Blood coagulation time measurement method

Abstract

Provided is a blood coagulation time measurement method. In this method, reactions X(i) are obtained through smoothing and zero-point adjustment of measured values P(i) for a blood sample coagulation reaction, and then accumulation ratios Z(i) of the reactions X(i) are obtained. These values are used to calculate an index for calculating a coagulation time Tc, and a determination is made as to whether the index meets a standard. This process is repeated successively until an index that meets the standard is obtained.

Description

[Technical field]

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

[0002] A blood coagulation test is a test to diagnose a patient's blood coagulation ability by adding a specific reagent to a blood sample from the patient and measuring the blood coagulation time. Typical examples of blood coagulation times include the prothrombin time (PT), activated partial thromboplastin time (APTT), and thrombin time. A blood coagulation test can examine a patient's hemostatic and fibrinolytic abilities. Abnormalities in blood coagulation ability mainly cause an increase in clotting time. For example, an increase in clotting time can be caused by the effects of coagulation inhibitors, a decrease in components involved in coagulation, a congenital deficiency of blood coagulation factors, or an acquired autoantibody that inhibits the coagulation reaction.

[0003] In recent years, automatic analyzers that perform automatic measurements of blood coagulation tests have been widely used, and blood coagulation tests can be easily performed. For example, in some automatic analyzers, a 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 scattered light obtained. In a normal blood coagulation reaction, the amount of scattered light increases rapidly due to the progress of coagulation after a certain amount of time has passed since the addition of the reagent, 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 change in the amount of scattered light over time. Several methods, such as a percentage detection method and a differential method, are used to calculate the coagulation time using an automatic analyzer. Among them, the percentage detection method allows the measurement of the coagulation time quite accurately even for abnormal samples such as low fibrinogen samples, chyle samples, and hemolysis samples by detecting the point where the amount of change in scattered light per unit time is large, for example, the point where the amount of maximum scattered light reaches 50%.

[0004] On the other hand, photometric data from an analyzer contains various noises due to the condition of the device, reagent, and specimen, which may result in erroneous detection of the clotting time. In the automatic analysis of blood samples, it is required to remove the adverse effects of noise and calculate a reliable clotting time. A method for avoiding erroneous detection of the clotting time due to noise in photometric data has been devised. Patent Document 1 describes a blood clotting time measurement method in which scattered light amount data obtained in real time from an analyzer is smoothed and an origin is adjusted to obtain reference data X, and reference integral data Y obtained by further integrating the reference data is calculated from the reference data, and reference ratio data Z, which is a ratio of an integrated value of the reference data at each adjacent minute time, is calculated, and a reference data value Xd is selected from among the points at which the reference ratio data Z becomes a predetermined constant reference ratio data value Zs, at a point after the peak of the reference ratio data Z and at a point at which the reference integral data Y becomes equal to or greater than a predetermined threshold value Ys, and the time from the mixing point to the point at which the value of 1 / N (N is a constant integer equal to or greater than 1) of the Xd corresponds is determined as the clotting time. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Japanese Patent Application Publication No. 6-249855 Summary of the Invention [Problem to be solved by the invention]

[0006] The present invention relates to a blood coagulation time measuring method capable of accurately measuring the coagulation time of blood samples showing various blood coagulation reaction curves. [Means for solving the problem]

[0007] That is, the present invention provides the following. <1> A method for measuring blood coagulation time, comprising: [1] Smoothing and zeroing the measured value P(i) of the coagulation reaction of the blood sample to obtain the reaction X(i); 〔2〕Obtaining the integrated ratio Z(i) of the reaction X(i), where the Z(i) is the ratio of the integrated value of X in the first measurement interval to the integrated value of X in the second measurement interval adjacent to the first interval. 〔3〕Calculating the parameters te[k], X(te[k]), tc[k], te[k - 1], X(te[k - 1]), and tc[k - 1]. Here, k is an integer greater than or equal to 2; te[k] and te[k - 1] are the measurement times of i that satisfy Z(i) < Zs[k] and Z(i) < Zs[k - 1], respectively; X(te[k]) and X(te[k - 1]) are the reactions X at te[k] and te[k - 1], respectively; tc[k] and tc[k - 1] are the measurement times of i that satisfy X(i) = {X(te[k]) × Q%} and {X(te[k - 1]) × Q%}, respectively; 1 < Q < 100. 〔4〕Calculating the index R[k] and / or the index TR[k]. Here, R[k] = X(te[k]) / X(te[k - 1]) (2) TR[k] = Δtc[k] / Δte[k] (5) Δtc[k] = tc[k] - tc[k - 1] (3) Δte[k] = te[k] - te[k - 1] (4) 〔5〕When the R[k] and / or TR[k] satisfy a predetermined condition, determining the tc[k] or the tc[k - 1] as the solidification time Tc. A method including the above. <2>The method according to <1>, wherein when the R[k] or TR[k] does not satisfy the predetermined condition in step [5], k = k + 1, and steps [3] to [5] are repeated. <3>The method according to <1> or <2>, wherein Zs[k] < Zs[k - 1], Zs[k] is greater than 1, and Zs[1] is less than or equal to 1.100. <4>The method according to any one of <1> to <3>, wherein k is less than or equal to 10. <5> The difference between Zs[k-1] and Zs[k] is 0.050 to 0.001. <1> ~ <4> The method according to any one of the preceding claims. <6> Z(i) is of the formula: Z(i) = {X(i+1)+X(i+2)+...+X(i+m)} / {X(im)+X(i-m+1)+...+X(i-1)} (1) (m=10~30) Represented by <1> ~ <5> The method according to any one of the preceding claims. <7> Step [3] is performed after i reaches a predetermined calculation start point s. <1> ~ <6> The method according to any one of the preceding claims. <8> The calculation start point s is a measurement point after the time when the rate of the coagulation reaction is maximum. <7> The method described. <9> Further, when the number of measurement points between two points where V(i)=Vs (V(i) is the differential value of the reaction X(i)) exceeds a predetermined value, the later measurement point where V(i)=Vs is detected as the calculation start point s. <7> or <8> The method described. <10> The calculation start point s is a measurement point after Z(i) obtained in step [2] reaches a peak. <7> The method described. <11> Steps (1) to (2) and steps (3) to (5) are carried out in parallel. <1> ~ <10> The method according to any one of the preceding claims. <12> When Tc is not determined through the repetition of steps [3] to [5], a result indicating that Tc was not normally determined is obtained. <2> ~ <11> The method according to any one of the preceding claims. Effect of the Invention

[0008] According to the method of the present invention, it is possible to accurately measure the clotting time of blood samples that show various blood clotting reaction curves, including normal and abnormal samples. Furthermore, when analyzing a large number of samples in real time using an automatic analyzer, the method of the present invention prevents erroneous detection of the clotting time and optimizes the analysis time per sample, thereby improving the analysis efficiency. [Brief description of the drawings]

