added to the cuvette, and after 171 seconds, 50 μL of calcium chloride solution was added to start the coagulation reaction. The reaction was performed while maintaining the temperature at about 37°C. The coagulation reaction was measured (photometrically) by irradiating the cuvette with light from an LED light with a wavelength of 660 nm as a light source and measuring the amount of scattered light at 90 degrees side at 0.1 second intervals. The maximum measurement time was 360 seconds (3600 data points, 0.1 second intervals).

[0047] 1.4) Obtaining reaction P(i) and reaction rate V(i) After smoothing the photometric data from each sample, including noise removal, the reaction P(i) was calculated by performing a zero-point adjustment so that the amount of scattered light at the start of photometry was 0. The first derivative V(i) was calculated from P(i).

[0048] 1.5) APTT measurement (percentage method) The APTT of each specimen was measured by the percentage method. That is, the time point when P(i) reached the maximum value Pmax within the measurement time was determined as the end point of the coagulation reaction, and the time point when the scattered light amount reached 50% of the scattered light amount at the end point of the coagulation reaction was determined as the APTT. Table 2 shows the type and number of test specimens, and the minimum and maximum values of APTT in the specimens of each type.

[0049]

Table 2

[0050] 1.6) Calculation of coagulation time Tc based on the maximum velocity ratio From V(i) obtained from each specimen, the maximum reaction velocity Vmax was detected. After the maximum value continuous width K(i) exceeded the maximum value continuous width threshold Ks, the provisional maximum value Vmax0 when the detection end point Tt (the time point when V(i) became 70% of Vmax) was reached was determined as Vmax. The velocity threshold Vs was a function expressed by the following formula according to the procedure of Reference Example 1 described later. Ks was set to 2.2. Vs(i)=-83+(158214÷i 2 ) [i is the measurement point] The time Te when V(i)=Vmax×S% was determined (S = 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 and 95). The time Tc when P(Tc)=P(Te)×50% was determined and used as the coagulation time. Te and Tc determined for each S were hereinafter referred to as Tes and Tcs, respectively, according to the corresponding S. For example, Te and Tc when S = 5 are referred to as Te5 and Tc5, respectively.

[0051] Fig. 6 shows the measured Te, Tc, and an example of APTT by the percentage method. In Figs. 6A and 6B, P(i) (top) and V(i) (bottom) of the specimens with the minimum (27.2 seconds) and maximum (141.2 seconds) APTT by the percentage method, respectively, are shown at the time point when Pmax is reached (diamond), the APTT by the percentage method based on Pmax (triangle), Te40 and Te 20 (square), and Tc 40 and Tc 20 (circle) are shown. In both FIGS. 6A and 6B, Tc marked with a circle 40 and Tc 20 is close to the APTT by the percentage method (conventional method) based on Pmax marked with a triangle and is shown to reflect the APTT. Also, in both FIGS. 6A and 6B, the time when P(i) becomes the maximum value Pmax is about 360 seconds, and the time difference between the time of Pmax and Te 40 and Te 20 is about 320 seconds in FIG. 6A and about 140 - 160 seconds in FIG. 6B. By obtaining Te and Tc based on the maximum speed ratio, it has been shown that the APTT can be calculated more than 140 seconds earlier compared to the conventional percentage method based on Pmax.

[0052] 2. Evaluation of the accuracy of Tc 2.1) Correlation analysis The correlation between the APTT by the percentage method and the APTT (Tc) based on the maximum speed ratio was evaluated. For each of Tc (Tc5 - Tc 95 ) when S = 5 - 95, a linear regression analysis with the APTT by the percentage method was performed to obtain the slope, intercept, and correlation coefficient of the regression line.

