Method for analyzing blood clotting ability of blood sample

Abstract

Provided is a method for analyzing the blood clotting ability of a blood sample. The method comprises the following steps: 1) obtaining a first differential V (i) of a solidification reaction curve of a detected sample, where i represents a measurement point number or time; and 2) the following a) or b): a) determining a minimum value pk of a point that becomes Xk or more when V (i) reaches a maximum value Vmax or before, and a maximum value qk of a point that becomes Xk or more when V (i) reaches Vmax or after, where k represents a set of integers from 1 to n, n is an integer of 2 or more, Xk is Vmax * Sk%, 0 < Sk < = 100; and b) determining the maximum value of the V (i) after relative valuation.

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 or functional defect of coagulation factor VIII (FVIII). 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 types 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 promotes the blood coagulation reaction by binding to activated coagulation factor IX (FIXa) at one antigen-binding site and coagulation factor X (FX) at the other antigen-binding site to replace the cofactor function of activated coagulation factor VIII (FVIIIa).

[0004] Emicizumab is regularly administered for the purpose of suppressing the bleeding tendency of 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 preoperative blood coagulation test, a physician who is not aware that the patient has hemophilia A fails to notice the reduced coagulation ability of the patient and cannot perform appropriate treatment, so there is a risk of significant bleeding during the operation.

[0005] In Patent Document 1, a method is described in which a blood coagulation start reagent containing active coagulation factor XI is added to a blood sample, the amount of thrombin generation is measured, and based on this, a blood coagulation reaction caused by a substance having FVIII substitution activity is evaluated. In Patent Documents 2 and 3, a method for evaluating the coagulation ability of a blood sample is described, which includes obtaining a parameter related to the differentiation of the coagulation waveform of a blood sample after administration of a substance having FVIII substitution 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 Patent Application Laid-Open No. 2017-106925 Summary of the Invention

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

[0012] The present invention provides the following.

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

[0014] 1) Obtaining the first derivative V(i) of the coagulation reaction curve of a test blood sample for which it is necessary to detect the presence or absence of a substance having factor VIII substitution activity, where i represents the measurement point number or time; and

[0015] 2) The following a) or b):

[0016] a) Determining the minimum value p of the points that become X k or more at or before the time when V(i) reaches the maximum value Vmax, k and the maximum value q of the points that become X k or more at or after the time when V(i) reaches Vmax, k where k represents a set of integers from 1 to n, n is an integer of 2 or more, and X k is Vmax × S k %, 0 < S k ≤ 100;

[0017] b) Determine the maximum value after relative value conversion of V(i).

[0018] 〔2〕According to the method described in 〔1〕, wherein the blood sample to be tested is a blood sample with normal activated partial thromboplastin time (APTT).

[0019] 〔3〕According to the method described in 〔1〕 or 〔2〕, wherein n is an integer from 5 to 50.

[0020] 〔4〕According to the method described in any one of 〔1〕 to 〔3〕, further comprising calculating a parameter for analyzing the coagulation ability of the blood sample to be tested according to the above p k or q k .

[0021] 〔5〕According to the method described in 〔4〕, wherein

[0022] the above parameter is selected from the mean difference before and after, CV, qp ratio, vB(S k ), sharpness rate vAB(S k ), time rate vTB(S k ), vAB(S k )×VmaxT, vAB(S k )×APTT, a parameter related to the relative values of p k and q k , and at least one of their standard deviation indices (SDI).

[0023] Here,

[0024] the mean difference before and after represents (the average of q a ~q b - the average of p a ~p b ) / (the average of p a ~p b and q a ~q b ), a, b are integers selected from 1 to n, n is defined as above, a < b.

[0025] CV represents the coefficient of variation for p a ~p b and q a ~q b ), a, b are integers selected from 1 to n, n is defined as above, a < b.

[0026] The qp ratio represents q k / p k .

[0027] vB(S k ) represents pk to q k spacing of,

[0028] VmaxT satisfies V(VmaxT)=Vmax,

[0029] APTT is the activated partial thromboplastin time of the above-mentioned blood sample to be tested, i.e., APTT,

[0030] vAB(S k )=vH(S k ) / vB(S k ),

[0031] vTB(S k )=vB(S k ) / VmaxT,

[0032] Here,

[0033] vH(S k ) is expressed by the following formula,

[0034] [Mathematical formula 1]

[0035]

[0036] [6] According to the method described in [5], wherein,

[0037] The parameter related to the relative values of the above-mentioned p k and q k is the slope of the first regression line of the curve of p a ~p b and q c ~q d of the above-mentioned blood sample to be tested relative to p a ~p b and q c ~q d of the reference sample. Here, a, b, c, and d are each independently integers selected from 1 to n, a < b, c < d, and n is defined as above,

[0038] The reference sample is a blood sample not administered with a medicament having FVIII replacement activity.

[0039] [7] According to the method described in any one of [1] to [3], wherein, further comprising calculating a parameter for analyzing the coagulation ability of the above-mentioned blood sample to be tested according to the maximum value of V(i) after being normalized by the above-mentioned relative value.

[0040] [8] According to the method described in [7], wherein,

[0041] The above parameters are at least one selected from modified Vmax, modified Vmax×VmaxT, modified Vmax×APTT, and their standard deviation index, i.e., SDI.

[0042] The modified Vmax is the maximum value of the above-mentioned V(i) after being made relative.

[0043] VmaxT satisfies V(VmaxT)=Vmax.

[0044] APTT is the activated partial thromboplastin time of the above-mentioned blood sample to be tested, i.e., APTT.

[0045] 〔9〕The method according to any one of 〔4〕~〔8〕, further comprising detecting the presence or absence of the substance having factor VIII replacement coagulation activity in the above-mentioned blood sample to be tested according to the above parameters.

[0046] 〔10〕The method according to 〔9〕, including: outputting the detection result of the presence or absence of the substance having factor VIII replacement coagulation activity.

[0047] 〔11〕The method according to any one of 〔1〕~〔10〕, further comprising:

[0048] measuring the activated partial thromboplastin time of the blood sample, i.e., APTT; and

[0049] selecting a blood sample with no prolonged APTT as the blood sample to be tested for detecting the presence or absence of the substance having factor VIII replacement coagulation activity.

[0050] 〔12〕The method according to any one of 〔1〕~〔11〕, wherein the substance having factor VIII replacement coagulation activity is a bispecific antibody that replaces the cofactor function of factor VIII for coagulation.

[0051] 〔13〕The method according to any one of 〔1〕~〔12〕, wherein the substance having factor VIII replacement coagulation activity is emicizumab.

[0052] 〔14〕A program for implementing the method according to any one of 〔1〕~〔13〕.

[0053] 〔15〕An apparatus for implementing the method according to any one of 〔1〕~〔13〕.

[0054] 〔16〕A system for implementing the method according to any one of 〔1〕~〔13〕, comprising a program for implementing the method according to any one of 〔1〕~〔13〕 and a device controlled by the program.

[0055]

[17] The device according to

[15] or the system according to

[16] , wherein it has an output unit for outputting the analysis result of the coagulation ability of the blood sample using any one of the methods of [1] to

[131] .

[0056] According to the method of the present invention, from a blood sample in which coagulation abnormalities could not be detected by the previous APTT assay, the measurement data of the coagulation reaction (coagulation reaction data) obtained during the APTT assay can be used to detect that the test sample is a blood sample after administration of a drug having FVIII replacement activity such as emicizumab. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 is an example of a coagulation reaction curve.

[0058] Figure 2 Is used to illustrate X k 、p k and q k Concept map.

[0059] Figure 3A This is an example of a blood coagulation analysis process using the method of the present invention.

[0060] Figure 3B This is a conceptual diagram showing the configuration of an apparatus for performing the analysis method utilizing blood coagulation ability of the present invention.

[0061] Figure 4 ΝΝ is the APTT of the sample group used in the Examples. NE is a control sample group, E1 is a sample from a hemophilia A patient who was given emicizumab, and E2 is a sample from a hemophilia A patient who was given emicizumab and had an inhibitor.

