Method for obtaining a product having an increased platelet concentration and a reduced Anti-a and / or Anti-b antibody content

Abstract

The invention relates to a method for obtaining a product having an increased blood platelet concentration and a reduced anti-A and / or anti-B antibody content, said method comprising: - assembling one or more leukocyte / platelet 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 assembly of leukocyte / platelet layers and of particles bound or not bound 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 bound to anti-A and / or anti-B antibodies, and a supernatant of a product having an increased platelet concentration and a reduced anti-A and / or anti-B antibody content, and - extracting said thus separated product having an increased platelet concentration and a reduced anti-A and / or anti-B antibody content.

Description

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 method 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 method.

[0002] It typically applies to the preparation of a blood component containing platelets such as a single platelet concentrate (SPC) or a pool of platelet concentrates (PC).

[0003] Whole blood is made up of two types of components: blood cells including red blood cells, white blood cells, 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, patients with thrombocytopenia, i.e., those with a low platelet count, are only transfused with platelet concentrates.

[0005] There are several methods for preparing platelet concentrates from whole blood, including the buffy coat method (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] In this preparation method, a unit of whole blood is centrifuged to separate the blood into three layers: a plasma supernatant layer, a leukoplatelet intermediate layer comprising a mixture of leukocytes, platelets, red blood cells and plasma, and a red blood cell pellet layer. The leukoplatelet layer is separated from the other layers. Between four and eight leukoplatelet layers of blood are thus prepared. isogroup are then combined and resuspended in plasma or in a platelet additive solution. This mixture is centrifuged again (gentle centrifugation) so as to obtain a layer of platelet concentrate supernatant and a layer of red blood cell pellet. 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 ABC compatibility rules to a platelet concentrate transfusion as to a plasma transfusion (minor compatibility). Thus, AB plasma can be used regardless of the recipient's group, and A or B plasma can be used for an O recipient. This rule prevents post-transfusion hemolysis, i.e., hemolysis of the recipient's red blood cells by the anti-A and anti-B hemolyzing antibodies present in the donor's plasma.

[0008] However, it is not uncommon to transfuse ABC-incompatible platelet concentrates, especially in emergencies or when ABC-compatible platelet concentrates are unavailable. In this case, 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 ABC-incompatible transfusion and facilitate the inventory of platelet concentrates in blood banks and hospitals.

[0010] Document WO 2021 / 144380 discloses a bag system for removing anti-A and / or anti-B antibodies from plasma, said system comprising a bag containing particles grafted with oligosaccharides capable of binding the anti-A and / or anti-B antibodies present in the plasma. The bag system further comprises a filter comprising a membrane with a porosity of less than 3 μm, in particular 0.65 μm, in order to separate the grafted particles from the plasma reduced in anti-A and / or anti-B antibodies.

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

[0012] 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 comprises a bag containing a suspension of gel beads in a buffer solution, a drip chamber with a porous mesh at the bottom of the drip chamber to retain the beads, a bag for collecting the filtrate 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 blood group-specific antibodies. This kit is used on a final platelet concentrate and requires additional steps of connecting and disconnecting the bags, which is inconvenient and increases the preparation time of the blood component.

[0013] The invention provides a simpler method 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 provides 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 leukoplatelet layers obtained by centrifugation of whole blood, - bringing said assembly of leukoplatelet 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 assembly of leukoplatelet layers and particles bound or not to anti-A and / or anti-B antibodies - separation by centrifugation of said mixture, so as to obtain a pellet comprising red blood cells and particles bound or not to antibodies anti-A and / or anti-B, and a supernatant of a platelet concentrate 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 bag 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 a tube to an outlet orifice of said assembly bag, said filtration unit comprising a porous element which acts as 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 filtrate collection bag, said bag being connected, via a tube, to an outlet orifice of the filtration unit.

[0016] The accompanying drawings illustrate the invention:

[0017] [Fig. 1] represents a schematic view of a filtration unit used in one embodiment of the method of the invention.

[0018] [Fig. 2] represents a schematic sectional view of a filtration unit of Figure 1.

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

[0020] [Fig. 4] represents a schematic view of another bag system for implementing the method for obtaining a product concentrated in platelets and reduced in anti-A and / or anti-B antibodies.

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

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

[0023] Such a platelet concentrate product is, for example, a single platelet concentrate (SPC) or a collection of platelet concentrates (PC) prepared from buffy coats.

[0024] A platelet concentrate essentially consists of platelets suspended in plasma. Depending on the preparation method, it also contains 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 group 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. A platelet concentrate from a group B donor includes plasma with anti-A antibodies, a platelet concentrate from a group O donor contains plasma with both anti-A and anti-B antibodies, and a platelet concentrate from a group AB donor contains 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 risks triggering hemolysis in a recipient who is not ABC compatible.