[0053] FIG. 7 shows the linear regression line of Tc5 against the APTT by the percentage method for 24 specimens. Tc5 had a high correlation with the APTT by the percentage method. The correlation between the APTT by the percentage method and Tc was evaluated. For each of Tc (Tc5 - Tc 95 ) when S = 5 - 95, a linear regression analysis with the APTT by the percentage method was performed to obtain the slope, intercept, and correlation coefficient of the regression line. FIG. 8 shows the slope, intercept, and correlation coefficient of the linear regression line of Tc5 - Tc 95 against the percentage method APTT. Tc5 - Tc 95Among them, the slope of the regression line was 0.86 - 1.00, the intercept was -0.1 - 1.6, and the correlation coefficient was 0.992 - 1.000 (Figs. 8A - C). It was confirmed that the APTT measurement method based on the maximum speed ratio had the performance of meeting the criteria for the approval of medical products for in vitro diagnosis by the Ministry of Health, Labour and Welfare, "the correlation coefficient is 0.9 or more and the slope of the regression line formula is 0.9 - 1.1 compared with the control measurement method".

[0054] 2.2) Measurement accuracy Tc5 - Tc for 24 specimens 95 was compared with the control (APTT by the percentage method). Each row in the tables of Figs. 9A and B represents Tc (from left to right, Tc5 - Tc 95 ) of each specimen. When the difference between Tc and the control was within ±5% (Fig. 9A) and within ±3% (Fig. 9B) of the control, it was shown in gray. Tc5 - Tc 40 of all specimens was consistent with the control within an error of ±5%, and Tc5 - Tc 20 of all specimens was consistent with the control within an error of ±3%.

[0055] 3. Accuracy evaluation of Tc - 2 In the same procedure as in 2.1), for each of Tc 0.5 - Tc5, a linear regression analysis with APTT by the percentage method was performed to obtain the slope, intercept, and correlation coefficient of the regression line. Fig. 10A shows the linear regression line of Tc 0.5 for 24 specimens with respect to APTT by the percentage method, and Figs. 10B - D show the slope, intercept, and correlation coefficient of the linear regression line of Tc 0.5 - Tc5 with respect to the percentage method APTT, respectively. Tc 0.5 - Tc5 all had a very high correlation with APTT by the percentage method. Fig. 11 is a table comparing Tc 0.5 - Tc5 for 24 specimens with the control (APTT by the percentage method) in the same procedure as in 2.2), and when the difference between Tc and the control was within ±1% of the control, it was shown in gray. Tc 0.5 - Tc5 of all specimens was consistent with the control within an error of ±1%.

[0056] Example 2 Prothrombin time (PT) measurement 1. Method 1.1) Sample A total of 23 test samples were used: 9 normal plasma samples and 14 abnormal plasma samples with prolonged PT. Plasma from healthy individuals was used as normal plasma. Plasma from patients administered the anticoagulant warfarin was used as abnormal plasma. Five samples with PT-INR values indicating blood warfarin concentrations of 1 to 2, five with PT-INR values of 2 to 3, and four with PT-INR values of 3 to 4 were used.

[0057] 1.2) Clotting reaction measurement The coagulation reaction was measured using an automatic blood coagulation analyzer CP3000 (manufactured by Sekisui Medical Co., Ltd.). 50 μL of the sample was dispensed into a cuvette (reaction vessel) and heated at 37°C for 45 seconds, and then 100 μL of thromboplastin solution heated to approximately 37°C was added to the cuvette to start the coagulation reaction. The reaction was carried out while maintaining the temperature at approximately 37°C. The coagulation reaction was measured (photometrically) by irradiating the cuvette with light from an LED light with a wavelength of 660 nm as a light source and measuring the amount of scattered light at 90 degrees side at 0.1 second intervals. The maximum measurement time was 300 seconds (3000 data points, 0.1 second intervals).

[0058] 1.3) Obtaining reaction P(i) and reaction rate V(i) After smoothing the photometric data from each sample, including noise removal, the reaction P(i) was calculated by performing a zero-point adjustment so that the amount of scattered light at the start of photometry was 0. The first derivative V(i) was calculated from P(i).

[0059] 1.4) PT measurement (percentage method) The PT of each sample was measured by the percentage method. The time when P(i) reached its maximum value Pmax within the measurement time was determined as the end point of the coagulation reaction, and the time when the scattered light amount reached 45% of the end point of the coagulation reaction was determined as PT. The types and numbers of samples tested, as well as the minimum and maximum PT values in each type of sample, are shown in Table 3.

