Reagent for measuring activated partial thromboplastin time, method for producing same, reagent kit for measuring activated partial thromboplastin time, measurement method, sensitivity adjustment method, and activated partial thromboplastin time adjustment agent

Abstract

A reagent for measuring activated partial thromboplastin time, the reagent comprising phosphatidylcholine (PC), phosphatidylserine (PS), and phosphatidylethanolamine (PE), wherein the ratio (PS / PC) of the concentration of the PS to the concentration of the PC is 0.26-2.00, and the concentration of the PS is 14-60 µg / mL.

Description

Reagent for Measuring Activated Partial Thromboplastin Time, Method for Producing the Same, Reagent Kit for Measuring Activated Partial Thromboplastin Time, Measuring Method, Sensitivity Adjustment Method, and Activated Partial Thromboplastin Time Adjusting Agent

[0001] The present invention relates to a reagent for measuring activated partial thromboplastin time, a method for producing the same, a reagent kit for measuring activated partial thromboplastin time, a method for measuring activated partial thromboplastin time, a method for adjusting the sensitivity of a reagent for measuring activated partial thromboplastin time, and an activated partial thromboplastin time adjusting agent. This application claims priority from Japanese Patent Application No. 2024-232773 filed in Japan on December 27, 2024, the content of which is incorporated herein by reference.

[0002] Blood coagulation tests are tests for evaluating a patient's blood coagulation ability by adding a predetermined reagent to a patient's blood sample and measuring the blood coagulation time and the like. Typical examples of blood coagulation time include prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT), and the like. Abnormalities in blood coagulation ability cause an extension of the coagulation time. Causes of the extension of the coagulation time include a decrease in coagulation-related components, congenital deficiency of blood coagulation factors (such as hemophilia), autoantibodies that inhibit the coagulation reaction (lupus anticoagulant; LA, anticoagulation factor antibodies, etc.), and coagulation inhibitors (such as heparin).

[0003] In blood coagulation tests, a reagent is added to a blood sample, and then the subsequent blood coagulation reaction is measured to measure the blood coagulation time. When an extension of the coagulation time is observed, it is determined that there is an abnormality in blood coagulation ability.

[0004] Reagents for blood coagulation tests contain components for promoting blood coagulation under certain conditions. For example, reagents for measuring APTT contain coagulation factor activators such as ellagic acid, phospholipids, buffers, and the like. On the other hand, there are various types of commercially available reagents for measuring APTT, and the sensitivities to various coagulation abnormalities differ among these reagents.

[0005] Patent Document 1 discloses methods to improve sensitivity to LA, such as lowering the concentration of phosphatidylserine (PS) in the APTT measurement reagent and increasing the ratio of PS concentration to phosphatidylcholine (PC) concentration (PS / PC). It also discloses a method to increase sensitivity to heparin, which involves lowering the PS / PC value.

[0006] Japanese Patent Publication No. 2020-56578

[0007] APTT is a coagulation time that reflects the function of intrinsic coagulation factors. APTT measurement is used not only for screening tests for LA and monitoring of heparin therapy, but also for screening tests for intrinsic coagulation factors. Therefore, as in Patent Document 1, if the concentration of PS is lowered to improve sensitivity to LA, the function that promotes the coagulation reaction is suppressed, and in the case of samples deficient in coagulation factors, the coagulation time may be excessively prolonged. If the coagulation time is excessively prolonged, it may exceed the measurement time of the device and become undetectable, or it may exceed the measurement time specified for the reagent and output an unreliable measurement result. In addition, it may prolong the Turn Around Time (TAT), which is the time until the test result is reported.

[0008] The inventors investigated the phospholipid composition of APTT measurement reagents and found that by adjusting the PS concentration and the PS / PC value, not only the sensitivity to LA but also the sensitivity to coagulation factor deficiency and coagulation factor inhibitors can be improved, thus completing the present invention.

[0009] The object of the present invention is to provide a reagent for measuring activated partial thromboplastin time that has improved sensitivity to LA and also improved sensitivity to coagulation factor deficiency and coagulation factor inhibitors, a method for producing the same, a reagent kit for measuring activated partial thromboplastin time, a method for measuring activated partial thromboplastin time, a method for adjusting the sensitivity of the reagent for measuring activated partial thromboplastin time, a blood coagulation time modifier for coagulation factor deficiency samples in activated partial thromboplastin time measurement, and a method for determining the type of abnormal sample.

[0010] The present invention encompasses the following embodiments: [1] A reagent for measuring activated partial thromboplastin time comprising phosphatidylcholine (PC), phosphatidylserine (PS), and phosphatidylethanolamine (PE), wherein the ratio of the concentration of PS to the concentration of PC in the reagent for measuring activated partial thromboplastin time (PS / PC) is 0.26 or more and 2.00 or less, and the concentration of PS is 14 μg / mL or more and 60 μg / mL or less. [2] The reagent for measuring activated partial thromboplastin time according to [1], wherein the concentration of PC is 30 μg / mL or more and 70 μg / mL or less. [3] The reagent for measuring activated partial thromboplastin time according to [1] or [2], wherein the concentration of PE is 20 μg / mL or more and 50 μg / mL or less. [4] The activated partial thromboplastin time measuring reagent according to any one of [1] to [3], wherein the PC, the PS, and the PE are in the form of liposomes. [5] The activated partial thromboplastin time measuring reagent according to [4], wherein the average particle size of the liposomes is 120 nm or more and 600 nm or less. [6] The activated partial thromboplastin time measuring reagent according to any one of [4] or [5], wherein the zeta potential of the measuring reagent is -120 mV or more and -20 mV or less. [7] A method for producing an activated partial thromboplastin time measuring reagent, comprising the steps of: obtaining a mixture of phosphatidylcholine (PC), phosphatidylserine (PS), and phosphatidylethanolamine (PE); and using the mixture to form liposomes containing the PC, the PS, and the PE, thereby obtaining an activated partial thromboplastin time measuring reagent containing the liposomes, wherein the ratio of the concentration of PS to the concentration of PC (PS / PC) in the activated partial thromboplastin time measuring reagent is 0.26 or more and 2.00 or less, and the concentration of PS is 14 μg / mL or more and 60 μg / mL or less.[8] A reagent kit for measuring activated partial thromboplastin time, comprising: a first reagent containing phosphatidylcholine (PC), phosphatidylserine (PS), and phosphatidylethanolamine (PE); and a second reagent containing calcium ions, wherein the ratio of the concentration of PS to the concentration of PC in the first reagent (PS / PC) is 0.26 or more and 2.00 or less; and the concentration of PS in the first reagent is 14 μg / mL or more and 60 μg / mL or less. [9] A method for measuring activated partial thromboplastin time, comprising the steps of: obtaining a mixed solution by mixing a reagent for measuring activated partial thromboplastin time with a target sample; and measuring the activated partial thromboplastin time of the mixed solution, wherein the reagent for measuring activated partial thromboplastin time comprises phosphatidylcholine (PC), phosphatidylserine (PS), and phosphatidylethanolamine (PE), and in the reagent for measuring activated partial thromboplastin time, the ratio of the concentration of PS to the concentration of PC (PS / PC) is 0.26 or more and 2.00 or less, and the concentration of PS is 14 μg / mL or more and 60 μg / mL or less.

