Patent Description:
Hemophilia is a hemorrhagic disease caused by congenital defect or dysfunction of coagulation factor VIII (FVIII) or coagulation factor IX (FIX). Hemophilia is called hemophilia A when it is caused by FVIII and called hemophilia B when it is caused by FIX. In hemophilic patients, bleeding symptoms are found in deep tissues such as intraarticular tissues or intramuscular tissues, and intracranial hemorrhage also occurs in severe cases.

The severity of hemophilia is classified based on FVIII activity or FIX activity in the blood. Specifically, patients with an activity of less than <NUM>% are classified as severe, patients with an activity of <NUM>% or more and less than <NUM>% are classified as moderate, and patients with an activity of <NUM>% or more and less than <NUM>% are classified as mild, based on the FVIII activity or FIX activity of a healthy subject as <NUM>%. Patients with severe hemophilia present bleeding symptoms at a significantly higher frequency compared to moderate and mild patients. However, replacement therapy with FVIII or FIX can dramatically reduce the frequency of bleeding by maintaining FVIII activity or FIX activity in the patient's blood at <NUM>% or more.

For replacement therapy, a coagulation factor preparation purified from plasma or prepared by genetic recombination technology is mainly used. In recent years, a bispecific antibody having a FVIII-substituting activity has been developed. This bispecific antibody substitutes for the function as a cofactor of activated coagulation factor VIII (FVIIIa). That is, this bispecific antibody can promote the activation of FX by FIXa by binding to both activated coagulation factor IX (FIXa) and coagulation factor X (FX). This promotes blood coagulation. Meanwhile, the process of blood coagulation promoted by the bispecific antibody does not require activation from FVIII to FVIIIa unlike the normal process by FVIII.

In replacement therapy using a coagulation factor preparation such as FVIII, the efficacy of the administered coagulation factor is monitored by activated partial thromboplastin time (APTT) measurement, coagulation waveform analysis, and the like. However, as described above, since blood coagulation by a bispecific antibody is different from normal blood coagulation by FVIII, it has been difficult to acquire data reflecting the actual efficacy of a bispecific antibody by the existing method for monitoring the efficacy of a coagulation factor preparation.

Under such circumstances, the present inventors have found so far that the efficacy of a substance having a FVIII-substituting activity can be evaluated with appropriate sensitivity using the thrombin generation amount in a blood specimen as an index (see Patent Literature <NUM>). However, this method requires a special measuring equipment, so it has not been widely spread as a clinical test. In addition, the present inventors have found that the factor VIII-substituting activity of the bispecific antibody can be measured by coagulation waveform analysis using a commercially available APTT measuring reagent (see Non Patent Literature <NUM>). However, with this method, the efficacy of the bispecific antibody could not be evaluated with appropriate sensitivity.

<CIT> relates to a method for evaluating blood coagulation reaction.

<NPL>, relates to anti-factor IXa/X bispecific antibody (ACE910): hemostatic potency against ongoing bleeds in a hemophilia A model and the possibility of routine supplementation.

<CIT> relates to a coagulation activity measuring apparatus, measuring chip, and measuring method.

<CIT> relates to a method for determining the total coagulation activity of a blood or plasma sample.

<CIT> relates to a character input device, character input method and character input program.

<CIT> relates to a blood coagulation analyzer.

<CIT> relates to an inspection method of APTT measuring reagent.

<NPL>, relates to a modified thrombin generation test for investigating very low levels of factor VIII activity in hemophilia A.

<CIT> relates to a blood coagulation analyzer and blood coagulation analyzing method.

<NPL>, relates to an optimal monitoring on bypassing therapy for hemophilia A patients with inhibitor using aPTT-based clot waveform analysis with the presence of minimal tf.

In order to use a substance having a FVIII-substituting activity such as the bispecific antibody for treatment of hemophilia or the like, it is important to establish a method capable of appropriately evaluating the efficacy of the substance. Accordingly, it is desirable to develop a means for appropriately evaluating coagulability of a blood specimen containing a substance having a FVIII-substituting activity. It is also desirable to develop a new means for evaluating the coagulability of a blood specimen. The present inventors have found conditions capable of appropriately evaluating coagulability of a blood specimen containing a substance having a FVIII-substituting activity, and a novel approach for evaluating the coagulability of a blood specimen, thereby the present invention has been completed.

A second embodiment of the present invention provides a method for evaluating coagulability of a blood specimen. This method comprises the steps of preparing a measurement sample from a blood specimen obtained from a subject to whom a substance having a coagulation factor VIII-substituting activity is administered, a coagulation factor XII-activating agent, phospholipids, tissue factor, and a calcium ion-containing aqueous solution, irradiating the measurement sample with light to acquire optical information on the light amount from the measurement sample, and evaluating coagulability of the blood specimen based on the acquired optical information, as defined in the claims.

A fifth embodiment of the present invention provides the use of a reagent for evaluating coagulability of a blood specimen obtained from a subject to whom a substance having a coagulation factor VIII-substituting activity is administered, containing a coagulation factor XII-activating agent, phospholipids, and tissue factor, as defined in the claims.

A sixth embodiment of the present invention provides a reagent kit for evaluating coagulability of a blood specimen obtained from a subject to whom a substance having a coagulation factor VIII-substituting activity is administered, comprising a first reagent containing a coagulation factor XII-activating agent, phospholipids and tissue factor, and a coagulation initiation reagent comprising a calcium ion-containing aqueous solution, as defined in the claims.

A seventh embodiment of the present invention provides a reagent kit for evaluating coagulability of a blood specimen obtained from a subject to whom a substance having a coagulation factor VIII-substituting activity is administered, comprising a first reagent, a second reagent, and a coagulation initiation reagent comprising a calcium ion-containing aqueous solution, wherein the first reagent contains a coagulation factor XII-activating agent and phospholipids and the second reagent contains tissue factor, the first reagent contains a coagulation factor XII-activating agent and phospholipids and the second reagent contains tissue factor and phospholipids, or the first reagent contains a coagulation factor XII-activating agent and tissue factor and the second reagent contains phospholipids, as defined in the claims.

A twelfth embodiment of the present invention provides a reagent kit for evaluating coagulability of a blood specimen obtained from a subject to whom a substance having a coagulation factor VIII-substituting activity is administered, comprising a first reagent and a second reagent, wherein the first reagent contains a coagulation factor XII-activating agent and phospholipids and the second reagent contains tissue factor and calcium ions, as defined in the claims.

According to the present invention, it is possible to appropriately evaluate coagulability of a blood specimen obtained from a subject to whom a substance having a factor VIII-substituting activity is administered.

The blood specimen is not particularly limited as long as it is derived from blood containing a substance having a FVIII-substituting activity. Examples of the type of specimen include whole blood and plasma. Among them, plasma is preferable, and platelet-removed plasma is particularly preferable. The platelets can be removed by a known method such as centrifugation or filter separation. A mixture obtained by adding a substance having a FVIII-substituting activity to commercially available plasma may be used as a blood specimen. Examples of the commercially available plasma include coagulation factor-deficient plasma and the like. A coagulation factor preparation such as FVIII may be added to the blood specimen as necessary.

In a preferred embodiment, blood collected from a subject to whom a substance having a FVIII-substituting activity is administered or blood plasma prepared from the blood is used as a blood specimen containing a substance having a FVIII-substituting activity. Examples of the subject include patients with hemorrhagic disease caused by defect or dysfunction of coagulation factor. Examples of the hemorrhagic disease include hemophilia A, hemophilia B, acquired hemophilia, von Willebrand disease, and the like. More preferably, the subject is a patient with hemorrhagic disease in which the activity of either or both of FVIII and FVIIIa is decreased or deficient. The activity value of FVIII in the patient is, for example, less than <NUM>%, <NUM>% or <NUM>%, preferably less than <NUM>%, <NUM>%, <NUM>%, <NUM>% or <NUM>%, more preferably less than <NUM>%, <NUM>%, <NUM>% or <NUM>%, and particularly preferably less than <NUM>%, based on the FVIII activity value of a healthy subject as <NUM>%. The method itself for measuring the activity value of FVIII in a blood specimen is known in the art, and examples thereof include a synthetic substrate method and the like.

The substance having a FVIII-substituting activity is a substance having cofactor activity similar to FVIIIa in blood. However, the substance having a FVIII-substituting activity does not include FVIII and FVIIIa. The substance having a FVIII-substituting activity is a substance capable of specifically binding to both FIX or FIXa and FX and capable of promoting activation of FX by FIXa (that is, production of FXa), wherein the substance is a bispecific antibodies that specifically bind to both FIX or FIXa and FX, as defined in the claims. The bispecific antibodies themselves are known in the art, and disclosed, for example, in <CIT>, <CIT>, and <CIT>. More specifically, examples of the substance having a FVIII-substituting activity include ACE910 (Q499-z121/J327-z119/L404-k) (Emicizumab) which is an anti-FIXa/FX bispecific antibody described in Patent Literature (<CIT>).

The origin of the bispecific antibody is not particularly limited, and may be an antibody derived from any mammal such as human, mouse, rat, hamster, rabbit, goat, horse or camel, and is preferably a human antibody. Incidentally, the method itself for acquiring a human antibody is known in the art, and for example, a method utilizing a non-human transgenic animal having a human antibody gene and the like are known. Fragments of bispecific antibodies and derivatives thereof may be used, and examples thereof include Fab fragments, F(ab')<NUM> fragments, diabodies, linear antibodies, single chain antibodies, and the like. In addition, the bispecific antibody may be a genetically modified antibody such as a chimeric antibody or a humanized antibody.

The coagulation factor XII (FXII)-activating agent is not particularly limited as long as it is a known substance that is known to activate FXII to promote the production of activated factor XII (FXIIa) and promote blood coagulation in vitro. Examples of the activating agent include substances having a negative charge. Examples of the substance include ellagic acid, kaolin, celite, silica, and the like. Among them, ellagic acid is preferred. As ellagic acid, ellagic acid in the state of forming a metal ion with a chelate may be added. The FXII-activating agent is preferably in the form of a liquid in which the FXII-activating agent is dissolved in a suitable solvent.

Examples of the phospholipid include phosphatidylethanolamine (PE), phosphatidylcholine (PC) and phosphatidylserine (PS). One preferably two, more preferably all kinds of phospholipids selected from PE, PC and PS can be added. The phospholipid may be a naturally occurring phospholipid or a synthetic phospholipid. Among them, synthetic phospholipids or naturally occurring phospholipids purified to have a purity of <NUM>% or more are preferred. The fatty acid side chains of PE, PC and PS are not particularly limited, and examples thereof include palmitic acid, oleic acid, stearic acid, and the like. Among them, oleic acid is preferable. The phospholipid is preferably in the form of a liquid in which the phospholipid is dissolved in a suitable solvent.

