JP3487615B2 - Blood test filter - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a filter for inspecting the properties of blood cells such as red blood cells and white blood cells in blood, particularly the blood cell deformability.

[0002]

2. Description of the Related Art Erythrocytes make up about 40 to 50% of blood constituents and supply oxygen to the whole body by the action of hemoglobin contained therein, which is indispensable for sustaining life. Cells. For example, the oxygen requirement of brain cells is extremely high, and when oxygen supply from red blood cells is interrupted due to cerebral circulation disorder, etc., cell destruction, so-called “brain death” occurs in just a few minutes. In the old days, vasoconstriction / dilatability, platelet aggregation, blood coagulation factors, etc. have been problems as factors that regulate circulatory dynamics, but recently, in addition to these, the "deformability" of red blood cells has attracted attention. ing. That is, normally mature erythrocytes have a diameter of 7 to 8 μm, a thickness of 2 to 3 μm, and have a disk shape with both sides depressed.
It has the ability to deform in various ways when subjected to external force (deformability), and can easily pass through capillaries smaller than its own diameter, thereby supplying oxygen to the whole body.
Therefore, when red blood cells do not have a normal deformability, or when the deformability is reduced, a fatal injury to the human body occurs. In many diseases such as diabetes, as well as congenital hemolytic anemia (such as sickle cell disease), the deterioration of red blood cell deformability has become a problem. In particular, the role of erythrocyte deformability in the microcirculatory dynamics is emphasized in connection with the remarkable increase in ischemic infarction diseases such as myocardial infarction and cerebral infarction accompanying the westernization of diet in recent years.

Reflecting the above situation, many studies have been conducted in recent years on the deformability of red blood cells, but the deformability of red blood cells is not clearly defined as a physical quantity. Therefore, the evaluation currently depends on the measurement method. It is the most widely used method for measuring deformability, and is also a valid method as a model of the microcirculatory system.
There is a filtration method using "Nuclepore Filter (a product of Nuclepore Inc., USA)". This filter was created by irradiating a polycarbonate thin film with charged particles in a nuclear reactor and adding an alkali etch to the formed track to expand the track, and the pores have a non-uniform distribution and fusion, There are many branches, and the edges are rough. Also, due to its material (polycarbonate), it is practically impossible to reuse. Therefore, the Nuclepore filtration method has a problem in its reproducibility and quantification property, and in particular, it cannot be said to be sufficiently effective as a diagnostic or determination method in clinical medicine which is a complex and versatile system.

The usual washing of red blood cells with a centrifuge cannot prevent the white blood cell from being contaminated with white blood cells.
Further, in the filtration method using the above-mentioned Nuclepore filter, blood cells are easily activated by mechanical stimulation due to branching of rough edges and holes of the Nuclepore filter, and clogging is likely to occur. . Therefore, in filtration of a red blood cell suspension using this filter, there may be a state in which it is not possible to distinguish whether the deformability of red blood cells is observed or the influence of clogging of white blood cells is observed. Moreover, in principle, it is neither possible to predict, nor to separate and analyze the actions of both. As a countermeasure against this, a method using a leukocyte removal filter is also used, but the leukocyte removal filter is also supplemented with red blood cells having a reduced deformability. Therefore, the deformability of whole red blood cells having a normal distribution (including red blood cells with reduced deformability) cannot be evaluated, which is a problem especially in clinical medicine. Further, the fusion of the holes causes the holes to be larger than the diameter of the red blood cells, the originally intended deformability cannot be effectively detected, and the detection sensitivity is lowered. Furthermore, it is self-evident that the above-mentioned conventional filters cannot be reused, which amplifies the defects of their quantification and reproducibility due to their geometrical non-uniformity.

