Method for analyzing blood specimen

Abstract

This method for analyzing a coagulation function of a blood specimen comprises: optically measuring a coagulation reaction of a sample containing a test blood specimen and an APTT measurement reagent to acquire coagulation reaction data; acquiring a value of an index representing a change in turbidity of the sample after the end stage of the coagulation reaction on the basis of the coagulation reaction data; and acquiring information related to fibrinolysis of the test blood specimen on the basis of the value of the index.

Description

Methods for analyzing blood samples

[0001] The present invention relates to a method for analyzing blood samples.

[0002] In the blood coagulation reaction, fibrin is produced from fibrinogen by the action of thrombin, which is generated in the blood. Subsequently, these fibrin molecules bind to each other to form fibrous fibrin polymers, and cross-linking bonds are formed between these fibrous fibrin polymers to create physically stable fibers (stabilized fibrin). The enzymatic breakdown reaction of fibrin is called the fibrinolysis reaction. Fibrinolysis reactions include the serine protease plasminogen activator (PA; vascular endothelial cell-derived tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA)) plasmin system, and other systems, such as those mediated by leukocyte-derived enzymes elastase and cathepsin.

[0003] Blood coagulation factor XIII (FXIII) is a transglutaminase that cross-links proteins and is a heterotetramer consisting of an A subunit dimer and a B subunit dimer. FXIII circulates in the blood and is activated by thrombin to cross-link fibrin, converting it into stabilized fibrin. FXIII also increases fibrin's resistance to plasmin by covalently binding the antifibrinolytic protein α2-antiplasmin to fibrin. In FXIII deficiency, fibrin cross-linking does not progress or progresses slowly, resulting in insufficient formation of stabilized fibrin and a tendency to bleed. Congenital FXIII deficiency is mainly characterized by bleeding, abnormal wound healing, and recurrent miscarriages, and is often first discovered after birth due to umbilical cord bleeding. Acquired FXIII deficiency, on the other hand, can be caused by excessive consumption or impaired production of FXIII due to massive bleeding during major surgery, or by autoimmune factors. In the latter case of acquired FXIII deficiency, severe bleeding symptoms may suddenly develop, requiring early treatment. However, FXIII deficiency cannot be detected by common coagulation screening tests such as PT and APTT tests, making diagnosis difficult. The establishment of a simple analytical method for FXIII deficiency is desired.

[0004] As methods for measuring FXIII activity, a latex agglutination reaction method using an antibody against FXIII and an absorbance method for detecting a color change caused by the reaction of activated FXIII with a substrate in a reagent are generally used. However, these methods are not suitable for screening tests.

[0005] Patent Document 1 describes a method for quantifying FXIII activity by utilizing the generation of ammonia released when active FXIII (transglutaminase) crosslinks fibrin. Patent Document 2 describes a method for analyzing a blood sample, which comprises: coagulating a blood sample in the presence of a fibrinolytic system activator to obtain a coagulation waveform; differentiating the obtained coagulation waveform to obtain values of a plurality of parameters relating to differentiation of the coagulation waveform; and obtaining information on the cause of abnormality in the fibrinolytic system of the blood sample based on the values of the plurality of parameters. As examples of causes of abnormality in the fibrinolytic system, deficiency of α2-antiplasmin (α2AP), deficiency of plasminogen activator inhibitor-1 (PAI-1), and deficiency of plasminogen are described.

[0006] Japanese National Publication of International Patent Application No. 2013-506429, Japanese Unexamined Patent Publication No. 2022-123129

[0007] A method for conveniently analyzing FXIII deficiency is desired.

[0008] The present invention provides the following as representative embodiments: (1) A method for analyzing a blood sample, comprising: optically measuring the coagulation reaction of a sample containing a blood sample and an APTT measuring reagent and obtaining coagulation reaction data; obtaining an index value representing the turbidity change of the sample after the end of the coagulation reaction based on the coagulation reaction data; and obtaining information regarding the fibrinolysis of the blood sample based on the index value. (2) The method according to (1), wherein the information regarding the fibrinolysis of the blood sample is information regarding the FXIII activity level of the blood sample. (3) The method according to (1) or (2), wherein the coagulation reaction data is data representing the change in the coagulation reaction over time. [4] The method according to [3], wherein the coagulation reaction data is a correction curve R(i) or a coagulation rate curve V(i), where R(i) is a curve obtained by relativeizing the coagulation reaction curve P(i) so that the value at the set time Te is 100%, where Te is 120 to 600 seconds, V(i) is the first derivative curve of the coagulation reaction curve P(i), and i is the number of measurement points. [5] The method according to any one of [1] to [4], wherein the index representing the turbidity change is an index representing the decrease in turbidity. [6] The index representing the turbidity transition is RmaxT, ΔR, rF, SFa, or SFb, where RmaxT is the time when R(i) is Rmax, ΔR = Rmax - 100, where Rmax is the maximum value of R(i), rF is the slope of the regression line of R(i) in the interval from the starting point St to the ending point Et, where St is a point after the starting point of the end stage of the coagulation reaction, and Et ≤ Te, SFa is the area under the curve of R(i) in the range from Ts to Te where R(i) exceeds 100, where Ts is the time when R(i) = 100 before RmaxT, and SFb is the area under the curve of R(i) in the range from RmaxT to Te where R(i) exceeds 100. [4] The method described.[7] The method described in [4], wherein the index representing the turbidity transition is rT, where rT is the number of i values ​​or the time length corresponding to the number of i values ​​that satisfy rV(i) ≥ rVs in the range from the starting point of the end of the coagulation reaction to the set time Tev, where rV(i) = V(i) / Vmax × 100, Vmax is the maximum value of V(i), Tev is 80 to 180 seconds, and rVs is 0.01 to 0.2. [8] The index representing the turbidity transition is tR or sR, where tR is the number of i values ​​or the time length corresponding to the number of i values ​​that satisfy rR(i) ≥ 100 in the range from the start of the final stage of the coagulation reaction to the set time Ter, sR is the slope of the regression line of R(i) from Ta to the set time Tes, where Ter is 90 to 600 seconds, Tes is 120 to 600 seconds, Ta is the time corresponding to the maximum value of i that satisfies R(i) ≥ 50 and rR(i) = sRs, rR(i) = {SumR(i) / SumR(i - k)} × 100, where SumR(i) is expressed by the following formula. The method according to [4], wherein i > k, k = 2 to 20, and sRs is 100.05 to 101.00. [9] The method according to any one of [1] to [8], further comprising calculating the APTT of the test blood sample based on the coagulation reaction data.

[10] The method according to any one of [1] to [9], wherein the test blood sample is a blood sample that does not show APTT extension.

[11] The method according to

[10] , wherein the test blood sample is a blood sample that does not show PT extension.

[12] The method according to any one of [1] to

[11] , further comprising estimating the fibrinogen concentration of the test blood sample based on the coagulation reaction data before obtaining the value of the index representing the turbidity transition.

[13] The method according to

[12] , wherein the coagulation reaction data is the maximum value of the coagulation reaction curve P(i), or P(i) at the starting point of the end stage of the coagulation reaction.

[14] The method according to

[13] , wherein if the maximum value of P(i) or the value of P(i) at the start of the final stage of the coagulation reaction is smaller than a predetermined value, the fibrinogen concentration of the blood sample is estimated to be lower than the lower limit of the normal range.

[15] The method according to

[14] , wherein if the estimated fibrinogen concentration is lower than the lower limit of the normal range, information indicating that the fibrinogen concentration of the blood sample is low is output, or the value of the index representing the turbidity change is not obtained.

[0009] The method of the present invention makes it possible to obtain information on fibrinolysis of blood samples using coagulation reaction data obtained from conventional APTT measurements performed in hospitals and clinics. For example, according to the method of the present invention, the FXIII activity level of a blood sample can be evaluated, thereby making it possible to easily detect blood samples with FXIII deficiency, which were previously often overlooked. Furthermore, the method of the present invention can be easily automated using an automated blood coagulation analyzer.

