JP2003149096A - Blood filter film and method therefor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for acquiring blood serum, blood plasma, blood corpuscles, or leukocytes from a small amount of whole blood. SOLUTION: This blood filter film is made of a film of hole diameters between 0.5-40 μm and of the coefficient of variation of dispersions of the hole diameters is 20% or less, and this blood filter method uses two such films or more of the same average hole diameters or different average hole diameters.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a filtration membrane for removing blood cells from whole blood, and a method for capturing and recovering red blood cells or white blood cells from whole blood using the filtration membrane.

[0002]

2. Description of the Related Art The types and concentrations of constituents in blood such as metabolites, proteins, lipids, electrolytes, enzymes, antigens and antibodies are usually measured using serum or plasma obtained by centrifugation of whole blood as a sample. Has been done. However, centrifugation requires labor and time, and especially when a small number of specimens are to be processed promptly or in the field, etc., the centrifugal separation method using a centrifuge powered by electricity is unsuitable. Therefore, a method of separating and collecting serum or plasma from whole blood by filtration has been studied.

A method for obtaining serum or plasma from whole blood by filtration is commercially available as "Plasma Filter PF" manufactured by Fuji Photo Film Co., Ltd.
As known from Japanese Patent No. 1432, there is a method in which 3 to 6 glass fiber filter papers are loaded in a plastic holder and whole blood is filtered by suction. However, this filtration method can filter about 150 μL of plasma, but requires about 3 mL of whole blood, and cannot filter a trace amount of whole blood.

A method for capturing blood cells from whole blood by filtration to obtain serum or plasma is disclosed in JP-A-10-18591.
There is a method of using a three-dimensional porous body described in Japanese Patent Laid-Open No. 0, and polysulfone membrane, cellulose acetate membrane and the like are known. However, it is difficult to make the pore size uniform in this membrane.

On the other hand, white blood cells are collected and collected from whole blood,
In recent years, it has been actively used to collect genes based on this and use them for gene analysis or gene diagnosis, and a technique for recovering leukocytes from whole blood is required.

A method for removing only white blood cells from whole blood is
Terumo Co., Ltd. "Immuno Guard (registered trademark) III-"
There are known a polyurethane porous filter which is practically used in "RC", a polyester filter which is practically used in "POL transfusion filter PL1J" sold by Nippon Pall Ltd., and the like. However, this filter is for removing white blood cells from whole blood for transfusion,
It is difficult to capture leukocytes from a small amount of whole blood and recover leukocytes.

[0007]

There is a need for a method of urgently obtaining serum or plasma from a small amount of whole blood, such as when a small amount of whole blood can be collected from a newborn baby. Further, in the method of capturing blood cells from whole blood by filtration, there is a demand for a method of fractionating and collecting only white blood cells for use in genetic diagnosis and the like.

[0008]

[Means for Solving the Problems] By using a thin membrane having a uniform pore size, it is possible to collect only blood cells from a small amount of whole blood and recover serum or plasma. It was found that only white blood cells can be captured and collected.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION As a material for a membrane, a polymer compound which is soluble in a non-water-soluble solvent such as poly-ε-caprolactone, poly-3-hydroxybutyrate, agarose, poly-2-hydroxyethyl acrylate, and polysulfone. Can be used, but it is preferable to use poly-ε-caprolactone.

Although a film having a honeycomb-like structure can be formed with only these materials, it is preferable to add an amphipathic material. Examples of the amphipathic material include amphipathic polyacrylamide.

The mixing ratio of the membrane material and the amphipathic material is
It is preferable to use the weight ratio in the range of 0: 1 to 1: 0. More preferably, the weight ratio is in the range of 5: 1 to 20: 1.

The solvent for casting may be any water-insoluble solvent such as chloroform, dichloromethane, carbon tetrachloride, cyclohexane, etc., which can dissolve the polymer compound as the material of the membrane. The polymer concentration at the time of casting may be any concentration that can form a polymer film,
For industrial mass production, it is desirable to form a film at a concentration as high as possible, which is 0.1 wt% or more.

