Centrifugation device and method for detecting microparticles in a biological sample

Abstract

The invention relates to a centrifugation method, wherein a solid support (10) is rotated, the solid support (10) containing one or more defined adsorption zones intended to be brought into contact with one or more biological samples, the solid support (10) being tilted during rotation at a predetermined angle α in relation to a horizontal plane, the predetermined angle α being between 55° and 84°.

Description

[0001] Description

[0002] Title: Centrifugation device and method for the detection of microparticles in a biological sample

[0003] technical field

[0004] The present invention relates to the field of in vitro detection and diagnostics, particularly using samples of biological origin. More specifically, the invention relates to a centrifugation device and an adapter, as well as a centrifugation method, for the purpose of detecting microparticles, such as cells or cell agglutinations.

[0005] The invention can be applied in particular to the detection of antigenic markers present on the surface of cells and / or antibodies present in biological samples (plasma, serum, supernatants, etc.). It can notably be applied, in a transfusion context, for the determination of blood groups and / or the detection of anti-erythrocyte antibodies.

[0006] The invention can also be used for the determination of cellular phenotypes such as platelet or lymphocyte groups.

[0007] It can also be used for the detection of antigens and antibodies of all kinds.

[0008] Previous technique

[0009] US patent 8,852,958 describes a method for detecting a specific complex formed by the reaction between an antibody in a solution and an antigen bound to a magnetic particle, with the application of a magnetic field. This method does not promote antigen / antibody interaction.

[0010] There is a need to facilitate in vitro detection and diagnosis, particularly using samples of biological origin. Specifically, there is a need to promote contact between microparticles, such as cells or agglutinates in a solution, and the surface of a solid support, such as the bottom of a well, particularly in a microplate.

[0011] There is therefore a need to develop new centrifugation devices and processes for the detection of microparticles in biological samples that remain applicable in clinical settings, blood banks, and medical analysis laboratories. There also remains a need to develop centrifugation devices and processes for the detection of microparticles in biological samples that are adaptable to automated high-throughput techniques, and more specifically, centrifugation devices and processes for the detection of microparticles in biological samples that allow for the multiplex detection of erythrocyte antigens and / or anti-erythrocyte antibodies.

[0012] There also remains a need to develop centrifugation devices and processes for the detection of microparticles in a biological sample that are acceptable from a regulatory point of view in medical biology.

[0013] The invention aims to meet all or part of these needs.

[0014] Description of the invention

[0015] Centrifugation process

[0016] The invention thus relates, according to a first of its aspects, to a method of centrifuging at least one biological sample, in which a solid support is rotated, the solid support containing one or more delimited adsorption zones intended to be brought into contact with one or more biological samples, the solid support being inclined during rotation at a predetermined angle a relative to a horizontal plane, the predetermined angle a being between 55° and 84°.

[0017] In one embodiment, the solid support is inclined at a predetermined angle α with respect to a horizontal plane only when driven in rotation by the rotor. The solid support may not be inclined at a predetermined angle α with respect to a horizontal plane when not driven in rotation by the rotor.

[0018] In one embodiment, the solid support is inclined at a predetermined angle α with respect to a horizontal plane, with or without rotational drive from the rotor. The solid support can be inclined at a predetermined angle α with respect to a horizontal plane even in the absence of rotational drive from the rotor.

[0019] In the invention, each solid support can advantageously have the same angle.

[0020] Choosing the specific tilt angle allows for better interaction between components of the biological sample, such as red blood cells, and the bottom surface of the solid support. Centrifugation allows particles, such as red blood cells, from the biological sample to slide onto the adsorption zone(s).

[0021] This configuration allows for the detection of specific interactions over non-specific interactions in the results obtained after using the centrifugation device. Sensitivity and specificity of detection are increased.

[0022] The angle of inclination is defined as the angle between a horizontal plane, perpendicular to a centrifugal rotation axis, and a general plane of the solid support. A general plane of the solid support is a plane in which the absorption zones are located.

[0023] The predetermined angle can be between 58° and 81°, or even between 60° and 75°, or even between 63° and 68°, being notably in the order of 64° or 65°.

[0024] The centrifugation speed can be between 130 g and 3000 g, or even between 500 g and 2000 g, or even between 530 g and 1500 g, being for example in the order of 500 g to 1000 g.

[0025] The centrifugation time can be longer than 30 seconds, or even longer than 1 minute, or longer than 2 minutes. The centrifugation time can be between 30 seconds and 10 minutes, or even between 1 minute and 7 minutes, or even between 1.5 minutes and 5 minutes, for example, on the order of 2 to 4 minutes.