[0010] Coagulation reaction curve (A) and correction curve (B) for a normal sample. Coagulation reaction curves (A and C) and correction curves (B and D) for a normal sample and an FXIII-deficient sample. A, B: data up to 120 seconds, C, D: data up to 600 seconds. Solid line and × mark: normal sample, dashed line and + mark: FXIII-deficient sample. Explanation of parameters used to calculate the turbidity transition index (TI) using an FXIII-deficient sample (A) and a normal sample (B). Example of coagulation rate ratio curve rV(i) for an FXIII-deficient sample (A) and a normal sample (B). rV(i) (gray, right axis) and a magnified curve near 0% (black, left axis). Example of R ratio curve rR(i) for an FXIII-deficient sample (A) and a normal sample (B). Explanation of parameters used to calculate the R regression slope sR using an FXIII-deficient sample (A) and a normal sample (B). One embodiment of the analytical apparatus according to the present invention. A: An example of the detection process for a sample potentially deficient in FXIII using the analytical method of the present invention. B: Details of S2 in process A. Coagulation reaction curves of the samples used in the examples. A: Normal samples (N1-N10), B: FXIII deficient series samples (A5, A10, A20, A40, A60). A, B: Correction curve (A) for normal samples (N1-N10) and a magnified view of the area around 100% (B). C, D: Correction curve (C) for FXIII deficient series samples (A5, A10, A20, A40, A60) and a magnified view of the area around 100% (D). RmaxT of the samples used in the examples. ΔR of the samples used in the examples. rF of the samples used in the examples. rF of the samples used in the examples. SFa and SFb of the samples used in the examples. rT of the samples used in the examples. tR of the samples used in the examples. sR of the samples used in the examples. A-C: Coagulation reaction curves (A) and correction curves (B) and enlarged view of the area around 100% (C) for normal samples (N1-N10) and low-concentration fibrinogen samples (LF1, LF2). Solid line: normal sample, dotted line: LF1, dashed line: LF2. × marks indicate Pmax. D: Relationship between fibrinogen concentration (Fbg) and coagulation reaction P (Tb). A: Relationship between FXIII activity level and rF for series A and B. B: Estimated results of FXIII activity level for series A and B.

[0011] All patent, non-patent, and other publications cited herein are incorporated herein by reference in their entirety.

[0012] In this specification, blood samples, blood coagulation factors, blood coagulation factor deficiencies, coagulation factor activity, blood coagulation reactions, and blood coagulation times may be simply referred to as "sample," "coagulation factor," "factor deficiency," "factor activity," "coagulation reaction," and "coagulation time," respectively. For example, in this specification, "tested blood sample" may be simply referred to as "tested sample." Also, in this specification, "FXIII activity" may be simply referred to as "activity," and "changes in turbidity over time" may be referred to as "turbidity transitions."

[0013] In this specification, "FXIII deficiency" means that FXIII activity is less than 70%. In this specification, "FXIII activity is X%" means that the FXIII activity is X% relative to the FXIII activity (100%) in a standard sample. The standard sample may be a pooled sample from healthy individuals or a commercially available product (for example, Factor Assay Control Plasma from George King Bio-Medical, Inc.). In this specification, "blood sample with FXIII deficiency" may also be simply referred to as "FXIII deficiency."

[0014] In blood coagulation tests, such as the activated partial thromboplastin time (APTT), the coagulation reaction of the reaction solution (test sample) obtained by adding a predetermined reagent to a blood sample is measured, and the coagulation time is calculated from the data showing the change in the obtained coagulation reaction over time (coagulation reaction data).

[0015] In a normal coagulation reaction, thrombin, which is produced in the blood, acts to convert fibrinogen into fibrin, which then cross-links to form stabilized fibrin, leading to blood coagulation. On the other hand, in cases of fibrinolysis abnormalities, such as FXIII deficiency, the formation of stabilized fibrin from fibrin does not proceed, resulting in a decreased resistance of the formed fibrin to plasmin, and thus a bleeding tendency. However, in cases of fibrinolysis abnormalities such as FXIII deficiency, the coagulation cascade is normal, so both PT (prothrombin time) and APTT, which represent the coagulation function of the coagulation cascade as coagulation time, are within the normal range. Therefore, fibrinolysis abnormalities such as FXIII deficiency cannot be detected from the results of coagulation time measurements, which examine hemostatic function.

[0016] For measuring coagulation reactions, common methods such as optical methods for measuring scattered light, transmittance, and absorbance are used, and the change in the amount of coagulation reaction (turbidity of the test sample associated with fibrin formation) over time (turbidity transition) is measured as coagulation reaction data. The measured coagulation reaction data is generally represented by a coagulation reaction curve. When the means of measuring the coagulation reaction is scattered light, the coagulation reaction data can be displayed as a coagulation reaction curve (hereinafter sometimes simply referred to as the "reaction curve") that represents the change in scattered light over time. The amount of scattered light from the test sample correlates with turbidity, and therefore the coagulation reaction curve based on scattered light is sigmoid-shaped. In the case of transmittance or absorbance, the coagulation reaction curve is inverse sigmoid-shaped.

[0017] The inventors conducted a detailed analysis of the coagulation reaction curve after the end of the coagulation reaction in APTT measurements and discovered that the coagulation reaction curve for FXIII deficiency differs from that of normal samples. It can be inferred that the coagulation reaction curve after the end of the coagulation reaction reflects the state of fibrinolysis. Specifically, after the end of the coagulation reaction, the coagulation reaction curve of normal samples shows a gradual upward trend or is almost horizontal, whereas in FXIII deficiency, which causes fibrinolysis abnormalities, the coagulation reaction curve reaches a maximum point and then shows a gradual downward trend. Furthermore, it was confirmed that the lower the FXIII activity, the earlier the point at which the coagulation reaction curve reaches its maximum, and the greater the subsequent decrease tends to be.

[0018] Figure 1 illustrates the coagulation reaction curve of a normal sample as an example of APTT measurement. In Figure 1A, the vertical axis represents the amount of scattered light, and the horizontal axis represents the reaction time (seconds). Point a is the reaction start point, and areas b and c are the end stages of the coagulation reaction. Here, the end stage of the coagulation reaction is the stage at which it is estimated that most of the fibrinogen in the test sample has been converted into fibrin. FXIII is activated by thrombin and cross-links fibrin molecules to form stabilized fibrin. Therefore, areas b and c are the stages of progressing fibrin stabilization. In area c of Figure 1A, the reaction curve continues to rise gradually, suggesting that fibrin densification is progressing (cross-linking between fibrin molecules is advancing).

[0019] Figure 1B shows the corrected reaction curve (hereinafter referred to as the corrected curve) obtained by correcting the reaction curve of Figure 1A so that its maximum value is 100%. Point d, indicated by the × mark, is the maximum value of the corrected curve. Figure 1B also shows the solidification time (APTT) calculated using the percentage method, which is the time it takes for the corrected curve to reach 50%.

[0020] The difference in reaction curves between normal and FXIII-deficient samples is explained using Figure 2. Figures 2A and 2B show reaction curves up to a reaction time of 120 seconds. Figure 2A is the reaction curve due to scattered light intensity, and Figure 2B is the corrected curve obtained by correcting the reaction curve of Figure 2A so that the maximum value is 100%. Figures 2C and 2D show the case where the reaction time in Figures 2A and 2B is extended to 600 seconds. In all of Figures 2A to D, the solid line represents the normal sample and the dashed line represents the FXIII-deficient sample, with the maximum value indicated by an "x" and a "+" mark, respectively. If APTT using the percentage method is defined as the time it takes for the reaction to reach 50% in Figure 2B or Figure 2D, then it is the same for both normal and FXIII-deficient samples: 29 seconds. The maximum value (marked with an "x") for the normal sample is located at the right end of the reaction curve (the end point of the reaction time) in both the 120-second and 600-second reaction times, and the reaction curve rises gradually after the coagulation reaction is complete. In contrast, the maximum value (indicated by a +) for the FXIII-deficient sample is located at 86 seconds for both the 120-second and 600-second reaction times, and the reaction curve gradually decreases after the maximum value.

[0021] Figure 2 shows that FXIII deficiency and normal samples can be distinguished by the time it takes to reach the maximum value of the reaction curve or the subsequent changes in the reaction curve. In normal samples, the reaction curve becomes horizontal or upward after the end of the coagulation reaction, and the maximum value shifts backward in proportion to the reaction measurement time. On the other hand, in FXIII deficiency samples, the maximum value is determined during the reaction time, and the reaction curve decreases after the maximum value. Thus, normal samples and FXIII deficiency can be distinguished by the difference in the reaction curve after the end of the coagulation reaction, or by the time it takes to reach the maximum value of the reaction curve.