Since the material of the water-insoluble membrane, for example, poly-ε-caprolactone, is dissolved in the water-insoluble solvent, for example, chloroform, when the solvent is evaporated by the high humidity air, the heat of vaporization causes Condensation of water, which gradually grows as the solvent evaporates and has a diameter of 0.5-40.
Water droplets of about μm size. Since the water-insoluble poly-ε-caprolactone cannot be dissolved in this water droplet, a film having pores in this portion can be obtained. For example, since it is cast two-dimensionally on a petri dish, the grown water droplets are regularly arranged like a two-dimensional close-packed structure of spheres,
As a result, a film having a honeycomb structure is obtained.

The pore diameter is controlled by controlling the temperature and humidity of the atmosphere or the air to be sprayed by adjusting the concentration and amount of the liquid to be cast and supplying it to a support layer such as a petri dish to control the evaporation speed and dew condensation speed of the solvent. By,
Can be controlled. Furthermore, by adding a trace amount of a surfactant to suppress and stabilize the fusion of water droplets, a more regular honeycomb structure film can be prepared.

The high-humidity air blown onto the membrane has a relative humidity of 3
0% and 80% are mainly used, but the humidity may be such that moisture in the air can be condensed on the surface of the film, and the relative humidity may be 20 to 100% depending on the temperature. Alternatively, a relatively inert gas such as nitrogen or argon may be used.

The flow rate of the high-humidity air blown onto the film may be any flow rate that allows the moisture in the air to be condensed on the surface of the film and allows the solvent used for casting to be evaporated.

The temperature of the atmosphere when the high-humidity air is blown may be any temperature at which the solvent used for casting can be evaporated, and is 15 to 32 ° C. at the laboratory level and 5 to 80 ° C. at the production level. Is desirable.

By varying the concentration of the solution to be cast, the amount of the solution, the type of solvent, and the relative humidity, temperature, and flow rate of the air to be blown, the dew condensation, the growth of water droplets, and the evaporation rate of the solvent are controlled, and various pore sizes are regular A film with a honeycomb-like structure can be obtained, which is a component in blood and has a diameter of about 3 μm.
Platelets, white blood cells having a diameter of about 15 μm, and red blood cells having a large deformability but having a diameter of about 7 μm can be used as filters that can be separated from each other by changing the pore size.

Further, by stacking membranes having almost the same pore diameter, the ability to separate as a filter can be enhanced. Furthermore, by stacking a plurality of membranes having different pore sizes, it is possible to simultaneously separate or fractionate some biological substances. For example, pore size 5.5-8.5μ
m membrane and a membrane with a pore diameter of 3.5 μm or less are stacked and whole blood is supplied from the membrane side with a large pore diameter, whereby leukocytes can be captured by a membrane with a large pore diameter and red blood cells can be captured by a membrane with a small pore diameter. Can be achieved at the same time.

Further, by setting the pore size to the submicron order, it can be expected as a selective separation and recovery technique for a target biological substance represented by blood purification such as dialysis.

In the present invention, the honeycomb-like structure means a structure in which a plurality of pores having a substantially constant pore diameter are regularly arranged and such pores penetrate the membrane. The cross section of the hole is not particularly limited, and may be circular, elliptical, hexagonal, rectangular, square or the like.

[0022]

The present invention will be described in more detail with reference to the following examples, but the scope of the present invention is not limited to the following examples.

Example 1 Preparation of honeycomb-like structure film (1) Poly-ε-caprolactone (compound 1) having an average molecular weight of 70,000 to 100,000 and amphipathic polyacrylamide (compound 2) in a weight ratio of 10: 1. Chloroform solution (0.1 to 2 wt% as polymer concentration) mixed at a ratio of 5 was cast on a petri dish with a diameter of 10 cm to obtain a relative humidity of 3
By blowing high humidity air of 0 to 80% at a flow rate of 1 to 20 L / min and evaporating the chloroform solvent, a film having a honeycomb-like structure having flexibility, elasticity, and strong mechanical strength was obtained.

