JPS6330888B2 - - Google Patents
Info
- Publication number
- JPS6330888B2 JPS6330888B2 JP54043585A JP4358579A JPS6330888B2 JP S6330888 B2 JPS6330888 B2 JP S6330888B2 JP 54043585 A JP54043585 A JP 54043585A JP 4358579 A JP4358579 A JP 4358579A JP S6330888 B2 JPS6330888 B2 JP S6330888B2
- Authority
- JP
- Japan
- Prior art keywords
- fibers
- average diameter
- captures
- platelets
- filled
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired
Links
- 239000000835 fiber Substances 0.000 claims description 47
- 210000004698 lymphocyte Anatomy 0.000 claims description 38
- 239000000126 substance Substances 0.000 claims description 27
- 210000000601 blood cell Anatomy 0.000 claims description 15
- 239000000725 suspension Substances 0.000 claims description 15
- 239000006285 cell suspension Substances 0.000 claims description 14
- 239000011324 bead Substances 0.000 claims description 11
- 239000012209 synthetic fiber Substances 0.000 claims description 4
- 229920002994 synthetic fiber Polymers 0.000 claims description 4
- 239000012784 inorganic fiber Substances 0.000 claims description 2
- 239000002184 metal Substances 0.000 claims description 2
- 210000000265 leukocyte Anatomy 0.000 description 46
- 210000001616 monocyte Anatomy 0.000 description 29
- 210000003714 granulocyte Anatomy 0.000 description 27
- 210000004369 blood Anatomy 0.000 description 26
- 239000008280 blood Substances 0.000 description 26
- 238000000034 method Methods 0.000 description 9
- 239000007788 liquid Substances 0.000 description 8
- 239000002504 physiological saline solution Substances 0.000 description 7
- 238000011109 contamination Methods 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- 238000010586 diagram Methods 0.000 description 4
- 239000011521 glass Substances 0.000 description 4
- 239000000427 antigen Substances 0.000 description 3
- 102000036639 antigens Human genes 0.000 description 3
- 108091007433 antigens Proteins 0.000 description 3
- 238000004140 cleaning Methods 0.000 description 3
- 239000000306 component Substances 0.000 description 3
- 210000003743 erythrocyte Anatomy 0.000 description 3
- 239000002657 fibrous material Substances 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- 239000004952 Polyamide Substances 0.000 description 2
- 239000012503 blood component Substances 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 229920002647 polyamide Polymers 0.000 description 2
- -1 polypropylene Polymers 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 229920000742 Cotton Polymers 0.000 description 1
- 239000004698 Polyethylene Substances 0.000 description 1
- 239000004743 Polypropylene Substances 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- 108090000190 Thrombin Proteins 0.000 description 1
- 230000004931 aggregating effect Effects 0.000 description 1
- 210000003719 b-lymphocyte Anatomy 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 230000002998 immunogenetic effect Effects 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 210000002381 plasma Anatomy 0.000 description 1
- 229920003217 poly(methylsilsesquioxane) Polymers 0.000 description 1
- 229920002239 polyacrylonitrile Polymers 0.000 description 1
- 229920000728 polyester Polymers 0.000 description 1
- 229920000573 polyethylene Polymers 0.000 description 1
- 229920001155 polypropylene Polymers 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- 229920000915 polyvinyl chloride Polymers 0.000 description 1
- 239000004800 polyvinyl chloride Substances 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 229920003002 synthetic resin Polymers 0.000 description 1
- 239000000057 synthetic resin Substances 0.000 description 1
- 239000004753 textile Substances 0.000 description 1
- 229960004072 thrombin Drugs 0.000 description 1
- 238000002054 transplantation Methods 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
Landscapes
- Investigating Or Analysing Biological Materials (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Description
【発明の詳細な説明】
本発明は血液、体液またはこれらを処理して得
られる血球浮遊液から、血小板混入の非常に少な
いリンパ球浮遊液を得るための装置に関するもの
である。DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an apparatus for obtaining a lymphocyte suspension with very little platelet contamination from blood, body fluids, or a blood cell suspension obtained by processing these.
さらに詳しく述べると、血球浮遊液から簡単な
操作で短時間に血小板の混入の少ないリンパ球浮
遊液を得るための、構成の異なる三つの部分から
なる装置に関するものである。 More specifically, the present invention relates to a device consisting of three parts with different configurations for obtaining a lymphocyte suspension containing few platelets from a blood cell suspension with simple operations and in a short time.
近年、免疫学，免疫遺伝学の進歩に伴ない、リ
ンパ球を血液から純粋に取り出し、このリンパ球
浮遊液を用いて、ヒトの主要組織適合抗原
（HLA）を測定することが多くなつてきた。これ
はHLAが移植の成否を決めるだけでなく、多く
の疾患がHLAの特定の型とよく相関することが
明らかにされてきたからである。 In recent years, with advances in immunology and immunogenetics, it has become increasingly common to extract pure lymphocytes from blood and use this lymphocyte suspension to measure human major histocompatibility antigens (HLA). . This is because not only does HLA determine the success or failure of transplantation, but it has also been shown that many diseases are well correlated with specific types of HLA.