[0026] In one embodiment, the centrifugation process is carried out at a speed of 1000 g, for a minimum duration of 1 minute, and with an angle of inclination between 58° and 68°.

[0027] In one embodiment, the centrifugation process is carried out at a speed between 500 g and 1000 g, for a minimum duration of 1 minute, and with an angle of inclination between 58° and 81°.

[0028] In one embodiment, the centrifugation process is carried out at a speed between 500 g and 2000 g, for a minimum duration of 2 minutes, and with an angle of inclination between 58° and 84°.

[0029] In one embodiment, a centrifugation method for the solid support particularly suitable for detecting microparticles is, for example, centrifugation at a speed of approximately 530 g for a duration of approximately 2 minutes, and with an angle of inclination of approximately 65°. The centrifugation method can be used in the following applications, although this list is not exhaustive: hemagglutination, detection and identification of cells such as red blood cells, detection and identification of microorganisms, viral particles, or artificial particles.

[0030] in vitro detection method

[0031] The centrifugation process can notably be used in an in vitro process for detecting antigens carried by a microparticle in a sample, such as an erythrocyte antigen.

[0032] The invention thus relates, independently or in combination with the foregoing, to an in vitro method for detecting antigens carried by microparticles, in particular erythrocytes, in a sample, comprising at least one centrifugation step carried out using the centrifugation method as described above.

[0033] The centrifugation process can notably be used in an in vitro process for detecting anti-erythrocyte antibodies in a sample, as described for example in patent application WO 2021 / 156391A1.

[0034] The invention further relates, independently or in combination with the above, to an in vitro method for detecting antibodies, in particular anti-erythrocyte antibodies, in a sample, comprising at least one centrifugation step carried out using the centrifugation method as described above.

[0035] Implementing the centrifugation step can accelerate and / or improve the detection process, and in particular eliminate an incubation step.

[0036] In one embodiment, the centrifugation step is preceded by a sedimentation step, which may in particular have a duration of at least 30 minutes.

[0037] The microparticles of the biological sample, in particular the erythrocytes, can be diluted with a dilution concentration of between 0.1% and 0.8%, or even between 0.2% and 0.7%, or even between 0.3% and 0.6%, being in particular about 0.5%, by volume.

[0038] The biological sample, particularly plasma, can be diluted to a concentration of less than 60%, or even less than 50%, preferably less than 40%, or even less than 30%, by volume. In this invention, the inventors propose to take advantage of the fact that the solid support containing the delimited adsorption zones, for example, composed of antibodies, can be used as a microparticle capture system. Indeed, the capture of microparticles on the antibody deposits is enhanced by centrifugation. More specifically, the duration, speed, and angle of inclination of the solid support during centrifugation positively influence the intensity of microparticle capture on the spots of the biochip.

[0039] Knowing the nature of the antibodies deposited, particularly monoclonal antibodies, on the solid support, as well as their specificity towards the antigens of interest, the inventors were able to determine the nature of the antigens present on the surface of the tested cells and therefore the cellular phenotype initially present in the tested sample.

[0040] This technology can be advantageously implemented in blood typing tests such as ABO / RhD typing, phenotyping, and extended phenotyping, as well as in the detection of regular anti-erythrocyte antibodies, such as anti-ABO antibodies, or irregular antibodies. This application is particularly relevant for performing the indirect plasma test, also known as the Simonin test, and for conducting an Irregular Agglutinin Screen (IAS).

[0041] This method offers reliability at least comparable to gel column tests usually implemented in immunohematology, while remaining compatible with multiplex applications.

[0042] Such multiplex applications may include or consist of, among other things, the detection of:

[0043] - of several erythrocyte antigens on the surfaces of red blood cells in a sample;

[0044] - of several erythrocyte antigens on the surfaces of red blood cells in several samples;

[0045] - of several types of anti-erythrocyte antibodies in a sample;

[0046] - of several types of anti-erythrocyte antibodies in several samples.

[0047] Advantageously, these applications can therefore allow the simultaneous detection of several parameters, in one or more samples.

[0048] Thus, the present invention relates to an in vitro method for detecting microparticles in a biological sample, which may include the following steps: a) bringing said sample into contact with a solid support containing one or more delimited adsorption zones having previously fixed antibodies or antibody fragments capable of binding antigenic determinants present on said microparticles; b) carrying out centrifugation according to defined parameters of time, speed and angle of inclination of the solid support; c) determining the presence or absence of a capture on at least one of said adsorption zones, so as to detect the presence or absence of microparticles.

