Method for determining blood coagulation factor xiii activity

Abstract

To provide a method for accurately determining factor XIII activity values in finished products and intermediate products in which factor XIII is purified or concentrated compared to plasma, without being affected by sample composition while also improving the workability required to measure physical properties corresponding to factor XIII activity values by using the FXIII-GLDH method.SOLUTION: Provided is a method for determining an unknown factor XIII activity value contained in a test sample by comparing the physical properties of the test sample with those of a known reference sample. The physical properties correspond to the amount of change per unit time of NAD(P)H or NAD(P)H analogues consumed or NAD(P) or NAD(P) analogues produced in the FXIII-GLDH method. The reference sample and test sample contain fibrinogen at a concentration of 2.0 to 4.0 mg / mL.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a method for determining the activity value of blood coagulation factor XIII (hereinafter also simply referred to as factor XIII), a method for preparing a test sample, a method for preparing a measurement point sample, and a method for producing a blood product. [Background technology]

[0002] Factor XIII is an important factor that contributes to the stabilization of fibrin in the hemostatic mechanism of blood.

[0003] Factor XIII is found mainly in plasma, platelets, and the placenta. Factor XIII preparations formulated from this substance are an important factor in hemostatic action in clinical applications, and are highly effective in supplementing congenital or acquired factor XIII deficiency or hypokalemia, as well as promoting wound healing after general surgery.

[0004] Factor XIII is also available as a pharmaceutical product, not only as a single-agent factor XIII preparation, but also as one of the active ingredients in biological tissue adhesives used for adhering and sealing tissues during surgery.

[0005] Representative methods for measuring (determining) factor XIII activity include a method based on the solubility of fibrin clots (clot lysis), a method based on the amount of amine incorporated (amine incorporation method), and a method based on the amount of ammonia produced. A method has also been reported in which fibrinogen is converted to fibrin by thrombin, and the fibrin is crosslinked by activated factor XIII to form a fibrin clot, and the resulting reaction is measured as a clotting time dependent on factor XIII activity (Patent Document 1).

[0006] However, among the three representative methods mentioned above, the method based on clot lysis (Non-Patent Document 1) can only provide qualitative results by evaluating whether clots (fibrin clots) formed by coagulating plasma samples with calcium chloride or thrombin are soluble in 5 mol / L urea solution, 2% acetic acid, or 1% monochloroacetic acid, and even if evaluation is performed after preparing a dilution series of the sample, only semi-quantitative results can be obtained. Therefore, the amine uptake method or a method based on the amount of ammonia produced is generally used.

[0007] The amine uptake method has been commercialized as the Iatro®-FL FXIII kit (Patent Document 2), but this kit is no longer on the market. Therefore, when determining the factor XIII activity value in a test sample, it is necessary to prepare a casein solution, a mixed solution of thrombin, monodansylcadaverine, and calcium, and a maleimide solution.

[0008] After preparation of each reagent, the test sample and casein solution are mixed and incubated at 56°C for 4 minutes to remove fibrinogen denaturation. After cooling on ice, a mixed solution of thrombin, monodansylcadaverine, and calcium is added and incubated at 37°C for 10 minutes to activate factor XIII and incorporate the fluorescent monodansylcadaverine into casein. The reaction is then terminated by the addition of maleimide, and the monodansylcadaverine-dimethylcasein complex is separated from the free monodansylcadaverine by gel filtration. The fluorescence intensity (emission 510 nm; excitation 335 nm) derived from the monodansylcadaverine incorporated into the complex is measured, and the activity is calculated by comparing it with a calibration curve based on fluorescence intensities measured using standard human plasma.

[0009] Thus, the amine incorporation method is a method for measuring the fluorescence intensity derived from monodansylcadaverine incorporated into the complex as a physical property corresponding to the factor XIII activity, and the factor XIII activity is determined based on this fluorescence intensity value obtained as a physical property.

[0010] Furthermore, the third method based on the amount of ammonia produced (Patent Document 3) has been commercialized by Siemens Healthineers as a kit specifically designed to measure plasma under the trade name "Berichrom (registered trademark) FXIII / Berichrom (registered trademark) FXIII" (Non-Patent Document 2).

[0011] In the ammonia-based method, factor XIII in plasma is first activated by thrombin in the presence of calcium ions (factor XIIIa), and fibrin polymerization is inhibited by a fibrin aggregation inhibitor (such as Gly-Pro-Arg-Pro).

[0012] In this state, when two substrates for Factor XIIIa, specifically a specific glutamine-containing peptide (synthetic substrate) and glycine ethyl ester, are present, Factor XIIIa undergoes a cross-linking reaction between the glycine ethyl ester and the synthetic substrate.

[0013] This cross-linking reaction generates ammonia, which is then incorporated into α-ketoglutarate by glutamate dehydrogenase, which consumes NADH.

[0014] The rate at which NADH is consumed, that is, the amount of change per unit time, increases as the amount of ammonia produced increases, and the amount of ammonia produced increases as the activity of factor XIIIa increases.

[0015] Therefore, the amount of NADH that changes during the reaction is measured by absorbance (A 340 ) to determine the change per unit time, and the absorbance (A 340 ) per unit time, the activity value is calculated and a decision is made.

[0016] In the study of reference materials for plasma sample measurements based on the amount of ammonia produced, there have been reports disclosing a measurement method in which standard plasma was diluted with factor XIII-deficient plasma to evaluate the dependency on factor XIII concentration (Patent Document 3, Patent Document 4).

[0017] Additionally, there have been reports of assay kits (Berichrome FXIII) that have been modified specifically for measuring the activity of recombinant factor XIII (Non-Patent Document 3: Kerlin B, Brand B, Inbal A, et al., Journal of Thrombosis and Haemostasis, vol. 12: 2038-2043, 2014). However, the samples and calibration curve were prepared using recombinant factor XIII, and bovine serum albumin and K2-EDTA were added to the diluent saline solution, so the kit is not suitable for use with a variety of samples.

[0018] As described above, the method based on the amount of ammonia uses the absorbance of NADH (A 340 This is a method for measuring the absorbance (A 340 In the following explanation, for convenience of explanation, a method based on the amount of ammonia, in which a predetermined substrate for factor XIII having a glutamine residue is reacted with glycine ethyl ester by factor XIII in the presence of calcium ions and thrombin to generate ammonia, and the ammonia is reacted with α-ketoglutaric acid by glutamate dehydrogenase in the presence of NAD(P)H or an NAD(P)H analog, and the amount of change per unit time in the amount of consumed NAD(P)H or an NAD(P)H analog, or the amount of generated NAD(P) or an NAD(P) analog, is determined, is also referred to as the FXIII-GLDH method. [Prior art documents] [Patent documents]

[0019] [Patent Document 1] Japanese Patent Application Publication No. 06-094726 [Patent Document 2] Japanese Unexamined Patent Publication No. 58-216959 [Patent Document 3] Japanese Patent Application Publication No. 01-309700 [Patent Document 4] Special table 2013-506429 publication [Non-patent literature]

[0020] [Non-Patent Document 1] Jennings I, Kitchen S, Woods TAL, and Preston FE. Problems relating to the laboratory diagnosis of factor XIII deficiency: a UK NEQAS study. J Thromb Haemost 2003; 1: 2603-2608. [Non-patent document 2] In vitro diagnostic reagent Berichrome FXIII package insert [Non-patent document 3] Kerlin B, Brand B, Inbal A, et al., Journal of Thrombosis and Haemostasis, vol.12: 2038-2043, 2014 Summary of the Invention [Problem to be solved by the invention]

[0021] As mentioned above, factor XIII is used as one of the active ingredients in factor XIII preparations and tissue adhesives, and during the manufacturing process, factor XIII is purified and concentrated.

[0022] Therefore, manufacturers and testing companies of such preparations determine factor XIII activity using products or intermediates in which factor XIII is purified or concentrated compared to plasma as test items.

[0023] In this regard, the above-mentioned amine incorporation method is useful as a method for measuring a physical property value corresponding to the factor XIII activity value for determining the activity value, i.e., the fluorescence intensity value derived from the monodansylcadaverine incorporated into the complex.

[0024] However, the amine uptake method is laborious because no kits are available and it is necessary to prepare a casein solution, a mixed solution of thrombin, monodansylcadaverine, and calcium, and a maleimide solution. In addition, regeneration of the gel filtration column requires a significant amount of work, so alternative methods are needed.

[0025] On the other hand, the absorbance of NADH (A 340 The FXIII-GLDH method, which measures the value of FXIII-GLDH, does not require the complicated regeneration of a gel filtration column, and commercially available kits are available if necessary.It is also compatible with automatic analyzers, making it an attractive method in terms of workability.

[0026] However, the FXIII-GLDH method is limited to plasma as the test item, and if the test item is a product in which factor XIII is more purified or concentrated than plasma, the calculated factor XIII activity value will be lower than the value predicted from the factor XIII activity value previously determined by the amine uptake method.

[0027] The present invention has been made in consideration of the above circumstances, and provides a method that improves the workability required for measuring physical properties corresponding to factor XIII activity values ​​by using the FXIII-GLDH method, while accurately determining the factor XIII activity values ​​of products or intermediates in which factor XIII is in a more purified or concentrated state than in plasma, without being affected by the sample composition.