[0027] To reduce this risk, the method of the invention comprises the following steps: - the assembly of one or more leukoplatelet layers obtained by centrifugation of whole blood, - bringing said assembly of leukoplatelet 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 assembly of leukoplatelet layers and particles bound or not to anti-A and / or anti-B antibodies, - 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 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.

[0028] The method of the invention is based on the process for preparing platelet concentrates from leukoplatelet layers.

[0029] One or more buffy coats are obtained by hard centrifugation of a unit of whole blood. After hard centrifugation of whole blood, a red blood cell pellet, a plasma supernatant, and an intermediate layer called the buffy coat, which includes plasma, platelets, leukocytes, and residual red blood cells, are obtained.

[0030] The assembly is achieved by combining between four and eight leukoplatelet layers. Advantageously, the assembly also 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 method then comprises contacting said assembly with a set of particles capable of binding anti-A and / or anti-B antibodies.

[0033] The particles capable of binding anti-A and / or anti-B antibodies are in particular 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 antibodies and / or anti-B antibodies. For example, these particles are ultrafine cellulose particles grafted with a group A or B antigenic oligosaccharide, such as those described in WO2016 / 177967. In another example, the particles are those described in EP3141558A1 or IsoClear beads marketed by Prometic Bioseparations.

[0035] For example, 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 buffy coat assembly for a time sufficient 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 bags each containing a buffy coat into an assembly bag comprising particles, as described in more detail below.

[0038] The method then comprises separating the mixture by centrifugation in order not only to prepare a platelet concentrate from leukoplatelet 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 immunoglobulins M (IgM) and immunoglobulins G (IgG). For each of the anti-A and anti-B antibodies, the platelet concentrate product contains, in particular, an IgM and / or IgG titre 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 an indirect one for the titration of IgG.

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

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

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

[0045] Leukocytes are retained by the porous leukocyte removal element by one or both of the following mechanisms. The first mechanism is adsorption of leukocytes to the surface of the porous leukocyte removal element. Leukocytes are adsorbed to cationic and moderately hydrophobic surfaces. The quality of adsorption also depends on the contact surface available for leukocyte adsorption. The second mechanism is sieving and depends mainly on the pore diameter of the porous leukocyte removal element.

[0046] The porous leukocyte removal element allows selective leukocyte removal of a platelet-concentrated product, i.e. it is capable of retaining leukocytes while allowing platelets to pass through.

[0047] In particular, when the fluid to be filtered is a platelet concentrate derived from leukoplatelet layers, the porous leukoreduction element is capable of obtaining a number of leukocytes in the filtered platelet concentrate which is less than 1.10 6 and to allow at least 70% of blood platelets to pass through. The number of platelets in the platelet concentrate after filtration is notably at least 2.10 11 platelets.

[0048] The porous leukocyte removal element comprises 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 makes it possible to retain leukocytes by mechanical filtration.

[0049] According to one embodiment, the porous leukoreduction element comprises one or more layers of nonwoven. In particular, the porous leukoreduction element comprises between 5 and 40 layers of nonwoven, and more particularly between 15 and 25 layers of nonwoven.

[0050] For example, the fiber diameter of the nonwoven layer(s) is between 0.3 and 7 pm, with an average between 1 and 3 pm.

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

[0052] Advantageously, the porous leukocyte removal element is impregnated with a biocompatible polymer preventing platelets from adhering to its surface.

[0053] A biocompatible polymer means a polymer compatible with blood or blood components, preventing or reducing the adsorption of proteins and platelets on the membrane coated with such a polymer.

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

[0055] According to one embodiment, the biocompatible monomer is 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.

[0056] According to one variant, the biocompatible polymer consists of at least one unit originating from a biocompatible monomer and at least one unit originating from a hydrophobic monomer.

[0057] According to one embodiment, the hydrophobic monomer is selected 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 helps maintain the biocompatible polymer on the membrane surface.

[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; a 2-hydroxyethyl methacrylate monomer unit and a diethylaminoethyl methacrylate monomer unit; or a vinyl pyrrolidone monomer unit and a vinyl acetate monomer unit.

[0060] According to a particular embodiment, the porous leukocyte removal element further comprises a pre-filter and / or post-filter which are produced in the form of at least one layer of non-woven fabric and which are arranged respectively on the upstream side and downstream side of the porous layer(s).

[0061] 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.

[0062] The pre-filters and post-filters are impregnated with a biocompatible polymer that facilitates the passage of 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 leukocyte removal 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] To further reduce the risk of particles in the final product, the process includes passing the concentrated product into reduced platelets in anti-A and / or anti-B antibodies through a porous barrier element to particles and / or erythrocytes 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.

[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 product concentrated in platelets and reduced in anti-A and / or anti-B antibodies is previously deleukocyted, in particular by passing through a porous deleukocyteization element as described above.

