Process for obtaining a platelet-concentrated product reduced in anti-A and / or anti-B antibodies

Abstract

Process for obtaining a platelet-concentrated product reduced in anti-A and / or anti-B antibodies. The invention relates to a method for obtaining a product concentrated in blood platelets and reduced in anti-A and / or anti-B antibodies, said method comprising: - the assembly of one or more buffy coats obtained by centrifugation of whole blood, - the contacting of said assembly with a set of particles capable of binding anti-A and / or anti-B antibodies, for a time sufficient to bind anti-A and / or anti-B antibodies to said particles, so as to obtain a mixture of assembled buffy coats and particles bound or not to anti-A and / or anti-B antibodies, - the separation by centrifugation of said mixture, so as to obtain a pellet comprising red blood cells and particles bound or not to anti-A and / or anti-B antibodies, and a supernatant of a product concentrated in platelets and reduced in anti-A and / or anti-B antibodies,and - the extraction of said concentrated product into platelets reduced in anti-A and / or anti-B antibodies thus separated. Figure for the abbreviation: Fig. 3,

Description

Title of the invention: Process for obtaining a concentrated platelet product reduced in anti-A and / or anti-B antibodies

[0001] The invention relates to a process for obtaining a product concentrated in blood platelets and reduced in anti-A and / or anti-B antibodies, as well as a bag system for implementing such a process.

[0002] It typically applies to the preparation of a blood component containing platelets such as 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 buffy coat layers of isogroup blood prepared in this way are then combined 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 thus obtained is finally 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] Since platelet concentrates contain plasma, some transfusion centers apply the same ABO compatibility rules when transfusing a platelet concentrate as when transfusing plasma (compatibility minor). Thus, AB plasma can be used regardless of the recipient's blood type, and A or B plasma can be used for an O recipient. This rule helps to avoid 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.

[0008] However, it is not uncommon to transfuse ABO-incompatible platelet concentrates, particularly 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.

[0009] Reducing anti-A and / or anti-B antibodies in platelet concentrates would reduce the risk of post-transfusion hemolysis in the event of ABO incompatible transfusion and facilitate the inventory of platelet concentrates in blood banks and hospitals.

[0010] A pouch system for removing anti-A and / or anti-B antibodies from plasma is known from document WO 2021 / 144380. The system comprises a pouch containing particles grafted with oligosaccharides capable of binding anti-A and / or anti-B antibodies present in the plasma. The pouch system further comprises a filter including a membrane with a porosity of less than 3 µm, in particular 0.65 µm, for separating the grafted particles from the plasma reduced to anti-A and / or anti-B antibodies.

[0011] Since platelets have a size of approximately 2 to 4 pm, this membrane, having a pore size of 0.65 pm, is not suitable for separating these grafted particles from a platelet concentrate.

[0012] Document WO 2021 / 225507 describes a kit for reducing anti-A and / or anti-B antibodies from a blood component such as plasma, platelet preparations, or immunoglobulins. This kit includes a bag containing a suspension of gel beads in a buffer solution, a dropper chamber with a porous mesh at the bottom to retain the beads, a filtrate collection bag, and a bag containing the blood component to be filtered. The beads comprise carbohydrate antigens, such as disaccharides, trisaccharides, tetrasaccharides, or higher oligosaccharides, with selectivity for antibodies specific to a blood group. This kit is used on a final platelet concentrate and requires additional steps of connecting and disconnecting the bags, which is inconvenient and lengthens the preparation time of the blood component.

[0013] The invention proposes a simpler process for obtaining a product concentrated in blood platelets and reduced in anti-A and / or anti-B antibodies, without an additional preparation step compared to a conventional preparation.

