JP6034238B2 - Blood coagulation test method - Google Patents
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Description
本発明は、血液や血漿の凝固能を測定する血液凝固検査方法に関するものである。 The present invention relates to a blood coagulation test method for measuring blood or plasma coagulation ability.
凝固活性は、臨床検査の重要な項目であり、凝固活性指標の１つにプロトロンビン時間（Prothrombin time：ＰＴ）がある。プロトロンビン時間を測定する血液凝固検査により、血液凝固活性（血液凝固能）を求めることができる。プロトロンビン時間は、外因系の凝固因子への感度が高いと考えられており、外因系の凝固因子の欠損のスクリーニングや、肝機能の異常、さらに経口投与による抗凝血薬療法のモニタリングに用いられる。 Coagulation activity is an important item in clinical examinations, and one of the indicators of coagulation activity is prothrombin time (PT). Blood clotting activity (blood clotting ability) can be determined by a blood clotting test that measures prothrombin time. Prothrombin time is thought to be highly sensitive to exogenous clotting factors and is used to screen for deficiencies in extrinsic clotting factors, abnormal liver function, and oral anticoagulant therapy .
従来の血液凝固時間の測定には、撹拌抵抗方式，光散乱方式，熱伝導方式，水晶振動子方式などの方法が開発されているが、一般に、撹拌抵抗方式および光散乱方式が多く用いられている（非特許文献１参照）。撹拌抵抗方式は、試料（検体）を活性化剤と一緒に導入してフィンで撹拌し、撹拌の抵抗の上昇から血液凝固時間を測定する方法である。 Conventional methods for measuring blood coagulation time, such as the stirring resistance method, light scattering method, heat conduction method, and crystal resonator method, have been developed. Generally, the stirring resistance method and the light scattering method are often used. (See Non-Patent Document 1). The stirring resistance method is a method in which a sample (specimen) is introduced together with an activator and stirred with fins, and the blood coagulation time is measured from an increase in stirring resistance.
光散乱方式は、測定容器内で、対象となる血漿に凝固活性化を促す成分を含む試薬を混合し、容器に対し光を入射させ、入射した光の散乱光の光量変化を測定して血液凝固時間を測定する方法である。散乱光量から血液凝固時間を得る方法としては、散乱光量をそのまま利用する方法、散乱光量の微分値を利用する方法、散乱光量がある一定値に達するまでの時間を求める方法がある。 In the light scattering method, a reagent containing a component that promotes coagulation activation is mixed into the target plasma in a measurement container, light is incident on the container, and the change in the amount of scattered light of the incident light is measured to measure blood. This is a method for measuring the coagulation time. As a method of obtaining the blood coagulation time from the scattered light amount, there are a method of using the scattered light amount as it is, a method of using a differential value of the scattered light amount, and a method of obtaining a time until the scattered light amount reaches a certain value.
屈折率変化を用いて測定する手法においては、マイクロ流路内における異なる地点の屈折率変化と、時間，溶液の移動距離から流速を算出し、算出した流速が血漿の凝固活性度の違いにより変化することを利用して血液凝固を検査する技術がある（特許文献１〜３参照）。マイクロ流路を用いた流速測定によれば、少量の検体で迅速かつ正確な凝固活性が測定できるという利点がある。 In the measurement method using the refractive index change, the flow rate is calculated from the refractive index change at different points in the microchannel, the time, and the distance traveled by the solution, and the calculated flow rate changes depending on the difference in plasma coagulation activity. There is a technique for examining blood coagulation by utilizing this (see Patent Documents 1 to 3). According to flow rate measurement using a microchannel, there is an advantage that rapid and accurate clotting activity can be measured with a small amount of sample.
しかしながら、マイクロ流路内を移送している検体の流速測定では、検体の流速が粘度に依存しているため、凝固能が同じでも粘度が異なる検体を測定する場合には、これらの凝固能を正しく区別できないという問題があった。 However, when measuring the flow rate of a sample transported in a microchannel, the flow rate of the sample depends on the viscosity. There was a problem that it could not be distinguished correctly.
本発明は、以上のような問題点を解消するためになされたものであり、凝固能が同じでも粘度が異なる検体であってもこれらを区別して正確に血液凝固検査ができるようにすることを目的とする。 The present invention has been made to solve the above-described problems, and is capable of accurately performing a blood coagulation test by distinguishing samples having the same coagulation ability but different viscosities. Objective.