このHLAタイピングには、HLA抗原をもつて
いるリンパ球と、抗HLA抗体を反応させて、補
体の存在下で死んだリンパ球を測定する方法がよ
く行われているが、HLA−Ａ，Ｂ，Ｃは血小板
もこの抗原を持つているため、血小板が多く混入
すると、このHLAタイピングに誤差を生じる結
果となる。このため現在は、フイコール・コンレ
イ法などで得た血小板を多く含むリンパ球浮遊液
にトロンビンなどの血小板凝集剤を加え、遠心分
離して血小板を沈降させる操作を数回くり返し
て、血小板混入の少ないリンパ球浮遊液を得てい
る。 HLA typing is often performed by reacting lymphocytes carrying HLA antigens with anti-HLA antibodies and measuring dead lymphocytes in the presence of complement. Platelets in B and C also contain this antigen, so if a large number of platelets are mixed in, this will result in errors in HLA typing. For this reason, current methods include adding a platelet aggregating agent such as thrombin to a lymphocyte suspension rich in platelets obtained by the Feucoll-Conray method, etc., and repeating the procedure several times to centrifuge and sediment the platelets to reduce platelet contamination. A lymphocyte suspension is obtained.
この方法は、遠心分離を数回くり返すため手間
がかかり、また、フイコール・コンレイ法では白
血球中の単球が除去されないなどの欠点を有す
る。 This method is time-consuming because centrifugation is repeated several times, and the Feicoll-Conray method has drawbacks such as not removing monocytes from white blood cells.
本発明者らは、これまで操作の容易なフイルタ
ーを用いるリンパ球の分離方法について種々検討
し、比較的血小板の混入の少ない方法を確立して
きたが、更に混入する血小板を減少させるため鋭
意研究したところ、血小板を吸着する物質を詰め
た部分を白血球捕捉部分、顆粒球捕捉部分ととも
に用いることにより、血小板の混入が非常に少な
く、純度・収率よくリンパ球を得ることができる
ことを発見し、本発明を完成させるに到つた。す
なわち本発明は、容器に平均直径が5μ以上、20μ
以下の繊維をかさ密度0.04ｇ／cm3以上、0.4ｇ／
cm3以下で詰めた部分と、平均直径が10μより大き
く60μ以下であり、かつ平均直径が前記部分の繊
維よりも大きい繊維を詰めた部分と、血小板を吸
着する物質を詰めた部分とを有し、平均直径が
5μ以上、20μ以下の繊維を詰めた部分が、これよ
りも大きい平均直径を有する繊維を詰めた部分よ
りも血球浮遊液の排出側に配置されていることを
特徴とするリンパ球浮遊液を得るための装置を要
旨とするものである。 The present inventors have so far investigated various methods for separating lymphocytes using filters that are easy to operate, and have established a method with relatively little platelet contamination.However, they have also conducted intensive research to further reduce the number of platelets contaminating. However, they discovered that by using a part filled with a substance that adsorbs platelets together with a white blood cell trapping part and a granulocyte trapping part, it was possible to obtain lymphocytes with very little platelet contamination and with high purity and yield. He has completed his invention. That is, in the present invention, the average diameter of the container is 5μ or more, 20μ
The following fibers have a bulk density of 0.04g/cm3 or more, 0.4g/cm3 or more.
It has a part packed with fibers of cm 3 or less, a part packed with fibers whose average diameter is greater than 10μ and 60μ or less and whose average diameter is larger than the fibers in the part, and a part packed with a substance that adsorbs platelets. and the average diameter is
Obtaining a lymphocyte suspension characterized in that a portion filled with fibers of 5 μ or more and 20 μ or less is arranged on the discharge side of the blood cell suspension than a portion packed with fibers having a larger average diameter. The gist is a device for this purpose.
本発明に係る装置は三つの構成の異なる部分か
らなるが、平均直径が５〜20μの繊維をつめた部
分は血球浮遊液中の白血球を捕捉するものであ
り、平均直径10〜60μの繊維をつめた部分は白血
球のうち、顆粒球・単球を捕捉するもの、血小板
を吸着する物質をつめた部分は血小板を捕捉する
ためのものである。 The device according to the present invention consists of three parts with different configurations. The part filled with fibers with an average diameter of 5 to 20 μ is used to capture white blood cells in the blood cell suspension, and the part filled with fibers with an average diameter of 10 to 60 μ is used to capture white blood cells in a blood cell suspension. The filled part is for capturing granulocytes and monocytes among white blood cells, and the part filled with a substance that adsorbs platelets is for capturing platelets.
本発明で繊維とは、合成繊維，半合成繊維，再
生繊維，天然繊維，無機繊維等の人造または天然
の繊維から選ばれるが、繊維自身あるいは繊維に
付着している物質が血液を変性させるようなもの
であつてはならない。 In the present invention, fibers are selected from artificial or natural fibers such as synthetic fibers, semi-synthetic fibers, regenerated fibers, natural fibers, and inorganic fibers, but the fibers themselves or substances attached to the fibers may denature blood. It should not be a thing.
繊維の平均直径（Ｄcm）とは一本の繊維の重さ
（ｘｇ），長さ（ｙcm）、材料の密度（ ｇ／cm3）
から、次式で定義されるものをいう。尚、この場
合
繊維の断面は、円形のものを標準とするが断面が
天然木綿繊維や人造繊維にみられる種々の非円形
断面であつてもよい。 The average diameter (Dcm) of a fiber is the weight (xg), length (ycm), and density of the material (g/cm 3 ) of a single fiber.