[0014] According to a first aspect, the invention proposes a process for obtaining a product concentrated in blood platelets and reduced in anti-A and / or anti-B antibodies, said process comprising: - the assembly of one or more leukocyte-platelet layers obtained by centrifugation of whole blood, - bringing said assembly of leukocyte-platelet layers into contact with a set of particles capable of binding anti-A and / or anti-B antibodies, for a time sufficient to bind anti-A and / or anti-B antibodies to said particles, so as to obtain a mixture of leukocyte-platelet layer assembly and particles bound or not to anti-A and / or anti-B antibodies - the separation by centrifugation of said mixture, so as to obtain a pellet comprising red blood cells and particles bound or not to anti-A and / or anti-B antibodies, and a supernatant of a platelet-concentrated product reduced in anti-A and / or anti-B antibodies, and - the extraction of said product concentrated in platelets and reduced in anti-A and / or anti-B antibodies thus separated, so as to isolate the product concentrated in platelets and reduced in anti-A and / or anti-B antibodies.

[0015] According to a second aspect, the invention relates to a pocket system for implementing the method according to the first aspect, comprising: - an assembly pocket comprising a set of particles capable of binding anti-A and / or anti-B antibodies, - a filtration unit, connected via tubing to an outlet of said assembly bag, said filtration unit comprising a porous element providing a barrier to particles and / or erythrocytes, said porous 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 and - a filtrate collection bag, said bag being connected, via a tube, to an outlet port of the filtration unit.

[0016] The attached drawings illustrate the invention:

[0017] [Fig-1] represents a schematic view of a filtration unit used in a implementation of the process of the invention.

[0018] [Fig.2] represents a schematic cross-sectional view of a filtration unit of the [Fig.l].

[0019] [Fig.3] represents a schematic view of a pocket system for implementing the process to obtain a product concentrated in platelets and reduced in anti-A and / or anti-B antibodies.

[0020] [Fig.4] represents a schematic view of another pocket system for putting in implements the process to obtain a product concentrated in platelets and reduced in anti-A and / or anti-B antibodies.

[0021] The invention proposes a process for obtaining a product concentrated in blood platelets and reduced in anti-A and / or anti-B antibodies.

[0022] A platelet concentrate product is a product whose platelet concentration is higher than the normal platelet concentration in blood. A platelet concentrate product is typically obtained by fractionating whole blood.

[0023] Such a platelet-concentrated product is, for example, a unit platelet concentrate (UPC) or an assembly of platelet concentrates (PC) prepared from leukocyte-platelet layers.

[0024] A platelet concentrate essentially comprises platelets suspended in plasma. Depending on the preparation method, it also includes residual leukocytes and erythrocytes, which represent an immunological risk for the transfused patient. The presence of plasma in the platelet concentrate also implies the presence of anti-A and / or anti-B antibodies, depending on the blood type of the donor from whom it is derived.

[0025] For example, a platelet concentrate from a group A donor includes plasma with anti-B antibodies. That from a group B donor includes plasma with anti-A antibodies, that from a group O donor contains plasma with anti-A and anti-B antibodies, and that from a group AB donor includes plasma without anti-A or anti-B antibodies.

[0026] The presence of anti-A and / or B antibodies in platelet concentrates from whole blood of group A, B or O donors may trigger hemolysis in an ABO-incompatible recipient.

[0027] To reduce this risk, the process of the invention comprises the following steps: - the assembly of one or more leukocyte-platelet layers obtained by centrifugation of whole blood, - bringing said assembly of leukocyte layers into contact with a set of particles capable of binding anti-A and / or anti-B antibodies, for a sufficient time to bind anti-A and / or anti-B antibodies to said particles, so as to obtain a mixture of assembly of leukocyte layers and particles bound or not to anti-A and / or anti-B antibodies, - the separation by centrifugation of said mixture, so as to obtain a pellet comprising red blood cells and particles bound or not to anti-A and / or anti-B antibodies, and a supernatant of a product concentrated in platelets and reduced in anti-A and / or anti-B antibodies, and - the extraction of said concentrated product in platelets reduced in anti-A and / or anti-B antibodies thus separated so as to isolate the concentrated product in platelets and reduced in anti-A and / or anti-B antibodies.

[0028] The process of the invention is based on the process of preparing plasma concentrates quetates from leukocyte-platelet layers.