[0062] Figure 5 The coagulation reaction curves R(i) (upper) and the first differential curves V(i) (lower) of NE, E1, and E2 are shown.

[0063] Figure 6 is the modified reaction rate curve of NE, E1 and E2.

[0064] Figure 7 A is the plot of Vmax versus APTT for each sample, and B is the distribution of Vmax for each sample group.

[0065] Figure 8 A to D in FIG are plots of VmaxT versus APTT for NE, E1, E2, and all samples. E is the distribution of VmaxT for each sample group.

[0066] Figure 9A therein is the plotted point of the corrected Vmax of each sample relative to APTT. The dashed line is the threshold line. B is the distribution of the corrected Vmax of each sample group.

[0067] Figure 10 A therein is the plotted point of [corrected Vmax × VmaxT] of each sample relative to APTT. B is the distribution of [corrected Vmax × VmaxT] of each sample group.

[0068] Figure 11 A therein is the plotted point of [corrected Vmax × APTT] of each sample relative to APTT. B is the distribution of [corrected Vmax × APTT] of each sample group.

[0069] Figure 12 A therein is the plotted point of vTB(30) (=vB(30) / VmaxT) of each sample relative to APTT. B is the distribution of vTB(30) of each sample group.

[0070] Figure 13 A therein is the plotted point of vAB(10) (=vH(10) / vB(10)) of each sample relative to APTT. The dashed line is the threshold line. B is the distribution of vAB(10) of each sample group.

[0071] Figure 14 A therein is the plotted point of [vAB(10) × VmaxT] of each sample relative to APTT. B is the distribution of [vAB(10) × VmaxT] of each sample group. C is the plotted point of [vAB(10) × APTT] of each sample relative to APTT. D is the distribution of [vAB(10) × APTT] of each sample group.

[0072] Figure 15 A therein is the plotted point of the qp ratio (q8 / p8) of each sample relative to APTT. B is the distribution of the pq ratio of each sample group.

[0073] Figure 16 A therein is the plotted point of q6-p6 of each sample relative to APTT. The dashed line is the threshold line. B is the distribution of q6-p6 of each sample group.

[0074] Figure 17 A therein is the plotted point of the average difference before and after of each sample relative to APTT. B is the distribution of the average difference before and after of each sample group. C is the plotted point of the lower average difference before and after of each sample relative to APTT. D is the distribution of the lower average difference before and after of each sample group. E is the plotted point of the middle average difference before and after of each sample relative to APTT. F is the distribution of the middle average difference before and after of each sample group.

[0075] Figure 18A in [the figure] is the plotting point of the overall CV of each sample relative to APTT. B is the distribution of the overall CV of each sample group. C is the plotting point of the lower CV of each sample relative to APTT. D is the distribution of the lower CV of each sample group.

[0076] Figure 19 is relative to p k and relative to q k (k = 1 to 20) is the slope of the regression line. A and B are the plotting points (A) of the slope of the regression line relative to APTT and the distribution (B) of each sample group when the NE sample with the minimum APTT is used as the reference sample (●). C and D are the plotting points (C) of the slope of the regression line relative to APTT and the distribution (D) of each sample group when the NE sample with the maximum APTT is used as the reference sample (●).

[0077] Figure 20 is relative to p k and relative to q k (k = 6 to 20) is the slope of the regression line. A and B are the plotting points (A) of the slope of the regression line relative to APTT and the distribution (B) of each sample group when the NE sample with the minimum APTT is used as the reference sample (●). C and D are the plotting points (C) of the slope of the regression line relative to APTT and the distribution (D) of each sample group when the NE sample with the maximum APTT is used as the reference sample (●). Detailed implementation mode

[0078] All patent documents, non-patent documents and other publications cited in this specification are incorporated herein by reference in their entirety.

[0079] In a blood coagulation test for activated partial thromboplastin time (APTT) determination, 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 calculated. In the measurement of the blood coagulation reaction, usual 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. 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 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.

[0080] 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 a patient with hemophilia A is administered a substance having FVIII replacement activity such as emicizumab (hereinafter, also referred to as "FVIII replacement substance"), the APTT of the sample of this patient fails to be prolonged and converges within or shortens beyond this reference range. It is difficult to distinguish whether a test sample is a sample from a hemophilia A patient administered an FVIII replacement substance based on the reference range of APTT by APTT measurement.

[0081] The present inventors have found that even when no prolongation of the coagulation time is observed in the APTT measurement for a blood sample from a hemophilia A patient administered emicizumab, the progress of the blood coagulation reaction (blood coagulation reaction rate curve) is different from that of a sample not administered emicizumab.

[0082] In the present invention, based on the coagulation reaction data of a test sample measured during APTT measurement, specifically, parameters derived from the blood coagulation rate curve obtained by differentiating the coagulation reaction curve once, the presence or absence of an FVIII replacement substance in the test sample is detected. In the present invention, in the conventional APTT measurement, it is possible to detect a sample administered an FVIII replacement substance from a sample in which no coagulation abnormality can be detected.

[0083] In the present specification, an FVIII replacement substance refers to a substance capable of substituting 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). Examples of FVIII replacement substances include bispecific antibodies that substitute the cofactor function of FVIII (for example, bispecific antibodies mimicking the FVIII structure). Examples of such bispecific antibodies include emicizumab. 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 substituting 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.

[0084] [Analysis Method for Coagulation Ability of Blood Samples]

[0085] In the method for analyzing the coagulation capacity of a blood sample according to the present invention (hereinafter referred to as the method of the present invention), differences in the coagulation energy of blood samples that are indistinguishable by the apoptotic clotting time (APTT) can be detected as differences in the progression of the coagulation reaction (coagulation reaction rate curve). In the method of the present invention, parameters for analyzing the coagulation capacity of the test sample can be obtained based on the first derivative of the coagulation reaction curve of the test sample.

[0086] Hereinafter, the method of the present invention will be described.

[0087] 1. Coagulation reaction measurement

[0088] In the method of the present invention, plasma from a subject whose blood has been collected for a coagulation test is preferably used as the test sample. This sample may be supplemented with an anticoagulant commonly used for coagulation tests. For example, plasma can be obtained by collecting blood using a sodium citrate-added blood collection tube and then centrifuging it.

[0089] 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, an 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.

[0090] 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.

[0091] 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.

[0092] 2. Obtaining the coagulation reaction curve and the first differential V(i)

[0093] Based on the measured coagulation reaction data, a coagulation reaction curve R(i) can be obtained. Here, "i" represents the number of measurement points or time from the start of the coagulation reaction (also referred to as the number of measurement points and time, respectively). For example, if the measurement (photometry) interval is 0.1 seconds, it is expressed as time = 0.1 × number of measurement points. That is, R(i) can be a function of the number of measurement points or a function of time. Generally speaking, the coagulation reaction curve R(i) is the measurement value obtained by measuring the coagulation reaction, which has been subjected to noise removal or smoothing by conventional means, or zero point adjustment for adjusting the initial value or relative value conversion of the curve as needed.

[0094] An example of a coagulation reaction curve is shown in Figure 1 . Figure 1 The horizontal axis represents time, and the vertical axis represents the coagulation reaction (scattered light amount), and the scattered light amount at the start of the measurement is adjusted to 0. As time passes, the coagulation reaction of the mixed solution progresses, and the scattered light amount increases. Figure 1 The coagulation reaction curve of the scattered light amount shown is generally S-shaped.

[0095] The first derivative V(i) is obtained from the above-mentioned R(i). The differential process of obtaining V(i) from R(i) can be implemented by any method. For example, it can be performed by calculating the average slope value within an interval. In the calculation of the average slope value within an interval, for a constant number of measurement points before and after each measurement point i, for example, 2K + 1 measurement points from i - K to i + K can be used. Here, K is an arbitrary integer. For example, when K is 2, 5 measurement points of the (i - 2)-th, (i - 1)-th, i-th, (i + 1)-th, and (i + 2)-th can be used. The average slope value refers to the slope value when these multiple measurement points are linearly approximated. For the operation method of linear approximation, conventional methods such as the least squares method can be used. The average slope value of these measurement points can be regarded as the first derivative V(i) of R(i) at the measurement point i. Alternatively, the first derivative of the relative value of R(i) can also be obtained as V(i). For example, it can also be transformed so that the maximum value of R(i) becomes a specified value, for example, 100, and the first derivative is obtained to get V(i). The first derivative V(i) constitutes a curve representing the rate of the solidification reaction or the reaction progress rate.

