JPS643850B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an apparatus for separating and recovering blood components from blood, body fluids, or a blood cell suspension obtained by processing these. More specifically, blood, body fluids, or a blood cell suspension obtained by treating these are brought into contact with a polyacrylonitrile-based fibrous material to cause granulocytes to selectively adhere to the fibers and to separate them from lymphocytes. The present invention relates to a blood component separation and recovery device including a leukocyte component separation and recovery device which is characterized by desorption and recovery of leukocyte components while minimizing activity loss. Blood is divided into blood cells and plasma, and blood cell components are divided into red blood cells, white blood cells, and platelets, among which white blood cells generally consist of granulocytes and lymphocytes. Recently, blood cells have decreased during chemotherapy for cancer, and various blood components have been transfused as a symptomatic treatment, as well as for severe infections, etc. due to complications such as infection due to lack of granulocytes and bleeding tendency due to lack of platelets. I became like that. Traditionally, blood components have been separated by adding a flocculant such as dextran, hydroxyethyl, or starch to precipitate red blood cells to obtain white blood cells.
The white blood cell purity is low, it takes time, and it is not possible to separate granulocytes and lymphocytes in the white blood cells. There are methods to collect white blood cells in the bathy coat using centrifugation, and methods to place blood on a density gradient solution and centrifuge it to separate each blood cell component based on the difference in specific gravity, but it is difficult to process large amounts. In particular, it is difficult to separate granulocytes and lymphocytes in white blood cells with high purity. Therefore, even when granulocytes are injected to prevent or treat infectious diseases, white blood cells are lost due to impurity lymphocytes. There were disadvantages such as side effects due to antigen incompatibility, such as fever and chills, and a gradual decrease in the effectiveness of the transfusion. On the other hand, lymphocytes are attracting attention as a raw material for various immunotherapies and interferon production, and social demands are increasing day by day. Next, a method of separating some blood cells by adhesion (also called adhesion) of leukocyte components to fibrous substances has been known for a long time, and as an application of this method, plasma and platelet suspensions are currently obtained by centrifugation. Filters filled with cotton, rayon, polyester fiber, nylon fiber, glass fiber, etc. have been put into practical use for the purpose of removing white blood cell components, which are impurities, from human blood. The centrifugal separator described above is extremely expensive and can only be installed in large hospitals with a large number of patients. Separated blood cells have a short lifespan of only a few hours, so blood is collected from the donor at the recipient's side, and the necessary blood components are separated and transfused to the recipient (patient). An easy method is desired, such as returning the components to blood donors. The adhesion separation method for fibrous substances is a simple method that allows separation with good purity and requires blood to flow through a column.
This is an area in which further improvement is expected, especially as the activity of granulocytes is decreasing. The present inventors have conducted intensive research on methods for adhesion and desorption of blood cell components to fibrous substances, and as a result, we have found that polyacrylonitrile fibers with an iodine orientation of 4 or less, which will be described later, can absorb granulocyte components in white blood cells. The present invention was achieved by discovering that granulocytes with highly selective adhesion and a significantly high activity retention rate can be recovered when the diameter of the fibers is larger than 10 μm. That is, the gist of the present invention is that from blood, body fluids, or a blood cell suspension obtained by processing these,
In an apparatus for separating and recovering part or all of blood components, iodine orientation is used to separate and recover the granulocyte components in white blood cells by substantially selectively adhering them and then desorbing them. This is a blood component separation and recovery device characterized by comprising a container filled with polyacrylonitrile-based fibrous material having a degree of 4 or less. The polyacrylonitrile fiber referred to in the present invention refers to 70 mol% or more of acrylonitrile and 100 mol% or less of acrylonitrile, and a vinyl compound that can be copolymerized with this, such as (meth)acrylic acid or its alkyl ester.
