JP4671823B2 - Blood coagulation factor inactivation method and blood coagulation factor inactivation sample - Google Patents
Blood coagulation factor inactivation method and blood coagulation factor inactivation sample Download PDFInfo
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Description
本発明は、血液凝固因子の不活化方法、及び不活化型の血液凝固因子を含有する血液凝固因子不活化試料に関する。また本発明は、前記血液凝固因子不活化試料を用いた血液凝固因子活性の測定方法、及び当該測定方法に用いる検体処理用血漿に関する。 The present invention relates to a blood coagulation factor inactivation method and a blood coagulation factor inactivated sample containing an inactivated blood coagulation factor. The present invention also relates to a blood coagulation factor activity measurement method using the blood coagulation factor inactivated sample, and a specimen processing plasma used in the measurement method.
先天性又は後天性血液凝固異常を検査するために血液凝固検査が行なわれる。ここで、先天的な凝固異常として有名な血友病は、血液凝固第ＶＩＩＩ因子又は第ＩＸ因子の欠乏によって起る疾患である。 A blood coagulation test is performed to check for congenital or acquired blood coagulation abnormalities. Here, hemophilia, which is famous as a congenital clotting abnormality, is a disease caused by a deficiency of blood coagulation factor VIII or factor IX.
血液凝固異常の検査としては、ＡＰＴＴ（活性化部分トロンボプラスチン時間）、ＰＴ（プロトロンビン時間）、フィブリノゲンの定量などがある。血液凝固異常の原因を明らかにするためには、各々の血液凝固因子を測定する必要がある。その方法として、ＡＰＴＴやＰＴに基づいて、血液凝固因子の活性を測定する方法が挙げられる。 Examples of the examination of blood coagulation abnormality include APTT (activated partial thromboplastin time), PT (prothrombin time), and fibrinogen quantification. In order to clarify the cause of abnormal blood coagulation, it is necessary to measure each blood coagulation factor. Examples of the method include a method of measuring the activity of a blood coagulation factor based on APTT or PT.
ここで、ＡＰＴＴやＰＴに基づく凝固因子の活性測定は、各凝固因子欠乏血漿に検査しようとする血液検体を加えて、凝固時間の延長の有無、程度を測定する方法であることから、血液凝固検査には、検査対象とする凝固因子が欠乏している血漿が必要とされる。 Here, the activity measurement of the coagulation factor based on APTT or PT is a method of adding the blood sample to be examined to each coagulation factor-deficient plasma and measuring the presence or absence of the extension of the coagulation time. The test requires plasma that is deficient in the clotting factor to be tested.
ＡＰＴＴやＰＴの測定に用いる凝固因子欠乏血漿としては、先天的欠乏症のヒト等のほ乳動物の血漿、あるいは正常血漿に所定の凝固因子と結合する抗体を用いた免疫吸着法により所定の凝固因子を除去した人工的欠乏血漿が用いられている。 As the coagulation factor-deficient plasma used for the measurement of APTT and PT, a predetermined coagulation factor is obtained by immunoadsorption using an antibody that binds to a predetermined coagulation factor in plasma of mammals such as humans with congenital deficiency or normal plasma. Removed artificial deficient plasma is used.
取得容易という点から、人工的欠乏血漿の利用が高まっているが、免疫吸着法による凝固因子の人為的除去は、使用する抗体の調製が困難であったり、第Ｖ因子又は第ＶＩＩＩ因子あるいはフォンヴィルブラント因子（ＶＷＦ）が除去されたり、大量の抗体を用いて長時間の吸着除去処理することが必要であるため、製造コストが高いという問題がある。 The use of artificially deficient plasma is increasing from the viewpoint of easy acquisition. However, artificial removal of coagulation factors by immunoadsorption method is difficult to prepare antibodies to be used, factor V or factor VIII or von There is a problem that the manufacturing cost is high because it is necessary to remove Wilvillet factor (VWF) or to perform adsorption removal treatment for a long time using a large amount of antibody.
免疫吸着法以外に、特許文献１（特開２００２−９０３６１）に、ヒト血漿と合成ポリマーとを混合させて、凝固因子を吸着除去する方法が提案されている。しかし、この方法では、第Ｖ因子又は第ＶＩＩＩ因子あるいはフォンヴィルブラント因子（ＶＷＦ）が除去されるという問題がある。 In addition to the immunoadsorption method, Patent Document 1 (Japanese Patent Application Laid-Open No. 2002-90361) proposes a method of adsorbing and removing a coagulation factor by mixing human plasma and a synthetic polymer. However, this method has a problem that factor V, factor VIII or von Willebrand factor (VWF) is removed.
本発明は、以上のような事情に鑑みてなされたものであり、その目的は、従来の血液凝固因子を吸着除去する方法とは全く異なる新規な方法、すなわち、試料中の血液凝固因子の不活化を、短時間で効率よく行なうことができる方法を提供することにある。また、本発明の目的は、従来の凝固因子欠乏血漿のような血液凝固因子が欠乏している試料とは全く異なる新規な試料、すなわち、不活化型の血液凝固因子を含有する血液凝固因子不活化試料を提供することにある。さらに、本発明の血液凝固因子不活化試料を用いた血液凝固因子の活性測定方法及び当該方法に用いる検体処理用血漿を提供する。 The present invention has been made in view of the circumstances as described above, and the object of the present invention is a novel method that is completely different from the conventional method of adsorbing and removing blood coagulation factors, that is, the absence of blood coagulation factors in a sample. An object of the present invention is to provide a method capable of efficiently performing activation in a short time. Another object of the present invention is to provide a novel sample that is completely different from a sample lacking a blood coagulation factor, such as a conventional coagulation factor-deficient plasma, that is, a blood coagulation factor-free sample containing an inactivated blood coagulation factor. It is to provide an activated sample. Furthermore, a blood coagulation factor activity measurement method using the blood coagulation factor inactivated sample of the present invention and a specimen processing plasma used in the method are provided.
本発明の血液凝固因子の不活化方法は、第Ｖ因子及び第ＶＩＩＩ因子の少なくともいずれか一方を含む試料をイミノジ酢酸基（−Ｎ（ＣＨ２ＣＯＯＲ）２、Ｒが水素又は金属イオンを示す）を有する化合物と接触させて、該試料中の第Ｖ因子及び第ＶＩＩＩ因子の少なくともいずれか一方を不活化型に変化させる工程を含む。 In the method for inactivating a blood coagulation factor of the present invention, a sample containing at least one of factor V and factor VIII is treated with an iminodiacetate group (—N (CH 2 COOR) 2 , R represents hydrogen or a metal ion). And a step of changing at least one of Factor V and Factor VIII in the sample to an inactivated form by contacting with a compound having the formula:
前記イミノジ酢酸基のＲは一価の金属イオンであることが好ましく、前記一価の金属イオンはナトリウムイオンであることが好ましい。 R of the iminodiacetic acid group is preferably a monovalent metal ion, and the monovalent metal ion is preferably a sodium ion.
前記イミノジ酢酸基を有する化合物は、前記イミノジ酢酸基を有する担体であってもよく、この場合、前記イミノジ酢酸基を有する陽イオン交換樹脂又は前記イミノジ酢酸基を有する顆粒状アガロースゲルであることが好ましい。また、担体を用いる場合の前記接触処理は、前記試料に対して前記担体を５ｗ／ｖ％以上となる割合で行なうことが好ましい。 The compound having the iminodiacetic acid group may be a carrier having the iminodiacetic acid group, and in this case, it may be a cation exchange resin having the iminodiacetic acid group or a granular agarose gel having the iminodiacetic acid group. preferable. Moreover, it is preferable to perform the said contact process in the case of using a support | carrier in the ratio which becomes 5 w / v% or more of the said support | carrier with respect to the said sample.
本発明の不活化方法で用いられる前記試料は、血漿であることが好ましい。 The sample used in the inactivation method of the present invention is preferably plasma.
本発明の血液凝固因子不活化試料は、不活化型の第Ｖ因子及び不活化型の第ＶＩＩＩ因子の少なくともいずれか一方を含有するものである。 The blood coagulation factor inactivated sample of the present invention contains at least one of inactivated factor V and inactivated factor VIII.
さらに、フォンヴィルブラント（ｖｏｎ ｗｉｌｌｅｂｒａｎｄｔ）因子を含有することが好ましく、前記フォンヴィルブラント因子は、リストセチンコファクター活性を有していることが好ましい。 Furthermore, it preferably contains a von Willebrand factor, and the von Willebrand factor preferably has ristocetin cofactor activity.
本発明の血液凝固因子活性の測定方法は、不活化型の第Ｖ因子及び不活化型の第ＶＩＩＩ因子を含有する血液凝固因子不活化血漿を含む検体処理用血漿を用いて、血液検体に含まれる血液凝固因子の活性を測定する方法であって、血液検体、凝固時間を測定するための測定試薬及び検体処理用血漿を混合して測定用試料を調製する工程；当該測定用試料における凝固時間を測定する工程；及び当該凝固時間に基づいて血液凝固因子の活性を算出する工程を含む。 The method for measuring blood coagulation factor activity of the present invention is included in a blood sample using a plasma for sample treatment containing blood coagulation factor inactivated plasma containing inactivated factor V and inactivated factor VIII. A method for measuring the activity of a blood coagulation factor, comprising: preparing a measurement sample by mixing a blood sample, a measurement reagent for measuring the coagulation time and plasma for sample treatment; coagulation time in the measurement sample Measuring the blood clotting factor based on the clotting time.
前記血液凝固因子不活化血漿は、イミノジ酢酸基（−Ｎ（ＣＨ２ＣＯＯＲ）２、Ｒは水素又は金属イオンを示す）を有する化合物と血漿を接触させる処理により得られたものであることが好ましく、前記血液検体が血漿であることが好ましい。 The blood coagulation factor inactivated plasma is preferably obtained by treatment of plasma with a compound having an iminodiacetic acid group (—N (CH 2 COOR) 2 , R represents hydrogen or a metal ion). The blood sample is preferably plasma.
本発明の血液凝固因子活性の測定方法の具体的態様としては、
（１）前記測定試薬がプロトロンビン時間測定試薬であって、前記血液凝固因子の活性を算出する工程において、第Ｖ因子の活性を算出する方法；
（２）前記検体処理用血漿は、さらに活性型に変化可能な第ＶＩＩＩ因子を含み、前記測定試薬がプロトロンビン時間測定試薬であり、前記血液凝固因子の活性を算出する工程において、第Ｖ因子の活性を算出する方法；
（３）前記検体処理用血漿は、さらに活性型に変化可能な第Ｖ因子を含み、前記測定試薬が、活性化部分トロンボプラスチン時間測定試薬であり、前記血液凝固因子の活性を算出する工程において、第Ｖ因子の活性を算出する方法；
（４）前記検体処理用血漿は、さらに活性型に変化可能な第ＶＩＩＩ因子を含み、前記測定試薬が活性化部分トロンボプラスチン時間測定試薬であり、前記血液凝固因子の活性を算出する工程において、第Ｖ因子の活性を算出する方法が挙げられる。
As a specific embodiment of the method for measuring blood coagulation factor activity of the present invention,
(1) The method in which the measurement reagent is a prothrombin time measurement reagent and the activity of factor V is calculated in the step of calculating the activity of the blood coagulation factor;
(2) The plasma for specimen processing further contains Factor VIII that can be changed to an active form, the measurement reagent is a prothrombin time measurement reagent, and in the step of calculating the activity of the blood coagulation factor, A method of calculating activity;
(3) In the step of calculating the activity of the blood coagulation factor, the specimen processing plasma further includes Factor V that can be changed to an active form, the measurement reagent is an activated partial thromboplastin time measurement reagent, A method for calculating the activity of factor V;
(4) In the step of calculating the activity of the blood coagulation factor, the sample processing plasma further includes Factor VIII that can be changed to an active form, and the measurement reagent is an activated partial thromboplastin time measurement reagent. A method for calculating the activity of factor V can be mentioned.
本発明の検体処理用血漿は、不活化型の第Ｖ因子及び不活化型の第ＶＩＩＩ因子を含有する血液凝固因子不活化血漿に、活性型変換可能な凝固第Ｖ因子又は活性型変換可能な凝固第ＶＩＩＩ因子が添加されてなるものである。 The plasma for specimen processing of the present invention can be converted into a coagulation factor V or an active form that can be converted into a blood coagulation factor inactivated plasma containing an inactivated factor V and an inactivated factor VIII. Coagulation factor VIII is added.
