FR3152409A1 - Process for the selective removal of leukocytes and erythrocytes from a platelet concentrate product - Google Patents

Abstract

Method for the selective removal of leukocytes and erythrocytes from a platelet concentrate product. The invention relates to a method for selectively removing leukocytes and erythrocytes from a blood platelet concentrate product, said method comprising passing a blood platelet concentrate and leukocyte-reduced product 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. Abstract: Fig. 3

Description

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

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

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

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.

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.

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)).

In this preparation method, a unit of whole blood is centrifuged to separate the blood into three layers: a plasma supernatant layer, a buffy coat layer consisting of a mixture of leukocytes, platelets, red blood cells, and plasma, and a packed red blood cell layer. The buffy coat is separated from the other layers. Four to eight buffy coats thus separated are then combined and resuspended in plasma or platelet additive solution. This mixture is centrifuged again (gentle centrifugation) to obtain a platelet supernatant layer and a packed red blood cell layer. The platelet concentrate 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.

However, leukocytes have very significant adverse effects, which has led to attempts 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. Leukocytes have also been shown to negatively affect platelet preservation.

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 transfusion recipient (Moncharmont P. et al. "Red blood cell alloimmunization after platelet transfusion: a 5-year study." Blood Transfusion 12.Suppl 1 (2014): s147).

Reckhaus et al. found that this risk of alloimmunization is greater with buffy-coat-derived platelet concentrates (BC-PC) than with apheresis-derived platelet concentrates (AP-PC), the latter containing on average fewer residual red blood cells. Indeed, BC-PC platelet concentrates contain on average 0.302.10 ⁹ red blood cells/L while AP-PC platelet concentrates contain 0.014.10 ⁹ /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.)

For removing leukocytes from platelet concentrates intended for transfusion, filtration units containing a leukocyte-removing medium are already known. In such units, the leukocyte-removing medium comprises one or more membranes and/or one or more non-woven layers made of a polymer material and treated so as to improve the leukocyte-removal rate and/or the recovery of platelets.

For example, document EP 2 783 717 A1 describes a filter for selectively removing leukocytes from a fluid containing platelets, said filter comprising a nonwoven coated with a polymer obtained from a methyl methacrylate monomer and a poly(ethylene oxide) methacrylate macromonomer.

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

In another example, US 5,152,905 describes a filter for removing leukocytes from platelet-rich plasma (PRP) prepared from a whole blood donation, the filter comprising polybutylene terephthalate (PBT) fibers grafted with a hydroxyethyl methacrylate (HEMA) and methacrylic acid (MAA) monomer. Alternatively, the filter comprises a higher-density porous medium upstream to block red blood cells. This porous medium acts as a valve by blocking the flow of PRP as soon as the red blood cells come into contact with this porous medium and aggregate on its surface. This document indicates that the retention of red blood cells is not due to the pore size of the porous medium or to mechanical filtration.

US 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 particularly, the porous body is a polyurethane foam of which more than 60% of the pores have a diameter greater than or equal to 4 µm.

These last two filters, marketed under the names Autostop (Haemonetics) and Imugard (Terumo BCT), do not allow a residual red blood cell concentration 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).

Indeed, red blood cells, although about 5 to 9 µm in size, are deformable and able to pass through pores smaller than 1 µm. Platelets are about 2 to 4 µm in size and leukocytes are about 6 to 19 µm.

The invention provides a method for obtaining a product concentrated in platelets reduced in leukocytes and containing in particular less than 80 erythrocytes per µL, more particularly less than 15 erythrocytes per µL.

According to a first aspect, the invention provides 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, 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.

According to a second aspect, the invention relates to a filtration unit for implementing the method of the invention comprising an outer casing provided with at least one inlet orifice and at least one outlet orifice, the casing enclosing from upstream to downstream:
- a porous leukocyte depletion element 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, and
- a 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.

According to a third aspect, the invention relates to a bag 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 orifice of the filtration unit.

A fourth aspect of the invention relates to a bag system for implementing the method according to the invention, comprising:
- a first filtration unit for the selective elimination of leukocytes, said first 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,
- 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 elimination 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 by means of a tube, to an outlet orifice of said first filtrate collection bag, and
- a second filtrate collection bag, said second collection bag being connected, via a tube, to an outlet orifice of said second filtration unit.

The accompanying drawings illustrate the invention:

FIG. 1 represents a schematic view of a filtration unit according to the invention.

FIG. 2 represents a schematic sectional view of a filtration unit of the FIG. 1 .

FIG. 3 represents a schematic view of a bag system for implementing the process for removing leukocytes and erythrocytes from a platelet concentrate product.

FIG. 4 represents a schematic view of another bag system for implementing the method for removing leukocytes and erythrocytes from a platelet concentrate product.