[0009] [Figure 1] 2 shows a basic flow chart of one embodiment of the blood coagulation time measuring method according to the present invention. [Diagram 2] A: Reaction X and differential value V, B: Reaction X and integral ratio Z. In both A and B, the left side is a normal sample, and the right side is an FVIII-deficient sample. [Diagram 3] 1 is a conceptual diagram of a method for detecting the calculation start point s based on the peak width of V. [Figure 4] A: Examples of Zs[1] and corresponding te[1], X(te[1]), tc[1] and X(tc[1]). B: Examples of Zs[1]~Zs[3] and corresponding te[1]~te[3], X(te[1])~X(te[3]), tc[1]~tc[3]. [Diagram 5] 2 is a detailed flow chart of one embodiment of the blood coagulation time measuring method according to the present invention. [Figure 6] X response curves for sample 1 (left) and sample 10 (right). [Figure 7] tc[k] (left) and te[k] (right) with different Zs[k]. [Figure 8] A-B: Plots of R[k](%) versus APTT and their enlargements. C-D: Plots showing the accuracy of Tc calculation by R[k](%) and their enlargements. E: X(te[k]) / X(te[8]) at different Zs[k]. [Figure 9] A: X(te[k]) / X(te[8]) at different Zs[k]. B-D: Plots of TR[k](ΔC / ΔE)(%), ΔE, and ΔC against the APTT. [Figure 10] A-B: tc[k] and te[k] calculated from samples 1-10. This method: Value when Tc was determined, 1.001: Value at Zs = 1.001, 1.031: Value at Zs = 1.031. C-D: Differences in tc[k] and te[k] between this method or Zs = 1.031 and Zs = 1.001. [Figure 11] A table showing the reaction curves of APTT-prolonged samples whose reaction did not finish within the measurement time, and the obtained parameters and indexes. [Figure 12] Response curves for low fibrinogen samples and tables showing the obtained parameters and indices. [Figure 13] Influence of the calculation starting point s detected under different conditions 1 to 3 on the parameters for Tc calculation. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] 1. Blood coagulation reaction measurement In blood coagulation tests, a specific reagent is added to a blood sample, the blood coagulation reaction is measured, and the blood coagulation time is measured from the coagulation reaction. The coagulation reaction is measured by a general means, such as an optical means for measuring the amount of scattered light, transmittance, absorbance, etc., or a mechanical means for measuring the viscosity of plasma. The coagulation reaction curve of a normal blood sample basically shows a sigmoid shape, depending on the measurement means. For example, the coagulation reaction curve based on the amount of scattered light of a normal sample usually rises sharply due to the progress of coagulation after a certain amount of time has passed since 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 sample having a coagulation abnormality shows various shapes depending on the cause of the abnormality, such as a delayed rise time of the curve or a gradual rise. The variety of coagulation reaction curves of abnormal samples makes it difficult to accurately measure the coagulation time with an automatic analyzer.

[0011] In a conventional general blood clotting time measurement, data is acquired at least until the end of the clotting reaction, and the clotting time is calculated based on the acquired data. For example, there is a method in which the end of the clotting reaction is judged to be the time when the scattered light amount is saturated, and then the clotting time is determined to be the time when the clotting reaction curve reaches the maximum speed between the time of adding the reagent and the time when the clotting reaction ends (differential method), and a method in which the clotting time is determined to be the time when the scattered light amount reaches 1 / Q of the time when the clotting reaction ends (percentage detection method, see Patent Document 1). However, due to the abnormal shape or noise of the clotting reaction curve of the abnormal sample as described above, erroneous detection of the end of the clotting reaction occurs, and for example, the end of the clotting reaction may be detected too early, or the end of the clotting reaction may not be detected. As a result of such erroneous detection of the end of the clotting reaction, an inaccurate clotting time is calculated.

[0012] In an automatic analyzer, in order to efficiently analyze a large number of samples, it is desirable to immediately end the measurement when it is determined that the coagulation reaction of one sample has ended, and to start the measurement of the next sample. However, such a method has the risk of ending the measurement even while the measurement is in progress due to the above-mentioned false detection of the end of the coagulation reaction at a too early time, resulting in missing necessary data. On the other hand, if the measurement time per sample is fixed to a sufficiently long time, it is possible to prevent missing data due to false detection of the end of the coagulation reaction, but such a method reduces the overall analysis efficiency because the measurement time is longer than necessary for many samples.

[0013] For example, when measuring the clotting time of a sample with a small change in the clotting reaction curve, such as a factor VIII (FVIII)-deficient sample, using the method described in Patent Document 1, the appropriate value of the reference ratio data value Zs is relatively small (e.g., 1.01), which means that a relatively long clotting reaction measurement is required to measure the clotting time of the sample. On the other hand, the appropriate Zs for a normal sample is a larger value (about 1.05), which means that the clotting time of a normal sample can be calculated with a shorter clotting reaction measurement. Therefore, when measuring the clotting time of all samples, including normal and abnormal samples, using an automatic analyzer according to the method described in Patent Document 1, the relatively long measurement time for some abnormal samples must also be applied to many normal samples. This is not an efficient analysis method.

[0014] The blood clotting time measuring method of the present invention prevents erroneous detection of the clotting time due to the abnormal shape of the clotting reaction curve as described above, and enables accurate measurement of the clotting time. Furthermore, according to the present invention, the measurement time can be optimized so that the minimum clotting reaction measurement time required for each clotting time measurement can be applied to various blood samples including normal samples and abnormal samples.

[0015] In the blood coagulation time measurement method of the present invention (hereinafter also referred to as the method of the present invention), the coagulation reaction of a test blood sample mixed with a reagent is measured. The blood coagulation time is measured based on the time series data of the coagulation reaction obtained by this measurement. Examples of blood coagulation times measured by the method of the present invention include prothrombin time (PT), activated partial thromboplastin time (APTT), and coagulation time in fibrinogen concentration (Fbg) measurement.

[0016] In the method of the present invention, the subject's plasma is preferably used as the test blood sample. In the following description, the blood sample is also simply referred to as the sample. An anticoagulant that is usually used in coagulation tests may be added to the sample. For example, blood is collected using a blood collection tube containing sodium citrate, and then centrifuged to obtain plasma.

[0017] A coagulation time measuring reagent is added to the test specimen to initiate a blood coagulation reaction. The coagulation reaction of the mixture after the addition of the reagent can be measured. The coagulation time measuring reagent used can be selected arbitrarily according to the purpose of measurement. Various reagents for measuring coagulation times are commercially available (for example, APTT measuring reagent Coagpia APTT-N; manufactured by Sekisui Medical Co., Ltd.). A general means, for example, an optical means for measuring the amount of scattered light, transmittance, absorbance, etc., or a mechanical means for measuring the viscosity of plasma, may be used to measure the coagulation reaction. The start time of the coagulation reaction can typically be defined as the time when the coagulation reaction is initiated by mixing the reagent with the specimen, but other timings may also be defined as the start time of the reaction. The time for which the measurement of the coagulation reaction is continued can be, for example, several tens of seconds to about 7 minutes from the time when the specimen and the reagent are mixed. This measurement time may be a fixed value determined arbitrarily, or may be until the end of the coagulation reaction of each specimen is detected. During the measurement time, the progress of the coagulation reaction can be repeatedly measured (photometrically if optically detected) at a predetermined interval. For example, the 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. In addition, various measurement conditions can be appropriately set depending on the test specimen, reagent, measurement means, etc.

[0018] A series of operations in the above-mentioned coagulation reaction measurement can be performed using an automatic analyzer. One example of an automatic analyzer is the blood coagulation automatic analyzer CP3000 (manufactured by Sekisui Medical Co., Ltd.). Alternatively, some operations may be performed manually. For example, the preparation of the test sample may be performed manually, and the subsequent operations may be performed by the automatic analyzer.

