Method for the selective removal of leukocytes and erythrocytes from a platelet concentrate product

Abstract

Method for the selective removal of leukocytes and erythrocytes from a platelet concentrate. The invention relates to a method for selectively removing leukocytes and erythrocytes from a platelet concentrate, said method comprising passing a platelet concentrate reduced to leukocytes through a porous erythrocyte barrier element, said porous erythrocyte barrier element comprising at least one porous membrane made of a hydrophilic material, the pores of said membrane having a nominal size of between 1 µm and 4 µm, and an absolute pore size of between 2 µm and 5 µm. Figure for the abstract: Fig. 3

Description

Title of the invention: Method for the selective removal of leukocytes and erythrocytes from a platelet concentrate product

[0001] The invention relates to a method for the selective removal of leukocytes and erythrocytes from a platelet-concentrated blood product, as well as a filtration unit and bag systems for implementing said method.

[0002] It typically applies to the filtration of blood or a blood component containing platelets such as platelet-rich plasma (PRP), a single platelet concentrate (SPC) or a platelet concentrate assembly (PC).

[0003] Whole blood consists of two types of components: blood cells including red blood cells, leukocytes and platelets, and plasma, a pale yellow liquid in which the blood cells are suspended.

[0004] Currently, in the vast majority of cases, patients are only transfused with the blood components necessary for their condition. For example, only platelet concentrates are transfused to patients with thrombocytopenia, that is, those whose platelet count in the blood is reduced.

[0005] There are several methods for preparing platelet concentrates from whole blood, among which we can mention the method of preparation from leukocyte-platelet layers (European Directorate for the Quality of Medicines. Healthcare (EDQM), Council of Europe: Guide to the preparation, use and quality assurance of blood components, (2019)).

[0006] According to this preparation method, a unit of whole blood is centrifuged to separate the blood into three layers: a plasma supernatant layer, an intermediate buffy coat layer comprising a mixture of leukocytes, platelets, red blood cells, and plasma, and a red blood cell pellet layer. The buffy coat layer is separated from the other layers. Between four and eight of these separated buffy coat layers are then recombined and resuspended in plasma or in a platelet additive solution. This mixture is centrifuged again (gentle centrifugation) to obtain a platelet concentrate supernatant layer and a red blood cell pellet layer. The platelet concentrate is then separated and stored for up to 4 to 7 days at room temperature. This platelet concentrate still contains residual leukocytes and red blood cells.

[0007] However, leukocytes have very significant adverse effects, which has led to efforts to eliminate them from blood components intended for transfusion. Indeed, leukocytes increase the risk of immune rejection such as graft-versus-host disease and promote the transmission of infectious agents. It has also been demonstrated that leukocytes negatively affected platelet preservation.

[0008] On the other hand, residual red blood cells and platelet-derived microparticles present in platelet concentrates lead to a risk of alloimmunization of red blood cells, i.e. the production of anti-erythrocyte antibodies in the transfused (Moncharmont P. et al. "Red blood cell alloimmu-nisation after platelet transfusion: a 5-year study." Blood Transfusion 12.Suppl 1 (2014): sl47).

[0009] Reckhaus et al. found that this risk of alloimmunization is greater with platelet concentrates prepared from an assembly of buffy-coat platelets (BC-PC) than with platelet concentrates obtained by apheresis (AP-PC), the latter containing on average fewer residual red blood cells. Indeed, BC-PC platelet concentrates contain on average 0.302 x 10⁹ red blood cells / L while AP-PC platelet concentrates contain 0.014 x 10⁹ / L (Reckhaus, Johanna, et al. "Platelet transfusion induces alloimmunization to D and non-D Rhesus antigens." Transfusion medicine and hemotherapy 45.3 (2018): 167-172.)

[0010] To remove leukocytes from platelet concentrates intended for transfusion, filtration units containing a leukoreduction medium are already known. In such units, the leukoreduction medium comprises one or more membrane(s) and / or one or more non-woven layers made of a polymer material and treated so as to improve the leukoreduction rate and / or platelet recovery.

[0011] For example, document EP 2 783 717 Al describes a filter for selectively removing leukocytes from a fluid containing platelets, said filter containing a non-woven fabric coated with a polymer obtained from a methyl methacrylate monomer and a poly(ethylene oxide) methacrylate macromonomer.

[0012] In document WO 2022 / 136138 A1, the filter for selectively removing leukocytes from a platelet-containing fluid comprises a non-woven fabric impregnated with a copolymer of 2-methoxyethyl methacrylate and a hydrophobic momomer such as methyl methacrylate or vinyl acetate.

[0013] In another example, US patent 5,152,905 describes a filter for removing leukocytes from platelet-rich plasma (PRP) prepared from a whole blood donation. The filter comprises poly(butylene terephthalate) (PBT) fibers grafted with a monomer of hydroxyethyl methacrylate (HEMA) and methacrylic acid (MAA). Alternatively, the filter includes upstream a higher-density porous medium to block red blood cells. This porous medium acts as a valve by blocking the flow of PRP as soon as red blood cells come into contact with it and aggregate on its surface. This patent indicates that the retention The presence of red blood cells is not due to the size of the pores in the porous medium nor to mechanical filtration.