In preparing the measurement sample, the order of mixing the blood specimen, the FXII-activating agent, and the phospholipids is not particularly limited. For example, the blood specimen and the FXII-activating agent are mixed, and then the phospholipids may be mixed therein. Alternatively, the blood specimen and the phospholipids are mixed, and then the FXII-activating agent may be mixed therein. Or, the FXII-activating agent and the phospholipids are mixed, and then the blood specimen may be mixed therein. Alternatively, the blood specimen, the FXII-activating agent, and the phospholipids may be substantially simultaneously mixed.

A commercially available APTT measuring reagent containing a FXII-activating agent and phospholipids may be used. In this case, an APTT measuring reagent may be used so that the respective final concentrations of the FXII-activating agent and the phospholipids in the measurement sample fall within the ranges as defined in the claims. However, a general commercially available APTT measuring reagent is not intended to be used with the respective final concentrations of the FXII-activating agent and the phospholipids in the measurement sample within the above range. Therefore, a commercially available APTT measuring reagent may be diluted and used as necessary. Examples of the diluent include physiological saline, buffer with a pH of <NUM> to <NUM>, water, and the like. Also, commercially available buffers may be used, and examples thereof include Owren's Veronal buffer (Sysmex Corporation), TC buffer (Sysmex Corporation), imidazole buffer (HYPHEN BioMed), and the like.

It is preferable to mix a blood specimen, a FXII-activating agent and phospholipids, incubate them under predetermined conditions, and then add the calcium-containing aqueous solution described later. Such a predetermined condition may be any known condition as long as it accelerates the reaction between the coagulation factor and the substance having a FVIII-substituting activity in the blood specimen, with the FXII-activating agent and the phospholipid, and examples thereof include a condition of incubating at a temperature of <NUM> or more and <NUM> or less for a time of <NUM> minutes or more and <NUM> minutes or less.

A calcium ion-containing aqueous solution may be used as a reagent for initiating blood coagulation. The calcium ion-containing aqueous solution is not particularly limited as long as it can provide calcium ions necessary for blood coagulation in the measurement sample. As the calcium ion-containing aqueous solution, an aqueous solution of a calcium salt is preferable, and examples thereof include an aqueous calcium chloride solution and the like. The calcium ion content in the measurement sample may be an amount sufficient to cause coagulation, and for example, it is usually <NUM> or more and <NUM> or less, and preferably <NUM> or more and <NUM> or less, in terms of calcium chloride concentration. Incidentally, the calcium ion-containing aqueous solution is also referred to as "calcium solution" hereinafter.

A blood specimen, a FXII-activating agent and phospholipids may be mixed, and then a calcium solution is added to obtain a measurement sample. Then, using the time when the calcium solution is added as the measurement starting point, optical information on the light amount described later may be acquired from the measurement sample.

The preparation of the measurement sample may be performed by a manual method or may be carried out by a fully automatic measurement device. Examples of the device include CS-<NUM> (Sysmex Corporation), CS-<NUM> (Sysmex Corporation), CS-2000i (Sysmex Corporation), and the like.

The measurement sample obtained as described above is irradiated with light to acquire optical information on the light amount from the measurement sample. The light to be irradiated to the measurement sample may be light which is usually used for measuring coagulation time, and examples thereof include light having a wavelength of around <NUM>, and preferably, light having a wavelength of <NUM>. A light source is not particularly limited, and examples thereof include a light emitting diode, a halogen lamp, and the like.

By irradiating the measurement sample with light from the above light source, scattered light and transmitted light are generated from the measurement sample. Examples of the optical information on the light amount include the amount of scattered light or the amount of transmitted light, and scattered light intensity, transmittance, absorbance and the like are preferable.

The measurement conditions are not particularly limited, but irradiation of light and acquisition of optical information on the light amount are continuously or intermittently from the start of measurement (at the time of adding a calcium solution) to the completion of the coagulation reaction (formation of fibrin clot). In this way, based on the optical information on the light amount (e.g., scattered light intensity, transmittance or absorbance) continuously or intermittently measured over the whole process of coagulation, it is possible to acquire the parameter on differentiation of coagulation waveform described later at any time point or time in the coagulation process. Incidentally, the irradiation of light and the acquisition of optical information on the light amount may be performed by a fully automatic measurement device. Examples of the devices include CS-<NUM> (Sysmex Corporation), CS-<NUM> (Sysmex Corporation), and CS-2000i (Sysmex Corporation) of a fully automated blood coagulation measuring apparatus, and the like.

The coagulation waveform may be a waveform representing a temporal change in the optical information on the light amount (e.g., amount of scattered light, transmittance or absorbance). With reference to <FIG>, coagulation waveform and its waveform analysis will be described. In the coagulation waveform (upper graph) in <FIG>, point a is a measurement starting point, point b is a fibrin precipitation (start of coagulation) point, and a-b shows coagulation time. Point c is a middle point of coagulation, point d is an end point of coagulation, and point e is an end point of measurement. When the coagulation waveform is differentiated (primarily differentiated), the coagulation rate is calculated (see the middle graph in <FIG>). The point c of the coagulation waveform corresponds to the maximum value of the first derivative. When the coagulation rate is differentiated (secondarily differentiated), the coagulation acceleration is calculated (see the lower graph in <FIG>). In the method, acquisition of coagulation time and coagulation waveform may be arbitrary.

At least one parameter on differentiation of coagulation waveform may be acquired based on the acquired optical information. The parameter on differentiation of coagulation waveform is not particularly limited as long as it is a value indicating at least one of coagulation rate, coagulation acceleration, and coagulation deceleration obtained on the basis of the acquired optical information. The value indicating the coagulation rate corresponds to a value that can be obtained from the first derivative of the coagulation waveform, and the value indicating the coagulation acceleration and the value indicating the coagulation deceleration correspond to values that can be obtained from the second derivative of the coagulation waveform.

Examples of the parameter on differentiation of coagulation waveform include |Min <NUM>|, |Min <NUM>|, Max <NUM>, AUC, and Slope. |Min <NUM>| is the absolute value of the minimum value of the first derivative of the coagulation waveform and represents the maximum coagulation rate. |Min <NUM>| is the absolute value of the minimum value of the second derivative of the coagulation waveform and represents the maximum coagulation acceleration. Max <NUM> is the maximum value of the second derivative of the coagulation waveform and represents the maximum coagulation deceleration. AUC is the area of a region surrounded by the coagulation waveform or a waveform obtained by first derivative or second derivative of the coagulation waveform. Slope is the magnitude of the slope of a tangent at an arbitrary point of the coagulation waveform or a waveform obtained by first derivative or second derivative of the coagulation waveform. Among them, |Min <NUM>|, |Min <NUM>| and Max <NUM> are preferable. |Min <NUM>|, |Min <NUM>| and Max <NUM> are also denoted as |min <NUM>|, |min <NUM>| and max <NUM>, respectively. The parameter on differentiation of coagulation waveform may be a value obtained by combining two or more of these values. Examples thereof include the sum, difference, product, and ratio of at least two values selected from |Min <NUM>|, |Min <NUM>|, Max <NUM>, AUC and Slope, and the like.

When the coagulation time has been also acquired, the parameter on differentiation of coagulation waveform may be a value obtained by combining the value acquired from the first derivative or second derivative of the coagulation waveform with the coagulation time. Examples of the value include the sum, difference, product, and ratio of at least one value selected from |Min <NUM>|, |Min <NUM>| and Max <NUM>, AUC and Slope and the value of coagulation time, and the like.

The coagulability of a blood specimen containing a substance having a FVIII-substituting activity may be evaluated, based on the value of the acquired parameter. The degree of promotion of the blood coagulation reaction by the substance having a FVIII-substituting activity can be quantitatively evaluated based on the parameter. Accordingly, it is possible to evaluate the activity as a cofactor of the substance, that is, the efficacy of the substance.

It is preferable to acquire a value indicating the coagulability of a blood specimen, from the value of the acquired parameter. As the value indicating coagulability, an index reflecting the activity of blood coagulation is preferable, and the FVIII activity value is particularly preferable. The FVIII activity value may be expressed as a percentage when the activity value of a healthy subject is taken as <NUM>%, or it may be expressed as an international unit (IU/dL or U/dL) in a predetermined amount of plasma.

For example, acquisition of the FVIII activity value from the value of the acquired parameter can be performed as follows. First, a FVIII preparation is added to commercially available FVIII-deficient plasma at various concentrations to prepare a specimen whose FVIII activity value is known. The FVIII activity value of these specimens may be determined by a known method such as a synthetic substrate method or may be determined from the addition amount of the preparation. Then, with respect to these specimens, a parameter on differentiation of coagulation waveform is acquired as described above, using the FXII-activating agent, the phospholipids and the calcium solution described above. For each specimen, a calibration curve is prepared by plotting the value of the parameter with respect to the FVIII activity value. Based on the resulting calibration curve, the value of the parameter for the blood specimen containing a substance having a FVIII-substituting activity is converted into FVIII activity value. The calibration curve may be created for each measurement of a blood specimen, or a predetermined calibration curve may be used. Alternatively, by accumulating data, an equation for directly converting from the value of the parameter to the value indicating coagulability of a blood specimen may be derived.

The coagulability of a blood specimen containing a substance having a FVIII-substituting activity can be evaluated, based on the acquired value indicating coagulability. For example, when the FVIII activity value has been acquired from the value of the acquired parameter, the efficacy of the substance having a FVIII-substituting activity can be evaluated in the same manner as the efficacy of the FVIII preparation. The appropriate range when converting the efficacy of the substance having a FVIII-substituting activity into FVIII activity value may be defined as <NUM> U/dL or more and <NUM> U/dL or less or FVIII per <NUM>µg/mL of ACE910. This range was calculated based on the effect of hemostatic effect and pharmacokinetics of porcine FVIII and ACE910 in the cynomolgus hemophilia model (see Non Patent Literature <NUM>). Comparing at the maximum blood concentration at each dose when showing equivalent hemostatic activity, ACE910 was <NUM>µg/mL, porcine FVIII was <NUM> U/dL, and the expected haemostatic effect at <NUM>µg/mL ACE910 was equivalent to <NUM> U/dL of the FVIII preparation. Meanwhile, comparing at the minimum blood concentration at each dose when showing equivalent hemostatic activity, ACE910 was <NUM>µg/mL, porcine FVIII was <NUM> U/dL, and the expected haemostatic effect at <NUM>µg/mL ACE910 was equivalent to <NUM> U/dL of the FVIII preparation. Here, it is confirmed that the range of this converted value does not also deviate from the result of the number of bleeding in ACE910 clinical trial in hemophilia A patients.