[0060] [Table 3]

[0061] 1.5) Calculation of solidification time Tc based on the maximum speed ratio From V(i) obtained from each specimen, the maximum reaction rate Vmax was detected in the same procedure as in Example 1. The speed threshold Vs was defined as a function expressed by the following formula according to the procedure of Reference Example 1 described later. Ks was set to 0.8. Vs(i)=-148+(51935÷i 2 ) [i is the measurement point] The time Te when V(i)=Vmax×S% and the time Tc when P(Tc)=P(Te)×45% were determined (S = 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 and 95), and Tc5~Tc 95 was obtained.

[0062] Fig. 12 shows the measured Te, Tc and an example of PT by the percentage method. In Figs. 12A and B, P(i) (top) and V(i) (bottom) of the specimens with the minimum (9.5 seconds) and maximum (51.6 seconds) PT by the percentage method are shown, respectively, at the point when Pmax is reached (diamond), PT by the percentage method based on Pmax (triangle), Te 40 and Te 15 (square), and Tc 40 and Tc 15 (circle). In both Figs. 12A and B, Tc marked with a circle 40 and Tc 15 is close to PT by the percentage method (conventional method) marked with a triangle based on Pmax, indicating that it reflects PT. Also, in both Figs. 12A and B, the time when P(i) reaches the maximum value Pmax is about 300 seconds, and the time difference between the time of Pmax and Te 40 and Te 15 is about 285 seconds in Fig. 12A and about 240 - 250 seconds in Fig. 12B. It was shown that by obtaining Te and Tc based on the maximum speed ratio, PT can be calculated more than 240 seconds earlier compared to the conventional percentage method based on Pmax.

[0063] 2. Accuracy evaluation of Tc 2.1) Correlation analysis In the same procedure as 2.1) of Example 1, for each of Tc5 to Tc 95 , a simple linear regression analysis with PT measured by the percentage method was performed to obtain the slope, intercept, and correlation coefficient of the regression line.

[0064] Fig. 13 shows the simple linear regression line of Tc5 with respect to PT by the percentage method for 23 specimens. Tc5 had a high correlation with PT by the percentage method. The correlation between PT by the percentage method and Tc was evaluated. For each of Tc (Tc5 to Tc 95 ) when S = 5 to 95, a simple linear regression analysis with PT by the percentage method was performed to obtain the slope, intercept, and correlation coefficient of the regression line. Fig. 14 shows the slope, intercept, and correlation coefficient of the simple linear regression line of Tc5 to Tc 95 with respect to PT by the percentage method. Between Tc5 and Tc 95 , the slope of the regression line was 0.91 to 1.00, the intercept was 0.0 to 0.2, and the correlation coefficient was all 1.000 (Figs. 14A to C). It was confirmed that the PT measurement method based on the maximum speed ratio had the performance of satisfying the performance criteria for medical products for in vitro diagnosis approved by the Ministry of Health, Labour and Welfare, "correlation coefficient of 0.9 or more and slope of the regression line equation of 0.9 to 1.1 compared with the control measurement method".

[0065] 2.2) Measurement accuracy Tc5 to Tc 95 for 23 specimens were compared with the control (PT by the percentage method). Each row in the table of Figs. 15A and B represents Tc (from left to right, Tc5 to Tc 95 ) of each specimen. When the difference between Tc and the control was within ±5% (Fig. 15A) and within ±3% (Fig. 15B) of the control, it was represented in gray. Tc5 to Tc 40 of all specimens were in agreement with the control with an error within ±5%, and Tc5 to Tc 15 of all specimens were in agreement with the control with an error within ±3%.