, it is defined by the following formula. Furthermore, in this case The cross section of the fiber is generally circular, but the cross section may be any of the various non-circular cross sections found in natural cotton fibers and man-made fibers.
また、容器は一般にカラムと呼ばれる円筒形
状、円錐台形状とするのが使用操作上一般的であ
るが他の形状の容器であつても支障はなく、繊維
の充填室とその両側に血球浮遊液の入口と被処理
液の出口を設ければよい。容器の材質はガラス
や、ポリプロピレン、ポリエチレン、ポリスチレ
ン、ポリ塩化ビニール等の合成樹脂を選ぶとよ
い。かさ密度とは、使用繊維重量（ｇ）を繊維の
かたまりが占める体積（cm3）で割つた値（ｇ／
cm3）である。 In addition, the container is generally cylindrical or truncated conical, which is called a column, for operational reasons, but there is no problem with containers having other shapes. What is necessary is to provide an inlet for the liquid to be treated and an outlet for the liquid to be treated. The material for the container should preferably be glass or synthetic resin such as polypropylene, polyethylene, polystyrene, or polyvinyl chloride. Bulk density is the value (g/
cm3 ).
容器内の繊維はなるべく均一な充填密度でつめ
られることが好ましい。 It is preferable that the fibers in the container be packed with as uniform packing density as possible.
容器内に収納する繊維は、一本一本の単繊維に
予め均しくほぐすのが好ましく、また各単繊維は
繊維相互に絡合して全体として繊維群の塊り形状
を保つ程度の長さであるのが望ましい。繊維が短
かいと分離される血液成分中に浮遊してくる危険
があり、このため通常は市販の紡織繊維程度以上
の長さの繊維を使用するのが好都合である。 It is preferable that the fibers to be stored in the container be uniformly loosened into individual single fibers in advance, and each single fiber should be long enough to intertwine with each other and maintain the shape of the fiber group as a whole. It is desirable that If the fibers are too short, there is a risk that they will float in the blood components being separated, so it is usually convenient to use fibers that are longer than commercially available textile fibers.
特に、切れ目のないフイラメントを30cm程度以
上の長さで切断し、容器につめたフイルターは、
繊維がもれ出ることが全くなく、用いるのに好ま
しい。繊維の充填量は、被処理血球浮遊液の量、
通過速度によつて任意に決定することができる。 In particular, filters made by cutting unbroken filament into lengths of approximately 30 cm or more and filling them in a container are
The fibers do not leak out at all, making it suitable for use. The amount of fiber packed depends on the amount of blood cell suspension to be treated,
It can be arbitrarily determined depending on the passing speed.
本発明は、基本的には白血球を捕捉する部分
と、顆粒球・単球を捕捉する部分で、血液中のリ
ンパ球を純度・収率よく回収し、また血小板を吸
着する物質を詰めた部分で血小板を吸着・除去す
るものである。白血球を捕捉する部分に詰める繊
維の平均直径は5μ以上、20μ以下である。本発明
者らのこれまでの検討によると、繊維の平均直径
が小さいほど、白血球の捕捉力が強く、従つて血
球浮遊液中の白血球を、白血球を捕捉する部分に
充分捕捉するためには、白血球を捕捉する部分内
の繊維の平均直径は20μ以下、好ましくは10μ以
下である必要がある。また、繊維の平均直径が小
さすぎると白血球の捕捉力が強すぎ、フイルター
から白血球を回収することが困難であり、白血球
を捕捉する部分内の繊維の平均直径は5μ以上、
好ましくは7μ以上が必要である。白血球を捕捉
する部分内のかさ密度は0.04ｇ／cm3以上、0.4
ｇ／cm3以下である。かさ密度が低すぎるとリンパ
球を充分捕捉できず、また、かさ密度が高すぎる
と、残存する赤血球が多くなり、またリンパ球が
回収されにくくなる。白血球を捕捉する部分内の
繊維の平均直径が10μ以下の場合は、かさ密度は
0.25ｇ／cm3以下が好ましい。 The present invention basically consists of a part that captures white blood cells, a part that captures granulocytes and monocytes, and a part that collects lymphocytes from blood with high purity and high yield, and a part that is filled with a substance that adsorbs platelets. It adsorbs and removes platelets. The average diameter of the fibers packed into the part that traps white blood cells is 5μ or more and 20μ or less. According to the studies conducted by the present inventors so far, the smaller the average diameter of the fibers, the stronger the leukocyte trapping power. The average diameter of the fibers within the part that traps leukocytes should be less than 20μ, preferably less than 10μ. In addition, if the average diameter of the fibers is too small, the leukocyte trapping force will be too strong, making it difficult to collect leukocytes from the filter.
Preferably, 7μ or more is required. The bulk density within the part that captures white blood cells is 0.04 g/ cm3 or more, 0.4
g/cm 3 or less. If the bulk density is too low, lymphocytes cannot be captured sufficiently, and if the bulk density is too high, more red blood cells will remain and lymphocytes will be difficult to collect. If the average diameter of the fibers within the part that traps leukocytes is less than 10μ, the bulk density is
It is preferably 0.25 g/cm 3 or less.
