JP7410652B2 - Method for quantifying fibrinogen - Google Patents

Description

The present invention relates to a method for quantitatively determining fibrinogen using a dry fibrinogen reagent.

Quantification of fibrinogen, along with prothrombin time and activated partial thromboplastin time, is a test to determine abnormality or normality of blood coagulation ability, and is widely performed in clinical settings, particularly in clinical laboratories. In recent years, the importance of fibrinogen quantification has been once again pointed out in perioperative medicine and perinatal medicine. In critical massive bleeding, the concentration of fibrinogen in the blood decreases significantly. Therefore, the fibrinogen concentration in the patient's blood is checked, and if it is less than 150 mg/dL, fresh frozen plasma or fibrinogen concentrates are administered to keep the patient alive. It is also necessary to confirm whether the blood fibrinogen concentration has returned to the normal range after administration of fresh frozen plasma or fibrinogen concentrate. This measurement must be particularly rapid, since if the fibrinogen concentration in the blood does not reach the normal range after the procedure, further treatment will be required to keep the patient alive. Since fibrinogen quantification is used for such purposes in perioperative medicine and perinatal medicine, there has been a desire for a system that can more quickly and accurately determine blood fibrinogen concentration.

A commonly used method for quantifying fibrinogen using a thrombin reagent solution is the thrombin time method discovered by Clauss VA (Clauss VA: Gerinnungsphysiologische schnellmethode zur bestimmung des fibrinogens, Acta Haematologica, 17, 237-246, 1957). be. The thrombin time method utilizes the fact that the rate of conversion of fibrinogen to fibrin by an excess amount of thrombin depends primarily on the fibrinogen concentration.

This quantitative method involves diluting plasma with an arbitrary buffer solution, prewarming this diluted solution, adding a reagent solution containing thrombin, measuring the clotting time, and using the obtained clotting time with a pre-prepared calibration curve. This method is used to convert the fibrinogen concentration into fibrinogen concentration. The clotting time in this quantitative method refers to the time from the addition of the thrombin reagent solution to the end point. The end point is detected by an optical measurement that detects an increase in turbidity or a physical measurement that detects an increase in viscosity.

This quantitative method and the thrombin reagent used in this quantitative method are widely accepted in the world and are practiced in clinical laboratories. However, the lyophilized thrombin reagent must be reconstituted with purified water each time it is used (reconstituted solutions cannot be stored for long periods of time), and whole blood must be centrifuged to form plasma. This method of quantification is difficult because it takes time to perform measurements and involves many steps, such as the need to dilute the plasma with a diluent and the need to pre-warm the diluted plasma. , it was not necessarily a quantitative method suitable for use in the perioperative and perinatal period.

An improved method of quantifying fibrinogen described above is a method of quantifying fibrinogen using a dry reagent containing thrombin. The method is shown in JP-A-06-094725 (Patent No. 2776488) and JP-A-06-141895 (Patent No. 2980468). The dry reagent containing thrombin used in this quantitative method is obtained by adding magnetic particles to a thrombin reagent solution, dispensing a certain amount of the mixed solution onto a reaction slide, and then freeze-drying.

In the quantitative method using the dry reagent, after adding the sample to the reagent, a combination of an oscillating magnetic field and a static permanent magnetic field is applied at predetermined intervals to move the magnetic particles contained in the dry reagent. The unique feature is that the motion signal of the object is captured as the amount of change in scattered light, and the end point is detected from the change over time. In this method, the time from the addition of the sample to the end point is defined as the coagulation time, and the obtained coagulation time is converted into the fibrinogen concentration using a calibration curve prepared in advance.

An example of an analyzer that can use this method is the product name CG02N (manufactured by A&T Co., Ltd.). In the case of the above device, a combination of an oscillating magnetic field and a stationary permanent magnetic field is applied at 0.5 second intervals, and magnetic particle motion signals are monitored at the same intervals.

When using the above device, the change in the motion signal of the magnetic particles over time corresponds inversely to the change in viscosity in the dry reagent (inverse correlation). The end point is detected as the point at which the magnetic particle motion signal has attenuated by 30% with respect to the peak value. The peak value of the magnetic particle movement signal obtained immediately after adding the sample is the point at which all the constituents in the dry reagent are dissolved and the viscosity of the dry reagent is at its minimum value. Here, if the peak value of the motion signal is X, and the signal value after an arbitrary time has elapsed is Y, then the viscosity increase when the signal intensity attenuation becomes (X-Y) x 100/X (%) is , corresponds to the point where the viscosity is X/Y times the minimum value. That is, the point where the magnetic particle motion signal attenuated by 30% with respect to the peak value corresponds to the point where the viscosity increased by 1.43 times with respect to the minimum value of viscosity after adding the specimen.

JP-A-06-141895 (Japanese Patent No. 2980468) discloses the above technique by mixing a sample with a fibrinogen quantitative dry reagent containing a protein having thrombin activity and magnetic particles, and measuring the coagulation time. In the method of quantifying fibrinogen in a specimen, the end point is the point at which the viscosity of the dry reagent increases 1.05 to 2.00 times its minimum value, and the time from the addition of the specimen to the end point is defined as the coagulation time. It is described as a method for quantifying fibrinogen.

This method is useful in that it is not necessary to reconstitute the lyophilized thrombin reagent into purified water or the like each time it is used, and there is no need to pre-warm the diluted specimen. However, this quantitative method requires that plasma and whole blood samples be diluted with a special diluent, which is not necessarily sufficient as a quantitative method used in perioperative and perinatal medical care.

When fibrinogen is quantified using the dry reagent containing thrombin shown in JP-A-06-094725 (Patent No. 2776488), the clotting time obtained is extremely short when measuring undiluted plasma or undiluted whole blood. The coagulation time corresponding to the blood fibrinogen concentration cannot be detected. Therefore, in order to obtain a coagulation time that corresponds to the blood fibrinogen concentration, it was necessary to extend the coagulation time.

JP-A-06-094725 (Patent No. 2776488) JP 06-141895 (Patent No. 2980468)

Clauss VA: Gerinnungsphysiologische schnellmethode zur bestimmung des fibrinogens, Acta Haematologica, 17, 237-246, 1957.

The present inventors added an amino acid or a salt thereof or a saccharide to improve the solubility of the dry reagent, and added highly active thrombin or a highly active thrombin-like protein to strongly promote the thrombin reaction. We have completed a fibrinogen quantitative drying reagent containing a fibrin monomer association inhibitor to inhibit the natural association of fibrinogen (Japanese Patent Application No. 2018-229919). Japanese Patent Application No. 2018-229919 describes that an extended coagulation time can be obtained without deteriorating the reproducibility of the coagulation time.

However, when the present inventors applied the dry reagent described in the above-mentioned Japanese Patent Application No. 2018-229919 to the quantitative method of the above-mentioned Japanese Patent Application Laid-Open No. 6-141895 (Patent No. 2980468), when undiluted plasma was measured. It was found that although undiluted whole blood can be quantified correctly, it may not be possible to quantitate correctly even after hematocrit correction when measuring undiluted whole blood. Although we do not wish to be bound by any particular theory, this is because when measuring undiluted whole blood, not only the amount of plasma components fluctuates due to differences in the hematocrit values of the samples, but also the differences in the viscosity of each sample. This is thought to be due to variations in the solubility of the dry fibrinogen quantitative reagent. It was also discovered that when measuring undiluted whole blood, fibrinogen cannot be quantified correctly unless the starting point (the starting point of the clotting reaction: the point at which clotting time is 0 seconds) is defined. This is a problem that has not been recognized in the prior art.

The problem that the invention seeks to solve is to at least partially solve the above problem. More specifically, the problem to be solved by the present invention is to use a new technology suitable for use in perioperative medicine and perinatal medicine, namely, to analyze plasma or whole blood specimens using a fibrinogen quantitative dry reagent. An object of the present invention is to provide a method for quantifying fibrinogen that does not require a dilution operation and can accurately quantify fibrinogen.

The present inventors have conducted intensive research to solve the above technical problems. As a result, they discovered that the starting point (the starting point of the coagulation reaction: the point at which the coagulation time is 0 seconds) can be determined by adding a sample to the dry fibrinogen quantitative reagent and analyzing the resulting magnetic particle movement signal. Specifically, the inventors discovered that the starting point can be detected as an arbitrary point in an interval in which the ratio of magnetic particle movement signals at a certain time interval is maintained within a certain range for a certain period of time by calculating a plurality of magnetic particle motion signal ratios at a certain time interval. completed.