[0066] Example 3 Fibrinogen concentration measurement 1. Method 1.1) Sample A sample (sample 10) with a fibrinogen concentration ([Fbg]) of 980 mg / dL was prepared by adding human fibrinogen (Human Fibrinogen, Enzyme Research Laboratories, product name: FIB 2) to human fibrinogen-depleted plasma (Fibrinogen Deficient Human Plasma, Affinity Biologicals Inc., product name: Fg Deficient Plasma). As standard samples for creating a calibration curve, sample 10 was mixed with saline in volume ratios of 1:9, 7:3, and 10:0 to prepare three samples with fibrinogen concentrations ([Fbg]) of 98 mg / dL, 686 mg / dL, and 980 mg / dL, respectively. In addition, sample 10 was mixed with the human fibrinogen-depleted plasma in volume ratios ranging from 1:9 to 10:0 to prepare 10 concentration series samples with gradually differing fibrinogen concentrations ([Fbg]) (Table 4).

[0067] [Table 4]

[0068] 1.2) Clotting reaction measurement The fibrinogen measurement reagent used was the thrombin reagent and specimen diluent attached to Coagpia Fbg (manufactured by Sekisui Medical Co., Ltd.). The coagulation reaction was measured using an automatic blood coagulation analyzer CP3000 (manufactured by Sekisui Medical Co., Ltd.). 10 μL of the specimen and 90 μL of the specimen diluent were dispensed into a cuvette and heated at 37°C for 45 seconds, after which 50 μL of thrombin reagent heated to about 37°C was added to the cuvette to start the coagulation reaction. The reaction was carried out while maintaining the temperature at about 37°C. The coagulation reaction was measured by irradiating the cuvette with light from an LED light with a wavelength of 660 nm as a light source and measuring the amount of scattered light at 90 degrees side at 0.1 second intervals. The maximum measurement time was 300 seconds (3000 data points, 0.1 second intervals). The coagulation reaction was measured twice for each of the three standard specimens and the 10 concentration series specimens.

[0069] 1.3) Create reaction P(i) and reaction rate V(i) After performing a smoothing process including noise removal on the photometric data from each specimen, zero-point adjustment processing was performed so that the scattered light amount at the start point of photometry became zero, and reaction P(i) was created. The first derivative value V(i) was calculated from P(i).

[0070] 1.4) Calculation of fibrinogen concentration by the percentage method Since the reaction curve of the low fibrinogen specimen may continue to rise gently until the end of measurement, it is not appropriate to use the maximum value Pmax of the reaction curve as the starting point for calculation by the percentage method. Therefore, in this example, the starting point for calculation by the percentage method was determined based on the integrated value ratio Z(i) of reaction P(i) (see Patent Document 1). The integrated value ratio Z(i) reflects the tendency of the change in P(i). That is, Z(i) is large at the initial stage of the reaction where P(i) rises, but gradually approaches 1 as P(i) approaches the plateau. In this example, Z(i) was calculated from P(i) by the following formula, and the time when Z(i) became smaller than the threshold value Zs was set as the starting point for calculation by the percentage method. Zs was set to 1.05. Z(i) = {P(i+1)+P(i+2)+...+P(i+m)} / {P(i-m)+P(i-m+1)+...+P(i-1)} (m = 20)

[0071] The clotting times of three standard specimens and ten specimens of the concentration series were measured by the percentage method. That is, when P(i) at the above-mentioned calculation starting point was set to 100%, the time point when P(i) reached 63% was determined as the clotting time. The clotting time of each specimen was measured twice based on two clotting reaction measurements. For the clotting times measured from the three standard specimens, the average value of the two measurements was calculated, and the logarithm of the average value was plotted against the logarithm of [Fbg] (mg / dL) of the standard specimen to create a calibration curve by the percentage method. According to the created calibration curve, the fibrinogen concentration ([Fbg] calculated value by the percentage method, mg / dL) of each concentration series specimen was calculated.

[0072] 1.5) Calculation of fibrinogen concentration based on the maximum velocity ratio From V(i) obtained from each specimen, the maximum reaction rate Vmax was detected in the same procedure as in Example 1. The velocity threshold Vs was defined as a function expressed by the following equation according to the procedure of Reference Example 1 described later. Ks was set to 1.1. Vs(i)=19+(9357÷i 2 ) [i is the measurement point] The time Te when V(i)=Vmax×S% and the time Tc when P(Tc)=P(Te)×63% were determined (S = 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 and 95), and Tc5 to Tc 95 were obtained. For each of the 10 concentration series specimens, Tc5 to Tc 95 was calculated twice to obtain data for 20 specimens. Also, for each of the 3 standard specimens, Tc5 to Tc 95 was calculated twice, and the average value of the two measurements was calculated. The logarithm of the average value was plotted against the logarithm of [Fbg] (mg / dL) of the standard specimen to create a calibration curve based on Tc. According to the created calibration curve, the fibrinogen concentration (calculated value of [Fbg] by the maximum velocity ratio, mg / dL) of each concentration series specimen was calculated.

