Method for analyzing blood clotting ability of blood sample

Abstract

The present invention provides a method for analyzing the blood clotting ability of a blood sample. The method comprises the following steps: 1) detecting a coagulation reaction end point Pe in a coagulation reaction curve of a detected blood sample in which whether a substance with coagulation VIII factor substitution activity exists or not needs to be detected; 2) calculating T (X), where T (X) represents the measurement point or time at which the coagulation reaction curve reaches X% of Pe, and X is a variable greater than 0 and equal to or less than 100; and 3) detecting a blood sample containing a substance having a coagulation factor VIII alternative activity on the basis of T (X).

Description

Technical Field

[0001] The present invention relates to a method for analyzing the coagulation ability of a blood sample. Background Art

[0002] Hemophilia A is a hemorrhagic disease caused by a deficiency of coagulation factor VIII (FVIII) or a functional defect. Hemophilia A is classified into congenital hemophilia A with congenital deficiency of FVIII and acquired hemophilia A caused by autoantibodies (inhibitors) against FVIII, and there are also cases where congenital hemophilia A patients have inhibitors. In addition, hemophilia A is classified into mild, moderate, and severe according to the FVIII activity level in the blood. The blood coagulation time of samples from hemophilia A patients is prolonged according to the severity in blood coagulation tests.

[0003] Emicizumab, a therapeutic agent for hemophilia A, is a recombinant humanized bispecific antibody that can promote the blood coagulation reaction. Emicizumab binds to activated coagulation factor IX (FIXa) at one antigen-binding site and to coagulation factor X (FX) at the other antigen-binding site to substitute the cofactor function of activated coagulation factor VIII (FVIIIa), thereby promoting the blood coagulation reaction.

[0004] Emicizumab is regularly administered for the purpose of suppressing the bleeding tendency in hemophilia A patients. The administered emicizumab remains in the blood for a certain period. During this period, since the blood coagulation time, which is a typical feature of coagulation abnormalities, is not observed in the blood sample obtained by collecting blood from a hemophilia A patient administered with emicizumab in a blood coagulation test, the result is as if there is no coagulation abnormality. On the other hand, the shortening (to below the upper limit value of the reference range) of the prolonged coagulation time of hemophilia A patients caused by such emicizumab may pose a significant risk to the patients. For example, in the case where a hemophilia A patient is urgently transported due to a traffic accident or the like and undergoes surgery, if the prolongation of the coagulation time (activated partial thromboplastin time; APTT) is not observed in the usual pre-operative blood coagulation test, a doctor who is not aware that the patient has hemophilia A fails to notice the reduction in the patient's coagulation ability and cannot perform appropriate treatment, so there is a risk of significant bleeding during the operation.

[0005] In Patent Document 1, there is described a method for evaluating a blood coagulation reaction caused by a substance having FVIII replacement activity by adding a blood coagulation starting reagent containing active coagulation factor XI to a blood sample and measuring the amount of thrombin generation. In Patent Documents 2 and 3, there are described methods for evaluating the coagulation ability of a blood sample, which include obtaining a parameter related to the differentiation of the coagulation waveform of a blood sample after administration of a substance having FVIII replacement activity, obtaining an FVIII activity value as a value representing the coagulation ability based on this parameter, and evaluating the coagulation ability of the blood sample based on the obtained FVIII activity value.

[0006] Prior Art Documents

[0007] Patent Documents

[0008] Patent Document 1: International Publication Gazette No. 2014 / 050926

[0009] Patent Document 2: International Publication Gazette No. 2016 / 170944

[0010] Patent Document 3: Japanese Unexamined Patent Application Publication No. 2017 - 106925 Summary of the Invention

[0011] The present invention provides a method for analyzing the coagulation ability of a blood sample, which can detect a blood sample after administration of a medicament having FVIII replacement activity.

[0012] The present invention provides the following.

[0013] 〔1〕A method, which is a method for analyzing the coagulation ability of a blood sample, comprising:

[0014] 1) Detecting the coagulation reaction end point Pe in the coagulation reaction curve of a test blood sample that needs to be detected for the presence or absence of a substance having factor VIII replacement activity;

[0015] 2) Calculating T(X), where T(X) represents the measurement point or time at which the coagulation reaction curve reaches X% of Pe, and X is a variable greater than 0 and 100 or less;

[0016] 3) Detecting a test blood sample containing a substance having factor VIII replacement activity based on T(X).

[0017] 〔2〕The method according to 〔1〕, wherein the test blood sample is a blood sample in which the activated partial thromboplastin time, i.e., APTT, is not prolonged.

[0018] 〔3〕The method according to 〔1〕 or 〔2〕, wherein the above (3) includes: calculating a parameter according to T(X), and detecting a test blood sample containing the substance having the coagulation factor VIII replacement activity according to the parameter.

[0019] The parameter is at least one selected from the change amount of T(X) within a given range of X, the ratio of T(X), the interval change rate of T(X), the parameter related to the coefficient of variation of T(X), and their standard deviation index (SDI).

[0020] 〔4〕The method according to 〔3〕, wherein

[0021] The change amount of T(X) within a given range of X is [(T(X2) / C) - (T(X1) / C)];

[0022] The ratio of T(X) within a given range of X is [T(X2) / T(X1)];

[0023] The interval change rate of T(X) within a given range of X is [(X2 - X1) / {(T(X2) / C) - (T(X1) / C)}];

[0024] Here, X1 and X2 are each independently 1 to 100, where X2 > X1, and C is a constant.

[0025] 〔5〕The method according to 〔3〕, wherein

[0026] The parameter related to the coefficient of variation of T(X) is CV(X1:X2), CV(X1 - J:X1 + J), or AVE{CV(X1 ± J):CV(X2 ± J)},

[0027] Here,

[0028] CV(X1:X2) is the coefficient of variation of T(X) in the range of X from X1 to X2, expressed by the following formula:

[0029] CV(X1:X2) = SD{T(X1):T(X2)} / AVE{T(X1):T(X2)} × 100 [%]

[0030] AVE{T(X1):T(X2)}: The average value of T(X1) to T(X2)

[0031] SD{T(X1):T(X2)}: The standard deviation of T(X1) to T(X2)

[0032] CV(X1 - J:X1 + J) is the coefficient of variation of T(X) in the range of (X1 - J) to (X1 + J),

[0033] AVE{CV(X1±J): CV(X2±J)} is the average value of CV(X−J: X+J) of X between X1 and X2,

[0034] wherein X1 and X2 are each independently 1 to 100, and X2 > X1, and J = 2 to 44.

[0035] 〔6〕The method according to any one of 〔3〕 to 〔5〕, wherein it includes detecting a test blood sample containing the substance having the coagulation factor VIII substitution activity by comparing the parameter of the above-mentioned T(X) with a given threshold value.

[0036] 〔7〕The method according to any one of 〔1〕 to 〔6〕, wherein it includes outputting the result of the above-mentioned detection.

[0037] 〔8〕The method according to any one of 〔1〕 to 〔7〕, further including:

[0038] measuring the activated partial thromboplastin time of the blood sample, i.e., APTT;

[0039] selecting a blood sample with no prolonged APTT as the test blood sample that needs to detect the presence or absence of the substance having the coagulation factor VIII substitution activity.