The present invention includes the following embodiments.
[1] A method for quantifying fibrinogen, comprising:
(i) a step of adding a specimen to a fibrinogen quantitative dry reagent containing magnetic particles;
(ii) moving the magnetic particles in the reagent and monitoring the magnetic particle movement signal after addition of the analyte; and
(iii) calculating a plurality of magnetic particle motion signal ratios at fixed time intervals for the magnetic particle motion signals monitored in step (ii);
including;
Starting point is any point in the interval where the magnetic particle motion signal ratio for the above fixed time interval is maintained within a certain range for a certain period of time, and 5 to 50% relative to the peak value of the magnetic particle motion signal after the starting point. An arbitrary point among the points attenuated by % is the end point, and the time from the starting point to the end point is the solidification time.
The method for quantifying fibrinogen.
[2] The method for quantifying fibrinogen according to Embodiment 1, wherein the time interval used to calculate the magnetic particle motion signal ratio is a fixed time interval selected from 0.1 seconds to 2 seconds.
[3] The method for quantifying fibrinogen according to Embodiment 1 or 2, wherein the time interval used to calculate the magnetic particle motion signal ratio is 0.5 seconds, 1 second, 1.5 seconds, or 2 seconds.
[4] The method for quantifying fibrinogen according to Embodiment 1 or 2, wherein the time interval used to calculate the magnetic particle motion signal ratio is 1 second.
[5] The method for quantifying fibrinogen according to Embodiment 1, wherein the fixed range of the magnetic particle motion signal ratio is 1.0±0.2.
[6] The method for quantifying fibrinogen according to Embodiment 2, wherein the fixed range of the magnetic particle motion signal ratio is 1.0±0.1.
[7] The method for quantifying fibrinogen according to any one of Embodiments 1 to 6, wherein the time interval during which the magnetic particle motion signal ratio is maintained within a certain range is 1.5 seconds.
[8] The method for quantifying fibrinogen according to any one of Embodiments 1 to 7, wherein the starting point is the first point of a time interval in which the magnetic particle motion signal ratio is maintained within a certain range.
[9] The method for quantifying fibrinogen according to any one of Embodiments 1 to 8, wherein the end point is any point among the points at which the peak value of the magnetic particle movement signal after the starting point is attenuated by 20 to 30%.
[10] The method for quantifying fibrinogen according to Embodiment 9, wherein the end point is a point at which the peak value of the magnetic particle movement signal after the starting point is attenuated by 30%.
[11] The method for quantifying fibrinogen according to Embodiment 9, wherein the end point is a point at which the peak value of the magnetic particle movement signal after the starting point is attenuated by 20%.
[12] A program for executing the method for quantifying fibrinogen according to any one of Embodiments 1 to 11.
[13] An information recording medium on which the program described in Embodiment 12 is recorded.
[14] A fibrinogen quantitative measurement device incorporating the program described in Embodiment 12 or storing the information recording medium described in Embodiment 13.

The present invention enables rapid and accurate fibrinogen quantification without requiring reagent preparation or specimen dilution operations.

This is an example of a typical reaction slide used for the dry fibrinogen quantitative reagent. Figure 2 is a partially exploded view of the reaction slide of Figure 1; This is a calibration curve obtained by a conventional quantitative method in Example 2 (Comparative Example 2). It is a calibration curve obtained by the quantitative method of the present invention in Example 2 (the present invention). These are the results of a correlation test between fibrinogen quantitative values determined by the Clauss method and fibrinogen quantitative values determined by the quantitative method of the present invention in Example 3. These are the results of a correlation test between the fibrinogen quantitative value when the measurement sample in Example 4 was citrated plasma and the fibrinogen quantitative value when the measurement sample was citrated whole blood. Regarding the quantitative method of the present invention, an example will be shown in which the monitoring cycle of the magnetic particle motion signal, the calculation cycle of the magnetic particle motion signal ratio, and the time interval used for calculating the magnetic particle motion signal ratio are the same. Regarding the quantitative method of the present invention, an example will be shown in which the monitoring cycle of the magnetic particle motion signal and the calculation cycle of the magnetic particle motion signal ratio are the same, and the time intervals used for calculating the magnetic particle motion signal ratio are different. Regarding the quantitative method of the present invention, an example will be shown in which the monitoring cycle of the magnetic particle motion signal, the calculation cycle of the magnetic particle motion signal ratio, and the time interval used for calculating the magnetic particle motion signal ratio are all different. Regarding the quantitative method of the present invention, an example in which the monitoring cycle of magnetic particle motion signals is changed will be shown. Regarding the quantitative method of the present invention, an example will be shown in which both the monitoring period of the magnetic particle movement signal and the calculation period of the magnetic particle movement signal ratio are changed. Regarding the quantitative method of the present invention, an example will be shown in which the ratio of magnetic particle movement signals is calculated continuously and then calculated intermittently. Regarding the quantitative method of the present invention, an example will be shown in which the ratio of magnetic particle motion signals is calculated intermittently and then continuously.

In certain embodiments, the invention provides a method for fibrinogen quantification that is amenable to perinatal and perioperative use. In another embodiment, the present invention provides a step of (i) adding a sample to a fibrinogen quantitative dry reagent containing magnetic particles, (ii) moving the magnetic particles in the reagent after adding the sample, and generating a magnetic particle movement signal. and (iii) calculating a magnetic particle motion signal ratio at a fixed time interval for the magnetic particle motion signals monitored in step (ii). A plurality of magnetic particle motion signal ratios at fixed time intervals can be calculated. At this time, an arbitrary point within the interval in which the magnetic particle motion signal ratio for the above fixed time interval is maintained within a certain range for a certain period of time is set as the starting point, and the peak value of the magnetic particle motion signal after the starting point is set as the starting point. Any point among the points attenuated by 5 to 50% can be set as the end point, and the time from the starting point to the end point can be set as the coagulation time. Steps (ii) and (iii) may be performed simultaneously.

As used herein, the term "magnetic particle movement signal" refers to the step (ii) in which a combination of an oscillating magnetic field and a stationary permanent magnetic field is applied at predetermined intervals after the addition of the sample to cause the magnetic particles contained in the reagent to move; It refers to the amount of change in scattered light when exposed to light (sometimes referred to as S _n in this specification). In this specification, for convenience, the magnetic particle movement signal observed at the time of reagent addition is referred to as S ₀ .

As used herein, the time at which a magnetic particle motion signal is monitored refers to a time point at which a magnetic particle motion signal is measured (sometimes referred to herein as mm _n ). Further, in the drawings, the time at which magnetic particle motion signals are monitored may be indicated by a black circle symbol. In this specification, for convenience, the time of sample addition is set to 0 seconds (mm ₀ ) as the time at which magnetic particle motion signals are monitored. Note that this is merely a convenience for establishing a standard, and as long as the clotting time is ultimately calculated by the method of the present invention, the time point of sample addition may be set appropriately, such as -5 seconds. . In certain embodiments, monitoring of magnetic particle motion signals may be continuous or intermittent.

As used herein, the term "monitoring cycle of magnetic particle motion signals" refers to the cycle of monitoring magnetic particle motion signals, that is, the time interval during which magnetic particle motion signals are monitored. For example, if the times at which magnetic particle motion signals S ₀ , S ₁ , S ₂ , S ₃ , S ₄ . . . are monitored are mm ₀ , mm ₁ , mm ₂ , mm ₃ , mm ₄ , . The monitoring period of the movement signal can be expressed as (mm ₁ - mm ₀ ), (mm ₂ - mm ₁ ), (mm ₃ -mm ₂ ), (mm ₄ -mm ₃ ), . . . In certain embodiments, the monitoring period of the magnetic particle motion signal can be constant. In another embodiment, the monitoring period of the magnetic particle motion signal may be varied. The monitoring period of the magnetic particle motion signal is sometimes indicated by an arrow symbol in the drawings (←→). In certain embodiments, the monitoring period for magnetic particle motion signals may be selected from 0.1 seconds to 2 seconds.

In the method of the present invention, in step (iii), a magnetic particle motion signal ratio at a fixed time interval is calculated for the monitored magnetic particle motion signal in step (ii). In this specification, the time at which the ratio of magnetic particle movement signals is calculated refers to the timing at which the ratio of magnetic particle movement signals is calculated (sometimes referred to as mr _n in this specification). Further, in the drawings, the time at which the ratio of magnetic particle motion signals is calculated may be indicated by an open circle symbol. To illustrate with the measuring device of the present invention, the magnetic particle motion signal at the time of sample addition is measured (S ₀ ), and then, when the second magnetic particle motion signal (S ₁ ) is measured, (S ₁ /S ₀ ) can be calculated as a ratio of magnetic particle motion signals. In this specification, the time point at which the ratio of magnetic particle motion signals can be calculated is referred to as the time at which the ratio of magnetic particle motion signals is calculated. In reality, there is a slight time difference because the device performs calculation processing, and strictly speaking, the time when S ₁ is measured and the time mr ₁ at which the ratio of magnetic particle motion signals is calculated are different. However, in this specification, for convenience, the time when the ratio of magnetic particle motion signals can be calculated is defined as the time when the ratio of magnetic particle motion signals is calculated. Note that this does not mean that the device must immediately calculate the ratio of magnetic particle motion signals as soon as S ₁ is measured, that is, as soon as the ratio of magnetic particle motion signals can be calculated. do not have. For example, the device of the present invention temporarily stores the measurement signals in memory after S ₀ and S ₁ are measured, that is, after the ratio of magnetic particle motion signals can be calculated, and then, after a predetermined period of time, A ratio of magnetic particle motion signals may be calculated.

In this specification, the period for calculating the ratio of magnetic particle motion signals refers to the period for calculating the ratio of magnetic particle motion signals, that is, the time for calculating the ratio of an arbitrary first magnetic particle motion signal and the time for calculating the ratio of an arbitrary second magnetic particle motion signal. is the time interval between the time at which the ratio of magnetic particle motion signals is calculated. For example, the times at which the magnetic particle motion signals are monitored are mm ₀ , mm ₁ , mm ₂ , mm ₃ , mm ₄ , etc., and the times at which the ratio of the magnetic particle motion signals is calculated are mr ₁ , mr ₂ , If mr ₃ , mr ₄ ... and mm ₁ = mr ₁ , mm ₂ = mr ₂ , mm ₃ = mr ₃ , mm ₄ = mr ₄ ..., then the calculation period of the ratio of magnetic particle motion signals is It can be expressed as (mr ₂ − mr ₁ ), (mr ₃ − _{mr 2} ), (mr ₄ − mr ₃ ), etc. In the drawings, the calculation cycle of the ratio of magnetic particle motion signals is sometimes indicated by a thick white arrow symbol. In some embodiments, the calculation period of the ratio of magnetic particle motion signals can be constant. In another embodiment, the calculation period of the ratio of magnetic particle motion signals may be varied. In certain embodiments, the calculation period of the ratio of magnetic particle motion signals may be selected from 0.1 seconds to 2 seconds.