[0028] The present invention also provides a method for preparing a test sample and a measurement point sample to be used in the method for determining factor XIII activity, as well as a method for producing a blood product comprising a step of determining the factor XIII activity of a semi-finished product by the method for determining factor XIII activity. [Means for solving the problem]

[0029] In order to solve the above-mentioned conventional problems, the method for determining factor XIII activity according to the present invention (1) is a method for determining the activity of unknown blood coagulation factor XIII (factor XIII) contained in a test sample other than plasma by comparing the physical property values ​​of the test sample with those of a reference sample with known factor XIII activity, wherein the physical property value corresponds to the change per unit time in the amount of NAD(P)H or NAD(P)H analog consumed or the amount of NAD(P) or NAD(P) analog produced when a predetermined substrate for factor XIII having a glutamine residue is reacted with glycine ethyl ester in the presence of calcium ions and thrombin by factor XIII in the test sample or reference sample to generate ammonia, and the ammonia is reacted with α-ketoglutaric acid in the presence of NAD(P)H or an NAD(P)H analog by glutamate dehydrogenase, and the reference sample and test sample contain fibrinogen at a concentration of 2.0 to 4.0 mg / mL.

[0030] In order to solve the above-mentioned conventional problems, the method for determining a factor XIII activity value according to the present invention includes: (2) a method for determining a factor XIII activity value in a test article (excluding plasma), by measuring a physical property value corresponding to an unknown blood coagulation factor XIII (factor XIII) activity value contained in a test sample (excluding plasma) prepared from the test article (excluding plasma), and comparing the measured physical property value with a physical property value corresponding to a known factor XIII activity value contained in a reference sample, wherein the physical property value corresponding to the factor XIII activity value in the test sample and the reference sample is determined by the following: a predetermined substrate for factor XIII having a glutamine residue is reacted with glycine ethyl ester by factor XIII in the test sample or the reference sample in the presence of calcium ions and thrombin to generate ammonia; and a reaction of the ammonia with α-ketoglutaric acid by glutamate dehydrogenase in the presence of NAD(P)H or an NAD(P)H analogue consumed or NAD(P)H generated in the reaction. ) is a physical property value corresponding to the change in the amount of the analogue per unit time, and the reference sample is composed of one measurement point sample having a known factor XIII activity value that matches a predetermined target activity value for the measurement point sample, or a plurality of measurement point samples having known factor XIII activity values ​​that match a plurality of different target activity values ​​for the measurement point sample, where the activity of factor XIII in 1 mL of normal plasma is taken as 1 unit, and the test article and test sample have a predicted factor XIII activity value in advance, and when there is one measurement point sample, the The factor XIII activity value of the measurement point sample was set as the target activity value for the test sample, and if there were multiple measurement point samples, the target activity value for the test sample was set as a value selected from the range from the activity value of the measurement point sample with the highest factor XIII activity to the activity value of the measurement point sample with the lowest factor XIII activity among the reference samples.The test sample was prepared so that the predicted value of factor XIII activity would be the target activity value for the test sample, and the reference sample and test sample contained fibrinogen at a concentration of 2.0 to 4.0 mg / mL.

[0031] The method for determining factor XIII activity according to the present invention is also characterized in the following respects. (3) The target activity value of the measurement point sample is set to 1.0 unit / mL, and the reference sample is composed of one measurement point sample with a factor XIII activity value of 1.0 unit / mL. (4) The target activity values ​​of the measurement point samples are set to 0.5 units / mL, 1.0 units / mL, and 1.5 units / mL, and the reference sample is composed of three measurement point samples with factor XIII activity values ​​of 0.5 units / mL, 1.0 units / mL, and 1.5 units / mL. (5) The reaction is carried out at 37°C. (6) The physical property value corresponding to the factor XIII activity value in the test sample and the reference sample is the change in absorbance of NADH consumed by the reaction at a wavelength of 340 nm per minute, and this change is calculated as the quotient obtained by dividing the difference in absorbance measured 5 minutes and 10 minutes after the start of the reaction by 5.

[0032] In addition, the method for preparing a test sample according to the present invention is (7) a method for preparing a test sample used in the method for determining factor XIII activity value described in (2), wherein the test item is a highly active test item having a predicted factor XIII activity value of more than 1.5 units / mL, and the test sample is prepared by diluting the highly active test item using a fibrinogen test solution containing fibrinogen at a concentration of 2.0 to 4.0 mg / mL in Owen-Veronal buffer solution.

[0033] The test sample preparation method according to the present invention is also characterized in the following respects. (8) If the predicted value of the factor XIII activity of the highly active test article exceeds 10.0 units / mL, prepare a test intermediate diluted sample by diluting it with a specimen diluent containing 0.5 w / v% HSA and / or 0.1 v / v% Tween 20 in Owen-Veronal buffer so that the predicted value of factor XIII activity is 10 times the target activity value of the test sample, and then prepare a test sample by diluting the test intermediate diluted sample 10 times with the fibrinogen test solution so that the predicted value of factor XIII activity is the target activity value of the test sample. (9) The fibrinogen concentration of the fibrinogen test solution is 2.5 mg / mL.

[0034] Furthermore, the method for preparing a measurement point sample according to the present invention is (10) a method for preparing the measurement point sample used in the method for determining factor XIII activity value described in (2), wherein the measurement point sample is prepared by diluting a standard substance having a known factor XIII activity value greater than the target activity value of the measurement point sample using a fibrinogen test solution containing fibrinogen at a concentration of 2.0 to 4.0 mg / mL in an Owen-Veronal buffer solution.

[0035] The method for preparing a measurement point sample according to the present invention is further characterized in that (11) when the factor XIII activity value of the standard substance exceeds 10 times the target activity value of the measurement point sample, the measurement point intermediate diluted sample is prepared by diluting the standard substance with a specimen dilution solution containing Owen-Veronal buffer containing 0.5 w / v% HSA and / or 0.1 v / v% Tween 20 so that the factor XIII activity value becomes 10 times the target activity value of the measurement point sample, and then the measurement point sample is prepared by diluting the measurement point intermediate diluted sample 10 times with the fibrinogen test solution so that the factor XIII activity value becomes the target activity value of the measurement point sample.

[0036] Furthermore, the method for producing a blood product according to the present invention comprises (12) a semi-finished product preparation step of subjecting the collected blood or a blood fraction to a predetermined pre-treatment to prepare a semi-finished product (excluding plasma), a factor XIII activity determination step of determining the factor XIII activity of the semi-finished product by the factor XIII activity determination method described in (2) above, using the semi-finished product or a processed product (excluding plasma) obtained by subjecting the semi-finished product to the treatment required for measurement as a test substance, and a product production step of determining the suitability of the semi-finished product based on the factor XIII activity determined in the factor XIII activity determination step, and, if the semi-finished product is determined to be suitable, subjecting it to a predetermined post-treatment as necessary to produce a blood product.

[0037] The method for producing a blood product according to the present invention is also characterized by the following points. (13) The test sample used in the method for determining the factor XIII activity value is prepared by the method for preparing a test sample according to any one of (7) to (9). (14) The measurement point sample used in the method for determining factor XIII activity value is prepared by the method for preparing a measurement point sample described in (10) or (11) above. [Effects of the Invention]

[0038] The method for determining factor XIII activity according to the present invention is a method for determining an unknown blood coagulation factor XIII (factor XIII) activity value contained in a test sample other than plasma by comparing the physical property values ​​of the test sample with those of a reference sample having known factor XIII activity, and the physical property value is determined by reacting a predetermined substrate of factor XIII having a glutamine residue with glycine ethyl ester in the presence of calcium ions and thrombin using factor XIII in the test sample or the reference sample to generate ammonia, and then reacting the generated ammonia with α-ketoglutaric acid using glutamate dehydrogenase in the presence of NAD(P)H or an NAD(P)H analog. The fibrinogen concentration in the reference sample and test sample is 2.0 to 4.0 mg / mL. This improves the workability of measuring physical properties corresponding to factor XIII activity values ​​based on the FXIII-GLDH method, while providing a method for accurately determining factor XIII activity values ​​of products and intermediates in which factor XIII is more purified or concentrated than in plasma, without being affected by the sample composition.

[0039] Furthermore, according to the method for determining the factor XIII activity value of the present invention, a test sample (excluding plasma) prepared from a test article (excluding plasma) can be used.and determining the activity of factor XIII in the test article or the test sample by measuring a physical property corresponding to an unknown activity of blood coagulation factor XIII (factor XIII) contained in the test article or the test sample and comparing it with a physical property corresponding to a known activity of factor XIII contained in a reference sample, wherein the physical property corresponding to the activity of factor XIII in the test sample and the reference sample is determined by reacting a predetermined substrate of factor XIII having a glutamine residue with glycine ethyl ester in the presence of calcium ions and thrombin with factor XIII in the test sample or the reference sample. The reference sample is a physical property value corresponding to the change per unit time of NAD(P)H or an NAD(P)H analog consumed or NAD(P) or an NAD(P) analog produced by reacting glutamate dehydrogenase with α-ketoglutaric acid in the presence of NAD(P)H or an NAD(P)H analog to generate ammonia, and then reacting the ammonia with α-ketoglutaric acid in the presence of NAD(P)H or an NAD(P)H analog, and the reference sample is one measurement point sample or a measurement point sample having a known factor XIII activity value that matches a predetermined target activity value for the measurement point sample, where the activity of factor XIII in 1 mL of normal plasma is defined as 1 unit. is composed of a plurality of measurement point samples having known factor XIII activity values ​​that are matched to a plurality of different measurement point sample activity target values, and the test article and test sample have predicted factor XIII activity values. When there is one measurement point sample, the factor XIII activity value of the measurement point sample is set as the test sample activity target value, while when there are multiple measurement point samples, a value selected from the range from the activity value of the measurement point sample having the maximum factor XIII activity to the activity value of the measurement point sample having the minimum factor XIII activity among the reference samples is set as the test sample activity target value. The test sample is prepared so that the predicted value of factor XIII activity is the target activity value of the test sample, and the reference sample and test sample contain fibrinogen at a concentration of 2.0 to 4.0 mg / mL.Therefore, by using the FXIII-GLDH method as a premise, it is possible to improve the workability required to measure physical properties corresponding to factor XIII activity values, while providing a method that can accurately determine the factor XIII activity values ​​of products and intermediates in which factor XIII is purified or concentrated compared to plasma, without being affected by the sample composition.