[0067] The porous barrier element to particles and / or erythrocytes is intended to retain the particles of a platelet-concentrated product while allowing the 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, i.e. the production of anti-erythrocyte antibodies in the transfused person. Removing residual erythrocytes from the platelet concentrate product is therefore particularly advantageous.

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

[0071] The porous barrier element for particles and / or erythrocytes comprises a hydrophilic membrane. According to 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, made hydrophilic by chemical coating.

[0072] In particular, the membrane is coated with a biocompatible polymer as described above preventing the adhesion of platelets to its surface. The biocompatible polymer is the same or different as that used to impregnate the porous leukoreduction element.

[0073] In 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 of between 1 pm and 4 pm.

[0075] Nominal pore size means the predominant pore size of a membrane. The membrane contains pores larger or smaller than this nominal size, but the number of pores with the nominal pore size is greater than the number of pores with any other size.

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

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

[0078] Absolute pore size refers to the size of the smallest particles completely retained by the membrane. The absolute pore size of a membrane is the maximum pore size of a membrane.

[0079] A membrane with an absolute pore size 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 retain sufficient particles and / or erythrocytes.

[0080] Such a membrane is manufactured, in particular, by a phase inversion process, a stretching 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 regular pore diameter.

[0081] For example, the pore density of the membrane is between 4.10 5 and 2.10 7 pores / cm 2 , notably of approximately 2.10 6 pores / cm 2 .

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

[0083] For example, the membrane has a nominal pore size of 2 pm and an absolute size 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 barrier element for particles and / or erythrocytes further comprises a pre-filter and / or post-filter which are produced in the form of at least one layer of non-woven fabric and which are arranged respectively on the upstream side and downstream side 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 facilitating 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 barrier element for particles and / or erythrocytes comprises several porous membranes made of a hydrophilic material of the same porosity or of decreasing porosity from upstream to downstream of the porous barrier element for particles and / or erythrocytes, in the filtration direction. 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 avoid clogging of such a stack of membranes, the porous element acts as a barrier to particles and / or erythrocytes includes interlayers with a porosity greater than that of the membranes. For example, these interlayers are layers of non-woven fabric with an average porosity of between 3 and 15 pm, particularly between 7 and 10 pm.

[0090] These interlayers are advantageously impregnated with a biocompatible polymer facilitating the passage of platelets as described above. The biocompatible polymer is the same or different as that used to impregnate the porous membrane.

[0091] Advantageously, to reduce the risk of particles being present in the final platelet-concentrated product, before contacting with the buffy coat assembly, all of the particles are passed through a sieve having a mesh size larger than the pore size of the porous leukoreduction 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 method for obtaining 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 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 μm, and - a porous barrier element for 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 of between 1 pm and 4 pm, and an absolute pore size of between 2 pm and 5 pm.

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

[0095] Each of the porous leukoreduction and barrier elements to particles and / or erythrocytes are notably those described above.

[0096] The porous leukocyte-reduction element is arranged upstream of the porous particle and / or erythrocyte barrier element. Thus, the platelet-concentrated product is first reduced to leukocytes, then reduced to particles and / or erythrocytes. This arrangement reduces the risk of clogging of the membrane forming the porous particle and / or erythrocyte barrier element.

[0097] We now describe in connection with figure 3, a bag system 6 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 bag 7 for collecting the filtrate, said bag 7 being connected, via a first tube 8, to an outlet orifice 3 of the filtration unit 1.

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

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

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

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

[0103] For the preparation of a buffy coat assembly, the assembly bag 9 is in fluid communication with a set of tubing in an “octopus” configuration consisting of a main tubing 11 and secondary tubing 12 connected to the main tubing. These secondary tubings 12 are intended to be connected in a sterile manner to at least four bags containing a buffy coat and possibly 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 the anti-A and / or anti-B antibodies. These particles are advantageously passed through a sieve as described above before arrangement in the assembly pocket.

[0105] According to a particular embodiment, the filtrate collection bag 7 is in fluid 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 comprises 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 fitted with a clamp to control the flow of fluids inside the bag system.

[0108] When the filtration unit comprises a flexible casing, the filtration unit 1 swells during filtration. In order to avoid this swelling, the filtration unit is advantageously placed in a rigid case (not shown).

[0109] In connection with the bag system of Figure 3, a particular use of the bag system 6 for the preparation of a platelet concentrate reduced in anti-A and / or anti-B antibodies, leukocytes and erythrocytes is now described.

[0110] Between four and eight bags containing a buffy coat are connected in a sterile manner, for example using a sterile connection device of the SCD type (Terumo) to the secondary tubes 12 of a bag system 6. A bag of preservation solution of the SSP+ type (Macopharma, France) is also connected in a sterile manner to one of the secondary tubes 12.