[0024]

[Chemical 1]

[0025]

[Chemical 2]

The structures of the films prepared under the above various conditions are
Observation with an optical microscope and a scanning electron microscope revealed that
The film had a honeycomb-like structure with a pore diameter of 5 to 40 μm, and had a structure in which a single layer of holes penetrated from the front surface to the back surface.
The pore size was a perfect circle over the entire cast surface, and the sizes were almost the same. The hole diameter is measured from the photographed image, and the distribution of the hole diameter is calculated.
It was found to be below%. In addition, since it was possible to observe diffracted light of 10th order or more over the entire surface of the cast film in the evaluation of light scattering using a laser, it was found that a porous film having extremely high regularity could be produced.

Example 2 Preparation of honeycomb-like structure film (2) Amphiphilic polyacrylamide consisting of compound 2 was used as a material for the film, and the solvent for casting was chloroform.
Change to benzene, toluene, xylene, and change the solution concentration to 1.
0 g / L, cast amount 30 μL, glass is used for the substrate to be cast, and the flow rate of high humidity air is 0.09 L / L.
Minutes, the relative humidity was set to 85%, and the temperature was set to 20 ° C. The morphological observation result at this time is shown in FIG. In FIG. 1, the pore diameter is 0.5 μm to 10 μm from the top.

Example 3 Preparation of Honeycomb-Like Structure Membrane (3) Poly-ε-caprolactone consisting of Compound 1 and amphipathic polyacrylamide consisting of Compound 2 were used as membrane substances at a weight ratio of 10: 1. Chloroform was used as the solvent, the solution concentration was 10.0 g / L, the casting amount was changed to 5, 10, 20 mL, glass was used as the substrate to be cast, and the flow rate of high-humidity air was 2.0 L / min. Then, the cast amount dependence of the film that can be produced when the relative humidity was set to 30% and the temperature was set to 20 ° C. was evaluated. The morphological observation result at this time is shown in FIG. In FIG. 2, the hole diameter is
It is 8 μm to 35 μm from the top.

Example 4 Preparation of Honeycomb-Like Membrane (4) Poly-ε-caprolactone consisting of Compound 1 and amphipathic polyacrylamide consisting of Compound 2 were used as materials for the membrane in a weight ratio of 10: 1. Chloroform was used as a solvent, the solution concentration was changed to 1, 5, 10, and 20 g / L, the casting amount was 10 mL, hydrogel was used as the substrate to be cast, and the flow rate of high-humidity air was 2.0 L / L.
Minutes, the relative humidity was set to 30%, and the temperature was set to 20 ° C. to evaluate the solution concentration dependence of the film that can be formed. The morphological observation result at this time is shown in FIG. In FIG. 3, the hole diameter is
The minimum is 15 μm and the maximum is 25 μm.

Example 5 Preparation of Honeycomb-Like Structure Membrane (5) Poly-ε-caprolactone consisting of Compound 1 was used as the substance forming the membrane, chloroform was used as the solvent, the solution concentration was 1.0 g / L, and casting was performed. Set the volume to 5 mL and cast the substrate on agarose gel, glass, mica,
Instead of PHEMA, the flow rate of high-humidity air was set to 2.0 L / min, the relative humidity was set to 30%, and the temperature was set to 20 ° C. The morphological observation result at this time is shown in FIG. In FIG. 4, the hole diameter is
The minimum is 7 μm and the maximum is 14 μm.

In FIGS. 1 to 4, the length of each bar is 20 μm.

Example 6 Evaluation of Filtration Performance (1) Prior to evaluating the blood filtration performance of the formed honeycomb-like structure membrane, polystyrene particles having a known particle diameter were subjected to a passage experiment and passed through by changing the pore diameter. The type of particles and the passage rate were investigated. The results are shown in Table 1. Pore size 5.5
It was possible for the first time to show that with the 8.5 μm membrane, particles with a diameter of 3 μm pass but particles with a diameter of 10 μm and above do not pass at all.