本発明の血液凝固因子の不活化方法は、試料とイミノジ酢酸基を有する化合物とを接触させる接触処理により、試料に含まれる第Ｖ因子及び第ＶＩＩＩ因子の少なくともいずれか一方を不活化型に変化させることができるので、簡便で効率的である。 In the method for inactivating a blood coagulation factor of the present invention, at least one of factor V and factor VIII contained in a sample is changed to an inactivated type by a contact treatment in which the sample is contacted with a compound having an iminodiacetic acid group. It is simple and efficient.
また、前記血液凝固因子の不活化方法を利用することで、少なくともＶＷＦに実質的に影響を与えることなく、第Ｖ因子又は第ＶＩＩＩ因子が不活化型に変化した状態で残存する試料を得ることができる。つまり、得られた血液凝固因子不活化試料には、従来の吸着除去により得られる凝固因子欠乏血漿とは異なり、第ＶＩＩＩ因子及び／又は第Ｖ因子が不活化型に変化した状態で含まれており、さらに、ＶＷＦについては実質的に影響を受けることなく、残存している。 Further, by using the blood coagulation factor inactivation method, a sample that remains in a state in which factor V or factor VIII is changed to an inactivated type without substantially affecting at least VWF is obtained. Can do. In other words, the obtained blood coagulation factor inactivated sample is different from the conventional coagulation factor-deficient plasma obtained by adsorption removal, and contains factor VIII and / or factor V in an inactivated form. Furthermore, VWF remains substantially unaffected.
さらに、このような血液凝固因子不活化試料を、従来の凝固因子欠乏血漿に代えて、血液凝固因子活性の測定方法に使用することができる。これにより、別途ＶＷＦを添加しなくても、フォンビルブラント病と血友病Ａを区別して判定することができる。 Further, such a blood coagulation factor inactivated sample can be used in a method for measuring blood coagulation factor activity in place of conventional coagulation factor-deficient plasma. Thereby, even if VWF is not added separately, von Willebrand disease and hemophilia A can be distinguished and determined.
〔血液凝固因子の不活化方法〕
本発明の血液凝固因子の不活化方法は、試料をイミノジ酢酸基（−Ｎ（ＣＨ２ＣＯＯＲ）２、Ｒが水素又は金属イオンを示す）を有する化合物と接触させて、該試料中の第Ｖ因子及び第ＶＩＩＩ因子の少なくともいずれか一方を、不活化型に変化させる工程を含む。
[Inactivation method of blood coagulation factor]
According to the method for inactivating a blood coagulation factor of the present invention, a sample is contacted with a compound having an iminodiacetic acid group (—N (CH 2 COOR) 2 , R represents hydrogen or a metal ion), and A step of changing at least one of factor and factor VIII to an inactivated form.
本発明で用いられる試料としては、第Ｖ因子又は第ＶＩＩＩ因子の少なくとも一方を含有するものであれば特に限定されず、例えば、ヒト等のほ乳動物の血漿、血清、全血などが挙げられる。さらに、第Ｖ因子、第ＶＩＩＩ因子、第ＶＩＩＩ因子−ＶＷＦ複合体等の血液凝固因子製剤を含む溶液が挙げられる。尚、血液凝固因子製剤には、血液から抽出又は精製されたものと遺伝子組み換え技術により調製されるものがある。 The sample used in the present invention is not particularly limited as long as it contains at least one of factor V or factor VIII, and examples thereof include plasma, serum, whole blood and the like of mammals such as humans. Furthermore, a solution containing a blood coagulation factor preparation such as Factor V, Factor VIII, and Factor VIII-VWF complex can be mentioned. Blood coagulation factor preparations include those extracted or purified from blood and those prepared by gene recombination techniques.
本発明で接触処理に用いられる前記イミノジ酢酸基（−Ｎ（ＣＨ２ＣＯＯＲ）２）は、式中のＲがH又は金属イオンであり、好ましくは金属イオンであり、より好ましくは一価の金属イオンである。一価の金属イオンとしては、例えば、ナトリウムイオンやカリウムイオンが挙げられる。 In the iminodiacetic acid group (—N (CH 2 COOR) 2 ) used for the contact treatment in the present invention, R in the formula is H or a metal ion, preferably a metal ion, more preferably a monovalent metal. Ion. Examples of monovalent metal ions include sodium ions and potassium ions.
前記化合物は、前記イミノジ酢酸基を官能基として有するものであれば特に限定されず、有機化合物であっても無機化合物であってもよい。また、前記化合物には、官能基を導入する種々の方法により、直接的に又はスペーサーやカップリング剤などを介して間接的にイミノジ酢酸基が結合している化合物も含まれる。要するに、接触処理に用いた際に第Ｖ因子及び第ＶＩＩＩ因子の少なくともいずれか一方を不活化することが可能な、イミノジ酢酸基を有する化合物であればよい。このような化合物としては、例えば担体が挙げられる。具体的には、陽イオン交換樹脂や顆粒状アガロースゲルなど有機化合物より構成される担体、磁性ビーズ、シリカ粒子、モノリス型シリカカラムなど無機化合物より構成される担体が挙げられる。 The compound is not particularly limited as long as it has the iminodiacetic acid group as a functional group, and may be an organic compound or an inorganic compound. In addition, the compound includes a compound in which an iminodiacetic acid group is bonded directly or indirectly through a spacer or a coupling agent by various methods for introducing a functional group. In short, any compound having an iminodiacetic acid group that can inactivate at least one of Factor V and Factor VIII when used in the contact treatment may be used. Examples of such a compound include a carrier. Specific examples include carriers composed of organic compounds such as cation exchange resins and granular agarose gels, and carriers composed of inorganic compounds such as magnetic beads, silica particles, and monolith type silica columns.
イミノジ酢酸基を有する陽イオン交換樹脂とは、水や溶媒に不溶な三次元網状樹脂に、官能基としてイミノジ酢酸基（−Ｎ（ＣＨ２ＣＯＯＨ）２）を含む陽イオン交換できる樹脂で、アルカリ金属、アルカリ土類金属、重金属等を選択的に吸着できる樹脂である。 A cation exchange resin having an iminodiacetic acid group is a resin capable of cation exchange containing an iminodiacetic acid group (—N (CH 2 COOH) 2 ) as a functional group in a three-dimensional network resin insoluble in water or a solvent. It is a resin that can selectively adsorb metals, alkaline earth metals, heavy metals and the like.
陽イオン交換樹脂は、多孔性球状粒子（ビーズタイプ）でもよいし、微視的空孔を有するフィルム状（イオン交換膜）であってもよい。陽イオン交換樹脂の基礎となる高分子母体は、特に限定しないが、一般にスチレン−ジビニルベンゼン共重合体が用いられる。イオン交換膜の場合には、陽イオン交換樹脂の微粉末を造膜性の結合剤（ポリエチレン、ポリスチレン、フェノール樹脂などの熱可塑性、熱硬化性樹脂）を用いてコロイド状に分散させた状態で膜状に成型した不均質型；膜状を維持するための高分子（ポリエチレン、ポリプロピレン、ポリ塩化ビニル、フッ素樹脂など）と陽イオン交換性基をもつ高分子（ポリスチレン誘導体、ポリアクリル酸、ポリエチレンイミン、ポリビニルピリジン誘導体など）とを混合したり、橋かけさせた半均質型；スチレン−ブタジエン共重合体膜に陽イオン交換基を導入したものや、陽イオン交換基を含む熱可塑性高分子を成型し成膜したものである均質型のいずれでもよい。要するに、短時間で原料となる試料に含まれる凝固因子に、活性型変換を不能とするような化学的変化を与え得るように、十分な接触面積が確保されていればよい。 The cation exchange resin may be porous spherical particles (bead type) or may be in the form of a film having microscopic pores (ion exchange membrane). The polymer matrix that is the basis of the cation exchange resin is not particularly limited, but generally a styrene-divinylbenzene copolymer is used. In the case of an ion exchange membrane, a fine powder of a cation exchange resin is dispersed in a colloidal form using a film-forming binder (thermoplastic or thermosetting resin such as polyethylene, polystyrene, or phenol resin). Heterogeneous type molded into a film; polymer (polyethylene, polypropylene, polyvinyl chloride, fluororesin, etc.) to maintain the film and a polymer with a cation exchange group (polystyrene derivative, polyacrylic acid, polyethylene) Semi-homogeneous type mixed with or crosslinked with imine, polyvinyl pyridine derivatives, etc .; styrene-butadiene copolymer membranes introduced with cation exchange groups or thermoplastic polymers containing cation exchange groups Any of a homogeneous type formed by forming and forming a film may be used. In short, it is sufficient that a sufficient contact area is ensured so that the coagulation factor contained in the sample as a raw material in a short time can be subjected to a chemical change that disables active conversion.
前記イミノジ酢酸基を有する顆粒状アガロースゲルとは、顆粒状に形成されたアガロースゲルであり、官能基としてイミノジ酢酸を有するアガロースゲルである。具体的には、Amersham Biosciences社製のセファロースやBio Rad社製のバイオゲルなどが挙げられる。 The granular agarose gel having an iminodiacetic acid group is an agarose gel formed in a granular form, and is an agarose gel having iminodiacetic acid as a functional group. Specific examples include Sepharose manufactured by Amersham Biosciences and Biogel manufactured by Bio Rad.
試料と上記イミノジ酢酸基を有する化合物とを接触させる接触処理の方法は、特に限定しないが、例えば、イミノジ酢酸基を有する担体の場合、容器中にイオン交換樹脂と試料をいれて、所定時間攪拌混合する方法、イオン交換樹脂を充填したカラムに試料を流す方法などが挙げられる。 The contact treatment method for bringing the sample into contact with the compound having an iminodiacetic acid group is not particularly limited. For example, in the case of a carrier having an iminodiacetic acid group, the ion exchange resin and the sample are placed in a container and stirred for a predetermined time. The method of mixing, the method of flowing a sample through the column filled with ion exchange resin, etc. are mentioned.
接触処理に使用するイミノジ酢酸基を有する化合物の量は、その種類に応じて適宜設定される。例えば、イミノジ酢酸基を有する化合物がイミノジ酢酸基を有する担体の場合、接触処理は、試料（体積）に対して担体（質量）が５ｗ／ｖ％以上となる量で行なうことが好ましい。５ｗ／ｖ％未満では、血液凝固因子の不活化型への変化が不十分であったり、処理に長時間を要する可能性がある。 The amount of the compound having an iminodiacetic acid group used for the contact treatment is appropriately set according to the type. For example, when the compound having an iminodiacetic acid group is a carrier having an iminodiacetic acid group, the contact treatment is preferably performed in such an amount that the carrier (mass) is 5 w / v% or more with respect to the sample (volume). If it is less than 5 w / v%, there is a possibility that the change of the blood coagulation factor to the inactivated type is insufficient or that the treatment takes a long time.
さらに、接触処理の時間は、使用するイミノジ酢酸基を有する化合物の種類、量によって適宜設定される。例えば、イミノジ酢酸基を有する化合物がイミノジ酢酸基を有する陽イオン交換樹脂の場合、試料に対する担体が５ｗ／ｖ％程度であれば２〜６時間、１０ｗ／ｖ％程度であれば１〜４時間程度で、接触処理しない場合と比べて、活性を１％以下にまで不活化することができる。また、イミノジ酢酸基（Ｒがナトリウムイオン）を有するセファロースでは、試料に対する担体が１０〜２０ｗ／ｖ％程度であれば３〜４時間で活性を１％以下にまで不活化することができる。 Furthermore, the time for the contact treatment is appropriately set depending on the type and amount of the compound having an iminodiacetic acid group to be used. For example, when the compound having an iminodiacetic acid group is a cation exchange resin having an iminodiacetic acid group, it is 2 to 6 hours if the carrier for the sample is about 5 w / v%, and 1 to 4 hours if it is about 10 w / v%. The activity can be inactivated to 1% or less as compared with the case where the contact treatment is not performed. Moreover, in the sepharose which has iminodiacetic acid group (R is a sodium ion), if the support | carrier with respect to a sample is about 10-20 w / v%, activity can be inactivated to 1% or less in 3 to 4 hours.
以上のような接触処理により、試料に含まれる第Ｖ因子及び第ＶＩＩＩ因子の少なくともいずれか一方を、不活化型に変化させることができる。試料として、第Ｖ因子及び第ＶＩＩＩ因子の双方を含む試料、例えば正常血漿を用いた場合は、第Ｖ因子及び第ＶＩＩＩ因子の双方が不活化型となる。ここで、不活化型の血液凝固因子とは、活性化型に変化することができない血液凝固因子のことである。 By the contact treatment as described above, at least one of Factor V and Factor VIII contained in the sample can be changed to an inactivated type. When a sample containing both factor V and factor VIII, for example, normal plasma, is used as the sample, both factor V and factor VIII are inactivated. Here, the inactivated blood coagulation factor is a blood coagulation factor that cannot be changed to an activated type.
