JPS643849B2 - - Google Patents

Description

Detailed Description of the Invention The present invention provides a granulocyte separation material that can easily separate granulocytes from blood cell suspensions such as blood and body fluids with high yield and high purity, and a granulocyte separation material filled with the separation material. It concerns a sampling device. In recent years, blood transfusion therapy has expanded to include not only whole blood transfusion, but also red blood cell transfusion, granulocyte transfusion, platelet transfusion, and plasma transfusion. Among these, granulocyte infusion has recently been actively applied to agranulocytemia, various leukemias, infections associated with aplastic anemia, and granulocytopenia caused by anticancer drug administration or radiation irradiation. Since the lifespan of peripheral blood granulocytes is extremely short, the granulocytes used for granulocyte transfusion must be collected from healthy blood donors by extracorporeal circulation. This method for collecting granulocytes can be roughly divided into two methods: continuous centrifugation method and adsorption/desorption method using fibers. Continuous centrifugation is an excellent method that causes little damage to blood cells, but it has problems in that the equipment is complicated and expensive, and the collected granulocyte suspension is often contaminated with lymphocytes, resulting in a low yield. On the other hand, the adsorption/desorption method using fibers has the advantage that the purpose can be achieved with a simple and inexpensive device, and there is less lymphocyte contamination. However, in the currently frequently used adsorption/desorption method using fibers, in order to collect the granulocytes attached to the fibers, it is essential to tap a container filled with fibers while running a collection solution, and the collected granulocytes are sterilized. Functional declines such as migratory and migratory abilities, as well as morphological degeneration, have been noted. There are also other problems, such as forcing medical personnel to tap the container. The present inventors focused on this problem and conducted a detailed study on the adhesion (or adsorption) behavior of granulocytes to solid surfaces. As a result, by pre-adhering a fatty acid derivative having 10 to 22 carbon atoms to the solid surface, the adhesive force of the granulocytes is weakened, the granulocytes can be easily recovered, and the collected granulocytes are less deformed. This discovery led to the completion of the present invention. That is, the present invention provides a granulocyte separation material in which a fatty acid derivative having a fatty acid having 10 to 22 carbon atoms is adhered to a carrier. This is a granulocyte collector filled in a container with an outlet. The granulocyte separation material of the present invention is produced, for example, as follows. That is, 0.1 to 10 g of a fatty acid derivative is dissolved in 100 ml of an organic solvent, and a carrier insoluble in the organic solvent and water, such as 1 to 10 g of fiber, is immersed in the solution.
After the immersion treatment for 1 to 60 minutes, the organic solvent is completely removed by drying to obtain a carrier to which the fatty acid derivative is attached at a ratio of 0.1 to 100 mg/g of fiber. The fatty acid derivatives consisting of fatty acids having 10 to 22 carbon atoms used in the present invention include alcohol esters and amides of fatty acids, such as alcohol esters and glycols of fatty acids with methanol, ethanol, probanol, long-chain alcohols, etc. Examples include esters, glycol esters such as glycerin esters and polyethylene glycol esters, esters with monosaccharides and polysaccharides, amides with α-, β-, and γ-amino acids, amides with sphingosine, and amides with amidosaccharides. The fatty acids constituting these fatty acid derivatives are preferably unsaturated fatty acids because they adhere well to the surface of the carrier. For example, preferred are myristoleic acid, palmitoleic acid, oleic acid, linoleic acid, linoleic acid, and the like. More preferably, oleic acid, which is an unsaturated fatty acid having 18 carbon atoms,
These include linoleic acid and linoleic acid. It is preferable that the phase transition temperature of the fatty acid derivative from a solid state to a liquid or liquid crystal state is 45° C. or lower, since this shows good adhesion to solid surfaces. The organic solvent for dissolving the fatty acid derivative may be any organic solvent as long as it can sufficiently dissolve the fatty acid derivative and does not cause dissolution denaturation of the support. For example, ethanol, diethyl ether, chloroform, carbon tetrachloride, ethylene dichloride, acetone, etc. are used. The carrier may be in any form such as membrane, granule, powder, or fiber, but it is preferable to use fibers in the form of cotton because they are easy to handle. As the material of the carrier, nylon, polyester, polypropylene, polyacrylonitrile, cotton, rayon, copper ammonia rayon, etc. can be used. Although the phenomenon underlying the present invention has not been fully elucidated, the following mechanism is considered. The granulocyte separation material attached with fatty acid derivatives is
Difficult to adsorb plasma proteins such as fibrinogen. Since these plasma proteins are involved in the strong adhesion of granulocytes, the above fact suppresses the adhesion of granulocytes to the surface of the separation material, allowing the collection of granulocytes without significantly damaging their functions. It seems that this is now possible. The surface treatment effect of the present invention is achieved by mechanical stimulation.
It is also noticeable when collecting granulocytes that have adhered to the separation material. In the conventional method of collecting granulocytes by knocking them out from the separation material, the results vary depending on the strength of the beating method, but when hitting the strongest, it is possible to recover more than 90% of the granulocytes that have stuck to the separation material. It is possible. However, in general, the goal is to collect granulocytes with less adhesive deformation and less functional loss, with a recovery of around 70%. A recovery method using pulsatile flow, which provides a weaker stimulation than knocking out, is also possible. However, with this method, generally only about 20 to 30% of the granulocytes adhered to the separation material can be recovered. This phenomenon suggests that only the portion of adherent granulocytes that is the weakest and has the least functional decline can be recovered. By using the granulocyte separation material of the present invention, it is possible to easily knock out granulocytes that have adhered to the separation material, and even when using a granulocyte collection method under milder conditions using a pulsatile flow pump, it is possible to easily knock out granulocytes that have adhered to the separation