According to a first aspect, the invention provides a method for selectively removing leukocytes and erythrocytes from a blood platelet concentrate product.

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 or by performing apheresis.

A platelet-concentrated product is, for example, a platelet-rich plasma (PRP), a single platelet concentrate (SPC) prepared from a PRP or a buffy coat, or a platelet concentrate assembly (PC).

In the case of a platelet concentrate assembly, this is prepared in particular by the method of preparation from leukoplatelet layers, using an additive solution for platelets.

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%).

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

The method of the invention makes it possible to selectively separate these residual leukocytes and erythrocytes from blood platelets.

In particular, in the case of a platelet concentrate derived from leukoplatelet layers, the process makes it possible to obtain a number of leukocytes in the platelet concentrate of less than 1.10 ⁶ , a number of platelets of at least 2.10 ¹¹ and a concentration of residual erythrocytes of less than 80 erythrocytes per µL.

The method of the invention comprises passing 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 of between 1 µm and 4 µm, and an absolute pore size of between 2 µm and 5 µm.

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

The porous erythrocyte barrier element is intended to retain residual erythrocytes from a platelet concentrate product previously reduced to leukocytes while allowing platelets to pass through.

The porous erythrocyte barrier element 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, made hydrophilic by chemical coating.

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

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.

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

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.

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.

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 hydrophobic monomer is methyl methacrylate or vinyl acetate.

This hydrophobic monomer helps maintain the biocompatible polymer on the membrane surface.

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.

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.

The membrane forming a porous barrier element to erythrocytes has a nominal pore size between 1 µm and 4 µm.

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.

A membrane with a nominal pore size greater than 4 µm does not retain sufficient residual erythrocytes from a platelet concentrate product. A membrane with a nominal pore size less than 1 µm does not allow sufficient recovery of platelets.

The membrane also has an absolute pore size of between 2 µm and 5 µm.

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.

A membrane with an absolute pore size less than 2 µm does not allow sufficient platelets to pass through. A membrane with an absolute pore size greater than 5 µm does not retain sufficient erythrocytes.

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.

For example, the pore density of the membrane is between 4.10 ⁵ and 2.10 ⁷ pores/cm², especially around 2.10 ⁶ pores/cm².

In particular, the membrane thickness is between 9 and 12 µm.

For example, the membrane has a nominal pore size of 2 µm and an absolute size of 2.5 µm.

In another example, the membrane has a nominal pore size of 3 µm and an absolute size of 4.2 µm.

According to a particular embodiment, the porous erythrocyte barrier 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 membrane.

These pre-filters or post-filters have an average pore size greater than that of the membrane, for example between 25 and 50 µm.

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

According to another embodiment, the porous erythrocyte barrier element comprises several porous membranes made of a hydrophilic material of the same porosity or of decreasing porosity from upstream to downstream of the porous erythrocyte barrier element, 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.

Advantageously, in order to avoid clogging of such a stack of membranes, the porous erythrocyte barrier element comprises interlayers with a porosity greater than that of the membranes. For example, these interlayers are non-woven layers having an average porosity of between 3 and 15 µm, in particular between 7 and 10 µm.

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.

According to the method of the invention, the platelet-concentrated product is reduced to leukocytes before passing through the porous erythrocyte barrier element. The reduction in leukocytes prevents clogging of the porous membrane forming the erythrocyte barrier.

Thus, the method for selectively removing leukocytes and erythrocytes from a product concentrated in blood platelets comprises the prior passage of the product concentrated in platelets through a porous element for leukocyte deleukocyte adsorption and/or filtration of the leukocytes, so as to obtain a product concentrated in platelets and reduced in leukocytes.

The passage of the concentrated blood platelet product through a porous leukocyte removal element is carried out by gravity or under pressure.

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 on cationic and moderately hydrophobic surfaces. The quality of adsorption also depends on the surface area available for leukocyte adsorption. The second mechanism is sieving and depends mainly on the pore diameter of the porous leukocyte removal element.

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

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 of allowing at least 70% of the blood platelets to pass through. The number of platelets in the platelet concentrate after filtration is in particular at least 2.10 ¹¹ platelets.

The porous leukocyte removal element comprises at least one porous layer whose pores have an average diameter of between 3 and 15 µm, in particular between 7 and 10 µm. This pore size makes it possible to retain leukocytes by mechanical filtration.

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

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

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

Advantageously, the porous leukocyte depletion element is impregnated 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 erythrocyte barrier element.

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).

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 µm.

The pre-filters and post-filters are impregnated with a biocompatible polymer that facilitates the passage of platelets as described above.

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

Indeed, since platelet concentrates contain plasma, some transfusion centers apply the same ABO compatibility rules when transfusing a platelet concentrate as they do when transfusing plasma (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 helps prevent 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.