上記不活化方法における接触処理は、第Ｖ因子、第ＶＩＩＩ因子に対する影響は互いに独立的である。従って、この不活化方法を採用することにより、他の因子に影響を与えることなく、第Ｖ因子のみ、第ＶＩＩＩ因子のみが不活化型に変化した試料（血液凝固因子不活化試料）を得ることができる。 The contact treatment in the inactivation method is independent of the influence on Factor V and Factor VIII. Therefore, by adopting this inactivation method, a sample (blood coagulation factor inactivated sample) in which only factor V or only factor VIII is changed to an inactivated type without affecting other factors is obtained. Can do.
第Ｖ因子とは、分子量３３万の糖タンパクで、Ｘａ因子、プロトロンビン、リン脂質、Ｃａ２＋とともにプロトロンビナーゼ複合体を形成し、Ｘａ因子によるプロトロンビンの活性化を上昇させる補酵素として働く。第Ｖ因子は、トロンビンによりＡｒｇ（７０９番）、Ａｒｇ（１０１８番）、Ａｒｇ（１５４５番）のＣ末端側が開裂して、活性型Ｖ因子となる。しかし、上記不活化方法で不活化された第Ｖ因子は、活性型へ変換できない。イミノジ酢酸基を有する化合物との接触処理により、第Ｖ因子がいかなる化学変化を受けるかは明らかではないが、おそらく、因子の活性型へ変換する部分が化学的又は立体構造的に変化したため、あるいは活性型へ変換する部分の一部が脱落したため、あるいはその他の部分の化学的変化若しくは一部脱落のために活性化しようとする部分が変化したためと考えられる。 Factor V is a glycoprotein with a molecular weight of 330,000, which forms a prothrombinase complex with factor Xa, prothrombin, phospholipid, and Ca 2+ and functions as a coenzyme that increases the activation of prothrombin by factor Xa. Factor V is cleaved by thrombin at the C-terminal side of Arg (709), Arg (1018), and Arg (1545) to become active factor V. However, the factor V inactivated by the inactivation method cannot be converted into an active form. It is not clear what chemical changes Factor V undergoes by contact with a compound having an iminodiacetic acid group, but probably because the portion of the factor that is converted to the active form has been chemically or conformally altered, or This is probably because part of the part to be converted to the active form has dropped out, or because the part to be activated has changed due to a chemical change or part of the other part.
また、第ＶＩＩＩ因子は、分子量３３万の糖タンパクで、ＩＸａ因子、Ｘ因子、リン脂質、Ｃａ２＋とともにテンナーゼ複合体を形成し、ＩＸａ因子によるＸ因子の活性化を上昇させる補酵素として働く。トロンビンによりＡｒｇ（３７２番）、Ａｒｇ（７４０番）、Ａｒｇ（１６８９番）のＣ末端側が開裂して、活性型ＶＩＩＩ因子となる。しかし、上記不活化方法で不活化された第ＶＩＩＩ因子は、活性型へ変換できない。イミノジ酢酸基を有する化合物との接触処理により、第ＶＩＩＩ因子がいかなる化学変化を受けるかは明らかではないが、おそらく、因子の活性型へ変換する部分が化学的又は立体構造的に変化したため、あるいは活性型へ変換する部分の一部が脱落したため、あるいはその他の部分の化学的変化若しくは一部脱落のために活性化しようとする部分が変化したためと考えられる。しかし、接触処理により、第ＶＩＩＩ因子におけるＶＷＦ結合能には影響を与えないと考えられる。試料として、ＶＷＦを含む血漿等を用いて、接触処理を行なった試料（処理済血漿）では、ＶＷＦが含まれ、そのＶＷＦは第ＶＩＩＩ因子が正常である血液と同様に、リストセチンコファクター活性（Ｒｃｏ活性）を示すことができるからである。 Factor VIII is a glycoprotein with a molecular weight of 330,000, forms a tennase complex with factor IXa, factor X, phospholipid, and Ca 2+ , and functions as a coenzyme that increases the activation of factor X by factor IXa. Thrombin cleaves the C-terminal side of Arg (No. 372), Arg (No. 740), and Arg (No. 1689) to form active factor VIII. However, factor VIII inactivated by the above inactivation method cannot be converted into an active form. It is not clear what chemical change of factor VIII is caused by contact treatment with a compound having an iminodiacetic acid group, but it is probably due to a chemical or conformational change in the portion of the factor that is converted to the active form, or This is probably because part of the part to be converted to the active form has dropped out, or because the part to be activated has changed due to a chemical change or part of the other part. However, it is considered that the contact treatment does not affect the VWF binding ability of factor VIII. As a sample, a sample subjected to contact treatment using plasma or the like containing VWF (treated plasma) contains VWF, which is similar to blood with normal factor VIII, like ristocetin cofactor activity ( This is because (Rco activity) can be exhibited.
このように、本発明の血液凝固因子不活化方法では、試料中に含まれる第Ｖ因子及び第ＶＩＩＩ因子を活性型に変換できない、所謂不活型に変化させることにより、試料中の第Ｖ因子、第ＶＩＩＩ因子が活性を示さなくなる。つまり、従来の免疫吸着法による第Ｖ因子、第ＶＩＩＩ因子の吸着除去とは異なり、第Ｖ因子、第ＶＩＩＩ因子が変化した状態で残存し、第Ｖ因子又は第ＶＩＩＩ因子に対する抗体が反応できる形で試料中に存在する。 Thus, in the blood coagulation factor inactivation method of the present invention, the factor V and VIII contained in the sample cannot be converted into an active form, so that the factor V in the sample is changed. Factor VIII no longer shows activity. That is, unlike the adsorption removal of factor V and factor VIII by the conventional immunoadsorption method, the form in which factor V and factor VIII remain changed and the antibody against factor V or factor VIII can react. Present in the sample.
〔血液凝固因子不活化試料〕
本発明の血液凝固因子不活化試料は、上記本発明の不活化方法を利用して得られるもので、第Ｖ因子及び第ＶＩＩＩ因子の少なくともいずれか一方が不活化型の試料である。
[Blood coagulation factor inactivated sample]
The blood coagulation factor inactivated sample of the present invention is obtained using the inactivation method of the present invention, and at least one of factor V and factor VIII is an inactivated sample.
試料として血漿を用いる場合、第Ｖ因子及び第ＶＩＩＩ因子の少なくともいずれか一方が不活化型に変化した血漿を得ることができる。また、試料として正常血漿（第Ｖ因子及び第ＶＩＩＩ因子を含む血漿）を用いた場合、第Ｖ因子及び第ＶＩＩＩ因子が不活化した血液凝固因子不活化血漿を得ることができる。正常血漿を試料として用いた血液凝固因子不活化血漿は、従来の免疫吸着除去法による第Ｖ因子、第ＶＩＩＩ因子の吸着除去とは異なり、正常血漿中に含まれていた第Ｖ因子及び第ＶＩＩＩ因子が不活化型に変化した状態で残存している。さらに、第Ｖ因子又は第ＶＩＩＩ因子に対する抗体が反応できる形で存在する。さらにまた、この血液凝固因子不活化血漿にはＶＷＦが残存しており、血液凝固因子不活化血漿中の第ＶＩＩＩ因子はＶＷＦと結合して複合体を形成することが可能であり、Ｒｃｏ活性を有している。 When plasma is used as a sample, plasma in which at least one of factor V and factor VIII is changed to an inactivated form can be obtained. When normal plasma (plasma containing factor V and factor VIII) is used as a sample, blood coagulation factor inactivated plasma in which factor V and factor VIII are inactivated can be obtained. The blood coagulation factor inactivated plasma using normal plasma as a sample is different from the adsorption removal of factor V and factor VIII by the conventional immunoadsorption removal method, and the factor V and factor VIII contained in normal plasma The factor remains in an inactivated form. Furthermore, it exists in the form which can react with the antibody with respect to factor V or VIII. Furthermore, VWF remains in the blood coagulation factor inactivated plasma, and factor VIII in the blood coagulation factor inactivated plasma can bind to VWF to form a complex, and has Rco activity. Have.
ここで、ＶＷＦとは、血小板膜タンパク質ＧＰＩｂと血管内皮下組織の間に介在して結合させる接着因子として、止血初期の血小板粘着にかかわる。また、循環血液中では第ＶＩＩＩ因子と非共有結合による複合体を形成し、第ＶＩＩＩ因子の安定化に寄与する。 Here, VWF is an adhesion factor that is intervened and bound between the platelet membrane protein GPIb and the subendothelial tissue, and is related to platelet adhesion in the early stage of hemostasis. Further, in the circulating blood, it forms a non-covalent complex with factor VIII, contributing to the stabilization of factor VIII.
尚、本発明の血液凝固因子不活化試料がＶＷＦを含む場合、第ＶＩＩＩ因子が正常である血漿と同様に、Ｒｃｏ活性を示すＶＷＦを含んでもよいし、ＶＷＦがＲｃｏ活性を有していなくてもよいし、ＶＷＦが化学的又は立体構造的に変化したものや一部脱落したものを含んでいるものも包含する。 In addition, when the blood coagulation factor inactivated sample of the present invention contains VWF, it may contain VWF showing Rco activity as well as plasma with normal factor VIII, and VWF does not have Rco activity. It may also include those in which VWF is chemically or three-dimensionally changed or partially lost.
〔血液凝固因子活性の測定方法〕
本発明の血液凝固因子不活化方法を正常血漿に適用して得られる、第Ｖ因子及び第ＶＩＩＩ因子の双方とも不活化された本発明の血液凝固因子不活化血漿を用いて、血液検体に含まれる血液凝固因子の活性を測定することができる。
すなわち、本発明の血液凝固因子活性の測定方法は、不活化型の第Ｖ因子及び不活化型の第ＶＩＩＩ因子を含有する血液凝固因子不活化血漿を含む検体処理用血漿を用いて、血液検体に含まれる血液凝固因子の活性を測定する方法であって、血液検体、凝固時間を測定するための測定試薬及び検体処理用血漿を混合して測定用試料を調製する工程；前記測定用試料における凝固時間を測定する工程；及び前記凝固時間に基づいて血液凝固因子の活性を算出する工程を含む。
[Method for measuring blood coagulation factor activity]
Included in blood samples using the blood coagulation factor inactivated plasma of the present invention obtained by applying the blood coagulation factor inactivation method of the present invention to normal plasma, wherein both factor V and factor VIII are inactivated The activity of blood clotting factors can be measured.
That is, the method for measuring blood coagulation factor activity according to the present invention uses a blood sample using plasma for sample treatment containing blood coagulation factor inactivated plasma containing inactivated factor V and inactivated factor VIII. A method for measuring the activity of a blood coagulation factor contained in a blood sample, a step of preparing a measurement sample by mixing a blood sample, a measurement reagent for measuring a clotting time, and a plasma for sample processing; Measuring a clotting time; and calculating a blood clotting factor activity based on the clotting time.
前記血液凝固因子の活性は、測定しようとする特定の凝固因子について正常な血液検体又は血液検体の標準品とを比較することにより算出することができる。正常な血液検体としては、例えば、健常人より採取した血液、該血液から調製された血漿などが挙げられる。また、血液検体の標準品としては、例えば、市販されている標準血液や標準血漿などが挙げられる。算出された血液凝固因子の活性の有無により、血液検体中の血液凝固因子の有無を知ることができる。また、血液凝固因子の活性の値により、血液検体中の血液凝固因子の存在量を知ることができる。さらに血液凝固因子の有無や存在量により、血液検体中の血液凝固因子の異常の有無を知ることができる。 The activity of the blood coagulation factor can be calculated by comparing a specific blood coagulation factor to be measured with a normal blood sample or a standard blood sample. Examples of normal blood samples include blood collected from healthy people, plasma prepared from the blood, and the like. Examples of standard blood samples include commercially available standard blood and standard plasma. The presence or absence of the blood coagulation factor in the blood sample can be known from the calculated presence or absence of the blood coagulation factor activity. Further, the amount of blood coagulation factor present in the blood sample can be known from the value of the activity of the blood coagulation factor. Furthermore, the presence or absence of a blood coagulation factor and the amount of the blood coagulation factor can be used to determine whether or not the blood coagulation factor is abnormal in the blood sample.
前記測定試薬としては、プロトロンビン時間（ＰＴ）を測定するＰＴ測定試薬、又は活性化部分トロンボプラスチン時間（ＡＰＴＴ）を測定するＡＰＴＴ測定試薬を用いることができる。 As the measurement reagent, a PT measurement reagent for measuring prothrombin time (PT) or an APTT measurement reagent for measuring activated partial thromboplastin time (APTT) can be used.