[0073] Figure 16 shows the measured Te, Tc, and an example of the clotting time by the percent method. In Figures 16A and B, P(i) (top) and V(i) (bottom) of the specimens with the minimum ([Fbg]=98 mg / dL) and maximum ([Fbg]=980 mg / dL) [Fbg] are shown, respectively, with the calculation starting point (diamond) by the integrated value ratio, the clotting time by the percent method based on the calculation starting point (triangle), Te 75 and Te 30 (square), and Tc 75 and Tc 30 (circle). In both Figures 16A and B, Tc 75 and Tc 30 marked with circles are close to the clotting time by the percent method (conventional method) marked with triangles, indicating that they reflect the clotting time.

[0074] 2. Accuracy Evaluation of Fibrinogen Concentration Measurement Based on Tc 2.1) Correlation Analysis Figure 17 shows the linear regression line of the calculated [Fbg] values by the percent method against the calculated [Fbg] values by Tc 30 for the data of the concentration series specimens (n = 10×2). The calculated [Fbg] values based on Tc 30 had a high correlation with the calculated [Fbg] values by the percent method. Figure 18 shows the slope, intercept, and correlation coefficient of the linear regression line of the calculated [Fbg] values calculated from Tc5 to Tc 95 against the calculated [Fbg] values by the percent method. Between Tc5 and Tc 95 , the slope of the regression line was 0.97 - 1.06, the intercept was -27.1 - 32.0, and the correlation coefficient was 0.992 - 0.999 (Figure 18A - C). It was confirmed that the fibrinogen concentration measurement method based on the maximum speed ratio yielded results equivalent to those of the standard method based on the percent method, and thus had performance satisfying the criteria for approval of medical products for in vitro diagnosis by the Ministry of Health, Labour and Welfare, "correlation coefficient of 0.9 or more and slope of the regression line equation of 0.9 - 1.1 when compared with the control measurement method".

[0075] 2.2) Accuracy of measurement The calculated [Fbg] values based on Tc5 to Tc 95 for the data of the concentration series specimens (10 specimens × 2 in Table 4) were compared with the expected values (Fbg concentrations of the specimens shown in Table 4). Each row in the tables of Figure 19AB represents the Tc of each specimen (from Tc5 to Tc 95 ) from left. When the error between Tc and the expected value was within ±10% (Figure 19A) and within ±5% (Figure 19B), it was represented in gray. For all specimens, Tc 20 to Tc 95 matched the expected value with an error within ±10%, and for all specimens, Tc 30 to Tc 75 matched the expected value with an error within ±5%.

[0076] Example 4 Accuracy evaluation The accuracy of the coagulation time measurement method based on the maximum speed ratio by this method was compared with the method described in Patent Document 1 (coagulation time measurement method using the integrated value ratio Z(i) of reaction P(i)). Z(i) is the ratio of the integrated value of P(i) in the first measurement interval to the integrated value of P(i) in the second measurement interval adjacent to the first interval, and reflects the tendency of the change of P(i). Z(i) is large at the initial stage of the reaction where P(i) increases, but gradually approaches 1 asymptotically as P(i) approaches the plateau. In the method described in Patent Document 1, after Z(i) becomes smaller than the threshold value Zs, when a predetermined value is satisfied, it is determined as the coagulation time. The coagulation time measurement method based on Z(i) is disclosed as a highly reliable coagulation time measurement method that can accurately measure the coagulation time not only of normal specimens but also of abnormal specimens such as low fibrinogen specimens. In this example, the accuracy of the coagulation time measurement method by this method based on the maximum speed ratio was evaluated by comparison with the coagulation time measurement method using the integrated value ratio.