The present inventors have newly found that tissue factor involved in the extrinsic coagulation pathway is further added, in addition to the FXII-activating agent and the phospholipids which are involved in the intrinsic coagulation pathway, whereby the coagulability of a blood specimen containing a substance having a FVIII-substituting activity can be evaluated. Hereinafter, a method for evaluating coagulability of a blood specimen according to the second embodiment that further uses tissue factor will be described (hereinafter, also referred to as "the method according to the second embodiment").

In the method according to the second embodiment, a measurement sample is prepared from a blood specimen containing a substance having a FVIII-substituting activity (preferably, a blood specimen obtained from a subject administered a substance having a FVIII-substituting activity), a FXII-activating agent, phospholipids, tissue factor, and a calcium ion-containing aqueous solution. The blood specimen, the substance having a FVIII-substituting activity, the FXII-activating agent, the phospholipids, and the calcium ion-containing aqueous solution may be the same as those described above.

In the method according to the second embodiment, by further using tissue factor, monitoring of coagulability becomes possible even when the blood specimen is a specimen obtained from a subject using a combination of a bypass preparation and a substance having a FVIII-substituting activity. The bypass preparation is a generic term for preparations that can promote blood coagulation even when FVIII and/or FIX is not present in the blood in sufficient amount (that is, can bypass a coagulation pathway involving FVIII and/or FIX). As the bypass preparation, for example, an activated coagulation factor VII (FVIIa) preparation, an activated prothombin complex concentrate (APCC) and the like are known.

The tissue factor may be a natural tissue factor derived from a rabbit brain, a human placenta or the like, or may be tissue factor prepared by a genetic recombination technique. The final concentration of the tissue factor in the measurement sample is <NUM> ng/mL or more and <NUM> ng/mL or less, and preferably <NUM> ng/mL or more and <NUM> ng/mL or less, as defined in the claims. The order of mixing the blood specimen, the FXII-activating agent, the phospholipids, and the tissue factor is not particularly limited. For example, either one of the FXII-activating agent, the phospholipids and the tissue factor may be mixed with the blood specimen, then the remaining two may be mixed simultaneously or sequentially. Or, the two selected from the FXII-activating agent, the phospholipids and the tissue factor may be mixed with the blood specimen, then the remaining one may be mixed. Alternatively, the FXII-activating agent, the phospholipids and the tissue factor may be mixed, then the blood specimen may be mixed.

In the method according to the second embodiment, the final concentration of the FXII-activating agent in the measurement sample is <NUM> or more and <NUM> or less, and preferably <NUM> or more and <NUM> or less, as defined in the claims. The final concentration of the phospholipids in the measurement sample is <NUM> or more and <NUM> or less, and preferably <NUM> or more and <NUM> or less, as defined in the claims.

In the method according to the second embodiment, the measurement sample obtained as described above is irradiated with light to acquire optical information on the light amount from the measurement sample. In the method according to the second embodiment, the coagulability of a blood specimen containing a substance having a FVIII-substituting activity is evaluated, based on the acquired optical information. In It is preferable to acquire at least one selected from the coagulation time and the parameter on differentiation of coagulation waveform from the acquired optical information, and evaluate the coagulability of a blood specimen, based on the acquired value. Here, the optical information and the type of parameter on differentiation of coagulation waveform and the procedure of acquisition are the same as those described above. The procedure for evaluating coagulability of a blood specimen is also the same as described above.

The present invention also includes the use of a reagent for evaluating coagulability of a blood specimen containing a substance having a FVIII-substituting to be used activity (hereinafter, also referred to simply as "reagent"). The reagentlaccording to the claims is suitable for use in the method described above, and contains a FXII-activating agent and phospholipids respectively at a predetermined concentration. The reagent accommodated in a first container <NUM> is shown in <FIG>, as an example of the appearance of the reagent. The types of the FXII-activating agent and the phospholipids are the same as those described above.

In the reagent to be used according to the claims, the concentration of the FXII-activating agent in the reagent is not particularly limited as long as the final concentration in the measurement sample can be adjusted to the range described above, and as defined in the claims. When preparing the measurement sample with a fully automatic coagulation time measuring apparatus, the concentration of the FXII-activating agent is, for example, <NUM> or more and <NUM> or less, in consideration of the amount of reagent that can be aspirated by the apparatus, and as defined in the claims.

The concentration of the phospholipids in the reagent is not particularly limited as long as the final concentration in the measurement sample can be adjusted to the range described in the method according to the first aspect. When preparing a measurement sample with a fully automatic coagulation time measuring apparatus, the concentration of the phospholipids is, for example, <NUM> or more and <NUM> or less, in consideration of the amount of reagent that can be aspirated by the apparatus, and as defined in the claims. When PE, PC and PS are contained as phospholipids, the sum of the concentrations of PE, PC and PS in the reagent may be within the above range.

When the concentration of the FXII-activating agent in the reagent is set at <NUM> or more and <NUM> or less, the concentration of the phospholipids in the reagent may be, for example, <NUM> or more and <NUM> or less.

Also, the present invention also includes a reagent kit for evaluating coagulability of a blood specimen containing a substance having a FVIII-substituting activity, as defined in the claims (hereinafter, also referred to simply as "reagent kit"). The reagent kit is suitable for use in the method described above and comprises a first reagent containing a FXII-activating agent and phospholipids respectively at a predetermined concentration and a coagulation initiation reagent comprising a calcium ion-containing aqueous solution. The reagent kit including a first reagent accommodated in a first container <NUM> and a coagulation initiation reagent accommodated in a second container <NUM> is shown in <FIG>, as an example of the appearance of the reagent kit. The types of the FXII-activating agent and the phospholipids are the same as those described above. In addition, each concentration of the FXII-activating agent and the phospholipids in the reagent is as described above.

The coagulation initiation reagent may be is the same as the calcium ion-containing aqueous solution used in the method according to the claims. The calcium ion content in the coagulation initiation reagent may be any amount as long as it can be adjusted to a final concentration capable of causing coagulation, and for example, it is usually <NUM> or more and <NUM> or less, and preferably <NUM> or more and <NUM> or less, in terms of calcium chloride concentration.

The reagent for evaluating coagulability of a blood specimen containing a substance having a FVIII-substituting activity may further contain tissue factor, in addition to the FXII-activating agent and the phospholipid. Hereinafter, the use of the reagent according to the fifth embodiment further using tissue factor will be described.

The use of the reagent according to the fifth embodiment is suitable for use in the method according to the second embodiment described above, and contains a FXII-activating agent, phospholipids, and tissue factor. The appearance of the reagent may be similar to that of the reagent to be used as described above, and is shown, for example, in <FIG>. Specifically, a reagent containing a FXII-activating agent, phospholipids, and tissue factor is accommodated in the first container <NUM>. The types of the FXII-activating agent, the phospholipids and the tissue factor are the same as those described above. In addition, the respective concentration of each of the FXII-activating agent and the phospholipids in the use of the reagent according to the fifth embodiment is not particularly limited, and for example, may be the same as that described above. In the use of the reagent according to the fifth embodiment, the concentration of the tissue factor in the reagent is <NUM> ng/mL or more and <NUM> ng/mL or less, as defined in the claims.

The reagent kit for evaluating coagulability of a blood specimen containing a substance having a FVIII-substituting activity may further contain tissue factor, in addition to the FXII-activating agent and the phospholipids. Hereinafter, the reagent kits according to the sixth and seventh embodiments further using tissue factor will be described.

The reagent kits according to the sixth and seventh embodiments are suitable for use in the method described above. The reagent kit according to the sixth embodiment comprises a first reagent containing a FXII-activating agent, phospholipids and tissue factor, and a coagulation initiation reagent comprising a calcium ion-containing aqueous solution. The appearance of the reagent kit according to the sixth embodiment may be similar to that of the reagent kit described above, and is shown, for example, in <FIG>. Specifically, a first reagent containing a FXII-activating agent, phospholipids and tissue factor is accommodated in a first container <NUM>, and a coagulation initiation reagent comprising a calcium ion-containing aqueous solution is accommodated in a second container <NUM>.

The reagent kit according to the seventh embodiment comprises a first reagent, a second reagent, and a coagulation initiation reagent comprising a calcium ion-containing aqueous solution. The reagent kit including a first reagent accommodated in a first container <NUM>, a second reagent accommodated in a second container <NUM>, and a coagulation initiation reagent accommodated in a third container <NUM> are shown in <FIG>, as an example of the appearance of the reagent kit according to the seventh embodiment. Each of the FXII-activating agent, the phospholipid, and the tissue factor may be contained in either the first reagent or the second reagent. For example, it is possible that the first reagent contains the FXII-activating agent and the phospholipids and the second reagent contains the tissue factor, the first reagent contains the FXII-activating agent and the phospholipids and the second reagent contains the tissue factor and the phospholipids, or the first reagent contains the FXII-activating agent and the tissue factor and the second reagent contains the phospholipids.

In the reagent kits according to the sixth and seventh embodiments, the types and concentrations of the FXII-activating agent, phospholipids and tissue factor in the reagent are not particularly limited, and are, for example, the same as the concentrations as those described for the reagents and uses as described above and as defined in the claims. In the reagent kit according to the seventh embodiment, when phospholipids are contained in both the first reagent and the second reagent, the concentration of the phospholipids in each reagent may be half the concentration as described for the use of the reagent described above. In addition, the coagulation initiation reagent comprising the calcium ion-containing aqueous solution may be the same as that described for the reagent kit as described above.

The reagent kit according to the twelfth embodiment is suitable for use in the method described above. The reagent kit according to the twelfth embodiment comprises a first reagent and a second reagent. The first reagent may contain a FXII-activating agent and phospholipids, and the second reagent may contain tissue factor and calcium ions. The second reagent may further contain phospholipids. The appearance of the reagent kit according to the twelfth embodiment may be similar to that of the reagent kit described above, and is shown, for example, in <FIG>. Specifically, a first reagent containing a FXII-activating agent and phospholipids is accommodated in a first container <NUM>, and a second reagent containing tissue factor and calcium ions is accommodated in a second container <NUM>.