On the other hand, white blood cells are as small as about 1/700 of red blood cells as a blood cell component of blood, but white blood cells have several kinds of cells having different functions and have various physiological functions. Functionally, it is roughly divided into phagocyte groups (granulocytes, monocytes) that are responsible for bactericidal action and immune cell groups (lymphocytes) that are responsible for cellular immune action. It is well known that, although the number is small, it is responsible for important physiological functions, for example, that death occurs when an immune cell group is invaded by an AIDS virus. Furthermore, recently, the role of white blood cells (mainly phagocytic group) in the microcirculatory system has been attracting attention as an expression mechanism of arteriosclerosis and thrombosis, and post-infarction recanalization of ischemic infarction diseases such as myocardial infarction. Tissue damage resulting from excessive production of reactive oxygen species (such as superoxide anion) by leukocytes and release of proteolytic enzymes has become a problem. Therefore, the incidence of these is also high, and the development of studies on the rheological properties of leukocytes in severe diseases, especially their deformability, has been earnestly desired. The frequently used method of measuring deformability of white blood cells is essentially the same as that of red blood cells. For example, in the filtration method, the measurement of deformation of red blood cells is mainly performed with a pore size of 3 to 5 μm.
m filters are used, whereas that of white blood cells is predominantly 5-8 μm. White blood cells (phagocytic group) are rapidly activated by mechanical stimulation, hypoxia (for example, ischemia), inflammation and the like. Therefore, in pathophysiology, it is extremely important to know the relationship between activation and deformability. However, the conventional micropore filter exerts a mechanical stimulus on leukocytes due to the irregularity of pores (rough edges and branching). That is, the filtration itself directly affects the activation of white blood cells. In addition, all the problems described in relation to red blood cell deformability such as the problem of reproducibility due to non-reusability also apply to white blood cells.

[0006] By the way, leukocytes have the ability (chemotacticity) not only to flow by blood flow but also to recognize and respond to stimuli of various physiologically active substances and to migrate themselves. Leukocyte chemotaxis is a direct indication of the physiological process of inflammation and the early response of the immune system. Today, there is a strong interest in the response and reactivity of immune cell groups, that is, chemotaxis, in connection with the epidemic of AIDS and the problem of organ transplantation. A method often used as a simple leukocyte chemotaxis test method is Nuclepore Filter (or Millipore Filter,
The pores are more irregular than those of the Nuclepore filter. The chamber (upper and lower chambers) divided by the upper and lower chambers contains the sample (physiologically active substance) and the upper chamber contains the leukocyte suspension, and the sample diffuses through the filter. Is a method of examining the degree of leukocyte migration in response to. Depending on the purpose of the experiment, normally, Nuclepore having a pore size of 3 to 8 μm is used. However, it is not always satisfactory in terms of quantification, such that the sample diffusion state cannot be kept constant due to the non-uniformity of the distribution of the pores, the irregularity of the pore diameter, and the fact that it cannot be reused.

Although the importance of the role played by red blood cells and white blood cells in microcirculation disorders has been described above, blood further contains coagulation factors such as platelets and fibrinogen, which influence micropore circulation dynamics. I can't wait for the matter. That is, the circulatory disorder in the living body is embodied as the sum of all these factors. Therefore, it is important to know the actions of individual factors as well as their interactions. For example, the largest loop of ADP, which is a typical platelet aggregation-inducing substance, is erythrocytes, and stenosis of blood vessels and damage destruction of erythrocytes at bifurcation have a direct effect on platelets. Therefore, in order to understand the microcirculatory dynamics in the living body, it is also important to know the ability of whole blood (blood itself, a term used in comparison with blood constituents such as red blood cells and platelets) to pass through micropores. Is. In this case too, the drawbacks of the prior art described above apply and are further amplified.

The same applies to leukocyte deformability, leukocyte chemotaxis (in this case, geometrical heterogeneity also leads to sample diffusion heterogeneity), and whole blood micropore passage ability. It also applies to measurements, the drawbacks of which are amplified.

The present invention has been made in view of the above-mentioned problems of the prior art, and is a filter that can be used in not only basic research but also quantitative measurement of blood cell deformability that can contribute to clinical medicine. The object of the present invention is to provide a blood test filter that can be repeatedly used.