[0077] 1. Method 1.1) Sample Specimen A: A specimen obtained by adding unfractionated heparin to normal plasma so that the unfractionated heparin concentration becomes 0.9 IU / mL (APTT 139.0 seconds). Specimen B: Among the heparin-containing plasma used in Example 1, the specimen with the maximum APTT (126.2 seconds).

[0078] 1.2) Coagulation reaction measurement The same as in Example 1.

[0079] 1.3) Creation of reaction P(i), reaction rate V(i), and integrated value ratio Z(i) After performing smoothing processing including noise removal on the photometric data from each specimen, zero-point adjustment processing was performed so that the scattered light amount at the start of photometry became 0 to create the reaction P(i). From P(i), the first derivative value V(i) was calculated. The integrated value ratio Z(i) of the reaction P(i) was calculated by the following formula. Z(i) = {P(i+1)+P(i+2)+...+P(i+m)} / {P(i-m)+P(i-m+1)+...+P(i-1)} (m = 20)

[0080] Figure 20 shows P(i) and V(i) (left), as well as P(i) and Z(i) (middle) and an enlarged view of Z(i) (right) for two specimens A (top) and B (bottom). In specimen A, immediately after the start of the measurement, no clear peak (initial peak) was observed in V(i), but multiple early peaks were observed in Z(i). These early peaks were presumed to reflect disturbances in turbidity when the mixture of specimen and reagent was mixed in the reaction vessel (cuvette). In specimen B, immediately after the start of the measurement, early peaks were observed in both V(i) and Z(i), which were presumed to reflect an abnormal early reaction (so-called early reaction). In both cases, the early peak of V(i) was smaller than the early peak of Z(i) and was considerably smaller than the peak (reaction peak) that appeared after the start of the coagulation reaction (the rise of P(i)). This showed that the reaction peak of V(i) could be easily distinguished from the early peak. On the other hand, since the initial peak of Z(i) was larger than the reaction peak, it was found that in order to correctly recognize the reaction peak of Z(i), it is necessary to set a judgment criterion that does not erroneously detect the initial peak. Therefore, it became clear that this method based on the maximum velocity ratio is more resistant to measurement noise than the method using the integrated value ratio, and enables more accurate coagulation time measurement.

[0081] Reference Example 1: Determination of Vs, Ks, and Ws The procedure for calculating the velocity threshold Vs, the maximum value duration threshold Ks, and the peak width threshold Ws based on the data of the reference specimen population will be described. As the reference specimen population, the data of the 24 specimens used in Example 1 (hereinafter, APTT group), the 23 specimens used in Example 2 (hereinafter, PT group), or the 20 specimens used in Example 3 (hereinafter, Fbg group) were used. Furthermore, the control PN I and control PN II of the coagpia control PN (manufactured by Sekisui Medical Co., Ltd.) used as quality control samples were also included in the reference specimen population of each group.

[0082] 1.1 Procedure for setting a fixed value Vs For each specimen in the reference specimen population, the maximum value Vmax of the reaction rate V(i) during the measurement period was determined. The minimum value among the Vmax values obtained from each specimen was determined, and 50% of the minimum Vmax was defined as Vs. The determined Vs values were 36 for the APTT group, 107 for the PT group, and 17 for the Fbg group.