[0029] One or more leukocyte-platelet layers are obtained by hard centrifugation of a unit of whole blood. After hard centrifugation of the whole blood, a pellet of red blood cells, a plasma supernatant, and an intermediate layer called the leukocyte-platelet layer, comprising plasma, platelets, leukocytes, and residual red blood cells, are obtained.

[0030] The assembly is notably achieved by joining between four and eight buffy coats. Advantageously, the assembly further includes the addition of an additive solution for SSP+ type platelets (Macoparma).

[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] The process then includes bringing said assembly into contact with a set of particles capable of binding anti-A and / or anti-B antibodies.

[0033] Particles capable of binding anti-A and / or anti-B antibodies include those described in document WO 2021 / 144380.

[0034] 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.

[0035] 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.

[0036] The particles are incubated with the assembly of buffy coats for a sufficient time to bind anti-A and / or anti-B antibodies to said particles. The incubation time is, for example, between 30 minutes and two hours, in particular approximately one hour. At the end of the contact time, a mixture of buffy coats and particles, bound or not to anti-A and / or anti-B antibodies, is obtained. The buffy coats essentially comprise platelets and plasma reduced in anti-A and / or anti-B antibodies.

[0037] Advantageously, the assembly and contacting steps are carried out simultaneously by transferring the contents of one or more pouches, each containing a leukocyte-platelet layer, into an assembly pouch comprising particles, as described in more detail below.

[0038] The process then includes separating the mixture by centrifugation in order not only to prepare a platelet concentrate from leukocyte-platelet layers but also to separate the particles of said platelet concentrate. After centrifugation, a pellet is obtained comprising red blood cells and particles bound or not to anti-A and / or anti-B antibodies, and a supernatant of a product concentrated in platelets and reduced in anti-A and / or anti-B antibodies.

[0039] In order to isolate the product concentrated in platelets and reduced in anti-A and / or anti-B antibodies, the product obtained in the previous step is extracted, for example using a press.

[0040] Anti-A and anti-B antibodies include immunoglobulin M (IgM) and immunoglobulin G (IgG). For each of the anti-A and anti-B antibodies, the platelet concentrate contains, in particular, an IgM and / or IgG titer of less than or equal to 1 / 16.

[0041] The determination of the antibody titer is carried out for example by a direct agglutination test for the titration of IgM and indirect for the titration of IgG.

[0042] In order to eliminate residual leukocytes from the platelet-concentrated product reduced in anti-A and / or anti-B antibodies, the process further comprises passing the platelet-concentrated product reduced in anti-A and / or anti-B antibodies through a porous leukoreduction element by adsorption and / or filtration of leukocytes, said porous leukoreduction element comprising at least one layer whose pores have an average diameter of between 3 and 15 pm.

[0043] The passage of the concentrated blood platelet product through a porous leukoreduction element is carried out by gravity or more particularly under pressure, for example using a press.

[0044] Advantageously, the passage through the porous leukoreducing element is carried out simultaneously with the extraction step of the platelet-concentrated product reduced in anti-A and / or anti-B antibodies, so as to obtain a platelet-concentrated product leukoreduced and reduced in anti-A and / or anti-B antibodies.

[0045] 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.

[0046] 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.

[0047] In particular, when the fluid to be filtered is a platelet concentrate derived from leukocyte-platelet layers, the porous leukoreduction element is capable of obtaining a number The leukocyte count in the filtered platelet concentrate must be less than 1 x 10⁶, and at least 70% of the platelets must pass through. The platelet count in the platelet concentrate after filtration must be at least 2 x 10¹¹ platelets.

[0048] The porous leukoreduction 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 the leukocytes to be retained by mechanical filtration.

[0049] 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.

[0050] 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.

[0051] In particular, the porous leukoreducing element comprises polyester fibers, for example polybutylene terephthalate or polyethylene terephthalate fibers.

[0052] Advantageously, the porous leukoreducing element is impregnated with a biocompatible polymer preventing the adhesion of platelets to its surface.

[0053] 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.

[0054] The biocompatible polymer consists of at least one unit from a biocompatible monomer.