[0019] 1. Measurement of blood clotting time The present invention will be described below with reference to the basic flow of one embodiment of the method for measuring blood coagulation time according to the present invention shown in FIG.

[0020] 1.1 Obtaining the reaction X, differential value V, and integral ratio Z In the method of the present invention, the analytical device sequentially obtains measurement values ​​P(i) (e.g., photometric values ​​of the amount of scattered light) of the coagulation reaction from the start of the reaction. Here, "i" represents the measurement point number. For example, if the measurement (photometric) interval is 0.1 seconds, P(i) represents the measurement value 0.1 x i seconds after the start of measurement.

[0021] The measured value P(i) contains noise during photometry and fluctuations unrelated to the reaction that appear immediately after the start of photometry, so the measured value is smoothed by a known method. When the coagulation reaction is measured by the amount of scattered light, a zero-point adjustment process is performed to subtract the amount of scattered light from the reaction liquid before the reaction. Thus, the acquired time-series measured value P(i) is successively smoothed and zero-point adjusted to obtain the reaction X(i) (step S1). The smoothing process of the measured value may use any of various known methods for noise removal. For example, the smoothing process may include a filtering process, or a process of integrating a differential value obtained by calculating a difference value or an average slope within a section, which will be described later. In the zero-point adjustment, for example, the smoothed measured value may be adjusted so that the value at the start of measurement is 0. Furthermore, the measured value P(i) may be subjected to an initial fluctuation elimination process. The initial fluctuation elimination process may be performed so that all values ​​from the start of photometry to a predetermined initial fluctuation elimination time are 0. Essentially, as shown in FIG. 2, the response X(i) constitutes a smoothed and zeroed clotting response curve.

[0022] Next, the integrated ratio Z(i) of the reaction X(i) is obtained from the reaction X(i) (step S2). Furthermore, a differential value V(i) of the reaction X(i) may be obtained. The differential value V(i) can be used to detect the calculation start point s, which will be described later.

[0023] The differential value V(i) is obtained by first-order differentiation of the reaction X(i). The differentiation process can be performed by any method, for example, by calculating the average slope value within the interval. In calculating the average slope value within the interval, a certain number of measurement points before and after each measurement point i, for example, 2K+1 measurement points from iK to i+K, can be used. For example, five measurement points i-2, i-1, i, i+1, and i+2 can be used. The average slope value means the slope value when these multiple measurement points are linearly approximated. A common method such as the least squares method can be used as a calculation method for linear approximation. The average slope value of these measurement points can be regarded as the first-order differential value at the measurement point i.

[0024] The integrated ratio Z(i) is the ratio between the integrated value of X in a first measurement section and the integrated value of the reaction X in a second measurement section adjacent to the first section. More specifically, Z(i) is the ratio between the integrated value of the reaction X in a first measurement section before the measurement point i (for example, X(im) to X(i-1) at measurement points [im] to [i-1]) and the integrated value of the reaction X in a second measurement section after the measurement point i (for example, X(i+1) to X(i+m) at measurement points [i+1] to [i+m]). Here, the first measurement section and the second measurement section are preferably of the same length. That is, when the reaction X at the measurement point i is X(i) and m is a constant, the integrated ratio Z(i) at the measurement point i is calculated by the following formula (1). Z(i) = {X(i+1)+X(i+2)+...+X(i+m)} / {X(im)+X(i-m+1)+...+X(i-1)} (1) In formula (1), m is preferably set so that the time width of the minute measurement section is 1 to 3 seconds. That is, if the measurement (photometry) time interval is 0.1 seconds, m is preferably 10 to 30, and if the measurement (photometry) time interval is 0.2 seconds, m is preferably 5 to 15. The optimal value of m can be selected so that an appropriate coagulation time can be calculated taking into account the measurement conditions. Therefore, the calculation of the integrated ratio Z(i) can be performed after X(i+m) at the measurement point i+m is acquired as the 2m+1th data for X. However, in order to avoid the influence of the fluctuation of X at the beginning of the reaction, it is preferable to start the calculation of Z(i) on the condition that X(im) to X(i-1) are all equal to or greater than a predetermined threshold value Xs. Since the reaction curve by the scattered light measurement is sigmoidal, usually, once X(i) exceeds Xs, X(i)>Xs remains the same thereafter. If X(i)>Xs and then X(i)≦Xs, the data can be regarded as abnormal. Xs is preferably 10% to 50% of the maximum value of the predicted reaction X. For example, if the maximum value of the reaction X is estimated to be about 700, Xs is preferably 70 to 350.

[0025] By the above procedure, the reaction X(i), differential value V(i), and integral ratio Z(i) at each measurement point i can be obtained. With reference to FIG. 2, the relative differences in the shapes of the reaction X, differential value V, and integral ratio Z for a normal sample with a normal APTT and an FVIII-deficient sample (hereinafter, a prolonged sample) with a prolonged APTT will be described. FIG. 2A shows the reaction X and differential value V, and FIG. 2B shows the reaction X and integral ratio Z. In both FIG. 2A and B, the left is a normal sample, and the right is a prolonged sample. The horizontal axis is converted into time from the start of the reaction. Reaction X) As shown in Figure 2A, X constitutes the smoothed and zero-adjusted clotting reaction curve. In normal specimens (Figure 2A, left), the rising point of X is early and the rising slope is steep. However, in prolonged specimens (Figure 2A, right), the rising point of X is late and the rising slope is shallow. In the normal specimen (Figure 2A, left), the top position of the V peak is high and the shape is almost symmetrical. However, in the extended specimen (Figure 2A, right), the top position of the V peak is low (in this example, it is smaller than one-sixth of that of the normal specimen), the shape is asymmetrical, and the peak is bimodal instead of single. In a normal sample (Figure 2B, left), Z reaches a maximum value of about 8 when the reaction curve rises, and then rapidly decreases toward 1. On the other hand, in an extended sample (Figure 2B, right), Z reaches a maximum value of about 2.5, and the decrease is also gradual. In either case, the cumulative ratio Z approaches 1 as the clotting reaction curve approaches the plateau (the blood clotting reaction approaches completion).

[0026] 1.2 Calculation of clotting time Tc 1.2.1 Determining the starting point for calculations In the method of the present invention, the clotting time Tc is calculated using the above reaction X(i), differential value V(i), and integrated ratio Z(i). In the method of the present invention, X, V, and Z can be acquired in parallel with the clotting reaction measurement procedure described below. First, the timing to start the calculation process of the clotting time Tc, that is, the calculation start point s, is determined based on the X, V, and Z acquired so far. In one example, any measurement point after Z reaches its peak can be set as the calculation start point s. For example, after Z reaches a threshold value, the measurement point at which Z reaches the threshold value again after passing through the peak can be set as the calculation start point s. Taking FIG. 2B as an example, after the integrated ratio Z reaches 2, the measurement point at which Z reaches 2 again after passing through the peak can be set as the calculation start point s. In another example, the measurement point at which the coagulation reaction rate reaches the maximum value Vmax is obtained and set as the calculation start point s. Most simply, the measurement point corresponding to the first peak top in the curve composed of sequentially acquired V(i) can be set as the calculation start point s corresponding to Vmax. On the other hand, when determining the calculation start point s, it is desirable to eliminate the influence of false detection of Vmax and fluctuations caused by noise that may occur when the coagulation reaction rate curve, which is often seen in coagulation factor-deficient specimens, becomes bimodal (for example, the right side of FIG. 2A). Therefore, in a preferred example, the measurement point when sequentially acquired V(i) exceeds a predetermined value, passes through the peak, and reaches a predetermined condition can be set as the calculation start point s. FIG. 3 shows a conceptual diagram of an example of the method for determining the calculation start point s. FIG. 3 shows V(i) and the parameters used for determining the calculation start point s. V(i) forms a curve with the peak value Vmax. Vs is the value of S% when Vmax is set to 100%. V(i) once reaches Vs, passes through the peak, and reaches Vs again at V(s). When the peak width Ws, which is the width (number of measurement points) between two points where V(i)=Vs, exceeds a predetermined value, the measurement point s at V(s) is detected as the calculation start point s.