[0049] In particular, the in vitro detection method according to the invention can be characterized in that said erythrocyte antigens to be detected are defined among the following systems or antigens: ABO, Rhesus, Kell, MNS, P, Lutheran, Lewis, Duffy, Kidd, Diego, Cartwright, Xg, Scianna, Dombrock, Colton, Landsteiner-Wiener, Chido / Rodgers, Hh, Kx, Gerbich, Cromer, Knops, Indian, OK, RAPH, John Milton Hagen, Li, Globoside, GIL, Rh-associated glycoprotein, Forssmann, JR, LAN, VEL, CD59.

[0050] In particular, the in vitro detection method according to the invention can be characterized in that said antibodies or antibody fragments in the adsorption zone are monoclonal or polyclonal antibodies or lectins selected from: anti-A antibodies (ABOI), anti-B antibodies (ABO2), anti-AB antibodies (ABO3), anti-H lectins (H1), anti-Al lectins, anti-D antibodies (RH1), anti-C antibodies (RH2), anti-c antibodies (RH4), anti-E antibodies (RH3), anti-e antibodies (RH5), anti-K antibodies (KEL1), anti-k antibodies (KEL2), anti-Kpb antibodies (KEL4), anti-Fya antibodies (FY1), anti-Fyb antibodies (FY2), anti-Jka antibodies (JK1), anti-Jkb antibodies (JK2), anti-S antibodies (MNS3), anti-s antibodies (MNS4), anti-Lea antibodies (LE1), anti-Leb antibodies (LE2), anti-M antibodies (MNS1), anti-N antibodies (MNS2), anti-PI antibodies, anti-Kpa antibodies (KEL3), anti-Lua antibodies (LUI),anti-Lub (LU2) antibodies, anti-Cw (RH8) antibodies.

[0051] In particular, the in vitro detection method according to the invention can be characterized in that the sample is a biological sample selected from: a human serum, a human plasma, a human whole blood sample, a human cell suspension, an animal biological sample, in particular an animal serum, an animal plasma, an animal whole blood sample, an animal cell suspension.

[0052] In particular, the in vitro detection method according to the invention can be characterized in that at least two of the adsorption zones of the solid support are spaced at least 100 pm apart, and / or that said adsorption zones define an area of ​​at least 10000 pm2.

[0053] In particular, the in vitro detection method according to the invention can be characterized in that said solid support is a plastic support, and said adsorption zones are hydrophilic.

[0054] Centrifugation device

[0055] The invention also relates, independently or in combination with the foregoing, to a centrifugation device for implementing a centrifugation process, particularly as defined above. The centrifugation device may, in particular, include the characteristics mentioned above. It may be used for the centrifugation of at least one biological sample.

[0056] The invention thus relates, independently or in combination with the foregoing, to a device for centrifuging at least one biological sample, in particular for implementing a process as described above, comprising:

[0057] - a rotating drive rotor,

[0058] - a receiving basket, fixed to the rotor for rotation,

[0059] - at least one solid support carried by the receiving basket, the solid support containing one or more delimited adsorption zones intended to be brought into contact with one or more biological samples, the device being configured so that, at least when the basket is driven in rotation by the rotor, the solid support is inclined at a predetermined angle a relative to a horizontal plane, the predetermined angle a being between 55° and 84°.

[0060] The basket may have a fixed inclination relative to the rotor. Its inclination may not be variable. The basket may have a fixed inclination relative to the horizontal plane.

[0061] In one embodiment, the solid support and the receiving basket are a single piece.

[0062] In one embodiment, the device can be configured so that the solid support is inclined at a predetermined angle α with respect to a horizontal plane, but only when the basket is rotated by the rotor. The device can also be configured so that the solid support is not inclined at a predetermined angle α with respect to a horizontal plane when the basket is not rotated by the rotor.

[0063] In one embodiment, the device can be configured so that the solid support is inclined at a predetermined angle α with respect to a horizontal plane, with or without the basket being rotated by the rotor. The device can be configured so that the solid support is inclined at a predetermined angle α with respect to a horizontal plane even in the absence of the basket being rotated by the rotor.

[0064] The centrifugation device can be configured to accommodate a plurality of solid supports, for example between 2 and 48 solid supports, in particular between 8 and 36 solid supports, or even between 10 and 24 solid supports, or even between 12 and 20 solid supports, for example about 12 solid supports.