[0096] For the acquisition of R(i) and V(i) of the above-mentioned test sample, it can be carried out in parallel with the measurement of the solidification reaction of the test sample, or it can also be carried out after the measurement of the solidification reaction is completed. The measurement of the solidification reaction of the test sample is preferably carried out until the solidification reaction ends. The end of the solidification reaction can be determined according to any criterion such as the moment when R(i) reaches a steady state, the moment when V(i) reaches the maximum value and then decreases to 0 or a certain value (for example, below S% of the maximum value). Alternatively, within the range where the measured values required for obtaining the parameters for analyzing the coagulation time (APTT) and the coagulation ability of the test sample described later can be obtained, the measurement of the solidification reaction can be ended at a time earlier than the end of the solidification reaction.

[0097] As described above, R(i) and V(i) in this specification can be functions of the number of measurement points respectively, or can be functions of time. The parameters for analyzing the coagulation ability of the test sample described later can also be data based on the number of measurement points, or can also be data based on the time reference. In the following of this specification, R(i) and V(i) are sometimes simply referred to as R and V respectively.

[0098] 3. APTT Calculation

[0099] The coagulation time (APTT) of the test sample can be calculated based on the coagulation reaction data obtained in the above-mentioned coagulation reaction measurement. The calculation method of APTT is not particularly limited. For example, APTT can be calculated based on the above R(i) or V(i) according to any method. As an example of the calculation method of APTT, a method of calculating the coagulation time as the time when R(E) representing the reaction R of R(i) at the coagulation reaction end point E reaches N% can be cited (percentage detection method); a method of calculating the coagulation time as the time when V(i) reaches the maximum value Vmax or reaches this N%; a method of calculating the coagulation time based on the time change of the cumulative ratio of R(i) in a minute time period (refer to Japanese Patent Laid-Open No. 6-249855 and WO2021 / 132552); a method of calculating the coagulation time based on the weighted average time of V(i) (refer to WO2021 / 177452); a method of calculating the coagulation time as the time when R(i) reaches N% of R(Te) with the time when it reaches a specified value as the calculation starting point Te after V(i) reaches the maximum value Vmax (refer to WO2021 / 206107), etc.

[0100] 4. Acquisition of parameters for analyzing coagulation ability

[0101] 4.1) Test sample for which parameters should be acquired

[0102] Next, in the method of the present invention, parameters for analyzing the coagulation ability of the test sample are acquired based on the above V(i). In the method of the present invention, it is sufficient to acquire the parameters for the test sample that needs to detect the presence or absence of FVIII replacement substance. The test sample that needs to detect the presence or absence of FVIII replacement substance refers to, generally speaking, a sample with no prolonged APTT. It is inferred that a sample with prolonged APTT is a sample with coagulation abnormality (i.e., high bleeding risk or thrombus risk) that does not require investigation of the presence or absence of FVIII replacement substance, and therefore it is not necessarily necessary to apply the method of the present invention.

[0103] In one embodiment of the method of the present invention, after calculating the APTT based on the above coagulation reaction data measured from an arbitrary test sample, a sample with no prolonged APTT is selected as the test sample for which it is necessary to detect the presence or absence of an FVIII replacement substance. For the selected test sample, V(i) calculated from the coagulation reaction data obtained by measuring using the above coagulation reaction can be obtained. This V(i) can be calculated together with the above R(i) etc. during APTT measurement, or this V(i) can also be measured for APTT, and after selecting a test sample with no prolonged APTT to which the method of the present invention should be applied, it can be calculated based on the R(i) of the selected test sample. In another embodiment of the method of the present invention, as the test sample for which it is necessary to detect the presence or absence of an FVIII replacement substance, a sample known to have no prolonged APTT based on existing coagulation reaction data is selected. V(i) can be obtained based on the existing coagulation reaction data.

[0104] 4.2) Parameters based on the peak shape of V

[0105] In one embodiment, the parameter for analyzing the blood coagulation ability of the above test sample is a parameter that reflects the progress of the coagulation reaction, that is, a parameter based on the peak shape of V. In obtaining this parameter, first, it is determined that V becomes a preset set value X k as described above (i.e., satisfying V(i) ≥ X k ) of the minimum value p k of the number of measurement points or time k and the maximum value q k . Since V usually has a peak shape with the maximum value Vmax as the peak top, p k and q k can exist at the time when V reaches Vmax or before and after that time, respectively. That is, V(p k -D) < X k ≤ V(p k ), V(q k ) ≥ X k > V(q k +D). Here, D is the measurement interval of the coagulation reaction data. When p k and q k are the number of measurement points, D = 1. When p k and q k are time, for example, when measuring at 0.1-second intervals, D = 0.1. In the method of the present invention, X k is a variable, and p k and q k corresponding to each X k are determined. For example, X n as (X1, ···, X nWhen there are n (where k represents a set of integers from 1 to n, and n is an integer greater than or equal to 2), determine the points p where V(i) reaches Vmax or becomes X before that k and the points q where V(i) reaches Vmax or becomes X after that k to obtain n p k (p1, ···, pn) and n q k (q1, ···, q k ). As described above, p k and q n can be data based on the measurement point standard or data based on the time standard. When the above solidification reaction data contains large noise, peaks of V caused by the noise appear, and p k and q k may be misdetected. By removing the noise of the solidification reaction data before obtaining V, or removing the peaks of V from the noise or p k and q k from this, misdetection of p k and q k can be prevented. k and q k

[0106] The value of the variable X k can be set arbitrarily. Preferably, X k is greater than 0 and less than or equal to Vmax. Preferably, X k can be set based on Vmax. For example, X k is defined as Vmax × S k (%), where S k only needs to be greater than 0 and less than or equal to 100, and preferably can be set in the range of 0.5 to 99. Preferably, S k = a + (k - 1) × b, where a > 0, b > 0, and k is defined as above. When a = 1 and b = 1, S k = k, and the maximum value of S k is 100. It should be noted that when S k is 100, X k = Vmax, p k = q k . For example, when setting n X k (X1, ···, X n ), set n S k (S1, ···, S n ) (where k, n are as described above, each Sk is greater than 0 and less than or equal to 100, preferably 0.5 to 99, more preferably 5 to 50). The set variable X kThe number is not particularly limited, preferably 5 to 50, more preferably 10 to 30, that is, n is preferably an integer of 5 to 50, more preferably 10 to 30. Preferably, n S k It consists of a set of values that change stepwise at equal intervals from their minimum value to their maximum value.

[0107] That is, p k , q k Satisfy the following formula.

[0108] V(p k −D) < X k ≤V(p k ), V(q k ) ≥ X k > V(q k +D), where D is the measurement interval,

[0109] Among them,

[0110] p k ≤VmaxT≤q k , where VmaxT is the measurement point number or time when V(VmaxT) = Vmax,

[0111] X k = Vmax × S k (%),

[0112] 0 < S k ≤100, preferably 0.5 ≤ S k ≤99, more preferably 5 ≤ S k ≤50,

[0113] k is an integer from 1 to n,

[0114] n is preferably an integer of 5 to 50, more preferably 10 to 30.

[0115] Refer to Figure 2 , for X k , p k and q k for description. In Figure 2 , the first derivative V of the solidification reaction curve is plotted against time. The peak of V is the maximum value Vmax (100%), and the time when V = Vmax is represented by VmaxT. Twenty X k (i.e., k is an integer from 1 to 20) are respectively specified as values that increase stepwise at equal intervals (every 5%) from Vmax × 3% (k = 1) to Vmax × 98% (k = 20). Lines representing each X k are drawn under the curve of V. For each X k , the time when V is X k (X kThere are two intersections of the line with V, one before VmaxT and the other after VmaxT. As a result, for 20 X k Detect the time when 40 points become V = X k Among these moments, the 20 before VmaxT (t[1] to t

[20] ) correspond to p k The 20 after VmaxT (t

[40] to t

[21] ) correspond to q k Correspond.

