JP4078460B2 - Plasma separation filter, plasma separation method and plasma separation apparatus using the same - Google Patents

Description

本願発明において、動水半径は下記の式（１）により計算される：
ＤＨ＝Ｒ×（ρ−ｒｍ）／４ｒｍ （１）
ＤＨ：装着された極細繊維の集合体の平均動水半径（μｍ）
Ｒ ：極細繊維の平均繊維直径（μｍ）
ρ ：極細繊維の密度（ｇ／cm³）
ｒｍ：装着された極細繊維の集合体の平均嵩密度（ｇ／cm³）
式（１）に示されるように、装着された極細繊維の集合体の平均動水半径ＤＨは、同じ素材の極細繊維を用いた場合（つまり、ρが一定の場合）、Ｒおよびｒｍにより決定される。
【００４０】
上記平均動水半径が3.0μｍを超える場合には、血球が繊維間隙を通過し易くなる。この場合は、血液から白血球を除去する白血球除去フィルターまたは白血球分離フィルターなどの従来のフィルターと同様の効果を与えるにすぎない。白血球（一部血小板を含む）のように特異的な粘着性をもつ成分のみが極細繊維の集合体に吸着除去されることになる。
【００４１】
平均動水半径が0.5μｍ未満の場合、フィルター内の繊維間隙、すなわち、血液の流路が狭くなりすぎて血球成分が目詰まりを起こし易い。また、通過する液量を増加しようとしてフィルターに圧力をかけると圧力損失が大きくなり、赤血球の溶血が生じ易くなる。
【００４２】
なお、平均動水半径０．５μｍ〜３．０μｍの範囲においては、平均動水半径が小さいほど血漿成分のように粒子径が小さいものの透過性に影響を与えることがない。他方、血球成分のような粒子径の大きいものはフィルター内を通過しにくくなる。従って、平均動水半径は０．５μｍ〜２．８μｍが好ましく、特に好ましくは、０．５μｍ〜２．０μｍである。
【００４３】
さらに、本願発明の極細繊維の集合体の動水半径は、血液の供給側から血漿の出口側に至る軸方向にわたって一定であり得、また、極細繊維の集合体の部分により変化させ得る。また、動水半径は、血液の入口から通過液の出口に向かって徐々に小さくされ得る。このことにより、血漿の出口付近での血球成分と血漿成分との分離効率が高くされ得る。
【００４４】
なお、本願発明において、平均動水半径というときは、入口と出口とを有する容器に装着された状態の極細繊維の集合体であって、実質的に血漿分離に関与し得る極細繊維の集合体における平均動水半径をいう。従って、例えば、図２の空隙１６を埋めるための基材１７として極細繊維の集合体を用いた場合（図３）は、基材１７の極細繊維の集合体は血漿分離に関与し得ないので、この部分を除いた部分の平均動水半径をいう。
【００４５】
このことを別の側面からみると、フィルターに装着された極細繊維の集合体を全体としてみたときに、上記好ましい範囲外の平均動水半径となる場合があることを示している。しかし、この場合であっても血漿を分離し得るということは、少なくとも装着された極細繊維の集合体の一部が、上記好適な範囲の平均動水半径を有することを示すことに他ならない。
【００４６】
本願発明においては、血漿分離用の極細繊維の集合体の前に血液中の異物を取り除くプレフィルターを設置し得る。これらのプレフィルターの平均細孔径や平均動水半径は当然、該極細繊維の集合体の平均動水半径より大きくなるが、フィルター全体としての平均動水半径にはプレフイルターの平均孔径は考慮せず、メインフィルター部分の平均動水半径を用いなければならない。
【００４７】
本願発明において、極細繊維の集合体は、極細繊維が不規則に集合した状態をいう。この状態は、例えば、綿状、不織布状、織布状、編布状の極細繊維を、単独で、あるいは組み合わせて、例えば圧縮することにより得られ得る。フィルターに装着する極細繊維は、成形性、加工性、取り扱いの容易さ、および成形後のチャネリングや偏流の起こり難さの点から、好ましくは、不織布状または綿状の極細繊維である。特に、不織布状であることが好ましい。極細繊維の集合体をフィルターケースに充填したとき、均一性が保たれ易く、また部分的な粗密が出来難く、血液の流れが均等になるからである。
【００４８】
極細繊維の素材は特に限定されず、例えば、ポリエステル、ポリプロピレン、ポリアミドまたはポリエチレンなどが挙げられる。好ましくは、疎水性のポリプロピレンやポリエステル系の素材（例えば、ポリエチレンテレフタレート）が使用され得る。
【００４９】
これらの素材は血液と接触するとき、血漿あるいは血清の成分を吸着したり、逆に血漿あるいは血清中に素材の一部が溶出することがなく好ましい。従来技術の項で述べたようにガラス繊維を用いる血漿あるいは血清分離フィルターを用いると、ガラス繊維中から金属イオンが溶出したり、リンや脂質がガラス繊維に吸着するため、これらの物質を測定することが出来なかった。
【００５０】
本願発明における血球分離層の長さは５ｍｍ以上が好ましい。血球分離層の長さとは極細繊維の集合体と血液とが接触するところから、血液（血漿あるいは血清）が極細繊維の集合体と離れるところまでの長さをいう。前述したように、本願発明は、極細繊維の集合体中における血液成分の移動速度の差を利用して、血球と血漿とを分離している。血球分離層の入口側からの加圧、あるいは出口側からの減圧、あるいは両方を同時に行うことにより、血液を極細繊維の集合体中で移動させる。このとき、血球成分は、極細繊維の間隙を極細繊維と衝突を繰り返す。粘着性をもつ白血球や血小板は極細繊維に吸着し、赤血球は粘着性を持たないので、変形を繰り返しながら移動する。他方、血漿あるいは血清は、液性成分であるので、赤血球より早く極細繊維中を移動し、出口に早く到達する。このとき、血球分離層の長さが５ｍｍ未満であると、血球と血漿の移動距離に充分な差が生じず、両者の分離が不十分となるので好ましくない。血球分離層の長さは長いほど、血球と血漿の分離効率は高くなるが、他方で、圧力損失が大きくなる、あるいは必要な極細繊維の量や血液量が増加するという問題が生じる。従って、必要とする血漿あるいは血清量、用いる血液量、フィルターの大きさの限度等により、血液分離層の長さは決定されるが、理論上の上限値は存在しない。
【００５１】
不織布を用いる場合、血液が不織布の平面（積層された不織布の平面）に対して平行に流動することが好ましい。一般に不織布をフィルターとして用いる場合、処理液体の流動方向は不織布の平面（積層された不織布の平面）に対して垂直方向とされている。しかし、本発明においては、不織布の面に対して平行に血液を流動させることにより、血球と血漿成分の分離効率が向上する。不織布の平面方向に対して平行に血液を流すと、血液が入口から出口まで流れるときに、全流路長にわたって、極細繊維の切れ目がなく、そのために、血液の流れの均一性が向上するためと考えられるが、この理由に限定されない。
【００５２】
極細繊維に親水化剤が固定されたものも、本願発明のフィルターとして用いられ得る。親水化剤の固定は、物理的あるいは化学的に行われ得る。親水化剤を繊維表面に固定することにより、極細繊維と血液との親和性が高まる。従って、血液を血球と血漿とに分離する際、圧力損失が低下し、分離速度が早められ得る。親水化剤としては、血漿あるいは血清に混入しても分析を妨害しないものであれば、特に限定されない。ポリビニルピロリドンが好ましい。ポリビニルピロリドンは、分子量が比較的大きいため溶出速度が比較的遅いものの、血液中に溶出する。しかし、血液成分の分析に影響しない。ポリビニルピロリドンの固定化方法は特に限定されなず、公知の方法が用いられ得る。例えばポリビニルピロリドンの水溶液を極細繊維の集合体に浸した後に乾燥することで容易に繊維表面に物理的に固定化できる。また、このようなポリビニルピロリドンを物理的に表面に固定化した極細繊維の集合体を加熱処理、放射線処理することにより容易にポリビニルピロリドンどうしを架橋させ得る。架橋することにより、血液中へのポリビニルピロリドンの溶出をより低く抑え得る。
【００５３】
加熱処理する場合、その方法は特に限定されない。例えば、オートクレーブ処理のように加圧下において加熱する方法、あるいは恒温槽内に放置する方法等が挙げられる。また、加熱処理の温度も特に限定されないが、７０℃以上が好ましく、１００℃以上がさらに好ましい。加熱温度は高いほど架橋の効率が向上する。上限温度は、用いる極細繊維の性質、ポリビニルピロリドン自体の耐熱性などにより、一義的に決まらないが、２００℃以下が好ましく、１５０℃以下がさらに好ましい。加熱時間は、架橋を充分に行うため、長い方が好ましいが、用いる極細繊維の性質、ポリビニルピロリドンの変性の面から制限を受ける。一般的には、２０分以上２時間以下が好ましい。また、加熱による架橋は、極細繊維が親水化剤水溶液に浸漬している場合（ＷＥＴ状態）、あるいは浸漬後乾燥した場合（ＤＲＹ状態）のいずれにおいても行われ得る。いずれの場合においても、ポリビニルピロリドンが極細繊維に固定され得る。未反応のポリビニルピロリドンは水で洗浄することにより取り除かれ得る。
【００５４】
放射線を用いて親水化剤を固定する方法も特に限定されない。例えば、γ線照射や電子線照射、コロナ放電等が挙げられる。処理できる厚みや、操作性の面からγ線照射が好ましい。照射量についても、ポリビニルピロリドンが充分に架橋される照射量であれば特に限定されない。放射線照射による極細繊維素材やポリビニルピロリドン自体の変性を起こさない範囲として１０ＫＧｙ以上、５０ＫＧｙ以下が好ましい。また、放射線照射はＷＥＴ状態あるいはＤＲＹ状態で行われ得る。未反応のポリビニルピロリドンは、水洗浄等で取り除かれ得る。
【００５５】
ポリビニルピロリドンは種々の分子量のものが入手され得る。血液中への溶出を避けるために、大きい分子量のものを用いることが特に好ましい。
【００５６】
この親水化剤が固定された極細繊維の集合体を用いてフィルターを作製する。
【００５７】
フィルターの作製は、綿状、不織布状、織布状、編布状の極細繊維を、単独で、あるいは組み合わせて、積層し、圧縮することにより行い得る。
【００５８】
本願発明において、フィルターの容器の形状は特に限定されない。直方体状、円盤状、円筒状、円錐台状、扇型状などが挙げられる。これらのうち、直方体状、円盤状あるいは扇形状のフィルターを用いて極細繊維の集合体の面に対して平行に流す場合分離性能が向上する。直方体状の場合、一方の端面から他方の端面に血液を流す、あるいは、円盤状に形成し、その周囲面から中心に向かって血液を流す。これらの形状の容器で不織布を押圧することにより、フィルターをシールし得る。従って、接着剤を用いる必要がなくなるので特に好ましい。中でも、円盤状あるいは扇形状の場合には血液が移動するに従って、血液の流路の断面積が徐々に小さくなるため、血液成分の横方向への移動のムラが小さくなるのでより好ましい。特に円盤状のフィルターは操作性に優れ、最も好ましい。なお、円盤状の容器の場合、装着される不織布も円盤状となる。この円盤状の不織布の円周部全面にわたって血液を供給し得るように血液の入口を構成することが好ましい。例えば、円盤状の不織布と円盤状の容器の円周部に隙間を設け、かかる隙間に連通する１個または複数個の入口が容器の側面、上面あるいは下面に開口し得る。
【００５９】
本願発明のフィルターに用いる極細繊維の平均繊維直径は、好ましくは、０．５〜３．５μｍであり、さらに好ましくは０．５〜２．８μｍであり、特に好ましくは０．５〜２．０μｍである。
【００６０】
上記の平均繊維直径を有する極細繊維は、メルトブローなどの通常の極細繊維紡糸法により得られ得る。
【００６１】
ここで、極細繊維の平均繊維直径は、極細繊維の集合体を２０００倍の電子顕微鏡で撮影した写真の中からランダムに選択した５０本の極細繊維の径をノギスまたはスケールルーペで計測して求めた値の平均値をいう。
【００６２】
繊維の平均繊維直径が３．５μｍを超えると、極細繊維の集合体の単位体積あたりの繊維の長さが短くなる結果、繊維と繊維との交絡箇所が少なくなり、繊維間隙が大きくなる。その結果、血球のように粒子径の大きな成分も極細繊維の集合体を通過しやすくなり、血球と血漿の分離が悪くなる。さらに白血球や血小板に対する吸着性が低下する。
【００６３】
極細繊維の平均繊維直径が０．５μｍ未満の場合には、単位体積あたりの繊維の長さが長くなる結果、繊維と繊維との交絡箇所が多くなり繊維間隙が小さくなる。その結果、血球がフィルターに詰まり易くなる。さらに極細繊維の集合体の圧力損失が大きくなるために赤血球の溶血が起こり易くなる。
【００６４】
本願発明に用いられる装着された極細繊維の集合体の平均嵩密度は、好ましくは０．１５〜０．６０ｇ／ｃｍ³であり、さらに好ましくは０．１８〜０．５０ｇ／ｃｍ³であり、特に好ましくは０．２５〜０．４０ｇ／ｃｍ³である。
【００６５】
ここで、極細繊維の集合体の平均嵩密度とは、極細繊維の集合体の重量を極細繊維の集合体の容積で除した値をいう。
【００６６】
平均嵩密度が０．１５ｇ／ｃｍ³より小さい場合、極細繊維の集合体の紡糸直後の平均嵩密度（たとえばメルトブロー紡糸法の場合、０．０８〜０．１０ｇ／ｃｍ³）との差が小さいため、極細繊維の集合体の圧縮率が小さくなる。従って、装着された極細繊維の集合体において部分的に疎密が発生し易く、血液の通過速度にむらが生じ得る。また、平均的に繊維間隙が大きいため、血液と血漿との分離が不十分になる。
【００６７】
装着された極細繊維の集合体の平均嵩密度が０．６０ｇ／ｃｍ³より大きい場合、極細繊維の集合体の製造に特殊な加熱圧縮などの工程を必要とし、圧縮加工工程が複雑になる。さらに、装着された極細繊維の集合体の繊維間隙が小さくなるために血球成分がフィルターに目詰まりを起こし易く、そして、極細繊維の集合体の圧力損失が大きくなるために、赤血球の溶血が起こり易くなる。
【００６８】
なお、平均嵩密度０．１５〜０．６０ｇ／ｃｍ³の範囲において、平均嵩密度を大きくすることにより、極細繊維の集合体の均一度は、より向上する。他方、加工性は低下するので、好ましくは０．１８〜０．５０ｇ／ｃｍ³であり、特に好ましくは０．２５〜０．４０ｇ／ｃｍ³である。
【００６９】
本願発明のフィルターに装着される極細繊維の集合体の嵩密度は部分的に変化させ得る。例えば、フィルターの容器の入口から出口に向かって、装着された極細繊維の集合体の嵩密度を徐々に大きくし得る。このようにすると、血液成分が出口に向かって移動するに従って血球と血漿との分離性が向上される。
【００７０】
本願発明の装置のフィルターに装着される極細繊維の集合体の血液成分の流路径（Ｄ）に対する流路長（Ｌ）の比（Ｌ／Ｄ）は、０．１５〜６である。好ましくは０．２５〜４であり、特に好ましくは０．５〜２である。
【００７１】
ここで、血液成分の流路長（Ｌ）とは、血液が極細繊維に接触してから、血漿が極細繊維を離脱するまでの極細繊維の集合体内部の直線距離（通常、極細繊維の集合体の長さ）をいう。また、血液成分の流路径（Ｄ）とは、流路長の方向と垂直方向をなす血液入口部の極細繊維の集合体表面の断面積の円相当直径をいう。
【００７２】
円相当直径は、断面積（Ａ）から、次式（２）により求める。
【００７３】
Ｄ＝２（Ａ／π）^1/2 （２）
なお、極細繊維の集合体表面は、厳密には極細繊維の折れ曲がりに起因する小さな凸凹を有するが、上記断面積はこの凸凹を無視して、平面として算出する。また、極細繊維の折れ曲がりに起因する凸凹以外に、極細繊維の集合体の表面加工などにより形成された大きな凸凹を有する場合は、上記断面積は、凸凹部を平均化した平面として算出する。
【００７４】
Ｌ／Ｄが０．１５より小さい場合、流路が短すぎて血液中の各成分の移動距離が短くなる。さらに、断面積が大きいため血液中の各成分の移動速度が横方向にムラを生じる。従って、血球成分と血漿成分との分離が不十分になる。
【００７５】
Ｌ／Ｄが６を超える場合、分離効率は向上するが、移動距離が長くなるために圧力損失が高くなり、赤血球の溶血が生じ得る。
【００７６】
本願発明のフィルターは、以下の特性を有する。
【００７７】
赤血球濃度が６〜８×１０⁹個／ｍｌである新鮮牛血液を０．２〜０．４Kg/ｃｍ²の圧力で分離し、分離開始から該装着された極細繊維の集合体の間隙体積の１０％に相当する量の血漿を回収したときに、
(a)血漿中の赤血球濃度が分離前の血液中の赤血球濃度に対して０．１％以下である；および
(b)実質的に赤血球が溶血していない。
【００７８】
さらに、分離された血漿中の電解質濃度が、遠心分離で得られた血漿中の電解質濃度と比較して９０％以上保持されている。
【００７９】
本願発明において、血漿分離フィルターにて分離された血漿中の電解質濃度が、通常の遠心分離して得られた血漿中の電解質濃度と比較して９０％以上保持されていることが好ましい。つまり、フィルター分離後の電解質濃度の減少が遠心分離した場合と比較して１０％以内であることが好ましい。５％以内であれば、より好ましい。血漿中の電解質濃度の減少が１０％を越えると、生化学診断の信頼性が低くなり好ましくない。生化学検査の測定精度からみて、両者の差が１０％以内であれば、事実上を問題を生じるレベルでなく、５％以内であればほとんど問題はない。ここで、血漿とは、採血で得られた血液に抗凝固剤を添加した後、遠心分離して得られた上清いう。なお、遠心分離は、1000Ｇ、１０分間行う。
【００８０】
本願発明においては、血漿分離フィルターにて分離された血漿の採取初期および採取終了時の蛋白質濃度が、９０％以上保持されていることが好ましい。つまり、フィルター分離後の蛋白質濃度の減少が遠心分離した場合と比較して１０％以内であることが好ましい。５％以内であれば、より好ましい。血漿中の蛋白質濃度の減少が１０％を越えると、生化学診断の信頼性が低下すると共に、採取初期と採取終了時の値が異なり正確な診断が出来なくなることがある。また、タンパク質濃度の差が１０％を越えると、血漿タンパク質の組成が大きく変動する事があり、病状の診断に用いることが出来なくなることがあり好ましくない。通常１０％以内の差であれば、臨床診断上、大きな問題はなく、５％以内の差であれば測定の誤差範囲内におさまるので好ましい。
【００８１】
本願発明のフィルターの容器の材質は特に限定されず、例えば、金属、ガラス、プラスチック類、たとえばポリエチレン、ポリプロピレン、ナイロン、ポリカーボネート、ポリスチレン、ポリエステル、ＡＢＳ樹脂などが挙げられる。内部の状態を観察する場合には、透明、半透明の材質を選択すればよい。加工性、割れにくさ、軽量の点からはプラスチック類が好ましい。
【００８２】
以下、本願発明の血漿分離装置に用いられるフィルターを、図面を用いて説明する。本願発明のフィルターは、最も単純には、図１に示される。図１は、入口１２、および出口１３を有する容器１５に、円筒形の扁平の極細繊維の集合体１４が装着されたフィルター１１を示す図である。このフィルター１１においては、極細繊維が圧縮されて、容器１５の内周部表面と極細繊維の集合体１４の外周部表面との間が適当な空隙１６を有するように装着されている。この装着された極細繊維の集合体１４の外周部は、血液が通過し得る適当な孔を有する、例えばプラスチックの板で構成され得る。円筒形に装着された極細繊維の集合体１４の外周部の上部にある容器１５の入口１２から血液を供給すると、空隙１６を通って、血液が装着された極細繊維の集合体１４に均一に供給され、極細繊維の集合体１４の外周部から中心部に向かって血液が流れ、その間に血漿と血球とに分離され、容器１５の中心部にある出口１３から血漿が回収される。以下、単に、極細繊維の集合体１４というときは、フィルターに装着された極細繊維の集合体を意味する。
【００８３】
別の実施態様として、図２のフィルター１１がある。このフィルター１１は、円筒形の容器１５に装着された円筒形の極細繊維の集合体１４を有する。容器１５の入口１２から血液を供給し、極細繊維の集合体１４で分離された血漿が、容器１５の出口１３から回収される。入口１２と出口１３は、それぞれ、任意の位置に配置され得る。
【００８４】
図１のフィルターと同様に、図２の円筒形の極細繊維の集合体１４においても、入口１２の近傍に、空隙１６を有し得る。空隙１６は、血液の供給、加圧を均等に行う場合等に配置され得る。
【００８５】
図３のフィルター１１は、図２のフィルター１１の空隙１６に、適当な基材１７を配置したフィルターである。配置される基材１７としては、極細繊維の集合体１４の繊維間隙よりも大きな繊維間隙を有する基材であり、血液中の全成分を通過させ得るものであれば、どのようなものでも使用され得る。この基材は、例えば、板状、紙状、繊維状等の形状の濾材であり得る。図１のフィルターにおいても、空隙１６には、基材１７が配置され得る。
【００８６】
図４のフィルター１１は、開口部１８と出口１３を有する容器１５に、極細繊維の集合体１４が装着されたフィルターである。
【００８７】
フィルター１１は、以下に述べる血液の供給手段および／または血漿回収手段とに着脱自在に取り付けられ得る。また、別の実施態様として、図５（ａ）および図５（ｂ）に例示すように、容器１５は、入口１２または開口部１８を有する部分（図５の１５ａ）と、極細繊維の集合体１４を有する部分と、出口１３を有する部分（図５の１５ｂ）との３部分とから構成され、各部分が着脱自在に構成され得る。この構成により、装着される極細繊維の集合体の部分のみが交換され得る。交換され得る極細繊維の集合体部分は、血液透過可能な膜などで被覆され得、着脱自在な形で嵌合可能なカセットの形態とされ得る。着脱自在な装着手段としては、嵌合、螺合などの手段、マグネットによる手段などがあり得る。これらの着脱は自動的に行われ得る。
【００８８】
上述のように、本願発明のフィルターが極細繊維を積層して作製される場合には、血液を流す方向は、フィルターは積層面に対してほぼ垂直か、あるいは積層面に対してほぼ平行であり得る。前者の場合は柱状あるいは錘状の形状が適している。例えば円柱状のフィルター（図２〜４）の上部から下部に向かって血液を流す場合、あるいは頂点部分を円錐状に切除した円錐台状のフィルターを用いて円錐の底面から頂点方向に向かって血液を流す場合等が該当する。後者の場合は、円盤状に積層したフィルター（例えば、図１）を用いて、積層面に平行に周囲から中心に向かって、あるいは平板状に積層したフィルターの一方の側面から対抗する側面方向に向かって、血液を流す場合が該当する。円錐台状、あるいは円盤状のフィルターは、血液が移動するに従って、血液の流路の断面積が徐々に小さくなるため、血液成分の移動速度が速くなり、横方向への血液成分の移動のムラが小さくなるので、血液の分離効率が向上する。
【００８９】
本願発明のフィルターは複数個連結され得る。直列に連結すると分離能が向上する。並列に連結すると処理する血液量が増大する。
【００９０】
さらに、フィルターと血液のサンプリング容器とを結合させて使用され得る。このようなサンプリング容器と結合したフィルターは、一定量の血液を採取してフィルターに供給する手段および使用したフィルターを自動的に交換する手段を有する自動分析装置に用いられ得る。
（血漿分離方法）
血漿を分離する方法は、出口における圧力損失が０．０３〜５ｋｇ／ｃｍ²である。この値は、フィルターの形状、血液の処理速度に依存する。
【００９１】
圧力損失が０．０３ｋｇ／ｃｍ²より小さい場合には、極細繊維の集合体内の血液に対する負荷が小さい。従って、疎水性が高い極細繊維を使用した場合には血液を極細繊維の集合体内に送ることができない、あるいは処理に時間がかかりすぎるという問題がある。また、極細繊維の集合体内の血球成分と血漿成分との移動速度に差が生じず、血漿の分離が不十分となる。
【００９２】
圧力損失が５ｋｇ／ｃｍ²より大きい場合、血液の流速が大きすぎる。従って、血漿と血球との通過時間の差が小さいため、血漿が分離されないことがある。さらに、圧力がかかるために、赤血球が溶血したり、フィルターあるいは装置が損傷する場合がある。
【００９３】
上記範囲内では圧力を大きくすると得られる血漿量が多くなり、血漿を得る時間が短縮される。他方で、溶血の可能性があるので、０．０５〜３ｋｇ／ｃｍ²の範囲が好ましい。０．１〜１ｋｇ／ｃｍ²の範囲がより好ましい。
【００９４】
本願発明の血漿分離方法においては、処理血液量と極細繊維の総面積との割合は、特に限定されないが、血液１ｍｌあたり、０．１〜３ｍ²が好ましい。処理血液量と極細繊維の総面積との割合血液１ｍｌあたり、０．１ｍ²より小さい場合、血漿の分離効率が悪くなる。処理血液量と極細繊維の総面積との割合が血液１ｍｌあたり３ｍ²より大きい場合は、血液量が少なすぎるため、血液の送液および血漿の回収が困難になる。さらに、極細繊維に血漿蛋白が吸着される結果、血漿成分中の蛋白成分が、元の血液中の蛋白成分を正確に反映しない場合が生じる。さらに、圧力損失も起こりやすくなる。
【００９５】
上記範囲内では極細繊維の表面積を大きくすると得られる血漿量が多くなる。他方で、繊維に吸着される蛋白量も増加する。従って、上記割合は、０．２〜２ｍ²の範囲が好ましい。０．３〜１．５ｍ²の範囲がより好ましい。
【００９６】
本願発明の血漿分離方法においては、血液の処理の線速度は、特に限定されるわけではないが、０．０５から５０ｃｍ／分程度である。線速度が、０．０５ｃｍ／分より小さいと、極細繊維の集合体中を血液成分が通過する時間が長くなる。その結果、分離した血漿と血球が移動中に拡散して、分離が不十分になる。
【００９７】
処理線速度が５０ｃｍ／分より大きいと、圧力損失が大きくなり、赤血球が溶血する。
【００９８】
なお、円盤状（例えば図１）の外周部から中心部に向かって送液する場合、あるいは円錐状の容器を用いて、円錐の頂点方向に向かって送液する場合には、場所により線速度が異なる。このような場合の線速度は、血液が極細繊維と接触してから離れるまでの平均処理線速度をいう。
【００９９】
本願発明の血漿分離方法においては、処理する血液量（Ｂ）と極細繊維の集合体の間隙体積（Ａ）との比（Ｂ／Ａ）は、０．２以上であることが好ましい。比が０．２より小さい（血液の容量が間隙体積の２０％より小さい）場合、血液量が少なすぎるため、血液の送液および血漿の回収が困難になる。さらに、極細繊維に血漿蛋白が吸着される結果、血漿成分中の蛋白成分が、元の血液中の蛋白成分を正確に反映しない場合が生じる。比は０．５以上が好ましく、０．７以上が特に好ましい。
【０１００】
本願発明の血漿分離方法においては、血液を供給、加圧する方法として、ピストン、加圧空気などを用い得る。また血液成分とは反応しない液体、例えば混合を防ぐために粘度の高い液体、パラフィン、グリセリンなどを用いて押し出す方法がある。加圧空気あるいはパラフィンなどを用いる方法は、処理する血液量が極細繊維の集合体の間隙体積よりも少ない容量である場合に適する。しかし、加圧空気の場合は、極細繊維の集合体中の間隙で生じる表面張力のために極細繊維の集合体中の血液が流動できなくなる場合がある。これに対して、パラフィンあるいはグリセリンを用いる方法は、表面張力の問題が生じないので、好ましい。
【０１０１】
（血漿分離装置）
本願発明の血漿分離装置は、血漿分離フィルターを含み、好ましくは該フィルターに血液を供給する血液供給手段、供給された血液を加圧する加圧手段、および分離された血漿を回収する血漿回収手段を含む。さらに、分離された血漿中の血球および／またはヘモグロビンを検出する検出手段、血球および／またはヘモグロビンが混入する血漿を分別するための切替手段、血球および／またはヘモグロビンが混入する血漿を回収する血球および／またはヘモグロビン含有血漿回収手段、血球および／またはヘモグロビンが混入しないことが確認された血漿を回収する血漿回収手段を含み得る。さらに、上記血液供給手段は一定量の血液を供給する血液供給手段であり得、血漿回収手段は一定量の血漿を回収する血漿回収手段であり得る。以下に上記の各手段について説明する。
【０１０２】
＜血液供給手段＞
本願発明の装置において、血液をフィルターに供給する手段としては、既知の手段が使用され得る。例えば、ポンプを用いて血液を供給する手段、血液の入った容器を加圧し、その圧力を利用して血液を供給する手段、フィルターの内部を減圧し、血液保存容器から圧力差を利用して血液を供給する手段（例えば、真空ポンプを用いる方法、あるいは図６（ｂ）において、ピストンを上方に移動してシリンダー内部を減圧し、入口１２より血液を導く方法）などが挙げられる。もちろん、ピペットなどの手動により血液を供給する手段も含まれ得る。血液をフィルターに供給する手段は、フィルターの入口に接続され得る。接続方法は、血液が漏出しない方法であればいかなる方法をも使用され得る。例えば、血液供給手段の血液の出口と、フィルターの入口とが嵌合、継合、あるいは螺合し得る構成とすることにより接続され得る。
【０１０３】
さらに、血液供給手段の出口を密閉し、フィルターの入口に穿孔可能な手段を配置する構成とし得る。逆に、フィルターの入口を密閉し、血液供給手段に穿孔可能な手段を配置する構成とし得る。穿孔可能な手段としては、中空で管状のもの、例えば、注射針などが挙げられる。いずれの場合も、穿孔により血液がフィルターに供給され得るようになる。このような穿孔可能な接続手段は、血液供給の自動化、血液との接触を避ける場合に特に有効である。近年用いられている真空採血管で採取した血液から血漿を分離する場合は、血液の入った真空採血管を直接、フィルターの密閉された入口に穿孔する。フィルターの容器内を減圧することにより、血液がフィルターに装着された極細繊維の集合体に供給される。血液が供給されたら真空採血管を引き抜き、フィルターの入口側から加圧するという装置も可能である。
【０１０４】
血液の供給手段は、一定量の血液を供給する供給手段であり得る。例えば、ローラーポンプ、シリンダーポンプ等の一定量の血液供給装置が用いられ得る。血液が一定流量供給されたときに、供給手段を停止させ得る。たとえば、血液の貯留装置にセンサーを設け、血液貯留槽の血液の減少量を測定して、供給手段を停止させ得る。
【０１０５】
本願発明の装置により処理される血液成分の容量（上記供給手段による血液の供給量）は、好ましくは、フィルターに装着された極細繊維の集合体の間隙体積の20％以上であり、さらに好ましくは50％以上であり、特に好ましくは70％以上である。20％未満である場合、装着された極細繊維の集合体内に血液を送ることが困難となり得、血漿成分の一部が間隙中に残留したまま回収されないおそれがある。ここで、装着された極細繊維の集合体の間隙体積は、装着された極細繊維の集合体の体積−（装着された極細繊維の集合体の総重量／装着された極細繊維の密度）として定義される。
【０１０６】
＜加圧手段＞
装着された極細繊維の集合体に保持または供給された血液は、この装着された極細繊維の集合体の血液側を加圧および／または透過液側を減圧することにより、この装着された極細繊維の集合体内を透過液側に移動し、血漿と血球との移動速度の差を利用して血漿と血球とに分離される。このとき、血球成分である白血球および血小板は装着された極細繊維の集合体に吸着される。加圧および減圧する方法は特に限定されない。ポンプによる加圧、気体例えば加圧空気による加圧、血液成分と反応および相溶しない液体（例えば、パラフィン、グリセリンなど）を加圧して押し出す方法、あるいは真空ポンプやシリンダーを用いて吸引する方法などが挙げられる。血液成分と相溶しないパラフィンなどで押し出す方法は、特に血液の量が少ないときに有効であり、血液成分の表面張力による流動性の問題を生じることがないので好ましい。
【０１０７】
加圧手段を有する分離装置の一例を図６（ａ）および図６（ｂ）に示す。図６（ａ）の装置では、フィルターの開口部１８から容器１５の内壁１３と気密に密接する密接手段２２を有する摺動可能な加圧手段２４を挿入して、開口部１８から供給した血液を加圧する。加圧された血液は、極細繊維の集合体１４を通過する間に分離され、出口１３から血漿が回収される。
【０１０８】
図６（ｂ）の装置では、フィルター１１の容器１５の内壁１９と気密に密接する密接手段２２を有する加圧手段２４が摺動可能にとりつけらている。この装置は、入口１２に弁２０が設けられている。そして加圧手段２４の上方への移動に伴い、内部が減圧されて弁２０が開き、血液が入口１２から供給される。さらに加圧手段２４の下方への移動に伴い、入口１２の弁２０を閉じると同時に血液が加圧され、血漿が分離される。この手順を繰り返すことにより、連続的に血漿が分離され得る。この場合、ピストンへの加圧手段としては公知の手段が用いられ得る。
【０１０９】
本願発明の装置においては、血液供給手段が加圧手段としても使用され得る。図７はその一例を示す図である。図７では、フィルター１１の入口１２と、血液供給手段２５とが接続されている装置である。血液供給手段２５は、その内壁２３と気密に密接する密接手段２２とを有する部材２４が配置されており、内部に血液２６が貯留されている。部材２４を下に押し下げると、血液２６がフィルター１１に供給され、加圧されて、血漿が分離される。従って、血液供給手段２５は、同時に加圧手段２４を有し、加圧手段としても使用され得る。例えば、注射用シリンジが、具体的な例である。
【０１１０】
加圧あるいは減圧手段を用いる場合、血漿および血球の移動速度を調節するため、加圧および減圧の程度を調整する圧力調整手段が備えられ得る。装着された極細繊維の集合体の前後にポンプを配設し、溶血が起きないように圧力を調節し得る。この場合、特に装着された極細繊維の集合体を通過する前の血液成分の圧力と、通過後の血漿成分の圧力とがそれぞれ独立して調節され得る。このとき、加圧および減圧の程度はコンピューターなどで自動制御され得る。
【０１１１】
上記加圧および／または減圧による圧力損失（フィルターの入口と出口の圧力差）は、極細繊維の集合体の形状や血液の処理速度に依存するが、好ましくは0.03〜５kg/cm²であり、さらに好ましくは0.05〜３kg/cm²であり、特に好ましくは0.1〜１kg/cm²である。差圧が0.03kg/cm²未満の場合、処理時間が長くなったり、血漿の分離が不十分となるおそれがある。このとき、疎水性の高い極細繊維を使用していると、極細繊維の集合体の中に血液を送ることが困難となり得る。差圧が５kg/cm²を超える場合、血漿が装着された極細繊維の集合体内を通過する時間と、血球が装着された極細繊維の集合体内を通過する時間との差が短くなるため、血球が混入し、血漿の回収が困難となり得る。さらに、赤血球が溶血したり、装置や装着された極細繊維の集合体に損傷が生じるおそれがある。
【０１１２】
＜回収手段＞
極細繊維の集合体によって分離され、透過液側に移動した血漿は、検査などに使用するために回収される。回収する手段としては、既知の手段をとり得る。例えば、フィルター内で血液に付与された差圧をそのまま利用して、あるいはポンプなどで透過液を吸い込んでまたは透過液を送り出して、分離された血漿が試料容器に回収され得る。分離された血漿は、フィルターの出口の直下において、試料容器に回収され得る。あるいは、中空の管を出口に接続して試料容器に導入し得る。フィルターの出口と回収手段とを接続する場合、上記の血液の供給手段とフィルターとの接続手段と同様の手段が用いられ得る。
【０１１３】
試料容器などに蓄えられた血漿は検査試料として供される。試料容器としては、チューブ、バイアルなどが挙げられ、その材質は得られた血漿の保存に影響がなければいずれの材料をも使用し得る。例えば、上記フィルターに使用する容器と同様の材料が使用され得る。
【０１１４】
極細繊維の集合体による血漿の分離に際して、時間経過とともに遅れて血球が透過してくるため、分離された血漿中に血球が混入することがある。さらに操作条件によっては赤血球が装着された極細繊維の集合体中で破壊され、溶出したヘモグロビンが透過液中に混入することがある。臨床検査などのために血漿を分離する際、得られる血漿に血球やヘモグロビンが混入することは避けなければならない。透過液の血漿中に血球やヘモグロビンがある一定量以上に混入すると、得られた血漿は臨床検査試料として使用し得ない。
【０１１５】
上記の問題点を解決するため、血球を含有しない最初の透過液（血漿）をあらかじめ選択された量で回収し得る。例えば、本願明細書中に記載される条件下において、装着された極細繊維の集合体の間隙体積の10％に相当する容量の血漿を回収することにより、実質的に血球を含有しない血漿が得られ得る。このような定量は、例えば、上記血液の供給量の測定と同様に、液量計を血漿回収容器に配置して行い得る。液量は、例えばセンサーで検出することにより測定され得る。血漿が一定量回収された後に、血漿回収容器を移動させて同一の血漿サンプルをいくつかに分取し得る。この移動は手動または自動により行われ得る。この場合、いったん血漿をプールして一定量を分注する手段が使用され得る。さらに、一定量の血漿が得られたときに、血液供給手段、加圧手段を停止するように装置を構成し得る。これらの制御はコンピューターを用いて行われ得る。あるいは、一定量を回収した後には、下記で説明する切替手段により、透過してくる血漿を廃棄または他の容器に回収し得るように配置し得る。なお、以下に述べる血球および／またはヘモグロビン検出手段を設けるときは、血漿定量装置は、血球および／またはヘモグロビン含有試料を回収する容器への出口付近に、あるいは血漿試料容器内に配置することが好ましい。
【０１１６】
＜血球および／またはヘモグロビン検出手段＞
上記のように血漿に血球などが混入すると検査試料として使用し得ない。従って、本願発明の装置は、好ましくは、透過液側に血球および／またはヘモグロビンを検出する血球および／またはヘモグロビン検出手段を配置し得る。透過液中に血球および／またはヘモグロビンが検出された際には、加圧、血液供給を停止し、血球および／またはヘモグロビンが混入する血漿を回収または廃棄する回収手段にラインを切替えるようにし得る。このことにより、血球やヘモグロビンの混入がなく、臨床検査試料として有用な血漿が確実に得られ得る。なお、血球が検出された血漿、または遅れて透過した血球成分は生体または被験体に戻し得る。
【０１１７】
血漿透過液中の血球および／またはヘモグロビン（血球および／またはヘモグロビン）の検出手段としては、光学的手段、化学反応による発色手段などが挙げられる。簡便には、血球および／またはヘモグロビンは直接、光学的手段により検出され得る。例えば、血漿中に血球が混入すると光透過性が低下する、あるいは、ヘモグロビンが混入すると血漿が赤色化するという現象がみられる。いずれの場合も光学的手段により検出され得る。例えば、光学方式により一定値以上の漏血を検出する漏血センサーなどが使用され得る。漏血センサーとは、溶液中の血球やヘモグロビンを検出するセンサーであり、例えば、分光光度計により溶液の一定波長の吸光度または透過度を測定する。具体的には、溶液中の血球による濁度（例えば、波長630nmの吸光度）、あるいは、ヘモグロビンの吸光度（例えば、波長430nmの吸光度）を測定することにより、溶液中の血球および／またはヘモグロビンの漏出が検出され得る。
【０１１８】
化学反応による発色手段としては、血球および／またはヘモグロビンとの化学反応による発色を利用する方法がある。この発色は光学的手段により検出し得る。例えば、ヘモグロビンのペルオキシダーゼ様作用を利用する方法が挙げられる。適切な過酸化物および色原体を選択することにより、感度が調節され得る。
【０１１９】
本願発明の目的を考慮すれば、血球および／またはヘモグロビンの検出が可能であれば、光学的手段以外の手段が用いられ得る。例えば、電気的細胞計数器であるセルカウンターにより血球の検出を行い得る。
【０１２０】
血球および／またはヘモグロビン検出手段のうち、光学的手段は、血漿の流路である出口から回収手段までの間に配置され得る。例えば、センサーを外部から挿入し、直接血漿と接触させて血球および／またはヘモグロビンを検出し得る。あるいは、血漿の流路である配管を挟むように配置し、一方から光、レーザーなどを発光させ、他方でこの発光光を受け、血球および／またはヘモグロビンを検出し得る。この場合、配管は光が透過できる透明性であるものが好ましい。光透過性が良好でない配管を用いるときは、配管のみの光透過性を予め測定しておけばよい。また、配管に微細なサンプリングによる検出手段を設け、血漿を採取して上記の発色試薬、例えば試験紙で血球および／またはヘモグロビンの混入が検出され得る。血球および／またはヘモグロビンを検出したとき、切替手段により血球および／またはヘモグロビン含有血漿を廃棄または回収し得る。さらに、この血球および／またはヘモグロビン検出手段は、血液供給手段、加圧手段、あるいは下記の切替手段と連結され得、供給または加圧の停止、切替手段による廃棄または回収などの処理と連動され得る。
【０１２１】
血漿は、一般に、溶血がない場合でも若干のヘモグロビンを含有しており、その濃度には個体差がある。また、近年多く使われている真空採血管を用いた採血では、採血時に多少の溶血が起きることが知られている。しかし、この程度の低濃度で血漿中にヘモグロビンが含まれていても、臨床検査データにさほど影響を与えないことがわかっている。従って、血漿中に含まれる血球あるいはヘモグロビン濃度には許容範囲があり、その範囲内で血球および／またはヘモグロビンが含まれていても特に問題がない。例えば、得られた血漿中の赤血球濃度は、分離前の血液中の赤血球濃度に対して0.1％以下であれば、許容され得る。
【０１２２】
従って、本願発明の装置においては、血球および／またはヘモグロビン検出手段が、一定の濃度を越えた血球および／またはヘモグロビン濃度を検出したときに、切替手段を作動させて、血球および／またはヘモグロビンが多く混入した血漿を廃棄し、あるいは血液の供給または加圧の停止するように、接続され得る。
【０１２３】
＜切替手段＞
血球および／またはヘモグロビンが混入する血漿を分別するための切替手段は、出口以降に配置される。この切替手段としては、切替コック、切替バルブなどが挙げられる。切替手段は自動または手動のいずれをも採用し得る。自動の場合、血漿が切替手段を通過する前の位置に、血球および／またはヘモグロビン検出手段を配置させる。切替手段は血球および／またはヘモグロビン検出手段と連結される。血漿中の血球および／またはヘモグロビンが予め設定した値を超えた時に、血球および／またはヘモグロビン検出手段からのシグナルにより切替手段が作動し、血漿を回収するラインから血球および／またはヘモグロビンが混入する血漿を回収または廃棄するラインに切り替える。回収、廃棄手段としては、上記の血漿回収手段が用いられ得る。
【０１２４】
（作用）
本願発明における血球と血漿の分離機構は、主に両成分の極細繊維の集合体中における移動速度の差を利用したものである。極細繊維の集合体中における移動速度は、血漿成分のほうが血球成分よりも大きい。従って、極細繊維の集合体に血液を供給し、圧力をかけることにより、血漿が血球より先に移動し、一定の距離を移動した後、両者は完全に分離する。従って、まず、血漿が流出し、ついで血球が流出するので、この時間差を利用して血漿を回収し得る。
【０１２５】
上記のように、本願発明は、血液成分の比重の差を利用する遠心分離法、あるいは血液成分のサイズの差を利用する膜分離法とは根本的に異なる。さらに、極細繊維に対する血液成分の吸着性のみを利用する白血球の分離法ともまた異なる。
【０１２６】
【発明の効果】
本願発明の血漿分離装置は、血漿と血球の移動速度の差を利用している。従って、得られる血漿成分は希釈を受けたり、成分が変わることなく、遠心分離から得られる血漿となんら変わることがない。さらに、透過液側に血球および／またはヘモグロビンの検出装置と、これに連動したコックを備えることにより、試料採取用血漿に血球および／またはヘモグロビンが混入することが防止される。このように本願発明の装置によれば、血球および／またはヘモグロビンの混入がない血漿試料を効率的、簡便、迅速かつ安全に得ることができる。従って、本願発明の装置は、臨床検査用など、少量の血液を短時間で血漿または血清に分離することが要求される分野において有用である。また、本願発明の血漿分離装置は、従来人手に頼っていた臨床検査用血漿試料の採取の自動化を可能とし、臨床検査の自動化、迅速化、安全性の向上に対して大きく寄与し得る。
【０１２７】
以下、実施例をあげて本願発明を具体的に説明するが、本願発明はこれに限定されるものではない。
【０１２８】
【実施例】
実施例においては、極細繊維の集合体として、通常のメルトブロー法により紡糸したポリエチレンテレフタレート製の極細繊維（密度：１．３８ｇ／ｃｍ³）からなる不織布を使用した。牛血液として、採血後８時間以内の新鮮牛血液を用いたが、採血直後に血液の凝固を防止するために、抗凝固剤（ＡＣＤ：クエン酸デキストロース）を加えた。この牛血液中の赤血球濃度は７．２×１０⁹個／ｍｌ、白血球濃度は６．８×１０⁶個／ｍｌ、血小板濃度は２．１×１０⁸個／ｍｌであった。
【０１２９】
血漿分離フィルターの性能を、血漿中の混入血球（赤血球、白血球、血小板）濃度、赤血球の溶血の有無、並びに、血液成分（血清アミラーゼ、総コレステロール、中性脂肪、尿素窒素、血糖、総蛋白質濃度、アルブミン）を分析して、評価した。なお、上記の牛血液を遠心分離して得た血漿を比較の対象とした。
【０１３０】
血漿中の混入血球濃度は、コールターカウンター法により測定した。各血球の検出限度は赤血球濃度で２×１０⁵個／ｍｌ、白血球濃度で１×１０⁵個／ｍｌ、血小板濃度で１×１０⁶個／ｍｌであった。
【０１３１】
溶血は、Ｏ−トルイジン法を用い、血漿中のへモグロビン濃度を測定して検出した。
【０１３２】
各血液成分の分析は以下の方法で行った。
【０１３３】
(i)血清アミラーゼ：Ｇ５ＣＮＰ法
(ii)総コレステロール：コレステロールオキシダーゼ・ペルオキシダーゼ発色法
(iii)中性脂肪：ＬＰＬ・グリセロール３リン酸オキシダーゼ・ペルオキシダーゼ発色法
(iv)尿素窒素：ウレアーゼ・インドフェノール法
(v)血糖：グルコースオキシダーゼ・ペルオキシダーゼ発色法
(vi)総蛋白質濃度：ビューレット法
(vii)アルブミン：ＢＣＧ（ブロムクレゾールグリーン）法
【０１３４】
（実施例１）
図１に示すような血漿分離フィルターを作製した。入口として容器の上面端部に直径１．０ｍｍの穴、出口として底面中央部に直径１．０ｍｍの穴を有し、容器内部の直径５２．０ｍｍ、厚さ２．０ｍｍのアクリル製の円盤状容器を準備した。この容器に、極細繊維の集合体として平均繊維直径１．８μｍのポリエチレンテレフタレート製の極細繊維からなる不織布（目付５０ｇ／ｍ²、厚さ約２ｍｍ）１．４ｇ（１４枚を重層）を、直径５０．０ｍｍ、厚さ２．０ｍｍとなるように重層した。同時に、極細繊維の集合体の外周部表面と容器の内周部表面との間に幅１．０ｍｍの隙間を設けた。作製されたフィルターは、極細繊維の集合体の容積：約３．９ｃｍ³、隙間の容積：約０．３２ｃｍ³であった。この血漿分離フィルター中の極細繊維の総表面積、極細繊維の集合体の平均嵩密度、平均動水半径、間隙体積及びＬ／Ｄはそれぞれ表１に記載した通りである。それぞれの値は、以下の式で計算した。ポリエチレンテレフタレートの密度は、上記のように、１．３８ｇ／ｃｍ³である。
【０１３５】