Vinyl chloride, vinyl bromide, vinylpyrrolidone, vinyl acetate, vinyl propionate, styrene, styrene sulfonic acid or its salts, (meth)allylsulfonic acid or its salts, sulfophenyl (meth)
A copolymer (hereinafter referred to as an acrylonitrile polymer) with one or more of allyl ether or a salt thereof, 2-acrylamido-2-methylpropanesulfonic acid or a salt thereof, and 0 to 30 mol% or less,
After dissolving in a solvent, it is spun using a wet, dry or dry-wet method, stretched and post-treated to form a fiber. In recent years, research has been conducted on polyacrylonitrile fibers as adhesive materials for leukocyte components. For example, Japanese Patent Application Laid-Open No. 119013/1984 discloses a method for separating and collecting all leukocytes in blood by adhering them to each other using fibers with an average diameter of 10 μ or less. Sticking is not allowed. However, we discovered that the polyacrylonitrile fibers produced by the present inventors selectively adhere to granulocytes in white blood cells, and after extensive study, we found that as the degree of fiber orientation increases, the adhesiveness of granulocytes decreases. It was found that the selectivity decreased. Although this causal relationship is unclear, it is thought that when the fiber structure becomes highly oriented, the cohesive force between polyacrylonitrile molecules increases, and the apparent polarizability decreases, resulting in a decrease in the adhesiveness of the granulocytes. It has been known that nylon fibers with high polarity selectively adhere to granulocytes, but polyester fibers and polypropylene fibers with low polarity have low selectivity for granulocytes and lymphocytes. The degree of orientation of the polyacrylonitrile fiber in the present invention was quantified using the following degree of iodine orientation.
In other words, 25 polyacrylonitrile fibers with length L ₀
℃ for 2 hours in an aqueous solution containing an iodine concentration of 1.2 mol/potassium iodine and a potassium iodine concentration of 1.5 mol/cm, the contracted length L was measured with a micrometer equipped with a microscope, and the L ₀ /L ratio was calculated as the degree of iodine orientation. And so. The degree of iodine orientation measured in this way is a physical property value that simultaneously represents the orientation of the crystalline region and the orientation of the amorphous region in polymers such as polyacrylonitrile where the crystalline regions are not clearly defined, and the It is often used in practice as a value representing the degree of orientation in the axial direction. In other words, when oriented fibers are immersed in an iodine/potassium iodide solution, iodine molecules penetrate into the molecules that make up the fibers, weakening the intermolecular forces, so the oriented fibers become non-oriented, and the fibers become oriented in the fiber axis direction. contraction occurs. Therefore, it can be said that fibers with a high degree of iodine orientation have a high polymer orientation in the fiber axis direction. The penetration force of iodine between oriented molecules also changes depending on the concentration of iodine in the solution in which the fiber is immersed.
Care must be taken because the length L after contraction changes depending on the measurement conditions. At a concentration of 1.2 mol/mol, there is no need to worry because iodine will penetrate into areas where intermolecular forces are strong. The degree of iodine orientation is thus also an index of the strength of the intermolecular force of polyacrylonitrile molecules, and the smaller this value is, the weaker the intermolecular force is and the easier the infiltration of molecules such as dyes results in a large amount of saturated dyeing. From this, it is thought that polyacrylonitrile with weak intermolecular forces causes granulocytes to adhere effectively. When measuring the degree of iodine orientation of commercially available standard polyacrylonitrile fibers, Bonnell
2.5, Trelon 3.7, Cashmiron 4.5, Exlan
It was 4.5. According to research conducted by the present inventors, it has become clear that when the degree of iodine orientation exceeds 4, the selectivity for granulocytes and lymphocytes decreases, making it impractical.
However, when the degree of iodine orientation is less than 1.5, the fiber loses its strength and becomes difficult to use. The degree of iodine orientation of the fiber is a manufacturing parameter in the fiber manufacturing process,
For example, it can be changed by changing the stretching temperature, stretching ratio, relaxation conditions, etc. The present inventors have found that when the average diameter of polyacrylonitrile fibers is larger than 10μ, the loss of granulocyte activity is extremely small compared to conventional nylon fibers or thin polyacrylonitrile fibers, making it extremely practical. I found it. That is, when the fibers are thin, it is observed under a microscope that the granulocytes spread out and adhere to the fibers. As a result, adhesive granulocytes are significantly deformed, enzymes are released more frequently, and granulocyte activity is greatly reduced as with nylon. Therefore, it is not preferable that the diameter of the fibers be smaller than that of granulocytes (10 to 16 μm in diameter), and when the diameter is larger than 10 μm, granulocytes can be recovered with no small loss of activity.