本発明に係る血液凝固検査方法は、流路の延在方向に直列に配列して隣り合う部分が接触して流路を流れる状態に緩衝液および血漿を含む検体を流路に導入する第１工程と、流路を流れる流体の流速を測定して第１流速値を得る第２工程と、流路に新たに凝固活性剤を供給し、検体および凝固活性剤が、流路の延在方向に直列に配列して隣り合う部分が接触して第１工程と同じ状態で流路を流れる状態とする第３工程と、凝固活性剤を供給した後で、流路を流れる流体の流速を測定して第２流速値を得る第４工程と、第１流速値で第２流速値を除することで規格化した値により検体の血液凝固能を判定する第５工程とを備える。 The blood coagulation test method according to the present invention is a first method for introducing a specimen containing a buffer solution and plasma into a flow path in a state where adjacent portions are in contact with each other and flow through the flow path in series in the extending direction of the flow path. A step, a second step of measuring the flow velocity of the fluid flowing in the flow path to obtain a first flow velocity value, a new coagulation activator being supplied to the flow path, and the specimen and the coagulation activator being in the direction in which the flow path extends A third step in which the adjacent parts are arranged in series and flow through the flow path in the same state as the first step, and after supplying the coagulation activator, the flow velocity of the fluid flowing through the flow path is measured. The fourth step of obtaining the second flow rate value and the fifth step of determining the blood coagulation ability of the specimen based on the value normalized by dividing the second flow rate value by the first flow rate value.
上記血液凝固検査方法において、流路に接続して設けられた吸引流路の毛管力により吸引することで流れる状態とすればよい。また、流路に接続して設けられた吸引ポンプにより吸引することで流れる状態としてもよい。 In the blood coagulation test method, the blood coagulation test method may be achieved by suctioning by the capillary force of a suction channel provided in connection with the channel. Moreover, it is good also as a state which flows by attracting | sucking with the suction pump provided connected to the flow path.
上記血液凝固検査方法において、流速は、表面プラズモン共鳴に基づく流速測定により測定すればよい。 In the blood coagulation test method, the flow rate may be measured by flow rate measurement based on surface plasmon resonance.
以上説明したことにより、本発明によれば、凝固能が同じでも粘度が異なる検体であってもこれらを区別して正確に血液凝固検査ができるようになるという優れた効果が得られる。 As described above, according to the present invention, it is possible to obtain an excellent effect that a blood coagulation test can be accurately performed by distinguishing samples having the same coagulation ability but different viscosities.
以下、本発明の実施の形態について図を参照して説明する。図１は、本発明の実施の形態における血液凝固検査方法を説明するフローチャートである。この方法は、まず、ステップＳ１０１で、流路の延在方向に直列に配列して隣り合う部分が接触して流路を流れる状態に緩衝液および血漿を含む検体を流路に導入する（第１工程）。次に、ステップＳ１０２で、流路を流れる流体の流速を測定して第１流速値を得る（第２工程）。 Hereinafter, embodiments of the present invention will be described with reference to the drawings. FIG. 1 is a flowchart for explaining a blood coagulation test method according to an embodiment of the present invention. In this method, first, in step S101, a specimen containing a buffer solution and plasma is introduced into the flow path in a state where the adjacent portions are arranged in series in the flow path extending direction and flow through the flow path (first step). 1 step). Next, in step S102, the flow velocity of the fluid flowing through the flow path is measured to obtain a first flow velocity value (second step).
次に、ステップＳ１０３で、上記流路に新たに凝固活性剤を供給し、検体および凝固活性剤が、流路の延在方向に直列に配列して隣り合う部分が接触して第１工程と同じ状態で流路を流れる状態とする（第３工程）。このようにして凝固活性剤を供給した後で、ステップＳ１０４で、流路を流れる流体の流速を測定して第２流速値を得る（第４工程）。以上のように、第１流速値および第２流速値を得たら、ステップＳ１０５で、第１流速値で第２流速値を規格化した値により検体の血液凝固能を判定する（第５工程）。 Next, in step S103, a coagulation activator is newly supplied to the flow path, and the specimen and the coagulation activator are arranged in series in the extending direction of the flow path and the adjacent portions come into contact with the first process. It is set as the state which flows through a flow path in the same state (3rd process). After supplying the coagulation activator in this way, in step S104, the flow velocity of the fluid flowing through the flow path is measured to obtain a second flow velocity value (fourth step). As described above, when the first flow velocity value and the second flow velocity value are obtained, in step S105, the blood coagulation ability of the specimen is determined based on the value obtained by normalizing the second flow velocity value with the first flow velocity value (fifth step). .