顆粒球・単球を捕捉する部分内の繊維状物質
は、平均直径が10μより大きく、60μ以下であり、
かつ白血球を捕捉する部分に用いた繊維状物質よ
りも平均直径が大きいものである。 The fibrous material in the part that traps granulocytes and monocytes has an average diameter of more than 10μ and less than 60μ,
In addition, the average diameter is larger than that of the fibrous material used in the part that traps white blood cells.
平均直径が10μ以下になると、顆粒状・単球は
よく捕捉・除去できるが、リンパ球も捕捉され、
リンパ球の回収率が低下する。また平均直径が
60μを越えると、顆粒状・単球捕捉力が小さく、
これらの血球が多く混入するようになる。また顆
粒球・単球を捕捉する部分に用いる繊維は白血球
を捕捉する部分のものより平均直径が大きい方
が、リンパ球を多量白血球を捕捉する部分に送る
ことができる。 When the average diameter is less than 10 μ, granular cells and monocytes can be easily captured and removed, but lymphocytes are also captured.
Lymphocyte recovery rate decreases. Also, the average diameter
If it exceeds 60 μ, the granule/monocyte capture force is small;
Many of these blood cells become mixed in. Furthermore, if the fibers used in the part that captures granulocytes and monocytes have a larger average diameter than those in the part that captures white blood cells, lymphocytes can be sent to the part that captures a large amount of white blood cells.
顆粒球・単球を捕捉する部分内の繊維状物質の
かさ密度は、血球浮遊液が顆粒球・単球を捕捉す
る部分を通過する際の速度や温度に著しく影響を
受けるが、0.05ｇ／cm3以上、0.5ｇ／cm3以下が好
ましく0.1ｇ／cm3以上、0.4ｇ／cm3以下であれば更
に好ましい。 The bulk density of the fibrous material in the part that captures granulocytes and monocytes is significantly affected by the speed and temperature at which the blood cell suspension passes through the part that captures granulocytes and monocytes, but it is 0.05 g/ It is preferably 0.1 g/cm 3 or more and 0.5 g/cm 3 or less, and more preferably 0.1 g/cm 3 or more and 0.4 g/cm 3 or less.
以上の、白血球を捕捉する部分と顆粒球・単球
を捕捉する部分を組合せることにより、血液など
の血球浮遊液からかなり血小板の除去されたリン
パ球浮遊液を得ることができるが、本発明者らは
血小板を吸着する物質を詰めた部分を白血球を捕
捉する部分、顆粒球・単球を捕捉する部分の少な
いリンパ球浮遊液を得ることができることを発見
した。この血小板を吸着する物質は、血液成分を
変性することなく、血小板を良く吸着し、白血球
成分はあまり吸着しないものならば基本的には使
用可能であるが、特にガラスビーズや他の無機物
ビーズ、金属ビーズなどが、用いるのに好まし
い。これらのビーズは、径が小さい方が表面積が
大きく、少量でもよく血小板を吸着できる、余り
径が小さいと、リンパ球なども非特異的に捕捉さ
れる。 By combining the above-mentioned part that captures leukocytes and the part that captures granulocytes/monocytes, it is possible to obtain a lymphocyte suspension from which platelets have been considerably removed from a blood cell suspension such as blood. They discovered that it was possible to obtain a lymphocyte suspension containing a portion packed with a substance that adsorbs platelets, a portion that captures leukocytes, and a portion that captures granulocytes and monocytes. The substance that adsorbs platelets can basically be used as long as it does not denature blood components, adsorbs platelets well, and does not adsorb leukocyte components very well, but glass beads and other inorganic beads, in particular, can be used. Metal beads and the like are preferred for use. The smaller the diameter of these beads, the larger the surface area, and can adsorb platelets even in small amounts.If the diameter is too small, lymphocytes and the like will be non-specifically captured.
本発明者らの検討では、ビーズの直径は50μ以
上、1000μ以下、好ましくは70μ以上、300μ以下
であれば、リンパ球を余り捕捉しないで、血小板
をよく吸着・除去できることがわかつた。 The present inventors have found that when the diameter of the beads is 50μ or more and 1000μ or less, preferably 70μ or more and 300μ or less, platelets can be well adsorbed and removed without trapping too many lymphocytes.
血小板を吸着する物質をつめた部分の大きさ
は、白血球を捕捉する部分、顆粒球・単球を捕捉
する部分と同様、処理血液量などに左右される
が、大体１〜100ml位の大きさであれば、少量か
ら多量の血液が処理できる。 The size of the part filled with the substance that adsorbs platelets, like the part that captures white blood cells and the part that captures granulocytes and monocytes, depends on the amount of blood processed, etc., but is approximately 1 to 100 ml in size. If so, small to large amounts of blood can be processed.
第１図は、本発明の装置の一例を示す模式図で
ある。血小板を吸着する物質を詰めた部分１は、
顆粒球・単球を捕捉する部分２の後に置かれ、白
血球を捕捉する部分３は、一番後に配置されてい
る。また、顆粒球・単球を捕捉する部分の前に
は、血球浮遊液の導入口である出入口４があり、
白血球を捕捉する部分の後には、血球浮遊液の出
口である、出入口５が取り付けられている。血小
板を吸着する物質を詰めた部分には、ビーズ状物
質７が入つており、またこの部分には、ビーズの
もれ出るのを防ぐためメツシユ６が入つている。
白血球を捕捉する部分と顆粒球・単球を捕捉する
部分には、それぞれ繊維状物質９，８がよく開繊
されて、詰められている。 FIG. 1 is a schematic diagram showing an example of the apparatus of the present invention. Part 1 is filled with a substance that adsorbs platelets.
