ES2369630T3 - PROCEDURE TO SEPARATE LEUKOCYTES FROM THE BLOOD. - Google Patents

Abstract

Procedure for reducing the number of leukocytes in blood by means of an in vitro treatment of blood in a structure consisting of a multiplicity of fibers based on organic polymers, wherein this multiplicity of fibers is contained in a housing with an input element and an outlet element, in which blood is introduced into the carcass through the inlet element, then circulates through the carcass, the blood circulating along the outer surfaces of the fibers and, finally, leaves the carcass at through the exit element, characterized in that fibers are used which, with the circulation of blood along its outer surface, determine a formation of the activating product of complement C5a at a concentration of at least 10 μm per m 2 of fiber surface , and because the arrangement of the fibers exhibits a high degree of organization, it being understood by a high degree of organization that a large part of the fibers are arranged adjacently along their direction of extension.

Description

The invention relates to a method for separating leukocytes from blood, as well as to a device for carrying out this procedure.

Blood is essentially composed of plasma and cellular elements. Among the latter are erythrocytes (red blood cells), thrombocytes (platelets) and leukocytes (white blood cells). Among the white blood cells are lymphocytes, monocytes and neutrophilic granulocytes (neutrophils, polymorphonuclear leukocytes (PMN)). Lymphocytes play a decisive action on specific immunity. Neutrophil monocytes and granulocytes are types of cells that are involved in nonspecific immune defenses or inflammatory reactions. Its mission is, for example, to cancel invading microorganisms, previously marked by certain proteins of the organism (for example, C3b of the complement system or the IgG immunoglobulin) as foreign organisms.

Once the cells have approached the invading microorganisms, they pour both oxygen radicals and proteases that destroy the microorganisms, to be subsequently phagocytosed. When this reaction does not occur in full, or goes unchecked and chronically, the release of oxygen radicals and proteases, of an aggressive nature, can damage the body's own tissues. During inflammation, intense communication and coordination is established among all cell types that are used, among others, by various cytokines. The reaction is very complex and has not yet been fully elucidated. However, ultimately, it results in clinically observable symptoms of inflammation, which include swelling, redness and fever. In this context, they are characteristic, among others, an increase in monocytes and neutrophilic granulocytes generated in the bone marrow, and circulating in the blood, as well as certain chemical messengers. In addition to the aforementioned proinflammatory cytokines, complement proteins C3a and C5a are also formed, which are generated by the activation of proteins C3 and C5, and which act as a parameter of complement activation during the inflammatory or acute phase reaction.

In a series of inflammatory diseases such as ulcerative colitis, Crohn's disease and rheumatoid arthritis, extracorporeal therapies are currently used. According to the current state of the art, a certain number of cells (probably monocytes and special neutrophilic granulocytes) are extracted from the patient, circulating the patient's blood extracorporeally and treating it with a cell filter. They are used as leukocyte filters, for example, columns filled with particles or beads of cellulose acetate. In this case, the separation is mainly produced by cellular adsorption on the surface of the beads. These products are already commercially available. Blood is passed through a column containing cellulose acetate spheres. The cellulose acetate spheres reduce adsorption, especially the granulocytes and monocytes contained in the blood.

US 6,489,007 discloses a method for separating leukocytes from blood by adsorption on a support. In this case, the blood is contacted with this support, preferably in the form of what is called pearls, where the support has a higher affinity for infected, activated or defective leukocytes compared to leukocytes that do not exhibit these characteristics.

Alternatively, felts or tissues are also used for extracorporeal separation of leukocyte filters. For example, for the separation of leukocytes from a blood can for transfusions (such as a concentrate of erythrocytes or platelets), felt-based products are used, in which cell separation takes place primarily by mechanical filtration through of said felt. For transfusion, batches of 500 ml of blood are typically filtered in less than half an hour. The process is carried out with the help of the force of gravity in a single step and not in the circulation. In order to use a cellular filter in the extracorporeal circulation, it must be possible to filter approximately 1 to 6 l of blood, driven by a pump, for a few hours. To this end, polypropylene felts with a cylindrical shell are available commercially, with an inlet connection in the head area and an outlet connection in the opposite end to the head area. With the felt used in this case, the leukocytes are retained thanks to the effects of filtration and adsorption.