[10] The preparation of a reagent for measuring activated partial thromboplastin time comprising phosphatidylcholine (PC), phosphatidylserine (PS), and phosphatidylethanolamine (PE) includes the steps of: adjusting the ratio of the concentration of PS to the concentration of PC in the reagent for measuring activated partial thromboplastin time (PS / PC) to 0.26 or more and 2.00 or less, and adjusting the concentration of PS to 14 μg / mL or more and 60 μg / mL or less; mixing the reagent for measuring activated partial thromboplastin time with the target sample and measuring the activated partial thromboplastin time, which is t seconds; and determining that the prepared reagent for measuring activated partial thromboplastin time has good sensitivity to the target sample when the activated partial thromboplastin time is within the range of 40 seconds or more and 200 seconds or less, or when the rate of variation of the activated partial thromboplastin time calculated by the following formula A is 110% or more. A method for adjusting the sensitivity of a reagent for measuring activated partial thromboplastin time. [Formula A: Percentage change in activated partial thromboplastin time (%) = (the t seconds which is the activated partial thromboplastin time of the target sample) / (the t seconds which is the activated partial thromboplastin time of a normal sample) × 100 … (A)

[11] The sensitivity adjustment method according to

[10] , wherein the target sample is at least one selected from the group consisting of a coagulation factor deficiency sample, a coagulation factor inhibitor-containing sample, an LA-containing sample, and a heparin-containing sample.

[12] A blood coagulation time modifier comprising phosphatidylcholine (PC), phosphatidylserine (PS), and phosphatidylethanolamine (PE), wherein the blood coagulation time modifier is added to a coagulation factor deficiency sample when measuring the activated partial thromboplastin time of the coagulation factor deficiency sample, wherein the ratio of the concentration of PS to the concentration of PC in the blood coagulation time modifier (PS / PC) is 0.26 or more and 0.81 or less, and the concentration of PS is 14 μg / mL or more and 42 μg / mL or less.

[0011] According to the above embodiment, it is possible to provide a reagent for measuring activated partial thromboplastin time that has improved sensitivity to LA and also improved sensitivity to coagulation factor deficiency and coagulation factor inhibitors, a method for producing the same, a reagent kit for measuring activated partial thromboplastin time, a method for measuring activated partial thromboplastin time, a method for adjusting the sensitivity of the reagent for measuring activated partial thromboplastin time, and an activated partial thromboplastin time adjusting agent.

[0012] Figure 1 is a graph showing the APTT of each sample measured using the APTT measuring reagent of the present invention. Figure 2 is a graph showing the ratio of the APTT of abnormal samples to the APTT of normal samples measured using the APTT measuring reagent of the present invention. Figure 3 is a graph showing the ratio of the APTT of factor VIII-deficient samples to the APTT of normal samples measured using the APTT measuring reagent of the present invention. Figure 4 is a graph showing the ratio of the APTT of factor VIII inhibitor-containing samples to the APTT of normal samples measured using the APTT measuring reagent of the present invention. Figure 5 is a graph showing the ratio of the APTT of factor IX-deficient samples to the APTT of normal samples measured using the APTT measuring reagent of the present invention. Figure 6 is a graph showing the ratio of the APTT of factor V-deficient samples to the APTT of normal samples measured using the APTT measuring reagent of the present invention. Figure 7 is a graph showing the ratio of the APTT of an LA-containing sample to the APTT of a normal sample, measured using the APTT measuring reagent of the present invention. Figure 8 is a graph showing the zeta potential of the APTT measuring reagent containing liposomes, which is included in the APTT measuring reagent of the present invention. Figure 9 is a graph showing the particle size of liposomes included in the APTT measuring reagent of the present invention. Figure 10 is a graph showing the rate of variation of coagulation time with respect to the particle size of liposomes included in the APTT measuring reagent of the present invention. Figure 11 is a graph showing the particle size of liposomes included in the APTT measuring reagent of the present invention.

[0013] In the following Specification, "blood clotting time" may be simply referred to as "clotting time."

[0014] <APTT Measurement Reagent> The APTT measurement reagent according to the first aspect of the present invention comprises phosphatidylcholine (PC), phosphatidylserine (PS), and phosphatidylethanolamine (PE). The ratio of the concentration of PS to the concentration of PC in the APTT measurement reagent (PS / PC) is 0.26 or more and 2.00 or less, and the concentration of PS is 14 μg / mL or more and 60 μg / mL or less.

[0015] The APTT measurement reagent of this embodiment contains PC, PS, and PE as phospholipids. The phospholipids contained in the APTT measurement reagent may be naturally derived phospholipids or synthetic phospholipids. Examples of naturally derived phospholipids include those derived from rabbit brain, bovine brain, human placenta, soybeans, and egg yolk.

[0016] There are no particular limitations on the fatty acid side chains (i.e., acyl groups) of PC, PS, and PE. For example, they are preferably acyl groups having 8 to 20 carbon atoms, and more preferably acyl groups having 14 to 18 carbon atoms. Examples of acyl groups with carbon atoms within the above range include lauroyl groups, myristoyl groups, palmitoyl groups, stearoyl groups, and oleoyl groups. PC, PS, and PE all have two fatty acid side chains in their molecules, and these two fatty acid side chains may be the same or different.

[0017] The concentrations of PC, PS, and PE in the APTT measuring reagent in this embodiment can be calculated by dividing the weight (or mass) of each of the PC, PS, and PE used in the preparation of the APTT measuring reagent by the volume of the prepared APTT measuring reagent. In other words, the concentrations of PC, PS, and PE in the APTT measuring reagent represent the ratio of the mass of each PC, PS, and PE to the total volume of the APTT measuring reagent.

[0018] There are no particular limitations on the type of polyphosphate (PC) included in the APTT measurement reagent. Examples include palmitoyloleoylphosphatidylcholine (POPC), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), and dioleoylphosphatidylcholine (DOPC). The APTT measurement reagent may contain one type of PC or two or more types.

[0019] The concentration of PC in the APTT measurement reagent is not particularly limited as long as the ratio of the concentration of PS to the concentration of PC (hereinafter sometimes referred to as "PS / PC") is within the above numerical range, preferably 30 μg / mL or more and 70 μg / mL or less, more preferably 51 μg / mL or more and 70 μg / mL or less, and even more preferably 51 μg / mL or more and 67 μg / mL or less. If the concentration of PC is above the above lower limit, the sensitivity to LA is further improved, and the APTT can be appropriately extended when measuring a sample containing LA, and excessive extension of APTT due to excessively high sensitivity to coagulation factor deficiencies such as factor V deficiency samples can be prevented. If the concentration of PC is below the above upper limit, the sensitivity to factor VIII inhibitor is further improved, and the APTT can be appropriately extended when measuring a sample containing factor VIII inhibitor. The upper and lower limits of the concentration of PC in the APTT measurement reagent described above can be arbitrarily combined.