[0010]

The blood test filter according to the present invention has an overall thickness of 5 to 5 formed by electrodeposition.
A thin metal plate of 30 μm with a hole diameter of 3 to 20 μm at the narrowest part,
Micropores having a funnel-shaped or pincushion-shaped cross-section are formed such that the diameter of the opening on the inlet side of blood cells is more than 1 and up to 10 times the diameter of the narrowest part. It is characterized by that.

The present invention will be described in detail below with reference to the accompanying drawings. FIG. 1 is a cross-sectional view of a blood test filter according to the present invention. The filter according to the present invention is composed of thin plates 10 formed by electrodeposition of a metal material that can be electrodeposited, for example, a metal material such as nickel, silver, gold, copper, etc., with micropores 11 formed substantially uniformly. Hole 11 in this example
Has a pincushion-shaped cross-section, and there is no distinction between the front and back when functioning as a filter. Also,
In the present invention, "electrodeposition" should be understood in a broad sense,
Although electroplating and electroforming are included, particularly wet electroplating can be preferably used. By constructing the filter with electrodeposited metal in this way, the problem of alignment of the pattern on the front surface that occurs when holes are formed in the metal plate by etching, and the maximum hole formation when etching from one side There are various advantages such as solving the problem that it is difficult to maintain the dimensional accuracy of the narrow portion.

The thin plate 10 made of electrodeposited metal has a thickness of 5 to 3
It is 0 μm. If the thickness is less than 5 μm, it becomes thinner than the body length of blood cells, and the blood cells easily slip through, which makes accurate measurement difficult. In addition, since blood cells pass through long blood vessels, it is considered possible that the thickness exceeds 30 μm, but it is difficult to make the thickness above 30 μm in terms of electrodeposition processing.
On the contrary, when the thickness is increased, it is expected that the passage of blood cells will be alienated due to the generation of static electricity.

Further, in the present invention, the micropores are distributed almost uniformly so that the diameter of the opening on the inlet side of blood cells is in the range of more than 1 time and up to 10 times with respect to the narrowest part of each hole 11. Is formed. When the diameter of the opening on the inlet side of blood cells (blood) is less than or equal to 1 times the diameter of the narrowest portion or exceeds 10 times, a good blood cell capturing effect cannot be obtained, and blood cells with normal deformability are obtained. It is not preferable because the passage property of is degraded.

In addition, it is important for improving the quantitativeness of the inspection that the fine holes as described above are formed in the thin plate 10 substantially uniformly. In addition, the sum of the cross-sectional areas in the plane direction of the micropores in one filter is calculated as the area of the narrowest part and is 0.1 mm ^{2 to 4} m.
The range of m ² is particularly preferable. Further, the formation density of the micropores is preferably 100 to 40,000 / mm ² .

FIG. 2 shows an example in which the micropore 11 has a funnel-shaped cross section.

FIG. 3 shows an example in which a metal coating layer 12 formed by electrodeposition is further formed on both the front and back surfaces of the thin metal plate 10 and the inner wall surfaces of the minute holes. As the metal of the metal coating layer, the same components as the metal used for electrodeposition of the thin plate can be used. The formation of such a metal coating layer is advantageous for making the cross-sectional shape of the micropores into a pincushion shape, and also smoothing the state of the inner wall or peripheral portion of the micropores to improve the passage characteristics of normal blood cells. It is advantageous in the above.

As described above, in the present invention, since the filter is made of the electrodeposited metal and the micropores having a pincushion shape or a funnel shape in cross section are uniformly formed, only the abnormal blood cells whose deformability is lowered are efficiently formed. It is also excellent in that it can be captured quantitatively, and is also excellent in durability to withstand repeated use.

The method of manufacturing the blood test filter of the present invention will be described below. Here, a method for manufacturing the filter of the embodiment shown in FIG. 3 will be described.

