JP3876022B2 - Method for quantifying substances that affect blood clotting time - Google Patents

Description

*いずれも血液凝固時間に影響を与える物質を含んでいない血漿におけるAPTT値であるが、ヘパリン、低分子ヘパリンの実験を行った日とデルマタン硫酸の実験を行った日が異なるため、これらの値に違いがある。従って、血液凝固時間には、使用する検体（血漿等）による相違が生じる。本発明は、また、このような検体による誤差をなくす定量方法も提供する（実施例２）。
【００６３】
上記物質を用いて得たAPTT値を用いて数値の変換を行い、数値の多項式回帰法による回帰により検量線の作成を行った。即ち、下記手順に従い数値の変換(Logit-log変換)、回帰を行った。
1)被験測定対象物質濃度(x)を常用対数値に変換する。(X=Logx)
2)各被験測定対象物質濃度(x)におけるAPTT値(T)に対する、被験当該物質の未添加時(0濃度)のAPTT値(T₀)の比率(Tr)を計算する(Tr=(T₀/T)×K;K=1.0)。
3)Trの値をロジット(Logit)変換する。本実施例は凝固時間を延長するケースであるので1>Trとなり、Y= Ln(Tr/(1-Tr))となる。従って、本実施例の場合は、Ln(T₀/T)/(1-T₀/T) = Ln(T₀/T-T₀) = Ln((T-T₀)/T₀)^-1 = - Ln(ΔAPTT/T₀)と変換される。ただしΔAPTT= T-T₀。
4)それぞれのX値と、それに対応するY値のデータを多項式回帰し、多項方程式で回帰曲線（一次式の場合は、直線）を算出する。Y=ΣC_kX^k、ただしK≧0、C_kは実数。
【００６４】
理論値は実際の検体中の測定対象物質濃度、計算値は回帰計算によって求めた検量線の式により該検体のAPTTの測定値から測定対象物質濃度を逆算した数値である。
【００６５】
測定によって得たAPTT値、算出された回帰曲線を示す多項式（一次、二次、三次、ただし、ヘパリンは一次、二次のみ）から得られた計算値、並びに理論値と計算値から得た真度(真度＝(計算値−理論値)/理論値×100%)の一覧を表２〜５（本発明方法）に示す。また上記の従来法で得た理論値を参考の為併記した。また、算出された回帰曲線を図７〜９（本発明方法）に示す。本実施例では、回帰式は三次式までを例示したが、四次以上の多項式での回帰曲線も用いることが可能である。
【００６６】
上記回帰曲線（検量線）を用いてAPTT値から未知当該物質濃度を求める場合は、未知濃度の当該測定対象物質を含む検体のAPTT値を測定しYを算出し、これを得られた多項式に代入しXを得、10^Xを計算し、最終的に未知濃度xを得る。表２〜５の結果から明らかなように、本発明によれば、広い濃度範囲でよい真度が得られる。
【００６７】
【表２】

従来法：Y=0.495X+1.311（但し、この場合のY=log（検体のAPTT値）、X=log（検体中のヘパリン濃度）（図４）
【００６８】
【表３】

従来法：Y=0.303X+1.217 （但し、この場合のY=log（検体のAPTT値）、X=log（検体中の低分子ヘパリン濃度）（図５）
【００６９】
【表４】

【表５】

従来法：Y=0.253X+0.898 （但し、この場合のY=log（検体のAPTT値）、X=log（検体中のデルマタン硫酸濃度）（図６）
【００７０】
【実施例２】
（本発明方法によるデルマタン硫酸の定量）
血漿の加熱処理がAPTTにおよぼす影響と、ラット血漿あるいは標準血漿（コアグトロール１）を血液凝固因子供給源として使用した影響を調べた。測定対象物質としてはデルマタン硫酸を用いた。
【００７１】
血漿中の血液凝固因子量の検体差がAPTTの測定値に影響し、さらに定量値に影響することが、実施例１の表１において使用した血漿が異なる検体であったためにグリコサミノグリカンの濃度が０のときの凝固時間が相違していることからも明らかであるため（表１参照）、デルマタン硫酸を含有する検体中の血液凝固因子を失活させ、APTT測定に使用する血液凝固因子は別に添加した。血液凝固因子の失活の手法として、サンプルの加熱処理を試みた。加熱処理のAPTTに対する影響を、デルマタン硫酸濃度0及び250μg/mlにおいて検討した。その後、血液凝固因子の供給源として、コアグトロール１及びラット血漿を使用した。
【００７２】
加熱処理による影響は、ラットから実施例１の方法と同様に採取した血漿に、生理食塩水に溶解したデルマタン硫酸を最終容量の1/10添加し、この状態での最終濃度を0及び250μg/mlとし、これを検体とした。加熱処理検体は、65℃で20分間加熱し、実施例１と同様のAPTT試薬とカルシウム溶液を100μlずつ添加してそれぞれのAPTT値を実施例１と同様に血液凝固自動測定装置で測定した。非加熱処理検体のデルマタン硫酸濃度0、250μl/mlの検体において、それぞれのAPTT値は14.2秒及び38.1秒であったが、加熱処理を行った検体は、いずれのデルマタン硫酸濃度において少なくとも120秒の間で凝固は認められなかった（表６）。
【００７３】
一方、加熱処理の後に血液凝固因子を別途添加した場合のAPTT値の変化は、上記加熱検体20μlに説明書に従って調製したコアグトロール１を80μl添加し、上記方法と同様にAPTT試薬とカルシウム溶液を添加し、APTT値を実施例１と同様に血液凝固自動測定装置で測定した。その結果、加熱処理を行った場合には凝固が起こらなかった検体で、コアグトロール１の添加によって凝固活性が復活し、さらにデルマタン硫酸によるAPTT値の延長作用が観察された（表６）。
【００７４】
また、データは示さないが、実施例１と同様の方法で用意されたラット血漿を上記コアグトロール１の代替として使用した場合にも上記結果と同様に凝固活性の復活とデルマタン硫酸によるAPTTの延長作用が起こることを確認している。
【００７５】
【表６】

【００７６】
【実施例３】
（本発明方法１と本発明方法２の組み合わせによるデルマタン硫酸の定量）
APTT法を用い、ラットの尿及び肝臓組織ホモジネイトを検体とした血液凝固時間を延長する物質の定量の検討を行った。測定対象物質としてデルマタン硫酸を用いた。
【００７７】
肝臓は、その一部(2.550g)に4倍量のリン酸緩衝液を添加し、氷冷下ポリトロン型ホモジナイザー(約15,000rpm)でホモジナイズし、その後、3,000rpm, 4℃の条件下において15分間遠心分離した上清を肝臓ホモジナイズサンプルとした。尿の場合は氷冷下で、ラットが一晩に排泄した尿を回収し、3,000rpm, 4℃の条件下において遠心分離した上清を尿サンプルとした。それぞれのサンプルは、-30℃で凍結保存した。
【００７８】
本発明方法２に従い不活化処理として融解後に56℃で20分間加熱処理した。生理食塩水に溶解したデルマタン硫酸をサンプルの全体量に影響を与えない少量添加し十分に攪拌して検体（肝臓ホモジネイト検体、尿検体）とした。各検体を20μl取り、コアグトロール１を80μl添加し、APTT測定に供試した。また、参考値として肝臓ホモジネイトサンプルや尿サンプルの代替として生理食塩水（生食）にデルマタン硫酸を各濃度で溶解した（対照生理食塩水検体）場合についてもAPTT測定を行い、本発明方法１に従って数値変換を行った（表７〜１０及び図１０〜１８）。
【００７９】
【表７】

【００８０】
【表８】

【００８１】
【表９】

【００８２】
【表１０】

【００８３】
【実施例４】
（本発明方法１及び本発明方法２の組み合わせによる各種GAGの定量）
実施例２及び３は測定検体の血液凝固因子を不活化し、デルマタン硫酸の量を測定したが、デルマタン硫酸以外の血液凝固時間を延長する物質が知られているので、実施例２と同様の熱処理（65℃、20分）では破壊されないと考えられるGAGであるヘパリン、低分子ヘパリン、コンドロイチン硫酸Eについても調べた。測定法は以下に記す方法で行った。
【００８４】
即ち、実施例１と同様にラット血漿を採取し、ラット血漿の全体量に影響を与えないごく少量の生理食塩水に溶解した各種GAGを各々加えた後、十分混和して検体とし、この検体を65℃で20分間加熱処理した（加熱処理検体）。加熱処理検体の20μlを80μlのコアグトロール１と混和後、実施例１と同様の方法によってそれぞれのAPTT値をデュプリケートで測定し、その平均を測定値とし、本発明方法１に従って数値変換した（表１１〜１４及び図１９〜２６）。
【００８５】
【表１１】

【００８６】
【表１２】

【００８７】
【表１３】

【００８８】
【表１４】

【００８９】
ヘパリンは活性単位で示されることが多いため、当該実施例中のヘパリンの例（表１２）について、標準検体のU/mlとして示される濃度の理論値と凝固時間の関係で示し、当該実施例中のヘパリンの例と同様に検量線の作成と曲線回帰を試みた（表１５および図２７、２８）。ただし、ヘパリンは、和光純薬工業（株）(Cat No.085-00134;182.5U/mg)を使用した。
【００９０】
【表１５】

【００９１】
【実施例５】
（本発明方法１によるスルファチドの定量）
血液凝固時間（APTT）を短縮させ得る物質について検討した。硫酸化糖脂質であるスルファチドをリン酸緩衝溶液に溶解し、該物質の濃度測定を本発明方法１で試みた。
【００９２】
スルファチドをリン酸緩衝溶液に種々の濃度で溶解し検体とし、この溶液20μlと標準血漿コアトロール1を80μlを混和し、実施例１と同様にAPTTをデュプリケートで測定し、平均値を測定値とし、本発明方法１に従って数値変換した（表１６〜１８及び図２９〜３５）。
【００９３】
【表１６】

【００９４】
スルファチドが0μg/mlの場合のAPTT平均値は32.3秒であった。図２９に示したように23.4μg/ml以上（区間A）では容量依存的にAPTTは延長した。一方11.7μg/ml以下、0.18μg/ml以上ではAPTTは短縮し、このうち、1.5μg/ml以下、 0.18μg/ml以上（区間B）ではこの短縮は容量逆相関性であった。そこで区間A、区間B各々についてLogit-log変換を行い、各々検量線を作成した。図３０〜３５に示した如く、延長区間のみならず、短縮区間でもこれらのデータは各々区間独立した多項式で表すことが可能である。
【００９５】
尚、本実施例ではスルファチドをリン酸緩衝溶液に溶かしたものそのままを用いているが、この物質は65℃、20分の処理や凍結融解の処理に安定である。従って、生体由来検体中のスルファチドを測定する場合、本発明方法２に従って不活化処理を行うことができる。
【００９６】
【表１７】