[0020] There are no particular limitations on the type of PS included in the APTT measurement reagent. Examples include palmitoyloleoylphosphatidylserine (POPS), dipalmitoylphosphatidylserine (DPPS), distearoylphosphatidylserine (DSPS), and dioleoylphosphatidylserine (DOPS). The APTT measurement reagent may contain one type of PS or two or more types.

[0021] The concentration of PS contained in the APTT measurement reagent is 14 μg / mL or more and 60 μg / mL or less, preferably 18 μg / mL or more and 60 μg / mL or less, and more preferably 18 μg / mL or more and 43 μg / mL or less. If the concentration of PS is above the lower limit, it is possible to prevent excessive prolongation of APTT due to an unnecessarily high sensitivity to coagulation factor deficiency samples. If the concentration of PS is below the upper limit, the sensitivity to LA is improved, and APTT can be appropriately prolonged when measuring samples containing LA, and excessive prolongation of APTT due to an unnecessarily high sensitivity to coagulation factor deficiencies such as V factor deficiency samples can be prevented. The upper and lower limits of the concentration of PS contained in the APTT measurement reagent described above can be arbitrarily combined.

[0022] There are no particular limitations on the type of PE contained in the APTT measurement reagent. Examples include palmitoyloleoylphosphatidylethanolamine (POPE), dipalmitoylphosphatidylethanolamine (DPPE), distearoylphosphatidylethanolamine (DSPE), and dioleoylphosphatidylethanolamine. The APTT measurement reagent may contain one type of PE or two or more types.

[0023] There are no particular limitations on the concentration of PE contained in the APTT measurement reagent, but it is preferably 20 μg / mL or more and 50 μg / mL or less, more preferably 24 μg / mL or more and 50 μg / mL or less, and even more preferably 24 μg / mL or more and 32 μg / mL or less. If the concentration of PE is above the lower limit, the sensitivity to LA is further improved, and the APTT can be appropriately extended when measuring a sample containing LA, and excessive extension of APTT due to excessively high sensitivity to coagulation factor deficiencies such as factor V deficiency samples can be prevented. If the concentration of PE is below the upper limit, the sensitivity to factor VIII inhibitor is further improved, and the APTT can be appropriately extended when measuring a sample containing factor VIII inhibitor. The upper and lower limits of the concentration of PE contained in the APTT measurement reagent described above can be arbitrarily combined.

[0024] In the APTT measurement reagent of this embodiment, the PS / PC is 0.26 or more and 2.00 or less, preferably 0.26 or more and 1.00 or less, and more preferably 0.27 or more and 0.83 or less. When the PS / PC is above the lower limit, it is possible to prevent excessive prolongation of APTT due to an unnecessarily high sensitivity to coagulation factor deficiency samples. By preventing excessive prolongation of APTT, it is possible to avoid problems such as mistakenly detecting samples that do not actually require treatment (for example, non-hemophilia A samples with factor VIII activity of 40% or more) as abnormal samples, or mistakenly determining that APTT is prolonged before surgery even though the sample has a low bleeding risk, thus preventing prompt surgery. Furthermore, when the PS / PC is below the upper limit, the sensitivity to LA is improved, and APTT can be appropriately prolonged when measuring samples containing LA. The upper and lower limits of PS / PC can be arbitrarily combined.

[0025] The solvent used in the APTT measurement reagent of this embodiment can be appropriately selected from aqueous solvents commonly used in the field of blood testing, and is not particularly limited. Examples of such aqueous solvents include water, physiological saline, and buffers. Therefore, the APTT measurement reagent of the present invention is an aqueous solution and may contain a buffer having appropriate buffering properties. The buffer can be appropriately selected from buffers commonly used in the field of blood testing, and examples include tris(hydroxymethyl)aminomethane buffer, phosphate buffer, borate buffer, or Good's buffer.

[0026] Examples of the aforementioned Good's buffer include piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES), N-(2-acetamide)-2-aminoethanesulfonic acid (ACES), 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES), N-[tris(hydroxymethyl)methyl]-2-hydroxy-3-aminopropanesulfonic acid (TAPSO), N-tris(hydroxymethyl)methyl-3- Aminopropanesulfonic acid (TAPS), piperazine-N,N'-bis(2-hydroxypropanesulfonic acid) (POPSO), 3-[4-(2-hydroxyethyl)-1-piperazinyl]propanesulfonic acid [(H)EPPS], N-cyclohexyl-2-aminoethanesulfonic acid (CHES), 2-morpholinoethanesulfonic acid (MES), bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane (Bis-Tris), N- (2-Acetamide)iminodiacetic acid (ADA), 3-morpholino-2-hydroxypropanesulfonic acid (MOPSO), N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), 3-morpholinopropanesulfonic acid (MOPS), N-[tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid (TES), 3-[N,N-bis(2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid (DIP Examples include N-SO, 3-[4-(2-hydroxyethyl)-1-piperazinyl]-2-hydroxypropanesulfonic acid (HEPPSO), N-[tris(hydroxymethyl)methyl]glycine (Tricine), N,N-bis(2-hydroxyethyl)glycine (Bicine), N-cyclohexyl-3-amino-2-hydroxypropanesulfonic acid (CAPSO), and N-cyclohexyl-3-aminopropanesulfonic acid (CAPS). These buffers can be used individually or in combination of two or more.

[0027] The hydrogen ion concentration of the buffer is not particularly limited as long as it exhibits buffering capacity, and is preferably pH 4 or higher and pH 9 or lower at 2 to 37°C, more preferably pH 6 or higher and pH 8 or lower. Similarly, the buffer content in the APTT measurement reagent is not particularly limited as long as it exhibits buffering capacity, and is preferably 0.1 mM or higher and 1000 mM or lower, more preferably 0.5 mM or higher and 500 mM or lower, even more preferably 1 mM or higher and 200 mM or lower, and particularly preferably 10 mM or higher and 100 mM or lower relative to the total volume of the APTT measurement reagent. The upper and lower limits of the buffer content in the APTT measurement reagent can be arbitrarily combined.

[0028] The APTT measurement reagent of this embodiment may further contain phospholipids other than PC, PS, and PE. The phospholipids further contained in the APTT measurement reagent are not particularly limited, and examples include phosphatidylinositol (PI), cardiolipin (CL), phosphatidylglycerol (PG), sphingomyelin (SM), and phosphatidic acid (PA). By using negatively charged phospholipids such as PI, CL, PG, and PA as the phospholipids further contained in the APTT measurement reagent, the dispersibility of liposomes can be improved.