The same is true for devices intended to separate blood components from EP 0 312 595, in which a cylindrical shell filled with cotton fibers is described, with an input element and an output element for blood. Cotton fibers are cut fibers which, according to the figures of EP 0 312 595, are arranged in two layers formed by fiber aggregates in random order. Blood components such as, for example, white blood cells or platelets, are retained by size exclusion in the intermediate spaces between the fibers of the fiber aggregates.

According to EP 1 230 940 a filter is also used to separate leukocytes from the blood. The filter can be a felt, a fabric or a membrane of flat or hollow fibers, of porous character. The blood to be treated is circulated through the felt, tissue or porous membrane. The filter material has a coating of a hydrophilic synthetic polymer. This coating allows the passage of thrombocytes through the filter, with the simultaneous separation of leukocytes by adsorption.

WO 95/18665 discloses a filter and a method for the separation of leukocytes and substances with virus inactivating potential from plasma or other blood fractions. The filter is based on a network of textile fibers. On the network are covalently linked ligands with high affinity for virus or leukocyte inactivating substances. In this case, it is a selective method, but technically very expensive, since the ligands must be attached to a polymer matrix directly or through a linker.

In US 4,925,572 a filter is described by which a blood product is circulated to separate the leukocytes, in which, according to their porous element composition, the leukocytes are separated both by adsorption and by filtration. The porous element may preferably be a fiber-formed element, wherein these porous elements may have a pore diameter in the range of approx. 4 to approx. 8 m. The porous element for separating the leukocytes can show a superficial modification in order to generate, in this way, a certain surface tension and, with it, a good ability to cross-link the element. The US document

4,925,572 aspires to a separation of leukocytes mainly by adsorption.

Leukocyte retention with this type of filter is based on a cellular entrapment in fiber felts, as well as a more or less intense adsorption of the cells on the fiber surface. In general, with this procedure, the different blood cells are subjected to a strong mechanical overload, which can lead to cell activation or even the destruction of blood cells.

An essential drawback of existing systems and procedures is that different types of cells cannot be adsorbed in a targeted or specific manner and that, in addition to monocytes and granulocytes, lymphocytes, thrombocytes and erythrocytes are also adsorbed. Depending on the indication, this may not be necessary or even harmful to the patient. Thrombocyte adsorption is a special case. After activation, thrombocytes play a central role in blood clotting. So that during the extracorporeal circulation the blood does not clot, the coagulation must be counteracted in a medicinal way by means of the administration of heparin as an anticoagulant. If, despite the anticoagulant, coagulation occurs, the consequence is the clogging of the filter.

An additional drawback of conventional leukocyte filters is the often difficult handling before clinical application, for example in regard to ventilation. In extracorporeal circulation, air bubbles represent a potential risk for the patient, so it is very desirable to avoid them. The simpler the elimination of the air present in the filter, the better its handling and the safer its use.

Therefore, it is the mission of the present invention to make available a method for reducing blood leukocytes in which at least the drawbacks of the prior art are reduced, in which the blood is treated carefully, and in which there are alternatives for the targeted separation of different types of cells. Additionally, it is the mission of the present invention to make available a device for carrying out such a procedure.

This task is solved by a procedure to reduce the number of leukocytes in blood by treating the blood in a structure formed by multiple fibers based on organic polymers, where the multiplicity of fibers is contained in a housing with an input element and an outlet element, in which blood enters the carcass through the inlet element, then circulates through the carcass, flowing around the outer surfaces of the fibers and, finally, leaves the carcass through of the outlet element, and that is distinguished by the fact that fibers are used in which, when blood circulates around its outer surfaces, they determine the formation of a complement activating product C5a at a concentration of at least 10 porg per m2 of fiber surface, and why the fiber structure shows a high degree of organization, meaning a high degree of organization than a high number or fiber is arranged in a coordinated manner along its extension direction.

The invention also comprises a device for reducing the number of leukocytes in the blood, comprising a multiplicity of fibers based on organic polymers in a housing with an input element and an output element, wherein the fibers have at least one of its ends incorporated in a filling mass fixed to the inner wall of the housing, so that around the fibers an outer space is formed through which the blood circulates, and which is distinguished by the fact that the structure of the fibers shows a high degree of organization, it is understood by high degree of organization that a high number of fibers is arranged in a coordinated manner along their extension direction, and why organic polymer based fibers determine the formation of the complement activating product C5a at a concentration of at least 10 porg per m2 of fiber surface.