【００９７】
【表１８】

【００９８】
【実施例６】
（本発明方法１及び本発明方法２の組み合わせによるデルマタン硫酸の測定）
病態血漿中の血液凝固時間を延長する物質の濃度測定を病態血漿を用いて行った。病態モデルとして、ラット汎発性血管内凝固症候群（Disseminated Intravascular Coagulation, DIC）モデルを、測定対象物質としては、デルマタン硫酸を選び、下記の添加回収実験を行った。
【００９９】
ラットDICモデルの３匹の血漿は PCT国際公開 WO95/09188号公報の実施例４に従って調製した。すなわち、50mg リポポリサッカライド（LPS）/kg/4時間点滴静注後の病的血漿である。これらの検体及び正常ラットの血漿を実施例１と同様に採取して-20℃で保存し、融解後に実施例３と同様に少量の生食に溶解した種々の濃度のデルマタン硫酸を各々加えて測定検体とし、実施例２と同様に65℃で20分間加熱処理した（加熱処理検体）。これら加熱処理検体の各々20μlに80μlのコアグトロール１を添加し、APTT法を用いた定量を実施例３と同様に行った（表１９）。
【０１００】
【表１９】

【０１０１】
上記の如く、本法で測定された値は、DICモデル血漿及び正常血漿とも、きわめて近いことが示された。正常ラットで得られた標準検体のAPTT値を基に得られた三つの検量線の回帰式の各々に上記のDICモデル血漿（測定検体）のAPTT値をそれぞれ代入して計算値及び真度を算出した（表２０）。
【０１０２】
【表２０】

【０１０３】
上記に示したとおり、50〜100μg/mlの間では、測定値の信頼性がやや劣る傾向にあるが、それ以降では、実用的範囲で測定対象物質濃度が測定されていることが判明した。
【０１０４】
【実施例７】
（本発明方法１及び本発明方法２の組み合わせによる複数のGAGを含む検体の定量）
血液凝固時間に影響を与える物質が複数入った検体における定量を行った。測定対象物質の例としてデルマタン硫酸を、上記測定対象物質以外の血液凝固時間に影響を与える物質としてヘパリンを例にとり、測定対象物質の入る溶媒としては、実施例１と同様に採取したラット血漿を選び以下の実験を行った。デルマタン硫酸を0〜90mg/ml、ヘパリンを900μg/mlとなるように各々生理食塩水に溶解した。血漿2.7mlに生理食塩水0.15mlを加えたものに、0.15mlの種々の濃度のデルマタン硫酸を溶解した生理食塩水を添加したもの、及び血漿2.7mlにヘパリン、900μg/mlを含む生理食塩水0.15mlを加えたものに、0.15mlの種々の濃度のデルマタン硫酸を溶解した生理食塩水を加えたものをそれぞれ用意した（測定検体）。これらを65℃で20分間加熱処理した後、ヘパリンを加えた群には凍結乾燥された酵素（ヘパリナーゼ、0.1U 生化学工業（株）製）を加え、ヘパリンを分解した（酵素処理検体）。その後、これらの酵素処理検体を20μlとり、80μlのコアグトロール１に加え、実施例１と同様にAPTT値をデュプリケートで測定し、平均値を測定値とした（表２１）。
【０１０５】
【表２１】

【０１０６】
測定対象物質(デルマタン硫酸)のみを含有する検体,及び,測定対象物質及び測定対象物質以外で凝固時間に影響を与える物質(ヘパリン)双方を含有しない溶媒である生理食塩水のAPTT値でLogit-log変換を行い回帰直線あるいは回帰曲線を作成した（表２２）。
【０１０７】
【表２２】

【０１０８】
次に、測定対象物質（デルマタン硫酸）を含有し、更に血液凝固時間に影響を与える物質(ヘパリン)も含有する検体を酵素処理した後得たAPTT値を上記数式に各々代入して得た計算値と理論値を比較した（表２３）。
【０１０９】
【表２３】

【０１１０】
上記の表に示したごとく、 酵素処理後のサンプルの濃度は、1500μg/ml以上の範囲で、理論値と誤差21.7%以下の精度で測定できる事が判明した。
【０１１１】
【実施例８】
（本発明方法１及び本発明方法２の組み合わせによるヘパリンの定量）
APTTと同様にPTを応用した。測定対象物質の例としては、ヘパリンを用いた。ヘパリンを生理食塩水に種々の濃度で溶解後、ラット血漿に加えた（検体）。この時、加えた容量が、全体の血漿容量に影響しないよう配慮した。検体を65℃で20分間加熱処理した（加熱処理検体）。20μlの加熱処理検体を80μlのコアグトロール１と混和後、説明書通りに調整したPT試薬（トロンボチェックPT：ミドリ十字）を用いてPTを測定し、APTTの時と同様に測定値でLogit-log変換を行い回帰直線あるいは回帰曲線を作成した（表２４）。
【０１１２】
【表２４】