[0029] There are no particular limitations on the form of the phospholipids contained in the APTT measurement reagent of this embodiment, but it is preferable that they be in the form of liposomes. When the phospholipids are in the form of liposomes, the physiological membrane environment is reproduced, and the accuracy and reproducibility of the reagent are ensured by appropriately activating the intrinsic coagulation pathway. In addition, by existing as stable vesicles in the reagent, it becomes stable during long-term storage.

[0030] There are no particular limitations on the average particle size of the liposomes, but it is preferably 120 nm or more and 600 nm or less, more preferably 150 nm or more and 500 nm or less, and even more preferably 300 nm or more and 450 nm or less. The upper and lower limits of the average particle size of the liposomes can be arbitrarily combined. When the average particle size of the liposomes is within the above range, the sensitivity to LA can be appropriately extended, and excessive extension of APTT due to excessively high sensitivity to coagulation factor deficiency and coagulation factor inhibitors can be better prevented. The average particle size of the liposomes is the median diameter (D ５０ ) and can be measured by dynamic light scattering. Measurement by dynamic light scattering is performed using a particle size distribution analyzer (for example, Zetasizer Nano ZS (Malvern Panalogical)).

[0031] The zeta potential of the APTT measurement reagent containing the liposomes is not particularly limited. For example, the zeta potential of the APTT measurement reagent containing the liposomes (particularly the first reagent (R1), details of which will be described later) is preferably -120 mV or higher and -20 mV or lower, more preferably -90 mV or higher and -35 mV or lower, and even more preferably -64 mV or higher and -48 mV or lower. The upper and lower limits of the zeta potential of the APTT measurement reagent containing the liposomes can be arbitrarily combined. If the zeta potential of the APTT measurement reagent is above the lower limit, the sensitivity to LA is further improved, and the APTT can be appropriately prolonged when measuring a sample containing LA. If the zeta potential of the APTT measurement reagent is below the upper limit, excessive prolongation of the APTT due to unnecessarily high sensitivity to coagulation factor deficiency samples can be prevented. The zeta potential of the APTT measurement reagent can be measured by light scattering electrophoresis. Measurements using light scattering electrophoresis are performed using a zeta potential analyzer (for example, Zetasizer Nano ZS (Malvern Panalogical)).

[0032] The APTT measurement reagent of this embodiment preferably further contains an activator. The activator is not particularly limited as long as it is a component that promotes the blood coagulation reaction, and is at least one selected from the group consisting of, for example, ellagic acid, kaolin, and celite.

[0033] The amount of the activator is not particularly limited as long as it is an amount that exerts an effect as an activator, and can be appropriately determined according to the type of activator used and the measurement conditions. For example, if the activator contained in the APTT measurement reagent is ellagic acid, the amount of ellagic acid in the APTT measurement reagent is preferably 0.001 mg / mL or more and 2 mg / mL or less, more preferably 0.001 mg / mL or more and 0.5 mg / mL or less, even more preferably 0.001 mg / mL or more and 0.1 mg / mL or less, and particularly preferably 0.005 mg / mL or more and 0.05 mg / mL or less, relative to the total volume of the APTT measurement reagent after preparation. The upper and lower limits of the ellagic acid content in the APTT measurement reagent can be arbitrarily combined.

[0034] The APTT measurement reagent of this embodiment preferably further contains a metal salt compound as a component that suppresses the formation of precipitate. The metal salt compound is at least one metal salt compound selected from the group consisting of zinc chloride, aluminum chloride, nickel chloride, manganese(II) chloride, yttrium chloride, copper chloride, potassium aluminum sulfate, ammonium iron(III) sulfate, copper sulfate (e.g., copper(II) sulfate pentahydrate), and their hydrates.

[0035] There are no particular limitations on the content of the metal salt compound in the APTT measurement reagent, and it can be appropriately determined depending on the type of metal salt compound used and the measurement conditions. For example, if the metal salt compound contained in the APTT measurement reagent is copper sulfate, the copper sulfate content in the APTT measurement reagent is preferably 0.0001 mg / mL or more and 0.15 mg / mL or less, more preferably 0.001 mg / mL or more and 0.05 mg / mL or less, even more preferably 0.005 mg / mL or more and 0.04 mg / mL or less, and particularly preferably 0.0075 mg / mL or more and 0.0225 mg / mL or less, relative to the total volume of the prepared APTT measurement reagent. The upper and lower limits of the copper sulfate content in the APTT measurement reagent can be arbitrarily combined.

[0036] The APTT measuring reagent of this embodiment may further contain, for example, calcium ions as a component that initiates blood coagulation. Examples of calcium ions include water-soluble calcium compounds such as calcium chloride, calcium lactate, calcium gluconate, calcium glucuronate, and calcium tartrate. These calcium compounds can be used individually or in combination of two or more. There are no particular limitations on the calcium compound content in the APTT measuring reagent; it is preferably 5 mM or more and 100 mM or less, more preferably 10 mM or more and 50 mM or less, relative to the total volume of the prepared APTT measuring reagent. The upper and lower limits of the calcium compound content in the APTT measuring reagent can be arbitrarily combined.

[0037] The APTT measurement reagent of this embodiment may further contain other additives to improve its shelf life and stability. Such additives may be any additives commonly used in reagents for measuring coagulation time, such as preservatives, antioxidants, and stabilizers. Examples of preservatives include sodium azide and known antibiotics such as Proclin® 300. Examples of antioxidants include butylhydroxyanisole. Examples of stabilizers include polyethylene glycol and polyvinylpyrrolidone.

[0038] When the APTT measurement reagent of this embodiment contains an ellagic acid compound, it is preferable to include a solubilizing agent for the purpose of preventing the precipitation of the ellagic acid compound (i.e., solubilizing the ellagic acid compound). The solubilizing agent is not particularly limited as long as it has the effect of preventing the precipitation of the ellagic acid compound, and examples include amino acids having aromatic rings, polyvinyl alcohol compounds, and phenols.

[0039] The content of the solubilizer in the APTT measurement reagent is not particularly limited as long as it is an effective amount for solubilizing the ellagic acid compound. For example, it is preferably 0.001 mg / mL or more and 0.1 mg / mL or less relative to the volume of the prepared APTT measurement reagent. If the content of the solubilizer in the APTT measurement reagent is above the above lower limit, precipitation of ellagic acid can be suppressed even when the APTT measurement reagent is stored for a long period of time.

[0040] The APTT measurement reagent in this embodiment may be in liquid form, a frozen form thereof, or a dry form. The dry reagent is dissolved in water, physiological saline, or buffer, etc., at the time of use to prepare a liquid reagent.