C5a is a dissociation product of the C5 plasma protein. The maximum value of the concentration of C5a in blood is therefore limited by the concentration of C5 in the blood plasma, where the concentration of C5 in plasma undergoes strong individual variations and can reach values of approximately 40 mg / l up to 150 mg / l Based on the ratio of the molar mass of C5 to C5a, a theoretical maximum concentration of C5a in blood of 9 mg / l is obtained.

The concentration of the complement activator product C5a in the blood plasma is calculated using a sandwich ELISA assay (enzyme coupled immunity detection) of the DRG Diagnostics Company, Marburg (Germany). After contact of the fibers with human donor blood (50 U / ml of heparin), 1.8 ml of blood is extracted at different times and stopped with 0.2 ml of 100 mM EDTA solution. Before the analysis, C5 is precipitated, according to the manufacturer's instructions (200 µl of plasma + 200 µl of precipitation reagent). In the determination they use 50 µl of the supernatant. The detection sensitivity of the assay is <0.02 g / l, the recovery rate of C5a in plasma is 86-114%, and the coefficient of variation is 5-8% (intra-assay) or 610% (between trials). The measured concentration of C5a depends on the volume of blood and the surface of the fibers. Therefore, for the calculation of the concentration of C5a referred to the outer surface of the fibers, the absolute content of C5a in the sample must be determined and expressed in relation to the outer surface of the fibers. In this case, a ratio of the volume of blood (V) to the surface of the fibers (A) (V / A) of 0.3 l / m2 should be observed. The calculation of the concentration of C5a referred to the surface of the fibers is carried out after a treatment period of 3 h, that is, the blood sample is circulated for 3 h along the outer surface of the fibers, maintaining a linear flow rate of 5 to 30 cm / min. Since the results of the measurement are subject to individual variations depending on the donor, the number of random tests N must be at least 2, indicating the average value of the random tests.

Without intending to adhere to any theory, it is assumed that in the treatment of inflammatory diseases an important significance is assigned to complement activation, and it is estimated that a reduction in the number of leukocytes, combined with complement activation, is essentially more effective than the decrease in the number of leukocytes alone. For this, the complement activation, determined by the concentration of C5a in blood, must be greater than the limit value according to the invention.

It is likely that the fact that certain leukocytes can be activated by C5a offers a relationship between the leukocyte number and C5a parameters. The activation by C5a and other factors determine that the cells develop a greater adhesiveness, consequently joining with greater intensity the surfaces that generate C5a.

Therefore, it is preferable that the fibers cause an additionally reinforced formation of the complement activator product C5a, at a concentration of at least 75 µl per m2 of fiber surface.

Especially preferably, the fibers determine a formation of the complement activator product C5a at a concentration of at least 100 µl per m2 of fiber surface.

To prevent the cells contained in the blood from being damaged, it is important that the blood does not penetrate significantly into the fibrous material, or that it does not circulate through this material. Therefore, it is preferable to use a fiber material and, consequently, fibers with an impermeable surface or, in the case of fibers with a porous surface, with a maximum pore size of 0.1 µm.

The fibers based on organic polymers can be multi-filament fibers, that is, fibers composed of multiple individual filaments, although preferably the fibers are mono-filament, that is, they are composed of a single filament.

Since the generation of C5a necessary for the process according to the invention depends not only on the polymer, but also on additions or on the degree of substitution of the polymer, the expression "based on organic polymers" comprises polymeric substances as such, substitutions, mixtures thereof, copolymers of these substances, as well as, possibly, adjuvants or additives added such as, for example, hydrophilizers.

By the term "high degree of organization" within the meaning of the present invention it should be understood that the fibers exhibit a similar arrangement or that, for the most part, they are arranged along their extension direction adjacently. In theory, a bundle of straight fibers, arranged parallel to each other, exhibits the highest degree of organization. In the sense of the present invention, a bundle of corrugated fibers also shows a high degree of organization, wherein the fibers of the fiber bundle exhibit the same extension direction in their entirety. Also in this case, and within the meaning of the present invention, a fiber bundle arranged in a loop is considered to exhibit a high degree of organization. In this situation, the arrangement of the fibers between them is similar. In the same way, by high degree of organization it should be understood that at least 30% of the fibers are arranged in parallel. Additionally, fibers arranged in multiple layers are included, wherein the fibers within a layer are arranged essentially parallel to each other. However, the parallel fibers of one layer can be crossed with the parallel fibers of another layer. These structures are described in EP 285 812. Structures according to the invention with a high degree of organization do not include felts or flat structures with randomly arranged fibers, in which the fibers are arranged in a completely disorganized manner and are mixed. each. In comparison with the felts, the structure according to the invention of the fibers with a high degree of organization exhibits a greater surface area and, in the application of the method according to the invention, a uniform thickness of the blood film. The high degree of organization is responsible for the blood circulating along the fibers showing comparatively reduced turbulence and that the cells contained in the blood are subjected to a minor shear overload. In addition, the high degree of organization greatly prevents the formation of dead spaces and preferred channels, also referred to as shunts. In this way, it is possible to treat the blood in a particularly careful way.