[0055] 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.

[0056] 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.

[0057] 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 monomer is methyl methacrylate or vinyl acetate.

[0058] This hydrophobic monomer contributes to maintaining the biocompatible polymer on the surface of the membrane.

[0059] 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; or a 2-hydroxyethyl methacrylate monomer unit and a me diethylaminoethyl thacrylate; or one vinyl pyrrolidone monomer unit and one vinyl acetate monomer unit.

[0060] 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 sides of the porous layer(s).

[0061] These pre-filters or post-filters have, in particular, a larger average pore size to that of the porous layer(s), for example between 25 and 50 pm.

[0062] The pre-filters and post-filters are impregnated with a biocompatible polymer that facilitates the passage of the platelets as described above. This biocompatible polymer may or may not be the same as that used to impregnate the non-woven layer(s).

[0063] According to one embodiment, the steps of separation by centrifugation and extraction, combined or not with the step of passing through a porous leukoreduction element, are sufficient to ensure that the final product concentrated in platelets and reduced in anti-A and / or anti-B antibodies no longer contains particles.

[0064] In order to further reduce the risk of the presence of particles in the final product, the process includes passing the platelet-reduced product containing anti-A and / or anti-B antibodies through a porous barrier element against particles and / or 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 2 pm and 5 pm.

[0065] The passage of the product concentrated in platelets and reduced in anti-A and / or anti-B antibodies through the porous barrier element to particles and / or erythrocytes is carried out by gravity or under pressure, for example using a press.

[0066] Advantageously, in order to reduce the risk of membrane clogging, the platelet-concentrated product reduced in anti-A and / or anti-B antibodies is pre-leukoreduced, in particular by passing through a porous leukoreduction element as described above.

[0067] The porous element, a barrier to particles and / or erythrocytes, is intended to retain particles of a platelet-concentrated product while allowing platelets to pass through.

[0068] This porous element is also capable of retaining residual erythrocytes from a platelet-concentrated product previously reduced to leukocytes while allowing the platelets to pass through.

[0069] Indeed, residual red blood cells present in platelet concentrates lead to a risk of alloimmunization of red blood cells, that is, the production of anti-erythrocyte antibodies in the transfused recipient. Eliminating residual erythrocytes from the platelet concentrate product is therefore particularly advantageous.

[0070] After passing through a porous barrier element (particles and / or erythrocytes), the platelet-concentrated product reduced in anti-A and / or anti-B antibodies includes a residual erythrocyte concentration of less than 80 erythrocytes per pL.

[0071] The porous element that acts as a barrier to particles and / or 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.

[0072] In particular, the membrane is coated 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 porous leukoreducing element.

[0073] 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.

[0074] The membrane forming a porous barrier element to particles and / or erythrocytes has a nominal pore size between 1 pm and 4 pm.

[0075] By nominal pore size, we mean the predominant pore size of a membrane. The membrane comprises pores larger or smaller than this nominal size, but the number of pores having the nominal pore size is greater than the number of pores having another size.

[0076] A membrane with a nominal pore size greater than 4 pm does not sufficiently retain the residual particles and / or erythrocytes from a platelet concentrate. A membrane with a nominal pore size less than 1 pm does not allow for sufficient platelet recovery.

[0077] The membrane also has an absolute pore size of between 2 pm and 5 pm.

[0078] 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.

[0079] 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 particles and / or erythrocytes.

[0080] 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.

[0081] For example, the pore density of the membrane is between 4.105 and 2.107 pores / cm2, in particular about 2.106 pores / cm2.

[0082] In particular, the thickness of the membrane is between 8 and 12 pm.

[0083] For example, the membrane has a nominal pore size of 2 pm and a size absolute of 2.5 pm.

[0084] In another example, the membrane has a nominal pore size of 3 pm and an absolute size of 4 pm.

[0085] According to a particular embodiment, the porous element barrier to particles and / or 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.

[0086] These pre-filters or post-filters have in particular an average pore size greater than that of the porous membrane, for example between 25 and 50 pm.