[0022] As shown in Figure 2, the difference in reaction curves between normal samples and FXIII-deficient samples after the completion of the coagulation reaction reflects the turbidity changes of the test samples. In other words, after the completion of the coagulation reaction, turbidity is maintained or tends to increase in normal samples, while turbidity tends to decrease in FXIII-deficient samples. Furthermore, the difference in reaction curves can be interpreted as reflecting the degree of fibrin density. That is, after the completion of the coagulation reaction, densification of the fibrin formed by the coagulation reaction (cross-linking between fibrins, conversion to stabilized fibrin) progresses in normal samples, while in FXIII-deficient samples, conversion to stabilized fibrin does not progress, and it can be inferred that fibrin degradation (fibrin weakening, fibrin dissolution due to fibrinolysis) occurs.

[0023] Therefore, by using coagulation reaction data (turbidity changes) from the end of the coagulation reaction stage onward, it is possible to infer the fibrin stability of the test sample or fibrin vulnerability due to FXIII deficiency. In other words, based on indicators related to the turbidity changes of the test sample from the end of the coagulation reaction stage onward, it becomes possible to estimate fibrinolytic abnormalities, including FXIII deficiency.

[0024] Therefore, the present invention makes it possible to evaluate the fibrinolysis of a test specimen or to detect specimens that may have fibrinolytic abnormalities by utilizing coagulation reaction data obtained by conventional APTT measurement. An example of a fibrinolytic abnormality evaluated or detected by the present invention is FXIII deficiency.

[0025] In one embodiment, the present invention provides a method for analyzing the coagulation function of a blood sample (hereinafter also referred to as "the method of the present invention"). The method of the present invention includes optically measuring the coagulation reaction of a sample containing a test sample and an APTT measuring reagent and obtaining coagulation reaction data; obtaining an index value representing the turbidity change of the sample after the end of the coagulation reaction based on the coagulation reaction data; and obtaining information regarding fibrinolysis of the test sample based on the index value.

[0026] The method of the present invention will be described in detail below.

[0027] 1. Test specimens Examples of test specimens in the method of the present invention include specimens suspected of having fibrinolytic abnormalities, specimens for testing hemostatic function as a preoperative examination, and specimens for hemostatic function screening to test coagulation time (APTT). An example of a specimen suspected of having fibrinolytic abnormalities is a specimen suspected of having FXIII deficiency. Examples of specimens suspected of having FXIII deficiency include specimens from patients with a family history or past history of FXIII deficiency, and specimens from patients who exhibit sudden severe bleeding symptoms despite having no family history or past history, and whose PT and APTT are normal, suggesting acquired FXIII deficiency. In the first embodiment of the present invention, a blood specimen in which PT and APTT are within the normal range is used as the test specimen. In the second embodiment of the present invention, a blood specimen in which PT, APTT and fibrinogen concentration (Fbg) are within the normal range is used as the test specimen. In another embodiment, it is also possible to use a sample in which an abnormality in the coagulation cascade is suspected due to prolonged coagulation time such as PT and APTT as the test sample, and to further investigate whether or not it has fibrinolytic abnormalities such as FXIII deficiency.

[0028] In the method of the present invention, the subject's plasma collected for coagulation testing is preferably used as the test sample. Anticoagulants commonly used in coagulation testing may be added to the sample. For example, after collecting blood using a blood collection tube containing sodium citrate, citrated plasma (hereinafter referred to as plasma) is obtained by centrifugation.

[0029] 2. Coagulation Time Measurement Reagents Commercially available APTT measurement reagents can be used as APTT measurement reagents in the method of the present invention. Examples of APTT measurement reagents include, but are not limited to, APTT reagent Coagupia APTT-F and Coagupia APTT-F calcium chloride solution (both manufactured by Sekisui Medical Co., Ltd.). A test sample is prepared by mixing the APTT measurement reagent with the test specimen. The prepared test sample is then subjected to coagulation reaction measurement.

[0030] 3. Acquisition of Turbidity Time Series Data 3.1. Coagulation Reaction Measurement Coagulation reaction data can be acquired according to the procedure for normal coagulation reaction measurement in APTT measurement. Any means capable of measuring the turbidity change of the test sample is acceptable as the means for measuring the coagulation reaction. In the method of the present invention, the change in turbidity over time in the test sample is optically measured and used as coagulation reaction data. As means for optical measurement, for example, scattered light amount, transmittance, absorbance, etc. In the following specification, the present invention will be described assuming that the measurement means is scattered light amount.

[0031] The start point of the coagulation reaction (reaction start point) can typically be defined as the time when the reagent (calcium chloride solution) is mixed with the sample to initiate the coagulation reaction, but other timings may also be defined as the reaction start point. The time for which the coagulation reaction is measured (sometimes referred to as the measurement time) can be, for example, at least 120 seconds, preferably at least 180 seconds, from the time of mixing of the sample and the reagent (calcium chloride solution) (reaction start point). For example, the measurement time may be about 2 to 10 minutes from the reaction start point. However, if APTT is also measured at the same time, it may be about 3 to 10 minutes. During the measurement time, the progress of the coagulation reaction (photometric measurement of scattered light) can be repeatedly performed at predetermined intervals. For example, measurements can be performed at 0.1-second intervals. The temperature of the sample during measurement is under normal conditions, for example, 30°C to 40°C, preferably 35°C to 39°C. Furthermore, various measurement conditions can be appropriately set depending on the sample, reagent, measurement means, etc.

[0032] The series of operations in the aforementioned coagulation reaction measurement can be performed using an automated analyzer. An example of an automated analyzer is the CP3000 automated blood coagulation analyzer (manufactured by Sekisui Medical Co., Ltd.). Alternatively, some operations may be performed manually. For example, a human can prepare the sample, and the subsequent operations can be performed by an automated analyzer.

[0033] 3.2. Acquisition of Coagulation Reaction Data Coagulation reaction data for the test sample is acquired by the measurement described above. In one embodiment, the coagulation reaction data can be represented by a coagulation reaction curve P(i). Here, "i" is a variable that represents the number of measurement points or time from the reaction start point (also simply called the number of measurement points and time, respectively). Here, time can be expressed as a function of the number of measurement points, and vice versa. That is, when the measurement (photometric) interval is w (seconds), time (seconds) = w × number of measurement points. For example, if the measurement interval is 0.1 seconds, then time = 0.1 × number of measurement points. In other words, P(i) may be a function of the number of measurement points or a function of time. Generally, P(i) is the measured value of the coagulation reaction measurement after noise reduction or smoothing processing by conventional means. Alternatively, P(i) may be the data after noise reduction or smoothing processing has been performed and the zero point has been adjusted. The zero point adjustment may be, for example, by shifting P(i) so that its initial value is 0.

[0034] The aforementioned P(i) may be converted to a relative value. The relative valued data R(i) can be calculated, for example, according to the formula: R(i) = [(P(i) - Pmin) / (P(t) - Pmin)] × A (where Pmin represents the minimum value of P(i), t is a predetermined point, and A is an arbitrary constant). R(i) is a corrected curve obtained by correcting (converting to a relative value) P(i) so that P(t) = A.

[0035] 3.3. Obtaining the (coagulation rate curve) V(i) The first derivative curve (coagulation rate curve) V(i) may be obtained from P(i) as described above. Differentiation of the coagulation reaction curve can be performed by any method, for example, by calculating the average slope value within the interval. Alternatively, V(i) may be expressed as a relative value.

[0036] 3.4. APTT Calculation The coagulation time (APTT) of a sample can be calculated from the coagulation reaction data of the sample obtained by the coagulation reaction measurement described above. The method for calculating APTT is not particularly limited. For example, APTT can be calculated according to any method based on P(i) or V(i). Examples of APTT calculation methods, but not limited to these, include: a method in which the coagulation time is calculated when P(i) reaches N% of its maximum value (percentage detection method); a method in which the coagulation time is calculated when V(i) reaches N% of its maximum value; a method in which the calculation starting point T is when the cumulative ratio of P(i) in a minute time period reaches a predetermined value, and the coagulation time is calculated when P(i) reaches N% of P(T) (Japanese Patent Publication No. Hei 6-249855); a method in which the coagulation time is calculated based on the change in the cumulative ratio of P(i) in a minute time period over time (see WO2021 / 132552). In the above methods, R(i) may be used instead of P(i).

