Reagent and method for measuring blood coagulation factor activity

Abstract

To provide a measurement reagent and a measurement method for coagulation factor activity with improved accuracy, which is not affected by a coagulation time measurement reagent in blood coagulation factor activity measurement.SOLUTION: A measurement reagent comprises: a standard product; a standard product diluent; a specimen diluent; plasma with deficient blood coagulation factors whose activity is to be measured; and a coagulation time measurement reagent. The standard product diluent contains the plasma with the deficient blood coagulation factors whose activity is to be measured.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a reagent for measuring blood coagulation factor activity and a method for measuring blood coagulation factor activity. [Background technology]

[0002] A widely used method for measuring blood coagulation factor activity is the one-stage coagulation method, which is based on the clotting time assay. The reagent composition is based on three components: a clotting time assay reagent, a sample diluent, and clotting factor-deficient plasma (see Japanese Patent Publication Nos. 2002-90361 and 2006-119126). One problem with coagulation factor activity assay reagents is that they are affected by the composition of the clotting time assay reagent (PT / APTT). Specifically, it has been observed that the accuracy (dilution linearity) of assays is impaired due to the influence of the matrix in the sample, depending on the reagent composition. This effect is particularly pronounced in the low range of coagulation factor activity. This low range presents a significant challenge because accuracy is critical for clinical significance. To address this issue, a method for reducing the sample volume, which avoids the influence of the matrix in the sample and improves dilution linearity, is known. However, this method falls below the lower limit of dispensing accuracy possible with automated analyzers, making it difficult to achieve highly accurate measurements. Therefore, a method for improving dilution linearity without changing the sample volume is needed. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Japanese Patent Application Laid-Open No. 2002-90361 [Patent Document 2] Japanese Patent Application Laid-Open No. 2006-119126 Summary of the Invention [Problem to be solved by the invention]

[0004] In view of such a situation, an object of the present invention is to provide a coagulation factor activity measurement reagent and a measurement method with improved accuracy that are not affected by a coagulation time measurement reagent in the measurement of coagulation factor activity.

Means for Solving the Problems

[0005] As a result of intensive studies to solve the above problems, the present inventors have found that the dilution linearity can be improved by using plasma lacking a coagulation factor in the standard diluent. However, usually, in the measurement of coagulation time performed by an automatic analyzer, the dilution of the standard for calibration curve creation is carried out in the reaction cuvette. Generally, a sample diluent is used for diluting the standard. While the standard is discharged into the reaction cuvette by the sample nozzle, the sample diluent is discharged into the reaction cuvette by the reagent nozzle. However, when using plasma lacking a coagulation factor for diluting the standard, if the plasma lacking a coagulation factor is aspirated and discharged by the reagent nozzle, there is a possibility that plasma components may interfere when the coagulation time measurement reagent is aspirated and discharged thereafter, resulting in a problem that the measurement accuracy is impaired. Therefore, by preparing a dilution series of the standard and the standard diluent with the sample nozzle in the sample rack in advance and measuring the mixed sample to create a calibration curve, it becomes possible to use even a solution that may affect the measurement reagent as the standard diluent, leading to the present invention.

[0006] That is, the present invention relates to the following inventions: [1] A standard diluent for a reagent for measuring coagulation factor activity, comprising plasma lacking the coagulation factor to be measured for activity measurement, said standard diluent. [2] A reagent for measuring coagulation factor activity, comprising a standard, a standard diluent, a sample diluent, plasma lacking the coagulation factor to be measured for activity measurement, and a coagulation time measurement reagent, wherein said standard diluent comprises plasma lacking the coagulation factor to be measured for activity measurement. [3] The reagent for measuring coagulation factor activity according to [2], wherein said standard diluent is plasma lacking the coagulation factor to be measured for activity measurement. [4] Prepare a mixed sample by mixing a standard product and a standard product diluent at a predetermined ratio. Measure the coagulation time of the mixed sample. Create a calibration curve based on the measured values of the mixed sample. Mix a specimen, a specimen diluent, plasma lacking a blood coagulation factor to be measured for activity, and a coagulation time measurement reagent, and measure the coagulation time. Calculate the activity of the blood coagulation factor in the specimen based on the calibration curve. A method for measuring the activity of a blood coagulation factor, including the steps above, wherein the standard product diluent contains plasma lacking the blood coagulation factor to be measured for activity. [5] The method for measuring the activity of a blood coagulation factor according to [4], wherein the standard product diluent is plasma lacking the blood coagulation factor to be measured for activity. [6] The method for measuring the activity of a blood coagulation factor according to [4] or [5], wherein the step of preparing the mixed sample is performed by a sample nozzle unit of an analyzer for measuring coagulation time.