The part 3 that captures leukocytes is placed after the part 2 that captures granulocytes and monocytes, and the part 3 that captures leukocytes is located at the rearmost position. In addition, in front of the part that captures granulocytes and monocytes, there is an entrance/exit port 4 that is the introduction port for the blood cell suspension.
An inlet/outlet 5, which is an outlet for the blood cell suspension, is installed after the part that traps white blood cells. A bead-like substance 7 is contained in the part filled with a substance that adsorbs platelets, and a mesh 6 is contained in this part to prevent the beads from leaking out.
Fibrous substances 9 and 8 are well-opened and packed in the part for capturing white blood cells and the part for capturing granulocytes/monocytes, respectively.
第２図も、本発明の装置の一例を示す模式図で
ある。この例では三つの部分の位置関係は第１図
と同じであるが、この三つの部分は別々の容器に
入れられ、顆粒球・単球を捕捉する部分と、血小
板を吸着する物質をつめた部分は結合部１０で、
また血小板を吸着する物質を詰めた部分と、白血
球を捕捉する部分は結合部１１で、それぞれ連結
されている。これらの結合部は、ただの導管でも
よくまた容易にとりはずせる構造のものでもよ
い。 FIG. 2 is also a schematic diagram showing an example of the apparatus of the present invention. In this example, the positional relationship of the three parts is the same as in Figure 1, but these three parts are placed in separate containers, with a part that captures granulocytes and monocytes and a substance that adsorbs platelets. The part is the joint part 10,
Further, the portion filled with a substance that adsorbs platelets and the portion that captures white blood cells are connected to each other by a connecting portion 11. These connections may be simple conduits or may be of a structure that allows them to be easily removed.
第３図も、本発明の装置の一例を示すものであ
るが、第２図と異なり、血小板を吸着する物質を
詰めた部分が最初におかれ、次が顆粒球・単球を
捕捉する部分となつている。また、血小板を吸着
する物質を詰めた部分が一番後に配置されている
例を示したものが第４図である。以上の例のよう
に、血小板を吸着する物質を詰めた部分は、他の
２つの部分の位置の如何んにかかわらず、どこに
でも配置することが可能であるが、白血球を捕捉
する部分と、顆粒球・単球を捕捉する部分の位置
関係では、顆粒球・単球を捕捉する部分が、白血
球を捕捉する部分の前、すなわち血球浮遊液の導
入側に配置されなければならない。このように配
置することにより、まず顆粒球・単球を捕捉する
部分で血液中の顆粒球・単球を除いてから、白血
球を捕捉する部分での残りの白血球、すなわちリ
ンパ球を捕捉することができる。 Figure 3 also shows an example of the device of the present invention, but unlike Figure 2, the part filled with the substance that adsorbs platelets is placed first, and then the part that captures granulocytes and monocytes. It is becoming. Further, FIG. 4 shows an example in which the part filled with a substance that adsorbs platelets is placed at the rearmost position. As in the above example, the part filled with the substance that adsorbs platelets can be placed anywhere, regardless of the position of the other two parts, but the part that captures white blood cells and Regarding the positional relationship of the parts that capture granulocytes and monocytes, the part that captures granulocytes and monocytes must be placed in front of the part that captures white blood cells, that is, on the side where the blood cell suspension is introduced. By arranging it in this way, the part that captures granulocytes and monocytes first removes the granulocytes and monocytes from the blood, and then the part that captures white blood cells captures the remaining white blood cells, that is, lymphocytes. Can be done.
また、第５図は、２つの部分が同一の容器に入
つた構造のものを示したものであり、特に第６図
のように、血小板を吸着する物質は、顆粒球・単
球を捕捉するための繊維とよく混合して入れても
よい。 In addition, Figure 5 shows a structure in which two parts are housed in the same container. In particular, as shown in Figure 6, the substance that adsorbs platelets captures granulocytes and monocytes. It may also be mixed well with the fibers for use.
なお、１０，１１の結合部をはじめ、１２，１
３，１４，１５，１６，１７などの結合部は、た
だの導管でもよくまた取りはずせて、そこから液
を入れることができる構造でもよく、また第７図
のような三方活栓であつてもよい。 In addition, including the joint parts 10 and 11, 12 and 1
The joints 3, 14, 15, 16, 17, etc. may be mere conduits, or may have a structure that allows them to be removed and liquid can be poured in there, or they may be three-way stopcocks as shown in Figure 7. good.