上記の血漿分離フィルターに牛血液４ｍｌを容器の入口から注入し、極細繊維の集合体の外周部表面と容器の内周部表面の隙間に充填した後、０．２ｋｇ／ｃｍ²の圧力をかけ牛血液を押し出した。牛血液は極細繊維の集合体の外周部表面から入り、極細繊維の集合体の内部を外周部から円心部に向かって水平方向に移動し、加圧後２０秒経過して、容器の出口から血漿が流出し始め、さらにその後４秒経過して流出液中に赤血球が混入し始めた。よって、流出し始めて約２秒間の血漿を採取し、０．２９ｍｌを得た。平均処理線速度は７５ｍｍ／分（＝２５ｍｍ／２０秒）であった。
【０１３６】
上記で得られた０．２９ｍｌの血漿中の赤血球、白血球、血小板濃度は検出限度以下であった。よって、分離前の牛血液中の赤血球濃度に対する得られた血漿中の赤血球濃度（以下赤血球混入率）は０．００３％（＝（２×１０⁵÷７．２×１０⁹）×１００）以下であった。また得られた血漿のヘモグロビン濃度は５ｍｇ／ｄｌであり、血漿成分の分析結果は表１の通りであった。
【０１３７】
（実施例２）
実施例１と同様に血漿分離フィルターを作製した。ただし、不織布の充填量は０．８ｇ（８枚を重層）とした。
【０１３８】
上記の血漿分離フィルター中の極細繊維の総表面積、極細繊維の集合体の平均嵩密度、平均動水半径、間隙体積及びＬ／Ｄはそれぞれ表１に記載した通りである。
【０１３９】
上記の血漿分離フィルターを用いて実施例１と同様に血漿分離を行った。血漿は加圧後１５秒経過してから流出し始め、さらにその後２秒経過してから赤血球か混入し始めた。よって、流出し始めて約１．５秒間の血漿を採取し、０．３３ｍｌを得た。平均処理線速度は１００ｍｍ／分であった。
【０１４０】
上記で得られた０．３３ｍｌの血漿中の白血球及び血小板濃度は検出限度以下であり、赤血球濃度は３×１０⁵個／ｍｌであった。よって、赤血球混入率は０．００４％であった。また、得られた血漿のヘモグロビン濃度は検出限度以下であり、血漿成分の分析結果は表１の通りであった。
【０１４１】
（実施例３）
図３に示すような血漿分離フィルターを作製した。入口として容器の上面端部に直径１．０ｍｍの穴、出口として底面中央部に直径１．０ｍｍの穴を有し、容器内部の直径２９．０ｍｍ、厚さ６．５ｍｍのポリプロピロレン製の円柱状容器を準備した。この容器に、極細繊維の集合体として平均繊維直径０．８μｍのポリエチレンテレフタレート製の極細繊維からなる不織布（目付５０ｇ／ｍ²、厚さ約３ｍｍ）１．２ｇ（１２枚を重層）を、直径２９．０ｍｍ、厚さ６．０ｍｍとなるように、極細繊維の集合体の上表面と容器の内面上部表面に厚さ０．５ｍｍの隙間を設けて充填し、隙間には平均繊維直径１０μｍのポリエチレンテレフタレート製の極細繊維よりなる不織布（目付５０ｇ／ｍ²、厚さ約１ｍｍ）０．３３ｇ（２枚を重層）を充填した血漿分離フィルターを作製した（極細繊維の集合体の容積：約４．０ｃｍ³、隙間の容積：約０．３３ｃｍ³）。
【０１４２】
上記の血漿分離フィルター中の極細繊維の総表面積、極細繊維の集合体の平均嵩密度、平均動水半径、間隙体積及びＬ／Ｄはそれぞれ表１に記載した通りである。
【０１４３】
上記の血漿分離フィルターに牛血液４ｍｌを容器の入口から注入し、極細繊維の集合体の上表面と容器の内面上部の隙間の極細繊維に充填した後、０．４ｋｇ／ｃｍ²の圧力をかけ牛血液を押し出した。牛血液は極細繊維の集合体の上表面から入り、極細繊維の集合体の内部を下方向に向かって移動し、加圧後３０秒経過して容器の出口から血漿が流出し始め、さらにその後５秒経過した時点で流出液中に赤血球が混入し始めた。よって、流出し始めて約３秒間の血漿を採取し、０．３１ｍｌを得た。平均処理線速度は１２ｍｍ／分であった。
【０１４４】
上記で得られた０．３１ｍｌの血漿中の血球濃度は全て検出限度以下であった。よって、赤血球混入率は０．００３％以下であった。また、得られた血漿のヘモグロビン濃震度は８ｍｇ／ｄｌであり、血漿成分の分析結果は表１の通りであった。
【０１４５】
（実施例４）
図１に示すような血漿分離フィルターを作製した。入口として容器の上面端部に直径１．０ｍｍの穴、出口として底面中央部に直径１．０ｍｍの穴を有し、容器内部の直径１３４ｍｍ、厚さ０．３０ｍｍのアクリル製の円盤状容器を準備した。この容器に、極細繊維の集合体として平均繊維直径３．０μｍのポリエチレンテレフタレート製の極細繊維からなる不織布（目付５０ｇ／ｍ²、厚さ２ｍｍ）２．０ｇ（３枚を重層）を、直径１３０ｍｍ、厚さ０．３０ｍｍとなるように重層した。同時に、極細繊維の集合体の外周部と容器の内周部との間に幅２．０ｍｍの隙間を設けて、図１に示すような血漿分離フィルターを作製した（極細繊維の集合体の容積：約４．０ｃｍ³、隙間の容積：約０．２５ｃｍ³）。
【０１４６】
上記の血漿分離フィルター中の極細繊維の総表面積、極細繊維の集合体の平均嵩密度、平均動水半径、間隙体積及びＬ／Ｄはそれぞれ表１に記載した通りである。
【０１４７】
上記の血漿分離フィルターを用いて実施例１と同様に血漿分離を行った。ただし、０．４ｋｇ／ｃｍ²の圧力をかけて牛血液を押し出した。血漿は加圧後１２０秒経過してから流出し始め、さらにその後１８秒経過してから赤血球が混入し始めた。よって、流出開始から１２秒間、血漿を採取し、０．２６ｍｌを得た。平均処理線速度は３２．５ｍｍ／分であった。
【０１４８】
上記で得られた０．２６ｍｌの血漿中の白血球及び血小板濃度は検出限度以下であり、赤血球濃度は２．９×１０⁸個／ｍｌであった。よって、赤血球混入率は０．０４％であった。また、得られた血漿のヘモグロビン濃度は４ｍｇ／ｄｌであり、血漿成分の分析結果は表１の通りであった。
【０１４９】
（実施例５）
図２に示すような血漿分離フィルターを作製した。入口として容器の上面端部に直径１．０ｍｍの穴、出口として底面中央部に直径１．０ｍｍの穴を有し、容器内部の直径１５，２ｍｍ、厚さ２４ｍｍのポリプロピレン製の円柱状容器を準備した。この容器に、極細繊維の集合体として平均繊維直径１．８μｍのポリエチレンテレフタレート製の極細繊維からなる綿１．４ｇを、直径１５．２ｍｍ、厚さ２２ｍｍとなるように、極細繊維の集合体の上表面と容器の内面上部表面に厚さ２．０ｍｍの隙間を設けて充填した血漿分離フィルターを作製した（極細繊維の集合体の容積：約４．０ｃｍ³、隙間の容積：約０．３６ｃｍ³）。
【０１５０】
上記の血漿分離フィルター中の極細繊維の総表面積、極細繊維の集合体の平均嵩密度、平均動水半径、間隙体積及びＬ／Ｄはそれぞれ表１に記載した通りである。
【０１５１】
上記の血漿分離フィルターに牛血液４ｍｌを容器の入口から注入し、極細繊維の集合体の上表面と容器の内面上部の隙間に充填した後、０．４ｋｇ／ｃｍ²の圧力をかけ牛血液を押し出した。生血液は極細繊維の集合体の上表面から入り、極細繊維の集合体の内部を下方向に向かって移動し、血漿は加圧後３００秒経過してから流出し始め、さらにその後７０秒経過してから赤血球が混入し始めた。よって、流出し始めから３０秒間の血漿を採取し、０．３０ｍｌを得た。平均処理線速度は４．４ｍｍ／分であった。
【０１５２】
上記で得られた０．３０ｍｌの血漿中の血球濃度は全て検出限度以下であった。よって、赤血球混入率は０．００３％以下であった。また、得られた血漿のヘモグロビン濃度は６ｍｇ／ｄｌであり、血漿成分の分析結果は表１の通りであった。
【０１５３】
（実施例６）
実施例３と同様に血漿分薙フィルターを作製した。ただし、極細繊維の集合体として極細繊維の集合体として平均繊維直径０．８μｍのポリエチレンテレフタレート製の極細繊維からなる綿を用いた。
【０１５４】
上記の血漿分離フィルター中の極細繊維の総表面積、極細繊維の集合体の平均嵩密度、平均動水半径、間隙体積及びＬ／Ｄは実施例３と同一であり、それぞれ表１に記載した通りである。
【０１５５】
上記の血漿分離フィルターを用いて実施例３と同様に血漿分離を行った。血漿は加圧後８８０秒経過してから流出し始め、その後さらに１１５秒経過してから赤血球が混入し始めた。よって、流出し始めて９０秒間の血漿を採取し、０．３１ｍｌを得た。平均処理線速度は０．４１ｍｍ／分であった。
【０１５６】
上記で得られた０．３１ｍｌの血漿中の血球濃度は全て検出限度以下であった。よって、赤血球混入率は０．００３％以下であった。また、得られた血漿のヘモグロビン濃度は１０ｍ ｇ／ｄｌであり、血漿成分の分析結果は表１の通りであった。
【０１５７】
（実施例７）
実施例５と同様に血漿分離フィルターを作製した。ただし、容器内部の直径１０．０ｍｍ、厚さ５５．０ｍｍの容器を用い、極細繊維の集合体として平均繊維直径３．２μｍの極細繊維からなる綿２．０ｇを、直径１０．０ｍｍ、厚さ５１．０ｍｍとなるように、極細繊維の集合体の上表面と容器内の上面部に厚さ４．０ｍｍの隙間を設けた（極細繊維の集合体の容積：約４．０ｃｍ³、隙間の容積：約０．３１ｃｍ³）。
【０１５８】
上記の血漿分離フィルター中の極細繊維の総表面積、極細繊維の集合体の平均嵩密度、平均動水半径、間隙体積及びＬ／Ｄはそれぞれ表１に記載した通りである。
【０１５９】
上記の血漿分離フィルターを用いて実施例５と同様に血漿分離を行った。血漿加圧後２６４秒経過してから流出し、その後さらに２５秒経過してから赤血球が混入し始めた。よって、流出し始めてから２２秒間の血漿を採取し、０．２６ｍｌを得た。平均処理線速度は１１．６ｍｍ／分であった。
【０１６０】
上記で得られた０．２６ｍｌの血漿中の白血球及び血小板濃度は検出限度以下であり、赤血球濃度は４．３×１０⁸個／ｍｌであった。よって、赤血球混入率は０．０６％であった。また、得られた血漿のヘモグロビン濃度は検出限度以下であり、血漿成分の分析結果は表１の通りであった。
【０１６１】
（実施例８）
実施例５と同様に血漿分離フィルターを作製した。ただし、極細繊維からなる綿の充填量を０．８ｇとした。このフィルターの極細繊維の総表面積、極細繊維の集合体の平均嵩密度、平均動水半径、間隙体積及びＬ／Ｄはそれぞれ表１に記載している。
【０１６２】
この血漿分離フィルターを用いて実施例５と同様に血漿分離を行った。血漿は加圧後約１５０秒経過してから流出し始め、その後さらに２０秒経過してから赤血球が混入し始めた。よって、流出し始めてから１７秒間、血漿を採取し、０．３４ｍｌを得た。平均処理線速度は８．８ｍｍ／分であった。
【０１６３】
上記で得られた０．３４ｍｌの血漿中の白血球及び血小板濃度は検出限度以下であり、赤血球濃度は２．２×１０⁸個／ｍｌであった。よって、赤血球混入率は０．０３％であった。また、得られた血漿のヘモグロビン濃度は検出限度以下であり、血漿成分の分析結果は表１の通りであった。
【０１６４】
【表１】