Advantages of the Invention

[0007] According to the coagulation factor activity measurement reagent and measurement method of the present invention, it is possible to improve the dilution linearity in the low-value region of coagulation factor activity measurement without changing the amount of specimen used for coagulation time measurement and coagulation factor activity measurement.

Brief Description of the Drawings

[0008]

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BEST MODE FOR CARRYING OUT THE INVENTION

[0009] The reagent for measuring blood coagulation factor activity according to the present invention includes at least (1) a standard product, (2) a standard product diluent, (3) a sample diluent, (4) plasma lacking the blood coagulation factor to be the subject of activity measurement (hereinafter sometimes referred to as deficient plasma), and (5) a coagulation time measurement reagent. Its characteristic is that the above - mentioned (2) standard product diluent contains plasma lacking the blood coagulation factor to be the subject of activity measurement. That is, in the conventional reagent for measuring blood coagulation factor activity, usually, the standard product is diluted with a sample diluent (buffer solution), but the present invention is characterized in that plasma lacking the blood coagulation factor to be the subject of activity measurement is used for diluting the standard product. In the present invention, (2) the standard product diluent and (4) the deficient plasma can be provided in separate containers, or can be provided in the same container and aliquoted at the time of use.

[0010] The blood coagulation factor to be the subject of activity measurement in the present invention is not particularly limited as long as it is a blood coagulation factor for which a method of measuring activity using a coagulation time measurement reagent is known. For example, when using an activated partial thromboplastin time (APTT) measurement reagent, factor VIII, factor IX, factor XI, and factor XII can be mentioned. When using a prothrombin time (PT) measurement reagent, factor II, factor V, factor VII, and factor X can be mentioned.

[0011] As the specimen in the present invention, a normal specimen used for measuring the activity of blood coagulation factors, for example, plasma added with an anticoagulant (particularly sodium citrate) can be used.

[0012] The measurement reagent of the present invention can have the same configuration as a conventionally known measurement reagent for blood coagulation factor activity, except that the standard diluent contains plasma lacking the blood coagulation factor to be measured for activity.

[0013] The standard product (standard for calibration curve creation) used in the present invention is not particularly limited as long as it contains a predetermined amount of the blood coagulation factor to be measured for activity. For example, in addition to commercially available standard blood and standard plasma, blood specimens such as blood collected from healthy individuals and plasma prepared from the blood can be mentioned. Also, it may be the blood coagulation factor itself to be measured for activity. For example, a natural product purified from blood, a recombinant prepared by genetic recombination technology, etc. can also be used.

[0014] As the specimen diluent used in the present invention, a buffer solution can usually be used, and if desired, for example, a stabilizer, a preservative, a sensitizer, etc. can be added.

[0015] The coagulation time measurement reagent used in the present invention can be appropriately selected according to the blood coagulation factor to be measured for activity. Each coagulation time measurement reagent can be added with, if desired, for example, a preservative, a stabilizer, a sensitizer, etc. in addition to the main components shown below.