【０１１３】
【実施例９】
（本発明キットの構成及び使用法）
本実施例のヘパリン測定キットは次の１〜７の要素からなる。
１．標準ヘパリン生理食塩水（ヘパリン60μg/ml(10.95U/ml)、1ml）１バイアル。
２．生理食塩水（ヘパリン希釈用、1ml）１バイアル。
３．標準血漿（抗体検査の結果HIV, HCV, HBV等の病原微生物に感染が認められない健常人10人からクエン酸ナトリウムを抗凝固剤として採血して得た新鮮血漿を等量ずつ混和しプールしたもの、0.5ml相当の凍結乾燥品、通常は2〜8℃保存）１バイアル。
４．蒸留水（標準血漿溶解用、0.5ml）１バイアル。
５．APTT試薬（ウサギ由来セファリン0.2mg/ml、エラグ酸0.03mg/ml含有、3ml、通常は冷蔵保存）１バイアル。
６．塩化カルシウム溶液（0.02M、4ml）１バイアル。
７．測定用ソフトウェア記憶媒体
【０１１４】
使用に際しては標準血漿を室温に戻した後、蒸留水１バイアルを全量泡立てないように穏やかに加え、穏やかに均一となるように攪拌し、20〜30分間程度放置する。この溶解した標準血漿80μlに標準ヘパリン生理食塩水を生理食塩水で3〜60μg/mlの範囲で段階希釈した溶液あるいは生理食塩水を20μlずつ気泡を作らぬよう注意して均一に添加する。当該溶液を3分間、37℃に保温後、あらかじめ37℃に加熱しておいたAPTT試薬を100μlずつ添加し、更に37℃に3分間保温する。当該溶液に塩化カルシウム溶液を100μlずつ添加して凝固を開始させ、凝固が完了するまでの時間を測定する。ヘパリン濃度の理論値とそれぞれの凝固時間を測定後、当該理論値と測定値、さらに標準ヘパリン生理食塩水に含まれるヘパリンの活性単位(U/mg)と、回帰方法とを測定用ソフトウェアに入力して検量線の作成を行う。検体は56℃で20分間の加熱処理後室温に戻し、その20μlを上記手順により蒸留水に溶解した標準血漿80μlに気泡を作らぬよう均一に添加する。当該溶液を37℃で3分間保温後、あらかじめ37℃に加熱しておいたAPTT試薬を100μlずつ添加し、該溶液を37℃で3分間保温後、塩化カルシウム溶液を100μl添加して凝固を開始させる。凝固が完了するまでの時間を測定し、測定用ソフトウェアで作成した上記検量線をメモリから呼び出し、検体の凝固時間を入力し、当該測定値から検体中の測定対象物質濃度を算出する。
【０１１５】
【発明の効果】
本発明によれば、血液凝固時間に影響を与える物質の精度が高く、操作が簡便であり多数の検体を処理することが可能である定量方法が提供される。
【０１１６】
すなわち、本発明の定量方法の一態様によれば、既知濃度の測定対象物質溶液（標準検体）の濃度の対数値と、対照凝固時間に対する相対凝固時間の比((T-T₀)/T₀)のロジット（Logit）値とを変数として検量線を求めているので、広い測定濃度範囲で精度の高い定量を行うことが可能である。
【０１１７】
また、本発明の定量方法の別の態様によれば、測定対象物質を含む検体に内在する血液凝固因子を前記測定対象物質を破壊しない条件下で不活化しているので、検体中の測定対象物質以外の物質の影響を受けずに精度の高い定量を行うことが可能である。
【図面の簡単な説明】
【図１】本発明の定量装置の構成の一例を示すブロック図である。
【図２】本発明のキットに含まれる媒体に記録されたプログラムの処理手順を示すフローチャートである。
【図３】ヘパリン、低分子ヘパリンおよびデルマタン硫酸の濃度(μg/ml)の対数値とAPTTの関係を示す。
【図４】ヘパリンの濃度(μg/ml)の対数値とAPTTの対数値の関係を示す。
【図５】低分子ヘパリン(LMWHep)の濃度(μg/ml)の対数値とAPTTの対数値の関係を示す。
【図６】デルマタン硫酸(DS)の濃度(μg/ml)の対数値とAPTTの対数値の関係を示す。
【図７】ヘパリンの濃度(μg/ml)の対数値とAPTTのロジット値の関係を示す。
【図８】低分子ヘパリン(LMWHep)の濃度(μg/ml)の対数値とAPTTのロジット値の関係を示す。
【図９】デルマタン硫酸(DS)の濃度(μg/ml)の対数値とAPTTのロジット値の関係を示す。
【図１０】生理食塩水中のデルマタン硫酸(DS)濃度(μg/ml)の対数値とAPTTのロジット値の関係と検量線の回帰式（一次式）を示す。
【図１１】生理食塩水中のデルマタン硫酸(DS)濃度(μg/ml)の対数値とAPTTのロジット値の関係と検量線の回帰式（二次式）を示す。
【図１２】生理食塩水中のデルマタン硫酸(DS)濃度(μg/ml)の対数値とAPTTのロジット値の関係と検量線の回帰式（三次式）を示す。
【図１３】肝臓ホモジネイト中のデルマタン硫酸(DS)濃度(μg/ml)の対数値とAPTTのロジット値の関係と検量線の回帰式（一次式）を示す。
【図１４】肝臓ホモジネイト中のデルマタン硫酸(DS)濃度(μg/ml)の対数値とAPTTのロジット値の関係と検量線の回帰式（二次式）を示す。
【図１５】肝臓ホモジネイト中のデルマタン硫酸(DS)濃度(μg/ml)の対数値とAPTTのロジット値の関係と検量線の回帰式（三次式）を示す。
【図１６】尿中のデルマタン硫酸(DS)濃度(μg/ml)の対数値とAPTTのロジット値の関係と検量線の回帰式（一次式）を示す。
【図１７】尿中のデルマタン硫酸(DS)濃度(μg/ml)の対数値とAPTTのロジット値の関係と検量線の回帰式（二次式）を示す。
【図１８】尿中のデルマタン硫酸(DS)濃度(μg/ml)の対数値とAPTTのロジット値の関係と検量線の回帰式（三次式）を示す。
【図１９】血漿中のヘパリン濃度(μg/ml)の対数値とAPTTのロジット値の関係と検量線の回帰式（一次式）を示す。
【図２０】血漿中のヘパリン濃度(μg/ml)の対数値とAPTTのロジット値の関係と検量線の回帰式（二次式）を示す。
【図２１】血漿中の低分子ヘパリン(LMWHep)濃度(μg/ml)の対数値とAPTTのロジット値の関係と検量線の回帰式（一次式）を示す。
【図２２】血漿中の低分子ヘパリン(LMWHep)濃度(μg/ml)の対数値とAPTTのロジット値の関係と検量線の回帰式（二次式）を示す。
【図２３】血漿中の低分子ヘパリン(LMWHep)濃度(μg/ml)の対数値とAPTTのロジット値の関係と検量線の回帰式（三次式）を示す。
【図２４】血漿中のコンドロイチン硫酸E(CSE)濃度(μg/ml)の対数値とAPTTのロジット値の関係と検量線の回帰式（一次式）を示す。
【図２５】血漿中のコンドロイチン硫酸E(CSE)濃度(μg/ml)の対数値とAPTTのロジット値の関係と検量線の回帰式（二次式）を示す。
【図２６】血漿中のコンドロイチン硫酸E(CSE)濃度(μg/ml)の対数値とAPTTのロジット値の関係と検量線の回帰式（三次式）を示す。
【図２７】血漿中のヘパリン濃度(U/ml)の対数値とAPTTのロジット値の関係と検量線の回帰式（一次式）を示す。
【図２８】血漿中のヘパリン濃度(U/ml)の対数値とAPTTのロジット値の関係と検量線の回帰式（二次式）を示す。
【図２９】スルファチド濃度(μg/ml)がAPTTにおよぼす影響を示す。区間Ａおよび区間Ｂは、それぞれ、検量線の作成を行ったAPTTが延長する区間および短縮する区間である。
【図３０】 APTT延長区間の血漿中のスルファチド濃度(μg/ml)の対数値とAPTTのロジット値の関係と検量線の回帰式（一次式）を示す。
【図３１】 APTT延長区間の血漿中のスルファチド濃度(μg/ml)の対数値とAPTTのロジット値の関係と検量線の回帰式（二次式）を示す。
【図３２】 APTT延長区間の血漿中のスルファチド濃度(μg/ml)の対数値とAPTTのロジット値の関係と検量線の回帰式（三次式）を示す。
【図３３】 APTT短縮区間の血漿中のスルファチド濃度(μg/ml)の対数値とAPTTのロジット値の関係と検量線の回帰式（一次式）を示す。
【図３４】 APTT短縮区間の血漿中のスルファチド濃度(μg/ml)の対数値とAPTTのロジット値の関係と検量線の回帰式（二次式）を示す。
【図３５】 APTT短縮区間の血漿中のスルファチド濃度(μg/ml)の対数値とAPTTのロジット値の関係と検量線の回帰式（三次式）を示す。[0001]
BACKGROUND OF THE INVENTION
The present invention reduces the concentration of substances such as drugs that affect blood clotting time such as partial thromboplastin time (PTT), activated partial thromboplastin time (APTT), prothrombin time (PT), and thrombin time (TT) for a short time. In particular, the present invention relates to a method for measuring accurately, conveniently and inexpensively. The present invention also relates to an apparatus for quantifying the substance in various specimens using the same principle, and a reagent kit used for this quantification method.
[0002]
[Prior art]
General methods such as high-performance liquid chromatography (HPLC) are used to measure substances that affect the blood coagulation system, such as glycosaminoglycans (hereinafter also referred to as GAG) such as heparin and dermatan sulfate, and argatroban. The measurement target substance can be directly measured using an instrument, or the biochemical activity specific to the measurement target substance can be measured indirectly as otherwise performed during heparin measurement. (Blood coagulation test handbook, published by Kakudo Yagi Shoten, Michio Fujimaki, edited by Katsuyuki Fukutake, 1992). However, the former method requires a specific measuring instrument, and further requires a simple pretreatment that takes a long time to make the measurement possible by the measuring apparatus, and there are drawbacks such as low detection sensitivity. To do. In the latter method, it is essential that the target substance has a specific activity. In order to detect the specific activity, in many cases, an expensive reagent that is difficult to obtain is required. It is difficult to measure a large number of samples at a time because it requires rapid processing to measure the activity, and long-term storage of the sample leads to a decrease in the activity and prevents accurate measurement. There are problems such as narrow range. Although an immunochemical measurement method using a specific antibody can be considered, there is a problem that detection is impossible when the measurement target substance is not immunogenic or low, and Not reached. There is also an attempt to measure the concentration of the target substance by measuring blood clotting time such as partial thromboplastin time (PTT), activated partial thromboplastin time (APTT), prothrombin time (PT), and thrombin time (TT). (In Heparin, pp. 393-415, Lane, DA and Lindahl, U. eds, Edward Arnold, 1989), in the conventional method, there is a mathematical correlation between the measured substance concentration and the coagulation time. It is difficult to directly quantify the concentration from the measurement value of the clotting time, the concentration range that can be quantified from the created calibration curve is narrow, and many clotting factors contained in the sample are used. Therefore, there is a problem that the difference in the specimen is large regardless of the concentration of the substance to be measured.
[0003]
[Problems to be solved by the invention]
There is a need for a simple and accurate quantitative method for substances that affect blood coagulation time, and a quantitative method that is easy to quantify. In particular, compared with the above-mentioned conventional quantification method, the measurement method can be quantified accurately and in a wide measurement range, and the measurement is not affected by substances such as blood coagulation factors other than the measurement target substance in the sample. There is a demand for a method for quantitatively quantifying a target substance specifically with high accuracy.
[0004]
[Means for Solving the Problems]
As a result of diligent research, the present inventors have obtained a calibration curve in a specific manner from the concentration of a substance to be measured having a known concentration and the coagulation time, so that it can be accurately quantified in a wide measurement range, and measured from a measurement value. It is possible to quantify the concentration of the target substance, and by pre-processing the specimen containing the target substance under specific conditions, the target substance can be specified with high accuracy without being affected by substances other than the target substance in the specimen. The present invention was completed.
[0005]
That is, the first gist of the present invention is a method for quantifying a substance that affects blood coagulation time, which is a clotting time in a standard specimen containing the substance having a known concentration and a specimen to be measured whose concentration of the substance is unknown. In addition, the coagulation time in the reference standard sample not containing the substance is measured, and the logarithmic value of the concentration of the substance affecting the blood coagulation time in the standard specimen and the relative coagulation time (Tr) represented by the following formula (1) A calibration curve is created using the logit value represented by the following formula (2) as a variable, and the concentration of the substance in the sample to be measured is determined based on the obtained calibration curve. (Hereinafter also referred to as method 1 of the present invention).
[0006]
▲ 1 ▼ Tr = (T ₀ / T) × K
(2) Logit (Tr) = Ln (Tr / | 1-Tr |)
(In the formula, Tr is the relative solidification time, T ₀ Is a clotting time in the control sample, T is a clotting time in the standard sample, K is an arbitrary constant (preferably 1.0) satisfying 0.8 ≦ K ≦ 1.2, and Ln is a natural logarithm. )
Preferably, the method for creating the calibration curve is a polynomial regression method.
[0007]
The second gist of the present invention is a method for quantifying a substance that affects the blood coagulation time, and does not destroy the blood coagulation factor present in a sample to be measured whose concentration of the substance is unknown. In a quantitative method characterized by inactivation under conditions, and then mixing the specimen and standard specimen with a certain amount of blood coagulation factor and measuring the clotting time of the resulting mixture (hereinafter referred to as the method of the present invention). 2 is also expressed, and the methods 1 and 2 of the present invention may be simply expressed as the method of the present invention).
[0008]
Preferably, the inactivation is by heat treatment, and the preferable condition is 50 to 70 ° C.
The method 2 of the present invention can be combined with the method 1 of the present invention.
[0009]
A substance that influences blood coagulation time preferable as a measurement target substance of the method of the present invention is a carbohydrate or a complex carbohydrate, and more preferably a glycosaminoglycan or a glycolipid.
[0010]
Furthermore, the present invention also provides a device for carrying out the method of the present invention (hereinafter also referred to as the device of the present invention) and a kit for performing the method of the present invention (hereinafter also referred to as the kit of the present invention).
[0011]
That is, a quantitative determination apparatus for a substance that affects blood coagulation time, a standard specimen containing the substance at a known concentration, a specimen to be measured whose concentration of the substance at an unknown concentration is unknown, and a reference standard specimen not containing the substance A means of measuring the coagulation time of the above, a logarithmic value of the known concentration and a logit value represented by the following formula (2) of the relative coagulation time (Tr) represented by the following formula (1) as a polynomial There is provided the quantification apparatus comprising means for calculating a calibration curve by a regression method, and means for calculating the concentration of the substance from the coagulation time of the specimen to be measured based on the calibration curve. .
[0012]
▲ 1 ▼ Tr = (T ₀ / T) × K
(2) Logit (Tr) = Ln (Tr / | 1-Tr |)
(In the formula, Tr is the relative solidification time, T ₀ Is a clotting time in the control sample, T is a clotting time in the standard sample, K is a constant satisfying 0.8 ≦ K ≦ 1.2 (preferably 1.0), and Ln is a natural logarithm. )
The kit of the present invention is a kit for performing a method for quantifying a substance that affects blood coagulation time, a standard specimen containing the substance at a known concentration, a control standard specimen not containing the substance, a blood coagulation factor, and an activity. It is a kit characterized by including a chemical agent. Preferably, a calibration curve is obtained by a polynomial regression method using the logarithm value of the known concentration and the logit value represented by the following formula (2) of the relative coagulation time (Tr) represented by the following formula (1) as variables. And a medium recording a program for causing a computer to execute a procedure for calculating the concentration of the substance from the coagulation time of the specimen to be measured based on the calibration curve. .
[0013]
▲ 1 ▼ Tr = (T ₀ / T) × K
(2) Logit (Tr) = Ln (Tr / | 1-Tr |)
(In the formula, Tr is the relative solidification time, T ₀ Is a clotting time in the control sample, T is a clotting time in the standard sample, K is a constant satisfying 0.8 ≦ K ≦ 1.2 (preferably 1.0), and Ln is a natural logarithm. )
[0014]
DETAILED DESCRIPTION OF THE INVENTION
Embodiments of the present invention will be described below.
1. Method 1 of the present invention
A sample to be measured whose concentration of the measurement target substance affecting the blood coagulation time is unknown, that is, a sample for which the concentration of the measurement target substance is to be measured (measurement sample), a reference sample that does not contain the measurement target substance, and further known With respect to a standard specimen containing a substance whose concentration is to be measured, the coagulation time is measured by a measurement system relating to blood coagulation time. In this case, if any of the measurement sample, reference standard sample, or standard sample does not contain blood clotting factor at all or lacks part of it, coagulation does not occur alone even if an activating factor is added The blood coagulation factors are mixed so that the blood coagulation factors of all these samples have the same concentration. Taking the logarithmic value of the concentration of the measurement target substance contained in the standard sample (logarithmic conversion), the relative clotting time represented by the following formula (1) is obtained from the clotting time of the standard specimen and the reference standard specimen not containing the measurement target substance. Then, the relative coagulation time is subjected to logit conversion represented by the following formula (2) to calculate a logit value, and a calibration curve is created using the logarithmic value and the logit value. The normal Logit conversion does not use an absolute value like the Logit conversion shown by the following formula (2) used in the method 1 of the present invention. In this specification, the conversion by the following formula (2) is referred to as a logit conversion. The conversion value obtained by the conversion method is referred to as a logit value. The Logit conversion after converting the concentration of the substance to be measured into a logarithmic value and converting the coagulation time into the relative coagulation time will be described as Logit-log conversion in the future. As an application of Logit conversion, the following equation (2) can be used as an alternative to Logit conversion. If necessary, the calibration curve is calculated by regressing the logarithmic value and the Logit value into a regression equation. Using the calibration curve, the Logit value calculated from the clotting time of the specimen is used to determine the concentration of the substance to be measured in the specimen, and the substances that affect the blood clotting time are quantified.
▲ 1 ▼ Tr = (T ₀ / T) × K
(2) Logit (Tr) = Ln (Tr / | 1-Tr |)
▲ 2 ▼ 'Logit (Tr) = Ln (Tr / | 1-Tr |) ^m + Ln (n)
(In the formula, Tr is the relative solidification time, T ₀ Is a coagulation time in the control standard sample, T is a coagulation time in the standard sample, K is a constant of 0.8 ≦ K ≦ 1.2, Ln is a natural logarithm, m and n are natural numbers, and particularly, n satisfies n> 0. K calculates the concentration of the measurement target substance in a known standard sample on a calibration curve, and changes it as a correction constant before calculating the concentration of the measurement target substance in the sample as necessary when an error occurs. Normally, K = 1.0 is sufficient. Further, the application conversion of (2) 'is a means for changing the slope and intercept of the calibration curve, and can be appropriately used when the numerical calculation method becomes simple by using the application conversion. Usually, a simple Logit conversion expressed by the formula (2) can be used sufficiently. Such application conversion is also included in the technical scope of the present invention. )
[0015]
The technique in each step of the method 1 of the present invention will be described in detail below.
(1) Specimen to measure the concentration of the substance to be measured that affects blood clotting time (Measurement specimen)
The substance to be measured that affects the blood coagulation time is not particularly limited as long as it is a substance that causes the blood coagulation time to be extended or shortened by its presence in a normal blood coagulation system, but preferably has a sugar chain structure. Examples of the substance having the sugar chain structure include saccharides, complex carbohydrates and the like, and examples of the saccharide include dermatan sulfate (DS), chondroitin sulfate E (CSE), heparin (Hep), and low molecular weight heparin (LMWHep). ) And the like, and examples of complex carbohydrates include glycolipids such as sulfatide. For example, dermatan sulfate, chondroitin sulfate E, heparin, or low molecular weight heparin is a substance that prolongs the blood coagulation time. For example, sulfatide is a substance that prolongs or shortens the blood coagulation time depending on the concentration condition. The sample can be used as a sample in the present invention as long as the solution can contain a substance that affects the blood coagulation time, and is not particularly limited. For example, a sample for a clinical test or a pharmaceutical product containing the substance can be used. These include body fluids for monitoring, solutions obtained from the process for producing the substance, and the like. Specifically, examples of body fluids include blood, plasma, serum, lymph, tissue fluid, joint fluid, cerebrospinal fluid, sweat, tear fluid, urine, and the like. Tissue extract and the like, physiological saline, various buffers, water A water-soluble solution such as can be used as a specimen in the same manner as a body fluid.
[0016]
(2) Control sample and standard sample
The control sample is not particularly limited as long as it is an aqueous liquid that does not contain a substance that affects the blood clotting time. For example, blood, plasma, serum, lymph, tissue fluid, joint fluid, cerebrospinal fluid, sweat, tears Fluid, body fluid such as urine, tissue extract, physiological saline, various buffers, water, and the like. Further, the standard specimen is the liquid containing the measurement target substance at a known concentration. The liquid used as the solvent for the sample, the standard sample, and the control sample is not necessarily the same, but is preferably the same.
[0017]
(3) Concentration of substance to be measured
The concentration of the substance to be measured in this specification is, for example, a concentration indicated by (weight / volume (mg / ml, μg / ml, etc.), (activity / volume (U / ml, etc.)), etc. .
[0018]
(4) Measurement system for blood clotting time
The measurement system related to the blood coagulation time is not particularly limited as long as it is a method utilizing the coagulation of the specimen.For example, partial thromboplastin time (PTT), activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time ( TT) (both clinical laboratory manuals published by Bunkodo, edited by Motoharu Kitamura, 1988).
[0019]
(5) Activator
The activator in the present invention is an activator for initiating the coagulation of a specimen by the above measurement system to be used, for example, an APTT measurement reagent in APTT measurement, a PT measurement reagent in PT measurement, or the like. For example, as an APTT measurement reagent, a combination of “APTT reagent” and calcium chloride solution contained in commercially available thrombocheck APTT (Midori Cross Co., Ltd .; trade name) etc. is preferable. APTT measurement kit containing a reagent having the following structure: a diatomaceous earth (for example, Celite (trade name)), a mineral such as kaolin mineral, a contact factor such as tannin such as ellagic acid or silicic anhydride, and crude cephalin It may be used in the same manner as described in the description of the above, and may be applied to APTT measurement in the method of the present invention. For example, the PT measurement reagent is preferably a “PT reagent” and a calcium chloride solution contained in commercially available Thrombocheck PT (Midori Cross; trade name), etc., but has the same configuration as the “PT reagent”. A reagent, that is, thromboplastin may be used in place of the “PT reagent” in the same manner as described in the instruction manual of the PT measurement kit and applied to the PT measurement in the method of the present invention. Similarly, PTT measurement and TT measurement are similar to 0.02M calcium chloride solution and commercially available kits for PTT and TT measurement, for example, crude cephalin as an alternative to PTT reagent for PTT measurement, TT reagent for TT measurement. Measurement can be performed by using thrombin as an alternative to the above.
[0020]
(6) Solidification time
The coagulation time in the present invention is, for example, a measurement value such as a measurement value of absorbance with an absorptiometer, a measurement value of turbidity with a turbidimeter or a turbidimetry, or a measurement value of viscosity with a viscometer. Indicates the time to reach a certain value indicative of clotting.
[0021]
(7) Blood coagulation factor