上述したように、実施の形態によれば、凝固活性剤を加えていない状態で測定した結果得られた第１流速値で、凝固活性剤を加えた状態で測定した第２流速値を規格化するようにしたので、検体（血漿）における粘性の影響を排除することができる。この結果、実施の形態によれば、凝固能が同じでも粘度が異なる検体であってもこれらを区別して正確に血液凝固検査ができるようになる。 As described above, according to the embodiment, the first flow rate value obtained as a result of measurement without adding the coagulation activator is normalized to the second flow rate value measured with the coagulation activator added. As a result, the influence of viscosity on the specimen (plasma) can be eliminated. As a result, according to the embodiment, it is possible to accurately perform a blood coagulation test by distinguishing between samples having the same coagulation ability but different viscosities.
次に、より詳細に説明する。まず、流速を測定するために用いる流路について説明する。測定には、図２に示すようなフローセルを用いればよい。図２は、フローセルの構成を示す平面図（ａ）および断面図（ｂ）である。このフローセルは、まず、ガラスなどの光を透過する下部基板２０１ａと、下部基板２０１ａの上に配置される上部基板２０２と、下部基板２０１ａと上部基板２０２との間に配置されるスペーサ部２０１ｂとを備える。 Next, it demonstrates in detail. First, the flow path used for measuring the flow velocity will be described. For the measurement, a flow cell as shown in FIG. 2 may be used. FIG. 2 is a plan view (a) and a cross-sectional view (b) showing the configuration of the flow cell. The flow cell includes a lower substrate 201a that transmits light such as glass, an upper substrate 202 that is disposed on the lower substrate 201a, and a spacer portion 201b that is disposed between the lower substrate 201a and the upper substrate 202. Is provided.
下部基板２０１ａは、例えば、ＢＫ７ガラスから構成することができる。また、下部基板２０１ａは、透明なプラスチックから構成してもよい。また、スペーサ部２０１ｂは、両面が接着面とされた樹脂のフィルムから構成されている。この場合、下部基板２０１ａ，スペーサ部２０１ｂ，上部基板２０２を重ねて接続することで、各々が接着固定されるものとなる。 The lower substrate 201a can be made of, for example, BK7 glass. Further, the lower substrate 201a may be made of a transparent plastic. The spacer portion 201b is made of a resin film whose both surfaces are adhesive surfaces. In this case, the lower substrate 201a, the spacer portion 201b, and the upper substrate 202 are overlapped and connected to each other to be bonded and fixed.
下部基板２０１ａは、例えば、平面形状が１６ｍｍ×１６ｍｍで板厚が１ｍｍとされている。スペーサ部２０１ｂおよび上部基板２０２も、平面視の外形が１６ｍｍ×１６ｍｍとされている。また、上部基板２０２は、板厚３ｍｍ程度とされている。 For example, the lower substrate 201a has a planar shape of 16 mm × 16 mm and a plate thickness of 1 mm. The spacer portion 201b and the upper substrate 202 also have an outer shape in plan view of 16 mm × 16 mm. Further, the upper substrate 202 has a thickness of about 3 mm.
上部基板２０２およびスペーサ部２０１ｂには、試料溶液が導入される導入口２０３が形成されている。導入口２０３は、上部基板２０２およびスペーサ部２０１ｂを貫通して形成されている。また、上部基板２０２と下部基板２０１ａとの間には、試料溶液が移送される流路２０４が配置されている。また、流路２０４の一端は、導入口２０３に接続している。流路２０４は、スペーサ部２０１ｂに形成された溝部により構成される。流路２０４は、例えば、幅１ｍｍ、高さ１０〜１００μｍ程度に形成されている。 In the upper substrate 202 and the spacer portion 201b, an introduction port 203 into which a sample solution is introduced is formed. The introduction port 203 is formed through the upper substrate 202 and the spacer portion 201b. In addition, a flow path 204 for transferring the sample solution is disposed between the upper substrate 202 and the lower substrate 201a. One end of the flow path 204 is connected to the introduction port 203. The flow path 204 is configured by a groove portion formed in the spacer portion 201b. The flow path 204 is formed with a width of 1 mm and a height of about 10 to 100 μm, for example.