[0116] Based on the above p k 、q k , parameters for analyzing the coagulation ability of the sample to be tested can be calculated. As an example of such a parameter, the difference between the respective averages of p k and q k can be cited (hereinafter, also referred to as the front-back average difference). In this specification, sometimes the average value of the point p k existing before the point where V reaches Vmax is called the front Ave, and similarly, sometimes the average value of the point q k existing after the point where V reaches Vmax is called the back Ave, and sometimes the average value of p k and q k as a whole is called the full Ave. For example, the front Ave, the back Ave, and the full Ave can be calculated from all p k (p1, ···, p n ) and all q k (q1, ···, q n ) (n is defined as above). Or the front Ave, the back Ave, and the full Ave can be calculated as the average values of p k and q k for any range (for example, (p a , ···, p b ) and (q a , ···, q b ), where a and b are each independently integers selected from 1 to n, a < b, and n is defined as above). In one embodiment, the front-back average difference is made relative, for example, it can be defined as the ratio of the difference between the front Ave and the back Ave to the full Ave ([(back Ave - front Ave) / full Ave]).

[0117] As another example of the above parameter, the coefficient of variation (hereinafter, also referred to as CV) for p k and q k can be cited. For example, CV can be defined as for all p k (p1, ···, p n ) and all q k (q1, ···, qn )(where n is defined as above) coefficient of variation (%). Alternatively, CV can be defined for any range of p k and q k (e.g., (p a , ···, p b ) and (q a , ···, q b ), where a and b are each independently integers selected from 1 to n, a < b, and n is defined as above) coefficient of variation (%). As shown in the examples described later, the average difference before and after and CV reflect the width of the peak width of V or the degree of asymmetry of the peak shape in a sample containing an FVIII substitute substance.

[0118] As another example of the above parameters, q k to p k ratio (i.e., q k / p k , hereinafter, also referred to as the qp ratio) (k is any integer selected from 1 to n, and n is defined as above). As another example of the above parameters, the baseline length vB(S k ) can be cited, which represents the interval (time length or number of measurement points) from p k to q k (k is any integer selected from 1 to n, and n is defined as above). When V has a unimodal peak shape as shown in Figure 2 , vB(S k ) corresponds to the interval from p k to q k (= [q k - p k , or = [q k - p k + D], where D is defined as above). As shown in the examples described later, the qp ratio and vB(S k ) also reflect the width of the peak width of V or the degree of asymmetry of the peak shape in a sample containing an FVIII substitute substance.

[0119] As another example of the above parameters, parameters related to the relative values of p k and q k can be cited. Hereinafter, the relative values of p k and q k will be respectively referred to as relative p k (or rp k ) and relative q k (or rq k ). In one embodiment, rp k = p k / C, rq k = q k / C, and obtain rp within the range of a to b for k k (rp a , ···, rp b ) and / or rq within the range of c to d for k k (rq c , ···, rq d )(where a, b, c, and d are each independently integers selected from 1 to n, a < b, c < d, and n is defined as above). Here, C is a function related to time or the number of measurement points. For example, VmaxT, APTT, or the conversion value for the number of measurement points, any p k and q k , or the weighted average time vT(S k ) shown below. The difference values reflecting the obtained (rp a , ···, rp b ) and / or (rq c , ···, rq d ) and those of the reference sample (rp a , ···, rp b ) and / or (rq c , ···, rq d ) are used as this parameter. For example, the slope δ of the first regression line of the curve of (rp a , ···, rp b ) and / or (rq c , ···, rq d ) of the test sample relative to those of the reference sample (rp a , ···, rp b ) and / or (rq c , ···, rq d ) can be used as this parameter. This parameter uses p k and q k representing the progress of the coagulation reaction (coagulation reaction rate curve), takes the progress of the sample without the FVIII replacement substance as the reference, and is thus numerically obtained in such a way as to know the different degrees of the progress of the coagulation reaction of the sample containing the FVIII replacement substance. This parameter also reflects the width of the peak width of V or the degree of asymmetry of the peak shape of the sample containing the FVIII replacement substance.

[0120] Thus, the above-mentioned p k , q k or the parameter calculated from them reflect the coagulation ability of the test sample and indicate the presence of the FVIII replacement substance in the test sample. By using these parameters, the presence or absence of the FVIII replacement substance in the test sample can be detected.

[0121] 4.3) Parameters Based on the Weighted Average of V

[0122] In one embodiment, the parameter for analyzing the blood coagulation ability of the above-mentioned sample to be tested is a parameter based on the weighted average of V. Specifically, the sharpness rate (vAB(S k )) and the time rate (vTB(S k )) in the specified region at V(i) (where i is time or the measurement point number) shown in the following formula can be cited.

[0123] vAB(S k ) = vH(S k ) / vB(S k )

[0124] vTB(S k ) = vB(S k ) / VmaxT

[0125] Here,

[0126] [Mathematical Formula 2]

[0127]

[0128] For p k , q k , S k , vB(S k ), as defined in 4.2) above,

[0129] That is,

[0130] V(p k - D) < Vmax × S k (%) ≤ V(p k ),

[0131] V(q k ) ≥ Vmax × S k (%) > V(q k + D),

[0132] Here, p k , q k represent time or the measurement point number, p k ≤ VmaxT ≤ q k , VmaxT satisfies V(VmaxT) = Vmax,

[0133] D is the measurement interval,

[0134] 0 < S k ≤ 100, preferably 0.5 ≤ S k ≤ 99, more preferably 5 ≤ S k ≤ 50,

[0135] k is an integer from 1 to n,

[0136] n is preferably an integer from 5 to 50, more preferably from 10 to 30,

[0137] vB(S k ) is p k to q k of the interval (time length or number of measurement points).

[0138] It should be noted that APTT or the conversion value of the number of measurement points, the weighted average time vT(S k ) etc. can be used to replace VmaxT as the denominator of the calculation formula of v TB(S k ). It should be noted that vT(S k ) depends on whether i is time or the number of measurement points, and can be obtained as a function of time or as a function of the number of measurement points.

[0139] [Mathematical formula 3]

[0140]

[0141] vH(S k ) corresponds to the Y coordinate of the weighted average point of the region satisfying V(i)≥Vmax×S k (%) under V(i), and vAB(S k ) and vTB(S k ) represent the sharpness rate and time rate of this region.

[0142] 4.4) Parameters based on the relative value of V

[0143] In one embodiment, the maximum value after relative value conversion of the first derivative curve is determined (hereinafter, referred to as corrected Vmax). For example, R is relatively valued so that the maximum value is a specified value (as an example, 100), and then the maximum value of the first derivative curve obtained by performing the first derivative is determined as the corrected Vmax. Alternatively, the corrected Vmax can also be obtained by multiplying Vmax by a correction coefficient (= specified value / maximum value of R). This corrected Vmax can be used as a parameter for analyzing the coagulation ability of the above-mentioned test sample.

[0144] 4.5) Other parameters

[0145] The above parameters can be multiplied or divided by values related to the time or number of measurement points of the coagulation reaction, such as VmaxT, vT(S k) Or APTT or the conversion value of the number of measurement points thereof, and use the obtained value as a parameter for analyzing the coagulation ability of the test sample in the present invention. As such parameters, [Corrected Vmax × VmaxT], [Corrected Vmax × APTT], and [vAB(S k ) × VmaxT] can be cited. The parameters of the present invention depend on i, p k , q k Whether it is time or the number of measurement points, it can be a parameter related to time or a parameter related to the number of measurement points. And as described above, in the case of multiplying or dividing parameters, it is preferable that the parameter related to time is multiplied or divided by a function of time, and the parameter related to the number of measurement points is multiplied or divided by a function of the number of measurement points.