material. An increase in granulocyte recovery of more than % is observed. As described above, by using the granulocyte separating material of the present invention, it has become possible for the first time to collect granulocytes practically not only by the knockout method but also by the pulsatile flow method. When separating granulocytes using the separation material of the present invention, the liquid to be treated contains citrate, which chelates calcium ions that play a major role in the adhesion of granulocytes, and is effective in maintaining the function of granulocytes. It is preferable that the serum protein contains serum proteins such as: When collecting granulocytes from a healthy person by extracorporeal circulation, it is preferable to use type AB serum as the above-mentioned serum protein. In addition, using a pulsatile flow pump instead of pumping out would also lead to less labor for medical personnel. Next, in the granulocyte collector of the present invention, the granulocyte separating material described above is filled into a container having a blood inlet and an outlet, so that the blood passes through while coming into contact with the granulocyte separating material. be. The packing density of the granulocyte separating material is preferably 0.05 to 0.20 g/cm ³ when the carrier is fibrous. A specific example of the granulocyte collector of the present invention will be explained based on FIGS. 1 and 2. A granulocyte separation material 1 is filled in a cylinder 2, and rings 3 are placed around the openings at both ends of the cylinder 2. A mesh 4 fixed by welding is provided, and is fixed by being tightened with silicone packings 5 and 6 provided with the ring 3 sandwiched therebetween. Then, a lid 8 having a blood inlet/outlet tube 7 provided in the center is placed over the mesh 4 from the outside, and is tightened and fixed together with the mesh 4 by tightening rings 9 screwed onto the outer periphery of both ends of the cylinder 2. The granulocyte separating material 1 is fixedly held within the cylinder 2 by a mesh 4, and the blood inlet/outlet tube 7 has a nozzle shape into which a tube can be inserted.
The blood flows through the granulocyte collector, and the granulocyte separation material 1
The granulocytes are adhered to and held on the granulocyte separating material 1 and separated from other components in the blood. The granulocyte collector of the present invention can also collect granulocytes from blood containing an anticoagulant taken out of the body.
It is also possible to collect granulocytes from blood that is continuously perfused by extracorporeal circulation. When the granulocyte collector of the present invention is used for extracorporeal circulation in large animals or humans, commercially available artificial kidney circuits are connected before and after the container. Fill the circuit and granulocyte collector with heparinized saline prior to use. Blood is continuously drawn from the body surface blood vessels of animals loaded with 100 to 200 units of heparin per kg of body weight, and perfused for 2 hours using a commercially available artificial kidney pump at a flow rate of usually 50 ml/min or less. After perfusion, 500 ml of heparin-added physiological saline is poured into the container to flush out non-adhesive red blood cells, lymphocytes, etc. Next, a pulsating pump (EP-B25 manufactured by Iwaki Co., Ltd.)
500 ml of collection solution [consisting of 300 ml of physiological saline, 150 ml of serum or plasma, and 50 ml of anticoagulant (ACD-A solution consisting of citric acid, sodium citrate, and glucose)] into a container and collect the granulocytes. It is preferable that the recovery liquid is flowed in the opposite direction to the direction in which the blood was flowed. The beat rate is preferably 40 to 120 pulses/min. Next, an example will be given and explained. Example 1 Oleic acid monoglycerin ester (melting point 35.5
℃) in 100 ml of diethyl ether,
g of polyester fiber (3 denier) was added to the solution.
Soaked for 30 minutes. After adsorbing excess solution with paper, it was dried under reduced pressure at room temperature for 3 hours to completely remove diethyl ether. 5 g of this granulocyte collection material was uniformly filled into a container with a diameter of 40 mm and a length of 40 mm (inner volume of 50 cm ³ ) shown in Figs. Filled with saline. Fresh bovine blood (granulocyte count 4.2 x 10 ³ cells/mm ³ , granulocyte purity 52%) 1 with heparin added at 1 unit/ml blood and heated to 37°C was pumped at a flow rate of 10 ml/min using a roller pump. Then, the inside of the granulocyte collector was washed with 100 ml of heparin-added physiological saline. Then, using a pulsating pump (electromagnetic metering pump EP-B25 type manufactured by Iwaki Co., Ltd.) at 60 pulses/min.
500 ml of the collection solution was poured into the tube to collect the granulocytes. The direction of flow was opposite to that of blood. Recovered liquid composition: 300 ml of physiological saline, 150 ml of autologous plasma.
ml, and 50 ml of anticoagulant solution (ACD-A solution). The results are shown in Table 1. Here, the granulocyte recovery rate is the number of granulocytes in the recovery solution/total number of granulocytes before entering the granulocyte collector x 100 (%). Granulocyte purity is the ratio of the number of granulocytes to the total number of white blood cells expressed as a percentage. The granulocyte survival rate is the percentage of viable granulocytes showing resistance to trypan blue staining among the granulocytes in the collected fluid. The amount of fatty acid derivatives attached to the fibers was determined by extracting with an organic solvent, removing the organic solvent by drying, and measuring the weight. Comparative Example 1 Table 1 shows the results of a comparative experiment conducted under the same conditions as in Example 1, except that the oleic acid monoglycerol ester adhesion treatment was not performed. Comparative Example 2 Table 1 shows the results of a comparative experiment conducted under the same conditions as in Example 1 using caprylic acid methyl ester (melting point -34°C). Comparative Example 3 Using ethyl lignocerate (melting point 54.8°C),
The results of a comparative experiment conducted under the same conditions as Example 1 are shown in the first example.
Shown in the table. Example 2 Stearic acid triglyceride was attached to nylon fibers (2 denier) in the same manner as in Example 1, and the same experiment as in Example 1 was conducted. Results first
Shown in the table. Example 3 Octadecyl laurate (melting point 37°C) was added to polyacrylonitrile fiber (5°C) in the same manner as in Example 1.
The same experiment as in Example 1 was conducted. The results are shown in Table 1. Example 4 Dodecyl palmitate (melting point 30°C) was added to Example 1.
The same experiment as in Example 1 was conducted by attaching it to polypropylene fiber (7 denier) in the same manner as in Example 1. The results are shown in Table 1. 【table】