Likewise, the high degree of organization allows the reduction in the number of leukocytes not to be essentially caused by a sieving effect, as would be the case, for example, on a felt, but by adsorption effects, whereby a especially careful treatment of blood. In the effect of screening that is verified in a felt, an involuntary reduction of other components of the blood, for example, of thrombocytes, is mandatory.

Not only solid fibers are suitable for the process according to the invention or the device according to the invention, but hollow fibers can also be used. The hollow fibers have an inner and an outer surface. When a hollow fiber is used, the circulation, as in solid fibers, takes place along the outer surface. Before or after circulation around the outer surfaces of the hollow fibers, blood can also circulate through its light. However, it has been shown that the additional circulation around the light does not provide any advantage. Obviously, it is possible to use hollow fibers as hollow fibers with an impermeable or porous structure as hollow fibers. In this case, care should be taken that the process according to the invention is carried out in such a way that the blood does not essentially penetrate the hollow fibers or the hollow fiber membrane, but instead circulates around the membrane wall.

Preferably, the number of fibers is in the range of 2,000 to 20,000 fibers, especially preferably, in the range of 4,000 to 14,000 fibers. The outer diameter of the fibers should be between 0.05 mm and 2 mm, preferably between 0.1 mm and 2 mm and, especially preferably, between 0.2 mm and 1 mm.

Preferably, the arrangement of the fibers has a specific surface for the treatment of blood of between 0.1 and 100 cm2 of fiber surface per ml of blood to be treated, preferably between 0.5 and 20 cm2 of fiber surface per ml of blood to treat. The amount of blood to be treated is obtained from the duration of the blood treatment and the flow rate.

As organic polymer fibers, natural polymer fibers or synthetic polymers synthesized can be taken into account. Natural polymer fibers are, in particular, those based on cellulosic polymers that also comprise fibers subjected to so-called polymer-analogous reactions. Examples of these cellulose-based fibers are those of regenerated cellulose, cellulose acetate or modified cellulose such as, for example, with cellulose esters, cellulose ethers, cellulose modified with benzyl (benzylcellulose) groups, or celluloses modified with diethylaminoethyl, or mixtures of these cellulosic polymers. With cellulosic polymer-based fibers the process according to the invention achieves a strong reduction in the number of leukocytes, obtaining a particularly high reduction with regenerated cellulose fibers. In addition, chitin or chitosan-based fibers can also be used.

Among the organic polymers are also those polymers manufactured by synthetic routes. As synthetic polymer fibers, those consisting of polyolefins, polyamides, polyacrylonitrile, polycarbonates or polyesters, as well as the modifications, mixtures, combinations or copolymers of these polymers can be used. Preferably, fibers based on sulfonic polymers such as, for example, polysulfone or polyethersulfone are used. These polymers can be combined with additional polymers such as, for example, polyethylene oxide, polyhydroxy ether, polyethylene glycol, polyvinyl alcohol or polycaprolactone by way of additives. Additionally, the fibers may be provided with a coating with an additive. Preferably, these fibers contain a hydrophilizer, for example, polyvinylpyrrolidone or, also, hydrophilic modifications of these polymers.

Obviously, the procedure is not only suitable for reducing the number of leukocytes in whole blood, but also for reducing the number of residual leukocytes in the blood plasma or other blood concentrates. Accordingly, in the context of the present invention, the term "blood" comprises whole blood, blood plasma or a blood concentrate.

It has been shown that, with the fibrous materials mentioned, leukocytes are reduced first. In particular, the number of granulocytes and monocytes is reduced with cellulose fiber materials. With cellulosic materials, lymphocytes undergo a small reduction.

Therefore, in the context of the process according to the invention it is possible to reduce in a directed manner a certain class of cell types; for example, from the class of leukocytes, reduce monocytes and granulocytes, but not lymphocytes. In addition, cellulose-based fibrous materials are distinguished in that thrombocyte retention is performed to a limited extent.