Blood coagulation factors are all blood coagulation factors contained in plasma that is healthy with respect to the blood coagulation system, and the method of adding the coagulation factor is to the measurement reaction system if the blood coagulation factor induces normal coagulation by the activation factor. The mixing method and the form of the reagent are not limited. The state of the solution is preferable when mixing, but it is not limited to this as long as it can be added accurately. For example, it is possible to mix the lyophilized product so that it is easily dissolved in the reaction solution. is there. When the blood coagulation factor is mixed with the specimen, the final concentration is preferably 5 to 200% of the concentration of these factors in the plasma of a healthy person. In addition, as the blood coagulation factor in the present invention, it is preferable to use a commercially available standard plasma containing a necessary blood coagulation factor such as Coagtrol 1 (Corporate Midori Cross; trade name). Platelet-rich plasma (PRP) containing a lot of platelets obtained by adding a blood coagulation inhibitor such as sodium citrate and centrifuging at a low rotation speed (for example, about 150 × g), or a high rotation speed ( For example, any poor platelet plasma (PPP) obtained at about 1,000 × g can be used. Furthermore, if the liquid contains all blood coagulation factors contained in normal plasma and the ability to cause clotting is not lost by the activation of the blood coagulation factors, for example, lyophilized untreated plasma can be lyophilized. A solution obtained by re-dissolving the obtained lyophilized product in distilled water is also possible.
[0022]
(8) Logarithmic conversion
The logarithmic conversion of the concentration of the substance to be measured in the present invention is not particularly limited. For example, a common logarithm value, a natural logarithm value, etc. are taken. It is preferable.
[0023]
(9) Regression method
The regression method in the present invention is not particularly limited as long as it is a regression method for expressing the relationship between the logarithmic value and the Logit value by a mathematical expression. However, since the error between the calculated numerical value and the theoretical value is small and the accuracy is high, it is preferable to return to the regression equation using the polynomial regression method, and considering practicality, it is possible to return to the quadratic equation or the cubic equation. More preferred.
[0024]
The present invention also provides the following method 2 of the present invention as a method for measuring a substance that affects blood coagulation time. An embodiment of the method 2 of the present invention will be described below.
[0025]
2. Method 2 of the present invention
When measuring the coagulation time in a measurement system related to blood coagulation time, if the sample (measurement sample) whose concentration should affect the blood coagulation time is a biological sample that already contains a blood coagulation factor Inactivate the blood clotting factor inherent in the measurement sample under conditions that do not destroy the target substance, and then mix the sample and standard sample with a certain amount of blood clotting factor, and then adjust the clotting time of the resulting mixture. taking measurement. The concentration of the substance to be measured can be calculated by a conventional method of creating a calibration curve using the measured coagulation time as it is or using the logarithmically converted value and the coagulation time as they are.
[0026]
The technique in each step of the method 2 of the present invention will be described in detail below. The measurement sample, the control standard sample and the standard sample, the measurement system relating to the blood coagulation time, the concentration of the substance to be measured, the coagulation time, the blood coagulation factor and the like are the same as those described for the method 1 of the present invention.
[0027]
(1) Treatment to inactivate blood coagulation factors under conditions that do not destroy the substance to be measured
Examples of the treatment for inactivating the blood coagulation factor inherent in the specimen without destroying the substance to be measured in the present invention include enzyme treatment, heat treatment, acid treatment, alkali treatment, denaturation treatment with an organic solvent, and freeze-thawing. Repeating treatment is preferable, and among them, heat treatment is simple and preferable. The temperature in the heat treatment is usually 30 to 100 ° C, preferably 40 to 80 ° C, more preferably 50 to 70 ° C, and most preferably 56 to 65 ° C. Although the time in the heat treatment is not limited as long as the measurement target substance is not destroyed by heating, it is usually several minutes to several tens of minutes, preferably 15 to 25 minutes, particularly preferably about 20 minutes. In the case of acid treatment or alkali treatment, it is necessary to appropriately adjust the hydrogen ion concentration (pH) with, for example, a buffer solution after the treatment. In the case of a modification treatment with an organic solvent, a treatment for removing the organic solvent is necessary after the modification treatment. Furthermore, for example, in the case of enzyme treatment using a proteolytic enzyme or the like, it is necessary to deactivate the enzyme after the treatment, for example, by heat treatment or the like. Moreover, as an enzyme treatment method, it is possible to employ a method of contacting with an immobilized enzyme. In this case, it is not always necessary to deactivate. In the inactivation treatment, the use of the above treatment alone or the combination of a plurality of treatments is not particularly limited.
[0028]
(2) Mixing with a certain amount of blood coagulation factor
The mixing with a certain amount of blood coagulation factor in the present invention means that a blood coagulation factor is added separately in place of the endogenous blood coagulation factor inactivated by the inactivation treatment. The amount added is the amount that the blood coagulation factor induces normal coagulation with the activator, and is appropriately determined by the measurement system.
[0029]
As described above, even when the method 1 and method 2 of the present invention are carried out independently, it is possible to obtain accurate measurement values in a wide concentration range as compared with the conventional method, but the method 1 and method of the present invention. When the method described in 2 is used in combination with the inactivation treatment of the blood coagulation factor inherent in the specimen and the numerical conversion of the measurement value, it is possible to obtain an accurate measurement value in a wider concentration range.
[0030]
That is, only the measurement sample and the standard sample or the measurement sample containing the measurement target substance described in the method 1 of the present invention are treated so as not to destroy the measurement target substance, and the endogenous blood coagulation factor is obtained. After inactivation, the method 1 of the present invention was described by adding all blood coagulation factors to the same concentration as in the method 1 of the present invention to each of the measurement sample, the control standard sample, and the standard sample. The coagulation time is measured in the same manner as in Method 1 of the present invention by adding an activator, and a calibration curve is created using the measured value of the measurement target substance concentration or activity unit and coagulation time in the control standard sample and standard sample. The concentration of the substance to be measured in the sample can be determined from the calibration curve and the coagulation time of the sample to be measured.
[0031]
In addition, the methods 1 and 2 of the present invention and the quantification method combining them can quantitate only the substance to be measured even when a plurality of substances that affect the blood coagulation time are included in the sample to be measured. . In this case, in the methods 1 and 2 of the present invention or a combination thereof, before starting the coagulation of the specimen, an operation for bringing the above substances other than the target substance into a state that does not affect the blood coagulation system is performed. There is a need to do. The operation is, for example, an enzyme treatment method. For example, when the substance to be measured and heparin coexist, treatment with a heparin-degrading enzyme such as heparinase decomposes heparin before starting the coagulation of the specimen, and then initiates coagulation to measure the coagulation time. Do.
[0032]
The present invention also provides a measuring instrument (the device of the present invention) and a kit (the kit of the present invention) using the method of the present invention. Hereinafter, embodiments of the device of the present invention and the kit of the present invention will be described.
[0033]
3. Device of the present invention
An apparatus for measuring a substance concentration that affects blood coagulation time by the method described in Method 1 of the present invention, an example of which will be described with reference to the block diagram of FIG. This apparatus of the present invention is equipped with a reaction execution unit 1 and a sample measurement unit 2 as means for measuring the coagulation time of a measurement sample, a reference standard sample, and a standard sample, a means for calculating a calibration curve, and a concentration of the substance. As a means for calculating, an input unit 3, a central processing unit 4, a data storage unit 5, a program storage unit 6 and an output unit 7 are provided.
[0034]
Hereinafter, each component will be described in detail.
(1) Reaction Department
The reaction execution unit 1 includes a measurement sample, a reference standard sample, and a reactor in which the standard sample coagulates. It is preferable that a heater capable of heating is provided in order to inactivate endogenous blood coagulation factors by heating, and the method of the present invention described after the explanation of the method 2 of the present invention by using this heater. It is also possible to perform a measurement combining 1 and the method 2 of the present invention.
[0035]
(2) Specimen measurement unit
The sample measurement unit 2 measures the coagulation time by measuring changes in the measurement sample, the reference standard sample, and the standard sample. Considering simplification of the apparatus, it is preferable to install the apparatus adjacent to the reaction execution unit 1.
[0036]
The means for measuring the coagulation time provided in the sample measuring unit 2 is not particularly limited as long as the measuring step of the method 1 of the present invention can be performed. For example, an absorbance measurement device using a spectrophotometer, a turbidity measurement device using a turbidimeter or turbidimetry, a viscosity detection device using a viscometer, etc. A method using absorbance is preferred. The time from the start of coagulation until the measured value reaches a certain value indicating coagulation is measured as the coagulation time.
[0037]
(3) Input section
Using the input unit 3, the measurement target substance concentration in the standard sample and the measurement value of the coagulation time at the concentration, if necessary, the activity unit (U / mg) of the measurement target substance in the standard sample, the coagulation time of the sample Enter the measured value and coefficient (K) for numerical conversion, and select the regression method for calculating the calibration curve. If necessary, the input unit 3 also inputs the heating temperature and heating time of the heater of the reaction execution unit 1. The input unit 3 includes, for example, a keyboard, a numeric keypad, a touch panel, etc., but is not necessarily limited thereto.
[0038]
(4) Central processing unit
The following processing is performed.
・ Measurement substance concentration or activity unit and regression equation information (regression equation selection information and coefficient (K)) in the standard sample input from the input unit 3 are stored in the data storage unit 5, and the sample measurement is performed. The coagulation times of the sample, the reference standard sample and the standard sample measured in the unit are acquired from the sample measurement unit and stored in the data storage unit 5.
A program for executing the calibration curve calculation procedure is read from the program storage unit 6, the calibration curve is calculated by executing the program, and stored in the data storage unit 5 as necessary.
-A program for executing the unknown concentration calculation procedure is read from the program storage unit 6 according to the information input in the input unit 3, and the program is executed to calculate the substance concentration or activity unit in the sample. Accordingly, the data is stored in the data storage unit 5. The calculated calibration curve, the concentration of the substance to be measured in the sample, and the data stored in the data storage unit 5 are output to the output unit 7.
If necessary, the heating temperature and heating time of the heater stored in the data storage unit 5 are read and the heater of the reaction execution unit 1 is controlled.
[0039]
The central processing unit 4 is composed of, for example, a microprocessor.
[0040]
(5) Data storage unit
The concentration of the measurement target substance in the standard sample and the regression equation information (regression equation selection information and coefficient (K)), and the measured measurement sample, control standard sample, and standard sample coagulation time are stored. For example, it is configured by a storage device such as a random access memory or a hard disk.
[0041]
(6) Program storage unit
A program for causing the central processing unit to execute a calibration curve calculation procedure, an unknown concentration calculation procedure, and a mutual conversion procedure of concentration description is stored. For example, it is composed of a read-only memory. Alternatively, the program is transferred from a storage medium to a random access memory.
[0042]
The calibration curve calculation procedure reads from the data storage unit 5 the measurement target substance concentration or activity unit in the standard sample, the selection information of the regression equation, and the coagulation time data of the measurement sample, the control standard sample, and the standard sample. Polynomial regression based on regression equation information using logarithmic value of concentration in specimen and logit value expressed by formula (2) of relative coagulation time (Tr) expressed by formula (1) below as variables. This is a procedure for calculating a calibration curve by the method.
[0043]
▲ 1 ▼ Tr = (T ₀ / T) × K
(2) Logit (Tr) = Ln (Tr / | 1-Tr |)
(In the formula, Tr is the relative solidification time, T ₀ Is a clotting time in the control sample, T is a clotting time in the standard sample, K is a constant satisfying 0.8 ≦ K ≦ 1.2 (preferably K = 1.0), and Ln is a natural logarithm. )
The unknown concentration calculation procedure is a procedure for calculating the concentration from the coagulation time of the measurement specimen read from the data storage unit 5 based on the calculated regression equation as a calibration curve.
[0044]
The mutual conversion procedure of concentration description is the mutual conversion procedure of the concentration indicated by (weight / volume) and the concentration indicated by (activity / volume), that is, the input active unit (number of units per 1 mg) and the above unknown concentration This is a procedure for converting the concentration indicated by (weight / volume) into the concentration indicated by (activity / volume), or vice versa, using the numerical values obtained by the calculation procedure.
[0045]
(7) Output section
The calculated substance concentration, regression equation, etc. are output. For example, it is configured by screen display means such as a liquid crystal display panel and printing means such as a printer.
[0046]
Each device that performs each of the above-described means provided in the device of the present invention is not necessarily included in the same housing, and the configuration is not particularly limited as long as information is appropriately processed.
[0047]
4. Kit of the present invention
The kit of the present invention is a kit for performing the method 1 of the present invention and the method 2 of the present invention singly or in combination. The standard specimen, blood coagulation factor and activation factor described for the method 1 of the present invention are more preferable. Is a kit containing a medium and / or a control sample in which a program for mathematically processing the measurement value of the clotting time obtained by the measurement is recorded.
[0048]
The technique in the kit of the present invention will be described in detail below.
(1) Control sample, standard sample, blood coagulation factor and activation factor
The control standard sample, standard sample, and blood coagulation factor are as described for the method 1 of the present invention. Examples of the kit of the present invention include a heparin measurement kit and a dermatan sulfate measurement kit. For example, in the case of a heparin measurement kit, a standard heparin sample containing a certain amount of heparin at a constant concentration is included as a standard sample. Is preferred.
[0049]
The activator in the kit of the present invention is a substance in which the activation of the blood coagulation factor is induced by mixing the activator, and examples include those described for the method 1 of the present invention. If it is a factor, it will not be specifically limited. The activator is determined by the blood coagulation system (APTT, PT, PTT, TT, etc.) used for measurement of the coagulation time. For example, the APTT reagent, PT reagent, etc. described in the activator section of the method 1 of the present invention And a solution containing calcium ions.
[0050]
It is desirable that the control standard sample, the standard sample, the blood coagulation factor and the activation factor are prepared in a form suitable for storage or use and included in the kit.
[0051]
(2) Medium on which a program for mathematical processing is recorded
The medium on which the program for performing mathematical processing in the kit of the present invention is written is a conversion method of various numerical values described in the method 1 of the present invention by inputting the measurement value of the coagulation time by a prescribed method. Further, a program for creating a calibration curve described in the above-described method 1 of the present invention, that is, performing regression of numerical values, calculating a concentration of a measurement target substance in a sample, and performing mutual conversion of concentration descriptions as necessary using the converted values Are recorded, the type of the storage medium is not limited, and the environment in which the program operates is not particularly limited.
[0052]
A procedure of an example of the program will be described with reference to the flowchart of FIG.
In S1, the concentration of the substance affecting the blood coagulation time contained in the standard sample, the coagulation time of the standard sample, and the coagulation time of the control standard sample are input. In addition, a coefficient (K) is input as necessary.
[0053]
In S2, a regression equation used to calculate a calibration curve is selected. For example, the primary expression, the secondary expression, and the tertiary expression are selected.
In S3, based on the information input in S1 and the information selected in S2, the logarithmic value of the concentration of the substance affecting the blood coagulation time in the standard sample and the relative coagulation represented by the following formula (1) A calibration curve is calculated using the logit value represented by the following formula (2) of time (Tr) as a variable.
[0054]
▲ 1 ▼ Tr = (T ₀ / T) × K
(2) Logit (Tr) = Ln (Tr / | 1-Tr |)
(In the formula, Tr is the relative solidification time, T ₀ Is a clotting time in the control sample, T is a clotting time in the standard sample, K is an arbitrary constant satisfying 0.8 ≦ K ≦ 1.2, and Ln is a natural logarithm. )
[0055]
In S4, the coagulation time of the specimen to be measured is input.
In S5, the concentration (unknown concentration) of the measurement target substance in the sample to be measured is calculated based on the calibration curve. Further, mutual conversion of density description is performed as necessary.
[0056]
In S6, the concentration calculated in S5 and the regression equation as the calibration curve calculated in S3 are displayed. These may be displayed separately or in a graph display.
[0057]
Among the above procedures, the order of S1, S2 and S4 may be arbitrary. S4 may be before S3, but in this case S1 and S2 must be performed before S3.
[0058]
S1 to S3 correspond to the procedure for calculating the calibration curve, and S4 to S5 correspond to the means for calculating the unknown concentration.
[0059]
【Example】
Examples of the present invention will be described below, but the present invention is not limited thereto.
[0060]
[Example 1]
(Quantification of various glycosaminoglycans by the method 1 of the present invention)
The effects of various substances that prolong the blood coagulation time in rat plasma on APTT, and the correlation between the numerical conversion value of APTT and the concentration value of the substance as the measurement target substance were examined. Glycosaminoglycan (GAG) heparin (hereinafter Hep), low molecular heparin (hereinafter LMWHep) and dermatan sulfate (DS) were used as the measurement target substances, and changes in APTT of untreated plasma collected. I investigated.
That is, a syringe containing a 3.8 wt% sodium citrate aqueous solution is prepared, and about 9 times as much blood as the sodium citrate aqueous solution is collected using the syringe. The mixture was centrifuged for 15 minutes at 4 ° C. and 1000 × G, and the supernatant was separated as plasma. APTT reagent (Thrombocheck APTT: Midori cross) that was added to 100 μl of the plasma, 100 μl of the above-mentioned various substances to be measured dissolved in physiological saline at each concentration, left at 37 ° C. for 1 minute, and then kept at 37 ° C. 100 μl and left at 37 ° C. for 2 minutes, then added 100 μl of 0.02M calcium chloride solution kept at 37 ° C. to start coagulation, and the coagulation time was measured by Baxter's blood coagulation automatic measuring device (AMELUNG KC10A) (Table 1, FIG. 3).
[0061]
With heparin, APTT is prolonged from 0.12 μg / ml, and coagulation does not occur at 12 μg / ml or higher, and with low molecular weight heparin, APTT is prolonged from 1.2 μg / ml, and coagulation does not occur at 40 μg / ml or higher. . In dermatan sulfate, APTT was extended from 1 μg / ml, and was extended to 65.7 sec at 3000 μg / ml. Using a known method (In Heparin, pp. 393-415, Lane, DA and Lindahl, U. eds, Edward Arnold, 1989) Was created (described later). However, the calibration curve has low accuracy, and the concentration range that can be practically measured was very narrow (Comparative Example; FIGS. 4 to 6).
[0062]
[Table 1]