[Brief explanation of the drawing]

FIGS. 1 and 2 show an example of the granulocyte collector of the present invention, with FIG. 1 showing a partially cutaway perspective view and FIG. 2 showing a longitudinal side view. 1... Granulocyte separation material, 2... Cylinder, 3... Ring, 4
... mesh, 5, 6... silicon packing, 7...
Blood lead-in/out tube, 8...lid, 9...tightening ring.

Claims (1)

[Scope of Claims] 1. A granulocyte separation material comprising a fatty acid derivative having a fatty acid having 10 to 22 carbon atoms attached to a carrier. 2. The granulocyte separation material according to claim 1, wherein the fatty acid derivative is a fatty acid derivative having an unsaturated fatty acid. 3. The granulocyte separation material according to claim 2, wherein the fatty acid derivative is a glycerin ester of an unsaturated fatty acid having 18 carbon atoms. 4. The granulocyte separation material according to any one of claims 1 to 3, wherein the fatty acid derivative has a phase transition temperature from solid to liquid or liquid crystal state in a range of 45°C or lower. 5. The granulocyte separation material according to any one of claims 1 to 4, wherein the carrier to which the fatty acid derivative is attached is fibrous. 6. A granulocyte collector, in which a container having a blood inlet and an outlet is filled with a granulocyte separating material in which a fatty acid derivative having a fatty acid having 10 to 22 carbon atoms is attached to a carrier. 7 The carrier is fibrous and the packing density in the container is
The granulocyte collector according to claim 6, wherein the granulocyte concentration is 0.05 to 0.20 g/cm ³ .