[0037] Based on the calculated APTT of the sample, the test sample to which the method of the present invention is applied can be selected. In one embodiment, after measuring the coagulation reaction of any sample and calculating the APTT, samples in which it cannot be definitively determined that there is an extension of APTT are selected as test samples of the present invention. In another embodiment, samples in which it is known from existing coagulation reaction data that there is no clear extension of APTT are selected as test samples of the present invention. Alternatively, samples in which an abnormality in the coagulation cascade is presumed due to an extension of APTT may be selected as test samples of the present invention in order to further investigate whether or not there is a fibrinolysis abnormality.

[0038] 4. Calculation of the Turbidity Trend Index (TI) In the method of the present invention, a decrease in the turbidity of the test sample after the completion of the coagulation reaction is detected from the coagulation reaction data of the test sample. Specifically, in the method of the present invention, a value of an index representing the turbidity trend of the test sample after the completion of the coagulation reaction is obtained from the coagulation reaction data of the test sample. Hereinafter, the index representing the turbidity trend will be called the turbidity trend index (TI). More specifically, TI may be an index representing the decrease in turbidity of the test sample. Based on the value of TI, it is possible to evaluate whether the test sample is a sample in which the turbidity has decreased after the completion of the coagulation reaction compared to a sample derived from a normal sample.

[0039] "The period after the completion of the coagulation reaction" is defined as the stage at which it is estimated that most of the fibrinogen in the test sample has been converted into fibrin. "The period after the completion of the coagulation reaction" can be determined based on the rate of change of the coagulation reaction (coagulation rate). In one embodiment, the time at which the coagulation rate decreases to a predetermined value after reaching its maximum can be defined as the starting point of the "completion of the coagulation reaction," and the time thereafter can be defined as "the period after the completion of the coagulation reaction."

[0040] In another embodiment, the "end stage of the coagulation reaction" can be defined as follows: The starting point (Tb) of the "end stage of the coagulation reaction" can be defined as the point in time when the integrated ratio Z(i) of P(i) reaches a predetermined value ZsH. The time after Tb can be defined as "after the end stage of the coagulation reaction". The ending point (Tc) of the "end stage of the coagulation reaction" can be defined as the point in time when the integrated ratio Z(i) reaches a predetermined value ZsL. Here, the integrated ratio Z(i) of P(i) is calculated by the following equation: Integrated ratio Z(i) = Pb(i) / Pa(i) Pa(i) = sum from P(i-m) to P(i-1) Pb(i) = sum from P(i+1) to P(i+m) m is preferably 5 to 50 (for example, 20). The preferred ranges for ZsH and ZsL are ZsH > ZsL, where ZsH is between 1.05 and 1.01, and ZsL is between 1.02 and 1.0005.

[0041] 4.1. TI Based on the Coagulation Reaction Curve Figure 3 illustrates the TI based on the coagulation reaction curve. Figures 3A and 3B show the corrected curve R(i) where P(i) is relative so that the value at the set time Te is 100%. Figure 3A shows data for an FXIII-deficient sample, and Figure 3B shows data for a normal sample. The set time Te can preferably be set in the range of 120 to 600 seconds (from the reaction start point, the same applies hereafter), and in Figure 3, Te is 180 seconds. Figure 3A shows points a (Ts, 100), b (RmaxT, Rmax), c (Te, 100), and d (RmaxT, 100). Here, Te is the end point of the data used to calculate TI. Rmax is the maximum value of R(i) before Te, RmaxT is the time point of Rmax, and Ts is the time point before RmaxT when R(i) = 100. In Figure 3A, a to d appear as separate points. In Figure 3B, R(i) is continuously increasing, so Rmaxes at Te, and therefore points b, c, and d have the same coordinates, and point a does not exist.

[0042] 4.1.1. Time of Maximum Turbidity Value RmaxT RmaxT is the point in time when R(i) reaches its maximum value. In the case of Figure 3A, RmaxT < Te, and in the case of Figure 3B, RmaxT = Te.

[0043] 4.1.2. Turbidity change ΔR ΔR = Rmax - 100. In Figure 3A, ΔR is represented by the difference in the vertical axis direction between point b and point c. In Figure 3B, ΔR = 0.

[0044] 4.1.3. Turbidity reduction rate rF rF is the slope of the regression line of R(i) in a predetermined section. When the start point of the predetermined section is defined as St and the end point is defined as Et, St is preferably a point after the start point (Tb) of the end stage of the coagulation reaction, more preferably St≧RmaxT, and preferably Et≦Te (where St<Et). More preferably, St=60 to 210 (seconds) and Et=180 to 350 (seconds), where preferably Et-St=60 to 290 (seconds). In a preferred embodiment, rF is the slope of the regression line of R(i) from RmaxT to Te. In this case, rF is the slope of the regression line of R(i) from point b to point c shown in FIG. 3A. If a regression line cannot be obtained as in FIG. 3B, then rF=0.

[0045] 4.1.4. Excess turbidity SF There are two types of SF: SFa and SFb. SFa is the area of the region where R(i) exceeds 100 in the area under the curve of R(i) in the range from Ts to Te. SFb is the area of the region where R(i) exceeds 100 in the area under the curve of R(i) in the range from RmaxT to Te. In the case of FIG. 3A below, SFa is the area of the region surrounded by R(i) and the 100% line in the range from point a to point c. SFb is the area of the region surrounded by R(i) and the 100% line in the range from point d to point c. If R(i) does not exceed 100% as in FIG. 3B, then SFa=0 and SFb=0.

[0046] 4.2. TI based on solidification rate curve Next, TI based on the solidification rate curve V(i) will be described. A solidification rate ratio curve rV(i)=V(i) / Vmax×100 is obtained. FIG. 4A is an example of rV(i) for FXIII deficiency, and FIG. 4B is an example of rV(i) for a normal specimen. These figures show rV(i) (gray, right axis) from the start of the reaction to a set time Tev (120 seconds), and a curve obtained by enlarging the vicinity of 0% thereof (black, left axis). The broken line parallel to the vertical axis represents VmaxT, the time at which Vmax is reached. When comparing FXIII deficiency (FIG. 4A) and a normal specimen (FIG. 4B), in FIG. 4B, rV(i) is always above the 0% line (positive side) from VmaxT to 120 seconds, whereas in FIG. 4A, rV(i) alternates between above the 0% line (positive side) and below the 0% line (negative side) after around 70 seconds, and remains almost on the negative side after around 80 seconds.

[0047] 4.2.1. Rate ratio arrival time rT rT is the number of i's satisfying rV(i)≧rVs (or the time length corresponding to the number of i's) in the range from the start point Tb of the end stage of the coagulation reaction to Tev. Tev and rVs can be set arbitrarily, preferably Tev=80 to 180 (seconds) (Tb<Tev), and rVs=0.01 to 0.2. In FIGS. 4A and 4B, as an example, Tev is set to 120 seconds, rVs is set to 0.1, and rVs is indicated by a dotted line parallel to the horizontal axis. Although rT can take values according to Tev and rVs, as shown in FIGS. 4A and 4B, rT is smaller in FXIII deficiency than in normal specimens.

[0048] 4.3. TI based on R ratio curve Next, TI using the R ratio curve rR(i) will be described. rR(i) is obtained by the following formula using the aforementioned R(i). rR(i)={SumR(i) / SumR(i−k)}×100 Here, SumR(i) is represented by the following formula. i is the number of measurement points, and i>k, k can be appropriately set according to the measurement conditions of the coagulation reaction, for example, k=2 to 20.

[0049] Figure 5A shows an example of rR(i) in FXIII deficiency when k=10, and Figure 5B shows an example of rR(i) in a normal sample. Comparing rR(i) in FXIII deficiency (Figure 5A) and a normal sample (Figure 5B), in Figure 5B, rR(i) is always above 100, whereas in Figure 5A, rR(i) falls below 100 from around 70 seconds onward. This difference indicates that R(i) after the end of the coagulation reaction tends to decrease in FXIII deficiency (Figure 5A) and increases in a normal sample (Figure 5B).

[0050] 4.3.1. R-ratio time tR tR is the number of i values ​​(or the time length corresponding to the number of i values) that satisfy rR(i) ≥ 100 in the range from the starting point Tb of the end stage of the coagulation reaction to the set time Ter. Ter can be set arbitrarily, preferably Ter = 90 to 600 (seconds) (Tb < Ter), and is 180 seconds in Figure 5. As shown in Figures 5A and B, tR is smaller in FXIII deficiency than in normal samples.