次に、本発明の装置の使用方法を説明する。第
１図の装置では、まずポンプなどの液を移動させ
る器具を５の出入口に取り付け、４の出入口から
血液などの血球浮遊液を装置に導入する。導入さ
れた血液はまず顆粒球・単球を捕捉する部分２で
顆粒球・単球を除去され、次に血小板を吸着する
物質をつめた部分１で血小板が吸着・除去され
る。次に血液は白血球を捕捉する部分３に入り、
ここでリンパ球が捕捉される。この後、白血球を
捕捉する部分内の赤血球や血漿を除去するため、
出入口５から生理食塩水などの生理的溶液からな
る洗浄液を入れ、出入口４から出す。次に白血球
を捕捉する部分内のリンパ球を回収するため、生
理的溶液からなる回収液を高速流で出入口４から
入れる。この場合、回収液の量は白血球を捕捉す
る部分の空間の体積の量ぐらいが好ましい。 Next, a method of using the apparatus of the present invention will be explained. In the apparatus shown in FIG. 1, a liquid moving device such as a pump is first attached to the inlet/outlet 5, and a blood cell suspension such as blood is introduced into the apparatus through the inlet/outlet 4. From the introduced blood, granulocytes and monocytes are first removed in part 2, which captures granulocytes and monocytes, and then platelets are adsorbed and removed in part 1, which is filled with a substance that adsorbs platelets. The blood then enters part 3 where it traps white blood cells;
Lymphocytes are captured here. After this, in order to remove red blood cells and plasma in the part that traps white blood cells,
A cleaning solution consisting of a physiological solution such as physiological saline is put in through the inlet/outlet 5 and taken out through the inlet/outlet 4. Next, in order to collect the lymphocytes in the area where the leukocytes are captured, a collection liquid consisting of a physiological solution is introduced from the inlet/outlet 4 in a high-speed flow. In this case, the amount of recovery liquid is preferably about the same volume as the space in which the leukocytes are captured.
第２図の装置では、血液を第１図と同様装置に
導入し、リンパ球を白血球を捕捉する部分に集め
る。次に、洗浄液を白血球を捕捉する部分に入れ
るのであるが、これには出入口５から入れる、
１１の部分をとりはずし、この部分から入れ
る、１１が第７図のような三方活栓１８のとき
は、導管１９を通つて入れる、などの方法があ
る。また回収時は回収液を高流速で白血球を捕捉
する部分に入れてもよいが、また回収液を流しな
がら、白血球を捕捉する部分の容器を木の棒など
でたたくこともできる。 In the device shown in FIG. 2, blood is introduced into the device in the same manner as in FIG. 1, and lymphocytes are collected in the area where white blood cells are captured. Next, the cleaning solution is poured into the part where the white blood cells are captured, and this is done through the entrance and exit port 5.
There are methods such as removing the part 11 and inserting it from this part, or if 11 is a three-way stopcock 18 as shown in FIG. 7, inserting it through the conduit 19. During collection, the collection liquid may be poured into the leukocyte trapping part at a high flow rate, or the container in the white blood cell trapping part can be struck with a wooden stick while the collection liquid is flowing.
第３図の装置は第２図とほぼ同様の操作で扱う
ことができるが、第４図では、最***液を装置に
入れ、顆粒球・単球を捕捉する部分と、白血球を
捕捉する部分を通つた血液は、１５の所から三方
活栓で装置外に出す方が、出入口５から出すより
も好ましい。次に洗浄液を１５の所か、又は１４
の所から白血球を捕捉する部分に入れ、最後に白
血球を捕捉する部分に血清成分を入れ、次に血清
成分を含む回収液を１４の所から白血球を捕捉す
る部分に入れ、リンパ球を回収し、これを血小板
を吸着する物質をつめた部分に通して、５の出入
口から出す。 The device in Figure 3 can be handled in the same way as in Figure 2, but in Figure 4, blood is first put into the device, and the part that captures granulocytes and monocytes and the part that captures white blood cells are shown. It is preferable to let the blood that has passed out of the device from the three-way stopcock at 15 than to let it out from the inlet/outlet port 5. Next, apply the cleaning solution to 15 or 14
Insert the white blood cells into the part that captures white blood cells from point 14, and finally put serum components into the part that captures white blood cells.Next, put the collection solution containing serum components into the part that captures white blood cells from point 14, and collect the lymphocytes. This is passed through a part filled with a substance that adsorbs platelets, and then exited through the entrance and exit port 5.
このようにして得たリンパ球浮遊液には、血小
板の混入が非常に少なく、また赤血球や顆粒球・
単球の混入も少ない。また装置の操作も非常に簡
単である。得られたリンパ球は種々の機能がよく
保たれており、バイアビリテイーも高い。またＴ
細胞・Ｂ細胞の比率も、フイルターの条件により
もとの血液とほぼ同じものや、変化したものを得
ることができる。 The lymphocyte suspension obtained in this way has very little platelet contamination, and also contains red blood cells, granulocytes, and
Contamination with monocytes is also low. The device is also very easy to operate. The obtained lymphocytes have various functions well maintained and have high viability. Also T
Depending on the filter conditions, it is possible to obtain blood with a cell/B cell ratio that is almost the same as the original blood or with a changed ratio.
以上のように、本発明の装置を用いることによ
り、血液等の血球浮遊液から簡単な操作で、純
度・収率よくリンパ球を分離でき、また無菌操作
も容易なため、各種医療施設・研究機関におい
て、リンパ球を用いた研究が容易に行えるもので
ある。 As described above, by using the device of the present invention, lymphocytes can be separated from blood cell suspensions such as blood with simple operations and with high purity and yield, and the sterile operation is also easy, so it can be used in various medical facilities and research facilities. Research using lymphocytes can be easily conducted at institutions.
