JPH04187206A - Leucocyte separator and production of leucocyte separating material - Google Patents

Abstract

PURPOSE:To obtain the device which efficiently and safely removes leucocytes and allows the permeation of platelets by constituting a part of the leucocyte separating material and/or the inside of a housing with a polymer of ethylene glycol. CONSTITUTION:This device is constituted of the leucocyte separating material consisting of an extremely fine non-woven fabric and a porous membrane, etc., and the housing. The polymer of the ethylene glycol is introduced into at least a part of the leucocyte separating material in contact with the blood, etc., and/or the inside of the housing. This introduction is executed by a method of first dissolving the diacrylate of the polymer of the ethylene glycol into a solvent, such as alcohol, immersing the separating material, etc., therein to deposit this material, etc., then evaporating the solvent. The material is then irradiated with radiations to polymerize the diacrylate of the ethylene glycol polymer on the surface by which the material is coated with this polymer. The unpolymerized matter is washed and extracted in the solvent and is dried. Only the leucocytes are efficiently and safely separated by using this material.

Description

Detailed Description of the Invention (Industrial Application Field) The present invention is an apparatus for removing leukocytes from blood or blood components. The present invention also relates to leukocyte separation materials and devices for safe selective separation and removal.

(Prior Art) In recent years, the approach to blood transfusion has been to replace the conventional whole blood transfusion with component transfusion, in which only the components required by the patient are transfused. For example, patients who tend to bleed or who have a decreased number of platelets in their blood for some reason are repeatedly transfused with a platelet preparation that has a high hemostasis effect. However, since platelet preparations are produced by centrifugation, the difference in density between platelets and white blood cells is small, so platelet preparations contain a large amount of leukocytes. White blood cells, which are originally useful to living organisms, become harmful substances in blood transfusions. In other words, anti-leukocyte antibodies are produced in the recipient's body due to repeated transfusions of other people's leukocytes, and these anti-leukocyte antibodies can cause transfusion side effects such as fever, chills, headache, nausea, and allergic reactions, and may cause the blood platelets that have been transfused to Survival rate decreases. Also, the transfused lymphocytes treat the recipient's internal tissues as foreign substances.

This can cause an important rejection reaction called G V HD, which recognizes and attacks G V HD. For the reasons mentioned above, the necessity of removing contaminated leukocytes with respect to platelet transfusion is strongly advocated.

Furthermore, it has been reported that clinical symptoms are improved by removing lymphocytes from the body fluids of patients with autoimmune diseases such as rheumatism. (For example, Funakoshi et al., Trial of thoracic duct drainage as immunotherapy for rheumatoid arthritis patients, Artificial Organs 17(2) 417(19E18)) Methods for removing lymphocytes from body fluids include the use of blood or lymph fluid. Collect,
Although there is a method for removing the lymphocytes contained therein, the former is preferable from the viewpoint of ease of operation and continuity.

However, when removing leukocytes from blood, there is a risk that coexisting platelets may also be removed at the same time. Therefore, there is a strong desire to develop a method for removing leukocytes from blood that does not supplement platelets.

Centrifugation is a method for removing white blood cells from blood, its components, or platelet preparations, but this method requires expensive equipment, low red blood cell and white blood cell removal rates, and low platelet recovery rates, and is difficult to operate. It is complicated, and there are problems such as the inability to perform online processing at the time of blood donation or transfusion.

On the other hand, in recent years, a leukocyte removal device using a microfiber nonwoven filter has been developed and is used to remove leukocytes from red blood cell preparations. (For example, Japanese Patent Application Laid-Open No. 61-276564) These leukocyte removal devices can efficiently remove leukocytes from red blood cell preparations, and are small and easy to operate. However, since platelets are also separated and removed at the same time, It cannot be used to remove white blood cells inside. There are also examples of using these devices for peripheral blood extracorporeal circulation to treat autoimmune diseases such as rheumatism (e.g., Taira et al., Ieucocytapheresis 1 using a leukocyte removal filter).
FI bed effect and safety of artificial organs 16(2) (1987
)) There was a problem that most of the platelets were captured.