前記血液検体としては、全血や血漿を用いることができる。全血と血漿のいずれを用いるかは、測定で使用する試薬や装置などに応じて適宜選択すればよい。なお、吸光度などの光学的情報に基づいて凝固時間を測定する場合、血液検体としては血漿を用いることが望ましい。 As the blood sample, whole blood or plasma can be used. Whether to use whole blood or plasma may be appropriately selected according to the reagent or apparatus used in the measurement. When measuring the coagulation time based on optical information such as absorbance, it is desirable to use plasma as a blood sample.
ＰＴとは、血液検体に組織トロンボプラスチンとカルシウムを加えて凝固するまでの時間で、組織トロンボプラスチンとカルシウムの添加により、第Ｖ因子、第ＶＩＩ因子、第Ｘ因子の関与のもと、プロトロンビンがトロンビンとなり、フィブリノーゲンをフィブリンに転化して凝塊が形成される。従って、プロトロンビン、第Ｖ因子、第ＶＩＩ因子、第Ｘ因子の欠損又は異常、フィブリノゲンの減少によって凝固時間が延長する。 PT is the time it takes for a blood sample to coagulate after adding tissue thromboplastin and calcium. By adding tissue thromboplastin and calcium, prothrombin becomes thrombin with the involvement of factor V, factor VII, and factor X. , Fibrinogen is converted to fibrin and a clot is formed. Therefore, prothrombin, factor V, factor VII, factor X deficiency or abnormality, and decrease in fibrinogen prolong clotting time.
ＰＴ測定試薬とは、主成分として、血液凝固誘因のための組織トロンボプラスチン及びカルシウムイオンを含んでいる試薬である。組織トロンボプラスチンとしては、ウサギ、ウシ、ヒトなどの脳、胎盤から抽出もしくは精製したもの又は遺伝子組み換え技術により調製したものが用いられ得る。また、組織トロンボプラスチン及びカルシウムの他、緩衝液、防腐剤、安定化剤などが添加されていてもよい。 The PT measurement reagent is a reagent containing tissue thromboplastin for triggering blood coagulation and calcium ions as main components. As tissue thromboplastin, those extracted or purified from the brain, placenta of rabbits, cows, humans, etc., or those prepared by genetic recombination techniques can be used. In addition to tissue thromboplastin and calcium, buffers, preservatives, stabilizers and the like may be added.
ＡＰＴＴとは、血液検体に、リン脂質、活性化剤及びカルシウムイオンを添加して凝固するまでの時間で、第ＶＩＩＩ因子、第Ｖ因子の欠損、異常により凝固時間が延長する。また、ＰＴの測定結果とあわせて、ＰＴが正常でＡＰＴＴが延長の場合には第ＶＩＩＩ因子欠損、ＰＴ及びＡＰＴＴの双方が延長の場合には第Ｖ因子欠損と判定することができる。 APTT is the time required to coagulate after adding phospholipid, activator and calcium ion to a blood sample, and the clotting time is prolonged due to deficiency or abnormality of factor VIII or factor V. Further, together with the measurement result of PT, when PT is normal and APTT is extended, it can be determined as factor VIII deficiency, and when both PT and APTT are extended, it can be determined as factor V deficiency.
ＡＰＴＴ測定試薬とは、主成分として、血液凝固誘因のためのリン脂質、活性化剤およびカルシウムイオンを含有している。リン脂質の由来としては、合成リン脂質、ウサギ、ウシなどの脳やヒトの胎盤などから有機溶媒で抽出したものや大豆由来のものを用いることができる。ＡＰＴＴ試薬における活性化剤とは、第XI因子や第XII因子を活性化するものであり、例えばカオリン、セライト、シリカ、エラジン酸を挙げることができる。また、上記主成分のほかに緩衝剤、防腐剤、安定化剤が添加されていてもよい。 The APTT measurement reagent contains, as main components, a phospholipid for inducing blood coagulation, an activator and calcium ions. As the origin of phospholipids, synthetic phospholipids, those extracted from brains such as rabbits and cows, human placenta and the like, and those derived from soybeans can be used. The activator in the APTT reagent is an agent that activates factor XI or factor XII, and examples thereof include kaolin, celite, silica, and ellagic acid. In addition to the above main components, a buffer, preservative, and stabilizer may be added.
本発明の測定方法の具体的態様としては、下記ａ）〜ｄ）の態様が挙げられる。なお、ここでは、血液検体として血漿を用いる場合の具体的態様を示す。
ａ）検体処理用血漿として、不活化型の第Ｖ因子及び不活化型の第ＶＩＩＩ因子を含有する血液凝固因子不活化血漿（第Ｖ＆ＶＩＩＩ不活化血漿）を使用し、前記測定試薬としてＰＴ測定試薬を用いて、第Ｖ因子の活性を測定する。
具体的には、第Ｖ＆ＶＩＩＩ不活化型血漿と正常血漿を混合し、この混合液に測定用試薬であるＰＴ測定試薬を添加し、凝固時間を測定する。この混合液において段階的に希釈した正常血漿を用いて、因子活性と凝固時間の関係を示す検量線を作成する。一方、被検血漿と前記第Ｖ＆ＶＩＩＩ不活化血漿とを混合してなる混合液（被検血漿含有混合液）に、ＰＴ測定試薬を添加し、凝固時間を測定する。先に作成した検量線と前記混合液の凝固時間に基づいて、被検血漿に含まれる第Ｖ因子の活性を算出することができる。
Specific embodiments of the measurement method of the present invention include the following embodiments a) to d). In addition, the specific aspect in the case of using plasma as a blood sample is shown here.
a) A blood coagulation factor inactivated plasma (factor V & VIII inactivated plasma) containing an inactivated factor V and an inactivated factor VIII is used as the sample processing plasma, and a PT measurement reagent as the measurement reagent Is used to measure factor V activity.
Specifically, the V & VIII inactivated plasma and normal plasma are mixed, a PT measurement reagent as a measurement reagent is added to this mixture, and the coagulation time is measured. Using normal plasma diluted stepwise in this mixture, a calibration curve showing the relationship between factor activity and clotting time is prepared. On the other hand, a PT measurement reagent is added to a mixed solution (test plasma-containing mixed solution) obtained by mixing the test plasma and the V & VIII inactivated plasma, and the clotting time is measured. Based on the previously prepared calibration curve and the coagulation time of the mixed solution, the activity of factor V contained in the test plasma can be calculated.
ｂ）検体処理用血漿として、第Ｖ＆ＶＩＩＩ不活化血漿に、活性型に変化可能な凝固第ＶＩＩＩ因子を添加してなる検体用処理血漿を使用し、測定試薬としてＰＴ測定試薬を用いて、第Ｖ因子の活性を測定する。
第Ｖ＆ＶＩＩＩ不活化血漿に、活性型に変化可能な凝固第ＶＩＩＩ因子を添加してなる検体処理用血漿は、第Ｖ因子不活化型で、第ＶＩＩＩ因子は活性型に変化可能な血漿である。この検体処理用血漿と被検血漿とを混合してなる混合液（被検血漿含有混合液）に、ＰＴ測定試薬を添加し、凝固時間を測定する。段階的に希釈した正常血漿を用いて作成した検量線及び被検血漿含有混合液の凝固時間に基づいて、被検血漿に含まれる第Ｖ因子の活性を算出することができる。
b) Sample-treated plasma is prepared by adding sample-treated plasma obtained by adding coagulation factor VIII that can be changed to active form to inactivated V & VIII plasma, and using PT measurement reagent as the measurement reagent. Measure the activity of the factor.
Sample processing plasma obtained by adding coagulation factor VIII that can be changed to an active form to inactivated V & VIII plasma is a factor V inactivated type, and factor VIII is a plasma that can be changed to an active type. A PT measurement reagent is added to a mixed solution (a mixed solution containing test plasma) obtained by mixing the sample processing plasma and the test plasma, and the clotting time is measured. The activity of factor V contained in the test plasma can be calculated based on a calibration curve prepared using normal plasma diluted stepwise and the coagulation time of the test plasma-containing mixed solution.
ｃ）検体処理用血漿として、第Ｖ＆ＶＩＩＩ不活化血漿に、活性型に変化可能な凝固第Ｖ因子を添加してなる検体処理用血漿を使用し、測定試薬としてＡＰＴＴ測定試薬を用いて、第ＶＩＩＩ因子の活性を測定する。
すなわち、第Ｖ＆ＶＩＩＩ不活化血漿に、活性型に変化可能な第Ｖ因子を添加してなる検体処理用血漿は、第ＶＩＩＩ因子が不活化型で、第Ｖ因子は活性型に変化可能な血漿である。この検体処理用血漿と被検血漿とを混合してなる混合液（被検血漿含有混合液）に、ＡＰＰＴ測定試薬を添加し、凝固時間を測定する。段階的に希釈した正常血漿を用いて作成した検量線及び被検血漿含有混合液の凝固時間に基づいて、被検血漿に含まれる第Ｖ因子の活性を算出することができる。
c) As the sample processing plasma, the sample processing plasma obtained by adding the coagulation factor V which can be changed to the active form to the V & VIII inactivated plasma is used, and the APTT measuring reagent is used as the measuring reagent. Measure the activity of the factor.
That is, the specimen processing plasma obtained by adding Factor V that can be changed to an active form to Factor V & VIII inactivated plasma is an inactive form of Factor VIII and a plasma that can be changed to an active form. is there. An APPT measurement reagent is added to a mixed solution (a mixed solution containing test plasma) obtained by mixing the sample processing plasma and the test plasma, and the clotting time is measured. The activity of factor V contained in the test plasma can be calculated based on a calibration curve prepared using normal plasma diluted stepwise and the coagulation time of the test plasma-containing mixed solution.
ｄ）検体処理用血漿として、第Ｖ＆ＶＩＩＩ不活化型血漿に、活性型に変化可能な凝固第ＶＩＩＩ因子を添加してなる検体処理用血漿を使用し、測定試薬としてＡＰＴＴ測定試薬を用いて、第Ｖ因子の活性を測定する。
すなわち、第Ｖ＆ＶＩＩＩ不活化型血漿に、活性型に変化可能な第ＶＩＩＩ因子を添加してなる検体処理用血漿は、第Ｖ因子が不活型で、第ＶＩＩＩ因子は活性型に変化可能な血漿である。この検体処理用血漿と被検血漿とを混合してなる混合液（被検血漿含有混合液）に、ＡＰＰＴ測定試薬を添加し、凝固時間を測定する。段階的に希釈した正常血漿を用いて作成した検量線及び被検血漿含有混合液の凝固時間に基づいて、被検血漿に含まれる第Ｖ因子の活性を算出することができる。
d) As the sample processing plasma, the sample processing plasma obtained by adding the coagulation factor VIII that can be changed to the active form to the V & VIII inactivated plasma, and using the APTT measurement reagent as the measurement reagent, The activity of factor V is measured.
That is, the specimen processing plasma obtained by adding factor VIII that can be changed to an active form to the V & VIII inactivated plasma is a plasma in which factor V is inactive and factor VIII can be changed to an active form. It is. An APPT measurement reagent is added to a mixed solution (a mixed solution containing test plasma) obtained by mixing the sample processing plasma and the test plasma, and the clotting time is measured. The activity of factor V contained in the test plasma can be calculated based on a calibration curve prepared using normal plasma diluted stepwise and the coagulation time of the test plasma-containing mixed solution.
以上のような態様の測定方法は、従来から使用されている凝固因子欠乏血漿を用いて血液凝固因子の活性測定方法に代えて、採用することができる。
本発明の測定方法で検体処理用血漿として使用する血液凝固因子不活化血漿又は検体処理用血漿の調製に使用する血液凝固因子不活化血漿においては、上述のように、第Ｖ因子、第ＶＩＩＩ因子が不活化型となっているが、第ＶＩＩＩ因子におけるＶＷＦ結合部位に関する実質的影響が少ない、ないしはほとんどないためか、ＶＷＦが残存し、しかもＲｃｏ活性を有している。従って、従来の凝固因子欠乏血漿を用いた活性測定方法と比べて、以下のような有利な点がある。
The measurement method of the above aspects can be employed instead of the blood coagulation factor activity measurement method using conventionally used coagulation factor-deficient plasma.
In the blood coagulation factor inactivated plasma used for the sample processing plasma in the measurement method of the present invention or the blood coagulation factor inactivated plasma used for the preparation of the sample processing plasma, as described above, factor V, factor VIII Is inactivated, but VWF remains and has Rco activity because of little or no substantial effect on the VWF binding site in Factor VIII. Therefore, there are the following advantages compared with the conventional method of measuring activity using clotting factor-deficient plasma.