[0027] 1.2.2 Parameter acquisition Next, the parameters necessary for Tc calculation are acquired (step S3). First, the measurement time (time from the reaction start time) te[1] at the measurement point when Z(i) first becomes less than a predetermined first threshold Zs[1] (Z(i)<Zs[1]) after the calculation start point s is detected. If necessary, the acquisition of Z(i) is continued until Z(i)<Zs[1] is satisfied. When te[1] is detected, the reaction X(te[1]) at te[1] is determined. Next, the measurement time tc[1] at the measurement point corresponding to X(i) that has a value of Q% of X(te[1]) or approximates it is detected (where tc[1] < te[1]). For example, the time tc[1] that satisfies X(tc[1]) = {X(te[1]) × Q%} can be detected. Also for example, the value closest to {X(te[1]) × Q%} can be set as X(tc[1]). Also for example, when X(tc’[1]) < {X(te[1]) × Q%} < X(tc”[1]) (tc’[1] and tc”[1] are the times at adjacent measurement points), either tc’[1] or tc”[1] can be detected as tc[1]. When the measurement (photometry) interval is sufficiently short (for example, 0.1 second, etc.), since there is no significant difference in the calculated value of the final Tc, either tc’[1] or tc”[1] can be selected. Q can be any value such that 0 < Q < 100, preferably 10 - 80, more preferably 20 - 70 (for example, 50). Figure 4A shows a conceptual diagram representing the relationship between Zs[1], te[1] and X(te[1]) on the solidification reaction curve (the curve composed of X(i)), and tc[1] and X(tc[1]).

[0028] Furthermore, when Z(i) first reaches less than a predetermined second threshold Zs[2] (Z(i) < Zs[2]), the measurement time te[2] at the measurement point is detected (where Zs[2] < Zs[1]). If necessary, the acquisition of Z(i) is continued until Z(i) < Zs[2] is satisfied. The X at te[2] is X(te[2]). Next, the time tc[2] at the measurement point corresponding to X(i) that has a value of Q% of X(te[2]) or approximates it is detected (at this time, tc[2] < te[2]).

[0029] The obtained te above is the time when Z(i) reaches the threshold value Zs, representing the provisional solidification end point. On the other hand, tc is the time when the value of X becomes Q% of X at the provisional solidification end point, representing the provisional solidification point. Figure 4B is a conceptual diagram showing Zs[1] to Zs[3], and the corresponding provisional solidification end points te[1] to te[3] on the reaction curve X, and X(te[1]) to X(te[3]) at that time, as well as the provisional solidification points tc[1] to tc[3]. As shown in Figure 4B, as the reaction curve approaches the plateau (the solidification reaction approaches the end), the increasing width of te becomes larger, while the increasing width of X(te) gradually becomes smaller. On the other hand, as the solidification reaction curve approaches the plateau, tc also gradually increases, but the change is smaller compared to te.

[0030] 1.2.3 Calculation of Tc In the method of the present invention, a Tc calculation step is performed to determine whether the provisional solidification point tc is the true solidification time Tc by using the changes in the provisional solidification end point te and the provisional solidification point tc accompanying the progress of the solidification reaction as described above. That is, in the method of the present invention, parameters such as te, tc, and X(te) derived from Zs obtained above are used to obtain an index for Tc calculation (step S4).

[0031] Therefore, in the method of the present invention, the measurement point when Z(i) first reaches less than the k-th threshold value Zs[k] (Z(i) < Zs[k]) is detected (where Zs[k] < Zs[k - 1], k is an integer of k≥2). If necessary, the acquisition of Z(i) is continued until Z(i) < Zs[k] is satisfied. Then, parameters derived from Zs[k] (for example, te[k], tc[k], X(te[k]), etc.) and parameters derived from the (k - 1)-th threshold value Zs[k - 1] (where Zs[k] < Zs[k - 1]) obtained previously (for example, te[k - 1], tc[k - 1], X(te[k - 1]), etc.) are acquired.

[0032] Preferably, the index can be obtained based on any one or more of te[k], tc[k], and X(te[k]) and any one or more of te[k - 1], tc[k - 1], and X(te[k - 1]).

[0033] In the Tc calculation step, if the index satisfies a predetermined criterion, the tentative freezing point tc[k] or tc[k-1] is determined as the coagulation time Tc. Therefore, the index may be obtained after te[2], tc[2], and X(te[2]) derived from the second threshold Zs[2] are obtained. Preferably, the first index may be obtained based on te[1], tc[1], or X(te[1]) derived from Zs[1] and te[2], tc[2], or X(te[2]) derived from Zs[2] when k=2.

[0034] In one embodiment, the index is a difference or ratio between X(te[k]) derived from the k-th threshold Zs[k] and X(te[k-1]) derived from the previous threshold Zs[k-1]. Preferably, R[k] according to the following formula (2) is used as the index for calculating Tc. R[k] = X(te[k]) / X(te[k-1]) (2) The R[k] may be expressed as a ratio or a percentage. If the R[k] is equal to or smaller than a predetermined reference value Rs, the tc[k] or the tc[k-1] can be determined as the clotting time Tc. In this case, the te[k] or the te[k-1] can be determined as the true end point of clotting.

[0035] In another embodiment, Δtc[k], Δte[k], and TR[k] are first calculated according to the following formulas (3) to (5). The TR[k] is used as an index for calculating Tc. Δtc[k]=tc[k]-tc[k-1] (3) Δte[k]=te[k]-te[k-1] (4) TR[k]=Δtc[k] / Δte[k] (5) The TR[k] may be expressed as a ratio or a percentage. If the TR[k] is equal to or smaller than a predetermined reference value TRs, the tc[k] or the tc[k-1] can be determined as the clotting time Tc. In this case, the te[k] or the te[k-1] can be determined as the true end point of clotting.

[0036] The reference values ​​Rs and TRs can be appropriately set so that the accuracy of Tc determined by the present invention is a desired value. For example, it is preferable to adjust Rs and TRs so that the Tc determined by the method of the present invention for various samples is 90% or more, preferably 95% or more, of the actually measured clotting time. The actually measured clotting time can be estimated from the end point of the clotting reaction of the visually measured clotting reaction curve. Alternatively, the Tc obtained when Zs is close to 1 (for example, Zs = 1.001) may be estimated as the actually measured clotting time. For example, Rs (%) is preferably 110% or less, more preferably 105% or less, and TRs (%) is preferably 15% or less, more preferably 10% or less.

[0037] In a preferred embodiment, when both of the above criteria are met, for example, when R[k]≦Rs and TR[k]≦TRs, the tc[k] or the tc[k-1] can be determined as the coagulation time Tc.

[0038] On the other hand, if the index does not satisfy the predetermined criterion, the above procedure is repeated with k = k + 1. This procedure is repeated while incrementing k until an index that satisfies the criterion is obtained.