In the method of the present invention, the period of monitoring the magnetic particle movement signal and the period of calculating the ratio of the magnetic particle movement signal may be the same or different. For example, in one embodiment, both the period of monitoring the magnetic particle movement signal and the period of calculating the ratio of the magnetic particle movement signal may be 0.5 seconds, but the present invention is not limited thereto. For example, in another embodiment, the period for monitoring the magnetic particle movement signal may be 0.1 seconds, and the period for calculating the ratio of the magnetic particle movement signal may be 0.5 seconds, but the invention is not limited thereto.

In this specification, the time interval used to calculate the magnetic particle motion signal ratio refers to the time interval from the time when S ₁ is monitored to the time when S ₂ is monitored when calculating the signal ratio S ₂ /S ₁ . say. For example, the times at which magnetic particle motion signals are monitored are mm ₀ , mm ₁ , mm ₂ , mm ₃ , mm ₄ , etc., and the magnetic particle motion signals monitored at mm ₀ are monitored at S ₀ , mm ₁ . S ₁ is the magnetic particle motion signal monitored in mm ₂ , S ₂ is the magnetic particle _motion signal monitored in mm ₃ , S ₄ is the magnetic particle motion signal monitored in mm ₄ , ..., and the times at which the ratio of magnetic particle motion signals is calculated are mr ₁ , mr ₂ , mr ₃ , mr ₄ , ..., mm ₁ = mr ₁ , mm ₂ = mr ₂ , mm ₃ = mr ₃ , mm ₄ = mr ₄ , the signal ratio calculated by mr ₁ is S ₁ /S ₀ , the signal ratio calculated by mr ₂ is S ₂ /S ₁ , and the signal ratio calculated by mr ₃ is S If the signal ratio calculated by ₃ /S ₂ and mr ₄ is S ₄ /S ₃ , the time intervals used to calculate the magnetic particle motion signal ratio are (mm ₁ - mm ₀ ), (mm ₂ - mm ₁ ) , (mm ₃ - mm ₂ ), (mm ₄ - mm ₃ ), etc. In the drawings, the time interval used to calculate the magnetic particle motion signal ratio may be indicated by a black thick arrow symbol. Note that there may be another signal between S ₀ and S ₁ that is not used in calculating the signal ratio (S ₁ /S ₀ ). That is, in some embodiments, it is not necessary to use all measurement points (measurement signals) for calculation. In the method of the present invention, the time interval used to calculate the magnetic particle motion signal ratio is constant. That is, to explain using the above example, it can be expressed as (mm ₁ - mm ₀ )=(mm ₂ -mm ₁ )=(mm ₃ -mm ₂ )=(mm ₄ -mm ₃ )... In certain embodiments, the time interval used to calculate the magnetic particle motion signal ratio is a fixed time interval selected from 0.1 seconds to 2 seconds, such as a 0.5 second, 1 second, 1.5 second or 2 second interval, preferably The interval is 1 second.

Fibrinogen quantitative reagents used in the present invention include highly active thrombin or highly active thrombin-like protein, magnetic particles, heparin neutralizers, fibrin monomer association inhibitors, calcium salts, fibrinogen containing amino acids or their salts, or saccharides. Examples include quantitative dry reagents.

To illustrate the method for preparing a dry fibrinogen quantitative reagent, first, a buffer containing a fibrin monomer association inhibitor and an amino acid or a salt thereof or a saccharide is prepared, and then highly active thrombin or a highly active thrombin-like protein is added to the buffer. A method can be adopted in which the particles are dissolved in a solution, then magnetic particles are added to the solution to obtain a final solution, and a fixed amount of the final solution is dispensed onto an arbitrary reaction slide, frozen, and then lyophilized. The buffer may further include a heparin neutralizing agent and/or an antifoaming agent.

The reaction slide used in the above preparation method is not particularly limited as long as it can optically monitor the viscosity increase in the dry reagent for fibrinogen quantitative determination as attenuation of the motion signal of the magnetic particles during fibrinogen measurement. An example is a reaction slide as shown in FIGS. 1 and 2. FIG. 1 is a top view of the reaction slide. The part surrounded by the dotted line in FIG. 1 is a reaction cell part consisting of a final solution dispensing port and a sample addition port for preparing a fibrinogen quantitative dry reagent. FIG. 2 shows details of the structure of the reaction cell section. First, a transparent polyester plate B is laminated to a white polyester plate C, and then a transparent polyester plate A is laminated on top of the laminated transparent polyester plate B to form a reaction cell part. Configure. First, a surfactant aqueous solution is filled through the dispensing port shown in FIG. 1 and removed by suction to make the portion D hydrophilic. Thereafter, by injecting the final solution for fibrinogen quantitative dry reagent through the dispensing port, the portion D is filled with the final solution. When using this type of reaction slide, it is usually possible to dispense 20 to 30 μL of the final solution for the fibrinogen quantitative dry reagent described above. For a method for quantifying fibrinogen using such magnetic particles, see, for example, Patent Document 2. The entire contents of which are incorporated herein by reference.

A reaction slide as shown in FIG. 1 is sometimes referred to herein as a dry reagent card. That is, in certain embodiments, the fibrinogen quantitative dry reagent can be applied to a dry reagent card.

In certain embodiments, the dry reagent layer of the fibrinogen quantitative dry reagent preferably (i) dissolves quickly after the specimen is added; In certain embodiments, the dry reagent layer of the fibrinogen quantitative dry reagent preferably has (ii) no or substantially no difference in dissolution rate between the reagents. In certain embodiments, the dry reagent layer of the fibrinogen quantitative dry reagent preferably has (iii) impact resistance (also referred to as impact resistance). In certain embodiments, the fibrinogen quantitative dry reagent preferably has (iv) a uniform dry reagent layer. In certain embodiments, the fibrinogen quantitative dry reagent is preferably such that (v) the substances added to satisfy (i) to (iv) above do not affect the reaction or do not substantially affect the reaction. do not have. In certain embodiments, the dry fibrinogen quantitative reagent satisfies all of (i) to (v).

The content of each component in the fibrinogen quantitative dry reagent described below indicates the weight and activity per mL of the final solution dispensed onto the reaction slides shown in Figures 1 and 2, unless otherwise specified.

In certain embodiments, the fibrinogen quantitative dry reagent is
(i) thrombin or a protein with thrombin activity;
(ii) magnetic particles;
(iii) a fibrin monomer association inhibitor;
(iv) calcium salts;
(v) a dry reagent layer solubility improver;
(vi) dry reagent layer reinforcing material, and
(vii) pH adjuster (pH buffer)
Contains as an essential ingredient. In another embodiment, the dry fibrinogen quantitative reagent may further optionally include a heparin neutralizing agent and/or an antifoaming agent. In certain embodiments, the fibrinogen quantitative dry reagent is for measuring undiluted plasma or whole blood specimens.

As used herein, "undiluted whole blood" refers to whole blood that has not been diluted, such as by adding a dilution buffer to the whole blood sample after it has been collected. Therefore, even if blood is diluted with citric acid contained in the blood collection tube during blood collection (such blood is generally referred to as citrated whole blood), special dilution procedures are not required for whole blood after blood collection. If not, it shall correspond to undiluted whole blood as referred to herein. Therefore, undiluted whole blood includes citrated whole blood that has not been diluted and heparinized whole blood. Furthermore, as used herein, "undiluted plasma" refers to plasma that is a supernatant obtained by centrifuging undiluted whole blood, without further dilution operations such as adding a dilution buffer. Therefore, undiluted plasma includes citrated plasma and heparinized plasma that have not been diluted. Note that in this specification, undiluted and undiluted have the same meaning.

In certain embodiments, the dry fibrinogen quantitative reagent comprises thrombin or a protein with thrombin activity. In this specification, a protein having thrombin activity is sometimes referred to as a thrombin-like protein. As used herein, thrombin activity promotes both (i) the conversion of fibrinogen to fibrin monomer, and (ii) the activation of factor XIII to XIIIa in the presence of calcium ions. It refers to the activity that can be performed. Furthermore, a protein having such activity is referred to as a protein having thrombin activity. However, this does not mean that a single protein must proceed with both reactions (i) and (ii) above. That is, in a specific embodiment, the thrombin activity involves (i) a first protein that promotes the conversion reaction of fibrinogen to fibrin monomer, and (ii) a second protein that promotes the activation reaction of factor XIII to XIIIa. Mixtures can be used. An example of the first protein is snake thrombin (snake-derived thrombin-like enzyme). The second protein is thought to be a protein that specifically cleaves between arginine at position 37 and glycine at position 38 counting from the N-terminus of the A subunit of factor XIII. Thrombin or proteins having thrombin activity include, but are not limited to, bovine thrombin, human thrombin, and recombinants thereof. In certain embodiments, the thrombin or protein with thrombin activity can be bovine thrombin. Bovine thrombin that is generally commercially available as a freeze-dried product can be used. In addition, as thrombin or a protein with thrombin activity, snake thrombin (snake-derived thrombin-like enzyme) and factor Examples include, but are not limited to, combinations with proteins that have a cleaving action. The activity of thrombin or a protein with thrombin activity to be contained in the dried fibrinogen quantitative reagent is not particularly limited, but the amount of bovine thrombin activity may be selected within the range of, for example, 100 to 500 NIHU/1 mL final solution, but 150 to 400 NIHU/1 mL. A range of final solutions is preferred.