[0111] The leukoplatelet layers flow into the assembly bag 9 comprising all of the particles. The bags having contained said leukoplatelet layers are then rinsed using the preservation solution. Then the remainder of the preservation solution is transferred to the assembly bag 9. The set of main and secondary tubes 11, 12 is then separated from the assembly bag 9 by welding.

[0112] The buffy coat mixture in the assembly pocket 9 is incubated with all of the particles 17 for a time sufficient to bind said antibodies to said particles, for example one hour.

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

[0114] The assembly bag 9 containing the red blood cell pellet and particles and the platelet concentrate supernatant is then pressed, for example using a Macopress Smart type press device (Macopharma, France) in order to send the platelet concentrate into the filtrate collection bag 7 via the filtration unit 1 of the invention. This produces a platelet concentrate reduced in anti-A and / or anti-B antibodies, leukocytes and erythrocytes.

[0115] A variant of the bag system for obtaining a concentrated platelet product reduced in anti-A and / or anti-B antibodies is shown in Figure 4. In this variant, the porous leukoreduction element and the porous particle and / or erythrocyte barrier element are each arranged in a separate filtration unit. The elements common to the bag system of Figure 3 retain the same numbering.

[0116] In connection with Figure 4, the bag system 18 comprises a filtration unit 22 comprising the porous erythrocyte barrier element and / or particles and another filtration unit 19 comprising the porous leukocyte removal element.

[0117] The pocket system 18 of Figure 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 orifice 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 orifice of the filtration unit 19 - a filtration unit 22 connected via a sixth tube 23 to an outlet orifice of the first filtrate collection bag 20, - the filtration unit 22 comprising a porous leukocyte removal element, said filtration unit 22 being connected via a sixth tube to an outlet orifice of the bag 20 for collecting the first filtrate, 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 connection with the bag system of Figure 4, a particular use of the bag system 18 for the preparation of a platelet concentrate reduced in anti-A and / or anti-B antibodies, leukocytes and erythrocytes is now described.

[0119] An assembly of 4 to 8 leukoplatelet 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 in order to obtain a layer of red blood cell pellet 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 device (Macopharma, France) in order to send the platelet concentrate into the bag 20 for collecting the first filtrate via the filtration unit 19 in order to obtain a platelet concentrate reduced in leukocytes. The platelet concentrate reduced in leukocytes is then sent using the press into the filtrate collection bag 7 via the second filtration unit 22 in order to obtain a platelet concentrate without particles and 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 leukoplatelet 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 was transferred into the assembly bag of a commercial bag system (TRV806L, Macopharma). This bag system essentially consisted of an assembly bag, a leukocyte removal filter for platelet concentrates (TXP), a bag for collecting the filtrate and storing the platelet concentrate, and a sampling bag.

[0125] For the three test samples, a commercial bag system (TRV806L, Macopharma) was used, modified by introducing into the assembly bag 480 mg of ultrafine cellulose particles grafted with a group B antigenic oligosaccharide, of ellipsoidal shape having an approximate size of 2.2 pm x 8 pm. This modified system is of the type shown in Figure 3. Each of the three assemblies was transferred into the assembly bag comprising the particles, then incubated with said particles under agitation 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 assembly bag 9 to filtrate collection bag 7 via the leukocyte removal filter TXP.

[0127] Analyses

[0128] Immunoglobulin G (IgG) and immunoglobulin M (IgM) antibody titers were determined by gel card on 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 of a factor of 2 (1 / 1, 1 / 2,..,, 1 / 64) and put in contact with red blood cells of blood group B. The titer returned corresponds to the last dilution where the agglutinates of red blood cells due to the presence of blood antibodies remain visible.

[0131] The results are shown below.

[0132] [Table 1]

[0133] The results show that incubation with anti-B antibody-binding particles results in a leukocyte-depleted platelet concentrate with reduced anti-B antibodies.

[0134] Further testing showed no substantial difference in 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: - the assembly of 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 assembly of leukoplatelet layers and particles bound or not to anti-A and / or anti-B antibodies, - 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 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 leukocyte depletion element by adsorption and / or by filtration of the leukocytes, said porous leukocyte depletion element 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 of 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.

6. Method according to any one of claims 1 to 4, characterized in that it further comprises the passage of the product concentrated in platelets reduced in anti-A and / or anti-B antibodies through a porous element barrier to particles and / or erythrocytes 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.

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 10, characterized in that the method comprises: 8, characterized in that said membrane has a nominal pore size of 2 pm and an absolute size of 2.5 pm.

10. A method according to any one of claims 6 to 10, characterized in that the method comprises: 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. A bag system for carrying out the method according to any one of claims 1 to 10 comprising: - an assembly pocket 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 acts as a barrier to particles and / or erythrocytes, said porous element comprising at least one porous membrane made in 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 filtrate collection bag, 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 depletion 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. A 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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