[0040] 〔9〕The method according to any one of 〔1〕 to 〔8〕, wherein the substance having the coagulation factor VIII substitution activity is a bispecific antibody that substitutes for the cofactor function of coagulation factor VIII.

[0041] 〔10〕The method according to any one of 〔1〕 to 〔9〕, wherein the substance having the coagulation factor VIII substitution activity is emicizumab.

[0042] 〔11〕A program for implementing the method according to any one of 〔1〕 to 〔10〕.

[0043] 〔12〕An apparatus for implementing the method according to any one of 〔1〕 to 〔10〕.

[0044] 〔13〕A system for implementing the method according to any one of 〔1〕 to 〔10〕, comprising a program for implementing the method according to any one of 〔1〕 to 〔10〕 and a device controlled by the program.

[0045] 〔14〕The apparatus according to 〔12〕 or the system according to 〔13〕, wherein it comprises an output unit for outputting the result of the detection of a test blood sample containing a substance having the coagulation factor VIII substitution activity by using the method according to any one of 〔1〕 to 〔10〕.

[0046] According to the method of the present invention, from a blood sample in which coagulation abnormalities could not be detected in previous APTT measurements, using the measurement data (coagulation reaction data) of the coagulation reaction obtained during APTT measurement, it is possible to detect that the test sample is a blood sample after administration of a medicament having FVIII replacement activity such as emicizumab. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 is an example of a coagulation reaction curve.

[0048] Figure 2 is an explanation of T(X). The times when reaching 10%, 50%, and 90% of Pe on the coagulation reaction curve are represented as T(10), T(50), and T(90), respectively.

[0049] Figure 3A is an example of a blood coagulation analysis process using the method of the present invention.

[0050] Figure 3B is a conceptual diagram showing the configuration of a device for performing an analysis method of coagulation ability using the present invention.

[0051] Figure 4 is the coagulation reaction curve P(i) of the sample group used in the examples. A - C: P(i) of NE, E1, and E2. D, E: A graph showing the average values of P(i) of E1 and E2 (aE1 and aE2; dashed lines) together with the average value of P(i) of NE (aNE; solid line).

[0052] Figure 5 is the APTT of the sample group used in the examples. NE: control sample group, E1: sample from a hemophilia A patient administered emicizumab. E2: sample from a hemophilia A patient administered emicizumab and containing an inhibitor.

[0053] Figure 6 is the modified reaction curve of the sample group used in the examples. A - C: modified reaction curves of NE, E1, and E2. D, E: A graph showing the average values of the modified reaction curves of E1 and E2 (aE1 and aE2; dashed lines) together with the average value of the modified reaction curve of NE (aNE; solid line).

[0054] Figure 7 A is the plotted points of the interval change rate of T(X) of each sample with respect to APTT. Figure 7 B is the distribution of the interval change rate of each sample group.

[0055] Figure 8A is the plotted point of the change in T(X) of each sample relative to APTT. Figure 8 B is the distribution of the change in T(X) of each sample group.

[0056] Figure 9 A is the plotted point of T70 / T30 of each sample relative to APTT. Figure 9 B is the distribution of T70 / T30 of each sample group.

[0057] Figure 10 is the CV curve of the sample groups used in the examples. A - C: CV curves of NE, E1, and E2. D, E: Graph showing the average values of the CV curves of E1 and E2 (aE1 and aE2 respectively; dashed lines) together with the average value of the CV curve of NE (aNE; solid line).

[0058] Figure 11 A is the plotted point of CV(30:50) of each sample relative to APTT. Figure 11 B is the distribution of CV(30:50) of each sample group.

[0059] Figure 12 A is the plotted point of AVE{CV(30±4):CV(50±4)} of each sample relative to APTT. Figure 12 B is the distribution of AVE{CV(30±4):CV(50±4)} of each sample group.

[0060] Figure 13 A is the plotted point of Vmax of each sample relative to APTT. Figure 13 B is the distribution of Vmax of each sample group.

[0061] Figure 14 A is the plotted point of VmaxT of each sample relative to APTT. Figure 14 B is the distribution of VmaxT of each sample group. Detailed implementation mode

[0062] All patent documents, non - patent documents, and other publications cited in this specification are incorporated herein by reference in their entirety.

[0063] In a blood coagulation test for activated partial thromboplastin time (APTT) measurement, a prescribed reagent is added to a test blood sample, and the subsequent blood coagulation reaction is measured. Based on the coagulation reaction data, the blood coagulation time (APTT) is determined. For the measurement of the blood coagulation reaction, common methods can be used, such as optical methods for measuring the amount of scattered light, transparency, absorbance, etc., or mechanical methods for measuring the viscosity of plasma. Generally, the blood coagulation reaction is represented by a coagulation reaction curve showing the time-dependent change in the amount of the coagulation reaction. In the following description of this specification, the blood sample, blood coagulation reaction, and blood coagulation time may sometimes be abbreviated as sample, coagulation reaction, and coagulation time, respectively.

[0064] In samples with coagulation abnormality factors, in most cases, the APTT is prolonged compared to normal samples without coagulation abnormalities. The prolongation of APTT is one of the main indicators of coagulation abnormalities related to bleeding risk or thrombosis risk. In samples from patients with hemophilia A, the APTT is usually prolonged compared to the reference range (the range in which samples with APTT within this range are regarded as "normal"). On the other hand, when samples from hemophilia A patients are administered with substances having FVIII replacement activity such as emicizumab (hereinafter, also referred to as "FVIII replacement substances"), the APTT of the samples of these patients is not prolonged and converges within or exceeds the reference range and shortens. It is difficult to distinguish whether the test sample is a sample from a hemophilia A patient administered with an FVIII replacement substance based on the reference range of APTT.

[0065] The present inventors found that even when no prolongation of the coagulation time is observed in the APTT measurement for blood samples from hemophilia A patients administered with emicizumab, the progress of the blood coagulation reaction (coagulation reaction curve) is different from that of samples not administered with emicizumab.

[0066] In the present invention, based on the coagulation reaction data of the test sample measured during APTT measurement, specifically based on the parameters derived from the coagulation reaction curve, the presence or absence of an FVIII factor replacement substance in the test sample is detected. The above parameters used in the present invention are parameters obtained without differentiating the coagulation reaction curve (coagulation waveform), and are different from the parameters related to the differentiation of the coagulation waveform used for evaluating coagulation ability in Patent Documents 2 and 3 above. In the present invention, samples administered with FVIII replacement substances can be detected from samples in which coagulation abnormalities could not be detected in previous APTT measurements.