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

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

To reduce the titration of anti-A and/or anti-B antibodies, the method comprises, before the platelet-concentrated product passes through the porous erythrocyte barrier element, bringing a blood platelet-concentrated product into contact with a set of particles capable of binding the anti-A and/or anti-B antibodies of said platelet-concentrated product, so as to obtain a mixture of a platelet-concentrated product reduced in anti-A and/or anti-B antibodies and particles bound or not to anti-A and/or anti-B antibodies.

The contact of the platelet-concentrated product with all the particles is carried out before or after the elimination of the leukocytes by passage through the porous leukocyte removal element, but before passage through the porous element forming barriers to the erythrocytes.

The particles capable of binding anti-A and/or anti-B antibodies are in particular those described in document WO 2021/144380.

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 the IsoClear beads marketed by Prometic Bioseparations.

For example, particles have an average size greater than 15 µm with a size distribution ranging from less than 2 µm up to about 100 µm.

The platelet concentrate product reduced in anti-A and/or anti-B antibodies is then passed through the porous erythrocyte barrier element or through the porous leukoreduction element and then the erythrocyte barrier in order to separate the particles from the platelet concentrate product reduced in anti-A and/or anti-B antibodies. The porous erythrocyte barrier element then forms a porous particle barrier element since it allows the retention of particles bound or not bound to the anti-A and/or anti-B antibodies of the platelet concentrate product reduced in anti-A and/or anti-B antibodies.

In a particular embodiment, to ensure that the set of particles does not comprise particles having a size smaller than that of the pores of the porous membrane forming a barrier element to the erythrocytes and/or to the particles, before contacting with the platelet concentrate product, the set of particles is passed through a sieve having meshes whose size is greater than the size of the pores of the porous membrane. For example, the sieve has meshes having a size of 5 µm, 10 µm or 20 µm.

According to another aspect and in connection with figures 1 and 2, the invention relates to a filtration unit 1 for implementing the method for selectively removing leukocytes and erythrocytes from a product concentrated in blood platelets, said filtration unit 1 comprising 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 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 µm and 4 µm, and an absolute pore size of between 2 µm and 5 µm.

The terms “upstream” and “downstream” are defined in relation to the direction of flow of the concentrated platelet product in the filtration unit.

Each of the porous leukodepletion and erythrocyte barrier elements are notably those described above.

The porous leukocyte depletion element is arranged upstream of the porous erythrocyte barrier element. Thus, the platelet-concentrated product is first reduced to leukocytes, then reduced to erythrocytes. This arrangement reduces the risk of clogging of the membrane forming the porous erythrocyte barrier element.

We now describe in connection with the FIG. 3 , a 6-bag system for the selective removal of leukocytes and erythrocytes from a platelet concentrate product.

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.

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 erythrocytes as described above.

Alternatively, the filtration unit comprises only one porous element 5 that acts as a barrier to erythrocytes.

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

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.

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, said set of tubing 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.

In a particular embodiment, the assembly bag 9 comprises a set of particles 17 capable of binding the anti-A and/or anti-B antibodies of said platelet-concentrated product. These particles are advantageously passed through a sieve before arrangement in the assembly bag.

According to another 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.

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).

The tubing is fitted with a clamp to control the flow of fluids inside the bag system.

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).

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

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.

The buffy coats flow into the assembly bag 9. The bags that contained said buffy coats are then rinsed using the preservation solution. The remainder of the preservation solution is then 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.

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

The assembly bag 9 containing the red blood cell pellet 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 leukocytes and erythrocytes.

In the variant according to 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 product, before centrifugation of the assembly bag 9, the mixture of leukoplatelet layers in the assembly bag 9 is incubated with all of the particles 17 for a time sufficient to bind said antibodies to said particles, for example one hour. 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. At the end of the process, a platelet concentrate reduced in leukocytes, erythrocytes and anti-A and/or anti-B antibodies is obtained.

A variant of a bag system for selectively removing leukocytes and erythrocytes from a blood platelet concentrate product is shown in FIG. 4 . In this variant, the porous leukoreduction element and the porous erythrocyte barrier element are each arranged in a separate filtration unit. This bag system is used to prepare a platelet concentrate from buffy coats according to the "train" method. The elements common to the bag system of the FIG. 3 keep the same numbering.

In connection with the FIG. 4 , the 18 pocket system includes:
- a first filtration unit 19 for the selective elimination of leukocytes, said first filtration unit 19 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,
- 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 elimination 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 22 being further connected via a sixth 23 tube, to an outlet orifice 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.

The bag system 18 further comprises a bag 9 for assembling leukoplatelet layers, connected or intended to be connected via a second tube 10 to the inlet orifice of the first filtration unit 19. In a variant not shown, this assembly bag 9 comprises a set of particles 17 capable of binding anti-A and/or anti-B antibodies.