[0065] In the invention, each solid support can advantageously have the same angle.

[0066] Choosing the specific angle of inclination allows for good interaction between the biological sample, for example red blood cells, and the bottom surface of the solid support.

[0067] This configuration allows for the highlighting of specific interactions over non-specific interactions in the results obtained after using the centrifugation device. The relevance of the measurements is improved, and their sensitivity and specificity are increased.

[0068] The predetermined angle can be between 58° and 81°, or even between 60° and 75°, or even between 63° and 68°, being notably in the order of 64° or 65°.

[0069] The device may include at least one locking lug for the solid support in an inclined position when the basket is driven in rotation by the rotor.

[0070] The locking lug can be placed on the receiving basket.

[0071] Alternatively, the locking lug can be positioned on the solid support.

[0072] The locking lug can be configured so as to allow the solid support to be held in an inclined position with the predetermined angle a.

[0073] The locking lug can bear against the centrifuge rotor. The angle of inclination can depend on the position of the locking lug, particularly its distance from the axis of rotation or from an edge of the basket. The basket may have two locking lugs.

[0074] Alternatively, the locking lug can be placed on an adapter that receives the solid support.

[0075] Adapter

[0076] Regardless of its form, the solid support can be placed directly in the basket or integrated into an adapter which is itself inserted into the basket, as described below.

[0077] In one embodiment, the solid support can be a single unit with the basket. This configuration simplifies the design of the centrifugation device. The basket may include a stop to limit its tilting when stationary.

[0078] Alternatively, the solid support is attached to the basket. It can be placed directly inside the basket.

[0079] Alternatively, the solid support can be inserted into an adapter which is itself attached to the basket. The adapter can be configured to tilt relative to the rotor when the basket is rotated by the rotor.

[0080] This configuration allows the tilt to occur only during rotation. Therefore, there is no tilt when stationary, which is advantageous for preventing the biological sample(s) from sinking.

[0081] The invention also relates, independently or in combination with the above, to an adapter for a centrifugation device, in particular as defined above.

[0082] The adapter may include the features mentioned above. The adapter may be attached to a basket of the centrifugation device.

[0083] The invention relates in particular, independently or in combination with the above, to an adapter for a centrifugation device, comprising a locking lug in an inclined position relative to a rotor of the centrifugation device, in particular of the centrifugation device as described above.

[0084] The locking lug can be configured to allow the adapter to be held in an inclined position with the predetermined angle a.

[0085] The locking lug can cooperate with the receiving basket by bearing against the centrifuge rotor. The angle of inclination can depend on the position of the locking lug, in particular its distance from the axis of rotation or its distance from an edge of the adapter.

[0086] The adapter may have two locking lugs.

[0087] The adapter may include a housing to receive at least one solid support containing one or more delimited adsorption zones intended to be brought into contact with one or more biological samples, in particular to receive a strip of several wells or a single well.

[0088] The solid support can be fixed in the adapter housing, so as to prevent its ejection during centrifugation.

[0089] The dwelling can be delimited by two parallel walls, extending vertically. The two walls can be separated by a gap of between 3 and 30 mm, or even between 4 and 25 mm, or even between 5 and 20 mm, or better yet between 6 and 15 mm, with a gap of approximately 9 mm.

[0090] The housing may also be defined by a bottom wall, as well as by at least one side wall, including a single side wall. The side wall may, in particular, connect two parallel walls. The housing may, in particular, lack a second side wall. Instead, it may include an opening for inserting the solid support.

[0091] A locking lug can be placed on one of the two parallel walls. The adapter can have two locking lugs, one on each of the two parallel walls.

[0092] The adapter may include a retaining portion within the receiving basket. This retaining portion may be flattened, extending, for example, along an elongation plane. The elongation plane may, for example, be perpendicular to a horizontal plane when the adapter is in place within the centrifugation device. The elongation plane may also extend vertically.

[0093] A locking lug can be placed on the retaining portion. The adapter may have two locking lugs on the retaining portion.

[0094] The retaining portion may include a curved notch or a keying feature, which can be configured to ensure correct positioning of the adapter and its rotation for centrifugation. The retaining portion may include one or more, including two, ports for assembly with the basket. These ports can be configured to cooperate with one or more studs on the receiving basket.

[0095] The receiving basket of the centrifuge can be configured to receive the holding portion. It may include a slot extending in a vertical plane for this purpose. The receiving basket may also have a single slot to receive a single adapter.