[0067] In this specification, an FVIII replacement substance refers to a substance that can replace the cofactor function of FVIII. The cofactor function of FVIII refers to the ability of activated FVIII (FVIIIa) to form a complex of activated coagulation factor IX (FIXa) and coagulation factor X (FX). As an example of an FVIII replacement substance, a bispecific antibody that replaces the cofactor function of FVIII (for example, a bispecific antibody that mimics the FVIII structure) can be cited. As an example of such a bispecific antibody, emicizumab can be cited. Emicizumab is a recombinant humanized bispecific antibody that binds to activated coagulation factor IX (FIXa) at one antigen-binding site and binds to coagulation factor X (FX) at the other antigen-binding site, thereby replacing the cofactor function of FVIII. Emicizumab is a medicament for suppressing bleeding tendency in congenital and acquired hemophilia A. Furthermore, next-generation FIXa / FX bispecific antibodies (Mim8, NXT007, etc.) having the same mechanism of action as emicizumab are also being developed.

[0068] [Analysis method for coagulation ability of blood sample]

[0069] In the analysis method for the coagulation ability of a blood sample based on the present invention (hereinafter, also referred to as the method of the present invention), the difference in the coagulation ability of blood samples that cannot be distinguished in the clotting time (APTT) can be detected as the difference in the progress of the coagulation reaction (coagulation reaction curve). In the method of the present invention, based on the coagulation reaction curve of the test sample, parameters for analyzing the coagulation ability of the test sample can be obtained without obtaining its differential curve.

[0070] Hereinafter, the method of the present invention will be described.

[0071] 1. Acquisition of coagulation reaction curve and detection of coagulation reaction end point Pe

[0072] 1.1. Coagulation reaction measurement

[0073] In the method of the present invention, as the test sample, plasma of a subject who has had blood drawn for a coagulation test is preferably used. For this sample, an anticoagulant commonly used for coagulation tests can be added. For example, plasma can be obtained by centrifuging after blood is drawn using a blood collection tube containing sodium citrate.

[0074] The coagulation reaction curve of the test sample used in the method of the present invention can be calculated from the coagulation reaction data obtained according to the steps of the coagulation reaction measurement in the conventional APTT assay. Specifically, in the coagulation reaction measurement, the APTT assay reagent is added to the test sample to start the blood coagulation reaction. The coagulation reaction in the mixed solution containing the reagent and the test sample is measured. APTT assay reagents are commercially available (for example, APTT reagent Coagpia APTT-N and Coagpia APTT-N calcium chloride solution; both are manufactured by Sekisui Medical Co., Ltd.). For the measurement of the coagulation reaction, conventional means can be used, such as optical means for measuring the amount of scattered light, transparency, absorbance, etc., or mechanical means for measuring the viscosity of plasma, etc. In the following description, the method of the present invention will be described using the coagulation reaction measurement based on the amount of scattered light as an example.

[0075] The reaction start time of the coagulation reaction can typically be defined as the moment when the reagent is mixed into the sample to start the coagulation reaction, but other opportunities can also be defined as the reaction start time. The time for continuing the measurement of the coagulation reaction can be, for example, tens of seconds to about 7 minutes from the moment the sample and the reagent are mixed. The measurement time can be an arbitrarily determined fixed value, or it can be the moment until the end of the coagulation reaction of each sample is detected. During the measurement time, the measurement of the progress of the coagulation reaction (photometry of the amount of scattered light) can be repeated at specified intervals. For example, it can be measured at intervals of 0.1 seconds. The temperature of the mixed solution in the measurement is a normal condition, for example, 30°C to 40°C, preferably 35°C to 39°C. In addition, the various conditions of the measurement can be appropriately set according to the sample to be tested, the reagent, the measuring means, etc.

[0076] The above-mentioned series of operations for measuring the coagulation reaction can be performed using an automatic analyzer. An example of an automatic analyzer is the blood coagulation automatic analyzer CP3000 (manufactured by Sekisui Medical Co., Ltd.). Alternatively, some operations can be performed manually. For example, a human operator may prepare the sample to be tested, and subsequent operations may be performed by the automatic analyzer.

[0077] 1.2. Obtaining the coagulation reaction curve

[0078] Based on the measured solidification reaction data, a solidification reaction curve P(i) can be obtained. Here, "i" represents the number of measurement points or time from the start of the solidification reaction (also simply referred to as the number of measurement points and time respectively). For example, if the measurement (light measurement) interval is 0.1 second, it is expressed as time = 0.1 × the number of measurement points. That is, P(i) can be a function of the number of measurement points or a function of time. In the following description of this specification, P(i) is sometimes simply referred to as P. Generally, the solidification reaction curve P is obtained by removing noise or smoothing the measured values of the solidification reaction using ordinary means, or performing zero adjustment for adjusting the initial value or relative value conversion of the curve as needed.

[0079] An example of the solidification reaction curve is shown in Figure 1 . Figure 1 The horizontal axis of [Figure] represents time, and the vertical axis represents the solidification reaction (scattering light amount), and is adjusted so that the scattering light amount at the start of measurement is 0. As time passes, due to the progress of the solidification reaction of the mixed solution, the scattering light amount increases. As Figure 1 shown, the solidification reaction curve based on the scattering light amount is generally S-shaped.

[0080] If necessary, the differential curve of this solidification reaction curve P can be obtained, such as the reaction rate curve (first derivative) or the reaction acceleration curve (second derivative). The differential processing of the solidification reaction curve can be implemented using any method. For example, it can be performed by calculating the average slope value within an interval. In this specification, the solidification reaction curve, the first derivative curves such as the reaction rate curve, and the second derivative curves such as the reaction acceleration curve are also respectively referred to as "0th curve", "1st curve", and "2nd curve".

[0081] 1.3. Detection of the solidification reaction end point Pe

[0082] In the method of the present invention, the solidification reaction end point Pe of the solidification reaction curve P of the test sample is detected. Pe can be determined based on any criterion such as the time when P reaches a steady state, the time when the first derivative curve of P reaches a peak and then decreases to 0 or a fixed value (refer to WO2021 / 206107), the earliest point at which the cumulative ratio of P(i) in a minute time period becomes less than a threshold value (e.g., 1.001) (refer to WO2021 / 132552 or the embodiments described later). The detection of Pe can be performed after obtaining P up to a specified measurement time, or the detection of Pe can also be performed in parallel with the acquisition of P, and the acquisition of P is ended after detecting Pe. In the latter case, the measurement time of one sample can be shortened to the necessary minimum. For example, the steps described in WO2021 / 206107 or WO2021 / 132552 can perform the detection of Pe in parallel with the acquisition of P (so-called detection of Pe in real time).

[0083] As an embodiment of the method of the present invention, an example of the detection step of Pe according to the method described in WO2021 / 132552 will be described in detail. The cumulative ratio of P(i) in a minute time period is defined as Z(i) and calculated using the following formula:

[0084] Cumulative ratio Z(i)=Pb(i) / Pa(i)

[0085] Pa(i)=Sum of P(i - m) to P(i - 1)

[0086] Pb(i)=Sum of P(i + 1) to P(i + m)

[0087] In the above formula, i represents the measurement point number, and m can be appropriately set according to the measurement conditions of the solidification reaction, analysis items, etc. For example, m = 10 to 30. Detect P(i) at the earliest measurement point or time when Z(i) becomes less than the threshold value Zs as the solidification reaction end point Pe. Zs can be appropriately set according to the analysis item, but is greater than 1 and 1.1 or less. For example, in the case of APTT measurement, it is preferably that Zs is 1.05 or less, and more preferably in the range of 1.01 to 1.001. In order to prevent misdetection of Pe due to the initial reaction or the like, the calculation of Z(i) is preferably performed after i reaches a specified calculation start point, that is, after P(i) becomes a specified value or more. In this step, it is possible to measure the solidification reaction while obtaining P(i) and calculating Z(i) in parallel, thereby detecting Pe.