[0146] 4.6) Standard Deviation Index (SDI)

[0147] The units of the respective parameters described in the above 4.2) to 4.5) can be converted into the Standard Deviation Index (SDI) relative to the reference value, and the obtained value can be used as a parameter for analyzing the coagulation ability of the test sample in the present invention. Specifically, the statistical value of a sample without FVIII replacement substance (that is, a sample without FVIII replacement substance and with no prolonged clotting time, hereinafter also referred to as non - administered (ND) sample) can be used, and the SDI calculated by the following formula can be used as a parameter. More preferably, the absolute value (|SDI|) of the SDI calculated by the following formula 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)|.

[0148] 5. Detection of Samples Containing FVIII Replacement Substance

[0149] As shown in the examples described later, the above - mentioned parameters for analyzing coagulation ability calculated in the present invention, the average difference before and after, the overall CV, the qp ratio, vB(S k ), vAB(S k ), vTB(S k ), the parameters related to the relative values of p k and q k 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. Based on these parameters, it is possible to detect samples containing FVIII replacement substance.

[0150] In the analysis of the coagulation ability of the present invention, as an example of the parameter for detecting the sample containing the FVIII replacement substance, examples of the parameter include the average difference before and after, total CV, qp ratio, vB(S k ), vAB(S k ), vTB(S k ), the parameter related to the relative value of p k and q k , and at least one of the modified Vmax. Multiply or divide this parameter by a value related to the time of the coagulation reaction or the number of measurement points, such as VmaxT, APTT, or the conversion value for the number of measurement points, or the value obtained by vT(S k ), for example, [modified Vmax × VmaxT], [modified Vmax × APTT], [vAB(S k ) × VmaxT], and [vAB(S k ) × APTT] can also be cited as examples of the parameters for detecting the sample containing the FVIII replacement substance. Alternatively, the SDI of each of the above parameters can also be cited as an example of the parameter for detecting the sample containing the FVIII replacement substance. In the analysis of the coagulation ability of the present invention, any one of these parameters can be used, or two or more can be used in combination. It is preferable to use at least one parameter selected from the average difference before and after, total CV, qp ratio, and vTB(S k ), the parameter related to the relative value of p k and q k , and any SDI of these parameters.

[0151] In the detection of the sample containing the FVIII replacement substance based on the above parameters, for example, when the parameters are the average difference before and after, total CV, qp ratio, vB(S k ), vTB(S k ), and the parameter related to the relative value of p k and q k , when the parameter calculated from the test sample is above a specified detection threshold (hereinafter, also simply referred to as "threshold") or greater than the threshold, the test sample can be detected as a sample containing the FVIII replacement substance. In addition, for example, when the parameters are vAB(S k ), [vAB(S k ) × VmaxT], [vAB(S k ) × APTT], modified Vmax, [modified Vmax × VmaxT], and [modified Vmax × APTT], when the parameter calculated from the test sample is below the threshold or less than the threshold, the test sample can be detected as a sample containing the FVIII replacement substance.

[0152] 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 parameters are the average difference before and after, the overall CV, the qp ratio, vTB(S k ) and the parameter related to the relative value of p k and q k , the maximum value or the average value + K × standard deviation (SD) (K is preferably 1.5 to 3.4) of the parameters calculated from the ND sample group can be set as the threshold value. On the other hand, when the parameters are [vAB(S k ) × VmaxT], [vAB(S k ) × APTT], [modified Vmax × VmaxT], and [modified Vmax × APTT], the minimum value or the average value - K × standard deviation (SD) (K is preferably 1.3 to 2.3) of the parameters calculated from the non-dosed sample group can be set as the threshold value.

[0153] Taking the threshold value (threshold line) that varies two-dimensionally instead of the fixed-value threshold as a reference, it is possible to distinguish a sample containing an FVIII replacement substance from an ND sample. For example, when plotting the value of the test sample on the two-dimensional plane of (APTT, vB(S k )) if it is above the threshold line (for example, [vB(S k ) = a × APTT + b]; a and b are arbitrary coefficients), the test sample can be detected as a sample containing an FVIII replacement substance. Additionally, for example, when plotting the value of the test sample on the plane of (APTT, vAB(S k )) or the plane of (APTT, modified Vmax), if it is below the threshold line (for example, [vAB(S k ) = a × APTT + b] or [modified Vmax = a × APTT + b]; a and b are arbitrary coefficients), the test sample can be detected as a sample containing an FVIII replacement substance. The threshold line can be obtained, for example, through the steps described in Example 1 below.

[0154] In the case of SDI with the above parameters, the above threshold can be set according to the SDI of this parameter pre-calculated from the sample group containing the FVIII replacement substance or the ND sample group. For example, the threshold for the above |SDI| can be set as follows: among the parameters calculated from the sample group containing the FVIII replacement substance, extract the parameter value with the value closest to the distribution area 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 with the value closest to the distribution area 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 (refer to Example 9 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. If the |SDI| calculated from the test sample is greater than this threshold, the test sample can be detected as a sample containing the FVIII replacement substance.

[0155] As the reference for the above detection, more than 2 of the above parameters can be used. In this case, thresholds (or threshold lines) are set for each parameter, and it is investigated whether each parameter calculated from the test sample meets the criteria based on their respective thresholds (or threshold lines). In one example, if all of the more than 2 parameters meet the threshold, the test sample is detected as a sample containing the FVIII replacement substance. In another example, if at least one of the more than 2 parameters meets the threshold, the test sample is detected as a sample containing the FVIII replacement substance.

[0156] The sample detected as a sample containing the FVIII replacement substance is a sample from a patient with abnormal blood coagulation ability who has been administered the FVIII replacement substance, and it is inferred that this patient is a patient with a high bleeding risk during surgery, etc. Clinically, as the FVIII replacement substance, emicizumab, a drug for suppressing the bleeding tendency of hemophilia A, is known. Therefore, it can be inferred that the sample containing the FVIII replacement substance detected by the method of the present invention is a sample from a hemophilia A patient who has been administered emicizumab.

[0157] In the above detection criteria, within the range where samples containing the 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.

[0158] 6. Application to Other Coagulation Reaction Measurement Methods

[0159] As described above, the method of the present invention has been described by taking the case of measuring the coagulation reaction based on the amount of scattered light as an example. 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 of the present invention. Therefore, such applications are included in the scope of the present invention. For example, for the reaction R(i) shown by an inverse S-shaped coagulation reaction curve based on transparency (the amount of transmitted light), the positive and negative are opposite to those based on the above-mentioned amount of scattered light. In such a case, those skilled in the art can understand that in the method of the present invention described above, the signs of R(i) and V(i) are reversed, and the minimum value Vmin is determined instead of the maximum value Vmax of V, p k and q k are respectively determined as the points where V(i) becomes X before and after reaching Vmin k and the like.

[0160] 7. Program and apparatus

[0161] The method for analyzing the blood 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 the blood 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 the blood 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 the blood coagulation ability of the present invention, and this system includes a device or program for performing the method for analyzing the blood coagulation ability of the present invention described above.

[0162] An example of the blood coagulation analysis process performed under the control of the program of the present invention and using the method of the present invention is described below and Figure 3A in the following.

[0163] S1: Obtain R(i) from the measured coagulation reaction data (arbitrary).

[0164] S2: Calculate APTT based on R(i).

[0165] S3: If APTT is not prolonged, go to S4; if APTT is prolonged, go to S8.

[0166] S4: Calculate V(i) based on R(i).

[0167] S5: Calculate one or more parameters based on V(i).

[0168] S6: Compare the parameter with a threshold value.

[0169] →When at least one of the parameters satisfies the threshold, go to S7.

[0170] →When all of the parameters do not satisfy the threshold, go to S8.

[0171] S7: Output the detection of APTT and the sample containing the FVIII replacement substance.

[0172] S8: Output APTT.

[0173] Hereinafter, an embodiment of the device of the present invention will be described. An embodiment of the device of the present invention is an Figure 3B automatic analysis device 1 as shown. The automatic analysis device 1 includes a control unit 10, an operation unit 20, a measurement unit 30, and an output unit 40.