[0041] <Method for Producing APTT Measurement Reagent> The second embodiment of the present invention provides a method for producing an APTT measurement reagent, comprising the steps of: obtaining a mixture of phosphatidylcholine (PC), phosphatidylserine (PS), and phosphatidylethanolamine (PE); and using the mixture to form liposomes containing PC, PS, and PE, thereby obtaining an activated partial thromboplastin time measurement reagent containing the liposomes. In the APTT measurement reagent, the ratio of the concentration of PS to the concentration of PC (PS / PC) is 0.26 or more and 2.00 or less, and the concentration of PS is 14 μg / mL or more and 60 μg / mL or less. The APTT measurement reagent of the first embodiment can be produced by the production method of this embodiment.

[0042] The concentrations of PC, PS, and PE in the APTT measuring reagent according to this embodiment can be calculated by dividing the mass of PC, PS, and PE used in the preparation of the APTT measuring reagent by the volume of the prepared APTT measuring reagent.

[0043] In the step of obtaining a mixture by mixing PC, PS and PE, the PS / PC in the reagent for measuring APTT to be produced is 0.26 or more and 2.00 or less, and the concentration of PS is 14 μg / mL or more and 60 μg / mL or less. Appropriately determine the mass of PS and PC to be used and the amount of the solvent.

[0044] In the reagent for measuring APTT produced in this embodiment, the preferable ranges of the contents of phospholipids such as PC, PS and PE, PS / PC, and other components are the same as those of the reagent for measuring APTT in the first embodiment.

[0045] The mixture of PC, PS and PE can be prepared, for example, by weighing predetermined amounts of solid PC, PS and PE respectively and dissolving them in an organic solvent. As the organic solvent, a solvent that can dissolve phospholipids and has a low boiling point is preferable. Examples of such an organic solvent include chloroform, methanol, and their mixtures.

[0046] PC, PS and PE are also commercially available in the state of solutions previously dissolved in an organic solvent. In this embodiment, solution-state PC, PS and PE previously dissolved in an organic solvent may be used.

[0047] In the step of using the mixture obtained by the above step to form liposomes containing PC, PS and PE and obtaining a reagent for measuring activated partial thromboplastin time containing liposomes, first, liposomes are formed.

[0048] The mixing of PC, PS and PE and the formation of liposomes are carried out, for example, as follows. From the mixture obtained by the above step, the organic solvent is evaporated by an evaporator or the like to obtain a thin film of phospholipid. An appropriate aqueous solvent (preferably a buffer) is added to this thin film of phospholipid. Thereby, the phospholipid is swollen by the aqueous solvent, and a liposome-containing solution can be obtained. Thereby, a reagent for measuring APTT in the first embodiment containing liposomes containing PC, PS and PE as phospholipids can be produced.

[0049] In this embodiment, in order to sufficiently swell the phospholipids, it is preferable to add a solvent to the phospholipid thin film and then stir the resulting solution. Stirring is performed using, for example, a stirrer. The stirring speed is not particularly limited as long as the liposomes are not disrupted, and may be, for example, 400 rpm or more and 650 rpm or less. The stirring time is preferably 45 minutes or more and 120 minutes or less, more preferably 60 minutes or more and 90 minutes or less.

[0050] In this embodiment, it is preferable to irradiate the liposome-containing solution with ultrasound in order to disperse the liposomes. The frequency of the ultrasound is preferably, for example, 35 kHz or higher and 40 kHz or lower. The irradiation time of the ultrasound is preferably, for example, 5 minutes or more and 20 minutes or less.

[0051] In this embodiment, in order to homogenize the particle size of the liposomes, the liposome-containing solution after sonication may be subjected to extruder treatment using a membrane filter and extruder having a desired pore size. TAPS buffer is used as the dispersion medium for the liposome dispersion. By reducing the average particle size of the liposomes through extruder treatment, it is expected that the excessive extension of APTT will be suppressed.

[0052] The method for producing the APTT measurement reagent according to this embodiment preferably includes a step of mixing a mixture of PC, PS, and PE, or the above-mentioned liposome-containing solution, with the above-mentioned activator.

[0053] <APTT Measurement Reagent Kit> The APTT measurement reagent kit, according to a third aspect of the present invention, comprises a first reagent containing phosphatidylcholine (PC), phosphatidylserine (PS), and phosphatidylethanolamine (PE), and a second reagent containing calcium ions, wherein the ratio of the concentration of PS to the concentration of PC in the first reagent (PS / PC) is 0.26 or more and 2.00 or less, and the concentration of PS in the first reagent is 14 μg / mL or more and 60 μg / mL or less. The first reagent of this aspect contains the APTT measurement reagent of the first aspect.

[0054] The concentrations of PC, PS, and PE in the APTT measurement reagent kit of this embodiment can be calculated by dividing the mass of PC, PS, and PE used in the preparation of the APTT measurement reagent kit by the volume of the first reagent after preparation.

[0055] The second reagent in this embodiment is a reagent that initiates a blood coagulation reaction when added to a mixture of the target sample and the first reagent. The second reagent contains calcium ions. As calcium ions, for example, water-soluble calcium compounds such as calcium chloride, calcium lactate, calcium gluconate, calcium glucuronate, and calcium tartrate can be used. Any one of these calcium compounds or any combination of two or more can be used. There are no particular limitations on the calcium compound content in the APTT measurement reagent, but it is preferably 5 mM or more and 100 mM or less, more preferably 10 mM or more and 50 mM or less, relative to the total volume of the APTT measurement reagent. The upper and lower limits of the calcium compound content in the APTT measurement reagent can be arbitrarily combined.

[0056] <APTT Measurement Method> The fourth aspect of the present invention is an APTT measurement method, which includes the steps of: obtaining a mixed solution (hereinafter sometimes referred to as "sample solution") by mixing an APTT measurement reagent with a target sample; and measuring the activated partial thromboplastin time of the mixed solution. The APTT measurement reagent contains phosphatidylcholine (PC), phosphatidylserine (PS), and phosphatidylethanolamine (PE), wherein the ratio of the concentration of PS to the concentration of PC (PS / PC) in the APTT measurement reagent is 0.26 or more and 2.00 or less, and the concentration of PS is 14 μg / mL or more and 60 μg / mL or less.

[0057] The concentrations of PC, PS, and PE in the APTT measuring reagent in this embodiment can be calculated by dividing the mass of PC, PS, and PE used in the preparation of the APTT measuring reagent by the volume of the APTT measuring reagent.

[0058] The aforementioned sample is preferably a blood sample. The blood sample is blood, plasma obtained from blood, or preparations thereof. As the blood sample, plasma obtained from blood collected from a subject is preferred. The method for obtaining plasma from blood is known in this technology. For example, plasma can be obtained by centrifuging the blood without hemolysis to remove blood cell components. In addition, the blood collected from the subject may have known anticoagulants commonly used in clinical tests of blood coagulation ability added to it. An example of such an anticoagulant is sodium citrate.