[0016] The APTT measurement reagent (kit) that can be used for measuring the activity of factor VIII, factor IX, factor XI, or factor XII is, for example, a combination of a synthetic phospholipid and an APTT reagent containing elagic acid or colloidal silica as an activator and a calcium chloride solution. The APTT reagent is added to the plasma specimen to activate the contact factor system, and then calcium chloride is added to initiate the coagulation reaction, and the time until fibrin precipitation can be determined. The APTT measurement reagent (kit) can usually be provided in a two-reagent system.

[0017] PT measurement reagents that can be used for measuring the activities of Factor II, Factor V, Factor VII, or Factor X include, for example, those containing human tissue thromboplastin and calcium chloride. By adding human tissue thromboplastin and calcium chloride to a plasma specimen to initiate a coagulation reaction, the time until fibrin precipitation can be determined. The PT measurement reagent is a single reagent system, but it can also be a two-reagent system consisting of a human tissue thromboplastin solution and a calcium chloride solution. Also, as the human tissue thromboplastin, natural human tissue thromboplastin can be used, or recombinant human tissue thromboplastin prepared by genetic recombination technology can also be used.

[0018] The "plasma lacking a blood coagulation factor to be measured for activity" used in the present invention means plasma that does not contain the blood coagulation factor to be measured for activity and contains sufficiently factors related to fibrin formation (blood coagulation) (excluding the above-mentioned blood coagulation factor to be measured for activity).

[0019] For example, when the factor to be measured for activity is Factor VIII, examples of plasma lacking Factor VIII (Factor VIII-deficient plasma) include plasma that sufficiently contains at least Factor IX, Factor XI, and Factor XII. More specifically, plasma derived from patients with congenital Factor VIII deficiency (hemophilia A) or plasma from which Factor VIII has been removed from normal plasma by immunological methods or the like can be mentioned. When a specimen plasma is added to Factor VIII-deficient plasma that sufficiently contains factors related to blood coagulation other than Factor VIII, a correction effect on APTT (coagulation time) is recognized in proportion to the Factor VIII activity in the specimen plasma. Therefore, by measuring the APTT of the specimen, the Factor VIII activity of the specimen can be calculated and determined based on a calibration curve prepared using a standard product.

[0020] For example, when the target of activity measurement is factor IX, examples of plasma lacking factor IX (factor IX-deficient plasma) include plasma that sufficiently contains at least factor VIII, factor XI, and factor XII. More specifically, it can include plasma from patients with congenital factor IX deficiency (hemophilia B), or plasma from which factor IX has been removed from normal plasma by immunological methods or the like. When the sample plasma is added to factor IX-deficient plasma that sufficiently contains factors related to blood coagulation other than factor IX, a correction effect on APTT (coagulation time) is recognized in proportion to the activity of factor IX in the sample plasma. Therefore, by measuring the APTT of the sample, the activity of factor IX in the sample can be calculated and determined based on the calibration curve prepared using the standard product.

[0021] For example, when the target of activity measurement is factor II, factor V, factor VII, or factor X, examples of plasma lacking factor II, factor V, factor VII, or factor X include plasma from patients with congenital factor II deficiency, congenital factor V deficiency, congenital factor VII deficiency, or congenital factor X deficiency, respectively. When the sample plasma is added to deficient plasma that sufficiently contains factors related to blood coagulation other than the blood coagulation factor to be measured, a correction effect on PT (coagulation time) is recognized in proportion to the activity of the factor to be measured in the sample plasma. Therefore, by measuring the PT of the sample, the activity of the factor to be measured in the sample can be calculated and determined based on the calibration curve prepared using the standard product.

[0022] The "plasma lacking the blood coagulation factor to be measured for activity measurement" used in the present invention can be added with preservatives, stabilizers, etc., if desired.

[0023] The standard product diluent used in the present invention, in one aspect, includes "plasma lacking the blood coagulation factor to be measured for activity", and in another aspect, consists of "plasma lacking the blood coagulation factor to be measured for activity" (that is, it is "plasma lacking the blood coagulation factor to be measured for activity"). Regarding the term "plasma lacking the blood coagulation factor to be measured for activity", the description regarding the above-mentioned "plasma lacking the blood coagulation factor to be measured for activity", which is one of the reagents constituting the measurement reagent of the present invention, can be applied.