In the reagent kit according to the twelfth embodiment, the types and concentrations of the FXII-activating agent, phospholipids and tissue factor in each reagent are not particularly limited, and for example, are the same as the concentrations as those described for the reagents and uses as described above. In the reagent kit according to the twelfth embodiment, when phospholipids are contained in both the first reagent and the second reagent, the concentration of the phospholipids in each reagent may be half the concentration as described for the reagent to be used according to the claims. In addition, the content of calcium ions in the second reagent is the same as that described for the coagulation initiation reagent of the reagent kit as described above. The second reagent may contain calcium ions that are a coagulation initiation reagent. Therefore, first, a first reagent and a blood specimen containing a substance having a coagulation factor VIII-substituting activity are mixed, and then a second reagent is added to the obtained mixture.

An example of a blood specimen analyzer suitable for carrying out the present invention will be described below, with reference to the drawings. The present invention itself is defined in the claims. As shown in <FIG>, a blood specimen analyzer <NUM> includes a measurement device <NUM> for preparing and optically measuring a measurement sample, a control device <NUM> for analyzing measurement data acquired by the measurement device <NUM> and giving an instruction to the measurement device <NUM>. The measurement device <NUM> includes a measurement unit <NUM> for acquiring optical information on the light amount from the measurement sample, and a specimen transporting unit <NUM> arranged in front of the measurement unit <NUM>.

The measurement unit <NUM> is provided with lids 20a and 20b, a cover 20c, and a power button 20d. A user can open the lid 20a and replace a reagent container <NUM> placed in reagent tables <NUM> and <NUM> (see <FIG>) with a new reagent container <NUM>, or a user can newly add another reagent container <NUM>. To the reagent container <NUM> is attached a barcode label 103a printed with a barcode including the type of the reagent to be accommodated and a reagent ID made up of serial number provided to the reagent.

The user can open the lid 20b and replace a lamp unit <NUM> (see <FIG>). The user can also open the cover 20c and replace a piercer 17a (see <FIG>). The specimen transporting unit <NUM> transports a specimen container <NUM> supported by a specimen rack <NUM> to an aspiration position by the piercer 17a. The specimen container <NUM> is hermetically sealed by a rubber lid 101a.

When using the blood specimen analyzer <NUM>, the user first presses the power button 20d of the measurement unit <NUM> to activate the measurement unit <NUM>, and presses a power button <NUM> of the control device <NUM> to activate the control device <NUM>. When the control device <NUM> is activated, a log-on screen is displayed on a display unit <NUM>. The user inputs the user name and the password on the log-on screen to log on to the control device <NUM>, and starts using the blood specimen analyzer <NUM>.

The configuration of the measurement device will be described below. As shown in <FIG>, the measurement unit <NUM> includes reagent tables <NUM> and <NUM>, a cuvette table <NUM>, a barcode reader <NUM>, a cuvette supply section <NUM>, a catcher <NUM>, a specimen dispensing arm <NUM>, a reagent dispensing arm <NUM>, an urgent specimen setting section <NUM>, an optical fiber <NUM>, a detecting section <NUM>, a cuvette transfer section <NUM>, a warming section <NUM>, a disposal port <NUM>, a fluid section <NUM>, and a lamp unit <NUM>.

Each of the reagent tables <NUM> and <NUM> and the cuvette table <NUM> has an annular shape and is configured rotatably. Each of the reagent tables <NUM> and <NUM> corresponds to a reagent storing section, onto which a reagent container <NUM> is placed. The barcode of the reagent container <NUM> placed on the reagent tables <NUM> and <NUM> is read by the barcode reader <NUM>. Information (type of reagent, reagent ID) read from the barcode is input to the control device <NUM> and stored in a hard disk <NUM> (see <FIG>).

In an apparatus suitable for carrying out the present invention, the reagent container <NUM> in which an APTT measuring reagent containing a FXII-activating agent and phospholipids, a diluent for diluting the reagent, a calcium solution, and the like are each accommodated is placed on the reagent tables <NUM> and/or <NUM>. In the apparatus, the reagent container <NUM> in which a FXII-activating agent, phospholipids, tissue factor, a calcium solution, and the like are each accommodated may be placed on the reagent tables <NUM> and/or <NUM>. Alternatively, the reagent container <NUM> in which the reagent used according to the fifth embodiment or each reagent of the kit according to the sixth or seventh embodiment is each accommodated may be placed. In any of the apparatuses, the reagent container <NUM> in which each of the FVIII preparation and the FVIII-deficient plasma is accommodated may be placed on the reagent tables <NUM> and/or <NUM>.

The cuvette table <NUM> is formed with a support portion 13a composed of a plurality of holes capable of supporting a cuvette <NUM>. A new cuvette <NUM> introduced into the cuvette supply section <NUM> by the user is sequentially transferred by the cuvette supply section <NUM>, and the cuvette <NUM> is placed on the support portion 13a of the cuvette table <NUM> by the catcher <NUM>.

A stepping motor is connected to each of the specimen dispensing arm <NUM> and the reagent dispensing arm <NUM> so as to be able to move up and down and rotatably. A piercer 17a of which a tip is sharply formed is provided at the tip of the specimen dispensing arm <NUM>, so that the lid 101a of the specimen container <NUM> can be punctured. A pipette 18a is provided at the tip of the reagent dispensing arm <NUM>. The tip of the pipette 18a is formed flat unlike the piercer 17a. An electrostatic capacitance type liquid level detection sensor <NUM> (see <FIG>) is connected to the pipette 18a.

When the specimen container <NUM> is transported to a predetermined position by the specimen transporting unit <NUM> (see <FIG>), the piercer 17a is positioned just above the specimen container <NUM> by the rotational movement of the specimen dispensing arm <NUM>. Then, the specimen dispensing arm <NUM> is moved downward, the piercer 17a penetrates the lid 101a of the specimen container <NUM>, and the blood specimen accommodated in the specimen container <NUM> is aspirated by the piercer 17a. When an urgent blood specimen is set in the urgent specimen setting section <NUM>, the piercer 17a intervenes in the specimen supplied from the specimen transporting unit <NUM> and aspirates the urgent blood specimen. The blood specimen aspirated by the piercer 17a is discharged into an empty cuvette <NUM> on the cuvette table <NUM>.

The cuvette <NUM> into which the blood specimen has been discharged is transferred from the support portion 13a of the cuvette table <NUM> to a support portion 24a of the warming section <NUM> by a catcher 23a of the cuvette transfer section <NUM>. The warming section <NUM> warms the blood specimen accommodated in the cuvette <NUM> placed in the support portion 24a at a predetermined temperature (for example, <NUM>) for a certain period of time. When the warming of the blood specimen by the warming section <NUM> is finished, the cuvette <NUM> is again gripped by the catcher 23a. Then, the cuvette <NUM> is positioned at a predetermined position while being gripped by the catcher 23a, and in this state, the reagent aspirated by the pipette 18a is discharged into the cuvette <NUM>.

In the dispensing of the reagent by the pipette 18a, first, the reagent tables <NUM> and <NUM> are rotated, and the reagent container <NUM> that accommodates the reagent corresponding to the measurement item is transported to an aspiration position by the pipette 18a. Then, after the position of the pipette 18a in the vertical direction is positioned at the origin position, the pipette 18a is lowered until the lower end of the pipette 18a comes into contact with the liquid level of the reagent by the liquid level detection sensor <NUM>. When the lower end of the pipette 18a comes into contact with the liquid level of the reagent, the pipette 18a is further lowered to an extent that a necessary amount of the reagent can be aspirated. Then, the lowering of the pipette 18a is stopped, and the reagent is aspirated by the pipette 18a. The reagent aspirated by the pipette 18a is discharged into the cuvette <NUM> gripped by the catcher 23a. Then, the blood specimen and the reagent in the cuvette <NUM> are agitated by the vibrating function of the catcher 23a. Thus, the measurement sample is prepared. Thereafter, the cuvette <NUM> that accommodates the measurement sample is transferred to a support portion 22a of the detecting section <NUM> by the catcher 23a.

The lamp unit <NUM> supplies light having plural kinds of wavelengths used for detection of an optical signal by the detecting section <NUM>. An example of the configuration of the lamp unit <NUM> will be described with reference to <FIG>. The lamp unit <NUM> corresponds to a light source, and includes a halogen lamp 27a, a lamp case 27b, condenser lenses 27c to 27e, a disk-shaped filter section 27f, a motor <NUM>, a light transmission type sensor <NUM>, and an optical fiber coupler 27i.

With reference to <FIG>, light from the lamp unit <NUM> is supplied to the detecting section <NUM> via the optical fiber <NUM>. A plurality of hole-shaped support portions 22a is provided in the detecting section <NUM>, and a cuvette <NUM> can be inserted into each of the support portions 22a. The end part of the optical fiber <NUM> is attached to each of the support portions 22a, and the cuvette <NUM> supported by the support portion 22a can be irradiated with light from the optical fiber <NUM>. The detecting section <NUM> irradiates the cuvette <NUM> with light supplied from the lamp unit <NUM> via the optical fiber <NUM> and detects the light amount of light to be transmitted through the cuvette <NUM> (or scattered light from the cuvette <NUM>).

<FIG> show an example of one configuration of the plurality of support portions 22a arranged in the detecting section <NUM>, and the other support portions 22a have the same configuration. With reference to <FIG>, the detecting section <NUM> is formed with a circular hole 22b into which the tip of the optical fiber <NUM> is inserted. The detecting section <NUM> is further formed with a circular communication hole 22c for communicating the hole 22b with the support portion 22a. The diameter of the hole 22b is larger than the diameter of the communication hole 22c. A lens 22d for condensing light from the optical fiber <NUM> is arranged at the end of the hole 22b. Further, on the inner wall surface of the support portion 22a, a hole 22f is formed at a position facing the communication hole 22c. A photodetector <NUM> is arranged at the back of the hole 22f. The photodetector <NUM> corresponds to a light receiving portion, and outputs an electric signal corresponding to the amount of received light. The light transmitted through the lens 22d is condensed on the light receiving surface of the photodetector <NUM>, through the communication hole 22c, the support portion 22a, and the hole 22f. The optical fiber <NUM> is prevented from falling off by a plate spring 22e in a state in which the end part of the optical fiber <NUM> is inserted into the hole 22b.

With reference to <FIG>, when the cuvette <NUM> is supported by the support portion 22a, the light condensed by the lens 22d is transmitted through the cuvette <NUM> and the sample accommodated in the cuvette <NUM>, and the transmitted light enters the photodetector <NUM>. As the blood coagulation reaction progresses in the sample, the turbidity of the sample increases. Along with this, the amount of light to be transmitted through the sample (the amount of transmitted light) decreases, and the level of the detection signal of the photodetector <NUM> decreases.