ＶＷＦの質的、量的異常に基づくフォンヴィルブラント病では、第ＶＩＩＩ因子の安定性が失われ、その血中濃度が低下するため、検査では、出血時間の延長、第ＶＩＩＩ因子活性、ＶＷＦ量の低下ないし異常、Ｒｃｏ活性の低下及びリストセチンを用いた血小板凝集能の低下といった症状を示し、第ＶＩＩＩ因子欠損、活性の低下を原因とする血友病Ａとは異なるものである。従って、第ＶＩＩＩ因子欠損による血友病の正確な判定のためには、フォンヴィルブラント因子が活性を失うことなく、血液凝固因子不活化血漿中に存在し、且つ第ＶＩＩＩ因子のみが欠乏又は活性型への変換不能であるものであることが好ましい。この点、免疫吸着除去法で製造された凝固因子欠乏血漿を凝固因子活性測定に用いられる検体処理用血漿として使用する場合、別途ＶＷＦを添加して測定に使用する必要があったのに対し、本発明の検体処理用血漿では、Ｒｃｏ活性を有するままでＶＷＦが残存しているので、別途ＶＷＦを添加しなくてもよい。 In von Willebrand disease based on qualitative and quantitative abnormalities of VWF, the stability of factor VIII is lost and its blood concentration is lowered. Therefore, the examination shows that the bleeding time is prolonged, factor VIII activity, VWF amount This is different from hemophilia A caused by factor VIII deficiency and decreased activity, such as decreased or abnormal levels, decreased Rco activity, and decreased platelet aggregation ability using ristocetin. Therefore, for accurate determination of hemophilia due to factor VIII deficiency, von Willebrand factor is present in blood coagulation factor inactivated plasma without losing activity, and only factor VIII is deficient or active. It is preferable that it cannot be converted into a mold. In this regard, when the coagulation factor-deficient plasma produced by the immunoadsorption removal method is used as the sample processing plasma used for the measurement of the coagulation factor activity, it was necessary to add VWF separately and use it for the measurement. In the sample processing plasma of the present invention, VWF remains with Rco activity, so that it is not necessary to add VWF separately.
尚、本発明の活性測定方法に用いられる検体処理用血漿又は検体処理用血漿の調製に使用する血液凝固因子不活化血漿としては、第Ｖ因子、第ＶＩＩＩ因子が１００％不活化されたものでなくてもよい。検体処理用血漿中の第V因子又は第ＸＩＩＩ因子の不活化の程度は、接触処理前の試料に含まれる活性型の因子を１００％としたとき、８０％以上であればよく、好ましくは９５％以上であり、より好ましくは９９％以上である。 In addition, the blood coagulation factor inactivated plasma used for the preparation of the sample processing plasma or the sample processing plasma used in the activity measuring method of the present invention is one in which factor V and factor VIII are 100% inactivated. It does not have to be. The degree of inactivation of factor V or factor XIII in the plasma for sample treatment may be 80% or more, preferably 95%, assuming that the active factor contained in the sample before the contact treatment is 100%. % Or more, and more preferably 99% or more.
さらに、本発明の活性測定方法に用いられる検体処理剤又は血液凝固因子不活化血漿と、従来のＰＴ試薬とを組合わせて、ＰＴ測定試薬キットとして使用することができる。また、本発明の活性測定方法に用いられる検体処理剤又は血液凝固因子不活化血漿と、従来のＡＰＴＴ試薬とを組み合わせ、ＡＰＴＴ時間測定試薬キットとして使用することができる。 Furthermore, the specimen treatment agent or blood coagulation factor inactivated plasma used in the activity measurement method of the present invention and a conventional PT reagent can be combined and used as a PT measurement reagent kit. Moreover, the specimen treatment agent or blood coagulation factor inactivated plasma used in the activity measurement method of the present invention and a conventional APTT reagent can be combined and used as an APTT time measurement reagent kit.
〔測定方法〕
（１）第Ｖ因子の活性（％）測定
測定試料５μｌ及びオーレンベロナール緩衝液４５μｌをキュベットにとり、これに、第Ｖ因子欠乏血漿（シスメックス社、神戸）を５０μｌ添加混合して、３７℃で１分間加温した。
〔Measuring method〕
(1) Measurement of factor V activity (%) 5 μl of a measurement sample and 45 μl of orenveronal buffer solution are placed in a cuvette, and 50 μl of factor V-deficient plasma (Sysmex Corp., Kobe) is added and mixed at 37 ° C. Warmed for 1 minute.
この混合物に、プロトロンビン時間測定試薬として、トロンボチェックＰＴ（シスメックス社、神戸）１００μｌを添加混合して、凝固時間を血液凝固分析装置コアグレックス８００（島津製作所、京都）で測定した。 To this mixture, 100 μl of thrombocheck PT (Sysmex Corp., Kobe) was added and mixed as a prothrombin time measurement reagent, and the coagulation time was measured with a blood coagulation analyzer Core Grex 800 (Shimadzu Corporation, Kyoto).
（２）第ＶＩＩＩ因子の活性（％）測定
測定試料１０μｌ及びオーレンベロナール緩衝液４０μｌをキュベットにとり、これに、第ＶＩＩＩ因子欠乏血漿（シスメックス社、神戸）５０μｌを添加混合して、３７℃、１分間加温した。
(2) Measurement of factor VIII activity (%) Take 10 μl of measurement sample and 40 μl of orenveronal buffer in a cuvette, add 50 μl of factor VIII-deficient plasma (Sysmex Corp., Kobe) and mix at 37 ° C. Warmed for 1 minute.
この混合物に、活性化部分トロンボプラスチン時間測定試薬として、トロンボチェックＡＰＴＴ−ＳＬＡ（シスメックス社、神戸）５０μｌを混合し、３７℃、３分間加温し、２０ｍＭのＣａＣｌ２を５０μｌ添加後、凝固時間をコアグレックス８００（島津製作所、京都）で測定した。 To this mixture, 50 μl of thrombocheck APTT-SLA (Sysmex Corp., Kobe) was mixed as an activated partial thromboplastin time measurement reagent, heated at 37 ° C. for 3 minutes, 50 μl of 20 mM CaCl 2 was added, and the coagulation time was adjusted. Measurements were made with a Core Grex 800 (Shimadzu Corporation, Kyoto).
（３）第ＶＩＩＩ因子の検出
第ＶＩＩＩ因子の存在有無の確認は、Ｂｒｉｔｉｓｈ Ｊｏｕｒｎａｌ ｏｆ Ｈａｅｍａｔｏｌｏｇｙ，１９９４,８６，１０６−１１１に記載された方法に従って行った。ここに記載されている方法では、第ＶＩＩＩ因子に対するポリクローナル抗体を用いたＥＬＩＺＡ法により、測定サンプル中の第ＶＩＩＩ因子を検出する。
(3) Detection of Factor VIII The presence or absence of Factor VIII was confirmed according to the method described in British Journal of Haematology, 1994, 86, 106-111. In the method described here, Factor VIII in a measurement sample is detected by the ELIZA method using a polyclonal antibody against Factor VIII.
（４）ＶＷＦの検出
測定試料１０μｌをキュベットにとり３７℃で３分加温した後、ＶＷＦ試薬緩衝液（ＲＩ）（Ｄａｄｅ−Ｂｅｈｒｉｎｇ社、ドイツ）を２００μｌ添加し、３７℃、５分加温した。次いで、ＶＷＦ試薬ラテック液（Ｒ２）（Ｄａｄｅ−Ｂｅｈｒｉｎｇ社、ドイツ）を１００μｌ添加して得られた混合液の吸光度７００ｎｍをコアグレックス８００（島津製作所、京都）で測定した。
(4) Detection of VWF After taking 10 μl of a measurement sample in a cuvette and heating at 37 ° C. for 3 minutes, 200 μl of VWF reagent buffer (RI) (Dade-Behring, Germany) was added and heated at 37 ° C. for 5 minutes. . Next, the absorbance 700 nm of the mixture obtained by adding 100 μl of VWF reagent Latec solution (R2) (Dade-Behring, Germany) was measured with Coagrex 800 (Shimadzu Corporation, Kyoto).
（５）ＶＷＦのＲｃｏ活性の測定
測定試料１００μｌをスライド判定板に採取し、これにＶＷＦリストセチンコファクター活性測定用試薬（Ｄａｄｅ−Ｂｅｈｒｉｎｇ社、ドイツ）２００μｌを滴下した。スライド判定板を３分間撹拌し、凝集を認めたときに陽性（活性有り）と判定する。
(5) Measurement of RWF activity of VWF 100 μl of a measurement sample was collected on a slide judgment plate, and 200 μl of a reagent for measuring VWF ristocetin cofactor activity (Dade-Behring, Germany) was added dropwise thereto. The slide judgment plate is stirred for 3 minutes, and when agglutination is observed, it is judged as positive (active).
ブランクには生理食塩水を用いた。標準品には、ヒト正常血漿ＳＨＰ（Ｄａｄｅ−Ｂｅｈｒｉｎｇ社、ドイツ）を用い、生理食塩水で順次２倍、４倍、８倍、および１６倍に希釈したときの凝集度合いを目視により観察し、以下の基準に従って判定した。すなわち、ヒト正常血漿ＳＨＰ原液と同程度の凝集を示したものを「４＋」、ヒト正常血漿ＳＨＰの２倍希釈液と同程度の凝集を示したものを「３＋」、ヒト正常血漿ＳＨＰの４倍希釈液と同程度の凝集を示したものを「２＋」、ヒト正常血漿ＳＨＰの８倍希釈液と同程度の凝集を示したものを「１＋」、ヒト正常血漿ＳＨＰの１６倍希釈液同程度もしくはそれ以下の凝集を示したものを「−」と判定した。 Saline was used for the blank. As a standard product, human normal plasma SHP (Dade-Behring, Germany) was used, and the degree of aggregation when diluted with physiological saline in order of 2, 4, 8 and 16 times was visually observed. The determination was made according to the following criteria. That is, “4+” indicates the same degree of aggregation as the human normal plasma SHP stock solution, “3+” indicates the same degree of aggregation as the 2-fold dilution of the human normal plasma SHP, and 4 of the human normal plasma SHP. “2+” indicates the same degree of aggregation as the 2-fold diluted solution, “1+” indicates the same level of aggregation as the 8-fold diluted human normal plasma SHP, and the 16-fold diluted human normal plasma SHP. Those exhibiting a degree of aggregation or less were judged as “−”.
〔陽イオン交換樹脂の種類と第Ｖ因子、第ＶＩＩＩ因子に対する影響〕
表１に示すような陽イオン交換樹脂を用いた。
[Cation exchange resin types and effects on Factor V and Factor VIII]
A cation exchange resin as shown in Table 1 was used.
ヒト新鮮血漿１０ｍｌを、表１に示す陽イオン交換樹脂（ビーズタイプ）の１種１ｇ（１０ｗ／ｖ％）と混合し、室温で２時間撹拌を行い、接触処理済血漿を得た。 10 ml of human fresh plasma was mixed with 1 g (10 w / v%) of a cation exchange resin (bead type) shown in Table 1 and stirred at room temperature for 2 hours to obtain contact-treated plasma.
この処理済血漿の第Ｖ因子の活性、第ＶＩＩＩ因子の活性を、上記方法に従って測定した。第Ｖ因子の活性、第ＶＩＩＩ因子の活性は、接触処理していない標準品（シスメックス社製のコアグトロールＮ）を測定した場合に得られる凝固時間に基づく活性を１００％としたときの割合として算出した。測定結果を図１に示す。図中、縦軸は、第ＶＩＩＩ因子の活性（％）を示す。 The activity of factor V and factor VIII in the treated plasma was measured according to the above method. Factor V activity and factor VIII activity are calculated as percentages when the activity based on the coagulation time obtained when measuring a non-contact-treated standard product (Coagtrol N manufactured by Sysmex Corporation) is 100%. did. The measurement results are shown in FIG. In the figure, the vertical axis represents the factor VIII activity (%).
図１から、スルホン酸型陽イオン交換樹脂では、第Ｖ因子、第ＶＩＩＩ因子いずれも活性が大部分残っていた。また、カルボン酸型陽イオン交換樹脂では、凝固第Ｖ因子については１０％程度まで活性を低減させることができたが、凝固第ＶＩＩＩ因子については４０％程度活性が残っていた。一方、イミノジ酢酸型陽イオン交換樹脂では、第Ｖ因子、第ＶＩＩＩ因子のいずれに対しても、活性がほとんど消失していた。 From FIG. 1, in the sulfonic acid type cation exchange resin, both factor V and factor VIII remained mostly active. In the carboxylic acid type cation exchange resin, the activity of coagulation factor V could be reduced to about 10%, but the activity of coagulation factor VIII remained about 40%. On the other hand, in the iminodiacetic acid type cation exchange resin, the activity was almost lost with respect to both factor V and factor VIII.