実施例 １
第３図のように、本発明による装置を構成し
た。即ち、内径15mm，長さ76mmの円筒容器に、直
径0.1mmのガラス・ビーズを一ぱいに詰めた血小
板を吸着する物質を詰めた部分１，内径10mm，長
さ75mmの円筒容器に、平均直径20.7μのポリアミ
ド繊維を、かさ密度0.15ｇ／cm3（重量にして
0.884ｇ）で詰めた顆粒球・単球を捕捉する部分
２，内径10mm，長さ25.5mmの円筒容器に、平均直
径8.2μのポリアクリロニトリル系繊維を、かさ密
度0.132ｇ／cm3（0.264ｇ）で詰めた白血球を捕捉
する部分３を、第３図のように配置した。また、
フイルターの結合部は容易に取りはずしができる
ようにした。Example 1 An apparatus according to the present invention was constructed as shown in FIG. That is, a cylindrical container with an inner diameter of 15 mm and a length of 76 mm is filled with glass beads of 0.1 mm in diameter and a substance that adsorbs platelets is filled in part 1.A cylindrical container with an inner diameter of 10 mm and a length of 75 mm is filled with an average diameter of 20.7 mm. μ polyamide fibers with a bulk density of 0.15 g/cm 3 (in terms of weight)
Polyacrylonitrile fibers with an average diameter of 8.2μ were placed in a cylindrical container with an inner diameter of 10 mm and a length of 25.5 mm, and a bulk density of 0.132 g/cm 3 (0.264 g). ) was placed as shown in FIG. 3 to trap the white blood cells. Also,
The filter joint can be easily removed.
この装置の出入口５にポンプをとり付け、37℃
に加温した健康人のヘパリン加血液５mlを、１
ml／minの流速で出入口４から吸い込み、血液が
すべて装置の導入された後、生理食塩水10mlを、
10ml／minの流速で出入口４から引き続き、吸い
込んだ。この操作によつて、血液は１を通り、つ
づいて２を経て３に入つた。この後、連結部１３
をとりはずし、１３から生理食塩水15mlを第１フ
イルターに４ml／minの流速で吸い込み、次に出
入口５に取り付けたポンプをはずし、生理食塩水
２mlを入れた注射器を結合部１３に取り付け、注
射器の吸子を強く押し、出入口５から２mlのリン
パ球浮遊液を回収した。この液にはリンパ球は元
の血液の40％の濃度で含まれ、顆粒球・単球は白
血球の5.8％含まれていた。また血小板は元の血
液の0.12％の濃度で含まれていた。 Attach a pump to the entrance/exit 5 of this device and keep the temperature at 37°C.
5ml of heparinized blood from a healthy person warmed to 1
Suction from the inlet/outlet port 4 at a flow rate of ml/min, and after all the blood has been introduced into the device, 10 ml of physiological saline is added.
Suction was continued from port 4 at a flow rate of 10 ml/min. By this operation, the blood passed through 1, then through 2, and then into 3. After this, the connecting part 13
, suck 15 ml of physiological saline from 13 into the first filter at a flow rate of 4 ml/min, then remove the pump attached to the inlet/outlet port 5, attach a syringe containing 2 ml of physiological saline to the coupling part 13, and remove the syringe from the syringe. The sucker was strongly pressed and 2 ml of lymphocyte suspension was collected from the inlet/outlet port 5. This fluid contained lymphocytes at 40% of the original blood concentration, and granulocytes and monocytes at 5.8% of white blood cells. Platelets were also present at a concentration of 0.12% of the original blood.
実施例 ２
第２図のように、本発明による装置を構成し
た。即ち、内径15mm，長さ40mmの円筒容器に、平
均直径16μのポリエステル繊維をかさ密度0.12
ｇ／cm3（重量にして0.848ｇ）で詰めた２、内径
15mm，長さ50mmの内筒容器に、直径0.1mmのガラ
スビーズを一ぱいに詰めた３、内径10mm，長さ28
mmの円筒容器に、平均直径7.5μのポリアミド繊維
をかさ密度0.13ｇ／cm3（0.286ｇ）で詰めた１を
第２図のように配置した。また、各部分の結合部
は、容易に取りはずしができるようにした。Example 2 An apparatus according to the present invention was constructed as shown in FIG. That is, a cylindrical container with an inner diameter of 15 mm and a length of 40 mm is filled with polyester fibers with an average diameter of 16 μm and a bulk density of 0.12.
2, inner diameter packed with g/ cm3 (0.848g in weight)
An inner tube container with a diameter of 15 mm and a length of 50 mm is filled with glass beads with a diameter of 0.1 mm.3, an inner diameter of 10 mm and a length of 28
Polyamide fibers 1 having an average diameter of 7.5 μm and having a bulk density of 0.13 g/cm 3 (0.286 g) were placed in a cylindrical container of 1 mm in diameter as shown in FIG. Additionally, the joints between each part can be easily removed.
この装置の出入口５にポンプをとり付け、37℃
に加温した健康人のヘパリン加血液５mlを、１
ml／minの流速で出入口４から吸い込み、血液が
すべて装置に導入された後、ヒトAB型血清５
ml、引き続いて生理食塩水５mlを、１ml／minの
流速で出入口４から吸い込んだ。この後、結合部
１１を取りはずし、１１から生理食塩水18mlを１
に５ml／minの流速で吸い込み、次に出入口５に
取り付けたポンプをはずし、生理食塩水２mlを入
れた注射器を出入口５に取り付け、注射器の吸子
を強く押し、結合部１１から２mlのリンパ球浮遊
液を回収した。この液にはリンパ球は元の血液の
40％の濃度で含まれ、顆粒球・単球は全白血球の
７％含まれていた。また血小板は元の血液の0.1
％の濃度で含まれていた。 Attach a pump to the inlet/outlet 5 of this device and keep the temperature at 37°C.