[0040] Furthermore, if the target activity value of the measurement point sample is set to 1.0 unit / mL and the reference sample is composed of a single measurement point sample with a factor XIII activity value of 1.0 unit / mL, the factor XIII activity value of the test sample can be reliably determined based on the reference sample consisting of a single measurement point sample with factor XIII activity close to that of normal plasma.

[0041] Furthermore, if the target activity values ​​of the measurement point samples are set to 0.5 units / mL, 1.0 units / mL, and 1.5 units / mL, and the reference sample is composed of three measurement point samples with factor XIII activity values ​​of 0.5 units / mL, 1.0 units / mL, and 1.5 units / mL, a calibration curve can be created based on the reference samples consisting of multiple measurement point samples with factor XIII activity ranges close to that of normal plasma, and the factor XIII activity value of the test sample can be determined.

[0042] Furthermore, if the reaction is carried out at 37°C, the measurement can be carried out under appropriate enzyme reaction conditions, and the factor XIII activity value of the test sample can be determined stably.

[0043] Furthermore, the physical property value corresponding to the factor XIII activity value in the test sample and the reference sample is the change in absorbance of NADH consumed by the reaction at a wavelength of 340 nm per minute. This change can be calculated by dividing the difference in absorbance measured 5 minutes and 10 minutes after the start of the reaction by 5, thereby enabling the stable determination of the factor XIII activity value of the test sample.

[0044] Furthermore, according to the method for preparing a test sample of the present invention, there is provided a method for preparing a test sample used in the method for determining factor XIII activity value of the present invention, wherein the test article is a highly active test article having a predicted factor XIII activity value of greater than 1.5 units / mL, and the test sample is prepared by diluting the highly active test article using a fibrinogen test solution containing fibrinogen at a concentration of 2.0 to 4.0 mg / mL in an Owen-Veronal buffer solution, thereby providing a method for preparing a test sample that can determine the factor XIII activity of a test article having higher activity than plasma using the FXIII-GLDH method.

[0045] Furthermore, if the predicted value of the factor XIII activity of the highly active test article exceeds 10.0 units / mL, a test intermediate diluted sample is prepared by diluting the sample with a specimen dilution solution containing 0.5 w / v% HSA and / or 0.1 v / v% Tween 20 in Owen-Veronal buffer so that the predicted value of factor XIII activity is 10 times the target activity value of the test sample, and then the test intermediate diluted sample is diluted 10 times with the fibrinogen test solution to prepare a test sample so that the predicted value of factor XIII activity is the target activity value of the test sample.This allows the use of a more economical dilution solution when performing high-fold dilutions, such as 50-fold or 100-fold.

[0046] Furthermore, if the fibrinogen concentration of the fibrinogen test solution is 2.5 mg / mL, the fibrinogen test solution can be suitable for dilution by 5 times or more.

[0047] Furthermore, according to the method for preparing a measurement point sample of the present invention, there is provided a method for preparing the measurement point sample used in the method for determining a factor XIII activity value of the present invention, wherein the measurement point sample is prepared by diluting a standard substance having a known factor XIII activity value greater than the target activity value of the measurement point sample using a fibrinogen test solution containing fibrinogen at a concentration of 2.0 to 4.0 mg / mL in an Owen-Veronal buffer solution. In this case, the measurement point sample can be prepared by diluting a non-plasma material having a known activity higher than that of plasma.

[0048] Furthermore, when the factor XIII activity value of the standard substance exceeds 10 times the target activity value of the measurement point sample, a measurement point intermediate diluted sample is prepared by diluting the sample with a specimen dilution solution containing 0.5 w / v% HSA and / or 0.1 v / v% Tween 20 in Owen-Veronal buffer so that the factor XIII activity value is 10 times the target activity value of the measurement point sample, and then the measurement point intermediate diluted sample is diluted 10 times with the fibrinogen test solution to prepare a measurement point sample so that the factor XIII activity value is the target activity value of the measurement point sample.In this way, when performing high-magnification dilutions such as 50-fold or 100-fold, a more economical dilution solution can be used.

[0049] Furthermore, the method for producing a blood product according to the present invention comprises a semi-finished product preparation step in which collected blood or a blood fraction is subjected to a predetermined pretreatment to prepare a semi-finished product (excluding plasma), a factor XIII activity determination step in which the semi-finished product or a processed product (excluding plasma) obtained by subjecting the semi-finished product to the necessary treatment for measurement is used as the test substance and the factor XIII activity of the semi-finished product is determined by the method for determining factor XIII activity according to the present invention, and a product production step in which the suitability of the semi-finished product is judged based on the factor XIII activity determined in the factor XIII activity determination step, and if the semi-finished product is judged to be suitable, a predetermined post-treatment is carried out as necessary to produce a blood product. By using the FXIII-GLDH method as a premise, it is possible to improve the workability required for measuring physical property values ​​corresponding to factor XIII activity, and to produce a blood product in which the factor XIII activity of the semi-finished product in a purified or concentrated state compared to plasma is determined.

[0050] Furthermore, if the test sample used in the method for determining factor XIII activity is prepared by the above-mentioned method for preparing a test sample according to the present invention, a test sample that can be determined by the FXIII-GLDH method can be prepared, and then the factor XIII activity of the semi-finished product, which has higher activity than plasma, can be determined to produce a blood product.

[0051] Furthermore, if the measurement point sample used in the method for determining factor XIII activity is prepared by the method for preparing a measurement point sample according to the present invention, a measurement point sample can be prepared by diluting a non-plasma substance that has a known higher activity than plasma, and the factor XIII activity of the semi-finished product can be determined based on the measurement point sample to produce a blood product. [Brief explanation of the drawings]

[0052] [Figure 1] FIG. 1 is an explanatory diagram showing the results of an investigation into a diluent for a highly active sample. DETAILED DESCRIPTION OF THE INVENTION

[0053] The present invention provides a method for determining the activity value of unknown blood coagulation factor XIII (factor XIII) contained in a test sample other than plasma by comparing the physical properties of the test sample with those of a reference sample with known factor XIII activity.The present invention is based on the FXIII-GLDH method, thereby improving the workability required to measure physical properties corresponding to factor XIII activity values, and provides a method that can accurately determine the factor XIII activity value of products or intermediates in which factor XIII is purified or concentrated compared to plasma, without being affected by the sample composition.

[0054] The present invention also provides a method for determining the factor XIII activity of a test article (excluding plasma) by measuring physical properties corresponding to an unknown blood coagulation factor XIII (factor XIII) activity contained in a test sample (excluding plasma) prepared from the test article (excluding plasma) and comparing the physical properties with physical properties corresponding to a known factor XIII activity contained in a reference sample. The method is based on the FXIII-GLDH method, thereby improving the workability required for measuring physical properties corresponding to factor XIII activity, while also providing a method that can accurately determine the factor XIII activity of products or intermediates in which factor XIII is more purified or concentrated than in plasma, without being affected by the sample composition. In the following description, the average factor XIII activity in 1 mL of normal plasma is defined as one unit.

[0055] In the method for determining a factor XIII activity value according to this embodiment, the test article is an article other than plasma that is the target of the determination of the factor XIII activity value, and the test sample is a sample derived from the test article that is not plasma and is the direct subject of testing.

[0056] As a first example, when accurately determining the factor XIII activity of a preparation in which the factor XIII activity is increased by approximately 100 times compared to plasma, this preparation is the target product for determining the factor XIII activity value and therefore becomes the test product.

[0057] Furthermore, the FXIII-GLDH method was originally designed to determine the factor XIII activity of plasma, and it is desirable that the sample actually subjected to the FXIII-GLDH method have a factor XIII activity value close to that of average normal plasma (approximately 1.0 units / mL). However, the preparation in this example is too active to be subjected to the FXIII-GLDH method.

[0058] Therefore, since this test article is predicted to have about 100 times the factor XIII activity of plasma, a sample prepared by diluting this preparation 100 times to a factor XIII activity value that is considered to be approximately equivalent to that of plasma is the sample that is the direct test subject, that is, the test sample. In the following explanation, a test article that has or is predicted to have such high activity that dilution is necessary in preparing the test sample, for example, an activity exceeding 1.5 units / mL, is referred to as a high-activity test article, and a test article that has or is predicted to have an activity exceeding 10.0 units / mL is referred to as a specific high-activity test article.

[0059] As a second example, a preparation with factor XIII activity comparable to that of plasma and containing fibrinogen at a concentration of 2.0 mg / mL or more, as described below, can be considered a test item but can also be a direct test subject, and therefore a test sample, if it does not contain any components that would interfere with measurement by the FXIII-GLDH method.