[0059] The APTT measurement reagent in this embodiment may be the APTT measurement reagent of the first embodiment or the APTT measurement reagent kit of the third embodiment. If the APTT measurement reagent is the APTT measurement reagent of the first embodiment, a sample solution is obtained by mixing the APTT measurement reagent with the target sample. If the APTT measurement reagent is the APTT measurement reagent kit of the third embodiment, a sample solution is obtained by mixing the first reagent with the target sample, and then mixing the solution obtained by mixing the first reagent and the target sample with the second reagent.

[0060] Next, the APTT of the sample solution obtained in the above-mentioned process is measured. The method for measuring APTT is not particularly limited as long as it is a method commonly used for APTT measurement. For example, when using an automated blood coagulation analyzer (e.g., CP3000 (manufactured by Sekisui Medical Co., Ltd.)) to measure APTT, light is irradiated onto the sample to be measured, and the amount of optical change due to the coagulation reaction (also called the amount of scattered light intensity change) is measured, and APTT is calculated from the amount of change.

[0061] <Method for adjusting the sensitivity of APTT measuring reagent> A fifth aspect of the present invention is a method for adjusting the sensitivity of an APTT measuring reagent, which includes the steps of: preparing an APTT measuring reagent containing phosphatidylcholine (PC), phosphatidylserine (PS), and phosphatidylethanolamine (PE), adjusting the ratio of the concentration of PS to the concentration of PC in the APTT measuring reagent (PS / PC) to 0.26 or more and 2.00 or less, and adjusting the concentration of PS to 14 μg / mL or more and 60 μg / mL or less; mixing the APTT measuring reagent and a target sample and measuring the APTT in t seconds; and determining that the adjusted APTT measuring reagent has good sensitivity to the target sample when at least one of the following conditions is met: the APTT is within the range of 40 seconds or more and 200 seconds or less, or the rate of change of the APTT calculated by the following formula A is 110% or more. Percentage variation of activated partial thromboplastin time (%) = (APTT of the target sample in t seconds) / (APTT of a normal sample in t seconds) × 100

[0062] The concentrations of PC, PS, and PE in the APTT measuring reagent according to this embodiment can be calculated by dividing the mass of PC, PS, and PE used in the preparation of the APTT measuring reagent by the volume of the prepared APTT measuring reagent.

[0063] In the step of adjusting the PS / PC and PS concentration in the APTT measurement reagent, liposomes are prepared so that the PS concentration reaches a predetermined concentration and added to the APTT measurement reagent. In this embodiment, the predetermined concentration refers to a PS concentration within the range of 0.26 to 0.81 for the ratio of PS concentration to PC concentration (PS / PC) contained in the APTT measurement reagent, and between 14 μg / mL and 42 μg / mL.

[0064] The preferred range of content of phospholipids such as PC, PS, and PE, PS / PC, and other components in the APTT measurement reagent adjusted by the sensitivity adjustment method of this embodiment is the same as that of the APTT measurement reagent of the first embodiment.

[0065] The step of mixing the reagent, whose PS concentration has been adjusted to a predetermined concentration, with the target sample and measuring the APTT (application time limit) in seconds is performed according to the APTT measurement method of the fourth embodiment.

[0066] There are no particular limitations on the specimens to be measured for APTT in this embodiment, but it is preferable that they be at least one selected from the group consisting of coagulation factor deficiency specimens, coagulation factor inhibitor-containing specimens, LA-containing specimens, and heparin-containing specimens.

[0067] When the APTT of a normal sample is measured using the APTT measurement reagent of the first embodiment or the APTT measurement reagent kit of the third embodiment, and the APTT measurement method of the fourth embodiment, measurement results comparable to those of conventional APTT measurement reagents can be obtained. For example, when the APTT of a predetermined normal sample is measured using the APTT measurement reagent of the first embodiment or the APTT measurement reagent kit of the third embodiment, it is preferable that the APTT is within the range of 20 seconds or more and less than 40 seconds. Furthermore, when the APTT of a predetermined abnormal sample is measured, it is preferable that the APTT is within the range of 40 seconds or more and 200 seconds or less. Note that if the APTT exceeds 200 seconds, it exceeds the measurement range of the device, so simultaneous reproducibility of the measurement results cannot be guaranteed, or there is a possibility of no detection.

[0068] In this embodiment, a specified normal sample is a sample in which the APTT measured using a conventional APTT measuring reagent (for example, Coagupia APTT-N (manufactured by Sekisui Medical Co., Ltd.)) is in the range of 20 seconds or more and less than 40 seconds. Such a sample may be a blood sample taken from a subject, or it may be a reagent further prepared from the blood sample (for example, Coagupia Control P-N I (manufactured by Sekisui Medical Co., Ltd.)). In this embodiment, a specified abnormal sample is a sample in which the APTT measured using a conventional APTT measuring reagent is in the range of 40 seconds or more and 200 seconds or less. Such a sample may be a blood sample taken from a subject, or it may be a reagent further prepared from the blood sample (for example, Coagupia Control P-N II (manufactured by Sekisui Medical Co., Ltd.)).

[0069] In the step of determining whether the prepared reagent has good sensitivity, the measured APTT (application time threshold) in t seconds is evaluated based on the following criteria 1 and 2. If the APTT in t seconds satisfies at least one of the following criteria 1 and 2, the prepared reagent is determined to have good sensitivity. Criterion 1: The APTT is within the range of 40 seconds or more and 200 seconds or less. Criterion 2: The APTT variability is 110% or more. The APTT variability is calculated using the following formula: APTT variability (%) = (APTT in t seconds of the target sample) / (APTT in t seconds of a normal sample) × 100

[0070] <APTT Adjuster for Coagulation Factor Deficiency Samples in APTT Measurement> The sixth aspect of the present invention is a blood coagulation time adjuster for coagulation factor deficiency samples in APTT measurement, which comprises phosphatidylcholine (PC), phosphatidylserine (PS), and phosphatidylethanolamine (PE). The blood coagulation time adjuster is added to the coagulation factor deficiency sample when measuring the APTT of the coagulation factor deficiency sample, and the ratio of the concentration of PS to the concentration of PC in the blood coagulation time adjuster (PS / PC) is 0.26 or more and 0.81 or less, and the concentration of PS is 14 μg / mL or more and 42 μg / mL or less.

[0071] The concentrations of PC, PS, and PE in the blood coagulation time regulator in this embodiment can be calculated by dividing the weights of PC, PS, and PE used in the preparation of the blood coagulation time regulator by the volume of the blood coagulation time regulator after preparation.

[0072] The coagulation factor deficiency specimens in this embodiment are not particularly limited, and examples include specimens deficient in factor V, factor VIII, factor IX, factor X, factor XI, and factor XII.

[0073] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0074] For the preparation of the APTT measurement reagent, liposomes, HEPES-Tris (pH 7.3), ellagic acid, copper(II) sulfate pentahydrate, Proclin® 300 (manufactured by Sigma-Aldrich), trisodium citrate, glycine, and phenol were used.