[0096] In one embodiment, the receiving basket of the device may have one or more slots for receiving one or more adapters. Each slot can receive one adapter. The receiving basket may have between 1 and 4 slots, and preferably 1 slot.

[0097] The centrifugation device can include between 2 and 48 adapters, in particular between 4 and 16 adapters, or even between 6 and 24 adapters, for example about 12 adapters.

[0098] The adapter can be made of a plastic or metallic material, for example polyethylene.

[0099] Each adapter can carry a recognition code, for example a QR code. The recognition code can be received on a tab of the adapter.

[0100] Solid support

[0101] The sturdy support can be placed directly in the basket or integrated into an adapter which is itself inserted into the basket.

[0102] The solid support can be in the form of a multi-well array or a single well. The solid support can include a plurality of wells, notably between 1 and 16 wells, or even between 4 and 12 wells, for example 8 wells.

[0103] Alternatively, the solid support can be without a well.

[0104] In one embodiment, the solid support is free of tubes. In another example embodiment, the solid support is free of plates, particularly microplates.

[0105] Alternatively, the solid support can be compatible with a gel card centrifuge.

[0106] A solid support particularly suitable for the invention may be a biochip; a biochip being a surface capable of containing one or more delimited adsorption zones, said adsorption zones being coated, for example, with lectins, antibodies, or antibody fragments, capable of binding, for example, antigenic determinants, cells, or microparticles coated with antigenic determinants. A biochip is a support capable of detecting one or more antigenic determinants, particularly erythrocyte determinants, for example, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, or more than fifteen, on one or more samples, simultaneously or not, during a multiplex analysis process.

[0107] A solid support can be in the form of a plate, microplate, slide, membrane, or a multi-well or single-well strip. Preferably, such a solid support can be in the form of a microplate or a multi-well or single-well strip.

[0108] A solid support can be made of any material suitable for carrying out the analysis method, such as a plastic or non-plastic support. Such a solid support is, for example, a support based on a polymer or a mixture of polymers. A suitable solid support according to the invention is, for example, a support made of polystyrene, polypropylene, poly(meth)acrylate, polybutadiene, polycarbonate, or combinations thereof. Preferably, a suitable solid support is polystyrene-based, such as a microplate, whether breakable or not, and with or without high adhesion.

[0109] Another type of suitable solid support according to the invention is, for example, an inorganic support, such as glass and / or silicon, and / or a metallic support.

[0110] Another example of a suitable solid support is a membrane, for example a nitrocellulose, PVDF (Polyvinylidene Fluoride), nylon membrane or combinations thereof.

[0111] In one embodiment, a solid support comprises a single compartment. This single compartment may be a compartment comprising one or more walls. An example of such a solid support comprising a single compartment, with or without one or more walls, is a blade or a membrane.

[0112] In a particular embodiment of the invention where a solid support (for example a slide or a membrane) comprises a single compartment, typically at least one (for example one or two) solid support is used per sample to be analyzed.

[0113] According to one embodiment, a solid support comprises a plurality of compartments. When a solid support comprises at least two compartments, said compartments are isolated from each other, so that they do not communicate with each other, or only in a limited way, i.e. such that the different compositions or solutions used for the analysis cannot circulate from one compartment to another during the analysis, or at least not freely.

[0114] Thus, according to one embodiment of the solid support, a solution (for example, a sample) added to one compartment does not, or only to a limited extent, pass into the other compartments. For example, the compartment(s) comprise or are made up of a base and one or more walls, the wall(s) isolating the compartment(s) from each other so that they do not communicate with one another.

[0115] An example of a compartment is a well. A solid support includes, for example, a plurality of wells, for example, a set of at least 2 wells, in particular between 2 and 16 wells, or even between 4 and 12 wells, for example 8 wells.

[0116] A solid support is, for example, a microplate. The microplate can be a 24, 48, or 96-well microplate, for example.

[0117] A suitable solid support for the process includes one or more defined adsorption zones. These defined adsorption zones may include, or even consist of, spots.

[0118] A delimited adsorption zone is not necessarily of a single defined shape. As such, a delimited adsorption zone may include one or more spots, spaced regularly or not, and comprising at least one antibody or antibody fragments or lectins capable of binding antigenic determinants of erythrocytes, bound to the surface of said support, and / or where applicable of said compartment, in particular by non-covalent physico-chemical interactions (in particular of the weak bond type, for example, ionic, van der Waals, hydrogen and / or hydrophobic) and / or by covalent bonds.