Therefore, attempts have been made to coat the surface of these ultrafine fiber nonwoven fabric filters with a hydrophilic substance to increase the permeability of platelets.

(For example, Kanmoto et al.; Development of leukocyte removal filter for platelet transfusion, Journal of the Japanese Society of Blood Transfusion, 35 (3), 370-3
74 (1! IE19), Table et al.: Leukocyte depletion therapy using coated filters - Immunological studies for RA patients - Artificial organs 18 (3) (1989)) According to this, the coating reduces the permeation of platelets. rate has increased and more than 99% of white blood cells have been removed. However, since this type of coating agent has 1011 groups introduced as hydrophilic groups, complement is activated.

In addition, the stability of coating materials and catalysts during polymerization is also an important factor in terms of safety, but with conventional coated leukocyte removal filters, the present inventors have found that eluate There are no leukocyte removal filters that pass the eluate test section of the current Ministry of Health and Welfare's approval standards for dialysis-type artificial kidneys, and the safety of these filters cannot be said to be sufficient.

(Problems to be Solved by the Invention) Conventional white blood cell removal filters using coated nonwoven fabric filters have problems such as activation of complement components in blood by the coating material and elution of the coating material into the processing solution. It had become.

An object of the present invention is to provide a device and a method for manufacturing the device that can efficiently and safely remove leukocytes from blood or blood components or platelet preparations, and that can permeate platelets.

(Means for Solving the Problems) That is, the present invention provides (1) a method in which a leukocyte separation material is housed in a housing and comes into contact with blood or blood components, and leukocytes are removed from the blood or blood components. (2) a leukocyte separation device in which at least a portion of the leukocyte separation material and/or the interior of the housing that contacts blood or blood components has an ethylene glycol polymer; This is a method for producing a leukocyte separation material in which a leukocyte separation material is supported on a filter material and then irradiated with radiation to polymerize an ethylene glycol polymer on the surface.

Conventionally, hydroxyethyl methacrylate (hereinafter abbreviated as IIEMA) has been mainly used as a coating material for leukocyte removal filters for treating platelet preparations. This aims to weaken the interaction between platelets and materials and improve the permeability of platelets by introducing the 011 group. However, although the initial goal of improving platelet permeability was achieved, a problem arose in that it activated a protein called complement in plasma, which controls the coagulation and immune systems. It was also found that there is a risk that the coating material may be leached into the processing solution. Therefore, the present inventors fabricated nonwoven fabric filters coated with various substances, processed blood and platelet products, and investigated the relationship between the coating material and leukocyte removal rate, platelet permeability, and complement activation. We conducted a thorough study. In addition, various catalysts and methods for coating without eluents were intensively investigated. As a result, they found that adding a multimer of ethylene glycol to the surface of the material reduces platelet permeability and does not activate complement. In addition, stability can be imparted to the coating material by supporting the leukocyte removal filter material with ethylene glycol polymer diacrylate and then irradiating the surface with radiation to polymerize the ethylene glycol polymer on the surface. This discovery led to the present invention. The present invention will be explained in detail below.

The leukocyte removal device used in the present invention has a leukocyte separation material and a housing as its main components, and the material and structure are not particularly limited as long as the separation material can remove leukocytes by contacting blood or blood components. However, examples include microfiber nonwoven fabrics, porous membranes, glass beads, and sponge structures.

The present invention is characterized in that at least a portion of the surface of the device that comes into contact with blood or blood components contains an ethylene glycol polymer. That is, this device usually consists of a material and a housing for installing the same, and the leukocyte separation material can also be divided into a pre-filter for removing minute particles contained in the processing liquid and a leukocyte-removing filter. Since the inside of the housing and the prefilter also come into contact with blood or blood components, it is necessary to introduce a polymer of ethylene glycol into these parts as well as the leukocyte removal filter. Most preferably, a multimer of ethylene glycol is introduced into the leukocyte removal filter.