[0060] The test item does not necessarily have to be a finished product, but may be, for example, an intermediate product produced during the manufacturing process.

[0061] Furthermore, the test article should have a predicted factor XIII activity value (predicted factor XIII activity value), as in the previous examples, such as "factor XIII activity increased by about 100 times compared to plasma" or "factor XIII activity is at the same level as plasma."

[0062] The predicted factor XIII activity value of the test product may be a value predicted based on manufacturing experience of the test product, or a value predicted from the factor XIII activity value of another lot of the test product measured by this method or another method (e.g., amine incorporation method), and the basis for the prediction is not particularly limited. However, since the dilution factor when preparing the test sample is determined based on this predicted factor XIII activity value, it is desirable for the value to be as accurate as possible.

[0063] Furthermore, the test sample does not necessarily need to be prepared with a target factor XIII activity value (approximately 1.0 units / mL) close to that of average normal plasma. For example, the target activity value may be 0.8 units / mL or 1.5 units / mL, or any other value within the range that ensures the accuracy of measurement by the FXIII-GLDH method. In the following explanation, the target activity value during preparation (dilution) is referred to as the target test sample activity value.

[0064] Like the test article, the test sample should also have a predicted factor XIII activity value. For example, when preparing a test sample by diluting the test article, the predicted factor XIII activity value of the test sample is the factor XIII activity value corresponding to the current dilution situation, predicted from the predicted factor XIII activity value of the test article. That is, it is the predicted factor XIII activity value in the test sample predicted in real time as the amount of diluent added during dilution of the test article is increased by twofold, threefold, fourfold, etc. The test article is diluted by adding diluent until the predicted factor XIII activity of the test sample reaches the target test sample activity value. In the first example above, the target test sample activity value was set to 1.0 unit / mL, and a test article with a predicted factor XIII activity of 100 units / mL was diluted to prepare a test sample with a predicted factor XIII activity of 1.0 unit / mL.

[0065] In the method for determining the factor XIII activity value according to this embodiment, the physical property value corresponding to the factor XIII activity value is the amount of change per unit time of NAD(P)H or NAD(P)H analogs consumed or NAD(P) or NAD(P) analogs produced in the FXIII-GLDH method, and is preferably the absorbance (A) of NADH at a wavelength of 340 nm. 340 ) per unit time.

[0066] This change per unit time can be calculated, for example, as the difference between the absorbance measured at a first timing after the start of the reaction and the absorbance measured at a second timing after the first timing. In this case, it is preferable to measure the first timing at least three minutes after the start of the reaction, since this allows the measurement to be performed after the reaction has stabilized. It is also preferable to measure the first and second timings at least one minute after the start of the reaction, since this allows for more accurate measurements.

[0067] More specifically, the difference between the absorbances measured 5 and 10 minutes after the start of the reaction (change per 5 minutes) can be used, or the difference can be divided by 5 to obtain the change per minute. This method of determining the change allows for more reliable determination of the factor XIII activity, since the measurement is performed after the reaction has stabilized.

[0068] Furthermore, the reaction in this FXIII-GLDH method is preferably carried out under a predetermined temperature condition, for example, 37° C. By using such a temperature, the measurement can be carried out under appropriate enzyme reaction conditions, and the factor XIII activity value of the test sample can be determined stably.

[0069] In the method for determining factor XIII activity according to this embodiment, the activity of a test article is determined based on the relative relationship between the physical properties of the test article and the physical properties of a reference sample with a known activity. In other words, the reference sample is a sample used as a measurement point for a calibration curve, such as a standard sample.

[0070] In the method for determining a factor XIII activity value according to this embodiment, the reference sample is composed of a single measurement point sample or a measurement point sample group consisting of a plurality of measurement point samples.

[0071] When a single measurement point sample constitutes a reference sample, the measurement point sample has a known factor XIII activity value that matches a predetermined target activity value for the measurement point sample. The target activity value for the measurement point sample in this case is not particularly limited as long as it is within a range that ensures the accuracy of measurement by the FXIII-GLDH method, but is preferably within a range of 0.5 units / mL to 1.5 units / mL.

[0072] Furthermore, when the reference sample is composed of a single measurement point sample, the factor XIII activity of the measurement point sample may be set to 1.0 unit / mL (the target activity of the measurement point sample may be set to 1.0 unit / mL). By adopting such a configuration, the factor XIII activity of the test sample can be reliably determined based on the reference sample composed of a single measurement point sample having a factor XIII activity close to that of normal plasma.

[0073] Furthermore, when the reference sample is composed of multiple measurement point samples, three measurement point samples may be used, each with a factor XIII activity of 0.5 units / mL, 1.0 units / mL, and 1.5 units / mL (target activity values ​​for the measurement point samples are set to 0.5 units / mL, 1.0 units / mL, and 1.5 units / mL). By using this configuration, a calibration curve can be created based on the reference sample composed of multiple measurement point samples with a factor XIII activity range close to that of normal plasma, and the factor XIII activity of the test sample can be determined.

[0074] When a reference sample is made up of multiple measurement-point samples, each measurement-point sample has a known factor XIII activity value that matches a predetermined target activity value for each of the multiple different measurement-point samples. The target activity value for the measurement-point sample is not particularly limited as long as it is within a range that ensures the accuracy of measurement by the FXIII-GLDH method, but is preferably within a range of 0.5 units / mL to 1.5 units / mL.

[0075] Furthermore, a characteristic feature of the method for determining a factor XIII activity value according to this embodiment is that the reference sample and the test sample contain fibrinogen at a concentration of 2.0 mg / mL or more.

[0076] The fibrinogen to be contained in the reference sample (each measurement point sample) and the test sample is not particularly limited as long as it is 2.0 mg / mL or more, but it is preferably in a range close to the fibrinogen concentration in normal human plasma, for example, in the range of 2.0 mg / mL to 4.0 mg / mL.

[0077] Furthermore, with this configuration, the FXIII-GLDH method is used as a premise, thereby improving the workability required to measure physical properties corresponding to factor XIII activity values, while also making it possible to accurately determine the factor XIII activity values ​​of products or intermediates in which factor XIII is in a more purified or concentrated state than plasma, without being affected by the sample composition.

[0078] The factor XIII activity value may be determined for either the test article or the test sample, or for both.

[0079] The factor XIII activity value is determined by measuring the physical property value corresponding to the factor XIII activity value of the test sample and comparing it with the physical property value corresponding to the factor XIII activity value of a reference sample.

[0080] Specifically, when the reference sample is composed of a single measurement point sample, the factor XIII activity value of the test sample can be determined by calculating how many times the physical property value corresponding to the factor XIII activity value of the test sample is larger than the physical property value corresponding to the factor XIII activity value of the reference sample, and multiplying this value by the known factor XIII activity value of the reference sample.

[0081] Furthermore, when a reference sample is constructed from multiple measurement point samples, the physical property values ​​corresponding to the factor XIII activity value of the test sample can be compared with a calibration curve constructed based on the physical property values ​​corresponding to the factor XIII activity value of the reference sample, and the corresponding activity value can be determined as the factor XIII activity value of the test sample.

[0082] In either case, it goes without saying that the values ​​may be corrected as necessary, for example, by subtracting a predetermined background or by multiplying the values ​​by a predetermined constant to match specific measurement conditions.

[0083] This specification also provides a method for preparing a test sample to be used in the above-described method for determining factor XIII activity value according to this embodiment, particularly when the test article is a highly active test article.

[0084] As mentioned above, the FXIII-GLDH method was originally designed to determine plasma factor XIII activity, and it is desirable that the sample actually used in the FXIII-GLDH method has a factor XIII activity value close to that of average normal plasma (approximately 1.0 unit / mL).

[0085] If the predicted factor XIII activity value of the test article is, for example, about 0.5 units / mL to 1.5 units / mL, it can be used as a test sample as long as it satisfies other requirements such as fibrinogen concentration, and therefore does not require dilution.

[0086] However, in the case of highly active test products exceeding 1.5 units / mL, or more particularly, those exceeding 2.0 units / mL or 5.0 units / mL, the accuracy of measurement using the FXIII-GLDH method is not necessarily clear. Therefore, it is desirable to appropriately set a target activity value for the test sample so that the predicted factor XIII activity of the test product is approximately 1.0 units / mL, which is close to that of average normal plasma, and to dilute the test product with a specified diluent.

[0087] The specified diluent used here can be factor XIII-deficient human plasma or a buffer solution containing fibrinogen at a specified concentration and having a buffer range of pH 7.5 (hereinafter referred to as the sample preparation diluent).

[0088] Examples of buffers with a buffer range of pH 7.5 include Tris-HCl buffer and barbital buffers (e.g., barbital·lithium·HCl buffer), with Owren's Veronal Buffer (OV) being particularly preferred.

[0089] The concentration of fibrinogen should be such that the final concentration in the diluted test sample is 2.0 mg / mL or more, preferably in the range of 2.0 mg / mL to 4.0 mg / mL, and can be adjusted appropriately depending on the dilution ratio.

[0090] For example, the test sample can be prepared by diluting the highly active test product B / C times using a diluent containing fibrinogen at a concentration of {(D×B / C)-A} / {(B / C)-1} mg / mL, where the fibrinogen concentration of the test product is A mg / mL, the Factor XIII activity value of the test product is B units / mL, the target test sample activity value of the test sample is C units / mL, and the fibrinogen concentration to be contained in the test sample is D mg / mL (where 0≦A≦D×B / C, B>0, B>C, 0.5≦C≦1.5, 2.0≦D≦4.0).