[0039] A specific example of the procedure for obtaining the index and determining Tc is described below. Preferably, an index for calculating Tc is first calculated using parameters te[1], tc[1], or X(te[1]) related to Zs[1] and parameters te[2], tc[2], or X(te[2]) related to Zs[2].

[0040] In one embodiment, the index is the difference or ratio between X(te[1]) and X(te[2]). Preferably, R[2] according to the following formula is used as the index for calculating Tc. R[2](%)={X(te[2]) / X(te[1])}(%) (2)'

[0041] In a preferred embodiment, first, Δtc[2], Δte[2], and TR[2] are calculated according to the following formula. The TR[2] is used as an indicator for calculating Tc. Δtc[2]=tc[2]-tc[1] (3)’ Δte[2]=te[2]-te[1] (4)’ TR[2](%)={Δtc[2] / Δte[2]}(%) (5)’

[0042] When the indicator meets a predetermined criterion, tc[1] or tc[2] is determined as the solidification time Tc. Preferably, tc[2] is determined as Tc. For example, if the above R[2] is equal to or less than a predetermined reference value Rs, the tc[2] or the tc[1] is determined as Tc. Also, for example, if the above TR[2] is equal to or less than a predetermined reference value TRs, the tc[2] or the tc[1] is determined as Tc. Preferably, when both of the above two criteria are met, for example, when R[2]≦Rs and TR[2]≦TRs, the tc[2] or the tc[1] is determined as Tc.

[0043] On the other hand, when the indicator does not meet a predetermined criterion, for a third threshold value Zs[3] (where Zs[3]<Zs[2]), te[3], tc[3] or X(te[3]) is obtained in the same manner as above, and an indicator is calculated from these and te[2], tc[2] or X(te[2]) by the same procedure. For example, when R[2] is greater than Rs and TR[2] is greater than TRs, for the third threshold value Zs[3], X(te[3]) is obtained in the same manner as above, and then R[3] is calculated according to the above formula (2), and / or te[3] and tc[3] are obtained, and then TR[3] is calculated according to the above formulas (3) to (5). If the calculated indicator meets the criterion, the tc[2] or the tc[3] is determined as Tc. Preferably, when R[3]≦Rs and / or TR[3]≦TRs, the tc[3] or the tc[2] is determined as Tc.

[0044] If the index does not meet the standard, repeat the same procedure for a fourth threshold value Zs[4] (where Zs[4] < Zs[3]).

[0045] In this way, while sequentially performing the measurement of the coagulation reaction and the acquisition of X, Y, and Z in parallel until Tc is determined, repeat the same procedure for the Nth threshold value Zs[N] (where Zs[N] < Zs[N - 1]; N ≥ 2). Finally, the tc[N] or the tc[N - 1] is determined as Tc. If the difference between Zs[N] and Zs[N - 1] is set within an appropriate range, since tc[N] and tc(N - 1) show relatively close values when R[N] ≤ Rs and / or TR[N] ≤ TRs are satisfied, whether tc[N] or tc(N - 1) is selected for Tc, no practical problem will occur in the clinical judgment using Tc. However, from the perspective of enhancing the reliability of the calculated value of Tc, it is desirable to select tc[N] for Tc.

[0046] The method of the present invention is basically an application of the percentage method that has been conventionally used as a general calculation method for blood coagulation time. In the percentage method, the time point when the coagulation reaction progresses to Q% (for example, 50%) at the end of the reaction is determined as the coagulation time Tc. Therefore, also in the method of the present invention, it is ideal that the time point when the coagulation reaction reaches Q% when the integrated ratio Z is 1 or close to it (when the blood coagulation reaction is almost finished) is calculated as the coagulation time Tc. If the calculation conditions are appropriate, since te[k] is the time point before the sigmoid-shaped coagulation reaction curve approaches the plateau, the coagulation time Tc determined by the method of the present invention will not be significantly different from the ideal value. Also in the percentage method, when the coagulation reaction is set as the coagulation time at a ratio C% (for example, 45% or less or 55% or more) away from 50% at the end of the reaction, after obtaining the 50% coagulation time with Q% = 50%, the coagulation time at C% may be calculated based on the same end point of the reaction.

[0047] 1.2.4 Threshold Zs As shown in FIG. 2, Z(i) approaches 1 as the coagulation reaction curve composed of X(i) approaches a plateau. Therefore, Zs[k] is a value greater than 1. When Zs[k] reaches a preset minimum value Zsmin, the repetition of the above procedure ends, and the tc[k] or the tc[k-1] calculated based on the Zs[k], preferably the tc[k], is determined as the coagulation time Tc. Zsmin may be greater than 1, and may be appropriately set according to the measurement conditions (e.g., reagent, analyzer, and analysis item). For example, in the case of APTT measurement, Zsmin is preferably 1.0001 to 1.05, more preferably 1.0002 to 1.01, and even more preferably 1.0005 to 1.002 (e.g., 1.001).

[0048] Alternatively, when the incremented integer k reaches a preset maximum number N, the repetition of the above procedure ends, and tc[N] or tc[N-1], preferably tc[N], calculated based on Zs[N] is determined as the coagulation time Tc. From the viewpoint of efficiency of calculating the coagulation time, N is preferably 20 or less, more preferably 10 or less, and further preferably 8 or less.

[0049] The first threshold value Zs[1] may be appropriately set according to the measurement conditions (e.g., reagent, analyzer, and analysis item). For example, in the case of APTT measurement, it is preferably 1.100 or less, more preferably 1.080 or less, and even more preferably 1.050 or less. For example, in the APTT measurement of an FVIII-deficient sample, the integrated ratio Z at the maximum coagulation reaction rate is often 1.03 to 1.04, so Zs[1] may be selected to be about 1.04.

[0050] The interval of the threshold Zs (the difference between Zs[k-1] and Zs[k]; ΔZs) can be determined depending on Zs[1], Zsmin, the maximum number N of k, or the measurement frequency (sampling period) of the coagulation reaction, but from the viewpoint of accuracy and efficiency of calculating the coagulation time, it is preferably in the range of 0.050 to 0.001, more preferably in the range of 0.020 to 0.002, and even more preferably in the range of 0.010 to 0.005.

[0051] It is preferable that each value is set so that Zs[1], Zsmin, the maximum number N of k, and ΔZs are within the appropriate ranges described above. For example, preferable Zsmin, ΔZs, and N can be determined, and Zs[1] can be determined accordingly. Alternatively, preferable Zs[1], Zsmin, and N can be determined, and ΔZs can be determined accordingly.

[0052] For example, when Zsmin = 1.001, k ≤ 8, and ΔZs = 0.005, Zs[1] = 1.036. However, the examples of the combinations of Zs[1], Zsmin, ΔZs, and the number of repetitions in the method of the present invention are not limited to these, and those skilled in the art can appropriately set appropriate values.

[0053] 1.2.5 Error handling If te[k] that satisfies Z(i) < Zs[k] is not found through repeated execution of the above-described procedure, or if the index does not meet the criteria, the Tc calculation step ends incompletely without Tc being determined. In such a case, in the method of the present invention, the last acquired tc[k] may be determined as Tc, or it can be regarded that Tc cannot be calculated. In either case, when Tc is not determined, a result indicating that Tc was not determined normally can be obtained.