【０１６５】
（比較例１）
実施例１と同様に血漿分離フィルターを作製した。ただし、極細繊維の集合体として平均繊維直径２．５μｍの極細繊維からなる不織布（目付５０ｇ／ｍ²、厚さ２ｍｍ）０．８ｇ（８枚を重層）を充填した。このフィルター中の極細繊維の総表面積、極細繊維の集合体の平均嵩密度、平均動水半径、間隙体積及びＬ／Ｄはそれぞれ表２に記載した通りである。
【０１６６】
上記の血漿分離フィルターを用いて実施例１と同様に血漿分離を行った。流出液は加圧後１０秒経過して出始めたが、流出液には初めから血球が混入していた。流出し始めて約１．０秒間の流出液を採取し、０．３３ｍｌを得た。
【０１６７】
上記で得られた０．３３ｍｌの流出液中の白血球及び血小板濃度は検出限度以下であったが、赤血球濃度は３．８×１０⁸個／ｍｌであった。よって、赤血球混入率は５．３％であった。また、得られた流出液の血漿中のヘモグロビン濃度は検出限度以下であった。血漿成分の分析は省略した。
【０１６８】
（比較例２）
実施例１と同様に血漿分離フィルターを作製した。但し、極細繊維の集合体として平均繊維直径１．０ｍｍの極細繊維からなる不織布（目付５０ｇ／ｍ²、厚さ２ｍｍ）２．０ｇ（２０枚を重層）を充填した。この血漿分離フィルター中の極細繊維の総表面積、極細繊維の集合体の平均嵩密度、平均動水半径、間隙体積及びＬ／Ｄはそれぞれ表２に記載した通りである。
【０１６９】
上記の血漿分離フィルターを用いて実施例１と同様に血漿分離を行った。但し、０．４ｋｇ／ｃｍ²で加圧した。流出液は加圧後１３６５秒経過して出始めたが、流出液は初めから赤色を呈していた。流出し始めて１６００秒間の流出液を採取し、０．２６ｍｌを得た。
【０１７０】
上記で得られた０．２６ｍｌの流出液中の血球濃度は全て検出限度以下であったが、流出液の血漿中のヘモグロビン濃度は９５ｍｇ／ｄｌであった。血漿成分の分析は省略した。
【０１７１】
（比較例３）
実施例３と同様に血漿分離フィルターを作製した。但し、容器内部の直径５０．０ｍｍ、厚さ２．３ｍｍのアクリル製の円柱状容器に、極細繊維の集合体として平均繊維直径１．８μｍのポリエチレンテレフタレート製の極細繊維からなる不織布（目付５０ｇ／ｍ²、厚さ約１ｍｍ）１．４ｇ（１４枚を重層）を、直径５０．０ｍｍ、厚さ２．０ｍｍとなるように、極細繊維の集合体の上表面と容器の内面上部表面に厚さ０．３ｍｍの隙間を設けて充填し、隙間には平均繊維直径１０μｍのポリエチレンテレフタレート製の極細繊維からなる不織布（目付５０ｇ／ｍ²、厚さ約１ｍｍ）０．１７ｇ（１枚）を充填した血漿分離フィルターを作製した（極細繊維の集合体の容積：３．９ｃｍ³、隙間の容積：０．５９ｃｍ³）。
【０１７２】
上記の血漿分離フィルター中の極細繊維の総表面積、極細繊維の集合体の平均嵩密度、平均動水半径、間隙体積及びＬ／Ｄはそれぞれ表２に記載した通りである。
【０１７３】
上記の血漿分離フィルターを用いて実施例３と同様に血漿分離を行った。流出液は加圧後１５秒経過して出始めたが、流出液には初めから血球が混入していた。流出し始めて約１．５秒の流出液を採取し、０．３０ｍｌを得た。
【０１７４】
上記で得られた０．３０ｍｌの流出液中の白血球及び血小板濃度は検出限度以下であったが、赤血球濃度は６．９×１０⁹個／ｍｌであった。よって、赤血球混入率は５５％であった。また、得られた流出液の血漿中のヘモグロビン濃度は検出限度以下であった。血液成分の分析は省略した。
【０１７５】
（比較例４）
実施例５と同様に血漿分離フィルターを作製した。但し、容器内部の直径８．０ｍｍ、厚さ８６．０ｍｍのポリプロピレン製容器に、極細繊維の集合体を直径８．０ｍｍ、厚さ８０．０ｍｍとなるように、極細繊維の集合体の上表面と容器内の上面部に厚さ６．０ｍｍの隙間を設けて充填した（極細繊維の集合体の容積：４．０ｃｍ³、隙間の容積：０．３０ｃｍ³）。この血漿分離フイルター中の極細繊維の総表面積、極細繊維の集合体の平均嵩密度、平均動水半径、間隙体積及びＬ／Ｄはそれぞれ表２に記載した通りである。
【０１７６】
上記の血漿分離フィルターを用いて実施例５と同様に血漿分離を行った。流出液は加圧後１９２０秒経過して出始めたが、流出液は初めから赤色を呈していた。流出し始めて３６００秒間の流出液を得たが、０．２５ｍｌしか得られなかった。
【０１７７】
上記で得られた０．２５ｍｌの流出液中の血球濃度は全て検出限度以下であったが、流出液の血漿中のへモグロビン濃度は７５ｍｇ／ｄｌであった。血漿成分の分析は省略した。
【０１７８】
（比較例５）
実施例１と同じ血漿分離フィルターを作製し、実施例１と同様に血漿分離を行った。但し、用いた牛血液の量を０．５ｍｌとした。
【０１７９】
血液を全て極細繊維内に送っても、透過液を得ることが出来なかった。そこで、さらに加圧空気を容器入口から送ったが、加圧空気は極細繊維内を血液より早く通過し、出口には空気を含んだ泡状の血液しか得られなかった。これは、極細繊維の集合体の間隙体積３．０ｍｌに比べ、処理する血液量が少なすぎて、均一に血液を送液出来なかったためと考えられた。
【０１８０】
（比較例６）
実施例３と同様に血漿分離フィルターを作製した。ただし、容器内部の直径３５．０ｍｍ、厚さ４．６ｍｍのポリプロピレン製容器に、極細繊維の集合体として平均繊維直径１．８μｍのポリエチレンテレフタレート製の極細繊維からなる不織布（目付５０ｇ／ｍ²、厚さ１ｍｍ）０．８７ｇ（１８枚を重層）を、直径３５．０ｍｍ、厚さ４．２ｍｍとなるように、極細繊維の集合体の上表面と容器内の上面部に厚さ０．４ｍｍの隙間を設けて充填し、隙間には平均繊維直径１０μｍのポリエチレンテレフタレート製の極細繊維からなる不織布（目付５０ｇ／ｍ²、厚さ１ｍｍ）０．８７ｇ（１枚を重層）を充填した（極細繊維の集合体の容積：４．０ｃｍ³、隙間の容積：０．３８ｃｍ³）。
【０１８１】
上記の血漿分離フィルター中の極細繊維の総表面積、極細繊維の集合体の平均嵩密度、平均動水半径、間隙体積及びＬ／Ｄはそれぞれ表２に記載した通りである。
【０１８２】
上記の血漿分離フィルターを用いて実施例３と同様に血漿分離を行った。流出液は加圧後１５秒経過して出始めたか、流出液には初めから赤血球が混入していた。流出し始めて約１．５秒間の流出液を採取し、０．３４ｍｌを得た。平均処理線速度は１６．８ｍｍ／分であった。
【０１８３】
上記で得られた０．３４ｍｌの流出液中の白血球及び血小板濃度は検出限度以下であったが、赤血球濃度は１．１×１０⁹個／ｍｌであった。よって、赤血球残存量は１５％であった。また、得られた流出液の血漿中のヘモグロビン濃度は検出限度以下であった。血漿成分の分析は省略した。
【０１８４】
【表２】

【０１８５】
（実施例９）
実施例１と同じ円盤状容器に、平均繊維直径１．５μｍのポリエチレンテレフタレート極細繊維不織布２ｇを直径５０ｍｍ、厚さ２．０ｍｍに切断し、容器の内周部表面との間に１．０ｍｍの隙間を設けて充填した。このときの平均動水半径は０．６４μｍである。
【０１８６】
これに抗凝固剤としてＡＣＤ液を加えた、ヘマトクリット４５％の牛血液を容器の外周部から内側へ、０．５ｋｇ／ｃｍ²の圧力で送液した。血液は容器内を不織布面に対して水平方向に極細繊維の集合体内を通過し、約３０秒後に出口から血漿が透過してきた。血漿の透過は約４５秒間続き、その後は透過液中に血球が混入し始めた。従って、血球の混入のない血漿は加圧後３０秒から４５秒間採取でき、その総量は０．７ｍｌであった。得られた血漿のへモグロビン濃度は３ｍｇ／ｄｌ以下で溶血は認められなかった。また、血球分析器で測定した赤血球、白血球、血小板も全て検出限界以下であった。得られた血漿の生化学分析値を表３に示す。採取初期の透過液、採取終了時の透過液、極細繊維の集合体の間隙体積の１０％にあたる0.25mlを集めたサンプル、および同一の血液を遠心分離して得られた血漿を比較したところ、それらの間で有意差が認められなかった。また、得られた血漿中のフィブリノーゲン量をセルロースアセテート電気泳動により定量したところ、遠心分離により得られた血漿中の濃度の約３０％に減少していたが、完全に除かれていなかった。一昼夜放置したとき、フィブリンの析出は認められなかった。
【０１８７】
（実施例１０）
実施例９で用いたものと同じフィルターを使用した。これに抗凝固剤を加えず採血直後のヘマトクリット４７％のヒト血液を、０．５ｋｇ／ｃｍ²の圧力で送液した。血液は容器内を不織布の面に対して水平方向に極細繊維の集合体内を通過し、約３５秒後に出口から透過液が得られた。透過液には約５５秒後から血球が混入し始めた。血球の混入のない透過液は加圧後３５秒から２０秒間採取でき、その総量は１．０ｍ１であった。得られた透過液のヘモグロビン濃度は３ｍｇ／ｄＬ以下で溶血は認められなかった。また、血球分析器で測定した赤血球、白血球、血小板も全て検出限界以下であった。得られた透過液の生化学分析値を表３に示す。採取初期の透過液、採取終了時の透過液、極細繊維の集合体の間隙体積の１０％にあたる0.25mlを集めたサンプル、および同一の血液を凝固後遠心分離して得られた血清を比較したところ、それらの間で有意差が認められなかった。
【０１８８】
また、得られた透過液中にフィブリノーゲンは検出されず、得られた液体は血漿でなく、血清であった。同一人からヘパリンを抗凝固剤として加えた血液を遠心分離して得られた血漿中のフィブリノーゲン濃度は２２０ｍｇ／ｄＬであった。
【０１８９】
ヒト血液を用いた以外は、実施例１と同一の実験を行ったにも係わらず、採取できた透過液量に違いが認められたのは、赤血球の大きさがヒトとウシとでは異なるためと考えられる。また、フィブリノーゲンが完全に除去されているのは、抗凝固剤を添加していないため、フィルターに吸着したためと考えられる。
【０１９０】
（実施例１１）
実施例１で用いたポリエチレンテレフタレートの極細繊維の不織布を０．１％のポリビニルピロリドンK-90（BASF社製のコリドンK-90、分子量３６万）溶液に浸漬し、ウエットの状態のまま50KGyのγ線照射を行った。γ線照射後の不織布を純水で洗浄し、架橋しなかったポリビニルピロリドンを除去し、乾燥し、表面にポリビニルピロリドンが固定された不織布を得た。この不織布を実施例１と同様にして容器に充填し、実施例１０と同様の実験を行った。血液は、容器内を不織布の積層面に対して平行に移動し、約１５秒後に血漿が出口からでてきた。約２５秒後から血球が混入し始めた。血球が混入していない血漿は、約１０秒間採取できた。その総量は1.2mlであった。得られた血漿中のヘモグロビン濃度は3mg/dl以下であり、溶血は認められなかった。また、血球分析器で測定した赤血球、白血球、血小板もすべて検出限界以下であった。得られた血漿の分析値を表３に示した。採取初期の透過液、採取終了時の透過液、極細繊維の集合体の間隙体積の１０％にあたる0.25mlを集めたサンプル、および同一の血液を凝固後遠心分離して得られた血清を比較したところ、それらの間で有意差が認められなかった。
【０１９１】
得られた血漿中のフィブリノーゲンの量をセルロースアセテート電気泳動により測定したところ、遠心分離により得られた血漿中の濃度の約２０％に減少していたが、完全に除かれていなかった。一昼夜放置したとき、フィブリンの析出は認められなかった。
【０１９２】
ポリビニルピロリドンを極細繊維に固定することにより、血液の極細繊維に対する親和性が向上し、血液が通過する際の抵抗が減少し、血漿が分離されてくるまでの時間が短縮された。
【０１９３】
（比較例７）
ガラス繊維濾紙（糸径０．８〜２．５μｍ）を約２ｍｍ角に裁断し、水を加えてミキサーにて撹拌し、脱水後、図４に示すような５ｍＬのシリンジ容器に充填率０．５ｇ／ｃｍ³となるように充填した。平均動水半径は１．４５μｍである（ガラス繊維の比重２．２）。これに、実施例１と同様に、抗凝固剤としてＡＣＤ液を加え、ヘマトクリット４５％の牛血液を、容器の上部から０．５ｋｇ／ｃｍ²の圧力で送液した。血液は容器内の上部から下部に向かって極細繊維の集合体内を通過し、約６０秒後に出口から血漿が透過してきた。血漿の透過は約９０秒後まで続き、その後は血漿中に血球が混入し始めた。血球の混入のない血漿は加圧後６０秒から３０秒間採取でき、その総量は０．２ｍｌであった。得られた血漿のヘモグロビン濃度は３ｍｇ／ｄＬ以下で溶血は認められなかった。また、血球分析器で測定した赤血球、白血球、血小板も全て検出限界以下であった。得られた血漿の生化学分析値を表３に示す。採取初期の透過液、採取終了時の透過液、得られた0.2mlのサンプルおよび同一の血液を遠心分離して得られた血漿を比較した。得られた0.2mlのサンプルを使用したのは極細繊維の集合体の間隙体積の１０％にあたる0.25mlを集めたサンプルが得られなかったからである。採取初期の透過液は、総タンパク質濃度が他に比べて有意に低かったほか、電解質や脂質の測定値が遠心分離血漿と測定値が異なった。これは、タンパク質、電解質、脂質がガラス繊維に吸着するほか、電解質の一部がガラス繊維から血漿中に溶出するためと考えられる。また、得られた血漿中のフィブリノーゲンの量をセルロースアセテート電気泳動法により求めたところ、遠心分離で得られた血漿中の濃度の約３０％に減少していたが、完全に除かれてはいなかった。一昼夜放置したときに、フィブリンの析出は認められなかった。結果を表３に示す。
【０１９４】
（比較例８）
実施例１と同様の容器に糸径３．５μｍのポリエチレンテレフタレート不織布を２．４ｇ充填した。このときの平均動水半径は１．１３μｍである。これに実施例１と同様の方法で血液を送液したところ、約４５秒後に透過液が得られたが、この透過液には初期から赤血球が含まれ、血漿の分離は出来なかった。この原因は、繊維径が３．５μｍと大きく、そのため、繊維間隙を赤血球が通り抜けるときの変形度が小さく、赤血球と血漿の移動速度差が生じなかったためと考えられる。結果を表３に示す。
【０１９５】
【表３】