In the present invention, the term "ethylenezo glycol multimer" refers to ethylene glycol in the form of a dimer or more. Although there is no particular restriction on the molecular weight of the ethylene glycol polymer in the present invention,
From the viewpoint of stability, it is better not to have too large a size, and preferably a polyethylene glycol with a molecular weight of 1500 or less,
More preferably, polyethylene glycol having a molecular weight of 600 or less is used.

Various methods of introducing the ethylene glycol polymer into the leukocyte-removing Lylter of the present invention are possible, such as graft polymerization and mixing into the material, but ethylene glycol- It is desirable to coat the surface of the material with a multimeric diacrylate by radiation polymerization. Coating methods are generally known and are not particularly limited, but for example, the following methods are known. The diacrylate of the ethylene glycol polymer is dissolved in a solvent (alcohol, water, etc.), the filter material is immersed in the solution, the diacrylate of the ethylene glycol polymer is supported on the material, and then the solvent is evaporated. Thereafter, radiation sufficient to polymerize the diacrylate of the ethylene glycol polymer on the surface is irradiated. The unpolymerized monomer is washed in a solvent such as alcohol or water, extracted, and dried.

The housing has an inlet and an outlet, and has the shape of a cylindrical column, tray, etc., and is made of plastic such as polyester or polycarbonate, and may be opaque, but it is transparent so that the internal filtration state can be observed. Plastic is preferred.

-l O- The effects of the present invention and more detailed explanation will be added below using Examples.

(Example) In the Examples and Comparative Examples of the present invention, an ultrafine fiber nonwoven fabric (thread diameter 1.5 μm) of polyethylene terephthalate obtained by melt blowing was used as a material for leukocyte separation material.
) was used.

In addition, the leukocyte removal rate and platelet permeability were measured using platelet-rich plasma (hereinafter abbreviated as PPP) prepared by centrifuging fresh bovine blood supplemented with ACD. The number of white blood cells and platelets were measured by a conventional method using a Coulter counter, and the leukocyte removal rate and platelet permeability were calculated from the number of white blood cells and platelets before and after passing through the filter. 'Biocompatibility evaluation was performed using AC as an anticoagulant in fresh human blood.
C3, which is one of the complement components, is used to add D solution.
The concentration of C3m, which is an activated form of C3m, was measured before and after passing through the filter. C,la concentration was measured by radioimmunoassay method.

The eluate test was carried out in accordance with the eluate test method of the Japanese Pharmacopoeia Test Method for Plastic Containers for Infusions, in which 1 g of the sample was eluted in 100% pure water at 121°C for 11 hours, and the resulting eluate was analyzed. went. Test items are properties, foam, and pH 1 ultraviolet absorption spectrum. In addition, the total organic carbon content of the eluate was also measured.

Example 1 A 2.5% methanol solution of diacrylate of polyethylene glycol (molecular weight 400) was prepared.

This ultra-fine fiber non-woven fabric with a thread diameter of 1.5μ produced by the melt-blowing method is After being immersed in Liquid 8 for 1 minute, it was dried in a vacuum dryer at 80°C for 112 hours. The dried nonwoven fabric was irradiated with an electron beam of 10 Mrad to perform a polymerization reaction. In order to remove unreacted particles, the product was washed with high-temperature pure water and then dried in a dryer at 80°C for 112 hours.

The weight change before and after coating showed that 30% of the weight of the nonwoven fabric was coated with polyethylene glycol.

This coated nonwoven fabric was weighed out in an amount of 0.7 g and was incorporated into a mini module having an area of 10 c+i, and was used in a bovine blood experiment. 100mj at a processing speed of 5mfi/min! When treated with PRP, the leukocyte removal rate was 99.8%.
, the platelet permeability was 98%. In addition, when human blood was poured into a module assembled in the same way and complement activity was examined, the C3a value was 240n before passing through the filter.
g/cell and 320 ng/mff1 after passing through the filter, and there was no significant difference between the values before and after passing.

Next, 1.0 g of this nonwoven fabric was added to 100 g of pure water, and elution was performed in an autoclave at 121° C. for 11 hours. Using the obtained eluate, an eluate test was conducted. The properties, ultraviolet absorbance, foaming test, and pH change were all below standards. In addition, the total organic carbon content of the eluate was 10 pp
It was m.