[0119] Non-exhaustively, the delimited adsorption zones of the solid support, in particular the deposits of antibodies or antibody fragments, can be obtained manually or by any device, in particular any device suitable for the preparation of microarrays, such as a contact or non-contact device, in particular any device enabling the deposit of volumes less than one microliter such as piezoelectric or solenoid valve systems.The term "spot" here refers to a delimited adsorption zone, or part of a delimited adsorption zone of the solid support, for example of a compartment of the solid support, comprising at least one compound of interest bound to the surface of said solid support (or, where applicable, of said compartment), in particular by non-covalent physicochemical interactions (in particular of the weak bond type, for example, ionic, van der Waals, hydrogen and / or hydrophobic) and / or by covalent bonds, generally obtained by the deposition of at least one drop of a solution containing a determined quantity of said compound(s) of interest at a precise location on the surface of said support and / or said compartment.

[0120] A spot can be discoidal, cylindrical, or approximately discoidal or cylindrical in shape, for example, oval, particularly when the support is a microplate or slide. Alternatively, a spot can be square or rectangular (it may be, in particular, a band), for example, when the support is a membrane, or any other shape.

[0121] A solid support can thus include at least two, or even three, delimited adsorption zones, for example three zones, four zones or five zones, or at least six zones.

[0122] A solid support can therefore include at least three spots, for example three spots, four spots or five spots, or at least six spots.

[0123] The presence or absence of capture in at least one of the adsorption zones can be directly observed if the microparticles to be captured are colored. In particular, the capture of human red blood cells results in the visualization of a red color in the adsorption zone.

[0124] Brief description of the drawings

[0125] The invention will be better understood upon reading the detailed description that follows, the non-limiting examples of its implementation, and upon examination of the attached drawing in which:

[0126] [Fig la] Figure la is a schematic and partial top view of a centrifugation device according to the invention.

[0127] [Fig 1b] Figure 1b is a schematic and partial side view of the centrifugation device of Figure 1a. [Fig 2a] Figure 2a is a schematic and partial top view of the rotating centrifugation device of Figure 1a.

[0128] [Fig 2b] Figure 2b is a schematic and partial side view of the centrifugal device of figure 1a in rotation.

[0129] [Fig 3a] Figure 3a is a perspective view of a solid support used with the centrifugation device of figure 1a.

[0130] [Fig 3b] Figure 3b is a perspective view of a receiving basket of the centrifugation device of figure 1a.

[0131] [Fig 3c] Figure 3c is a perspective view of an adapter for the centrifugation device of figure 1a.

[0132] [Fig 4a] Figure 4a is a schematic and partial perspective view of an adapter that can be used with the centrifugation device of figure 1a.

[0133] [Fig 4b] Figure 4b is a schematic and partial side view of the adapter in Figure 4a.

[0134] [Fig 4c] Figure 4c is a schematic and partial bottom view of the adapter in Figure 4a.

[0135] [Fig 4d] Figure 4d is a schematic and partial side view of the adapter in Figure 4a.

[0136] [Fig 4e] Figure 4e is a schematic and partial front view of the adapter in Figure 4a.

[0137] [Fig 5] Figure 5 is a schematic and partial view of an alternative embodiment of a centrifugation device.

[0138] [Fig 6] Figure 6 is a schematic and partial side view illustrating the centrifugation device of Figure 5 at rest and in rotation.

[0139] Detailed description

[0140] Figures 1a to 2b illustrate a centrifugation device 1 for implementing the process according to the invention. This process of centrifuging at least one biological sample involves rotating a solid support 10 containing one or more defined adsorption zones intended to be brought into contact with one or more biological samples. The solid support 10 is inclined during rotation at a predetermined angle α with respect to a horizontal plane between 55° and 84°. In the invention, each solid support may advantageously have the same angle. The angle of inclination α is formed between a horizontal plane, perpendicular to an axis of rotation X of the centrifugation, and a general plane Z of the solid support 10, in which the adsorption zones are located.

[0141] In the illustrated example, the centrifugation process is implemented at a speed of approximately 530 g for a duration of approximately 2 minutes, and with an angle of inclination of approximately 65°. Such a centrifugation process for the solid support is particularly suitable for facilitating the detection of microparticles.

[0142] This centrifugation method can be used, in particular, in an in vitro method for detecting antigens carried by a microparticle in a sample, such as an erythrocyte antigen. The in vitro method for detecting anti-erythrocyte antibodies in a sample then includes at least one centrifugation step performed using the centrifugation method described above.