[0051] 4.3.2. R-regression slope sR sR is the slope of the regression line of R(i) from Ta to the set time Tes, as shown in Figures 6A and 6B. Ta is the time corresponding to the maximum value of i that satisfies R(i) ≥ 50 and rR(i) = sRs, and sRs is ≥ 100, preferably 100.05 to 101.00. Tes can be set arbitrarily, preferably Tes = 120 to 600 (seconds) (Tb < Tes), and is 180 seconds in Figure 6. In Figures 6A and B, sRs is 100.10 and is represented by a dashed line parallel to the horizontal axis. Ta is the time when the dashed line and rR(i) intersect around 50 seconds, and is represented by a dashed line parallel to the vertical axis in the figure. The starting and ending points of R(i) for obtaining sR are represented by coordinates as (Ta, R(Ta)) and (Tes, R(Tes)), respectively. After Ta, R(i) tends to decrease in FXIII deficiency (Figure 6A), as rR(i) is below 100, while in normal samples (Figure 6B), it tends to increase, as rR(i) is above 100. Therefore, sR will be smaller in FXIII deficiency than in normal samples.

[0052] 5. Evaluation of fibrinolysis based on TI The TI calculated above is used to obtain information about the fibrinolysis of the test specimen. Examples of information about the fibrinolysis of the test specimen include information about the FXIII activity level of the test specimen, more specifically, whether the FXIII activity of the test specimen is at a normal level, and whether the test specimen may be FXIII deficient.

[0053] In one embodiment, information regarding the fibrinolysis of a test sample is obtained by comparing the TI value calculated from the test sample with a threshold value. The threshold value can be predetermined based on the TI value calculated from a group of normal samples. As mentioned above, the TI of FXIII deficiency is greater or less than that of normal samples, depending on the type of deficiency. More specifically, compared to normal samples, RmaxT, rF, rT, tR, and sR are smaller in FXIII deficiency, while ΔR, sFa, and sFb are larger. Therefore, in one embodiment of the method of the present invention, information regarding the fibrinolysis of a test sample, such as whether the FXIII activity of the test sample is at a normal level or whether the test sample may be FXIII deficient, is detected based on the TI value.

[0054] The fibrinolysis information obtained above can be output in any format. For example, information on whether or not the test sample may have FXIII deficiency can be output in any format. For example, if there is a possibility of FXIII deficiency, more detailed output could include text or flags indicating information such as "FXIII activity is suspected to be low" or "FXIII activity testing is necessary." In addition, the APTT of the test sample can be output along with the above information.

[0055] In some samples where PT and APTT are normal and FXIII deficiency is suspected based on TI, there may be samples with low Fbg (fibrinogen concentration). Here, low means a value lower than the lower limit of the normal range. The Fbg of a sample can be measured by methods such as the thrombin time method (Clauss method). Alternatively, the Fbg of a sample can be estimated using the coagulation reaction curve of PT or APTT. The maximum reaction amount of the coagulation reaction curves of PT and APTT is known to correlate with Fbg, and the PT-derived method is a known method for measuring Fbg using the PT coagulation reaction curve. Similarly, although the accuracy is not as high as the thrombin time method, it is also possible to estimate whether Fbg is low or not using the APTT coagulation reaction curve.

[0056] Even if a low Fbg level suggests the possibility of FXIII deficiency based on TI, the sample may actually be from a patient with hyperfibrinolysis rather than FXIII deficiency. An example of such a patient is hyperfibrinolytic DIC. Therefore, in the method of the present invention, the analysis (procedure) can be changed depending on whether the Fbg level is low or not. More specifically, when the PT and APTT of the test sample are normal, the estimated Fbg value (low or not low) estimated from the coagulation reaction curve of PT or APTT can be processed as follows: If the Fbg level is not low, the sample can be estimated to have a possible FXIII deficiency; on the other hand, if the Fbg level is low, the possible pathological conditions are wasting acquired FXIII deficiency due to massive bleeding and hyperfibrinolysis. In the latter case, based on the test results of fibrinolytic parameters such as FDP and D-dimer, the sample may be judged to be in a hyperfibrinolytic state (rather than FXIII deficiency).

[0057] Therefore, in the method of the present invention, the Fbg of a test sample may be estimated based on the coagulation reaction data of the test sample before obtaining the TI. For example, the Fbg of a sample can be estimated based on the maximum value of P(i) (Pmax). Alternatively, the Fbg of a sample can be estimated based on the reaction amount at the start (Tb) or end (Tc) of the aforementioned "end stage of coagulation reaction," i.e., P(Tb) or P(Tc). For example, if the Pmax, or P(Tb) or P(Tc) of a test sample is smaller than a predetermined value, the Fbg of the sample can be estimated to be low.

[0058] In the method of the present invention, if the Fbg of a test sample is estimated to be low, information indicating that the Fbg of the test sample is low may be output along with the output of the fibrinolysis information of the test sample described above. For example, a sentence or flag indicating information such as "Suspected low concentration of fibrinogen" may be output. Alternatively, information such as "Suspected low Fbg" or "Please perform fibrinolysis tests" may be output to prompt further testing for Fbg. Or, as another method of handling cases where the Fbg of a test sample is estimated to be low, the sample may be determined not to be a normal sample and treated as a sample to be excluded from this analysis.

[0059] 6. Application to Other Coagulation Reaction Measurement Methods The method of the present invention has been described above using the case of coagulation reaction measurement based on scattered light intensity as an example. However, those skilled in the art will know that it is possible to apply other coagulation reaction measurement methods (for example, coagulation reaction measurement based on transmittance, absorbance, etc.) to the coagulation reaction measurement of the present invention, and such applications are therefore included within the scope of the present invention.

[0060] 7. Program and Analytical Apparatus The series of processes of the method of the present invention described above can be performed automatically by an automated analytical apparatus. For example, an automated analytical apparatus controlled by a computer program can be used. Therefore, one aspect of the present invention is a program for performing the method of the present invention described above. Another aspect of the present invention is an analytical apparatus (hereinafter sometimes abbreviated as "apparatus") for performing the method of the present invention described above. The execution of the method of the present invention by the apparatus can be controlled by the program of the present invention. A further aspect of the present invention is a system for performing the method of the present invention described above, the system comprising an analytical apparatus or program for performing the method of the present invention described above.

[0061] One embodiment of the analytical apparatus of the present invention will be described below with reference to Figure 7. In one embodiment of the apparatus of the present invention, the automatic analytical apparatus 1 comprises a control unit 10, an operation unit 20, a measurement unit 30, and an output unit 40. The configuration of the automatic analytical apparatus 1 will be described below.

[0062] The control unit 10 controls the overall operation of the automatic analyzer 1. The control unit 10 may be composed of a computer (e.g., a personal computer). The control unit 10 is equipped with a CPU, memory, storage, communication interface (I / F), etc., and can process commands from the operation unit 20, control the operation of the measurement unit 30, save and analyze measurement data received from the measurement unit 30, save analysis results, and control the output of analysis results by the output unit 40. Furthermore, the control unit 10 may be connected to other devices such as external media and a host computer. Note that in the control unit 10, the computer that controls the operation of the measurement unit 30 and the computer that performs analysis of the data measured by the unit 30 may be the same or different.

[0063] The operation unit 20 receives input from the operator and transmits the obtained input information to the control unit 10. For example, the operation unit 20 is equipped with a user interface (UI) such as a keyboard or touch panel. The output unit 40, under the control of the control unit 10, outputs information related to the fibrinolysis of the sample, and, if necessary, coagulation reaction data of the sample measured by the measurement unit 30, and the resulting TI, APTT, etc. For example, the output unit 40 is equipped with a display device such as a display.

[0064] The measurement unit 30 acquires measurement data of the coagulation reaction of the test sample. The measurement unit 30 includes various equipment and analysis modules necessary for measurement, such as a sample container for blood samples, a reagent container for test reagents, a reaction vessel for the reaction between the sample and reagents, a probe for dispensing blood samples and reagents into the reaction vessel, a light source, a detector for detecting scattered or transmitted light from the sample in the reaction vessel, a data processing circuit for sending data from the detector to the control unit 10, and a control circuit that controls the processing of the measurement unit 30 in response to commands from the control unit 10. Alternatively, if R(i), V(i), APTT, etc. are to be calculated using already acquired coagulation reaction data, the measurement unit 30 is not necessary.