[0054] 2. Exemplary procedure for determining Tc Hereinafter, an example of a more specific procedure for determining the solidification time Tc according to the present invention will be described in more detail with reference to the detailed flow shown in FIG. 5. The procedure shown in FIG. 5 is an example, and ordinary changes by those skilled in the art, such as changing the order of operations, are possible.

[0055] S001: Set Zs stepwise from a high value to a low value in advance as shown in Table 1 below, and create an array Zs[k] so that each Zs can be sequentially selected by a variable k (counter). k is an integer, and 1 is set as the initial value.

[0056] [Table 1]

[0057] S002: After the number of data points P(i) (where i represents the measurement point number) regarding the solidification reaction exceeds the number of data points required for the smoothing process, the initial fluctuation removal process, and the zero point adjustment process (for example, 50 data points), start acquiring the reaction X(i), the differential value V(i), and the integration ratio Z(i).

[0058] S003: While continuing to acquire the photometric data and X(i), V(i), and Z(i), search for the time te[1] at the earliest i after the calculation start point s that satisfies Z(i) < Zs[1]. From the searched te[1], obtain parameters such as X(te[1]) and tc[1] as described in 1.2.2 above.

[0059] S004: If it is impossible to obtain the time te[1] at i that satisfies Z(i) < Zs[1] in S003, branch to S020 when i reaches the upper limit value, determine that "reaction not detected", and make it impossible to calculate the solidification time. For example, when the measurement (photometric) time is set at 0.1 - second intervals for 360 seconds, the maximum number of measurement points is 3600, which is the upper limit value of i. On the other hand, if te[1] can be obtained, branch to S005.

[0060] S005: Increment k.

[0061] S006: When the previous process is S005, k = 2, and when the previous process is S010, k is an integer of 3 or more. Then, while continuing to acquire P(i), X(i), V(i), and Z(i), search for the time te[k] at i that satisfies Z(i) < Zs[k].

[0062] S007: If it is impossible to obtain te[k] in S006, branch to S030 when i reaches the upper limit value, determine that "reaction in progress", and make it impossible to calculate the solidification time. The upper limit value of i is the same as in S004. If it can be obtained, branch to S008.

[0063] S008: If te[k] can be obtained in S006, as described in 1.2.2 above, parameters such as X(te[k]) and tc[k] are determined. From te[k - 1] that satisfies Z(i) < Zs[k - 1], parameters such as X(te[k - 1]) and tc[k - 1] are determined, and an index for Tc determination is calculated. Examples of such an index include R[k] represented by the above formula (2) and TR[k] represented by the above formula (5). Either R[k] or TR[k] may be used as the index, or both may be used as the index. Next, it is examined whether the index satisfies the criterion. The criteria include the following (a) or (b), and preferably, it is required that both the following (a) and (b) are satisfied. Criterion (a): R[k] ≤ Rs Criterion (b): TR[k] ≤ TRs If the above criteria are satisfied, it branches to S040, and in other cases, it branches to S009.

[0064] S009: If neither criterion (a) nor (b) holds in S008, k is incremented.

[0065] S010: When the incremented k exceeds a preset maximum value, it branches to S011. When k is less than or equal to the maximum value, it branches to S006, k is incremented, and the search for a new te[k] continues.

[0066] S011: Since neither criterion (a) nor (b) holds in S008 and k has exceeded the maximum value, the Tc determination procedure ends incompletely. When proceeding to this step, Tc may or may not be output. When outputting Tc, the last obtained tc (i.e., tc[k - 1]) is determined as Tc. The output of Tc may be automatic or manual by button operation or the like.

[0067] S020: This is the case where the coagulation reaction measurement ends without finding te[1] that satisfies Z(i) < Zs[1], and Tc cannot be calculated. If the process proceeds to this step, since it can be determined that there is no coagulation reaction for some reason, a "reaction not detected" flag may be output.

[0068] S030: Since te[k] that satisfies Z(i) < Zs[k] was not found, Tc cannot be calculated. If the process proceeds to this step, since it can be determined that the measurement ended during the coagulation reaction, a "reaction in progress" flag may be output.

[0069] S040: The coagulation end point can be confirmed, and tc[k - 1] or tc[k] is output as Tc. Preferably, tc(k) is output as Tc. There is no flag, or a "normal end" flag may be output.

[0070] 3. Application to Other Coagulation Reaction Measurement Methods The method of the present invention is basically a blood coagulation time measurement method using a blood coagulation reaction measurement based on the amount of scattered light. However, those skilled in the art can apply the method of the present invention to other measurement methods, for example, a blood coagulation time measurement method using a blood coagulation reaction measurement based on transmittance, absorbance, etc.

Examples

[0071] 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.

[0072] Example 1 1. Method For a plurality of mixed samples obtained by mixing a blood specimen (abnormal specimen) derived from a subject with an abnormal coagulation reaction due to blood coagulation factors and a normal blood specimen (normal specimen) at different ratios, the coagulation time was measured according to the method of the present invention.

[0073] 1) Samples As an abnormal sample, Factor VIII Deficient Plasma (manufactured by George King Bio-Medical, Inc.) (hereinafter, FVIII-deficient plasma) with a factor VIII (FVIII) concentration of 0.1% or less was used. As a normal sample, normal pooled plasma (hereinafter, normal plasma) with a FVIII concentration considered to be 100% was used. Mixed plasmas (samples 1 to 9, respectively) with FVIII concentrations of 50%, 25%, 10%, 5%, 2.5%, 1%, 0.75%, 0.5%, and 0.25% were prepared by changing the mixing ratio of FVIII-deficient plasma and normal plasma. In addition, sample 10 was prepared from FVIII-deficient plasma alone (FVIII concentration of 0.1% or less).

[0074] 2) Reagents The APTT measurement reagent used was Coagpia APTT-N (manufactured by Sekisui Medical Co., Ltd.) The calcium chloride solution used was Coagpia APTT-N calcium chloride solution (manufactured by Sekisui Medical Co., Ltd.)

[0075] 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 measurement reagent heated to about 37°C was added to the cuvette, and after 171 seconds, 50 μL of 25 mM 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).

[0076] 4) Preparation of coagulation reaction curve A photometric curve P was obtained from the photometric data, and smoothing processing including noise removal was performed on the P. After that, a zero-point adjustment processing was performed so that the amount of scattered light at the start of photometry was 0, and the reaction X was calculated.

[0077] 5) Obtaining the differential value V and the integral ratio Z From the reaction X, the first derivative value V and the integrated ratio Z were calculated according to the following formula (1). Z(i) = {X(i+1)+X(i+2)+...+X(i+m)} / {X(im)+X(i-m+1)+...+X(i-1)} (1) m = 20 The calculation of Z was started on the condition that X(im) through X(i-1) were all equal to or greater than Xs. Xs was set to 350.

[0078] 6) Calculation starting point The calculation start point s was set to the point where Z(i) exceeded a predetermined value and then fell below that predetermined value, which was set to 1.2.

[0079] 7) Setting the threshold Zs Zs[k] was set as shown in Table 2 below. [Table 2]

[0080] 8) Parameters te[k], tc[k], X(te[k]), te[k-1], tc[k-1], X(te[k-1]) (k=integer from 2 to 8).