[0091] Although the fibrinogen concentration of the diluent for sample preparation can basically be adjusted in this way, a fibrinogen concentration of 2.5 mg / mL is preferable because it allows the final concentration of fibrinogen in the test sample to be 2.0 mg / mL or more when diluted by a dilution factor of 5 or more. In the following explanation, the Owen-Veronal buffer solution containing fibrinogen at a concentration of 2.5 mg / mL will be referred to as the fibrinogen test solution when referring to the buffer for sample preparation.

[0092] Incidentally, the dilution solution for sample preparation can be used for test articles that require dilution by about 10 times, or by about 1000 times, regardless of the dilution factor.

[0093] However, since the cost per unit volume of the diluent for sample preparation can be relatively high, it is possible to use a cheaper diluent in addition to the diluent for sample preparation, mainly from the viewpoint of economy.

[0094] For example, as in the first example described above, if the predicted factor XIII activity of the test article is 100 units / mL and the target activity of the test sample is 1.0 units / mL, an intermediate dilution target value (e.g., 10 units / mL, hereinafter referred to as the intermediate diluted sample target value) can be set, and an intermediate diluted sample can be prepared from 100 units / mL to 10 units / mL using a separate, inexpensive diluent for high-activity samples. The final dilution step, from 10 units / mL to 1 unit / mL, leading to the preparation of the test sample, can then be performed using the sample preparation diluent. In the following description, the dilution method in which an intermediate diluted sample is prepared by diluting with the diluent for high-activity samples as described above, and then the intermediate diluted sample is diluted with the sample preparation diluent to prepare the test sample, is referred to as the heterogeneous buffer dilution method.

[0095] The diluent for highly active samples can be a buffer solution containing 0.5 w / v% human serum albumin (HSA) and / or 0.1 v / v% Tween 20 and having a pH of 7.5 in the buffer range. Examples of buffer solutions containing a pH of 7.5 in the buffer range include Tris-HCl buffer and barbital buffers (e.g., barbital-lithium-HCl buffer), with Owren's Veronal Buffer (OV) being particularly preferred. In the following description, Owren's Veronal Buffer containing 0.1 v / v% Tween 20 will be referred to as the "specimen diluent" when referring to the diluent for highly active samples.

[0096] Furthermore, when diluting a test item, the decision as to whether to dilute it using only a sample preparation diluent (e.g., fibrinogen test solution) or to use a heterogeneous buffer dilution method and also use a diluent for a highly active sample (e.g., specimen diluent) is not particularly strictly defined. However, for example, if the predicted factor XIII activity of the test item is approximately 10.0 units / mL to 15.0 units / mL, one value within this range (e.g., 10.0 units / mL) can be used as the boundary value, and if the activity is below this boundary value, the test item can be diluted with only a sample preparation diluent, and if the activity is above the boundary value, the heterogeneous buffer dilution method can be used, thereby enabling the test sample to be prepared more inexpensively.

[0097] Furthermore, when a heterogeneous buffer dilution method is used to dilute the test article, the target value for the intermediate dilution sample can be set appropriately taking into account economic efficiency, and can be, for example, approximately 5.0 units / mL to 15.0 units / mL.

[0098] In addition, the test sample preparation method according to this embodiment involves diluting a highly active test article using a fibrinogen reagent solution to prepare a test sample. However, "using a fibrinogen reagent solution" does not necessarily mean that all of the diluents used to dilute the highly active test article must be fibrinogen reagent solution. In other words, it should be interpreted as including cases where, as in the heterogeneous buffer dilution method, a sample preparation diluent is used for the final dilution and a different diluent is used for the previous dilution steps. Additionally, in the test sample preparation method according to this embodiment, the test sample may be prepared by diluting a highly active test article using at least a fibrinogen reagent solution. More specifically, the test sample may be prepared by diluting an active test article while using a fibrinogen reagent solution for the final dilution step.

[0099] This specification also provides a method for preparing measurement point samples used in the method for determining factor XIII activity according to the present embodiment, particularly a method for preparing measurement point samples from a standard substance having such high activity that dilution is required. In the following description, a standard substance having such high activity that dilution is required for the preparation of the measurement point sample, for example, a standard substance having a factor XIII activity value exceeding a predetermined target measurement point sample activity, used when preparing a measurement point sample having a target measurement point sample activity, is referred to as a high-activity standard substance. More specifically, a standard substance having a factor XIII activity value exceeding 1.0 units / mL, which is used when preparing a measurement point sample having a target measurement point sample activity of 1.0 units / mL, has such high activity that dilution is required for the preparation of the measurement point sample, and can be considered a high-activity standard substance.

[0100] The method for preparing measurement point samples according to this embodiment is characterized in that the measurement point samples are prepared by diluting a highly active standard substance with the aforementioned sample preparation diluent. Furthermore, fibrinogen test solution is also suitable as the sample preparation diluent for preparing measurement point samples. Furthermore, factor XIII-deficient human plasma can also be used instead of the sample preparation diluent.

[0101] Furthermore, when the standard substance is a high-activity standard substance (hereinafter also referred to as a specific high-activity standard substance) whose factor XIII activity value exceeds 10 times the target activity value of the measurement point sample, the measurement point sample may be prepared by first setting the target value of the intermediate diluted sample using a diluent for high activity samples, as in the preparation of the test sample described above, using the heterogeneous buffer dilution method, and then performing the final dilution using a diluent for sample preparation. Furthermore, even when preparing measurement point samples using the heterogeneous buffer dilution method, the specimen diluent is also suitable as a diluent for high activity samples.

[0102] In addition, the method for preparing a measurement point sample according to this embodiment involves diluting a standard substance using a fibrinogen test solution to prepare a test sample, but "using a fibrinogen test solution" does not necessarily mean that all of the diluting liquid used to dilute the standard substance should be fibrinogen test solution, as in the method for preparing a test sample according to this embodiment described above.

[0103] This specification also provides a method for producing a blood product, particularly a method for producing a blood product comprising a step of determining the factor XIII activity value of a semi-finished product by the method for determining the factor XIII activity value of this embodiment.

[0104] Here, blood products are medicines whose active ingredient is human blood or a substance derived from human blood, and are a concept that includes both blood products for transfusion and plasma fraction products.

[0105] The method for producing a blood product according to this embodiment includes a semi-finished product preparation step, a factor XIII activity value determination step, and a product production step.

[0106] The semi-finished product preparation step is a step in which the collected blood or a blood fraction is subjected to a predetermined pretreatment to prepare a semi-finished product.

[0107] The term "semi-finished product" refers to a product that is in a state before it can be shipped as a finished product, and includes not only intermediate products in the manufacturing process (including products in the middle of processing), but also products that are almost complete and only require confirmation of factor XIII activity. For the sake of convenience, hereinafter, semi-finished products other than plasma will be referred to as "target semi-finished products."

[0108] Pretreatment is a process carried out in the process of preparing semi-finished products from blood or blood fractions (hereinafter also referred to as raw materials), and includes any process such as separating a predetermined component from the raw materials or intermediate products, adding a predetermined component to the raw materials or intermediate products, thermal treatment of heating or cooling the raw materials or intermediate products, dynamic treatment of vibrating, stirring or causing convection in the raw materials or intermediate products, filling the raw materials or intermediate products into containers or attaching labels, etc., or a combination of these processes.

[0109] The raw material blood to be subjected to pretreatment is blood that has been collected and separated from the circulatory system of a living body. Furthermore, blood fractions to be subjected to pretreatment are blood products obtained after certain components have been removed from the blood or products separated from the blood, such as red blood cell products, plasma products, and platelet products. While blood fractions themselves are separated from the circulatory system of a living body, the blood itself before fractionation is not necessarily separated from the circulatory system of a living body, as in the case of component blood donations, for example.

[0110] The factor XIII activity value determination step is a step in which the target semi-finished product or a processed product (excluding plasma) obtained by subjecting the target semi-finished product to the processing necessary for measurement by the FXIII-GLDH method is used as the test substance, and the factor XIII activity value of the semi-finished product is determined by the factor XIII activity value determination method of this embodiment.

[0111] The test sample and measurement point sample used for measurement by the FXIII-GLDH method in this factor XIII activity determination step can be prepared by the test sample preparation method or measurement point sample preparation method according to the present embodiment. This configuration allows a test sample that can be determined by the FXIII-GLDH method to determine the factor XIII activity of a semi-finished product with higher activity than plasma, thereby producing a blood product. Alternatively, a non-plasma product known to have higher activity than plasma can be diluted to prepare a measurement point sample, and the factor XIII activity of the semi-finished product can be determined based on this sample to produce a blood product.

[0112] Semi-finished products that can be used as test substances include those that can be used as test samples as they are, and those that can be used to prepare test samples by diluting the test substance with the aforementioned sample preparation diluent or high activity sample diluent. To give an example for ease of understanding, in the case of manufacturing a factor XIII preparation, which is a plasma fraction preparation as a blood product, intermediate products, raw fractions, final bulk, and small products in a state before commercialization, in which factor XIII has been purified to a certain extent during the manufacturing process, are examples of semi-finished products that can be used as test substances as they are, and those that can be used as test samples by diluting the test substance with the aforementioned sample preparation diluent or high activity sample diluent.

[0113] Furthermore, even if the target semi-finished product cannot be used as a test substance as is or after dilution, this processed product can be used as a test substance by performing the necessary processing for measurement using the FXIII-GLDH method.