[0065] The control unit 10 analyzes the coagulation reaction of the sample based on the coagulation reaction data. This analysis may include the calculation of R(i), V(i), and APTT, the calculation of TI, and the acquisition of information on fibrinolysis using TI. The R(i) and V(i) data may be created in the control unit 10 based on measurement data from the measurement unit 30, or they may be created in another device, such as the measurement unit 30, and sent to the control unit 10. The control unit 10 may store APTT, turbidity transition index TI, thresholds used to acquire information on fibrinolysis for normal samples, or the control unit 10 may acquire these values ​​stored in an external device or on a network for the purpose of detection.

[0066] The above analysis can be performed by a program for carrying out the method of the present invention. Therefore, the control unit 10 may be equipped with a program for carrying out the method of the present invention.

[0067] The analysis results from the control unit 10 are sent to the output unit 40 and output. The output can take any form, such as display on a screen, transmission to a host computer, or printing. The output information from the output unit may include APTT of the test sample, information on fibrinolysis, information on estimated fibrinogen concentration, etc. The type of output information from the output unit can be controlled by the program of the present invention.

[0068] 8. Analysis Flow Figure 8 illustrates one embodiment of the process for detecting a sample potentially deficient in FXIII using the method of the present invention, which is carried out under the control of the program of the present invention.

[0069] First, the main process is explained using Figure 8A and the following. S1: Obtain reaction curve data of the test sample. S2: Determine whether to exclude the sample from analysis based on the reaction curve data, and proceed to S7 if exclusion is necessary. S3: If exclusion is not necessary in S2, obtain the turbidity transition index TI. S4: If the obtained index meets the criteria, proceed to S5; otherwise, proceed to S6. S5: Output information that a sample potentially deficient in FXIII has been detected. S6: Output information that no sample potentially deficient in FXIII has been detected. S7: Output information that the sample is excluded from analysis because it has been determined to be excluded from analysis. Note that APTT may also be output in the output of S5, S6, and S7.

[0070] Next, an embodiment of the S2 process for performing exclusion determination will be explained with reference to Figure 8B. S2-1: If Fgb is low, proceed to S2-4. S2-2: If APTT exceeds the threshold, proceed to S2-4. S2-3: Determine that exclusion is not necessary, proceed to S3. S2-4: Determine that exclusion is necessary, proceed to S7.

[0071] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples. The coagulation reaction measurement in the following examples is performed according to the APTT measurement procedure.

[0072] 1. Method 1.1) Samples - Normal samples (NP): Plasma from healthy individuals (N1-N10, n=10). - Normal pooled plasma (PNP): George King Bio-Medical, Inc. (PA and PB, n=2) - FXIII-deficient samples (dFXIII): FXIII-deficient plasma (George King Bio-Medical, Inc.; plasma from FXIII-deficient patients with FXIII activity levels less than 5%) (Lots A and B, 1 each). - FXIII-deficient series samples, from Lots A and B of dFXIII (n=1 each) Normal pooled plasma (PNP) was added to the FXIII-deficient samples from each lot to prepare FXIII-deficient series samples with calculated FXIII activity levels of 10%, 20%, 40%, and 60% (A10-A60 and B10-B60). However, the FXIII activity level was calculated by setting the FXIII activity of FXIII-deficient samples (dFXIII) to 0% and the FXIII activity of normal pooled plasma (PNP) to 100%, and then calculating it from the volume ratio when the two are mixed. Samples from Lot A and Lot B were added to the FXIII-deficient series of samples as samples A5 and B5, respectively, with FXIII activity levels of less than 5%.

[0073] Table 1 shows the sample type, FXIII activity (%), sample identification code, and n number. Note that samples A5, A10, A20, A40, and A60 in Table 1 may be collectively referred to as F13 deficiency series sample A (or series A), and samples B5, B10, B20, B40, and B60 may be collectively referred to as F13 deficiency series sample B (or series B).

[0074]

[0075] 1.2) For measuring the coagulation reaction, Coagupia APTT-F and calcium chloride solution (both manufactured by Sekisui Medical Co., Ltd.) were used. The coagulation reaction of the test sample, including the specimen, was measured using the CP3000 automated blood coagulation analyzer (manufactured by Sekisui Medical Co., Ltd.). 50 μL of the specimen was heated in a cuvette at 37°C for 45 seconds, then 50 μL of APTT reagent at approximately 37°C was added. After another 171 seconds, 50 μL of calcium chloride solution was added to initiate the coagulation reaction. The reaction was carried out at 37°C. To measure the coagulation reaction, light with a wavelength of 660 nm from an LED light source was irradiated onto the side of the cuvette, and the amount of scattered light scattered 90 degrees to the side at 0.1-second intervals was measured. The measurement time was 600 seconds.

[0076] 1.3) Acquisition of Coagulation Reaction Curves After smoothing the measurement data of the coagulation reaction from each test sample at 0.1-second intervals, including noise reduction, a zero-point adjustment process was performed so that the amount of scattered light at the start of measurement was 0 to obtain P(i). P(i) was converted to a relative value so that it was 100 (%) at the set time Te, and the correction curve R(i) was calculated.

[0077] 1.4) Acquisition of the solidification rate curve V(i) V(i) was obtained as the slope (average slope value within the interval) at time i, using 21 consecutive reaction data from P(i-10) to P(i+10).

[0078] 1.5) The maximum values ​​of P(i), R(i), and V(i) within the range from the start of the reaction to the set time Te (seconds) for acquiring Pmax, Rmax, and Vmax were defined as Pmax, Rmax, and Vmax, respectively. In addition, the time P(i) reaches Pmax (PmaxT (seconds)), the time R(i) reaches Rmax (RmaxT (seconds)), and the time V(i) reaches Vmax (VmaxT (seconds)) were determined.

[0079] 1.6) APTT calculation was calculated using a time setting Te of 240 seconds, with APTT defined as the point at which R(i) reached 50% of Rmax. Table 2 shows the VmaxT and APTT (seconds) for each sample. At the bottom of Table 2, the mean (M), standard deviation (S), M-3S, and M+3S for normal samples (N1 to N10) for each value are shown. It was shown that the APTT and VmaxT for all samples in series A and series B did not exceed M±3S. Furthermore, the APTT for these samples was below the upper limit of the normal range (39 seconds).

[0080] 1.7) Calculation of Tb The starting point Tb of the final stage of the coagulation reaction was calculated. Tb was calculated as the point at which the cumulative ratio Z(i) of P(i) reaches a predetermined value ZsH. The cumulative ratio Z(i) was calculated with m=20 in the following formula: Cumulative ratio Z(i) = Pb(i) / Pa(i) Pa(i) = Sum from P(i-m) to P(i-1) Pb(i) = Sum from P(i+1) to P(i+m) When Tb was calculated in the range of ZsH from 1.05 to 1.01, Tb was earliest when ZsH was 1.05.

[0081] Table 2 shows Tb, as well as the time points Ta (Example 7), Ts (Example 4), and RmaxT (Examples 1, 2, 3, 4, and 6) used to calculate the turbidity transition index TI in the examples described below. In Table 2, Tb was calculated using the above formula with ZsH = 1.05, Ta was calculated using Example 7 with sRs = 101.00, Ts was calculated using Example 4 with Te = 240 (seconds), and RmaxT was calculated using 1.5) above with Te = 240 (seconds). In all samples, Ta, Ts, and RmaxT were at time points later than the starting point Tb of the end stage of the coagulation reaction.

[0082]

[0083] 2. Coagulation reaction Figure 9 shows the coagulation reaction curves of the samples. Figure 9A shows the coagulation reaction curves of normal samples (N1 to N10), and Figure 9B shows the coagulation reaction curves of series A (A5, A10, A20, A40, A60). The × marks on the coagulation reaction curves represent Pmax. Pmax T was 500 seconds or more for normal samples, while it was 206 seconds or less for A5, A10, A20, A40 and A60 of series A.

[0084] Figure 10 shows the corrected curve R(i) = P(i) / P(Te) × 100 with Te set to 597 seconds. Figure 10A shows R(i) for normal samples (N1 to N10), and Figure 10C shows R(i) for series A (A5, A10, A20, A40, A60). Figures 10B and 10D show magnified views of the area around 100% on the vertical axis of Figures 10A and 10C, respectively. The × marks on the coagulation reaction curves represent Rmax. From Figure 10D, it is shown that the slope of R(i) after Rmax T for series A (A5, A10, A20, A40, A60) is inversely proportional to the FXIII activity, and Rmax is inversely proportional to the FXIII activity. In other words, in series A, the lower the activity, the greater the rate of decrease in R(i) after RmaxT. From this, it was inferred that the dissolution (fibrinolysis) of unstabilized fibrin was gradually progressing due to the deficiency of FXIII.