As shown in FIG. 4A, first, the thickness of 0.
A resist layer 22 is formed on a stainless plate 21 having a side of 1 to 0.3 mm and a side of 121 mm. This resist layer 22 is
It can be formed by uniformly applying a resist on the stainless steel plate 21 by a method such as a spin coating method, a roll coating method, or a dip pulling method, and performing a heat treatment. The heat treatment is usually 40, although it depends on the type of resist used.
Perform at about 50 ° C for about 30 minutes. As the type of resist, it is preferable to use rubber-based negative resist, PVA negative resist, or casein negative resist. In addition to stainless steel, copper, nickel or the like can be used as the substrate.

Next, as shown in FIG. 3B, a desired pattern is drawn on the resist layer 22 by ionizing radiation 23 using an exposure device to form an exposed portion 24. Subsequently, it is developed with a predetermined developing solution and rinsed with a predetermined rinsing solution to form a resist pattern having a thickness of 1 μm to 6 μm and a hole diameter of 15 μm as shown in FIG.

Next, as shown in FIG. 3D, nickel 25 is plated on one surface by electroplating to a thickness of 5 to 10 μm. At this time, the stainless steel 21 is plated, but the resist pattern 24 is not plated, so that an opening having a hole diameter of 8 to 10 μm is formed.

Next, after heat-drying treatment, the plated nickel 25 is peeled off ((e) in the figure).

Subsequently, nickel 26 is plated on both surfaces by electroplating so as to have a thickness of 5 to 10 μm, thereby completing the filter 6 having a pore diameter of 3 to 5 μm as shown in FIG. The shape of the holes can be appropriately controlled by the amount of plating.

As described above, in the blood test filter according to the present invention, the filter thickness is thin and constant, the distribution and shape of the pores of the filter are very uniform, and the pore diameter is constant, and there is no fusion or branching of pores. Moreover, such inconvenience does not occur during use. Further, since the filter is formed by electrodeposition using a metal such as nickel, the inlet part of the hole can be formed in a funnel shape, and the edge becomes extremely smooth. Furthermore, since it has excellent durability according to the present invention, it can be reused at least 100 times by performing ultrasonic cleaning for several seconds after use.

[0025]

[Example] Manufacturing example A stainless steel plate (SU with a thickness of 0.1 mm and a side of 121 mm) (SU
S304 material), a casein-based negative resist FR No. 15 manufactured by Fuji Yakuhin Co., Ltd. was applied by a dip pulling method, and 4
Heat treatment was performed at 5 ° C. for 30 minutes to obtain a uniform resist film.

Next, a pattern was drawn on this resist film by a conventional method. At this time, ultraviolet rays with a wavelength of 365 nm were exposed for 2 to 3 minutes by a 3 kW mercury lamp.

Subsequently, the exposed resist film is replaced with 40 to 5
After developing with hot water of 0 ° C., rinsing with water and spin drying were performed to form a resist pattern having a thickness of 3 μm and a hole diameter of 15 μm.

Next, this was baked at 200 to 250 ° C. for several minutes, immersed in a nickel sulfamate bath, and nickel was plated on one side by electroplating to a thickness of 10 μm. The composition and plating conditions of this nickel sulfamate bath are shown below. Nickel sulfamate 400 g / l Boric acid 30 g / l pH 4.0 Bath temperature 50 ° C. Current density 2 A / dm ² hours 10 minutes Subsequently, after heat-drying at 80 ° C. for 5 minutes, the plated nickel was peeled off. Thickness 10μm, Pore diameter 8μ
It became m.

Subsequently, after heat-drying treatment, nickel is plated on both sides to obtain a thickness of 14 μm and a hole diameter of 5 μm.
m filters according to the invention have been completed. The composition of the nickel sulfamate bath and the plating conditions at this time are as follows. Nickel sulfamate 400 g / l Boric acid 30 g / l pH 4.0 Bath temperature 50 ° C. Current density 4 A / dm ² hours 2 to 3 minutes Next, the planar form of the filter manufactured in the above-mentioned examples will be described.

FIG. 8 is a plan view of an example in which a total of 64 filters, each having 8 sides, are manufactured on the substrate at the same time.
A filter surrounded by a resist pattern 81 is shown on a substrate 80 made of stainless steel having a side length of 121 mm.