また、このフローセルは、下部基板２０１ａの上部基板２０２側表面に、金属薄膜２０９が形成されている。金属薄膜２０９は、例えば、下部基板２０１ａ側のＴｉ層とチタン層の上に形成されたＡｕ層とから構成されている。例えば、よく知られたスパッタ法や真空蒸着法などにより、下地基板２０１ａの上に上記金属を堆積することで、金属薄膜２０９が形成できる。下部基板２０１ａの全域に金属薄膜２０９が形成されているので、流路２０４の下面（下部基板２０１ａ側）は、金属表面となる。 In the flow cell, a metal thin film 209 is formed on the surface of the lower substrate 201a on the upper substrate 202 side. The metal thin film 209 is composed of, for example, a Ti layer on the lower substrate 201a side and an Au layer formed on the titanium layer. For example, the metal thin film 209 can be formed by depositing the metal on the base substrate 201a by a well-known sputtering method, vacuum deposition method, or the like. Since the metal thin film 209 is formed over the entire area of the lower substrate 201a, the lower surface of the flow path 204 (on the lower substrate 201a side) is a metal surface.
また、フローセルには、吸引流路２０６が形成され、吸引流路２０６には、流路２０４の他端が接続している。吸引流路２０６は、フローセルの流路２０４の両脇に展開して配置され、流路２０４の両脇の各々において、流路２０４に接続する連結部２０６ａおよび主吸引部２０６ｂを備える。導入口２０３と吸引流路２０６（連結部２０６ａ）とが、流路２０４により連通している。 Further, a suction channel 206 is formed in the flow cell, and the other end of the channel 204 is connected to the suction channel 206. The suction flow path 206 is disposed so as to be developed on both sides of the flow path 204 of the flow cell, and includes a connecting portion 206 a and a main suction portion 206 b connected to the flow path 204 on each side of the flow path 204. The introduction port 203 and the suction channel 206 (connecting portion 206a) communicate with each other through the channel 204.
また、吸引流路２０６の形成領域の上部基板２０２には、複数の貫通孔２０７が形成されている。貫通孔２０７は、例えば円筒形状（円管）である。例えば、連結部２０６ａにおいては、連結部２０６ａの幅方向には１つの貫通孔２０７が配置され、主吸引部２０６ｂにおいては、下部基板２０１ａ（上部基板２０２）の平面方向に、複数の貫通孔２０７が２次元的に配列されている。下部基板２０１ａと上部基板２０２の対向する方向に垂直な方向の吸引流路２０６の幅は、流路２０４より広く形成され、この幅方向に、複数の貫通孔２０７の列が配置され、この列が、幅方向に垂直な流路方向に配列されている。このような構造の上部基板２０２は、例えば、ポリジメチルシロキサンやアクリル樹脂などから形成すればよい。 A plurality of through holes 207 are formed in the upper substrate 202 in the formation region of the suction flow path 206. The through hole 207 has, for example, a cylindrical shape (circular tube). For example, in the connecting portion 206a, one through hole 207 is disposed in the width direction of the connecting portion 206a, and in the main suction portion 206b, a plurality of through holes 207 are provided in the planar direction of the lower substrate 201a (upper substrate 202). Are arranged two-dimensionally. The width of the suction channel 206 in the direction perpendicular to the direction in which the lower substrate 201a and the upper substrate 202 face each other is formed wider than the channel 204, and a plurality of through holes 207 are arranged in the width direction. Are arranged in the channel direction perpendicular to the width direction. The upper substrate 202 having such a structure may be formed from, for example, polydimethylsiloxane or acrylic resin.
ここで、流路２０４は、液体に対して毛細管現象が発現する範囲の断面寸法とされ、同様に、貫通孔２０７は、液体に対して毛細管現象が発現する範囲の管径とされている。また、吸引流路２０６の下部基板２０１ａおよび上部基板２０２の対向する方向（上下方向）の間隔は、流路２０４より液体が浸入したときに、上下方向に隙間が形成されない範囲とされている。言い換えると、浸入した液体が、吸引流路２０６の上下の両面に同時に接触可能な状態とされていればよい。 Here, the flow path 204 has a cross-sectional dimension in a range where a capillary phenomenon occurs with respect to the liquid, and similarly, the through hole 207 has a tube diameter within a range where the capillary phenomenon appears with respect to the liquid. In addition, the interval in the direction (vertical direction) in which the lower substrate 201a and the upper substrate 202 face each other in the suction channel 206 is set to a range in which no gap is formed in the vertical direction when liquid enters the channel 204. In other words, it is only necessary for the liquid that has entered to be in contact with both the upper and lower surfaces of the suction channel 206 at the same time.