[0014] US patent 5,234,593 describes another type of filter for selectively removing leukocytes from a platelet concentrate using a porous body having a continuous, three-dimensional reticular texture, containing continuous open pores having an average diameter of 6 to 12 µm. More specifically, the porous body is a polyurethane foam in which more than 60% of the pores have a diameter greater than or equal to 4 µm.

[0015] These last two filters, marketed under the names Autostop (Haemonetics) and Imugard (Terumo BCT), do not allow a concentration of residual red blood cells to be reached as low as in an apheresis platelet concentrate (Van der Meer P. et al. "Leucoreduced platelet concentrates in additive solution: an evaluation of filters and storage containers." Vox sanguinis 81.2 (2001): 102-107).

[0016] Indeed, red blood cells, although approximately 5 to 9 µm in size, are deformable and able to pass through pores smaller than 1 µm. Platelets, on the other hand, are approximately 2 to 4 µm in size, and leukocytes are approximately 6 to 19 µm in size.

[0017] The invention proposes a process for obtaining a platelet-concentrated product reduced in leukocytes and containing in particular less than 80 erythrocytes per pL, more particularly less than 15 erythrocytes per pL.

[0018] According to a first aspect, the invention proposes a method for selectively removing leukocytes and erythrocytes from a blood platelet concentrate product, said method comprising passing a blood platelet concentrate product reduced in leukocytes through a porous erythrocyte barrier element, said porous erythrocyte barrier element comprising at least one porous membrane made of a hydrophilic material, the pores of said membrane having a nominal size between 1 pm and 4 pm, and an absolute pore size between 2 pm and 5 pm.

[0019] According to a second aspect, the invention relates to a filtration unit for implementing the process of the invention comprising an outer casing provided with at least one inlet orifice and at least one outlet orifice, the casing containing from upstream to downstream: - a porous element for leukocyte adsorption and / or filtration, said porous leukocyte deleukocyte element comprising at least one layer whose pores have an average diameter between 3 and 15 pm, and - a porous barrier element to erythrocytes comprising at least one porous membrane made of a hydrophilic material, the pores of said membrane having a nominal size between 1 pm and 4 pm, and an absolute pore size between between 2 pm and 5 pm.

[0020] According to a third aspect, the invention relates to a pouch system for the selective removal of leukocytes and erythrocytes from a platelet concentrate product, comprising: - a filtration unit according to the second aspect of the invention, and - a filtrate collection bag, said bag being connected, via a tube, to an outlet port of the filtration unit.

[0021] A fourth aspect of the invention relates to a pocket system for implementing the method according to the invention, comprising: - a first filtration unit for the selective removal of leukocytes, said first filtration unit comprising a porous element for leukocyte deleukoreduction by adsorption and / or filtration, said porous deleukoreduction element comprising at least one layer whose pores have an average diameter between 3 and 15 pm, - a first filtrate collection bag, said first bag being connected, via a tube, to an outlet orifice of said first filtration unit, - a second filtration unit for the selective removal of erythrocytes, said second filtration unit comprising a porous erythrocyte barrier element, said porous erythrocyte barrier element comprising at least one porous membrane made of a hydrophilic material, the pores of said membrane having a nominal size of between 1 µm and 4 µm, and an absolute pore size of between 2 µm and 5 µm, said second filtration unit being further connected, or intended to be connected, via tubing, to an outlet of said first filtrate collection bag, and - a second filtrate collection bag, said second collection bag being connected, via a tube, to an outlet port of said second filtration unit.

[0022] The attached drawings illustrate the invention:

[0023] [Fig-1] represents a schematic view of a filtration unit according to the invention.

[0024] [Fig.2] represents a schematic cross-sectional view of a filtration unit of the [Fig.l].

[0025] [Fig.3] represents a schematic view of a pocket system for implementing the process for removing leukocytes and erythrocytes from a platelet-concentrated product.

[0026] [Fig.4] represents a schematic view of another pocket system for putting in works the process to remove leukocytes and erythrocytes from a platelet-concentrated product.

[0027] According to a first aspect, the invention proposes a method for selectively removing leukocytes and erythrocytes from a product concentrated in blood platelets.

[0028] A platelet-rich plasma product is a product whose platelet concentration is higher than the normal platelet concentration in blood. A platelet-rich plasma product is typically obtained by fractionating whole blood or by performing apheresis.

[0029] A platelet-concentrated product is, for example, platelet-rich plasma (PRP), a unit platelet concentrate (UPC) prepared from PRP or a buffy coat, or an assembly of platelet concentrates (PC).

[0030] In the case of an assembly of platelet concentrates, this is prepared in particular by the method of preparation from leukocyte-platelet layers, using an additive solution for platelets.

[0031] When using a platelet additive solution, the final platelet concentrate generally consists of platelets resuspended in a mixture of plasma (20-40%) and additive solution (60-80%).

[0032] According to this preparation method, the platelet concentrate includes leukocytes but also residual erythrocytes which represent an immunological risk for the transfused patient.

[0033] The process of the invention makes it possible to selectively separate these residual leukocytes and erythrocytes from the blood platelets.

[0034] In particular, in the case of a platelet concentrate derived from leukocyte-platelet layers, the process makes it possible to obtain a number of leukocytes in the platelet concentrate of less than 1.106, a number of platelets of at least 2.1011 and a concentration of residual erythrocytes of less than 80 erythrocytes per pL.