[0081] 9) Index for calculating coagulation time Tc Index (a): R[k](%)(= X(te[k]) / X(te[k-1]) × 100) Index (b): TR[k](%)(= ΔC / ΔE × 100) ΔC = tc[k]-tc[k-1] ΔE = te[k]-te[k-1]

[0082] 10) Calculation process From the photometric data, the reaction X, the integrated ratio Z, and, if necessary, the differential value V were sequentially obtained, and from the calculation start point s onwards, the parameters: te[k], tc[k], X(te[k]), te[k-1], tc[k-1], and X(te[k-1]) were calculated using Zs[k] shown in Table 1, and further, the index (a): R[k] (%) and the index (b): TR[k] (%) were calculated. The tc[k] when the following criteria (a) and (b) were met was taken as the clotting time Tc. The initial value of k was k=1, and the above process was repeated while increasing it by 1 up to a maximum of 8 until the following criteria (a) and (b) were met. If the criteria were not met at k=8, tc[8] was taken as Tc. Criteria (a): R[k](%)≦Rs, Rs=105% Criteria (b): TR[k](%)≦TRs, TRs=10% In this embodiment and the following embodiments, in order to evaluate the determined Tc, the coagulation reaction measurement was continued up to the maximum measurement time even after the Tc determination criteria were met (both criteria (a) and (b) below were met), and parameters and indexes were obtained.

[0083] 2. Evaluation of Tc calculation criteria 1) Changes in the provisional solidification end point te and provisional solidification point tc over time Figure 6 shows the reaction curves of X for sample 1 (left, FVIII concentration 50%) and sample 10 (right, FVIII concentration 0.1% or less) against the measurement time. The markers on the curves are the provisional coagulation end point te[k] (white square) and the provisional coagulation point tc[k] (white circle) (k=2-8, Zs[k]=1.031-1.001). For both samples 1 and 10, tc[2] and te[2] were the smallest, and tc[8] and te[8] were the largest. For both samples 1 and 10, the provisional coagulation end point te[8] (Zs[8]=1.001) was a value that could be visually determined as the true coagulation reaction end point. The values ​​of tc[k] were almost the same for sample 1, but were different for sample 10.

[0084] The time point when Z(i) < Zs[8](=1.001) is satisfied was defined as the coagulation end point, and the time point when X was 50% of the coagulation end point was defined as APTT. Fig. 7 shows plots of tc[k] (left) and te[k] (right) against APTT for Zs[k] (k = 1 to 8, Zs[k] = 1.036 to 1.001) for Samples 1 to 10. As APTT increased, the distribution width of the pseudo coagulation point tc[k] widened. A similar tendency was confirmed for the pseudo coagulation end point te[k]. However, the slowest point te[8] was located at a position farther from the other points.

[0085] 2) Tc determination criteria for index (a) Figs. 8A and 8B are plots of the index (a): R[k](%) against APTT (A) and an enlarged view thereof (B) obtained for Samples 1 to 10. Even though R[k] was derived from the same Zs value, its distribution differed depending on APTT. From Fig. 8B, it was found that by setting the reference value Rs for Tc determination to 105%, R[k] ≤ Rs in approximately 96% of the cases, and the coagulation time Tc(APTT) could be accurately calculated.

[0086] Figures 8C and 8D are plots (C) and their enlarged views (D) showing the accuracy of Tc calculation by R[k] (%) from samples 1 to 10, by the corresponding Zs[k]. The vertical axis shows the relative Tc calculation value at Zs[1] to [7], where the calculated Tc value at Zs=1.001 (Zs[8]) for each sample is taken as 100%, and indicates the accuracy (%) of the calculated value. There were no Zs values ​​with an accuracy of 96% or more for sample 10 (APTT=about 130 seconds) (even Zs[7]=1.006 did not reach 96%), and for sample 9 (APTT=about 90 seconds), Zs[7]=1.006 and Zs[6]=1.011 were applicable. Figure 8E is a plot showing X(te[k]) / X(te[8]) (%) by the corresponding Zs[k]. The vertical axis is the relative value of X(te[1])~X(te[7]) at Zs[1]~[7] when X(te[8]) at Zs=1.001(Zs[8]) for each sample is 100%, and represents the relative height (%) of the reaction curve X. The relative height is distributed according to the size of the APTT. In comparison with Figure 8A, it can be seen that as the relative height of the reaction curve approaches 100%, R[k] also decreases toward 100%.

[0087] From the above, it was shown that when determining the clotting time Tc, [R[k](%)≦105%] can be set as the criterion indicating that the relative height of the reaction curve has reached 90% (i.e., the clotting reaction is in the terminal stage).

[0088] 3) Tc determination criteria for indicator (b) Figure 9A is a plot of X(te[k]) / X(te[8]) (%) for each corresponding Zs[k] (same as Figure 8E), and Figures 9B-D are plots of the index (b): TR[k] (=ΔC / ΔE) (%), ΔE, and ΔC against APTT. ΔE and ΔC tended to increase with the APTT value, and the amount of change was larger for ΔE than for ΔC (Figures 9C-D). As a result, as shown in Figure 9B, TR[k] (=ΔC / ΔE), which is the ratio of the amount of change between the two, also increased with APTT. The distribution of TR[k] against APTT showed the same behavior as when the vertical axis of Figure 9A (X(te[k])) was reversed. Therefore, it was shown that TR[k] indirectly reflects the relative height of the response curve at X(te[k]).

[0089] 3.Tc determination The parameters and indices obtained for samples 1 to 10 are shown in Tables 3 to 6. The Tc calculation process was completed without reaching the maximum measurement time for any sample. The columns for parameters and indices after Tc was determined are shaded. The gray columns indicate that the indices met criteria (a) or (b).

[0090] [Table 3]

[0091] [Table 4]

[0092] [Table 5]

[0093] [Table 6]

[0094] In Figures 10A and 10B, the provisional coagulation point tc[k] (A) and the provisional coagulation end point te[k] (B) when the coagulation time Tc was determined are plotted against the APTT (in the figure, this method). In the figure, tc[k] and te[k] when Zs = 1.001 (k = 8) as a control and Zs = 1.031 (k = 2) as a comparative example are also plotted. In addition, the difference obtained by subtracting the values ​​of tc[k] and te[k] in the control (Zs = 1.001, k = 8) from the values ​​in the present method or Comparative Example 1 is shown in Figures 10C and D, respectively. When Tc was determined by this method, tc[k] (= determined Tc) overlapped with tc[k] (almost equal to APTT) in the control. The difference in tc[k] between this method and the control was within 1 second. On the other hand, te[k] in this method was close to the comparative example when the APTT was short, but overlapped with the control after APTT 90 seconds. te[k] was 33 seconds at the shortest (right figure). This indicates that when the APTT is less than 90 seconds, the coagulation reaction measurement time required to obtain Tc by this method is about 20 to 30 seconds shorter than the control. On the other hand, between the comparative example and the control, the difference in tc[k] (= determined Tc) was within 1 second when the APTT was up to 50 seconds, but increased with the extension of the APTT, reaching a maximum of 29 seconds, while the difference in te[k] was a minimum of 26 seconds and a maximum of 142 seconds. That is, in the comparative example, although the coagulation reaction measurement time was shorter than that of the control, when the APTT was less than 50 seconds, the coagulation time was equivalent to that of the control, but when the APTT was greater than 50 seconds, the calculated coagulation time was shorter than that of the control. From the above, it was demonstrated that the method of the present invention can achieve both accurate calculation of the coagulation time and shortening of the measurement time.