5ml of heparinized blood from a healthy person warmed to 1
After all the blood has been introduced into the device by suctioning from the inlet/outlet port 4 at a flow rate of ml/min, human AB type serum 5
ml followed by 5 ml of physiological saline was sucked in through port 4 at a flow rate of 1 ml/min. After this, remove the joint 11 and pour 18 ml of physiological saline from 11.
Next, remove the pump attached to port 5, attach a syringe containing 2 ml of physiological saline to port 5, press the sucker of the syringe firmly, and remove 2 ml of lymphocytes from junction 11. The suspension was collected. This fluid contains lymphocytes from the original blood.
It was contained at a concentration of 40%, and granulocytes and monocytes comprised 7% of the total white blood cells. Also, platelets are 0.1 of the original blood.
Contained at a concentration of %.
第１図，第２図，第３図，第４図，第５図，第
６図は本発明の装置の例を示す模式図、第７図は
詰めた各部分間の結合部の説明図である。
１……血小板を吸着する物質を詰めた部分、２
……顆粒球・単球を捕捉する部分、３……白血球
を捕捉する部分、４……出入口、５……出入口、
６……メツシユ、７……血小板を吸着する物質、
８……繊維状物質、９……繊維状物質、１０，１
１，１２，１３，１４，１５，１６，１７……結
合部、１８……三方活栓、１９……導管。
Figures 1, 2, 3, 4, 5, and 6 are schematic diagrams showing examples of the device of the present invention, and Figure 7 is an explanatory diagram of the joints between the packed parts. It is. 1... Part filled with a substance that adsorbs platelets, 2
... Part that captures granulocytes/monocytes, 3... Part that captures white blood cells, 4... Entrance/exit, 5... Entrance/exit,
6...Metushiyu, 7...Substance that adsorbs platelets,
8... Fibrous substance, 9... Fibrous substance, 10,1
1, 12, 13, 14, 15, 16, 17...joining part, 18...three-way stopcock, 19...conduit.
Claims (1)
かさ密度0.04ｇ／cm3以上、0.4ｇ／cm3以下で詰め
た部分と、平均直径が10μより大きく、60μ以下
であり、かつ平均直径が前記部分の繊維より大き
い繊維を詰めた部分と、血小板を吸着する物質を
詰めた部分とを有し、平均直径が5μ以上、20μ以
下の繊維を詰めた部分が、これよりも大きい平均
直径を有する繊維を詰めた部分よりも血球浮遊液
の排出側に配置されていることを特徴とするリン
パ球浮遊液を得るための装置。 ２ 繊維が合成繊維，半合成繊維，再生繊維，天
然繊維，無機繊維から選ばれた少なくとも１種で
ある特許請求の範囲第１項記載のリンパ球浮遊液
を得るための装置。 ３ 血小板を吸着する物質がカラスビーズ、金属
ビーズ、無機物ビーズから選ばれた少なくとも１
種である特許請求の範囲第１項記載のリンパ球浮
遊液を得るための装置。[Scope of Claims] 1. A container filled with fibers with an average diameter of 5 μ or more and 20 μ or less at a bulk density of 0.04 g/cm 3 or more and 0.4 g/cm 3 or less, and a portion with an average diameter of more than 10 μ and 60 μ or less. and has a part filled with fibers with an average diameter larger than the fibers in the part, and a part filled with a substance that adsorbs platelets, and the part filled with fibers with an average diameter of 5 μ or more and 20 μ or less, A device for obtaining a lymphocyte suspension, characterized in that the device is disposed on the discharge side of the blood cell suspension from a portion filled with fibers having an average diameter larger than this. 2. The device for obtaining a lymphocyte suspension according to claim 1, wherein the fiber is at least one selected from synthetic fibers, semi-synthetic fibers, regenerated fibers, natural fibers, and inorganic fibers. 3 At least one substance that adsorbs platelets is selected from crow beads, metal beads, and inorganic beads.
An apparatus for obtaining a lymphocyte suspension according to claim 1, which is a seed lymphocyte suspension.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP4358579A JPS55135749A (en) | 1979-04-12 | 1979-04-12 | Unit to obtain lymphocyte floating liquid |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP4358579A JPS55135749A (en) | 1979-04-12 | 1979-04-12 | Unit to obtain lymphocyte floating liquid |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS55135749A JPS55135749A (en) | 1980-10-22 |
| JPS6330888B2 true JPS6330888B2 (en) | 1988-06-21 |
Family
ID=12667851
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP4358579A Granted JPS55135749A (en) | 1979-04-12 | 1979-04-12 | Unit to obtain lymphocyte floating liquid |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS55135749A (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0068042A1 (en) * | 1981-07-01 | 1983-01-05 | Hans Dr. Selzer | Drug from the lymphs of mammals |
| JP2673567B2 (en) * | 1987-12-10 | 1997-11-05 | 株式会社日本抗体研究所 | Method for removing granulocytes in blood and granulocyte removing apparatus used therefor |
-
1979
- 1979-04-12 JP JP4358579A patent/JPS55135749A/en active Granted
Also Published As
| Publication number | Publication date |
|---|---|
| JPS55135749A (en) | 1980-10-22 |
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