In certain embodiments, the dry fibrinogen quantitative reagent comprises magnetic particles. As the magnetic particles used in the fibrinogen quantitative drying reagent, any known magnetic particles can be used without any restrictions. Examples of the magnetic particles include, but are not limited to, triiron tetroxide particles, iron sesquioxide particles, iron particles, cobalt particles, nickel particles, and chromium oxide particles. In some embodiments, the magnetic particles can be triiron tetroxide microparticles. That is, in a specific embodiment, triiron tetroxide fine particles are preferably used in terms of the strength of the motion signal of the magnetic particles obtained. The particle size of the magnetic particles is not particularly limited, but may have an average particle size of 0.05 to 5 μm, 0.1 to 3.0 μm, for example, 0.25 to 0.5 μm, but is not limited thereto. In certain embodiments, the magnetic particles can have an average particle size of 0.1-3.0 μm. In this specification, the average particle diameter refers to the particle diameter (D50) at 50% of the integrated value in the particle size distribution determined by laser diffraction/scattering method, unless otherwise specified. The amount of magnetic particles contained in the dried fibrinogen quantitative reagent is not particularly limited, and is preferably in the range of 4 to 40 mg/1 mL final solution, for example.

In certain embodiments, the dry fibrinogen quantitative reagent may include a heparin neutralizing agent as an optional component. As the heparin neutralizing agent, any known agent can be used without any restriction, and examples thereof include, but are not limited to, polybrene, protamine sulfate, heparinase, and the like. In one embodiment, polybrene can be preferably used as the heparin neutralizing agent due to its good storage stability and cost. The amount of the heparin neutralizing agent contained in the fibrinogen quantitative drying reagent may be appropriately set and is not particularly limited. In one embodiment, when polybrene is used as a heparin neutralizing agent, the amount of polybrene contained in the dry quantitative fibrinogen reagent is preferably in the range of, for example, 50 to 300 μg/1 mL final solution.

The dry fibrinogen quantitative reagent contains a fibrin monomer association inhibitor. As the fibrin monomer association inhibitor contained in the fibrinogen quantitative drying reagent, any known inhibitor can be used without any restriction. Examples of fibrin monomer association inhibitors include GPRP (glycine-proline-arginine-proline) peptide and its derivatives, such as GPRP-amide, GHRP (glycine-histidine-arginine-proline) peptide and its derivatives, such as GHRP-amide, etc. Examples include, but are not limited to. In another embodiment, the fibrin monomer association inhibitor can be GPRPA (glycine-proline-arginine-proline-alanine) peptide and derivatives thereof, such as GPRPA-amide. In one embodiment, GPRP peptide and its derivatives are suitable as fibrin monomer association inhibitors in terms of their affinity for fibrinogen. The peptide is an analog of knob 'A' which is exposed by the release of fibrinopeptide A from the alpha chain of fibrinogen when thrombin reacts with fibrinogen, and the peptide is present in the gamma chain instead of knob 'A'. It inhibits the association of fibrin monomers by binding to hole 'a' (John WW: Mechanisms of fibrin polymerization and clinical implications, Blood, 121(10), 1712-1719, 2013).

The amount of the fibrin monomer association inhibitor to be contained in the dry fibrinogen quantitative reagent may be set as appropriate and is not particularly limited. When GPRP amide is used as a fibrin monomer association inhibitor, the amount of GPRP amide to be included in the fibrinogen quantitative dry reagent is preferably in the range of 100 to 300 μg/1 mL final solution.

In certain embodiments, the fibrinogen quantitative dry reagent comprises a calcium salt. As the calcium salt used in the dry reagent, any known calcium salt can be used without any restriction. Examples of salts of inorganic acids and calcium include calcium chloride, calcium nitrite, calcium sulfate, and calcium carbonate. Further, examples of salts of organic acids and calcium include calcium lactate and calcium tartrate. In certain embodiments, calcium chloride is suitable as the calcium salt. The amount of calcium salt contained in the dry reagent for quantitative determination of fibrinogen may be appropriately set and is not particularly limited. When calcium chloride dihydrate is used as the calcium salt, the amount of calcium chloride dihydrate contained in the dry quantitative fibrinogen reagent is preferably in the range of 0.2 to 2 mg/1 mL final solution.

In certain embodiments, the fibrinogen quantitative dry reagent includes a dry reagent layer solubility enhancer. Examples of the dry reagent layer solubility improver include amino acids, salts thereof, and saccharides. The amino acid, salt thereof, or saccharide used may be any of neutral amino acids or salts thereof, acidic amino acids or salts thereof, basic amino acids or salts thereof, monosaccharides, and polysaccharides. Representative acidic amino acids or salts thereof include glutamic acid, sodium glutamate, aspartic acid, sodium aspartate, and the like. Representative neutral amino acids or salts thereof include glycine, glycine hydrochloride, alanine, and the like. Typical basic amino acids or salts thereof include lysine, lysine hydrochloride, arginine, and the like. Furthermore, examples of monosaccharides include glucose, fructose, and the like. Further, examples of polysaccharides include sucrose, lactose, dextrin, and the like. Among these, glycine is the most preferred because of its good solubility when the sample is added to the dry fibrinogen quantitative reagent, its good reproducibility of the motion signal of the obtained magnetic particles, and its good impact resistance. preferable. That is, in certain embodiments, the dry reagent layer solubility enhancer can be glycine.

The amount of the dry reagent layer solubility improver, such as an amino acid or a salt thereof, or a saccharide, to be contained in the dry fibrinogen quantitative dry reagent may be appropriately set and is not particularly limited. In one embodiment, when glycine is used as the dry reagent layer solubility improver, the amount of glycine contained in the fibrinogen quantitative dry reagent is 1.5% by weight or more, 1.6% by weight or more, 1.7% by weight or more, 1.8% by weight or more, 1.9% by weight or more, 2.0% by weight or more, 2.1% by weight or more, 2.2% by weight or more, 2.3% by weight or more, 2.4% by weight or more, 2 .5% by weight or more, 2.6% by weight or more, 2.7% by weight or more, 2.8% by weight or more, 2.9% by weight or more, 3.0% by weight or more, 3.1% by weight or more, 3. 2% by weight or more, 3.3% by weight or more, 3.4% by weight or more, 3.5% by weight or more, 3.6% by weight or more, 3.7% by weight or more, 3.8% by weight or more, 3.9 weight% or more, 4.0 weight% or more, 4.1 weight% or more, 4.2 weight% or more, 4.3 weight% or more, 4.4 weight% or more, 4.5 weight% or more, 4.6 weight% % or more, 4.7 weight % or more, 4.8 weight % or more, 4.9 weight % or more, for example, 5.0 weight %. In one embodiment, when glycine is used as the dry reagent layer solubility improver, the amount of glycine contained in the fibrinogen quantitative dry reagent is 5.0% by weight or less, 4.9% by weight or less, 4.8% by weight or less, 4.7% by weight or less, 4.6% by weight or less, 4.5% by weight or less, 4.4% by weight or less, 4.3% by weight or less, 4.2% by weight or less, 4.1% by weight or less, 4 .0 weight% or less, 3.9 weight% or less, 3.8 weight% or less, 3.7 weight% or less, 3.6 weight% or less, 3.5 weight% or less, 3.4 weight% or less, 3. 3% by weight or less, 3.2% by weight or less, 3.1% by weight or less, 3.0% by weight or less, 2.9% by weight or less, 2.8% by weight or less, 2.7% by weight or less, 2.6 Weight% or less, 2.5% by weight or less, 2.4% by weight or less, 2.3% by weight or less, 2.2% by weight or less, 2.1% by weight or less, 2.0% by weight or less, 1.9% by weight % or less, 1.8% by weight or less, 1.7% by weight or less, 1.6% by weight or less, for example, 1.5% by weight. In this specification, the amount of glycine contained in the fibrinogen quantitative drying reagent includes all combinations in which the lower limit and upper limit are set to any of the above values. For example, in some embodiments, the amount of glycine contained in the dry fibrinogen quantitative reagent is 1.5 to 5.0% by weight, 2.0 to 5.0% by weight, 2.5 to 5.0% by weight, 3.0 to 5.0% by weight. 5.0% by weight, 3.5-5.0% by weight, 4.0-5.0% by weight, 4.5-5.0% by weight, 1.5-4.5% by weight, 2.0- 4.5% by weight, 2.5-4.5% by weight, 3.0-4.5% by weight, 3.5-4.5% by weight, 4.0-4.5% by weight, 1.5- 4.0% by weight, 2.0-4.0% by weight, 2.5-4.0% by weight, 3.0-4.0% by weight, 3.5-4.0% by weight, 1.5- 3.5% by weight, 2.0-3.5% by weight, 2.5-3.5% by weight, 3.0-3.5% by weight, 1.5-3.0% by weight, 2.0- It may be 3.0% by weight, 2.5-3.0% by weight, 1.5-2.5% by weight, 2.0-2.5% by weight, or 1.5-2.0% by weight. In one embodiment, when glycine is used as the dry reagent layer solubility improver, the amount of glycine contained in the dry reagent for fibrinogen quantitative determination is preferably in the range of 1.5 to 4.0% by weight. In another embodiment, when glycine is used as the dry reagent layer solubility improver, the amount of glycine contained in the dry reagent for quantitative determination of fibrinogen is preferably in the range of 2.0 to 3.0% by weight. When measuring undiluted plasma, if glycine is used as a solubility enhancer in the dry reagent layer, the amount of glycine to be included in the dry reagent for fibrinogen quantitative determination is within the above range, for example, 1.5% to 4.0% by weight. be able to. When measuring undiluted whole blood, when glycine is used as a solubility enhancer in the dry reagent layer, the amount of glycine contained in the dry fibrinogen quantitative dry reagent can be within the above range, for example, 1.5% by weight or more. . For example, when measuring undiluted whole blood and using glycine as a solubility enhancer in the dry reagent layer, the amount of glycine contained in the dry reagent for fibrinogen quantitative determination is 1.5 to 5.0% by weight, 1.5 to 4% by weight. .5% by weight, for example 1.5 to 4.0% by weight. If measurement is possible with undiluted plasma or undiluted whole blood, and if glycine is used as a solubility enhancer in the dry reagent layer, the amount of glycine contained in the dry fibrinogen quantitative dry reagent may be in various combinations within these ranges. . Note that in this specification, % by weight refers to the concentration in the final solution, that is, the final concentration, unless otherwise specified.