[0035] The process of the invention comprises the passage of a product concentrated in blood platelets and reduced in leukocytes through a porous erythrocyte barrier element, said porous erythrocyte barrier element comprising at least one porous membrane made of a hydrophilic material, the pores of said membrane having a nominal size between 1 pm and 4 pm, and an absolute pore size between 2 pm and 5 pm.

[0036] The passage of the product concentrated in blood platelets and reduced in leukocytes through a porous barrier element to erythrocytes is carried out by gravity or more particularly under pressure, for example using a press.

[0037] The porous barrier element to erythrocytes is intended to retain residual erythrocytes from a platelet-concentrated product previously reduced to leukocytes while allowing the platelets to pass through.

[0038] The porous barrier element to erythrocytes comprises a hydrophilic membrane. In one embodiment, the membrane is made of a naturally hydrophilic material such as polyester, polyester sulfone, cellulose acetate, or nylon. Alternatively, the membrane is made of a hydrophobic material such as polycarbonate, rendered hydrophilic by chemical coating.

[0039] In particular, the membrane is coated with a biocompatible polymer.

[0040] By biocompatible polymer is meant a polymer compatible with blood or blood components, preventing or reducing the adsorption of proteins and platelets onto the membrane coated with such a polymer.

[0041] The biocompatible polymer consists of at least one unit from a biocompatible monomer.

[0042] According to one embodiment, the biocompatible monomer is chosen from the list consisting of the monomers of N-vinylpyrrolidone, 2-methoxyethyl acrylate, 2-methacryloyloxyethylphosphorylcholine, 2-hydroxyethyl methacrylate, ethylene glycol and mixtures of these monomers.

[0043] According to one variant, the biocompatible polymer consists of at least one unit from a biocompatible monomer and at least one unit from a hydrophobic monomer.

[0044] According to one embodiment, the hydrophobic monomer is chosen from the group consisting of an alkyl acrylate, an alkyl methacrylate, and a vinyl alkylate. For example, this hydrophobic monomer is methyl methacrylate or vinyl acetate.

[0045] This hydrophobic monomer contributes to maintaining the biocompatible polymer on the surface of the membrane.

[0046] Particular examples of biocompatible polymers are polymers consisting of a 2-methoxyethyl acrylate monomer unit and a methyl methacrylate or vinyl acetate monomer unit; a poly(ethylene oxide) methacrylate monomer unit and a methyl methacrylate monomer unit; a 2-hydroxyethyl methacrylate monomer unit and a diethylaminoethyl methacrylate monomer unit; or a vinyl pyrrolidone monomer unit and a vinyl acetate monomer unit.

[0047] According to one embodiment, the membrane is a naturally hydrophilic membrane coated with a biocompatible polymer. More particularly, the membrane is a polyester membrane coated with a biocompatible polymer consisting of a 2-methoxyethyl acrylate monomer unit and a methyl methacrylate monomer unit.

[0048] The membrane forming a porous barrier element to erythrocytes has a nominal pore size between 1 pm and 4 pm.

[0049] Nominal pore size means the predominant pore size of a membrane. The membrane comprises pores larger or smaller than this size. nominal, but the number of pores having the nominal pore size is greater than the number of pores having another size.

[0050] A membrane with a nominal pore size greater than 4 pm does not retain a sufficient number of residual erythrocytes from a platelet concentrate. A membrane with a nominal pore size less than 1 pm does not allow for sufficient platelet recovery.

[0051] The membrane also has an absolute pore size of between 2 pm and 5 pm.

[0052] By absolute pore size, we mean the size of the smallest particles completely retained by the membrane. The absolute pore size of a membrane corresponds to the maximum pore size of a membrane.

[0053] A membrane with an absolute pore size of less than 2 pm does not allow sufficient platelets to pass through. A membrane with an absolute pore size greater than 5 pm does not sufficiently retain erythrocytes.

[0054] Such a membrane is manufactured in particular by a phase inversion process, a drawing process, or an ion bombardment process followed by etching (track-etching). The track-etching process is advantageous in that it allows for a high pore density and a uniform pore diameter.

[0055] For example, the pore density of the membrane is between 4.105 and 2.107 pores / cm2, in particular about 2.106 pores / cm2.

[0056] In particular, the thickness of the membrane is between 9 and 12 pm.

[0057] For example, the membrane has a nominal pore size of 2 pm and a size absolute of 2.5 pm.

[0058] In another example, the membrane has a nominal pore size of 3 pm and an absolute size of 4.2 pm.

[0059] According to a particular embodiment, the porous barrier element to erythrocytes further comprises a pre-filter and / or post-filter which are made in the form of at least one layer of non-woven fabric and which are arranged respectively on the upstream and downstream sides of the porous membrane.

[0060] These pre-filters or post-filters have in particular an average pore size greater than that of the membrane, for example between 25 and 50 pm.

[0061] The pre-filters and post-filters are advantageously impregnated with a bio-polymer compatible facilitating the passage of the pads as described above.

[0062] According to another embodiment, the erythrocyte barrier porous element comprises several porous membranes made of a hydrophilic material having the same porosity or decreasing porosity from upstream to downstream of the erythrocyte barrier porous element, in the direction of filtration. The nominal size of these membranes is between 2 and 7 µm, for example 2 µm, 3 µm and 5 µm.