[0075] Coagulation time was measured using an optical method. The optical measurement was performed using a CP3000 automated blood coagulation analyzer (manufactured by Sekisui Medical Co., Ltd.), under the following conditions.

[0076] [Phospholipids] The following phospholipids were used as liposomes: DOPE (dioleoylphosphatidylethanolamine, molecular weight: 744.03), DOPC (dioleoylphosphatidylcholine, molecular weight: 786.11), DOPS (dioleoylphosphatidylserine, molecular weight: 810.03). In the following examples, DOPE may be referred to as PE, DOPC as PC, and DOPS as PS.

[0077] [Liposomes] Liposomes were prepared by the following method. Phospholipid / chloroform solutions containing predetermined amounts of DOPE, DOPC, and DOPS were prepared and added to a round-bottom flask. The round-bottom flask was rotated in a rotary vaporizer to evaporate the chloroform and form a thin film of phospholipid on the inner wall of the flask. 1000 mL of buffer solution (containing TAPS, HEPES, and glycine, pH 7.4) was added to the round-bottom flask to swell the thin film of phospholipid to obtain a liposome-containing buffer solution. The liposome-containing buffer solution was stirred with a stirrer at 500 rpm for 60 minutes. Then, the liposome-containing buffer solution was irradiated with 37 kHz ultrasound for 15 minutes using a water bath type ultrasonic device UT-306H (Sharp Corporation) to disperse the liposomes. The liposome-containing buffer was subjected to extruder treatment using a 0.2 μm polycarbonate membrane (Millipore) and EmulsiFlex-C50 (Avestin) to homogenize the particle size of the liposomes.

[0078] Reagents 1 to 5 were prepared as APTT measurement reagents containing the aforementioned phospholipids. Here, reagents 1 to 5 each consist of a first reagent (R1) and a second reagent (R2). Table 1 shows the proportion of each phospholipid in the liposomes contained in each first reagent (R1) of reagents 1 to 5.

[0079] The DOPS content in the APTT measurement reagents is 15 parts by mass for reagent 1, 20 parts by mass for reagent 2, 25 parts by mass for reagent 3, 30 parts by mass for reagent 4, and 35 parts by mass for reagent 5, per 100 parts by mass of liposomes. Furthermore, the content of DOPC, DOPE, and DOPS was determined and prepared so that the DOPE / DOPC value was constant for each reagent.

[0080]

[0081] Table 2 shows the total phospholipid concentration and the concentration of each phospholipid contained in reagents 1 to 5. Here, the concentration of each phospholipid is shown as the value obtained by dividing the mass of PC, PS, and PE used in the preparation of reagent 1 (R1) by the volume of reagent 1 (R1) after preparation.

[0082]

[0083] Table 3 shows the ratios of each phospholipid in the liposomes contained in each of the first reagents (R1) of reagents 1 to 5.

[0084]

[0085] [APTT Measurement Reagents] The compositions of the first reagent (R1) and the second reagent (R2) contained in the APTT measurement reagents are shown in Tables 4 and 5, respectively.

[0086]

[0087]

[0088] [Samples] The following samples were used as specimens. L1 sample: Normal sample (Coagupia control P-N I (manufactured by Sekisui Medical, product code: 405RBU)) F8DP sample: Factor VIII deficiency sample (CONGENITAL FACTOR VIII DEFIENT PLASMA (manufactured by GEORGE KING BIO-MEDICAL, product code: 7569)) F8INH sample: Factor VIII inhibitor-containing sample (CONGENITAL FACTOR VIII DEFIENT PLASMA WITH INHIBITOR (manufactured by GEORGE KING BIO-MEDICAL, product code: 7199)) F9DP sample: Factor IX deficiency sample (CONGENITAL FACTOR IX DEFIENT PLASMA (manufactured by GEORGE KING BIO-MEDICAL, catalog number: 7305) F5DP sample: Factor V deficiency sample (CONGENITAL FACTOR V DEFIENT PLASMA (manufactured by GEORGE KING BIO-MEDICAL, catalog number: 7101)) LA sample: LA-containing sample (POSITIVE LUPUS ANTICOAGULANT PLASMA (manufactured by GEORGE KING BIO-MEDICAL, catalog number: 6891)) A normal sample is a sample in which, when APTT is measured using a conventional APTT measuring reagent, the APTT is within the range of 20 seconds or more and less than 40 seconds. A factor VIII inhibitor-containing sample is a normal sample to which factor VIII inhibitor has been further added.

[0089] [APTT Measurement Method] APTT was measured according to the APTT measurement method described in the fourth aspect. APTT measurement was performed using the CP3000 automated blood coagulation analyzer (manufactured by Sekisui Medical Co., Ltd.) under the following conditions: 50 μL of the sample was heated in a cuvette at 37°C for 45 seconds, then 50 μL of the first reagent (R1) was added and heated for a further 171 seconds. 50 μL of the second reagent (R2) was added to the heated mixture to initiate the coagulation reaction. The reaction was carried out at 37°C. Light with a wavelength of 660 nm was irradiated onto the sample (sample solution) to which R2 had been added, and the amount of optical change due to the coagulation reaction (change in scattered light intensity) was measured. APTT was determined from the measured coagulation reaction.

[0090] <Example 1> APTT was evaluated for each sample when reagents 1 to 5 were used. The results are shown in Figure 1 and Table 6 below.

[0091]

[0092] Figure 1 and Table 6 show that in normal samples, the APTT is between 20 seconds and 40 seconds for all reagents. Therefore, it is shown that the APTT of normal samples can be measured correctly using the APTT measurement reagent of the present invention.

[0093] In abnormal samples, APTT was significantly prolonged with all reagents compared to normal samples, demonstrating that the APTT measuring reagent of the present invention is sensitive to factor V, VIII, and IX deficiency samples, factor VIII inhibitor-containing samples, and LA-containing samples. Furthermore, the APTT of these abnormal samples was within the range of 40 seconds to 200 seconds. Therefore, it was demonstrated that measuring the APTT of the above abnormal samples using the APTT measuring reagent of the present invention allows for differentiation from the APTT of normal samples and does not result in excessive APTT prolongation.

[0094] The PS contained in APTT measuring reagents acts as a scaffold for blood coagulation factors in the blood coagulation reaction, thereby promoting the blood coagulation reaction. Therefore, if the PS content in the APTT measuring reagent is low, for example, less than 14 μg / mL, it is possible that the APTT will be excessively prolonged. Similarly, if the PS / PC ratio in the APTT measuring reagent is less than 0.26, it is possible that the APTT will be excessively prolonged.

[0095] <Example 2> The ratio of the APTT of each abnormal sample to the APTT of a normal sample was calculated. The results are shown in Figures 2 to 7 and Table 7 below.