In certain embodiments, the dry fibrinogen quantitative reagent includes a pH buffer (also referred to as a pH adjuster). Prior to freeze-drying, a buffer containing a protein with thrombin activity, magnetic particles, a heparin neutralizer, a fibrin monomer association inhibitor, a calcium salt, and a dry reagent layer solubility enhancer is prepared at a pH between 6.0 and 8.0. It is not particularly limited as long as it has a buffering effect. In certain embodiments, the pH adjusting agent (pH buffering agent) can adjust the pH of the reagent from pH 6.0 to pH 8.0, such as about pH 7.35 or about pH 7.5. Suitable examples of the buffer include 40mM HEPES buffer (pH=7.35) and 40mM Tris-HCl buffer (pH=7.5).

In some embodiments, the dry quantitative fibrinogen reagent of the present invention includes a dry reagent layer reinforcement. Examples of the dry reagent layer reinforcing material include, but are not limited to, bovine serum albumin, human serum albumin, and the like. When bovine serum albumin is used as the dry reagent layer reinforcing material, the amount of the dry reagent layer reinforcing material contained in the quantitative dry reagent is preferably in the range of 0.6 to 2.0 mg/1 mL final solution.

In one embodiment, the dry fibrinogen quantitative reagent according to the present invention may include an antifoaming agent as an optional component. Examples of antifoaming agents include, but are not limited to, sorbitan monolaurate, silicone antifoaming agents, and polypropylene glycol antifoaming agents. When sorbitan monolaurate is used as the antifoaming agent, the amount of the antifoaming agent contained in the quantitative drying reagent is preferably in the range of about 0.001 to about 0.010% by weight.

As a method for drying a buffer solution containing the above-mentioned components, a freeze-drying method is preferable from the viewpoint of the solubility of the fibrinogen quantitative drying reagent, the strength of the motion signal of the obtained magnetic particles, and the reproducibility. When drying by air drying, the solubility of the reagent is poor, so the motion signal of the magnetic particles is weak, making it difficult to detect the end point. Furthermore, in the case of an air-dried reagent, even if an end point is found, the coagulation time determined from the end point may not correspond to the fibrinogen concentration.

Freezing and freeze-drying methods are not particularly limited. For example, after dispensing the final solution for fibrinogen quantitative dry reagent onto a reaction slide from the dispensing port shown in Figure 1, the reaction slide is stored overnight in a freezer kept below -40°C and frozen, or frozen on a shelf. General freezing methods can be used, such as setting the reaction slide in a freeze dryer at a temperature of -40°C or lower and storing it overnight to freeze, or flash-freezing the reaction slide with liquid nitrogen. Furthermore, the method of freeze-drying frozen reaction slides is not particularly limited. To illustrate the freeze-drying method, frozen reaction slides are ramped linearly in vacuum from -30°C to -20°C in 24 hours, then linearly from -20°C to 30°C in 20 hours. One method is to raise the temperature, hold it at 30°C for 3 hours, and then release the vacuum using dry air.

It is preferable that the freeze-dried fibrinogen quantitative drying reagent is immediately sealed with an aluminum film in a dehumidified environment. The dehumidified environment is not particularly limited, but an environment with a room temperature of 22 to 27°C and a relative humidity of 35% or less is preferable. In addition, the specifications of the aluminum film are not particularly limited, but include polyester film (thickness 12 μm), polyethylene resin (thickness 15 μm), aluminum foil (thickness 9 μm), polyethylene resin (thickness 20 μm), polyethylene film (thickness 30 μm). ) is preferably a 5-layer aluminum film (thickness: 86 μm) bonded with an AC coating agent. The entire dry fibrinogen quantitative reagent is enclosed in the aluminum film and sealed by heat welding. The dry fibrinogen quantitative determination reagent is preferably stored refrigerated in a sealed state until it is used to quantify fibrinogen.

In the quantitative method of the present invention, the range of the magnetic particle motion signal ratio is not particularly limited. For example, the certain range of magnetic particle motion signal ratio is 1.0±0.05 to 1.0±0.2, such as 1.0±0.2, 1.0±0.19, 1.0±0.18, 1.0±0.17, 1.0±0.16, 1.0±0.15, 1.0±0.14, It can be 1.0±0.13, 1.0±0.12, 1.0±0.11, 1.0±0.1, 1.0±0.09, 1.0±0.08, 1.0±0.07, 1.0±0.06, 1.0±0.05. In one embodiment, the certain range of magnetic particle motion signal ratio is preferably 1.0±0.05 to 1.0±0.15, with 1.0±0.1 being particularly preferable due to the good reproducibility of the resulting clotting times. . In other words, the certain ranges of magnetic particle motion signal ratio are 0.8-1.2 range, 0.81-1.19 range, 0.82-1.18 range, 0.83-1.17 range, 0.84-1.16 range, 0.85-1.15 range, 0.86 to 1.14 range, 0.87 to 1.13 range, 0.88 to 1.12 range, 0.89 to 1.11 range, 0.9 to 1.1 range, 0.91 to 1.09 range, 0.92 to 1.08 range, 0.93 to 1.07 range , 0.94 to 1.06, 0.95 to 1.05, etc., but a range of 0.9 to 1.1 is particularly preferred from the viewpoint of good reproducibility of the obtained coagulation time.

In the quantitative method of the present invention, the time (interval) during which the magnetic particle motion signal ratio is maintained within a certain range is not particularly limited. For example, the times (intervals) during which the magnetic particle motion signal ratio is maintained within a certain range are, for example, 1 to 5 seconds, 1 to 4 seconds, 1 to 3 seconds, 5 seconds, 4.5 seconds, 4 seconds, 3.5 seconds, 3 The duration may be 2 seconds, 2.5 seconds, 2 seconds, 1.5 seconds, 1 second, etc., but is not limited thereto. In one embodiment, it is preferable that the time (interval) during which the magnetic particle motion signal ratio is maintained within a certain range is 1 to 3 seconds, but it is particularly preferable because the reproducibility of the obtained coagulation time is good. It is 1.5 seconds.

In the quantification method of the present invention, the starting point refers to an arbitrary point within an interval in which a plurality of magnetic particle motion signal ratios at fixed time intervals are monitored and the ratio is maintained within a fixed range for a fixed period of time. The magnetic particle motion signal ratio over a period of time can be monitored continuously or intermittently. In certain embodiments, the starting point can be the beginning of an interval in which the ratio remains within a certain range for a certain period of time. In another embodiment, the starting point is set to a point other than the first point in an interval in which the ratio is maintained within a certain range for a certain period of time, for example, in an interval in which the ratio is maintained within a certain range for a certain period of time. It may also be the second point, third point, fourth point, etc. within. Such embodiments are also essentially included in the present invention. Note that in this specification, the starting point is a convenient starting point defined by the method of the present invention in order to avoid initial fluctuations in the signal after sample addition; Although this will be explained as a point (sec), this does not mean that the coagulation reaction does not actually start at all until this point.

Regarding the quantification method of the present invention, the peak value refers to the peak value of the magnetic particle motion signal after the starting point, unless otherwise specified, and this is the maximum value among the magnetic particle motion signals after the starting point. This is different from the peak value known from the prior art. That is, in the method disclosed in Japanese Patent Application Laid-Open No. 06-141895 (Patent No. 2980468), the maximum value among all the measured signals was simply taken as the peak value. However, when the present inventors applied the dry reagent described in Japanese Patent Application No. 2018-229919 to the quantitative method of Japanese Patent Application Laid-Open No. 6-141895 (Patent No. 2980468), magnetic particle movement signals were detected in the early stage of measurement after sample addition. There were large variations in fibrinogen, and fibrinogen could not be accurately quantified by using the maximum value among all measured signals as the peak value. Accordingly, the present invention aims to more accurately quantify fibrinogen in an undiluted sample by defining the starting point and correctly understanding the peak value of the magnetic particle movement signal after the starting point.

In the quantitative method of the present invention, the end point refers to any point among the points at which the peak value of the magnetic particle motion signal after the starting point determined by the above method is attenuated by 5 to 50%. For example, assuming that the peak value of the magnetic particle movement signal after the starting point is 100%, if the magnetic particle movement signal has a signal value corresponding to 70% of the peak value, this is referred to as a 30% attenuated point in this specification. For example, the end point is a point attenuated by 5 to 50% with respect to the peak value of the magnetic particle movement signal after the starting point, a point attenuated by 10 to 45%, a point attenuated by 15 to 40%, a point attenuated by 20 to 35%, The points may be 20 to 30% attenuated, for example, 20% attenuated, 25% attenuated, or 30% attenuated, but are not limited thereto. Particularly preferred is a point attenuated by 30% from the peak value of the magnetic particle motion signal in view of the good reproducibility of the obtained coagulation time. In one embodiment, different conditions for determining the end point can be used depending on whether the blood being measured is undiluted whole blood or undiluted plasma. That is, for example, if the blood to be measured is undiluted whole blood, the end point is the point at which the peak value of the magnetic particle movement signal after the starting point is attenuated by 20%; for example, if the blood to be measured is undiluted plasma, The end point can be a point at which the peak value of the magnetic particle motion signal after the starting point is attenuated by 30%. The respective end points to be used can be appropriately selected from points at which the peak value of the magnetic particle movement signal after the above-mentioned starting point is attenuated by 5 to 50%. In this specification, the peak value of the magnetic particle movement signal after the starting point refers to the maximum signal (C) among the magnetic particle movement signals measured after the starting point, and this includes the starting point itself. sell. That is, if the magnetic particle motion signal at the starting point is the largest signal among the magnetic particle motion signals measured after the starting point, that becomes the peak value of the magnetic particle motion signals after the starting point.