[0087] The pre-filters and post-filters 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 leukoreduction element.

[0088] According to another embodiment, the porous element that acts as a barrier to particles and / or erythrocytes comprises several porous membranes made of a hydrophilic material with the same porosity or with decreasing porosity from upstream to downstream of the porous element that acts as a barrier to particles and / or erythrocytes, 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.

[0089] Advantageously, in order to prevent clogging of such a stack of membranes, the porous element that acts as a barrier to particles and / or 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 between 3 and 15 pm, in particular between 7 and 10 pm.

[0090] 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.

[0091] Advantageously, to reduce the risk of particles being present in the final platelet concentrate, before contact with the assembly of leukoreduced platelet layers, all the particles are passed through a sieve having a mesh size larger than the pore size of the leukoreducing porous element and / or the porous membrane. For example, the sieve has a mesh size of 5 µm, 10 µm, or 20 µm.

[0092] In connection with Figures 1 and 2, a filtration unit 1 is described below for implementing a process to obtain a product concentrated in blood platelets and reduced in anti-A and / or anti-B antibodies.

[0093] The filtration unit 1 comprises 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 particles and / or 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.

[0094] The terms "upstream" and "downstream" are defined with respect to the direction of flow of the concentrated product in platelets in the filtration unit.

[0095] Each of the porous leukoreduction and particle and / or erythrocyte barrier elements includes those described above.

[0096] The leukoreduction porous element is positioned upstream of the porous element that acts as a barrier to particles and / or erythrocytes. Thus, the platelet-concentrated product is first reduced to leukocytes, then to particles and / or erythrocytes. This arrangement reduces the risk of clogging of the membrane forming the porous element that acts as a barrier to particles and / or erythrocytes.

[0097] We now describe in connection with [Fig.3], a 6-pocket system for obtaining a product concentrated in platelets and reduced in anti-A and / or anti-B antibodies.

[0098] 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.

[0099] The filtration unit 1 comprises from upstream to downstream a porous element 4 for leukoreduction by adsorption and / or filtration of leukocytes, and a porous element 5 barrier to particles and / or erythrocytes as described above.

[0100] Alternatively, the filtration unit comprises only one porous element 5 barrier to particles and / or erythrocytes.

[0101] The outer casing of the filtration unit 1 is flexible, rigid or semi-rigid.

[0102] The pocket system 6 further includes a connected assembly pocket 9 or intended to be connected via a second tube 10 to the filtration unit 1.

[0103] For the preparation of an assembly of buffy coats, the assembly pocket 9 is in fluidic communication with a set of tubing in confi The "octopus" configuration consists of a main tube 11 and secondary tubes 12 connected to the main tube. These secondary tubes 12 are intended to be connected in a sterile manner to at least four bags containing a buffy coat and optionally to a bag containing an additive solution for preserving platelets or plasma.

[0104] According to the invention, the assembly pocket 9 comprises a set of particles 17 capable of binding anti-A and / or anti-B antibodies. These particles are advantageously passed through a sieve as described above before being arranged in the assembly pocket.

[0105] According to a 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.

[0106] 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).

[0107] The tubing is provided with a clamp to control the flow of fluids inside the bag system.

[0108] 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).

[0109] In relation to the 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 anti-A and / or anti-B antibodies, leukocytes and erythrocytes.

[0110] 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.

[0111] The buffy coats flow into the assembly pouch 9, which contains all the particles. The pouches that contained the buffy coats are then rinsed with the preservation solution. The remaining preservation solution is then transferred to the assembly pouch 9. The main and secondary tubing assembly 11, 12 is then separated from the assembly pouch 9 by welding.

[0112] The mixture of leukocyte and platelet layers in the assembly pocket 9 is incubated with the set of particles 17 for a sufficient time to bind said antibodies to said particles, for example one hour.

[0113] The assembly bag 9 is centrifuged to obtain a pellet layer of red blood cells and particles and a supernatant layer of platelet concentrate. After centrifugation of the assembly bag 9, the pellet layer includes not only red blood cells but also particles bound or unbound to anti-A and / or anti-B antibodies. The resulting platelet concentrate is reduced to leukocytes, erythrocytes, and anti-A and / or anti-B antibodies.