Furthermore, if the fibers are thick and the average diameter exceeds 500μ, it will be difficult to fill the fiber into a column, and the surface area will also become small, which is not preferable. The fibers used in the present invention are prepared in advance from plasma, serum,
Plasma albumin, gelatin, modified gelatin, heparin, N-hydroxyethylpiperazine-N'-
ethanesulfonate (HEPES), polyethylene glycol and its derivatives, polypropylene glycol and its derivatives, polypropylene glycol-polyethylene glycol block polymer,
Pretreatment can be performed with one or more of polyvinylpyrrolidone, polyvinyl alcohol, and the like. The device of the present invention is not limited to any particular form. That is, any material may be used as long as it allows efficient contact between the polyacrylonitrile fiber and blood, body fluid, or a blood cell suspension obtained by treating these. A preferred form is one in which the fibers are packed in a cylindrical column at an appropriate density, which allows continuous adhesion and desorption of leukocyte components. Also, instead of a cylindrical column, a conical container is also effective. One in which the fibers are filled in a closed space having an inlet and an outlet is preferable from the viewpoint of portability and sterilization. FIG. 1 shows an example of the apparatus of the present invention, in which a polyacrylonitrile fiber 3 with an iodine orientation degree of 4 or less is introduced into a column 2 provided with an inlet 1 and an outlet 4 for a blood cell suspension.
It is a simple thing filled with. A blood cell suspension is sent into the column from an inlet 1 by gravity or by the action of a pump, and granulocytes among white blood cells are selectively adhered to the fibers, and a blood cell suspension containing few granulocytes is collected from an outlet 4. Next, the inside of the column is washed with an appropriate washing solution, and a desorption solution is allowed to flow to desorb and recover the granulocytes.
It is also possible to further selectively separate red blood cells and white blood cells depending on the properties of the blood cell suspension by installing other suitable filters or separation devices before the inlet 1 and after the outlet 4. In the present invention, blood, body fluid, or a blood cell suspension obtained by processing these refers to not only whole blood but also ascites fluid, bone marrow fluid, etc., as well as blood cells from which some blood cells have been removed using a hemagglutinating agent such as dextran. Blood cell suspension, blood cell suspension obtained by centrifugation, etc.
Refers to a blood cell suspension obtained by some processing operation, such as a blood cell suspension obtained by cell electrophoresis. When these blood, body fluids, or the blood cell suspension obtained by processing them are processed with the apparatus of the present invention, they can be used as they are or in physiological saline, heparin-added physiological saline, HEPES-added physiological saline, magnesium-added physiological saline, etc. It may be used after being diluted with a saline solution containing a cell adsorption promoter of a salt, a metal salt such as a calcium salt, a PH buffer, or the like. For example, in the case of a column device, the treatment time is preferably a short time in which the ink is allowed to flow down as it is, but in some cases, the ink bait may be heated or cooled and allowed to stay in the column for an appropriate time. Next, when desorbing the adherent white blood cell components, use any desorbent such as plasma, serum, PH buffer, chelating agent such as citric acid or EDTA, other known drugs, physical factors such as temperature change, mechanical stimulation, etc. Or a combination of two or more can be carried out. In the device of the present invention, when adhesion-separating granulocytes and lymphocytes in white blood cell components, the degree of iodine orientation is 4.
The system is characterized by a column packed with the following polyacrylonitrile fibers, but other known devices can be combined to form a blood component separation and recovery system. Next, a concrete explanation will be given using examples. Example 1 A total of 100 parts of acrylonitrile, vinyl acetate, and sodium methallylsulfonate was suspended and polymerized at 40°C with potassium persulfate and sodium bisulfite in 500 parts of water under a nitrogen stream to obtain acrylonitrile 93.
%, 6.4% vinyl acetate, and 0.6% sodium methallylsulfonate. After washing and drying this copolymer, it was dissolved in dimethylacetamide to a stock solution concentration of 20%, spun in 35% dimethylacetamide water at 40°C, stretched 4 times in boiling water, and dried on a heating roller. The fibers were crimped and fixed in a humid heat environment of 130°C to obtain a 5 denier (diameter 24.6 μm) polyacrylonitrile fiber.
This was washed with ethanol for 4 hours using a Soxhlet extractor, dried, and then thoroughly washed with distilled water.