また、イミノジ酢酸型陽イオン交換樹脂との接触処理済血漿については、第ＶＩＩＩ因子の活性測定、第ＶＩＩ因子の検出、ＶＷＦの検出、Ｒｃｏ活性の測定を行なった。第ＶＩＩＩ因子の活性は、接触処理していないヒト血漿を測定した場合の凝固時間に基づく活性を１００％として、各接触処理済血漿の活性を算出した。また、第ＶＩＩＩ因子の検出及びＶＷＦの検出においては、接触処理していないヒト血漿を測定した場合に得られる各測定結果を１００％含有の場合として、接触処理済血漿の第Ｖ因子の含有率（％）、ＶＷＦ含有率（％）を示した。測定結果を表２に示す。 In addition, plasma treated with an iminodiacetic acid type cation exchange resin was subjected to factor VIII activity measurement, factor VII detection, VWF detection, and Rco activity measurement. Regarding the activity of factor VIII, the activity of each plasma treated with contact was calculated with the activity based on the clotting time when measuring human plasma not subjected to contact treatment as 100%. In addition, in the detection of factor VIII and the detection of VWF, each measurement result obtained when measuring human plasma not subjected to contact treatment is assumed to contain 100%, and the content rate of factor V in the contact-treated plasma (%), VWF content (%). The measurement results are shown in Table 2.
表２から、第ＶＩＩＩ因子活性がほとんど失われているのに対し、第ＶＩＩＩ因子は残存していた。またＶＷＦも残存し、しかもＲｃｏ活性を示していた。 From Table 2, factor VIII activity was almost lost while factor VIII remained. VWF also remained and showed Rco activity.
〔イミノジ酢酸イオン交換型樹脂の濃度と接触時間の関係〕
ヒト新鮮血漿１００ｍｌと、イミノジ酢酸型イオン交換樹脂（ムロマック Ａ−１）を、イオン交換樹脂濃度０ｗ／ｖ％、２．５ｗ／ｖ％、５．０ｗ／ｖ％、７．５ｗ／ｖ％、１０．０ｗ／ｖ％となるように混合し、処理前、接触処理時間を０．５時間、１．０時間、１．５時間、２．０時間とした場合の、第ＶＩＩＩ因子の活性、第Ｖ因子の活性を調べた。第ＶＩＩＩ因子の活性、第Ｖ因子の活性は、接触処理していない標準品（シスメックス社製のコアグトロールＮ）を測定した場合に得られる凝固活性を１００％として算出した。それぞれ測定結果を図２及び図３に示す。図中、縦軸は活性（％）、横軸は接触処理時間を示す。
[Relationship between concentration of iminodiacetate ion exchange resin and contact time]
100 ml of human fresh plasma and iminodiacetic acid type ion exchange resin (Muromac A-1), ion exchange resin concentrations 0 w / v%, 2.5 w / v%, 5.0 w / v%, 7.5 w / v%, Factor VIII activity when mixed to 10.0 w / v% and the contact treatment time is 0.5 hours, 1.0 hour, 1.5 hours, and 2.0 hours before treatment, Factor V activity was examined. The activity of factor VIII and the activity of factor V were calculated with the coagulation activity obtained when measuring a standard product not subjected to contact treatment (Coagtrol N, manufactured by Sysmex Corporation) as 100%. The measurement results are shown in FIGS. In the figure, the vertical axis represents activity (%), and the horizontal axis represents contact treatment time.
図２及び図３から、５ｗ／ｖ％以上の条件で、３０分以上、好ましくは２時間以上処理すると、第Ｖ因子及び第ＶＩＩＩ因子のいずれの活性も１％以下にまで低下した。 2 and 3, when the treatment was performed for 30 minutes or more, preferably 2 hours or more under the condition of 5 w / v% or more, both the activities of Factor V and Factor VIII were reduced to 1% or less.
〔接触処理における第Ｖ因子、第ＶＩＩＩ因子の独立性の確認〕
先天的に第Ｖ因子のみ欠乏した血漿中の第ＶＩＩＩ因子、先天的に第ＶＩＩＩ因子のみ欠乏の血漿中の第Ｖ因子が、イミノジ酢酸型陽イオン交換樹脂との接触処理により直接的に不活化されるかどうかを検討した。
[Confirmation of independence of factor V and factor VIII in contact treatment]
Factor VIII in plasma congenitally deficient only in factor V and factor V in plasma congenitally deficient only in factor VIII are directly inactivated by contact treatment with iminodiacetic acid type cation exchange resin We examined whether to be done.
第Ｖ因子欠乏血漿（４例：患者１〜４）ならびに第ＶＩＩＩ因子欠乏血漿（４例：患者１〜４）は、ジョージ キングバイオメディカル（ＵＳＡ）より購入した。 Factor V-deficient plasma (4 cases: patients 1 to 4) and factor VIII-deficient plasma (4 cases: patients 1 to 4) were purchased from George King Biomedical (USA).
各血漿サンプル１ｍｌに、イミノジ酢酸型イオン交換樹脂（ムロマック Ａ−１）を１０ｗ／ｖ％となるように混合して、２時間攪拌処理した。 1 ml of each plasma sample was mixed with iminodiacetic acid type ion exchange resin (Muromac A-1) at 10 w / v% and stirred for 2 hours.
得られた処理済血漿について、第ＶＩＩＩ因子の活性、第Ｖ因子の活性を測定した。第Ｖ因子の活性、第ＶＩＩＩ因子の活性は、接触処理していない標準品（シスメックス社製のコアグトロールＮ）の凝固時間を活性１００％の場合として算出した。測定結果をそれぞれ図４及び図５に示す。 About the obtained processed plasma, the activity of factor VIII and the activity of factor V were measured. Factor V activity and factor VIII activity were calculated by assuming that the clotting time of a standard product (Coagtrol N manufactured by Sysmex Corporation) not subjected to contact treatment was 100% active. The measurement results are shown in FIGS. 4 and 5, respectively.
図４から、第Ｖ因子欠乏血漿中の第ＶＩＩＩ因子活性は接触処理１時間で正常レベル（１００％）から１０％付近まで低下し、以後２時間から４時間で１％未満となった。また、図５からわかるように、第ＶＩＩＩ因子欠乏血漿中の第Ｖ因子活性は接触処理１時間で正常レベル（１００％）から１０％付近まで低下し、以後４時間までにはすべての血漿試料で１％未満となった。
従って、本発明のイミノジ酢酸型陽イオン交換樹脂との接触処理による方法では、第Ｖ因子、第ＶＩＩＩ因子を独立的に不活化型に変化することがわかった。
From FIG. 4, the factor VIII activity in the factor V-deficient plasma decreased from the normal level (100%) to around 10% after 1 hour of the contact treatment, and then decreased to less than 1% after 2 hours to 4 hours. In addition, as can be seen from FIG. 5, the factor V activity in the factor VIII-deficient plasma decreases from the normal level (100%) to around 10% after 1 hour of the contact treatment, and then all plasma samples by 4 hours thereafter. It was less than 1%.
Therefore, it was found that in the method of contact treatment with the iminodiacetic acid type cation exchange resin of the present invention, Factor V and Factor VIII are independently changed to the inactivated type.
〔イミノジ酢酸型イオン交換樹脂との接触処理による第Ｖ因子、第ＶＩＩＩ因子残存の確認〕
純品の第ＶＩＩＩ因子（Ｋｏｇｅｎａｔｅ ＦＳ：バイエル薬品：２５０Ｕ／ｍｌ）を生理活性食塩水に溶解させてなる溶液（２５０００％）、及びこの生理活性食塩水溶液を２０％イミノジ酢酸型陽イオン交換樹脂で４時間、接触処理した溶液（処理済溶液）を、還元下及び非還元下の条件でＳＤＳ電気泳動にかけた。
[Confirmation of remaining factor V and factor VIII by contact treatment with iminodiacetic acid type ion exchange resin]
A pure factor VIII solution (Kogenate FS: Bayer Yakuhin: 250 U / ml) dissolved in physiologically active saline (25000%) and this physiologically active saline solution with 20% iminodiacetic acid type cation exchange resin The solution treated for 4 hours (treated solution) was subjected to SDS electrophoresis under reducing and non-reducing conditions.
また、ヒト第Ｖ因子（５０μｇ／ｍｌ：Ｈｅｍａｔｏｌｏｇｉｃ Ｔｅｃｈｎｏｌｏｇｙ Ｉｎｃ．，米国）を生理活性食塩水で溶解させてなる溶液（１５０００％）、及びこの第Ｖ因子含有溶液を２０％イミノジ酢酸型陽イオン交換樹脂で４時間処理して得られた溶液（処理済溶液）を、それぞれ還元下及び非還元下の条件で、ＳＤＳ電気泳動にかけた。 In addition, a solution (15000%) obtained by dissolving human factor V (50 μg / ml: Hematological Technology Inc., USA) with physiologically active saline, and a 20% iminodiacetic acid cation exchange solution containing this factor V-containing solution The solution (treated solution) obtained by treating with resin for 4 hours was subjected to SDS electrophoresis under the conditions of reduction and non-reduction, respectively.
ＳＤＳポリアクリルアミドゲル電気泳動（以下、ＳＤＳ―ＰＡＧＥ）はＭＩＮＩ−ＰＲＯＴＥＩＮ ＩＩ電気泳動装置（日本バイオラッド）を用いて行なった。還元下の条件では、試料を１ｗ／ｖ％ＳＤＳ溶液で４倍に希釈した試料溶液を、５ｗ／ｖ％２−メルカプトエタノール溶液と共に、５６℃で３０分間処理し、０．１ｗ／ｖ％ＳＤＳを含む７．５％ポリアクリルアミドゲル上で６０Ｖ、２．５時間展開させた。一方、非還元下の条件では、同様に試料を１ｗ／ｖ％ＳＤＳ溶液で４倍に希釈した溶液を、０．１ｗ／ｖ％ＳＤＳを含む７．５％ポリアクリルアミドゲル上で同様に６０Ｖ、２．５時間展開させた。緩衝液には０．０２ｗ／ｖ％ＳＤＳを含む２５ｍＭトリス−１９２ｍＭグリシン緩衝液（ｐＨ７．５）を用いた。染色には２Ｄ銀染色試薬ＩＩ（第一化学薬品）を用いた。 SDS polyacrylamide gel electrophoresis (hereinafter referred to as SDS-PAGE) was performed using a MINI-PROTEIN II electrophoresis apparatus (Nippon Bio-Rad). Under the conditions under reduction, a sample solution obtained by diluting the sample 4 times with 1 w / v% SDS solution was treated with 5 w / v% 2-mercaptoethanol solution at 56 ° C. for 30 minutes, and 0.1 w / v% SDS The gel was developed on a 7.5% polyacrylamide gel containing 60 V for 2.5 hours. On the other hand, under non-reducing conditions, similarly, a solution obtained by diluting the sample 4 times with 1 w / v% SDS solution was similarly applied to 60 V, 7.5% polyacrylamide gel containing 0.1 w / v% SDS. Developed for 2.5 hours. The buffer used was 25 mM Tris-192 mM glycine buffer (pH 7.5) containing 0.02 w / v% SDS. For staining, 2D silver staining reagent II (Daiichi Kagaku) was used.
図６（ａ）は非還元下の条件で電気泳動して得られた電気泳動写真であり、図６（ｂ）は還元下の条件で電気泳動して得られた電気泳動写真である。
写真において、レーン１は分子量マーカー、レーン２は接触処理前の第ＶＩＩＩ因子（Ｋｏｇｅｎａｔｅ ＦＳ）を含む溶液、レーン３及びレーン４は第ＶＩＩＩ因子含有溶液の処理済溶液、レーン５は接触処理前のヒト第Ｖ因子を含む溶液、レーン６はヒト第Ｖ因子溶液の処理済溶液である。
FIG. 6 (a) is an electrophoretic photograph obtained by electrophoresis under non-reducing conditions, and FIG. 6 (b) is an electrophoretic photograph obtained by electrophoresis under reducing conditions.
In the photograph, lane 1 is a molecular weight marker, lane 2 is a solution containing Factor VIII (Kogenate FS) before contact treatment, lane 3 and lane 4 are treated solutions of a Factor VIII-containing solution, and lane 5 is before contact treatment. A solution containing human factor V, lane 6, is a treated solution of human factor V solution.