[0174] The control unit 10 controls the overall operation of the automatic analysis device 1. The control unit 10 may 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 measurement data received from the measurement unit 30 and perform data analysis, save analysis results, control the analysis results output by the output unit 40, etc. Further, 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 analyzes the data measured by the measurement unit 30 may be the same or different.

[0175] 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 a user interface (UI) 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, R and V based on this, parameters for analyzing the blood coagulation ability, and analysis results such as the coagulation time (APTT) as needed. For example, the output unit 40 includes a display device such as a display.

[0176] The measurement unit 30 performs a series of operations for blood coagulation analysis and obtains measurement data on the coagulation reaction of a specimen containing a blood sample. The measurement unit 30 is equipped with various instruments and analysis modules required for blood coagulation analysis, 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 detector 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, etc. Or use known coagulation reaction data to obtain V. In the case of calculating parameters, the measurement unit 30 is not required.

[0177] The control unit 10 analyzes the coagulation reaction of the sample based on the coagulation reaction data. In this analysis, it may include the acquisition of the above-mentioned R and V, the calculation of APTT, the calculation of various parameters, and the detection of a sample containing an FVIII substitute substance using the parameter, etc. Or R and V 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, a threshold value for detecting a sample containing an FVIII substitute substance can be stored, or the control unit 10 can read the threshold value stored in an external device or network for this detection.

[0178] The above-mentioned analysis can be executed by a program for performing the method of the present invention. Therefore, the control unit 10 can be equipped with a program for performing the analysis method of the blood coagulation ability of the present invention.

[0179] The analysis result obtained by the control unit 10 is transmitted to the output unit 40 for output. The output can be in any form such as display on a screen, sending to a host computer, printing, etc. The output information output from the output unit may include R or V, APTT, parameters for analyzing blood coagulation ability, the detection result of a sample containing an FVIII substitute substance, etc. And the output information can also include other information obtained in the APTT measurement (detection of the initial reaction, possibility of a chylous sample, etc.). The type of the output information output from the output unit can be controlled by the program of the present invention.

[0180] Examples

[0181] Hereinafter, examples are given to explain the present invention in further detail, but the present invention is not limited to these examples.

[0182] 1. Method

[0183] 1.1) Sample

[0184] 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).

[0185] 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.

[0186] 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.

[0187] 1.2) Coagulation reaction measurement

[0188] 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.

[0189] 1.3) Obtaining R(i) and V(i)

[0190] After smoothing the measurement data obtained from the coagulation reaction of each sample, including noise removal, zero-point adjustment is performed so that the scattered light intensity at the start of measurement is zero. The coagulation reaction R(i) is generated (where i represents the time from the start of the coagulation reaction). Based on R(i), the primary differential (coagulation reaction rate) V(i) is calculated. The maximum value Vmax of V(i) and the time VmaxT at which V(i) reaches the maximum value Vmax are calculated for each sample.

[0191] 1.4) APTT calculation

[0192] The APTT of each sample was calculated by the percentage detection method. That is, the time point when R(i) reaches the maximum value Rmax within the measurement time was determined as the coagulation reaction end point E, and the time point when R(i) reaches 50% of R(E) was taken as the APTT. Figure 4 The APTT of NE, E1, and E2 is shown. Regarding the APTT, it is 27.1 - 39.3 seconds for NE, 21.2 - 37.9 seconds for E1, and 21.3 - 32.3 seconds for E2. The distribution ranges of the APTT of E1 and E2 overlap with the distribution range of NE. Also, in E1 and E2, an 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-administered groups (E1 and E2) and the non-emicizumab-administered group (NE) cannot be distinguished solely by the APTT.

[0193] 1.5) Production of the corrected reaction rate curve

[0194] To compare the shapes of the rate curves of each sample, the corrected reaction rate curve was produced according to the following steps. The relative values were made so that the maximum value of V(i) is 100, and it was shifted in the time axis direction so that the time to reach the maximum value is 40 seconds. The curve thus obtained was taken as the corrected reaction rate curve. The name of the horizontal axis when representing this corrected reaction rate curve was set as the corrected time, and the name of the vertical axis was set as the corrected reaction rate.

[0195] 1.6) Calculation of the corrected Vmax

[0196] The corrected Vmax (= Vmax × F) was calculated by multiplying the Vmax of each sample by the coefficient F (= 100 / Rmax).

[0197] 1.7) Calculation of the parameters related to V(i)

[0198] The parameters related to V(i) of each sample were calculated. It was stipulated that X k = Vmax × S k (%)(k is an integer from 1 to 20, and S k increases from 3% every 5% to 98%, that is, S1 = 3%, S 20 = 98%). The 20 p k constituted by the minimum values of i that satisfy V(i) ≥ X k in the time before VmaxT were obtained, and the 20 q 20 constituted by the maximum values of i that satisfy V(i) ≥ X k in the time after VmaxT were obtained (refer to k Figure 2 ). According to the obtained p 20 Figure 2 ) and q kand q k Calculate the following parameters.

[0199] Previous Ave: p k The average value (k is any range from 1 to 20)

[0200] Subsequent Ave: q k The average value (k is as described above)

[0201] Total Ave: p k and q k The average value

[0202] Average difference between before and after: (Subsequent Ave - Previous Ave) / Total Ave

[0203] CV: Regarding p k and q k The coefficient of variation (%)

[0204] q / p ratio: q k / p k

[0205] Sharpness ratio vAB(S k ) = vH(S k ) / vB(S k )), or

[0206] vAB(S k ) = vH(S k ) / vB(S k 〖ID=53]]) × VmaxT, or

[0207] vAB(S k ) = vH(S k ) / vB(S k ) × APTT

[0208] Time ratio vTB(S k ) = vB(S k ) / VmaxT

[0209] It should be noted that vH(S k ) and vB(S k ) are as follows:

[0210] vB(S k ) : q k - p k + 0.1 (seconds)

[0211] [Mathematical formula 4]

[0212]

[0213] (p k 、q k、S k , k as above)

[0214] In the following, the parentheses on both sides of the parameter name may be omitted. For example, vB(30) may be represented as vB30.

[0215] 2. Coagulation reaction

[0216] Figure 5 The coagulation reaction curves R(i) (scattered light intensity after adjustment at 0 point) (top) and the first differential curves V(i) (bottom) for NE, E1, and E2, plotted against measurement time, are shown. The reaction curves for E1 and E2 rise earlier than those for NE, but the rise tends to be slower. Figure 6 The corrected reaction rate curves of NE, E1, and E2 are plotted against the corrected time. The corrected reaction rate curves of E1 and E2 tend to have wider peak widths and more asymmetric peak shapes (slower descent from the peak top) compared to NE.

[0217] 3. Parameter comparison between sample groups

[0218] 3.1) Comparative Example 1 Vmax

[0219] Figure 7 It represents the distribution of the maximum value Vmax of V(i) for NE, E1 and E2. Figure 7 A represents the plot of Vmax versus APTT for each sample. Figure 7 B shows the distribution of Vmax for each sample group. The Vmax of E1 and E2 tended to be smaller than that of NE, but the distribution area overlapped with that of NE. It was difficult to distinguish the emicizumab-treated groups (E1 and E2) from the non-treated group (NE) based on Vmax alone.

[0220] 3.2) Comparative Example 2 VmaxT

[0221] Figure 8 A to D represent plots of VmaxT versus APTT for NE, E1, E2, and all samples, respectively. Figure 8 E represents the distribution of VmaxT for each sample group. VmaxT for E1 and E2 tended to be smaller than that for NE, but the distribution overlapped with that for NE. VmaxT alone was insufficient to distinguish the emicizumab-treated groups (E1 and E2) from the non-treated group (NE).