[0088] 1.4. APTT Calculation

[0089] The coagulation time (APTT) of the test sample can be calculated from the coagulation reaction data obtained in the above-mentioned coagulation reaction measurement. The calculation method of APTT is not particularly limited. For example, it can be mentioned that APTT can be calculated based on the above P(i) or its first derivative curve according to an arbitrary method. As an example of the calculation method of APTT, a method of calculating the coagulation time by taking the time when P(i) reaches N% of the coagulation reaction end point Pe (percentage detection method) can be mentioned; a method of calculating the coagulation time by taking the time when the first curve of P reaches N% of its maximum value; a method of calculating the coagulation time by taking the cumulative ratio of P(i) in a minute time period reaching a specified value as the calculation starting point Te and taking the time when P(i) reaches N% of P(Te) (Japanese Patent Laid-Open No. 6-249855); a method of calculating the coagulation time based on the time-dependent change of the cumulative ratio of P(i) in a minute time period (refer to WO2021 / 132552); a method of calculating the coagulation time based on the weighted average time of the first curve of P (refer to WO2021 / 177452); a method of calculating the coagulation time by taking the time when the first curve of P reaches a specified value after reaching the maximum value as the calculation starting point Te and taking the time when P(i) reaches N% of P(Te) (refer to WO2021 / 206107), etc.

[0090] 2. Calculation of T(X)

[0091] After detecting Pe, T(X) representing the number of measurement points or time when the coagulation reaction curve P(i) reaches X% of Pe is calculated. More specifically, T(X) is the number of measurement points or time from the start point (time 0) of the coagulation reaction measurement to when P reaches X% of Pe. That is, the following formula holds for T(X).

[0092] P(T(X)) = Pe × X%

[0093] Here, X is a variable greater than 0 and less than or equal to 100. Therefore, T(X) is a function of the variable X. In the method of the present invention, X is preferably 1 to 100, more preferably 5 to 95. For example, each X can be a value deviated from 1 or more (i.e., the increment of X ≥ 1), or can also be a value deviated from 5 or more (i.e., the increment of X ≥ 5). It is preferred that X increases from the initial value with an increment J (J is an integer from 1 to 5). In one example, X is a variable that changes with an increment of 1 within the range of 1 to 100 (i.e., X = {1, 2, 3, ···, 97, 98, 99, 100}). In another example, X is a variable that changes with an increment of 5 within the range of 5 to 95 (i.e., X = {5, 10, 15, ···, 90, 95}).

[0094] Alternatively, T(X) can be expressed as the average value of T(X−K) to T(X+K). In this case, K is preferably less than the increment J of X described above. For example, if K = 2, then T(X) can be expressed as the average value of T(8) to T(12) when X = 10, or as the average value of T(13) to T(17) when X = 15.

[0095] Figure 2 is a diagram for explaining T(X). In the figure, a solidification reaction curve showing the points reaching 10 to 90% of Pe is shown, and the times reaching 10%, 50%, and 90% of Pe are represented as T(10), T(50), and T(90), respectively. The time tPe of Pe corresponds to T(100). It should be noted that in this specification and the drawings, for T(X), the parentheses of X may sometimes be removed and labeled as "TX".

[0096] 3. Acquisition of parameters for analyzing coagulation ability

[0097] 3.1. Test sample for which parameters should be acquired

[0098] Next, in the method of the present invention, parameters for analyzing the coagulation ability of a test sample are acquired based on the above T(X). In the method of the present invention, it is sufficient to acquire such parameters for a test sample that needs to be tested for the presence or absence of an FVIII replacement substance. A test sample that needs to be tested for the presence or absence of an FVIII replacement substance is, generally speaking, a sample in which the APTT is not prolonged. It is inferred that a sample with a prolonged APTT is a sample with coagulation abnormalities (i.e., a high bleeding risk or thrombus risk) that does not require investigation of the presence or absence of an FVIII replacement substance, and therefore the method of the present invention is not necessarily applicable.

[0099] In one embodiment of the method of the present invention, the solidification reaction of an arbitrary test sample is measured, and after calculating the APTT, a sample in which the APTT is not prolonged is selected as a test sample that needs to be tested for the presence or absence of an FVIII replacement substance. For the selected test sample, T(X) can be acquired from the solidification reaction data (such as corrected P(i), Pe) obtained in the measurement of the solidification reaction described above. This T(X) can be calculated together with the above P(i) etc. when calculating the APTT, or this T(X) can also be calculated as follows: After calculating the APTT and selecting a test sample in which the APTT is not prolonged for which the method of the present invention should be applied, it is calculated based on the solidification reaction data (such as corrected P(i) and Pe) of the selected test sample. In another embodiment of the method of the present invention, as a test sample that needs to be tested for the presence or absence of an FVIII replacement substance, a sample known to have a non-prolonged APTT is selected from existing solidification reaction data. T(X) can be acquired from this existing solidification reaction data.

[0100] 3.2. Acquisition of parameters based on T(X)

[0101] Based on the above-mentioned T(X), parameters for analyzing the blood coagulation ability of the test sample can be calculated. As examples of such parameters, the change amount of T(X) within a given range of X, the ratio of T(X), and the interval change rate of T(X) can be cited. Preferably, the change amount of T(X) is represented by [T(X2) - T(X1)], the ratio of T(X) is represented by [T(X2) / T(X1)], and the interval change rate of T(X) is represented by [(X2 - X1) / (T(X2) - T(X1))]. Alternatively, the change amount of T(X) and the interval change rate of T(X) can be calculated respectively as the change amount and the interval change rate of the relative value of T(X). For example, the change amount of T(X) can be represented by [(T(X2) / C) - (T(X1) / C)], and the interval change rate of T(X) can be represented by [(X2 - X1) / {(T(X2) / C) - (T(X1) / C)}]. Here, X1 and X2 are each independently preferably 1 to 100, more preferably 5 to 95, where X2 > X1. More specifically, in the case of the ratio of T(X), preferably X1 = 5 to 55, X2 = 30 to 95, and X2 - X1 = 10 to 90. In the case of the change amount of T(X), preferably X1 = 5 to 55, X2 = 35 to 95, and X2 - X1 = 10 to 90. In the case of the interval change rate of T(X), preferably X1 = 5 to 55, X2 = 30 to 90, and X2 - X1 = 10 to 85. C is a constant, and examples thereof can include APTT, VmaxT (the time when the reaction rate becomes maximum, see below), any T(X) within the range of X = 40 to 60, etc.

[0102] As another example of the above parameters, a parameter related to the coefficient of variation CV of T(X) can be cited. In one example, the parameter related to this CV is CV(X1:X2) represented by the following formula, which represents the coefficient of variation CV of T(X) when X is within the range of X1 to X2. Here, X1 and X2 are each independently preferably 1 to 100, where X2 > X1.