* All are APTT values in plasma that does not contain substances that affect blood clotting time, but these values are different because the day when heparin and low molecular weight heparin experiments were conducted and the day when dermatan sulfate experiments were conducted. There is a difference. Therefore, the blood clotting time varies depending on the specimen (plasma or the like) used. The present invention also provides a quantitative method that eliminates such an error caused by the specimen (Example 2).
[0063]
Numerical values were converted using the APTT values obtained using the above substances, and a calibration curve was prepared by regression of the numerical values using a polynomial regression method. That is, numerical conversion (Logit-log conversion) and regression were performed according to the following procedure.
1) Convert the test substance concentration (x) to the common logarithm value. (X = Logx)
2) APTT value (T concentration) when the test substance is not added (0 concentration) to APTT value (T) at each test substance concentration (x) ₀ ) Ratio (Tr) (Tr = (T ₀ / T) × K; K = 1.0).
3) Convert the Tr value to Logit. In this embodiment, since the solidification time is extended, 1> Tr, and Y = Ln (Tr / (1-Tr)). Therefore, in this embodiment, Ln (T ₀ / T) / (1-T ₀ / T) = Ln (T ₀ / TT ₀ ) = Ln ((TT ₀ ) / T ₀ ) ^-1 =-Ln (ΔAPTT / T ₀ ) And converted. However, ΔAPTT = TT ₀ .
4) Polynomial regression is performed on each X value and the corresponding Y value data, and a regression curve (a straight line in the case of a linear expression) is calculated using a polynomial equation. Y = ΣC _k X ^k , But K ≧ 0, C _k Is a real number.
[0064]
The theoretical value is the concentration of the substance to be measured in the actual sample, and the calculated value is a numerical value obtained by back-calculating the concentration of the substance to be measured from the measured value of APTT of the sample by the equation of the calibration curve obtained by regression calculation.
[0065]
APTT value obtained by measurement, calculated value obtained from a polynomial (first-order, second-order, third-order, heparin is only first-order and second-order) indicating the calculated regression curve, and true value obtained from theoretical value and calculated value A list of degrees (trueness = (calculated value−theoretical value) / theoretical value × 100%) is shown in Tables 2 to 5 (method of the present invention). The theoretical values obtained by the above conventional method are also shown for reference. Moreover, the calculated regression curve is shown to FIGS. 7-9 (method of this invention). In this embodiment, the regression equation is exemplified up to a cubic equation, but a regression curve using a polynomial of a fourth or higher order can also be used.
[0066]
When calculating the unknown substance concentration from the APTT value using the above regression curve (calibration curve), measure the APTT value of the sample containing the substance to be measured having an unknown concentration, calculate Y, and use the resulting polynomial Substituting to get X, 10 ^X And finally, an unknown concentration x is obtained. As is clear from the results in Tables 2 to 5, according to the present invention, good accuracy can be obtained in a wide concentration range.
[0067]
[Table 2]