[0063] Advantageously, in order to prevent clogging of such a stack of membranes, the porous barrier element to the erythrocytes comprises intercalated layers with a porosity greater than that of the membranes. For example, these intercalated layers are non-woven layers having an average porosity of between 3 and 15 pm, in particular between 7 and 10 pm.

[0064] These interlayers are advantageously impregnated with a biocompatible polymer that facilitates the passage of platelets as described above. The biocompatible polymer may or may not be the same as that used to impregnate the porous membrane.

[0065] According to the process of the invention, the platelet-concentrated product is reduced to leukocytes before passing through the porous barrier element to the erythrocytes. The reduction to leukocytes prevents clogging of the porous membrane forming the barrier to the erythrocytes.

[0066] Thus, the process for selectively removing leukocytes and erythrocytes from a blood platelet concentrate product includes prior passage of the platelet concentrate product through a porous leukoreduction element by adsorption and / or filtration of leukocytes, so as to obtain a platelet concentrate product reduced in leukocytes.

[0067] The passage of the concentrated blood platelet product through a porous leukoreduction element is carried out by gravity or under pressure.

[0068] Leukocytes are retained by the porous leukoreduction element by one or both of the following mechanisms. The first mechanism is the adsorption of leukocytes onto the surface of the porous leukoreduction element. Leukocytes are adsorbed onto cationic and moderately hydrophobic surfaces. The quality of adsorption also depends on the available contact surface area for leukocyte adsorption. The second mechanism is sieving and depends primarily on the diameter of the pores in the porous leukoreduction element.

[0069] The porous leukoreducing element allows selective leukoreducing of a platelet-concentrated product, i.e. it is able to retain leukocytes while allowing platelets to pass through.

[0070] In particular, when the fluid to be filtered is a platelet concentrate derived from buffy coats, the porous leukoreducing element is capable of obtaining a leukocyte count in the filtered platelet concentrate that is less than 1 x 10⁶ and of allowing at least 70% of the blood platelets to pass through. The platelet count in the platelet concentrate after filtration is, in particular, at least 2 x 10¹¹ platelets.

[0071] The porous leukoremoval element comprising at least one porous layer whose pores have an average diameter of between 3 and 15 pm, in particular between 7 and 10 pm. This pore size allows leukocytes to be retained by mechanical filtration.

[0072] According to one embodiment, the porous leukoreduction element comprises one or more layers of non-woven fabric. In particular, the porous leukoreduction element comprises between 5 and 40 layers of non-woven fabric, and more particularly between 15 and 25 layers of non-woven fabric.

[0073] For example, the diameter of the fibers of the non-woven layer(s) is between 0.3 and 7 pm, with an average between 1 and 3 pm.

[0074] In particular, the porous leukoreducing element comprises polyester fibers, for example polybutylene terephthalate or polyethylene terephthalate fibers.

[0075] Advantageously, the porous leukoreducing element is impregnated with a biocompatible polymer as described above, preventing platelet adhesion to its surface. The biocompatible polymer may or may not be the same as that used to impregnate the erythrocyte barrier porous element.

[0076] According to a particular embodiment, the porous leukoreduction element further comprises a pre-filter and / or post-filter which are made in the form of at least one layer of non-woven material and which are arranged respectively on the upstream and downstream side of the porous layer(s).

[0077] These pre-filters or post-filters have in particular an average pore size greater than that of the porous layer(s), for example between 25 and 50 pm.

[0078] The pre-filters and post-filters are impregnated with a biocompatible polymer facilitating the passage of platelets as described above.

[0079] According to a particular embodiment, the platelet-concentrated product is further reduced in anti-A and / or anti-B antibodies.

[0080] Indeed, since platelet concentrates contain plasma, some transfusion centers apply the same ABO compatibility rules for platelet concentrate transfusions as for plasma transfusions (minor compatibility). Thus, AB plasma can be used regardless of the recipient's blood type, and A or B plasma can be used for a recipient with blood type O. This rule helps prevent post-transfusion hemolysis, that is, hemolysis of the recipient's red blood cells by the hemolytic anti-A and anti-B antibodies present in the donor's plasma.

[0081] However, it is not uncommon to transfuse ABO-incompatible platelet concentrates in emergencies or when ABO-compatible platelet concentrates are unavailable. In such cases, platelet concentrates with low anti-A and / or anti-B antibody titers are selected.

[0082] Reducing anti-A and / or anti-B antibodies in platelet concentrates not only reduces the risk of post-transfusion hemolysis in the event of ABO incompatible transfusion but also facilitates the inventory of platelet concentrates in blood banks and hospitals.

[0083] To reduce the titration of anti-A and / or anti-B antibodies, the process includes, before the passage of the platelet concentrate product through the porous barrier element to the erythrocytes, bringing a blood platelet concentrate product into contact with a set of particles capable of binding the anti-A and / or anti-B antibodies to said platelet concentrate product, so as to obtain a mixture of a platelet concentrate product reduced in anti-A and / or anti-B antibodies and particles bound or not to anti-A and / or anti-B antibodies.

[0084] The contact of the platelet-concentrated product with all the particles is carried out before or after the elimination of leukocytes by passing through the porous leukocytation element, but before passing through the porous element forming barriers to the erythrocytes.