The term "coagulation time" as used in the present invention refers to the time from the above-mentioned starting point to the above-mentioned end point. That is, in the fibrinogen quantitative determination method of the present invention, the clotting time is calculated as the time from the above-mentioned starting point to the above-mentioned end point. The resulting clotting time is correlated to the fibrinogen concentration. Examples of devices to which the fibrinogen quantitative method of the present invention can be applied include the product name CG02N (manufactured by A&T Co., Ltd.), but the devices that can be used are not limited to this.

CG02N is a device suitable for the conventional fibrinogen quantitative method (Japanese Unexamined Patent Publication No. 06-141895 (Patent No. 2980468)). After adding the sample to the fibrinogen quantitative dry reagent, it is applied with an oscillating magnetic field and a stationary permanent magnetic field at 0.5 second intervals. combinations are applied and the magnetic particle motion signals are monitored at equal intervals. In order to carry out the fibrinogen quantification method of the present invention using the apparatus, in addition to the above, for example, in a specific embodiment, the magnetic particle motion signal ratio at 1 second intervals is continuously calculated, and the ratio is 1.0±0.1. It is possible to detect the first point of the interval that is maintained within the range for 1.5 seconds as the starting point. At this time, the end point is a predetermined value, for example, a value selected from 5 to 50%, for example, a point attenuated by 30% from the peak value of the magnetic particle movement signal after the starting point, and the time from the starting point to the ending point is calculated as the solidification time. can do. However, this is just an example, and the present invention is not limited to this.

A series of operations including these calculation processes may be performed by controlling the device using a program or software. The program or software may be built into the device or recorded on an information recording medium. In one embodiment, the present invention provides a program or software for carrying out the fibrinogen quantification method of the present invention. In one embodiment, the present invention provides an information recording medium on which the program or software of the present invention is recorded. In one embodiment, the present invention provides a fibrinogen quantitative measurement device incorporating a program or software for executing the fibrinogen quantitative measurement method of the present invention, or storing the information recording medium. In one embodiment, the fibrinogen quantitative measurement device of the present invention includes a CG02N device incorporating the program of the present invention.

Table 1 shows examples in which arbitrary whole blood samples were measured using the fibrinogen quantification method of the present invention. In this method, magnetic particle motion signals are monitored at 0.5 second intervals immediately after the addition of the analyte. That is, the monitoring period of the magnetic particle motion signal is 0.5 seconds. Then, the magnetic particle motion signal ratio at 1 second intervals is calculated continuously. In other words, the time interval used to calculate the magnetic particle motion signal ratio is 1 second. That is, (magnetic particle movement signal with a monitoring time of 1.0 seconds) / (magnetic particle movement signal with a monitoring time of 0 seconds), (magnetic particle movement signal with a monitoring time of 1.5 seconds) / (magnetic particle movement signal with a monitoring time of 0.5 seconds), It is calculated as follows: (Magnetic particle movement signal with a monitoring time of 2.0 seconds)/(Magnetic particle movement signal with a monitoring time of 1.0 seconds)... The period in which the ratio is maintained within the range of 1.0±0.1 for 1.5 seconds is the period in which the monitoring time is 5.0 to 6.5 seconds. Since the first point is when the monitoring time is 5.0 seconds, that point can be used as the starting point (the starting point of the coagulation reaction: the point at which the coagulation time is 0 seconds). The peak value of the magnetic particle motion signal after the starting point is 2726c at a monitoring time of 7.0 seconds. The magnetic particle motion signal that is 30% lower than the peak value of the magnetic particle motion signal after the origin is calculated to be 1908c. That is, the end point is the point where the magnetic particle motion signal becomes 1908c, and the coagulation time is calculated to be 20.1 seconds. Since the magnetic particle motion signal 1908c is a calculated value, its corresponding monitoring time and ratio of magnetic particle motion signals at 1 second intervals are not shown in the table. That is, the clotting time obtained by the method of the present invention does not need to be at any of the actual measurement points (actual monitoring time).

Note that the fibrinogen quantitative determination method of the present invention is not limited to the above. The monitoring period of the magnetic particle motion signal, the calculation period of the magnetic particle motion signal ratio, and the time interval used for calculating the magnetic particle signal ratio may be all the same (see, for example, FIG. 7) or may be different (see, for example, FIG. 8, 9). Also, the monitoring period of the magnetic particle motion signal may be constant (see, eg, FIGS. 7, 8, 9) or variable (see, eg, FIG. 10). Further, the monitoring cycle of the magnetic particle motion signal and the calculation cycle of the magnetic particle motion signal ratio may be constant (see, for example, FIGS. 7, 8, and 9), or may vary (see, for example, FIG. 11). Further, the ratio of magnetic particle motion signals may be calculated continuously (see, for example, FIGS. 7 and 8), or may be calculated intermittently (see, for example, FIG. 9). It may be calculated periodically (for example, see FIG. 12), or it may be calculated intermittently and then continuously (see, for example, FIG. 13). The monitoring period of the magnetic particle motion signal, the calculation period of the magnetic particle motion signal ratio, and the time interval used for calculating the magnetic particle motion signal ratio may be set to various conditions. However, it is preferable that the conditions for creating the calibration curve and the conditions for measuring the specimen are the same. Various other embodiments that will become apparent from the description herein are also encompassed by the present invention.

The method for quantifying fibrinogen in citrated plasma using the clotting time is not particularly limited. To give a typical example, first, three types of citrated plasma with known fibrinogen concentrations and different concentrations were measured using the above method as samples, and the clotting time corresponding to each citrated plasma was obtained. After that, a calibration curve is created in advance based on this. Next, after measuring any citrated plasma using the above method as a sample and obtaining the clotting time, the fibrinogen concentration of any citrated plasma can be found using the calibration curve created above. It will be done. The calibration curve used in this method is preferably a linear regression equation in which the Y axis is LN (fibrinogen concentration) and the X axis is LN (coagulation time). The resulting linear regression equation is a linear equation (Y=A×X+B), and the fibrinogen concentration of any citrated plasma can be calculated using the following equation based on the slope (A) and intercept (B) of the linear equation. be done.
[Number 1]
Fibrinogen concentration in any citrated plasma = e ^B × (clotting time) ^A

The fibrinogen concentration in the specimen is usually expressed as the fibrinogen concentration in citrated plasma. Since a whole blood sample contains not only plasma components but also blood cell components, when fibrinogen is quantified using a whole blood sample as a sample, it is necessary to consider the hematocrit value of the sample. That is, when a whole blood specimen is used as a sample, it is necessary to correct the fibrinogen concentration converted from the clotting time obtained by whole blood measurement using a hematocrit correction formula to calculate the fibrinogen concentration in the specimen. In addition, in the case of citrated whole blood, the measurement sample is obtained by adding and mixing 9 volumes of whole blood to 1 volume of sodium citrate solution, whereas in the case of heparinized whole blood, heparin sodium Alternatively, since a measurement sample is obtained by adding and mixing whole blood to lithium heparin powder, the hematocrit correction formula to be applied differs depending on whether citrated whole blood is used or heparinized whole blood is used. Specifically, the fibrinogen concentration in the sample when citrated whole blood is used as a sample is calculated using the following correction formula.
[Number 2]
Fibrinogen concentration in specimen = Fibrinogen concentration in citrated whole blood x (100/(100 - hematocrit value x 0.9))

In addition, the fibrinogen concentration in the sample when heparinized whole blood is used as a sample is calculated using the following correction formula.
[Number 3]
Fibrinogen concentration in specimen = fibrinogen concentration in heparinized whole blood x 0.9 x (100/(100 - hematocrit value))

Note that when citrated whole blood is used as a measurement sample and the hematocrit value is determined using citrated whole blood, the fibrinogen concentration in the sample is calculated using the following correction formula.
[Number 4]
Fibrinogen concentration in specimen = Fibrinogen concentration in citrated whole blood x (100/(100 - hematocrit value))

The results of fibrinogen quantification using the method of the present invention and the results of fibrinogen quantification using the conventional Clauss method agree extremely well. Furthermore, good results were obtained in terms of reproducibility, making reliable quantification possible even when undiluted whole blood was used as a sample. Reliable quantification is also possible even when undiluted plasma is used as a sample.

While the invention has been generally illustrated and illustrated, it may be further understood by reference to the following specific examples. The examples presented herein are for illustrative purposes only and are not intended to be limiting unless otherwise specified.

[Example 1 Clotting time measurement using the fibrinogen quantitative method of the present invention]
First, the fibrinogen quantitative drying reagent described in Japanese Patent Application No. 2018-229919 was produced by the following method.
Contains 10mM _CaCl2.2H2O , 2.0(wt/v)% glycine, 160μg/ _mL polybrene, 1.2mg/mL bovine serum albumin, 0.005(wt/v)% sorbitan monolaurate, and 200μg/mL GPRPamide. 40 mM HEPES buffer (pH 7.35) was added to lyophilized bovine thrombin (manufactured by Oriental Yeast) and dissolved to obtain a reagent solution having a thrombin activity of 333 NIHU/mL. To 35 mL of the reagent solution, 0.47 g of triiron tetroxide (product name AAT-03; average particle size 0.35 μm; manufactured by Toda Kogyo) was added and suspended to obtain a final solution. 25 μL of the final solution was dispensed onto the reaction slide shown in FIG. The reaction slide was stored overnight in a freezer kept at -40°C and frozen. The frozen reaction slides were then lyophilized. The conditions for freeze-drying were to linearly increase the temperature from -30℃ to -20℃ in 24 hours in a vacuum, then linearly increase the temperature from -20℃ to 30℃ in 20 hours, and finally to 30℃. After holding it for 3 hours, the vacuum was released using dry air. The lyophilized reagents were immediately sealed in aluminum film in a dehumidified environment.