[0114] For example, in the case of the FXIII-GLDH method, in which absorbance is measured as a physical property corresponding to factor XIII activity, a cloudy target semi-finished product containing dispersants that affect absorbance cannot be used as a test substance because it may be difficult to accurately determine the factor XIII activity. However, if the target semi-finished product is subjected to a process required for measurement, such as centrifugation or filtration, to remove the dispersants without affecting the factor XIII activity, the treated product can be used as a test substance. Hereinafter, such treated products that are not plasma will also be referred to as target treated products.

[0115] The product processing step is a step in which the suitability of the target semi-finished product is judged based on the factor XIII activity value determined in the factor XIII activity determination step, and if the target semi-finished product is judged to be suitable, it is subjected to predetermined post-processing as necessary to produce a blood product.

[0116] "The suitability of the target semi-finished product is determined based on the factor XIII activity value determined in the factor XIII activity value determination step" means that the determined factor XIII activity value is compared with a predetermined activity value (suitability standard value) for determining suitability of the target semi-finished product to determine whether it is suitable or not.

[0117] The factor XIII activity determined in the factor XIII activity determination step may be the factor XIII activity of a test article or a test sample. The test article may be a target semi-finished product or a target processed product.

[0118] In other words, the judgment of whether a target semi-finished product is suitable may be based on the factor XIII activity value determined for the target semi-finished product itself, or may be based on the factor XIII activity value determined for the target processed product or for a dilution (test sample) of the target semi-finished product or the target processed product (test article). However, it is assumed that the judgment of suitability of the factor XIII activity value determined for the target processed product or each dilution coincides with the suitability of the target semi-finished product.

[0119] According to the first example mentioned above, the quality of the formulation of the first example is controlled (suitability judged) based on the factor XIII activity value of the formulation (activity value before dilution) obtained by multiplying the determined factor XIII activity value of the test sample by 100. This also includes controlling (suitability judged) the quality of the formulation based on the factor XIII activity value of the formulation diluted to 1 / 100 (the state of the diluted product that is the test sample) without having to perform the calculation of multiplying by 100 again.

[0120] If the treatment required for the measurement of the target processed material affects the factor XIII activity value, the activity value of the target semi-finished product may be calculated taking into account the activity affected by that treatment.

[0121] Furthermore, according to the method for producing a blood product according to this embodiment as described above, by using the FXIII-GLDH method as a premise, it is possible to improve the workability required for measuring physical properties corresponding to factor XIII activity values, while producing a blood product in which the factor XIII activity value of a semi-finished product in which factor XIII is more purified or concentrated than in plasma has been determined.

[0122] The method for determining factor XIII activity value, the method for preparing test samples, and the method for preparing measurement point samples according to this embodiment will be described in detail below with reference to examples of determining factor XIII activity value and the process for determining the concentrations of various components.

[0123] [1] Example of determining factor XIII activity In this example, the factor XIII activity of the factor XIII fraction (FXIII fraction), an intermediate product of plasma fractionation, was determined. The factor XIII activity of the FXIII fraction was estimated by the amine uptake assay and was approximately 1280 units / mL.

[0124] (1-1) Preparation of diluent for sample preparation In this example, a fibrinogen test solution was used as the diluent for sample preparation. The fibrinogen test solution was prepared by diluting a fibrinogen solution with a known fibrinogen concentration with an Owlen-Veronal buffer solution (Sysmex Corporation) to prepare an Owlen-Veronal buffer solution containing 2.5 mg / mL of fibrinogen.

[0125] (1-2) Preparation of dilution solution for highly active samples In this example, a specimen dilution solution was prepared as a dilution solution for highly active samples. Specifically, an Owen Veronal buffer solution containing 0.1 v / v% Tween 20 (Nacalai Tesque) was used as the specimen dilution solution.

[0126] (1-3) Preparation of test samples The test product, FXIII original, is a specific high activity test product with a predicted factor XIII activity of approximately 1280 units / mL, so in this example, it was diluted using the heterogeneous buffer dilution method.

[0127] Specifically, the target value for the intermediate diluted sample was set to approximately 10.0 units / mL, and the target activity value for the test sample was set to approximately 1.0 units / mL.First, the FXIII original was diluted 128-fold with the sample dilution solution to prepare an intermediate diluted sample with a predicted factor XIII activity value of approximately 10.0 units / mL, which was the target value for the intermediate diluted sample.

[0128] Next, the intermediately diluted sample was diluted 10-fold with fibrinogen test solution to prepare a test sample with a predicted factor XIII activity of approximately 1.0 unit / mL, which was the target activity value for the test sample.

[0129] (1-4) Preparation of reference samples In this example, the reference sample consisted of multiple (three) measurement point samples. Each measurement sample was prepared by the heterogeneous buffer dilution method using a factor XIII-containing solution with a known factor XIII activity value, which was a specific high activity standard substance.

[0130] Specifically, for the first measurement point sample, the intermediate diluted sample target value was set to 5.0 units / mL and the measurement point sample activity target value was set to 0.5 units / mL; for the second measurement point sample, the intermediate diluted sample target value was set to 10.0 units / mL and the measurement point sample activity target value was set to 1.0 units / mL; and for the third measurement point sample, the intermediate diluted sample target value was set to 15.0 units / mL and the measurement point sample activity target value was set to 1.5 units / mL.

[0131] Next, the factor XIII-containing solution was diluted with a specimen dilution solution to prepare first to third intermediately diluted samples whose predicted factor XIII activity values ​​reached the target values ​​for the intermediately diluted samples.

[0132] The first to third intermediately diluted samples were then diluted 10-fold with fibrinogen test solution to prepare the first to third measurement point samples whose predicted factor XIII activity values ​​were equal to the target activity values ​​for the test samples.

[0133] In addition, as a reference sample for comparison (reference sample for comparison), first to third comparative measurement point samples were prepared using factor XIII-deficient human plasma at concentrations of 0.5 units / mL, 1.0 units / mL, and 1.5 units / mL.

[0134] (1-5) Measurement by FXIII-GLDH method In this example, the physical properties corresponding to the factor XIII activity of the test sample and the reference sample were measured by the FXIII-GLDH method, which was calculated as the change in absorbance per minute at a wavelength of 340 nm of NADH consumed in the reaction. In particular, in this example, the factor XIII coagulation factor kit, Verichrome FXIII (Sysmex), was used and the measurement was performed on the fully automated blood coagulation analyzer CS-2000i (Sysmex).

[0135] (1-6) Determination of Factor XIII Activity of Test Samples and Test Articles The factor XIII activity was also determined by comparing the measurement results of the test sample with those of the reference sample using the fully automated blood coagulation analyzer CS-2000i and software.

[0136] Specifically, a calibration curve was created using the measurement results of the samples at the first to third measurement points, and the Factor XIII activity value of the test sample was determined by comparing it with the measurement results of the test sample, and then the Factor XIII activity value of the test article was determined by multiplying this value by 1280. The results are shown in Table 1. [Table 1]

[0137] As shown in Table 1 as Example 1, the factor XIII activity value determined by comparing the calibration curve based on the measurement results of a reference sample prepared by diluting a standard substance, a factor XIII-containing solution, with a specimen diluent and a fibrinogen test solution using the heterogeneous buffer dilution method with the measurement results of a test sample prepared by diluting a test substance, a factor XIII original, with a specimen diluent and a fibrinogen test solution using the heterogeneous buffer dilution method, was approximately 1250 units / mL, which was close to the predicted value of approximately 1280 units / mL previously estimated using the amine incorporation method.

[0138] Here, Comparative Example 1 shown in Table 1 shows the factor XIII activity value determined by comparing the calibration curve based on the measurement results of a reference sample prepared by diluting a factor XIII-containing solution, which is a standard substance, with an Owren-Veronal buffer solution, with the measurement results of a test sample prepared by diluting the test substance, original FXIII solution, with an Owren-Veronal buffer solution.

[0139] As can be seen from the results of Comparative Example 1, when Owen-Veronal buffer was used for dilution, the reference sample and test sample did not contain fibrinogen at concentrations of 2.0 mg / mL or higher, and the predicted activity value of 1380 units / mL was significantly lower (450 units / mL).

[0140] In addition, Example 2 shown in Table 1 shows the factor XIII activity value determined by comparing the calibration curve based on the measurement results of a reference sample prepared by diluting a standard substance, a factor XIII-containing solution, with factor XIII-deficient human plasma, with the measurement results of a test sample prepared by diluting the test substance, original factor XIII, with factor XIII-deficient human plasma (George King Bio-Medical).

[0141] As can be seen from the results of Example 2, when factor XIII-deficient human plasma was used for dilution, the reference sample and test sample contained fibrinogen at a concentration of 2.0 mg / mL or more, resulting in an activity of 1300 units / mL, which is close to the predicted activity of 1280 units / mL.

[0142] Moreover, Examples 3 and 4 shown in Table 1 show examples in which the reference sample was composed of a single measurement point sample and the factor XIII activity value was determined.

[0143] Specifically, Example 3 shows the factor XIII activity value determined by comparing the measurement results of the standard human plasma for blood coagulation tests (SIEMENS), which is a standard substance, as the measurement point sample (reference sample) with the measurement results of the test sample, which is the test item FXIII original diluted with factor XIII-deficient human plasma.