[0085] Particles capable of binding anti-A and / or anti-B antibodies include those described in document WO 2021 / 144380.

[0086] In particular, these particles are grafted with oligosaccharides, such as oligosaccharides capable of binding antibodies such as anti-A and / or anti-B antibodies. For example, these particles are ultrafine cellulose particles grafted with an antigenic oligosaccharide of group A or B, such as those described in document WO2016 / 177967. In another example, the particles are those described in document EP3141558A1 or the IsoClear beads marketed by Prometic Bioseparations.

[0087] For example, the particles have an average size greater than 15 pm with a size distribution ranging from less than 2 pm up to about 100 pm.

[0088] The platelet concentrate reduced in anti-A and / or anti-B antibodies is then passed through the erythrocyte barrier porous element or through the leukoreduction porous element and then the erythrocyte barrier porous element in order to separate the particles from the platelet concentrate reduced in anti-A and / or anti-B antibodies. The erythrocyte barrier porous element then forms a particle barrier porous element since it allows the retention of particles bound or unbound to the anti-A and / or anti-B antibodies of the platelet concentrate reduced in anti-A and / or anti-B antibodies.

[0089] In a particular embodiment, to ensure that the set of particles does not include any particles smaller than the pores of the porous membrane forming a barrier to erythrocytes and / or particles, before contact with the platelet concentrate, the set of particles is passed through a sieve having a mesh size larger than the pores of the porous membrane. For example, the sieve has a mesh size of 5 µm, 10 µm, or 20 µm.

[0090] According to another aspect and in connection with Figures 1 and 2, the invention relates to a unit filtration unit 1 for implementing the process for selectively removing leukocytes and erythrocytes from a blood platelet concentrate product, said filtration unit 1 comprising an outer casing having at least one inlet orifice 2 and at least one outlet orifice 3, the casing containing from upstream to downstream: - a porous leukoreduction element 4 by adsorption and / or filtration of leukocytes, said porous leukoreduction element comprising at least one layer whose pores have an average diameter between 3 and 15 pm, and - a porous barrier element to erythrocytes 5 comprising at least one porous membrane made of a hydrophilic material, the pores of said membrane having a nominal size between 1 pm and 4 pm, and an absolute pore size between 2 pm and 5 pm.

[0091] The terms "upstream" and "downstream" are defined with respect to the direction of flow of the concentrated product in platelets in the filtration unit.

[0092] Each of the porous leukoreduction and erythrocyte barrier elements includes those described above.

[0093] The leukoreduction porous element is positioned upstream of the erythrocyte barrier porous element. Thus, the platelet concentrate is first reduced to leukocytes, then to erythrocytes. This arrangement reduces the risk of clogging of the membrane forming the erythrocyte barrier porous element.

[0094] A 6-pocket system for the selective removal of leukocytes and erythrocytes from a platelet-concentrated product is now described in connection with [Fig.3].

[0095] The bag system 6 comprises a filtration unit 1 and a filtrate collection bag 7, said bag 7 being connected, via a first tube 8, to an outlet port 3 of the filtration unit 1.

[0096] The filtration unit 1 comprises, upstream to downstream, a porous element 4 for leukoreduction by adsorption and / or filtration of leukocytes, and a porous element 5 barrier to erythrocytes as described above.

[0097] Alternatively, the filtration unit comprises only one porous element 5 barrier to erythrocytes.

[0098] The outer casing of the filtration unit is flexible, rigid or semi-rigid.

[0099] The bag system 6 further includes an assembly bag 9 connected or intended to be connected via a second tube 10 to the filtration unit 1.

[0100] For the preparation of an assembly of leukocyte-platelet layers, the assembly pocket 9 is in fluidic communication with a set of tubing in an "octopus" configuration, said set of tubing being made up of a tubing main 11 and secondary tubing 12 connected to the main tubing. These secondary tubing 12 are intended to be connected in a sterile manner to at least four bags containing a buffy coat and possibly to one bag containing an additive solution for preserving platelets or plasma.

[0101] In a particular embodiment, the assembly pouch 9 comprises a set of particles 17 capable of binding anti-A and / or anti-B antibodies to said platelet concentrate. These particles are advantageously passed through a sieve before being arranged in the assembly pouch.

[0102] According to another particular embodiment, the filtrate collection bag 7 is in fluidic communication via a third tube 13 to a satellite bag 14 intended to receive the air present in the filtrate collection bag 7 and / or to collect a sample of the fluid contained in the filtrate collection bag 7.

[0103] In order to take samples of the fluid contained in the bag 7, the bag system includes a fourth tube 15 connected to the third tube 13, said fourth tube 15 being provided at its end with a sampling means 16. The sample taken by this means is used for the detection of bacterial contamination (Bact / Alert® system for example).

[0104] The tubing is provided with a clamp to control the flow of fluids inside the bag system.

[0105] When the filtration unit includes a flexible casing, the filtration unit 1 swells during filtration. To avoid this swelling, the filtration unit is advantageously placed in a rigid case (not shown).

[0106] In relation to the "octopus" type pocket system of [Fig.3], a particular use of the pocket system 6 is now described for the preparation of a platelet concentrate reduced in leukocytes and erythrocytes.