With reference to <FIG>, the configuration of the detecting section <NUM> when scattered light is used will be described. On the inner side surface of the support portion 22a, a hole <NUM> is provided at a position which is the same height as the communication hole 22c. A photodetector 22i is arranged at the back of the hole <NUM>. When the cuvette <NUM> is inserted into the support portion 22a and light is emitted from the optical fiber <NUM>, the light scattered by the measurement sample in the cuvette <NUM> is irradiated to the photodetector 22i via the hole <NUM>. In this example, the detection signal from the photodetector 22i indicates the intensity of scattered light by the measurement sample. Also, as shown in <FIG>, both the light to be transmitted through the measurement sample and the light to be scattered by the measurement sample may be detected.

As described above, the detecting section <NUM> irradiates the cuvette <NUM> with light supplied from the lamp unit <NUM>, and acquires optical information from the measurement sample. The acquired optical information is transmitted to the control device <NUM>. The control device <NUM> performs analysis based on the optical information and displays the analysis result on a display unit <NUM>.

After completion of the measurement, the cuvette <NUM> that has become unnecessary is transported by the cuvette table <NUM>, and discarded to the disposal port <NUM> by the catcher <NUM>. During the measurement operation, the piercer 17a and the pipette 18a are appropriately washed with a liquid such as a cleaning liquid supplied from the fluid section <NUM>.

The hardware configuration of the measurement device will be described below. As shown in <FIG>, the measurement unit <NUM> includes a control section <NUM>, a stepping motor section <NUM>, a rotary encoder section <NUM>, a liquid level detection sensor <NUM>, a sensor section <NUM>, a mechanism section <NUM>, an optical information acquiring section <NUM>, and a barcode reader <NUM>.

With reference to <FIG>, the control section <NUM> includes a CPU <NUM>, a memory <NUM>, a communication interface <NUM>, and an I/O interface <NUM>. The CPU <NUM> executes a computer program stored in the memory <NUM>. The memory <NUM> is composed of a ROM, a RAM, a hard disk, and the like. The CPU <NUM> drives the specimen transporting unit <NUM> via the communication interface <NUM> and also transmits and receives instruction signals and data with the control device <NUM>. The CPU <NUM> controls each section in the measurement unit <NUM> via the I/O interface <NUM>, and also receives signals outputted from each section.

The stepping motor section <NUM> includes stepping motors for driving the reagent tables <NUM> and <NUM>, the cuvette table <NUM>, the catcher <NUM>, the specimen dispensing arm <NUM>, the reagent dispensing arm <NUM>, and the cuvette transfer section <NUM>, respectively. The rotary encoder section <NUM> includes a rotary encoder that outputs a pulse signal corresponding to the amount of rotational displacement of each stepping motor included in the stepping motor unit <NUM>.

The liquid level detection sensor <NUM> is connected to the pipette 18a provided at the tip of the reagent dispensing arm <NUM>, and detects that the lower end of the pipette 18a has come into contact with the liquid level of the reagent. The sensor section <NUM> includes a sensor for detecting that the vertical position of the pipette 18a is positioned at the origin position and a sensor for detecting that the power button 20d is pressed. The mechanism section <NUM> includes a mechanism for driving the cuvette supply section <NUM>, the urgent specimen setting section <NUM>, the warming section <NUM> and the fluid section <NUM>, and an air pressure source which supplies pressure to the piercer 17a and the pipette 18a so that dispensing operation by the piercer 17a and the pipette 18a can be performed. With reference to <FIG>, the optical information acquiring section <NUM> includes at least the lamp unit <NUM>, the optical fiber <NUM>, and the detecting section <NUM>.

The configuration of the control device <NUM> will be described below. As shown in <FIG>, the control device <NUM> includes the display unit <NUM>, an input unit <NUM>, and a computer body <NUM>. The control device <NUM> receives optical information from the measurement unit <NUM>. Then, a processor of the control device <NUM> calculates a parameter on differentiation of coagulation waveform based on the optical information. The processor of the control device <NUM> may also calculate coagulation time based on the optical information. Then, the processor of the control device <NUM> executes a computer program for evaluating coagulability of a blood specimen. Accordingly, the control device <NUM> also functions as an apparatus for evaluating coagulability of a blood specimen.

As to the functional configuration of the control device <NUM>, as shown in <FIG>, the control device <NUM> includes an acquisition unit <NUM>, a storage unit <NUM>, a calculation unit <NUM>, and an output unit <NUM>. The acquisition unit <NUM> is communicably connected to the measurement unit <NUM> via a network. The output unit <NUM> is communicably connected to the display unit <NUM>.

The acquisition unit <NUM> acquires the optical information transmitted from the measurement unit <NUM>. The storage unit <NUM> stores an expression for calculating values of various parameters on differentiation of coagulation waveform and the like. The storage unit <NUM> may also store an expression for calculating coagulation time, an expression for converting a parameter on differentiation of coagulation waveform into a FVIII activity value or the like, and a threshold value for the value of the parameter or a converted value thereof. Using the information acquired by the acquisition unit <NUM>, the calculation unit <NUM> calculates the values of the various parameters, according to the expression stored in the storage unit <NUM>. Further, the calculation unit <NUM> may convert the value of the calculated parameter into the FVIII activity value. The output unit <NUM> outputs the values of the parameters calculated by the calculation unit <NUM> or a converted value thereof, as reference information regarding the blood specimen.

As shown in <FIG>, the computer body <NUM> of the control device <NUM> includes a CPU <NUM>, a ROM <NUM>, a RAM <NUM>, a hard disk <NUM>, a reading device <NUM>, an input/output interface <NUM>, a communication interface <NUM>, an image output interface <NUM>, and a power button <NUM>. The CPU <NUM>, the ROM <NUM>, the RAM <NUM>, the hard disk <NUM>, the reading device <NUM>, the input/output interface <NUM>, the communication interface <NUM>, the image output interface <NUM>, and the power button <NUM> are communicably connected by a bus <NUM>.

The CPU <NUM> executes a computer program stored in the ROM <NUM> and a computer program loaded in the RAM <NUM>. Each of the above-described functional blocks is realized by the CPU <NUM> executing an application program. Thus, the computer system functions as a terminal serving as a determination device for evaluating coagulability of a blood specimen.

The ROM <NUM> includes a mask ROM, PROM, EPROM, EEPROM, and the like. In the ROM <NUM>, a computer program executed by the CPU <NUM> and data used for the computer program are recorded.

The RAM <NUM> includes SRAM, DRAM, and the like. The RAM <NUM> is used for reading the computer program recorded in the ROM <NUM> and the hard disk <NUM>. The RAM <NUM> is also used as a work area of the CPU <NUM> when executing these computer programs.

The hard disk <NUM> has installed therein an operating system, a computer program such as an application program (a computer program for evaluating coagulability of a blood specimen) to be executed by the CPU <NUM>, data used for executing the computer program, and setting contents of the control device <NUM>.

The reading device <NUM> includes a flexible disk drive, a CD-ROM drive, a DVD-ROM drive, and the like. The reading device <NUM> can read a computer program or data recorded on a portable recording medium <NUM> such as a CD or a DVD.

The input/output interface <NUM> includes, for example, a serial interface such as USB, IEEE1394 or RS-232C, a parallel interface such as SCSI, IDE or IEEE1284, and an analog interface including a D/A converter, an A/D converter and the like. The input unit <NUM> such as a keyboard and a mouse is connected to the input/output interface <NUM>. The user inputs an instruction via the input unit <NUM>, and the input/output interface <NUM> receives a signal input via the input unit <NUM>.

The communication interface <NUM> is, for example, an Ethernet (registered trademark) interface or the like. The control device <NUM> can transmit print data to a printer through the communication interface <NUM>. The communication interface <NUM> is connected to the measurement unit <NUM>, and the CPU <NUM> transmits and receives an instruction signal and data with the measurement unit <NUM> via the communication interface <NUM>.

The image output interface <NUM> is connected to the display unit <NUM> including an LCD, a CRT, and the like. The image output interface <NUM> outputs a video signal corresponding to image data to the display unit <NUM>, and the display unit <NUM> displays an image based on the video signal outputted from the image output interface <NUM>.

With reference to <FIG>, during the measurement operation, the CPU <NUM> of the measurement unit <NUM> temporarily stores in the memory <NUM> the data (optical information) obtained by digitizing the detection signal outputted from the detecting section <NUM> (see <FIG>). The storage area of the memory <NUM> is divided into areas for each support portion 22a. In each area, the data (optical information) which are acquired when the cuvette <NUM> supported by the corresponding support portion 22a is irradiated with light having a predetermined wavelength are sequentially stored. Thus, the data are sequentially stored in the memory <NUM> over a predetermined measurement time. When the measurement time elapses, the CPU <NUM> stops storing the data in the memory <NUM>, and transmits the stored data to the control device <NUM> via the communication interface <NUM>. The control device <NUM> processes and analyzes the received data, and displays the analysis result on the display unit <NUM>.

The processing in the measurement unit <NUM> is mainly performed under the control of the CPU <NUM> of the measurement unit <NUM>, and the processing in the control device <NUM> is mainly performed under the control of the CPU <NUM> of the control device <NUM>. With reference to <FIG>, when the measurement processing is started, the measurement unit <NUM> aspirates a predetermined amount of a test plasma from the specimen container <NUM> transported by the specimen transporting unit, and dispenses the aspirated blood specimen into an empty cuvette <NUM> on the cuvette table <NUM>. When also measuring FVIII-deficient plasma as a control, the measurement unit <NUM> aspirates a predetermined amount of FVIII-deficient plasma from the reagent container <NUM> containing FVIII-deficient plasma accommodated in the reagent accommodating section, and dispenses it into an empty cuvette <NUM>. When also measuring FVIII-deficient plasma to which FVIII preparation is added, the measurement unit <NUM> aspirates a predetermined amount of FVIII-deficient plasma from the reagent container <NUM> in which the FVIII-deficient plasma is accommodated, and dispenses it into an empty cuvette <NUM>. Then, the measurement unit <NUM> aspirates a predetermined amount of the FVIII preparation from the reagent container <NUM> in which the FVIII preparation is accommodated, dispenses it into the cuvette <NUM> containing the FVIII-deficient plasma, and stirs it.