As a variant shown on the FIG. 4 , a set of particles 17 capable of binding anti-A and/or anti-B antibodies of a platelet-concentrated product is placed in the pocket 20 for collecting the first filtrate.

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

An assembly of four to eight leukoplatelet layers suspended in a platelet preservation solution is obtained in an assembly bag 9 by the “train” method. This assembly bag 9 is connected in a sterile manner to the rest of the bag system 18 and then centrifuged in order to obtain a layer of red blood cell pellet and a layer of platelet concentrate supernatant.

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 reduced in leukocytes and erythrocytes.

In the variant according to which a set of particles capable of binding anti-A and/or anti-B antibodies is arranged in the assembly pocket 9, the buffy coat assembly 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 a buffy coat assembly reduced in anti-A and/or anti-B antibodies.

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

Example 1 Impregnation polymer solution

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 A1

Example 2: Impregnation

A polyester (PETE) membrane having a nominal pore size of 2 µm and an absolute pore size of 2.5 µm is impregnated by padding and drying with the polymer solution prepared above.

A polyester membrane (PETE) having a nominal pore size of 5 µm is impregnated by padding and drying with the polymer solution prepared above.

A nonwoven layer of polybutylene terephthalate (42 g/m², average pore diameter of 8.5 µm) is impregnated by padding and drying with the polymer solution prepared above.

Example 3: Test of filtration of erythrocytes in a miniature filter

A miniature filter is made by placing a filter medium with the following composition in a rigid box:

Composition 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 1 layer of impregnated non-woven fabric 8.5 µm 1 impregnated membrane 2 µm

The filtration surface of each of the layers and membranes is approximately 14 cm².

A platelet concentrate is prepared by combining four leukoplatelet layers 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.

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

Before filtration After filtration Erythrocyte concentration (erythrocytes/µL) 1092 80 Leukocyte concentration
(/µL) 0.067 0 Platelet count 2.5.10 ¹¹ 2.2.10 ¹¹

The platelet recovery rate is around 88%. The red blood cell filtration rate is around 93%.

Example 4: Full-scale erythrocyte filtration test

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

Composition 1 Composition 2 1 2 µm impregnated membrane 1 impregnated membrane with 5 µm porosity 1 layer of impregnated non-woven fabric with a porosity of 8.5 µm 1 impregnated membrane with porosity of 2 µm

The filtration surface area of each of the layers and membranes is approximately 53 cm².

A platelet concentrate is prepared by combining four leukoplatelet layers 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.

The leukocyte-reduced platelet concentrate (265 mL) is then filtered through a filter having one of the compositions 1 or 2 above (see Table 3).

Composition 1 Composition 2 Erythrocyte concentration (erythrocytes/µL) Before filtration 1349 1357 After filtration
Day 1 21 27 After filtration
Day 7 9 8 Leukocyte concentration
(10 ³ /µL) Before filtration 0.0001 0 After filtration
Day 1 0 0 After filtration
Day 7 0.0009 0.0009 Platelet count
Before filtration 2.2.10 ¹¹ 2.2.10 ¹¹

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

Example 5: Leukocyte and erythrocyte filtration test

A filter is made by placing a filter medium having the following composition in a rigid box:

Composition porosity 20 layers of impregnated non-woven fabric 8.5 µm 1 impregnated membrane 2 µm

The filtration surface area of each of the layers and membranes is approximately 53 cm².

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

The filtration results are as follows:

Before filtration After filtration Erythrocyte concentration (erythrocytes/µL) 1,58.10 ⁶ 26 Leukocyte concentration
(10 ³ /µL) 10.71 0 Platelet count 3.9.10 ¹¹ 2.2.10 ¹¹

Pre-filtration concentrations are the concentrations of cells in the buffy coat assembly and additive solution, before centrifugation.

Claims (15)

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 µm and 4 µm, and an absolute pore size of between 2 µm and 5 µm. Method according to claim 1, characterized in that said membrane is obtained by ion bombardment followed by etching. Method according to one of claims 1 or 2, characterized in that said membrane is made of polycarbonate or polyester. Method according to any one of claims 1 to 3, characterized in that said membrane has a nominal pore size of 2 µm and an absolute size of 2.5 µm. 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. 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 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. Method according to claim 6, characterized in that the porous leukocyte removal element comprises one or more layers of non-woven fabric. Method according to one of claims 6 or 7, characterized in that the porous leukocyte removal element comprises polybutylene terephthalate or polyethylene terephthalate fibers. 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 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. 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. 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 µm, 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 µm and 4 µm, and an absolute pore size of between 2 µm and 5 µm. 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). 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 of said platelet-concentrated product. 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 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 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 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, via a tube (8), to an outlet orifice of said second filtration unit (22). 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 to 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).