[0114] The assembly bag 9 containing the pellet of red blood cells and particles and the platelet concentrate supernatant 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 anti-A and / or anti-B antibodies, leukocytes, and erythrocytes.

[0115] A variant of a pocket system for obtaining a platelet concentrate reduced in anti-A and / or anti-B antibodies is shown in [Fig. 4]. In this variant, the leukoreducing porous element and the porous element that acts as a barrier to particles and / or erythrocytes are each arranged in a separate filtration unit. The elements common to the pocket system of [Fig. 3] retain the same numbering.

[0116] In relation to [Fig.4], the pocket system 18 includes a filtration unit 22 comprising the porous barrier element to erythrocytes and / or particles and another filtration unit 19 comprising the porous leukoreduction element.

[0117] The pocket system 18 of [Fig.4] comprises: - an assembly pocket 9 comprising a set of particles 17 capable of binding anti-A and / or anti-B antibodies, - the other filtration unit 19, connected via a tube 10 to an outlet of said assembly bag 9, - a collection bag 20 for the first filtrate, said bag being connected, via a fifth tube 21, to an outlet of the filtration unit 19 - a filtration unit 22 connected via a sixth tube 23 to an outlet of the collection bag for the first filtrate 20, - the filtration unit 22 comprising a porous leukoreduction element, said filtration unit 22 being connected via a sixth tube to an outlet orifice of the 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 filtration unit 22.

[0118] 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 anti-A and / or anti-B antibodies, leukocytes and erythrocytes.

[0119] An assembly of 4 to 8 leukocyte-platelet layers suspended in a platelet preservation solution is transferred into an assembly bag 9 comprising particles capable of binding anti-A and / or anti-B antibodies. The assembly is incubated with the particles for approximately one hour.

[0120] The assembly bag 9 is then centrifuged to obtain a layer of pellet of red blood cells and particles bound or not to anti-A and / or anti-B antibodies and particles and a layer of supernatant of platelet concentrate reduced in anti-A and / or anti-B antibodies.

[0121] 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 filtrate collection bag 7 via the second filtration unit 22 to obtain a particle-free platelet concentrate reduced in leukocytes and erythrocytes.

[0122] Example 1: Reduction of anti-B antibodies in a platelet concentrate

[0123] In order to carry out anti-B antibody reduction tests on homogeneous samples, three "control" samples and three "test" samples are prepared from six assemblies of four leukocyte-platelet layers of the same blood group and 250 ml of platelet preservation solution (SSP+, Macopharma) combined and then divided again into six homogeneous assemblies.

[0124] For the three control samples, each of the three assemblies is transferred into the assembly bag of a commercial bag system (TRV806L, Macopharma). This bag system essentially comprises an assembly bag, a platelet concentrate de-leukocyte filter (TXP), a filtrate collection and platelet concentrate storage bag, and a sampling bag.

[0125] For the three test samples, a commercially available pouch system (TRV806L, Macopharma) is used, modified by introducing into the assembly pouch 480 mg of ultrafine cellulose particles grafted with a group B antigenic oligosaccharide, ellipsoidal in shape and approximately 2.2 µm x 8 µm in size. This modified system is of the type shown in [Fig. 3]. Each of the three assemblies is transferred into the assembly pouch containing the particles and then incubated with said particles under shaking for 60 minutes.

[0126] For all six samples, the bag system is then centrifuged and placed on a press (Macopress, Macopharma) so as to transfer the platelet concentrate from the assembly bag 9 to the filtrate collection bag 7 via the TXP leukocyte filter.

[0127] Analyses

[0128] Immunoglobulin G (IgG) and immunoglobulin M (IgM) antibody titers were determined by gel card on the control and test samples.

[0129] The determination of the antibody titer is carried out by a direct agglutination test for the titration of IgM and indirect for the titration of IgG.