[0033]

[Table 1] The particle passage rate is measured with a particle counter.

Example 7 Evaluation of Filtration Performance (2) A leukocyte trapping experiment in human whole blood was carried out by using the prepared membrane having a honeycomb-like structure. Pore size 5.2μm, 9.8μ
Using a membrane of m, human whole blood collected with a heparin blood collection tube was filtered, and blood was cast on a hemocytometer to measure the number of white blood cells. The number of white blood cells was 4800 cells / μL before filtration. After that, it was found that the number was 0 / μL (Table 2). Also, the filtered filtrate is rotated at 3000 rpm for 10 times.
When the color of the supernatant obtained by centrifugal separation was visually confirmed, no hemolysis was confirmed.

[0035]

[Table 2] White blood cell count before filtration 4800 / μL

Example 8 Evaluation of Filtration Performance (3) An erythrocyte trapping experiment in human whole blood was carried out using the prepared membrane having a honeycomb-like structure. Using a membrane having a pore size of 3.5 to 5.5 μm, three pieces each having a diameter of 5 mm were punched out, laminated, and allowed to stand on a nitrocellulose membrane. Human whole blood having a hematocrit value of 40% collected by a heparin blood collection tube was diluted with plasma obtained by centrifuging the same whole blood to obtain whole blood prepared to have a hematocrit value of 2.5%, and the whole blood was laminated. When 5 μL was spotted on the membrane and left for 10 seconds, then the laminated membrane was lifted up, and it was evaluated whether or not the red color of red blood cells remained in the plasma transferred to the nitrocellulose membrane by filtration. No red color was observed in the membrane having a pore size of 3.5 μm, confirming that red blood cells could be captured (Table 3).

[0037]

[Table 3]

[0038]

Industrial Applicability According to the present invention, blood cells can be separated from serum or plasma from a trace amount of whole blood, and further only white blood cells can be captured and separated.

[Brief description of drawings]

FIG. 1 shows the solvent dependence of the pore size when forming a honeycomb-like film.

[Fig. 2] Fig. 2 shows the cast amount dependency of the pore size when a honeycomb-like film is formed.

[Fig. 3] Fig. 3 shows the concentration dependence of the pore diameter when forming a honeycomb-like film.

[Fig. 4] Fig. 4 shows dependence of a pore size on a substrate to be cast when a honeycomb-like film is formed.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) B01D 69/12 B01D 69/12 71/48 71/48 G01N 33/48 G01N 33/48 H (72) Invention Person Sakano Yoshiki 3-11-46, Izumisui, Asaka-shi, Saitama, Fuji Shashin Film Co., Ltd. (72) Inventor Toshiko Ito 3--11, Sensui, Asaka-shi, Saitama Prefecture, Fujifilm Shin-Film Co., Ltd. (72) Inventor Kaoru Terashima 3-11-46 Izumisui, Asaka-shi, Saitama Prefecture F-term in Fuji Shashin Film Co., Ltd. (reference) 2G045 CA25 HA06 HA14 JA07 2G052 AA30 AD26 CA40 EA03 EA11 JA16 4C077 AA12 BB02 EE01 KK15 4N006 PP13 LL02 LL02 LL02 LL02 LL006 GA02 GA13 MA04 MA08 MA22 MB06 MC48X MC54X NA10 NA34 NA41 PB44 PB45 PC41 PC80

Claims (4)

[Claims] 1. A blood filtration membrane having a pore size of 0.5 to 40 μm and a variation in the pore size of 20% or less in variation coefficient. 2. The blood filtration membrane according to claim 1, which has a honeycomb-like structure. 3. The blood filtration membrane according to claim 1, which is composed of a substance mainly containing poly-ε-caprolactone. 4. A method for hemofiltration by using two or more membranes having the same average pore diameter or different membranes having different average pore diameters according to any one of claims 1 to 3.