CC plastic syringe with a constant surface area (2500
cm ² ) was obtained. in this column
After adding 20 mmol HEPES-buffered saline (HBS) and expelling the air, the red blood cell concentrate containing the batty coat obtained by separation immediately after blood collection was added to HBS containing 1 mmol magnesium chloride and calcium chloride, respectively. The mixture was suspended at a hematocrit of 50% and passed through the column at room temperature at a constant flow rate of 12 ml/min. Cells that adhere to the column
Using 10 mmol phosphate buffered saline containing 17 mmol citric acid and a pH of 5.6, the column was desorbed and collected while hitting the column. The number of white blood cells was measured using a Coulter counter, and the granulocyte/lymphocyte content was measured from the particle size distribution pattern using a Coulter channelizer. The loss of granulocyte activity due to adhesion to the fibers was determined by incubating the granulocytes with each fiber at 37°C for 30 minutes, and then quantifying the enzymes (in this case, lysozyme and lactate dehydrogenase [LDH]) that leaked out of the cells. The results are summarized in Table 1.
It can be seen that many of the adherent cells are granulocytes, and enzyme loss is very small. Example 2 In the same manner as in Example 1, acrylonitrile 93
%, 5 denier (diameter 24.6μ) made of polyacrylonitrile copolymer made of 7% vinyl acetate.
fibers were obtained. Using this fiber, blood components were separated in the same manner as in Example 1. The results are shown in Table 1. Example 3 In the same manner as in Example 1, acrylonitrile 93
%, 6% methyl acrylate and 1% sodium vinylbenzenesulfonate, polyacrylonitrile fibers are molded using dimethylacetamide,
Leukocyte differentiation was performed in the same manner. The results are summarized in Table 1. Example 4 Acrylic fiber (2.5 denier) molded under almost the same conditions as in Example 1 but using dimethyl sulfoxide instead of dimethyl acetamide.
A column was filled with the mixture, and a leukocyte separation experiment was conducted in the same manner as in Example 1. The results are shown in Table 1. Example 5 The polyacrylonitrile copolymer produced in Example 2 was molded to a size of 0.12 denier (3.8 μm in diameter), and leukocytes were separated and recovered in the same manner as in Example 1. The results are summarized in Table 1. Although the adhesion of granulocytes and lymphocytes is the same as in Examples 1 and 2, it can be seen that there is a large amount of enzyme leakage and that the degree of deactivation of granulocytes is large. Comparative Example 1 Acrylonitrile 92 was prepared by the method of Example 1.
%, 7.5% methyl acrylate, and 0.5% sodium methallylsulfonate, and mixed this in 60% sodium rhodanate water for 11 hours.
% concentration, spun in 13% sodium rhodanate water at 0°C, stretched 12 times in boiling water, crimped and heat treated at 120°C for 30 minutes to obtain acrylonitrile fiber. A leukocyte adhesion experiment was conducted in the same manner as in Example 1, and the results were as shown in Table 1. The purity of the recovered granulocytes (the proportion of granulocytes contained in white blood cells) is 70%, which is not much different from the proportion contained in white blood cells before separation, and it can be seen that there is no difference in the adhesiveness of granulocytes and lymphocytes. . 【table】

[Brief explanation of the drawing]

FIG. 1 shows a blood component separation and recovery apparatus of the present invention. 1... Column inlet, 2... Column, 3... Fibrous material, 4... Column outlet.

Claims (1)

[Scope of Claims] 1. In an apparatus for separating or recovering part or all of blood components from blood, body fluids, or a blood cell suspension obtained by processing these, the apparatus substantially eliminates granulocyte components in white blood cells. It is characterized by a container filled with a polyacrylonitrile-based fibrous material having an iodine orientation degree of 4 or less as defined below, for separating and recovering the leukocyte component by selectively adhering it and then desorbing it. Blood component separation and recovery equipment. Polyacrylonitrile fiber with length L ₀ is heated at 25℃.
The L ₀ /L ratio is determined by measuring the contracted length L using a micrometer with a microscope after immersion in an aqueous solution containing an iodine concentration of 1.2 mol/and a potassium iodine concentration of 1.5 mol/ for 2 hours. 2. The blood component separation and recovery device according to claim 1, wherein the average diameter of the polyacrylonitrile-based fibrous material is larger than 10μ.