Subsequently, the measurement unit <NUM> transfers the cuvette <NUM> into which the plasma is dispensed to the warming section <NUM>, and warms the plasma in the cuvette <NUM> to a predetermined temperature (for example, <NUM>). Thereafter, the measurement unit <NUM> adds a reagent and a calcium solution to the cuvette <NUM> to prepare a measurement sample (step S11). Here, in the apparatus, when a usual APTT measuring reagent containing a FXII-activating agent and phospholipids is placed, the measurement unit <NUM> can dilute the APTT measuring reagent with a predetermined ratio with a diluent to prepare a reagent for blood coagulation analysis. Specifically, a reagent for blood coagulation analysis can be prepared as follows. The measurement unit <NUM> aspirates a predetermined amount of the usual APTT measuring reagent from the reagent container <NUM> in which the reagent is accommodated and dispenses it into an empty reagent container <NUM>. Then, the measurement unit <NUM> aspirates a predetermined amount of the diluent from the reagent container <NUM> in which the diluent is accommodated, adds it to the reagent container <NUM> into which the usual APTT measuring reagent is dispensed, and stirs it. The dilution ratio can be determined as appropriate depending on the APTT measuring reagent to be used, and is, for example, <NUM> times or more and <NUM> times or less, and preferably <NUM> times or more and <NUM> times or less, in the case of a general commercially available APTT measuring reagent. In the apparatus a reagent obtained by may be diluting a general commercially available APTT measuring reagent may be used as a reagent for blood coagulation analysis.

Preferably, the measurement unit <NUM> prepares a measurement sample using the diluted APTT measuring reagent, so that the final concentration of the FXII-activating agent in the measurement sample is <NUM> or more and <NUM> or less and the final concentration of the phospholipids is <NUM> or more and <NUM> or less, or the final concentration of the FXII-activating agent in the measurement sample is <NUM> or more and <NUM> or less and the final concentration of phospholipids is <NUM> or more and <NUM> or less.

Thereafter, the measurement unit <NUM> transfers the cuvette <NUM> to which the reagent is added to the detecting section <NUM>, and irradiates the cuvette <NUM> with light to measure the measurement sample (step S12). The measurement unit <NUM> starts measuring time from the time when the calcium solution is added to the cuvette <NUM>. In this measurement, data (the amount of scattered light or the amount of transmitted light) based on the light with a wavelength of <NUM> is sequentially stored in the memory <NUM> during the measurement time. At this time, the data is stored in the memory <NUM> in a state associated with the elapsed time from the addition time point of the calcium solution. Then, when the measurement time elapses, the measurement unit <NUM> stops the measurement, and transmits the measurement result (data) stored in the memory <NUM> to the control device <NUM> (step S13). Accordingly, when the control device <NUM> receives the measurement result (data) from the measurement unit <NUM> (step S21: YES), the control device <NUM> executes analysis processing on the received measurement result (step S22). That is, the control device <NUM> converts from the parameters on differentiation of coagulation waveform (|Min <NUM>|, |Min <NUM>|, Max <NUM>, AUC and Slope) into a value indicating coagulability of a blood specimen (for example, FVIII activity value), for a measurement sample. The control device <NUM> may also calculate the coagulation time and coagulation waveform of the measurement sample. After performing the analysis processing, the control device <NUM> executes display processing of the analysis result (step S23).

With reference to <FIG>, the processing flow when using one parameter on differentiation of coagulation waveform will be described. Here, a case where the value of |min <NUM>| as the value of the parameter on differentiation of coagulation waveform is acquired from the optical information on the light amount from the measurement sample, the acquired value is converted to acquire a FVIII value, and this FVIII value is outputted as reference information on coagulability of a blood specimen will be described as an example. In this example, values of |min <NUM>|, max <NUM>, AUC or Slope may be acquired, in place of |min <NUM>|. Alternatively, a plurality of parameters may be acquired, and the FVIII value may be acquired from the value of each parameter. In this case, as the reference information, the average value, the minimum value, the maximum value and the like of the FVIII value converted from the values of the plurality of parameters may be outputted.

First, in step S101, the acquisition unit <NUM> of the control device <NUM> acquires optical information (scattered light intensity, or transmittance or absorbance), based on the data (the amount of scattered light or the amount of transmitted light) received from the measurement unit <NUM>. Next, in step S102, the calculation unit <NUM> calculates a value of |min <NUM>| from the optical information acquired by the acquisition unit <NUM>, according to the equation for calculating a parameter on differentiation of coagulation waveform stored in the storage unit <NUM>. Although the coagulation time and the coagulation waveform are not used for the determination processing described later, the calculation unit <NUM> may further calculate the coagulation time and the coagulation waveform from the optical information acquired by the acquisition unit <NUM>.

In step S103, the calculation unit <NUM> calculates the FVIII activity value from the calculated value of |min <NUM>|, according to the conversion formula stored in the storage unit <NUM>. In step S103, the calculation unit <NUM> transmits the acquired FVIII activity value to the output unit <NUM>. In step S104, the output unit <NUM> outputs the FVIII activity value, and displays it on the display unit <NUM>, or makes a printer to print it. Alternatively, the FVIII activity value may be outputted by voice. As a result, the FVIII activity value can be provided to the user as reference information on coagulability of the test plasma.

As an example of a screen displaying the analysis result, a screen for displaying the result of analyzing the coagulation process of the test plasma using a reagent containing a FXII-activating agent and phospholipids will be described with reference to <FIG>. A screen D1 includes an area D11 for displaying a specimen number, an area D12 for displaying a measurement item name, a button D13 for displaying a detailed screen, an area D14 for displaying measurement data and time, an area D15 for displaying measurement results, an area D16 for displaying analysis information, and an area D17 for displaying a coagulation waveform and a graph obtained by differentiating it.

In the area D15, measurement items and measurement values are displayed. In the area D15, the "APTT sec" is the activated partial thromboplastin time. |Min <NUM>|, |Min <NUM>|, Max <NUM> and the like may be displayed as values of parameters on differentiation of coagulation waveform in the region D15.

In the area D16, analysis items and reference information are displayed. In the area D16, the "Index" is the value of the parameter on differentiation of the coagulation waveform used for the calculation of FVIII activity value. The "FVIII converted value (reference)" is the value of the FVIII activity value converted from the value of Index. The evaluation of the efficacy of a substance having a FVIII-substituting activity is desirably performed not only based on this result, but also information such as other examination results and physical findings of the subject. Accordingly, it is displayed as "(reference)" to indicate that the FVIII converted value by the blood specimen analyzer is reference information.

Whether or not the coagulability of a blood specimen containing a substance having a FVIII-substituting activity could be appropriately evaluated by diluting a commercially available APTT measuring reagent and using it as a reagent for blood coagulation analysis whose concentrations of a FXII-activating agent and phospholipids were adjusted was investigated.

As the commercially available APTT measuring reagent, Thrombocheck APTT-SLA (Sysmex Corporation) was used. This reagent is composed of ellagic acid (<NUM>) as a FXII-activating agent and synthetic phospholipids (<NUM>). The APTT measuring reagent and Owren's Veronal buffer (Sysmex Corporation) were mixed in ratios of reagent : buffer of <NUM> : <NUM>, <NUM> : <NUM>, <NUM> : <NUM>, <NUM> : <NUM> and <NUM> : <NUM> expressed by volume ratios to prepare reagents for blood coagulation analysis (corresponding to reagents of <NUM>-fold, <NUM>-fold, <NUM>-fold, <NUM>-fold and <NUM>-fold dilutions of Thrombocheck APTT-SLA, respectively). As a coagulation initiation reagent containing calcium ions, a <NUM> calcium chloride solution (Sysmex Corporation) was used. Also, Thrombocheck APTT-SLA was used without dilution as a control.

ACE910 (Q499-z121/J327-z119/L404-k) which is an anti-FIXa/FX bispecific antibody described in Patent Literature (<CIT>) was used as a substance having a FVIII-substituting activity. This antibody was acquired by the methods described in <CIT>, <CIT> and <CIT>. Specifically, the antibody was acquired as follows. First, the antibody gene described in <CIT> was incorporated into a vector for animal cell expression, and the resulting construct was transfected into HEK293 cells to express an anti-FIXa/FX bispecific antibody. Then, the bispecific antibody contained in the culture supernatant of the cells was purified by Protein A and gel filtration. It was confirmed that the resulting bispecific antibody had FXa production promoting activity, by the method described in <CIT>, <CIT>, or the like.

The bispecific antibody ACE910 was added to FVIII-deficient human plasma (George King Bio-Medical) to a concentration of <NUM>, <NUM>, <NUM>, <NUM> or <NUM>µg/mL to prepare blood specimens containing a substance having a FVIII-substituting activity. Also, as a specimen for preparing a calibration curve, blood specimens obtained by adding recombinant human FVIII (rhFVIII, Bayer Co. ) to FVIII-deficient human plasma (George King Bio-Medical) so as to have a concentration of <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> or <NUM> IU/dL were prepared.

For preparing and measuring a measurement sample, a fully automated blood coagulation measurement device CS-<NUM> (Sysmex Corporation) was used. A reagent for blood coagulation analysis (<NUM>µL) was added to a blood specimen (<NUM>µL), and the mixture was incubated at <NUM> for <NUM> minutes. Then, a measurement sample was prepared by adding a <NUM> calcium chloride solution (<NUM>µL). The transmittance of the measurement sample was continuously measured for <NUM> seconds from the addition of the calcium chloride solution. The respective final concentrations of ellagic acid and phospholipids in the measurement sample are as shown in Table <NUM>.

The coagulation time and |Min <NUM>|, |Min <NUM>| and Max <NUM> were calculated as parameters on differentiation of coagulation waveform, based on the temporal change in the resulting transmittance, and plotted on a graph. The graphs of the coagulation time, |Min <NUM>|, |Min <NUM>| and Max <NUM> obtained from the blood specimens containing ACE910 and the blood specimens containing rhFVIII are shown in <FIG>, <FIG>, <FIG> and <FIG>, respectively. The coagulation time, |Min <NUM>|, |Min <NUM>| and Max <NUM> of the blood specimens containing ACE910 were converted into FVIII activity values, using the graphs of the blood specimens containing rhFVIII as calibration curves, to create graphs from the resulting converted values. The resulting graphs are shown in <FIG>, <FIG>, <FIG> and <FIG>, respectively.