[0107] Between four and eight bags containing a leukocyte-platelet layer are connected asterilely, for example using a sterile SCD type connection device (Terumo) to the secondary tubing 12 of a bag system 6. A bag of SSP+ type preservation solution (Macopharma, France) is also connected asterilely to one of the secondary tubing 12.

[0108] The buffy coats flow into the assembly bag 9. The bags that contained said buffy coats are then rinsed with the preservation solution. The remaining preservation solution is then transferred to the assembly bag 9. The main and secondary tubing assembly 11, 12 is then separated from the assembly bag 9 by welding.

[0109] The assembly bag 9 is centrifuged to obtain a layer of red blood cell pellet and a layer of platelet concentrate supernatant.

[0110] The assembly bag 9 containing the red blood cell pellet and the supernatant of The platelet concentrate is then pressed, for example using a Macopress Smart type press (Macopharma, France), to send the platelet concentrate into the filtrate collection bag 7 via the filtration unit 1 of the invention. This yields a platelet concentrate reduced in leukocytes and erythrocytes.

[0111] In the embodiment in which the assembly bag 9 comprises a set of particles 17 capable of binding anti-A and / or anti-B antibodies to said platelet concentrate, before centrifugation of the assembly bag 9, the mixture of buffy coats in the assembly bag 9 is incubated with the set of particles 17 for a sufficient time to bind said antibodies to said particles, for example, one hour. After centrifugation of the assembly bag 9, the pellet layer comprises not only red blood cells but also particles bound or not to anti-A and / or anti-B antibodies. At the end of the process, a platelet concentrate reduced in leukocytes, erythrocytes, and anti-A and / or anti-B antibodies is obtained.

[0112] A variant of a pocket system for selectively removing leukocytes and erythrocytes from a platelet concentrate is shown in [Fig. 4]. In this variant, the leukoremoval porous element and the erythrocyte barrier porous element are each arranged in a separate filtration unit. This pocket system is used to prepare a platelet concentrate from buffy coats according to the "train" method. The elements common to the pocket system of [Fig. 3] retain the same numbering.

[0113] In relation to [Fig.4], the pocket system 18 comprises: - a first filtration unit 19 for the selective removal of leukocytes, said first filtration unit 19 comprising a porous element for leukocyte deleukoreduction by adsorption and / or filtration, said porous deleukoreduction element comprising at least one layer whose pores have an average diameter between 3 and 15 pm, - a collection bag for the first filtrate 20, said first collection bag 20 being connected, via a fifth tube 21, to an outlet orifice of said first filtration unit 19, - a second filtration unit 22 for the selective removal of erythrocytes, said second filtration unit comprising a porous erythrocyte barrier element, said porous erythrocyte barrier element comprising at least one porous membrane made of a hydrophilic material, the pores of said membrane having a nominal size between 1 µm and 4 µm, and an absolute pore size between 2 µm and 5 µm, said second filtration unit 22 being further connected, via a sixth tube 23, to an outlet port of said first filtrate collection bag 20, and - a filtrate collection bag 7, said collection bag being connected, via a first tube 8, to an outlet orifice of said second filtration unit 22.

[0114] The pocket system 18 further includes a pocket 9 for assembling leukocyte and platelet layers, connected or intended to be connected via a second tube 10 to the inlet port of the first filtration unit 19. In an alternative not shown, this assembly pocket 9 includes a set of particles 17 capable of binding anti-A and / or anti-B antibodies.

[0115] In an alternative shown in [Fig.4], a set of particles 17 capable of binding anti-A and / or anti-B antibodies from a platelet-concentrated product is placed in the collection bag 20 of the first filtrate.

[0116] In relation to the pocket system of [Fig.4], a particular use of the pocket system 18 is now described for the preparation of a platelet concentrate reduced in leukocytes and erythrocytes.

[0117] An assembly of four to eight leukocyte-platelet layers suspended in a platelet preservation solution is obtained in an assembly bag 9 by the "train" method. This assembly bag 9 is connected aseptically to the rest of the bag system 18 and then centrifuged to obtain a layer of red blood cell pellet and a layer of platelet concentrate supernatant.

[0118] This bag is then pressed, for example using a Macopress Smart type press (Macopharma, France), to send the platelet concentrate into the first filtrate collection bag 20 via the filtration unit 19 to obtain a leukocyte-reduced platelet concentrate. The leukocyte-reduced platelet concentrate is then sent using the press into the second filtrate collection bag 7 via the filtration unit 22 to obtain a platelet concentrate reduced in leukocytes and erythrocytes.

[0119] In the variant in which a set of particles capable of binding anti-A and / or anti-B antibodies is arranged in the assembly pocket 9, the assembly of leukocyte-platelet layers is brought into contact with said particles for a time sufficient to bind said antibodies to said particles, for example one hour, in order to obtain an assembly of leukocyte-platelet layers reduced in anti-A and / or anti-B antibodies.

[0120] In the other embodiment in which the set of particles 17 capable of binding anti-A and / or anti-B antibodies is arranged in the first filtrate collection bag 20, the leukocyte-reduced platelet concentrate is then brought into contact with said particles for a sufficient time to bind said antibodies to said particles, for example one hour, before being transferred to the filtrate collection bag 7 via the second filtration unit 22, in order to obtain a platelet concentrate reduced to leukocytes, erythrocytes and anti-A and / or anti-B antibodies.