FIG. 9 is an enlarged view of a portion which constitutes one filter. A portion surrounded by the resist pattern 81 constitutes a filter, a mesh portion 82 is constituted in a diagonally shaded portion of a circular center, and a filter is provided around the mesh portion 82. A frame portion 83 for fixing the is formed. The supporting portion 84 is provided for facilitating the peeling of the filter, which is attached on multiple surfaces after electrodeposition, as a single film from the substrate. When using individual filters, the support portion 84 is used separately.

FIG. 10 is an enlarged plan view showing the positional relationship of the minute holes 11 formed in the mesh portion 82. In this embodiment, the positional relationship is such that the three minute holes form the vertices of a triangle. Are arranged in. The distance between the micropores in this case is 30 μm.

An example of measuring the erythrocyte deformability measured by using the thus-produced erythrocyte test filter is shown below. Measurement of red blood cell deformability and its evaluation The deformability of red blood cells was investigated using the quantitative vertical glass tube method. The outline of this method is shown in FIG. That is, the filter 31 according to the present invention is attached to the glass tube 30 which is vertically stood,
The erythrocyte suspension was filtered using gravity. A red blood cell suspension is flown in from the upper part of the glass tube 30, a pressure transducer 32 installed at the zero level of the glass tube 30 continuously detects a pressure drop, and data is taken into a personal computer through an interface.
The flow rate was calculated. The metric is obtained by taking the derivative at a certain height (pressure) of the height (pressure) -time curve and multiplying it by the cross-sectional area of the glass tube. The flow rate is calculated by a personal computer, and the obtained pressure-flow rate curve is shown in FIG. 6 as an example of the effect of the hematocrit value (the ratio of red blood cells in the red blood cell suspension). (In FIG. 6, the uppermost straight line represents a NaCl solution having a hematocrit value of 0%, and becomes a downwardly convex curve as the hematocrit value increases.) This shows a decrease in the flow rate with an increase in the hematocrit value. Moreover, it is very similar to the experiment performed using a living blood vessel perfusion sample (frog perfusion of hindlimbs, etc.). The deformability of red blood cells was quantitatively analyzed using such a pressure-flow rate relationship. The representative results obtained so far are listed below.

1. In the above erythrocyte deformability test method using the filter according to the present invention, due to the shaping of the filter pores, the influence of white blood cells that cannot be mixed with normal washing red blood cell fluid (as described above, this is due to the irregularity of pores). Caused by mechanical irritation) was found not to be received at all. As mentioned above, this is a remarkable result not only in establishing quantitativeness in measuring red blood cell deformability but also in applying to clinical medicine.

2. Since the pore size of the filter is constant and there is no fusion or bifurcation, the degree of detection of changes in erythrocyte deformability is constant, and compared to conventional Nuclepore filters (for example, as mentioned above, the fusion sensitivity of the pores increases the detection sensitivity. Decrease), and a remarkable increase in detection sensitivity was observed. A congenital disease in which hemoglobin is oxidized and denatured to form a precipitate condensate (Heinz bodies) in red blood cells due to amino acid substitution of the hemoglobin molecule and erythrocyte breakdown is accelerated to cause hemolytic anemia is called unstable hemoglobinosis. However, such a decrease in deformability of red blood cells is something that anyone can imagine. However,
In the past, there was a case where it was reported by a deformability test using a Nuclepore filter that there was no decrease in deformability, but by using the filter according to the present invention, a clear decrease in deformability of Heinz body-forming red blood cells ( In particular, the problem was solved by the finding that the pore size of 3 μm was remarkable).