[0085] Figure 10 shows that it is possible to estimate the FXIII activity level by using indicators related to FXIII activity based on reaction curve data, such as RmaxT, the slope of R(i) after RmaxT, and R(RmaxT).

[0086] Example 1 The setting time Te was set to 120, 180, 240, and 597 seconds, and the maximum turbidity time RmaxT was obtained as the time at which R(i) was maximum within the range of 0 to Te (seconds). The results are shown in the table in Figure 11. In the table in Figure 11, the minimum value for normal samples (NP) is shown in bold, and when the RmaxT of samples other than NP is less than the minimum value for NP, the value is shown in bold with a gray background. The bottom row of the table is the coefficient of determination (square of the correlation coefficient) of the regression line between FXIII activity and RmaxT for series A and series B. As shown in Figure 11, the RmaxT of normal samples (NP) and PNP tended to be around Te or just before Te. On the other hand, for samples with activity of 20% or less, the RmaxT was within 93 seconds for series A and within 82 seconds for series B for all Te values, indicating that they can be distinguished from NP. In samples with 40% activity, RmaxT was within 112.6 seconds for A40 and within 122.6 seconds for B40. The FXIII activity level required to distinguish series A and B as NP was shown to be 20% or less when Te was 120 seconds, and 40% or less when Te was 180 seconds or more. Furthermore, the coefficient of determination was 0.79 or higher, indicating a correlation between RmaxT and FXIII activity.

[0087] Example 2 The setting time Te was set to 120, 180, and 240 seconds, and the turbidity change ΔR (= Rmax - 100) was obtained. The results are shown in the table in Figure 12. This index is 0 when R(Te) is equal to Rmax. When this index is greater than the maximum value of ΔR for normal samples (NP), it is shown with a gray background and in bold. The bottom row of the table is the coefficient of determination (square of the correlation coefficient) of the regression line between FXIII activity and ΔR for series A and series B. The FXIII activity levels that could distinguish series A and series B from NP were 20% or less when Te was 120 seconds, 60% or less when Te was 180 seconds, and 40% or less when Te was 240 seconds. Furthermore, the coefficient of determination was 0.82 or higher, indicating that ΔR correlates with FXIII activity.

[0088] Example 3: The setting time Te was set to 120, 180, and 240 seconds, and the turbidity reduction rate rF was obtained as the slope of the regression line of R(i) from RmaxT to Te. The results are shown in the table in Figure 13A. Cases where this index is negative are shown in bold, and cases where it is smaller than the minimum value of NP are shown with a gray background. The bottom row of the table is the coefficient of determination (square of the correlation coefficient) of the regression line between FXIII activity and rF for series A and series B. The FXIII activity levels of series A and B, where rF is negative (a decreasing trend in R(i)), were 20% or less, 40% or less, and 60% or less at Te 120, 180, and 240 seconds, respectively. The coefficient of determination was 0.79 or higher, indicating that rF correlates with FXIII activity.

[0089] The slope of the regression line for R(i) in various intervals was determined and obtained as rF. The starting point of each interval was defined as St and the ending point as Et, with St set to 60-210 seconds and Et to 180-350 seconds. The results are shown in the table in Figure 13B. The table in Figure 13B also shows RmaxT at Te = 350 seconds, and the time point Ts at which R(i) is 100 before RmaxT. In the table, negative rF values ​​are shown in bold, and values ​​smaller than the minimum value of NP are shown with a gray background. The bottom row of the table shows the coefficient of determination (square of the correlation coefficient) of the regression line between FXIII activity and rF for series A and series B. Since the coefficient of determination was 0.81 or higher in all cases, it was shown that rF correlated with FXIII activity in all the intervals shown in the table.

[0090] Example 4: The setting time Te was set to 120, 180, and 240 seconds, and the excess turbidity SF was obtained. The time point Ts where R(i) is 100 before RmaxT was determined, and the area under the curve of R(i) between Ts and Te where R(i) is greater than 100 was defined as SFa. Similarly, the area under the curve of R(i) between RmaxT and Te where R(i) is greater than 100 was defined as SFb. The results are shown in the table in Figure 14. In Figure 14, Table A is SFa and Table B is SFb. Cases where this index is greater than the maximum value of NP are shown in bold with a gray background. The bottom row of the table shows the coefficient of determination (square of the correlation coefficient) of the regression line between the FXIII activity of series A and series B and SFa and SFb. In Table A, the FXIII activity levels that distinguished normal samples (NP) from normal samples were ≤20% for Te at 120 seconds, ≤60% for Te at 180 seconds, and ≤40% for Te at 240 seconds. Table B showed a similar trend, but the coefficient of determination was higher in Table A than in Table B for all Te values.

[0091] Example 5 The setting time Te was set to 120 seconds, and rV(i) = V(i) / Vmax was calculated. For setting speed ratios rVs of 0.17, 0.12, 0.08, 0.04, and 0.02, the time to reach the speed ratio rT was obtained as the time length corresponding to the number of i values ​​in the range from Tb (ZsH = 1.01) to Te for which rV(i) is greater than or equal to rVs. The results for VmaxT and rT for each rVs are shown in the table in Figure 15. The mean (M), standard deviation (S), M-3S, and M+3S of normal samples (N1 to N10) are shown at the bottom of the table, and the values ​​are set to bold with a gray background when this index exceeds M±3S. The "coefficient of determination" in the table is the square of the correlation coefficient of the regression line between the FXIII activity of series A and series B and rT. The Tb values ​​for series A and B were within M±1.8S, but rT exceeded M±3S only when rVs was 0.17 or less at an activity of 5%, and when rVs was 0.12 or less at an activity of 20% or less. When rVs was 0.04 or less, series A exceeded M±3S when its activity was 40% or less. The coefficient of determination tended to increase as rVs decreased, and was 0.90 or higher when rVs was 0.04 or less, indicating that rT correlates with FXIII activity.

[0092] Example 6 The rR(i) value described in 4.3 above was calculated as the R ratio every second. However, with k=10, in order to exclude noise until the reaction curve reached the upward stage, rR(i) was set to 100 (fixed value) when R(i-9) was less than 1. The setting time Te was set in 60-second intervals from 120 seconds to 540 seconds, and the R ratio time tR was obtained as the number of i values ​​(corresponding to the time length (seconds)) in which rR(i) was 100 or more within the range from Tb(ZsH=1.01) to Te. The results are shown in the table in Figure 16. The mean (M), standard deviation (S), M-2S, and M-3S of normal samples (N1 to N10) are shown at the bottom of the table. If this index is smaller than M-2S, it is shown in bold, and if it is smaller than M-3S, the background is further set to gray. The "Coefficient of Determination" in the table is the square of the correlation coefficient between the regression lines of FXIII activity and tR for series A and B. In almost all cases of series A and B, tR was smaller than M-3S. The coefficient of determination tended to increase as Te increased, but all were above 0.71, indicating that tR correlates with FXIII activity. It was shown that FXIII deficiency can be distinguished from normal samples (NP) by using tR.

[0093] Example 7 For the rR(i) obtained in Example 6, the R regression slope sR was obtained as the slope of R(i) within the range from the starting point Ta to the ending point Te, where Ta corresponds to the maximum value of i for which R(i) is 50 or greater and rR(i) is equal to the set value sRs. The table in Figure 17 shows the sR when the set time Te is 180 seconds and the set values ​​sRs are 100.05, 100.10, 100.20, 100.30, 100.40, and 101.00, respectively. The mean (M), standard deviation (S), and M-3S of normal samples (N1 to N10) are shown at the bottom of the table, and bold text and a gray background are used when sR is smaller than M-3S. The "Coefficient of Determination" in the table is the square of the correlation coefficient of the regression line between the FXIII activity of series A and series B and sR. In series A and B, the FXIII activity level that is lower than M-3S is 40% or less for all setpoints. Furthermore, the coefficient of determination was 0.91 or higher for all setpoints of sRs, indicating that sR correlates with FXIII activity.

[0094] In normal samples (NP), the increase in sRs is due to the upward trend in R(i) after the completion of the coagulation reaction in NP. On the other hand, the lower sR values ​​in series A and B compared to NP are due to the downward trend in R(i) after the completion of the coagulation reaction. This demonstrates that FXIII deficiency can be distinguished from normal samples (NP) by using sR.