[0222] 3.3) Example 1 Correction of Vmax

[0223] Figure 9 The distribution of modified Vmax for NE, E1 and E2 is shown. Figure 9 A represents the plot of the corrected Vmax relative to APTT for each sample.Figure 9 B represents the distribution of the corrected Vmax for each sample group. Figure 9 The dashed line of A is obtained according to the following steps: 1) Obtain the regression line y = ax + b of the plotted points of each sample of NE, 2) Obtain the plotted point (the outermost point) among the plotted points of NE that is on the side of the distribution region of E1 and E2 from the regression line and is the farthest from the regression line, 3) Obtain the plotted point (the closest point) among the plotted points of E1 and E2 that is the closest to the regression line, 4) Calculate the line that passes through the midpoint of the line connecting the outermost point and the closest point and is parallel to the regression line. The distribution region of the corrected Vmax is different between the emicizumab-administered groups (E1 and E2) and the non-administered group (NE), and the two groups can be Figure 9 divided by the dashed line of A, indicating that the dashed line obtained according to the above steps can be used as a two-dimensional threshold (threshold line) for distinguishing between the emicizumab-administered groups (E1 and E2) and the non-administered group (NE). It can be seen that by setting such a threshold line, the emicizumab-administered groups (E1 and E2) and the non-administered group (NE) can be distinguished based on APTT and the corrected Vmax.

[0224] Figure 10 A represents the plotted points of [corrected Vmax × VmaxT] of each sample of NE, E1, and E2 with respect to APTT. Figure 10 B represents the distribution of [corrected Vmax × VmaxT] for each sample group. It can be seen that the distribution region of [corrected Vmax × VmaxT] is different between the emicizumab-administered groups (E1 and E2) and the non-administered group (NE), and based on this value, the emicizumab-administered groups (E1 and E2) and the non-administered group (NE) can be distinguished.

[0225] Figure 11 A represents the plotted points of [corrected Vmax × APTT] of each sample of NE, E1, and E2 with respect to APTT. Figure 11 B represents the distribution of [corrected Vmax × APTT] for each sample group. The distribution region of [corrected Vmax × APTT] is different between the emicizumab-administered groups (E1 and E2) and the non-administered group (NE), and based on this value, it can be seen that the emicizumab-administered groups (E1 and E2) and the non-administered group (NE) can be distinguished. According to Figure 10 and 11 the results, it can be seen that if values obtained by multiplying the corrected Vmax by parameters related to the reaction time such as VmaxT and APTT are used, even without setting a two-dimensional threshold (threshold line), based on a specific threshold, the emicizumab-administered groups (E1 and E2) and the non-administered group (NE) can be distinguished.

[0226] 3.4) Example 2 vTB(S k )

[0227] Figure 12 A represents the plotted points of vTB(30) of each sample of NE, E1, and E2 relative to APTT. Figure 12 B represents the distribution of vTB(30) for each sample group. In the figure, vTB(30) is represented as vB(30) / VmaxT. The distribution region of vTB(30) is different between the emicizumab-administered groups (E1 and E2) and the non-administered group (NE). From this value, it is known that the emicizumab-administered groups (E1 and E2) and the non-administered group (NE) can be distinguished.

[0228] 3.5) Example 3 vAB(S k )

[0229] Figure 13 A represents the plotted points of vAB(10) = vH(10) / vB(10) of each sample of NE, E1, and E2 relative to APTT. Figure 13 B represents the distribution of vAB(10) for each sample group. In the figure, vAB(10) is represented as vH(10) / vB(10). Figure 13 The dotted line of A is calculated in the same way as in Example 1 (3.3 above). The distribution region of vAB(10) is different between the emicizumab-administered groups (E1 and E2) and the non-administered group (NE), and the two groups can be Figure 13 divided by the dotted line of A. It is known that by setting a two-dimensional threshold (threshold line) like this dotted line, according to APTT and vAB(S k ) the emicizumab-administered groups (E1 and E2) and the non-administered group (NE) can be distinguished.

[0230] Figure 14 A represents the plotted points of [vAB(10) × VmaxT] of each sample of NE, E1, and E2 relative to APTT. Figure 14 B represents the distribution of [vAB(10) × VmaxT] for each sample group. Figure 14 C represents the plotted points of [vAB(10) × APTT] of each sample of NE, E1, and E2 relative to APTT. Figure 14 D represents the distribution of [vAB(10) × APTT] for each sample group. In the figure, vAB(10) is represented as vH(10) / vB(10). The [vAB(10) × VmaxT] and [vAB(10) × APTT] of the emicizumab-administered groups (E1 and E2) are lower compared to the non-administered group (NE). It is known that if the value obtained by multiplying vAB(S k ) by VmaxT or APTT is used, even without setting a two-dimensional threshold (threshold line), based on a specific threshold, the emicizumab-administered groups (E1 and E2) and the non-administered group (NE) can be distinguished.

[0231] 3.6) Example 4 qp ratio

[0232] Calculate p for k = 8 k and q k , and obtain the qp ratio = q k / p k . Figure 15 A represents the plotted points of the qp ratio (q8 / p8) of each sample of NE, E1, and E2 relative to APTT. Figure 15 B represents the distribution of the qp ratio for each sample group. The distribution region of the pq ratio is different between the emicizumab-administered groups (E1 and E2) and the non-administered group (NE). From this value, it is known that the emicizumab-administered groups (E1 and E2) and the non-administered group (NE) can be distinguished. As Figure 5 and Figure 6 shown, it is speculated that the width of the peak width of V(i) and the peak shape (degree of asymmetry of the peak) in the emicizumab-administered groups (E1 and E2) relative to the non-administered group (NE) reflect the distribution difference of the qp ratio between the emicizumab-administered groups (E1 and E2) and the non-administered group (NE).

[0233] 3.7) Example 5 vB(S k )

[0234] Figure 16 A represents the plotted points of q6 - p6 (vB(6)) of each sample of NE, E1, and E2 relative to APTT. Figure 16 B represents the distribution of q6 - p6 for each sample group. Figure 16 The dotted line of A is calculated in the same way as in Example 1 (3.3) above. The distribution region of q6 - p6 is different between the emicizumab-administered groups (E1 and E2) and the non-administered group (NE), and the two groups can be Figure 16 divided by the dotted line of A. It is known that by setting a two-dimensional threshold (threshold line) such as this dotted line, the emicizumab-administered groups (E1 and E2) and the non-administered group (NE) can be distinguished according to APTT and q k -p k (vB(S k )) Figure 5 and Figure 6 shown, it is speculated that the width of the peak width of V(i) in the emicizumab-administered groups (E1 and E2) relative to the non-administered group (NE) reflects the distribution difference of vB(S k ) between the emicizumab-administered groups (E1 and E2) and the non-administered group (NE).

[0235] 3.8) Example 6 Mean difference before and after

[0236] Figure 17A to F represent the average difference before and after for each sample of NE, E1, and E2. Figure 17 A, C, and E represent the plotted points of the average difference before and after relative to APTT. ​ B, D, and F represent the distribution of the average difference before and after for each sample group. ​ A and B are the average differences before and after when k = 1 to 20, that is, for p k , q k detected from the entire peak of V, ​ C and D are the average differences before and after when k = 1 to 10, that is, for p k , q k detected from the lower side of the peak of V (lower average difference before and after), ​ E and F are the average differences before and after when k = 6 to 15, that is, for p k , q k detected from the middle of the peak of V (middle average difference before and after). In any case, the distribution region of the average difference before and after is different between the emicizumab-administered groups (E1 and E2) and the non-administered group (NE), and it can be seen that the emicizumab-administered groups (E1 and E2) and the non-administered group (NE) can be distinguished based on the average difference before and after. As ​ and ​ shown, it is speculated that the peak shape (degree of peak asymmetry) of V(i) in the emicizumab-administered groups (E1 and E2) relative to the non-administered group (NE) reflects the distribution difference of the average difference before and after between the emicizumab-administered groups (E1 and E2) and the non-administered group (NE).