【０１９６】
（実施例１２）
図８は、本願発明の装置の構成の一例を示す模式図である。容器３１は血液試料を収納する容器である。容器３１は、血液を供給するためのライン（またはチューブ）３２を介して血漿分離フィルター１１に接続されている。また、容器３１は、加圧空気の供給ライン３３と接続されている。
【０１９７】
容器３１には、密閉できる着脱自在な蓋３４が取り付けられている。容器３１はさらに攪拌機能または振盪機能を有し得る。容器３１またはライン３２には抗血液凝固剤を供給するラインが取り付けられ得る（図示せず）。
【０１９８】
容器３１中の血液２６は、コンプレッサー３５からライン３３を通じて送られた加圧空気により押し出され、ライン３２を通ってフィルター１１に供給される。加圧空気の圧力はコンプレッサー３５により調節される。
【０１９９】
フィルター１１に供給された血液は、容器３１へ送られた加圧空気による圧力で極細繊維の集合体１４内を通過し、この中で、血漿と血球に分離される。フィルター１１の透過液側より流出した血漿は、三方コック３６を経て血漿回収のためのライン（またはチューブ）３７を通り、試料血漿を収納する容器３８に回収される。
【０２００】
フィルター１１の透過液側には、血球および／またはヘモグロビンの混入を検出する血球および／またはヘモグロビン検出手段４０が取り付けられている。血球および／またはヘモグロビン検出手段４０に加え、さらに化学反応による方法あるいはセルカウンターによる血球および／またはヘモグロビンの混入を検出するのためのサンプリング口を設け得る。透過液の血漿中に血球および／またはヘモグロビンが検出されると、三方コック３６（例えば、電磁作用により開閉し得る３ポートバルブであり得る）を切り替え、透過液を廃液（または回収）ライン４１を経て廃液（または回収）タンク４２へ送る。同時に、容器３１への加圧空気の供給を停止し、血漿分離の操作を終了する。
【０２０１】
これらの手段は、血球および／またはヘモグロビン検出手段と連結され得、たとえば、血球および／またはヘモグロビンが検出されると自動的に血漿分離操作を終了するように連結され得る。
【０２０２】
さらに、血液送液ライン３２および血漿送液ライン３７には、洗浄水としての純粋を供給する純水供給ライン４３、および装置を乾燥するための乾燥空気を供給する乾燥空気供給ライン４４が接続され得る。この他に、本願発明の装置を洗浄する方法としては、回転ブラシでガラス器具などを機械的にブラッシングする方法、ジェット噴流による洗浄器であってノズルを通して高圧の水流を流す方法、超音波による方法などが挙げられる。これらの洗浄方法は、単独であるいは組み合わせて用いられ得る。上記の方法で、血漿分離操作終了後のラインの洗浄および乾燥が速やかに行われ、連続して次の血液試料が処理され得る。
【０２０３】
なお、血漿分離装置を構成する各ラインには、上記三方コック３６の他に、流体の流れ方向を切り替えるためのコック、チャンバー、圧力センサーのような各種部材が配設され得る（図示せず）。
【０２０４】
上記フィルター１１はホルダーなどに着脱自在に取り付けられ得る（例えば、図５参照）。マグネットによる保持および解除が適用され得る。フィルター１１は、入口側部分と（図５、１５ａ）、出口側部分（図５、１５ｂ）と、極細繊維の集合体（図５、１４）を含む部分との３つの部分で、それぞれが別個に着脱自在に構成され得る。装着される極細繊維の集合体を含む部分を着脱自在とすることにより、使い捨てとされ得、汚染が防止され得る。このように装着される極細繊維の重合体を含む部分が着脱自在であれば、ラインの洗浄および乾燥も効率的であり得る。
【０２０５】
本願発明の装置はまた、一定量の血液試料を自動的に血漿分離用のフィルターに流入させる機構を有し得る。これには例えば上記の定量ポンプが用いられ得る。さらに、フィルターまたはその各構成部分（装着される極細繊維の集合体を含む部分など）を自動的に交換する機構を有し得る。また、従来の血漿分離装置の血漿分離器（極細繊維の集合体）部分を上記の装着される極細繊維の集合体を含む部分またはフィルターで置換した構造でもあり得る。さらに、分離性能の向上が所望であれば該フィルターを直列に連結し得、処理すべき血液量を増加したい場合には並列に連結し得る。
【０２０６】
（実施例１３）
本願発明の装置は血液の供給から回収まで自動化され得、大量の数量の血液試料から血漿を分離し得る。例えば、図９は、本願発明の装置を自動化する構成の一例を示す模式図である。自動化により、多種類の血液サンプルが同時に処理され得る。
【０２０７】
図９において、血液サンプルはラック４８に並べられ、血液供給ライン３２の入口が血液サンプル２６のほぼ底面にくるように配置される。フィルター１１はホルダー４５に固定されている。血液供給ライン３２の末端は、フィルター１１の入口と気密に嵌合するように構成されており、血液供給ライン３２の先端が機械的圧力で押圧され、フィルターの入口と気密に嵌合される。同様に、血漿回収ライン３７の先端は、フィルター１１の出口と気密に嵌合するように構成されており、血漿回収ライン３７の先端は機械的圧力で押圧され、フィルター１１の出口と気密に嵌合される。次に、ラインの出口には血漿の回収手段である血漿を受けるサンプル容器３８がラック５０に並べられ、自動的に配置される。
【０２０８】
このようにしてラインが連結され、血漿を受けるサンプル容器が配置されると、血液供給手段である血液供給ポンプ４６が作動する。一定量の血液が流れた時に血液供給ポンプ４６が停止する。血液供給ポンプ４６が停止すると、バルブ４７が血液供給ライン３２を遮断する。全部の血液供給ポンプ４６が停止するとコンプレッサー３５が作動し、コンプレッサー３５から圧縮空気がフィルター１１に供給される。血液は空気圧により極細繊維の集合体１４を通過し、血漿が分離される。
【０２０９】
フィルター１１の出口には、血球および／またはヘモグロビン検出手段４０が配置されている。血球および／またはヘモグロビンが検出されると、その下流にある血球および／またはヘモグロビン切替手段である三方コック３６が切り替わり、血球および／またはヘモグロビンを含む血漿を血球および／またはヘモグロビン混入血漿の回収ライン４１に導く。同時に、空気供給バルブ４７が切り替わり、加圧を停止する。血球および／またはヘモグロビンが検出されると、そのサンプル容器にはマークがつけられ、次のサンプルの準備がなされる。
【０２１０】
血漿回収ライン３７の末端には液量計４９が配置され、一定量が回収されると、切替手段３６を切り替え、不要な血漿を血球および／またはヘモグロビン混入血漿の回収ライン４１に導く。同時に加圧空気の供給バルブ４７を切り替えて加圧を停止する。すべてのバルブが切り替わるとコンプレッサー３５は停止する。
【０２１１】
血漿を回収したサンプル容器３８から、血漿回収手段の末端であるラインを引き抜く。サンプル容器３８には蓋がされ、保存される。
【０２１２】
フィルター１１の入口と気密に嵌合している血液供給ライン３２の末端は、機械的圧力で引き離される。フィルター１１の出口と気密に嵌合した血漿回収ライン３７の先端も機械的圧力で引き離される。こうして各手段から切り離されたフィルター１１はホルダーから離され、廃棄される。
【０２１３】
次いで、ラインの洗浄を行う。フィルター１１の代わりに、血液供給ライン３２の末端および血漿回収ライン３７の先端と気密に嵌合し得る中空状の容器が配置される。洗浄液を受ける容器が、血漿回収手段の末端であるライン３７の出口に自動的にセットされる。血液の供給が終了した血液サンプル供給容器５１を並べたラック４８は移動し、血液サンプル供給容器５１は廃棄される。代わりに、洗浄液を入れた容器がラック４８に並べられ、血液供給ライン３２の入口が洗浄液のほぼ底面にくるようにセットされる。
【０２１４】
次に、バルブ４７が、洗浄液を供給するように切り替えられ、血液供給ポンプ４６が作動してライン３２、３７、４１を洗浄する。切替手段のバルブ３６を、それぞれ血漿回収ライン３７および血球および／またはヘモグロビン混入血漿回収ライン４１に切り替え、洗浄する。
【０２１５】
洗浄終了後、コンプレッサー３５が作動して空気を供給し、一定時間ラインを乾燥する。その後、洗浄水を並べたラックおよび、洗浄液を受けた容器を並べたラックが移動し、血液サンプルを並べたラック４８、血漿回収のための容器を並べたラックが所定の位置に配置される。さらに、洗浄時に用いる中空状の容器の代わりに、フィルター１１が上記の方法で配置される。そして、上記操作が繰り返される。
【０２１６】
これらの操作の順序は、任意に変更され得、また、洗浄液を回収する手段は上記に限定されることなく、個々の容器でなくともよい。いずれにせよ、このような自動化の装置の任意の改変もまた、本願発明に包含される。
【０２１７】
（実施例１４）
本願発明の装置は、通常少なくとも、（Ａ）フィルター、血液供給手段、血液加圧手段、および血漿回収手段を有する。これらの手段の他には、（ａ）一定量の血液を供給するための手段、（ｂ）一定量の血漿を回収するための手段、（ｃ）血球および／またはヘモグロビンを検出するための手段、（ｄ）切替手段、（ｅ）血球および／またはヘモグロビンが混入する血漿を回収する手段などが任意に組み合わされ得る。本願発明の装置において、各手段は互いに連動可能とし得る。さらに、各手段は、それらの作動を停止する手段と連動し得るように構成され得る。これらの手段の組み合わせは、血漿の使用目的、所望の血漿レベル、装置の規模などによって任意に変更し得る。例えば、以下の手段を組み合わせた装置が代表的である。（１）（Ａ）に加えて（ａ）を有する装置。（２）（Ａ）に加えて、（ｂ）を有する装置。（３）（Ａ）に加えて、（ａ）および（ｂ）を有する装置；この装置では、使用するフィルターの種類などによって予め選択した量の血液の供給および血漿の回収を行うことにより、迅速かつ簡便に試料が得られる。（４）（Ａ）に加えて、（ｃ）、（ｄ）および（ｅ）を有する装置；このとき（ｃ）、（ｄ）および（ｅ）は通常互いに連動可能である。（５）Ａに加えて、（ｂ）〜（ｅ）の全てを有する装置。（６）Ａに加えて、（ａ）〜（ｅ）の全てを有する装置；これは、本願発明の装置のうち、最も精度の高い血漿分離を行い得る装置の一例である。
【０２１８】
本願発明の血漿分離装置による血液の平均処理線速度（フィルターの入口から出口までの平均処理線速度）は、特に限定されるわけではないが、好ましくは0.5〜500mm／分である。処理線速度が遅い場合は多数の検体を処理するのに不都合であり、速すぎる場合には、差圧の増加により溶血するおそれがある。
【０２１９】
（実施例１５：自動分析システムとの接続）
自動分析システムとは、血液分析の各過程における操作を、順序良く統合してシステム化することである。すなわち、検体の採量から分析をへてデータの記録に至る全過程を自動化したシステムのことである。自動分析システムは、分析方式によって、フローシステムおよびディスクリートシステムの２つのシステムに分けられる。本願発明の血漿分離装置はこれらのシステムに接続することができる。すなわち、フローシステムにおいては、いくつかの部品がポリエチレン管で連結されており、分析されるべき試料がこの連結しているチューブを流れていくうちに分析および記録されるが、本願発明の装置はこのシステムのサンプラー部分と接続することができる。一方、ディスクリートシステムは個々の検体をそれぞれ独立した反応管で分析する方式であり、フローシステムと同様に、サンプラー部分に本願発明の装置が接続され得る。あるいは、本願発明の装置により別個に得られた血漿をサンプルとして用い、これらの自動分析システムに供給してもよい。この場合、従来の自動分析システムをそのまま使用することができる。自動分析システムのサンプラー部分と本願発明の血漿分離装置、例えば、図９の装置の血漿回収ライン３７と接続され得る。
【０２２０】
（実施例１６）
実施例１の血漿分離フィルターに、血液供給手段および加圧手段を有するシリンジを取り付け、図７の血漿分離装置を作製した。
【０２２１】
牛血液４ｍｌを容器の入口から注入し、フィルターに装着した極細繊維の集合体の外周部表面と容器の内周部表面との空隙に牛血液を充填した後、０．２ｋｇ／ｃｍ²の圧力をかけて牛血液を押し出した。牛血液は装着された極細繊維の集合体の外周部表面から入り、この極細繊維の集合体の内部を外周部から円心部に向かって水平方向に移動した。加圧後２０秒経過して、容器の出口から血漿が流出し始めた。この分離された血漿を、連続的にサンプリングした。赤血球の混入のない血漿が０．５ｍｌ得られた。平均処理線速度は７５ｍｍ／分（＝２５ｍｍ／２０秒）であった。
【０２２２】
流出初期にサンプリングした血漿を０．４ｍｌ集め、血漿中の混入血球（赤血球、白血球、血小板）濃度、赤血球の溶血の有無、並びに、血液成分（血清アミラーゼ、総コレステロール、中性脂肪、尿素窒素、血糖）を分析して、装置を評価した。表４に結果を示した。
【０２２３】
【表４】

【０２２４】
分離前の牛血液中の赤血球濃度に対する得られた血漿中の赤血球濃度（以下赤血球混入率）は上記検出限度を考慮すると、０．００３％（＝２×１０⁵÷７．２×１０⁹）×１００）以下であった。赤血球の溶血はなく、得られた血漿のヘモグロビン濃度は５ｍｇ／ｄｌであり、許容範囲であった。また、血液成分の分析結果も従来の遠心分離法と変わらず、本願発明の装置の有効性が証明された。
【図面の簡単な説明】
【図１】図１は、本願発明の血漿分離装置に使用されるフィルターの一例を示す図である。
【図２】図２は、本願発明の血漿分離装置に使用されるフィルターの一例を示す図である。
【図３】図３は、図２のフィルターの空隙部分に基材を配置したフィルターである。
【図４】図４は、本願発明の血漿分離装置に使用されるフィルターの一例を示す図である。
【図５】図５は、本願発明の血漿分離装置に使用されるフィルターの一例を示す図である。
【図６】図６は、図４のフィルターに、加圧手段を加えた装置である。
【図７】図７は、図１のフィルターに、血液の供給および加圧手段を接続した装置である。
【図８】図８は、本願発明の血漿分離装置の一例を示す模式図である。
【図９】図９は、本願発明の血漿分離装置を自動化する構成の一例を示す模式図である。
【符号の説明】
１１：フィルター
１２：入口
１３：出口
１４：極細繊維の集合体
１５：容器
１６：空隙
１７：基材
１８：開口部
１９：容器の内壁
２２：密接手段
２４：加圧手段
２５：血液供給手段
２６：血液
３１：血液供給手段の容器
３２：血液供給ライン
３３：加圧空気供給ライン
３４：蓋
３５：コンプレッサー
３６：三方コックまたは切替バルブ
３７：血漿回収ライン
３８：試料血漿回収容器
４０：血球および／またはヘモグロビン検出手段
４１：廃液回収ライン
４２：廃液回収タンク
４３：純水供給ライン
４４：乾燥空気供給ライン
４５：フィルターホルダー
４６：血液供給ポンプ
４７：バルブ
４８：血液サンプル供給容器ラック
４９：液量計
５０：試料血漿回収容器ラック
５１：血液サンプル供給容器[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a plasma separation filter, a blood separation method using the filter, and a plasma separation apparatus provided with the filter. More specifically, a small amount of plasma necessary for blood tests and the like, which is substantially free of blood cells and / or hemoglobin and has almost the same protein and electrolyte composition as blood, can be collected easily, quickly and safely. The present invention relates to a plasma separation filter, a blood separation method using the filter, and a plasma separation apparatus equipped with the filter.
[0002]
[Prior art]
So-called biochemical tests that measure blood components are widely used for diagnosis and follow-up of various diseases, and occupy an important position as clinical tests. The analytical technique has made significant progress in recent years. For example, with the development of various automatic analyzers, many specimens can be analyzed quickly and accurately.
[0003]
However, in the field of biochemical tests, plasma or serum previously separated from blood is used because contamination of red blood cells or the like interferes with analysis of the target substance. Plasma or serum is obtained in advance by collecting blood from a patient or subject and then aggregating the blood cell components once and centrifuging them. Coagulation and centrifugation take time to operate, which not only prevents shortening of clinical examination time but also requires a large centrifuge. Therefore, with the exception of large hospitals, clinical tests are currently outsourced to outside contractors. As a result, it takes several days to obtain the test results.
[0004]
As described above, in spite of the fact that many clinical examination processes are automated, plasma separation still relies on human hands. Accordingly, the plasma separation operation not only makes clinical tests inefficient, but also has a problem that employees are exposed to dangers such as infection by contact with blood.
[0005]
As a means for solving the above problems, a technique generally called dry chemistry is known. This is because when a small amount of blood is dropped on a small plate consisting of a serum separation layer consisting of an ultrafine fiber filter such as glass fiber and a reaction layer located below it, the serum is separated in the serum separation layer. The reaction layer reacts and develops color, which is measured with a spectrophotometer. This dry chemistry is a simple method that does not use a liquid coloring reagent and does not require cumbersome serum collection by centrifugation, but the items that can be measured are compared to general biochemical analysis and immunoassay using liquid reagents. The number of plates is limited, and since one inspection item is used for one plate, a large number of plates must be used to inspect a plurality of items. Therefore, it has not been widely spread.
[0006]
As a means for rapidly obtaining plasma, there is a membrane separation method. Japanese Patent Application Laid-Open No. 53-72691 discloses a method for separating plasma from blood using a fine tube-shaped filter element (pore size 0.05 to 0.5 μm) closed at one end as a filter medium. However, in this method, blood cells adhere to the surface of the filter element, so that not only a very long time is required for filtration of plasma, but also the permeability of components such as proteins contained in plasma is poor. On the other hand, when the filtration pressure is increased in order to increase the filtration rate, there is a problem that hemolysis (the erythrocyte membrane is broken and hemoglobin inside is released) occurs. Furthermore, Japanese Patent Application Laid-Open No. 60-11166 proposes a method for separating plasma from blood using a filtration cartridge (pore diameter 0.05 to 1 μm) using a hollow fiber bundle. However, in this method, it is necessary to perform a priming operation (wet the hollow fiber membrane with a physiological saline solution) as a preparation for the plasma separation work. However, there is a problem in that accurate analysis data cannot be obtained because diluted plasma is diluted with physiological saline or the like.
[0007]
Since the separation method using a membrane is a separation method based on a molecular size, the permeability of a substance having a relatively large molecular weight such as a protein in blood is lowered. Therefore, there arises a problem that the composition of the protein contained in the plasma does not accurately reflect the composition of the protein before separation. Moreover, if the pore size of the membrane is too large, there is a problem of hemolysis due to clogging of red blood cells.
[0008]
Apart from the above, various serum or plasma separation techniques for clinical tests using a fibrous filter have been proposed. Japanese Patent Laid-Open No. 61-38608 discloses a solid-liquid separation device made of a fiber using a volume filtration effect. This solid-liquid separation device can obtain plasma by pressurizing and flowing blood into fibers. However, since the pressure loss is large and the resistance of the filter medium is large, it takes several minutes to obtain plasma. Moreover, since protein in the blood is adsorbed to the fiber and the protein concentration of the plasma obtained at the initial stage is lowered, it has not yet been put into practical use.
[0009]
JP-A-4-208856 and JP-A-5-196620 disclose a separation filter having a glass fiber containing a polyacrylic ester derivative and polyethylene glycol, and a lectin-impregnated layer, and a method for separating and recovering serum or plasma components using the filter And a serum or plasma separation device using the separation filter. Although these methods and instruments can collect serum or plasma for clinical tests without using centrifugation, the amount of serum or plasma obtained is as small as about 100 μl, and the time required for separation is about 2 minutes. Therefore, it cannot be said that the time is sufficiently shortened compared with the centrifugation. Furthermore, since these technologies use glass fibers as a separating material, the concentration of electrolytes, phosphorus, and lipids in the resulting plasma is necessary for elution of metal ions from the glass fibers and adsorption of blood components to the fibers. However, it has a disadvantage that it is greatly different from the concentration in blood before separation. For this reason, these techniques have not been widely spread.
[0010]
[Problems to be solved by the invention]
As described above, there is currently no filter having sufficient performance as a filter for separating plasma / serum components efficiently and safely from a small amount of blood in a short time as a clinical test application.
[0011]
[Means for Solving the Problems]
It is an object of the present invention to provide a plasma or serum separation filter that can be separated from blood simply, quickly and safely and has excellent assemblability. According to the present invention, plasma or serum components having the same component composition as blood can be obtained without damaging blood cells in blood. The present invention provides separation of blood components using an aggregate of ultrafine fibers. The mechanism of separation of plasma or serum by the aggregate of ultrafine fibers of the present invention is to cause a difference between the moving speed of red blood cells moving in the aggregate of ultrafine fibers and plasma or serum. This difference in moving speed is due to factors such as the improvement of the ultrafine fiber material, average fine diameter and fiber gap size, blood cell separation layer length, ultrafine fiber morphology, blood flow direction, and fiber surface condition. Is achieved by optimizing As a result, plasma or serum in the blood is separated from blood cell components such as red blood cells and collected. Moreover, since the pressure loss of the filter of the present invention is low, blood is rapidly processed, and the concentration of the obtained plasma or serum electrolyte or protein is substantially the same as the concentration before blood is separated. Therefore, according to the present invention, it is possible to obtain plasma or serum equivalent to plasma or serum obtained by ordinary centrifugation. The present invention is described in detail below.
[0012]
The present invention relates to a plasma separation filter. The filter of the present invention is a filter in which an assembly of ultrafine fibers is attached to a container having an inlet and an outlet, and the filter has the following characteristics:
(1) The ratio (L / D) of the blood flow path diameter (D) to the blood flow path length (L) of the attached microfiber assembly is 0.15 to 6; and
(2) The average hydrodynamic radius of the aggregate of the attached ultrafine fibers is 0.5 to 3.0 μm.
[0013]
In a preferred embodiment, the plasma separation filter of the present invention has an erythrocyte concentration of 6-8 × 10 6. ⁹ 0.2-0.4Kg / cm of fresh cow blood that is pcs / ml ² When the amount of plasma corresponding to 10% of the interstitial volume of the attached microfiber assembly is recovered from the start of separation,
(a) the concentration of red blood cells in plasma is not more than 0.1% of the concentration of red blood cells in blood before separation; and
(b) It has the property that erythrocytes are not substantially hemolyzed.
[0014]
Furthermore, when the plasma of the present invention collects plasma under the above conditions, the electrolyte concentration in the separated plasma is maintained at 90% or more compared to the electrolyte concentration in plasma obtained by centrifugation. Or the protein concentration in the separated plasma is maintained at 90% or more compared to the protein concentration in plasma obtained by centrifugation.
[0015]
In a preferred embodiment, the microfiber is polyester, polypropylene, polyamide, or polyethylene.
[0016]
Furthermore, in a preferred embodiment, the aggregate of ultrafine fibers is a nonwoven fabric.
[0017]
In a preferred embodiment, the non-woven fabric is attached to the container alone or in a laminated manner, and the non-woven fabric surface of the attached ultrafine fiber assembly or the flat surface of the laminated non-woven fabric It is configured so that blood flows almost in parallel.
[0018]
In a further preferred embodiment, the container and the nonwoven fabric are disk-shaped, the inlet is configured to supply blood over the entire circumferential side surface of the disk-shaped nonwoven fabric, and the outlet is disk-shaped. It is comprised so that plasma may flow out from the center part of a nonwoven fabric.
[0019]
The present invention also relates to a filter in which a hydrophilizing agent is fixed to ultrafine fibers.
[0020]
In a preferred embodiment, the hydrophilizing agent is fixed on the surface of the ultrafine fiber.
[0021]
In a further preferred embodiment, the hydrophilizing agent is polyvinyl pyrrolidone. And this filter has the following characteristics:
(1) The ratio (L / D) of the blood flow path diameter (D) to the blood flow path length (L) of the attached microfiber assembly is 0.15 to 6; and
(2) An average hydrodynamic radius of the aggregate of the attached ultrafine fibers is 0.5 to 3.0 μm.
[0022]
In a preferred embodiment, the filter in which the hydrophilizing agent is fixed to the ultrafine fibers has the following characteristics:
Red blood cell concentration is 6-8x10 ⁹ 0.2-0.4Kg / cm of fresh cow blood that is pcs / ml ² When the amount of plasma corresponding to 10% of the interstitial volume of the attached microfiber assembly is recovered from the start of separation,
(a) the concentration of red blood cells in plasma is not more than 0.1% of the concentration of red blood cells in blood before separation; and
(b) It has the characteristic that erythrocytes are not hemolyzed substantially.
[0023]
Furthermore, the filter in which the hydrophilizing agent is fixed to the ultrafine fiber of the present invention is such that when plasma is collected under the above conditions, the electrolyte concentration in the separated plasma is the electrolyte in plasma obtained by centrifugation. The protein concentration in the separated plasma is maintained at 90% or more compared to the concentration of the plasma, or the protein concentration in the separated plasma is maintained at 90% or more compared with the protein concentration in the centrifuged plasma.
[0024]
In a preferred embodiment, the microfiber is polyester, polypropylene, polyamide, or polyethylene.
[0025]
Furthermore, in a preferred embodiment, the aggregate of ultrafine fibers is a nonwoven fabric.
[0026]
In a preferred embodiment, the non-woven fabric is attached to the container alone or in a laminated manner, and the non-woven fabric surface of the attached ultrafine fiber assembly or the flat surface of the laminated non-woven fabric It is configured so that blood flows almost in parallel.
[0027]
In a further preferred embodiment, the container and the nonwoven fabric are disk-shaped, the inlet is configured to supply blood over the entire circumferential side surface of the disk-shaped nonwoven fabric, and the outlet is disk-shaped. It is comprised so that plasma may flow out from the center part of a nonwoven fabric.
[0028]
The present invention also relates to a method for separating plasma using a plasma separation filter having the above characteristics or a plasma separation filter in which ultrafine fibers are fixed with a hydrophilizing agent.
[0029]
In a preferred embodiment, a filter is used in which the non-woven fabric is singly or laminated and attached to the container.
[0030]
In a more preferred embodiment, the linear velocity of the blood to be treated is 0.05 to 50 cm / min.
[0031]
Furthermore, the present invention relates to a plasma separation device having a plasma separation filter having the above characteristics or a plasma separation filter having ultrafine fibers fixed with a hydrophilizing agent.
[0032]
In a preferred embodiment, the device of the present invention further comprises blood supply means for supplying blood to the filter, and pressurization for pressurizing the blood supplied to the filter to separate plasma from the supplied blood. Means, and plasma collection means for collecting the separated plasma.
[0033]
In a further preferred embodiment, the device of the present invention comprises a blood cell and / or hemoglobin detection means for detecting blood cells and / or hemoglobin in separated plasma, and a switching for separating blood cells and / or hemoglobin-contaminated plasma. Means and a blood cell and / or hemoglobin-contaminated plasma recovery means for recovering plasma contaminated with sorted blood cells and / or hemoglobin.
[0034]
In a preferred embodiment, in the apparatus of the present invention, the filter is detachably attached between the blood supply means and the plasma collection means.
[0035]
In a preferred embodiment, the device of the present invention has a blood supply means for supplying a constant amount of blood and / or a plasma recovery means for recovering a constant amount of plasma, or both means.
[0036]
DETAILED DESCRIPTION OF THE INVENTION
The blood used in the present invention generally refers to a blood containing components such as blood cells and plasma. The blood may be derived from humans, cows, goats, dogs, rabbits and the like. Blood can be used as it is, and blood to which additives such as anticoagulants and hemagglutinating agents are added can also be used. Furthermore, when the blood is left without adding an additive or when a coagulant is added, fibrinogen in the blood changes to fibrin and the blood coagulates. Can be used. Further, blood that has been subjected to chemical treatment after centrifugation or the like can also be used.
[0037]
In addition, the plasma as used in this invention means the plasma which does not contain a blood cell component substantially. Therefore, it is not limited to plasma strictly containing no blood cell component. Furthermore, when the solid content in the blood is separated and removed after the blood is coagulated, serum without fibrinogen is obtained. In the present invention, the term “plasma” includes serum unless otherwise specifically used.
[0038]
Hereinafter, the plasma separation filter, the plasma separation method, and the plasma separation device will be described in this order.
[0039]
(Plasma separation filter)
In the plasma separation filter of the present invention, an assembly of ultrafine fibers is attached to a container having an inlet and an outlet, and the average hydrodynamic radius of the attached ultrafine fibers is preferably 0.5 μm to 3 μm. 0.0 μm. More preferably, it is 0.5-2.5 micrometers, Most preferably, it is 0.5-2.0 micrometers. Here, the average hydrodynamic radius is expressed as a concept that replaces the diameter when the gap of the attached microfiber assembly is non-circular, and is defined as follows:

In the present invention, the hydrodynamic radius is calculated by the following equation (1):
DH = R × (ρ−rm) / 4 rm (1)
DH: Average hydrodynamic radius (μm) of the aggregate of attached ultrafine fibers
R: Average fiber diameter (μm) of ultrafine fibers
ρ: density of ultrafine fibers (g / cm ^Three )
rm: average bulk density (g / cm) of the aggregate of attached ultrafine fibers ^Three )
As shown in the equation (1), the average hydrodynamic radius DH of the attached aggregate of ultrafine fibers is determined by R and rm when the ultrafine fibers of the same material are used (that is, when ρ is constant). Is done.
[0040]
When the average hydrodynamic radius exceeds 3.0 μm, blood cells easily pass through the fiber gap. In this case, only the same effect as a conventional filter such as a leukocyte removal filter or leukocyte separation filter that removes leukocytes from blood is provided. Only components having specific adhesive properties such as leukocytes (including some platelets) are adsorbed and removed by the assembly of ultrafine fibers.
[0041]
When the average hydrodynamic radius is less than 0.5 μm, the fiber gap in the filter, that is, the blood flow path becomes too narrow, and blood cell components are likely to be clogged. Further, if pressure is applied to the filter in an attempt to increase the amount of liquid passing therethrough, pressure loss increases and red blood cell hemolysis is likely to occur.
[0042]
In addition, in the range of an average hydrodynamic radius of 0.5 μm to 3.0 μm, the smaller the average hydrodynamic radius is, the smaller the particle size is as in the plasma component, but the permeability is not affected. On the other hand, those having a large particle size such as blood cell components are difficult to pass through the filter. Therefore, the average hydrodynamic radius is preferably 0.5 μm to 2.8 μm, particularly preferably 0.5 μm to 2.0 μm.
[0043]
Further, the hydrodynamic radius of the ultrafine fiber assembly of the present invention can be constant over the axial direction from the blood supply side to the plasma outlet side, and can be changed by the portion of the ultrafine fiber assembly. The hydrodynamic radius can be gradually decreased from the blood inlet toward the outlet of the passing liquid. This can increase the separation efficiency between the blood cell component and the plasma component in the vicinity of the plasma outlet.
[0044]
In the present invention, the average hydrodynamic radius is an aggregate of ultrafine fibers in a state of being attached to a container having an inlet and an outlet, and is an aggregate of ultrafine fibers that can substantially participate in plasma separation. The mean hydrodynamic radius at. Therefore, for example, when an aggregate of ultrafine fibers is used as the base material 17 for filling the gap 16 in FIG. 2 (FIG. 3), the aggregate of ultrafine fibers of the base material 17 cannot participate in plasma separation. The mean hydrodynamic radius of the part excluding this part.
[0045]
When this is seen from another aspect, when the aggregate of the ultrafine fibers attached to the filter is viewed as a whole, it may indicate that the average hydrodynamic radius outside the above preferred range may be obtained. However, even in this case, the fact that plasma can be separated is nothing but that it indicates that at least a part of the aggregate of attached ultrafine fibers has an average hydrodynamic radius in the above-mentioned preferable range.
[0046]
In the present invention, a prefilter for removing foreign substances in blood can be installed in front of an aggregate of ultrafine fibers for plasma separation. The average pore diameter and the average hydrodynamic radius of these prefilters are naturally larger than the average hydrodynamic radius of the aggregate of the ultrafine fibers, but the average pore radius of the prefilter is not considered in the average hydrodynamic radius of the filter as a whole. First, the average hydrodynamic radius of the main filter must be used.
[0047]
In the present invention, the aggregate of ultrafine fibers refers to a state in which the ultrafine fibers are irregularly aggregated. This state can be obtained, for example, by compressing, for example, cotton-like, non-woven fabric, woven fabric, or knitted fabric ultrafine fibers alone or in combination. The ultrafine fibers attached to the filter are preferably non-woven or cotton-like ultrafine fibers from the viewpoints of moldability, processability, ease of handling, and difficulty in channeling and drift after molding. In particular, the nonwoven fabric is preferable. This is because, when an assembly of ultrafine fibers is filled in a filter case, uniformity is easily maintained, partial roughness is difficult to achieve, and blood flow becomes uniform.
[0048]
The material for the ultrafine fiber is not particularly limited, and examples thereof include polyester, polypropylene, polyamide, and polyethylene. Preferably, hydrophobic polypropylene or polyester-based material (for example, polyethylene terephthalate) can be used.
[0049]
These materials are preferable because they do not adsorb plasma or serum components when they come into contact with blood, and conversely, some of the materials do not elute in plasma or serum. When using plasma or serum separation filters that use glass fibers as described in the section of the prior art, metal ions are eluted from the glass fibers, and phosphorus and lipids are adsorbed on the glass fibers. I couldn't.
[0050]
The length of the blood cell separation layer in the present invention is preferably 5 mm or more. The length of the blood cell separation layer refers to the length from the point where the aggregate of ultrafine fibers and blood come into contact with the place where blood (plasma or serum) is separated from the aggregate of ultrafine fibers. As described above, in the present invention, blood cells and plasma are separated by utilizing the difference in the moving speed of blood components in the assembly of ultrafine fibers. By performing pressurization from the inlet side of the blood cell separation layer, decompression from the outlet side, or both at the same time, blood is moved in the assembly of ultrafine fibers. At this time, the blood cell component repeatedly collides with the ultrafine fiber through the gap between the ultrafine fibers. Adhesive white blood cells and platelets are adsorbed to ultrafine fibers, and red blood cells do not have adhesive properties, and thus move while repeating deformation. On the other hand, since plasma or serum is a liquid component, it travels through ultrafine fibers faster than red blood cells, and reaches the outlet earlier. At this time, if the length of the blood cell separation layer is less than 5 mm, a sufficient difference in the moving distance between the blood cells and the plasma does not occur, and the separation between the two becomes insufficient. The longer the blood cell separation layer is, the higher the separation efficiency of blood cells and plasma is. On the other hand, there is a problem that the pressure loss increases, or the amount of necessary ultrafine fibers and the amount of blood increase. Therefore, although the length of the blood separation layer is determined by the amount of plasma or serum required, the amount of blood used, the size limit of the filter, etc., there is no theoretical upper limit.
[0051]
When using a nonwoven fabric, it is preferable that the blood flows parallel to the plane of the nonwoven fabric (the plane of the laminated nonwoven fabric). In general, when a nonwoven fabric is used as a filter, the flow direction of the treatment liquid is perpendicular to the plane of the nonwoven fabric (the plane of the laminated nonwoven fabric). However, in the present invention, separation efficiency of blood cells and plasma components is improved by flowing blood parallel to the surface of the nonwoven fabric. When blood flows parallel to the plane direction of the nonwoven fabric, when blood flows from the inlet to the outlet, there is no break in the ultrafine fibers over the entire flow path length, which improves the blood flow uniformity. However, it is not limited to this reason.
[0052]
Those in which a hydrophilizing agent is fixed to ultrafine fibers can also be used as the filter of the present invention. The immobilization of the hydrophilizing agent can be performed physically or chemically. By fixing the hydrophilizing agent to the fiber surface, the affinity between ultrafine fibers and blood is increased. Therefore, when separating blood into blood cells and plasma, the pressure loss is reduced, and the separation speed can be increased. The hydrophilizing agent is not particularly limited as long as it does not interfere with the analysis even if it is mixed with plasma or serum. Polyvinyl pyrrolidone is preferred. Polyvinyl pyrrolidone has a relatively high molecular weight and thus has a relatively low elution rate, but is eluted in blood. However, it does not affect the analysis of blood components. The method for immobilizing polyvinylpyrrolidone is not particularly limited, and a known method can be used. For example, it can be physically fixed on the fiber surface easily by immersing an aqueous solution of polyvinylpyrrolidone in an assembly of ultrafine fibers and then drying. Moreover, polyvinylpyrrolidone can be easily cross-linked by subjecting an aggregate of ultrafine fibers in which such polyvinylpyrrolidone is physically fixed to the surface to heat treatment and radiation treatment. By crosslinking, elution of polyvinylpyrrolidone into blood can be suppressed to a lower level.
[0053]
In the case of heat treatment, the method is not particularly limited. For example, the method of heating under pressure like an autoclave process, the method of leaving in a thermostat, etc. are mentioned. Further, the temperature of the heat treatment is not particularly limited, but is preferably 70 ° C. or higher, more preferably 100 ° C. or higher. The higher the heating temperature, the more efficient the crosslinking. The upper limit temperature is not uniquely determined depending on the properties of the ultrafine fibers used and the heat resistance of polyvinylpyrrolidone itself, but is preferably 200 ° C. or lower, and more preferably 150 ° C. or lower. The heating time is preferably longer for sufficient crosslinking, but is limited by the properties of the ultrafine fibers used and the modification of polyvinylpyrrolidone. Generally, 20 minutes or more and 2 hours or less are preferable. Moreover, the crosslinking by heating can be carried out either when the ultrafine fibers are immersed in the hydrophilizing agent aqueous solution (WET state) or when they are dried after immersion (DRY state). In any case, polyvinylpyrrolidone can be fixed to the ultrafine fibers. Unreacted polyvinylpyrrolidone can be removed by washing with water.
[0054]
A method for fixing the hydrophilizing agent using radiation is not particularly limited. Examples include γ-ray irradiation, electron beam irradiation, corona discharge, and the like. Γ-ray irradiation is preferable from the viewpoint of the thickness that can be treated and operability. The irradiation amount is not particularly limited as long as the polyvinylpyrrolidone is sufficiently crosslinked. The range of 10 KGy or more and 50 KGy or less is preferable as the range in which the ultrafine fiber material or polyvinylpyrrolidone itself is not denatured by irradiation. Further, radiation irradiation can be performed in a WET state or a DRY state. Unreacted polyvinyl pyrrolidone can be removed by washing with water or the like.
[0055]
Polyvinyl pyrrolidone is available in various molecular weights. In order to avoid elution into blood, it is particularly preferable to use one having a large molecular weight.
[0056]
A filter is produced using an aggregate of ultrafine fibers to which the hydrophilizing agent is fixed.
[0057]
The filter can be produced by laminating and compressing cotton-like, non-woven fabric, woven fabric, and knitted fabric-like ultrafine fibers alone or in combination.
[0058]
In the present invention, the shape of the filter container is not particularly limited. Examples include a rectangular parallelepiped shape, a disc shape, a cylindrical shape, a truncated cone shape, and a fan shape. Among these, the separation performance is improved when a parallelepiped, disk-shaped or fan-shaped filter is used to flow parallel to the surface of the aggregate of ultrafine fibers. In the case of a rectangular parallelepiped shape, blood is allowed to flow from one end face to the other end face, or it is formed in a disk shape, and blood is allowed to flow from its peripheral face toward the center. The filter can be sealed by pressing the nonwoven fabric with a container of these shapes. Therefore, it is particularly preferable because it is not necessary to use an adhesive. Among these, a disk shape or a fan shape is more preferable because the cross-sectional area of the blood flow path gradually decreases as the blood moves, so that unevenness in the movement of blood components in the lateral direction is reduced. In particular, a disk-shaped filter is most preferable because of its excellent operability. In addition, in the case of a disk-shaped container, the nonwoven fabric to be mounted also has a disk shape. It is preferable to configure the blood inlet so that blood can be supplied over the entire circumference of the disk-shaped nonwoven fabric. For example, a gap may be provided in the circumferential portion of the disk-shaped nonwoven fabric and the disk-shaped container, and one or a plurality of inlets communicating with the gap may open on the side surface, upper surface, or lower surface of the container.
[0059]
The average fiber diameter of the ultrafine fibers used in the filter of the present invention is preferably 0.5 to 3.5 μm, more preferably 0.5 to 2.8 μm, and particularly preferably 0.5 to 2.0 μm. It is.
[0060]
The ultrafine fibers having the above average fiber diameter can be obtained by a usual ultrafine fiber spinning method such as melt blow.
[0061]
Here, the average fiber diameter of the ultrafine fibers is obtained by measuring the diameters of 50 ultrafine fibers randomly selected from photographs obtained by photographing an aggregate of ultrafine fibers with an electron microscope of 2000 times with a caliper or a scale loupe. The average value of the measured values.
[0062]
When the average fiber diameter of the fibers exceeds 3.5 μm, the length of the fibers per unit volume of the aggregate of ultrafine fibers is shortened. As a result, the number of entangled portions between the fibers decreases, and the fiber gap increases. As a result, components having a large particle size such as blood cells are likely to pass through the assembly of ultrafine fibers, and separation of blood cells and plasma is worsened. Furthermore, the adsorptivity to leukocytes and platelets is reduced.
[0063]
When the average fiber diameter of the ultrafine fibers is less than 0.5 μm, the length of the fibers per unit volume is increased. As a result, the number of entangled portions between the fibers increases and the fiber gap decreases. As a result, blood cells tend to clog the filter. Furthermore, since the pressure loss of the aggregate of ultrafine fibers is increased, red blood cell hemolysis is likely to occur.
[0064]
The average bulk density of the aggregate of attached ultrafine fibers used in the present invention is preferably 0.15 to 0.60 g / cm. ^Three More preferably, 0.18 to 0.50 g / cm ^Three And particularly preferably 0.25 to 0.40 g / cm. ^Three It is.
[0065]
Here, the average bulk density of the aggregate of ultrafine fibers refers to a value obtained by dividing the weight of the aggregate of ultrafine fibers by the volume of the aggregate of ultrafine fibers.
[0066]
Average bulk density is 0.15 g / cm ^Three If smaller, the average bulk density immediately after spinning of the aggregate of ultrafine fibers (for example, 0.08 to 0.10 g / cm in the case of the melt blow spinning method) ^Three ) Is small, the compression ratio of the aggregate of ultrafine fibers is small. Therefore, partial density is likely to occur in the attached ultrafine fiber assembly, and blood passage speed may be uneven. Moreover, since the fiber gap is large on average, the blood and plasma are not sufficiently separated.
[0067]
The average bulk density of the attached ultrafine fiber assembly is 0.60 g / cm ^Three If it is larger, a special process such as heat compression is required to produce an assembly of ultrafine fibers, and the compression process is complicated. Furthermore, since the fiber gap of the attached microfiber assembly is small, blood cell components are likely to clog the filter, and because the pressure loss of the microfiber assembly is large, red blood cell hemolysis occurs. It becomes easy.
[0068]
In addition, average bulk density 0.15-0.60 g / cm ^Three In this range, by increasing the average bulk density, the uniformity of the aggregate of ultrafine fibers is further improved. On the other hand, the workability is lowered, and preferably 0.18 to 0.50 g / cm. ^Three And particularly preferably 0.25 to 0.40 g / cm. ^Three It is.
[0069]
The bulk density of the aggregate of ultrafine fibers attached to the filter of the present invention can be partially changed. For example, the bulk density of the aggregate of attached ultrafine fibers can be gradually increased from the inlet to the outlet of the filter container. In this way, the separation between blood cells and plasma is improved as the blood component moves toward the outlet.
[0070]
The ratio (L / D) of the channel length (L) to the channel diameter (D) of the blood component of the aggregate of ultrafine fibers attached to the filter of the device of the present invention is 0.15 to 6. Preferably it is 0.25-4, Most preferably, it is 0.5-2.
[0071]
Here, the flow path length (L) of the blood component is the linear distance inside the aggregate of ultrafine fibers from the time when blood contacts the ultrafine fibers until the plasma leaves the ultrafine fibers (usually a collection of ultrafine fibers). Body length). Further, the flow diameter (D) of the blood component refers to a circle-equivalent diameter of the cross-sectional area of the aggregated surface of the ultrafine fibers at the blood inlet portion that is perpendicular to the flow path length direction.
[0072]
The equivalent circle diameter is determined by the following equation (2) from the cross-sectional area (A).
[0073]
D = 2 (A / π) ^1/2 (2)
In addition, strictly speaking, the surface of the aggregate of ultrafine fibers has small irregularities due to the bending of the ultrafine fibers, but the cross-sectional area is calculated as a plane ignoring the irregularities. In addition to the unevenness caused by the bending of the ultrafine fiber, in the case of having a large unevenness formed by surface processing of the aggregate of ultrafine fibers, the cross-sectional area is calculated as a plane obtained by averaging the unevenness.
[0074]
When L / D is smaller than 0.15, the flow path is too short and the moving distance of each component in the blood is shortened. Furthermore, since the cross-sectional area is large, the moving speed of each component in the blood is uneven in the lateral direction. Therefore, separation of blood cell components and plasma components becomes insufficient.
[0075]
When L / D exceeds 6, the separation efficiency is improved, but since the moving distance becomes long, the pressure loss becomes high, and hemolysis of erythrocytes may occur.
[0076]
The filter of the present invention has the following characteristics.
[0077]
Red blood cell concentration is 6-8x10 ⁹ 0.2-0.4Kg / cm of fresh cow blood that is pcs / ml ² When the amount of plasma corresponding to 10% of the interstitial volume of the attached microfiber assembly is recovered from the start of separation,
(a) the concentration of red blood cells in plasma is not more than 0.1% of the concentration of red blood cells in blood before separation; and
(b) The red blood cells are not substantially hemolyzed.
[0078]
Furthermore, the electrolyte concentration in the separated plasma is maintained at 90% or more compared to the electrolyte concentration in the plasma obtained by centrifugation.
[0079]
In the present invention, it is preferable that the electrolyte concentration in plasma separated by the plasma separation filter is maintained at 90% or more compared to the electrolyte concentration in plasma obtained by normal centrifugation. That is, it is preferable that the decrease in the electrolyte concentration after the filter separation is within 10% as compared with the case where the centrifugation is performed. It is more preferable if it is within 5%. If the decrease in plasma electrolyte concentration exceeds 10%, the reliability of biochemical diagnosis is lowered, which is not preferable. In view of the measurement accuracy of the biochemical test, if the difference between the two is within 10%, it is not a level causing a problem in practice, and if it is within 5%, there is almost no problem. Here, plasma refers to a supernatant obtained by adding an anticoagulant to blood obtained by blood collection and then performing centrifugation. Centrifugation is performed at 1000 G for 10 minutes.
[0080]
In the present invention, it is preferable that the protein concentration at the beginning of collection and at the end of collection of plasma separated by the plasma separation filter is maintained at 90% or more. That is, the decrease in the protein concentration after the filter separation is preferably within 10% as compared with the case of centrifugation. It is more preferable if it is within 5%. If the decrease in plasma protein concentration exceeds 10%, the reliability of biochemical diagnosis decreases, and the values at the beginning of collection and at the end of collection differ, and accurate diagnosis may not be possible. On the other hand, if the difference in protein concentration exceeds 10%, the composition of the plasma protein may fluctuate greatly, and it may not be possible to use for diagnosis of a disease state, which is not preferable. Usually, a difference of 10% or less is not problematic in clinical diagnosis, and a difference of 5% or less is preferable because it falls within the measurement error range.
[0081]
The material of the container of the filter of the present invention is not particularly limited, and examples thereof include metal, glass, and plastics such as polyethylene, polypropylene, nylon, polycarbonate, polystyrene, polyester, and ABS resin. When observing the internal state, a transparent or translucent material may be selected. From the viewpoints of workability, resistance to cracking and light weight, plastics are preferred.
[0082]
Hereinafter, the filter used in the plasma separator of the present invention will be described with reference to the drawings. The filter of the present invention is shown most simply in FIG. FIG. 1 is a view showing a filter 11 in which a cylindrical flat microfiber assembly 14 is attached to a container 15 having an inlet 12 and an outlet 13. In the filter 11, the ultrafine fibers are compressed, and the filter 15 is mounted so that an appropriate gap 16 is provided between the inner peripheral surface of the container 15 and the outer peripheral surface of the aggregate 14 of the ultrafine fibers. The outer peripheral portion of the attached microfiber assembly 14 may be formed of, for example, a plastic plate having appropriate holes through which blood can pass. When blood is supplied from the inlet 12 of the container 15 at the upper part of the outer periphery of the assembly 14 of the microfibers mounted in a cylindrical shape, the blood is uniformly passed through the gap 16 to the microfiber assembly 14 mounted with blood. Then, blood flows from the outer peripheral portion of the aggregate 14 of ultrafine fibers toward the central portion, while being separated into plasma and blood cells, and the plasma is recovered from the outlet 13 in the central portion of the container 15. Hereinafter, the aggregate 14 of ultrafine fibers simply means an aggregate of ultrafine fibers attached to a filter.
[0083]
Another embodiment is the filter 11 of FIG. The filter 11 has a collection 14 of cylindrical ultrafine fibers attached to a cylindrical container 15. Blood is supplied from the inlet 12 of the container 15, and plasma separated by the microfiber assembly 14 is collected from the outlet 13 of the container 15. Each of the inlet 12 and the outlet 13 may be disposed at an arbitrary position.
[0084]
Similar to the filter of FIG. 1, the cylindrical ultrafine fiber assembly 14 of FIG. 2 may have a gap 16 in the vicinity of the inlet 12. The gap 16 may be disposed when blood is supplied and pressurized evenly.
[0085]
The filter 11 of FIG. 3 is a filter in which an appropriate base material 17 is disposed in the gap 16 of the filter 11 of FIG. As the base material 17 to be arranged, any base material that has a fiber gap larger than the fiber gap of the aggregate 14 of ultrafine fibers and can pass through all components in blood is used. Can be done. This base material may be a filter medium having a plate shape, paper shape, fiber shape, or the like. Also in the filter of FIG. 1, a base material 17 can be disposed in the gap 16.
[0086]
The filter 11 of FIG. 4 is a filter in which an assembly 14 of ultrafine fibers is attached to a container 15 having an opening 18 and an outlet 13.
[0087]
The filter 11 can be detachably attached to blood supply means and / or plasma recovery means described below. As another embodiment, as illustrated in FIGS. 5A and 5B, the container 15 includes a portion having an inlet 12 or an opening 18 (15a in FIG. 5), and a collection of ultrafine fibers. It is comprised from three parts, the part which has the body 14, and the part (15b of FIG. 5) which has the exit 13, Each part can be comprised detachably. With this configuration, only a part of the assembly of attached ultrafine fibers can be exchanged. The aggregate portion of the ultrafine fibers that can be exchanged can be covered with a blood permeable membrane or the like, and can be in the form of a cassette that can be detachably fitted. As the detachable mounting means, there can be means such as fitting and screwing, means using a magnet, and the like. These attachments and detachments can be performed automatically.
[0088]
As described above, when the filter of the present invention is produced by laminating ultrafine fibers, the direction of blood flow is substantially perpendicular to the laminated surface or substantially parallel to the laminated surface. obtain. In the former case, a columnar or spindle shape is suitable. For example, when blood flows from the upper part to the lower part of a cylindrical filter (FIGS. 2 to 4), or using a truncated cone filter with the apex portion cut into a conical shape, the blood flows from the bottom of the cone toward the apex. This applies to the case of flowing In the latter case, a filter laminated in a disk shape (for example, FIG. 1) is used, parallel to the laminated surface, from the periphery to the center, or in the side direction opposite from one side of the filter laminated in a flat plate shape. This is the case for blood flow. A frustoconical or disk-shaped filter gradually reduces the cross-sectional area of the blood flow path as the blood moves, so the moving speed of the blood component increases and the uneven movement of the blood component in the lateral direction occurs. , The blood separation efficiency is improved.
[0089]
A plurality of the filters of the present invention can be connected. Separation is improved when connected in series. When connected in parallel, the amount of blood to be processed increases.
[0090]
Furthermore, it can be used in combination with a filter and a blood sampling container. The filter combined with such a sampling container can be used in an automatic analyzer having means for collecting a certain amount of blood and supplying it to the filter and means for automatically replacing the used filter.
(Plasma separation method)
In the method of separating plasma, the pressure loss at the outlet is 0.03 to 5 kg / cm. ² It is. This value depends on the filter shape and blood processing speed.
[0091]
Pressure loss is 0.03kg / cm ² If it is smaller, the load on the blood in the assembly of microfibers is small. Accordingly, when ultrafine fibers having high hydrophobicity are used, there is a problem that blood cannot be sent into the aggregate of ultrafine fibers, or the processing takes too much time. In addition, there is no difference in the moving speed between the blood cell component and the plasma component in the ultrafine fiber assembly, resulting in insufficient plasma separation.
[0092]
Pressure loss is 5kg / cm ² If it is larger, the blood flow rate is too high. Therefore, the plasma may not be separated because the difference in the transit time between plasma and blood cells is small. Furthermore, due to the pressure applied, red blood cells may be hemolyzed or the filter or device may be damaged.
[0093]
When the pressure is increased within the above range, the amount of plasma obtained increases, and the time for obtaining plasma is shortened. On the other hand, there is a possibility of hemolysis, so 0.05-3 kg / cm ² The range of is preferable. 0.1-1kg / cm ² The range of is more preferable.
[0094]
In the plasma separation method of the present invention, the ratio of the treated blood volume and the total area of the ultrafine fibers is not particularly limited, but is 0.1 to 3 m per 1 ml of blood. ² Is preferred. Proportion of treated blood volume and total area of ultrafine fibers 0.1 ml per 1 ml of blood ² If it is smaller, the separation efficiency of plasma is deteriorated. The ratio of the treated blood volume to the total area of ultrafine fibers is 3m / ml of blood ² If it is larger, the blood volume is too small, making it difficult to send blood and collect plasma. Furthermore, as a result of the plasma protein being adsorbed on the ultrafine fibers, the protein component in the plasma component may not accurately reflect the protein component in the original blood. Furthermore, pressure loss is likely to occur.
[0095]
Within the above range, the amount of plasma obtained increases when the surface area of the ultrafine fibers is increased. On the other hand, the amount of protein adsorbed on the fiber also increases. Therefore, the above ratio is 0.2-2 m. ² The range of is preferable. 0.3-1.5m ² The range of is more preferable.
[0096]
In the plasma separation method of the present invention, the linear velocity of blood treatment is not particularly limited, but is about 0.05 to 50 cm / min. When the linear velocity is less than 0.05 cm / min, the time for blood components to pass through the aggregate of ultrafine fibers becomes longer. As a result, the separated plasma and blood cells diffuse while moving, resulting in insufficient separation.
[0097]
When the treatment linear velocity is higher than 50 cm / min, the pressure loss increases and the red blood cells are hemolyzed.
[0098]
When liquid is fed from the outer periphery of the disk shape (for example, FIG. 1) toward the center, or when liquid is fed toward the apex of the cone using a conical container, the linear velocity depends on the location. Is different. The linear velocity in such a case refers to an average processing linear velocity until blood leaves after contacting with the ultrafine fibers.
[0099]
In the plasma separation method of the present invention, the ratio (B / A) between the blood volume to be processed (B) and the interstitial volume (A) of the aggregate of ultrafine fibers is preferably 0.2 or more. If the ratio is less than 0.2 (the blood volume is less than 20% of the interstitial volume), the blood volume is too small, making it difficult to pump blood and collect plasma. Furthermore, as a result of the plasma protein being adsorbed on the ultrafine fibers, the protein component in the plasma component may not accurately reflect the protein component in the original blood. The ratio is preferably 0.5 or more, particularly preferably 0.7 or more.
[0100]
In the plasma separation method of the present invention, a piston, pressurized air, or the like can be used as a method for supplying and pressurizing blood. In addition, there is a method of extruding using a liquid that does not react with blood components, for example, a liquid with high viscosity, paraffin, glycerin or the like to prevent mixing. The method using pressurized air or paraffin is suitable when the volume of blood to be processed is smaller than the interstitial volume of the aggregate of ultrafine fibers. However, in the case of pressurized air, the blood in the aggregate of ultrafine fibers may not be able to flow due to the surface tension generated in the gaps in the aggregate of ultrafine fibers. On the other hand, the method using paraffin or glycerin is preferable because the problem of surface tension does not occur.
[0101]
(Plasma separator)
The plasma separation device of the present invention includes a plasma separation filter, and preferably includes blood supply means for supplying blood to the filter, pressure means for pressurizing the supplied blood, and plasma recovery means for recovering the separated plasma. Including. Furthermore, detection means for detecting blood cells and / or hemoglobin in the separated plasma, switching means for separating blood cells and / or plasma mixed with hemoglobin, blood cells for collecting blood cells and / or plasma mixed with hemoglobin, and It may include / or a hemoglobin-containing plasma recovery means, or a plasma recovery means for recovering plasma confirmed to be free from blood cells and / or hemoglobin. Further, the blood supply means can be a blood supply means for supplying a constant amount of blood, and the plasma recovery means can be a plasma recovery means for recovering a constant amount of plasma. The above means will be described below.
[0102]
<Blood supply means>
In the apparatus of the present invention, known means can be used as means for supplying blood to the filter. For example, a means for supplying blood using a pump, a means for pressurizing a container containing blood, a means for supplying blood using the pressure, a pressure inside the filter, and a pressure difference from the blood storage container Examples include a means for supplying blood (for example, a method using a vacuum pump, or a method in which in FIG. 6B, the piston is moved upward to depressurize the inside of the cylinder and lead blood from the inlet 12). Of course, means for manually supplying blood, such as a pipette, may also be included. Means for supplying blood to the filter may be connected to the inlet of the filter. Any connection method can be used as long as blood does not leak. For example, the blood outlet of the blood supply means and the inlet of the filter can be connected by being configured to be fitted, connected, or screwed together.
[0103]
Further, the outlet of the blood supply means may be sealed, and a means capable of being pierced at the inlet of the filter may be arranged. On the contrary, the filter inlet may be sealed and the blood supply means may be provided with a pierceable means. Examples of means that can be perforated include hollow and tubular means such as an injection needle. In either case, perforation allows blood to be supplied to the filter. Such a piercable connecting means is particularly effective in automating blood supply and avoiding contact with blood. When separating plasma from blood collected with a vacuum blood collection tube used in recent years, the vacuum blood collection tube containing the blood is directly drilled at the sealed inlet of the filter. By reducing the pressure inside the container of the filter, blood is supplied to the aggregate of ultrafine fibers attached to the filter. It is also possible to use a device that pulls out the vacuum blood collection tube and pressurizes it from the inlet side of the filter when blood is supplied.
[0104]
The blood supply means may be a supply means for supplying a certain amount of blood. For example, a certain amount of blood supply device such as a roller pump or a cylinder pump can be used. When the blood is supplied at a constant flow rate, the supply means can be stopped. For example, a sensor may be provided in the blood storage device, the amount of blood decrease in the blood storage tank may be measured, and the supply means may be stopped.
[0105]
The volume of blood components processed by the apparatus of the present invention (the amount of blood supplied by the supply means) is preferably 20% or more of the interstitial volume of the aggregate of ultrafine fibers attached to the filter, more preferably It is 50% or more, and particularly preferably 70% or more. If it is less than 20%, it may be difficult to send blood into the attached microfiber assembly, and some of the plasma components may remain in the gap and not be collected. Here, the interstitial volume of the attached ultrafine fiber assembly is defined as the volume of the attached ultrafine fiber assembly− (total weight of attached ultrafine fiber assembly / attached ultrafine fiber density). Is done.
[0106]
<Pressurizing means>
The blood held or supplied to the attached microfiber aggregate is pressurized on the blood side and / or depressurized on the permeate side of the attached ultrafine fiber assembly to thereby remove the attached ultrafine fiber. Is moved to the permeate side, and is separated into plasma and blood cells using the difference in moving speed between plasma and blood cells. At this time, leukocytes and platelets, which are blood cell components, are adsorbed to the attached aggregate of ultrafine fibers. The method of pressurizing and depressurizing is not particularly limited. Pressurization with a pump, pressurization with a gas such as pressurized air, a method of pressurizing and extruding a liquid that does not react with or compatible with blood components (eg, paraffin, glycerin, etc.), or a method of suction using a vacuum pump or cylinder Is mentioned. The method of extruding with paraffin or the like that is incompatible with blood components is particularly effective when the amount of blood is small, and is preferable because it does not cause fluidity problems due to the surface tension of blood components.
[0107]
An example of a separation apparatus having a pressurizing means is shown in FIGS. 6 (a) and 6 (b). In the apparatus of FIG. 6 (a), blood slidable pressurizing means 24 having close contact means 22 in airtight contact with the inner wall 13 of the container 15 is inserted from the opening 18 of the filter, and blood supplied from the opening 18 is inserted. Pressurize. The pressurized blood is separated while passing through the assembly 14 of ultrafine fibers, and plasma is collected from the outlet 13.
[0108]
In the apparatus of FIG. 6 (b), a pressurizing means 24 having a close contact means 22 in airtight contact with the inner wall 19 of the container 15 of the filter 11 is slidably mounted. This device is provided with a valve 20 at the inlet 12. As the pressurizing means 24 moves upward, the inside is depressurized, the valve 20 is opened, and blood is supplied from the inlet 12. Further, with the downward movement of the pressurizing means 24, the blood is pressurized and plasma is separated at the same time as the valve 20 of the inlet 12 is closed. By repeating this procedure, plasma can be continuously separated. In this case, a known means can be used as a means for pressurizing the piston.
[0109]
In the device of the present invention, the blood supply means can also be used as the pressurizing means. FIG. 7 is a diagram showing an example. FIG. 7 shows an apparatus in which the inlet 12 of the filter 11 and the blood supply means 25 are connected. The blood supply means 25 is provided with a member 24 having an inner wall 23 and an intimately close contact means 22, in which blood 26 is stored. When the member 24 is pushed down, the blood 26 is supplied to the filter 11 and pressurized to separate the plasma. Therefore, the blood supply means 25 has the pressurizing means 24 at the same time and can be used as the pressurizing means. For example, a syringe for injection is a specific example.
[0110]
In the case of using pressurization or decompression means, a pressure adjustment means for adjusting the degree of pressurization and decompression may be provided in order to adjust the moving speed of plasma and blood cells. A pump can be provided in front of and behind the assembly of attached ultrafine fibers to adjust the pressure so that hemolysis does not occur. In this case, in particular, the pressure of the blood component before passing through the assembly of attached ultrafine fibers and the pressure of the plasma component after passing can be adjusted independently. At this time, the degree of pressurization and decompression can be automatically controlled by a computer or the like.
[0111]
The pressure loss due to the above pressurization and / or depressurization (pressure difference between the inlet and outlet of the filter) depends on the shape of the microfiber aggregate and the blood processing speed, but is preferably 0.03 to 5 kg / cm. ² More preferably 0.05 to 3 kg / cm ² And particularly preferably 0.1-1 kg / cm ² It is. Differential pressure is 0.03kg / cm ² If the ratio is less than 1, the treatment time may be prolonged, or the plasma may be insufficiently separated. At this time, if ultrafine fibers having high hydrophobicity are used, it may be difficult to send blood into the aggregate of ultrafine fibers. Differential pressure is 5kg / cm ² Exceeding the time, the difference between the time required to pass through the assembly of microfibers attached with plasma and the time required to pass through the assembly of ultrafine fibers attached with blood cells is shortened. Can be difficult. Further, red blood cells may be hemolyzed, or damage may be caused to the device or the assembly of attached ultrafine fibers.
[0112]
<Recovery means>
Plasma separated by an aggregate of ultrafine fibers and moved to the permeate side is collected for use in examinations and the like. As a means for recovering, a known means can be taken. For example, the separated plasma can be collected in the sample container by using the differential pressure applied to the blood in the filter as it is, or sucking the permeate with a pump or the like and sending the permeate. The separated plasma can be collected in a sample container just below the outlet of the filter. Alternatively, a hollow tube can be connected to the outlet and introduced into the sample container. When connecting the outlet of the filter and the recovery means, the same means as the means for connecting the blood supply means and the filter can be used.
[0113]
Plasma stored in a sample container or the like is used as a test sample. Examples of the sample container include tubes and vials, and any material can be used as long as the material does not affect the storage of the obtained plasma. For example, the same material as the container used for the filter can be used.
[0114]
When plasma is separated by an assembly of ultrafine fibers, blood cells permeate with a lapse of time, so that blood cells may be mixed into the separated plasma. Furthermore, depending on the operating conditions, the cells may be broken in an assembly of ultrafine fibers equipped with red blood cells, and the eluted hemoglobin may be mixed into the permeate. When separating plasma for clinical examinations and the like, it is necessary to avoid mixing blood cells and hemoglobin into the resulting plasma. If a certain amount or more of blood cells or hemoglobin is mixed in the plasma of the permeate, the obtained plasma cannot be used as a clinical test sample.
[0115]
In order to solve the above problems, the first permeate (plasma) containing no blood cells can be collected in a preselected amount. For example, by collecting a volume of plasma corresponding to 10% of the interstitial volume of the attached microfiber assembly under the conditions described herein, plasma substantially free of blood cells is obtained. Can be. Such quantification can be performed, for example, by placing a liquid meter in the plasma collection container in the same manner as the measurement of the blood supply amount. The liquid volume can be measured, for example, by detecting with a sensor. After a certain amount of plasma has been collected, the plasma collection container can be moved to fractionate the same plasma sample. This movement can be done manually or automatically. In this case, means for once pooling the plasma and dispensing a certain volume can be used. Furthermore, the apparatus can be configured to stop the blood supply means and the pressurizing means when a certain amount of plasma is obtained. These controls can be performed using a computer. Or after collect | recovering a fixed quantity, it can arrange | position so that the permeated plasma can be discarded or collect | recovered to another container by the switching means demonstrated below. When the blood cell and / or hemoglobin detection means described below is provided, the plasma quantification device is preferably disposed in the vicinity of the outlet to the container for collecting the blood cell and / or hemoglobin-containing sample or in the plasma sample container. .
[0116]
<Blood cell and / or hemoglobin detection means>
As described above, if blood cells or the like are mixed into plasma, it cannot be used as a test sample. Therefore, the apparatus of the present invention can preferably be provided with blood cell and / or hemoglobin detection means for detecting blood cells and / or hemoglobin on the permeate side. When blood cells and / or hemoglobin is detected in the permeate, pressurization and blood supply may be stopped, and the line may be switched to a collection means for collecting or discarding plasma mixed with blood cells and / or hemoglobin. As a result, plasma useful as a clinical test sample can be obtained without any contamination of blood cells or hemoglobin. It should be noted that plasma from which blood cells have been detected or a blood cell component that has passed through later can be returned to a living body or a subject.
[0117]
Examples of the means for detecting blood cells and / or hemoglobin (blood cells and / or hemoglobin) in the plasma permeate include optical means, color developing means by chemical reaction, and the like. Conveniently, blood cells and / or hemoglobin can be detected directly by optical means. For example, when blood cells are mixed in plasma, the light transmittance is lowered, or when hemoglobin is mixed, the plasma turns red. In either case, it can be detected by optical means. For example, a blood leakage sensor that detects blood leakage of a certain value or more by an optical method can be used. A blood leak sensor is a sensor that detects blood cells and hemoglobin in a solution. For example, the absorbance or transmittance of a solution at a certain wavelength is measured with a spectrophotometer. Specifically, by measuring the turbidity (for example, absorbance at a wavelength of 630 nm) by blood cells in a solution or the absorbance of hemoglobin (for example, absorbance at a wavelength of 430 nm), leakage of blood cells and / or hemoglobin in the solution Can be detected.
[0118]
As a means for coloring by chemical reaction, there is a method utilizing color development by chemical reaction with blood cells and / or hemoglobin. This color development can be detected by optical means. For example, a method using the peroxidase-like action of hemoglobin can be mentioned. By selecting appropriate peroxides and chromogens, the sensitivity can be adjusted.
[0119]
In view of the object of the present invention, means other than optical means can be used as long as blood cells and / or hemoglobin can be detected. For example, blood cells can be detected by a cell counter which is an electric cell counter.
[0120]
Of the blood cell and / or hemoglobin detection means, the optical means may be disposed between the outlet which is a plasma flow path and the recovery means. For example, a sensor can be inserted from the outside and contacted directly with plasma to detect blood cells and / or hemoglobin. Or it arrange | positions so that piping which is a flow path of plasma may be pinched | interposed, light, a laser, etc. may be light-emitted from one side, and this emitted light may be received on the other, and a blood cell and / or hemoglobin may be detected. In this case, the pipe is preferably transparent so that light can be transmitted. When using a pipe with poor light transmission, the light transmission of only the pipe may be measured in advance. In addition, detection means by fine sampling is provided in the pipe, and plasma can be collected and contamination of blood cells and / or hemoglobin can be detected with the above-described coloring reagent, for example, test paper. When blood cells and / or hemoglobin is detected, the blood cells and / or hemoglobin-containing plasma can be discarded or collected by the switching means. Further, the blood cell and / or hemoglobin detection means can be connected to blood supply means, pressurization means, or the following switching means, and can be linked with processing such as supply or pressurization stop, discard or recovery by the switching means. .
[0121]
Plasma generally contains some hemoglobin even in the absence of hemolysis, and its concentration varies from individual to individual. In addition, it has been known that blood collection using a vacuum blood collection tube, which has been widely used in recent years, causes some hemolysis during blood collection. However, it has been found that the presence of hemoglobin in plasma at such a low concentration does not significantly affect clinical laboratory data. Therefore, the blood cell or hemoglobin concentration contained in plasma has an allowable range, and there is no particular problem even if blood cells and / or hemoglobin are contained within the range. For example, it is acceptable if the red blood cell concentration in the obtained plasma is 0.1% or less with respect to the red blood cell concentration in the blood before separation.
[0122]
Therefore, in the device of the present invention, when the blood cell and / or hemoglobin detecting means detects a blood cell and / or hemoglobin concentration exceeding a certain concentration, the switching means is operated to increase the amount of blood cells and / or hemoglobin. It can be connected to discard the contaminated plasma or to stop the blood supply or pressurization.
[0123]
<Switching means>
Switching means for separating blood cells and / or plasma mixed with hemoglobin is disposed after the outlet. Examples of the switching means include a switching cock and a switching valve. The switching means can be either automatic or manual. In the case of automatic, the blood cell and / or hemoglobin detection means is arranged at a position before the plasma passes through the switching means. The switching means is connected to blood cells and / or hemoglobin detection means. When blood cells and / or hemoglobin in plasma exceeds a preset value, the switching means is activated by a signal from the blood cells and / or hemoglobin detection means, and plasma in which blood cells and / or hemoglobin is mixed from the line for collecting plasma Switch to the line to collect or discard. As the recovery and disposal means, the above plasma recovery means can be used.
[0124]
(Function)
The separation mechanism of blood cells and plasma in the present invention mainly utilizes the difference in moving speed in the assembly of ultrafine fibers of both components. The moving speed in the assembly of ultrafine fibers is higher in the plasma component than in the blood cell component. Accordingly, when blood is supplied to the aggregate of ultrafine fibers and pressure is applied, the plasma moves ahead of the blood cells, and after a certain distance, the two are completely separated. Therefore, first, plasma flows out, and then blood cells flow out. Therefore, plasma can be collected using this time difference.
[0125]
As described above, the present invention is fundamentally different from a centrifugal separation method using a difference in specific gravity of blood components or a membrane separation method using a difference in size of blood components. Furthermore, it is also different from the leukocyte separation method that uses only the adsorptivity of blood components to ultrafine fibers.
[0126]
【The invention's effect】
The plasma separation device of the present invention utilizes the difference in the moving speed between plasma and blood cells. Therefore, the plasma component obtained does not undergo any dilution or change in the component, and does not change at all from the plasma obtained from centrifugation. Furthermore, by providing a blood cell and / or hemoglobin detection device on the permeate side and a cock linked to this, it is possible to prevent blood cells and / or hemoglobin from being mixed into the sample-collecting plasma. As described above, according to the device of the present invention, a plasma sample free of blood cells and / or hemoglobin can be obtained efficiently, simply, quickly and safely. Therefore, the device of the present invention is useful in fields where a small amount of blood is required to be separated into plasma or serum in a short time, such as for clinical examinations. In addition, the plasma separation apparatus of the present invention can automate the collection of plasma samples for clinical tests that have been conventionally relied on, and can greatly contribute to the automation, speed-up, and safety of clinical tests.
[0127]
EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated concretely, this invention is not limited to this.
[0128]
【Example】
In the examples, an ultrafine fiber (density: 1.38 g / cm) made of polyethylene terephthalate spun by an ordinary melt blow method as an aggregate of ultrafine fibers. ^Three ) Was used. As bovine blood, fresh bovine blood within 8 hours after blood collection was used, but an anticoagulant (ACD: dextrose citrate) was added to prevent blood coagulation immediately after blood collection. The red blood cell concentration in this bovine blood is 7.2 × 10 ⁹ / Ml, white blood cell concentration is 6.8 × 10 ⁶ / Ml, platelet concentration is 2.1 × 10 ⁸ Pieces / ml.
[0129]
The performance of the plasma separation filter includes the concentration of mixed blood cells (red blood cells, white blood cells, platelets), the presence or absence of red blood cell hemolysis, and blood components (serum amylase, total cholesterol, neutral fat, urea nitrogen, blood glucose, total protein concentration) , Albumin) was analyzed and evaluated. The plasma obtained by centrifuging the above bovine blood was used as a comparison target.
[0130]
The concentration of contaminating blood cells in plasma was measured by the Coulter counter method. The detection limit of each blood cell is 2 × 10 in red blood cell concentration ^Five Cells / ml, leukocyte concentration of 1 × 10 ^Five Cells / ml, platelet concentration 1 × 10 ⁶ Pieces / ml.
[0131]
Hemolysis was detected by measuring the hemoglobin concentration in plasma using the O-toluidine method.
[0132]
Each blood component was analyzed by the following method.
[0133]
(i) Serum amylase: G5CNP method
(ii) Total cholesterol: Cholesterol oxidase / peroxidase coloring method
(iii) Neutral fat: LPL, glycerol 3-phosphate oxidase, peroxidase coloring method
(iv) Urea nitrogen: urease / indophenol method
(v) Blood glucose: glucose oxidase / peroxidase coloring method
(vi) Total protein concentration: Burette method
(vii) Albumin: BCG (Bromcresol Green) method
[0134]
Example 1
A plasma separation filter as shown in FIG. 1 was prepared. An acrylic disk with a diameter of 5 mm and a thickness of 2.0 mm inside the container with a hole with a diameter of 1.0 mm at the top end of the container as the inlet and a hole with a diameter of 1.0 mm in the center of the bottom as the outlet. A container was prepared. In this container, a non-woven fabric composed of ultrafine fibers made of polyethylene terephthalate having an average fiber diameter of 1.8 μm as an aggregate of ultrafine fibers (weight per unit: 50 g / m ² The thickness was about 2 mm) and 1.4 g (14 layers were stacked) so that the diameter was 50.0 mm and the thickness was 2.0 mm. At the same time, a gap having a width of 1.0 mm was provided between the outer peripheral surface of the aggregate of ultrafine fibers and the inner peripheral surface of the container. The produced filter is a volume of an assembly of extra fine fibers: about 3.9 cm. ^Three , Gap volume: about 0.32cm ^Three Met. The total surface area of the ultrafine fibers in this plasma separation filter, the average bulk density of the aggregate of ultrafine fibers, the average hydrodynamic radius, the pore volume and L / D are as described in Table 1, respectively. Each value was calculated by the following formula. The density of polyethylene terephthalate is 1.38 g / cm as described above. ^Three It is.
[0135]