[0095] Example 8 Using two samples (LF1 and LF2) that were confirmed to have low fibrinogen levels, a method for excluding low-concentration fibrinogen samples was investigated. The fibrinogen concentrations of LF1 and LF2 were 110 and 87 (mg / dL), respectively. Figure 18 shows the reaction curves P(i) for normal samples (N1 to N10) and low-concentration fibrinogen samples (LF1 and LF2). In Figure 18, the solid line represents the normal samples, and the dotted and dashed lines represent the low-concentration fibrinogen samples LF1 and LF2, respectively. The × marks on the coagulation reaction curves represent the maximum value Pmax. In Figure 18A, the Pmax of normal samples (N1 to N10) is between 4000 and 8000, while the Pmax of low-concentration fibrinogen samples (LF1 and LF2) is below 2000. It is known to those skilled in the art that Pmax is proportional to the fibrinogen concentration of a sample. Figure 18A shows that low-concentration fibrinogen samples can be detected by comparing the Pmax of a test sample with that of a normal sample.

[0096] Figure 18B shows the corrected curves R(i) for LF1 (dotted line) and LF2 (dashed line) of normal samples (N1-N10, solid line) and low-concentration fibrinogen samples. R(i) in Figure 18B was created with P(Te) set to 100% (Te = 600 (sec)). Figure 18C shows an enlarged view of the area around 100% on the vertical axis of Figure 18B. As shown in Figures 18B and C, LF1 (dotted line) showed a trend similar to FXIII deficiency, and LF2 (dashed line) showed a trend similar to NP.

[0097] Figure 18D shows the plot of P(Tb) against fibrinogen concentration (Fbg) (mg / dL) and the regression line for normal samples (N1-N10) and low-concentration fibrinogen samples (LF1, LF2). Tb was calculated as described in 1.7) above, with ZsH = 1.01. The correlation coefficient of the regression line between Fbg and P(Tb) was 0.988, indicating that P(Tb) correlates with Fbg. The slope of the regression line was 25.4 and the intercept was -1250. Based on the regression line, P(Tb) at the lower limit of the normal range of Fbg, 200 (mg / dL), was calculated to be 3833. From these results, it was shown that if the P(Tb) of a sample is less than 3833, the sample can be judged to have low fibrinogen levels.

[0098] Table 3 is a classification table of specimens based on coagulation time (APTT), TI (turbidity reduction rate rF), and fibrinogen concentration (Pmax). Among specimens with low TI (rF), there were FXIII deficiency (classification 1) and low-concentration fibrinogen specimens (classification 2). On the other hand, there were also low-concentration fibrinogen specimens (classification 4) with TI (rF) no different from normal specimens (classification 3). Therefore, it was shown that information indicating whether or not a specimen is low-concentration fibrinogen is useful information for estimating FXIII deficiency in the test specimen. An example of a specimen in classification 2 is a patient specimen in which FXIII was excessively consumed due to massive bleeding, resulting in decreased fibrinogen concentration and decreased FXIII activity.

[0099]

[0100] Example 9 The FXIII activity level of the sample was estimated using the turbidity reduction rate rF from RmaxT to Te (240 seconds) obtained in Example 3. Figure 19A shows the relationship between the FXIII activity level (calculated value in 1.1 above) and rF for each sample in series A and B. For both series A and B, the relationship between the FXIII activity level and rF was linear when the FXIII activity level was between 20% and 60% (A20 to A60 and B20 to B60). A regression line was obtained from the data A20 to A60 and used as calibration curve A for FXIII activity based on rF. Similarly, calibration curve B was created from the data B20 to B60. The FXIII activity of series A was estimated using calibration curve B, and the FXIII activity of series B was estimated using calibration curve A. However, if the estimated value was negative, it was considered to be below the lower limit of the calibration curve (<20%).

[0101] Figure 19B shows the estimated FXIII activity levels for series A and B. When the calculated FXIII activity level was 10% or less, the estimated value for A10 was 6%, while the rest were "<20%". The estimated values ​​for A20, B20, A40, and B40 were 19%, 19%, 41%, and 43%, respectively, with a difference of ±3% between the estimated and calculated values. The estimated values ​​for A60 and B60 were 66% and 53%, respectively, with a difference of ±7% between the estimated and calculated values. From the above, it was shown that the FXIII activity level of a sample can be estimated using the turbidity reduction rate rF.

Claims

1. A method for analyzing a blood sample, comprising: optically measuring the coagulation reaction of a sample containing a blood sample and an APTT measuring reagent and obtaining coagulation reaction data; obtaining an index value representing the turbidity change of the sample after the end of the coagulation reaction based on the coagulation reaction data; and obtaining information regarding fibrinolysis of the blood sample based on the index value.

2. The method according to claim 1, wherein the information relating to the fibrinolysis of the blood sample is information relating to the FXIII activity level of the blood sample.

3. The method according to claim 1, wherein the coagulation reaction data is data representing the change in the coagulation reaction over time.

4. The method according to claim 3, wherein the coagulation reaction data is a correction curve R(i) or a coagulation rate curve V(i), where R(i) is a curve obtained by relativeizing the coagulation reaction curve P(i) so that the value at a set time Te becomes 100%, where Te is 120 to 600 seconds, V(i) is the first derivative curve of the coagulation reaction curve P(i), and i is the number of measurement points.

5. The method according to claim 1, wherein the index representing the turbidity change is an index representing a decrease in turbidity.

6. The index representing the turbidity transition is RmaxT, ΔR, rF, SFa, or SFb, where RmaxT is the time when R(i) is Rmax, ΔR = Rmax - 100, where Rmax is the maximum value of R(i), rF is the slope of the regression line of R(i) in the interval from the starting point St to the ending point Et, where St is a point after the starting point of the end stage of the coagulation reaction, and Et ≤ Te, SFa is the area under the curve of R(i) in the range from Ts to Te where R(i) exceeds 100, where Ts is the time when R(i) = 100 before RmaxT, and SFb is the area under the curve of R(i) in the range from RmaxT to Te where R(i) exceeds 100. The method according to claim 4.

7. The method according to claim 4, wherein the index representing the turbidity transition is rT, where rT is the number of i values ​​or the time length corresponding to the number of i values ​​that satisfy rV(i) ≥ rVs in the range from the starting point of the end of the coagulation reaction to the set time Tev, where rV(i) = V(i) / Vmax × 100, Vmax is the maximum value of V(i), Tev is 80 to 180 seconds, and rVs is 0.01 to 0.

2.

8. The index representing the turbidity transition is tR or sR, where, tR is the number of i values ​​or the time length corresponding to the number of i values ​​that satisfy rR(i) ≥ 100 in the range from the start of the final stage of the coagulation reaction to the set time Ter, sR is the slope of the regression line of R(i) from Ta to the set time Tes, where, Ter is 90 to 600 seconds, Tes is 120 to 600 seconds, Ta is the time corresponding to the maximum value of i that satisfies R(i) ≥ 50 and rR(i) = sRs, rR(i) = {SumR(i) / SumR(i - k)} × 100, where SumR(i) is expressed by the following formula, The method according to claim 4, wherein i > k, k = 2 to 20, and sRs is 100.05 to 101.

00.

9. The method according to claim 1, further comprising calculating the APTT of the test blood sample based on the coagulation reaction data.

10. The method according to claim 1, wherein the blood sample to be tested is a blood sample that does not have APTT prolongation.

11. The method according to claim 10, wherein the blood sample to be tested is a blood sample that does not have PT prolongation.

12. The method according to claim 1, further comprising estimating the fibrinogen concentration of the test blood sample based on the coagulation reaction data before obtaining the value of the index representing the turbidity transition.

13. The method according to claim 12, wherein the coagulation reaction data is the maximum value of the coagulation reaction curve P(i), or P(i) at the starting point of the end stage of the coagulation reaction.

14. The method according to claim 13, further comprising estimating that the fibrinogen concentration of the blood sample is lower than the lower limit of the normal range when the maximum value of P(i) or the value of P(i) at the starting point of the end stage of the coagulation reaction is smaller than a predetermined value.

15. The method according to claim 14, wherein, if the estimated fibrinogen concentration is lower than the lower limit of the normal range, information indicating that the fibrinogen concentration of the blood sample is low is output, or the value of the index representing the turbidity change is not obtained.

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