Conventional method: Y = 0.495X + 1.311 (in this case, Y = log (APTT value of specimen), X = log (heparin concentration in specimen) (FIG. 4)
[0068]
[Table 3]

Conventional method: Y = 0.303X + 1.217 (in this case, Y = log (APTT value of the specimen), X = log (low molecular heparin concentration in the specimen) (FIG. 5)
[0069]
[Table 4]

[Table 5]

Conventional method: Y = 0.253X + 0.898 (in this case, Y = log (APTT value of specimen), X = log (dermatan sulfate concentration in specimen) (FIG. 6)
[0070]
[Example 2]
(Quantification of dermatan sulfate by the method of the present invention)
The effects of plasma heat treatment on APTT and the effects of using rat plasma or standard plasma (Coagtrol 1) as a blood coagulation factor supply source were examined. As a substance to be measured, dermatan sulfate was used.
[0071]
The difference in the amount of blood coagulation factor in plasma affects the measured value of APTT, and further affects the quantitative value. Because the plasma used in Table 1 of Example 1 was a different sample, the glycosaminoglycan Since it is clear from the difference in coagulation time when the concentration is 0 (see Table 1), the blood coagulation factor in the specimen containing dermatan sulfate is inactivated and used for APTT measurement. Was added separately. As a method for inactivating the blood coagulation factor, a heat treatment of the sample was attempted. The effect of heat treatment on APTT was examined at dermatan sulfate concentrations of 0 and 250 μg / ml. Thereafter, coagtrol 1 and rat plasma were used as blood coagulation factor sources.
[0072]
The influence of the heat treatment was such that 1/10 of the final volume of dermatan sulfate dissolved in physiological saline was added to plasma collected from rats in the same manner as in Example 1, and the final concentration in this state was 0 and 250 μg / This was used as a sample. The heat-treated specimen was heated at 65 ° C. for 20 minutes, 100 μl of the same APTT reagent and calcium solution as in Example 1 were added, and each APTT value was measured with an automatic blood coagulation measurement apparatus as in Example 1. In the non-heat-treated specimens having a dermatan sulfate concentration of 0, 250 μl / ml, the APTT values were 14.2 seconds and 38.1 seconds, respectively, but the heat-treated specimens were at least 120 seconds at any dermatan sulfate concentration. No coagulation was observed (Table 6).
[0073]
On the other hand, when the blood coagulation factor is added separately after the heat treatment, the APTT value changes by adding 80 μl of coagtrol 1 prepared according to the instructions to 20 μl of the heated sample and adding APTT reagent and calcium solution in the same manner as above. Then, the APTT value was measured with the blood coagulation automatic measuring device in the same manner as in Example 1. As a result, in the sample in which coagulation did not occur when the heat treatment was performed, the coagulation activity was restored by the addition of Coagtrol 1, and further, the APTT value extending action by dermatan sulfate was observed (Table 6).
[0074]
In addition, although data are not shown, when rat plasma prepared in the same manner as in Example 1 was used as an alternative to Coagtrol 1, the coagulation activity was restored and APTT was prolonged by dermatan sulfate as in the above results. Make sure that happens.
[0075]
[Table 6]

[0076]
[Example 3]
(Quantification of dermatan sulfate by the combination of the method 1 of the present invention and the method 2 of the present invention)
Using the APTT method, we examined the quantification of substances that prolong the blood clotting time using rat urine and liver tissue homogenates. Dermatan sulfate was used as a measurement target substance.
[0077]
To the liver (2.550 g), add 4 times the amount of phosphate buffer, homogenize with a polytron homogenizer (about 15,000 rpm) under ice-cooling, and then add 15 parts under conditions of 3,000 rpm and 4 ° C. The supernatant after centrifugation for 5 minutes was used as a liver homogenized sample. In the case of urine, the urine excreted overnight by the rat was collected under ice-cooling, and the supernatant obtained by centrifugation under conditions of 3,000 rpm and 4 ° C. was used as a urine sample. Each sample was stored frozen at -30 ° C.
[0078]
According to the method 2 of the present invention, as an inactivation treatment, it was heated at 56 ° C. for 20 minutes after melting. A small amount of dermatan sulfate dissolved in physiological saline was added so as not to affect the total amount of the sample, and the sample was sufficiently stirred to prepare a specimen (liver homogenate specimen, urine specimen). 20 μl of each specimen was taken, 80 μl of Coagtrol 1 was added, and used for APTT measurement. As a reference value, APTT measurement was also performed when dermatan sulfate was dissolved in physiological saline (saline) at various concentrations (control saline sample) as an alternative to liver homogenate sample or urine sample, and according to the method 1 of the present invention. Numerical conversion was performed (Tables 7 to 10 and FIGS. 10 to 18).
[0079]
[Table 7]

[0080]
[Table 8]

[0081]
[Table 9]

[0082]
[Table 10]