[0096]

[0097] Figures 2 to 7 and Table 7 show a correlation between the PS / PC ratio in the APTT measurement reagent and the APTT values ​​of each abnormal sample relative to those of normal samples. Furthermore, it was shown that the PS concentration and PS / PC ratio in the APTT measurement reagent affect the sensitivity for factor V, VIII, and IX deficiency samples, as well as for factor VIII inhibitor-containing samples.

[0098] <Example 3> The relationship between the zeta potential of APTT measurement reagents containing liposomes, the liposome particle size, and APTT was evaluated. For reagents 6 to 9 used in Example 3, the concentrations of PE, PC, and PS were appropriately set to fall within the composition range of reagents 1 to 5, and liposome-containing buffers were obtained in the same manner as reagents 1 to 5. Reagents 6 to 9 with different liposome particle sizes were prepared by extruding the obtained liposome-containing buffers while adjusting the pore size of the membrane. The zeta potential was measured for the first reagent (R1) containing liposomes. The zeta potential of the liposomes in reagents 1 to 5 and the average particle size of the liposomes in reagents 1 to 9 were measured using a Zetasizer Nano ZS (Malvern Panalogical). APTT was measured using the same procedure as in Example 1. The results are shown in Figures 8 to 11.

[0099] Figures 8 and 9 show that as the PS concentration in the APTT measurement reagent increases, the zeta potential of the APTT measurement reagent becomes more negative, and the particle size increases.

[0100] Figures 8 to 11 show that the zeta potential of the APTT measurement reagent containing liposomes (first reagent (R1)) and the particle size of the liposomes affect the sensitivity to factor V, VIII, and IX deficient samples, as well as samples containing factor VIII inhibitor.

[0101] According to the above embodiments of this disclosure, it is possible to provide a reagent for measuring activated partial thromboplastin time that has improved sensitivity to LA and also improved sensitivity to coagulation factor deficiency and coagulation factor inhibitors, a method for producing the same, a reagent kit for measuring activated partial thromboplastin time, a method for measuring activated partial thromboplastin time, a method for adjusting the sensitivity of the reagent for measuring activated partial thromboplastin time, and an activated partial thromboplastin time adjusting agent.

Claims

1. A reagent for measuring activated partial thromboplastin time comprising phosphatidylcholine (PC), phosphatidylserine (PS), and phosphatidylethanolamine (PE), wherein the ratio of the concentration of PS to the concentration of PC in the reagent for measuring activated partial thromboplastin time (PS / PC) is 0.26 or more and 2.00 or less, and the concentration of PS is 14 μg / mL or more and 60 μg / mL or less.

2. The reagent for measuring activated partial thromboplastin time according to claim 1, wherein the concentration of the PC is 30 μg / mL or more and 70 μg / mL or less.

3. The reagent for measuring activated partial thromboplastin time according to claim 1, wherein the concentration of PE is 20 μg / mL or more and 50 μg / mL or less.

4. The reagent for measuring activated partial thromboplastin time according to claim 1, wherein the PC, the PS, and the PE are in the form of liposomes.

5. The reagent for measuring activated partial thromboplastin time according to claim 4, wherein the average particle size of the liposomes is 120 nm or more and 600 nm or less.

6. The reagent for measuring activated partial thromboplastin time according to claim 4, wherein the zeta potential of the measuring reagent is -120 mV or higher and -20 mV or lower.

7. A method for producing an activated partial thromboplastin time measuring reagent, comprising the steps of: obtaining a mixture of phosphatidylcholine (PC), phosphatidylserine (PS), and phosphatidylethanolamine (PE); and using the mixture to form liposomes containing the PC, the PS, and the PE, thereby obtaining an activated partial thromboplastin time measuring reagent containing the liposomes, wherein the ratio of the concentration of PS to the concentration of PC (PS / PC) in the activated partial thromboplastin time measuring reagent is 0.26 or more and 2.00 or less, and the concentration of PS is 14 μg / mL or more and 60 μg / mL or less.

8. A reagent kit for measuring activated partial thromboplastin time, comprising: a first reagent containing phosphatidylcholine (PC), phosphatidylserine (PS), and phosphatidylethanolamine (PE); and a second reagent containing calcium ions, wherein the ratio of the concentration of PS to the concentration of PC in the first reagent (PS / PC) is 0.26 or more and 2.00 or less; and the concentration of PS in the first reagent is 14 μg / mL or more and 60 μg / mL or less.

9. A method for measuring activated partial thromboplastin time, comprising the steps of: obtaining a mixed solution by mixing an activated partial thromboplastin time measuring reagent with a target sample; and measuring the activated partial thromboplastin time of the mixed solution, wherein the activated partial thromboplastin time measuring reagent comprises phosphatidylcholine (PC), phosphatidylserine (PS), and phosphatidylethanolamine (PE), and in the activated partial thromboplastin time measuring reagent, the ratio of the concentration of PS to the concentration of PC (PS / PC) is 0.26 or more and 2.00 or less, and the concentration of PS is 14 μg / mL or more and 60 μg / mL or less.

10. The preparation of an activated partial thromboplastin time measuring reagent containing phosphatidylcholine (PC), phosphatidylserine (PS), and phosphatidylethanolamine (PE) includes the steps of: adjusting the ratio of the concentration of PS to the concentration of PC in the activated partial thromboplastin time measuring reagent (PS / PC) to 0.26 or more and 2.00 or less, and adjusting the concentration of PS to 14 μg / mL or more and 60 μg / mL or less; mixing the activated partial thromboplastin time measuring reagent and the target sample, and measuring the activated partial thromboplastin time, which is t seconds; and determining that the prepared activated partial thromboplastin time measuring reagent has good sensitivity to the target sample when the activated partial thromboplastin time is within the range of 40 seconds or more and 200 seconds or less, or when the rate of variation of the activated partial thromboplastin time calculated by the following formula A is 110% or more. A method for adjusting the sensitivity of a reagent for measuring activated partial thromboplastin time. Variance of activated partial thromboplastin time (%) = (the activated partial thromboplastin time of the target sample, t seconds) / (the activated partial thromboplastin time of a normal sample, t seconds) × 100 … (A) 11. The sensitivity adjustment method according to claim 10, wherein the target sample is at least one selected from the group consisting of coagulation factor deficiency samples, coagulation factor inhibitor-containing samples, LA-containing samples, and heparin-containing samples.

12. A blood coagulation time modifier comprising phosphatidylcholine (PC), phosphatidylserine (PS), and phosphatidylethanolamine (PE), wherein the blood coagulation time modifier is added to a coagulation factor deficiency sample when measuring the activated partial thromboplastin time of the coagulation factor deficiency sample, the ratio of the concentration of PS to the concentration of PC in the blood coagulation time modifier (PS / PC) is 0.26 or more and 0.81 or less, and the concentration of PS is 14 μg / mL or more and 42 μg / mL or less.

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