上述したフローセルによれば、導入口２０３より導入された試料溶液は、毛細管現象により流路２０４を流れて吸引流路２０６に浸入する。吸引流路２０６に到達した試料溶液は、今度は、貫通孔２０７の毛細管現象により吸い上げられる。このように、導入口２０３に導入された試料溶液は、吸引流路２０６における複数の貫通孔２０７に吸い上げられることで、流路２０４を所定の流速で吸引流路２０６の方向に流れていくことになる。 According to the flow cell described above, the sample solution introduced from the introduction port 203 flows through the flow path 204 by capillary action and enters the suction flow path 206. The sample solution that has reached the suction channel 206 is then sucked up by the capillary phenomenon of the through-hole 207. As described above, the sample solution introduced into the introduction port 203 is sucked into the plurality of through holes 207 in the suction channel 206 and flows in the direction of the suction channel 206 at a predetermined flow rate. become.
上記フローセルによれば、複数の貫通孔２０７が設けられている吸引流路２０６が、導入口２０３より導入される試料溶液を、所定の流速（流量）で流路２０４に流すための吸引ポンプとして機能する。流路２０４が、測定領域となる。また、このフローセルによれば、流路２０４の側方（両側）に、吸引流路２０６（主吸引部２０６ｂ）が展開して配置されているようにしたので、フローセル全体の面積を大きく広げることなく、吸引流路２０６の領域を拡大した状態となっている。 According to the flow cell, the suction flow path 206 provided with a plurality of through holes 207 serves as a suction pump for flowing the sample solution introduced from the introduction port 203 into the flow path 204 at a predetermined flow rate (flow rate). Function. The flow path 204 becomes a measurement region. Further, according to this flow cell, since the suction flow path 206 (main suction portion 206b) is deployed and arranged on the side (both sides) of the flow path 204, the area of the entire flow cell is greatly expanded. The area of the suction flow path 206 is enlarged.
次に、上述したフローセルを用いた血液凝固検査方法について図３を用いて説明する。なお、図３では、フローセル２００の導入口２０３，流路２０４，吸引流路２０６を、仮想的に直線状に配列した状態として示している。 Next, a blood coagulation test method using the above-described flow cell will be described with reference to FIG. In FIG. 3, the inlet 203, the flow path 204, and the suction flow path 206 of the flow cell 200 are shown as being virtually arranged in a straight line.
はじめに、導入口２０３に緩衝液としてオーレンベロナール緩衝液を１μＬ滴下する。この結果、図３の（ａ）に示すように、緩衝液２１１が、毛細管力によって流路２０４に導入されて流れていき、流路２０４を充填して吸引流路２０６に到達し、複数の貫通孔に吸い上げられていく。この後、ある程度の量の緩衝液２１１が、複数の貫通孔に吸い上げられたところで全体として釣り合い、流れが停止する。この結果、流路２０４が緩衝液２１１で充填された状態となる。 First, 1 μL of auren veronal buffer solution is dropped into the introduction port 203 as a buffer solution. As a result, as shown in FIG. 3A, the buffer 211 is introduced into the flow path 204 by the capillary force and flows, fills the flow path 204, reaches the suction flow path 206, and has a plurality of It is sucked up by the through hole. Thereafter, a certain amount of the buffer solution 211 is balanced as a whole when the buffer solution 211 is sucked into the plurality of through holes, and the flow stops. As a result, the flow path 204 is filled with the buffer solution 211.
次に、図３の（ｂ）に示すように、７５％に希釈した血漿を含む検体２１２を、スポイト２０５により導入口２０３に１μＬ滴下する。このようにして新たに導入口２０３に検体２１２が供給されたことにより、吸引流路２０６の複数の貫通孔により緩衝液２１１が吸い上げられ、図３の（ｃ）に示すように、流路２０４の延在方向に、緩衝液２１１および検体２１２が直列に配列して隣り合う部分３２１が接触する状態で、流路２０４を流れる状態となる。この状態で、流速を測定して第１流速値を得る。 Next, as shown in FIG. 3B, 1 μL of a specimen 212 containing plasma diluted to 75% is dropped into the inlet 203 by the dropper 205. As the sample 212 is newly supplied to the introduction port 203 in this way, the buffer solution 211 is sucked up by the plurality of through holes of the suction channel 206, and as shown in FIG. In the extending direction, the buffer solution 211 and the specimen 212 are arranged in series, and the adjacent portion 321 is in contact with each other, so that the flow path 204 flows. In this state, the flow velocity is measured to obtain the first flow velocity value.