In order to be able to ensure in a more secure way that all the fibers come into contact uniformly with the blood circulating along the fibers, they can be arranged with each other with a certain separation, for example by means of the so-called separation fibers. ("Spaceryarn"). The separation between fibers is especially advantageous to avoid the sifting effect. These separation fibers are especially convenient, since, through them, the existence of a regular space between the fibers arranged essentially parallel is guaranteed. Structures with this type of separation fibers are described, for example, in EP 732 141 or EP 285 812. Preferably, the separation fibers are composed of the same material as most parallel fibers. However, it is also possible to reduce the number of other types of cells present in the blood using separation fibers of a different material.

In certain applications, it may be convenient to specifically separate the thrombocytes from the blood. For these cases, polyethylene terephthalate (PET), polysulfone or polyethersulfone fibers are suitable. If a reduction of thrombocytes and leukocytes is desired, the process according to the invention can be carried out with a combination, for example, of cellulose fibers and PET fibers.

As described, a multiplicity of fibers based on organic polymers is preferably arranged in a housing with an input element and an output element. In this case, the fibers are normally arranged in such a way that at least one of its two ends is included in a filling mass fixed to the inner wall of the housing, so that an outer space is generated around the fibers by the Blood circulates For example, the fibers can have their ends included separately in filling masses, extending essentially linearly between these filling masses. Structures of this type are used, for example, in the mode usually referred to as "cross-flow" in hollow fiber modules and are present, among others, in commercially available dialysis modules. In modules of this type, the performance of the method according to the invention involves the circulation of blood through the outer space of the hollow fibers. However, the fibers also have only one of their ends, or have both ends included in the same filling mass, while circulation occurs around the other end, or free loop. These fiber structures are described, for example, in EP 732 142 or EP 371 189.

The degree of filling of the housing with the organic polymer fibers according to the invention should be between 10% and 70%, preferably between 30% and 60%. Since the fibers, depending on the fibrous material, can swell to a different extent upon coming into contact with the liquid, the determination of the degree of filling of the shell with fibers must be made with the fibers in a swollen state. In the case of strongly swellable fibers, as happens for example in those with cellulose as a base, it is possible to observe clear differences in the diameter of the fiber in a swollen and non-swollen state. Thus, depending on the swelling, different degrees of filling are achieved when the fibers are present in the dry state. On the contrary, fibers that do not swell, or that do so to a limited extent, such as, for example, those based on polysulfone, exhibit zero or few differences in determining the degree of filling in a swollen or non-swollen state.

The degree of filling of the carcass should be limited to the intervals previously mentioned in order, on the one hand, to make available a sufficiently large fiber surface and, on the other hand, to avoid the appearance of a sifting effect in the reduction of leukocytes during the performance of the process according to the invention.

The residence time of the blood in the housing should be at least 0.5 min, without exceeding a duration of 5 min. In case of a residence time greater than 5 min, the tendency of the blood to clot increases and the risk of coagulation increases despite the use of an anticoagulant. In case of residence times of less than 0.5 min, on the contrary, leukocyte adsorption is insufficient. Therefore, a residence time of the blood in the housing of 1 min to 3 min is preferable.

The linear flow rate of blood through the shell with multiple fibers based on organic polymers should be between 5 and 30 cm / min. Linear flow rate means the average speed with which blood circulates through the housing from the input element to the output element. In the calculation of the linear flow rate, the so-called free circulation cross section is included, which is composed of the surface of the cross sections inside the housing, deducting the sum of the surfaces of the cross sections of all the fibers in the housing In this context, attention should be paid to the fact that the outer diameter of the fiber must be taken into account in the calculation of the surface of the cross sections of a fiber; in the case of fibrous materials that swell in contact with blood, the outside diameter in a swollen state is decisive. Linear flow rates less than 5 cm / min favor thrombocyte aggregation and blood clotting. Flow rates greater than 30 cm / min make it difficult to adsorb the cellular components of the blood to the fiber surfaces.

As mentioned above, organic polymer based fibers have a high degree of organization in their structure. In a preferred embodiment of the process according to the invention or of the device according to the invention, the organic polymer based fibers are arranged in the form of essentially parallel fiber bundles. In a further preferred embodiment of the process according to the invention, the organic polymer based fibers are arranged in one or multiple layers, wherein the fibers, within a layer, show an essentially parallel arrangement.

The invention will be explained in more detail by the following examples and figures.