[0024] In the present invention, the above-mentioned "standard diluent" and the above-mentioned "plasma lacking the blood coagulation factor to be measured for activity", which is one of the reagents constituting the measurement reagent of the present invention, may be completely identical in terms of the types of contained components and their blending ratios, or at least one of them may be different. In terms of substantially reducing the types of reagents constituting the measurement reagent of the present invention, it is preferable that they are completely identical in terms of the types of contained components and their blending ratios.

[0025] In addition, when the types of contained components and their blending ratios are completely identical, the above-mentioned "standard diluent" and the above-mentioned "plasma lacking the blood coagulation factor to be measured for activity", which is one of the reagents constituting the measurement reagent of the present invention, can be provided in separate containers, or can be provided in the same container and aliquoted at the time of use. In the former case, there is an advantage that it is not necessary to aliquot at the time of use. In the latter case, there is an advantage that the number of reagents constituting the measurement reagent of the present invention can be reduced.

[0026] The standard product diluent used in the present invention can be added with preservatives, stabilizers, sensitizers, etc., if desired, in addition to the main component "plasma lacking the blood coagulation factor to be measured for activity".

[0027] One aspect of the blood coagulation factor activity measurement reagent according to the present invention, the measurement reagent for blood coagulation factor VIII, includes a standard product (blood coagulation factor VIII), a standard product diluent, a sample diluent, factor VIII-deficient plasma, and an APTT measurement reagent (kit), and the standard product diluent contains factor VIII-deficient plasma. The APTT measurement reagent (kit) generally consists of an APTT reagent and a calcium chloride solution.

[0028] Another aspect of the blood coagulation factor activity measurement reagent according to the present invention, the measurement reagent for blood coagulation factor IX, includes a standard product (blood coagulation factor IX), a standard product diluent, a sample diluent, factor IX-deficient plasma, and an APTT measurement reagent (kit), and the standard product diluent contains factor IX-deficient plasma. The APTT measurement reagent (kit) generally consists of an APTT reagent and a calcium chloride solution.

[0029] The activity of blood coagulation factor VIII or blood coagulation factor IX can be determined by preparing a mixed sample by mixing a standard product and a standard product diluent at a predetermined ratio, measuring the clotting time of the mixed sample using factor-deficient plasma and an APTT measurement reagent (kit) to create a calibration curve, mixing a sample, a sample diluent, factor-deficient plasma, and an APTT measurement reagent (kit) to measure the clotting time of the sample, and converting it to activity based on the calibration curve.

[0030] The method for measuring blood coagulation factor activity according to the present invention includes a step of preparing a mixed sample by mixing a standard product and a standard product diluent at a predetermined ratio (mixed sample preparation step), a step of measuring the clotting time of the mixed sample (mixed sample measurement step), a step of creating a calibration curve based on the measured value of the mixed sample (calibration curve creation step), a step of mixing a sample, a sample diluent, plasma lacking the blood coagulation factor to be measured for activity, and a clotting time measurement reagent, and measuring the clotting time (sample measurement step), a step of calculating the activity of the blood coagulation factor of the sample based on the calibration curve (blood coagulation factor activity calculation step) including, wherein the standard sample diluent contains plasma lacking a blood coagulation factor to be measured for activity.

[0031] In the measurement method of the present invention, with respect to the terms "blood coagulation factor", "standard sample", "standard sample diluent", "specimen", "specimen diluent", "plasma lacking a blood coagulation factor to be measured for activity", and "coagulation time measurement reagent", the descriptions previously mentioned in the measurement reagent of the present invention can be directly applied as they are.