[0083]
[Example 4]
(Quantitative determination of various GAGs by the combination of the present method 1 and the present method 2)
In Examples 2 and 3, the amount of dermatan sulfate was measured by inactivating the blood clotting factor in the measurement sample. However, since substances other than dermatan sulfate that prolong the blood clotting time are known, they are the same as in Example 2. We also investigated heparin, low molecular weight heparin, and chondroitin sulfate E, which are GAGs that are not destroyed by heat treatment (65 ° C, 20 minutes). The measuring method was performed by the method described below.
[0084]
That is, rat plasma was collected in the same manner as in Example 1, and various GAGs dissolved in a very small amount of physiological saline that did not affect the total amount of rat plasma were added to each sample, and then mixed thoroughly to prepare a sample. Was heat-treated at 65 ° C. for 20 minutes (heat-treated specimen). After 20 μl of the heat-treated specimen was mixed with 80 μl of Coagtrol 1, each APTT value was measured in duplicate by the same method as in Example 1, and the average was taken as the measured value, and numerical conversion was performed according to the method 1 of the present invention (Table 11). -14 and Figures 19-26).
[0085]
[Table 11]

[0086]
[Table 12]

[0087]
[Table 13]

[0088]
[Table 14]

[0089]
Since heparin is often expressed in activity units, the example of heparin in the example (Table 12) is shown in relation to the theoretical value of the concentration shown as U / ml of the standard sample and the coagulation time. A calibration curve and curve regression were tried in the same manner as in the case of heparin (Table 15 and FIGS. 27 and 28). However, Wako Pure Chemical Industries, Ltd. (Cat No. 085-00134; 182.5 U / mg) was used as heparin.
[0090]
[Table 15]

[0091]
[Example 5]
(Quantification of sulfatide by the method 1 of the present invention)
Substances that can shorten the blood clotting time (APTT) were investigated. The sulfated glycolipid sulfatide was dissolved in a phosphate buffer solution, and the concentration of the substance was measured by the method 1 of the present invention.
[0092]
Sulfatide is dissolved in phosphate buffer solution at various concentrations to prepare specimens, 20 μl of this solution and 80 μl of standard plasma coretrol 1 are mixed, APTT is measured in duplicate as in Example 1, and the average value is taken as the measured value. The numerical values were converted according to the method 1 of the present invention (Tables 16 to 18 and FIGS. 29 to 35).
[0093]
[Table 16]

[0094]
When the sulfatide was 0 μg / ml, the average APTT value was 32.3 seconds. As shown in FIG. 29, APTT was prolonged in a dose-dependent manner at 23.4 μg / ml or more (section A). On the other hand, APTT was shortened at 11.7 μg / ml or less and 0.18 μg / ml or more, and among these, at 1.5 μg / ml or less and 0.18 μg / ml or more (section B), this shortening was a capacity inverse correlation. Therefore, Logit-log conversion was performed for each of section A and section B, and a calibration curve was created for each. As shown in FIGS. 30 to 35, these data can be expressed by independent polynomials not only in the extended section but also in the shortened section.
[0095]
In this example, sulfatide dissolved in a phosphate buffer solution is used as it is, but this substance is stable at 65 ° C. for 20 minutes and freeze-thaw treatment. Therefore, when measuring sulfatide in a biological specimen, inactivation treatment can be performed according to the method 2 of the present invention.
[0096]
[Table 17]

[0097]
[Table 18]

[0098]
[Example 6]
(Measurement of dermatan sulfate by the combination of Method 1 and Method 2 of the present invention)
The concentration of a substance that prolongs the blood coagulation time in the diseased plasma was measured using the diseased plasma. A rat disseminated intravascular coagulation (DIC) model was selected as the pathological model, dermatan sulfate was selected as the measurement target substance, and the following addition and recovery experiment was performed.
[0099]
Three plasmas of the rat DIC model were prepared according to Example 4 of PCT International Publication No. WO95 / 09188. That is, pathological plasma after intravenous infusion of 50 mg lipopolysaccharide (LPS) / kg / 4 hours. These specimens and normal rat plasma were collected in the same manner as in Example 1 and stored at −20 ° C. After thawing, each of various concentrations of dermatan sulfate dissolved in a small amount of raw food was added and measured as in Example 3. A sample was heat-treated at 65 ° C. for 20 minutes as in Example 2 (heat-treated sample). 80 μl of Coagtrol 1 was added to 20 μl of each of these heat-treated samples, and quantification using the APTT method was performed in the same manner as in Example 3 (Table 19).
[0100]
[Table 19]

[0101]
As described above, it was shown that the values measured by this method are very close to both DIC model plasma and normal plasma. Substituting the APTT value of the above DIC model plasma (measurement sample) into each of the regression equations of the three calibration curves obtained based on the APTT value of the standard sample obtained in normal rats, the calculated value and the accuracy are calculated. Calculated (Table 20).
[0102]
[Table 20]

[0103]
As shown above, the reliability of the measured value tends to be slightly inferior between 50 and 100 μg / ml, but after that, it was found that the concentration of the measurement target substance was measured within a practical range.
[0104]
[Example 7]
(Quantification of specimen containing a plurality of GAGs by the combination of the present method 1 and the present method 2)
Quantification was performed on specimens containing multiple substances that affect blood clotting time. Taking dermatan sulfate as an example of a measurement target substance, heparin as an example of a substance other than the measurement target substance that affects the blood coagulation time, and rat plasma collected in the same manner as in Example 1 as a solvent for the measurement target substance. The following experiments were performed. Dermatan sulfate was dissolved in physiological saline so as to be 0 to 90 mg / ml and heparin to 900 μg / ml. 2.7 ml of plasma supplemented with 0.15 ml of physiological saline, 0.15 ml of physiological saline in which various concentrations of dermatan sulfate are dissolved, and 2.7 ml of plasma containing physiological saline containing heparin and 900 μg / ml Each sample was prepared by adding 0.15 ml of physiological saline in which 0.15 ml of various concentrations of dermatan sulfate was dissolved (measurement sample). These were heat-treated at 65 ° C. for 20 minutes, and then the freeze-dried enzyme (heparinase, 0.1 U Seikagaku Corporation) was added to the group to which heparin was added to decompose heparin (enzyme-treated specimen). Thereafter, 20 μl of these enzyme-treated specimens were taken, added to 80 μl of coagtrol 1, APTT values were measured in duplicate as in Example 1, and the average value was taken as the measured value (Table 21).
[0105]
[Table 21]

[0106]
Logit- with the APTT value of a sample containing only the measurement target substance (dermatan sulfate) and physiological saline that is a solvent that does not contain both the measurement target substance and the substance other than the measurement target substance that affects the clotting time (heparin) Log transformation was performed to create a regression line or regression curve (Table 22).
[0107]
[Table 22]

[0108]
Next, calculation obtained by substituting APTT values obtained after enzymatic treatment of specimens that contain the substance to be measured (dermatan sulfate) and substances that further affect blood coagulation time (heparin) into the above formulas. Values and theoretical values were compared (Table 23).
[0109]
[Table 23]

[0110]
As shown in the above table, it was found that the concentration of the sample after the enzyme treatment can be measured with the accuracy of the theoretical value and the error of 21.7% or less in the range of 1500 μg / ml or more.
[0111]
[Example 8]
(Quantification of heparin by the combination of the method 1 of the present invention and the method 2 of the present invention)
PT was applied in the same way as APTT. As an example of the substance to be measured, heparin was used. Heparin was dissolved in physiological saline at various concentrations and then added to rat plasma (specimen). At this time, consideration was given so that the added volume did not affect the total plasma volume. The sample was heat-treated at 65 ° C. for 20 minutes (heat-treated sample). After mixing 20 μl of heat-treated specimen with 80 μl of Coagtrol 1, measure PT using PT reagent (thrombocheck PT: green cross) prepared as described in the manual, and logit-log with the measured value as in APTT. Conversion was performed to create a regression line or regression curve (Table 24).
[0112]
[Table 24]