They show:

Figure 1: Reduction of the number of leukocytes in a blood sample by the method according to the invention, using hollow fibers of regenerated cellulose, as a function of time.

Figure 2: Reduction of the number of granulocytes, lymphocytes, monocytes and thrombocytes in a blood sample treated by the method according to the invention, using hollow fibers of regenerated cellulose, as a function of time.

Figure 3: Concentration of the complement activating product C5a in a blood sample treated by the process according to the invention, using hollow fibers of regenerated cellulose, as a function of time.

Figure 4: Reduction of the number of granulocytes, lymphocytes, monocytes and thrombocytes in a blood sample treated by the method according to the invention, using hollow regenerated cellulose fibers and additional use of PET fibers, as a function of time.

Figure 5: Reduction of the number of leukocytes in a blood sample treated by the method according to the invention, using hollow fibers of modified cellulose, as a function of time.

Figure 6: Concentration of the complement activating product C5a in a blood sample treated by the method according to the invention, using hollow fibers of modified cellulose, as a function of time.

Figure 7: Reduction of the number of leukocytes in a blood sample treated by the method according to the invention, using hollow polyethersulfone fibers, as a function of time.

Figure 8: Reduction of the number of leukocytes in a blood sample treated by the method according to the invention, using hollow polyethersulfone fibers and additional use of PET fibers, as a function of time.

Figure 9: Concentration of the complement activating product C5a in a blood sample treated by the method according to the invention, using hollow polyether sulfone fibers and additional use of PET fibers, as a function of time.

Example 1

Blood samples were treated by the method according to the invention, using hollow regenerated cellulose fibers and the number of leukocytes, the number of certain types of leukocytes, the number of thrombocytes, as well as the formation of the complement activating product C5a, were determined. function of time. Figure 1 shows the relative percentage reduction in white blood cell count (WBC) in a blood sample treated by the method according to the invention. For this, a set of hollow regenerated cellulose fibers was used, with an outer surface of 1.36 m2 and an inner surface of 1.27 m2. The set of hollow fibers, composed of 7400 fibers with a fiber diameter of 242 µm in swollen state, and a length of 260 mm, was arranged in a cylindrical housing with an internal diameter of 33 mm. The degree of filling of the housing was approx. 40% As a blood sample, 410 ml of human blood was used, which was circulated for a maximum period of 3 h, with a flow rate of 50 ml / min, corresponding to a linear flow rate of 10 cm / min. The residence time of the blood in the carcass was 2.5 min. The experimental results depicted in Figure 1 clearly demonstrate that the circulation of blood along the outer surface of the fibers determines a marked reduction in the number of leukocytes. Circulation around the interior only results in a small reduction in the number of cells of approx. 15% of the initial amount. The internal and external circulations do not provide any advantage over the exclusively external circulation. Nor is the internal circulation along 2 consecutive modules leading to a reduction in leukocytes equivalent to that which can be achieved with the external circulation, even though the surface of contact of the blood with the fibrous material, of 2.54 m2, is manifestly greater than with the external circulation (1.36 m2). From Figure 1 it follows that it is only possible to reduce the number of cells when blood circulates along the outside of the hollow fibers. The hollow fibers through which blood has circulated inside exhibit zero or poor cell adhesion. The same is true when the internal circulation is combined with the external circulation.

Figure 2 shows the extent to which the number of different types of leukocytes is reduced, and the effect that the method according to the invention has on the number of thrombocytes. Figure 2 shows the relative percentage reduction in the number of monocytes, granulocytes, lymphocytes and thrombocytes as a function of blood treatment time, calculated using hollow fibers of regenerated cellulose with an outer surface of 0.56 m2 and 240 ml of human blood. For this purpose, 5400 fibers were arranged with a diameter of fibers in a swollen state of 275 µm and a length of 120 mm in a cylindrical housing with an internal diameter of 38 mm. The degree of filling of the housing was approx. 29% The blood sample was circulated for a maximum period of 3 h, with a flow rate of 117 ml / min, corresponding to a linear flow rate of 14 cm / min. The residence time of the blood in the carcass was approx. 0.8 min. After 3 h, a separation of monocytes and granulocytes greater than 95% was verified, although less for lymphocytes and thrombocytes, which experienced only a reduction of 20% and 15%, respectively. Therefore, with the use of regenerated cellulose, monocytes and granulocytes can be separated from the blood.

Figure 3 shows the concentration of complement activator product C5a in a blood sample treated by the method according to the invention. Experiments were calculated under the same conditions as indicated in Figure 1. After 3 hours of blood treatment, an external circulation was calculated.