3. The constant pore size of the filter according to the present invention not only increases the detection sensitivity, but also provides a quantitative method for the specificity of the regulatory factor of red blood cell deformability. The main defining factors of erythrocyte deformability are geometric factors of erythrocytes (cell surface area / volume ratio, morphology), intracellular viscosity, and membrane viscoelasticity. Should also be more sensitively detected by the 3 μm pores. In fact, we investigated the deformability of erythrocytes of patients with hereditary spherocytosis (HS) before and after splenectomy, and clearly confirmed that the importance of the involvement of the spleen in in vivo hemolysis of this disease was clarified. There is. That is, before the splenectomy, only a slight decrease in deformability was observed in the 5 μm hole, but a remarkable decrease in deformability was observed in the 3 μm hole, and after the splenectomy (elimination of microcytic spherical red blood cells, improvement of anemia). Have seen a dramatic improvement in deformability until it can be said that even a 3 μm hole is almost normal. As a pathophysiological mechanism of this disease,
It was found that the spleen not only acts on red blood cell depletion, but in this disease, so-called “conditioning” promotes microspherical spheroidization and causes hemolysis. Thus, the filtration with the 3 μm filter according to the present invention is extremely useful as a simulation of microcirculation in the spleen and therefore in predicting the effect of splenectomy. In the test method using the conventional filter, the clear cut does not give a meaningful result, and in fact, it has not been reported.

4. The deformability of erythrocytes is an important factor that influences microcirculatory dynamics. The necessity and importance of the research have been described above, but it is undeniable that the research is currently stagnant. The biggest cause is attributed to the conventional methods of measuring deformability and sensitivity. That is, the methods so far have not necessarily been effective for clinical medical research, which is a complex and diverse system. 2 and 3 above
The example described in the above section is also an example showing the clinical medical effectiveness of the filter according to the present invention, but more typical clinical examples are shown below.

An example of a typical death-related microcirculatory disorder due to reduced erythrocyte deformability is that due to sickle cells (a typical disease of inherited congenital hemolytic anemia). The hemoglobin of sickle cells undergoes significant polymerization under hypoxia (peripheral circulation), sicklings the morphology of erythrocytes, resulting in a marked decrease in deformability. Recently, treatment of hydroxyurea and erythropoietin for severely ill patients has been attempted, and good hematological results have been obtained. I was able to do it. As a matter of course, the improvement of clinical symptom by this treatment was expected to improve the deformability of sickle cell, but it is not only the conventional test method by Nuclepore but also other typical deformability test method. No clear improvement in deformability could be found by the micropipet method or ecytocytometry. However, 3
By measuring the μm pores with the filter of the invention, a significant improvement in the deformability test was found, which corresponds to an improvement in blood findings and thus to an improvement in clinical symptoms. This is an example showing that the fill ratio method using the filter of the present invention is also extremely effective for the determination of therapeutic effect. In other words, the filter of the present invention breaks through the delay in deformability research. It suggests that you can do it. By the way,
There are many reports on the deterioration of deformability, but there are no reports on improvement of deformability. In that sense, according to the filter of the present invention, including the above-mentioned examples of spherocytosis,
It is excellent in that it can effectively and quantitatively test the change performance.

5. As described above, the deformability test method using the filter according to the present invention has been described mainly with emphasis on clinical medical results in order to clearly show the effectiveness based on the quantitative property. It is naturally expected that they will also be effective in medical research, and the results of their basic medical research will contribute to the development of clinical medicine. This point will be described.

By the filtration method using the filter of the present invention, it was possible to quantitatively detect all the above-mentioned main defining factors of erythrocyte deformability. For example, when red blood cells are exposed to changes in osmotic pressure, they change their volume as if they were an osmometer, and in the hypotonic region they changed from dome-shaped to spherical, and in the hypertonic region they flattened and contracted with spinous red blood cell-like changes. Changes in morphology to shape or correspondingly, the deformability of erythrocytes is reduced mainly in the hypotonic region by a decrease in surface area to volume ratio and in the hypertonic region by an increase in internal viscosity and morphological change. It was possible to detect clearly with a quantitativeness not reported. By the way, the decrease in deformability caused by hypertonic solution is a main cause of the side effect of the water-soluble iodine contrast medium used for angiography, which is indispensable for today's medical examinations (the frequently used conventional contrast medium is extremely hypertonic). That is, the present inventor quantitatively showed that the effect of the osmotic pressure of the contrast agent on the deformability of red blood cells is much larger than that of the viscosity and the iodine amount of the contrast agent, and the recently developed so-called low The effectiveness of osmotic contrast agent was clarified. Therefore, the filtration method using the filter of the present invention is excellent as a simple and quantitative method for examining the effectiveness of a newly developed contrast agent. The same applies to various drugs related to microcirculatory dynamics.