例えば、フローセル２００は、流路２０４の下側に金属薄膜２０９を備えており、図４に示すような表面プラズモン共鳴測定装置（ＳＰＲ）を用いることで流速が測定できる。この装置は、光源４０１，偏光板４０２，入射側レンズ４０３，プリズム４０４，測定面４０５，光検出部４０６，データ処理部４０７を備える。光源４０１は、例えば、発光ダイオードから構成すればよい。また、偏光板４０２は、ｐ偏光を通過させる。 For example, the flow cell 200 includes a metal thin film 209 below the flow path 204, and the flow velocity can be measured by using a surface plasmon resonance measuring apparatus (SPR) as shown in FIG. This apparatus includes a light source 401, a polarizing plate 402, an incident side lens 403, a prism 404, a measurement surface 405, a light detection unit 406, and a data processing unit 407. The light source 401 may be composed of a light emitting diode, for example. The polarizing plate 402 allows p-polarized light to pass through.
測定においては、フローセル２００の下面（下部基板２０１ａ）を測定面４０５に設置する。プリズム４０４の測定面４０５と、フローセル２００との間には、マッチングオイル（不図示）を配置する。また、光源４０１からの光の光軸に、測定領域が重なるようにフローセル２００を載置する。 In the measurement, the lower surface (lower substrate 201a) of the flow cell 200 is placed on the measurement surface 405. Matching oil (not shown) is disposed between the measurement surface 405 of the prism 404 and the flow cell 200. In addition, the flow cell 200 is placed so that the measurement region overlaps the optical axis of the light from the light source 401.
測定においては、まず、光源４０１から出射された光を、偏光板４０２を通過させて入射側レンズ４０３で集光してプリズム４０４に入射させ、プリズム４０４の測定面４０５に密着させているフローセル２００の測定領域に照射する。図２，図３を用いて説明したように、フローセル２００の測定領域となる流路２０４には金属薄膜２０９が形成されており、金属薄膜２０９の裏面に、フローセル２００を透過してきた集光光が照射される。 In the measurement, first, the flow cell 200 in which the light emitted from the light source 401 passes through the polarizing plate 402, is collected by the incident side lens 403, enters the prism 404, and is in close contact with the measurement surface 405 of the prism 404. Irradiate the measurement area. As described with reference to FIGS. 2 and 3, the metal thin film 209 is formed in the flow path 204 serving as the measurement region of the flow cell 200, and the condensed light transmitted through the flow cell 200 on the back surface of the metal thin film 209. Is irradiated.
このようにして照射された集光光は、金属薄膜２０９の裏面で反射し、いわゆるＣＣＤイメージセンサなどの撮像素子よりなる光検出部４０５で光電変換されて強度（光強度）が得られる。このようにして得られた強度においては、流速測定対象の流体が接触した金属薄膜２０９の表面における、エバネッセント波と表面プラズモン波との共鳴が起こる角度で反射率が低くなる谷が観測される。この共鳴が起こる表面プラズモン共鳴角度は、金属薄膜２０９に接する測定対象の流体の屈折率に依存するため、光強度の変化により、屈折率の変化が求められる。データ処理部４０７では、上述した屈折率の変化から、流路２０４の測定領域を流れる流体の流速を算出する。得られた第１流速値は、７．５ｍｍ／ｓであった。 The condensed light irradiated in this manner is reflected by the back surface of the metal thin film 209 and is photoelectrically converted by the light detection unit 405 including an imaging element such as a so-called CCD image sensor to obtain intensity (light intensity). In the intensity obtained in this way, a trough whose reflectance decreases at an angle at which resonance between the evanescent wave and the surface plasmon wave occurs on the surface of the metal thin film 209 in contact with the fluid to be measured for flow velocity. Since the surface plasmon resonance angle at which this resonance occurs depends on the refractive index of the fluid to be measured in contact with the metal thin film 209, a change in the refractive index is required due to a change in the light intensity. The data processing unit 407 calculates the flow velocity of the fluid flowing through the measurement region of the flow path 204 from the above-described change in refractive index. The obtained first flow velocity value was 7.5 mm / s.
上述した流路２０４を緩衝液２１１および検体２１２が流れる状態は、例えば、図３の（ｄ）に示すように、検体２１２が流路２０４を充填して吸引流路２０６に到達し、ある程度、複数の貫通孔に吸い上げられたところで釣り合い、停止する。 The state in which the buffer solution 211 and the specimen 212 flow through the flow path 204 described above is, for example, as shown in FIG. 3D, the specimen 212 fills the flow path 204 and reaches the suction flow path 206. It balances and stops when it is sucked up by multiple through holes.