図６（ａ）及び図６（ｂ）の写真中のレーン３、４及び６において、蛋白質のバンドを確認できた。従って、接触処理によっても、第Ｖ因子、第ＶＩＩＩ因子が残存していることが確認できた。 In lanes 3, 4 and 6 in the photographs of FIGS. 6 (a) and 6 (b), protein bands could be confirmed. Therefore, it was confirmed that Factor V and Factor VIII remained even after the contact treatment.
〔イミノジ酢酸型陽イオン交換樹脂処理による第ＶＩＩＩ因子の活性への影響〕
生理食塩水に、第ＶＩＩＩ因子とＶＷＦ複合体である化血研のＣｏｆａｃｔｏｒ Ｆ（１００Ｕ／ｍｌ）を溶解させた第ＶＩＩＩ因子複合体溶液（１００００％、１Ｕ／ｍｌが１００％に相当）を、イミノジ酢酸型イオン交換樹脂２０％と４時間接触させた。処理済溶液について、第ＶＩＩＩ因子の活性測定、第ＶＩＩＩ因子の検出、ＶＷＦの検出、Ｒｃｏ活性の測定を、上記測定方法に従って行なった。
[Influence on the activity of factor VIII by iminodiacetic acid type cation exchange resin treatment]
Factor VIII complex solution (10000%, 1 U / ml is equivalent to 100%) in which factor VIII and VWF complex, Kakkenken Cofactor F (100 U / ml) is dissolved in physiological saline, Contact with 20% iminodiacetic acid type ion exchange resin for 4 hours. With respect to the treated solution, factor VIII activity measurement, factor VIII detection, VWF detection, and Rco activity measurement were performed according to the above measurement method.
一方、ヒトの正常血漿について、同様の処理を行ない、処理済血漿について第ＶＩＩＩ因子の活性測定、第ＶＩＩＩ因子の検出、ＶＷＦの検出、Ｒｃｏ活性の測定を行なった。これらの結果を併せて表３に示す。 On the other hand, human normal plasma was subjected to the same treatment, and the treated plasma was subjected to factor VIII activity measurement, factor VIII detection, VWF detection, and Rco activity measurement. These results are shown together in Table 3.
ここで、第ＶＩＩＩ因子複合体溶液の第ＶＩＩＩ因子活性は、接触処理していない第ＶＩＩＩ因子複合体溶液を測定した場合に得られる凝固活性を１００％として、接触処理した第ＶＩＩＩ因子複合体溶液中の第ＶＩＩＩ因子の活性（％）を算出し、その活性値が１％以上の場合は「＋」、１％未満の場合は「−」と判定した。ヒト正常血漿の第ＶＩＩＩ因子活性は、接触処理していないヒト正常血漿を測定した場合に得られる凝固活性を１００％として、接触処理したヒト正常血漿中の第ＶＩＩＩ因子の活性（％）を算出し、その活性値が１％以上の場合は「＋」、１％未満の場合は「−」と判定した。また、第ＶＩＩＩ因子複合体溶液の第ＶＩＩＩ因子及びＶＷＦは、接触処理していない第ＶＩＩＩ因子複合体溶液を測定した場合に得られる各測定結果を１００％として、接触処理した第ＶＩＩＩ因子複合体溶液の第ＶＩＩＩ因子含有率（％）及びＶＷＦ含有率（％）を算出し、それらの値が７０％以上の場合は「＋」、７０％未満の場合は「−」と判定した。ヒト正常血漿の第ＶＩＩＩ因子及びＶＷＦは、接触処理していないヒト正常血漿を測定した場合に得られる各測定結果を１００％として、接触処理したヒト正常血漿の第ＶＩＩＩ因子含有率（％）及びＶＷＦ含有率（％）を算出し、それらの値が７０％以上の場合は「＋」、７０％未満の場合は「−」と判定した。 Here, the factor VIII activity of the factor VIII complex solution is the factor VIII complex solution subjected to contact treatment, with the coagulation activity obtained when measuring the factor VIII complex solution not subjected to contact treatment as 100%. The activity (%) of the factor VIII was calculated, and when the activity value was 1% or more, it was determined as “+”, and when it was less than 1%, it was determined as “−”. Factor VIII activity of normal human plasma is calculated as factor VIII activity (%) in human normal plasma subjected to contact treatment, with the clotting activity obtained when measuring normal human plasma not subjected to contact treatment as 100%. When the activity value was 1% or more, “+” was determined, and when the activity value was less than 1%, “−” was determined. In addition, factor VIII and VWF of the factor VIII complex solution are the factor VIII complex subjected to contact treatment with each measurement result obtained when measuring the factor VIII complex solution not subjected to contact treatment as 100%. The factor VIII content (%) and VWF content (%) of the solution were calculated, and when these values were 70% or more, “+” was determined, and when they were less than 70%, “−” was determined. Factors VIII and VWF of human normal plasma are expressed as 100% of each measurement result obtained when human normal plasma not subjected to contact treatment is measured, and factor VIII content (%) of human normal plasma subjected to contact treatment and The VWF content (%) was calculated, and when those values were 70% or more, “+” was determined, and when the values were less than 70%, “−” was determined.
表３から、第ＶＩＩＩ因子活性は失われているが、第ＶＩＩＩ因子が残存していることがわかる。また、ＶＷＦについては残存しており、そのＶＷＦがＲｃｏ活性を有していることがわかった。つまり、第ＶＩＩＩ因子とＶＷＦの複合体に対する影響は、正常ヒト血漿による影響と同様の挙動を示すことを確認できた。 Table 3 shows that factor VIII activity is lost, but factor VIII remains. Further, it was found that VWF remained and the VWF had Rco activity. That is, it was confirmed that the influence on the complex of factor VIII and VWF showed the same behavior as the influence by normal human plasma.
〔イヌの血友病モデル〕
血液試料として、健常のビーグル犬より採血した血漿を用いた。
このイヌ正常血漿を、イミノジ酢酸型陽イオン交換樹脂（ムロマック Ａ−１）（２０ｗ／ｖ％）と４．５時間接触処理させた。
[Dog hemophilia model]
As a blood sample, plasma collected from a healthy beagle dog was used.
This dog normal plasma was contact-treated with iminodiacetic acid type cation exchange resin (Muromac A-1) (20 w / v%) for 4.5 hours.
得られた処理済血漿の第ＶＩＩＩ因子活性（ＦＶＩＩＩ因子：Ｃ）、及び第Ｖ因子活性（ＦＶ因子：Ｃ）を上記測定方法で測定した。接触処理していない標準品(シスメックス社製のコアグトロールＮ）を測定した場合に得られる凝固活性を１００％として、各測定サンプル中の第ＶＩＩＩ因子の活性（％）又は第Ｖ因子の活性（％）を算出した。正常血漿（原料血漿）の結果を図７、処理済血漿の結果を図８に示す。各図の縦軸は各血液凝固因子の活性（％）を示す。 Factor VIII activity (FVIII factor: C) and factor V activity (FV factor: C) of the obtained treated plasma were measured by the measurement method described above. Factor VIII activity (%) or factor V activity (%) in each measurement sample, assuming that the coagulation activity obtained when measuring a standard product (Coagtrol N manufactured by Sysmex Corporation) not subjected to contact treatment is 100% ) Was calculated. The result of normal plasma (raw material plasma) is shown in FIG. 7, and the result of processed plasma is shown in FIG. The vertical axis of each figure shows the activity (%) of each blood coagulation factor.
正常イヌ血漿の第ＶＩＩＩ因子活性は５６０％、第Ｖ因子活性は８７６％を示し、その活性はヒトの場合と比較して、それぞれ約６倍および約９倍高かった。しかしながら、処理済血漿では、第ＶＩＩＩ因子活性５．７％、第Ｖ因子活性１５．９％まで低下した。
以上のことから、ヒトの血漿に限らず、イヌの血漿においても、接触処理によって第ＶＩＩＩ因子や第Ｖ因子が不活化型に変化することがわかった。
Normal dog plasma showed factor VIII activity of 560% and factor V activity of 876%, which were about 6 times and about 9 times higher compared to humans, respectively. However, the treated plasma decreased factor VIII activity to 5.7% and factor V activity to 15.9%.
From the above, it was found that factor VIII and factor V change to inactivated form by contact treatment not only in human plasma but also in dog plasma.
〔イミノジ酢酸基を有するセファロースを用いた接触処理〕
生理食塩水に、第ＶＩＩＩ因子とＶＷＦの複合体である化血研のＣｏnｆａｃｔ Ｆ（財団法人化学及血清治療研究所：１００Ｕ／ｍｌ）を溶解させた第ＶＩＩＩ因子複合体溶液（１００００％、１Ｕ／ｍｌが１００％に相当）を、イミノジ酢酸基を有するセファロース２０％（ｗ／ｖ）と４時間接触処理させた。この接触処理により得られた溶液について、第ＶＩＩＩ因子の活性測定、第ＶＩＩＩ因子の検出、ＶＷＦの検出及びＲｃｏ活性の測定を、前記測定方法に従って行なった。一方、ヒトの正常血漿について、同様の接触処理を行ない、得られた血漿について第ＶＩＩＩ因子の活性測定、第ＶＩＩＩ因子の検出、ＶＷＦの検出及びＲｃｏ活性の測定を行なった。
[Contact treatment using sepharose having iminodiacetic acid groups]
A factor VIII complex solution (10000%, 1 U) obtained by dissolving Chemical Factor Serf's Confact F (100 U / ml), a complex of factor VIII and VWF, in physiological saline. / Ml is equivalent to 100%) and contact treatment with Sepharose 20% (w / v) having iminodiacetic acid groups for 4 hours. About the solution obtained by this contact treatment, the activity measurement of factor VIII, the detection of factor VIII, the detection of VWF and the measurement of Rco activity were carried out according to the measurement method. On the other hand, human normal plasma was subjected to the same contact treatment, and the obtained plasma was subjected to factor VIII activity measurement, factor VIII detection, VWF detection and Rco activity measurement.
第ＶＩＩＩ因子複合体溶液の第ＶＩＩＩ因子活性は、接触処理していない第ＶＩＩＩ因子複合体溶液を測定した場合に得られる凝固活性を１００％として、接触処理した第ＶＩＩＩ因子複合体溶液中の第ＶＩＩＩ因子の活性（％）を算出し、その活性値が１％以上の場合は「＋」、１％未満の場合は「−」と判定した。ヒト正常血漿の第ＶＩＩＩ因子活性は、接触処理していないヒト正常血漿を測定した場合に得られる凝固活性を１００％として、接触処理したヒト正常血漿中の第ＶＩＩＩ因子の活性（％）を算出し、その活性値が１％以上の場合は「＋」、１％未満の場合は「−」と判定した。また、第ＶＩＩＩ因子複合体溶液の第ＶＩＩＩ因子及びＶＷＦの検出は、接触処理していない第ＶＩＩＩ因子複合体溶液を測定した場合に得られる各測定結果を１００％として、接触処理した第ＶＩＩＩ因子複合体溶液の第ＶＩＩＩ因子含有率（％）及びＶＷＦ含有率（％）を算出し、それらの値が７０％以上の場合は「＋」、７０％未満の場合は「−」と判定した。ヒト正常血漿の第ＶＩＩＩ因子及びＶＷＦの検出は、接触処理していないヒト正常血漿を測定した場合に得られる各測定結果を１００％として、接触処理したヒト正常血漿の第ＶＩＩＩ因子含有率（％）及びＶＷＦ含有率（％）を算出し、それらの値が７０％以上の場合は「＋」、７０％未満の場合は「−」と判定した。測定結果を表４に示す。 The factor VIII activity of the factor VIII complex solution is defined as that in the contact-treated factor VIII complex solution, where the coagulation activity obtained when the factor VIII complex solution not subjected to contact treatment is measured is 100%. The activity (%) of factor VIII was calculated, and when the activity value was 1% or more, “+” was determined, and when it was less than 1%, “−” was determined. Factor VIII activity of normal human plasma is calculated as factor VIII activity (%) in human normal plasma subjected to contact treatment, with the clotting activity obtained when measuring normal human plasma not subjected to contact treatment as 100%. When the activity value was 1% or more, “+” was determined, and when the activity value was less than 1%, “−” was determined. In addition, the detection of the factor VIII and VWF in the factor VIII complex solution is carried out by using each measurement result obtained when the factor VIII complex solution not subjected to contact treatment is measured as 100%, and the factor VIII subjected to contact treatment. The factor VIII content (%) and VWF content (%) of the complex solution were calculated. When these values were 70% or more, “+” was determined, and when the values were less than 70%, “−” was determined. The detection of factor VIII and VWF in human normal plasma was carried out with each measurement result obtained when measuring human normal plasma not subjected to contact treatment as 100%, and the factor VIII content (% ) And VWF content (%) were calculated, and when those values were 70% or more, “+” was determined, and when they were less than 70%, “−” was determined. Table 4 shows the measurement results.