As a further molecular-based study example, a decrease in deformability due to a change in erythrocyte membrane protein caused by superoxide anion (O ₂ ⁻ ) is shown. O ₂ ^- is
Although it is indispensable for physiological functions of the body such as rapid rotation of intracellular electron transfer system and bactericidal action of leukocytes, its overproduction causes various oxidative disorders in cells and organs, such as aging and aging. It is well known that it triggers carcinogenesis. When O ₂ ⁻ is exposed to erythrocytes, intracellular hemoglobin is oxidized to methemoglobin, and O ₂ ⁻ is overproduced by this oxidation, and the band 3 protein, which is a high binding site of hemoglobin, is rapidly decomposed and disappears, In addition, degradation of membrane proteins forming so-called cytoskeleton such as ankyrin, band, 4.2 protein, spectrin, etc.
It has been found by examination with the filter of the present invention that disappearance occurs and the deformability of red blood cells is reduced in response to these changes. That is, the deformability decreased due to changes in membrane water permeability (increase in cell volume) due to structural changes in band 3 protein and changes in membrane viscoelasticity and morphology due to changes in membrane skeletal proteins such as spectrin. . This finding not only demonstrates the importance of the interaction of membrane proteins and their hemoglobin relates deformability, recanalization time after in infarct ischemic circulatory disorders O ₂ ^- excessive production by oxidative disorders (One recent This is to provide new basic medical information regarding major topics).

6. The present inventor also theoretically investigated the quantitative property of the filtration method using the filter according to the present invention. That is, the present inventor conducts a stochastic analysis using a lattice model based on the geometrical uniformity of the filter of the present invention, and further develops a theoretical analysis that has been performed by Nuclepore filtration until now. , Succeeded in generalizing. By the way, the theoretical analysis of Nuclepore is also performed assuming a filter having a very geometrically uniform shape and distribution of holes, such as nickel mesh. In the pressure-flow rate curve shown in FIG. 6, the solid line is the curve obtained from the theoretical formula, and the agreement with the experimental value (white circle) is surprising. This also guarantees the quantitativeness of the filtration method using the filter of the present invention. Leukocyte deformability test The leukocyte deformability test was performed in the same manner as for the red blood cells. In this case, the measurement was performed using a filter having a pore size of 5 to 8 μm.