[0121] Example 1 Polymer impregnation solution

[0122] An impregnation solution is prepared by solubilizing in ethanol a polymer obtained from a methyl methacrylate monomer and a poly(ethylene oxide) methacrylate macromer as described in document EP 2 783 717 Al

[0123] Example 2: Impregnation

[0124] A polyester (PETE) membrane having a nominal pore size of 2 pm and an absolute pore size of 2.5 pm is impregnated by scarf-drying with the polymer solution prepared above.

[0125] A polyester (PETE) membrane having a nominal pore size of 5 pm is impregnated by swaging and drying with the polymer solution prepared above.

[0126] A non-woven layer of polybutylene terephthalate (42 g / m2, average pore diameter of 8.5 pm) is impregnated by scrim-drying with the polymer solution prepared above.

[0127] Example 3: Test of erythrocyte filtration in a miniature filter

[0128] A miniature filter is made by placing a filtering medium with the following composition in a rigid housing:

[0129] [Tables 1] Composition and porosity: 1 impregnated membrane 5 µm, 1 layer of impregnated non-woven fabric 8.5 µm, 1 impregnated membrane 5 µm, 1 layer of impregnated non-woven fabric 8.5 µm, 1 impregnated membrane 2 µm, 1 layer of impregnated non-woven fabric 8.5 µm, 1 impregnated membrane 2 µm, 1 layer of impregnated non-woven fabric 8.5 µm, 1 impregnated membrane 2 µm, 1 layer of impregnated non-woven fabric 8.5 µm, 1 impregnated membrane 2 µm

[0130] The filtration surface area of ​​each of the layers and membranes is approximately 14 cm2.

[0131] A platelet concentrate is prepared by combining four leukocyte-platelet layers of the same blood group with 250 ml of platelet preservation solution (SSP+, Macopharma). The mixture is then centrifuged and filtered through a filter to selectively remove leukocytes (TXP, Macopharma) to obtain a leukocyte-reduced platelet concentrate.

[0132] The platelet concentrate is then filtered through the miniature filter above (see Table 1).

[0133] [Tables2] Before filtration After filtration Erythrocyte concentration (erythrocytes / pL) 1092 80 Leukocyte concentration ( / pL) 0.067 0 Platelet count 2.5 x 10¹¹ 2.2 x 10¹¹

[0134] The platelet recovery rate is approximately 88%. The red blood cell filtration rate is approximately 93%.

[0135] Example 4: Full-scale erythrocyte filtration test

[0136] Two types of filter are made by placing a filtering medium with the following composition in a flexible envelope:

[0137] [Tables3] Composition 1 Composition 2 1 impregnated membrane 2 µm 1 impregnated membrane with 5 µm porosity 1 layer of impregnated non-woven fabric with 8.5 µm porosity 1 impregnated membrane with 2 µm porosity

[0138] The filtration surface area of ​​each of the layers and membranes is approximately 53 cm2.

[0139] A platelet concentrate is prepared by combining four layers of leukocyte-platelets of the same blood group and 250 ml of platelet preservation solution (SSP+, Macopharma). The mixture is then centrifuged and filtered through a filter to selectively remove leukocytes (TXP, Macopharma) to obtain a leukocyte-reduced platelet concentrate.

[0140] The platelet concentrate reduced to leukocytes (265 mL) is then filtered through a filter having one of the compositions 1 or 2 above (see Table 3).

[0141] [Tables4] Composition 1 Composition 2 Erythrocyte concentration (erythrocytes / pL) Before filtration 1349 1357 After filtration Day 1 21 27 After filtration Day 7 9 8 Leukocyte concentration (100pL) Before filtration 0.0001 0 After filtration Day 1 0 0 After filtration Day 7 0.0009 0.0009 Platelet count Before filtration 2.2 x 10¹¹ 2.2 x 10¹¹

[0142] The platelet recovery rate is approximately 89% for composition 1 and 80% for composition 2. The red blood cell filtration rate is approximately 99% for composition 1 and 98% for composition 2.

[0143] Example 5: Leukocyte and erythrocyte filtration test

[0144] A filter is made by placing a filtering medium having the following composition in a rigid housing:

[0145] [Tables5] Composition: Porosity: 20 layers of impregnated non-woven fabric (8.5 µm), 1 impregnated membrane (2 µm)

[0146] The filtration surface area of ​​each of the layers and membranes is approximately 53 cm2.

[0147] A platelet concentrate is prepared by combining four layers of leukocytes and platelets of the same blood group with 250 ml of platelet preservation solution (SSP+, Macopharma). The mixture is then centrifuged and filtered through the filter to obtain a platelet concentrate reduced in leukocytes and erythrocytes.

[0148] The filtration results are as follows:

[0149] [Tableauxô] Before filtration After filtration Erythrocyte concentration (erythrocytes / pL) 1.58 x 10⁶ 26 Leukocyte concentration (10³ / pL) 10.71 0 Platelet count 3.9 x 10¹¹ 2.2 x 10¹¹

[0150] The concentrations before filtration are the concentrations of cells in the assembly of leukocyte and platelet layers and additive solution, before centrifugation.