[0083] 1.2 Procedure for setting the function Vs 1) Control P-N I and Control P-N II were used as "normal control" and "abnormal control", respectively. For the normal control, the APTT and Fbg concentrations were near the center within the reference range. For the abnormal control, the APTT exceeded the reference range and was prolonged, and the Fbg concentration was lower than the reference range. The reference range refers to the range of the central 95% (excluding the 2.5% on both sides) of the APTT or Fbg concentration distribution in the existing healthy population. 2) The coefficients of the function formula were determined using the measurement data of the normal control and the abnormal control. The maximum velocity time VmaxT of the normal control was designated as X1, and the maximum velocity Vmax was designated as Y1. Similarly, for the abnormal control, VmaxT was designated as X2 and Vmax as Y2. 3) To calculate Vs that is smaller than Vmax in the reference specimen population, X1 and X2 were multiplied by a coefficient C (preferably 50% ≤ C ≤ 90%) to obtain P1 and P2. In this reference example, C was set to 60%. 4) The function formula was set as y = b + a ÷ x 2 and a and b were determined from the simultaneous equations obtained by substituting P1 and Y1, and P2 and Y2 into x and y of the formula. 5) Vs was calculated from the determined a and b as Vs = b + a ÷ i 2 The calculation formulas for Vs obtained for the APTT group, PT group, and Fbg group were as follows. APTT group: Vs(i) = -83 + (158214 ÷ i 2 ) PT group: Vs(i) = -148 + (51935 ÷ i 2 ) Fbg group: Vs(i) = 19 + (9357 ÷ i 2 ) However, when Vs obtained from the above formula was less than the fixed value obtained in 1.1, Vs was set to the fixed value.

[0084] Figure 21A shows, from left to right, the distributions of Vmax of the APTT group, PT group, and Fbg group and the calculated Vs. Vmax is plotted against VmaxT. The circles in the figure indicate the data of normal controls, and the triangles indicate the data of abnormal controls. The dotted line represents the fixed value of Vs, and the solid line represents the function Vs. In the figure, the function Vs is expressed as a function of time t (seconds). In any group, as VmaxT increases, Vmax tends to decrease. As a result, the function Vs is represented as a curve that decreases with time and is finally replaced by the fixed value of Vs.

[0085] 2.1 Procedure for Setting Ks The maximum value (Kmax) of K(i) varies depending on the detection end point Tt, that is, the value of the V(Tt) / Vmax0 ratio. As Tt increases (the V(Tt) / Vmax0 ratio decreases), K(i) moves away from VmaxT, so Kmax increases. As Tt decreases (the V(Tt) / Vmax0 ratio increases), K(i) approaches VmaxT, so Kmax decreases. Using the measurement data of the reference specimen population, the distribution of Kmax was examined by changing the setting of the V(Tt) / Vmax0 ratio (%) from 50% to 90% in 10% increments. As shown in Table 5, the minimum and maximum values of the distribution of Kmax were confirmed to have the tendency as described above under the influence of the V(Tt) / Vmax0 ratio.

[0086]

Table 5

[0087] Figure 21B shows the distribution of Kmax and the calculated Ks when the V(Tt) / Vmax0 ratio is set to 70% in the APTT group, PT group, and Fbg group from left to right. Kmax is plotted against VmaxT. From these results, it was shown that Kmax depends on the time of the reaction rate peak and thus the clotting time, that is, as the clotting time increases, Kmax also increases, and as the clotting time decreases, Kmax also decreases. Thus, since Kmax tends to depend on time, it was shown that Ks may be a function of time or the measurement point. From the data in the figure, the calculation formula: Ks(t) = constant b + coefficient a × t 2 (the solid line in the figure) could be obtained. The constant b was set as the fixed value of Ks (the dotted line in the figure) and as the value of 60% of the minimum Kmax, which was 2.2 seconds in the APTT group, 0.8 seconds in the PT group, and 1.1 seconds in the Fbg group. The coefficient a was appropriately set so as not to overlap with the distribution of Kmax, and it was 0.0004 in all groups.

[0088] 3.1 Setting procedure of Ws The maximum value of W(i) (Wmax) also showed the same tendency as Kmax, that is, a tendency to depend on time. As shown in Table 6, the minimum and maximum values of the distribution of Wmax were examined by changing the set value of the V(Tt) / Vmax0 ratio (%) from 50% to 90% in 10% increments.

[0089]

Table 6

[0090] Figure 21C shows the distribution of Wmax and the calculated Ws when the V(Tt) / Vmax0 ratio is set to 70% in the APTT group, PT group, and Fbg group from left to right. Wmax is plotted against VmaxT. It was shown that as VmaxT increases, Wmax also increases, and as VmaxT decreases, Wmax also decreases. From the data in the figure, the calculation formula: Ws(t) = constant b + coefficient a × t 2(The solid line in the figure) could be obtained. The constant b was set as a fixed value of Ws and as a value of 60% of the minimum Wmax, being 3.4 seconds in the APTT group, 1.6 seconds in the PT group, and 1.9 seconds in the Fbg group. The coefficient a was appropriately set so as not to overlap with the distribution of Wmax, and was 0.0005 in any group.