C5a content in the treated sample of 403 /g / l, which corresponds to a formation of C5a of 165 g in the treated blood sample, or 121 g of C5a per m2 of fiber surface.

Example 2

A hollow fiber module similar to that described in Example 1, Figure 2 was used, although the hollow fiber bundle additionally contained polyethylene terephthalate (PET) fibers. Figure 4 shows the relative percentage reduction, as a function of time, of the number of monocytes, granulocytes, lymphocytes and thrombocytes with the additional use of polyethylene terephthalate fibers. The experimental conditions correspond, moreover, with those described in Example 1 with respect to Figure 2. By the additional use of polyethylene terephthalate fibers a much faster separation of monocytes and granulocytes is achieved, compared to exclusive use of regenerated cellulose fibers. After 15 minutes, more than 95% of monocytes and 75% of granulocytes had been separated from the sample. The lymphocyte separation in the sample was approx. 60% and that of thrombocytes reached approx. 75%

Example 3

Using commercially modified hollow cellulose fiber membranes with benzyl (benzylcellulose) groups, blood samples were treated by the process according to the invention and both the number of leukocytes and the formation of the complement activating product C5a were determined as a function weather. Figure 5 shows the relative percentage reduction in the number of leukocytes (WBC - see above) as a function of time with respect to the initial amount in a blood sample treated by the method according to the invention. In this case, hollow fibers with an inner surface of 1.36 m2 and an outer surface of 1.47 m2 are used. The treated blood sample consisted of 440 ml of human blood. For this, a set of 7900 fiber fibers was used, with a fiber diameter of 246 mm in swollen state and a length of 240 mm, included in a cylindrical housing with an internal diameter of 34 mm. The degree of filling of the housing was approx. 41% The blood sample was circulated for a maximum period of 3 h, with a flow rate of 50 ml / min, corresponding to a linear flow rate of 9.4 cm / min. The residence time of the blood in the carcass was approx. 2.5 min. Similarly to the experiments depicted in Figure 1, it is evident that a marked reduction in leukocytes occurs only with circulation along the outer surface. Even when the inner surface has a size similar to that of the outer surface, the circulation through the interior only leads to a small reduction of approx. 15% of the initial amount. The internal and external circulations do not suppose any advantage with respect to the exclusively external circulation. Circulation along the outer surface, parallel to the fibers, determines a reduction in leukocytes contained in the blood sample of 80%

The formation of the complement activator product C5a was analyzed under the same experimental conditions and with the same hollow fiber membrane as indicated in Figure 5. The results are represented in Figure 6. Similarly to Figure 5, it is It also observes in this case a clear difference between internal and external circulation. Although the contact surfaces are practically the same size, the formation of C5a with the external circulation is approx. 2.5 times greater than with the internal circulation. After 3 h of external circulation, a C5a concentration of 365 g / l, corresponding to 161 g in the blood sample, or 109 g of C5a per m2 of fiber surface is observed in the blood sample.

Example 4

As shown in Figure 7, it is also possible to reduce leukocytes using hollow polyethersulfone fibers. In this case, hollow fibers with an outer diameter of 300 µm, an effective length of 275 mm, an inner surface of 1.6 m2 and an outer surface of 2.2 m2 were used in a module with an inner diameter of 38.5 mm coated with 8500 fibers, that is, with a filling degree of approx. 52% 381 ml of human blood was circulated through the module, with a flow rate of 50 ml / min. The linear flow rate was approx. 9 cm / min and the residence time, 3.1 min. It is also shown here that hollow polyethersulfone (PES) fibers with external circulation produce a reduction in the number of leukocytes of approximately 70%. Under the conditions required to calculate the content of C5a in relation to the outer surface of the fibers, a formation of C5a based on the surface area of 30 g / m2 was determined for the hollow polyether sulfone fibers.

Example 5

In Example 5, in addition to hollow polyethersulfone fibers, PET fibers are used to carry out the process according to the invention. Figure 8 shows the relative percentage reduction of leukocytes (WBC - see above) in a blood sample treated by the method according to the invention. For this purpose, hollow polyethersulfone (PES) fibers combined with polyethylene terephthalate (PET) fibers were used, with an inner surface of 1.31 m2 and an outer surface of 1.57 m2. In a cylindrical housing with an inner diameter of 34 mm, 8000 fibers with a fiber diameter of 260 mm and a length of 240 mm were arranged. The degree of filling of the housing was approx. 47% As a blood sample, 470 ml of human blood was used, which was circulated for a maximum period of 3 h with a flow rate of 50 ml / min, corresponding to a linear flow rate of 10 cm / min. The residence time of the blood in the carcass was 2.3 min. In this case, it is shown that the hollow polyether sulfone fibers, combined with polyethylene terephthalate fibers, produce, with the external circulation, a reduction in the number of leukocytes of approximately 50%. The internal circulation only results in a small reduction of approx. 18% of the initial amount.