An arbitrary whole blood specimen was measured using the fibrinogen quantitative determination method of the present invention using the above dry reagent for fibrinogen quantitative determination. In this method, magnetic particle motion signals were monitored at 0.5 second intervals immediately after the addition of the analyte. That is, the monitoring period of the magnetic particle motion signal is 0.5 seconds. Then, the magnetic particle motion signal ratio was calculated continuously at 1 second intervals. In other words, the time interval used to calculate the magnetic particle motion signal ratio is 1 second. That is, (magnetic particle movement signal with a monitoring time of 1.0 seconds) / (magnetic particle movement signal with a monitoring time of 0 seconds), (magnetic particle movement signal with a monitoring time of 1.5 seconds) / (magnetic particle movement signal with a monitoring time of 0.5 seconds), Calculations were performed as follows: (magnetic particle movement signal with a monitoring time of 2.0 seconds)/(magnetic particle movement signal with a monitoring time of 1.0 seconds). The period in which the ratio was maintained within the range of 1.0±0.1 for 1.5 seconds was the period in which the monitoring time was 5.0 to 6.5 seconds. Since the first point was when the monitoring time was 5.0 seconds, that point was taken as the starting point (coagulation reaction starting point: point at which coagulation time was 0 seconds). The peak value of the magnetic particle motion signal after the starting point is 2726c at a monitoring time of 7.0 seconds. The magnetic particle motion signal that is 30% lower than the peak value of the magnetic particle motion signal after the origin was calculated to be 1908c. That is, the end point was the point where the magnetic particle motion signal was 1908c, and the coagulation time was calculated to be 20.1 seconds. The results are shown in Table 2.

[Example 2 Comparison between the conventional fibrinogen quantitative method (quantitative method of Patent No. 2980468) and the fibrinogen quantitative method of the present invention (the present invention) when using undiluted whole blood as a sample and using a fibrinogen quantitative dry reagent]
Fibrinogen quantitative dry reagent was prepared as described above.
First, a calibration curve was calculated using a conventional quantitative method (quantitative method disclosed in Patent No. 2980468). The calibration curve was calculated as follows. Human plasma containing 304 mg/dL of fibrinogen and fibrinogen-deficient plasma (manufactured by Clinisys Associates) were used to prepare a dilution series of seven types of human plasma ranging from 37 to 304 mg/dL. Next, the fibrinogen quantitative dry reagent was set in a blood coagulation analyzer CG02N (manufactured by A&T Co., Ltd.), 25 μL of diluted samples were added, and the coagulation time of each sample was determined. Finally, the data were plotted with the Y axis as LN (fibrinogen concentration) and the X axis as LN (coagulation time), and a regression equation was obtained to calculate a calibration curve using the conventional quantitative method.

As a result, the calibration curve in the conventional quantitative method is
[Number 5]
LN (fibrinogen concentration) = -0.8223 x LN (coagulation time) + 7.4718
(Figure 3).

Based on the above calibration curve formula, the fibrinogen concentration conversion formula was determined as follows.
[Number 6]
Fibrinogen concentration in a sample type = e ^7.4718 × (clotting time) ^-0.8223

Next, a calibration curve for the quantitative method of the present invention was calculated. The calibration curve was calculated as follows. Human plasma containing 304 mg/dL of fibrinogen and fibrinogen-deficient plasma (manufactured by Clinisys Associates) were used to prepare a dilution series of 7 types of human plasma ranging from 37 to 304 mg/dL. Next, set the fibrinogen quantitative dry reagent in a blood coagulation analyzer CG02N (manufactured by A&T Co., Ltd.), incorporate the software of the present invention, add 25 μL of diluted samples, and determine the clotting time of each sample. Ta. Finally, the data were plotted with the Y axis as LN (fibrinogen concentration) and the X axis as LN (coagulation time), and a regression equation was obtained to calculate a calibration curve for the quantitative method of the present invention.

As a result, the calibration curve in the quantitative method of the present invention is
[Number 7]
LN (fibrinogen concentration) = -0.7636 x LN (coagulation time) + 7.2234
(Figure 4).

Based on the above calibration curve formula, the fibrinogen concentration conversion formula was determined as follows.
[Number 8]
Fibrinogen concentration in a sample type = e ^7.2234 × (clotting time) ^-0.7636

Blood was collected from one healthy person using seven sodium citrate vacuum blood collection tubes (2 mL specification) to obtain 14 mL of citrated whole blood. The seven blood collection tubes were centrifuged at 4°C, 3000 rpm, 15 minutes. 4 mL of citrated plasma A was obtained by extracting 1 mL of supernatant (plasma) from each of the 4 tubes, leaving 3 out of the 7 centrifuged tubes, and dispensing them into PP containers. After adding 2.80 mL of citrated plasma A to one of the three remaining blood collection tubes, the tube was sealed tightly and mixed by inversion to obtain citrated whole blood B. Furthermore, 0.40 mL of citrated plasma A was added to one of the three remaining blood collection tubes, and the tube was sealed tightly and mixed by inversion to obtain citrated whole blood C. Furthermore, after removing 0.56 mL of supernatant (plasma) from one of the three remaining blood collection tubes, the tube was sealed tightly and mixed by inversion to obtain citrated whole blood D.

The hematocrit values of citrated whole blood B, citrated whole blood C, and citrated whole blood D were measured using a hematocytometer MYTHIC22 (J) (manufactured by A&T Co., Ltd.). As a result, citrated whole blood B was 15%, citrated whole blood C was 30%, and citrated whole blood D was 50%.

First, fibrinogen concentrations in citrated plasma A, citrated whole blood B, citrated whole blood C, and citrated whole blood D were investigated using a conventional quantitative method (Patent No. 2980468).

After setting the dry fibrinogen quantitative reagent in CG02N and setting it to plasma measurement mode, 25 μL of citrated plasma A was added to determine the clotting time. I did that 5 times. Using the above conversion formula for the obtained clotting time (fibrinogen concentration in a certain sample type = e ^7.4718 × (clotting time) ^-0.8223 ), the fibrinogen concentration in citrated plasma A using the conventional quantitative method can be calculated. I asked for it.

After setting the dry fibrinogen quantitative reagent in CG02N and setting it to whole blood measurement mode, 25 μL of citrated whole blood B was added to determine the clotting time. I did that 5 times. The obtained coagulation time was converted to fibrinogen concentration using the same conversion formula as above. Furthermore, the fibrinogen concentration of citrated whole blood B was determined using the conventional quantitative method using the following formula.
[Number 9]
Fibrinogen concentration in citrated whole blood B using conventional quantitative method = Converted fibrinogen concentration x (100/(100-15))

After setting the dry fibrinogen quantitative reagent in CG02N and setting it to whole blood measurement mode, 25 μL of citrated whole blood C was added to determine the clotting time. I did that 5 times. The obtained coagulation time was converted to fibrinogen concentration using the same conversion formula as above. Furthermore, the fibrinogen concentration of citrated whole blood C was determined using the conventional quantitative method using the following formula.
[Number 10]
Fibrinogen concentration in citrated whole blood C using conventional quantitative method = Converted fibrinogen concentration x (100/(100-30))

After setting the dry fibrinogen quantitative reagent in CG02N and setting it to whole blood measurement mode, 25 μL of citrated whole blood D was added to determine the clotting time. I did that 5 times. The obtained coagulation time was converted to fibrinogen concentration using the same conversion formula as above. Furthermore, the fibrinogen concentration of citrated whole blood D was determined using the conventional quantitative method using the following formula.
[Number 11]
Fibrinogen concentration in citrated whole blood D using conventional quantitative method = Converted fibrinogen concentration x (100/(100-50))

Next, fibrinogen concentrations in citrated plasma A, citrated whole blood B, citrated whole blood C, and citrated whole blood D were examined using the quantitative method of the present invention.

The dry reagent for fibrinogen quantitative determination was set in CG02N, however, software for executing the fibrinogen quantitative method of the present invention was installed, and after setting the system to plasma measurement mode, 25 μL of citrated plasma A was added to determine the clotting time. I did that 5 times. Using the above conversion formula for the obtained clotting time (fibrinogen concentration in a certain sample type = e ^7.2234 × (clotting time) ^-0.7636 ), the fibrinogen concentration of citrated plasma A in the assay method of the present invention can be calculated. I asked for

Set the dry reagent for fibrinogen quantitative determination in CG02N, incorporate the software that executes the fibrinogen quantitative method of the present invention, set it to whole blood measurement mode, then add 25 μL of citrated whole blood B and determine the clotting time. Ta. I did that 5 times. The obtained coagulation time was converted to fibrinogen concentration using the same conversion formula as above. Furthermore, the fibrinogen concentration of citrated whole blood B was determined using the quantitative method of the present invention using the following formula.
[Number 12]
Fibrinogen concentration in citrated whole blood B using the quantitative method of the present invention = converted fibrinogen concentration x (100/(100-15))

Set the dry reagent for fibrinogen quantitative determination in CG02N, incorporate the software that executes the fibrinogen quantitative method of the present invention, set it to whole blood measurement mode, and then add 25 μL of citrated whole blood C to determine the clotting time. Ta. I did that 5 times. The obtained coagulation time was converted to fibrinogen concentration using the same conversion formula as above. Furthermore, the fibrinogen concentration of citrated whole blood C was determined using the quantitative method of the present invention using the following formula.
[Number 13]
Fibrinogen concentration in citrated whole blood C using the quantitative method of the present invention = converted fibrinogen concentration x (100/(100-30))

Set the dry reagent for fibrinogen quantitative determination in CG02N, incorporate the software that executes the fibrinogen quantitative method of the present invention, set it to whole blood measurement mode, then add 25 μL of citrated whole blood D and determine the clotting time. Ta. I did that 5 times. The obtained coagulation time was converted to fibrinogen concentration using the same conversion formula as above. Furthermore, the fibrinogen concentration of citrated whole blood D was determined using the quantitative method of the present invention using the following formula.
[Number 14]
Fibrinogen concentration in citrated whole blood D using the quantitative method of the present invention = converted fibrinogen concentration x (100/(100-50))

Furthermore, citrated plasma A was quantified using the Clauss method. Fibrinogen was quantified using the Clauss method using Dataphy Fibrinogen (manufactured by Sysmex) as the reagent and KC4 Delta (manufactured by Tcoag Ireland Ltd) as the measuring device, according to the method indicated in the package insert of Dataphy Fibrinogen. It was measured five times, and the average value of 224 mg/dL was taken as the fibrinogen concentration of citrated plasma A according to the Clauss method. The results are shown below.