From the measurement results, it was found that high-sensitivity and high-accuracy measurement, which is completely incomparable with the conventional micropore filter, was possible. Especially, in the experiment of the phagocyte group sensitive to mechanical stimulation, the difference was remarkable. In addition, a filter having a pore size of 8 to 15 μm was used in such an experiment, because the agglutination phenomenon is also exhibited by the marked activation of leukocytes. Basic experiments such as the effect of leucocrit value (percentage of leukocytes in leukocyte suspension), temperature effect, bactericidal action of leukocytes and role of deformability and aggregating ability in immune response are described in Leukocyte chemotaxis and It is also extremely effective in experiments on the ability of whole blood to pass through micropores. Leukocyte Chemotaxis Test As shown in FIG. 7, a leukocyte chemotaxis was measured by inserting a filter 71 having a pore diameter of 3 to 8 μm between chemotaxis measurement chambers 70 composed of two chambers, an upper chamber and a lower chamber. For example, the sample chamber 72
When Escherichia coli-derived substances (or E. coli itself) are put in, the white blood cells recognize and respond to them, and chemotaxis is increased. White blood cells have microfilaments (polymerized actin) and microtubules (polymerized tubulin) that form the membrane lining structure and intracellular fibrous structure, and are responsible for the motility and deformability of white blood cells. ing. For example, it was reconfirmed by the inventor that colchicine, a drug that alienates tubulin polymerization, reduces chemotaxis and deformability. Interesting findings such as specific lymphocyte (T cell) responsiveness to monoclonal antibodies CD3, CD4, and CD8, and difference in responsiveness of red leukocyte cancer cells (K562 cells) and drug-resistant mutant cells thereof to anticancer agents (adriamycin) Is getting The chemotaxis test method can be applied not only to leukocytes but also to all cells having migratory properties such as fibroblasts and cancer cells. It was confirmed that the use of the filter according to the present invention markedly improves the quantification / reproducibility of the chemotaxis test method. The ability of whole blood to pass through micropores The ability of whole blood to pass through pores was measured by the quantitative vertical glass tube method described above using a filter having a pore size of 10 to 20 μm. This measurement is also a method for measuring the aggregating ability of whole blood. The addition of the platelet aggregation stimulating substance ADP was found to decrease the ability of whole blood to pass through micropores, that is, increase the aggregation ability in a concentration-dependent manner. This was confirmed by the concentration of the platelet aggregation inhibitor prostaglandin E ₁ . Was suppressed by sex. Using erythrocyte suspension by sonication instead of ADP (erythrocytes are a large pool of ADP), we obtained exactly the same results, demonstrating the direct effect on the platelets by the damage destruction of erythrocytes. What is of most interest today regarding whole blood agglutination is the effect of leukocytes on whole blood agglutination during infarct recanalization. It was confirmed that the representative leukocyte stimulating substance PMA markedly enhanced the whole blood aggregating ability. At the same time, the agglutinating ability of erythrocytes and leukocytes is also independently investigated, and the deformability of erythrocytes is reduced by the overproduction of O ₂ accompanying the activation of leukocytes. It was found that it was further enhanced. The filter according to the present invention was effective even when quantitatively performing such an experiment.

[Brief description of drawings]

FIG. 1 is a cross-sectional view of a blood test filter according to the present invention.

FIG. 2 is a cross-sectional view of a blood test filter according to the present invention.

FIG. 3 is a cross-sectional view of a blood test filter according to the present invention.

FIG. 4 is a process sectional view showing a manufacturing example of a blood test filter according to the present invention.

FIG. 5 is a schematic diagram of a measuring device for measuring red blood cell deformability using the blood test filter according to the present invention.

6 is a pressure-flow rate curve obtained by measurement with the measuring device of FIG.

FIG. 7 is a cross-sectional view of a chemotaxis measuring device for inspecting leukocyte chemotaxis.

FIG. 8 is a plan view showing a planar form of a filter manufactured in the example of the present invention.

FIG. 9 is an enlarged view of a portion that constitutes one filter in FIG.

FIG. 10 is an enlarged plan view showing the arrangement relationship of the micropores of the filter manufactured in the example of the present invention.

[Explanation of symbols]

10 thin metal plates 11 micropores 12 Metal coating layer 21 board 22 Resist layer 25 Nickel electrodeposition layer 30 glass tubes 31 Filter holder 32 Pressure Transducer 80 substrates 81 resist pattern 82 mesh section 83 frame 84 Support

Claims (6)

(57) [Claims] 1. A thickness of 5 to 3 formed entirely by electrodeposition.
A 0 μm thin metal plate having a pore diameter of 3 to 20 μm at the narrowest part, and a diameter of the opening on the inlet side of blood cells larger than 1 times and up to 10 times the narrowest part, or a funnel-shaped cross section. A blood test filter, which is characterized in that pincushion-shaped micropores are formed in a substantially uniform distribution. 2. The blood test filter according to claim 1, wherein a metal coating layer formed by electrodeposition is further formed on both front and back surfaces of the metal thin plate and inner wall surfaces of the micropores. 3. Micropores 100 to 40,000 / mm ²
The blood test filter according to claim 1, wherein the filter is formed with a density of. 4. The blood test filter according to claim 1, wherein the thin metal plate is nickel. 5. The blood test filter according to claim 1, wherein the metal coating layer is made of nickel. 6. The blood test filter according to claim 1, wherein the electrodeposition is electroplating.