Similarly to the results represented in Figure 8, depending on the circulation there are also large differences in relation to the formation of C5a. The results shown in Figure 9 are calculated

5 rum with the same membrane as described in Figure 8, and under the same conditions required for the calculation of the C5a content with respect to the fiber surface. With the internal circulation, a C5a concentration of only 30 g / ml was determined and, with the external circulation, a C5a concentration of 192 g / ml was obtained. This last value corresponds to a generation of C5a of 90 eng in the treated blood sample, or 58 porg per m2 of fiber surface.

10

Claims (15)

one.

 Procedure for reducing the number of leukocytes in blood by means of an in vitro treatment of blood in a structure consisting of a multiplicity of fibers based on organic polymers, wherein this multiplicity of fibers is contained in a housing with an input element and an outlet element, in which blood is introduced into the carcass through the inlet element, then circulates through the carcass, the blood circulating along the outer surfaces of the fibers and, finally, leaves the carcass at through the outlet element, characterized in that fibers are used which, with the circulation of blood along its outer surface, determine a formation of the complement activating product C5a at a concentration of at least 10 µm per m2 of fiber surface , and because the arrangement of the fibers exhibits a high degree of organization, it being understood by a high degree of organization that a large part of the fibers are arranged adjacently along their direction of extension.

2.

 Method according to claim 1, characterized in that the fibers determine a blood formation of the complement activating product C5a at a concentration of at least 75 µg per m2 of fiber surface.

3.

 Method according to claim 1 or 2, characterized in that the fibers based on organic polymers are hollow fibers, and that the blood circulates along the outer surface of the hollow fibers.

Four.

 Process according to one or more of claims 1 to 3, characterized in that the number of fibers based on organic polymers is in the range of 2,000 to 20,000 fibers.

5.

 Process according to one or more of claims 1 to 4, characterized in that the fiber diameter of the fibers based on organic polymers is between 0.05 mm and 2 mm.

6.

 Method according to one or more of claims 1 to 5, characterized in that the specific surface area of the organic polymer based fiber structure is between 0.1 and 100 cm2 per ml of blood to be treated.

7.

 Process according to one or more of claims 1 to 6, characterized in that the fibers based on organic polymers are composed of regenerated cellulose, cellulose acetate or cellulose modified with benzyl groups (benzyl cellulose).

8.

 Process according to claim 1, characterized in that the fibers based on organic polymers are essentially composed of polyethersulfone or polysulfone.

9.

 Method according to claim 7 or 8, characterized in that the structure of the multiplicity of fibers additionally also contains polyethylene terephthalate fibers.

10.

 Method according to one or more of claims 1 to 9, characterized in that the degree of filling of the housing with fibers is within the range of 10% to 70%.

eleven.

 Method according to one or more of claims 1 to 10, characterized in that the residence time of the blood in the housing is 0.5 min to 5 min.

12.

 Method according to one or more of claims 1 to 11, characterized in that the linear flow rate with which the blood circulates through the housing is between 5 and 30 cm / min.

13.

 Process according to one or more of claims 1 to 12, characterized in that the fibers based on organic polymers are present in the form of fiber bundles with essentially parallel fibers.

14.

 Process according to one or more of claims 1 to 13, characterized in that the fibers based on organic polymers are arranged in one or multiple layers, and that the fibers within a layer are arranged essentially parallel.

fifteen.

 Device for the reduction of blood leukocytes, comprising a multiplicity of fibers based on organic polymers in a housing with an input element and an output element, wherein the fibers have at least one of their ends included in a mass padding attached to the inner face of the housing, so that around the fibers an outer space is formed through which the blood circulates, characterized in that the arrangement of the fibers exhibits a high degree of organization, where by degree high organization is understood that a large part of the fibers are arranged adjacently along their direction of extension, and that the organic polymer-based fibers determine a formation of the complement activating product C5a at a concentration of at minus 10 porg per m2 of fiber surface.