Claims

Claims

1. A method for selectively removing leukocytes and erythrocytes from a product concentrated in blood platelets, said method comprising passing a product concentrated in blood platelets and reduced in leukocytes through a porous erythrocyte barrier element, characterized in that said porous erythrocyte barrier element comprises at least one porous membrane made of a hydrophilic material, the pores of said membrane having a nominal size of between 1 pm and 4 pm, and an absolute pore size of between 2 pm and 5 pm.

2. Method according to claim 1, characterized in that said membrane is obtained by ion bombardment followed by etching.

3. Method according to one of claims 1 or 2, characterized in that said membrane is made of polycarbonate or polyester.

4. A method according to any one of claims 1 to 3, characterized in that said membrane has a nominal pore size of 2 pm and an absolute size of 2.5 pm.

5. Method according to any one of claims 1 to 4, characterized in that said membrane is coated with a biocompatible polymer derived from at least one monomer chosen from the list consisting of monomers of N-vinylpyrrolidone, 2-methoxyethyl acrylate, 2-methacryloyloxyethylphosphorylcholine, 2-hydroxyethyl methacrylate, ethylene glycol and mixtures of these monomers.

6. Method according to any one of claims 1 to 5, characterized in that it comprises: - the prior passage of a product concentrated in platelets through a porous element for leukocyte depletion by adsorption and / or by filtration of the leukocytes, so as to obtain a product concentrated in platelets and reduced in leukocytes, said porous element for leukocyte depletion comprising at least one layer whose pores have an average diameter of between 3 and 15 μm.

7. A method according to claim 6, characterized in that the porous leukocyte removal element comprises one or more layers of non-woven fabric.

8. Method according to one of claims 6 or 7, characterized in that the porous leukocyte removal element comprises polybutylene terephthalate or polyethylene terephthalate fibers.

9. Method according to any one of claims 6 to 8, characterized in that the porous leukocyte removal element is impregnated with a biocompatible polymer derived from at least one monomer selected from the list consisting of the monomers of N-vinylpyrrolidone, 2-methoxyethyl acrylate, 2-methacryloyloxyethylphosphorylcholine, 2-hydroxyethyl methacrylate, ethylene glycol and mixtures of these monomers.

10. Method according to any one of claims 1 to 9, characterized in that it comprises, before the passage of a product concentrated in platelets through a porous element barrier to erythrocytes, the contacting of a product concentrated in blood platelets with a set of particles capable of binding the anti-A and / or anti-B antibodies of said product concentrated in platelets, so as to obtain a mixture of a product concentrated in platelets reduced in anti-A and / or anti-B antibodies and of particles bound or not to anti-A and / or anti-B antibodies.

11. Filtration unit (1) for implementing the method according to any one of claims 1 to 10, characterized in that it comprises an outer casing provided with at least one inlet orifice (2) and at least one outlet orifice (3), the casing enclosing from upstream to downstream: - a porous leukocyte removal element (4) by adsorption and / or by filtration of leukocytes, said porous leukocyte removal element comprising at least one layer whose pores have an average diameter of between 3 and 15 pm, and - a porous erythrocyte barrier element (5) comprising at least one porous membrane made of a hydrophilic material, the pores of said membrane having a nominal size of between 1 pm and 4 pm, and an absolute pore size of between 2 pm and 5 pm.

12. Bag system (6) for the selective removal of leukocytes and erythrocytes from a platelet-concentrated product, characterized in that it comprises: - a filtration unit (1) according to claim 11, and - a filtrate collection bag (7), said bag being connected, via a tube (8), to an outlet orifice (3) of the filtration unit (1).

13. Bag system according to claim 12, characterized in that it further comprises an assembly bag (9) connected via a tube (10) to an inlet orifice (2) of the filtration unit (1), said assembly bag (9) comprising a set of particles (17) capable of binding anti-A and / or anti-B antibodies to said platelet-concentrated product.

14. Bag system (18) for implementing the method according to any one of claims 1 to 10, characterized in that it comprises: - a first filtration unit (19) for the selective elimination of leukocytes, said first filtration unit comprising a porous element for leukocyte depletion by adsorption and / or by filtration of the leukocytes, said porous element for leukocyte depletion comprising at least one layer whose pores have an average diameter of between 3 and 15 μm, - a collection bag (20) for the first filtrate, said collection bag being connected, via a tube (21), to an outlet orifice of said first filtration unit (19), - a second filtration unit (22) for the selective elimination of erythrocytes, said second filtration unit (22) comprising a porous element that acts as a barrier to the erythrocytes,said porous erythrocyte barrier element comprising at least one porous membrane made of a hydrophilic material, the pores of said membrane having a nominal size of between 1 pm and 4 pm, and an absolute pore size of between 2 pm and 5 pm, said second filtration unit being furthermore on the one hand connected or intended to be connected by means of a tube (23), to an outlet orifice of said collection bag (20) of the first filtrate, and - a filtrate collection bag (7), said collection bag (7) being connected, by means of a tube (8), to an outlet orifice of said second filtration unit (22).,

15. Bag system according to claim 14 characterized in that it further comprises a set of particles (17) capable of binding anti-A and / or anti-B antibodies of said platelet-concentrated product, said set of particles being placed in said bag (20) for collecting the first filtrate or in an assembly bag (9) connected via a tube (10) to an inlet orifice of the first filtration unit (19).

Images