[0144] Furthermore, Example 4 shows the factor XIII activity value determined by comparing the measurement results of standard human plasma for blood coagulation tests, which is a standard substance, as a measurement point sample (reference sample) with the measurement results of a test sample prepared by diluting the test item, a factor XIII-containing solution, with factor XIII-deficient human plasma.

[0145] As can be seen from the results of Examples 3 and 4, when standard human plasma for blood coagulation tests was used as the reference sample and factor XIII-deficient human plasma was used for dilution of the test sample, the reference sample and the test sample contained fibrinogen at a concentration of 2.0 mg / mL or more, and the determined activity values ​​in both Examples were the same as or very close to the predicted activity values.

[0146] Thus, the results of Example 1, Comparative Example 1, and Examples 2 to 4 show that the method for determining factor XIII activity according to this embodiment improves the workability required for measuring physical properties corresponding to factor XIII activity by using the FXIII-GLDH method, while also enabling the factor XIII activity of products or intermediates in which factor XIII is more purified or concentrated than in plasma to be determined more accurately than when using reference samples or test samples with fibrinogen concentrations of less than 2.0 mg / mL.

[0147] [2] Various considerations The method for determining the factor XIII activity value, the method for preparing the test sample, and the method for preparing the measurement point sample according to the present embodiment described above have been perfected through various studies and trial and error.

[0148] In this section, we will explain the studies and evaluations that were conducted prior to the completion of these diluents for highly active samples (specimen diluents) and diluents for sample preparation (fibrinogen test solution), including experimental results.

[0149] (2-1) Study of diluents for highly active samples and specimen diluents First, a diluent for a highly active sample used in the method for determining the factor XIII activity value, the method for preparing a test sample, and the method for preparing a measurement point sample according to this embodiment was investigated.

[0150] Specifically, four dilutions were prepared: (a) Owren-Veronal buffer, (b) Owren-Veronal buffer + 0.5 w / v% HSA, (c) Owren-Veronal buffer + 0.1 v / v% Tween 20, and (d) Owren-Veronal buffer + 0.5 w / v% HSA + 0.1 v / v% Tween 20, and their effect on the measurement of the FXIII original was evaluated.

[0151] The absorbance change rate and linearity of the original FXIII solution prepared at approximately 0.5 to 2.0 units / mL using the dilutions (a) to (d) above were evaluated. The evaluation results are shown in Figure 1.

[0152] As shown in Figure 1, the linearity of the diluted solutions (a) to (d) was good. In addition, the measured value of the absorbance change rate for (a) was generally low, but no clear difference was observed for (b) to (d).

[0153] From these results, it was concluded that any of (b) to (d) can be used as a specimen diluent for the method for determining factor XIII activity value, the method for preparing test samples, and the method for preparing measurement point samples according to the present embodiment. Furthermore, it was determined that it is desirable to add either HSA or Tween 20, or both, to the Owen-Veronal buffer solution to prevent protein adsorption during dilution.

[0154] In addition, taking into consideration the ease of commercial availability and the simplicity of the preparation procedure, we decided to use (c) Owen Veronal buffer + 0.1 v / v% Tween 20, which contains only Tween 20, as the sample diluent for this test.

[0155] (2-2) Examination of fibrinogen concentration in the specimen Variations in fibrinogen concentration in test samples and reference samples affect the measurement results. Therefore, in this section, we investigated methods to make the fibrinogen concentration in test samples and reference samples uniform.

[0156] (a) Defibrinogenation We investigated a method for homogenizing each test sample and reference sample (hereinafter referred to as test samples, etc.) by defibrinogenating them and removing fibrinogen. This defibrinogenation method is also used for test samples in the amine uptake method.

[0157] However, to state the conclusion first, when carrying out the method for determining factor XIII activity value, the method for preparing test samples, and the method for preparing measurement point samples according to this embodiment, it has been determined that defibrinogenation is inappropriate for homogenizing the fibrinogen in test samples, etc.

[0158] Specifically, as with the amine uptake method, the test sample was heated to 55°C, the resulting fibrinogen aggregates were filtered through a syringe filter with a pore size of 10 μm, and the resulting filtrate was subjected to measurement.However, it was observed that the absorbance response curve was not linear for some test items and standard substances.

[0159] Furthermore, it was thought that this phenomenon was due to tiny aggregated particles that could not be removed with a 10 μm syringe filter affecting the measurement of the absorbance change rate. Therefore, when the sample was filtered with a filter with a smaller pore size of 5 μm, the problem of linearity of the absorbance response curve was resolved.

[0160] However, it was confirmed that the removal of fibrinogen resulted in a clear decrease in the rate of change in absorbance of the test samples.

[0161] In addition, the correlation coefficient of the regression line, R 2 Since the fibrinogen concentration also tended to decrease, it was thought that the linear range was likely to narrow due to defibrinogenation, and the measurement range was also likely to narrow.

[0162] Based on these results, it was decided that the fibrinogen concentration in test samples and the like would be homogenized by adding fibrinogen rather than by defibrinogenation.

[0163] (b) Study of sample preparation diluent (fibrinogen test solution) In order to homogenize fibrinogen in test samples, etc., it is convenient to remove fibrinogen from the test samples, etc. However, in view of the above-mentioned reasons, the method for determining a factor XIII activity value, the method for preparing a test sample, and the method for preparing a measurement point sample according to this embodiment are highly distinctive in that they employ a technique in which fibrinogen is added to the test sample, etc., by diluting the test sample with a fibrinogen-containing diluent for sample preparation to homogenize the concentration.

[0164] It is possible to use FXIII-deficient human plasma as an alternative to the diluent for sample preparation to dilute the test items and standard substances, but there are concerns about cost and stable supply, so the composition of the diluent for sample preparation was examined.

[0165] As a result, since the fibrinogen concentration in normal human plasma is approximately 2 to 3 mg / mL, we decided to use a sample preparation dilution solution containing 2.5 mg / mL of fibrinogen in Owen-Veronal buffer as the fibrinogen test solution.

[0166] Furthermore, because it is necessary to maintain a uniform fibrinogen concentration in the standard and all samples, the mixing ratio with fibrinogen test solution is fixed at 1:9 as an example. Specifically, the standard is prepared with sample diluent to 5, 10, or 15 units / mL, and then mixed with fibrinogen test solution at a 1:9 ratio to prepare concentrations of 0.5, 1, or 1.5 units / mL. The test article can be diluted to approximately 10 units / mL with sample diluent, and then mixed with fibrinogen test solution at a 1:9 ratio to prepare a concentration of approximately 1.0 unit / mL (heterogeneous buffer dilution method).

[0167] (c) Effect of sample and diluent on factor XIII activity Here, we used a factor XIII-containing solution or standard human plasma as the standard substance, and a factor XIII-containing solution, FXIII extract, or human standard plasma as the test substance. We investigated the effects on factor XIII activity when using human standard plasma, which does not require dilution, as the standard or test substance, or when using a factor XIII-containing solution or FXIII extract as the test substance and diluting it with fibrinogen test solution (the final dilution step in the case of the heterogeneous buffer dilution method). The results are shown in Table 2. For the human standard plasma in the table, the lyophilized powder was dissolved in 1 mL of water for injection and used for measurement without dilution. [Table 2]

[0168] As can be seen from Table 2, the factor XIII activity values ​​determined in Examples 1, 5, and 6 were not found to be extremely lower than the predicted activity values, compared to Comparative Example 1 shown in Table 1, where the activity value determined using Oren-Veronal buffer as the diluent was less than half of the predicted activity value.

[0169] To obtain values ​​as accurate as possible without being affected by sample composition, for example, values ​​close to the predicted activity value by the amine uptake method, it is desirable to use the same or equivalent diluents for diluting the standard and the test substance. Specifically, it is desirable to use the same fibrinogen test solution (and the test substance diluent and fibrinogen test solution, if necessary).

[0170] (2-3) Examination of fibrinogen test solution concentration Dilutions containing different concentrations of fibrinogen were prepared, and the influence of dilution with each dilution on the factor XIII activity value was evaluated.

[0171] Specifically, we determined the factor XIII activity of fibrinogen in the Owen-Veronal buffer solution at five concentrations: 1, 2, 2.5, 3, and 4 mg / mL. As shown in Table 3, we confirmed that there was almost no difference in the factor XIII activity at concentrations of at least 2 mg / mL. [Table 3]

[0172] As described above, the method for determining a factor XIII activity value according to this embodiment is a method for determining an unknown blood coagulation factor XIII (factor XIII) activity value contained in a test sample other than plasma by comparing the physical property values ​​of the test sample with those of a reference sample having known factor XIII activity, and the physical property values ​​are determined by reacting a predetermined substrate of factor XIII having a glutamine residue with glycine ethyl ester in the presence of calcium ions and thrombin using factor XIII in the test sample or the reference sample to generate ammonia, and then catalyzing the reaction of the ammonia with α-ketoglutaric acid by glutamate dehydrogenase in the presence of NAD(P)H or an NAD(P)H analog. The factor XIII activity value is a physical property value corresponding to the change per unit time in the amount of NAD(P)H or NAD(P)H analogs consumed or the amount of NAD(P) or NAD(P) analogs produced by the reaction with an enzyme. The reference sample and test sample contained fibrinogen at a concentration of 2.0 to 4.0 mg / mL. Therefore, by using the FXIII-GLDH method as a premise, it is possible to improve the workability required for measuring physical property values ​​corresponding to factor XIII activity values, while providing a method that can accurately determine the factor XIII activity values ​​of products or intermediates in which factor XIII is in a purified or concentrated state compared to plasma, without being affected by the sample composition.