[0103] CV(X1:X2) = SD{T(X1):T(X2)} / AVE{T(X1):T(X2)} × 100 [%]

[0104] Here,

[0105] AVE{T(X1):T(X2)}: The average value of T(X1) to T(X2).

[0106] SD{T(X1):T(X2)}: The standard deviation of T(X1) to T(X2).

[0107] Preferably, X1 = 5 to 55, X2 = 30 to 95, and X2 - X1 = 10 to 90.

[0108] In another example, the parameter related to this CV is the coefficient of variation CV(X1 - J:X1 + J) (also denoted as CV(X±J)) of T(X) within the range of (X1 - J) to (X1 + J). Here, preferably, X1 is 25 to 65 and J is 2 to 44.

[0109] In another example, the parameter related to this CV is the average value [AVE{CV(X1±J):CV(X2±J)}] of CV(X±J) when X is between X1 and X2. Preferably, X1 = 10 to 65, X2 = 25 to 95, and X2 - X1 = 5 to 85 (J is defined as above).

[0110] The values of the above parameters can be transformed into standard deviation indices (SDI) relative to a reference value, and the obtained values can be used as parameters for analyzing the coagulation ability of the test sample of the present invention. Specifically, the absolute value (|SDI|) of the SDI calculated by the following formula using the statistical values of a sample without FVIII replacement substance (i.e., a sample whose coagulation time without FVIII replacement substance is not prolonged, hereinafter also referred to as a non - administered (ND) sample) can be used as a parameter: |SDI| = |{(parameter value calculated from the test sample)-(average value of parameter values calculated from the ND sample group)} / (standard deviation of parameter values calculated from the ND sample group)|.

[0111] 4. Detection of Samples Containing FVIII Replacement Substance

[0112] As shown in the examples described later, the parameter values calculated for analyzing the coagulation ability in the present invention described above show different tendencies between the samples containing FVIII replacement substance (emicizumab) obtained from patients administered with FVIII replacement substance and the samples without FVIII replacement substance (emicizumab). Based on these parameters, samples containing FVIII replacement substance can be detected.

[0113] In the analysis of the blood coagulation ability of the present invention, as examples of the parameters for detecting the sample containing the FVIII replacement substance, at least one selected from the change amount of T(X), the ratio of T(X), the interval change rate of T(X), and the parameter related to the coefficient of variation of T(X) within the given range of X described above can be cited. As a more preferred example of this parameter, at least one selected from [(T(X2) / C) - (T(X1) / C)], [T(X2) / T(X1)], [(X2 - X1) / {(T(X2) / C) - (T(X1) / C)}], CV(X1:X2), CV(X±J), and AVE{CV(X1±J):CV(X2±J)} described above can be cited (where the definitions of X1, X2, C, and J are as described above). Alternatively, as an example of the parameter for detecting the sample containing the FVIII replacement substance, the SDI of each of the above parameters can also be cited. In the analysis of the blood coagulation ability of the present invention, any one of these parameters can be used, or two or more of them can be used in combination.

[0114] In the detection of the sample containing the FVIII replacement substance based on the above parameters, for example, in the case of the interval change rate of T(X) [(X2 - X1) / {(T(X2) / C) - (T(X1) / C)}], when the parameter calculated from the test sample is below or less than a specified detection threshold (hereinafter, also simply referred to as "threshold"), the test sample can be detected as a sample containing the FVIII replacement substance. On the other hand, in the case where the parameter is the change amount of T(X) [(T(X2) / C) - (T(X1) / C)], the ratio of T(X) [T(X2) / T(X1)], CV(X1:X2), CV(X±J), or AVE{CV(X1±J):CV(X2±J)}, when the parameter calculated from the test sample is above or greater than the threshold, the test sample can be detected as a sample containing the FVIII replacement substance.

[0115] As the above-mentioned threshold value, a value can be set that can distinguish a sample containing an FVIII replacement substance from a sample not containing an FVIII replacement substance (ND sample). For example, when the parameter is [(X2 - X1) / (T(X2) / C - T(X1) / C)], a statistical value (minimum value, average value, standard deviation, etc.) of the parameter calculated based on the ND sample group (for example, average value - K × standard deviation (SD), K is preferably 1.2 to 2.4) can be set as the threshold value. On the other hand, when the parameter is [T(X2) / C - T(X1) / C], [T(X2) / T(X1)], CV(X1:X2), CV(X±J), or AVE{CV(X1±J):CV(X2±J)}, a statistical value (maximum value, average value, standard deviation, etc.) of the parameter calculated based on the ND sample group (for example, average value + K × standard deviation (SD), K is preferably 1.3 to 3.4) can be set as the threshold value.

[0116] When the parameter is the SDI of each of the above-mentioned parameters, the above-mentioned threshold value can be set according to the SDI of the parameter pre-calculated by the sample group containing the FVIII replacement substance or the ND sample group. For example, the threshold value for the above-mentioned |SDI| can be set as follows: Among the parameters calculated from the sample group containing the FVIII replacement substance, extract the parameter value having the value closest to the distribution region of the ND sample group, and calculate its |SDI| according to the above formula; on the other hand, among the parameters calculated from the ND sample group, extract the parameter value having the value closest to the distribution region of the sample group containing the FVIII replacement substance, and calculate its |SDI| according to the above formula; the intermediate value between the |SDI| of the former and the |SDI| of the latter can be set as the threshold value (refer to Example 5 described later). Or in addition to this intermediate value, any value that can distinguish the distribution of the |SDI| of the former from the |SDI| of the latter can be set as the threshold value. If the |SDI| calculated from the test sample is greater than this threshold value, the test sample can be detected as a sample containing an FVIII replacement substance.

[0117] As the above-mentioned detection criterion, two or more of the above-mentioned parameters can be used. In this case, a threshold value is set for each parameter, and it is investigated whether each parameter calculated from the test sample satisfies the criterion based on its respective threshold value. In one example, if all of the two or more parameters satisfy the threshold value, the test sample is detected as a sample containing an FVIII replacement substance. In another example, when at least one of the two or more parameters satisfies the threshold value, the test sample is detected as a sample that may contain an FVIII replacement substance.

[0118] The sample tested as a sample containing an FVIII replacement substance is a sample from a patient with abnormal coagulation ability who has been administered an FVIII replacement substance, and it is inferred that the patient is a patient with a high bleeding risk during surgery or the like. Clinically, as an FVIII replacement substance, emicizumab, which is a medicament for suppressing the bleeding tendency of hemophilia A, is known. Therefore, it can be inferred that the patient from whom the sample containing the FVIII replacement substance detected by the method of the present invention is derived is a hemophilia A patient who has been administered emicizumab.

[0119] In the above detection criteria, within the range where a sample containing an FVIII replacement substance can be detected, those skilled in the art should understand that the criteria of "greater than the threshold" and "above the threshold" can be used interchangeably, and similarly, the criteria of "less than the threshold" and "below the threshold" can be used interchangeably.