[0113]
[Example 9]
(Configuration and use of the kit of the present invention)
The heparin measurement kit of this example is composed of the following elements 1 to 7.
1. One vial of standard heparin saline (heparin 60 μg / ml (10.95 U / ml), 1 ml).
2. 1 vial of normal saline (for dilution of heparin, 1 ml).
3. Standard plasma (equal amount of fresh plasma obtained by collecting sodium citrate as an anticoagulant from 10 healthy individuals who are not infected with pathogenic microorganisms such as HIV, HCV, HBV as a result of antibody tests, and pooled Lyophilized product equivalent to 0.5ml, usually stored at 2-8 ° C) 1 vial.
4). One vial of distilled water (for standard plasma lysis, 0.5 ml).
5). 1 vial of APTT reagent (containing 0.2 mg / ml of rabbit-derived cephalin, 0.03 mg / ml of ellagic acid, 3 ml, usually refrigerated).
6). 1 vial of calcium chloride solution (0.02M, 4ml).
7). Software storage medium for measurement
[0114]
For use, after returning the standard plasma to room temperature, gently add 1 vial of distilled water without foaming, gently agitate to be uniform, and leave it for about 20 to 30 minutes. A solution obtained by serially diluting standard heparin physiological saline with physiological saline in a range of 3 to 60 μg / ml or physiological saline to 80 μl of the dissolved standard plasma is added uniformly so as not to form bubbles by 20 μl. After the solution is kept at 37 ° C. for 3 minutes, 100 μl of APTT reagent heated to 37 ° C. in advance is added, and the solution is further kept at 37 ° C. for 3 minutes. 100 μl of calcium chloride solution is added to the solution to start coagulation, and the time until coagulation is completed is measured. After measuring the theoretical value of heparin concentration and the respective clotting time, input the theoretical value and measured value, the heparin activity unit (U / mg) contained in standard heparin saline, and the regression method to the measurement software. Then create a calibration curve. The sample is heated at 56 ° C. for 20 minutes and then returned to room temperature, and 20 μl thereof is uniformly added to 80 μl of standard plasma dissolved in distilled water according to the above procedure so as not to generate bubbles. Incubate the solution at 37 ° C for 3 minutes, then add 100 µl of APTT reagent preheated to 37 ° C. Incubate the solution at 37 ° C for 3 minutes, then add 100 µl of calcium chloride solution to start coagulation. Let The time until the coagulation is completed is measured, the calibration curve created by the measurement software is called from the memory, the coagulation time of the specimen is input, and the concentration of the substance to be measured in the specimen is calculated from the measured value.
[0115]
【The invention's effect】
ADVANTAGE OF THE INVENTION According to this invention, the precision of the substance which affects the blood coagulation time is high, the operation is simple, and the quantification method which can process many specimens is provided.
[0116]
That is, according to one aspect of the quantification method of the present invention, the logarithmic value of the concentration of the measurement target substance solution (standard sample) having a known concentration and the ratio of the relative clotting time to the control clotting time ((TT ₀ ) / T ₀ Since the calibration curve is obtained using the logit value of) as a variable, it is possible to carry out highly accurate quantification over a wide measurement concentration range.
[0117]
According to another aspect of the quantification method of the present invention, the blood coagulation factor inherent in the sample containing the measurement target substance is inactivated under conditions that do not destroy the measurement target substance. Accurate quantification is possible without being affected by substances other than substances.
[Brief description of the drawings]
FIG. 1 is a block diagram showing an example of the configuration of a quantitative device of the present invention.
FIG. 2 is a flowchart showing a processing procedure of a program recorded on a medium included in the kit of the present invention.
FIG. 3 shows the relationship between APTT and logarithmic values of heparin, low molecular weight heparin, and dermatan sulfate concentration (μg / ml).
FIG. 4 shows the relationship between the logarithm of heparin concentration (μg / ml) and the logarithm of APTT.
FIG. 5 shows the relationship between the logarithmic value of the concentration (μg / ml) of low molecular weight heparin (LMWHep) and the logarithmic value of APTT.
FIG. 6 shows the relationship between the logarithmic value of dermatan sulfate (DS) concentration (μg / ml) and the APTT logarithm value.
FIG. 7 shows the relationship between the logarithmic value of heparin concentration (μg / ml) and the logit value of APTT.
FIG. 8 shows the relationship between the logarithmic value of the concentration (μg / ml) of low molecular weight heparin (LMWHep) and the logit value of APTT.
FIG. 9 shows the relationship between the logarithmic value of dermatan sulfate (DS) concentration (μg / ml) and the logit value of APTT.
FIG. 10 shows the relationship between the logarithmic value of dermatan sulfate (DS) concentration (μg / ml) in physiological saline and the logit value of APTT, and the regression equation (primary equation) of the calibration curve.
FIG. 11 shows the relationship between the logarithmic value of the dermatan sulfate (DS) concentration (μg / ml) in physiological saline and the logit value of APTT, and the regression equation (secondary equation) of the calibration curve.
FIG. 12 shows the relationship between the logarithmic value of dermatan sulfate (DS) concentration (μg / ml) in physiological saline and the logit value of APTT, and the regression equation (third order) of the calibration curve.
FIG. 13 shows the relationship between the logarithmic value of dermatan sulfate (DS) concentration (μg / ml) in liver homogenate and the logit value of APTT, and the regression equation (primary equation) of the calibration curve.
FIG. 14 shows the relationship between the logarithmic value of dermatan sulfate (DS) concentration (μg / ml) in liver homogenate and the logit value of APTT, and the regression equation of the calibration curve (secondary equation).
FIG. 15 shows the relationship between the logarithmic value of dermatan sulfate (DS) concentration (μg / ml) in liver homogenate and the logit value of APTT, and the regression equation (third order equation) of the calibration curve.
FIG. 16 shows the relationship between the logarithmic value of urine dermatan sulfate (DS) concentration (μg / ml) and the logit value of APTT and the regression equation (primary equation) of the calibration curve.
FIG. 17 shows the relationship between the logarithmic value of dermatan sulfate (DS) concentration (μg / ml) in urine and the logit value of APTT, and the regression equation (secondary equation) of the calibration curve.
FIG. 18 shows the relationship between the logarithmic value of urine dermatan sulfate (DS) concentration (μg / ml) and the logit value of APTT, and the regression equation (third order) of the calibration curve.
FIG. 19 shows the relationship between the logarithmic value of heparin concentration (μg / ml) in plasma and the logit value of APTT and the regression equation (primary equation) of the calibration curve.
FIG. 20 shows the relationship between the logarithmic value of plasma heparin concentration (μg / ml) and the logit value of APTT, and the regression equation (secondary equation) of the calibration curve.
FIG. 21 shows the relationship between the logarithmic value of low molecular weight heparin (LMWHep) concentration (μg / ml) in plasma and the logit value of APTT and the regression equation (primary equation) of the calibration curve.
FIG. 22 shows the relationship between the logarithmic value of low molecular heparin (LMWHep) concentration (μg / ml) in plasma and the logit value of APTT and the regression equation (secondary equation) of the calibration curve.
FIG. 23 shows the relationship between the logarithmic value of low molecular weight heparin (LMWHep) concentration (μg / ml) in plasma and the logit value of APTT, and the regression equation (third order) of the calibration curve.
FIG. 24 shows the relationship between the logarithmic value of chondroitin sulfate E (CSE) concentration (μg / ml) in plasma and the logit value of APTT, and the regression equation (primary equation) of the calibration curve.
FIG. 25 shows the relationship between the logarithmic value of chondroitin sulfate E (CSE) concentration (μg / ml) in plasma and the logit value of APTT, and the regression equation of the calibration curve (secondary equation).
FIG. 26 shows the relationship between the logarithmic value of chondroitin sulfate E (CSE) concentration (μg / ml) in plasma and the logit value of APTT, and the regression equation (third order) of the calibration curve.
FIG. 27 shows the relationship between the logarithmic value of heparin concentration (U / ml) in plasma and the logit value of APTT and the regression equation (primary equation) of the calibration curve.
FIG. 28 shows the relationship between the logarithmic value of heparin concentration (U / ml) in plasma and the logit value of APTT and the regression equation (secondary equation) of the calibration curve.
FIG. 29 shows the effect of sulfatide concentration (μg / ml) on APTT. The section A and the section B are a section where the APTT that has created the calibration curve is extended and a section where it is shortened, respectively.
FIG. 30 shows the relationship between the logarithmic value of plasma sulfatide concentration (μg / ml) and APTT logit value in the APTT prolongation interval, and the regression equation (primary equation) of the calibration curve.
FIG. 31 shows the relationship between the logarithmic value of plasma sulfatide concentration (μg / ml) and APTT logit value in the APTT prolongation interval, and the regression equation (secondary equation) of the calibration curve.
FIG. 32 shows the relationship between the logarithmic value of plasma sulfatide concentration (μg / ml) and APTT logit value in the APTT prolongation interval, and the regression equation (third order) of the calibration curve.
FIG. 33 shows the relationship between the logarithmic value of the plasma sulfatide concentration (μg / ml) in the APTT shortened section and the logit value of APTT, and the regression equation (primary equation) of the calibration curve.
FIG. 34 shows the relationship between the logarithmic value of the plasma sulfatide concentration (μg / ml) in the APTT shortened section and the logit value of APTT and the regression equation (secondary equation) of the calibration curve.
FIG. 35 shows the relationship between the logarithmic value of plasma sulfatide concentration (μg / ml) in the APTT shortened section and the logit value of APTT, and the regression equation of the calibration curve (third-order equation).

Claims (16)

A method for quantifying a substance that affects blood coagulation time, wherein the coagulation time is measured in a standard specimen containing the substance with a known concentration and the specimen to be measured whose concentration of the substance is unknown, and the coagulation in a reference standard specimen not containing the substance. Logit expressed by the following equation [2] of the logarithm of the concentration of the substance that affects the blood clotting time in the standard sample and the relative clotting time (Tr) expressed by the following equation [1] (Logit) A quantification method characterized in that a calibration curve is created using a value as a variable, and the concentration of the substance in the sample to be measured is obtained based on the obtained calibration curve.
[1] Tr = (T ₀ / T) x K
[2] Logit (Tr) ＝ Ln (Tr / | 1-Tr |)
(In the formula, Tr is a relative coagulation time, T ₀ is a coagulation time in the control sample, T is a coagulation time in the standard sample, K is an arbitrary constant satisfying 0.8 ≦ K ≦ 1.2, and Ln is a natural logarithm. )   The quantification method according to claim 1, wherein the constant K is 1.0.   The quantification method according to claim 1 or 2, wherein the method of creating the calibration curve is a polynomial regression method.   The measurement of the clotting time inactivates an intrinsic blood coagulation factor by treating only the specimen to be measured and the standard specimen or the specimen to be measured under conditions that do not destroy the substance, The quantification method according to any one of claims 1 to 3, which is performed by mixing both samples after inactivation with a certain amount of blood coagulation factor and measuring the coagulation time of the obtained mixture.   A method for quantifying a substance that affects blood coagulation time, wherein a blood coagulation factor inherent in a specimen to be measured whose concentration of the substance is unknown is inactivated under conditions that do not destroy the substance, and then the specimen And a standard sample containing the substance of a known concentration is mixed with a certain amount of blood coagulation factor, and the coagulation time of the obtained mixture is measured.   The method according to claim 4 or 5, wherein the inactivation is by heat treatment.   The quantitative determination method according to claim 6, wherein the condition of the heat treatment is 50 to 70 ° C.   The quantification method according to any one of claims 1 to 7, wherein the substance affecting the blood coagulation time is a saccharide or a complex carbohydrate.   The method according to claim 5, wherein the substance affecting the blood coagulation time is glycosaminoglycan or glycolipid. Quantitative device for substances that affect blood clotting time, measuring the clotting time of a standard sample containing a known concentration of the substance, a sample to be measured whose concentration of the substance is unknown, and a control standard specimen not containing the substance A calibration curve by a polynomial regression method using a logarithmic value of the known concentration and a logit value represented by the following equation [2] of the relative coagulation time (Tr) represented by the following equation [1] as variables. And a means for calculating the concentration of the substance from the coagulation time of the specimen to be measured based on the calibration curve. [1] Tr = (T ₀ / T) x K
[2] Logit (Tr) ＝ Ln (Tr / | 1-Tr |)
(In the formula, Tr is a relative coagulation time, T ₀ is a coagulation time in the control sample, T is a coagulation time in the standard sample, K is a constant satisfying 0.8 ≦ K ≦ 1.2, and Ln is a natural logarithm.)   The quantitative device according to claim 10, wherein the constant K is 1.0.   The quantitative device according to claim 10 or 11, wherein the substance affecting the blood coagulation time is a carbohydrate or a complex carbohydrate.   The quantitative device according to claim 10 or 11, wherein the substance affecting the blood coagulation time is glycosaminoglycan or glycolipid. A kit for performing a method for quantifying a substance that affects blood coagulation time, comprising a standard specimen containing a known concentration of the substance, a blood coagulation factor , an activator, a standard specimen containing the known concentration of the substance, and the above By entering the clotting time in a control sample that does not contain a substance, the logarithmic value of the concentration of the substance that affects the blood clotting time in the standard specimen and the relative clotting time ( Tr ) represented by the following formula [1] by the procedure for calculating the calibration curve and logit (logit) value as a variable represented by the following formula [2], and the concentration of the substance to enter the coagulation time of the sample to be measured having an unknown, to be measured A kit comprising a medium recording a program for causing a computer to execute a procedure for calculating the concentration of the substance in a sample based on the calibration curve .
[1] Tr = (T ₀ / T) × K
[2] Logit (Tr) = Ln (Tr / | 1-Tr |)
(In the formula, Tr is a relative clotting time, T ₀ is a clotting time in the control sample, T is a clotting time in the standard sample, K is an arbitrary constant satisfying 0.8 ≦ K ≦ 1.2 , and Ln is a natural logarithm. ) The kit according to claim 14, wherein the substance affecting the blood coagulation time is a carbohydrate or a complex carbohydrate . The kit according to claim 14, wherein the substance affecting the blood coagulation time is a glycosaminoglycan or a glycolipid .

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