表４から、イミノジ酢酸基を有するセファロースを用いた接触処理により得られた第ＶＩＩＩ因子複合体溶液及びヒト正常血漿においても、第ＶＩＩＩ因子活性は失われているが、第ＶＩＩＩ因子が残存していることがわかった。また、ＶＷＦについては残存しており、それらＶＷＦがＲｃｏ活性を有していることがわかった。つまり、第ＶＩＩＩ因子とＶＷＦの複合体に対する接触処理の影響は、ヒト正常血漿に対する影響と同様の挙動を示すことを確認できた。 From Table 4, the factor VIII activity was lost in the factor VIII complex solution and human normal plasma obtained by contact treatment with sepharose having iminodiacetic acid groups, but factor VIII remained. I found out. Moreover, it was found that VWF remained and these VWF had Rco activity. That is, it was confirmed that the influence of the contact treatment on the complex of factor VIII and VWF showed the same behavior as the influence on human normal plasma.
〔イミノジ酢酸基を有するセファロースを用いた接触処理：接触処理時間と活性の変化〕
イミノジ酢酸基を有する化合物として、イミノジ酢酸基を有するセファロースであるChelating Sepharose Fast Flow（Amersham Biosciences社）を水酸化ナトリウム溶液で前処理したものを用いて、ヒト正常血漿を接触処理した。
水酸化ナトリウム水溶液の前処理により、セファロースが有するイミノジ酢酸基（−N（ＣＨ２ＣＯＯＲ）２）のＲがＨからナトリウムイオンに変わる。
[Contact treatment using sepharose having iminodiacetic acid group: change in contact treatment time and activity]
As a compound having an iminodiacetic acid group, human normal plasma was subjected to a contact treatment using a pretreatment of Chelating Sepharose Fast Flow (Amersham Biosciences), which is a sepharose having an iminodiacetic acid group, with a sodium hydroxide solution.
By pretreatment with an aqueous sodium hydroxide solution, R of iminodiacetic acid group (—N (CH 2 COOR) 2 ) possessed by Sepharose is changed from H to sodium ion.
接触処理は、ヒト正常血漿１００ｍｌと、イミノジ酢酸基を有するセファロースを、セファロースの濃度が０ｗ／ｖ％、５．０ｗ／ｖ％、１０．０ｗ／ｖ％、２０．０ｗ／ｖ％となるように混合して行った。そして、接触処理前及び各処理時間（接触処理の時間が１．０、２．０、４．０時間）における、第ＶＩＩＩ因子の活性及び第Ｖ因子の活性を前記測定方法に従って測定した。接触処理していない標準品（シスメックス社製のコアグトロールＮ）を測定した場合に得られる凝固活性を１００％として、各測定サンプル中の第ＶＩＩＩ因子の活性（％）又は第Ｖ因子の活性（％）を算出した。 In the contact treatment, 100 ml of human normal plasma and sepharose having iminodiacetic acid groups are added so that the concentration of sepharose is 0 w / v%, 5.0 w / v%, 10.0 w / v%, 20.0 w / v%. And mixed. And the activity of factor VIII and the activity of factor V were measured according to the said measuring method before contact processing and in each processing time (contact processing time is 1.0, 2.0, 4.0 hours). Factor VIII activity (%) or factor V activity (%) in each measurement sample, assuming that the coagulation activity obtained when measuring a standard product (Coagtrol N manufactured by Sysmex Corporation) not subjected to contact treatment is 100% ) Was calculated.
第ＶＩＩＩ因子の活性の測定結果を図９に示し、第Ｖ因子の活性の測定結果を図１０に示す。なお、図中、縦軸は、第ＶＩＩＩ因子又は第Ｖ因子の活性（％）を示し、横軸は接触処理した時間を示す。 The measurement result of the activity of factor VIII is shown in FIG. 9, and the measurement result of the activity of factor V is shown in FIG. In the figure, the vertical axis represents factor VIII or factor V activity (%), and the horizontal axis represents the contact treatment time.
図９において、第ＶＩＩＩ因子活性は接触処理１時間で１０％付近まで低下し、以後２時間から４時間で１〜５％までに低下した。また、図１０において、第ＶＩＩＩ因子欠乏血漿中の第Ｖ因子活性は接触処理１時間で２０〜６０％付近まで低下し、以後４時間までにはセファロースの濃度が５．０ｗ／ｖ％の場合には２０％にまで低下し、１０．０ｗ／ｖ％及び２０．０ｗ／ｖ％の場合には１％未満にまで低下した。これにより、イミノジ酢酸基を有するセファロースとの接触処理により、血漿中の第Ｖ因子、第ＶＩＩＩ因子の活性が低下することがわかった。 In FIG. 9, the factor VIII activity decreased to about 10% after 1 hour of the contact treatment, and then decreased to 1-5% after 2 hours to 4 hours. In FIG. 10, the factor V activity in the factor VIII-deficient plasma decreases to about 20 to 60% after 1 hour of contact treatment, and the sepharose concentration is 5.0 w / v% by 4 hours thereafter. In the case of 10.0 w / v% and 20.0 w / v%, it decreased to less than 1%. Thus, it was found that the activity of factor V and factor VIII in plasma was reduced by contact treatment with sepharose having an iminodiacetic acid group.
次に、水酸化ナトリウム溶液による前処理を行なわずに、すなわちイミノジ酢酸基のＲがＨの場合のセファロースを用いて、上記と同様に接触処理し、第ＶＩＩＩ因子の活性及び第Ｖ因子の活性を測定した。前処理したイミノジ酢酸基を有するセファロースを用いて接触処理した場合に得られる測定結果と、前処理していないイミノジ酢酸基を有するセファロースを用いて接触処理した場合に得られる測定結果とを比較すると、第Ｖ因子及び第ＶＩＩＩ因子のいずれの活性も、前処理する場合のほうがより短時間でより低いレベルに活性を低下させる（図示せず）。以上のことから、接触処理に用いるイミノジ酢酸基を有するセファロースについては、イミノジ酢酸基（−N（ＣＨ２ＣＯＯＲ）２）のRがHであるものよりもナトリウムイオンであるもののほうが、接触処理においてより効果的に第Ｖ因子及び第ＶＩＩＩ因子を不活化型へ変化することができることがわかる。 Next, without pretreatment with sodium hydroxide solution, that is, using Sepharose when R of iminodiacetic acid group is H, contact treatment is performed in the same manner as described above, and the activity of factor VIII and the activity of factor V Was measured. Comparing the measurement results obtained when the contact treatment is performed using the pretreated Iminodiacetic acid group sepharose and the measurement results obtained when the contact treatment is performed using the pretreatment iminodiacetic acid group sepharose Both the activities of Factor V and Factor VIII are reduced to a lower level in a shorter time when pretreated (not shown). From the above, for sepharose having an iminodiacetic acid group to be used for the contact treatment, the one in which R of the iminodiacetic acid group (—N (CH 2 COOR) 2 ) is sodium ion is more suitable in the contact treatment. It can be seen that Factor V and Factor VIII can be more effectively changed to the inactivated form.
本発明の不活化方法によれば、第Ｖ因子、第ＶＩＩＩ因子の活性型への変換を不能とするだけで、第Ｖ因子、第ＶＩＩＩ因子は実質的に残存しているので、第ＶＩＩＩ因子と共同で安定して存在するＶＷＦに影響を与えずに済む。従って、第Ｖ因子又は第ＶＩＩＩ因子だけを不活化した血液凝固因子不活化試料を製造できる。 According to the inactivation method of the present invention, since the conversion of factor V and factor VIII into an activated form is simply disabled, factor V and factor VIII remain substantially. It is not necessary to affect the VWF that exists stably in cooperation with the company. Accordingly, a blood coagulation factor inactivated sample in which only factor V or factor VIII is inactivated can be produced.
しかも本発明の不活化方法では、原料としての血漿をイミノジ酢酸型陽イオン交換樹脂と接触処理するだけで不活化できるので、従来の免疫吸着法による凝固因子欠乏血漿の製造方法と比べても、短時間で効率的に血液凝固因子不活化試料を製造できる。 Moreover, in the inactivation method of the present invention, since the plasma as a raw material can be inactivated simply by contact treatment with an iminodiacetic acid type cation exchange resin, compared with the conventional method for producing coagulation factor-deficient plasma by immunoadsorption method, A blood coagulation factor inactivated sample can be produced efficiently in a short time.
さらにまた、従来の因子吸着除去の方法により製造される第Ｖ因子、第ＶＩＩＩ因子欠乏血漿では、ＶＷＦを別途添加する必要があったが、本発明の不活化方法を用ることにより、ＶＷＦが活性可能な状態で残存している血液凝固因子不活化試料を得ることができる。 Furthermore, in the factor V and factor VIII deficient plasma produced by the conventional factor adsorption removal method, it was necessary to add VWF separately. However, by using the inactivation method of the present invention, VWF is reduced. A blood coagulation factor inactivated sample remaining in an active state can be obtained.
従って、本発明の血液凝固因子不活化試料を用いることにより、従来よりも効率的に、しかも低コストで、血液検体における第Ｖ因子、第ＶＩＩＩ因子の有無、活性を測定でき、ひいては凝固因子欠損・異常症の診断測定に利用できる。 Therefore, by using the blood coagulation factor inactivated sample of the present invention, it is possible to measure the presence / absence and activity of factor V and factor VIII in a blood sample more efficiently and at a lower cost than before.・ Can be used for diagnostic measurement of abnormalities.
Claims (19)
血液検体、凝固時間を測定するための測定試薬及び検体処理用血漿を混合して測定用試料を調製する工程；
前記測定用試料における凝固時間を測定する工程；及び
前記凝固時間に基づいて血液凝固因子の活性を算出する工程；
を含み、
前記検体処理用血漿が、請求項１〜６のいずれかに記載の不活化方法により得られた、不活化型の第Ｖ因子及び不活化型の第ＶＩＩＩ因子を含有する血液凝固因子不活化血漿を含む血液凝固因子活性の測定方法。 A method for measuring the activity of a blood coagulation factor contained in a blood sample using the plasma for sample processing,
A step of preparing a measurement sample by mixing a blood sample, a measurement reagent for measuring a coagulation time, and plasma for sample processing;
Measuring a clotting time in the measurement sample; and calculating a blood coagulation factor activity based on the clotting time;
Including
7. The blood coagulation factor inactivated plasma containing the inactivated factor V and the inactivated factor VIII obtained by the inactivation method according to any one of claims 1 to 6 A method for measuring blood coagulation factor activity.
前記血液凝固因子の活性を算出する工程において、第Ｖ因子の活性を算出する請求項１２又は１３の測定方法。 The measurement reagent is a prothrombin time measurement reagent,
The method according to claim 12 or 13, wherein the activity of factor V is calculated in the step of calculating the activity of the blood coagulation factor.
前記測定試薬がプロトロンビン時間測定試薬であり、
前記血液凝固因子の活性を算出する工程において、第Ｖ因子の活性を算出する請求項１２又は１３に記載の測定方法。 The sample processing plasma further comprises Factor VIII that can be converted into an active form,
The measurement reagent is a prothrombin time measurement reagent,
The measurement method according to claim 12 or 13, wherein in the step of calculating the activity of the blood coagulation factor, the activity of the factor V is calculated.
前記測定試薬が、活性化部分トロンボプラスチン時間測定試薬であり、
前記血液凝固因子の活性を算出する工程において、第Ｖ因子の活性を算出する請求項１２又は１３に記載の測定方法。 The sample processing plasma further includes a factor V that can be changed to an active form,
The measurement reagent is an activated partial thromboplastin time measurement reagent,
The measurement method according to claim 12 or 13, wherein in the step of calculating the activity of the blood coagulation factor, the activity of the factor V is calculated.
前記測定試薬が、活性化部分トロンボプラスチン時間測定試薬であり、
前記血液凝固因子の活性を算出する工程において、第Ｖ因子の活性を算出する請求項１２又は１３に記載の測定方法。 The sample processing plasma further comprises Factor VIII that can be converted into an active form,
The measurement reagent is an activated partial thromboplastin time measurement reagent,
The measurement method according to claim 12 or 13, wherein in the step of calculating the activity of the blood coagulation factor, the activity of the factor V is calculated.
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