After injecting 4 ml of bovine blood into the plasma separation filter from the inlet of the container and filling the gap between the outer peripheral surface of the aggregate of ultrafine fibers and the inner peripheral surface of the container, 0.2 kg / cm ² The pressure of was pushed out the cow blood. The bovine blood enters from the outer peripheral surface of the microfiber assembly, moves in the horizontal direction from the outer periphery to the center of the microfiber assembly, and 20 seconds after pressurization, the outlet of the container The plasma began to flow out of the erythrocytes, and further, 4 seconds later, red blood cells began to be mixed in the effluent. Therefore, plasma was collected for about 2 seconds after starting to flow out to obtain 0.29 ml. The average processing linear velocity was 75 mm / min (= 25 mm / 20 seconds).
[0136]
The concentration of red blood cells, white blood cells, and platelets in 0.29 ml of plasma obtained above was below the detection limit. Therefore, the erythrocyte concentration (hereinafter referred to as erythrocyte contamination rate) in the obtained plasma relative to the erythrocyte concentration in the bovine blood before separation is 0.003% (= (2 × 10 ^Five ÷ 7.2 × 10 ⁹ ) × 100) or less. Further, the hemoglobin concentration of the obtained plasma was 5 mg / dl, and the analysis results of the plasma components were as shown in Table 1.
[0137]
(Example 2)
A plasma separation filter was produced in the same manner as in Example 1. However, the filling amount of the non-woven fabric was 0.8 g (8 layers were stacked).
[0138]
The total surface area of the ultrafine fibers in the plasma separation filter, the average bulk density of the aggregate of ultrafine fibers, the average hydrodynamic radius, the pore volume, and L / D are as described in Table 1.
[0139]
Plasma separation was performed in the same manner as in Example 1 using the above plasma separation filter. Plasma started to flow after 15 seconds after pressurization, and further 2 seconds later, red blood cells began to be mixed. Therefore, plasma was collected for about 1.5 seconds after starting to flow out to obtain 0.33 ml. The average processing linear velocity was 100 mm / min.
[0140]
The white blood cell and platelet concentrations in the 0.33 ml plasma obtained above were below the detection limit, and the red blood cell concentration was 3 × 10. ^Five Pieces / ml. Therefore, the red blood cell contamination rate was 0.004%. Moreover, the hemoglobin concentration of the obtained plasma was below the detection limit, and the analysis results of the plasma components were as shown in Table 1.
[0141]
(Example 3)
A plasma separation filter as shown in FIG. 3 was prepared. A circle made of polypropylene having a diameter of 1.0 mm at the top end of the container as an inlet and a hole with a diameter of 1.0 mm at the center of the bottom as an outlet and having a diameter of 29.0 mm and a thickness of 6.5 mm inside the container A columnar container was prepared. In this container, a non-woven fabric composed of ultrafine fibers made of polyethylene terephthalate having an average fiber diameter of 0.8 μm as an aggregate of ultrafine fibers (weight per unit: 50 g / m ² , About 3 mm thick) 1.2 g (12 layers) are formed on the upper surface of the assembly of ultrafine fibers and the upper surface of the inner surface of the container so that the diameter is 29.0 mm and the thickness is 6.0 mm. A non-woven fabric made of ultrafine fibers made of polyethylene terephthalate having an average fiber diameter of 10 μm (measuring 50 g / m) ² A plasma separation filter filled with 0.33 g (two layers) having a thickness of about 1 mm was prepared (volume of the assembly of ultrafine fibers: about 4.0 cm) ^Three , Gap volume: about 0.33cm ^Three ).
[0142]
The total surface area of the ultrafine fibers in the plasma separation filter, the average bulk density of the aggregate of ultrafine fibers, the average hydrodynamic radius, the pore volume, and L / D are as described in Table 1.
[0143]
After injecting 4 ml of bovine blood into the plasma separation filter from the inlet of the container and filling the ultrafine fibers in the gap between the upper surface of the assembly of ultrafine fibers and the upper surface of the inner surface of the container, 0.4 kg / cm ² The pressure of was pushed out the cow blood. Bovine blood enters from the upper surface of the microfiber assembly, moves downward in the microfiber assembly, and after 30 seconds after pressurization, plasma begins to flow out from the outlet of the container, and then When 5 seconds passed, red blood cells began to be mixed in the effluent. Therefore, plasma was collected for about 3 seconds after starting to flow out to obtain 0.31 ml. The average processing linear velocity was 12 mm / min.
[0144]
The blood cell concentration in the 0.31 ml plasma obtained above was all below the detection limit. Therefore, the red blood cell contamination rate was 0.003% or less. Moreover, the hemoglobin concentration of the obtained plasma was 8 mg / dl, and the analysis results of the plasma components were as shown in Table 1.
[0145]
Example 4
A plasma separation filter as shown in FIG. 1 was prepared. An acrylic disk-shaped container with a diameter of 134 mm and a thickness of 0.30 mm inside the container having a hole with a diameter of 1.0 mm at the top end of the container as an inlet and a hole with a diameter of 1.0 mm in the center of the bottom as an outlet. Got ready. In this container, a non-woven fabric composed of ultrafine fibers made of polyethylene terephthalate having an average fiber diameter of 3.0 μm as an aggregate of ultrafine fibers (weight per unit: 50 g / m ² , 2 mm in thickness) 2.0 g (3 layers) were layered to have a diameter of 130 mm and a thickness of 0.30 mm. At the same time, a plasma separation filter as shown in FIG. 1 was prepared by providing a gap having a width of 2.0 mm between the outer periphery of the assembly of ultrafine fibers and the inner periphery of the container (volume of the assembly of ultrafine fibers). : About 4.0cm ^Three , Gap volume: about 0.25cm ^Three ).
[0146]
The total surface area of the ultrafine fibers in the plasma separation filter, the average bulk density of the aggregate of ultrafine fibers, the average hydrodynamic radius, the pore volume, and L / D are as described in Table 1.
[0147]
Plasma separation was performed in the same manner as in Example 1 using the above plasma separation filter. However, 0.4 kg / cm ² Extruded cow blood under the pressure of. Plasma began to flow out after 120 seconds had passed after pressurization, and then began to contaminate red blood cells after 18 seconds had passed. Therefore, plasma was collected for 12 seconds from the start of outflow to obtain 0.26 ml. The average processing linear velocity was 32.5 mm / min.
[0148]
The white blood cell and platelet concentrations in the 0.26 ml plasma obtained above were below the detection limit, and the red blood cell concentration was 2.9 × 10. ⁸ Pieces / ml. Therefore, the red blood cell contamination rate was 0.04%. Moreover, the hemoglobin concentration of the obtained plasma was 4 mg / dl, and the analysis results of the plasma components were as shown in Table 1.
[0149]
(Example 5)
A plasma separation filter as shown in FIG. 2 was prepared. A cylindrical cylindrical container made of polypropylene having a hole with a diameter of 1.0 mm at the top end of the container as an inlet and a hole with a diameter of 1.0 mm at the center of the bottom as an outlet and having a diameter of 15,2 mm and a thickness of 24 mm inside the container. Got ready. In this container, 1.4 g of cotton made of polyethylene terephthalate ultrafine fibers having an average fiber diameter of 1.8 μm as an aggregate of ultrafine fibers was placed in an ultrafine fiber aggregate so that the diameter was 15.2 mm and the thickness was 22 mm. A plasma separation filter was prepared by filling a gap of 2.0 mm in thickness between the upper surface and the upper surface of the inner surface of the container (the volume of the aggregate of ultrafine fibers: about 4.0 cm) ^Three , Gap volume: about 0.36cm ^Three ).
[0150]
The total surface area of the ultrafine fibers in the plasma separation filter, the average bulk density of the aggregate of ultrafine fibers, the average hydrodynamic radius, the pore volume, and L / D are as described in Table 1.
[0151]
After injecting 4 ml of bovine blood into the plasma separation filter from the inlet of the container and filling the gap between the upper surface of the assembly of ultrafine fibers and the upper part of the inner surface of the container, 0.4 kg / cm ² The pressure of was pushed out the cow blood. Live blood enters from the top surface of the microfiber assembly, moves downward inside the microfiber assembly, and plasma begins to flow out after 300 seconds after pressurization, and then 70 seconds Then red blood cells started to get mixed. Therefore, plasma was collected for 30 seconds from the start of outflow to obtain 0.30 ml. The average processing linear velocity was 4.4 mm / min.
[0152]
The blood cell concentrations in the 0.30 ml plasma obtained above were all below the detection limit. Therefore, the red blood cell contamination rate was 0.003% or less. Moreover, the hemoglobin concentration of the obtained plasma was 6 mg / dl, and the analysis results of the plasma components were as shown in Table 1.
[0153]
(Example 6)
A plasma fractionation filter was prepared in the same manner as in Example 3. However, cotton composed of ultrafine fibers made of polyethylene terephthalate having an average fiber diameter of 0.8 μm was used as the aggregate of ultrafine fibers.
[0154]
The total surface area of the ultrafine fibers in the plasma separation filter, the average bulk density of the aggregate of ultrafine fibers, the average hydrodynamic radius, the gap volume, and the L / D are the same as those in Example 3, and as described in Table 1, respectively. It is.
[0155]
Plasma separation was performed in the same manner as in Example 3 using the above plasma separation filter. Plasma began to flow out after 880 seconds after pressurization, and then began to contaminate red blood cells after another 115 seconds. Therefore, 90 seconds of plasma was collected after starting to flow out to obtain 0.31 ml. The average processing linear velocity was 0.41 mm / min.
[0156]
The blood cell concentration in the 0.31 ml plasma obtained above was all below the detection limit. Therefore, the red blood cell contamination rate was 0.003% or less. Further, the hemoglobin concentration of the obtained plasma was 10 mg / dl, and the analysis results of the plasma components were as shown in Table 1.
[0157]
(Example 7)
A plasma separation filter was produced in the same manner as in Example 5. However, using a container with a diameter of 10.0 mm and a thickness of 55.0 mm inside the container, 2.0 g of cotton made of ultrafine fibers having an average fiber diameter of 3.2 μm as an aggregate of ultrafine fibers was obtained with a diameter of 10.0 mm and a thickness of A gap having a thickness of 4.0 mm was provided between the upper surface of the assembly of ultrafine fibers and the upper surface portion in the container so as to be 51.0 mm (volume of the assembly of ultrafine fibers: about 4.0 cm) ^Three , Gap volume: about 0.31cm ^Three ).
[0158]
The total surface area of the ultrafine fibers in the plasma separation filter, the average bulk density of the aggregate of ultrafine fibers, the average hydrodynamic radius, the gap volume, and L / D are as described in Table 1.
[0159]
Plasma separation was performed in the same manner as in Example 5 using the above plasma separation filter. After 264 seconds had passed after the plasma pressurization, it flowed out, and after another 25 seconds had passed, red blood cells began to be mixed. Therefore, 22 seconds of plasma was collected after starting to flow out to obtain 0.26 ml. The average processing linear velocity was 11.6 mm / min.
[0160]
The white blood cell and platelet concentrations in the 0.26 ml plasma obtained above are below the detection limit, and the red blood cell concentration is 4.3 × 10 4. ⁸ Pieces / ml. Therefore, the red blood cell contamination rate was 0.06%. Moreover, the hemoglobin concentration of the obtained plasma was below the detection limit, and the analysis results of the plasma components were as shown in Table 1.
[0161]
(Example 8)
A plasma separation filter was produced in the same manner as in Example 5. However, the filling amount of cotton made of ultrafine fibers was 0.8 g. Table 1 shows the total surface area of the ultrafine fibers, the average bulk density of the aggregate of ultrafine fibers, the average hydrodynamic radius, the gap volume, and L / D of this filter.
[0162]
Plasma separation was performed in the same manner as in Example 5 using this plasma separation filter. The plasma began to flow out after about 150 seconds after pressurization, and then after another 20 seconds, red blood cells began to be mixed. Therefore, plasma was collected for 17 seconds after starting to flow out to obtain 0.34 ml. The average processing linear velocity was 8.8 mm / min.
[0163]
The white blood cell and platelet concentrations in the 0.34 ml plasma obtained above are below the detection limit, and the red blood cell concentration is 2.2 × 10 4. ⁸ Pieces / ml. Therefore, the red blood cell contamination rate was 0.03%. Moreover, the hemoglobin concentration of the obtained plasma was below the detection limit, and the analysis results of the plasma components were as shown in Table 1.
[0164]
[Table 1]