Example 2 A 1.0% methanol solution of diacrylate of polyethylene glycol (molecular weight 200) was prepared.

The same microfiber nonwoven fabric as in Example 1 was immersed in this solution for 1 minute, and then dried in a vacuum dryer at 80'C for 12 hours. The dried nonwoven fabric was irradiated with 5 Mrad of gamma rays to perform a polymerization reaction. Unreacted monomers were removed and dried in the same manner as in Example 1.

From the weight change before and after coating, it was found that 12% of the weight of the nonwoven fabric was coated with polyethylene glycol.

When it was incorporated into the same mini module as in Example 1 and subjected to a bovine blood experiment, the leukocyte removal rate was 99.5%, and the platelet permeability was 99%. In addition, when complement activity was examined, the value of C3a was 240 ny before passing through the filter, /
ml and 310 ng/d after passing through the filter, and there was no significant difference between the values before and after passing.

All eluate tests were below standards. Further, the total organic carbon content of the eluate was 8 ppm.

Comparative Example 1 The same ultrafine fiber nonwoven fabric as in Example 1 was used without being coated.

PPP using the same module as in Example 1 and under the same conditions.
The leukocyte removal rate was 99.5%, but the platelet permeability was 30%.

In addition, when the throughput exceeds 50 precepts, clogging occurs, and the pressure loss before and after passing through the filter increases, and the amount was 150 mm/g at the time of 100 mfi treatment.

In a human blood complement activation test, the C3a value was 240 ng/if before passing through the filter and 300 ng/ml after passing through the filter, and no significant difference was observed in the C3a value before and after passing through the filter.

In the eluate test at 121°C, all items were below standard values. The total organic carbon content of the eluate was 5 ppm.

Comparative Example 2 The same ultrafine fiber nonwoven fabric as in Example 1 was coated with 2-hydroxyethyl acrylate in the same manner as in Example 1. The coating amount was 25% by weight of the nonwoven fabric.

' When a bovine blood experiment was conducted using the same module and conditions as in Example 1, the leukocyte removal rate was 99.8% and the platelet penetration rate was 9.
0%, and stable operation was possible without clogging of the filter.

In a complement activation test based on human blood experiments, the QCaa value was 240 ng/alt before passing through the filter, and 1300 ng/Idl after passing through the filter, and the Caa value significantly increased before and after passing through the filter. This was considered to be because 2-hydroxyethyl acrylate has 0 groups.

In the eluate test at 121℃, the ultraviolet absorbance was 0.350.
exceeded the standard value. Although other items were below standard values, the total organic carbon content of the eluate was 48 pp111. The reason why the ultraviolet absorbance and total organic carbon content are higher than that of Example 1 is that 2-hydroxyethyl acrylate has one acrylate group, and PI! G400
This is thought to be due to the weaker binding force compared to diacrylate.

(Effects of the Invention) The present invention is constructed as described above, and by introducing a multimer of ethylene glycol onto the surface of the leukocyte removal filter, platelets from blood or blood components are permeated and only leukocytes are efficiently removed. selective separation and removal was possible in a safe and effective manner. In addition, after supporting leukocyte removal filter material with ethylene glycol polymer diacrylate,
By irradiating the surface with radiation to polymerize a multimer of edge length = 15 = recall on the surface, a stable coating with less eluate was possible.

Patent applicant: Toyobo Co., Ltd. ・-17=

Claims (2)

[Claims] (1) In an apparatus for removing leukocytes from blood or blood components by contacting with blood or blood components, the leukocyte separating material is housed in a housing, and/or the leukocyte separating material and/or A leukocyte separation device in which at least a portion of the interior of the housing has an ethylene glycol polymer. (2) The leukocyte separation material according to claim 1, wherein the leukocyte removal filter material is supported with diacrylate, which is an ethylene glycol polymer, and then irradiated with radiation to polymerize the ethylene glycol polymer on the surface. Production method.