The present inventors have previously found a relationship between ACE910 blood concentration and FVIII activity value from animal models of hemophilia. Specifically, based on the hemostatic effect of ACE910 and rhFVIII in cynomolgus monkeys exhibiting symptoms similar to acquired hemophilia A by administration of anti-FVIII-neutralizing antibody, ACE910 was defined to be converted into a FVIII activity value of <NUM> to <NUM> IU/dL per <NUM>µg/mL. In addition, the present inventors have confirmed that this converted value does not deviate from the result of clinical trial (the number of bleeding) in hemophilia A patients. Two broken lines in <FIG>, <FIG>, <FIG> and <FIG> show graphs obtained by converting the ACE910 concentration into a FVIII activity value of <NUM> or <NUM> IU/dL per <NUM>µg/mL. The region sandwiched between these two broken lines was defined as the ideal range, and whether or not the reagent for blood coagulation analysis described above in which FVIII converted from each parameter is within this ideal range enables appropriate evaluation of coagulability of the blood specimen containing ACE910 was investigated. In particular, the results of the range of <NUM> to <NUM>µg/mL considered clinically important as the blood concentration of ACE910 was investigated.

As shown in <FIG>, the FVIII activity values converted from the coagulation time were largely deviated from the ideal range even when any of the reagents was used. This indicates that the FVIII activity value converted from the coagulation time deviates from the activity value predicted from the animal models. Accordingly, it was shown that the coagulability of the blood specimen containing ACE910 cannot be appropriately evaluated also based on the coagulation time. As shown in <FIG>, <FIG> and <FIG>, in the case of using undiluted APTT reagent, the FVIII activity values each converted from |Min <NUM>|, |Min <NUM>| and Max <NUM> were not within the ideal range. Accordingly, it was shown that the coagulability of the blood specimen containing ACE910 cannot be appropriately evaluated with the commercially available APTT reagent. Meanwhile, when the reagents diluted <NUM> to <NUM>-folds were used, among the FVIII activity values converted from |Min <NUM>|, at least one FVIII activity value was included in the ideal range, in the range of an ACE910 concentration of <NUM> to <NUM>µg/mL. In addition, when the reagents diluted <NUM> to <NUM>-folds were used, among the FVIII activity values converted from |Min <NUM>|, at least one FVIII activity value was included in the ideal range, in the range of an ACE910 concentration of <NUM> to <NUM>µg/mL. In addition, when the reagents diluted <NUM> to <NUM>-folds were used, among the FVIII activity values converted from Max <NUM>, at least one FVIII activity value was included in the ideal range, in the range of an ACE910 concentration of <NUM> to <NUM>µg/mL. Accordingly, it was shown that the coagulability of a blood specimen containing a substance having a FVIII-substituting activity can be appropriately evaluated by adjusting the concentrations of the activators of the APTT measuring reagent and the phospholipids by dilution.

Also as to commercially available APTT measuring reagents which were different from the reagent used in Example <NUM>, whether the coagulability of a blood specimen containing a substance having a FVIII-substituting activity could be appropriately evaluated by diluting and using them was investigated in the same manner.

As the commercially available APTT measuring reagents, Thrombocheck APTT-SLA (Sysmex Corporation), Actin FSL (Sysmex Corporation), and Data Phi · APTT (FS) (Sysmex Corporation) were used. Actin FSL is composed of ellagic acid and phospholipids derived from soybean and rabbit brain. Data Phi • APTT (FS) is composed of ellagic acid and soybean-derived phospholipids. Each APTT measuring reagent and Owren's Veronal buffer (Sysmex Corporation) were mixed in a ratio of reagent : buffer of <NUM> : <NUM> expressed by a volume ratio to prepare a reagent for blood coagulation analysis (corresponding to a reagent of <NUM>-fold dilution of each reagent). As a coagulation initiation reagent containing calcium ions, a <NUM> calcium chloride solution (Sysmex Corporation) was used. Also, each reagent was used without dilution as a control.

The bispecific antibody ACE910 was added to FVIII-deficient human plasma (George King Bio-Medical) to a concentration of <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> or <NUM>µg/mL to prepare blood specimens containing a substance having a FVIII-substituting activity. Also, as a specimen for preparing a calibration curve, blood specimens obtained by adding recombinant human FVIII (rhFVIII, Bayer Co. ) to FVIII-deficient human plasma (George King Bio-Medical) so as to have a concentration of <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> or <NUM> IU/dL were prepared.

Preparation and measurement of the measurement sample were carried out in the same manner as in Example <NUM>.

The coagulation time and |Min <NUM>|, |Min <NUM>| and Max <NUM> were calculated as parameters on differentiation of coagulation waveform, based on the temporal change in the resulting transmittance, and plotted on a graph. The coagulation time, |Min <NUM>|, |Min <NUM>| and Max <NUM> of the blood specimens containing ACE910 were converted into FVIII activity values, using the graphs of the blood specimens containing rhFVIII as calibration curves, to create graphs from the resulting converted values. <FIG> show the converted values of each parameter obtained with the <NUM>-fold diluted reagent, respectively. In these figures, the graphs indicated by the broken lines are the ideal ranges defined from the animal models as in Example <NUM>.

The FVIII activity values converted from the coagulation time obtained using each undiluted reagent were largely deviated from the ideal range. Also, as shown in <FIG>, the FVIII activity values converted from the coagulation time obtained by using each of <NUM>-fold diluted reagents were also largely deviated from the ideal range. In addition, many of the FVIII activity values from |Min <NUM>|, |Min <NUM>| and Max <NUM> obtained using each undiluted reagent were deviated from the ideal range at an ACE910 concentration of <NUM> to <NUM>µg/mL. Accordingly, it was shown difficult to appropriately evaluate the coagulability of the blood specimen containing ACE910 with the commercially available APTT reagent. Meanwhile, as shown in <FIG>, many of the FVIII activity values from |Min <NUM>|, |Min <NUM>| and Max <NUM> obtained by using each of <NUM>-fold diluted reagents were included in the ideal range at an ACE910 concentration of <NUM> to <NUM>µg/mL. Accordingly, it was shown that the coagulability of a blood specimen containing a substance having a FVIII-substituting activity can be appropriately evaluated by diluting the commercial APTT reagent.

Whether or not the coagulability could be appropriately evaluated, even a blood specimen containing both a substance having a FVIII-substituting activity and FVIII, by the diluted APTT measuring reagent was investigated.

As the commercially available APTT measuring reagent, Thrombocheck APTT-SLA (Sysmex Corporation) was used. This APTT measuring reagent and Owren's Veronal buffer (Sysmex Corporation) were mixed in a ratio of reagent:buffer of <NUM> : <NUM> expressed by a volume ratio to prepare a reagent for blood coagulation analysis (corresponding to a reagent of <NUM>-fold dilution of Thrombocheck APTT-SLA). As a coagulation initiation reagent containing calcium ions, a <NUM> calcium chloride solution (Sysmex Corporation) was used.

The bispecific antibody ACE910 was added to FVIII-deficient human plasma (George King Bio-Medical) to a concentration of <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> or <NUM>µg/mL to prepare blood specimens containing a substance having a FVIII-substituting activity. Three additional sets of series of blood specimens containing ACE910 at various concentrations were prepared, and recombinant human FVIII (rhFVIII, Bayer Co. ) was added to each set so as to have a concentration of <NUM> or <NUM> IU/dL to prepare blood specimens containing both ACE910 and FVIII. Also, as a specimen for preparing a calibration curve, blood specimens obtained by adding recombinant human FVIII (rhFVIII, Bayer Co. ) to FVIII-deficient human plasma (George King Bio-Medical) so as to have a concentration of <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> or <NUM> IU/dL were prepared.

Preparation and measurement of the measurement sample were carried out in the same manner as in Example <NUM>. In the measurement sample, the final concentration of ellagic acid is <NUM>, and the final concentration of phospholipids is <NUM>.

|Min <NUM>|, |Min <NUM>| and Max <NUM> were calculated as parameters on differentiation of coagulation waveform, based on the temporal change in the resulting transmittance, and plotted on a graph. The resulting graphs are shown in <FIG>, <FIG> and <FIG>, respectively. |Min <NUM>|, |Min <NUM>| and Max <NUM> of each blood specimen were converted into FVIII activity values, using the graphs of the blood specimens containing rhFVIII as calibration curves, to create graphs from the resulting converted values. The resulting graphs are shown in <FIG>, <FIG> and <FIG>, respectively. As shown in these graphs, even in the presence of FVIII, a change in parameter value dependent on ACE910 concentration was observed. Accordingly, it was shown that the coagulability can be appropriately evaluated, even a blood specimen containing both a substance having a FVIII-substituting activity and FVIII, by adjusting the concentrations of the activators of the APTT measuring reagent and the phospholipids by dilution.

To <NUM>µL of factor VIII (FVIII) deficient human plasma (George King Bio-Medical, Lot No. <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>) added with bispecific antibody ACE910 or recombinant human FVIII (rhFVIII, Bayer Co. ) was added <NUM>µL of each of reagents <NUM> to <NUM> for blood coagulation analysis composed of ellagic acid and phospholipids. After incubation for <NUM> minutes, <NUM>µL of a <NUM> mol/L calcium chloride solution was added thereto to start a coagulation reaction, and measurement was performed with an automatic blood coagulation measuring device CS-2000i (Sysmex Corporation). The respective final concentrations of ellagic acid and phospholipids in the measurement sample were shown in Table <NUM> below. Incidentally, reagents <NUM> to <NUM> for blood coagulation analysis are denoted as Reagent <NUM> to Reagent <NUM> in <FIG>, respectively.

Each concentration of ellagic acid and phospholipids in Table <NUM> was described as the final concentration after mixing the plasma, the reagent for blood coagulation analysis, and the calcium chloride solution (final concentration in the measurement sample).

To <NUM>µL of FVIII-deficient human plasma (George King Bio-Medical, Lot No. <NUM>-<NUM>, <NUM>-<NUM>) added with ACE910 or recombinant human FVIII (rhFVIII, Bayer Co. ) was added <NUM>µL of each of reagents <NUM> to <NUM> for blood coagulation analysis composed of tissue factor, ellagic acid and phospholipids. After incubation for <NUM> minutes, <NUM>µL of a <NUM> mol/L calcium chloride solution was added thereto to start a coagulation reaction, and measurement was performed with an automatic blood coagulation measuring device CS-2000i (Sysmex Corporation). The respective final concentrations of tissue factor, ellagic acid and phospholipids in the measurement sample were shown in Table <NUM> below. Incidentally, reagents <NUM> to <NUM> for blood coagulation analysis are denoted as Reagent <NUM> to Reagent <NUM> in <FIG>, respectively.