[0143] The biological sample, in particular the erythrocytes, can be diluted beforehand with a dilution concentration of approximately 0.5%.

[0144] We will now describe the centrifugation device 1 in more detail.

[0145] The centrifugation device 1 comprises a rotating drive rotor 2, a receiving basket 5, which is rotationally fixed to the rotor 2, and the solid support carried by the receiving basket 5. The centrifugation device can be configured to accommodate a plurality of solid supports, for example, 12 solid supports. The solid support 10 is integrated into an adapter 20, which is itself inserted into the basket 5. The solid support is in the form of a multi-well bar, in particular 8 wells, as illustrated in Figure 3a.

[0146] The device is configured so that, at least when the basket is rotated by the rotor, the solid support is inclined at a predetermined angle α with respect to a horizontal plane. To this end, the device 1 includes a locking lug 12 for the solid support in the inclined position when the basket 5 is rotated by the rotor 2. The locking lug 12 is located on the receiving basket 5, which receives the solid support 10, as illustrated in Figure 3b.

[0147] In variants not shown, the locking lug could be located on the adapter 20 receiving the solid support 10 or on the solid support itself. The locking lug 12 can bear against the rotor 2 of the device 1. The locking lug 12 is configured to allow the solid support 10 to be held in an inclined position at a predetermined angle α, as shown in Figures 2a and 2b. The angle of inclination may depend on the position of the locking lug, in particular its distance from the axis of rotation or its distance from an edge of the basket or adapter.

[0148] The device also includes, in the example described, a second locking lug 14, intended to lock the solid support in a horizontal position when the basket 5 is not driven in rotation by the rotor 2, as shown in figures 1a and 1b. The second locking lug 14 can be arranged on the receiving basket 5 which receives the solid support 10.

[0149] Thus, the solid support 10 is inserted into the adapter 20, which is itself mounted on the basket 5. The adapter is configured to tilt relative to the rotor 2 when the basket 5 is driven in rotation by the rotor 2. This configuration allows the tilt to occur only during rotation. Consequently, there is no tilt when stationary, which is advantageous for preventing the biological sample(s) from leaking.

[0150] The adapter 20, shown separately in figure 3c, has a housing 24 to receive the solid support 10.

[0151] The dwelling 24 is delimited by two walls 26 parallel to each other, extending vertically.

[0152] Furthermore, the adapter 20 includes a retaining portion 30 in the receiving basket 5. The retaining portion is flattened, extending along a vertical elongation plane perpendicular to a horizontal plane when the adapter 20 is in place in the centrifugation device 1. The retaining portion 30 includes a curved notch or key, which is configured to promote the correct positioning of the adapter and its rotational drive for centrifugation.

[0153] The receiving basket 5 of the centrifugation device 1 is configured to receive the holding portion 30. It may have for this purpose a slot 6 to receive an adapter 20. Each slot 6 of the device 1 can receive an adapter 20.

[0154] We will now describe in more detail another example of an adapter 20 that can be used in a device according to the invention and for implementing a method according to the invention. The adapter 20 illustrated in Figures 4a to 4e also includes a housing 24 for receiving the solid support 10.

[0155] The housing 24 is delimited by two parallel walls 26 extending vertically. The two walls 26 are separated from each other by a gap of, for example, approximately 9 mm.

[0156] The housing 24 is also defined by a bottom wall 27 and a single side wall 28. The side wall 28 connects the two parallel walls 26. The housing 24 lacks a second side wall. Instead, it has an opening 29 for inserting the solid support 10.

[0157] In this example, a locking lug 12 is placed on one of the two parallel walls 26. The adapter 20 can have two locking lugs 12, one on each of the two parallel walls 26.

[0158] Furthermore, the adapter 20 includes a retaining portion 30 in the receiving basket 5. The retaining portion is flattened, extending along a vertical elongation plane perpendicular to a horizontal plane when the adapter 20 is in place in the centrifugation device 1.

[0159] The retaining portion 30 has a curved notch or keying feature, which is configured to promote the correct positioning of the adapter and its rotational drive for centrifugation.

[0160] In addition, the retaining portion 30 has two orifices 32 for assembly with the basket 5. They can be configured to cooperate with one or more studs of the receiving basket.

[0161] The receiving basket 5 of the centrifugation device 1 is configured to receive the holding portion 30. It may have for this purpose a slot 6 to receive an adapter 20. Each slot 6 of the device 1 can receive an adapter 20.