[0120] 5. Application to Other Coagulation Reaction Measurement Methods

[0121] Above, the case of measuring the coagulation reaction based on the amount of scattered light has been used as an example to illustrate the method of the present invention. However, those skilled in the art can apply other coagulation reaction measurement methods (such as coagulation reaction measurement based on transparency, absorbance, viscosity, etc.) to the coagulation reaction measurement in the present invention. Therefore, such an application is included in the scope of the present invention. For example, the sign of P(i) based on the inverted S-shaped coagulation reaction curve of transparency (amount of transmitted light) is opposite to that of P(i) based on the above-mentioned amount of scattered light. In such a case, it is obvious to those skilled in the art that the sign of P(i) is reversed in the method of the present invention described above.

[0122] 6. Program and Device

[0123] The method for analyzing coagulation ability of the present invention can be automatically performed using a computer program. Therefore, one aspect of the present invention is a program for performing the method for analyzing coagulation ability of the present invention described above. In addition, a series of steps of the method of the present invention described above can be automatically performed using an automatic analysis device. Therefore, one aspect of the present invention is a device for performing the method for analyzing coagulation ability of the present invention described above. The execution of the method of the present invention using this device can be controlled by the program of the present invention. Another aspect of the present invention is a system for performing the method for analyzing coagulation ability of the present invention, and this system includes a device or program for performing the method for analyzing coagulation ability of the present invention described above.

[0124] An example of the blood coagulation analysis process using the method of the present invention implemented under the control of the program of the present invention is described below and Figure 3A in

[0125] S1: Obtain P(i) based on the measured coagulation reaction data (arbitrary).

[0126] S2: Calculate APTT based on P(i).

[0127] S3: If there is no APTT prolongation, go to S4; if APTT is prolonged, go to S8.

[0128] S4: Calculate T(X) based on P(i).

[0129] S5: Calculate one or more parameters based on T(X).

[0130] S6: Compare the parameters with the threshold values.

[0131] → If at least one parameter meets the threshold value, go to S7.

[0132] → If all parameters do not meet the threshold value, go to S8.

[0133] S7: Output the detection of APTT and the sample containing the FVIII replacement substance.

[0134] S8: Output APTT.

[0135] Hereinafter, an embodiment of the device of the present invention will be described. One embodiment of the device of the present invention is the automatic analysis device 1 as Figure 3B shown. The automatic analysis device 1 includes a control unit 10, an operation unit 20, a measurement unit 30, and an output unit 40.

[0136] The control unit 10 controls the overall operation of the automatic analysis device 1. The control unit 10 can be constituted by a computer (e.g., a personal computer). The control unit 10 includes a CPU, a memory, a storage device, a communication interface (I / F), etc., and can process instructions from the operation unit 20, control the operation of the measurement unit 30, save the measured data received from the measurement unit 30 and perform data analysis, save the analysis results, control the analysis results output by the output unit 40, etc. Also, the control unit 10 can be connected to other devices such as an external medium and a host computer. It should be noted that in the control unit 10, the computer that controls the operation of the measurement unit 30 and the computer that performs the analysis of the data measured by the measurement unit 30 can be the same or different.

[0137] The operation unit 20 obtains the input from the operator and transmits the obtained input information to the control unit 10. For example, the operation unit 20 includes user interfaces (UIs) such as a keyboard and a touch panel. The output unit 40 outputs the detection result of the sample containing the FVIII replacement substance under the control of the control unit 10, and outputs the coagulation reaction data measured by the measurement unit 30 as needed, P(i), Pe, T(X), the parameters for analyzing the blood coagulation ability based on this, and the analysis results such as the coagulation time (APTT) as needed. For example, the output unit 40 includes a display device such as a display.

[0138] The measurement unit 30 performs a series of operations for blood coagulation tests and obtains the measurement data of the coagulation reaction of the specimen containing the blood sample. The measurement unit 30 includes various instruments and analysis modules required for blood coagulation tests, such as a sample container for accommodating the blood sample, a reagent container for storing the test reagent, a reaction container for the reaction of the sample and the reagent, a probe for dispensing the blood sample and the reagent into the reaction container, a light source, a detector for detecting the scattered light or transmitted light from the specimen in the reaction container, a data processing circuit for transmitting the data from the detector to the control unit 10, and a control circuit for controlling the operation of the measurement unit 30 under the instruction of the control unit 10. Or known coagulation reaction data is used to obtain P(i), Pe, and T(X). In the case of calculating the parameters, the measurement unit 30 is not required.

[0139] The control unit 10 analyzes the coagulation reaction of the sample based on the coagulation reaction data. In this analysis, it may include obtaining the above-mentioned P(i) and Pe, calculating the APTT, calculating T(X) and the parameters, and detecting the sample containing the FVIII replacement substance using these parameters. Or P(i), Pe, and T(X) can be produced by the control unit 10 based on the measurement data obtained from the measurement unit 30, or can be produced by other devices, for example, can be produced by the measurement unit 30 and then transmitted to the control unit 10. In the control unit 10, the threshold value for detecting the sample containing the FVIII replacement substance can be saved, or the control unit 10 can read this threshold value saved in an external device or network for this detection.

[0140] The above analysis can be executed by a program for performing the method of the present invention. Therefore, the control unit 10 can include a program for performing the analysis method of the blood coagulation ability of the present invention.

[0141] The analysis results obtained by the control unit 10 are transmitted to the output unit 40 for output. The output can be in any form such as display on a screen, transmission to a host computer, printing, etc. The output information output from the output unit can include P(i), Pe, APTT, T(X), parameters for analyzing coagulation ability, test results of samples containing FVIII surrogate substances, etc. In addition, the output information can also include other information obtained in the APTT measurement (detection of the initial reaction, the possibility of chylomicrons, etc.). The type of output information output from the output unit can be controlled by the program of the present invention.

[0142] Example

[0143] The present invention will be described in further detail below with reference to Examples, but the present invention is not limited to these Examples. In the following Examples, coagulation reaction measurement was performed according to the APTT assay procedure.

[0144] 1. Methods

[0145] 1.1) Sample

[0146] Control sample group (NE): plasma with an APTT within the reference range (24 to 39 seconds) of Coagpia APTT-N (manufactured by Sekisui Medical Co., Ltd.) (n=30).

[0147] Test sample group (E1): Plasma obtained from hemophilia A patients who were administered emicizumab (n=54). The emicizumab concentration was 12.2 to 79.2 μg / mL.

[0148] Test sample group (E2): Plasma obtained from hemophilia A patients with antibodies (inhibitors) to coagulation factor VIII (FVIII) who were given emicizumab (n=15). The emicizumab concentration ranged from 12.0 to 64.0 μg / mL, and the inhibitor titer ranged from 0.8 to 202 BU / mL.