Each concentration of tissue factor, ellagic acid and phospholipids in Table <NUM> was described as the final concentration after mixing the plasma, the reagent for blood coagulation analysis, and the calcium chloride solution (final concentration in the measurement sample).

To <NUM>µL of a sample obtained by adding ACE910 to FVIII-deficient human plasma (George King Bio-Medical, Lot No. <NUM>-<NUM>) added with <NUM>, <NUM> or <NUM> U/dL recombinant human FVIII (rhFVIII, Bayer Co. ) was added <NUM>µL of a reagent for blood coagulation analysis (reagent <NUM> for blood coagulation analysis in Table <NUM>) composed of ellagic acid and phospholipids. After incubation for <NUM> minutes, <NUM>µL of a <NUM> mol/L calcium chloride solution was added thereto to start a coagulation reaction, and measurement was performed with an automatic blood coagulation measuring device CS-2000i (Sysmex Corporation).

To <NUM>µL of a sample obtained by adding ACE910 to FVIII-deficient human plasma (George King Bio-Medical, Lot No. <NUM>-<NUM>) added with <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or <NUM> U/dL recombinant human FVIII (rhFVIII, Bayer Co. ) was added <NUM>µL of a reagent for blood coagulation analysis (reagent <NUM> or <NUM> for blood coagulation analysis in Table <NUM>) composed of tissue factor, ellagic acid and phospholipids. After incubation for <NUM> minutes, <NUM>µL of a <NUM> mol/L calcium chloride solution was added thereto to start a coagulation reaction, and measurement was performed with an automatic blood coagulation measuring device CS-2000i (Sysmex Corporation).

Each concentration of ellagic acid and phospholipids contained in reagent <NUM> for blood coagulation analysis is the same as that of ellagic acid and phospholipids contained in Thrombocheck which is a commercially available APTT measuring reagent. Reagent <NUM> for blood coagulation analysis is a reagent in which the phospholipid concentration is diluted to <NUM>/<NUM> without changing the ellagic acid concentration, and reagent <NUM> for blood coagulation analysis is a reagent in which the ellagic acid concentration is diluted to <NUM>/<NUM> without changing the phospholipid concentration. In any of reagents <NUM>, <NUM> and <NUM> for blood coagulation analysis, the coagulation rate in FVIII-deficient plasma each containing rhFVIII and ACE910, and coagulation acceleration were increased (see <FIG> and <FIG>, <FIG> and <FIG>, and <FIG> and <FIG>). Meanwhile, the case where a value obtained by converting each of |Min <NUM>| and |Min <NUM>| of the specimen containing <NUM>, <NUM> or <NUM>µg/mL ACE910 from the calibration curves of |Min <NUM>| and |Min <NUM>| of the specimen containing rhFVIII into FVIII activity fell in the expected ideal range (<NUM> to <NUM> U/dL FVIII per <NUM>µg/mL ACE910) for its haemostatic effect was only when reagent <NUM> for blood coagulation analysis was used (see <FIG> and <FIG>, <FIG> and <FIG>, and <FIG> and <FIG>. In the figures, a region sandwiched between two broken lines shows the ideal range). When using reagent <NUM> for blood coagulation analysis, two or more of the <NUM>, <NUM>, or <NUM>µg/mL FVIII activity values converted from |Min <NUM>| of the specimen containing ACE910 fell in the ideal range. From these results, it was shown that the commercially available APTT reagent (reagent <NUM> for blood coagulation analysis) overestimates the efficacy of ACE910, whereas reagent <NUM> for blood coagulation analysis can properly monitor the efficacy of ACE910.

Furthermore, as an example, when the effect of ACE910 in FVIII-deficient plasma containing a FVIII preparation was evaluated using reagent <NUM> for blood coagulation analysis, the coagulation rate and the coagulation acceleration increased by addition of ACE910 (see <FIG>). From these results, it was shown that the efficacy of ACE910 can be monitored even in the presence of FVIII, by using a reagent for blood coagulation analysis composed of ellagic acid and phospholipids at concentrations like reagent <NUM> for blood coagulation analysis.

Reagents <NUM> to <NUM> for blood coagulation analysis are reagents prepared so that the tissue factor, ellagic acid and phospholipids each have various concentrations. The coagulation time, coagulation rate and acceleration of ACE910 increased, even under conditions using any of reagents <NUM> to <NUM> for blood coagulation analysis (see <FIG>, <FIG>, <FIG>, and <FIG>). Based on the above, it was shown that the efficacy of ACE910 can be monitored by using a reagent for blood coagulation analysis composed of tissue factor, ellagic acid and phospholipids. Here, as an example, it was shown that a value obtained by converting the coagulation rate of <NUM>, <NUM> or <NUM>µg/mL ACE910 from the calibration curve of |Min <NUM>| of the specimen containing rhFVIII into FVIII activity falls in the expected ideal range (<NUM> to <NUM> U/dL FVIII per <NUM>µg/mL ACE910) for its haemostatic effect, by using reagents <NUM>, <NUM>, <NUM>, <NUM> and <NUM> for blood coagulation analysis (see <FIG>. In the figures, a region sandwiched between two broken lines shows the ideal range).

Furthermore, as an example, when the effect of ACE910 in FVIII-deficient plasma containing a FVIII preparation was evaluated each using reagents <NUM> and <NUM> for blood coagulation analysis, the coagulation rate and the coagulation acceleration increased by addition of ACE910 (see <FIG> and <FIG>). From these results, it was shown that the efficacy of ACE910 can be monitored even in the presence of FVIII, by using a reagent for blood coagulation analysis composed of tissue factor, ellagic acid and phospholipids.

Coagulation waveform analysis of bispecific antibody in FVIII-deficient plasma using reagent kit for blood coagulation analysis containing tissue factor, ellagic acid and phospholipids.

In Example <NUM>, reagent kits <NUM> to <NUM> for blood coagulation analysis composed of a first reagent containing ellagic acid and phospholipids and a second reagent containing tissue factor and a calcium chloride solution (<NUM> mol/L) were used. <NUM>µL of the first reagent was added to <NUM>µL of FVIII-deficient human plasma (George King Bio-Medical) added with ACE910 or recombinant human FVIII (rhFVIII, Bayer Co. After incubation for <NUM> minutes, <NUM>µL of the second reagent was added thereto to start a coagulation reaction, and measurement was performed with an automatic blood coagulation measuring device CS-<NUM> (Sysmex Corporation). The respective final concentrations of each of tissue factor, ellagic acid and phospholipids in the measurement sample prepared using reagent kits <NUM> to <NUM> for blood coagulation analysis were shown in Table <NUM> below. Reagent kits <NUM> to <NUM> for blood coagulation analysis are denoted as Reagent <NUM> to Reagent <NUM> in <FIG>, respectively.

Each concentration of tissue factor, ellagic acid, and phospholipids in Table <NUM> was described as the final concentrations after mixing the plasma, the first reagent, and the second reagent (final concentrations in the measurement sample).

The coagulation rate and acceleration of ACE910 increased, even under conditions using any of reagent kits <NUM> to <NUM> for blood coagulation analysis (see <FIG>, <FIG>, and <FIG>). Based on the above, it was shown that the efficacy of ACE910 can be monitored by using a reagent for blood coagulation analysis composed of tissue factor, ellagic acid and phospholipids. Here, as an example, it was shown that a value obtained by converting the coagulation rate of <NUM>, <NUM> or <NUM>µg/mL ACE910 from the calibration curve of |Min <NUM>| of the specimen containing rhFVIII into FVIII activity falls in the expected ideal range (<NUM> to <NUM> U/dL FVIII per <NUM>µg/mL ACE910) for its haemostatic effect, by using reagent kits <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> and <NUM> for blood coagulation analysis (see <FIG> and <FIG>. In the figures, a region sandwiched between two broken lines shows the ideal range).

In addition, as an example, it was shown that a value obtained by converting the coagulation rate of <NUM>, <NUM> or <NUM>µg/mL ACE910 from the calibration curve of |Min <NUM>| of the specimen containing rhFVIII into FVIII activity falls in the expected ideal range (<NUM> to <NUM> U/dL FVIII per <NUM>µg/mL ACE910) for its haemostatic effect, by using reagent kits <NUM>, <NUM> and <NUM> for blood coagulation analysis (see <FIG> and <FIG>. In the figures, a region sandwiched between two broken lines shows the ideal range).

Coagulation waveform analysis of bispecific antibody in FVIII-deficient plasma in the presence of bypass preparation using reagent kit for blood coagulation analysis containing tissue factor, ellagic acid and phospholipids.

In Example <NUM>, reagent kits <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> and <NUM> for blood coagulation analysis composed of a first reagent containing ellagic acid and phospholipids and a second reagent containing tissue factor and a calcium chloride solution (<NUM> mol/L) were used. Reagent kits <NUM>, <NUM>, <NUM>, <NUM> and <NUM> are the same as in Example <NUM>. To <NUM>µL of a sample obtained by adding ACE910 to FVIII-deficient human plasma (George King Bio-Medical) added with <NUM> or <NUM> U/mL APCC (Fiber (registered trademark), Baxter Limited) or FVIII-deficient human plasma (George King Bio-Medical) added with <NUM> or <NUM>µg/mL FVIIa (NovoSeven (registered trademark), Novo Nordisk Pharma Ltd. ) was added <NUM>µL of the first reagent. After incubation for <NUM> minutes, <NUM>µL of the second reagent was added thereto to start a coagulation reaction, and measurement was performed with an automatic blood coagulation measuring device CS-2000i (Sysmex Corporation). The respective final concentrations of the tissue factor, ellagic acid and phospholipids in the measurement sample prepared using each of the above reagent kits were shown in Table <NUM> below.

Claim 1:
A method for evaluating coagulability of a blood specimen, the method comprising the steps of
preparing a measurement sample from a blood specimen obtained from a subject to whom a substance having a coagulation factor VIII-substituting activity is administered, a coagulation factor XII-activating agent, phospholipids, tissue factor, and a calcium ion-containing aqueous solution;
irradiating the measurement sample with light to acquire optical information on the light amount from the measurement sample; and
evaluating coagulability of the blood specimen based on the acquired optical information,
wherein the substance having a coagulation factor VIII-substituting activity is a bispecific antibody specifically binding to (i) FIX or FIXa and (ii) FX,
wherein:
A) the final concentration of the FXII-activating agent in the measurement sample is <NUM> or more and <NUM> or less;
B) the final concentration of the phospholipids in the measurement sample is <NUM> or more and <NUM> or less; and
C) the final concentration of the tissue factor in the measurement sample is <NUM> ng/mL or more and <NUM> ng/mL or less.