[0032] In the mixed sample preparation step of the method of the present invention, mixed samples with various dilution ratios (for example, 100% / 50% / 25% / 10% / 5% / 2% / 1%) are prepared by mixing the standard sample and the standard sample diluent at a predetermined ratio. Since the standard sample diluent contains (or is) deficient plasma, when measuring the activity of the blood coagulation factor using an automatic analyzer, instead of a reagent nozzle unit that collects the reagent from a reagent bottle or the like and discharges it into a cuvette, a sample nozzle unit that collects the sample from a blood collection tube or the like and discharges it into a cuvette is used to aspirate and discharge the standard sample diluent. The reason for this is that if the standard sample diluent and the coagulation time measurement reagent described later are aspirated and discharged with the same nozzle, the reaction will start in the nozzle.

[0033] In the mixed sample measurement step of the method of the present invention, each mixed sample prepared in the mixed sample preparation step, the specimen diluent, the plasma lacking the blood coagulation factor to be measured for activity, and the coagulation time measurement reagent are mixed, and the coagulation time is measured.

[0034] In the calibration curve creation step of the method of the present invention, a calibration curve is created based on the measurement values obtained in the mixed sample measurement step.

[0035] In the specimen measurement step of the method of the present invention, the specimen, the specimen diluent, the plasma lacking the blood coagulation factor to be measured for activity, and the coagulation time measurement reagent are mixed, and the coagulation time is measured.

[0036] In the blood coagulation factor activity calculation step in the method of the present invention, the activity of the blood coagulation factor in the sample is calculated by converting each measured value of the sample obtained in the sample measurement step into the activity of the blood coagulation factor in the sample based on the calibration curve.

Example

[0037] Hereinafter, the present invention will be specifically described by way of examples, but these do not limit the scope of the present invention. All the measurements shown in the examples and comparative examples were performed using the blood coagulation test system STACIA CN10 (manufactured by LSI Medience Corporation).

[0038] 《Example 1: Preparation of calibration curve for measuring factor VIII coagulation》 STACIA CN10 has a multi-point dilution function for preparing a dilution series in a sample cup installed in a sample rack using a sample nozzle unit. Using this function, samples were prepared by mixing CoagGenesis Factor VIII (manufactured by LSI Medience Corporation) (hereinafter referred to as CG-FVIII), which is plasma lacking factor VIII coagulation, and CoagGenesis Calibrator (manufactured by LSI Medience Corporation), which is normal plasma, at various dilution rates (100% / 50% / 25% / 10% / 5% / 2% / 1%).

[0039] For the measurement of factor VIII coagulation activity, CoagGenesis APTT (manufactured by LSI Medience Corporation) was used as the APTT time measurement reagent. 5 μL of the prepared sample and 20 μL of CoagGenesis common diluent (manufactured by LSI Medience Corporation) were placed in a cuvette, to which 25 μL of CG-FVIII and 50 μL of APTT reagent were added, and after heating at 37°C for 3.5 minutes, 50 μL of calcium chloride solution was added, and the change in absorbance was measured at a wavelength of 660 nm while heating at 37°C to determine the time until fibrin precipitation. The measurement was performed with a multiplicity of 2, and a calibration curve was created using the average value of the results. The regression of the calibration curve used a broken line.

[0040] 《Comparative Example 1: Preparation of calibration curve for measuring factor VIII coagulation》 The calibration was performed according to the method of Example 1, except that the coagulation genesis common diluent (manufactured by LSI Medience Corporation) (HEPES 50 mmol / L, NaCl 150 mmol / L, pH 7.5) was used for diluting the calibrator.

[0041] 《Dilution linearity of factor VIII activity》 Coagulation genesis control I (manufactured by LSI Medience Corporation), which is normal control plasma, was diluted in 10 steps using CG-FVIII, and the factor VIII activity was measured at a multiplicity of 2 for each. Using the calibration curves prepared in Example 1 and Comparative Example 1, the respective factor VIII activities were calculated and the results of comparing the dilution linearity are shown in Fig. 1. Also, the measured value / theoretical value (%) at each dilution rate is shown in Fig. 2. In Comparative Example 1, the dissociation between the measured value and the theoretical value increased as the activity decreased, but in Example 1, the linearity was good. From this, it was found that by diluting the calibrator using plasma lacking coagulation factor VIII, it is possible to accurately measure the factor VIII activity up to the low value range.