次に、図３の（ｅ）に示すように、凝固活性剤２１３を、スポイト２０５により導入口２０３に滴下する。このようにして新たに導入口２０３に凝固活性剤２１３が供給されたことにより、吸引流路２０６の複数の貫通孔により緩衝液２１１および一部の検体２１２が吸い上げられ、図３の（ｆ）に示すように、流路２０４の延在方向に、検体２１２および凝固活性剤２１３が直列に配列して隣り合う部分３２２が接触する状態で、流路２０４を流れる状態となる。 Next, as shown in (e) of FIG. 3, the coagulation activator 213 is dropped into the introduction port 203 by the dropper 205. As the coagulation activator 213 is newly supplied to the introduction port 203 in this way, the buffer solution 211 and a part of the sample 212 are sucked up by the plurality of through holes of the suction channel 206, and (f) of FIG. As shown in FIG. 4, the specimen 212 and the coagulation activator 213 are arranged in series in the extending direction of the flow path 204, and the state where the adjacent portions 322 are in contact with each other is in a state of flowing through the flow path 204.
この凝固活性剤２１３を加えた段階での流路２０４における流れは、吸引流路２０６における毛細管力により発生させており、図３の（ｃ）を用いて説明した検体２１２を加えた段階と、同じフローセルの同じ流路２０４における状態である。従って、凝固活性剤２１３を加えた段階での流路２０４における流れは、検体２１２を加えた段階での流路２０４における流れと、同じ状態（条件）となる。 The flow in the channel 204 at the stage of adding the coagulation activator 213 is generated by the capillary force in the suction channel 206, and the stage of adding the specimen 212 described with reference to (c) of FIG. This is a state in the same flow path 204 of the same flow cell. Therefore, the flow in the flow path 204 when the coagulation activator 213 is added is in the same state (condition) as the flow in the flow path 204 when the sample 212 is added.
ここで、凝固活性剤２１３が検体２１２に部分３２２で接触した時点で凝固反応が開始し、この後、凝固反応を起こしながら部分３２２は流路２０４を吸引流路２０６の方向に移動していく。この移動の速度が第２流速値として測定されるものとなる。 Here, the coagulation reaction starts when the coagulation activator 213 comes into contact with the specimen 212 at the portion 322, and then the portion 322 moves in the direction of the suction channel 206 through the channel 204 while causing the coagulation reaction. . The speed of this movement is measured as the second flow velocity value.
この状態で、前述同様に表面プラズモン共鳴測定装置により流速を測定して第２流速値を得る。測定の結果得られた第２流速値は、６．０ｍｍ／ｓであった。なお、流路２０４を検体２１２および凝固活性剤２１３が流れる状態は、例えば、図３の（ｆ）に示すように、検体２１２が、ある程度、複数の貫通孔に吸い上げられ、一方で、導入口２０３における凝固活性剤２１３がほぼなくなったところで全体が釣り合い、停止する。 In this state, the flow velocity is measured by the surface plasmon resonance measuring device as described above to obtain the second flow velocity value. The second flow velocity value obtained as a result of the measurement was 6.0 mm / s. Note that the state in which the specimen 212 and the coagulation activator 213 flow through the flow path 204 is, for example, as shown in FIG. 3F, the specimen 212 is sucked up to a plurality of through holes to some extent. When the coagulation activator 213 in 203 is almost gone, the whole balances and stops.
以上のようにして得られた検体のみのときの第１流速値（７．５ｍｍ／ｓ）で、凝固反応中の第２流速値（６．０ｍｍ／ｓ）を規格化すると０．８となる。この規格化した値を凝固検査の指標として使用することで、検体（血漿）における粘性の影響を排除した状態で、検体の血液凝固能を判定することができる。 When the first flow rate value (7.5 mm / s) for only the specimen obtained as described above is normalized, the second flow rate value (6.0 mm / s) during the coagulation reaction is normalized to 0.8. . By using this normalized value as an index for the coagulation test, the blood coagulation ability of the sample can be determined in a state where the influence of the viscosity on the sample (plasma) is eliminated.
以上に説明したように、本発明によれば、緩衝液および血漿を含む検体が流路を流れる時の第１流速値で、検体に凝固活性剤を加えた状態で流路を流れる時の第２流速値を規格化するようにしたので、凝固能が同じでも粘度が異なる検体であってもこれらを区別して正確に血液凝固検査ができるようになる。 As described above, according to the present invention, the first flow velocity value when the specimen containing the buffer solution and plasma flows through the flow path is the first flow velocity value when flowing through the flow path with the coagulant activator added to the specimen. Since the two flow rate values are standardized, even blood samples having the same coagulation ability but different viscosities can be distinguished from each other to accurately perform a blood coagulation test.