[0149] 1.2) Coagulation reaction measurement

[0150] Coagpia APTT-N (manufactured by Sekisui Medical Co., Ltd.), an APTT assay reagent, was used as the assay reagent, and Coagpia APTT-N calcium chloride solution (manufactured by Sekisui Medical Co., Ltd.) was used as the calcium chloride solution. The coagulation reaction of the sample containing the specimen was measured using the blood coagulation automatic analyzer CP3000 (manufactured by Sekisui Medical Co., Ltd.). After 50 μL of the sample was heated at 37°C in a reaction cup for 45 seconds, 50 μL of the assay reagent at approximately 37°C was added, and after 171 seconds, 50 μL of the calcium chloride solution was added to initiate the coagulation reaction. The reaction was carried out at 37°C. To measure the coagulation reaction, the reaction cup was irradiated with light of a wavelength of 660 nm using an LED as the light source, and the amount of scattered light at 90 degrees was measured at 0.1 second intervals. The measurement time was 360 seconds.

[0151] 1.3) Obtaining the coagulation reaction curve

[0152] After smoothing processing including noise removal is performed on the measurement data of the coagulation reaction obtained from each sample, a zero-point adjustment process is performed so that the scattered light amount at the start of measurement is 0, and a coagulation reaction curve P(i) is prepared.

[0153] 1.4) Detection of the coagulation end point Pe and determination of APTT

[0154] As the coagulation reaction end point Pe, detect the earliest time when the cumulative ratio Z(i) of the coagulation reaction curve P(i) becomes less than the cumulative ratio threshold Zs (see WO2021 / 132552). The cumulative ratio threshold Zs is 1.001. Calculate the time T(50) when P(i) reaches 50% of Pe and determine it as the APTT. The cumulative ratio Z(i) at time i is calculated as follows:

[0155] Cumulative ratio Z(i) = Pb(i) / Pa(i)

[0156] Pa(i) = the sum of P(i-20) to P(i-1)

[0157] Pb(i) = the sum of P(i+1) to P(i+20)

[0158] 1.5) Calculation of T(X)

[0159] For each sample, the time T(X) required for P(i) to reach X% of Pe (measured in 1.4) was calculated using the following formula. X ranged from 1 to 100, with 1 set to 100, and T(1) to T(100) were calculated. Note that T(50) when X = 50 corresponds to the APTT.

[0160] P(T(X))=Pe×X%

[0161] 2. Coagulation reaction

[0162] 2.1) Coagulation reaction curve P(i)

[0163] Figure 4 The coagulation reaction curves P(i) of each sample are shown. The horizontal axis represents time, and the vertical axis represents the coagulation reaction (scattered light amount). Figure 4 A to C respectively represent P(i) of each sample of NE, E1, and E2. Figure 4 In D and E, the average values of P(i) of E1 and E2 (aE1 and aE2 respectively; dashed lines) and the average value of P(i) of NE (aNE; solid line) are shown together. Compared with the non-emicizumab administration group (NE), the P(i) of the emicizumab administration groups (E1 and E2) has an earlier rising time of the curve, but the rise of the curve has a slow tendency.

[0164] 2.2) APTT calculation

[0165] Figure 5 The APTT of NE, E1, and E2 is shown. The APTT is 27.1 - 39.3 seconds in NE, 21.2 - 37.9 seconds in E1, and 21.3 - 32.3 seconds in E2. The distribution ranges of APTT in E1 and E2 overlap with the distribution range of NE. And in E1 and E2, APTT shorter than the lower limit (24 seconds) of the reference range is sometimes shown. From the results, it can be seen that the emicizumab administration groups (E1 and E2) and the non-emicizumab administration group (NE) cannot be distinguished only by APTT.

[0166] 2.3) Modified reaction curve

[0167] Figure 6 The data obtained by relativizing the magnitude and reaction time of the coagulation reaction of the sample is shown. Hereinafter, it is also referred to as the modified reaction curve. In Figure 6 the horizontal axis is set as the time ratio of T(X) of each sample (T(X) / T(50)), and the vertical axis is set as X (%). Figure 6 A to C respectively represent the data of each sample of NE, E1, and E2. Figure 6 In D and E, the average values of the data of E1 and E2 (aE1 and aE2 respectively; dashed lines) and the average value of NE (aNE; solid line) are shown together. Similar to the P(i) shown in Figure 4 , in the emicizumab administration groups (E1 and E2), compared with the non-emicizumab administration group (NE), the rising time of the curve is earlier, but the rise of the curve has a slow tendency.

[0168] 3. Parameter comparison between sample groups

[0169] 3.1) Example 1: Interval change rate of T(X)

[0170] For each sample, calculate the interval change rate of T(X) when X ranges from 30 to 50, [(X2 - X1) / {(T(X2) / T(50)) - (T(X1) / T(50))}] (X1 = 30, X2 = 50). Figure 7 A represents the plotting points of the above interval change rate of each sample with respect to APTT, Figure 7 B represents the distribution of the above interval change rate of each sample group. The above interval change rates of E1 and E2 are less than NE. It can be seen that according to the interval change rate of T(X), the emicizumab administration groups (E1 and E2) and the non - administration group (NE) can be distinguished.

[0171] 3.2) Example 2: Change amount of T(X)

[0172] For each sample, calculate the change amount of T(X) when X ranges from 30 to 70, [{T(X2) - T(X1)} / T(50)] (X1 = 30, X2 = 70). Figure 8 A represents the plotting points of the above change amount of T(X) of each sample with respect to APTT, Figure 8 B represents the distribution of the above change amount of T(X) of each sample group. The above change amounts of T(X) of E1 and E2 are greater than NE. It can be seen that according to the change amount of T(X), the emicizumab administration groups (E1 and E2) and the non - administration group (NE) can be distinguished.

[0173] 3.3) Example 3: Ratio of T(X)

[0174] For each sample, set X as X1 = 30 and X2 = 70, and calculate the ratio of T(X), [T(X2) / T(X1)]. Figure 9 A represents the plotting points of T(70) / T(30) of each sample with respect to APTT, Figure 9 B represents the distribution of T(70) / T(30) of each sample group. The T(70) / T(30) of E1 and E2 are greater than NE. It can be seen that according to the ratio of T(X), the emicizumab administration groups (E1 and E2) and the non - administration group (NE) can be distinguished.

[0175] 3.4) Example 4: Parameter related to the coefficient of variation CV of T(X)

[0176] Calculate CV(X ± 4) according to the following formula.

[0177] CV(X ± 4) = {SD of T(X - 4) to T(X + 4)} / {AVE of T(X - 4) to T(X + 4)} × 100 [%]

[0178] Figure 10A to C respectively represent curves showing the coagulation reactions of samples of NE, E1, and E2, with the horizontal axis being CV(X±4) and the vertical axis being X (%). Hereinafter, these will also be referred to as CV curves. Figure 10 In D and E, the average values of the CV curves of E1 and E2 (aE1 and aE2 respectively; dashed lines) are shown together with the average value of NE (aNE; solid line). The CV(X±4) of E1 and E2 is larger than that of NE.

[0179] For each sample, the coefficient of variation CV(30:50) of T(X) belonging to the range of X = 30 to 50 is calculated. Figure 11 A represents the plotted points of CV(30:50) of each sample against APTT. Figure 11 B represents the distribution of CV(30:50) for each sample group. The CV(30:50) of E1 and E2 is greater than that of NE. It can be seen that according to CV(30:50), the emicizumab-administered groups (E1 and E2) and the non-administered group (NE) can be distinguished.