[0042] 《Example 2: Preparation of calibration curve for measuring coagulation factor IX》 Samples were prepared by mixing coagulation genesis factor IX (manufactured by LSI Medience Corporation) (hereinafter referred to as CG-FIX), which is plasma lacking coagulation factor IX, and coagulation genesis calibrator (manufactured by LSI Medience Corporation), which is normal plasma, at various dilution rates (125% / 100% / 50% / 25% / 10% / 5% / 2% / 1%).

[0043] For the measurement of factor IX coagulant activity, Coaggenesis APTT (manufactured by LSI Medience) was used as the APTT time measurement reagent. 5 μL of the prepared sample and 20 μL of Coaggenesis common diluent (manufactured by LSI Medience) were placed in a cuvette, to which 25 μL of CG-FIX and 50 μL of APTT reagent were added. After heating at 37°C for 3.5 minutes, 50 μL of calcium chloride solution was added, and the change in absorbance was measured at a wavelength of 660 nm while heating at 37°C to determine the time until fibrin precipitation. The measurement was performed with a multiplicity of 2, and a calibration curve was created using the average value of the results. The regression of the calibration curve used a broken line.

[0044] 《Comparative Example 2: Preparation of a calibration curve for measuring factor IX coagulant》 The procedure of Example 2 was followed except that Coaggenesis common diluent (LSI Medience) (HEPES 50 mmol / L, NaCl 150 mmol / L, pH 7.5) was used for diluting the calibrator.

[0045] 《Dilution linearity of factor IX activity》 Coaggenesis Control I (manufactured by LSI Medience), which is normal control plasma, was diluted 10-fold using CG-FIX, and the factor IX activity was measured with a multiplicity of 2 for each. Using the calibration curves created in Example 2 and Comparative Example 2, the factor IX activities were calculated for each, and the results of comparing the dilution linearity are shown in Figure 3. Also, the measured value / theoretical value (%) at each dilution rate is shown in Figure 4. In Comparative Example 2, the dissociation between the measured value and the theoretical value increased as the activity decreased, but in Example 2, the linearity was good. From this, it was found that by diluting the calibrator using plasma lacking factor IX coagulant, it is possible to accurately measure the factor IX activity even in the low-value region.

Industrial applicability

[0046] The present invention can be used for measuring blood coagulation factor activity.

Claims

1. (1) In an analyzer for measuring coagulation time, a step of preparing a plurality of mixed samples with various dilution ratios in which a standard product and a standard product diluent are mixed at a predetermined ratio, using a sample nozzle unit for collecting a sample instead of a reagent nozzle unit for collecting a coagulation time measurement reagent; (2) A step of measuring the coagulation time of the mixed sample; (3) A step of creating a calibration curve based on the measured values of the mixed sample; (4) A step of mixing (a) a specimen, (b) a specimen diluent, (c) a plasma lacking a blood coagulation factor to be measured for activity, which is provided in a container different from the standard product diluent, and (d) a coagulation time measurement reagent, and measuring the coagulation time; (5) A step of calculating the activity of the blood coagulation factor of the specimen based on the calibration curve The method for measuring blood coagulation factor activity, comprising: wherein the standard product diluent contains plasma lacking the blood coagulation factor to be measured for activity; The method for measuring blood coagulation factor activity according to claim 1, wherein the step (1) of preparing the mixed sample is performed on a sample rack on which a plurality of sample cups used for mixing at a predetermined ratio in an analyzer for measuring coagulation time can be installed.

2. The method for measuring blood coagulation factor activity according to claim 1, wherein the standard product diluent is plasma lacking the blood coagulation factor to be measured for activity.

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