[0095] Example 2 The specimens used were Factor XII Deficient Plasma (manufactured by George King Bio-Medical, Inc.) with a factor XII concentration of less than 0.1% (hereinafter referred to as FXII deficient plasma, FXII concentration 0%) and normal pooled plasma (hereinafter referred to as normal plasma) with an FXII concentration of 100%, and a mixed plasma (sample 11) with an FXII concentration of 0.25% was prepared by mixing FXII deficient plasma and normal plasma in a mixing ratio of 1:39.

[0096] For the prepared sample 11, a Tc satisfying the criteria (a) and (b) was searched for in the same procedure as in 1.10) of Example 1. However, for this sample, although te[k] and te[k] were obtained up to k=6 (Zs=1.011), the maximum measurement time was reached before k=7 (Zs=1.006) was obtained, and the Tc calculation process was terminated. As a result, in this example, the clotting time Tc was not output, and a flag indicating "reaction in progress" was output. The reaction curve X (te[k] and te[k] are shown) for sample 11, and the calculated parameters and indicators are shown in FIG. 11.

[0097] Example 3 For a specimen (sample 12) with a low fibrinogen concentration (62 mg / dL), Tc satisfying criteria (a) and (b) was searched for in the same procedure as in 1.10) of Example 1. Criteria (a) and (b) were satisfied when k=2 (Zs=1.031), and the clotting time Tc was determined to be 30.9 seconds. The reaction curve X (te[k], te[k] for k=1 to 8 are shown) for sample 12, and the calculated parameters and indicators are shown in FIG. 12. The gray columns in the table in FIG. 12 indicate that the indicator satisfied criteria (a) or (b). Compared with the left side of FIG. 6, where the fibrinogen concentration is at a normal level, the maximum value of the clotting reaction in FIG. 12 was about one-eighth, but it was confirmed that the clotting time Tc can be calculated by the method of the present invention even in specimens with such low fibrinogen concentrations.

[0098] Example 4 The conditions for the calculation start point s for Tc calculation were evaluated. A specimen (sample 13) in which the reaction curve continued to rise slowly at the beginning of the reaction was used. The calculation start point s was determined according to the following conditions, and a Tc that satisfied the criteria (a) and (b) was searched for in the same procedure as in 1.10) of Example 1. Condition 1 (same as the conventional technique): The calculation start point s was the point where the integrated ratio Z peaked, that is, Z exceeded a predetermined value and then fell below that predetermined value. The predetermined value of Z was 1.2. Condition 2: When the number of measurement points between two points where V(i)=Vs on either side of a peak exceeded Ws, the later measurement point where V(i)=Vs was set as the calculation start point s (see Figure 3). Vs was set to 90% Vmax and Ws was set to 10 (1 second). Condition 3: Same as condition 2, except that Vs was set to 50% Vmax and Ws was set to 10 (1 second).

[0099] Figure 13 shows the parameters and indices obtained under conditions 1 to 3. The left figure shows the response curve X and the differential value V, and the right figure shows X and the integrated ratio Z. In the figure, the white square markers represent Te[k] and the white circle markers represent Tc[k]. The right end is a table of the parameters and indices. The gray columns in the table indicate that the indices meet criteria (a) or (b). Under condition 1, Z fell below the specified value during the gradual rise of the reaction curve X at the beginning of the reaction, so Tc calculation began before V(i) reached its maximum, and Te[k] and Tc[k] were obtained for k = 1 to 4. However, the Tc calculation process continued without satisfying criteria (a) and (b), and finally Tc of 163.6 seconds was determined for k = 8 (Zs = 1.001). In conditions 2 and 3, Tc calculation started after V(i) exceeded its peak. The black square in the figure is the calculation start point s. In both conditions 2 and 3, criteria (a) and (b) were met at k=8 (Zs=1.001), and Tc of 163.6 seconds was determined. However, in condition 3, since Te[k] at k=1-4 was close, the indexes at k=1-4 were not used in the Tc calculation.

Claims

1. A method for measuring blood coagulation time, comprising: [1] smoothing and zeroing the measured value P(i) of the coagulation reaction of the blood sample to obtain the reaction X(i); [2] Obtaining an integrated ratio Z(i) of the reaction X(i), where Z(i) is the ratio of an integrated value of X in a first measurement section to an integrated value of X in a second measurement section adjacent to the first measurement section; [3] Calculating parameters te[k], X(te[k]), tc[k], te[k-1], X(te[k-1]), and tc[k-1], where k is an integer of 2 or greater; te[k] and te[k-1] are the measurement time at i that satisfies Z(i)<Zs[k] and the measurement time at i that satisfies Z(i)<Zs[k-1], respectively; where Zs[k] and Zs[k−1] are thresholds for Z(i), and Zs[k]<Zs[k−1]; X(te[k]) and X(te[k-1]) are reactions X at te[k] and te[k-1], respectively; tc[k] and tc[k-1] are the measurement time at i that satisfies X(i)={X(te[k])×Q%} and the measurement time at i that satisfies {X(te[k-1])×Q%}, respectively; 1<Q<100; [4] Calculating the index R[k] and / or the index TR[k]; Where: R[k]=X(te[k]) / X(te[k-1]) (2) TR[k]=Δtc[k] / Δte[k] (5) Δtc[k]=tc[k]-tc[k-1] (3) Δte[k]=te[k]-te[k-1] (4) [5] When the R[k] and / or the TR[k] satisfy a predetermined condition, the tc[k] or the tc[k-1] is determined as the coagulation time Tc; Where: the predetermined condition for R[k] is that R[k] is a predetermined reference value Rs or a value smaller than the predetermined reference value Rs; the predetermined condition for the TR[k] is that the TR[k] is a predetermined reference value TRs or a value smaller than the predetermined reference value TRs; A method comprising:

2. The method according to claim 1, wherein if R[k] or TR[k] does not satisfy the predetermined condition in step [5], k=k+1 is set and steps [3] to [5] are repeated.

3. The method of claim 1, wherein Zs[k] is greater than 1 and Zs[1] is less than or equal to 1.

100.

4. The method according to any one of claims 1 to 3, wherein k is 10 or less.

5. The method according to any one of claims 1 to 4, wherein the difference between Zs[k-1] and Zs[k] is 0.050 to 0.

001.

6. Z(i) is of the formula: Z(i) = [X(i+1)+X(i+2)+...+X(i+m)] / [X(im)+X(i-m+1)+...+X(i-1)] (1) （m＝10～30） The method according to any one of claims 1 to 5, wherein the formula is

7. The method according to any one of claims 1 to 6, wherein step [3] is performed after i reaches a predetermined calculation starting point s.

8. The method according to claim 7 , wherein the calculation start point s is a measurement point after the time at which the rate of the coagulation reaction is maximized.

9. The method further includes detecting, when the number of measurement points between two points where V(i)=Vs exceeds a predetermined value, the later measurement point where V(i)=Vs is detected as a calculation start point s; Where: V(i) is the derivative of the reaction X(i), Vs is the value when V(i) is S% of its peak value; 9. The method according to claim 7 or 8.

10. 8. The method according to claim 7, wherein the calculation start point s is a measurement point after Z(i) obtained in step [2] reaches a peak.

11. The method according to any one of claims 1 to 10, wherein steps [1] to [2] and steps [3] to [5] are carried out in parallel.

12. The method according to any one of claims 2 to 11, further comprising obtaining a result that Tc was not successfully determined when Tc was not determined through the repetition of steps [3] to [5].

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