なお、本発明は以上に説明した実施の形態に限定されるものではなく、本発明の技術的思想内で、当分野において通常の知識を有する者により、多くの変形および組み合わせが実施可能であることは明白である。例えば、流速は、表面プラズモン共鳴測定装置を用いた測定に限るものではない。 The present invention is not limited to the embodiment described above, and many modifications and combinations can be implemented by those having ordinary knowledge in the art within the technical idea of the present invention. It is obvious. For example, the flow velocity is not limited to measurement using a surface plasmon resonance measurement apparatus.
例えば、フローセルを透明な材料で構成すれば、流路を流れる流体の状態を、ビデオカメラなどにより撮像した結果により、流速測定を行うことができる。例えば、図２を用いて説明したフローセルの吸引流路２０６における貫通孔２０７を上昇する液面の移動状態をビデオカメラで撮像すれば、この移動距離と移動に要した時間とを求めることができる。この移動距離と移動に要した時間とから、流速を求めることができる。 For example, if the flow cell is made of a transparent material, the flow velocity can be measured based on the result of imaging the state of the fluid flowing through the flow path with a video camera or the like. For example, if the moving state of the liquid level rising through the through hole 207 in the suction flow path 206 of the flow cell described with reference to FIG. 2 is imaged with a video camera, the moving distance and the time required for the movement can be obtained. . The flow velocity can be obtained from the moving distance and the time required for the movement.
また、上述では、毛細管力を用いた吸引により、流路に流れる状態を形成するようにしたが、これに限るものではなく、流路に吸引ポンプを接続して用いて流れる状態を形成するようにしてもよい。この場合、吸引ポンプの吸引力を、測定の間は一定の状態としておけばよい。 Further, in the above description, the state of flowing in the flow path is formed by suction using capillary force. However, the present invention is not limited to this, and a state of flowing using a suction pump connected to the flow path is formed. It may be. In this case, the suction force of the suction pump may be kept constant during the measurement.
２０１ａ…下部基板、２０１ｂ…スペーサ部、２０２…上部基板、２０３…導入口、２０４…導入口、２０６…吸引流路、２０６ａ…連結部、２０６ｂ…主吸引部、２０７…貫通孔、２０９…金属薄膜。 201a ... lower substrate, 201b ... spacer portion, 202 ... upper substrate, 203 ... introduction port, 204 ... introduction port, 206 ... suction channel, 206a ... coupling portion, 206b ... main suction portion, 207 ... through hole, 209 ... metal Thin film.
Claims (4)
前記流路を流れる流体の流速を測定して第１流速値を得る第２工程と、
前記流路に新たに凝固活性剤を供給し、前記検体および前記凝固活性剤が、前記流路の延在方向に直列に配列して隣り合う部分が接触して前記第１工程と同じ状態で前記流路を流れる状態とする第３工程と、
前記凝固活性剤を供給した後で、前記流路を流れる流体の流速を測定して第２流速値を得る第４工程と、
前記第１流速値で前記第２流速値を除することで規格化した値により前記検体の血液凝固能を判定する第５工程と
を備えることを特徴とする血液凝固検査方法。 A first step of introducing a sample containing a buffer solution and plasma into the flow path in a state in which adjacent portions are arranged in series in the extending direction of the flow path and contact each other and flow through the flow path;
A second step of obtaining a first flow velocity value by measuring a flow velocity of the fluid flowing through the flow path;
The coagulation activator is newly supplied to the flow path, and the specimen and the coagulation activator are arranged in series in the extending direction of the flow path and the adjacent portions are in contact with each other in the same state as in the first step. A third step for causing the flow path to flow;
A fourth step of obtaining a second flow velocity value by measuring the flow velocity of the fluid flowing through the flow path after supplying the coagulation activator;
And a fifth step of determining the blood coagulation ability of the specimen by a value normalized by dividing the second flow velocity value by the first flow velocity value.
前記流路に接続して設けられた吸引流路の毛管力により吸引することで前記流れる状態とすることを特徴とする血液凝固検査方法。 The blood coagulation test method according to claim 1,
A blood coagulation test method, wherein the flow state is achieved by suction by a capillary force of a suction channel provided in connection with the channel.
前記流路に接続して設けられた吸引ポンプにより吸引することで前記流れる状態とすることを特徴とする血液凝固検査方法。 The blood coagulation test method according to claim 1,
A blood coagulation test method, wherein the flow state is achieved by suction with a suction pump connected to the flow path.
前記流速は、表面プラズモン共鳴に基づく流速測定により測定することを特徴とする血液凝固検査方法。 In the blood coagulation test method according to any one of claims 1 to 3,
The blood coagulation test method is characterized in that the flow velocity is measured by flow velocity measurement based on surface plasmon resonance.
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