[0165]
(Comparative Example 1)
A plasma separation filter was produced in the same manner as in Example 1. However, a non-woven fabric composed of ultrafine fibers having an average fiber diameter of 2.5 μm as an aggregate of ultrafine fibers (weight per unit: 50 g / m ² , 2 mm in thickness) was filled with 0.8 g (8 layers). The total surface area of the ultrafine fibers in this filter, the average bulk density of the aggregate of ultrafine fibers, the average hydrodynamic radius, the gap volume, and L / D are as described in Table 2, respectively.
[0166]
Plasma separation was performed in the same manner as in Example 1 using the above plasma separation filter. The effluent started to flow 10 seconds after pressurization, but blood cells were mixed in the effluent from the beginning. The effluent was collected for about 1.0 seconds after starting to flow out to obtain 0.33 ml.
[0167]
The white blood cell and platelet concentrations in the 0.33 ml effluent obtained above were below the detection limit, but the red blood cell concentration was 3.8 × 10. ⁸ Pieces / ml. Therefore, the red blood cell contamination rate was 5.3%. Further, the hemoglobin concentration in the plasma of the obtained effluent was below the detection limit. Analysis of plasma components was omitted.
[0168]
(Comparative Example 2)
A plasma separation filter was produced in the same manner as in Example 1. However, a non-woven fabric made of ultrafine fibers having an average fiber diameter of 1.0 mm as an aggregate of ultrafine fibers (weight per unit: 50 g / m ² , 2 mm thick) was filled with 2.0 g (20 layers). The total surface area of the ultrafine fibers in this plasma separation filter, the average bulk density of the aggregate of ultrafine fibers, the average hydrodynamic radius, the pore volume, and L / D are as shown in Table 2, respectively.
[0169]
Plasma separation was performed in the same manner as in Example 1 using the above plasma separation filter. However, 0.4 kg / cm ² Was pressurized. The effluent began to come out after 1365 seconds from the pressurization, but the effluent was red from the beginning. The effluent was collected for 1600 seconds after starting to flow out to obtain 0.26 ml.
[0170]
The blood cell concentrations in the 0.26 ml effluent obtained above were all below the detection limit, but the hemoglobin concentration in the plasma of the effluent was 95 mg / dl. Analysis of plasma components was omitted.
[0171]
(Comparative Example 3)
A plasma separation filter was prepared in the same manner as in Example 3. However, a non-woven fabric composed of ultrafine fibers made of polyethylene terephthalate having an average fiber diameter of 1.8 μm as an aggregate of ultrafine fibers in an acrylic cylindrical container having a diameter of 50.0 mm and a thickness of 2.3 mm inside the container (weight per unit: 50 g / m ² , About 1 mm thick) 1.4 g (14 layers) are formed on the upper surface of the assembly of ultrafine fibers and the inner upper surface of the container so that the diameter is 50.0 mm and the thickness is 2.0 mm. A non-woven fabric made of ultrafine fibers made of polyethylene terephthalate having an average fiber diameter of 10 μm (mesh of 50 g / m) ² A plasma separation filter filled with 0.17 g (one sheet) having a thickness of about 1 mm was prepared (volume of the aggregate of ultrafine fibers: 3.9 cm) ^Three , Volume of gap: 0.59cm ^Three ).
[0172]
Table 2 shows the total surface area of the ultrafine fibers, the average bulk density, the mean hydrodynamic radius, the pore volume, and the L / D of the ultrafine fibers in the plasma separation filter.
[0173]
Plasma separation was performed in the same manner as in Example 3 using the above plasma separation filter. The effluent started to flow 15 seconds after pressurization, but blood cells were mixed in the effluent from the beginning. The effluent was collected for about 1.5 seconds after starting to flow out to obtain 0.30 ml.
[0174]
The white blood cell and platelet concentrations in the 0.30 ml effluent obtained above were below the detection limit, but the red blood cell concentration was 6.9 × 10 6. ⁹ Pieces / ml. Therefore, the red blood cell contamination rate was 55%. Further, the hemoglobin concentration in the plasma of the obtained effluent was below the detection limit. Analysis of blood components was omitted.
[0175]
(Comparative Example 4)
A plasma separation filter was produced in the same manner as in Example 5. However, the upper surface of the ultrafine fiber aggregate is adjusted to a diameter of 8.0 mm and a thickness of 80.0 mm in a polypropylene container having a diameter of 8.0 mm and a thickness of 86.0 mm inside the container. And filled with a gap of 6.0 mm in thickness on the upper surface of the container (volume of aggregate of ultrafine fibers: 4.0 cm ^Three , Gap volume: 0.30cm ^Three ). The total surface area of the ultrafine fibers, average bulk density, average hydrodynamic radius, pore volume, and L / D of the ultrafine fibers in the plasma separation filter are as described in Table 2.
[0176]
Plasma separation was performed in the same manner as in Example 5 using the above plasma separation filter. The effluent started to be discharged after 1920 seconds from pressurization, but the effluent was red from the beginning. The effluent for 3600 seconds was obtained after starting to flow out, but only 0.25 ml was obtained.
[0177]
The blood cell concentrations in the 0.25 ml effluent obtained above were all below the detection limit, but the hemoglobin concentration in the plasma of the effluent was 75 mg / dl. Analysis of plasma components was omitted.
[0178]
(Comparative Example 5)
The same plasma separation filter as in Example 1 was prepared, and plasma separation was performed in the same manner as in Example 1. However, the amount of bovine blood used was 0.5 ml.
[0179]
A permeate could not be obtained even when all the blood was sent into the ultrafine fibers. Then, although pressurized air was further sent from the container inlet, the pressurized air passed through the ultrafine fiber earlier than the blood, and only foamy blood containing air was obtained at the outlet. This was thought to be because the amount of blood to be treated was too small compared with the pore volume of 3.0 ml of the aggregate of ultrafine fibers, and blood could not be fed uniformly.
[0180]
(Comparative Example 6)
A plasma separation filter was prepared in the same manner as in Example 3. However, in a polypropylene container having a diameter of 35.0 mm and a thickness of 4.6 mm inside the container, a non-woven fabric composed of ultrafine fibers made of polyethylene terephthalate having an average fiber diameter of 1.8 μm as an aggregate of ultrafine fibers (weight per unit: 50 g / m ² , 1 mm in thickness) 0.87 g (18 layers) having a thickness of 0. 5 on the upper surface of the assembly of ultrafine fibers and the upper surface of the container so that the diameter is 35.0 mm and the thickness is 4.2 mm. A 4 mm gap is provided for filling, and the gap is a nonwoven fabric made of ultrafine fibers made of polyethylene terephthalate having an average fiber diameter of 10 μm (weight per unit: 50 g / m ² , 1 mm thick) 0.87 g (one layer is layered) was filled (volume of the assembly of ultrafine fibers: 4.0 cm) ^Three , Gap volume: 0.38cm ^Three ).
[0181]
Table 2 shows the total surface area of the ultrafine fibers, the average bulk density, the mean hydrodynamic radius, the pore volume, and the L / D of the ultrafine fibers in the plasma separation filter.
[0182]
Plasma separation was performed in the same manner as in Example 3 using the above plasma separation filter. The effluent started to flow after 15 seconds from pressurization, or red blood cells had been mixed into the effluent from the beginning. The effluent was collected for about 1.5 seconds after starting to flow out to obtain 0.34 ml. The average processing linear velocity was 16.8 mm / min.
[0183]
The white blood cell and platelet concentrations in the 0.34 ml effluent obtained above were below the detection limit, but the red blood cell concentration was 1.1 × 10. ⁹ Pieces / ml. Therefore, the remaining amount of red blood cells was 15%. Further, the hemoglobin concentration in the plasma of the obtained effluent was below the detection limit. Analysis of plasma components was omitted.
[0184]
[Table 2]

[0185]
Example 9
In the same disk-like container as in Example 1, 2 g of polyethylene terephthalate ultrafine fiber nonwoven fabric having an average fiber diameter of 1.5 μm was cut into a diameter of 50 mm and a thickness of 2.0 mm, and 1.0 mm between the inner peripheral surface of the container. Filled with a gap. The average hydrodynamic radius at this time is 0.64 μm.
[0186]
To this was added ACD solution as an anticoagulant, and hematocrit 45% bovine blood was introduced from the outer periphery of the container to the inside, 0.5 kg / cm ² The liquid was fed at a pressure of. Blood passed through the assembly of ultrafine fibers in the horizontal direction with respect to the nonwoven fabric surface through the container, and after about 30 seconds, plasma permeated from the outlet. The permeation of plasma lasted for about 45 seconds, after which blood cells began to get mixed in the permeate. Therefore, plasma without blood cell contamination could be collected for 30 to 45 seconds after pressurization, and the total amount was 0.7 ml. The hemoglobin concentration of the obtained plasma was 3 mg / dl or less, and no hemolysis was observed. In addition, red blood cells, white blood cells, and platelets measured with a hemocytometer were all below the detection limit. Table 3 shows the biochemical analysis values of the obtained plasma. When comparing the permeate at the beginning of collection, the permeate at the end of collection, a sample of 0.25 ml corresponding to 10% of the interstitial volume of the microfiber assembly, and the plasma obtained by centrifuging the same blood, There was no significant difference between them. Further, when the amount of fibrinogen in the obtained plasma was quantified by cellulose acetate electrophoresis, it was reduced to about 30% of the concentration in plasma obtained by centrifugation, but it was not completely removed. When left standing for a whole day and night, no fibrin deposition was observed.
[0187]
(Example 10)
The same filter as used in Example 9 was used. Without adding anticoagulant, hematocrit 47% human blood immediately after blood collection, 0.5 kg / cm ² The liquid was fed at a pressure of. The blood passed through the container of ultrafine fibers in the horizontal direction with respect to the surface of the nonwoven fabric, and a permeate was obtained from the outlet after about 35 seconds. Blood cells started to mix into the permeate after about 55 seconds. The permeate without blood cell contamination could be collected from 35 seconds to 20 seconds after pressurization, and the total amount was 1.0 ml. The hemoglobin concentration of the obtained permeate was 3 mg / dL or less, and no hemolysis was observed. In addition, red blood cells, white blood cells, and platelets measured with a hemocytometer were all below the detection limit. Table 3 shows the biochemical analysis values of the obtained permeate. Comparison was made between the permeate at the beginning of collection, the permeate at the end of collection, a sample of 0.25 ml corresponding to 10% of the interstitial volume of ultrafine fibers, and the serum obtained by centrifuging the same blood after centrifugation. However, there was no significant difference between them.
[0188]
Further, fibrinogen was not detected in the obtained permeate, and the obtained liquid was not plasma but serum. Fibrinogen concentration in plasma obtained by centrifuging blood with heparin added as an anticoagulant from the same person was 220 mg / dL.
[0189]
Although the same experiment as in Example 1 was performed except that human blood was used, the difference in the amount of permeate collected was recognized because the size of red blood cells was different between humans and cattle. it is conceivable that. Moreover, it is thought that fibrinogen was completely removed because it was adsorbed on the filter because no anticoagulant was added.
[0190]
(Example 11)
The non-woven fiber of polyethylene terephthalate used in Example 1 was dipped in a 0.1% polyvinylpyrrolidone K-90 (BASF Kollidon K-90, molecular weight 360,000) solution, and the wet state was 50 kgy. Gamma irradiation was performed. The nonwoven fabric after γ-ray irradiation was washed with pure water to remove uncrosslinked polyvinylpyrrolidone and dried to obtain a nonwoven fabric having polyvinylpyrrolidone fixed on the surface. This non-woven fabric was filled in a container in the same manner as in Example 1, and the same experiment as in Example 10 was performed. The blood moved in the container in parallel to the laminated surface of the nonwoven fabric, and after about 15 seconds, plasma came out from the outlet. Blood cells started to mix after about 25 seconds. Plasma without blood cells could be collected for about 10 seconds. The total amount was 1.2 ml. The hemoglobin concentration in the obtained plasma was 3 mg / dl or less, and no hemolysis was observed. In addition, red blood cells, white blood cells, and platelets measured with a hemocytometer were all below the detection limit. The analysis value of the obtained plasma is shown in Table 3. Comparison was made between the permeate at the beginning of collection, the permeate at the end of collection, a sample of 0.25 ml corresponding to 10% of the interstitial volume of ultrafine fibers, and the serum obtained by centrifuging the same blood after centrifugation. However, there was no significant difference between them.
[0191]
When the amount of fibrinogen in the obtained plasma was measured by cellulose acetate electrophoresis, it was reduced to about 20% of the plasma concentration obtained by centrifugation, but it was not completely removed. When left standing for a whole day and night, no fibrin deposition was observed.
[0192]
By fixing polyvinylpyrrolidone to ultrafine fibers, the affinity of blood for ultrafine fibers was improved, the resistance when blood passed through decreased, and the time until plasma was separated was shortened.
[0193]
(Comparative Example 7)
Glass fiber filter paper (yarn diameter 0.8-2.5 μm) is cut into about 2 mm square, water is added and stirred with a mixer, and after dehydration, a 5 mL syringe container as shown in FIG. 5g / cm ^Three It filled so that it might become. The average hydrodynamic radius is 1.45 μm (specific gravity of glass fiber is 2.2). In the same manner as in Example 1, an ACD solution was added as an anticoagulant, and hematocrit 45% bovine blood was added from the top of the container to 0.5 kg / cm. ² The liquid was fed at a pressure of. The blood passed through the assembly of microfibers from the upper part to the lower part in the container, and plasma was permeated from the outlet after about 60 seconds. The permeation of plasma continued until about 90 seconds, after which blood cells began to be mixed into the plasma. Plasma without blood cell contamination could be collected for 60 to 30 seconds after pressurization, and the total volume was 0.2 ml. The hemoglobin concentration of the obtained plasma was 3 mg / dL or less, and no hemolysis was observed. In addition, red blood cells, white blood cells, and platelets measured with a hemocytometer were all below the detection limit. Table 3 shows the biochemical analysis values of the obtained plasma. The permeate at the beginning of collection, the permeate at the end of collection, the obtained 0.2 ml sample, and plasma obtained by centrifuging the same blood were compared. The obtained 0.2 ml sample was used because a sample in which 0.25 ml corresponding to 10% of the pore volume of the microfiber aggregate was not obtained. In the permeate at the beginning of collection, the total protein concentration was significantly lower than the others, and the measured values of electrolytes and lipids were different from those of centrifuge plasma. This is probably because proteins, electrolytes, and lipids are adsorbed on the glass fiber, and a part of the electrolyte is eluted from the glass fiber into the plasma. Further, the amount of fibrinogen in the obtained plasma was determined by cellulose acetate electrophoresis, and it was reduced to about 30% of the concentration in plasma obtained by centrifugation, but it was not completely removed. It was. When left standing for a whole day and night, no fibrin deposition was observed. The results are shown in Table 3.
[0194]
(Comparative Example 8)
A container similar to Example 1 was filled with 2.4 g of a polyethylene terephthalate nonwoven fabric having a yarn diameter of 3.5 μm. At this time, the average hydrodynamic radius is 1.13 μm. When blood was fed in the same manner as in Example 1, a permeate was obtained after about 45 seconds, but this permeate contained red blood cells from the beginning, and plasma could not be separated. This is probably because the fiber diameter is as large as 3.5 μm, and therefore, the degree of deformation when red blood cells pass through the fiber gap is small, and there is no difference in the moving speed between red blood cells and plasma. The results are shown in Table 3.
[0195]
[Table 3]