[0091] Reference Example 2 Figure 22 shows the relationships of the reaction rate V(t) and the velocity threshold Vs (upper), the maximum value continuous width K(t) and Ks (middle), and the peak width W(t) and Ws (lower) in the Vmax detection process for the specimen (A) with the minimum APTT and the specimen (B) with the maximum APTT in Example 1. In Figure 22, V(t) is shown by a solid line up to the point when it becomes 70% of Vmax after Vmax (detection end point Tt), and by a dotted line thereafter. W(t) indicates the 70% peak width. Vs was calculated by the following formula according to the aforementioned Reference Example 1, and was set as 36 when the calculated value was less than 36. Ks and Ws used the following fixed values. Vs(t)=-83+158214÷t 2 [t is time (seconds)] Ks=2.2 Ws=3.4

[0092] As shown in Fig. 22, Vs (dashed-dotted line) according to the above equation is very large at the initial stage of the reaction but decreases with time. It has been shown that by setting Vs that varies in this way, false detection of the initial peak due to early reactions or noise at the initial stage of the reaction can be prevented. Also, as shown in Fig. 22, K(t) shown by the dotted line shows a sharp single triangle exceeding the threshold value Ks in Fig. A, and varies up and down corresponding to the peak appearance of V(t) and exceeds the threshold value Ks (dashed-dotted line) in Fig. B. As shown at the bottom of Fig. 22, W(t) shown by the dotted line increases with time in both Fig. A and Fig. B, exceeds the threshold value Ws (dashed-dotted line), and reaches the maximum value Wmax when V(t) exceeds VmaxT and reaches 70% of Vmax. Wmax is 5.7 seconds in Fig. A because the peak shape is sharp, and 82 seconds in Fig. B because the peak shape is a wide double-peaked shape. In both cases of A and B, K(t) exceeded Ks and W(t) exceeded Ws at the detection end point Tt. From these results, it has been shown that not only when V(t) has one peak reaching Vmax as in A, but also when a prep-peak appears before the reaction rate V reaches Vmax as in B, Vmax can be correctly detected by using K(t) or W(t).

Claims

1. A method for measuring blood coagulation time, comprising: obtaining a coagulation reaction P(i) for a test specimen and a reaction rate V(i) which is the differential value of the reaction P(i), where i represents a measurement point, and the reaction P(i) is obtained by smoothing and zero-point adjustment of measurement data R(i) obtained from measurement of the blood coagulation reaction of the test specimen; determining a maximum value Vp of V(i) from after V(i) exceeds a threshold Vs to the latest V(i); determining a maximum value continuous width K(i), where the K(i) represents the time or the number of measurement points during which Vp remains a constant value at i; when the K(i) exceeds a threshold Ks, detecting the Vp as a provisional maximum value Vmax0; after detection of the Vmax0, when the latest V(i) decreases to a predetermined value while K(i)>Ks, determining the Vmax0 as the maximum reaction rate Vmax; calculating the time or measurement point at which V(i) becomes S% of Vmax after the point in time when V(i)=Vmax as a calculation start point Te of the blood coagulation time, where S is a predetermined value in the range of 5 to 95; and calculating a time Tc at which P(Tc)=P(Te)×N% (0<N<100) and determining the Tc as the blood coagulation time. A method comprising the above.

2. The method according to claim 1, wherein the obtaining of the reaction P(i) and the reaction rate V(i) is performed in parallel with the measurement of the blood coagulation reaction of the test specimen.

3. A method for measuring a coagulation factor concentration, comprising measuring the coagulation factor concentration of a test specimen based on the blood coagulation time of the test specimen measured by the method according to claim 1 or 2.

4. The method according to claim 3, wherein the coagulation factor is fibrinogen.

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