[0237] 3.9) Example 7 Total CV

[0238] ​ A to D represent the CV of p k and q k for each sample of NE, E1, and E2. ​ A and C represent the plotted points of the CV relative to APTT. ​ B and D represent the distribution of the CV for each sample group. ​ A and B are the CV (total CV) of p1 to p 20 and q1 to q 20 when k = 1 to 20, ​ C and D are the CV (lower CV) of p1 to p 10 and q1 to q 10 when k = 1 to 10. The CV of p k and q k has a different distribution region between the emicizumab-administered groups (E1 and E2) and the non-administered group (NE), and it can be seen that based on p k and q kThe CV can distinguish between the emicizumab-administered groups (E1 and E2) and the non-administered group (NE). As ​ and ​ shown, it is speculated that the peak width and peak shape (degree of peak asymmetry) of V(i) in the emicizumab-administered groups (E1 and E2) reflect the distribution difference in the CV of p k and q k between the emicizumab-administered groups (E1 and E2) and the non-administered group (NE).

[0239] 3.10) Example 8 p k and q k Ratio relative to VmaxT

[0240] The p k and q k of NE, E1, and E2 were compared. The p k and q k (k = 1 to 20 or k = 6 to 20) of each sample of NE, E1, and E2 were divided by VmaxT to obtain relative p k (= p k / VmaxT) and relative q k (= q k / VmaxT). For the reference sample, an NE sample with the minimum (27.1 seconds) or maximum (39.3 seconds) APTT was used. In the relative p k and relative q k data groups of the reference sample and the relative p k and relative q k data groups of each sample, a first-order regression line was obtained.

[0241] ​ A to D and ​ A to D represent the distribution of the slopes of the above regression lines for each sample of NE, E1, and E2. ​ represents the slope of the regression line for relative p k and relative q k (k = 1 to 20), ​ represents the slope of the regression line for relative p k and relative q k (k = 6 to 20). For the reference sample, in ​ A, B, and ​ A, B, an NE sample with the minimum APTT was used, and in ​ C, D, and ​ C, D, an NE sample with the maximum APTT was used. ​ A, C, and ​ A, C represent the plotted points of the above slopes for each sample relative to APTT, ​B, D, and ​ B and D represent the distributions of the above slopes for each sample group. In ​ A, C, and ​ A and C, the plotted points of the reference sample (reference NE) are indicated by black circles ●. In ​ any of the cases, the slope of the regression line tended to be greater in the emicizumab-administered groups (E1 and E2) than in the non-administered group (NE). From this, it can be seen that the emicizumab-administered group and the non-administered group can be distinguished based on this parameter.

[0242] 3.11) SDI of the parameter in Example 9

[0243] The proximity of the parameter (P) between the emicizumab-administered group (Emi; including E1 and E2) and the non-administered group (NE) was evaluated. As the parameter (P), [modified Vmax × VmaxT] shown in ​ , [modified Vmax × APTT] shown in ​ , vTB(30) shown in ​ , [vAB(10) × VmaxT] and [vAB(10) × APTT] shown in ​ , q8 / p8 shown in ​ , the front-back mean difference, the lower front-back mean difference, and the middle front-back mean difference shown in ​ , the overall CV and the lower CV shown in ​ and the 4 regression line slopes shown respectively in ​ AB, ​ CD, ​ AB, and ​ CD were used. Samples (NE-close samples) in which P in NE was closest to Emi (i.e., P was the largest or smallest) and samples (Emi-close samples) in which P in Emi was closest to NE (i.e., P was the smallest or largest) were extracted. The absolute value (|SDI|) of the standard deviation index of the average value of P of the two extracted samples with respect to the P of NE was calculated.

[0244] |SDI| = |{(P of the NE-close sample or the Emi-close sample) - (average value of P of NE)} / (standard deviation of P of NE)|

[0245] The |SDI| of the NE proximity samples and the Emi proximity samples for each parameter is shown in Table 1. The difference in |SDI| between the NE proximity samples and the Emi proximity samples, as shown in Examples 1 to 8, reflects that each parameter has different distributions in Emi and NE. From these results, it can be seen that by using the |SDI| of the parameters shown in Examples 1 to 8, the emicizumab-administered group and the non-administered group can be distinguished. For example, by calculating the |SDI| of the parameter of the test sample with respect to the reference value and comparing it with the threshold value, it is possible to detect whether the test sample is in the emicizumab-administered group. As the reference value, the average value of the parameters of NE as described above can be used. As an example of this threshold value, the intermediate value between the |SDI| of the NE proximity samples and the |SDI| of the Emi proximity samples as described above can be set.

[0246] [Table 11]

[0247]

Claims

1. A method, which is an analysis method for the coagulation ability of a blood sample, comprising: 1) Obtaining the first derivative V(i) of the coagulation reaction curve of a test blood sample that needs to be tested for substances having factor VIII replacement activity, where i represents the measurement point number or time; and 2) The following a) or b): a) Determine when V(i) reaches its maximum value Vmax or before it becomes X k The minimum value p of the above points k , and when V(i) reaches Vmax or becomes X after that k The maximum value q of the point above k , where k represents a set of integers from 1 to n, n is an integer greater than 2, and X k = Vmax×S k %, 0<S k ≤100; b) Determining the maximum value after relative value conversion of V(i).

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

3. The method according to claim 1, wherein The n is an integer from 5 to 50.

4. The method according to claim 1, wherein, Further includes calculating a parameter for analyzing the clotting ability of the blood sample to be tested according to said p k or q k ​ 5. The method according to claim 4, wherein, The parameter is selected from the front-back average difference, CV, qp ratio, vB(S k ), sharpness rate vAB(S k ), time rate vTB(S k ), vAB(S k )×VmaxT, vAB(S k )×APTT, and a parameter related to the relative values of p k and q k , and at least one of their standard deviation indices, i.e., SDI. Here, The average difference before and after is expressed as (q a ~q b average value of - p a ~p b average value of) / (p a ~p b and q a ~q b average value of), a and b are integers selected from 1 to n, n is defined as above, a < b, CV represents for p a ~p b and q a ~q b The coefficient of variation for, a and b are integers selected from 1 to n, n is defined as above, a < b The qp ratio represents q k / p k , vB(S k ) represents p k to q k of the interval VmaxT satisfies V(VmaxT)=Vmax, APTT is the activated partial thromboplastin time, i.e., APTT, of the test blood sample, vAB(S k ) = vH(S k ) / vB(S k ) vTB(S k ) = vB(S k ) / VmaxT, Here, vH(S k ) is expressed by the following formula 6. The method according to claim 5, wherein, The parameter related to the relative value of p k and q k is the slope of the first regression line of the curve of p a ~p b and q c ~q d of the test blood sample relative to the p a ~p b and q c ~q d of the reference sample. Here, a, b, c, and d are each independently integers selected from 1 to n, a < b, c < d, and n is defined as above. The reference sample is a blood sample not administered with a medicament having FVIII replacement activity.

7. The method according to claim 1, wherein Further comprising calculating a parameter for the analysis of the coagulation ability of the test blood sample according to the maximum value of the relative value-converted V(i).

8. The method according to claim 7, wherein, The parameter is at least one selected from modified Vmax, modified Vmax×VmaxT, modified Vmax×APTT, and their standard deviation index, i.e., SDI, Modified Vmax is the maximum value of the relative value-converted V(i), VmaxT satisfies V(VmaxT)=Vmax, APTT is the activated partial thromboplastin time, i.e., APTT, of the test blood sample.

9. The method according to any one of claims 4 to 8, wherein, Further comprising detecting the presence or absence of the substance having factor VIII replacement activity in the test blood sample according to the parameter.

10. The method according to claim 9, wherein, Comprising: Outputting the detection result of the presence or absence of the substance having factor VIII replacement activity.

11. The method according to claim 1, wherein, Further comprising: Measuring the activated partial thromboplastin time, i.e., APTT, of the blood sample; Selecting a blood sample in which APTT is not prolonged as the test blood sample that needs to be tested for the presence or absence of the substance having factor VIII replacement activity.

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

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

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

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

16. 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.

17. The device according to claim 15 or the system according to claim 16, wherein It comprises an output unit, and the output unit is used to output the analysis result of the coagulation ability of the blood sample by using the method according to claim 1.

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