[0196]
(Example 12)
FIG. 8 is a schematic diagram showing an example of the configuration of the apparatus of the present invention. The container 31 is a container for storing a blood sample. The container 31 is connected to the plasma separation filter 11 via a line (or tube) 32 for supplying blood. The container 31 is connected to a pressurized air supply line 33.
[0197]
A removable lid 34 that can be sealed is attached to the container 31. The container 31 may further have a stirring function or a shaking function. A line for supplying an anticoagulant can be attached to the container 31 or the line 32 (not shown).
[0198]
The blood 26 in the container 31 is pushed out by the pressurized air sent from the compressor 35 through the line 33 and supplied to the filter 11 through the line 32. The pressure of the pressurized air is adjusted by the compressor 35.
[0199]
The blood supplied to the filter 11 passes through the microfiber assembly 14 with the pressure of the pressurized air sent to the container 31, and is separated into plasma and blood cells. Plasma that has flowed out from the permeate side of the filter 11 passes through a three-way cock 36, passes through a line (or tube) 37 for plasma collection, and is collected in a container 38 that stores sample plasma.
[0200]
On the permeate side of the filter 11, a blood cell and / or hemoglobin detecting means 40 for detecting contamination of blood cells and / or hemoglobin is attached. In addition to the blood cell and / or hemoglobin detection means 40, a sampling port for detecting contamination of blood cells and / or hemoglobin by a chemical reaction method or a cell counter may be provided. When blood cells and / or hemoglobin is detected in the plasma of the permeate, the three-way cock 36 (for example, it may be a three-port valve that can be opened and closed by electromagnetic action) is switched, and the permeate is passed through the waste liquid (or recovery) line 41. Then, it is sent to the waste liquid (or recovery) tank 42. At the same time, the supply of pressurized air to the container 31 is stopped, and the plasma separation operation is terminated.
[0201]
These means may be coupled to blood cell and / or hemoglobin detection means, for example, to automatically terminate the plasma separation operation when blood cells and / or hemoglobin is detected.
[0202]
Further, a pure water supply line 43 for supplying pure as washing water and a dry air supply line 44 for supplying dry air for drying the apparatus are connected to the blood liquid supply line 32 and the plasma liquid supply line 37. obtain. In addition, as a method for cleaning the apparatus of the present invention, a method of mechanically brushing glassware or the like with a rotating brush, a method of cleaning a jet jet and flowing a high-pressure water flow through a nozzle, a method of ultrasonic Etc. These washing methods can be used alone or in combination. In the above-described method, the line after the plasma separation operation is finished is quickly washed and dried, and the next blood sample can be processed continuously.
[0203]
In addition to the three-way cock 36, various members such as a cock, a chamber, and a pressure sensor for switching the fluid flow direction may be provided in each line constituting the plasma separator (not shown). .
[0204]
The filter 11 can be detachably attached to a holder or the like (see, for example, FIG. 5). Magnet retention and release may be applied. The filter 11 is divided into three parts, an inlet side part (FIGS. 5 and 15a), an outlet side part (FIGS. 5 and 15b), and a part including an aggregate of ultrafine fibers (FIGS. 5 and 14), each of which is separate. It can be configured to be detachable. By making the portion including the assembly of attached ultrafine fibers detachable, it can be made disposable and contamination can be prevented. If the part containing the polymer of the ultrafine fibers attached in this way is detachable, cleaning and drying of the line can be efficient.
[0205]
The device of the present invention may also have a mechanism for automatically flowing a certain amount of blood sample into a plasma separation filter. For example, the above metering pump can be used. Furthermore, it may have a mechanism for automatically exchanging the filter or its constituent parts (such as a part including an assembly of attached ultrafine fibers). Moreover, it may be a structure in which the plasma separator (aggregate of ultrafine fibers) portion of the conventional plasma separator is replaced with a portion including the aggregate of ultrafine fibers to be mounted or a filter. Furthermore, the filters can be connected in series if an improvement in separation performance is desired, and can be connected in parallel if it is desired to increase the amount of blood to be processed.
[0206]
(Example 13)
The device of the present invention can be automated from blood supply to collection and can separate plasma from large quantities of blood samples. For example, FIG. 9 is a schematic diagram showing an example of a configuration for automating the apparatus of the present invention. With automation, multiple blood samples can be processed simultaneously.
[0207]
In FIG. 9, the blood samples are arranged in a rack 48 and are arranged so that the inlet of the blood supply line 32 is substantially at the bottom surface of the blood sample 26. The filter 11 is fixed to the holder 45. The distal end of the blood supply line 32 is configured to fit in an airtight manner with the inlet of the filter 11, and the tip of the blood supply line 32 is pressed by a mechanical pressure to be fitted in an airtight manner with the inlet of the filter. Similarly, the tip of the plasma collection line 37 is configured to fit in an airtight manner with the outlet of the filter 11, and the tip of the plasma collection line 37 is pressed by a mechanical pressure to fit in an airtight manner with the outlet of the filter 11. Combined. Next, at the outlet of the line, a sample container 38 for receiving plasma, which is a means for collecting plasma, is arranged in a rack 50 and automatically arranged.
[0208]
When the lines are connected in this way and a sample container for receiving plasma is arranged, the blood supply pump 46, which is a blood supply means, is activated. When a certain amount of blood flows, the blood supply pump 46 stops. When the blood supply pump 46 stops, the valve 47 shuts off the blood supply line 32. When all the blood supply pumps 46 are stopped, the compressor 35 is operated, and compressed air is supplied from the compressor 35 to the filter 11. The blood passes through the microfiber assembly 14 by air pressure, and the plasma is separated.
[0209]
A blood cell and / or hemoglobin detecting means 40 is disposed at the outlet of the filter 11. When blood cells and / or hemoglobin is detected, the blood cells and / or hemoglobin switching means downstream of the three-way cock 36 is switched, and the blood cell and / or hemoglobin-containing plasma is recovered from the blood cell and / or hemoglobin-containing plasma collection line 41. Lead to. At the same time, the air supply valve 47 is switched to stop pressurization. When blood cells and / or hemoglobin is detected, the sample container is marked and the next sample is prepared.
[0210]
A liquid meter 49 is disposed at the end of the plasma recovery line 37. When a fixed amount is recovered, the switching means 36 is switched to guide unnecessary plasma to the recovery line 41 of blood cells and / or hemoglobin-containing plasma. At the same time, the pressurized air supply valve 47 is switched to stop the pressurization. The compressor 35 stops when all the valves are switched.
[0211]
A line that is the end of the plasma collecting means is pulled out from the sample container 38 from which the plasma has been collected. The sample container 38 is covered and stored.
[0212]
The end of the blood supply line 32 that is airtightly fitted to the inlet of the filter 11 is pulled apart by mechanical pressure. The tip of the plasma collection line 37 that is airtightly fitted to the outlet of the filter 11 is also pulled away by mechanical pressure. The filter 11 thus separated from each means is separated from the holder and discarded.
[0213]
Next, the line is cleaned. Instead of the filter 11, a hollow container that can be airtightly fitted to the end of the blood supply line 32 and the tip of the plasma collection line 37 is disposed. A container for receiving the washing solution is automatically set at the outlet of the line 37 which is the end of the plasma collecting means. The rack 48 on which the blood sample supply containers 51 for which blood supply has been completed is moved, and the blood sample supply container 51 is discarded. Instead, the containers containing the cleaning liquid are arranged in the rack 48 and set so that the inlet of the blood supply line 32 is substantially at the bottom of the cleaning liquid.
[0214]
Next, the valve 47 is switched to supply the cleaning solution, and the blood supply pump 46 is operated to clean the lines 32, 37, and 41. The valve 36 of the switching means is switched to the plasma recovery line 37 and the blood cell and / or hemoglobin mixed plasma recovery line 41, respectively, and washed.
[0215]
After the cleaning is completed, the compressor 35 is operated to supply air, and the line is dried for a certain time. Thereafter, the rack in which the wash water is arranged and the rack in which the containers that have received the washing liquid are moved move, and the rack 48 in which the blood samples are arranged and the rack in which the containers for collecting plasma are arranged are arranged at predetermined positions. Furthermore, instead of the hollow container used at the time of cleaning, the filter 11 is arranged by the above method. Then, the above operation is repeated.
[0216]
The order of these operations can be arbitrarily changed, and the means for collecting the cleaning liquid is not limited to the above, and may not be an individual container. In any case, any modification of such automated equipment is also encompassed by the present invention.
[0217]
(Example 14)
The apparatus of the present invention usually has at least (A) a filter, blood supply means, blood pressurization means, and plasma recovery means. In addition to these means, (a) means for supplying a certain amount of blood, (b) means for collecting a certain amount of plasma, (c) means for detecting blood cells and / or hemoglobin , (D) switching means, (e) means for collecting plasma mixed with blood cells and / or hemoglobin, and the like may be arbitrarily combined. In the apparatus of the present invention, each means can be interlocked with each other. Furthermore, each means may be configured to be able to work with a means for stopping their operation. The combination of these means can be arbitrarily changed according to the intended use of plasma, the desired plasma level, the scale of the apparatus, and the like. For example, a device that combines the following means is typical. (1) A device having (a) in addition to (A). (2) A device having (b) in addition to (A). (3) In addition to (A), an apparatus having (a) and (b); in this apparatus, by supplying a blood in a preselected amount according to the type of filter used and collecting plasma, And a sample is obtained simply. (4) A device having (c), (d) and (e) in addition to (A); at this time, (c), (d) and (e) are usually capable of interlocking with each other. (5) A device having all of (b) to (e) in addition to A. (6) A device having all of (a) to (e) in addition to A; this is an example of a device capable of performing plasma separation with the highest accuracy among the devices of the present invention.
[0218]
The average processing linear velocity of blood (average processing linear velocity from the inlet to the outlet of the filter) by the plasma separator of the present invention is not particularly limited, but is preferably 0.5 to 500 mm / min. When the processing linear velocity is low, it is inconvenient for processing a large number of specimens. When the processing linear velocity is too high, hemolysis may occur due to an increase in differential pressure.
[0219]
(Example 15: Connection with automatic analysis system)
The automatic analysis system is a system in which operations in each process of blood analysis are integrated in order. In other words, it is a system that automates the entire process from sample collection to analysis and data recording. The automatic analysis system is divided into two systems, a flow system and a discrete system, depending on the analysis method. The plasma separator of the present invention can be connected to these systems. That is, in the flow system, several parts are connected by polyethylene pipes, and the sample to be analyzed is analyzed and recorded as it flows through the connected tubes. Can be connected with the sampler part of this system. On the other hand, the discrete system is a method of analyzing individual specimens with independent reaction tubes, and the apparatus of the present invention can be connected to the sampler portion in the same manner as the flow system. Alternatively, plasma obtained separately by the apparatus of the present invention may be used as a sample and supplied to these automatic analysis systems. In this case, a conventional automatic analysis system can be used as it is. The sampler portion of the automatic analysis system can be connected to the plasma separation apparatus of the present invention, for example, the plasma collection line 37 of the apparatus of FIG.
[0220]
(Example 16)
A syringe having a blood supply means and a pressurizing means was attached to the plasma separation filter of Example 1 to produce the plasma separation apparatus of FIG.
[0221]
After injecting 4 ml of bovine blood from the inlet of the container and filling the gap between the outer peripheral surface of the assembly of ultrafine fibers attached to the filter and the inner peripheral surface of the container, 0.2 kg / cm ² Extruded cow blood under the pressure of. The bovine blood entered from the outer peripheral surface of the attached ultrafine fiber assembly, and moved in the horizontal direction from the outer periphery to the center of the inside of the ultrafine fiber assembly. 20 seconds after the pressurization, plasma began to flow out from the outlet of the container. The separated plasma was sampled continuously. 0.5 ml of plasma without erythrocyte contamination was obtained. The average processing linear velocity was 75 mm / min (= 25 mm / 20 seconds).
[0222]
0.4 ml of plasma sampled at the beginning of outflow is collected, the concentration of blood cells (red blood cells, white blood cells, platelets) in plasma, the presence or absence of hemolysis of red blood cells, and blood components (serum amylase, total cholesterol, neutral fat, urea nitrogen, Blood glucose) was analyzed to evaluate the device. Table 4 shows the results.
[0223]
[Table 4]

[0224]
The red blood cell concentration (hereinafter referred to as the red blood cell contamination rate) in the obtained plasma relative to the red blood cell concentration in the bovine blood before separation is 0.003% (= 2 × 10) in consideration of the above detection limit. ^Five ÷ 7.2 × 10 ⁹ ) × 100) or less. There was no hemolysis of erythrocytes, and the hemoglobin concentration of the obtained plasma was 5 mg / dl, which was an acceptable range. Moreover, the analysis result of the blood component is not different from the conventional centrifugation method, and the effectiveness of the device of the present invention has been proved.
[Brief description of the drawings]
FIG. 1 is a diagram showing an example of a filter used in the plasma separation device of the present invention.
FIG. 2 is a diagram showing an example of a filter used in the plasma separation device of the present invention.
FIG. 3 is a filter in which a base material is arranged in a gap portion of the filter of FIG. 2;
FIG. 4 is a diagram showing an example of a filter used in the plasma separation device of the present invention.
FIG. 5 is a diagram showing an example of a filter used in the plasma separation device of the present invention.
6 is a device in which a pressurizing means is added to the filter of FIG. 4;
7 is a device in which blood supply and pressurizing means are connected to the filter of FIG. 1;
FIG. 8 is a schematic view showing an example of a plasma separation device of the present invention.
FIG. 9 is a schematic diagram showing an example of a configuration for automating the plasma separator according to the present invention.
[Explanation of symbols]
11: Filter
12: Entrance
13: Exit
14: Aggregation of ultrafine fibers
15: Container
16: Air gap
17: Base material
18: Opening
19: Inner wall of container
22: Close means
24: Pressurizing means
25: Blood supply means
26: Blood
31: Container for blood supply means
32: Blood supply line
33: Pressurized air supply line
34: Lid
35: Compressor
36: Three-way cock or switching valve
37: Plasma collection line
38: Sample plasma collection container
40: Blood cell and / or hemoglobin detection means
41: Waste liquid recovery line
42: Waste liquid recovery tank
43: Pure water supply line
44: Dry air supply line
45: Filter holder
46: Blood supply pump
47: Valve
48: Blood sample supply container rack
49: Liquid meter
50: Sample plasma collection container rack
51: Blood sample supply container

Claims (22)

A plasma separation filter having a collection of microfibers attached to a container having an inlet and an outlet, the filter having the following characteristics:
(1) The ratio (L / D) of the blood flow path diameter (D) to the blood flow path length (L) of the aggregate of the attached ultrafine fibers is 0.15 to 6;
(2) The average hydrodynamic radius of the attached ultrafine fiber aggregate is 0.5 to 3.0 μm;
(3) Separating fresh bovine blood with a red blood cell concentration of 6-8 × 10 ⁹ cells / ml at a pressure of 0.2-0.4 Kg / cm ² , When an amount of plasma corresponding to 10% of the interstitial volume is collected, (a) the red blood cell concentration in the plasma is 0.1% or less with respect to the red blood cell concentration in the blood before separation; and (b) parenchyma Erythrocytes are not hemolyzed
(4) The average fiber diameter of the ultrafine fibers is 0.5 or more and less than 3.5 μm;
(5) the ultrafine fiber is polyethylene terephthalate ;
(6) The aggregate of the ultrafine fibers is non-woven or cotton. The assembly of ultrafine fibers is attached to the container alone or laminated, and blood is substantially parallel to the surface of the nonwoven fabric or the plane of the laminated nonwoven fabric of the attached assembly of ultrafine fibers. The plasma separation filter according to claim 1, wherein the plasma separation filter is configured to flow through the plasma. The container and the assembly of ultrafine fibers are disk-shaped, and the inlet is configured to be able to supply blood over the entire circumferential side surface of the disk-shaped ultrafine fiber assembly, and the outlet is disk-shaped. The plasma separation filter according to claim 1 or 2, wherein the plasma is configured to flow out from the center of the nonwoven fabric. The plasma separation filter according to any one of claims 1 to 3, wherein a hydrophilizing agent is fixed to the ultrafine fibers. The plasma separation filter according to claim 4, wherein the hydrophilizing agent is fixed to a surface of the ultrafine fiber. The plasma separation filter according to claim 4 or 5, wherein the hydrophilizing agent is polyvinylpyrrolidone. The plasma separation filter according to claim 1, wherein the container and the assembly of the ultrafine fibers are configured to have a columnar shape or a truncated cone shape. The assembly of ultrafine fibers is attached to the container alone or in a stacked manner, and blood is configured to flow substantially perpendicular to the plane of the assembly of attached ultrafine fibers. Item 2. The plasma separation filter according to Item 1. The filter separates fresh bovine blood having an erythrocyte concentration of 6 to 8 × 10 ⁹ cells / ml at a pressure of 0.2 to 0.4 Kg / cm ² , and an assembly of the attached ultrafine fibers from the start of separation. When an amount of plasma corresponding to 10% of the interstitial volume of the plasma is collected, the electrolyte concentration in the separated plasma is maintained at 90% or more compared to the electrolyte concentration in the plasma obtained by centrifugation. The plasma separation filter according to any one of claims 1 to 8. The filter separates fresh bovine blood having an erythrocyte concentration of 6 to 8 × 10 ⁹ cells / ml at a pressure of 0.2 to 0.4 Kg / cm ² , and an assembly of the attached ultrafine fibers from the start of separation. When the amount of plasma corresponding to 10% of the interstitial volume is recovered, the protein concentration in the separated plasma is maintained at 90% or more compared to the protein concentration in the plasma obtained by centrifugation. The plasma separation filter according to any one of claims 1 to 9. further,
(7) The length of the blood cell separation layer is 5 mm or more;
(8) The ratio between the total area of the ultrafine fibers and the treated blood volume is 0.1 to ³ m ² per ml of blood;
(9) The ratio (B / A) of the interstitial volume (A) of the aggregate of ultrafine fibers to the blood volume (B) to be treated is 0.2 or more. The plasma separation filter according to any one of the items. A method of separating plasma using a plasma separation filter in which a collection of microfibers is attached to a container having an inlet and an outlet, the method comprising: supplying a blood sample to the plasma separation filter; and A method comprising the step of pressurizing the filter, wherein the pressure loss at the outlet is 0.03 to 5 kg / cm ² : Here, the plasma separation filter has the following characteristics: (1) Assembly of the attached ultrafine fibers The ratio (L / D) of the body blood sample channel diameter (D) to the blood sample channel length (L) is 0.15-6;
(2) An average hydrodynamic radius of the attached ultrafine fiber aggregate is 0.5 to 3.0 μm. (3) An average fiber diameter of the ultrafine fiber is 0.5 or more and less than 3.5 μm;
(4) the ultrafine fiber is polyethylene terephthalate ;
(5) ultrafine fiber aggregate of all SANYO of non-woven fabric or flocculent;
(6) Fresh bovine blood having an erythrocyte concentration of 6-8 × 10 ⁹ cells / ml is separated at a pressure of 0.2-0.4 Kg / cm ² , and the assembly of the attached ultrafine fibers is separated from the start of separation. When an amount of plasma corresponding to 10% of the interstitial volume is collected, (a) the red blood cell concentration in the plasma is 0.1% or less with respect to the red blood cell concentration in the blood before separation; and (b) parenchyma A method for separating plasma, wherein red blood cells are not hemolyzed . The plasma separation method according to claim 12, wherein a hydrophilizing agent is fixed to the ultrafine fibers of the plasma separation filter. The plasma separation method according to claim 12 or 13, wherein the blood sample to be processed has a linear velocity of 0.05 to 50 cm / min. further,
(6) The length of the blood cell separation layer is 5 mm or more;
(7) The ratio between the total area of the ultrafine fibers and the amount of treated blood sample is 0.1 to ³ m ² per ml of blood sample ;
(8) The ratio (B / A) between the gap volume (A) of the aggregate of ultrafine fibers and the amount of blood sample (B) to be treated is 0.2 or more, characterized in that 4. The method for separating plasma according to any one of the items. A plasma separator having a plasma separation filter in which a collection of ultrafine fibers is mounted in a container having an inlet and an outlet, the filter having the following characteristics:
(1) The ratio (L / D) of the blood flow path diameter (D) to the blood flow path length (L) of the aggregate of the attached ultrafine fibers is 0.15 to 6;
(2) The average hydrodynamic radius of the attached ultrafine fiber aggregate is 0.5 to 3.0 μm;
(3) The average fiber diameter of the ultrafine fibers is 0.5 or more and less than 3.5 μm;
(4) the ultrafine fiber is polyethylene terephthalate ;
(5) The aggregate of the ultrafine fibers is non-woven or cotton-like;
(6) Fresh bovine blood having an erythrocyte concentration of 6-8 × 10 ⁹ cells / ml is separated at a pressure of 0.2-0.4 Kg / cm ² , and the assembly of the attached ultrafine fibers is separated from the start of separation. When an amount of plasma corresponding to 10% of the interstitial volume is collected, (a) the red blood cell concentration in the plasma is 0.1% or less with respect to the red blood cell concentration in the blood before separation; and (b) parenchyma Erythrocytes are not hemolyzed
A plasma separator. The plasma separator according to claim 16, wherein a hydrophilizing agent is fixed to the ultrafine fibers of the plasma separation filter. Blood supply means for supplying blood to the plasma separation filter, pressurization means for pressurizing blood supplied to the filter to separate the supplied blood, and plasma recovery means for recovering the separated plasma The plasma separator according to claim 16 or 17, which has Furthermore, blood cells and / or hemoglobin detecting means for detecting blood cells and / or hemoglobin in the separated plasma, switching means for separating plasma mixed with blood cells and / or hemoglobin, and separated blood cells and / or hemoglobin The plasma separator according to claim 18 , further comprising a blood cell and / or hemoglobin mixed plasma collecting means for collecting plasma mixed with blood. The plasma separator according to claim 18 or 19 , wherein the filter is detachably attached between the blood supply means and the plasma collection means. It said apparatus, plasma separation apparatus according to any one of claims claims 18 2 0 of which has a plasma collecting means for collecting the blood supply means and / or a certain amount of plasma for supplying a constant amount of blood. further,
(6) The length of the blood cell separation layer is 5 mm or more;
(7) The ratio of the total area of the ultrafine fibers to the treated blood volume is 0.1 to ³ m ² per ml of blood;
(8) a gap volume (A) and treated blood amount of aggregate of ultrafine fibers (B) and the ratio of (B / A) is equal to or less than 0.2, according to claim 1 6-2 2. The plasma separator according to any one of items 1 .

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