Table 3 shows the measurement results using the conventional quantitative method, and Table 4 shows the measurement results using the quantitative method of the present invention. Specificity was evaluated by the recovery rate for the fibrinogen concentration of citrated plasma A (224 mg/dL) determined by the Clauss method. Samples with higher hematocrit values have higher viscosity. In Table 3, the values for high viscosity whole blood D are generally higher than for plasma A. In other words, from the results in Table 3, the conventional quantitative method cannot accurately quantify the fibrinogen concentration in whole blood samples with high hematocrit values, but the quantitative method of the present invention can accurately quantify the fibrinogen concentration even in whole blood samples with high hematocrit values. It is clear that the fibrinogen concentration can be quantified.

[Example 3 Correlation between fibrinogen quantitative value by Clauss method and fibrinogen quantitative value by quantitative method of the present invention]
Using 104 citrated plasma samples, the correlation between the results of fibrinogen quantification using the Clauss method and the results of fibrinogen quantification using the quantification method of the present invention was investigated. Fibrinogen quantification using the quantification method of the present invention was carried out by the following method.

The dry reagent for fibrinogen quantitative determination was set in CG02N, however, software for executing the fibrinogen quantitative method of the present invention was installed, and after setting the system to plasma measurement mode, 25 μL of citrated plasma was added to determine the clotting time. The obtained clotting time is calculated using the above conversion formula (fibrinogen concentration in a certain sample type = e ^7.2234 × (clotting time) ^-0.7636 ).
This was converted to fibrinogen concentration and used as the fibrinogen concentration in the quantitative method of the present invention.

Quantification of fibrinogen by the Clauss method was carried out using HemosIEL Fib CXL (manufactured by LSI Medience) as a reagent and STACIA (manufactured by LSI Medience) as a measuring device. It was quantified by the method indicated in the package insert of Hemos IEL Fib/CXL.

FIG. 5 shows a correlation diagram between fibrinogen quantification values by the Clauss method and fibrinogen quantification values by the quantitative method of the present invention. From FIG. 5, it is clear that the fibrinogen quantification value by the quantitative method of the present invention is in good agreement with the fibrinogen quantification value by the Clauss method, and the correlation is high.

[Example 4 Using the method of the present invention, correlation between the fibrinogen quantitative value when the measurement sample is citrated plasma and the fibrinogen quantitative value when the measurement sample is citrated whole blood]
The results of fibrinogen quantification using the assay method of the present invention on 80 samples of citrated whole blood and the results of fibrinogen assay using the assay method of the present invention on 80 samples of citrated plasma obtained by centrifuging the same samples. We investigated the correlation between

First, the hematocrit values of 80 citrated whole blood samples were determined using a blood cell counter MYTHIC22 (J) (manufactured by A&T Co., Ltd.). Next, set the dry reagent for fibrinogen quantitative determination in CG02N, install the software that executes the fibrinogen quantitative method of the present invention, set it to whole blood measurement mode, and then add 25 μL of citrated whole blood to each sample. The coagulation time was determined. The obtained coagulation time is converted to the above conversion formula.
[Number 15]
(Fibrinogen concentration in a certain sample type = e ^7.2234 × (clotting time) ^-0.7636 )
was used to convert to fibrinogen concentration.

Finally, the fibrinogen concentration in the sample was determined using the following formula when the measurement sample was citrated whole blood.
[Number 16]
Fibrinogen concentration in sample = converted fibrinogen concentration x (100/(100-hematocrit value))

The 80 samples of citrated whole blood for which the above measurements had been completed were centrifuged at 4°C, 3000 rpm, for 15 minutes, and the supernatant was collected to obtain 80 samples of citrated plasma. Next, set the dry reagent for fibrinogen quantitative determination in CG02N, incorporate the software that executes the fibrinogen quantitative method of the present invention, set it to plasma measurement mode, and then add 25 μL of citrated plasma to coagulate each sample. I asked for time. The obtained coagulation time is converted to the above conversion formula.
[Number 17]
(Fibrinogen concentration in a certain sample type = e ^7.2234 × (clotting time) ^-0.7636 )
was used to convert to fibrinogen concentration. The converted fibrinogen concentration was taken as the fibrinogen concentration in the sample when citrated plasma was used as the measurement sample.

Figure 6 shows a correlation diagram between the fibrinogen quantitative value when the measurement sample is citrated plasma and the fibrinogen quantitative value when the measurement sample is citrated whole blood using the quantitative method of the present invention. . From FIG. 6, when using the method of the present invention, the fibrinogen quantitative value when the measurement sample is citrated whole blood is in good agreement with the fibrinogen quantitative value when the measurement sample is citrated plasma, It is clear that there is a high correlation.

According to the present invention, fibrinogen can be quantitatively measured without dilution.
All documents mentioned herein are incorporated by reference in their entirety.

A Transparent resin plate B Transparent resin plate C White resin plate D Reagent filling section

Claims (14)

A method for quantifying fibrinogen, comprising the following steps (i), (ii) and (iii), namely:
(i) a step of adding a specimen to a fibrinogen quantitative dry reagent containing magnetic particles;
Here, the fibrinogen quantitative dry reagent is
(A) thrombin or a protein with thrombin activity;
(B) magnetic particles,
(C) Fibrin monomer association inhibitor,
(D) Calcium salt,
(E) dry reagent layer solubility improver,
(F) Dry reagent layer reinforcement material, and
(G) pH adjuster (pH buffer)
A fibrinogen quantitative dry reagent containing
The thrombin activity is an activity capable of promoting both (a) the conversion of fibrinogen to fibrin monomer, and (b) the activation of factor XIII to XIIIa in the presence of calcium ions. be,
(ii) moving the magnetic particles in the reagent after addition of the analyte and monitoring the magnetic particle movement signal;
Here, the magnetic particle movement signal is generated in step (ii) by applying a combination of an oscillating magnetic field and a stationary permanent magnetic field at predetermined intervals after adding the sample to move the magnetic particles contained in the reagent, and then exposing the reagent to light. is the amount of change in scattered light when
as well as
(iii) Regarding the magnetic particle motion signals monitored in step (ii) above, the magnetic particle motion signal ratio at a certain time interval , that is, the magnetic particle motion signal at one time separated by a certain time interval and the magnetic particle motion signal at another time separated by a certain time interval. calculating a plurality of ratios to magnetic particle motion signals ;
including;
Starting point is any point in the interval where the magnetic particle motion signal ratio for the above fixed time interval is maintained within a certain range for a certain period of time, and 5 to 50% relative to the peak value of the magnetic particle motion signal after the starting point. The fibrinogen quantification method described above, wherein an arbitrary point among the points at which % attenuation occurs is taken as the end point, and the time from the starting point to the end point is taken as the coagulation time. 2. The method for quantifying fibrinogen according to claim 1, wherein the time interval used to calculate the magnetic particle motion signal ratio is a constant time interval selected from 0.1 seconds to 2 seconds. 3. The fibrinogen quantitative determination method according to claim 1, wherein the time interval used for calculating the magnetic particle motion signal ratio is 0.5 seconds, 1 second, 1.5 seconds, or 2 seconds. 3. The method for quantifying fibrinogen according to claim 1 or 2, wherein the time interval used for calculating the magnetic particle motion signal ratio is 1 second. 2. The method for quantifying fibrinogen according to claim 1, wherein the predetermined range of the magnetic particle motion signal ratio is 1.0±0.2. 3. The method for quantifying fibrinogen according to claim 2, wherein the predetermined range of the magnetic particle motion signal ratio is 1.0±0.1. The method for quantifying fibrinogen according to any one of claims 1 to 6, wherein the time interval during which the magnetic particle motion signal ratio is maintained within a certain range is 1.5 seconds. The method for quantifying fibrinogen according to any one of claims 1 to 7, wherein the starting point is the first point of a time interval in which the magnetic particle motion signal ratio is maintained within a certain range. The method for quantifying fibrinogen according to any one of claims 1 to 8, wherein the end point is any point among the points at which the peak value of the magnetic particle movement signal after the starting point is attenuated by 20 to 30%. 10. The method for quantifying fibrinogen according to claim 9, wherein the end point is a point at which the peak value of the magnetic particle movement signal after the starting point is attenuated by 30%. 10. The method for quantifying fibrinogen according to claim 9, wherein the end point is a point at which the peak value of the magnetic particle movement signal after the starting point is attenuated by 20%. A program for executing the method for quantifying fibrinogen according to any one of claims 1 to 11. An information recording medium on which the program according to claim 12 is recorded. A fibrinogen quantitative measurement device in which the program according to claim 12 is incorporated or the information recording medium according to claim 13 is stored.

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