[0130] Samples of final platelet concentrate are diluted in PBS buffer, by successive dilution by a factor of 2 (1 / 1, 1 / 2, ..., 1 / 64) and brought into contact with erythrocytes of blood group B. The titer yielded corresponds to the last dilution where the agglutinations of red blood cells due to the presence of blood antibodies remain visible.

[0131] The results are shown below.

[0132] [Tables 1] IgG titer IgM titer Control 1 8 8 Control 2 8 8 Control 3 16 8 Test 1 2 Negative Test 2 4 Negative Test 3 2 Negative

[0133] The results show that incubation with particles binding anti-B antibodies makes it possible to obtain a leukoreduced platelet concentrate reduced in anti-B antibodies.

[0134] Further tests showed no substantial difference in terms of platelet recovery or platelet activation.

Claims

Claims

1. A method for obtaining a product concentrated in blood platelets and reduced in anti-A and / or anti-B antibodies, said method comprising: - assembling one or more leukoplatelet layers obtained by centrifugation of whole blood, - bringing said assembly into contact with a set of particles capable of binding anti-A and / or anti-B antibodies, for a time sufficient to bind anti-A and / or anti-B antibodies to said particles, so as to obtain a mixture of leukoplatelet layer assembly and particles bound or not to anti-A and / or anti-B antibodies, - separating said mixture by centrifugation, so as to obtain a pellet comprising red blood cells and particles bound or not to anti-A and / or anti-B antibodies, and a supernatant of a product concentrated in platelets and reduced in anti-A and / or anti-B antibodies,and - the extraction of said product concentrated in platelets reduced in anti-A and / or anti-B antibodies thus separated so as to isolate the product concentrated in platelets and reduced in anti-A and / or anti-B antibodies.,

2. Method according to claim 1, characterized in that it further comprises passing the product concentrated in platelets and reduced in anti-A and / or anti-B antibodies through 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.

3. A method according to claim 2, characterized in that the porous leukocyte removal element comprises one or more layers of non-woven fabric.

4. Method according to one of claims 2 or 3, characterized in that the porous leukocyte removal element comprises polybutylene terephthalate or polyethylene terephthalate fibers.

5. Method according to any one of claims 2 to 4, characterized in that the porous leukocyte removal element is impregnated 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 4, characterized in which further comprises the passage of the product concentrated in platelets reduced in anti-A and / or anti-B antibodies through a porous element which is a barrier to particles and / or erythrocytes and 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.

7. Method according to claim 6, characterized in that said barrier membrane to particles and / or erythrocytes is a membrane obtained by ion bombardment.

8. Method according to claim 6 or 7, characterized in that said membrane is made of polycarbonate or polyester.

9. A method according to any one of claims 6 to 8, characterized in that said membrane has a nominal pore size of 2 pm and an absolute size of 2.5 pm.

10. Method according to any one of claims 6 to 9, 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.

11. Bag system for implementing the method according to any one of claims 1 to 10 comprising: - an assembly bag comprising a set of particles capable of binding anti-A and / or anti-B antibodies, - a filtration unit, connected via a tube to an outlet orifice of said assembly bag, said filtration unit comprising a porous element which is a barrier to particles and / or erythrocytes, said porous 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 and - a bag for collecting the filtrate, said bag being connected, via a tube, to an outlet orifice of the filtration unit.

12. Bag system according to claim 11, characterized in that the filtration unit further comprises, upstream of the porous barrier element for particles and / or erythrocytes, a porous element for leukocyte deleukocyteization by adsorption and / or by filtration of leukocytes, said porous leukocyte depletion element comprising at least one layer whose pores have an average diameter of between 3 and 15 μm.

13. Bag system according to claim 11, characterized in that it further comprises: - another filtration unit for the selective elimination of leukocytes, said other filtration unit comprising a porous element for leukocyte depletion by adsorption and / or by filtration of leukocytes, said porous element for leukocyte depletion comprising at least one layer whose pores have an average diameter of between 3 and 15 μm, said other filtration unit being arranged upstream of the filtration unit comprising a porous element providing a barrier to particles and / or erythrocytes.

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