[0162] The centrifugation device in the described example includes 12 adapters.

[0163] The invention is not limited to the examples just described.

[0164] Regardless of its shape, the solid support can be placed directly in the basket or integrated into an adapter which is itself inserted into the basket.

[0165] In one embodiment, the solid support can be integral with the basket. This configuration simplifies the design of the centrifugation device. Alternatively, the solid support is attached to the basket, positioned directly within it without an adapter, as illustrated in Figures 5 and 6.

[0166] In the embodiment described with reference to Figures 1a to 2b, the solid support is inclined at a predetermined angle α with respect to a horizontal plane only when the basket is rotated by the rotor. The solid support is not inclined at a predetermined angle α with respect to a horizontal plane when the basket is not rotated by the rotor.

[0167] In the embodiment illustrated in Figures 5 and 6, the solid support is inclined at a predetermined angle α with respect to a horizontal plane, with or without the basket being rotated by the rotor. In this example, the solid support is inclined at a predetermined angle α with respect to a horizontal plane even when the basket is not rotated by the rotor.

Claims

Demands 1. A method for centrifuging at least one biological sample, in which a solid support (10) is rotated, the solid support (10) containing one or more delimited adsorption zones intended to be brought into contact with one or more biological samples, the solid support (10) being inclined during rotation at a predetermined angle α with respect to a horizontal plane, the predetermined angle α being between 55° and 84°.

2. Method according to the preceding claim, the predetermined angle a being between 58° and 81°, or even between 60° and 75°, or even between 63° and 68°, being in particular of the order of 64° or 65°.

3. A method according to one of the two preceding claims, the centrifugation speed being between 130 g and 3000 g, or even between 500 g and 2000 g, or even between 530 g and 1500 g, being for example in the order of 500 g to 1000 g.

4. A method according to any one of claims 1 to 3, the centrifugation time being between 30 s and 10 minutes, or even between 1 minute and 7 minutes, or even between 1.5 minutes and 5 minutes, being for example in the order of 2 minutes to 4 minutes.

5. In vitro method for detecting antigens carried by microparticles, in particular erythrocytes, in a sample, comprising at least one centrifugation step carried out using the centrifugation method according to any one of the preceding claims.

6. In vitro method for detecting antibodies, in particular anti-erythrocyte antibodies, in a sample, comprising at least one centrifugation step carried out using the centrifugation method according to any one of claims 1 to 4.

7. Centrifugation device (1) for at least one biological sample, for carrying out a process according to any one of the preceding claims, comprising: - a rotating drive rotor (2), - a receiving basket (5), fixed in rotation to the rotor (2), - at least one solid support (10) carried by the receiving basket (5), the solid support (10) containing one or more delimited adsorption zones intended to be brought into contact with one or more biological samples, the device (1) being configured so that, at least when the basket (5) is driven in rotation by the rotor, the solid support (10) is inclined at a predetermined angle α with respect to a horizontal plane, the predetermined angle α being between 55° and 84°.

8. Device according to the preceding claim, the predetermined angle a being between 58° and 81°, or even between 60° and 75°, or even between 63° and 68°, being in particular of the order of 64° or 65°.

9. Device according to one of the two preceding claims, comprising at least one locking lug (12) of the solid support (10) in inclined position when the basket (5) is driven in rotation by the rotor (2).

10. Device according to any one of the three preceding claims, the solid support (5) being in the form of a bar of several wells or a single well.

11. Device according to any one of the four preceding claims, the solid support (5) comprises a plurality of wells, in particular between 1 and 16 wells, or even between 4 and 12 wells, for example 8 wells.

12. Device according to any one of claims 7 to 9, the solid support being compatible with a gel card centrifuge.

13. Device according to any one of claims 1 to 12, the solid support (10) being inserted into an adapter (20) itself attached to the basket (5).

14. Device according to the preceding claim, the adapter (20) being configured to tilt relative to the rotor (2) when the basket (5) is driven in rotation by the rotor (2).

15. Adapter (20) for centrifugation device, comprising a locking lug (12) in an inclined position relative to a rotor (2) of the centrifugation device according to one of the two preceding claims.

16. Adapter (20) according to the preceding claim, comprising a housing (24) for receiving at least one solid support (10) containing one or more delimited adsorption zones intended to be brought into contact with one or more biological samples, in particular for receiving a strip of several wells or a single well.

17. Adapter (20) according to one of the two preceding claims, comprising a retaining portion (30) in the receiving basket (5).

Images