[0173] Furthermore, according to the method for determining the factor XIII activity value of this embodiment, a test sample (excluding plasma) prepared from a test article (excluding plasma) can be used.and determining the activity of factor XIII in the test article or the test sample by measuring a physical property corresponding to an unknown activity of blood coagulation factor XIII (factor XIII) contained in the test article or the test sample and comparing it with a physical property corresponding to a known activity of factor XIII contained in a reference sample, wherein the physical property corresponding to the activity of factor XIII in the test sample and the reference sample is determined by reacting a predetermined substrate of factor XIII having a glutamine residue with glycine ethyl ester in the presence of calcium ions and thrombin with factor XIII in the test sample or the reference sample. The reference sample is a physical property value corresponding to the change per unit time in the amount of NAD(P)H or an NAD(P)H analog consumed or the amount of NAD(P) or an NAD(P) analog produced by reacting ammonia with α-ketoglutaric acid in the presence of NAD(P)H or an NAD(P)H analog with glutamate dehydrogenase in the presence of NAD(P)H or an NAD(P)H analog to generate ammonia, and the reference sample is a measurement point sample having a known factor XIII activity value that matches a predetermined target activity value for the measurement point sample, where the activity of factor XIII in 1 mL of normal plasma is defined as 1 unit. The test article and the test sample are each composed of a test article and a plurality of test point samples having known factor XIII activity values ​​that are matched to a plurality of different target values ​​for the test point samples, and the test article and the test sample have a predicted factor XIII activity value. When there is one test point sample, the target value for the test sample is the factor XIII activity value of the test point sample. When there are multiple test point samples, the target value for the test sample is a value selected from the range from the activity value of the test point sample having the highest factor XIII activity to the activity value of the test point sample having the lowest factor XIII activity among the reference samples. The test sample was prepared so that the predicted value of factor XIII activity would be the target activity value of the test sample, and the reference sample and test sample contained fibrinogen at a concentration of 2.0 to 4.0 mg / mL.Therefore, by assuming the FXIII-GLDH method, it is possible to improve the workability required to measure physical properties corresponding to factor XIII activity values, while providing a method that can accurately determine the factor XIII activity values ​​of products and intermediates in which factor XIII is purified or concentrated compared to plasma, without being affected by the sample composition.

[0174] Finally, the above-described embodiments are merely examples of the present invention, and the present invention is not limited to the above-described embodiments. Therefore, even if the embodiments are different from those described above, various modifications can be made depending on the design, etc., as long as they do not deviate from the technical concept of the present invention.

Claims

1. A method for determining an unknown blood coagulation factor XIII (factor XIII) activity value contained in a test sample other than plasma by comparing the physical property values ​​of the test sample with those of a reference sample having known factor XIII activity, comprising: the physical property value corresponds to the change per unit time in the amount of NAD(P)H or the NAD(P)H analog consumed or the amount of NAD(P) or the NAD(P) analog produced, when a predetermined substrate for factor XIII having a glutamine residue is reacted with glycine ethyl ester by factor XIII in the test sample or the reference sample in the presence of calcium ions and thrombin to generate ammonia, and the ammonia is reacted with α-ketoglutaric acid by glutamate dehydrogenase in the presence of NAD(P)H or an NAD(P)H analog, A method for determining factor XIII activity, wherein the reference sample and the test sample contain fibrinogen at a concentration of 2.0 to 4.0 mg / mL.

2. A method for determining the activity of blood coagulation factor XIII (factor XIII) in a test article (excluding plasma), by measuring a physical property value corresponding to an unknown activity of blood coagulation factor XIII contained in a test sample (excluding plasma) prepared from the test article (excluding plasma), and comparing the measured physical property value with a physical property value corresponding to a known activity of factor XIII contained in a reference sample, comprising: The physical property values ​​corresponding to the factor XIII activity values ​​in the test sample and the reference sample are physical property values ​​corresponding to the change per unit time in the amount of NAD(P)H or the NAD(P)H analog consumed or the amount of NAD(P) or the NAD(P) analog produced, when a predetermined substrate for factor XIII having a glutamine residue is reacted with glycine ethyl ester by factor XIII in the test sample or the reference sample in the presence of calcium ions and thrombin to generate ammonia, and the ammonia is reacted with α-ketoglutaric acid by glutamate dehydrogenase in the presence of NAD(P)H or an NAD(P)H analog, The reference sample is composed of one measurement point sample having a known factor XIII activity value that matches a predetermined target activity value for the measurement point sample, where the activity of factor XIII in 1 mL of normal plasma is defined as 1 unit, or a plurality of measurement point samples having known factor XIII activity values ​​that match a plurality of different target activity values ​​for the measurement point sample, The test article and test sample have predicted factor XIII activity values, and when there is one measurement point sample, the factor XIII activity value of the measurement point sample is used as the target test sample activity value. When there are multiple measurement point samples, the target test sample activity value is a value selected from the range from the activity value of the measurement point sample having the highest factor XIII activity to the activity value of the measurement point sample having the lowest factor XIII activity among the reference samples. The test sample is prepared so that the predicted value of factor XIII activity becomes the target test sample activity value. A method for determining factor XIII activity, wherein the reference sample and the test sample contain fibrinogen at a concentration of 2.0 to 4.0 mg / mL.

3. The target activity of the sample at the measurement point is set to 1.0 units / mL; 3. The method for determining factor XIII activity according to claim 2, wherein the reference sample is a single measurement point sample having a factor XIII activity of 1.0 unit / mL.

4. The target activity values ​​of the sample at the measurement points are set to 0.5 units / mL, 1.0 units / mL, and 1.5 units / mL; 3. The method for determining factor XIII activity according to claim 2, wherein the reference sample is composed of three measurement point samples having factor XIII activity values ​​of 0.5 units / mL, 1.0 units / mL, and 1.5 units / mL.

5. 3. The method for determining factor XIII activity according to claim 2, wherein the reaction is carried out at 37°C.

6. the physical property value corresponding to the factor XIII activity value in the test sample and the reference sample is the amount of change in absorbance of NADH consumed by the reaction at a wavelength of 340 nm per minute; The method for determining factor XIII activity according to claim 2, wherein the change is calculated as the quotient obtained by dividing the difference in absorbance measured 5 minutes and 10 minutes after the start of the reaction by 5.

7. A method for preparing a test sample to be used in the method for determining factor XIII activity according to claim 2, comprising: the test article is a high activity test article having a predicted factor XIII activity of greater than 1.5 units / mL; A method for preparing a test sample, characterized in that the test sample is prepared by diluting the highly active test item using a fibrinogen test solution containing fibrinogen at a concentration of 2.0 to 4.0 mg / mL in Oren-Veronal buffer solution.

8. If the predicted factor XIII activity of the high-activity test article is greater than 10.0 units / mL, a test intermediate diluted sample is prepared by diluting the test sample with a sample diluent containing 0.5 w / v % HSA and / or 0.1 v / v % Tween 20 in Oren-Veronal buffer so that the predicted value of factor XIII activity is 10 times the target activity value of the test sample; The method for preparing a test sample according to claim 7, further comprising the step of diluting the intermediate diluted test sample 10 times with the fibrinogen test solution to prepare a test sample so that the predicted value of factor XIII activity becomes the target activity value of the test sample.

9. 8. The method for preparing a test sample according to claim 7, wherein the fibrinogen concentration of the fibrinogen test solution is 2.5 mg / mL.

10. 3. A method for preparing the measurement sample used in the method for determining factor XIII activity according to claim 2, comprising: The method for preparing a measurement point sample is characterized in that the measurement point sample is prepared by diluting a standard substance having a known factor XIII activity value greater than the target activity value of the measurement point sample using a fibrinogen test solution containing fibrinogen at a concentration of 2.0 to 4.0 mg / mL in an Owen-Veronal buffer solution.

11. If the factor XIII activity value of the standard substance is more than 10 times the target activity value of the measurement point sample, a sample dilution solution containing 0.5 w / v % HSA and / or 0.1 v / v % Tween 20 in Oren-Veronal buffer so that the factor XIII activity value is 10 times the target activity value of the sample at the measurement point, thereby preparing an intermediate diluted sample at the measurement point; The method for preparing a measurement point sample according to claim 10, further comprising the steps of: diluting the measurement point intermediate diluted sample 10 times with the fibrinogen test solution to prepare a measurement point sample so that the factor XIII activity value becomes the target activity value of the measurement point sample.

12. a semi-finished product preparation step in which the collected blood or blood fraction is subjected to a predetermined pretreatment to prepare a semi-finished product (excluding plasma); a step of determining the factor XIII activity value of the semi-finished product by the method of determining the factor XIII activity value of claim 2, using the semi-finished product or a processed product (excluding plasma) obtained by subjecting the semi-finished product to a necessary treatment for measurement as a test substance; a step of determining whether the semi-finished product is suitable or not based on the factor XIII activity value determined in the step of determining the factor XIII activity value, and if the semi-finished product is determined to be suitable, subjecting it to predetermined post-treatment as necessary to produce a blood product; A method for producing a blood product, comprising:

13. The method for producing a blood product according to claim 12, wherein the test sample used in the method for determining factor XIII activity is prepared by the method for preparing a test sample according to any one of claims 7 to 9.

14. 13. The method for producing a blood product according to claim 12, wherein the measurement point sample used in the method for determining factor XIII activity is prepared by the method for preparing a measurement point sample according to claim 10 or 11.

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