[0180] For each sample, the average value AVE{CV(30±4):CV(50±4)} of CV(X±4) in the range of X = 30 to 50 is calculated. Figure 12 A represents the plotted points of AVE{CV(30±4):CV(50±4)} of each sample against APTT. Figure 12 B represents the distribution of AVE{CV(30±4):CV(50±4)} for each sample group. The AVE{CV(30±4):CV(50±4)} of E1 and E2 is greater than that of NE. It can be seen that according to AVE{CV(30±4):CV(50±4)}, the emicizumab-administered groups (E1 and E2) and the non-administered group (NE) can be distinguished.

[0181] 3.5) Comparative Example 1 Vmax

[0182] Figure 13 Represents the distribution of the maximum value Vmax (maximum rate) of the first derivative (reaction rate) of P(i) for NE, E1, and E2. Figure 13 A represents the plotted points of Vmax of each sample against APTT. Figure 13 B represents the distribution of Vmax for each sample group. The Vmax of E1 and E2 tends to be less than that of NE, but the distribution range overlaps with that of NE. Just by Vmax, the emicizumab-administered groups (E1 and E2) and the non-administered group (NE) cannot be distinguished.

[0183] 3.6) Comparative Example 2 VmaxT

[0184] Figure 14Represents the distribution of the time VmaxT at which the first derivative (reaction rate) of P(i) with respect to NE, E1, and E2 reaches Vmax. Figure 14 A represents the plotted points of VmaxT of each sample relative to APTT. Figure 14 B represents the distribution of VmaxT for each sample group. The VmaxT of E1 and E2 tend to be less than that of NE, but the distribution regions overlap with NE. It is not possible to distinguish between the emicizumab-administered groups (E1 and E2) and the non-administered group (NE) based solely on VmaxT.

[0185] 3.7) SDI of the parameters in Example 5

[0186] Evaluate the proximity of the parameter (P) between the emicizumab-administered groups (Emi; including E1 and E2) and the non-administered group (NE). As the parameter (P), use the interval change rate of T(X) obtained in Example 1, the change amount of T(X) obtained in Example 2, T(70) / T(30) obtained in Example 3, CV(30:50) obtained in Example 4, and AVE{CV(30±4):CV(50±4)}. Extract the sample in NE where P is closest to Emi (i.e., P is the largest or smallest) (NE-close sample) and the sample in Emi where P is closest to NE (i.e., P is the smallest or largest) (Emi-close sample). Calculate the absolute value of the standard deviation index (|SDI|) of the P of the two extracted samples relative to the average value of P of NE.

[0187] |SDI| = |{(P of the NE-close sample or Emi-close sample) - (average value of P of NE)} / (standard deviation of P of NE)|

[0188] The |SDI| of the NE-close samples and Emi-close samples for each parameter is shown in Table 1. The difference in |SDI| between the NE-close samples and Emi-close samples, as shown in Examples 1 to 4, reflects that each parameter has a different distribution in Emi and NE. From these results, it can be seen that using the |SDI| of the parameters shown in Examples 1 to 4, it is possible to distinguish between the emicizumab-administered group and the non-administered group. For example, by calculating the |SDI| of the parameter of the test sample relative to the reference value and comparing it with the threshold value, it is possible to detect whether the test sample is an emicizumab-administered group. For this reference value, the average value of the parameters of NE described above can be used. As an example of this threshold value, the intermediate value between the |SDI| of the NE-close samples and the |SDI| of the Emi-close samples described above can be set.

[0189] Table 1

[0190]

Claims

1. A method for analyzing the coagulation ability of a blood sample, comprising: 1) Detecting the coagulation reaction end point Pe in the coagulation reaction curve of a test blood sample that needs to be tested for the presence of a substance with factor VIII replacement activity; 2) Calculating T(X), where T(X) represents the measurement point or time at which the coagulation reaction curve reaches X% of Pe, and X is a variable greater than 0 and less than or equal to 100; 3) Detecting a test blood sample containing a substance with factor VIII replacement activity based on T(X).

2. The method according to claim 1, wherein The test blood sample is a blood sample with a normal activated partial thromboplastin time (APTT).

3. The method according to claim 1, wherein The step 3) includes: calculating a parameter based on T(X), and detecting a test blood sample containing the substance with factor VIII replacement activity according to the parameter. The parameter is at least one selected from the change amount of T(X) within a given range of X, the ratio of T(X), the interval change rate of T(X), the parameter related to the coefficient of variation of T(X), and their standard deviation index (SDI).

4. The method according to claim 3, wherein The change amount of T(X) within the given range of X is [(T(X2) / C) - (T(X1) / C)], The ratio of T(X) within the given range of X is [T(X2) / T(X1)], The interval change rate of T(X) within the given range of X is [(X2 - X1) / {(T(X2) / C) - (T(X1) / C)}], where X1 and X2 are each independently 1 to 100, with X2 > X1, and C is a constant.

5. The method according to claim 3, wherein The parameter related to the coefficient of variation of T(X) is CV(X1:X2), CV(X1 - J:X1 + J), or AVE{CV(X1 ± J):CV(X2 ± J)}, where CV(X1:X2) is the coefficient of variation of T(X) when X is in the range of X1 to X2, expressed by the following formula: CV(X1:X2) = SD{T(X1):T(X2)} / AVE{T(X1):T(X2)} ×100[％] AVE{T(X1):T(X2)}: The average value of T(X1) to T(X2) SD{T(X1):T(X2)}: The standard deviation of T(X1) to T(X2) CV(X1 - J:X1 + J) is the coefficient of variation of T(X) in the range of (X1 - J) to (X1 + J), AVE{CV(X1 ± J):CV(X2 ± J)} is the average value of CV(X - J:X + J) when X is between X1 and X2, where X1 and X2 are each independently 1 to 100, with X2 > X1, and J = 2 to 44.

6. The method according to claim 3, wherein, It includes detecting a test blood sample containing the substance with factor VIII replacement activity by comparing the parameter based on T(X) with a given threshold.

7. The method according to claim 1, wherein, It includes outputting the result of the detection.

8. The method according to claim 1, wherein It further includes: Measuring the activated partial thromboplastin time (APTT) of the blood sample. As the test blood sample for detecting the presence or absence of the substance having the coagulation factor VIII replacement activity, a blood sample with no prolonged APTT is selected.

9. The method according to claim 1, wherein, The substance having the coagulation factor VIII replacement activity is a bispecific antibody that replaces the cofactor function of coagulation factor VIII.

10. The method according to claim 1, wherein, The substance having the coagulation factor VIII replacement activity is emicizumab.

11. A program for implementing the method according to claim 1.

12. An apparatus for implementing the method according to claim 1.

13. A system for implementing the method according to claim 1, comprising a program for implementing the method according to claim 1 and an apparatus controlled by the program.

14. The apparatus according to claim 12 or the system according to claim 13, wherein, It includes an output unit for outputting the test result of the test blood sample containing the substance having the coagulation factor VIII replacement activity by using the method according to claim 1.

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